CN108047086A

CN108047086A - Composition containing alpha-non-natural amino acid and polypeptide, be related to the method for alpha-non-natural amino acid and polypeptide with and application thereof

Info

Publication number: CN108047086A
Application number: CN201810031829.6A
Authority: CN
Inventors: 苗振伟; 刘俊杰; 赛·诺曼; 罗素·德瑞弗
Original assignee: Ambrx Inc
Current assignee: Ambrx Inc
Priority date: 2004-12-22
Filing date: 2005-12-21
Publication date: 2018-05-18
Anticipated expiration: 2025-12-21
Also published as: CN108047086B; ZA200704925B; CN101341118A

Abstract

Alpha-non-natural amino acid disclosed herein and polypeptide comprising at least one alpha-non-natural amino acid and the method for preparing these alpha-non-natural amino acids and polypeptide.Alpha-non-natural amino acid (itself or the part as polypeptide) can include a variety of possible functional groups, but usually have at least one oximido, carbonyl, dicarbapentaborane and/or azanol base.The non-natural amino acid polypeptides through further modifying after translating, the method realized the method for these modifications and purify these polypeptides are also disclosed herein.In general, modified non-natural amino acid polypeptides include at least one oximido, carbonyl, dicarbapentaborane and/or azanol base.The method using these natural amino acid polypeptides and modified natural amino acid polypeptide is further disclosed, it includes treatment, diagnosis and other biological technological uses.

Description

Composition containing alpha-non-natural amino acid and polypeptide is related to alpha-non-natural amino acid and more The method of peptide with and application thereof

Division explanation

Present application is the applying date on December 21st, 2005, and Application No. 201510093838.4 is entitled " to contain Have the composition of alpha-non-natural amino acid and polypeptide, be related to the method for alpha-non-natural amino acid and polypeptide with and application thereof " patent Shen Divisional application please, the patent application of Application No. 201510093838.4 are the applyings date on December 21st, 2005, application number For 200580044328.2 (PCT/US2005/046618), the entitled " combination containing alpha-non-natural amino acid and polypeptide The division of the patent application of object, the method for being related to alpha-non-natural amino acid and polypeptide and the purposes of alpha-non-natural amino acid and polypeptide " Application.

The cross reference of related application

Present application advocate United States provisional application the 60/638,418th filed in 22 days December in 2004,2004 years 12 United States provisional application the 60/638,527th filed in months 22 days, United States provisional application filed in 22 days December in 2004 the No. 60/639,195, United States provisional application the 60/696,210th, application on July 1st, 2005 filed in 1 day July in 2005 United States provisional application the 60/696,302nd and on July in 2005 1 filed in United States provisional application the 60/696,068th Number interests, wherein the disclosure of the application case is in being hereby incorporated by reference in their entirety.

Background technology

The ability that nongenetic coded amino acid (that is, " alpha-non-natural amino acid ") is incorporated in protein is allow to introduce Chemical functional group can provide the naturally occurring functional group (ε-NH of such as lysine₂, cysteine sulfydryl-SH, histidine Imino group etc.) valuable substitute.Known some chemical functional groups in 20 kinds of common inherited coded amino acids to existing Functional group to be inert, but with can be combined in the cleaning of the functional group on alpha-non-natural amino acid and effectively react to form stabilization It is bonded.

Nowadays methods availalbe is selectively introduced the chemical functional group being not present in protein, to 20 kinds of common genetics Property coded amino acid in the presence of all functional groups be chemically inert and it can be used for reagent with including some functional groups It effectively and selectively reacts to form stable covalent bond.

The content of the invention

Described herein for manufacture, purifying, characterize and using alpha-non-natural amino acid, non-natural amino acid polypeptides and through repairing Method, composition, technology and the strategy of the non-natural amino acid polypeptides of decorations.On the one hand it is derivative alpha-non-natural amino acid and/or non- Method, composition, technology and the strategy of natural amino acid polypeptide.In one embodiment, these methods, composition, technology and plan Slightly be related to it is chemically derived, be related in other embodiments it is biologically-derived, be related in other embodiments physics derivative, in other realities Apply derivative combination involved in example.In other or Additional examples of composition, these, which derive, has regioselectivity.At other or additionally In embodiment, these, which derive, has regiospecificity.In other or Additional examples of composition, these derive at ambient temperature to be fast Speed.In other or Additional examples of composition, these derivatives occur in aqueous solution.In other or Additional examples of composition, these spread out Life occurs under the pH value between about 4 and about 10.In other or Additional examples of composition, by adding accelerating agent, containing In the reagent and derivative reagent of alpha-non-natural amino acid, these are derived as stoichiometry, close to stoichiometry or class stoichiometry. It is provided in other or Additional examples of composition and allows required group stoichiometry, close to stoichiometry or class by adding accelerating agent The method being incorporated into learning metering on non-natural amino acid polypeptides.It is provided in other or Additional examples of composition by adding accelerating agent The plan allowed required group stoichiometry, be stoichiometrically incorporated into close to stoichiometry or class on non-natural amino acid polypeptides Summary, reaction mixture, synthesis condition.

On the one hand it is for the alpha-non-natural amino acid based on the chemically derived peptide of oxime key and protein.At other or additional implement In example, alpha-non-natural amino acid is incorporated in polypeptide, i.e. these embodiments are non-natural amino acid polypeptides.It is at other or additionally real It applies in example, alpha-non-natural amino acid is functionalized on its side chain so that it is reacted with derived molecules generates oxime key.Other or It is that the non-natural amino acid polypeptides for generating the non-natural amino acid polypeptides containing oxime can be reacted with derived molecules in Additional examples of composition.At it In he or Additional examples of composition, alpha-non-natural amino acid is to be selected from the amino acid with carbonyl, dicarbapentaborane, acetal, azanol or oxime side chain. In other or Additional examples of composition, alpha-non-natural amino acid be selected from have through protection or masked carbonyl, dicarbapentaborane, azanol or The amino acid of oxime side chain.In other or Additional examples of composition, alpha-non-natural amino acid includes the side chain sheltered through oxime.In other or volume In outer embodiment, alpha-non-natural amino acid includes carbonyl or dicarbapentaborane side chain, and wherein carbonyl or dicarbapentaborane is selected from ketone or aldehyde.Another It is the alpha-non-natural amino acid containing the functional group that oxime can be formed after useful appropriate functionalization co-reactant processing in one embodiment. In another or Additional examples of composition, alpha-non-natural amino acid is structurally similar to natural amino acid, but containing in foregoing functional group It is a kind of.In another or other embodiment, alpha-non-natural amino acid is similar to phenylalanine or tyrosine (aromatic amino acid)；And In another embodiment, alpha-non-natural amino acid is similar to alanine and leucine (hydrophobic amino acid).In one embodiment, non-natural Amino acid has the property different from those properties of natural amino acid.In one embodiment, these heterogeneitys are side chain Chemical reactivity, this different chemical reactivity allows the side chain of alpha-non-natural amino acid to undergo reaction in another embodiment, and makees For the unit of polypeptide, the side chain of the naturally occurring Amino Acid Unit even if in same polypeptide does not suffer from previous reaction yet.Another In embodiment, the side chain of alpha-non-natural amino acid has the chemistry orthogonal with the side chain of naturally occurring amino acid.In another embodiment In, the side chain of alpha-non-natural amino acid includes the part containing electrophilic reagent；In another embodiment, the side chain of alpha-non-natural amino acid On the part containing electrophilic reagent can undergo nucleophilic attack to generate protein derived from oxime.The foregoing implementation in this paragraph In any one of example, alpha-non-natural amino acid can exist as independent molecule or may be incorporated into the polypeptide of any length；If The latter, then the polypeptide can further merge naturally occurring or alpha-non-natural amino acid.

On the other hand it is the molecule substituted through azanol for being used to prepare the derivative non-natural amino acid polypeptides based on oxime key. It is for by derived molecules and containing being formed between carbonyl or the non-natural amino acid polypeptides of dicarbapentaborane in another embodiment The molecule substituted through azanol of non-natural amino acid polypeptides of the oxime key derivative containing carbonyl or dicarbapentaborane.In other embodiments, The foregoing non-natural amino acid polypeptides containing carbonyl or dicarbapentaborane are the non-natural amino acid polypeptides containing ketone group.In other or volume In outer embodiment, the alpha-non-natural amino acid containing carbonyl or dicarbapentaborane includes the side chain selected from ketone or aldehyde.It is at other or additionally real It applies in example, the molecule through azanol substitution includes the group selected from following object：Mark；Dyestuff；Polymer；Water-soluble polymer；It is poly- The derivative of ethylene glycol；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactivityization Close object；Resin；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Fat Acid；Carbohydrate；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, life Object material；Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other molecules The group covalently or non-covalently to interact；Light cage covers part；Can actinic radiation excitation part, ligand, can photoisomerization Part；Biotin；Biotin analog；It is combined with the part of heavy atom；The group chemically cracked；Can photodestruciton group； Extended side chain；The bonded sugar of carbon；Redox active agent；Aminothio acid；Toxin part；Part through isotope marks； Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electron dense group；Magnetic group；It is inserted into group；Chromophore；Energy Transfer agent；Bioactivator；Detectable label；Small molecule；Inhibition ribonucleic acid, radioactive nucleotides；Neutron capture agent；It is raw The derivative of object element；Quantum dot；Nanometer transfers element；Radioactivity transfers element；Abzyme, activation composite activating agent, virus, adjuvant, Aglycone, anaphylactogen, angiostatin, antihormones, antioxidant, aptamer, guide RNA, saponin, shuttle vector, Macromolecular intends epitope, receptor, reverse micelle and any combination thereof.In other or Additional examples of composition, substitute through azanol Molecule be through azanol substitute polyethylene glycol (PEG) molecule.In another embodiment, the side chain of alpha-non-natural amino acid have with The orthogonal chemistry of those side chains of naturally occurring amino acid allows alpha-non-natural amino acid and the molecule selectivity through azanol substitution Reaction.In another embodiment, the side chain of alpha-non-natural amino acid is included with the molecule selective reaction containing azanol containing electrophilic The part of sub- reagent；In another embodiment, the part containing electrophilic reagent on the side chain of alpha-non-natural amino acid can undergo parent Nuclear attack is to generate protein derived from oxime.The another aspect related with the embodiment described in this paragraph is by derivative point The modified non-natural amino acid polypeptides that son is generated with non-natural amino acid polypeptides reaction.Other embodiment is included to having modified Any further modification of non-natural amino acid polypeptides.

On the other hand it is to be used to prepare substituting through carbonyl or dicarbapentaborane for the derivative non-natural amino acid polypeptides based on oxime key Molecule.It is to spread out for passing through the oxime exchange reaction between derived molecules and peptide or protein matter containing oxime in another embodiment The molecule substituted through carbonyl or dicarbapentaborane of the raw non-natural amino acid polypeptides containing oxime.In another embodiment for can between In pH value range between about 4 and about 8 experience oxime with the non-natural amino acid polypeptides containing oxime exchange through carbonyl or two carbonyls The molecule of base substitution.It is for by (therefore being reacted in derived molecules with containing oxime by switch type in another embodiment Form new oxime key) or non-natural amino acid polypeptides containing azanol between form oxime key derivative containing oxime or containing the non-of azanol The molecule substituted through carbonyl or dicarbapentaborane of natural amino acid polypeptide.In another embodiment, substitute through carbonyl or dicarbapentaborane Molecule is the molecule substituted through aldehyde.In other embodiments, the molecule substituted through carbonyl or dicarbapentaborane is included selected from following object Group：Mark；Dyestuff；Polymer；Water-soluble polymer；The derivative of polyethylene glycol；Photocrosslinking agent；Cytotoxic compound； Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Resin；Second protein or polypeptide or polypeptide analog； Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbohydrate；Polynucleotide；DNA；RNA；Antisense poly-nuclear Thuja acid；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial；Nano-particle；Spin labeling；Fluorogen, containing metal Part；Radioactive segment；Novel functional group；With other molecule covalents or the group of noncovalent interaction；Light cage covers part； Can actinic radiation excitation part, ligand, can photoisomerization part；Biotin；Biotin analog；It is combined with heavy atom Part；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active agent；Ammonia Base thio-acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electronics Dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；Small molecule；Suppression Property ribonucleic acid processed, radioactive nucleotides；Neutron capture agent；The derivative of biotin；Quantum dot；Nanometer transfers element；Radioactivity Transfer element；Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, antihormones, Antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecular, intend epitope, receptor, reverse micelle and its What is combined.In other or Additional examples of composition, the molecule through aldehyde substitution is polyethylene glycol (PEG) molecule substituted through aldehyde.Another In one embodiment, the side chain of alpha-non-natural amino acid has the chemistry orthogonal with those side chains of naturally occurring amino acid, allows Alpha-non-natural amino acid and the molecule selective reaction substituted through carbonyl or dicarbapentaborane.In another embodiment, alpha-non-natural amino acid Side chain include and the part containing carbonyl or the molecule selective reaction of dicarbapentaborane, such as oximido or azanol base；In another reality It applies in example, the nucleophilic moiety on the side chain of alpha-non-natural amino acid can undergo electrophilic attack to generate protein derived from oxime.With The relevant another aspect of embodiment described in this paragraph is to be generated by derived molecules and non-natural amino acid polypeptides reaction Modified non-natural amino acid polypeptides.Other embodiment is included to having modified any further of non-natural amino acid polypeptides Modification.

On the other hand it is for generating the simple functions of the derivative non-natural amino acid polypeptides based on oxime key, difunctionality and more Function connexon.It is available for non-natural amino acid polypeptides of the connection containing carbonyl or dicarbapentaborane and other points in one embodiment The linker (bifunctional and multifunctional) of son.It is available for non-day of the connection containing oxime or azanol in another embodiment The linker (bifunctional and multifunctional) of right amino acid polypeptide and other molecules.In another embodiment, containing carbonyl Or the non-natural amino acid polypeptides of dicarbapentaborane include ketone and/or aldehyde side chain.Utilizing the alpha-non-natural amino acid containing oxime or azanol In the embodiment of polypeptide, linker contains carbonyl or dicarbapentaborane in one end；In other embodiments, carbonyl or two carbonyls Base is selected from aldehyde radical or ketone group.In other or Additional examples of composition, the connexon molecule through azanol substitution is through azanol substitution Polyethylene glycol (PEG) connexon molecule.In other or Additional examples of composition, the connexon molecule substituted through carbonyl or dicarbapentaborane is Polyethylene glycol (PEG) the connexon molecule substituted through carbonyl or dicarbapentaborane.In other embodiments, phrase " other molecules " includes (only for example) protein, other polymers and small molecule.In other or Additional examples of composition, the connexon containing azanol point Son on all ends include identical or equivalent group, so as to the non-natural amino acid polypeptides containing carbonyl or dicarbapentaborane After reaction, products therefrom is the same poly compound of the non-natural amino acid polypeptides containing carbonyl or dicarbapentaborane.In other embodiment In, turn to homodimerization with poly.In other or Additional examples of composition, the linker containing carbonyl or dicarbapentaborane is all On end include identical or equivalent group, so as to containing oxime or azanol non-natural amino acid polypeptides reaction after, gained Product is the same poly compound of the non-natural amino acid polypeptides containing oxime or azanol.In other embodiments, turned to together with poly Dimerization.In another embodiment, the side chain of alpha-non-natural amino acid has orthogonal with those side chains of naturally occurring amino acid Chemistry allows alpha-non-natural amino acid and the connexon molecule selective reaction substituted through azanol.In another embodiment, non-day The side chain of right amino acid has the chemistry orthogonal with those side chains of naturally occurring amino acid, allows alpha-non-natural amino acid and warp Carbonyl or the connexon molecule selective reaction of dicarbapentaborane substitution.In another embodiment, the side chain of alpha-non-natural amino acid includes With the part containing electrophilic reagent of the connexon molecule selective reaction containing azanol；In another embodiment, non-natural ammonia The part containing electrophilic reagent on the side chain of base acid can undergo the nucleophilic attack of the connexon molecule containing azanol to generate oxime Derivative protein.It is by connexon molecule and non-natural ammonia with the relevant another aspect of embodiment described in this paragraph Bonded " through modification or unmodified " non-natural amino acid polypeptides that base acid polypeptides reactive generates.Other embodiment include pair Any further modification of bonded " through modification or unmodified " non-natural amino acid polypeptides.

On the one hand it is that oximido product is generated come derived protein by the condensation of carbonyl or dicarbapentaborane and azanol reaction object Method.It includes in this aspect and is generated for being condensed based on the reactant containing carbonyl or dicarbapentaborane with the reactant containing azanol The method of protein adduct derived protein derived from oxime.It is with functionalized poly- through azanol in additional or other embodiment The method of ethylene glycol (PEG) molecule derivative protein containing ketone.It is additional or other in terms of in be by containing carbonyl or dicarbapentaborane Derived molecules and peptide or protein matter containing oxime between oxime exchange reaction derivative protein containing oxime method.It is additional or its In his aspect, the molecule substituted through azanol can include protein, other polymers and small molecule.

On the other hand the method for the molecule through azanol substitution of the derivative protein through ketone substitution is used for for chemical synthesis.Separately On the one hand the method for the molecule through azanol substitution of the derivative protein through aldehyde substitution is used for for chemical synthesis.In an embodiment In, the molecule substituted through azanol may include peptide, other polymers (non-branch and branch) and small molecule.It is in one embodiment system It is standby that suitable for the derivative non-natural amino acid polypeptides containing carbonyl or dicarbapentaborane, (only for example, it includes the non-natural ammonia containing ketone Base acid polypeptide) through azanol substitution molecule method.In another or Additional examples of composition, alpha-non-natural amino acid is in protein In vivo site is specifically incorporated between translation phase.In another or Additional examples of composition, the molecule through azanol substitution by carbonyl or The nucleophilic attack of dicarbapentaborane allows the locus specificity derivative that this contains the alpha-non-natural amino acid of carbonyl or dicarbapentaborane with site Specificity pattern forms polypeptide derived from oxime.In another or Additional examples of composition, the method for preparing the molecule through azanol substitution carries Derive the method for polypeptide for obtaining a variety of locus specificities.It is functionalized poly- through azanol for synthesis in another or Additional examples of composition The method of ethylene glycol (PEG) molecule.

Another aspect is for chemical synthesis for the derivative non-natural amino acid polypeptides through oxime substitution through carbonyl or dicarbapentaborane The method of substituted molecule.In one embodiment, the molecule substituted through carbonyl or dicarbapentaborane is the molecule substituted through ketone.It is real one It applies in example, the molecule substituted through carbonyl or dicarbapentaborane is the molecule substituted through aldehyde.In another embodiment, through carbonyl or dicarbapentaborane Substituted molecule includes protein, polymer (non-branch and branch) and small molecule.In another or Additional examples of composition, these sides Method supplement can between the in vivo translation phase of protein site specific incorporation of non-natural amino acids technology.In another or volume It is suitable for reacting to provide non-natural ammonia derived from locus specificity with non-natural amino acid polypeptides containing oxime to prepare in outer embodiment The conventional method of the molecule substituted through carbonyl or dicarbapentaborane of base acid polypeptide.It is to synthesize through carbonyl in another or Additional examples of composition Or the method for polyethylene glycol (PEG) molecule of dicarbapentaborane substitution.

On the other hand it is using the chemically derived non-day substituted through carbonyl or dicarbapentaborane of the bifunctional linker containing azanol The method of right amino acid polypeptide.It is that oxime key is generated by condensation reaction to connect the connexon substituted through azanol in one embodiment With the method for the protein through carbonyl or dicarbapentaborane substitution.In other or Additional examples of composition, substitute through carbonyl or dicarbapentaborane Alpha-non-natural amino acid is the alpha-non-natural amino acid substituted through ketone.In other or Additional examples of composition, double officials containing azanol are used Energy connexon, derivative and/or three-dimensional structure is precisely controlledly derivative through locus specificity for non-natural amino acid polypeptides. In one embodiment, these methods are used for linker (including (but not limited to) simple function, difunctionality and multifunctional connection Son) it is connected on the non-natural amino acid polypeptides containing carbonyl or dicarbapentaborane (including (but not limited to) containing ketone and containing aldehyde), wherein Azanol base is contained at least one connexon end, and the alpha-non-natural amino acid containing carbonyl or dicarbapentaborane can be bonded to by oxime key On polypeptide.In another or Additional examples of composition, these connexons are for alpha-non-natural amino acid of the connection containing carbonyl or dicarbapentaborane Polypeptide and other molecules, it includes (for example) protein, other polymers (branch and non-branch) and small molecules.

In some embodiments, non-natural amino acid polypeptides are bonded on water-soluble polymer.In some embodiments, water Soluble polymer includes polyalkylene glycol moiety.In some embodiments, peg molecule is double functional copolymer.In some realities It applies in example, double functional copolymer is bonded on the second polypeptide.In some embodiments, the second polypeptide is identical with the first polypeptide, In other embodiment, the second polypeptide is different polypeptide.In some embodiments, non-natural amino acid polypeptides include at least two The amino acid being bonded on water-soluble polymer (including polyalkylene glycol moiety).

In some embodiments, non-natural amino acid polypeptides include affinity of the increase non-natural amino acid polypeptides to receptor Substitution, addition or missing.In some embodiments, non-natural amino acid polypeptides include the steady of increase non-natural amino acid polypeptides Qualitatively substitute, add or lack.In some embodiments, non-natural amino acid polypeptides include increase non-natural amino acid polypeptides Water-soluble substitution, addition or missing.In some embodiments, non-natural amino acid polypeptides include non-day caused by increase Right deliquescent substitution, addition or missing of the amino acid polypeptide in host cell.In some embodiments, compared with without The amino acid polypeptide of substitution, addition or missing, non-natural amino acid polypeptides include regulatory protein enzyme resistance, serum half-life, exempt from The substitution of epidemic focus and/or expression, addition or missing.

In some embodiments, non-natural amino acid polypeptides are agonist, partial agonist, antagonist, partial antagonist Or reversed agonist.In some embodiments, agonist, partial agonist, antagonist, partial antagonist or reversed agonist bag Include the alpha-non-natural amino acid bonded with water-soluble polymer.In some embodiments, water-soluble polymer includes polyethylene glycol portion Point.In some embodiments, can (for example) be prevented including the polypeptide to alpha-non-natural amino acid that water-soluble polymer is bonded corresponding The dimerization of receptor.In some embodiments, the polypeptides for modulating including the alpha-non-natural amino acid bonded with water-soluble polymer is more Peptide and the combination for combining collocation object, ligand or receptor.In some embodiments, including the non-day bonded with water-soluble polymer One or more kinds of properties or activity of the polypeptides for modulating polypeptide of right amino acid.

In some embodiments, selection codon is selected from by amber codon (amber codon), ochre codon (ochre codon), opal codon (opal codon), unique codon (unique codon), rare codon (rare codon), unnatural codons (unnatural codon), five base codons (five-base codon) and The group of four base codons (four-base codon) composition.

Manufacture also described herein and the method for the bonded non-natural amino acid polypeptides of water-soluble polymer.In some implementations In example, the described method includes make to include alpha-non-natural amino acid through isolated polypeptide and the part including being reacted with alpha-non-natural amino acid Water-soluble polymer contact.In some embodiments, the alpha-non-natural amino acid pair being incorporated to and appointing in 20 kinds of common amino acids A kind of water-soluble polymer for not having reactivity has reactivity.In some embodiments, aqueous-based polymers include polyethylene glycol Part.The molecular weight of polymer can in broad range, it includes (but not limited to) between about 100Da and about 100,000Da or Scope between higher.The molecular weight of polymer can be between about 100Da and about 100,000Da, and it includes (but not limited to) 100,000Da、95,000Da、90,000Da、85,000Da、80,000Da、75,000Da、70,000Da、65,000Da、60, 000Da、55,000Da、50,000Da、45,000Da、40,000Da、35,000Da、30,000Da、25,000Da、20, 000Da、15,000Da、10,000Da、9,000Da、8,000Da、7,000Da、6,000Da、5,000Da、4,000Da、3, 000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da. In some embodiments, the molecular weight of polymer is between about 100Da and 50,000Da.In some embodiments, polymer Molecular weight is between about 100Da and 40,000Da.In some embodiments, the molecular weight of polymer is in about 1,000Da and 40, Between 000Da.In some embodiments, the molecular weight of polymer is between about 5,000Da and 40,000Da.In some embodiments In, the molecular weight of polymer is between about 10,000Da and 40,000Da.In some embodiments, peg molecule is branch Polymer.The molecular weight of branch PEG can between about 1,000Da and about 100,000Da, it includes (but not limited to) 100, 000Da、95,000Da、90,000Da、85,000Da、80,000Da、75,000Da、70,000Da、65,000Da、60, 000Da、55,000Da、50,000Da、45,000Da、40,000Da、35,000Da、30,000Da、25,000Da、20, 000Da、15,000Da、10,000Da、9,000Da、8,000Da、7,000Da、6,000Da、5,000Da、4,000Da、3, 000Da, 2,000Da and 1,000Da.In some embodiments, the molecular weight of branch PEG about 1,000Da and 50,000Da it Between.In some embodiments, the molecular weight of branch PEG is between about 1,000Da and 40,000Da.In some embodiments, prop up The molecular weight of chain PEG is between about 5,000Da and 40,000Da.In some embodiments, the molecular weight of branch PEG is about 5, Between 000Da and 20,000Da.

It is also described herein including including at least one alpha-non-natural amino acid described herein and pharmaceutically acceptable Supporting agent composition.In some embodiments, alpha-non-natural amino acid is bonded on water-soluble polymer.Bag also described herein The medical composition of pharmaceutically acceptable supporting agent and polypeptide is included, wherein at least one amino acid takes through alpha-non-natural amino acid Generation.In some embodiments, alpha-non-natural amino acid includes sugar moieties.In some embodiments, water-soluble polymer passes through sugared portion It is point bonded on polypeptide.Alpha-non-natural amino acid also described herein, non-natural amino acid polypeptides and modified non-natural amino The prodrug of sour polypeptide；Composition described further herein including these prodrugs and pharmaceutically acceptable supporting agent.Herein In the metabolin of alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides is also described；These Metabolin can have supplement or collaboration to strengthen alpha-non-natural amino acid, non-natural amino acid polypeptides and modified alpha-non-natural amino acid The required activity of the activity of polypeptide.Alpha-non-natural amino acid described herein also described herein, non-natural amino acid polypeptides and Modified non-natural amino acid polypeptides to organism (comprising need this metabolin patient) provide needed for metabolin purposes.

The cell of polynucleotide also described herein including coded polypeptide and including selection codon.In some embodiments In, cell includes substituting into alpha-non-natural amino acid into orthogonal RNA synzyme and/or the orthogonal tRNA in polypeptide.In some realities Apply in example, cell be in cell culture, and in other embodiments for multicellular organism (comprising amphibian, creep it is dynamic Object, birds and mammal) part cell.In any one of cell embodiment, other embodiment, which includes, generates non-day The expression of the polynucleotide of right amino acid polypeptide.It is to be produced using alpha-non-natural amino acid described herein in other embodiments The organism of raw non-natural amino acid polypeptides (including modified non-natural amino acid polypeptides).In other embodiments be containing The biology of alpha-non-natural amino acid described herein, non-natural amino acid polypeptides and/or modified non-natural amino acid polypeptides Body.These organisms include unicellular organism and multicellular organism, and it includes amphibian, reptile, birds and lactations to move Object.In some embodiments, non-natural amino acid polypeptides are in vitro to generate.In some embodiments, alpha-non-natural amino acid Polypeptide is generated in cell lysates.In some embodiments, non-natural amino acid polypeptides are translated by ribosomes and generated.

The method also described herein for manufacturing the polypeptide for including alpha-non-natural amino acid.In some embodiments, the method It is included in and allows to cultivate the poly-nuclear glycosides for including coded polypeptide, orthogonal RNA synzyme and/or orthogonal tRNA under conditions of expression of polypeptides The cell of acid；With the purified polypeptide from cell and/or culture medium.

The storehouse of alpha-non-natural amino acid described herein also described herein or alpha-non-natural amino acid described herein are more The storehouse or its combinatorial libraries of the storehouse of peptide or modified non-natural amino acid polypeptides described herein.It is also described herein containing At least one alpha-non-natural amino acid, at least one non-natural amino acid polypeptides and/or at least one modified non-natural amino The array of acid.The array of polynucleotide also described herein containing at least one coded polypeptide and including selection codon.This Array described herein can be used for the generation of non-natural amino acid polypeptides in screening organism (by the poly-nuclear for detecting coded polypeptide The transcription of thuja acid or the translation by detecting polypeptide).

The method of the required activity in screening also described herein storehouse described herein uses array described herein The method of screening storehouse described herein or other compounds and/or the required activity in the storehouse of polypeptide and/or polynucleotide.Herein In also describe this activity data from storehouse screening for develop and find new therapeutic agent purposes and therapeutic agent in itself.

Treatment half-life period, serum half-life or the method for circulation time of increase polypeptide also described herein.In some realities Apply in example, method include at least one alpha-non-natural amino acid substitute one or more amino acid in naturally occurring polypeptide and/ Or polypeptide is made to be coupled with water-soluble polymer.

The method also described herein that this patient treated is needed with the treatment of a effective amount of medical composition, wherein the doctor Drug composition includes the polypeptide comprising alpha-non-natural amino acid and pharmaceutically acceptable supporting agent.In some embodiments, non-day Right amino acid is coupled with water-soluble polymer.

It is the method for the treatment of illness, symptom or disease in other or alternate embodiment, the described method includes administration treatments A effective amount of non-natural amino acid polypeptides for including at least one alpha-non-natural amino acid, wherein the alpha-non-natural amino acid is to be selected from By containing oxime alpha-non-natural amino acid, alpha-non-natural amino acid containing carbonyl, alpha-non-natural amino acid containing dicarbapentaborane and non-natural amino containing azanol The group of acid composition.In other embodiments, these alpha-non-natural amino acids by biosynthesis be incorporated herein described in polypeptide In.In other or alternate embodiment, these non-natural amino acid polypeptides include at least one selected from Formulas I-XVIII, Formula X XX- The alpha-non-natural amino acid of the amino acid of XXXIV (A and B) or Formula X XXX-XXXXIII.

It is the method for the treatment of illness, symptom or disease in other or alternate embodiment, the described method includes administration treatments A effective amount of non-natural amino acid polypeptides including at least one alpha-non-natural amino acid containing oxime, and compared with homologous naturally occurring ammonia For base acid polypeptide, the biological usability of the enhancing polypeptide of non-natural amino acid polypeptides containing oxime of gained biosynthesis.

It is the method for the treatment of illness, symptom or disease in other or alternate embodiment, the described method includes administration treatments A effective amount of non-natural amino acid polypeptides including at least one alpha-non-natural amino acid containing oxime, and compared with homologous naturally occurring ammonia For base acid polypeptide, the non-natural amino acid polypeptides containing oxime of gained biosynthesis increase the safety profile of polypeptide.

It is the method for the treatment of illness, symptom or disease in other or alternate embodiment, the described method includes administration treatments A effective amount of non-natural amino acid polypeptides including at least one alpha-non-natural amino acid containing oxime, and compared with homologous naturally occurring ammonia For base acid polypeptide, the water solubility of the increase polypeptide of non-natural amino acid polypeptides containing oxime of gained biosynthesis.

It is the method for the treatment of illness, symptom or disease in other or alternate embodiment, the described method includes administration treatments A effective amount of non-natural amino acid polypeptides including at least one alpha-non-natural amino acid containing oxime, and compared with homologous naturally occurring ammonia For base acid polypeptide, the treatment half-life period of the increase polypeptide of non-natural amino acid polypeptides containing oxime of gained biosynthesis.

It is the method for the treatment of illness, symptom or disease in other or alternate embodiment, the described method includes administration treatments A effective amount of non-natural amino acid polypeptides including at least one alpha-non-natural amino acid containing oxime, and compared with homologous naturally occurring ammonia For base acid polypeptide, the serum half-life of the increase polypeptide of non-natural amino acid polypeptides containing oxime of gained biosynthesis.

It is the method for the treatment of illness, symptom or disease in other or alternate embodiment, the described method includes administration treatments A effective amount of non-natural amino acid polypeptides including at least one alpha-non-natural amino acid containing oxime, and compared with homologous naturally occurring ammonia For base acid polypeptide, the non-natural amino acid polypeptides containing oxime of gained biosynthesis extend the circulation time of polypeptide.

It is the method for the treatment of illness, symptom or disease in other or alternate embodiment, the described method includes administration treatments A effective amount of non-natural amino acid polypeptides including at least one alpha-non-natural amino acid containing oxime, and compared with homologous naturally occurring ammonia For base acid polypeptide, the non-natural amino acid polypeptides containing oxime of gained biosynthesis adjust the bioactivity of polypeptide.

It is the method for the treatment of illness, symptom or disease in other or alternate embodiment, the described method includes administration treatments A effective amount of non-natural amino acid polypeptides including at least one alpha-non-natural amino acid containing oxime, and compared with homologous naturally occurring ammonia For base acid polypeptide, the non-natural amino acid polypeptides containing oxime of gained biosynthesis adjust the immunogenicity of polypeptide.

Should be appreciated that method and composition described herein be not limited to ad hoc approach described herein, scheme, cell line, Construct and reagent and can equally it change.It is equally understood that mesh of the term used herein only for description specific embodiment , and the scope of method and composition described herein is not intended to limit, it should only be limited by appended claims.

Unless the context clearly indicates otherwise, otherwise as used in herein and appended claims, singulative " one " and " described " refers to comprising plural number.

Unless otherwise defined, otherwise all technical terms and scientific terminology used herein have as described in this article The identical meanings that those skilled in the art in the invention are usually understood.It is although similar or equivalent with those described herein Any method, apparatus and material can be used in the implementation or test of the present invention described herein, but preferred side will now be described Method, device and material.

All publication and patent mentioned in this article are to describe and disclose constructing described in (for example) publication During the purpose of body and method is hereby incorporated by reference in their entirety, it can make together with the present invention described herein With.Publication discussed herein only provides its disclosure before the date of application of present application.Anything herein It should not be construed as recognizing that present inventor described herein haves no right this exposure ahead of time due to prior inventions or for any other The date of content.

As used herein, the term " affinity marker " refer to it is reversible or irreversibly combined with another molecule with It is modified, is destroyed or the mark of formed compound.For example, affinity marker includes enzyme and its base Matter or antibody and its antigen.

Term " alkoxy ", " alkyl amino " and " alkylthio group " is used with its conventional meaning, and refers to leading to respectively Cross the bonded alkyl on molecule of oxygen atom, amino, sulphur atom.

Unless otherwise noted, otherwise term " alkyl " (itself or the part as another molecule) means linear chain or branch chain, Or cyclic hydrocarbon group or its combination, can be fully saturated, single insatiable hunger and/or how unsaturated and can include to have and specify carbon atom The bivalent group and multivalence group of number (i.e. C1-C10 means 1 to 10 carbon).The example of saturated hydrocarbyl including (but not limited to) Following group, such as methyl, ethyl, n-propyl, isopropyl, normal-butyl, tertiary butyl, isobutyl group, sec-butyl, cyclohexyl, (hexamethylene Base) methyl, Cvclopropvlmethvl, the (for example) homologue of n-pentyl, n-hexyl, n-heptyl, n-octyl and its similar group and different Structure body.Unsaturated alkyl is the alkyl with one or more double or triple bonds.The example of unsaturated alkyl include (but Be not limited to) vinyl, 2- acrylic, crotyl, 2- isopentene groups, 2- (butadienyl), 2,4- pentadienyls, 3- (1,4- penta Dialkylene), acetenyl, the homologue and isomers of 1- propinyls and 3- propinyls, 3- butynyls and higher level.Unless in addition It points out, otherwise term " alkyl " is also meaned comprising those derivatives of alkyl defined in more detail herein, such as " miscellaneous alkyl ", " alkylhalide group " and " high alkyl ".

Term " alkylidene " (itself or the part as another molecule) means the bivalent group as derived from alkane, such as by (- CH₂-) n illustrations, wherein n can be 1 to about 24.Only for example, these groups are including (but not limited to) with 10 or less carbon The group of atom, such as structure-CH₂CH₂- and-CH₂CH₂CH₂CH₂-." low alkyl group " or " low-grade alkylidene " is usually with 8 The relatively short-chain alkyl or alkylidene of a or less carbon atom.Unless otherwise noted, otherwise term " alkylidene " is also meaned comprising this Described in the text is those groups of " sub- miscellaneous alkyl ".

Term " amino acid " refers to naturally occurring amino acid and alpha-non-natural amino acid and to be similar to naturally occurring ammonia The amino acid analogue and amino acid simulant that the mode of base acid works.Natural coded amino acid is 20 kinds of common amino acids It is (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, different bright Propylhomoserin, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and figured silk fabrics ammonia Acid) and burnt lysine and selenocystein.Amino acid analogue is referred to identical with naturally occurring amino acid basic The compound of chemical constitution (α-carbon only for example, combined with hydrogen, carboxyl, amino and R group).These analogs can have There is modified R group (for example, nor-leucine) or there can be modified peptide backbone, and still retain and naturally occurring ammonia The identical basic chemical structure of base acid.The non-limiting examples of amino acid analogue include homoserine, nor-leucine, first sulphur ammonia Sour oxysulfide, methionine methyl sulfonium.

Amino acid herein can be by its title, its commonly known three letter symbols or by IUPAC-IUB biochemistries The one-letter symbol that naming committee (IUPAC-IUB Biochemical Nomenclature Commission) is recommended claims It exhales.In addition, nucleotide can be called by the single letter code that it usually receives.

" amino terminal modification group " refers to any molecule on terminal amino group.For example, these ends Amino End Group can be in the end of polymerizable molecular, and wherein these polymerizable moleculars are including (but not limited to) polypeptide, polynucleotide and more Sugar.Terminal modifying groups are including (but not limited to) various water-soluble polymers, peptide or protein matter.Only for example, it is end modified Group includes polyethylene glycol or seralbumin.Terminal modifying groups can be used for the treatment feature for changing polymerizable molecular, it includes (but not limited to) increases the serum half-life of peptide.

" antibody fragment " means any form of the antibody in addition to overall length form.Antibody fragment herein is included as depositing It is the antibody of the smaller group point in full length antibody and the antibody through Engineering Design.Antibody fragment including (but not limited to) Fv, Fc, Fab and (Fab') 2, scFv (scFv), double-chain antibody, three chain antibodies, four chain antibodies, difunctional hybrid antibody, Combination, variable region, framework region, constant region, heavy chain, light chain and the variable region of CDR1, CDR2, CDR3, CDR and alternative bone Frame non antibody molecule, bispecific antibody and the like (Maynard and Georgiou, 2000, Annu.Rev.Biomed.Eng.2:339-76；Hudson,1998,Curr.Opin.Biotechnol.9:395-402).It is another Functional substructure is scFv (scFv), including the heavy chain immunoglobulin being covalently attached by peptide connexon and light chain can Become area (S-z Hu et al., 1996, Cancer Research, 56,3055-3061).These small (Mr 25,000) protein lead to It often keeps the specificity and compatibility to the antigen in single polypeptide and appropriate structure can be provided for larger antigentic specificity molecule Build unit.Unless in addition particularly pointing out, otherwise clearly " antibody fragment " is included using the statement of term " antibody " and claim.

As used herein, term " aromatics " or " aryl " are referred to at least one with conjugated pi electron system Ring and closed-loop construct comprising isocyclic aryl Yu heterocyclic aryl (or " heteroaryl " or " heteroaromatic ").Carbocyclic ring or heterocyclic aromatic Group can contain 5 to 20 annular atoms.The term includes covalently bonded monocyclic or condensed polycyclic (that is, shared adjacent carbons The ring of atom pair) group.Aromatic group can be unsubstituted or be substituted.The non-limiting examples bag of " aromatics " or " aryl " group Containing phenyl, 1- naphthalenes, 2- naphthalenes, 4- xenyls, anthryl and phenanthryl.Each of above-mentioned aryl and heteroaryl ring-member take Dai Ji is the group selected from acceptable substituent group described herein.

For simplicity being applied in combination when with other terms (including (but not limited to) aryloxy group, aryl sulphur epoxide, virtue Alkyl) when, term " aromatics " or " aryl " include both aryl rings as defined above and heteroaryl ring.Therefore, term " aralkyl Base " or " alkaryl " mean those groups for being connected to comprising wherein aryl on alkyl, and (it includes (but not limited to) benzyl, benzene second Base, pyridylmethyl and its similar group), wherein the alkyl includes wherein, carbon atom (including (but not limited to) methylene) has been Those alkyl substituted by hetero atom (only for example, oxygen atom).The example of these aryl is including (but not limited to) phenoxy group Methyl, 2- pyridyloxymethyls, 3- (1- naphthalenes oxygroup) propyl and its similar group.

As used herein, term " arlydene " refers to divalent aryl.The non-limiting examples of " arlydene " include Phenylene, sub-pyridyl group, sub- pyrimidine radicals and sub- thienyl.The substituent group of arlydene is selected from described herein acceptable take The group of Dai Ji.

" double functional copolymer ", also referred to as " bifunctional linker ", referring to including two can be with other parts Specific reaction is to form the polymer of the functional group of covalent bond or non-covalent bond.It these parts can be including (but not limited to) natural Side group on amino acid or alpha-non-natural amino acid or peptide containing these natural amino acids or alpha-non-natural amino acid.Only citing comes It says, bifunctional linker can have the functional group for having reactivity with the group on the first peptide and have with the group on the second peptide Another functional group of reactivity thereby forms the binding element for including the first peptide, bifunctional linker and the second peptide.Become known for The numerous programs and connexon molecule that multiple compounds are connected with peptide.Referring to (for example) European patent application the 188,256th Number；U.S. Patent No. 4,671,958, No. 4,659,839, No. 4,414,148, No. 4,699,784；4,680th, No. 338；And the 4th, 569, No. 789, it is in being hereby incorporated by reference in their entirety." multifunctional polymer " is also referred to as " multifunctional connexon " refers to the polymer for including two or more functional groups that can be reacted with other parts. These parts including (but not limited to) natural amino acid or alpha-non-natural amino acid or can contain these natural amino acids or non-natural Side group (including (but not limited to) amino acid side group) on the peptide of amino acid is to form covalent bond or non-covalent bond.Difunctionality polymerization Object or multifunctional polymer can be it is any needed for length or molecular weight, and may be selected with bonded to one of compound or one Specific required interval or conformation are provided between a above molecule molecule in connection or compound.

As used herein, term " biological usability " refer to substance or its active part from pharmaceutical dosage form delivering and The rate and degree being made available by site of action or in general Xun Huan.The increase of biological usability refers to increasing object The rate and journey that matter or its active part are delivered from pharmaceutical dosage form and be made available by site of action or in general Xun Huan Degree.For example, the increase of biological usability can refer to be shown as when compared with other substances or active part in blood substance or its The increase of active moiety concentrations.The non-limiting examples of the assessment increased method of biological usability provide in example 88-92.This Kind method can be used for the biological usability for assessing any polypeptide.

As used herein, term " bioactive molecule ", " biologically-active moiety " or " bioactivator " means It can influence biosystem, path, any physics of molecule or biochemical property or times with the relevant interaction of organism What substance, the organism including (but not limited to) virus, bacterium, bacteriophage, transposon, prion, insect, fungi, plant, Animal and the mankind.Specifically, as used herein, bioactive molecule including (but not limited to) be intended for diagnosis, Healing, the disease alleviated, treat or prevent the mankind or other animals or the body or spirit for being additionally useful for the enhancing mankind or animal Any substance of health.The example of bioactive molecule is including (but not limited to) peptide, protein, enzyme, small-molecule drug, hardness poison Product, soft drugs, carbohydrate, inorganic atoms or molecule, dyestuff, lipid, nucleosides, radioactive nucleus, oligonucleotides, toxin, Cell, virus, liposome, particle and micella.Suitable for the bioactivity used with method and composition described herein The species of agent is including (but not limited to) drug, prodrug, radioactive nucleus, developer, polymer, antibiotic, fungicide, antiviral Agent, antiphlogistic, antitumor agent, cardiovascular agents, antianxiety agent, hormone, growth factor, steroid agent, microbe-derived toxin and Its analog.

" adjusting bioactivity " means raising or reduces the reactivity of polypeptide, change the selectivity of polypeptide, enhancing or reduction The substrate selective of polypeptide.Analyzing bioactivity change can be by comparing the bioactivity of non-native polypeptide and the life of natural polypeptides Object activity carries out.

As used herein, term " biomaterial " refers to the material of biological source, it includes (but not limited to) from Bioreactor and/or the material obtained by recombination method and technology.

As used herein, term " biophysics probe " refers to the spy of structure change that is detectable or monitoring molecule Pin.These molecules can be used for detecting or monitoring protein with other greatly including (but not limited to) protein and " biophysics probe " The interaction of molecule.The example of biophysics probe is including (but not limited to) spin labeling, fluorogen and can photoactivation base Group.

As used herein, term " biosynthesis " refers to any side using translation system (cell or acellular) Method, it includes use at least one of following components：Polynucleotide, codon, tRNA and ribosomes.For example, it is non- Part VIII " in vivo generating the polypeptide for including alpha-non-natural amino acid " and non-limiting examples 14 can be used in natural amino acid Described in methods and techniques " be incorporated to by biosynthesis " in non-natural amino acid polypeptides.In addition, selection " can be closed by biology Into being incorporated to " method for being applicable in alpha-non-natural amino acid in non-natural amino acid polypeptides is described in non-limiting examples 15-16.

As used herein, term " biotin analog " (or being referred to as " biotin mimetics ") is except biotin Outside any molecule for being combined with high-affinity with avidin and/or streptavidin.

As used herein, the term " carbonyl " refer on part contain be selected from by-C (O)-,-S (O)-,-S (O)₂- and-C (S)-composition group group, it includes (but not limited to) to contain at least one ketone group and/or at least one aldehyde Base and/or at least one ester group and/or the group of at least one carboxylic acid group and/or at least one thioester substrate.These carbonyl bags Containing ketone, aldehyde, carboxylic acid, ester and thioesters.In addition, these groups can be the part of linear, branch or ring molecule.

Term " carboxyl terminal modification group " refers to any molecule on terminal carboxyl group.For example, this A little terminal carboxyl groups can in the end of polymerizable molecular, wherein these polymerizable moleculars including (but not limited to) polypeptide, polynucleotide and Polysaccharide.Terminal modifying groups are including (but not limited to) various water-soluble polymers, peptide or protein matter.Only for example, end is repaiied It adorns group and includes polyethylene glycol or seralbumin.Terminal modifying groups can be used for the treatment feature for changing polymerizable molecular, bag Increase the serum half-life of peptide containing (but not limited to).

As used herein, term " chemically cracking group " (referred to as " chemically unstable ") is referred to sudden and violent It is exposed to the group for decomposing or cracking after acid, alkali, oxidant, reducing agent, chemical initiator or radical initiator.

As used herein, term " chemiluminescent groups " is referred in the case where not heating additionally due to chemical anti- It answers and luminous group.Only for example, in the presence of alkali and metallic catalyst, luminol (luminol) (5- amino -2, 3- dihydro -1,4- phthalazine diketones) and oxidant (such as hydrogen peroxide (H₂O₂)) reaction generation excitation state product (3- amino neighbours benzene two Formates, 3-APA).

As used herein, term " chromophore " refers to absorbing the light of visible wavelength, UV wavelength or IR wavelength Molecule.

As used herein, atom or molecule necessary to the effect that term " co-factor " refers to macromolecular.Co-factor Including (but not limited to) some other factors necessary to the activity of inorganic ions, coenzyme, protein or enzyme.Example includes hemochrome In heme, the metal ion of the magnesium in chlorophyll and protein.

As used herein, the molecule for referring to using at least two interaction between each other " is folded " altogether and so that is not rolled over The molecular conversion of folded or improper folding is refolding process, reaction or the method for the molecule suitably folded.Only for example, " altogether Fold " use at least two interaction between each other polypeptides and so that the polypeptide of unfolded or improper folding be changed into it is natural, suitable When the polypeptide of folding.These polypeptides can contain natural amino acid and/or at least one alpha-non-natural amino acid.

As used herein, " comparison window " refers to being used to compare same number after optimal comparison in two sequences The section of any one of the sequence of adjoining position and the adjoining position of canonical sequence.These adjoining positions including (but not limited to) by The group of about 20 to about 600 sequential cells compositions, it includes about 50 to about 200 sequential cells and about 100 to about 150 A sequential cells.Only for example, these sequences include polypeptide and the polypeptide containing alpha-non-natural amino acid, wherein sequential cells bag Natural amino acid containing (but not limited to) and alpha-non-natural amino acid.In addition, only for example, these sequences include polynucleotide, Nucleotide is corresponding sequential cells.Sequence alignment method for comparing is well known in the art.It can be by with lower section Method carries out the optimal sequence compared comparison, and it includes (but not limited to) Smith and Waterman (1970) Adv.Appl.Math.2:Local homology algorithm, Needleman and Wunsch (1970) J.Mol.Biol.48 of 482c:443 Homology alignment algorithm, Pearson and Lipman (1988) Proc.Nat'l.Acad.Sci.USA 85:2444 similitude Searching method, the computerized of these algorithms realize (wisconsin genetics software bag (Wisconsin Genetics Software Package) GAP in (Genetics Computer Group, 575Science Dr., Madison, WI), BESTFIT, FASTA and TFASTA) or manual alignment and visual inspection (referring to (for example) Ausubel et al., Current Protocols in Molecular Biology (1995 supplementary issue)).

For example, it is BLAST and BLAST available for the algorithm for measuring percentage of sequence identity and sequence similarity 2.0 algorithms are described in Altschul et al. (1997) Nuc.Acids Res.25:3389-3402 and Altschul et al. (1990) J.Mol.Biol.215:In 403-410.The software for carrying out BLAST analyses can be by national biology Technical information center (National Center for Biotechnology Information) is open to be obtained.BLAST algorithm Parameter W, T and X determine the sensitivity compared and speed.BLASTN programs (for nucleotide sequence) are 11, phase using word length (W) Prestige value (E) is 10, M=5, N=-4 and two chains are worth more by default.For amino acid sequence, BLASTP programs make It is 3 with word length, desired value (E) is 10 and BLOSUM62 score matrix (referring to Henikoff and Henikoff (1992) Proc.Natl.Acad.Sci.USA 89:10915) comparison (B) is 50, desired value (E) is 10, M=5, N=-4 and two Chain is worth more by default.BLAST algorithm is usually carried out in the case where " low-complexity " filter is closed.

BLAST algorithm also carry out similitude between two sequences statistical analysis (referring to (for example) Karlin and Altschul(1993)Proc.Natl.Acad.Sci.USA 90:5873-5787).The similitude provided by BLAST algorithm One measurement is minimum sum probability (P (N)), and offer two can accidentally occur matched general between nucleotide or amino acid sequence The instruction of rate.For example, if in the comparison of test nucleic acid and reference nucleic acid, minimum sum probability is less than about 0.2 or is less than About 0.01 or less than about 0.001, then it is similar to reference sequences to be considered as nucleic acid.

Term " variant through conservative sex modification " is applied to natural and non-natural amino acid sequence and natural and non-natural Nucleotide sequence, and combinations thereof.On specific nucleic acid sequence, " variant through conservative sex modification " refers to encoding identical or substantially Identical natural and non-natural amino acid sequence or wherein natural and unnatural nucleic acids do not encode natural and alpha-non-natural amino acid sequence Arrange those natural and unnatural nucleic acids (for substantially the same sequence).For example, due to the degeneracy of genetic code, A large amount of functionally identical nucleic acid encode any given protein.For example, codon GCA, GCC, GCG and GCU encode amino Sour alanine.Therefore, each position that alanine is specified by codon wherein, codon can change into any corresponding password Son is without changing encoded polypeptide.These variances are " silent variant ", are one in the variation through conservative sex modification Kind.Therefore such as coding is natural or each natural or non-native nucleic acid sequence herein of non-native polypeptide also describe it is natural or Each possible silent variant of unnatural nucleic acids.It is it will be understood by one of ordinary skill in the art that each in natural or unnatural nucleic acids (in addition to AUG and TGG, wherein AUG is generally only the codon of methionine to codon, and TGG is generally only the password of tryptophan Son) functionally identical molecule can be generated through modifying.Therefore, natural and non-native polypeptide natural and unnatural nucleic acids are encoded Each silent variant imply in each sequence.

On amino acid sequence, to changing, adding or lacking single natural and alpha-non-natural amino acid or small in coded sequence Nucleic acid, peptide, polypeptide or the indivedual of protein sequence of the natural and alpha-non-natural amino acid of percentage substitute, lack or are added to " variant through conservative sex modification " causes the missing of amino acid, the addition of amino acid or natural and non-natural amino wherein changing 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of the acid through being chemically similar.The known conservative replacement that functionally similar natural amino acid is provided in fields Table.These variants through conservative sex modification are different from polymorphie variant, inter-species homologue and method and composition described herein Allele and be not excluded for these substances.Each contain each other for the amino acid of conservative replaces for eight groups below：

1) alanine (A), glycine (G)；

2) aspartic acid (D), glutamic acid (E)；

3) asparagine (N), glutamine (Q)；

4) arginine (R), lysine (K)；

5) isoleucine (I), leucine (L), methionine (M), valine (V)；

6) phenylalanine (F), tyrosine (Y), tryptophan (W)；

7) serine (S), threonine (T)；And

8) cysteine (C), methionine (M)

(referring to (for example) Creighton, Proteins:Structures and Molecular Properties(W H Freeman&Co.；Second edition (in December, 1993)).

Unless otherwise noted, otherwise term " cycloalkyl " and " Heterocyclylalkyl " (itself is combined with other terms) distinguish table Show the annular form of " alkyl " and " miscellaneous alkyl ".Therefore, it is unsaturated and completely not to include saturation, part for cycloalkyl or Heterocyclylalkyl The ring of saturation is bonded.In addition, for Heterocyclylalkyl, hetero atom can occupy the position that heterocycle is connected to the rest part of molecule.It is miscellaneous Atom can be including (but not limited to) oxygen, nitrogen or sulphur.The example of cycloalkyl is including (but not limited to) cyclopenta, cyclohexyl, 1- hexamethylenes Alkenyl, 3- cyclohexenyl groups, suberyl and its similar to group.The example of Heterocyclylalkyl is including (but not limited to) 1- (1,2,5,6- Tetrahydro pyridyl), 1- piperidyls, 2- piperidyls, 3- piperidyls, 4- morpholinyls, morpholinyl, tetrahydrofuran -2- bases, tetrahydrochysene furan Mutter -3- bases, thiophane -2- bases, thiophane -3- bases, 1- piperazinyls, 2- piperazinyls and its similar to group.In addition, term Cover polycyclic structures, it includes (but not limited to) double-ring ring structures and three annular ring structures.Similarly, term " sub- heterocycle Alkyl " (itself or the part as another molecule) means the bivalent group derived from Heterocyclylalkyl, and term " cycloalkylidene " (itself or the part as another molecule) means the bivalent group derived from cycloalkyl.

As used herein, term " cyclodextrin " refers to being made of at least 6 to 8 glucose molecules in ring is formed Cyclic carbohydrates.Contain water soluble group in the outside of ring；It is the relatively non-pole that can accommodate small molecule at the center of ring Property hole.

As used herein, term " cytotoxicity " refers to the compound of injury cell.

As used herein, " denaturant " refers to any compound that reversible polymer can be caused to stretch or object Matter.Only for example, " denaturant " can cause the invertibity of protein to stretch.The intensity of denaturant will be by the property of specific denaturant Matter and concentration determine.For example, denaturant is including (but not limited to) chaotropic agent, detergent, water miscible organic solvent, phosphorus Fat or its combination.The non-limiting examples of chaotropic agent are including (but not limited to) urea, guanidine and sodium sulfocyanate.The non-limit of detergent Property example processed can be including (but not limited to)：Strong detergent, such as lauryl sodium sulfate or polyoxyethylene ether (such as Tween or Triton detergents), sarcosyl (Sarkosyl)；Mild nonionic detergent is (for example, digitonin (digitonin))；Mild cationic detergent, such as N->2,3- (two oily alkenyloxy groups)-propyl-N, N, N- trimethyl ammonium；Temperature With zwitterionic detergent (such as sodium taurocholate or NaTDC)；Or zwitterionic detergent, it includes (but not limited to) sulfonic acid sweet teas Dish alkali (Zwiftergent), 3- (3- courages amidopropyl) dimethylamino -1- propane sulfate (CHAPS) and 3- (3- courage acyls Aminocarbonyl propyl) dimethylamino -2- hydroxyl -1- propane sulfonates (CHAPSO).The non-limiting examples of water miscible organic solvent Including (but not limited to) acetonitrile, low-grade alkane alcohol (especially C₂-C₄Alkanol, such as ethyl alcohol or isopropanol) or lower alkyl glycol (C₂-C₄ Alkane glycol, such as ethylene glycol) it can be used as denaturant.The non-limiting examples of phosphatide are including (but not limited to) naturally occurring phosphorus Fat, such as phosphatidyl-ethanolamine, lecithin, phosphatidylserine and phosphatidylinositols；Or synthetic phospholipid derivative or variant, it is all Such as two caproyl lecithin or two heptanoyl group lecithin.

As used herein, term " detectable label " refers to the mark that analytical technology can be used to observe, these Analytical technology is including (but not limited to) fluorescence, chemiluminescence, electron spin resonance, ultraviolet/visible absorption spectra, mass spectrum, nuclear-magnetism Resonance, magnetic resonance and electrochemical method.

As used herein, the term " dicarbapentaborane " refer to containing at least two be selected from by-C (O)-,-S (O)-,-S (O)₂- and-C (S)-composition group part group, it includes (but not limited to) 1,2- dicarbapentaborane, 1,3- dicarbapentaborane and 1, 4- dicarbapentaborane and contain at least one ketone group and/or at least one aldehyde radical and/or at least one ester group and/or at least one The group of carboxylic acid group and/or at least one thioester substrate.These dicarbapentaborane include diketone, keto-aldehyde, ketone acid, ketone ester and ketone sulphur Ester.In addition, these groups can be the part of linear, branch or ring molecule.Two parts in dicarbapentaborane may be the same or different, And can include can be in the substituent group of any one place's generation (only for example) ester, ketone, aldehyde, thioesters or amide of two parts.

As used herein, term " drug " refers in prevention, diagnosis, alleviates, treats or cure disease or symptom The middle any substance used.

As used herein, term " dyestuff " refers to the solvable coloring matter containing chromophore.

As used herein, term " effective quantity " refers to that one or more kinds of controlled can be mitigated to a certain extent The disease for the treatment of or the medicament of institute's administration of the symptom of symptom or the sufficient amount of compound.As a result can be the disease of disease, symptom or Change needed for the reduction of the cause of disease and/or any other of alleviation or biosystem.For example, the medicament or chemical combination of institute's administration Object is including (but not limited to) natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or through repairing The non-amino acid polypeptide of decorations.Can administration contain these natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural ammonia The composition of base acid polypeptide or modified non-natural amino acid polypeptides is for preventative, enhancing and/or therapeutic treatment.It is in office Such as dosage can be used to increase the technology of research to determine for appropriate " effective " amount under what individual cases.

As used herein, term " electron dense group " refers to the base of the scattered electron when being irradiated with electron beam Group.These groups are including (but not limited to) ammonium molybdate, basic bismuth nitrate, cadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate, six Methenamine, 98.5%, anhydrous indium chloride, lanthanum nitrate, three acetate hydrate lead, three citrate hydrate lead plumbates, plumbi nitras, high iodine Acid, phosphomolybdic acid, phosphotungstic acid, the potassium ferricyanide, potassium ferrocyanide, ammoniated ruthenium oxychloride, silver nitrate, (the Ag calibratings of albumen silver：8.0-8.5%) " strong ", tetraphenylporphines silver (S-TPPS), sodium chloraurate, sodium tungstate, thallium nitrate, thiosemicarbazides (TSC), uranyl acetate, nitric acid Uranyl and vanadic sulfate.

As used herein, term " energy transfer agent " refers to contribute or receiving the energy from another molecule Molecule.Only for example, fluorescence resonance energy transfer (FRET) is dipole-dipole coupled processes, is supplied by this process fluorescence It is transferred to the excited energy non-radiation type of body molecule and does not excite on acceptor molecule, then obtained with longer wavelength fluorescent emission The energy given.

Term " enhancing " means the effect of effect or duration needed for increase or extension.For example, " enhancing " is treated The effect of agent refers to increase or the effect of extended treatment agent or duration, effect during treatment disease, illness or symptom Ability.As used herein, " enhancing effective quantity " refers to being enough to enhance treatment during treatment disease, illness or symptom The amount of the effect of agent.When in for patient, for this purposes, effectively amount should depend on the seriousness of disease, illness or symptom With process, previous treatment, the health status of patient and to the reaction of drug and the judgement of attending physician.

As used herein, term " eucaryote " refers to belonging to Eukarya (domain in phylogenetics Eucarya organism), it includes (but not limited to) animal (including (but not limited to) mammal, insect, reptile, bird Class etc.), infusorian, plant (including (but not limited to) monocotyledon, dicotyledon and algae), fungi, yeast, flagellum Worm, microsporidian and protist.

As used herein, term " fatty acid " " refers to the carboxylic acid with about C6 or longer hydrocarbon side chains.

As used herein, term " fluorogen " refers to emitting photon and and then the molecule that fluoresces after excitation.

As used herein, term " functional group ", " active part ", " activated group ", " leaving group ", " reactivity Site ", " chemically reactive group " and " chemical reactivity part " refer to part or the list of the molecule chemically reacted Member.These terms in chemical field some it is synonymous and be used to indicate herein perform some functions or activity and with other points Son has the part of the molecule of reactivity.

Term " halogen " includes fluorine, chlorine, iodine and bromine.

As used herein, term " halogen acyl group " refers to the acyl group containing halogen moiety, it includes (but not limited to)- C(O)CH₃、-C(O)CF₃、-C(O)CH₂OCH₃And it is similar to group.

As used herein, term " alkylhalide group " refers to the alkyl containing halogen moiety, it includes (but not limited to)- CF₃With-CH₂CF₃And it is similar to group.

As used herein, term " miscellaneous alkyl " refers to linear chain or branch chain or cyclic hydrocarbon group or its combination, by alkane Base and at least one be selected from are made of the hetero atom of O, N, Si and S group formed, and the wherein visual feelings of nitrogen-atoms and sulphur atom Condition is oxidized and nitrogen heteroatom can be optionally through quaternized.Hetero atom O, N and S and Si can be located at any interior position of miscellaneous alkyl It puts or is connected to positioned at alkyl at the position of rest part of molecule.Example is including (but not limited to)-CH₂-CH₂-O-CH₃、- CH₂-CH₂-NH-CH₃、-CH₂-CH₂-N(CH₃)-CH₃、-CH₂-S-CH₂-CH₃、-CH₂-CH₂,-S(O)-CH₃、-CH₂-CH₂-S (O)₂-CH₃,-CH=CH-O-CH₃、-Si(CH₃)₃、-CH₂- CH=N-OCH₃And-CH=CH-N (CH₃)-CH₃.In addition, at most Two hetero atoms can be it is continuous, such as ,-CH₂-NH-OCH₃With-CH₂-O-Si(CH₃)₃。

As used herein, term " sub- miscellaneous alkyl " refers to the bivalent group as derived from miscellaneous alkyl, such as by-CH₂- CH₂-S-CH₂CH₂- and-CH₂-S-CH₂-CH₂-NH-CH₂- illustrated (but not limiting).For sub- miscellaneous alkyl, it is identical or Different hetero atoms can also occupy the one or both ends of chain (including (but not limited to) alkylidene oxygroup, alkylenedioxy group, alkylene Base amino, alkylenediamino, amino oxygroup alkylidene and its similar to group).Further, it is miscellaneous for alkylidene and Asia For alkyl bond symbasis group, the direction of bonded group is not to be implied by writing the direction of the formula of bonded group.For example, formula-C (O)₂R'- expressions-C (O)₂R'- and-R'C (O)₂- the two.

As used herein, term " heteroaryl " or " heteroaromatic " are referred to containing at least one miscellaneous selected from N, O and S The aryl of atom；Wherein nitrogen-atoms and sulphur atom can be optionally oxidized, and nitrogen-atoms can be optionally through quaternized.Heteroaryl can It is substituted or is unsubstituted.Heteroaryl can be connected to by hetero atom on the rest part of molecule.The nonrestrictive reality of heteroaryl Example comprising 1- pyrrole radicals, 2- pyrrole radicals, 3- pyrrole radicals, 3- pyrazolyls, 2- imidazole radicals, 4- imidazole radicals, pyrazinyl, 2- oxazolyls, 4- oxazolyls, 2- phenyl -4- oxazolyls, 5- oxazolyls, 3- isoxazolyls, 4- isoxazolyls, 5- isoxazolyls, 2- thiazolyls, 4- thiazolyls, 5- thiazolyls, 2- furyls, 3- furyls, 2- thienyls, 3- thienyls, 2- pyridyl groups, 3- pyridyl groups, 4- pyrroles Piperidinyl, 2- pyrimidine radicals, 4- pyrimidine radicals, 5- benzothiazolyls, purine radicals, 2- benzimidazolyls, 5- indyls, 1- isoquinolyls, 5- isoquinolyls, 2- quinoxalinyls, 5- quinoxalinyls, 3- quinolyls and 6- quinolyls.

" high alkyl " refers to the alkyl as alkyl as used herein, the term.

As used herein, term " consistent " refers to identical two or more sequences or subsequence.Separately Outside, as used herein, term " substantially consistent " is referred to when through comparison window or as using comparison algorithm or by craft When the specified region of comparison and visual inspection measurement compares and compares maximum correspondence, there is the identical continuous list of certain percentage Two or more sequences of member.Only for example, if sequential cells are about 60% consistent, about on region is specified 65% is consistent, about 70% consistent, about 75% consistent, about 80% consistent, about 85% consistent, about 90% consistent or about 95% is consistent, So two or more sequences can be " substantially consistent ".These percentages describe two or more sequences " uniformity percentage ".The uniformity of sequence may be present on region of the length at least about 75-100 sequential cells, length Be on the region of about 50 sequential cells or (if not specified) entire sequence on.This definition is directed to the mutual of cycle tests Complementary series.Only for example, when amino acid residue is identical, two or more peptide sequences are consistent, and if Amino acid residue be on region is specified it is about 60% consistent, about 65% consistent, about 70% consistent, about 75% consistent, about 80% 1 It causes, is about 85% consistent, is about 90% consistent or about 95% is consistent, then two or more peptide sequences are " substantial one It causes ".Uniformity may be present on region of the length at least about 75-100 amino acid, the area that length is about 50 amino acid On domain or in the entire sequence of (if not specified) peptide sequence.In addition, only for example, when nucleic acid is identical, two Or more than two polynucleotide sequences are consistent, and if nucleic acid is about 60% consistent, about on region is specified 65% is consistent, about 70% consistent, about 75% consistent, about 80% consistent, about 85% consistent, about 90% consistent or about 95% is consistent, So two or more polynucleotide sequences are " substantially consistent ".Uniformity may be present in length as at least about On the region of 75-100 nucleic acid, on the region that length is about 50 nucleic acid or (if not specified) polynucleotide sequence it is whole In a sequence.

Compare on sequence, a usual sequence serves as reference sequences of the cycle tests compared with it.When using sequence During row comparison algorithm, by cycle tests and reference sequences input computer, subsequence coordinates, (if necessary) and specified sequence are specified Algorithm routine parameter.Default program parameters can be used or may specify alternate parameter.Subsequent sequence comparison algorithm is based on program parameter Calculate percentage of sequence identity of the cycle tests compared with reference sequences.

As used herein, term " immunogenicity " refers to the antibody for having reaction to administration medicine.For controlling The immunogenicity of the property treated non-natural amino acid polypeptides can be used for biological fluid moderate resistance non-natural amino acid polypeptides antibody Qualitatively and quantitatively calibrating obtain.These calibratings are including (but not limited to) radioimmunoassay (RIA), Enzyme-linked Immunosorbent Assay Examine and determine (ELISA), electrochemiluminescent immunoassay calibrating (LIA) and fluorescent immunoassay (FIA).For therapeutic non-natural amino acid polypeptides The analysis of immunogenicity include comparing the antibody response after the therapeutic non-natural amino acid polypeptides of administration and the therapeutic day of administration Antibody response after right amino acid polypeptide.

As used herein, term " inserting agent " (also referred to as " insertion group ") refers to can be inserted into the molecule of molecule The chemicals in intermolecular space between interior space or molecule.Only for example, inserting agent or insertion group can be insertion DNA Molecule in the accumulation base of double helix.

As used herein, term " separation " refers to that group of interest is separated and removed from the component being not concerned with Point.Separated substance can be dry or partial desiccation state or be solution form, and it includes (but not limited to) aqueous solutions.Through dividing From component can be homogeneous state or separated component can be to include the upper acceptable supporting agent of additional pharmaceutical and/or excipient Medical composition part.Technique of analytical chemistry can be used to measure in purity and homogenieity, wherein the technology includes (but not It is limited to) polyacrylamide gel electrophoresis or high performance liquid chromatography.In addition, when component of interest is through separating and to exist in preparation Main matter when, the component is described herein as through substantially purifying.As used herein, term " purifying " refers to Be it is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or purer component of interest.Only for example, when Nucleic acid or protein are concentrated without at least some cellular components to associate under native state with it or nucleic acid or protein For the concentration in vivo or in vitro generated than it high level when, these nucleic acid or protein are " separated ".Equally, For example, when on the outside of the gene and when coding protein rather than the open reading frame of gene of interest separate, the gene To be separated.

As used herein, term " mark " refers to being incorporated in compound and is easy to detect, thereby can detect and/or Monitor the substance of its physical distribution.

As used herein, term " bonded " refers to anti-by the chemistry between the functional group of connexon and another molecule The key or chemical part that should be formed.These keys can be including (but not limited to) covalent bond and non-covalent bond, and these chemical parts can Including (but not limited to) ester, carbonic ester, imines, phosphate, hydrazone, acetal, ortho esters, peptide bond and oligonucleotides key.In hydrolysis It is stable it is bonded mean it is described bonded substantially stable in water and in the case where being applicable in pH value (it includes (but not limited to) in physiology item Under part) long-term (perhaps or even indefinitely) do not react with water.In hydrolysis it is unstable or degradable it is bonded mean it is described bonded It can the degradation in water or aqueous solution (including such as blood).Unstable or degradable bonded of enzymatic mean it is described it is bonded can be by one Or more than one enzyme degradation.Only for example, PEG and related polymer can be included in main polymer chain or in polymerization owner It is degradable bonded in connection subbase group between one or more of chain and polymer molecule terminal functional group.These can It degrades and bonded reacts the ester formed including (but not limited to) by the alcohol radical on PEG carboxylic acids or activation PEG carboxylic acids and bioactivator Key, wherein these ester groups are usually hydrolyzed in physiological conditions with release bioactive agent.Other hydrolysis it is degradable it is bonded comprising (but It is not limited to) carbonic acid ester bond；The imine linkage formed by amine and aldehyde reaction；By alcohol the phosphoric acid ester bond formed is reacted with phosphate-based；Make For hydrazides and the hydrazone key of the reaction product of aldehyde；As aldehyde and the acetal bonds of the reaction product of alcohol；Reaction as formates and alcohol The original acid ester key of product；It is formed by amino (including (but not limited to) on the end of the polymer of such as PEG) and the carboxyl of peptide Peptide bond；And it is formed by phosphoramidite base (including (but not limited to) on the end of polymer) and the 5' hydroxyls of oligonucleotides Oligonucleotides key.

As used herein, term " culture medium " refers to growing and collect cell and/or by these cell tables Any culture medium for the product for reaching and/or secreting.These " culture mediums " are including (but not limited to) solution, solid, semisolid or can Support or the rigid carrier containing any host cell, the host cell is including (for example) bacterial host cell, yeast host Cell, insect host cell, plant host cell, eukaryotic host cell, mammalian host cell, Chinese hamster ovary celI, prokaryotic hosts Cell, Escherichia coli (E.coli) or pseudomonad (Pseudomonas) host cell and cell inclusion.These " cultures Base " has been grown on polypeptide including (but not limited to) wherein host cell and has secreted in culture medium therein, and it includes walked in multiplication Culture medium before or after rapid.These " culture mediums " are also including (but not limited to) the buffer solution containing host cell lysate Or reagent, such as the polypeptide and the host cell dissolving that generate into the cell or divide to discharge polypeptide.

As used herein, term " metabolin " refers to compound (such as natural amino acid polypeptide, non-natural amino Sour polypeptide, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides) derivative, be in compound (such as natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural ammonia Base acid polypeptide) metabolism when formed.Term " medicinal activity metabolin " or " active metabolite " refer to that compound is (such as natural Amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides) Biologically active derivatives, be (such as natural amino acid polypeptide, non-natural amino acid polypeptides, modified in this compound Natural amino acid polypeptide or modified non-natural amino acid polypeptides) metabolism when formed.

As used herein, term " metabolism " refer to predetermined substance by organism transform process sum.These mistakes Reaction of the journey including (but not limited to) hydrolysis and by enzymatic.Other information on metabolism is available from The Pharmacological Basis of Therapeutics, the 9th edition, McGraw-Hill (1996).Only for example, naturally Amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides Metabolin can be by by natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or through repairing The non-natural amino acid polypeptides administration hosts of decorations and tissue samples of the analysis from host differentiate or by by natural amino acid Polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides and liver are thin Born of the same parents in vitro cultivate and analyze gained compound to differentiate together.

As used herein, term " metal-chelator " refers to being formed the molecule of metal complex with metal ion. For example, these molecules can form two or more coordinate bonds with central metallic ions and can form ring structure.

As used herein, term " containing metal part " refers to the group containing metal ion, atom or particle.This A little parts are including (but not limited to) cis-platinum (cisplatin), chelated metal ions (such as nickel, iron and platinum) and metal nano Particle (such as nickel, iron and platinum).

As used herein, term " part for being combined with heavy atom " refers to being combined with atom usually heavier than carbon Ion group.These ions or atom are including (but not limited to) silicon, tungsten, gold, lead and uranium.

As used herein, term refers to existing to natural amino acid, alpha-non-natural amino acid, natural ammonia " through modification " The change of base acid polypeptide or non-natural amino acid polypeptides.These change or modification can by natural amino acid, alpha-non-natural amino acid, It is modified after the synthesis of natural amino acid polypeptide or non-natural amino acid polypeptides or by natural amino acid, alpha-non-natural amino acid, day The modification of the common translation of right amino acid polypeptide or non-natural amino acid polypeptides or posttranslational modification obtain.Form is " through modification or not Through modification " mean that discussed natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides regard Situation is through modification, that is, natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or the non-natural amino acid polypeptides discussed It can be through modification or unmodified.

As used herein, term " adjusted serum half-life " refers to that modified bioactive molecule is opposite Positive variation or negative variation in the circulating half-life of its non-unmodified form.For example, modified bioactive molecule Including (but not limited to) natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides.Citing comes It says, serum half-life is to take blood by multiple time points after administration bioactive molecule or modified bioactive molecule Liquid sample and the concentration of that molecule in each sample is measured to measure.The correlation of serum-concentration and time allow to count Calculate serum half-life.For example, adjusted serum half-life can be that serum half-life increases, and may be such that can be changed Good dosage regimen avoids poisonous effect.This serum increase can be at least about twice, at least about three times, at least about five times or At least about ten times.The non-limiting examples of the assessment increased method of serum half-life provide in example 88-92.This method can For assessing the serum half-life of any polypeptide.

As used herein, term " adjusted treatment half-life period " refers to living through modified biological for therapeutically effective amount Property positive variation or negative variation of the molecule compared with the half-life period of its unmodified form.For example, modified bioactivity Molecule is including (but not limited to) natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides.It lifts For example, pharmacokinetics and/or pharmacodynamic properties of the half-life period by multiple point in time measurement molecules after dispensing are treated To measure.Increased treatment half-life period may be such that and can obtain specific beneficial dosage regimen, specific beneficial accumulated dose or avoid not Work as effect.For example, increased treatment half-life period can be increased by the combination of increased effect, modified molecule and its target Or reduce, another parameter of unmodified molecule or mechanism of action increase or reduce or molecule (such as, is only illustrated by enzyme For, protease) decompose increase or decrease generation.The non-limiting examples of assessment treatment half-life period increased method are in example It is provided in 88-92.This method can be used for the treatment half-life period for assessing any polypeptide.

As used herein, term " nano-particle " refers to the grain to the granularity between about 1nm with about 500nm Son.

As used herein, term refers to that the molar ratio of the compound of participation chemical reaction is " close to stoichiometry " About 0.75 to about 1.5.

As used herein, term " non-eucaryote " refers to non-most eukaryotes.For example, non-eucaryote Body can belong to eubacteria (Eubacteria) and (it includes (but not limited to) Escherichia coli (Escherichia coli), thermophilic dwell It is hot bacterium (Thermus thermophilus) or bacillus stearothermophilus (Bacillus stearothermophilus), glimmering Light pseudomonad (Pseudomonas fluorescens), pseudomonas aeruginosa (Pseudomonas aeruginosa), stench Pseudomonad (Pseudomonas putida)), the domain in phylogenetics or Archimycetes (Archaea), it includes (but not Be limited to) Methanococcus jannaschii (Methanococcus jannaschii), hot autotrophic methane bacteria (Methanobacterium Thermoautotrophicum ancient green-ball bacterium (Archaeoglobus fulgidus), strong red-hot coccus), are flickered (Pyrococcus furiosus), Pyrococcus horikoshii (Pyrococcus horikoshii), Aeropyrum pernix (Aeuropyrum pernix) or thermophilic salt bacillus (Halobacterium), the richly endowed bacterium (Haloferax of the thermophilic salt of such as walsh ) and the domain in thermophilic salt bacillus specie NRC-1 or phylogenetics volcanii.

" alpha-non-natural amino acid " is referred to not as one kind in 20 kinds of common amino acids or burnt lysine or selenocystein Amino acid.Can other terms used synonymous with term " alpha-non-natural amino acid " be " non-naturally encoded amino acids ", " non-natural Amino acid ", " non-naturally-occurring amino acid " and its it is various carry hyphen and the form without hyphen.Term " non-natural Amino acid " is including (but not limited to) by modifying natural coded amino acid (including (but not limited to) 20 kinds of common amino acids or coke Lysine and selenocystein) it is naturally occurring but be incorporated to amino in the polypeptide chain of growth not by translation compound itself Acid.It is not the example of naturally occurring amino acid that naturally encodes including (but not limited to) N- Acetylglucos amino-Serine, N- Acetylglucos amino-L-threonine and O- phosphate tyrosine.In addition, term " alpha-non-natural amino acid " including (but not limited to) And non-naturally-occurring and can by synthesize obtain or can by modify alpha-non-natural amino acid obtain amino acid.

As used herein, term " nucleic acid " refer to deoxynucleotide, deoxyribonucleoside, nucleosides or nucleotide and its The polymer of single-stranded or double-stranded form.Only for example, these nucleic acid and nucleic acid polymers are including (but not limited to) (i) natural nucleus The analog of thuja acid has the binding property similar with reference nucleic acid and the generation in a manner of being similar to naturally occurring nucleotide It thanks；(ii) oligonucleotide analogs, it includes the similar of the DNA used in (but not limited to) PNA (peptidyl nucleic acid), antisense technology Object (thiophosphate, phosphoramidate, with and the like)；(iii) its variant through conservative sex modification is (comprising (but unlimited In) degenerate codon substitution) and complementary series and the sequence that explicitly points out.For example, degenerate codon substitution can pass through Generate one of them or more than one selected by (or all) codon the third place through mixing base and/or deoxyinosine residue Substituted sequence realizes (Batzer et al., Nucleic Acid Res.19:5081(1991)；Ohtsuka et al., J.Biol.Chem.260:2605-2608(1985)；And Rossolini et al., Mol.Cell.Probes 8:91-98 (1994))。

As used herein, term " oxidant " refers to remove the compound of electronics from the compound aoxidized Or substance.For example, oxidant is including (but not limited to) oxidized form of glutathione, cystine, cystamine, two sulphur threose of oxidized form Alcohol, oxidized form antierythrite (oxidized erythreitol) and oxygen.A variety of oxidants are suitable for side described herein In method and composition.

As used herein, term " pharmaceutically acceptable " is referred to including (but not limited to) salt, supporting agent or dilution The substance of agent does not eliminate the bioactivity or property of compound, and relative nontoxic, i.e. the substance can administration individual without Cause improper biological effect or with harmful way with it includes any one of the component in composition therein interactions.

As used herein, term " photoaffinity mark " is referred to after having in light is exposed to marking to it Affinity molecule forms the mark of bonded group.Only for example, this is bonded for covalently or non-covalently key connection.

As used herein, term " light cage covers part " is referred to after irradiating at a particular wavelength covalently or non-covalently With reference to the group of other lewis' acids.

As used herein, the group of fracture after term " can photodestruciton group " is referred in light is exposed to.

As used herein, term " photocrosslinking agent " refers to including two or more after being exposed in light It can be with two or more monomers or polymerizable molecular reaction and the compound for forming the functional group of covalently or non-covalently key connection.

As used herein, term " can photoisomerization part " is referred to wherein after light irradiates from a kind of isomeric form Become the group of another isomeric form.

As used herein, term " polyalkylene glycol " refers to linear or branch polyhydroxyl polyether polyalcohol.These Polyalkylene glycol is including (but not limited to) polyethylene glycol, polypropylene glycol, polytetramethylene glycol and its derivative.Other illustrative realities Example is applied to be listed in (for example) commercial supplier catalogue, the catalogue of such as Shearwater Corporation " polyethylene glycol and For the derivative of biomedical applications " (" Polyethylene Glycol and Derivatives for Biomedical Applications”)(2001).Only for example, these polyhydroxyl polyether polyalcohols have about 0.1kDa between about 100kDa Average molecular weight.For example, these polyhydroxyl polyether polyalcohols including (but not limited to) about 100Da and about 100,000Da or Between higher.The molecular weight of polymer can between about 100Da and about 100,000Da, it includes (but not limited to) 100, 000Da、95,000Da、90,000Da、85,000Da、80,000Da、75,000Da、70,000Da、65,000Da、60, 000Da、55,000Da、50,000Da、45,000Da、40,000Da、35,000Da、30,000Da、25,000Da、20, 000Da、15,000Da、10,000Da、9,000Da、8,000Da、7,000Da、6,000Da、5,000Da、4,000Da、3, 000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da. In some embodiments, the molecular weight of polymer is between about 100Da and 50,000Da.In some embodiments, polymer Molecular weight is between about 100Da and 40,000Da.In some embodiments, the molecular weight of polymer is in about 1,000Da and 40, Between 000Da.In some embodiments, the molecular weight of polymer is between about 5,000Da and 40,000Da.In some embodiments In, the molecular weight of polymer is between about 10,000Da and 40,000Da.In some embodiments, peg molecule is branch Polymer.The molecular weight of branch PEG can between about 1,000Da and about 100,000Da, it includes (but not limited to) 100, 000Da、95,000Da、90,000Da、85,000Da、80,000Da、75,000Da、70,000Da、65,000Da、60, 000Da、55,000Da、50,000Da、45,000Da、40,000Da、35,000Da、30,000Da、25,000Da、20, 000Da、15,000Da、10,000Da、9,000Da、8,000Da、7,000Da、6,000Da、5,000Da、4,000Da、3, 000Da, 2,000Da and 1,000Da.In some embodiments, the molecular weight of branch PEG about 1,000Da and 50,000Da it Between.In some embodiments, the molecular weight of branch PEG is between about 1,000Da and 40,000Da.In some embodiments, prop up The molecular weight of chain PEG is between about 5,000Da and 40,000Da.In some embodiments, the molecular weight of branch PEG is about 5, Between 000Da and 20,000Da.

As used herein, term " polymer " " refers to the molecule formed by repeating subunit.These molecules include (but not limited to) polypeptide, polynucleotide or polysaccharide or polyalkylene glycol.

Term " polypeptide ", " peptide " and " protein " is used interchangeably herein to refer to the polymer of amino acid residue. That is, the description for being directed to polypeptide is equally applicable to the description to peptide and the description to protein, and vice versa.These terms It is alpha-non-natural amino acid suitable for naturally occurring amino acid polymer and one of them or more than one amino acid residue Amino acid polymer.In addition, these " polypeptides ", " peptide " and " protein " include the amino acid chain of any length, it includes overall lengths Protein, wherein amino acid residue are bonded by covalent peptide bonds.

Term " posttranslational modification " refers to sending out afterwards in translated be incorporated in polypeptide chain of natural or alpha-non-natural amino acid Any modification of the raw amino acid.These modifications are in vivo modified including (but not limited to) common translation, common translation in vitro Modification (such as in cell free translation system), translation after in vivo modify and translate after in vitro modify.

As used herein, term " prodrug " or " pharmaceutically acceptable prodrug " refer in vivo or live body It is changed into the medicament of parent drug outside, wherein it does not eliminate the bioactivity of drug or property, and relative nontoxic, i.e. the object Matter can administration individual without cause improper biological effect or with harmful way with it includes in the component of composition therein Any one interaction.Prodrug is usually drug predecessor, after absorbing in administration person under inspection and then, passes through some processes (such as being changed by metabolic pathway) is changed into active or with higher active substance.Some prodrugs, which have, is present in prodrug On chemical group, cause prodrug activity smaller and/or assign drug solubility or some other properties.Once chemical group It cracks and/or through modification, active medicine is just generated from prodrug.Prodrug is changed in vivo by enzymatic reaction or non-enzymatic reaction For active medicine.Prodrug can provide the physicochemical properties of improvement, and such as better dissolubility, the delivery characteristics of enhancing are (such as Selectively targeted specific cells, tissue, organ or ligand) and improvement drug therapy value.The benefit bag of these prodrugs Containing (but not limited to)：(i) compared with parent drug, it is easy to offer medicine；(ii) prodrug can be by oral administration medicine supplying biological utilisation, and parent It cannot；And (iii), compared with parent drug, prodrug can also have the dissolubility in medical composition of improvement.Prodrug Pharmacology comprising active medicine is nonactive or the derivative of active reduction.Prodrug can be designed for adjusting drug or biology is living The amount of property molecule, by the property (such as physicochemical properties, biological medicine property or the pharmacokinetics that manipulate drug Matter) site of action needed for arrival.The example (without stint) of prodrug can be in the form of ester (" prodrug ") administration in favor of wearing The non-natural amino acid polypeptides of cell membrane transmission, wherein water-soluble be harmful to mobility, but once water-soluble wherein is to have The cell interior of benefit, just then metabolism is hydrolyzed to carboxylic acid (active entities).Prodrug can be designed to reversible pharmacological derivative, with It is transmitted as modifying agent with the drug enhanced to site-specific tissue.

As used herein, term " prevention effective dose " refers to prophylactically being applied to containing at least one in patient The amount of the composition of kind non-natural amino acid polypeptides or at least one modified non-natural amino acid polypeptides, can be in certain journey Mitigate the symptom of one or more kinds of disease, symptom or illnesss treated on degree.In these prophylactic uses, the amount Visually depending on the health status of patient, weight and its similar factor.Fully believe that those skilled in the art can pass through routine Experiment (increasing clinical test including (but not limited to) dosage) determines these prevention effective doses.

As used herein, term refers to existing " through protection " prevents chemical reactivity functional group in some reaction items " protecting group " reacted under part or part.Protecting group can change according to the type for the chemically reactive group protected.Only lift For example, if (i) chemically reactive group is amine or hydrazides, then protecting group may be selected from tertbutyloxycarbonyl (t-Boc) and 9- Fluorenylmethoxycarbonyl groups (Fmoc)；(ii) if chemically reactive group is mercaptan, then protecting group can be two sulphur of adjacent pyridyl group Compound；And (iii) if chemically reactive group be carboxylic acid (such as butyric acid or propionic acid) or hydroxyl, then protecting group can be benzyl Or alkyl, such as methyl, ethyl or tertiary butyl.

Only for example, obstruction/protecting group (blocking/protecting group) also selected from：

In addition, protecting group is including (but not limited to) including the group (such as Nvoc and MeNvoc) to photo-labile and institute Other known protecting groups in category field.Other protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, the 3rd edition, be complete by reference in John Wiley＆Sons, New York, NY, 1999 Portion is incorporated herein.

As used herein, term " radioactive segment " refers to that its core is spontaneous and sends nuclear radiation (such as α particles, β Son or γ particles) group；Wherein, α particles are helion, and β particles are electronics, and γ particles are high-energy photon.

As used herein, term " reactive compounds " refer under proper condition to another atom, molecule or Compound has the compound of reactivity.

Term " recombinant host cell " (also referred to as " host cell ") refers to including the cell of exogenous polynucleotide, In be used for by the method that exogenous polynucleotide is inserted into cell including (but not limited to) it is direct absorb, transduction, f- are matched or generated Known other methods in recombinant host cell fields.Only for example, the exogenous polynucleotide can be that nonconformity carries Body (including (but not limited to) plasmid) can be integrated into host genome.

As used herein, term " redox active agent " refers to aoxidizing or reduces another molecule, thereby aoxidizes The molecule that reduction activation agent becomes to be reduced or aoxidize.The example of redox active agent including (but not limited to) ferrocene, quinone, Ru2+/3+ complex compounds, Co2+/3+ complex compounds and Os2+/3+ complex compounds.

As used herein, term " reducing agent " refers to add the compound of electronics to the compound reduced Or substance.For example, reducing agent including (but not limited to) dithiothreitol (DTT) (DTT), 2 mercapto ethanol, dithioerythritol, Cysteine, cysteamine (2- aminoothyl mercaptans) and reduced glutathione.These reducing agents (only for example) can be used Sulfydryl is remained reducing condition and reduces intramolecular or intermolecular disulfide bond.

Improper folding or unfolded state are changed into nature or appropriate folding by " refolding " description as used herein Any process, reaction or the method for folded conformation.Only for example, refolding by the polypeptide containing disulfide bond by it is improper folding or not Collapsed state is the nature on disulfide bond or appropriate folded conformation.These polypeptides for containing disulfide bond can be native amino Sour polypeptide or non-natural amino acid polypeptides.

As used herein, term " resin " refers to high molecular weight insoluble polymer bead.Only for example, this A little beads can be used as the carrier or the site of attachment molecules before purification for Solid phase peptide synthesis.

As used herein, term " carbohydrate " refers to a series of carbohydrate, and it includes (but not limited to) sugar, lists Sugar, oligosaccharides and polysaccharide.

As used herein, term " security " or " security overview " refer to the number compared with administration drug For may be with the relevant side effect of drug administration.For example, by administration is multiple and only mild real estate is into side effect or nothing The drug of side effect is known as having outstanding security overview.The non-limiting examples of the method for security overview are assessed in example It is provided in 92.This method can be used for the security overview for assessing any polypeptide.

As used herein, phrase " selective cross " or " specific hybrid " refer to depositing when specific nucleotide sequence When being in complex mixture (including (but not limited to) total cell or storehouse DNA or RNA), molecule and institute under stringent hybridization conditions State the combination of sequence, in pairs or hybridization.

As used herein, term " spin labeling " refers to detect and can connect by ESR spectrum The molecule containing the atom or atomic group (stablizing paramagnetic group) for showing unpaired electron spin on to another molecule.These Spin labeling molecule can be single spin labeling or dual spin labeling including (but not limited to) nitroxyl and nitroxide.

As used herein, the molar ratio that term " stoichiometry " refers to participating in the compound of chemical reaction is about 0.9 to about 1.1.

As used herein, term " class stoichiometry " refers to becoming after reaction condition changes or in the presence of additive Chemical reaction for stoichiometry or close to stoichiometry.The variation of these reaction conditions including (but not limited to) temperature raise or PH value changes.These additive accelerating agents containing (but not limited to).

Phrase " stringent hybridization condition " referred under conditions of low ion concns and high-temperature, DNA, RNA, PNA or its The hybridization of his nucleic acid mimics or the sequence of its combination.For example, probe can be with the COMPLEX MIXED of nucleic acid under strict conditions In object (including (but not limited to) total cell or storehouse DNA or RNA) its target subsequences hybridization, without in complex mixture Other sequences hybridize.Stringent condition for sequence dependent and in varied situations can be different.For example, longer sequence compared with Specific hybrid under high-temperature.Stringent hybridization condition including (but not limited to)：(i) than special under definite ion concentration and pH value The heat fusion joint (Tm) of property sequence is about 5 DEG C -10 DEG C low；(ii) it is about 0.01M to about in about pH 7.0 to 8.3 times salinity of about pH 1.0M, and for short probe (including (but not limited to) 10 to 50 nucleotide), temperature is at least about 30 DEG C, and for long probe (including (but not limited to) more than 50 nucleotide) temperature is at least about 60 DEG C；(iii) destabilizing agent is added, it includes (but not It is limited to) formamide；(iv) 50% formamide, 5X SSC and 1%SDS are cultivated or 5X SSC, 1%SDS at 42 DEG C, 65 It is cultivated at DEG C, wherein between being washed about 5 minutes to about 120 minutes in 0.2X SSC and 0.1%SDS at 65 DEG C.Only lift For example, the detection of selectivity or specific hybrid is at least twice of background including (but not limited to) positive signal.On core The detailed guide of acid hybridization sees Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes,"Overview of principles In of hybridization and the strategy of nucleic acid assays " (1993).

As used herein, term " person under inspection " refers to the animal as treatment, observation or experiment object.Only illustrate For, person under inspection can be (but not limited to) mammal, and it includes the (but not limited to) mankind.

As used herein, refer to can be substantially or substantially free of before purification for term " substantially purified " Usually with component of interest or the component of interest of the other components to interact with component of interest.Only citing comes Say, when the preparation of component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, be less than about 10%th, less than about 5%, less than about the 4%, pollution components less than about 3%, less than about 2% or less than about 1% (in terms of dry weight) When, component of interest can be " substantially purified ".Therefore, the component of interest of " substantially purified " can have about 70%th, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or higher is pure Degree is horizontal.Only for example, in the case of the natural amino acid polypeptide or non-natural amino acid polypeptides that are generated in restructuring, natural ammonia Base acid polypeptide or non-natural amino acid polypeptides can be purified from n cell or host cell.For example, when preparation contain it is small In about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, be less than About 3%, less than about 2% or less than about 1% (in terms of dry weight) polluter when, natural amino acid polypeptide or alpha-non-natural amino acid The preparation of polypeptide can be " substantially purified ".For example, when natural amino acid polypeptide or non-natural amino acid polypeptides by When host cell restructuring generates, natural amino acid polypeptide or non-natural amino acid polypeptides can with about the 30% of dry cell weight, about 25%th, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1% or less amount exists.Citing comes It says, when natural amino acid polypeptide or non-natural amino acid polypeptides are recombinated by host cell to be generated, natural amino acid polypeptide or non- Natural amino acid polypeptide can be with the about 5g/L of dry cell weight, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about l00mg/L, about 50mg/L, about 10mg/L or about 1mg/L or less amount are present in culture medium In.For example, the natural amino acid polypeptide of " substantially purified " or non-natural amino acid polypeptides can have such as by appropriate side Method (it includes (but not limited to) SDS/PAGE analyses, RP-HPLC, SEC and Capillary Electrophoresis) measure about 30%, about 35%th, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%th, about 95%, about 99% or higher purity level.

Term " substituent group " (also referred to as " non-interference substituent group ") refers to another group that can be used on displacer molecule Group.These groups are including (but not limited to) halogen, C₁-C₁₀Alkyl, C₂-C₁₀Alkenyl, C₂-C₁₀Alkynyl, C₁-C₁₀Alkoxy, C₅-C₁₂Aralkyl, C₃-C₁₂Cycloalkyl, C₄-C₁₂Cycloalkenyl group, phenyl, be substituted phenyl, tolyl, xylyl, xenyl, C₂-C₁₂Alkoxyalkyl, C₅-C₁₂Alkoxy aryl, C₅-C₁₂Aryloxy alkyl, C₇-C₁₂Oxygroup aryl, C₁-C₆Alkyl sulfenyl Base, C₁-C₁₀Alkyl sulphonyl ,-(CH₂)_m-O-(C₁-C₁₀Alkyl) (wherein m be 1 to 8), aryl, substituted aryl, be substituted alkane Oxygroup, fluoroalkyl, heterocycle, substituted heterocyclic, nitro alkyl ,-NO₂、-CN、-NRC(O)-(C₁-C₁₀Alkyl) ,-C (O)- (C₁-C₁₀Alkyl), C₂-C₁₀Alkylthio alkyl ,-C (O) O- (C₁-C₁₀Alkyl) ,-OH ,-SO₂,=S ,-COOH ,-NR₂, carbonyl ,-C (O)-(C₁-C₁₀Alkyl)-CF₃、-C(O)-CF₃、-C(O)NR₂、-(C₁-C₁₀Aryl)-S- (C₆-C₁₀Aryl) ,-C (O)-(C₆-C₁₀ Aryl) ,-(CH₂)_m-O-(CH₂)_m-O-(C₁-C₁₀Alkyl) (wherein m is respectively 1 to 8) ,-C (O) NR₂、-C(S)NR₂、- SO₂NR₂、-NRC(O)NR₂、-NRC(S)NR₂, its salt and its similar to group.Each R group in previous list include (but Be not limited to) H, alkyl or substituted alkyl, aryl or substituted aryl or alkaryl.When substituent group is write from left to right by it Conventional chemical formulas specify when, be likewise covered by can by write from right to left structure generation chemically identical substituent group, example Such as ,-CH₂O- is equal to-OCH₂-。

Only for example, alkyl and miscellaneous alkyl (are included referred to as alkylidene, alkenyl, sub- miscellaneous alkyl, miscellaneous thiazolinyl, alkynyl, ring Alkyl, Heterocyclylalkyl, those groups of cycloalkenyl group and heterocycloalkenyl) substituent group including (but not limited to)：- OR ,=O ,=NR, =N-OR ,-NR₂,-SR ,-halogen ,-SiR₃、-OC(O)R、-C(O)R、-CO₂R、-CONR₂、-OC(O)NR₂、-NRC(O)R、-NRC (O)NR₂、-NR(O)₂R、-NR-C(NR₂)=NR ,-S (O) R ,-S (O)₂R、-S(O)₂NR₂、-NRSO₂R ,-CN and-NO₂.It is foregoing Each R group in list including (but not limited to) hydrogen, the miscellaneous alkyl for being substituted or being unsubstituted, be substituted or be unsubstituted Aryl (including (but not limited to) the aryl through 1-3 halogen substitution), the alkyl, alkoxy or the sulfane that are substituted or are unsubstituted Oxygroup or aralkyl.When two R groups are connected on same nitrogen-atoms, can combine to form 5 Yuans rings, 6 members with nitrogen-atoms Ring or 7 Yuans rings.For example ,-NR₂It means including (but not limited to) 1- pyrrolidinyls and 4- morpholinyls.

For example, the substituent group of aryl and heteroaryl is including (but not limited to)-OR ,=O ,=NR ,=N-OR ,-NR₂、- SR ,-halogen ,-SiR₃、-OC(O)R、-C(O)R、-CO₂R、-CONR₂、-OC(O)NR₂、-NRC(O)R、-NRC(O)NR₂、-NR (O)₂R、-NR-C(NR₂)=NR ,-S (O) R ,-S (O)₂R、-S(O)₂NR₂、-NRSO₂R、-CN、-NO₂、-R、-N₃、-CH(Ph)₂、 Fluorine (C₁-C₄) alkoxy and fluorine (C₁-C₄) alkyl, the scope of the sum of opening valence state of the number on zero to aromatic ring systems It is interior；And each R group wherein in previous list is including (but not limited to) hydrogen, alkyl, miscellaneous alkyl, aryl and heteroaryl.

As used herein, term " therapeutically effective amount " refer to administration suffered from disease, symptom or illness patient, It is enough to cure or at least partly prevents or mitigate to a certain extent the symptom of at least one treated disease, illness or symptom Composition containing at least one non-natural amino acid polypeptides and/or at least one modified non-natural amino acid polypeptides Amount.The effect of these compositions, it includes the seriousness and mistake of (but not limited to) disease, illness or symptom depending on the following conditions Journey, previous treatment, the health status of patient and to the reaction of drug and the judgement of attending physician.Only for example, treat Effective quantity can be determined by routine experiment (increasing clinical test including (but not limited to) dosage).

As used herein, term " thioalkoxy group " is referred to through oxygen atom and molecular binding containing sulfanyl.

Term " heat fusion joint " or Tm are in the state of the equilibrium 50% probe and the target sequence hybridization with target complementation Temperature (under definite ion concentration, pH value and nucleic acid concentration).

As used herein, term " toxin part " refers to damage or dead compound.

As used herein, term " treatment ", which includes, alleviates, mitigates or improves disease or symptom symptom, prevents other diseases Shape, improves or the potential metabolic disease of prevention symptom is because inhibiting disease or symptom, for example, preventing the development of disease or symptom, mitigating Disease or symptom so that disease or symptom return, and mitigate symptom or the disease of termination disease or symptom as caused by disease or symptom Shape.Term " treatment " is including (but not limited to) preventative and/or therapeutic treatment.

As used herein, term " water-soluble polymer " refers to dissolving in any polymer in aqueous solvent. These water-soluble polymers are including (but not limited to) polyethylene glycol, methoxy PEG-propionaldehyde, its list C₁-C₁₀Alkoxy or aryloxy group spread out Biological (being described in U.S. Patent No. 5,252,714, be to be incorporated herein by reference), mono methoxy-poly- second Glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyaminoacid, divinyl ether maleic anhydride, N- (2- hydroxypropyls)-methyl-prop Acrylamide, glucan, glucan derivative (including dextran sulfate), polypropylene glycol, polypropylene oxide/ethylene oxide copolymerization Object, polyoxyethylated polyols, heparin, heparin fragment, polysaccharide, oligosaccharides, glycan, cellulose and cellulose derivative (include (but not limited to) methylcellulose and carboxymethyl cellulose), seralbumin, starch and starch derivatives, polypeptide, poly- alkylene The copolymer, polyvinyl ethyl ether and alpha-beta-poly- of base glycol and its derivative, polyalkylene glycol and its derivative [(2- ethoxys)-DL- asparagines, with and the like or its mixture.Only for example, these water-soluble polymers with The coupling of natural amino acid polypeptide or non-native polypeptide can generate change, it includes (but not limited to) compared with unmodified shape Formula, water solubility increases, serum half-life increases or regulated, treatment half-life period increases or regulated, biological usability increases Greatly, bioactivity is regulated, circulation time extends, immunogenicity is regulated, physical association characteristic is regulated, it includes (but not limited to) is assembled and polymer is formed, receptor Binding change and one or more kinds of combinations for combining collocation object change Change and the effect of Receptor dimerization or multimerization change.In addition, these water-soluble polymers can have or can not have its own Bioactivity.

Unless otherwise instructed, otherwise using in fields mass spectrum, NMR, HPLC, protein chemistry, biochemistry, again Group DNA technique and pharmacological conventional method.

Compound presented herein is (including (but not limited to) alpha-non-natural amino acid, non-natural amino acid polypeptides and through repairing The non-natural amino acid polypeptides of decorations and the reagent for being used to prepare aforesaid compound) compound through isotope marks is included, It is identical with those compounds described in various formulas presented herein and structure, but one or more atoms are through having The atom substitution of the atomic mass or mass number different from the atomic mass or mass number being generally found in nature.It may be incorporated into this The example of isotope in the compound of invention includes the isotope of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, respectively such as²H、³H、¹³C、¹⁴C、¹⁵N、¹⁸O、¹⁷O、³⁵S、¹⁵F、³⁶Cl.Some compounds through isotope marks described herein are (such as such as³H With¹⁴Those compounds that the radio isotope of C is incorporated in) it is suitable for drug and/or substrate tissue distribution calibrating.In addition, With such as deuterium (i.e.²H isotope substitution) can provide some treatment advantages for thanking to stability generation by higher generation, such as in vivo Half-life period increases or reduction of volume requirements.

Some compounds herein are (including (but not limited to) alpha-non-natural amino acid, non-natural amino acid polypeptides and through repairing The non-natural amino acid polypeptides of decorations and the reagent for being used to prepare aforesaid compound) have asymmetric carbon atom and can therefore with Enantiomter or diastereomeric form exist.Based on its physical chemical differences, by known method (for example, chromatography and/or Fractional crystallization) diastereomeric mixtures can be separated into its indivedual diastereoisomer.Can by by with appropriate optical activity Enantiomeric mixture is changed into diastereomeric mixtures by compound (for example, alcohol) reaction, separate diastereoisomer and By indivedual diastereoisomers transformation (for example, hydrolysis) for corresponding pure enantiomter and by stage enantiomer separation.Comprising non- All these isomers of enantiomter, enantiomter and its mixture are considered the portion of composition described herein Point.

In additional or other embodiment, compound described herein is (including (but not limited to) alpha-non-natural amino acid, non- Natural amino acid polypeptide and modified non-natural amino acid polypeptides and the reagent for being used to prepare aforesaid compound) it is in the past The form of medicine uses.In additional or other embodiment, compound described herein is (including (but not limited to) non-natural amino Acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and the reagent for being used to prepare aforesaid compound) Administration needs the organism for generating metabolin to be metabolized afterwards, and the metabolin is used subsequently to generate required effect, and it includes required Response to treatment.It is alpha-non-natural amino acid and " through modification or unmodified " alpha-non-natural amino acid in other or Additional examples of composition The active metabolite of polypeptide.

Method described herein and formula are included using alpha-non-natural amino acid, non-natural amino acid polypeptides and modified N- oxides, crystal form (also referred to as polymorph) or the pharmaceutically acceptable salt of non-natural amino acid polypeptides. In some embodiments, alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides can be mutual Tautomeric forms thereof exists.All tautomers are both contained in alpha-non-natural amino acid presented herein, alpha-non-natural amino acid In the scope of polypeptide and modified non-natural amino acid polypeptides.In addition, alpha-non-natural amino acid described herein, non-natural ammonia Base acid polypeptide and modified non-natural amino acid polypeptides can with non solvate form and with it is pharmaceutically acceptable The solvate form thereof that solvent (such as water, ethyl alcohol with and the like) is formed exists.Alpha-non-natural amino acid presented herein, The solvate form thereof of non-natural amino acid polypeptides and modified non-natural amino acid polypeptides is also considered as disclosing in this article.

Some compounds herein are (including (but not limited to) alpha-non-natural amino acid, non-natural amino acid polypeptides and through repairing The non-natural amino acid polypeptides of decorations and the reagent for being used to prepare aforesaid compound) can exist with several tautomeric forms.Institute There is the part that these tautomeric forms are considered composition described herein.Equally, such as any chemical combination herein Object (including (but not limited to) alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and Be used to prepare the reagent of aforesaid compound) all enol-keto forms be considered the part of composition described herein.

Some compounds herein are (including (but not limited to) alpha-non-natural amino acid, non-natural amino acid polypeptides and through repairing The non-natural amino acid polypeptides of decorations and the reagent of any one for being used to prepare aforesaid compound) have it is acid and can be with medicine and pharmacology Upper acceptable salt forming cation.Some compounds herein are (including (but not limited to) alpha-non-natural amino acid, non-natural ammonia Base acid polypeptide and modified non-natural amino acid polypeptides and the reagent for being used to prepare aforesaid compound) can have alkalescence and because This can be with pharmaceutically acceptable anion forming salt.The composition that all these salt comprising disalt are described herein In scope and it can be prepared by conventional method.For example, salt can be by aqueous, non-aqueous or part aqueous medium Acidic entity is made to contact to prepare with basic entities.Salt is recycled by using at least one of following technology：Filtering；With non- Solvent deposition then filters；Evaporate solvent；Or (in the case of aqueous solution) is lyophilized.

The acid proton present in the parent non-natural amino acid polypeptides is through metal ion (such as alkali metal ion, alkaline earth Metal ion or aluminium ion) displacement or when being coordinated with organic base, the doctor of non-natural amino acid polypeptides disclosed herein can be formed Pharmaceutically acceptable salt.In addition, initial substance or intermediate can be used in the salt form of disclosed non-natural amino acid polypeptides Salt prepare.Non-natural amino acid polypeptides described herein can be by making non-natural amino acid polypeptides described herein Free alkali form is prepared as pharmaceutically acceptable acid-addition salts with pharmaceutically acceptable inorganic acid or organic acid reaction (it is the type of pharmaceutically acceptable salt).Alternatively, non-natural amino acid polypeptides described herein can be by making herein Described in non-natural amino acid polypeptides free acid form and pharmaceutically acceptable inorganic base or organic base reaction prepare For pharmaceutically acceptable base addition salts (it is the type of pharmaceutically acceptable salt).

The type of pharmaceutically acceptable salt including (but not limited to)：(1) acid formed with inorganic acid or organic acid adds Into salt, wherein the inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and its similar acid；The organic acid is such as Acetic acid, propionic acid, caproic acid, pentamethylene propionic acid, glycolic, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, rich horse Acid, tartaric acid, citric acid, benzoic acid, 3- (4- hydroxy benzoyls) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2- ethionic acids, 2- ethylenehydrinsulfonic acids, benzene sulfonic acid, 2- naphthalene sulfonic acids, 4- methyl bicycles-[2.2.2] oct-2-ene -1- carboxylic acids, Portugal Heptonic acid, 4,4' methylene bis-(3- hydroxyl -2- alkene -1- carboxylic acids), 3- phenylpropionic acids, trimethylace tonitric, butylacetic acid, the moon Osmanthus base sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, viscous furancarboxylic acid and its similar acid；(2) parent is worked as Acid proton present in compound is replaced through metal ion (such as alkali metal ion, alkaline-earth metal ions or aluminium ion)；Or The salt formed when being coordinated with organic base.Acceptable organic base includes ethanolamine, diethanol amine, triethanolamine, trometamol, N- Methylglucosamine with and the like.Acceptable inorganic base includes aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, hydrogen Sodium oxide molybdena with and the like.

A variety of methods can be used to analyze for the corresponding counter ion counterionsl gegenions of the pharmaceutically acceptable salt of non-natural amino acid polypeptides And discriminating, these methods are including (but not limited to) ion-exchange chromatography, ion chromatography, Capillary Electrophoresis, inductive couple plasma Body, atomic absorption spectrum, mass spectrum or any combination thereof.In addition, the salt that these non-natural amino acid polypeptides are pharmaceutically acceptable Therapeutic activity technology described in example 87-91 and method can be used to test.

It will be appreciated that salt is referred to comprising its solvent addition form or crystal form, especially solvate or polymorph.It is molten Agent closes object and contains the solvent of stoichiometry or the amount of non-stoichiometry, and usually in crystallization process with it is pharmaceutically acceptable Solvent (such as water, ethyl alcohol with and the like) is formed.Hydrate is formed when solvent is water or forms alcohol when solvent is alcohol Compound.Polymorph includes the different crystal assembled arrangement of the compound of identical element composition.Polymorph usually has different X Ray diffraction pattern, infrared spectrum, fusing point, density, hardness, crystal shape, optical property and electrical properties, stability and dissolving Property.The various factors for such as recrystallizing solvent, crystalline rate and storage temperature may be such that single crystal form is dominant.

The screening of the pharmaceutically acceptable salt polymorph of non-natural amino acid polypeptides and/or solvate and characterization can Realized using multiple technologies, the technology including (but not limited to) heat analysis, X-ray diffraction, spectroscopy, vapor sorption and Microexamination.Heat analysis method is absorbed in heat chemistry degradation or thermal physical process (turns including (but not limited to) polymorph Become), and using these methods analyze polymorphic forms between relation, measure weight loss with find out glass transition temperature or For excipient Study on Compatibility.These methods are including (but not limited to) differential scanning calorimetry (DSC), modulation differential scanning amount Hot method (MDSC), thermogravimetric analysis (TGA) and thermogravimetric and infrared analysis (TG/IR).Method of X-ray diffraction includes (but unlimited In) monocrystalline and Powder Diffractometer and synchrotron source.The various spectral techniques used including (but not limited to) Raman, FTIR, UVIS and NMR (liquid and solid-state).Various microscopies are including (but not limited to) polarization microscope, adjoint Energy dispersion-type X-ray analysis (EDX) scanning electron microscope (SEM), with EDX environmental scanning electron microscope ( In gas or steam atmosphere), IR microscopes and Raman microscope.

Description of the drawings

Described in detail below by referring to proposition illustrative embodiment is obtained to the method and composition of the present invention The more preferably understanding of feature and advantage, using the principle of our method, composition, equipment and device in the embodiment, and it is attached It is with schema：

Fig. 1 presentations method described herein, composition, strategy and technology some aspects relation diagrammatic representation Figure.

Illustrative, the non-limiting examples of the type of Fig. 2 presentations alpha-non-natural amino acid described herein.These non-naturals Amino acid can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino In any one of the method for sour polypeptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Illustrative, the non-limiting examples of the type of Fig. 3 presentations alpha-non-natural amino acid described herein.These non-naturals Amino acid can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino In any one of the method for sour polypeptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Fig. 4 presentations are used to prepare illustrative, the nonrestrictive reality of the synthetic method of alpha-non-natural amino acid described herein Example.These alpha-non-natural amino acids can be used for or be incorporated to being used to prepare, purifying, characterizing and using non-natural amino described herein Any in acid, the method for non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, composition, technology and strategy In person.

Fig. 5 presentations are used to prepare illustrative, the nonrestrictive reality of the synthetic method of alpha-non-natural amino acid described herein Example.These alpha-non-natural amino acids can be used for or be incorporated to being used to prepare, purifying, characterizing and using non-natural amino described herein Any in acid, the method for non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, composition, technology and strategy In person.

Fig. 6 presentations are used to prepare illustrative, the nonrestrictive reality of the synthetic method of alpha-non-natural amino acid described herein Example.These alpha-non-natural amino acids can be used for or be incorporated to being used to prepare, purifying, characterizing and using non-natural amino described herein Any in acid, the method for non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, composition, technology and strategy In person.

Fig. 7 presentations are modified to be formed with non-natural amino acid polypeptides of the reagent posttranslational modification containing carbonyl containing azanol The illustrative of the non-natural amino acid polypeptides containing oxime, non-limiting examples.These non-natural amino acid polypeptides can be used for or be incorporated to use In preparation, purifying, characterization and use alpha-non-natural amino acid described herein, non-natural amino acid polypeptides and modified non-day In any one of method, composition, technology and strategy of right amino acid polypeptide.

Fig. 8 presentations can be used for the reaction of non-natural amino acid polypeptides of the enhancing containing carbonyl and the reagent containing azanol to form warp Illustrative, the non-limiting examples of the additive of the non-natural amino acid polypeptides containing oxime of modification.

Fig. 9 presentations are with non-natural amino acid polypeptides of the reagent posttranslational modification containing oxime containing carbonyl to form modified contain Illustrative, the non-limiting examples of oxime non-natural amino acid polypeptides.These non-natural amino acid polypeptides can be used for or be incorporated to be used for It prepares, purify, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides and modified non-natural In any one of method, composition, technology and strategy of amino acid polypeptide.

Figure 10 presentations with non-natural amino acid polypeptides of the reagent posttranslational modification containing azanol containing carbonyl with formed through modification The illustrative of the non-natural amino acid polypeptides containing oxime, non-limiting examples.These non-natural amino acid polypeptides can be used for or be incorporated to It is used to prepare, purifies, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides and modified non- In any one of method, composition, technology and strategy of natural amino acid polypeptide.

Figure 11 presentations can be used for modification non-natural amino acid polypeptides to form the alpha-non-natural amino acid bonded through oxime containing PEG The illustrative of the reagent containing PEG of polypeptide, non-limiting examples.

Figure 12 presentations can be used for modification non-natural amino acid polypeptides to form the alpha-non-natural amino acid bonded through oxime containing PEG Illustrative, the non-limiting examples of the synthesis of the reagent containing PEG of polypeptide.

Figure 13 presentations can be used for modification non-natural amino acid polypeptides to form the alpha-non-natural amino acid bonded through oxime containing PEG Illustrative, the non-limiting examples of the synthesis containing amide groups PEG reagents of polypeptide.

Figure 14 presentations can be used for modification non-natural amino acid polypeptides to form the alpha-non-natural amino acid bonded through oxime containing PEG Illustrative, the non-limiting examples of the synthesis containing carbamate groups PEG reagents of polypeptide.

Figure 15 presentations can be used for modification non-natural amino acid polypeptides to form the alpha-non-natural amino acid bonded through oxime containing PEG Illustrative, the non-limiting examples of the synthesis containing carbamate groups PEG reagents of polypeptide.

Figure 16 presentations can be used for modification non-natural amino acid polypeptides to form the alpha-non-natural amino acid bonded through oxime containing PEG Illustrative, the non-limiting examples of the synthesis containing simple PEG reagents of polypeptide.

Figure 17 presentations can be used for modification non-natural amino acid polypeptides to form the alpha-non-natural amino acid bonded through oxime containing PEG A kind of the illustrative of reagent, non-limiting examples and this reagent of the PEG containing branch of polypeptide is for modification based on the non-of carbonyl The purposes of natural amino acid polypeptide.

Figure 18 presentations can be used for saying for the synthesis of bifunctional linker's group of modification and bonded non-natural amino acid polypeptides Bright property, non-limiting examples.

Figure 19 presentations can be used for the illustrative, non-of the multifunctional connection subbase group of modification and bonded non-natural amino acid polypeptides Limitative examples.

Figure 20 presentation bifunctional linkers group is for modifying non-natural amino acid polypeptides and by non-natural amino acid polypeptides Illustrative, the nonrestrictive representative graph of the bonded purposes to PEG group.

Figure 21 presentation bifunctional linkers group is for modifying non-natural amino acid polypeptides and by non-natural amino acid polypeptides The illustrative of the bonded purposes to PEG group, non-limiting examples.

Figure 22 presentation bifunctional linker groups are bonded together to form homotype two by two non-natural amino acid polypeptides Illustrative, the nonrestrictive representative graph of the purposes of aggressiveness.

Figure 23 presentation bifunctional linker groups are bonded together different to be formed by two different non-natural amino acid polypeptides Illustrative, the nonrestrictive representative graph of the purposes of matter dimer.

Illustrative, the nonrestrictive representative graph of the synthesis of alpha-non-natural amino acid of Figure 24 presentations containing carbonyl.These non-naturals Amino acid can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino In any one of the method for sour polypeptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Illustrative, the nonrestrictive representative graph of the synthesis of alpha-non-natural amino acid of Figure 25 presentations containing dicarbapentaborane.These non-days Right amino acid can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural ammonia In any one of the method for base acid polypeptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Illustrative, the nonrestrictive representative graph of the synthesis of alpha-non-natural amino acid of Figure 26 presentations containing dicarbapentaborane.

Illustrative, the nonrestrictive representative graph of the synthesis of alpha-non-natural amino acid of Figure 27 presentations containing dicarbapentaborane.These non-days Right amino acid can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural ammonia In any one of the method for base acid polypeptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Illustrative, the nonrestrictive representative graph of the synthesis of alpha-non-natural amino acid of Figure 28 presentations containing dicarbapentaborane.These non-days Right amino acid can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural ammonia In any one of the method for base acid polypeptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Illustrative, the nonrestrictive representative graph of the synthesis of alpha-non-natural amino acid of Figure 29 presentations containing dicarbapentaborane.These non-days Right amino acid can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural ammonia In any one of the method for base acid polypeptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Illustrative, the nonrestrictive representative graph of the synthesis of alpha-non-natural amino acid of Figure 30 presentations containing dicarbapentaborane.These non-days Right amino acid can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural ammonia In any one of the method for base acid polypeptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Illustrative, the nonrestrictive representative graph of the synthesis of alpha-non-natural amino acid of Figure 31 presentations containing dicarbapentaborane.These non-days Right amino acid can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural ammonia In any one of the method for base acid polypeptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Illustrative, the nonrestrictive representative graph of the synthesis of alpha-non-natural amino acid of Figure 32 presentations containing dicarbapentaborane.These non-days Right amino acid can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural ammonia In any one of the method for base acid polypeptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Illustrative, the nonrestrictive representative graph of the synthesis of alpha-non-natural amino acid of Figure 33 presentations containing dicarbapentaborane.These non-days Right amino acid can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural ammonia In any one of the method for base acid polypeptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Illustrative, the nonrestrictive representative graph of the synthesis of alpha-non-natural amino acid of Figure 34 presentations containing dicarbapentaborane.These non-days Right amino acid can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural ammonia In any one of the method for base acid polypeptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Alpha-non-natural amino acid of Figure 35 presentations containing carbonyl and the alpha-non-natural amino acid containing dicarbapentaborane it is illustrative, nonrestrictive Representative graph.These alpha-non-natural amino acids can be used for or be incorporated to being used to prepare, purifying, characterizing and using non-natural described herein In amino acid, the method for non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, composition, technology and strategy In any one.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 36 presentation alpha-non-natural amino acids.These alpha-non-natural amino acids can For or be incorporated to be used to prepare, purify, characterize and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides and In any one of method, composition, technology and strategy of modified non-natural amino acid polypeptides.

The synthesis of alpha-non-natural amino acid of Figure 37 presentations containing carbonyl and the alpha-non-natural amino acid containing dicarbapentaborane it is illustrative, non- Restricted representative graph.These alpha-non-natural amino acids can be used for or be incorporated to being used to prepare, purifying, characterizing and using described herein Alpha-non-natural amino acid, the method for non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, composition, technology and plan In any one of summary.

The synthesis of alpha-non-natural amino acid of Figure 38 presentations containing carbonyl and the alpha-non-natural amino acid containing dicarbapentaborane it is illustrative, non- Restricted representative graph.These alpha-non-natural amino acids can be used for or be incorporated to being used to prepare, purifying, characterizing and using described herein Alpha-non-natural amino acid, the method for non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, composition, technology and plan In any one of summary.

The synthesis of alpha-non-natural amino acid of Figure 39 presentations containing carbonyl and the alpha-non-natural amino acid containing dicarbapentaborane it is illustrative, non- Restricted representative graph.These alpha-non-natural amino acids can be used for or be incorporated to being used to prepare, purifying, characterizing and using described herein Alpha-non-natural amino acid, the method for non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, composition, technology and plan In any one of summary.

Illustrative, the nonrestrictive representative graph of alpha-non-natural amino acid of Figure 40 presentations containing dicarbapentaborane.These non-natural aminos Acid can be used for or be incorporated to being used to prepare, purifying, characterizing and more using alpha-non-natural amino acid described herein, alpha-non-natural amino acid In any one of the method for peptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Illustrative, the nonrestrictive representative graph of alpha-non-natural amino acid of Figure 41 presentations containing dicarbapentaborane.These non-natural aminos Acid can be used for or be incorporated to being used to prepare, purifying, characterizing and more using alpha-non-natural amino acid described herein, alpha-non-natural amino acid In any one of the method for peptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Illustrative, the nonrestrictive representative graph of alpha-non-natural amino acid of Figure 42 presentations containing dicarbapentaborane.These non-natural aminos Acid can be used for or be incorporated to being used to prepare, purifying, characterizing and more using alpha-non-natural amino acid described herein, alpha-non-natural amino acid In any one of the method for peptide and modified non-natural amino acid polypeptides, composition, technology and strategy.

Figure 43 presentations (a) are through protection or the alpha-non-natural amino acid of the keto-aldehyde Han 1,3- and (b) ketone containing 1-3- without protection Illustrative, the nonrestrictive representative graph of the alpha-non-natural amino acid of carboxyl (sulphur) ester.These alpha-non-natural amino acids can be used for or be incorporated to It is used to prepare, purifies, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides and modified non- In any one of method, composition, technology and strategy of natural amino acid polypeptide.

Illustrative, the nonrestrictive representative graph of alpha-non-natural amino acid of Figure 44 presentations containing hydrazides.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of alpha-non-natural amino acid of Figure 45 presentations containing hydrazides.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of alpha-non-natural amino acid of Figure 46 A and the 46B presentation containing oxime, and Figure 46 C presentations contain Illustrative, the nonrestrictive representative graph of the alpha-non-natural amino acid of hydrazine.These alpha-non-natural amino acids can be used for or be incorporated to be used to prepare, Purifying characterizes and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides and modified non-natural amino In any one of method, composition, technology and strategy of sour polypeptide.

One step of Figure 47 presentations is bonded to non-natural amino acid polypeptides and two steps are bonded to the explanation of non-natural amino acid polypeptides Property, nonrestrictive representative graph.For example, these engagements are PEGylated comprising non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 48 presentation mPEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 49 presentation mPEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 50 presentation mPEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 51 presentation mPEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 52 presentation mPEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 53 presentation mPEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 54 presentation mPEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 55 presentation mPEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 56 presentation mPEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 57 presentation mPEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 58 presentation mPEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 59 presentation mPEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 60 presentation hydroxylamine compounds.These alpha-non-natural amino acids can use In or be incorporated to and be used to prepare, purify, characterize and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides and warp In any one of method, composition, technology and strategy of the non-natural amino acid polypeptides of modification.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 61 presentation mPEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 62 A presentation hydroxylamine compounds；Figure 62 B presentation mPEG compounds Synthesis illustrative, nonrestrictive representative graph.These alpha-non-natural amino acids can be used for or be incorporated to being used to prepare, purifying, characterizing With the side for using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides In any one of method, composition, technology and strategy.

Figure 63 presentations (A) are modified by chemical transformation for (including containing dicarbapentaborane) non-natural amino acid polypeptides containing carbonyl Non-natural amino acid polypeptides and (B) modify alpha-non-natural amino acid by chemical transformation for the non-natural amino acid polypeptides containing azanol Illustrative, the non-limiting examples of polypeptide.These non-natural amino acid polypeptides can be used for or be incorporated to being used to prepare, purifying, characterizing With the side for using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides In any one of method, composition, technology and strategy.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 64 presentation PEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Illustrative, the nonrestrictive representative graph of the synthesis of Figure 65 presentation PEG- hydroxylamine compounds.These alpha-non-natural amino acids It can be used for or be incorporated to being used to prepare, purifying, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides In any one of method, composition, technology and strategy with modified non-natural amino acid polypeptides.

Specific embodiment

I. introduction

In recent years, the brand new technical in reporter protein matter science wishes to overcome numerous with protein site-specific The relevant limitation of sex modification.Specifically, by new component be added to prokaryotes Escherichia coli (E.coli) (for example, L.Wang et al., (2001),Science292:498-500) and eucaryote saccharomyces cerevisiae (Sacchromyces Cerevisiae) (S.cerevisiae) (for example, J.Chin et al.,Science301:964-7 (2003)) protein life In object synthesis mechanism, make it possible to that in vivo alpha-non-natural amino acid is incorporated in protein.Using the method, there is novelty Chemistry, many amino acids of physics or biological property are (comprising photoaffinity mark and can photoisomerization amino acid, keto amino acid And glycosylated amino acid) effectively and hi-fi it is close to respond amber in the protein that is incorporated in Escherichia coli and yeast Numeral TAG.Referring to (for example) J.W.Chin et al., (2002),Journal of the American Chemical Society124:9026-9027 (is fully incorporated) by reference；J.W.Chin and P.G.Schultz, (2002),ChemBioChem3(11):1135-1137 (is fully incorporated) by reference；J.W.Chin, et al., (2002),PNAS United States of America99(17):11020-11024 (is fully incorporated) by reference；And L.Wang And P.G.Schultz, (2002),Chem.Comm.,1-11 (is fully incorporated) by reference.These researchs have had proven to can Energy is selective and is routinely introduced into the chemical functional group being not present in protein, in 20 kinds of common inherited coded amino acids Existing all functional groups are chemically inert, and it can be used for effectively and selectively reacting to form stable covalent bond.

II. summarize

General introduction of Fig. 1 presentations to composition described herein, methods and techniques.In a way, use described herein Non-natural in generating and using including at least one alpha-non-natural amino acid or being modified through carbonyl, dicarbapentaborane, oximido or azanol base The means (method, composition, technology) of the polypeptide of amino acid.These alpha-non-natural amino acids can contain other functional groups, it includes (but not limited to)：Mark；Dyestuff；Polymer；Water-soluble polymer；The derivative of polyethylene glycol；Photocrosslinking agent；Cytotoxicity Close object；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Resin；Second protein or polypeptide or polypeptide Like object；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbohydrate；Polynucleotide；DNA；RNA；Antisense Polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial；Nano-particle；Spin labeling；Fluorogen；Contain The part of metal；Radioactive segment；Novel functional group；With other molecule covalents or the group of noncovalent interaction；Light cage is covered Part；Can actinic radiation excitation part；Ligand；It can photoisomerization part；Biotin；Biotin analog；It is combined with weight The part of atom；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active Agent；Aminothio acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups； Electron dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；(in the case, biology is living for bioactivator Property agent can include with therapeutic activity medicament and non-natural amino acid polypeptides or modified alpha-non-natural amino acid may act as with The common co-therapeutic agent of adjunctive therapy agent or as the means that therapeutic agent is delivered to required site in organism)；Detectable mark Note；Small molecule；Inhibition ribonucleic acid；Radioactive nucleotides；Neutron capture agent；The derivative of biotin；Quantum dot；Nanometer passes Pass element；Radioactivity transfers element；Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiogenesis suppression Make element, antihormones, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecular, intend epitope, receptor, instead Micella and any combination thereof.It should be noted that various foregoing functional groups are not the member's unclassified for meaning and implying a kind of functional group For the member of another functional group.Certainly, can be overlapped according to particular case.Only for example, water-soluble polymer is with gathering The derivative of ethylene glycol is overlapped in scope, however not fully and therefore two kinds of functional groups are listed in above for overlapping.

As shown in fig. 1, on the one hand to be selected and design is intended to repair using method described herein, composition and technology The method of the polypeptide of decorations.From the beginning new polypeptide can design, it includes (only for example) to be used as high-throughput screening methods (in this feelings Under condition, can design, synthesize, characterize and/or test numerous polypeptides) part or according to the interest of researcher design.New polypeptide Also can be designed according to the structure of known or characterizing part polypeptide.Only for example, growth hormone gene superfamily (Growth Hormone Gene Superfamily) (seeing below) has become the widely studied theme of scientific community；It is novel more Peptide can be designed according to the structure of the member of this gene superfamilies.The original for selecting which amino acid to substitute and/or modify It then individually describes in this article.The selection modified using which kind of is also described in this article, and available for meeting experimenter or most The demand of whole user.These demands can be including (but not limited to) the therapeutic efficiency for manipulating polypeptide；The security for improveing polypeptide is general Condition；Adjust pharmacokinetics, pharmacology and/or the drug effect of polypeptide；Such as, (only for example) increase water-soluble, biology can use Property；Increase serum half-life；Increase treatment half-life period；Adjust immunogenicity；Adjust bioactivity；Or extend circulation time.Separately Outside, these modifications include (only for example) provides other functional groups to polypeptide；Label, mark or detectable are incorporated into polypeptide Signal；The separating property of easyization polypeptide and any combinations of foregoing modification.

It is also described herein to have modified or be through modifying with the non-natural containing oximido, carbonyl, dicarbapentaborane or azanol base Amino acid.This aspect is comprising being used to prepare, purify, characterizing and the method using these alpha-non-natural amino acids.It is described herein Another aspect be method, strategy and the technology in polypeptide of being incorporated at least one alpha-non-natural amino acid.This aspect is same Sample includes and is used to prepare, purifies, characterizing and the method using these polypeptides containing at least one alpha-non-natural amino acid.This A aspect is equally comprising being used to prepare, purify, characterizing and using available for preparing (at least partly) containing at least one non-day The composition and method of the oligonucleotides (including DNA and RNA) of the polypeptide of right amino acid.This aspect equally includes to make Standby, purifying, characterization and use, which can be expressed, can be used for manufacturing the polypeptide of (at least partly) containing at least one alpha-non-natural amino acid These oligonucleotides cell composition and method.

Therefore, provided herein and description includes at least one alpha-non-natural amino acid or through carbonyl, dicarbapentaborane, oximido or hydroxyl The polypeptide of the alpha-non-natural amino acid of amido modification.In certain embodiments, have at least one alpha-non-natural amino acid or through carbonyl, The polypeptide of the alpha-non-natural amino acid of dicarbapentaborane, oximido or the modification of azanol base is included on some positions on polypeptide at least once Posttranslational modification.In some embodiments, common translation or posttranslational modification by cell mechanism (for example, glycosylation, acetylation, Acylated, lipid-modified, palmitoylation, palmitate addition, phosphorylation, glycolipids key modification and its similar to mechanism) occur, In many cases, these common translations or posttranslational modification based on cell mechanism betide the naturally occurring amino acid position on polypeptide At point, however, in certain embodiments, common translation or posttranslational modification based on cell mechanism are happened at the non-natural on polypeptide On amino acid sites.

In other embodiments, posttranslational modification does not utilize cell mechanism, but by using chemistry described herein Method or suitable for specific reactivity group other methods by the molecule including the second reactive group (comprising (but unlimited In) mark；Dyestuff；Polymer；Water-soluble polymer；The derivative of polyethylene glycol；Photocrosslinking agent；Cytotoxic compound；Medicine Object；Affinity marker；Photoaffinity marks；Reactive compounds；Resin；Second protein or polypeptide or polypeptide analog；It is anti- Body or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbohydrate；Polynucleotide；DNA；RNA；Antisense poly-nuclear glycosides Acid；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial；Nano-particle；Spin labeling；Fluorogen, containing metal Part；Radioactive segment；Novel functional group；With other molecule covalents or the group of noncovalent interaction；Light cage covers part；It can The part of actinic radiation excitation；Ligand；It can photoisomerization part；Biotin；Biotin analog；It is combined with the portion of heavy atom Point；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active agent；Amino Thio-acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electronics causes Close group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；Small molecule；Inhibit Property ribonucleic acid, radioactive nucleotides；Neutron capture agent；The derivative of biotin；Quantum dot；Nanometer transfers element；Radioactivity passes Pass element；It is abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, antihormones, anti- Oxidant, aptamer, saponin, shuttle vector, macromolecular, intend epitope, receptor, reverse micelle and its is any at guide RNA Combination) at least one first reactive group that includes is connected to (including (but not limited to) containing ketone, aldehyde, acetal, hemiacetal, oxime Or the alpha-non-natural amino acid of azanol functional group) alpha-non-natural amino acid functional group is provided.In certain embodiments, common translation or Posttranslational modification is in vivo carried out in eucaryote or non-eucaryote.In certain embodiments, posttranslational modification is not It is in vitro carried out using cell mechanism.This aspect, which equally includes, to be used to prepare, purifies, characterizing and using containing at least one The method of these polypeptides of these alpha-non-natural amino acids through common translation or posttranslational modification.

Method described herein, composition, strategy and technology scope in also comprising can be with the part as polypeptide Alpha-non-natural amino acid (containing carbonyl or dicarbapentaborane, oximido, azanol base or its masking or forms of protection) reaction it is any to generate The reagent modified after foregoing translation.In general, the alpha-non-natural amino acid of modification should contain at least one oxime after gained is translated Base；The modified alpha-non-natural amino acid containing oxime of gained can be subjected to subsequent modification reaction.This aspect, which also includes, to be used to prepare, is pure The method of change, characterization and the reagent using any posttranslational modification that can carry out the alpha-non-natural amino acid.

In certain embodiments, polypeptide includes a kind of at least one common translation in vivo carried out by host cell or turns over It is modified after translating, wherein posttranslational modification is not carried out usually by another host cell species.In certain embodiments, polypeptide includes At least one common translation in vivo carried out by eucaryote or posttranslational modification, wherein common translation or posttranslational modification are usual It is not carried out by non-eucaryote.These common translations or the example of posttranslational modification are including (but not limited to) glycosylation, acetylation, acyl Change, lipid-modified, palmitoylation, palmitate addition, phosphorylation, glycolipids key modification and its similar modification.In an embodiment In, common translation or posttranslational modification include by GlcNAc- asparagines it is bonded by oligosaccharides be connected to asparagine (include (but It is not limited to), wherein oligosaccharides includes (GlcNAc-Man)₂- Man-GlcNAc-GlcNAc, with and the like).In another implementation In example, common translation or posttranslational modification include by GalNAc- serines, GalNAc- threonines, GlcNAc- serines or GlcNAc- threonines are bonded oligosaccharides is connected to (including (but not limited to) Gal-GalNAc, Gal-GlcNAc etc.) serine or Threonine.In certain embodiments, protein or polypeptide may include secretion or positioning sequence, epitope label, FLAG marks Label, polyhistidyl tags, GST fusions and/or its analog.This aspect, which equally includes, to be used to prepare, purify, characterizes and uses The method of these polypeptides containing this at least one common translation or posttranslational modification.In other embodiments, non-natural is glycosylated Amino acid polypeptide is prepared with nonglycosylated form.This nonglycosylated form for glycosylating alpha-non-natural amino acid can be by following methods Or the combination of these any methods generates, the method is included from through separation or substantially purified or not purified glycosylation Chemistry or enzymatic remove oligosaccharides group in non-natural amino acid polypeptides；In not glycosylating in the host of this non-natural amino acid polypeptides (host includes prokaryotes or eucaryote through being engineered or being mutated not glycosylate this polypeptide) generates non-natural ammonia Base acid, the non-natural amino acid polypeptides are the cell culture generated by that would generally glycosylate the eucaryote of this polypeptide thereto Glycosylation inhibitor is introduced in base.(usually glycosylation means works as usual glycosylation non-natural amino acid polypeptides also described herein Can glycosylated polypeptide when naturally occurring polypeptide is through preparing under the conditions of glycosylated) these nonglycosylated forms.Certainly, lead to Often these nonglycosylated forms (or any polypeptide actually described herein) of glycosylation non-natural amino acid polypeptides can be Not purified form, substantial purified form or separated form.

In certain embodiments, non-natural amino acid polypeptides include at least one translation carried out in the presence of accelerating agent After modify, wherein posttranslational modification be stoichiometry, class stoichiometry or close to stoichiometry.In other embodiments, exist Contact polypeptide and the reagent of formula (XIX) in the presence of accelerating agent.In other embodiments, accelerating agent is selected from by following object The group of composition：

Polypeptide containing alpha-non-natural amino acid can contain it is at least one, at least two, at least three, at least four, at least Five, at least six, at least seven, at least eight, at least nine or ten or ten or more contain carbonyl or dicarbapentaborane, oxime Base, azanol base or its alpha-non-natural amino acid through forms of protection.Alpha-non-natural amino acid may be the same or different, for example, including 1, 2nd, 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 20 or more different alpha-non-natural amino acids Protein in may be present 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 20 with Upper different loci.In certain embodiments, by protein it is naturally occurring in the form of existing at least one of specific amino acids (but all or less than) substitutes through alpha-non-natural amino acid.

Provided herein and description method and composition include it is at least one containing carbonyl or dicarbapentaborane, oximido, Azanol base or its polypeptide through protecting or sheltering the alpha-non-natural amino acid of form.At least one alpha-non-natural amino acid is introduced into polypeptide In may be allowed application include specified chemical reaction (including (but not limited to) with one or more alpha-non-natural amino acids reaction, without with Usually existing 20 kinds of amino acid reaction) engagement chemistry.Once being incorporated to, then this can also be used in non-naturally-occurring amino acid side chain Described in the text or modify suitable for specific functional group present in natural coded amino acid or the chemical method of substituent group.

Alpha-non-natural amino acid method and composition described herein is provided with a variety of functional groups, substituent group or partial The binding element of substance and other substances, other described substances including (but not limited to)：Mark；Dyestuff；Polymer；Water-soluble polymeric Object；The derivative of polyethylene glycol；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reaction Property compound；Resin；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor； Aliphatic acid；Carbohydrate；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, ring paste Essence, biomaterial；Nano-particle；Spin labeling；Fluorogen；Part containing metal；Radioactive segment；Novel functional group；With it The group of his molecule covalent or noncovalent interaction；Light cage covers part；Can actinic radiation excitation part；Ligand；It can light Isomerized part；Biotin；Biotin analog；It is combined with the part of heavy atom；The group chemically cracked；It can photodestruciton Group；Extended side chain；The bonded sugar of carbon；Redox active agent；Aminothio acid；Toxin part；Through isotope marks Part；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electron dense group；Magnetic group；It is inserted into group；Color development Group；Energy transfer agent；Bioactivator；Detectable label；Small molecule；Inhibition ribonucleic acid；Radioactive nucleotides；Neutron is captureed Obtain agent；The derivative of biotin；Quantum dot；Nanometer transfers element；Radioactivity transfers element；Abzyme, activation composite activating agent, disease Poison, adjuvant, anaphylactogen, angiostatin, antihormones, antioxidant, aptamer, guide RNA, saponin, are worn aglycone Shuttle carrier, macromolecular intend epitope, receptor, reverse micelle and any combination thereof.

In certain embodiments, the alpha-non-natural amino acid of the compound described herein comprising formula (I)-(XXXIII), Non-natural amino acid polypeptides, connexon and reagent are under appropriate acid condition (including (but not limited to) pH 2 to 8) in aqueous solution Middle stabilization.In other embodiments, these compounds are in appropriate stable under acidic conditions at least one moon.In other embodiment In, these compounds were in appropriate stable under acidic conditions at least 2 weeks.In other embodiments, these compounds are in appropriate acidity Under the conditions of stablize at least 5 days.

Composition described herein, method, the another aspect of technology and strategy are for studying or using foregoing " through repairing Decorations are unmodified " methods of any one of non-natural amino acid polypeptides.(only for example) meeting is included in this aspect The treatment that benefits from the polypeptide including " through modification or unmodified " non-natural amino acid polypeptides or protein, diagnosis, base In calibrating, industry, cosmetics, phytobiology, environment, energy production, consumer products and/or military purposes.

III. in polypeptide alpha-non-natural amino acid positioning

Method and composition described herein includes and one or more alpha-non-natural amino acids is incorporated in polypeptide.One or more Alpha-non-natural amino acid can be incorporated in one or more specific locations for not destroying polypeptide active.This measure can be by carrying out " conservative " Substitution is (including (but not limited to) with non-natural or hydrophobic nature 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor hydrophobic amino acid, with non-natural or natural The huge huge amino acid of 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, with non-natural or natural hydrophilic 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor hydrophilic amino acid) and/or will be non- Natural amino acid insertion is not required to realize in the position of activity.

A variety of biochemistries and structural approach can be used to select the required position substituted in polypeptide through alpha-non-natural amino acid Point.Any position of polypeptide chain is suitably selected for being incorporated to alpha-non-natural amino acid, and for any or without specific required purpose, it can root It is selected according to rational design or by randomly choosing.The selection in required site can be based on generate have it is any needed for property or Activity ((is combined including (but not limited to) agonist, super agonist, partial agonist, reversed agonist, antagonist, receptor and adjusted Agent, receptor activity modulators, with combine collocation object combine conditioning agent, with reference to collocation object active regulator, with reference to collocation object structure As conditioning agent, dimer or polymer are formed), the non-natural amino acid polypeptides that change of inactive compared with natural molecule or property (it can be through further modifying or keeping unmodified) or manipulate polypeptide it is any physically or chemically, it is such as dissolubility, poly- Collection or stability.For example, can be used including (but not limited to) point mutation analysis, Alanine-scanning or homologue scanning method Method discriminating needs the position in the polypeptide of the bioactivity of polypeptide.Except included (but not limited to) alanine or homologue scan The method that mutation is formed differentiates to be possible according to for sought by polypeptide to the residue outside those residues of bioactivity key It is required activity be with alpha-non-natural amino acid substitute good candidate.Alternatively, through differentiating as the site to bioactivity key It may also be again according to the good candidate substituted for the required activity sought by polypeptide with alpha-non-natural amino acid.Another generation Can be the work of continuous substitution and observation to polypeptide simply carried out with alpha-non-natural amino acid on polypeptide chain in each position for method The influence of property.Any means, the techniques or methods for the position that selection is substituted with alpha-non-natural amino acid in any polypeptide are suitable for Method described herein, technology and composition.

Also the structure and activity that the naturally occurring mutant of the polypeptide containing missing can be studied may be allowed with measuring with non- The protein domain of natural amino acid substitution.Once remove the residue that may allow to be substituted with alpha-non-natural amino acid, so that it may use It is researched and proposed including (but not limited to) related polypeptide and the method for any associated ligands or protein-bonded three-dimensional structure The influence being substituted in each rest position.Protein Data Bank (Protein Data Bank) (PDB,www.rcsb.org) can obtain perhaps in (integrated data store of the three-dimensional structure data of the macromolecular containing protein and nucleic acid) The more the X-ray crystallization of peptide and NMR structures can be used for differentiating the amino acid position that with alpha-non-natural amino acid can be substituted.In addition, If three-dimensional structure data is unavailable, then can make model to study the secondary structure of polypeptide and tertiary structure.Therefore, may be used It is easily obtained the amino acid position of available alpha-non-natural amino acid substitution in itself.

Be incorporated to the illustrative site of alpha-non-natural amino acid including (but not limited to) exclude potential receptorbinding region those Site or the region that is combined with binding protein or ligand solvent can expose completely or partially, with neighbouring residue have it is minimum or Without interaction of hydrogen bond, it can minimally be exposed to neighbouring reactive residue and/or such as can mutually formed with it by particular polypeptide Receptor, ligand or protein-bonded three-dimensional crystalline structure is closed to be predicted as in the region of highly flexible.

A variety of alpha-non-natural amino acids be may replace or be incorporated in the given position in polypeptide.It for example, can be according to polypeptide Research with its associated ligands, receptor and/or protein-bonded three-dimensional crystalline structure selects the specific non-natural being incorporated to Amino acid, preferably conservative replaces.

In one embodiment, method described herein includes and alpha-non-natural amino acid, wherein non-natural is incorporated into polypeptide Amino acid includes the first reactive group；And make polypeptide with the molecule including the second reactive group (including (but not limited to) mark Note；Dyestuff；Polymer；Water-soluble polymer；The derivative of polyethylene glycol；Photocrosslinking agent；Cytotoxic compound；Drug；Parent With property mark；Photoaffinity marks；Reactive compounds；Resin；Second protein or polypeptide or polypeptide analog；Antibody is anti- Body segment；Metal-chelator；Co-factor；Aliphatic acid；Carbohydrate；Polynucleotide；DNA；RNA；Antisense polynucleotide；Sugar Class, water-soluble dendritic, cyclodextrin, biomaterial；Nano-particle；Spin labeling；Fluorogen, the part containing metal； Radioactive segment；Novel functional group；With other molecule covalents or the group of noncovalent interaction；Light cage covers part；It can be photochemical The part of radiation excitation；Ligand；It can photoisomerization part；Biotin；Biotin analog；It is combined with the part of heavy atom； The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active agent；Aminothio Acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electron dense base Group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；Small molecule；Inhibition core Ribosomal ribonucleic acid, radioactive nucleotides；Neutron capture agent；The derivative of biotin；Quantum dot；Nanometer transfers element；Radioactivity transfers element； It is abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, antihormones, anti-oxidant Agent, aptamer, guide RNA, saponin, shuttle vector, macromolecular, plan epitope, receptor, reverse micelle and its any group Close) contact.In certain embodiments, the first reactive group is carbonyl or dicarbonyl moiety and the second reactive group is azanol Part thereby forms oxime key.In certain embodiments, the first reactive group is hydroxylamine Part and the second reactive group is carbonyl Base or dicarbonyl moiety thereby form oxime key.In certain embodiments, the first reactive group for carbonyl or dicarbonyl moiety and Second reactive group is oxime part, and oxime exchange reaction thereby occurs.In certain embodiments, the first reactive group is oxime portion Divide and the second reactive group is carbonyl or dicarbonyl moiety, oxime exchange reaction thereby occurs.

In some cases, alpha-non-natural amino acid substitute or be incorporated to by in polypeptide other addition, substitution or missing group It closes to influence other chemistry, physics, pharmacology and/or biological natures.In some cases, other additions, substitution or missing can increase It adds the stability (including (but not limited to) the resistance to proteolytic degradation) of peptide or increases polypeptide to its appropriate receptor, coordination Body and/or protein-bonded compatibility.In some cases, other additions, substitution or missing can increase the solubility (bag of polypeptide Containing (but not limited to), when with Escherichia coli or other host cell expressions).In some embodiments, selection is used for natural Coding or the site of alpha-non-natural amino acid substitution, in addition, another site is selected for being incorporated to alpha-non-natural amino acid, to realize big Increase the deliquescent purpose of polypeptide after being expressed in enterobacteria or other recombinant host cells.In some embodiments, polypeptide includes It adjusts the compatibility to associated ligands, binding protein and/or receptor, adjust (including (but not limited to) increasing or decreasing) receptor Dimerization, stablize receptor dimer, adjust circulating half-life, adjust release or biological usability, beneficial to purifying improvement or change Become another addition, substitution or the missing of specific dosing way.Similarly, non-natural amino acid polypeptides may include the inspection for improveing polypeptide It surveys (including (but not limited to) GFP), purify, transmission penetrates tissue or cell membrane, prodrug are discharged or activated, size reduces or other The chemistry or enzymatic lysis sequence of characteristic, protease cleavage sequence, reactive group, antibody binding domain (including (but not limited to) FLAG or poly-His) or other sequences (including (but not limited to) FLAG, poly-His, GST etc.) or key based on compatibility Join molecule (including (but not limited to) biotin).

IV. the growth hormone supergene family as example

Method described herein, composition, strategy and technology be not limited to specific polypeptide or protein types, species or Family.Really, substantially any polypeptide can through design or modify with comprising it is at least one it is described herein " through modification or without Modification " alpha-non-natural amino acid.Only for example, polypeptide can be same with the treatment albumen matter selected from the group being made of following object Source：α -1 antitrypsins, angiostatin, anti-hemolytic factor, antibody, antibody fragment, apolipoprotein, apoprotein, Atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, C-X-C chemotactic factor (CF)s, T39765, NAP-2, ENA-78, gro-a, gro- B, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligands, cell factor, CC chemotactics The factor, monocyte chemoattractant protein-1, monocyte chemoattractant protein -2, monocyte chemoattractant protein-3, monocyte inflammatory egg In vain -1 α, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligands, collagen, colony stimulating factor (CSF), complement factor 5a, complement Inhibitor, complement receptor 1, cell factor, epithelium neutrophilic granulocyte activation peptide -78, MIP-16, MCP-1, epidermal growth factor (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin(EPO) (EPO), exfoliative toxin,or exfoliatin, factors IX, factor Ⅴ II, the factor VIII, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helix bundle albumen, G- CSF, glp-1, GM-CSF, glucocerebrosidase, promoting sexual gland hormone, growth factor, growth factor receptors, grf, Hedgelog protein, Hemochrome, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptors, LFA-1, LFA-1 Receptors, insdri, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-α, IFN-β, IFN-γ, interleukins (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactotransferrin, leukaemia inhibit because Son, luciferase, neural element, neutrophil inhibitory factor (nif) (NIF), oncostatin M, osteogenic protein, oncoprotein, The outer poison of paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, multi-effector, albumin A, Protein G, pth, pyrogenicity Plain A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxain, feritin, SCF, atom synthetic proteins, Soluble complement receptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, Growth hormone (somatotropin), streptokinase, super antigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic-shock syndrome toxin, thymosin α1, tectotype are fine It is the plasminogen-activating factor, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor α, tumor necrosis factor β, swollen Tumor necrosis factor receptor (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, Ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, testosterone receptor, aldehyde are solid Ketone receptor, ldl receptor and cortisone.

Therefore, growth hormone (GH) supergene family is described below for illustrative purpose and only carrying as example For, and it is not intended as the limitation of the scope to method described herein, composition, strategy and technology.In addition, in present application The example for using general terms as any member in GH supergene families is intended to referring to for GH polypeptides.It is therefore to be understood that On GH polypeptides or protein modification described herein and chemistry be equally applicable in GH supergene families it is any into Member, it includes specific those members listed herein.

Following protein include by the gene code of growth hormone (GH) supergene family those protein (Bazan, F.,Immunology Today 11:350-354(1990)；Bazan,J.F.Science 257:410-411(1992)； Mott, H.R. and Campbell, I.D., Current Opinion in Structural Biology 5:114-121 (1995)；Silvennoinen, O. and Ihle, J.N., Signalling by the Hematopoietic Cytokine Receptors(1996))：Growth hormone, prolactin, human placental lactogen, erythropoietin(EPO) (EPO), blood platelet life Cheng Su (TPO), interleukin 2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 unit), IL-13, IL-15, oncostatin M, ciliary neurotrophic factor, LIF ELISA, alpha interferon, β interference Element, ε interferon, interferon, omega interferon, τ interferon, G-CSF (G-CSF), granulocyte-macrophage are thin Born of the same parents' colony stimulating factor (GM-CSF), macrophage colony stimulatory factor (M-CSF) and cardiotrophin-1 (CT-1) (" GH Supergene family ").It is expected that other members of this gene family are differentiated in the future by cloning and sequencing gene.GH hyper-bases Because the member of family has similar secondary structure and tertiary structure, although they usually have limited amino acid or DNA sequence dna Uniformity.Common structure feature causes the newcomer of the gene family to be easy to differentiate and is applied similarly described herein Alpha-non-natural amino acid method and composition.

Include G-CSF (Zink et al., FEBS Lett.314:435(1992)；Zink et al., Biochemistry 33: 8453(1994)；Hill et al., Proc.Natl.Acad.Sci.USA 90:5167(1993))、GM-CSF(Diederichs, K. et al., Science 154:1779-1782(1991)；Walter et al., J.Mol.Biol.224:1075-1085 (1992)), IL-2 (Bazan, J.F. and McKay, D.B., Science 257:410-413(1992))；IL-4(Redfield Et al., Biochemistry 30:11029-11035(1991)；Powers et al., Science 256:1673-1677 And IL-5 (Milburn et al., Nature 363 (1992)):172-176 (1993)) numerous cell factors structure by X-ray diffraction and NMR researchs measure and show the surprising conservative with GH structures, but lack significant primary sequence homology. Think member (Lee et al., the J.Interferon Cytokine that IFN is this family according to model and other researchs Res.15:341(1995)；Murgolo et al., Proteins 17:62(1993)；Radhakrishnan et al., Structure 4:1453(1996)；Klaus et al., J.Mol.Biol.274:661(1997)).Comprising ciliary neurotrophic factor (CNTF), LIF ELISA (LIF), thrombopoietin (TPO), oncostatin M, macrophage colony stimulatory factor (M-CSF), IL- 3rd, IL-6, IL-7, IL-9, IL-12, IL-13, IL-15 and G-CSF (G-CSF) and such as α, β, ω, Other a large amount of cell factors and growth factor of τ, ε and the IFN of interferon belong to this family (in Mott and Campbell, Current Opinion in Structural Biology 5:114-121(1995)；Silvennoinen and Ihle (1996) looked back in Signalling by the Hematopoietic Cytokine Receptors).Nowadays think all Above-mentioned cell factor and growth factor include a big gene family.

In addition to shared similar secondary structure and tertiary structure, the member of this family, which shares it, must make cell surface Receptor oligomerization is with the property of signal transduction pathway in activating cell.Some GH family members (including (but not limited to) GH and EPO) combined with the receptor of single type and form it into homodimer.Including (but not limited to) its of IL-2, IL4 and IL-6 His family member is combined with receptor more than a type and these receptors is made to form heterodimer or the aggregation of higher level (Davis et al., (1993) Science 260:1805-1808；Paonessa et al., (1995) EMBO is J.14:1942- 1951；Mott and Campbell, Current Opinion in Structural Biology 5:114-121(1995)).It lures Become research and have shown that as GH, these other cell factors and growth factor contain multiple receptor binding sites (usual two It is a), and (Mott and Campbell, Current Opinion in Structural are combined with its homoreceptor successively Biology 5:114-121(1995)；Matthews et al., (1996) Proc.Natl.Acad.Sci.USA 93:9471- 9476).As GH, the major receptors binding site of these other family members is primarily present in four α spirals and A-B rings In.The specific amino acids participated in the helical bundle that receptor combines are different in family member.With member's phase of GH supergene families Related and second big multigene family of composition in the most cells surface receptor structure of interaction.Referring to the (for example) U.S. Patent the 6th, 608,183 is in being hereby incorporated by reference in their entirety.

The common conclusions obtained by the mutation research to a variety of GH supergene families members usually become for the ring of connection α spirals To in be not involved in receptor combine.Specifically, for the receptor in most of (if not all) family members combines, short B- C rings seem not necessarily.For this reason, in the member of GH supergene families, B-C rings can be through non-natural as described herein 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor.A-B rings, C-D rings (and D-E rings of the class IL-10 member of interferon/GH superfamilies) also can be through non-natural ammonia Base acid substitution.Closest to spiral A and the amino acid away from last spiral also tends to be not involved in receptor combination and be alternatively to be used for Introduce the site of alpha-non-natural amino acid.In some embodiments, alpha-non-natural amino acid is including (but not limited to) A-B, B-C, C-D Or preceding 1,2,3,4,5,6,7 or 7 of D-E rings is with substitution at any position in the ring structure of upper amino acid.In some implementations In example, alpha-non-natural amino acid is at rear 1,2,3,4,5,6,7 or 7 of A-B, B-C, C-D or D-E ring to substitute in upper amino acid.

GH families some members (including (but not limited to) EPO, IL-2, IL-3, IL-4, IL-6, IFN, GM-CSF, TPO, IL-10, IL-12p35, IL-13, IL-15 and beta interferon) contain the sugar that N- is bonded and/or O- is bonded.In protein Glycosylation site almost all is present in ring region and is not present in α helical bundles.Because ring region be usually not involved in by Can be to draw for alpha-non-natural amino acid to be substituted during body combines and because it is for being covalently attached the site of glycosyl group Enter the applicable site in protein.Protein includes the amino acid of the bonded glycosylation sites of N- and the bonded glycosylation sites of O- Can be the site of alpha-non-natural amino acid substitution, because these amino acid are surface exposure.Therefore, native protein may be allowed The huge glycosyl being connected at these sites on protein is rolled into a ball and glycosylation site is intended to be located remotely from receptor binding site Position.

It is possible that other members of GH gene families are found in future.The newcomer of GH supergene families can be by predicting egg Computer-aided secondary structure and the tertiary structure analysis of white matter sequence, and by being designed to differentiate the molecule combined with specific target Selection technique differentiate.There are four the member of GH supergene families usually tools or five by non-helical shape amino acid (ring region) The amphiphatic molecule spiral of connection.Protein can contain hydrophobic signal sequence to promote to secrete from cell in its N- end.After these GH supergene family members to find are also contained in method and composition described herein.

V. alpha-non-natural amino acid

The alpha-non-natural amino acid used in method and composition described herein has in following four property at least One：(1) functional group on the side chain of at least one alpha-non-natural amino acid has at least one and 20 kinds of common inherited codings Amino acid (that is, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, group Propylhomoserin, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, junket Propylhomoserin and valine) chemical reactivity it is orthogonal or at least with it is naturally occurring present in the polypeptide comprising alpha-non-natural amino acid The orthogonal feature of the chemical reactivity of amino acid and/or activity and/or reactivity；(2) alpha-non-natural amino acid through introducing is to 20 The common inherited coded amino acid of kind is essentially chemically inert；(3) alpha-non-natural amino acid can be steadily incorporated in polypeptide, excellent Selection of land with stability comparable with naturally occurring amino acid or under the conditions of characteristic physiological with stability, and further preferably Ground, described be incorporated to can be occurred by vivo system；And (4) alpha-non-natural amino acid include oxime functional group or can by with reagent Reaction is changed into the functional group of oximido, preferably under conditions of the biological property of the polypeptide comprising alpha-non-natural amino acid is not destroyed (unless the destruction of this biological property is the purpose of modification/transformation certainly) or wherein transformation can be between about 4 and about 8 Occur under aqueous conditions under pH value or the reactive site wherein on alpha-non-natural amino acid is electrophilic site.Available for this The composition of described in the text and the satisfaction of method are on the illustrative, non-of the amino acid of four kinds of property of alpha-non-natural amino acid Limitative examples are in be shown in Fig. 2, Fig. 3, Figure 35 and Figure 40-43.Any number of alpha-non-natural amino acid can be introduced into polypeptide. Alpha-non-natural amino acid can also include the oxime through protecting or sheltering or can deprotect blocking group or go to shelter by masking group Be changed into afterwards oximido through protecting or sheltering group.Alpha-non-natural amino acid can also include carbonyl or two carbonyls through protecting or sheltering Blocking group can deprotected or masking group is gone to be changed into carbonyl or dicarbapentaborane after masking and and then can be used for by base It reacts to form oximido with azanol or oxime.

Available for the alpha-non-natural amino acid in method and composition described herein including (but not limited to) including can light The amino acid of activatable crosslinking agent, the amino acid through spin labeling, Fluorescent amino acid, metal combination amino acid, containing metal-amino acid, Radioactive amino acids, the amino acid with novel functional group, amino acid, light with other molecule covalents or noncovalent interaction Cage cover and/or can photoisomerization amino acid, the amino acid including biotin or biotin analog, glycosylated amino acid (such as Through sugar-substituted serine), other amino acid through carbohydrate modification, containing keto amino acid, amino acid containing aldehyde, including poly- second The amino acid of glycol or other polyethers, through heavy atom substitution amino acid, chemically cracking and/or can photodestruciton amino acid, (including (but not limited to) polyethers or long chain hydrocarbons, it includes (but not for amino acid with the extended side chain compared with natural amino acid Be limited to) be greater than about 5 carbon or greater than about 10 carbon), containing the amino acid through the bonded sugar of carbon, redox active amino acids, contain The amino acid of aminothio acid and the amino acid including one or more toxin parts.

In some embodiments, alpha-non-natural amino acid includes carbohydrate fraction.The example of these amino acid include N- acetyl group- L- glucose amido-Serine, N- acetyl group-L- galactolipins amido-Serine, N- acetyl group-L- glucose amidos-L- Soviet Unions ammonia Acid, N- acetyl group-L- glucose amido-altheine and O- mannoses amido-Serine.The example of these amino acid Comprising the naturally occurring N- keys wherein between amino acid and sugar or O- keys by nature there is usually no covalent bond (include (but not limited to) alkene, oxime, thioether, amide with and the like) replace example.The example of these amino acid is also comprising natural There are in protein there is usually no carbohydrate, such as 2-DG, 2- deoxy-galactoses with and the like.

By the chemical part for being incorporated in these polypeptides to be incorporated in polypeptide by alpha-non-natural amino acid provide a variety of advantages and The operation of polypeptide.For example, the uniqueness reactivity of carbonyl or dicarbapentaborane functional group (including ketone functional group or aldehyde functional group) are held Perhaps repaiied with numerous selectivity that protein in vivo and is in vitro carried out containing any one of hydrazine reagent or reagent containing azanol Decorations.Heavy atom alpha-non-natural amino acid is (for example) applicable to determine phase x-ray structure data.Use alpha-non-natural amino acid site-specific Property introduce heavy atom also provide for selection heavy atom position when selectivity and flexibility.Photoreactivity alpha-non-natural amino acid (including (but not limited to) the amino with Benzophenone and aromatic yl azide (including (but not limited to) phenyl azide) side chain Acid) (for example) so that polypeptide in vivo and in vitro photo-crosslinking is effective.The example of photoreactivity alpha-non-natural amino acid include (but It is not limited to) to azido-phenylalanine and to benzoyl-phenylalanine.Polypeptide with photoreactivity alpha-non-natural amino acid It then can be by the excitation (offer Instantaneous Control) of photoreactive group come arbitrary crosslinking.In non-limiting examples, non-natural The methyl of amino acid can substitute through isotope marks and (substitute including (but not limited to) through methyl), as partial structurtes and dynamics Probe (including (but not limited to) by means of nuclear magnetic resonance and vibrational spectrum).

A. alpha-non-natural amino acid：Carbonyl, class carbonyl, masked carbonyl and structure and synthesis through protecting carbonyl

Amino acid with electrophilic reactivity group allow a variety of reactions with by various chemical reactions (comprising (but not It is limited to) nucleophilic addition) bonded molecule.These electrophilic reactivity groups include carbonyl or dicarbapentaborane (includes ketone group or aldehyde Base), class carbonyl or class dicarbapentaborane group (its have the reactivity similar to carbonyl or dicarbapentaborane and in structure with carbonyl or two Carbonyl is similar), masked carbonyl or masked dicarbapentaborane (it can be easy to be changed into carbonyl or dicarbapentaborane) or through protection Carbonyl or the dicarbapentaborane through protection (it has the reactivity similar with carbonyl or dicarbapentaborane after deprotection).These amino acid bags Amino acid containing the structure with formula (I)：

Wherein：

A is optional, and is when it is present low-grade alkylidene, is substituted low-grade alkylidene, rudimentary cycloalkylidene, is substituted Rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, rudimentary sub- miscellaneous alkyl, be substituted sub- miscellaneous alkyl, Rudimentary Asia Heterocyclylalkyl is substituted rudimentary sub- Heterocyclylalkyl, arlydene, is substituted arlydene, inferior heteroaryl, is substituted sub- heteroaryl Base, alkarylene are substituted alkarylene, sub- aralkyl or are substituted sub- aralkyl；

B is optional, and is the connexon selected from the group being made of following group when it is present：Low-grade alkylidene, Low-grade alkylidene, lower alkenylene are substituted, lower alkenylene, rudimentary sub- miscellaneous alkyl is substituted, is substituted the rudimentary miscellaneous alkane in Asia Base ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)_k- (wherein K for 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-NS (O)₂-、-OS(O)₂- ,-C (O)-(alkylene Base is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidenes Or be substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, Wherein R' is each independently H, alkyl or substituted alkyl；

J is

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

During R " being each independently H, alkyl, substituted alkyl or protecting group or when there are more than one R " group, two A R " optionally forms Heterocyclylalkyl；

R₁For H, amino protecting group, resin, amino acid, polypeptide or polynucleotide；And

R₂For OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide；

R₃With R₄It is each independently H, halogen, low alkyl group or is substituted low alkyl group or R₃With R₄Or two R₃Group Optionally form cycloalkyl or Heterocyclylalkyl；

Or-A-B-J-R groups are collectively formed including at least one carbonyl (comprising dicarbapentaborane), through protecting carbonyl (comprising warp Protect dicarbapentaborane) or masked carbonyl (include masked dicarbapentaborane) bicyclic or three annular cycloalkyl or Heterocyclylalkyl；

Or-J-R groups are collectively formed including at least one carbonyl (comprising dicarbapentaborane), through carbonyl is protected (to include through protection Dicarbapentaborane) or masked carbonyl (include masked dicarbapentaborane) monocyclic or bicyclic cycloalkyl or Heterocyclylalkyl；

Its restrictive condition is when A is phenylene and R₃During respectively H, B exists；And when A is-(CH₂)₄- and R₃Respectively H When, B is not -NHC (O) (CH₂CH₂)-；And when A and B are not present and R₃During respectively H, R is not methyl.These non-natural aminos Acid can be the form of salt or may be incorporated into non-natural amino acid polypeptides, polymer, polysaccharide or polynucleotide and optionally translated After modify.

In certain embodiments, formula (I) compound stablizes at least one moon in aqueous solution under appropriate acid condition. In some embodiments, formula (I) compound was in appropriate stable under acidic conditions at least 2 weeks.In certain embodiments, formula (I) chemical combination Object was in appropriate stable under acidic conditions at least 5 days.In certain embodiments, the acid condition is pH 2 to 8.

In some embodiments of formula (I) compound, B is low-grade alkylidene, is substituted low-grade alkylidene ,-O- (alkylidenes Or be substituted alkylidene)-,-C (R')=N-N (R')-,-N (R') CO- ,-C (O)-,-C (R')=N- ,-C (O)-(alkylidene or Be substituted alkylidene)-,-CON (R')-(alkylidene is substituted alkylidene)-,-S (alkylidene is substituted alkylidene)-,-S (O) (alkylidene is substituted alkylidene)-or-S (O)₂(alkylidene is substituted alkylidene)-.In some of formula (I) compound In embodiment, B is-O (CH₂)-,-CH=N- ,-CH=N-NH- ,-NHCH₂-、-NHCO-、-C(O)-、-C(O)-(CH₂)-、- CONH-(CH₂)-、-SCH₂- ,-S (=O) CH₂- or-S (O)₂CH₂-.In some embodiments of formula (I) compound, R C_1-6Alkane Base or cycloalkyl.In some embodiments of formula (I) compound, R is-CH₃、-CH(CH₃)₂Or cyclopropyl.In formula (I) compound Some embodiments in, R₁For H, tertbutyloxycarbonyl (Boc), 9- fluorenylmethoxycarbonyl groups (Fmoc), N- acetyl group, four acetyl fluorides Base (TFA) or benzyloxycarbonyl group (Cbz).In some embodiments of formula (I) compound, R₁For resin, amino acid, polypeptide or poly-nuclear Thuja acid.In some embodiments of formula (I) compound, R₂For OH, O- methyl, O- ethyls or O- tertiary butyls.In formula (I) compound Some embodiments in, R₂For resin, amino acid, polypeptide or polynucleotide.In some embodiments of formula (I) compound, R₂For Polynucleotide.In some embodiments of formula (I) compound, R₂For ribonucleic acid (RNA).In some realities of formula (I) compound It applies in example, R₂For tRNA.In some embodiments of formula (I) compound, tRNA specific recognitions selection codon.At formula (I) In some embodiments of compound, select codon be selected from by amber codon, ochre codon, opal codon, solely The group that special codon, rare codon, unnatural codons, five base codons and four base codons form.In formula (I) in some embodiments of compound, R₂To inhibit tRNA.

In some embodiments of formula (I) compound,It is selected from the group being made of following object：

(i) A is to be substituted low-grade alkylidene, C₄- arlydene is substituted arlydene, inferior heteroaryl, is substituted sub- heteroaryl Base, alkarylene are substituted alkarylene, sub- aralkyl or are substituted sub- aralkyl；

B is optional, and is the divalent linker selected from the group being made of following group when it is present：Lower alkylene Base, be substituted low-grade alkylidene, lower alkenylene, being substituted lower alkenylene ,-O- ,-O-, (alkylidene is substituted alkylene Base)-,-S- ,-S (O)-,-S (O)₂-、-NS(O)₂-、-OS(O)₂- ,-C (O)-,-C (O)-(alkylidene is substituted alkylene Base)-,-C (S)-,-N (R')-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-N (R') C (S)-,-S (O) N (R') ,-S (O)₂N (R')、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)N(R')-、-N(R')S(O)₂N(R')-、-N (R')-N=,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂-N(R')-N(R')-；

(ii) A is optional, and is to be substituted low-grade alkylidene, C when it is present₄- arlydene is substituted arlydene, Asia Heteroaryl is substituted inferior heteroaryl, alkarylene, is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；

B is the divalent linker selected from the group being made of following group：Low-grade alkylidene is substituted lower alkylene Base, lower alkenylene, be substituted lower alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S (O)-,-S (O)₂-、-NS(O)₂-、-OS(O)₂- ,-C (O)-,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-N (R')-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-, (alkylidene is substituted-N (R') CO- Alkylidene)-,-N (R') C (O) O- ,-N (R') C (S)-,-S (O) N (R') ,-S (O)₂N(R')、-N(R')C(O)N(R')-、-N (R')C(S)N(R')-、-N(R')S(O)N(R')-、-N(R')S(O)₂N (R')-,-N (R')-N=,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂-N(R')-N(R')-；

(iii) A is low-grade alkylidene；

B is optional, and is the divalent linker selected from the group being made of following group when it is present：Lower alkylene Base, be substituted low-grade alkylidene, lower alkenylene, being substituted lower alkenylene ,-O- ,-O-, (alkylidene is substituted alkylene Base)-,-S- ,-S (O)-,-S (O)₂-、-NS(O)₂-、-OS(O)₂- ,-C (O)-,-C (O)-(alkylidene is substituted alkylene Base)-,-C (S)-,-N (R')-,-C (O) N (R')-,-CSN (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-N (R')C(O)O-、-N(R')C(S)-、-S(O)N(R')、-S(O)₂N(R')、-N(R')C(O)N(R')-、-N(R')C(S)N (R')-、-N(R')S(O)N(R')-、-N(R')S(O)₂N (R')-,-N (R')-N=,-C (R')=N-N (R')-,-C (R')= N-N=,-C (R')₂- N=N- and-C (R')₂-N(R')-N(R')-；And

(iv) A is phenylene；

B is the divalent linker selected from the group being made of following group：Low-grade alkylidene is substituted lower alkylene Base, lower alkenylene, be substituted lower alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S (O)-,-S (O)₂-、-NS(O)₂-、-OS(O)₂- ,-C (O)-,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-N (R')-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-, (alkylidene is substituted-N (R') CO- Alkylidene)-,-N (R') C (O) O- ,-N (R') C (S)-,-S (O) N (R') ,-S (O)₂N(R')、-N(R')C(O)N(R')-、-N (R')C(S)N(R')、-N(R')S(O)N(R')-、-N(R')S(O)₂N (R')-,-N (R')-N=,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂-N(R')-N(R')-；

J is

R' is each independently H, alkyl or substituted alkyl；

R₁To be optional, and it is H, amino protecting group, resin, amino acid, polypeptide or polynucleotide when it is present；And

R₂To be optional, and it is OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide when it is present；And

R₃With R₄It is each independently H, halogen, low alkyl group or is substituted low alkyl group；

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

In addition, include the amino acid of the structure with formula (II)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

Its restrictive condition is when A is phenylene, and B exists；And when A is-(CH₂)₄- when, B is not -NHC (O) (CH₂CH₂)-；And in the absence of A and B, R is not methyl.These alpha-non-natural amino acids can be the form of salt or may be incorporated into non-day Modification after in right amino acid polypeptide, polymer, polysaccharide or polynucleotide and optionally translated.

In addition, include the amino acid of the structure with formula (III)：

Wherein：

B is the connexon selected from the group being made of following group：Low-grade alkylidene is substituted low-grade alkylidene, is low Grade alkenylene is substituted lower alkenylene, rudimentary sub- miscellaneous alkyl, is substituted rudimentary sub- miscellaneous alkyl ,-O- ,-O- (alkylidene or warp Substituted alkylene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_k(alkylene Base is substituted alkylidene)-,-C (O)-,-NS (O)₂-、-OS(O)₂,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted Asia Alkyl)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-S (O)_kN(R')-、-N(R')C(O)N (R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, wherein R' be each independently H, Alkyl or substituted alkyl；

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R_aIt is each independently selected from the group being made of following group：H, halogen, alkyl, substituted alkyl ,-N (R')₂、-C(O)_kR'(wherein k for 1,2 or 3) ,-C (O) N (R')₂,-OR' and-S (O)_kR', wherein R' are each independently H, alkyl or substituted alkyl.These alpha-non-natural amino acids can be the form of salt or may be incorporated into non-natural amino acid polypeptides, polymerization Modification after in object, polysaccharide or polynucleotide and optionally translated.

In addition, include following amino acid：

These alpha-non-natural amino acids can be optionally group through protection of group of the amino through protection, carboxyl and/or be salt Form or may be incorporated into non-natural amino acid polypeptides, polymer, polysaccharide or polynucleotide and it is optionally translated after modification.

In addition, include the amino acid of the structure below with formula (IV)：

Wherein

-NS(O)₂-、-OS(O)₂-, it is optional, and be the connection selected from the group being made of following group when it is present Son：Low-grade alkylidene is substituted low-grade alkylidene, lower alkenylene, is substituted lower alkenylene, rudimentary sub- miscellaneous alkyl, through taking For rudimentary sub- miscellaneous alkyl ,-O- ,-O- (alkylidene is substituted alkylidene)-, (alkylidene is substituted alkylene by-S- ,-S- Base)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene Or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidene or Be substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, Wherein R' is each independently H, alkyl or substituted alkyl；

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R_aIt is each independently selected from the group being made of following group：H, halogen, alkyl, substituted alkyl ,-N (R')₂、-C(O)_kR'(wherein k for 1,2 or 3) ,-C (O) N (R')₂,-OR' and-S (O)_kR', wherein R' are each independently H, alkyl or substituted alkyl；And n is 0 to 8；

Its restrictive condition is when A is-(CH₂)₄- when, B is not -NHC (O) (CH₂CH₂)-.These alpha-non-natural amino acids can be The form of salt may be incorporated into non-natural amino acid polypeptides, polymer, polysaccharide or polynucleotide and be repaiied after optionally translated Decorations.

In addition, include following amino acid：

Wherein these compounds optionally amino through protection, optionally carboxyl through protection, optionally amino through protection and carboxylic Base is through protecting or for its salt or may be incorporated into non-natural amino acid polypeptides, polymer, polysaccharide or polynucleotide and optionally pass through Posttranslational modification.

In addition, include the amino acid of the structure below with formula (VIII)：

Wherein,

R₂For OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide.

These alpha-non-natural amino acids can be the form of salt or may be incorporated into non-natural amino acid polypeptides, polymer, polysaccharide or poly- Modification after in nucleotide and optionally translated.

In addition, include the amino acid of the structure below with formula (IX)：

Wherein,

B is optional, and is the connexon selected from the group being made of following group when it is present：Low-grade alkylidene, Low-grade alkylidene, lower alkenylene are substituted, lower alkenylene, rudimentary sub- miscellaneous alkyl is substituted, is substituted the rudimentary miscellaneous alkane in Asia Base ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)_k- (wherein K for 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-NS (O)₂-、-OS(C))₂- ,-C (O)-(alkylene Base is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidenes Or be substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN (R') ,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, Wherein R' is each independently H, alkyl or substituted alkyl；

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

Wherein R_aIt is each independently selected from the group being made of following group：H, halogen, alkyl, substituted alkyl ,-N (R')₂、-C(O)_kR'(wherein k for 1,2 or 3) ,-C (O) N (R')₂,-OR' and-S (O)_kR', wherein R' are each independently H, alkyl or substituted alkyl.

In addition, include following amino acid：

In addition, include the amino acid of the structure below with formula (X)：

Wherein,

B is optional, and is the connexon selected from the group being made of following group when it is present：Low-grade alkylidene, Low-grade alkylidene, lower alkenylene are substituted, lower alkenylene, rudimentary sub- miscellaneous alkyl is substituted, is substituted the rudimentary miscellaneous alkane in Asia Base ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)_k- (wherein K for 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-NS (O)₂-、-OS(O)₂- ,-C (O)-(alkylene Base is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidenes Or be substituted alkylidene)-,-C (O) N (R') ,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, Wherein R' is each independently H, alkyl or substituted alkyl；

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R_aIt is each independently selected from the group being made of following group：H, halogen, alkyl, substituted alkyl ,-N (R')₂、-C(O)_kR'(wherein k for 1,2 or 3) ,-C (O) N (R')₂,-OR' and-S (O)_kR', wherein R' are each independently H, alkyl or substituted alkyl；And n is 0 to 8.

In addition, include following amino acid：

In addition to mono carbonyl structure, alpha-non-natural amino acid described herein can include such as dicarbapentaborane, class dicarbapentaborane, Masked dicarbapentaborane and the group through protecting dicarbapentaborane.

For example, the amino acid of the structure below with formula (V) is included：

Wherein,

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

In addition, include the amino acid of the structure below with formula (VI)：

Wherein,

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

In addition, include following amino acid：

Wherein these compounds optionally amino through protection and carboxyl through protecting or for its salt.These non-natural aminos Acid can be the form of salt or may be incorporated into non-natural amino acid polypeptides, polymer, polysaccharide or polynucleotide and optionally translated After modify.

In addition, include the amino acid of the structure below with formula (VII)：

Wherein,

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

In addition, include following amino acid：

Wherein optionally amino through protection or for its salt or may be incorporated into non-natural to these compounds through protection and carboxyl Modification after in amino acid polypeptide, polymer, polysaccharide or polynucleotide and optionally translated.

In addition, include the amino acid of the structure below with formula (XXX)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

X₁For C, S or S (O)；And L is alkylidene, is substituted alkylidene, N (R') (alkylidene) or N (R') and (is substituted Asia Alkyl), wherein R' is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl.

In addition, include the amino acid of the structure below with formula (XXX-A)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

L is alkylidene, be substituted alkylidene, N (R') (alkylidene) or N (R') (being substituted alkylidene), wherein R' be H, Alkyl, substituted alkyl, cycloalkyl are substituted cycloalkyl.

In addition, include the amino acid of the structure below with formula (XXX-B)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R₁For H, amino protecting group, resin, amino acid, polypeptide or polynucleotide；

And R₂For OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide；

In addition, include the amino acid of the structure below with formula (XXXI)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

X₁For C, S or S (O)；And n is 0,1,2,3,4 or 5；And every CR⁸R⁹R on group⁸And R⁹It is each independently selected from The group be made of H, alkoxy, alkylamine, halogen, alkyl, aryl or any R⁸And R⁹=O or cycloalkyl can be collectively formed, Or any and adjacent R⁸Cycloalkyl can be collectively formed in group.

In addition, include the amino acid of the structure below with formula (XXXI-A)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

N is 0,1,2,3,4 or 5；And every CR⁸R⁹R on group⁸And R⁹It is each independently selected from by H, alkoxy, alkyl Amine, halogen, alkyl, the group of aryl composition or any R⁸And R⁹=O or cycloalkyl or any and adjacent R can be collectively formed⁸Base Cycloalkyl can be collectively formed in group.

In addition, include the amino acid of the structure below with formula (XXXI-B)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

In addition, include the amino acid of the structure below with formula (XXXII)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

In addition, include the amino acid of the structure below with formula (XXXII-A)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

In addition, include the amino acid of the structure below with formula (XXXII-B)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

In addition, include the amino acid of the structure with formula (XXXX)：

Wherein：

M is-C (R₃)-,

Wherein (a) instruction is bonded with A groups bond and (b) instruction with carbonyl out of the ordinary, R₃With R₄Independently selected from H, halogen, Alkyl, substituted alkyl, cycloalkyl are substituted cycloalkyl or R₃With R₄Or two R₃Group or two R₄Group optionally shape Into cycloalkyl or Heterocyclylalkyl；

R is H, halogen, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

T₃For a key, C (R) (R), O or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkanes Base；

In addition, include the amino acid of the structure with formula (XXXXI)：

Wherein：

M is-C (R₃)-,

R_aIt is each independently selected from the group being made of following group：H, halogen, alkyl, substituted alkyl ,-N (R')₂、-C(O)_kR'(wherein k for 1,2 or 3) ,-C (O) N (R')₂,-OR' and-S (O)_kR', wherein R' are each independently H, alkyl or substituted alkyl.

In addition, include the amino acid of the structure with formula (XXXXII)：

Wherein：

R is H, halogen, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；And

T₃For O or S.

In addition, include the amino acid of the structure with formula (XXXXIII)：

Wherein：

R is H, halogen, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl.

In addition, include the amino acid of the structure below with formula (XXXXIII)：

Carbonyl or dicarbapentaborane functional group can be selectively anti-with the reagent containing azanol in aqueous solution in a mild condition The corresponding oxime key that should be in physiological conditions stablized with formation.Referring to (for example) Jencks, W.P., J.Am.Chem.Soc.81, 475-481(1959)；Shao, J. and Tam, J.P., J.Am.Chem.Soc.117 (14):3893-3899(1995).In addition, carbonyl Unique reactivity of base or dicarbapentaborane is allowed in the selective modification in the presence of other amino acid side chains.Referring to (for example) Cornish, V.W. et al., J.Am.Chem.Soc.118:8150-8151(1996)；Geoghegan, K.F. and Stroh, J.G.,Bioconjug.Chem.3:138-146(1992)；Mahal, L.K. et al., Science 276:1125-1128 (1997)。

The synthesis of acetyl group-(+/-)-phenylalanine and acetyl group-(+/-)-phenylalanine is described in reference The Zhang that mode is incorporated to, Z. et al., Biochemistry 42:In 6735-6746 (2003).It similar can prepare other and contain carbonyl The amino acid of base or dicarbapentaborane.In addition, the nonrestrictive illustrative synthesis of the alpha-non-natural amino acid included herein is in be shown in figure 4th, in Figure 24-Figure 34 and Figure 36-Figure 39.

In some embodiments, the polypeptide including alpha-non-natural amino acid through chemical modification to generate reactive carbonyl or two carbonyls Base functional group.It for example, can be by having the functional group of neighbouring amino and hydroxyl generation suitable for the aldehyde functional group of engagement reaction.Wherein Bioactive molecule is polypeptide, for example, N- terminal serines or threonine can be used, (it usually may be present or can be disappeared by chemistry Change or enzymatic digestion expose) under mild oxidation cracking condition aldehyde functional group is generated using periodate.Referring to (for example) Gaertner et al., Bioconjug.Chem.3:262-268(1992)；Geoghegan, K. and Stroh, J., Bioconjug.Chem.3:138-146(1992)；Gaertner et al., J.Biol.Chem.269:7224-7230(1994). However, known method is confined to the amino acid in the N- ends of peptide or protein matter in fields.

In addition, alpha-non-natural amino acid for example with neighbouring hydroxyl and amino can " masked " aldehyde functional group form simultaneously Enter in polypeptide.For example, 5- oxylysines have the hydroxyl of adjacent ε amine.The reaction condition for generating aldehyde is typically included in temperature The sodium metaperiodate of molar excess is added under the conditions of to aoxidize to avoid at other sites in polypeptide.The pH value of oxidation reaction Typically about 7.0.Typical reaction includes adding the sodium metaperiodate of about 1.5 molar excess into the buffer solution of polypeptide, connects It and cultivates in the dark about 10 minutes.Referring to (for example) U.S. Patent No. 6,423,685.

B. alpha-non-natural amino acid：The structure of amino acid containing azanol and synthesis

Alpha-non-natural amino acid containing azanol (also referred to as amino oxygroup) group allow with a variety of electrophilic groups react with Formed binding element (including (but not limited to) with PEG or other water-soluble polymers).As hydrazine, hydrazides are as semicarbazides, The nucleophilicity of the enhancing of amino oxygroup make its effectively and selectively with containing carbonyl or dicarbapentaborane (including (but not limited to) ketone, Aldehyde or with other similar chemically reactive functional groups) different kinds of molecules reaction.Referring to (for example) Shao, J. and Tarn, J., J.Am.Chem.Soc.117:3893-3899(1995)；H.Hang and C.Bertozzi, Ace.Chem.Res.34 (9):727- 736(2001).And be corresponding hydrazone to the reaction result of diazanyl, however, oxime usually by amino oxygroup and contains carbonyl or dicarbapentaborane Group (such as, ketone, aldehyde or with other similar chemically reactive functional groups) reaction generate.

Therefore, it is the non-natural amino with the side chain including following group in some embodiments being described herein Acid, the side chain includes azanol base, (it has the reactivity similar to azanol base and similar in construction to azanol class azanol base Base), masked azanol base (it can be easy to be changed into azanol base) or through protect azanol base (it is with similar after deprotection In the reactivity of azanol base).These amino acid include the amino acid of the structure with following formula：

Wherein：

K is-NR₆R₇Or-N=CR₆R₇；

R₆With R₇It is each independently selected from the group being made of following group：H, alkyl, substituted alkyl, alkenyl, through taking For alkenyl, alkoxy, it is substituted alkoxy, polyoxyalkylene, is substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, warp Substituted heteroaryl, alkaryl are substituted alkaryl, aralkyl and are substituted aralkyl ,-C (O) R " ,-C (O)₂R"、-C(O) N(R")₂, wherein R " is each independently hydrogen, alkyl, substituted alkyl, alkenyl, is substituted alkenyl, alkoxy, is substituted alcoxyl Base, aryl, heteroaryl, alkaryl, are substituted alkaryl, aralkyl or are substituted aralkyl substituted aryl；Or R₆Or R₇For L-X, wherein

X is selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymer；Polyethylene glycol Derivative；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Tree Fat；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbon water Compound；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial； Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other molecule covalents or The group of noncovalent interaction；Light cage covers part；It can photoisomerization part；Biotin；Biotin analog；It is combined with weight original The part of son；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active Agent；Aminothio acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups； Electron dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；And its Any combinations；And

L is optional, and is the connexon selected from the group being made of following group when it is present：Alkylidene, through taking For alkylidene, alkenylene, be substituted alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene or Be substituted alkylidene)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,- NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R')C(O)O-、-S(O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN(R')-、-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂-N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl.

In some embodiments of formula (XIV) compound, A is phenylene or is substituted phenylene.In formula (XIV) compound Some embodiments in, B is-(alkylidene is substituted alkylidene)-,-O- (alkylidene is substituted alkylidene)-,-S- it is (sub- Alkyl is substituted alkylidene)-or-C (O)-(alkylidene is substituted alkylidene)-.In some implementations of formula (XIV) compound In example, B is-O (CH₂)₂、-S(CH₂)₂-、-NH(CH₂)₂-、-CO(CH₂)₂- or-(CH₂)_n-, wherein n is 1 to 4.At formula (XIV) In some embodiments of compound, R₁For H, tertbutyloxycarbonyl (Boc), 9- fluorenylmethoxycarbonyl groups (Fmoc), N- acetyl group, four Acetyl fluoride base (TFA) or benzyloxycarbonyl group (Cbz).In some embodiments of formula (XIV) compound, wherein R₁For resin, amino Acid, polypeptide or polynucleotide.In some embodiments of formula (XIV) compound, wherein R₂For OH, O- methyl, O- ethyls or O- Tertiary butyl.In some embodiments of formula (XIV) compound, wherein R₂For resin, amino acid, polypeptide or polynucleotide.In formula (XIV) in some embodiments of compound, wherein R₂For polynucleotide.In some embodiments of formula (XIV) compound, wherein R₂For ribonucleic acid (RNA).In some embodiments of formula (XIV) compound, wherein R₂For tRNA.In formula (XIV) compound In some embodiments, wherein tRNA specific recognitions select codon.In some embodiments of formula (XIV) compound, wherein Selection codon is selected from by amber codon, ochre codon, opal codon, unique codon, rare codon, non- The group of native codon, five base codons and four base codons composition.In some embodiments of formula (XIV) compound In, wherein R₂To inhibit tRNA.In some embodiments of formula (XIV) compound, R₆With R₇It is each independently selected from by following The group of group composition：H, alkyl, substituted alkyl, alkoxy, be substituted alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, Aryl, substituted aryl, substituted heteroaryl, alkaryl, are substituted alkaryl, aralkyl and are substituted aralkyl heteroaryl Base.In some embodiments of formula (XIV) compound, R₆With R₇It is each independently selected from the group being made of following group：H、 Methyl, phenyl and-[(alkylidene is substituted alkylidene)-O- (hydrogen, alkyl or substituted alkyl)]_x, wherein x is 1-50. In some embodiments of formula (XIV) compound, K is-NR₆R₇。

In some embodiments of formula (XIV) compound, X is selected from by peptide, protein, enzyme, antibody, drug, dyestuff, fat The bioactivator for the group that matter, nucleosides, oligonucleotides, cell, virus, liposome, particle and micella form.In formula (XIV) in some embodiments of compound, X be selected from by antibiotic, fungicide, antivirotic, antiphlogistic, antitumor agent, The drug for the group that cardiovascular agents, antianxiety agent, hormone, growth factor and steroid agent form.In certain of formula (XIV) compound In a little embodiments, X is selected from by horseradish peroxidase, alkaline phosphatase, beta galactosidase and glucose oxidase group Into group enzyme.In some embodiments of formula (XIV) compound, X is selected from by fluorescence part, phosphorescent moieties, chemistry hair Light part, chelating moiety, electron dense part, magnetic part, insertion portion, radioactive segment, chromophoric moiety and energy The detectable label of the group of transfer part composition.

In certain embodiments, formula (XIV) compound stablizes at least one moon in aqueous solution under appropriate acid condition. In certain embodiments, formula (XIV) compound was in appropriate stable under acidic conditions at least 2 weeks.In certain embodiments, formula (XIV) compound was in appropriate stable under acidic conditions at least 5 days.In certain embodiments, the acid condition is pH 2 to 8.

These amino acid include the amino acid of the structure with formula (XV)：

Wherein

B is optional, and is the connexon selected from the group being made of following group when it is present：Low-grade alkylidene, Low-grade alkylidene, lower alkenylene are substituted, lower alkenylene, rudimentary sub- miscellaneous alkyl is substituted, is substituted the rudimentary miscellaneous alkane in Asia Base ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)_k- (wherein K for 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-NS (O)₂-、-OS(O)₂,-C (O)-(alkylidene Or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidene or Be substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, Wherein R' is each independently H, alkyl or substituted alkyl；

R₃With R₄It is each independently H, halogen, low alkyl group or is substituted low alkyl group or R₃With R₄Or two R₃Group Optionally form cycloalkyl or Heterocyclylalkyl.

It is nonrestrictive, represent acidic amino acid and have following structure：

Amino acid containing amino oxygroup can be by the amino acid predecessor (homoserine, serine and the Soviet Union that are easily obtained Propylhomoserin) it prepares.Referring to (for example) M.Carrasco and R.Brown, J.Org.Chem.68:8853-8858(2003).It is some Amino acid (such as L-2- amino -4- (amino oxygroup) butyric acid) containing amino oxygroup separates from natural origin (Rosenthal, G. et al., Life Sci.60:1635-1641(1997)).Other amino acid for containing amino oxygroup can be similar It prepares.In addition, the nonrestrictive illustrative synthesis of alpha-non-natural amino acid described herein is in shown in Figure 5.

C. alpha-non-natural amino acid：The chemical synthesis of the amino acid containing oxime

Alpha-non-natural amino acid containing oximido allow with containing some reactive carbonyls or dicarbapentaborane (including (but not limited to) Ketone, aldehyde or the other groups with similar reactivity) plurality of reagents react to form the new non-natural amino including new oximido Acid.This oxime exchange reaction allows non-natural amino acid polypeptides to be further functionalized.In addition, the original non-natural amino containing oximido Acid itself can be what is be applicable in, if oxime key necessary to amino acid is incorporated in polypeptide under the conditions of be stable (for example, live body It is interior, in vitro and chemical synthesis process described herein).

Therefore, it is the non-natural amino with the side chain including following group in some embodiments being described herein Acid, the side chain include oximido, class oximido (its have similar to oximido reactivity and similar in construction to oximido), it is masked Oximido (it can be easy to be changed into oximido) or through protect oximido (after deprotection its have similar to oximido reactivity). These amino acid include the amino acid of the structure with formula (XI)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R₅For H, alkyl, substituted alkyl, alkenyl, alkenyl, alkynyl are substituted, it are substituted alkynyl, alkoxy, are substituted alkane Oxygroup, alkyl alkoxy, polyoxyalkylene, are substituted polyoxyalkylene, aryl, are substituted substituted alkyl alkoxy Aryl, heteroaryl, alkaryl, are substituted alkaryl, aralkyl, are substituted aralkyl ,-(alkylidene or warp substituted heteroaryl Substituted alkylene)-ON (R ")₂,-(alkylidene is substituted alkylidene)-C (O) SR " ,-(alkylidene is substituted alkylidene)- S-S- (aryl or substituted aryl) ,-C (O) R " ,-C (O)₂R " or-C (O) N (R ")₂, wherein R " is each independently hydrogen, alkane Base, substituted alkyl, alkenyl, be substituted alkenyl, alkoxy, be substituted alkoxy, aryl, substituted aryl, heteroaryl, alkane virtue Base is substituted alkaryl, aralkyl or is substituted aralkyl；

Or R₅For L-X, wherein

X is selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymer；Polyethylene glycol Derivative；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Tree Fat；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbon water Compound；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial； Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other molecule covalents or The group of noncovalent interaction；Light cage covers part；It can photoisomerization part；Biotin；Biotin analog；It is combined with weight original The part of son；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active Agent；Aminothio acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups； Electron dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；And its Any combinations；And L is optional, and be the connexon selected from the group being made of following group when it is present：Alkylidene, warp Substituted alkylene, alkenylene, be substituted alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidenes Or be substituted alkylidene)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,- C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,- NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-(alkylidene is substituted alkylidene)-O-N=CR'- ,-(alkylidene is substituted alkylidene)-C (O) NR'- (alkylidene is substituted alkylidene)-,-(alkylidene is substituted alkylidene)-S (O)_k(alkylidene is substituted alkylidene)- S- ,-(alkylidene is substituted alkylidene)-S-S- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N (R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N =,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl；

Its restrictive condition is in the absence of A and B, and R is not methyl.

In some embodiments of formula (XI) compound, B for-O- (alkylidene is substituted alkylidene)-.Change in formula (XI) In some embodiments for closing object, B is-O (CH₂)-.In some embodiments of formula (XI) compound, R C_1-6Alkyl.In formula (XI) in some embodiments of compound, R is-CH₃.In some embodiments of formula (XI) compound, R₁For H, tertbutyloxycarbonyl (Boc), 9- fluorenylmethoxycarbonyl groups (Fmoc), N- acetyl group, tetrafluoro acetyl group (TFA) or benzyloxycarbonyl group (Cbz).At formula (XI) In some embodiments of compound, R₁For resin, amino acid, polypeptide or polynucleotide.In some implementations of formula (XI) compound In example, R₂For OH, O- methyl, O- ethyls or O- tertiary butyls.In some embodiments of formula (XI) compound, R₂For resin, amino Acid, polypeptide or polynucleotide.In some embodiments of formula (XI) compound, R₂For polynucleotide.

In some embodiments of formula (XI) compound, R₂For ribonucleic acid (RNA).In some realities of formula (XI) compound It applies in example, R₂For tRNA.In some embodiments of formula (XI) compound, tRNA specific recognitions selection codon.In formula (XI) in some embodiments of compound, selecting codon is selected from by amber codon, ochre codon, opal password The group that son, unique codon, rare codon, unnatural codons, five base codons and four base codons form. In some embodiments of formula (XI) compound, R₂To inhibit tRNA.In some embodiments of formula (XI) compound, R₅For alkane Base alkoxy, substituted alkyl alkoxy, are substituted polyoxyalkylene or-C (O) at polyoxyalkylene₂R".At formula (XI) In some embodiments of compound, R₅For-[(alkylidene is substituted alkylidene)-O- (hydrogen, alkyl or substituted alkyl)]_x, Middle x is 1-50.In some embodiments of formula (XI) compound, R₅For-(CH₂CH₂)-O-CH₃Or-COOH.

The amino acid of formula (XI) includes the amino acid of the structure with formula (XII)：

Wherein,

B is optional, and is the connexon selected from the group being made of following group when it is present：Low-grade alkylidene, warp Substitution low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, rudimentary sub- miscellaneous alkyl, be substituted rudimentary sub- miscellaneous alkyl ,- O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)_k- (wherein k is 1st, 2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-NS (O)₂-、-OS(O)₂- ,-C (O)-(alkylidene or Be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidene or warp Substituted alkylene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')- (alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-S (O)_kN (R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')= N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, wherein R' It is each independently H, alkyl or substituted alkyl；

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R₅For H, alkyl, substituted alkyl, alkenyl, alkenyl, alkynyl are substituted, it are substituted alkynyl, alkoxy, are substituted alkane Oxygroup, alkyl alkoxy, polyoxyalkylene, are substituted polyoxyalkylene, aryl, are substituted substituted alkyl alkoxy Aryl, heteroaryl, alkaryl, are substituted alkaryl, aralkyl, are substituted aralkyl ,-(alkylidene or warp substituted heteroaryl Substituted alkylene)-ON (R ")₂,-(alkylidene is substituted alkylidene)-C (O) SR " ,-(alkylidene is substituted alkylidene)- S-S- (aryl or substituted aryl) ,-C (O) R " ,-C (O)₂R " or-C (O) N (R ")₂；Wherein R " is each independently hydrogen, alkane Base, substituted alkyl, alkenyl, be substituted alkenyl, alkoxy, be substituted alkoxy, aryl, substituted aryl, heteroaryl, alkane virtue Base is substituted alkaryl, aralkyl or is substituted aralkyl；

Or R₅For L-X, wherein

X is selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymer；Polyethylene glycol Derivative；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Tree Fat；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbon water Compound；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial； Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other molecule covalents or The group of noncovalent interaction；Light cage covers part；It can photoisomerization part；Biotin；Biotin analog；It is combined with weight original The part of son；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active Agent；Aminothio acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups； Electron dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；And its Any combinations；And L is optional, and be the connexon selected from the group being made of following group when it is present：Alkylidene, warp Substituted alkylene, alkenylene, be substituted alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidenes Or be substituted alkylidene)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,- C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,- NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-(alkylidene is substituted alkylidene)-O-N=CR'- ,-(alkylidene is substituted alkylidene)-C (O) NR'- (alkylidene is substituted alkylidene)-,-(alkylidene is substituted alkylidene)-S (O)_k- (alkylidene is substituted alkylidene)- S- ,-(alkylidene is substituted alkylidene)-S-S- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N (R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N =,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl.

These amino acid include the amino acid with formula (XIII) structure：

Wherein,

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

Or R₅For L-X, wherein

Other non-limiting examples of these amino acid include the amino acid having following structure：

In addition, these amino acid include the amino acid of the structure with formula (XIV)：

Wherein：

K is-NR₆R₇Or-N=CR₆R₇；

R₆With R₇It is each independently selected from the group being made of following group：H, alkyl, substituted alkyl, alkenyl, through taking For alkenyl, alkoxy, it is substituted alkoxy, polyoxyalkylene, is substituted polyoxyalkylene, aryl, substituted aryl, miscellaneous Aryl, substituted heteroaryl, are substituted alkaryl, aralkyl and are substituted aralkyl ,-C (O) R " ,-C (O) alkaryl₂R"、-C(O)N(R")₂, wherein R " is each independently hydrogen, alkyl, substituted alkyl, alkenyl, is substituted alkenyl, alkoxy, warp Substituted alkoxy, aryl, heteroaryl, alkaryl, are substituted alkaryl, aralkyl or are substituted aralkyl substituted aryl；Or R₆Or R₇For L-X, wherein

X is selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymer；Polyethylene glycol Derivative；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Tree Fat；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbon water Compound；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial； Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other molecule covalents or The group of noncovalent interaction；Light cage covers part；It can photoisomerization part；Biotin；Biotin analog；It is combined with weight original The part of son；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active Agent；Aminothio acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups； Electron dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；And its Any combinations；And L is optional, and be the connexon selected from the group being made of following group when it is present：Alkylidene, warp Substituted alkylene, alkenylene, be substituted alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidenes Or be substituted alkylidene)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,- C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,- NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R')C(O)O-、-S(O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN(R')-、-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂N=N- and-C (R')₂-N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl.

These amino acid further include the amino acid of the structure with formula (XVI)：

Wherein：

X is selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymer；Polyethylene glycol Derivative；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Tree Fat；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbon water Compound；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial； Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other molecule covalents or The group of noncovalent interaction；Light cage covers part；It can photoisomerization part；Biotin；Biotin analog；It is combined with weight original The part of son；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active Agent；Aminothio acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups； Electron dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；And its Any combinations；And L is optional, and be the connexon selected from the group being made of following group when it is present：Alkylidene, warp Substituted alkylene, alkenylene, be substituted alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidenes Or be substituted alkylidene)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,- C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,- NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R')C(O)O-、-S(O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN(R')-、-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂-N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl.

In addition, these amino acid include the amino acid of the structure with formula (XVII)：

Wherein：

X is selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymer；Polyethylene glycol Derivative；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Tree Fat；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbon water Compound；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial； Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other molecule covalents or The group of noncovalent interaction；Light cage covers part；It can photoisomerization part；Biotin；Biotin analog；It is combined with weight original The part of son；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active Agent；Aminothio acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups； Electron dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；And its Any combinations；And L is optional, and be the connexon selected from the group being made of following group when it is present：Alkylidene, warp Substituted alkylene, alkenylene, be substituted alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidenes Or be substituted alkylidene)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,- C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,- NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R')C(O)O-、-S(O)_kN(R')、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN(R')-、-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂-N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl.

The non-limiting examples of these amino acid include the amino acid having following structure：

In addition, these amino acid include the amino acid of the structure with formula (XVIII)：

Wherein：

B is optional, and is the connexon selected from the group being made of following group when it is present：Low-grade alkylidene, Low-grade alkylidene, lower alkenylene are substituted, lower alkenylene, rudimentary sub- miscellaneous alkyl is substituted, is substituted the rudimentary miscellaneous alkane in Asia Base ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)_k- (wherein K for 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-NS (O)₂-、-OS(O)₂- ,-C (O)-(alkylene Base is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidenes Or be substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, Wherein R' is each independently H, alkyl or substituted alkyl；

X is selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymer；Polyethylene glycol Derivative；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Tree Fat；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbon water Compound；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial； Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other molecule covalents or The group of noncovalent interaction；Light cage covers part；It can photoisomerization part；Biotin；Biotin analog；It is combined with weight original The part of son；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active Agent；Aminothio acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups； Electron dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；And its Any combinations；And L is optional, and be the connexon selected from the group being made of following group when it is present：Alkylidene, warp Substituted alkylene, alkenylene, be substituted alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidenes Or be substituted alkylidene)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,- C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,- NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R')C(O)O-、-S(O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN(R')-、-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂-N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl；And

R_aIt is each independently selected from the group being made of following group：H, halogen, alkyl, substituted alkyl ,-N (R')₂、-C(O)_kR'(wherein k for 1,2 or 3) ,-C (O) N (R')₂,-OR' and-S (O)_kR'；Wherein R' is each independently H, alkyl or substituted alkyl and n are 0 to 8.

Alpha-non-natural amino acid based on oxime can by the method that has been described in fields or by method described herein come Synthesis, it includes：(a) alpha-non-natural amino acid containing azanol and the reagent reacting containing carbonyl or dicarbapentaborane are made；(b) make containing carbonyl The alpha-non-natural amino acid of base or dicarbapentaborane and the reagent reacting containing azanol；Or (c) makes the alpha-non-natural amino acid containing oxime contain with some There is the reagent of carbonyl or dicarbapentaborane (comprising such as reagent containing ketone or containing aldehyde reagent) reaction.Fig. 5 and these synthetic methods of Fig. 6 presentations Representativeness, non-limiting examples.

D. the cell of alpha-non-natural amino acid absorbs

When designing and during selection alpha-non-natural amino acid (including (but not limited to) for being incorporated in polypeptide), eukaryocyte it is non- Natural amino acid is absorbed as usually consider problem.For example, the high charge density of a-amino acid implies these chemical combination Object is less likely through cell.Natural amino acid is to be absorbed in eukaryocyte by the set of the Transmission system based on protein In.It can carry out assessing which alpha-non-natural amino acid (if present) is absorbed faster fast screening (example herein by cell The non-limiting examples for the test that 15 and 16 explanations can carry out alpha-non-natural amino acid).Referring to (for example) entitled " Protein In the U.S. Patent Publication case the 2004/198637th (during this case is hereby incorporated by reference in their entirety) of Arrays " Toxicological detection and Liu, D.R. and Schultz, P.G. (1999) Progress toward the evolution of an organism with an expanded genetic code.PNAS United States96:4780-4785.Although it inhales Receipts are easy to analyze by various calibratings, but design meets the alternative solution of the alpha-non-natural amino acid of cell absorption features to provide work The biosynthesis pathway of amino acid is generated in vivo.

In general, it is to be sufficient for effective albumen by the alpha-non-natural amino acid that cell as described herein absorption generates The concentration (including (but not limited to) n cell amount) of matter biosynthesis generates, but not up to such as influences the dense of other amino acid Spend or exhaust the degree of cellular resources.The typical concentration generated by this method is about 10mM to about 0.05mM.

E. the biosynthesis of alpha-non-natural amino acid

Many biosynthesis pathways are present in generating in amino acid and the cell of other compounds.It is although specific non- The biological synthesis method of natural amino acid may be not present in nature (including (but not limited to) cell), but described herein Method and composition these methods are provided.For example, the biosynthesis pathway of alpha-non-natural amino acid can be by adding newly Enzyme or the existing host cell approach of modification generate in host cell.Other new enzymes include naturally occurring enzyme or manually develop Enzyme.For example, biosynthesis (such as entitled " the In vivo incorporation of of p-Aminophenylalanine Example institute presentation in the WO 2002/085923 of unnatural amino acids ") addition is depended on from other biological body Known enzyme combination.The gene of these enzymes can be by introducing eukaryocyte to include the plasmid-transformed cells of these genes In.When being expressed in cell, these genes provide the enzymatic route of compound needed for synthesis.It is provided herein optionally to add Enzyme type example.Other enzyme sequences are seen in (for example) gene pool (Genbank).The enzyme manually to develop can phase Tongfang Formula is added in cell.By this method, cell mechanism and the source of cell are manipulated to generate alpha-non-natural amino acid.

A variety of methods can be used for preparing the novel enzymes for biosynthesis pathway or the existing approach that develops.For example, wrap Containing (but not limited to) such as by Maxygen, Inc. (can on WWW (world wide web) withwww.maxygen.comIt obtains ) exploitation recurrence restructuring can be used for develop novel enzyme and approach.Referring to (for example) Stemmer (1994), Rapid evolution of a protein in vitro by DNA shuffling,Nature370(4):389-391；And Stemmer,(1994),DNA shuffling by random fragmentation and reassembly:In vitro recombination for molecular evolution,Proc.Natl.Acad.Sci.USA.,91:10747-10751。 By Genencor (can on WWW withgenencor.comObtain) exploitation Similarly DesignPath^TMOptionally use In metabolic pathway engineering design, the way of alpha-non-natural amino acid is generated in cell for engineering design it includes (but not limited to) Footpath.This technology is using the combination of new gene (including (but not limited to) what is differentiated by functional genomics, molecular evolution and design Those) transform the existing approach in host organisms.Diversa Corporation (can on WWW withdiversa.comObtain) also provide Rapid Screening gene and gene approach storehouse technology, it includes (but not limited to) to generate use In the new way that alpha-non-natural amino acid is generated by biosynthesis.

In general, it is with foot with the alpha-non-natural amino acid that biosynthesis pathway engineered as described herein generates To carry out the concentration of effective Protein synthesis (including (but not limited to) n cell amount) generation, but not up to such as influence Other amino acid concentrations or the degree for exhausting cellular resources.The typical concentration in vivo generated by this method is about 10mM to about 0.05mM.Once to include generating the plasmid-transformed cells of the gene of the enzyme needed for particular approach and generate non-natural amino Acid then optionally further optimizes non-natural amino using in vivo selecting to be directed to ribosomal protein synthesis with cell growth The generation of acid.

F. other synthetic methods

Alpha-non-natural amino acid described herein can be used method described in fields or using described herein Technology is synthesized by its combination.As auxiliary, the various startings that following table offer may be combined to generate required functional group are electrophilic Sub- reagent and nucleopilic reagent.The information provided is intended for illustrative and not limits synthetic technology described herein.

Table 1：The example of covalent bond and its predecessor

In general, carbon electrophilic reagent is sensitive for the attack of complementary nucleopilic reagent (including carbon nucleophile), wherein The nucleopilic reagent of attack provides electronics to form new keys between nucleopilic reagent and carbon electrophilic reagent to carbon electrophilic reagent.

The non-limiting examples of carbon nucleophile are including (but not limited to) alkyl Grignard reagent (alkyl Grignard), alkene Base Grignard Reagent, aryl grignard reagent and alkynyl Grignard, organolithium, organic zinc, alkyl tin reagent, alkenyl tin reagent, Aryl tin reagent and alkynyl-tin reagents (Organotin), alkyl borane reagent, alkenyl borane reagent, aryl borane reagent and Alkynyl-borane reagents (organo-borane and organic borate)；These carbon nucleophiles have in water or polar organic solvent The advantages of kinetically stablizing.Other non-limiting examples of carbon nucleophile include phosphorus inner salt, enol and enolate Reagent；These carbon nucleophiles, which have, is relatively easy to the predecessor generation as known to the technical staff in synthetic organic chemistry field The advantages of.When being used in conjunction with carbon electrophilic reagent, carbon nucleophile shape between carbon nucleophile and carbon electrophilic reagent The carbon-carbon bond of Cheng Xin.

Suitable for the non-limiting examples of non-carbon nucleophile that are coupled with carbon electrophilic reagent including (but not limited to) primary amine With secondary amine, mercaptan, mercaptides and thioether, alcohol, alkoxide, azide, semicarbazides with and the like.When with carbon electrophilic When reagent is used in conjunction with, these non-carbon nucleophiles generally produce heteroatomic bond (C-X-C), and wherein X is hetero atom, it includes (but not limited to) oxygen, sulphur or nitrogen.

VI. there is the polypeptide of alpha-non-natural amino acid

For convenience, the form of the compound described in this part, property and other features are general and/or with spy Example is determined to describe.However, the form, property and other features described in this part should not be limited only to provided in this part one As property description or particular instance, but form, property and other features described in this part are similary well suited in formula All compounds in I-XVIII, XXX-XXXIV (A and B) and the scope of XXXX-XXXXIII, it includes in herein Specification, Formulas I-XVIII, XXX-XXXIV (A and B) described in claim and schema and XXXX-XXXXIII Any minor or specific compound in scope.

At least one alpha-non-natural amino acid is incorporated in polypeptide by composition described herein and method offer.Non-natural ammonia Base acid may be present at any position on polypeptide, any terminal position or any interior location it includes polypeptide.Compared with For homologous naturally occurring amino acid polypeptide, preferably alpha-non-natural amino acid does not destroy the activity and/or tertiary structure of polypeptide, unless This of activity and/or tertiary structure destroy a purpose being incorporated to alpha-non-natural amino acid in polypeptide.In addition, compared with homologous For naturally occurring amino acid polypeptide, by alpha-non-natural amino acid be incorporated in polypeptide can modified polypeptide to a certain extent activity (for example, manipulating the therapeutic efficiency of polypeptide；Improve the security overview of polypeptide；Adjust the pharmacokinetics of polypeptide, pharmacology and/ Or drug effect is (for example, increase is water-soluble, biological usability；Increase serum half-life；Increase treatment half-life period；Adjust immunogenicity； Adjust bioactivity；Or extend circulation time)；Other functional groups are provided to polypeptide；Label, mark are incorporated into polypeptide or can be examined Survey signal；The separating property of easyization polypeptide；And any combinations of foregoing modification) and/or tertiary structure, and do not cause to live completely The destruction of property and/or tertiary structure.It is usually to realize these mesh being incorporated to these of activity and/or tertiary structure modification Mark, although for homologous naturally occurring amino acid polypeptide, alpha-non-natural amino acid is incorporated in polypeptide also can be to polypeptide Activity and/or tertiary structure do not have an impact.Correspondingly, it is believed that non-natural amino acid polypeptides, including non-natural amino acid polypeptides Composition, for manufacture these polypeptides and peptide composition method, for purifying, separate and characterize these polypeptides and polypeptide The method and the method for these polypeptides of use and peptide composition of composition are in the scope of this disclosure.In addition, herein Described in non-natural amino acid polypeptides can be connected to another polypeptide and (including (for example) non-natural amino acid polypeptides or naturally deposit In amino acid polypeptide) on.

Non-natural amino acid polypeptides described herein can pass through biosynthesis or abiotic synthetically prepared.Biosynthesis is anticipated For meaning using any method of translation system (cell or acellular), it includes use at least one of following components：Poly-nuclear glycosides Acid, codon, tRNA and ribosomes.Abiotic synthesis means any method for not utilizing translation system：This method can be into One step, which is divided into, utilizes solid-state peptide symthesis method, the method for Solid-phase peptide synthesis；Utilize the method for at least one enzyme；It is and unfavorable With the method for at least one enzyme；In addition, any part in this subdivided portions can be overlapped and many methods can utilize these thin The combination of branch point.

Method described herein, composition, strategy and technology be not limited to the specific type of polypeptide or protein, species or Family.Really, substantially any polypeptide can include at least one alpha-non-natural amino acid described herein.Only for example, it is more Peptide can be homologous with the treatment albumen matter selected from the group being made of following object：α -1 antitrypsins, angiostatin, Anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, C-X-C become Change the factor, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, Calcitonin, c-kit ligands, cell factor, CC chemotactic factor (CF)s, monocyte chemoattractant protein-1, monocyte chemoattractant protein- 2nd, monocyte chemoattractant protein-3, -1 α of monocyte inflammatory protein, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligands, collagen, Colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cell factor, epithelium neutrophilic granulocyte activation Peptide -78, MIP-16, MCP-1, epidermal growth factor (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin(EPO) (EPO), Exfoliative toxin,or exfoliatin, factors IX, factor Ⅴ II, Factor IX, factor X, fibroblast growth factor (FGF), fibrin Original, fibronectin, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, promoting sexual gland hormone, growth The factor, growth factor receptors, grf, Hedgelog protein, hemochrome, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 Receptors, insdri, insulin-like growth because Sub (IGF), IGF-I, IGF-II, interferon (IFN), IFN-α, IFN-β, IFN-γ, interleukins (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), breast Siderophillin, LIF ELISA, luciferase, neural element, neutrophil inhibitory factor (nif) (NIF), oncostatin M, into Bone protein, oncoprotein, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, multi-effector, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxain, feritin, SCF, atom Synthetic proteins, Soluble complement receptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, life It is long adjust element, Somat, growth hormone, streptokinase, super antigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic-shock syndrome toxin, extrasin alpha 1st, histiotype plasminogen activation factor, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor α, tumour are bad Necrosis factor β, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urine Kinases, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, testosterone Receptor, aldosterone receptor, ldl receptor and cortisone.In related or other embodiment, non-natural amino acid polypeptides also can be with Any polypeptide member of growth hormone supergene family is homologous.

Non-natural amino acid polypeptides can be such as the further modification of the warp elsewhere or non-natural amino in this disclosure Sour polypeptide can be used without further modification.Alpha-non-natural amino acid can be incorporated in polypeptide for a variety of purposes, it includes (but Be not limited to) change of customization protein structure and/or function, change the size of Protease target point, acidity, nucleophilicity, hydrogen bond, Hydrophobicity, accessibility, targeting moiety (including (but not limited to) for polypeptide array) etc..Include the more of alpha-non-natural amino acid Peptide can have enhance or even completely new catalysis or bio-physical property.Only for example, following property can be by will be non- Natural amino acid is contained in polypeptide to be transformed：Toxicity, bio distribution, structural property, spectral quality, chemistry and/or light Chemical property, catalytic capability, half-life period (including (but not limited to) serum half-life), with the reaction of other molecules (comprising (but unlimited In) covalently or non-covalently) ability and its similarity.Combination with the polypeptide comprising at least one alpha-non-natural amino acid Object is suitable for (include, but are not limited to) novel therapeutic, diagnosis, catalyzing enzyme, industrial enzyme, binding protein (including (but not limited to) anti- Body) and study (including (but not limited to) the research to protein structure and function).Referring to (for example) Dougherty, (2000)Unnatural Amino Acids as Probes of Protein Structure and Function,Current Opinion in Chemical Biology,4:645-652。

In addition, the side chain of the non-natural amino acid constituents of polypeptide can provide various other functional groups to polypeptide；Only citing comes It says, and is not in a limitative way, the side chain of the non-natural amino acid moieties of polypeptide can include any one of following substance：Mark； Dyestuff；Polymer；Water-soluble polymer；The derivative of polyethylene glycol；Photocrosslinking agent；Cytotoxin compounds；Drug；Compatibility Mark；Photoaffinity marks；Reactive compounds；Resin；Second protein or polypeptide or polypeptide analog；Antibody or antibody piece Section；Metal-chelator；Co-factor；Aliphatic acid；Carbohydrate；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water Dissolubility dendritic, cyclodextrin, biomaterial；Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactivity Part；Novel functional group；With other molecule covalents or the group of noncovalent interaction；Light cage covers part；Can actinic radiation swash The part of hair；Ligand；It can photoisomerization part；Biotin；Biotin analog；It is combined with the part of heavy atom；Chemically The group of cracking；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active agent；Aminothio acid；Poison Property part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electron dense group；Magnetic Property group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；Small molecule；Inhibition ribose core Acid, radioactive nucleotides；Neutron capture agent；The derivative of biotin；Quantum dot；Nanometer transfers element；Radioactivity transfers element；Antibody It is enzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, antihormones, antioxidant, suitable Body, guide RNA, saponin, shuttle vector, macromolecular, plan epitope, receptor, reverse micelle and any combination thereof.

On the one hand, composition includes at least one at least one (including (but not limited to) at least two, at least three It is a, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten or ten or more) The polypeptide of alpha-non-natural amino acid.These alpha-non-natural amino acids may be the same or different.In addition, including 1,2,3,4,5,6,7,8,9, It 10th, can in the polypeptide of the similar and different alpha-non-natural amino acid of 11,12,13,14,15,16,17,18,19,20 or 20 or more It can be there are the different loci of 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 20 or more. On the other hand, composition includes (but all or less than) specific amino acids at least one wherein present in polypeptide through non-natural amino The polypeptide of acid substitution.For the given polypeptide with more than one alpha-non-natural amino acid, these alpha-non-natural amino acids can phase It is same or different that (such as, only for example, polypeptide can include two or more different types of alpha-non-natural amino acid or can Include two identical alpha-non-natural amino acids).For the given polypeptide with more than two alpha-non-natural amino acids, non-natural Amino acid can the alpha-non-natural amino acid of identical, different or for multiple numbers identical types and the different non-natural aminos of at least one The combination of acid.

Although the embodiment of non-natural amino acid polypeptides described herein can be by Solid-phase peptide synthesis (such as, only For example, on hard resin), solution phase peptide synthetic method and/or carry out chemical synthesis without the help of enzyme, it is but described herein The other embodiments of non-natural amino acid polypeptides allow through cell membrane, cell extract or lysate system or pass through Vivo system (such as, only for example, using prokaryotic cell or the cell mechanism of eukaryocyte) synthesizes.In other or volume In outer embodiment, a kind of key feature of non-natural amino acid polypeptides described herein is that it can be closed using ribosomes Into.In other or Additional examples of composition of the non-natural amino acid polypeptides being described herein, non-natural amino acid polypeptides can pass through The combination of method is (including (but not limited to) hard resin, without the help of enzyme, by means of ribosomes and/or pass through vivo system Combination) synthesize.

By ribosomes and/or vivo system synthesize non-natural amino acid polypeptides have on hard resin or not borrowing Help enzymatic synthesis non-natural amino acid polypeptides it is different the advantages of and feature.These advantages and features include different impurity point Cloth：There should be the impurity for coming from utilized biosystem using the system of ribosomes and/or vivo system, it includes hosts Cell protein, membrane part and lipid, and from utilizing hard resin and/or can without the help of the Impurity Distribution of the system of enzyme Include the other chemicals used in organic solvent, protecting group, resin material, coupling reagent and synthesis program.In addition, pass through The isotopic pattern of the non-natural amino acid polypeptides synthesized using ribosomes and/or vivo system can reflect the original that cell utilizes The isotopic pattern of material；On the other hand, on hard resin and/or without the help of enzymatic synthesis non-natural amino acid polypeptides it is same The plain pattern in position can reflect the isotopic pattern of the amino acid utilized in synthesis.In addition, it is by using ribosomes and/or in vivo Integration into alpha-non-natural amino acid can be substantially free of the D- isomers of amino acid and/or can be readily able to internal cysteines Amino acid is incorporated in the structure of polypeptide and/or may seldom provide internal amino acid deletion polypeptide.On the other hand, solid is passed through Resin and/or without using enzymatic synthesis non-natural amino acid polypeptides can have high level amino acid D- isomers and/or The internal cysteines amino acid of lower content and/or the internal amino acid deletion polypeptide of higher percent.In addition, fields Technical staff should be able to distinguish by using ribosomes and/or vivo system synthesis non-natural amino acid polypeptides with passing through Hard resin and/or the non-natural amino acid polypeptides without using enzymatic synthesis.

VII. the composition and method of nucleic acid and oligonucleotides are included

A. with general recombinant nucleic acid method in this article

In the numerous embodiments for the method and composition being described herein, polypeptide of interest will be encoded (comprising for example GH polypeptides) nucleic acid separation, clone and usually using recombination method transform.These embodiments are used for (include, but are not limited to) egg White matter is expressed or used during the variant from polypeptide, derivative, expression cassette or other sequences are generated.In some realities It applies in example, the sequence of coded polypeptide is operatively connected on allogeneic promoter.

Encoding the nucleotide sequence for the polypeptide for including alpha-non-natural amino acid can synthesize according to the amino acid sequence of parent polypeptide, And then change nucleotide sequence with realize the introducing of relevant amino acid residue (that is, be incorporated to or substitute) or remove (that is, lack or Substitution).Nucleotide sequence can form easily to modify by rite-directed mutagenesis according to conventional methods.Alternatively, nucleotide sequence can It is prepared by chemical synthesis, it includes (but not limited to) by using oligonucleotide synthesizer, wherein oligonucleotides is basis The amino acid sequence design of required polypeptide, and preferably select recombinant polypeptide by result from host cell therein preference those are close Numeral.For example, several small oligonucleotides of the part of polypeptide needed for coding can be closed by PCR, connection or connection chain reaction Into and assemble.Referring to (for example) Barany et al., Proc.Natl.Acad.Sci.88:189-193(1991)；U.S.6,521, 427, it is to be incorporated herein by reference.

Alpha-non-natural amino acid method and composition described herein utilizes the routine techniques in genetic recombination field.It takes off Dew includes Sambrook etc. for the basic works of the universal method of alpha-non-natural amino acid method and composition described herein People, Molecular Cloning, A Laboratory Manual (the 3rd edition, 2001)；Kriegler,Gene Transfer and Expression:A Laboratory Manual(1990)；And Current Protocols in Molecular Biology (Ausubel et al. volumes, 1994).

The general works for describing Protocols in Molecular Biology includes Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, Academic Press, Inc., San Diego,CA(Berger)；Sambrook et al., Molecular Cloning-A Laboratory Manual (second edition), the 1-3 volumes, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 (" Sambrook ") and Current Protocols in Molecular Biology, F.M.Ausubel et al. volume, The conjunction of Current Protocols, Greene Publishing Associates, Inc. and John Wiley＆Sons, Inc. Enterprise is provided, (1999 annual supplement) (" Ausubel ")).These works description mutation formed, carrier, promoter purposes and Many other related subjects are related to including (but not limited to) comprising the choosing for being used to prepare the protein comprising alpha-non-natural amino acid Select codon, orthogonal tRNA, orthogonal synthesis enzyme and its to gene or polynucleotide generation.

Various types of mutation, which are formed, to be used in alpha-non-natural amino acid method and composition described herein with for realization A variety of purposes, it includes (but not limited to) for generating novel synzyme or tRNA, for making tRNA molecular mutations, for making volume Code synzyme, the mutant polynucleotide in tRNA storehouses for generating synzyme storehouse, select codon for generating, are compiled for being inserted into The selection codon of alpha-non-natural amino acid in code protein or polypeptide of interest.It includes (but not limited to) rite-directed mutagenesis shapes Into, random point mutation formation, homologous recombination, DNA reorganization or other recurrence mutation forming method, chimeric construct, using containing urine The mutation formation of the template of pyrimidine, oligonucleotides directed mutagenesis, the DNA mutation through phosphorothioate are formed, lacked using band The mutation of the double-stranded DNA or its analog of mouth is formed, or any combination thereof.Other appropriate methodologies include point mispairing reparation, use Lack mutation formation, limitation-selection and the limitation-purifying of the host strain repaired, deletion mutation is formed, is closed by total gene Into Mutation induction, double-strand break reparation and its similar approach.It is formed including (but not limited to) the mutation including being fitted together to construct It is also contained in alpha-non-natural amino acid method and composition described herein.In one embodiment, mutation formation can be by natural There are molecule or become exclusive or mutation naturally occurring molecule Given information (compare including (but not limited to) sequence, physical property, Crystal structure or its analog) guidance.

Existing works and example describe these and other relative programs herein.Other information see following discloses case and In references cited therein：Ling et al., Approaches to DNA mutagenesis:an overview,Anal Biochem.254(2):157-178(1997)；Dale et al., Oligonucleotide-directed random mutagenesis using the phosphorothioate method,Methods Mol.Biol.57:369-374 (1996)；Smith,In vitro mutagenesis,Ann.Rev.Genet.19:423-462(1985)；Botstein and Shortle,Strategies and applications of in vitro mutagenesis,Science 229:1193- 1201(1985)；Carter,Site-directed mutagenesis,Biochem.J.237:1-7(1986)；Kunkel, The efficiency of oligonucleotide directed mutagenesis, in Nucleic Acids＆ In Molecular Biology (Eckstein, F. and Lilley, D.M.J. are compiled, Springer Verlag, Berlin)) (1987)；Kunkel,Rapid and efficient site-specific mutagenesis without phenotypic selection,Proc.Natl.Acad.Sci.USA 82:488-492(1985)；Kunkel et al., Rapid and efficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol.154,367-382(1987)；Bass et al., Mutant Trp repressors with new DNA- binding specificities,Science 242:240-245(1988)；Methods in Enzymol.100:468- 500(1983)；Methods in Enzymol.154:329-350(1987)；Zoller and Smith, Oligonucleotide- directed mutagenesis using Ml3-derived vectors:an efficient and general procedure for the production of point mutations in any DNA fragment,Nucleic Acids Res.10:6487-6500(1982)；Zoller and Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors,Methods in Enzymol.100: 468-500(1983)；Zoller and Smith, Oligonucleotide-directed mutagenesis:a simple method using two oligonucleotide primers and a single-stranded DNA template, Methods in Enzymol.154:329-350(1987)；Taylor et al., The use of phosphorothioate- modified DNA in restriction enzyme reactions to prepare nicked DNA,Nucl.Acids Res.13:8749-8764(1985)；Taylor et al., The rapid generation of oligonucleotide- directed mutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res.13:8765-8785(1985)；Nakamaye and Eckstein, Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis,Nucl.Acids Res.14:9679- 9698(1986)；Sayers et al., 5'-3'Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis,Nucl.Acids Res.16:791-802(1988)；Sayers etc. People, Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence ofethidium bromide,(1988) Nucl.Acids Res.16:803-814；Kramer et al., The gapped duplex DNA approach to oligonucleotide-directed mutation construction,Nucl.Acids Res.12:9441-9456 (1984)；Kramer and Fritz, Oligonucleotide-directed construction of mutations via gapped duplex DNA,Methods in Enzymol.154:350-367(1987)；Kramer et al., Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations,Nucl.Acids Res.16:7207 (1988)；Fritz et al., Oligonucleotide-directed construction of mutations:a gapped duplex DNA procedure without enzymatic reactions in vitro,Nucl.Acids Res.16: 6987-6999(1988)；Kramer et al., Point Mismatch Repair, Cell 38:879-887(1984)；Carter Et al., Improved oligonucleotide site-directed mutagenesis using M13vectors, Nucl.Acids Res.13:4431-4443(1985)；Carter,Improved oligonucleotide-directed mutagenesis using Ml3vectors,Methods in Enzymol.154:382-403(1987)； Eghtedarzadeh and Henikoff, Use of oligonucleotides to generate large deletions, Nucl.Acids Res.14:5115(1986)；Wells et al., Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin,Phil.Trans.R.Soc.Lond.A 317:415-423(1986)；Nambiar et al., Total synthesis and cloning of a gene coding for the ribonuclease S protein,Science 223:1299-1301(1984)；Sakmar and Khorana, Total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein(transducin),Nucl.Acids Res.14:6361-6372(1988)；Wells et al., Cassette mutagenesis:an efficient method for generation of multiple mutations at defined sites,Gene 34:315-323(1985)； Grundstrom et al., Oligonucleotide-directed mutagenesis by microscale'shot-gun' gene synthesis,Nucl.Acids Res.13:3305-3316(1985)；Mandecki,Oligonucleotide- directed double-strand break repair in plasmids of Escherichia coli:a method for site-specific mutagenesis,Proc.Natl.Acad.Sci.USA,83:7177-7181(1986)； Arnold,Protein engineering for unusual environments,Current Opinion in Biotechnology4:450-455(1993)；Sieber et al., Nature Biotechnology, 19:456-460 (2001).W.P.C.Stemmer,Nature 370,389-91(1994)；And I.A.Lorimer, I.Pastan, Nucleic Acids Res.23,3067-8(1995).Other details on these many methods are found in Methods in In Enzymology volumes 154, also describe with various mutation forming methods to leading to the problem of effective control of failure.

Method and composition described herein also includes and uses eukaryotic host cell, non-eukaryotic host cell and biology Body is by orthogonal tRNA/RS to being in vivo incorporated to alpha-non-natural amino acid.Host cell is passed through corresponding to polypeptide described herein Polynucleotide and comprising the polynucleotide corresponding to polypeptide described herein construct (including (but not limited to) corresponding to this The carrier of the polypeptide of described in the text can be (for example) cloning vector and expression vector) genetically engineered (including (but not limited to) Conversion, transduction or transfection).For example, by the code area of protein derived from orthogonal tRNA, orthogonal tRNA/synthetase and desire Domain is operably connected to have on functional gene expression control elements in required host cell.Carrier can be (for example) matter Grain, clay, bacteriophage, bacterium, virus, exposed polynucleotide and the form for engaging polynucleotide.It (is worn with standard method comprising electricity Hole (Fromm et al., Proc.Natl.Acad.Sci.USA 82,5824 (1985)), by viral vector infection, by beads Or the small particles high velocity ballistic that the Medium Culture of particle has nucleic acid penetrates) carrier is introduced into cell and/or microorganism or led to (Klein et al., Nature 327,70-73 (1987)) and/or its analog on surface.

Host cell through Engineering Design can in it is improved be suitable for it is all such as (e.g.) screening step, activating promoters or It selects to cultivate in the conventional nutrient culture of the behavior of transformant.These cells can be cultivated optionally as transgenic organism.Its He includes including (but not limited to) the applicable bibliography on Cell isolation and culture (for example, being separated on subsequent nucleic acid) Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, the 3rd edition, Wiley-Liss, New York and references cited therein；Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley&Sons,Inc.New York,NY；Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture；Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.

Can obtain several well-known process target nucleic acid being introduced into cell, either of which can be used for described herein Method and composition in.It includes：Recipient cell and the bacterial protoplast fusion containing DNA, electroporation, projectile are banged It hits and with viral vector infection (being discussed further herein) etc..Bacterial cell can be used for amplification to contain and correspond to this paper Described in polypeptide DNA construct plasmid number.Make the plasmid in bacterial growth to the log stages and bacterium that can pass through institute Known a variety of method separation are (see, e.g., Sambrook) in category field.In addition, numerous kits can buy on the market with For the plasmid purification from bacterium, (referring to (for example) EasyPrep^TM、FlexiPrep^TM, both from Pharmacia Biotech；StrataClean^TM, from Stratagene；And QIAprep^TM, from Qiagen).Through what is separated and purify Plasmid for transfectional cell or is incorporated to for the relevant carriers of infection biological body then after further treatment to generate other plasmids In.Typical carrier contains transcription and translation terminator, transcription and translation homing sequence and suitable for adjusting specific objective core The promoter of the expression of acid.Carrier optionally includes the General Expression box containing at least one independent terminator sequence, allows sequence Sequence (including (but not limited to) shuttle vector) that row box replicates in eucaryote or prokaryotes or both and for original The selected marker of both core system and eukaryotic system.Carrier be suitable in prokaryotes, eucaryote or preferred the two replicate and It integrates.Referring to Gillam and Smith, Gene 8:81(1979)；Roberts et al., Nature, 328:731(1987)； Schneider, E. et al., Protein Expr.Purif.6 (1):10-14(1995)；Ausubel,Sambrook,Berger (as above).The catalogue of bacterium and bacteriophage suitable for clone is provided by (for example) ATCC, for example, the The published by ATCC ATCC Catalogue of bacteria and bacteriophage (1992) Gherna et al. (eds.).For being sequenced, gram Other grand and molecular biology otherwise base programs and potential theoretical considerations also see Watson et al. (1992) in Recombinant DNA Second Edition Scientific American Books, NY.It is in addition, basic Upper any nucleic acid (and substantially any labeled nucleic acid, no matter standard or non-standard) can from a variety of commercial sources any one Customization or standard are ordered, these commercial sources such as the Midland Certified Reagent Company (Midland, TX mcrc.com), The Great American Gene Company (Ramona, CA, on the world wide web (www withgenco.com Obtain), ExpressGen Inc. (Chicago, IL, on the world wide web (www withexpressgen.comObtain), Operon Technologies Inc. (Alameda, CA) and many other sources.

B. codon is selected

Protein synthesis mechanism is described in detail in the selection codon being covered by method and composition described herein Genetic code subframe.For example, codon is selected including (but not limited to) unique three base codons, nonsense codon (such as terminator codon, it includes (but not limited to) amber codon (UAG) or opal codons (UGA)), non-natural are close Numeral, the codon of four or more base, rare codon or its analog.Gene or polynucleotide needed for can introducing In selection codon number there are broad range, encoding at least part of single poly-nuclear glycosides of polypeptide of interest In acid including (but not limited to) one or more, two or more, three or more, 4,5,6,7,8, 9,10 or 10 or more.

In one embodiment, the described method includes the selection codons used as terminator codon for being in vivo incorporated to One or more alpha-non-natural amino acid.For example, generate identification terminator codon (including (but not limited to) UAG) O-tRNA and it is aminoacylated with required alpha-non-natural amino acid by O-RS.This O-tRNA is not by the aminoacyl of naturally occurring host Base-tRNA synzyme identifies.Routine rite-directed mutagenesis can be used to be formed at the site of interest in polypeptide of interest to introduce eventually Only codon (including (but not limited to) UAG).Referring to (for example) Sayers, J.R. et al. (1988), 5', 3'Exonuclease in phosphorothioate-based oligomicleotide-directed mutagenesis.Nucleic Acids Res,16(3):791-802.It is non-when the nucleic acid of polypeptide O-RS, O-tRNA and coding of interest is in vivo combined Natural amino acid responds UAG codons and is able to be incorporated to obtain the polypeptide for containing alpha-non-natural amino acid in specified location.

The notable confusion being incorporated to without causing eukaryotic host cell of alpha-non-natural amino acid can in vivo be carried out.Citing comes It says, because the inhibition efficiency of UAG codons depends on O-tRNA, (inhibiting tRNA including (but not limited to) amber) discharges with eucaryon Between the factor (including (but not limited to) eRF) (it is combined with terminator codon and the peptide in growth is caused to be discharged from ribosomes) Competition, the expression of O-tRNA and/or inhibition tRNA can be increased to adjust by (include, but are not limited to) by inhibiting efficiency.

Codon is selected also to include extended codon, it includes the passwords of four or more base of (but not limited to) Sub (codon of such as, four, five, six or six or more bases).The example of four base codons includes (but unlimited In) AGGA, CUAG, UAGA, CCCU with and the like.The example of five base codons including (but not limited to) AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC with and the like.The feature of method and composition described herein includes Inhibit extended codon using according to frameshit.The codon of four or more base (can will include, but are not limited to) one Or in multiple alpha-non-natural amino acid insertion same proteins.For example, with anticodon loop (for example, at least 8- 10nt anticodon loops) saltant type O-tRNA (including (but not limited to) specific frameshift suppressor tRNA) in the presence of, four or The codon of four or more bases is read as single amino acid.In other embodiments, anticodon loop decodable code, it includes At least one four base codon of (but not limited to), at least one five base codon or at least one six base codon or more It is more.Because there are 256 possible four base codons, four or four can be used in a variety of alpha-non-natural amino acids in same cell The codon coding of a Yi Shang base.Referring to Anderson et al., (2002) Exploring the Limits of Codon and Anticodon Size,Chemistry and Biology,9:237-244；Magliery,(2001)Expanding the Genetic Code:Selection of Efficient Suppressors of Four-base Codons and Identification of"Shifty"Four-base Codons with a Library Approach in Escherichia coli,J.Mol.Biol.307:755-769。

For example, four base codons have been used for that alpha-non-natural amino acid is incorporated to egg using in vitro biological synthesis method In white matter.Referring to (for example) Ma et al., (1993) Biochemistry, 32:7939-7945；And Hohsaka et al., (1999)J.Am.Chem.Soc,121:34-40.With two chemical acylation frameshift suppressor tRNAs, using CGGG and AGGU in live body The outer NBU derivatives by 2- naphthylalanines and lysine are incorporated in streptavidin simultaneously.Referring to (for example) Hohsaka etc. People, (1999) J.Am.Chem.Soc, 121:12194-12195.In in vivo studying, Moore et al. researchs are anti-with NCUA The tRNALeu derivatives of codon inhibit the ability of UAGN codons (N can be U, A, G or C), and discovery tetrad group UAGA can be by TRNALeu with UCUA anticodons is decoded with 13% to 26% efficiency, wherein only a little decoding in 0 or -1 frame. Referring to, Moore et al., (2000) J.Mol.Biol., 298:195-205.In one embodiment, based on rare codon or nothing The extension codon of adopted codon can be used in method and composition described herein, can reduce in other unwanted positions Missense at point is readed over to be inhibited with frameshit.

For given system, select codon that can also include one of natural three base codon, wherein endogenous Sexual system is without using (or being rarely employed) natural base codon.For example, it includes lack to identify natural three bases password The system of the tRNA of son and/or the system that wherein three base codons are rare codon.

Codon is selected optionally to include nonnatural base pair.These nonnatural bases are accorded with to further expanding existing heredity Number system.One Extra bases by 64 by the number of triplet codon to increasing to 125.The property of 3rd base-pair includes steady Fixed and selective base pairing is incorporated to the effective enzymatic of high fidelity in DNA and by what polymerase carried out in nascent non-natural Effectively continue primer extension after base-pair synthesis.The description of nonnatural base pair to being suitably adapted for method and composition includes (for example) Hirao et al., (2002) An unnatural base pair for incorporating amino acid analogues into protein,Nature Biotechnology,20:177-182, and referring also to Wu, Y. et al. (2002)J.Am.Chem.Soc.124:14626-14630.Other related publication are listed in herein.

For in vivo using, non-natural nucleoside is permeable and phosphorylated to form corresponding triguaiacyl phosphate for film. In addition, increased hereditary information is stable and do not destroyed by cellular enzymes.Benner and other people previous make great efforts utilization and allusion quotation The different Hydrogen Bond Types of those Hydrogen Bond Types in type Watson-Crick base-pair (Watson-Crick pair), wherein being most worth The example that must be paid attention to is iso- C:Iso- G pairs.Referring to (for example) Switzer et al., (1989) J.Am.Chem.Soc, 111:8322- 8322；And Piccirilli et al., (1990) Nature, 343:33-37；Kool,(2000) Curr.Opin.Chem.Biol.,4:602-608.These bases are usually to a certain extent with natural base mispairing and being unable to enzyme Promote to replicate.Kool and partner confirm that the hydrophobic stacking interaction between base may replace hydrogen bond to drive the shape of base-pair Into.Referring to, Kool, (2000) Curr.Opin.Chem.Biol., 4:602-608；And Guckian and Kool, (1998) Angew.Chem.Int.Ed.Engl.,36(24):2825-2828.Making great efforts to develop the non-natural alkali for meeting all above-mentioned requirements During base pair, Schultz, Romesberg and partner systematically synthesize and have studied a series of unnatural hydrophobics Base.It was found that PICS:PICS self pairs are more more stable than natural base-pair, and it can pass through e. coli dna polymerase I's Klenow segments (KF) are effectively incorporated in DNA.Referring to (for example) McMinn et al., (1999) J.Am.Chem.Soc, 121: 11585-11586；And Ogawa et al., (2000) J.Am.Chem.Soc, 122:3274-3278.3MN-3MN self pairs can To be synthesized for the enough efficiency of biological function and selectivity by KF.Referring to (for example) Ogawa et al., (2000) J.Am.Chem.Soc,122:8803-8804.However, two kinds of bases serve as the chain terminator further replicated.Develop recently Go out and can be used for the mutant DNA polymerases for replicating PICS self pairs.In addition, 7AI self pairs can be replicated.Referring to (for example) Tae et al., (2001) J.Am.Chem.Soc, 123:7439-7440.Also developed and formed stable match somebody with somebody afterwards in combination Cu (II) To novel metal base-pair Dipic:Py.Referring to, Meggers et al., (2000) J.Am.Chem.Soc, 122:10714- 10715.It is described herein non-because extended codon and unnatural codons are inherently orthogonal with native codon Natural amino acid method can utilize this property to generate associated orthogonal tRNA.

Translation bypath system can also be used for alpha-non-natural amino acid being incorporated in required polypeptide.It, will in bypath system is translated Big sequence is incorporated in gene, but is not translated as protein.The sequence contain serve as induce ribosomes cross the sequence and Continue the structure of the signal of translation in insertion downstream.

In certain embodiments, protein of interest in method described herein and/or composition or polypeptide (or its Part) it is to be encoded by nucleic acid.In general, nucleic acid includes at least one selection codon, at least two selection codons, at least three Select codon, at least four selection codons, at least five selection codons, at least six selection codons, at least seven Select the selection codon of codon, at least eight selection codons, at least nine selection codons, ten or ten or more.

The gene for encoding protein of interest or polypeptide can be in " Mutagenesis and Other Molecular It uses those skilled in the art known under Biology Techniques " guidances and method described herein is lured Become with including (for example) for being incorporated to the one or more selection codon of alpha-non-natural amino acid.For example, by institute The nucleic acid mutagenesis of concern of albumen matter is comprising one or more selection codon, to provide one or more non- Natural amino acid is incorporated to.Method and composition described herein includes any this variant, and it includes (but not limited to) to appoint The mutant form of what protein (for example, including at least one alpha-non-natural amino acid).Similarly, method and group described herein It closes object and includes corresponding nucleic, i.e. there are one or more codings or allow one or more alpha-non-natural amino acid Be in vivo incorporated to selection codon any nucleic acid.

The nucleic acid molecules for encoding polypeptide of interest (comprising (only for example) GH polypeptides) can be easy to mutation in polypeptide It is any needed for cysteine is introduced at position.Cysteine is widely used in reactive molecule, water-soluble polymer, protein Or various other molecules are introduced on protein of interest.Suitable for cysteine to be incorporated to the method in the required position of polypeptide It is well known in the art, being such as described in U.S. Patent No. 6,608,183, (it is to be fully incorporated by reference Those methods and standard mutagenesis in herein) form technology.The use of this technology for introducing and utilizing cysteine can It is used with introducing described herein and using the technical tie-up of alpha-non-natural amino acid.

VIII. the in vivo generation of the polypeptide including alpha-non-natural amino acid

For convenience, the in vivo generation of the polypeptide including alpha-non-natural amino acid described in this part it is general and/ Or it is described with particular instance.However, the in vivo generation of the polypeptide including alpha-non-natural amino acid described in this part should not The general description or particular instance provided in this part is provided, but includes the more of alpha-non-natural amino acid described in this part The in vivo generation of peptide is similary well suited in the model in Formulas I-XVIII, XXX-XXXIV (A and B) and XXXX-XXXXIII All compounds in farmland, it includes described in description herein book, claim and the schema Formulas I-XVIII, XXX-XXXIV (A and B) and any minor or specific compound in the scope of XXXX-XXXXIII.

Polypeptide described herein can be used through modifying to add or substitute the amino not encoded in naturally occurring system Acid tRNA and tRNA synzyme and in vivo generate.

It generates and is described in using the method for the tRNA and tRNA synzyme for the amino acid not encoded in naturally occurring system (for example) Patent Application Publication 2003/0082575 (sequence number 10/126,927) and 2003/0108885 (sequence number 10/ 126,931) it is in being hereby incorporated by reference in their entirety in.These methods include generating independently of to translation system and Say the translating mechanism to function for the synzyme and tRNA of endogenous (and being therefore sometimes referred to as " orthogonal ").In an embodiment In, translation system includes the polynucleotide of coded polypeptide；Polynucleotide can be can be from from the corresponding DNA mRNA transcribed or mRNA It is generated in rna virus vector；In addition polynucleotide includes corresponding to the selection in the predesignated site for being incorporated to alpha-non-natural amino acid Codon.Translation system further comprises being directed to and (as appropriate) also includes the tRNA of alpha-non-natural amino acid, wherein the tRNA There is the foregoing selection codon of specificity/specific recognition to foregoing selection codon；In other embodiments, non-natural amino Acid is through aminoacylated.Alpha-non-natural amino acid include with Formulas I-XVIII described herein, XXX-XXXIV (A and B) and Those alpha-non-natural amino acids of the structure of any one of XXXX-XXXXIII.In other or Additional examples of composition, translation system Including having the amino acyl synthetase of specificity to the tRNA, and in embodiment at other or in addition, translation system includes Orthogonal tRNA and orthogonal aminoacyl tRNA synzyme.In other or Additional examples of composition, translation system is included in following object At least one：Plasmid (such as, only for example, in the form of DNA) including foregoing polynucleotide, including foregoing poly-nuclear glycosides The genomic DNA (such as, only for example, in the form of DNA) or foregoing polynucleotide of acid are integrated in genomic DNA therein (in other embodiments, described to be integrated into stable integration).In other or Additional examples of composition of translation system, codon is selected It is to be selected from by amber codon, ochre codon, opal codon, unique codon, rare codon, non-natural password The group of son, five base codons and four base codons composition.In other or Additional examples of composition of translation system, tRNA To inhibit tRNA.In other or Additional examples of composition, non-natural amino acid polypeptides are synthesized by ribosomes.

In other or Additional examples of composition, translation system includes orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA and closes Into enzyme (O-RS).In general, O-RS is preferentially with the aminoacylated O-tRNA of at least one alpha-non-natural amino acid and O- in translation system At least one selection codon not identified by other tRNA in system of tRNA identifications.Translation system is therefore by non-natural amino In the polypeptide generated in sour insertion system, to respond encoded selection codon, and then alpha-non-natural amino acid " substitution " is entered In position in encoded polypeptide.

A variety of orthogonal tRNA and aminoacyl tRNA synthetase for specific synthesizing amino acid to be inserted into polypeptide exist Described in fields, and it is commonly available in method described herein to generate alpha-non-natural amino acid described herein more Peptide.For example, ketone specificity O-tRNA/ aminoacyl-tRNA synthetases are described in Wang, L. et al., Proc.Natl.Acad.Sci.USA 100(1):56-61 (2003) and Zhang, Z. et al., Biochem.42 (22): In 6735-6746 (2003).Illustrative O-RS or part thereof is encoded by polynucleotide sequence and is included U.S. Patent Application Publication The ammonia disclosed in case 2003/0082575 and 2003/0108885 (each of which is in being hereby incorporated by reference in their entirety) Base acid sequence.Patent Application Publication 2003/0082575 is also described in the corresponding O-tRNA molecules that O-RS is used together It is to be fully incorporated this by reference in (sequence number 10/126,927) and 2003/0108885 (sequence number 10/126,931) Wen Zhong.In addition, Mehl et al. is in J.Am.Chem.Soc.2003；125:In 935-939 and Santoro et al. .Nature Biotechnology in October, 2002；20:1044-1048 (it is in being hereby incorporated by reference in their entirety) discusses screening Method and aminoacyl tRNA synthetase and tRNA molecules for being incorporated to p-Aminophenylalanine in polypeptide.

Illustrative O-tRNA sequences suitable for method described herein are including (but not limited to) such as United States Patent (USP) Shen Core that please be disclosed in publication 2003/0108885 (sequence number 10/126,931) (it is to be incorporated herein by reference) Nucleotide sequence SEQ ID NO:1-3.There is the O-tRNA/ aminoacyl-tRNA synthetases of specificity to specific alpha-non-natural amino acid To other examples be described in Patent Application Publication 2003/0082575 (sequence number 10/126,927), be with quote Mode be fully incorporated herein.Combined in saccharomyces cerevisiae O-RS containing both keto amino acid and amino acid containing azide and O-tRNA is described in Chin, J.W. et al., Science 301:In 964-967 (2003).

The purposes of O-tRNA/ aminoacyl-tRNA synthetases includes the specific codon of selection coding alpha-non-natural amino acid. Although any codon can be used, selection is usually required seldom or never in O-tRNA/ aminoacyl-tRNA synthetases wherein The codon used in the cell of expression.Only for example, illustrative codon includes nonsense codon, such as terminator codon The codon of (amber codon, ochre codon and opal codon), four or more base and seldom make With or without using natural three base codons of others.

Known mutation forming method in fields can be used (to be formed including (but not limited to) rite-directed mutagenesis, boxlike is dashed forward It is deformed into, limits selection mutation formation etc.) specific selection codon is introduced into the appropriate location of polynucleotide coding sequence.

It generates and can be used for the Protein synthesis mechanism for being incorporated to alpha-non-natural amino acid (such as O-RS, O-tRNA and just Hand over O-tRNA/O-RS to) the method for component be described in Wang, L. et al., Science 292:498-500(2001)；Chin, J.W. et al., J.Am.Chem.Soc.124:9026-9027(2002)；Zhang, Z. et al., Biochemistry 42:6735- In 6746 (2003).U.S. Patent Application Publication is described in for the method and composition being in vivo incorporated to of alpha-non-natural amino acid It is in being hereby incorporated by reference in their entirety in case 2003/0082575 (sequence number 10/126,927).For selecting to be used for The method of orthogonal tRNA-tRNA synzyme pair in the in vivo translation system of organism is also described in U.S. Patent Application Publication It is the side with reference in case 2003/0082575 (sequence number 10/126,927) and 2003/0108885 (sequence number 10/126,931) During formula is fully incorporated herein.In addition, entitled " Site Specific Incorporation of Keto Amino Acids PCT Publication case WO 04/035743 (it is the to be fully incorporated by reference) descriptions of into proteins " are for simultaneously Enter keto amino acid orthogonal RS and tRNA pairs.The PCT of entitled " Expanding the Eukaryotic Genetic Code " Publication WO 04/094593 (during it is hereby incorporated by reference in their entirety) descriptions are for will be non-naturally encoded Amino acid is incorporated to orthogonal RS and tRNA couples in eukaryotic host cell.

Method for generating at least one restructuring orthogonal aminoacyl-tRNA synzyme (O-RS) includes：(a) source is generated In (optionally mutant) the RS storehouses of at least one aminoacyl-tRNA synthetase (RS) from the first organism, wherein first Organism is including (but not limited to) prokaryotes body, such as, only for example, Methanococcus jannaschii, hot autotrophic methane bacteria, thermophilic Salt bacillus, Escherichia coli, the ancient green-ball bacterium of flicker, strong red-hot coccus, Pyrococcus horikoshii, Aeropyrum pernix, thermus thermophilus Or its analog or most eukaryotes；(b) select in (and/or screening) RS (optionally mutant RS) storehouse in non-natural amino The member of aminoacylated orthogonal tRNA (O-tRNA) in the presence of acid and natural amino acid, and then activity is provided and (is optionally mutated Type) RS pond；And/or there is no the situations of alpha-non-natural amino acid in (c) selection (optionally passing through Solid phase) described pond The active RS (including (but not limited to) mutant RS) of lower preferential aminoacylated O-tRNA, and then at least one restructuring O- is provided RS；Wherein at least one recombinates O-RS preferentially with the aminoacylated O-tRNA of alpha-non-natural amino acid.

In one embodiment, RS is nonactive RS.Nonactive RS can be by generating active RS mutation.Only citing comes Say, nonactive RS can by make at least about a kind, at least about 2 kinds, at least about 3 kinds, at least about 4 kinds, at least about 5 kinds, at least about 6 Kind or at least about 10 kinds or 10 kinds or more of amino acid mutation are different aminoacids (including (but not limited to) alanine) to generate.

Known various technologies can be used in fields to generate, it includes (but not limited to) according to egg in mutant RS storehouses The mutation of RS nucleotide is formed in the rational design of white matter three-dimensional RS structures or random or rational design technology.Only for example, Mutant RS can be by mutation site-specific, random mutation, the multifarious recombination mutation of generation, chimeric construct, rational design And known other methods generate in described herein or fields.

In one embodiment, select (and/or screening) RS (optionally mutant RS) storehouse in active members (it includes Those members of (but not limited to) aminoacylated orthogonal tRNA (O-tRNA) in the presence of alpha-non-natural amino acid and natural amino acid) Including (but not limited to)：By the positive selection or screening mark (including (but not limited to) antibiotics resistance gene or its analog) with And (optionally mutant) RS storehouses are introduced into multiple cells, wherein positive selection and/or screening mark include at least one selection Codon, it includes (but not limited to) amber codon, ochre codon, opal codon, unique codon, rare codons Son, unnatural codons, five base codons and four base codons；Give birth to the multiple cell in the presence of selective agent It is long；It is deposited by inhibiting at least one of positive selection or screening mark selection codon to differentiate in selection and/or screening agent In the cell of lower survival (or displaying specific reaction), and then provide the positive choosing in the pond containing active (optionally mutant) RS Select the subset of cell.Optionally, selection and/or screening agent concentration can change.

On the one hand, positive selectable marker is chloramphenicol acetyltransferase (CAT) gene and selects codon as CAT genes In Amber stop codon.Optionally, positive selectable marker is beta-lactam enzyme gene and selects codon as beta-lactam Amber stop codon in enzyme gene.On the other hand, positive screening mark includes fluorescence or luminous screening mark or based on parent Screening with property is marked (including (but not limited to) cell surface marker).

In one embodiment, the active RS (optionally mutant) in Solid phase or screening pond (including (but not limited to) Those activity RS of preferential aminoacylated O-tRNA in the case of there is no alpha-non-natural amino acid) including (but not limited to)：With Solid phase or screening mark are introduced the second biology by the pond of activity (optionally mutant) RS from positive selection or screening Multiple cells of body, wherein Solid phase or screening mark include at least one selection codon (including (but not limited to) antibiosis Plain resistant gene, it includes (but not limited to) chloramphenicol acetyltransferase (CAT) genes)；And differentiate and be supplemented with non-natural It survives in first culture medium of amino acid and screening or selective agent or shows specific screening reaction, but be not supplemented with non-day It cannot survive in right amino acid and the second culture medium of selection or screening agent or show the cell of specific reaction, and then provide With at least one restructuring survivaling cell of O-RS or screening cell.Only for example, CAT authentication schemes optionally serve as definite The positive selection and/or negative screening of appropriate O-RS recombinants.For example, clone pool is optionally with or without one kind or one Kind or more replicate on the growth plate containing CAT (it include at least one select codon) of alpha-non-natural amino acid.Containing non- Therefore the bacterium colony uniquely grown on the plate of natural amino acid is considered containing restructuring O-RS.On the one hand, change selection (and/or sieve Inspection) agent concentration.In certain aspects, the first organism is different from the second organism.Therefore, the first organism and/or second Organism optionally includes：Prokaryotes, eucaryote, mammal, Escherichia coli, fungi, yeast, archeobacteria, eubacteria, Plant, insect, protist etc..In other embodiments, screening mark includes fluorescence or luminous screening mark or based on affine Property screening mark.

In another embodiment, the activity of screening or selection (including (but not limited to) Solid phase) in pond (is optionally dashed forward Variant) RS including (but not limited to)：The pond of active mutant RS of the separation in positive selection step (b)；By Solid phase Or (wherein Solid phase or screening mark include at least one selection codon (including (but not limited to) toxicity mark to screening mark Remember gene, it includes (but not limited to) ribalgilase barnase genes, and codon is selected including at least one)) and activity The pond of (optionally mutant) RS is introduced into multiple cells of the second organism；And differentiate and do not supplementing the of alpha-non-natural amino acid It survives in one culture medium or shows specific screening reaction, but cannot be deposited in the second culture medium for being supplemented with alpha-non-natural amino acid The cell of the specific screening reaction of living or displaying, and then survivaling cell or screening of the offer at least one restructuring O-RS are thin Born of the same parents, wherein at least one recombinates O-RS has specificity to alpha-non-natural amino acid.On the one hand, at least one selection codon includes About two or more selection codons.These embodiments, which can optionally include wherein at least one selection codon, to be included Two or more selection codons, and wherein the first organism is different from the second organism (including (but not limited to) often One organism is optionally including (but not limited to) prokaryotes, eucaryote, mammal, Escherichia coli, fungi, yeast, Gu Bacterium, eubacteria, plant, insect, protist etc.).Equally, some aspects, which include wherein negative selection marker, includes ribose core Sour enzyme barnase gene (it includes at least one selection codon).Other aspects optionally include comprising wherein screening mark Fluorescence or luminous screening mark or the screening mark based on compatibility.In embodiment herein, screening and/or selection regard feelings Condition includes screening and/or selects the variation of stringency.

In another embodiment, the method for generating at least one restructuring orthogonal aminoacyl-tRNA synzyme (O-RS) can be into One step includes：(d) at least one restructuring O-RS of separation；(e) generate from it is at least one restructuring O-RS second group of O-RS (depending on Situation is mutated)；And (f) repeats step (b) and (c) until obtaining the prominent of the ability including preferential aminoacylated O-tRNA Modification O-RS.Optionally, step (d)-(f) (include, but are not limited to) is repeated at least about twice.On the one hand, from least one Second group of saltant type O-RS of kind restructuring O-RS (can be formed, site spy by being mutated to be formed including (but not limited to) random mutation Specific mutation formation, restructuring or its combination) it generates.

The stringency of selection/screening step is including (but not limited to) positive selection/screening step (b), Solid phase/screening Step (c) or both positive and Solid phase/screening step (b) and (c), in the above-mentioned methods, optionally comprising change selection/ Screening stringency.In another embodiment, positive selection/screening step (b), Solid phase/screening step (c) or positive and cloudy Both Sexual behavior mode/screening step (b) and (c) include the use of reporter gene, and wherein reporter gene is by fluorescence activated cell sorts Technology (FACS) detects or wherein reporter gene is by the detection that shines.Optionally, reporter gene is shown on cell surface, bites It is selected on phage display or its analog and according to the compatibility or catalytic activity for being related to alpha-non-natural amino acid or the like. In one embodiment, saltant type synzyme is shown on cell surface, on bacteriophage display or its analog.

Generate recombinate the method for orthogonal tRNA (O-tRNA) including (but not limited to)：(a) generate from least one The storehouse of the mutant tRNA of tRNA (including (but not limited to) the inhibition tRNA from the first organism)；(b) select (comprising (but Be not limited to) Solid phase) or screening in the case where the RS from the first organism is not present by the ammonia from the second organism The storehouse of aminoacylated (optionally mutant) tRNA of acyl group-tRNA synzyme (RS), and then tRNA is provided and (is optionally mutated Body) pond；And orthogonal RS (O-RS) ammonia in the pond of tRNA (optionally mutant) described in (c) selection or screening by introducing The member of acylation, and then at least one restructuring O-tRNA is provided；Wherein at least one recombinates O-tRNA identification selection codons And not by the RS from the second organism effectively identification and it is preferentially aminoacylated by O-RS.In some embodiments, at least one Kind tRNA is inhibition tRNA and/or including natural and/or nonnatural base unique three base codons or is nonsense password Son, rare codon, unnatural codons, the codon including at least four base, amber codon, ochre codon or egg White stone terminator codon.In one embodiment, recombinating O-tRNA has the raising of orthogonality.It will be appreciated that in some embodiments, O-tRNA is optionally introduced in the first organism from the second organism without modification.In various embodiments, the first life Object and the second organism it is identical or different and optionally selected from (include, but are not limited to) prokaryotes (including (but not limited to) Methanococcus jannaschii, hot autotrophic methane bacteria, Escherichia coli, thermophilic salt bacillus etc.), eucaryote, mammal, fungi, yeast, Archeobacteria, eubacteria, plant, insect, protist etc..In addition, restructuring tRNA is optionally aminoacylated through alpha-non-natural amino acid, Wherein alpha-non-natural amino acid is natively or by genetic manipulation biosynthesis in vivo.Alpha-non-natural amino acid optionally adds Into at least growth medium of the first organism or the second organism, wherein alpha-non-natural amino acid can reach appropriate intracellular Concentration is to allow to be incorporated in non-natural amino acid polypeptides.

On the one hand, selection (including (but not limited to) Solid phase) or in screening storehouse by aminoacyl-tRNA synthetase (step (b)) aminoacylated (optionally mutant) tRNA is included：By toxicity markers' gene, (wherein toxicity markers' gene is included at least It is a kind of that codon is selected (or to cause gene necessary to the gene for generating toxic agents or static agent or organism, wherein this mark Gene includes at least one selection codon)) and (optionally mutant) tRNA storehouse introducing from the multiple of the second organism In cell；And selection survivaling cell, wherein the survivaling cell contains including at least one orthogonal tRNA or non-functional The pond of (optionally mutant) tRNA of tRNA.For example, survivaling cell can be examined and determine by using compa-ratios cell density To select.

On the other hand, toxicity markers' gene can include two or more selection codons.It is described herein In another embodiment of method, toxicity markers' gene be ribalgilase barnase gene, wherein ribalgilase barnase Gene includes at least one amber codon.Optionally, ribalgilase barnase gene can include two or more Amber codon.

In one embodiment, orthogonal RS (O-RS) ammonia in the pond of selection or screening (optionally mutant) tRNA by introducing The member of acylation can include：By positive selection or screening marker gene, (wherein positive marker genes include drug resistance gene (including (but not limited to) beta-lactam enzyme gene, codon is selected including at least one, such as at least one amber terminates close Numeral) or organism necessary to gene or cause the gene of toxic agents removing toxic substances) and O-RS and (optionally mutant) tRNA Pond is introduced into multiple cells from the second organism；And differentiate in selection or screening agent (including (but not limited to) antibiosis Element) in the presence of the survivaling cell that grows or screening cell, and then the cell pool at least one restructuring tRNA is provided, wherein extremely A kind of restructuring tRNA is aminoacylated by O-RS and amino acid is inserted into the translation product by positive marker genes' coding less, with Respond at least one selection codon.In another embodiment, the concentration of selection and/or screening agent is changed.

The method for generating O-tRNA/O-RS pairs of specificity is provided.Method including (but not limited to)：(a) generate from next From the storehouse of the mutant tRNA of at least one tRNA of the first organism；(b) given birth in Solid phase or screening storehouse from first The RS of object be not present in the case of by the aminoacyl-tRNA synthetase (RS) from the second organism it is aminoacylated (depending on feelings Condition mutant) tRNA, and then the pond of (optionally mutant) tRNA is provided；(c) selection or screening (optionally mutant) tRNA Pond in by the member aminoacylated orthogonal RS (O-RS) introduced, and then provide at least one restructuring O-tRNA.It is described at least It is a kind of to recombinate O-tRNA identification selections codon and not by the effectively identifications of the RS from the second organism and by the preferential ammonia of O-RS It is acylated.The method is also generated comprising (d) from least one aminoacyl-tRNA synthetase from the 3rd organism (RS) storehouse of (optionally mutant) RS；(e) selection or screening are dashed forward in the presence of alpha-non-natural amino acid and natural amino acid The member of preferential aminoacylated at least one restructuring O-tRNA in the storehouse of variant RS, and then activity is provided and (is optionally mutated Body) RS pond；It is and preferential aminoacylated in the case of there is no alpha-non-natural amino acid in (f) Solid phase or screening pond Activity (optionally mutant) RS of at least one restructuring O-tRNA, and then at least one specificity O-tRNA/O- is provided RS pairs, wherein at least one specificity O-tRNA/O-RS is at least one to alpha-non-natural amino acid and with special to including Property restructuring O-RS and at least one restructuring O-tRNA.The specific O-tRNA/O-RS generated by method described herein To being included herewith in the scope and method of description.For example, it can include and (include, but not for specific O-tRNA/O-RS pairs Be limited to) mutRNATyr-mutTyrRS to (such as mutRNATyr-SS12TyrRS to), mutRNALeu-mutLeuRS to, MutRNAThr-mutThrRS to, mutRNAGlu-mutGluRS pairs or its analog.In addition, these methods include wherein first Organism is identical (including (but not limited to) Methanococcus jannaschii) with the 3rd organism.

For selecting the method for the orthogonal tRNA-tRNA synzyme pair in the in vivo translation system for the second organism It is also contained in method described herein.Method including (but not limited to)：By marker gene, tRNA and from the first organism Separation or first group cell of aminoacyl-tRNA synthetase (RS) introducing from the second organism from the first organism In；Marker gene and tRNA are introduced into the duplicate groups of cells from the second organism；And selection cannot be in duplicate groups of cells What survivaling cell or the specific screening of screening displaying in first group of middle survival reacted cannot generate this in duplicate groups of cells The cell of kind of reaction, wherein first group and duplicate groups of cells are grown in the presence of selection or screening agent, wherein survivaling cell or Screening cell includes the orthogonal tRNA-tRNA synzyme pair in the in vivo translation system for the second organism.Implement one In example, compare and select or screening includes in vivo complementary calibrating.The concentration of selection or screening agent can change.

Organism described herein includes a variety of organisms and multiple combinations.In one embodiment, organism regards feelings Condition be prokaryotes body, it includes (but not limited to) Methanococcus jannaschii, hot autotrophic methane bacteria, thermophilic salt bacillus, Escherichia coli, The ancient green-ball bacterium of flicker, strong red-hot coccus, Pyrococcus horikoshii, Aeropyrum pernix, thermus thermophilus or its analog.Alternatively, Organism is most eukaryotes, it includes (but not limited to) plant (including (but not limited to) complicated plant, such as monocotyledon Or dicotyledon), algae, protist, fungi (including (but not limited to) yeast etc.), animal is (including (but not limited to) the food in one's mouth Newborn animal, insect, arthropod etc.) or its analog.

A. the expression in non-eucaryote and eucaryote

The technology disclosed in this part can be applied to non-natural amino acid polypeptides described herein non-eucaryote and Expression in eucaryote.

To obtain the high level expression of cloned polynucleotide, usually the polynucleotide of polypeptide needed for coding is subcloned To the strong promoter containing guidance transcription, transcription/translation termination expression vector in, and if for coding protein For nucleic acid, then the ribosome bind site for translation initiation is usually subcloned.Be suitble to promoters description (for example) in In Sambrook et al. and Ausubel et al..

For expressing the bacterial expression system of polypeptide available for (include, but are not limited to) Escherichia coli, bacillus (Bacillus sp.), Pseudomonas fluorescens, pseudomonas aeruginosa, pseudomonas putida and salmonella (Salmonella) In (Palva et al., Gene 22:229-235(1983)；Mosbach et al., Nature 302:543-545(1983)).These The kit of expression system can be bought on the market.For the eukaryotic expression system of mammalian cell, yeast and insect cell It can buy on the market.It is more that expression (is described) elsewhere herein using orthogonal tRNA and aminoacyl tRNA synthetase wherein In the case of peptide, the host cell for expression is to be selected according to it using the ability of orthogonal components.Illustrative host cell Comprising gram-positive bacteria (Gram-positive bacteria) (including (but not limited to) bacillus brevis (B.brevis) or Bacillus subtilis (B.subtilis) or streptomycete (Streptomyce)) and Gram-negative bacteria (Gram-negative Bacteria) (Escherichia coli or Pseudomonas fluorescens, pseudomonas aeruginosa, pseudomonas putida) and yeast are true with other Nucleus.It can be used including O-tRNA/O-RS pairs of cell according to described herein.

Eukaryotic host cell as described herein or non-eukaryotic host cell, which provide synthesis, includes the non-of big dosage The ability of the polypeptide of natural amino acid.On the one hand, composition is optionally including (but not limited to) at least 10 micrograms, at least 50 micro- Gram, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least 1 gram or 1 gram or more of polypeptide including alpha-non-natural amino acid can be by vivo more The amount that peptide production method obtains (details for generating and purifying on recombinant protein provides herein).On the other hand, polypeptide Optionally (to be included in (include, but are not limited to) cell lysates, buffer solution, medical buffer solution or other liquid suspensions (but not limited to) about 1nl is to the volume between about 100L) in (include, but are not limited to) and often rise to few 10 microgram polypeptides, often rise to Few 50 microgram polypeptides, often rise to few 75 microgram polypeptides, often rise to few 100 microgram polypeptides, often rise to few 200 microgram polypeptides, every liter At least 250 microgram polypeptides often rise to few 500 microgram polypeptides, often rise to few 1 milligram of polypeptide or often rise to few 10 milligrams of polypeptides or every The concentration for rising 10 milligrams or more of polypeptide is present in composition.In the eukaryocyte comprising at least one alpha-non-natural amino acid Generate it is a large amount of (including (but not limited to) more than by other methods obtained (including (but not limited to) in vitro translating) it is usual can Can amount) protein be method described herein, technology and composition feature.

Eukaryotic host cell as described herein or non-eukaryotic host cell, which provide biosynthesis, includes big dosage Alpha-non-natural amino acid protein ability.For example, the polypeptide including alpha-non-natural amino acid can in cell extract, It (include, but are not limited to) at least 10 micrograms per litres, at least 50 in cell lysates, culture medium, buffer solution and/or its analog Micrograms per litre, at least 75 micrograms per litres, at least 100 micrograms per litres, at least 200 micrograms per litres, at least 250 micrograms per litres are at least 500 micro- G/l, at least 1 mg/litre, at least 2 mg/litres, at least 3 mg/litres, at least 4 mg/litres, at least 5 mg/litres, at least 6 Mg/litre, at least 7 mg/litres, at least 8 mg/litres, at least 9 mg/litres, at least 10 mg/litres, at least 20,30,40,50, 60th, 70,80,90,100,200,300,400,500,600,700,800,900 mg/litres, 1 g/l, 5 g/l, 10 g/l Or the concentration of 10 g/l or more of polypeptide generates.

1.Expression system, culture and separation

The technology disclosed in this part can be applied to the expression system of non-natural amino acid polypeptides described herein, culture And in separation.Non-natural amino acid polypeptides can be with any number of suitable expression system (including (but not limited to) yeast, insect Cell, mammalian cell and bacterium) it expresses.The description of illustrative expression system provides herein.

YeastAs used herein, term " yeast ", which includes, can express the gene that encodes non-natural amino acid polypeptides Any one of each primary yeast.These yeast including (but not limited to) ascosporogenous yeast (Endomycetale (Endomycetales), Basidiomycetes yeast and the ferment for belonging to Deuteromycotina (Fungi imperfecti) (gemma guiding principle (Blastomycetes)) group It is female.Ascosporogenous yeast is divided into two sections, Spermophthoraceae (Spermophthoraceae) and Saccharomycetaceae (Saccharomycetaceae).The latter includes four subfamilies, Schizosaccharomycoideae (Schizosaccharomycoideae) (for example, Schizosaccharomyces (genus Schizosaccharomyces)), Nadsonioideae (Nadsonioideae), fat ferment Female subfamily (Lipomycoideae) and yeast subfamily (Saccharomycoideae) are (for example, pichia (genus Pichia), Kluyveromyces (genus Kluyveromyces) and Blastocystis (genus Saccharomyces)). Basidiomycetes yeast includes Leucosporidium (genus Leucosporidium), Rhodosporidium (genus Rhodosporidium Sporobolomyces (genus Sporidiobolus), basidiomycetes (genus Filobasidium)), are locked And lock load bacterium (genus Filobasidiella).The yeast for belonging to Deuteromycotina (gemma guiding principle) group is divided into two sections, throws spore Saccharomycetaceae (Sporobolomycetaceae) is (for example, Sporobolomyces (genus Sporobolomyces) and cloth Le throw spore Saccharomyces (genus Bullera)) and Cryptococcaceae (Cryptococcaceae) (for example, candida (genus Candida))。

In certain embodiments, tieed up in the method, technology and the composition that are described herein using pichia, Crewe Saccharomyces, Blastocystis, Schizosaccharomyces, Hansenula (Hansenula), Torulopsis (Tondopsis) and vacation Species in silk saccharomyces are (including (but not limited to) pichia pastoris (P.pastoris), P.guillerimondii, wine brewing Yeast (S.cerevisiae), brewer's yeast (S.carlsbergensis), saccharomyces diastaticus (S.diastaticus), Doug are drawn This yeast (S.douglasii), S.khiyveri, S.norbensis, ellipsoideus yeast (S.oviformis), Kluyveromyces Lactis dimension ferment Female (K.lactis), Kluyveromyces fragilis (K.fragilis), Candida albicans (C.albicans), maltose vacation silk ferment Female (C.maltosa) and multiple-shaped nuohan inferior yeast (H.polymorpha)).

The suitable yeast for selecting to express non-natural amino acid polypeptides is the technical ability model in those skilled in the art In enclosing.In yeast host of the selection for expression, suitable host can have (for example) good point including (but not limited to) displaying Secrete those hosts of ability, low proteolytic activity and overall activity.Yeast usually available from a variety of sources, it includes (but It is not limited to) the Yeast Genetics reserve center of University of California's (Berkeley, CA) biophysics and medical physics system, (Yeast Genetic Stock Center,Department of Biophysics and Medical Physics, University of California (Berkeley, CA)) and American type culture collection (American Type Culture Collection)(“ATCC”)(Manassas,VA)。

Term " yeast host " or " yeast host cell ", which include, can be used as or have been used as recombinant vector or other transfer DNAs Receptor yeast.The term includes the offspring for having received recombinant vector or the original yeast host cell of other transfer DNAs. It will be appreciated that due to accidental or sense mutations, the offspring of single parental cell is in form or genome or complementary with original parent It is completely the same on total DNA.It is fully similar to and is intended to (such as there is the nucleotides sequence of coding non-natural amino acid polypeptides by relevant nature Row) offspring of parental cell of parent of characterization is contained in the offspring for thus defining and meaning.

Expression and conversion carrier (comprising extra-chromosomal replicon or integration vector) have been developed for converting to many yeast In host.For example, expression vector has been developed for saccharomyces cerevisiae (Sikorski et al., GENETICS (1998) 112:19； Ito et al., J.BACTERIOL. (1983) 153:163；Hinnen et al., PROC.NATL.ACAD.SCI.USA (1978) 75: 1929)；Candida albicans (Kurtz et al., M_OL.C_ELL.B_IOL.(1986)6:142)；Candida maltosa (Kunze etc. People, J.BASIC MICROBIOL. (1985) 25:141)；Multiple-shaped nuohan inferior yeast (Gleeson et al., J.GEN.MICROBIOL. (1986)132:3459；Roggenkamp et al., M_OL.GEN.GENET.(1986)202:302)；Kluyveromyces fragilis (Das Et al., J.BACTERIOL. (1984) 158:1165)；Kluyveromyces lactis (De Louvencourt et al., J.BACTERIOL.(1983)154:737；Van den Berg et al., BIO/TECHNOLOGY (1990) 8:135)； P.guillerimondii (Kunze et al., J.BASIC MICROBIOL. (1985) 25:141)；Pichia pastoris (the U.S. Patent the 5,324,639th；No. 4,929,555；And No. 4,837,148；Cregg et al., MOL.CELL.BIOL. (1985)5:3376)；Schizosaccharomyces pombe (Schizosaccharomyces pombe) (Beach and Nurse, NATURE (1981)300:706)；And Yarrowia lipolytica (Y.lipolytica) (Davidow et al., CURR.GENET. (1985) 10:380(1985)；Gaillardin et al., CURR.G_ENET.(1985)10:49)；Aspergillus nidulans (A.nidulans) (Ballance et al., BIOCHEM.BIOPHYS.RES.COMMUN. (1983) 112:284-89；Tilburn et al., G_ENE (1983)26:205-221；And Yelton et al., PROC NATL.ACAD.SCI.USA (1984) 81:1470-74)；Black song Mould (A.niger) (Kelly and Hynes, EMBO J. (1985) 4:475-479)；T.reesia(EP 0 244 234)；And Filamentous fungi, such as, mould (the Tolypocladium) (WO of neurospora (Neurospora), mould (Penicillium), curved neck 91/00357), each is in being hereby incorporated by reference in their entirety.

The control sequence of yeast vector is including (but not limited to) from such as alcohol dehydrogenase (ADH) (EP 0 284 044)； Enolase；Glucokinase；Glucose-6-phosphate isomerase；Glyceraldehyde-3-phosphate dehydrogenase (GAP or GAPDH)；Hexokinase； Phosphofructokinase；3-phoshoglyceric acid mutase；And the startup of the gene of pyruvate kinase (PyK) (EP 0 329 203) Subregion.The yeast PH05 genes of encoding acid phosphatase can also provide applicable promoter sequence (Miyanohara et al., PROC.NATL.A_CAD.SCI.USA(1983)80:1).It is suitble to promoter sequence that can include 3- phosphoric acid for other of yeast host Glycerate kinase (Hitzeman et al., J.BIOL.CHEM. (1980) 255 (4):12073-12080)；With other glycolytic ferments, Such as pyruvate decarboxylase, phosphotriose isomerase and glucose phosphate isomerase (Holland et al., BIOCHEMISTRY (1978)17(23):4900-4907；Hess et al., J.ADV.ENZYME REG. (1969) 7:Promoter 149-167).Tool The inducible Yeast promoter for having other advantages of the transcription by growth conditions control can include alcohol dehydrogenase 2；Different cell pigment C；Acid phosphatase；Metallothionein；Glyceraldehyde-3-phosphate dehydrogenase；With the relevant degrading enzyme of nitrogen metabolism；And responsible malt The promoter region of the enzyme of sugar and galactose utilization.EP is further described in for the suitable carrier in Yeast expression and promoter In 0073 657.

Yeast enhancers can be also used together with Yeast promoter.In addition, synthetic promoter also acts as Yeast promoter. For example, the upstream activating sequence (UAS) of Yeast promoter can be connected with the transcription activating region of another Yeast promoter, Generate synthesis hybrid promoter.The example of these hybrid promoters includes the ADH being connected with GAP transcription activatings region and adjusts sequence Row.It is in being hereby incorporated by reference in their entirety referring to U.S. Patent No. 4,880, No. 734 and the 4th, 876, No. 197.It is miscellaneous Close promoter other examples include by combined with the transcription activating region of glycolytic enzyme gene (such as GAP or PyK) ADH2, The promoter of the regulatory sequence composition of GAL4, GAL10 or PHO5 gene.Referring to EP 0 164 556.In addition, Yeast promoter can The naturally occurring promoter in the non-yeast source comprising the ability with combining yeast RNA polymerase and initiation transcription.

Other control elements of the part of Yeast expression carrier be may make up including (for example) from GAPDH or enolase gene Terminator (Holland et al., J.BlOL.C_HEM.(1981)256:1385).In addition, the replication orgin from 2 μ plasmid origins Suitable for yeast.Gene is selected as trpl genes present in yeast plasmid for suitable in yeast.Referring to Tschumper etc. People, GENE (1980) 10:157；Kingsman et al., GENE (1979) 7:141.Trpl genes provide to lack in tryptophan The selected marker of the mutants which had of the yeast of the ability of middle growth.Similarly, Leu2 defective yeasts bacterial strain (ATCC 20, 622 or be 38,626) complementary by the known plasmid with Leu2 genes.

By exogenous DNA be introduced into method in yeast host including (but not limited to) conversion spheroplast or through alkali metal sun from The Whole yeast host cell of subprocessing.For example, the conversion of yeast can according to Hsiao et al., P_ROC.N_ATL.A_CAD.S_CI.USA(1979)76:3829 and Van Solingen et al., J.BACT. (1977) 130:It is retouched in 946 The method stated carries out.However, the other methods that DNA is introduced into cell (are such as injected, electroporation or protoplast by core Fusion) it also can be according to S_AMBROOKEt al., M_OLECULAR C_LONING:A L_AB.M_ANUAL(2001) it is usually described in use.Yeast place Chief cell may then use that standard technique known to those skilled in the art to cultivate.

The other methods of expressing heterologous protein are described in U.S. Patent Publication case in yeast host cell No. 20020055169, U.S. Patent No. 6,361,969；No. 6,312,923；No. 6,183,985；6,083,723rd Number；No. 6,017,731；No. 5,674,706；No. 5,629,203；No. 5,602,034；With No. 5,089,398；It is beautiful State's review patent No. RE37,343 and No. RE35,749；PCT Publication patent application case WO 99/07862；WO 98/ 37208；With WO 98/26080；European patent application EP 0 946 736；EP 0 732 403；EP 0 480 480；WO 90/10277；EP 0 460 071；EP 0 340 986；EP 0 329 203；EP 0 324 274；And EP 0 164 556 In.Referring also to Gellissen et al., ANTONIE VAN LEEUWENHOEK (1992) 62 (l-2):79-93；Romanos etc. People, YEAST (1992) 8 (6):423-488；Goeddel,M_ETHODSIN ENZYMOLOGY(1990)185:3-7, each of which It is in being hereby incorporated by reference in their entirety.

During the amplification stage of standard feed batch fermentation process is used, yeast host bacterial strain can give birth in fermenter It is long.Due to the carbon utilization ways of specific yeast host or the difference of expression control model, fermentation process can be adjusted.Only lift For example, the fermentation of Saccharomyces cerevisiae host may need single glucose to feed, and compound nitrogen source is (for example, casein hydrolysis Product) and supplement multivitamin, and for optimum growh and expression, methylotrophic yeast pichia pastoris may need Glycerine, methanol and trace quantity mineral charging, but only need simple ammonium (nitrogen) salt.Referring to (for example) U.S. Patent No. 5,324,639 Number；Eniott et al., J.PROTEIN CHEM. (1990) 9:95；And Fieschko et al., BIOTECH.BlOENG. (1987)29:1113, each of which is in being hereby incorporated by reference in their entirety.

However, these fermentation process can have some common traits independent of used yeast host bacterial strain.It lifts For example, limiting growth nutrient (being usually carbon) can be added in fermenter to allow maximum growth during the amplification stage. In addition, fermentation process (is tieed up usually using the carbon containing sufficient amount, nitrogen, basic salt, phosphorus and other secondary nutrients are designed to Raw element, trace mineral and salt etc.) fermentation medium.U.S. is described in suitable for the example of the fermentation medium of Pichia pastoris In state's patent the 5th, 324, No. 639 and the 5th, 231, No. 178, each of which is in being hereby incorporated by reference in their entirety.

Infect the insect cell of baculoviralTerm " insect host " or " insect host cell " refer to can be used as or The insect of receptor as recombinant vector or other transfer DNAs.The term includes the protentomon host cell transfected Offspring.It will be appreciated that due to accidental or sense mutations, the offspring of single parental cell is in form or genome or and original parent It need not be completely the same on complementary total DNA.It is fully similar to and is intended to (such as there are coding non-natural amino acid polypeptides by relevant nature Nucleotide sequence) offspring of parental cell of parent of characterization is contained in the offspring for thus defining and meaning.

Those skilled in the art knows to express the selection of the suitable insect cell of polypeptide.Several insect species are filled Divide and be described in fields and can buy on the market, it includes (but not limited to) Aedes aegypti (Aedes aegypti), silkworms (Bombyx mori), drosophila (Drosophila melanogaster), fall army worm (Spodoptera frugiperda) with And cabbage looper (Trichoplusia ni).In insect host of the selection for expression, suitable host can include (but unlimited In) displaying especially has good secretion capacity, low proteolytic active and those hosts of overall activity.Insect can usually be obtained From a variety of sources, it includes (but not limited to) University of California (Berkeley, CA) biophysics and medical physics systems Insect heredity reserve center (the Insect Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA)) and American Type Tissue Culture Center (American Type Culture Collection) (" ATCC ") (Manassas, VA).

In general, the component of the insect expression system of infection baculoviral includes transfer vector, it is usually bacterial plasmid, contains The segment for having Baculovirus Gene group and the facility restriction site for the heterologous gene to be expressed of insertion；With with shifting load (this allows heterologous gene homologous recombination to enter to the wild-type baculovirus of the homologous sequence of baculoviral specific fragment in body In Baculovirus Gene group)；And appropriate insect host cell and growth medium.In carrier construction, transfectional cell, select bacterium Spot makes the material used in cell growth and its similar procedure, methods and techniques in the art in culture Handbook know and that these technologies of description can be obtained.

After heterologous gene is inserted into transfer vector, carrier and wild-type virus genome are transfected into insect host In cell, carrier recombinates wherein with viral genome.The recombinant virus of packaging is through expressing and differentiating and purifying restructuring spot.For The material and method of baculoviral/insect cell expression system can be purchased from (for example) Invitrogen Corp. in a kit form (Carlsbad,CA).Illustrative technique is described in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT It is to be incorporated herein by reference in STATION BULLETIN the 1555th (1987).Referring also to RICHARDSON, 39METHODS IN MOLECULAR BIOLOGY:BACULOVIRUS EXPRESSION PROTOCOLS(1995)；AUSUBEL Et al., CURRENT PROTOCOLS IN MOLECULAR B_IOLOGY16.9-16.11(1994)；KING and POSSEE, THE BACULOVIRUS SYSTEM:A LABORATORY GUIDE(1992)；And O'REILLY et al., BACULOVIRUS EXPRESSION VECTORS:A LABORATORY MANUAL(1992)。

Using baculoviral/insect cell expression system generate various heterologous proteins be described in below with reference in document and this A little technologies may be adapted to prepare non-natural amino acid polypeptides described herein.Referring to (for example) U.S. Patent No. 6,368,825； No. 6,342,216；No. 6,338,846；No. 6,261,805；No. 6,245,528,6,225,060；6,183,987th Number；No. 6,168,932；No. 6,126,944；No. 6,096,304；No. 6,013,433；No. 5,965,393；5th, No. 939,285；No. 5,891,676；No. 5,871,986；No. 5,861,279；No. 5,858,368；5,843,733rd Number；No. 5,762,939；No. 5,753,220；No. 5,605,827；No. 5,583,023；No. 5,571,709；5th, No. 516,657；No. 5,290,686；WO 02/06305；WO 01/90390；WO 01/27301；WO 01/05956；WO 00/55345；WO 00/20032WO 99/51721；WO 99/45130；WO 99/31257；WO 99/10515；WO 99/ 09193；WO 97/26332；WO 96/29400；WO 96/25496；WO 96/06161；WO 95/20672；WO 93/ 03173；WO 92/16619；WO 92/03628；WO 92/01801；WO 90/14428；WO 90/10078；WO 90/ 02566；WO 90/02186；WO 90/01556；WO 89/01038；WO 89/01037；WO 88/07082, each of which are In being hereby incorporated by reference in their entirety.

Carrier suitable for baculoviral/insect cell expression system is including (but not limited to) from baculoviral lucerne The insect expression of Mu three-spotted plusia (Autographacalifornica) nucleopolyhedrosis virus (AcNPV) and transfer vector are Virus expression carrier independent of helper virus.Virus expression carrier from this system is usually using strong virus polyhedral body Gene promoter is to drive the expression of heterologous gene.Usually referring to Reilly et al., BACULOVIRUS EXPRESSION VECTORS:A LABORATORY MANUAL(1992)。

Before foreign gene is inserted into rod-shaped viral genome, be typically included promoter, leader (if necessary), Coded sequence of interest and the said components of transcription terminator are assembled into intermediate indexable construct (transfer vector).In Between indexable construct be usually maintained in replicon, it is outer first can such as to stablize the chromosome maintained in host (such as bacterium) Part (for example, plasmid).Replicon should have dubbing system, thus maintain it in the suitable host that clones and expand. More particularly, plasmid can contain polyhedrin polyadenylation signal (Miller et al., ANN.REV.MICROBIOL. (1988)42:177) with protokaryon ampicillin (ampicillin) resistance (amp) gene and for being selected in Escherichia coli The replication orgin selected and bred.

It is a kind of to be used to the common transfer vector that foreign gene is introduced into AcNPV be pAc373.The technology people of fields Many other carriers known to member have also been designed including (for example) pVL985, by polyhedrin initiation codon by ATG ATT is changed into, and it introduces BamHI cloning sites at 32 base-pairs in ATT downstreams.Referring to Luckow and Summers, VIROLOGY 170:31-39(1989).Other commercial vectors are including (for example) PBlueBac4.5/V5-His； pBlueBacHis2；pMelBac；pBlueBac4.5(Invitrogen Corp.,Carlsbad,CA).

After heterologous gene is inserted into, transfer vector and wild-type baculovirus genome cotransfection are entered into insect cell place In master.The illustrative method that allogeneic dna sequence DNA is introduced into the required site in baculoviral is described in SUMMERS and SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN the 1555th (1987)；Smith et al., MOL.CELL.BIOL.(1983)3:2156；Luckow and Summers, VIROLOGY (1989) 170:In 31-39.Citing comes It says, in the gene that insertion such as polyhedron gene can be recombinated by homologous double cross；Also it can be inserted into and operated through engineering Into in the restriction enzyme sites in required Baculovirus Gene.Referring to Miller et al., BlOESSAYS (1989) 4:91.

TROTTER and W can be used in transfection_OOD,39METHODS IN MOLECULAR BIOLOGY(1995)；Mann and King,J.G_EN.V_IROL.(1989)70:Method described in 3501 is realized by electroporation.Alternatively, can be used liposome with Recombinant expression carrier and baculovirus transfection insect cell.Referring to (for example) Liebman et al., BlOTECHNlQUES (1999) 26(1):36；Graves et al., BIOCHEMISTRY (1998) 37:6050；Nomura et al., J.BIOL.CHEM. (1998) 273(22):13570；Schmidt et al., PROTEIN EXPRESSION AND PURIFICATION (1998) 12:323； Siffert et al., NATURE GENETICS (1998) 18:45；T_ILKINSEt al., CELL BIOLOGY:A LABORATORY HANDBOOK 145-154(1998)；Cai et al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10: 263；Dolphin et al., NATURE GENETICS (1997) 17:491；Kost et al., GENE (1997) 190:139； Jakobsson et al., J.B_IOL.CHEM.(1996)271:22203；Rowles et al., J.BIOL.CHEM. (1996) 271 (37):22376；Reversey et al., J.B_IOL.CHEM.(1996)271(39):23607-10；Stanley et al., J.B_IOL.CHEM.(1995)270:4121；Sisk et al., J.VIROL. (1994) 68 (2):766；And Peng et al., BIOTECHNIQUES(1993)14.2:274.Commercially available liposome including (for example)With (Invitrogen,Corp.,Carlsbad,CA).In addition, calcium phosphate transfection can be used.Referring to T_ROTTERAnd W_OOD,39METHODS IN MOLECULAR BIOLOGY(1995)；Kitts,NAR(1990)18(19):5667；And Mann and King, J.GEN.VIROL.(1989)70:3501。

Rhabdovirus expression vector usually contains bacilliform virus promoter.Bacilliform virus promoter is that can combine rod-shaped disease The downstream of malicious RNA polymerase and start code sequence (for example, structural gene) is (3') transcribed into any DNA sequence dna of mRNA.It opens Mover should have the transcription initiation region being usually located near the 5' ends of coded sequence.This transcription initiation region generally comprises RNA polymerizations Enzyme binding site and transcription initiation site.Bacilliform virus promoter can also have the second domain referred to as enhancer, (if depositing ) it is typically remote from structural gene.In addition, expression can be modulability or composition.

The structural gene that later stage in infection cycles largely transcribes provides especially suitable promoter sequence.Example includes From gene (FRIESEN et al., The Regulation of the Baculovirus Gene of coding viral polyhedron protein Expression in THE MOLECULAR BIOLOGY OF BACULOVIRUSES(1986)；EP 0 127 839 and 0 155 476) and coding p10 albumen gene (Vlak et al., J.GEN.VlROL. (1988) 69:765) sequence.

The rhabdovirus expression vector newly formed is wrapped up in infectious recombinant baculovirus and then growth spot can be by Such as Miller et al., BIOESSAYS (1989) 4:91；SUMMERS and SMITH, TEXAS AGRICULTURAL Those technologies described in EXPERIMENT STATION BULLETIN NO.1555 (1987) purify.

It has developed to infect the recombination rhabdovirus expression vector in several insect cells.For example, opened It sends particularly for the recombinant baculovirus in following insect：Aedes aegypti (ATCC CCL-125), silkworm (No. ATCC CRL-8910), drosophila (No. ATCC 1963), fall army worm and cabbage looper.Referring to WO 89/046,699；Wright, NATURE(1986)321:718；Carbonell et al., J.VlROL. (1985) 56:153；Smith et al., MOL.CELL.BIOL.(1983)3:2156.Usually referring to Fraser et al., IN VITRO CELL.DEV.BIOL. (1989) 25:225.More particularly, the cell line for rod string design generally comprises (but not limited to) Sf9 (meadows Noctuid) (ATCC CRL-1711), Sf21 (fall army worm) (Invitrogen Corp., catalog number (Cat.No.) 11497-013 (Carlsbad, CA)), Tri-368 (cabbage looper) and High-Five^TMBTI-TN-5B1-4 (cabbage looper).

Cell and culture medium for directly expression and amalgamation and expression heterologous polypeptide in baculoviral/expression can be in markets On buy.

BacteriumBacterial expression techniques are well known in the art.Variety carrier can be used in bacterial host.Carrier can Low or high multi-copy vector is copied to be single.Carrier can be used for cloning and/or expressing.Due to the abundant document on carrier, The commercial applicability of many carriers and carrier and its estriction map and the handbook of feature are even described, be not required to herein It to discuss extensively.It is well known that carrier generally includes to allow the mark of selection, these marks can provide cytotoxic agent resistance, Prototrophy or immunity.Multiple marks are typically, there are, different characteristic is provided.

Promoters are the downstream that can combine bacterial RNA polymerase and start code sequence (for example, structural gene) (3 ") are transcribed into any DNA sequence dna of mRNA.Promoter should have the transcription initiation being usually located near the 5' ends of coded sequence Area.This transcription initiation region generally comprises RNA polymerase binding site and transcription initiation site.Promoters, which can also have, to be claimed Make the second domain of operator, the place that beginning can be synthesized in RNA is Chong Die with adjacent R NA polymerase binding site points.Manipulate base Because allow negative regulator it is (inducible) transcription because gene repressor protein can combine operator and and then inhibit specific gene turn Record.Constructive expression can occur in the case of there is no negative regulation element (such as operator).In addition, positive regulator can pass through Gene activation protein binding sequence (if it is present, (5') it is typically closest to RNA polymerase binding sequence) obtains.Base Because the example of activated protein is metabolite activated protein (CAP), help to originate the transcription of lac operons in Escherichia coli [Raibaud et al., ANNU.REV.GENET. (1984) 18:173].Therefore modulated expression can be positivity or negativity, and then Enhancing weakens transcription.

The sequence of encoding metabolic pathway enzyme provides especially suitable promoter sequence.Example is included from such as gala Sugar, lactose (lac) [Chang et al., NATURE (1977) 198:1056] and the promoter sequence of the glycometabolism enzyme of maltose.Its His example includes the promoter sequence from biosynthetic enzyme, such as tryptophan (trp) (Goeddel et al., NUC.ACIDS RES.(1980)8:4057；Yelverton et al., NUCL.ACIDS RES. (1981) 9:731；U.S. Patent No. 4,738,921 Number；IFNPub. the 036th No. 776 and the 121st No. 775), each of which is in being hereby incorporated by reference in their entirety.β- Galactosidase (bla) promoter systems [Weissmann (1981) " The cloning of interferon and other Mistakes. " In Interferon 3 (I.Gresser volumes)], phageλ PL [Shimatake et al., NATURE (1981) 292:128] and T5 [U.S. Patent No. 4,689,406], each of which is in being hereby incorporated by reference in their entirety, to open Subsystem also provides applicable promoter sequence.The preferred method being contemplated herein utilizes strong promoter (such as T7 promoters) It is generated with inducing polypeptide with high level.The example of these carriers is serial including (but not limited to) the pET29 from Novagen, with And the pPOP carriers described in WO99/05297 (it is in being hereby incorporated by reference in their entirety).These expression systems are in place High-caliber polypeptide is generated in master without jeopardizing host cell viability or growth parameter(s).

In addition, the synthetic promoter being not present in nature also functions as promoters.It for example, can be thin by one The transcription-activating sequence of bacterium or phage promoter is connected with the operon sequence of another bacterium or phage promoter, generates conjunction Into hybrid promoter [U.S. Patent No. 4,551,433].For example, tac promoters are with being hindered by lac by trp promoters Hold back heterozygosis trp-lac promoters [Amann et al., the GENE (1983) 25 that both lac operon sequences of object adjusting are formed: 167；De Boer et al., P_ROC.NATL.ACAD.SCI.(1983)80:21].It is combined in addition, promoters can include to have The naturally occurring promoter in the non-bacterial source of bacterial RNA polymerase and the ability of starting transcription.The natural of non-bacterial source is deposited Also the high level expression of some genes in prokaryotes can be generated in promoter with the coupling of compatibility RNA polymerase.Bacteriophage T7 rna polymerase/promoter systems are example [Studier et al., the J.M of coupling promoter systems_OL.B_IOL.(1986)189: 113；Tabor et al., Proc Natl.Acad.Sci. (1985) 82:1074].In addition, hybrid promoter may also comprise bacteriophage Promoter and Escherichia coli operator region (IFNPub. the 267th 851).

In addition to function on subsequence, effective ribosome bind site is also applied for foreign gene in prokaryotes Expression.In Escherichia coli, ribosome bind site is referred to as Shine-Dalgarno (SD) sequences and includes initiation codon (ATG) length and at 3-11 nucleotide of upstream from start codon for 3-9 nucleotide sequence [Shine et al., NATURE(1975)254:34].Think that SD sequences pass through the base between making the 3' ends of SD sequences and Escherichia coli 16S rRNA It matches to promote mRNA and ribosomal combination [Steitz et al., " Genetic signals and nucleotide sequences in messenger RNA",In Biological Regulation and Development:Gene Expression (R.F.Goldberger is compiled, 1979)].To express eukaryotic gene and protokaryon with weak ribosome bind site Gene [Sambrook et al. " Expression of cloned genes in Escherichia coli ", Molecular Cloning:A Laboratory Manual,1989]。

Term " bacterial host " or " bacterial host cell " refer to can be used as have been used as recombinant vector or other transfer The bacterium of the receptor of DNA.The term includes the offspring of the primitive bacteria host cell transfected.It will be appreciated that due to accidental Or sense mutations, the offspring of single parental cell need not be complete on form or genome or total DNA with original parent complementation Unanimously.Fully it is similar to the parent for being intended to the parent as relevant nature (nucleotide sequence of polypeptide needed for such as there is coding) characterization The offspring of cell is contained in the offspring for thus defining and meaning.

Those skilled in the art knows to express the selection of the suitable host bacteria of required polypeptide.It is used in selection During the bacterial host of expression, suitable host can especially have at least one of following characteristics and excellent including (but not limited to) displaying Those hosts of choosing at least the two in following characteristics：Good inclusion body Forming ability, low proteolytic activity are well divided Secrete ability, good soluble protein generates ability and overall activity.Bacterial host usually available from a variety of sources, it includes (but not limited to) University of California (Berkeley, CA) biophysics and the bacterial genetic reserve center of medical physics system (the Bacterial Genetic Stock Center,Department of Biophysics and Medical Physics,University of California(Berkeley,CA))；And American type culture collection (American Type Culture Collection)(“ATCC”)(Manassas,VA).Industry/medicine fermentation is usually used Bacterium (such as W3110) from K bacterial strains or the bacterium (such as BL21) from B bacterial strains.Because its growth parameter is extremely It is known and to be active, so these bacterial strains are especially suitable.In addition, these bacterial strains are avirulence, for safety and environment The reason for, it is commercially important.In one embodiment of the method for being described herein and covering, escherichia coli host includes The bacterial strain of (but not limited to) BL21, DH10B or derivatives thereof.In another embodiment for the method for being described herein and covering, Escherichia coli host is deproteinized enzyme bacterial strain, and it includes (but not limited to) OMP- and LON-.In another embodiment, bacterial host For the species of pseudomonad, such as Pseudomonas fluorescens, pseudomonas aeruginosa and pseudomonas putida.Pseudomonad expresses bacterium The example of strain is Pseudomonasfluorescens biotype I, bacterial strain MB101 (Dow Chemical).

Once recombinant host cell strain has been established (that is, to express construct to have been introduced into host cell and there will be appropriate table Host cell up to construct separates), just recombinant host cell strain is being cultivated under conditions of polypeptide suitable for preparing.Recombinant host is thin Born of the same parents strain cultural method should depend on utilized expression construct property and host cell in itself.Recombinant host strain is usual It is cultivated using well known method in fields.Recombinant host cell usually comes containing absorbable carbon, nitrogen and inorganic salts Source, and optionally containing other well known protein culture replenishers in vitamin, amino acid, growth factor and fields It is cultivated in fluid nutrient medium.Fluid nutrient medium for cultivating host cell, which can optionally contain, prevents unwanted microorganism from giving birth to Long antibiotic or antifungal agent and/or including (but not limited to) for select the host cell containing expression vector antibiotic Compound.

Recombinant host cell can form culture in batches or continuously, wherein in the form of in batches or continuously collect cell (at it In in the case that required polypeptide accumulates in the cell) or collect culture supernatants.It is excellent for being prepared in prokaryotic host cell Batch culture and cell is selected to collect.

In one embodiment, non-natural amino acid polypeptides described herein are pure after being expressed in recombination system Change.Polypeptide can be purified by known a variety of methods in fields from host cell or culture medium.It is in general, thin in bacterial host The multiple polypeptides prepared in born of the same parents are soluble poor or insoluble (in the form of inclusion body).In one embodiment, recombinated for increase The deliquescent purpose of the polypeptide of preparation, can using those known methods in method and fields disclosed herein It is easy to carry out 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in seleced polypeptide.In the case of insoluble polypeptide, the polypeptide can by centrifugation or Filtering collects from host cell lysate and further can then carry out homogenizing for cell.In the polypeptide of poorly soluble In the case of, the compound including (but not limited to) polyethyleneimine (PEI) can be added to induce the precipitation of part soluble polypeptide.It is heavy The polypeptide in shallow lake then can easily be collected by centrifuging or filtering.A variety of methods known to those skilled in the art can be used Recombinant host cell is crushed or is homogenized to discharge inclusion body from intracellular.Host cell crushes or homogenizes usable known Technology carries out, and the technology homogenizes (dounce including (but not limited to) enzymatic clasmatosis, ultrasonic degradation, Dounce Homogenization) or high pressure release is broken.In one embodiment of the method for being described herein and covering, released using high pressure Discharge technique crushes e. coli host cell to discharge the inclusion body of polypeptide.It has found by merely with e. coli host cell The yield of insoluble polypeptide in inclusion bodies can be increased by a passage of homogenizer.When the inclusion body of processing polypeptide When, due to such as dissolving, the factor of mechanical shearing or protein hydrolysis, minimize and repeat time that homogenizes and forgive to maximize The yield of body and free of losses are favourable.

It may then use that any one of known numerous suitable lytic agents make insoluble or precipitation more in fields Peptide dissolves.For example, with urea or guanidine hydrochloride dissolution polypeptide.The volume of dissolving polypeptide should be minimized to use and can facilitated The batch size of processing is prepared on a large scale.The extensive business that the batch that recombinant host can be using volume as upper kilolitre wherein is grown This factor can be important in industry device.In addition, when manufacturing polypeptide in large-scale commercial applications device, particularly with human medical For purposes, if it would be possible, should avoid that the harsh chemicals of machine and container or polypeptide product in itself can be damaged.At this It has shown that and is can use compared with mild denaturant urea instead of more compared with causticity denaturant guanidine hydrochloride dissolution in described in the text and the method covered Peptide inclusion body.The use of urea significantly reduces the wind damaged stainless steel equipment used in the manufacture of polypeptide and purification process Danger, while effectively dissolving polypeptide inclusion body.

In the case of soluble polypeptide, peptide can be secreted into periplasmic space or culture medium.In addition, soluble peptide may be present In the cytoplasm of host cell.Soluble peptide can be concentrated before purification step is carried out.Standard technique can be used (to include (but not limited to) those described herein technology) from (for example) cell lysates or culture medium concentrate soluble peptide.Separately Outside, standard technique (including (but not limited to) those described herein technology) can be used to crush host cell and from host cell Cytoplasm or periplasmic space in discharge soluble peptide.

When polypeptide is prepared as fusion protein, fusion sequence is preferably removed.The removal of fusion sequence can be by comprising (but not It is limited to) method of enzymatic lysis or chemical cracking realizes, wherein it is preferred that enzymatic lysis.Those skilled in the art can be used Well known method come realize the enzymatic of fusion sequence remove.It is true by the characteristic merged for removing the selection of the enzyme of fusion sequence It is fixed, and reaction condition is specified by the selection of enzyme.It can be used including (but not limited to) cyanogen bromide, TEV protease and other reagents Reagent realize chemical cracking.The polypeptide of cracking is optionally purified by well-known process from the fusion sequence of cracking.These Method will be determined by the characteristic and property of fusion sequence and polypeptide.Purification process can be including (but not limited to) size exclusion color Spectrum, hydrophobic interaction chromatograph, ion-exchange chromatography or dialysis or any combination thereof.

Polypeptide is also optionally purified to remove DNA from protein solution.DNA can be by known any in fields Appropriate methodology removes, and it includes (but not limited to) precipitation or ion-exchange chromatographies.In one embodiment, by being precipitated with nucleic acid Agent (such as (but not limited to) protamine sulfate) precipitates to remove DNA.Can be used standard well-known process (including (but not limited to) Centrifugation or filtering) polypeptide is separated with the DNA precipitated.In the environment of polypeptide is used to treat the mankind, host nucleic acids molecule It removes as a key factor, and host cell DNA is reduced to pharmaceutically acceptable degree by method described herein.

Small-scale or large scale fermentation method can also be used in protein expression, it includes (but not limited to) fermenter, Shake flask, fluidized bed aerosol generator, hollow-fiber bioreactor, spinner culture system and tank diameter bioreactor System.Each of these methods can be carried out with batch-type, stream plus formula or continuous mode method.

The method standard in fields usually can be used in the human form of non-natural amino acid polypeptides described herein Recycling.For example, culture medium or cell lysates can remove cell debris through centrifuging or filtering.Supernatant can be concentrated Or it is diluted to required volume or is percolated in suitable buffer solution to adjust preparation for being further purified.Non- day described herein Being further purified for right amino acid polypeptide is including (but not limited to) the deamidation and clipped form for making polypeptide variants and accordingly complete Form separates.

Any one of property program illustrated below can be used for purifying non-natural amino acid polypeptides described herein：Affine color Spectrum；Anion or cation-exchange chromatography (using (include, but are not limited to) DEAE SEPHAROSE)；Silica chromatography；Reverse phase HPLC；Gel filtration (uses (include, but are not limited to) SEPHADEX G-75)；Hydrophobic interaction chromatograph；Size exclusion color Spectrum；Immobilized metal ion afinity chromatography；Ultrafiltration/diafiltration；Ethanol precipitation；Ammonium sulfate precipitation；Chromatofocusing；Displcement chromatography；Electrophoretic procedures (include The isoelectric focusing of (but not limited to) preparative), differential dissolubility (including (but not limited to) ammonium sulfate precipitation), SDS-PAGE, extraction Or any combination thereof.

According to known to those skilled in the art and the standardization program that uses, in method and composition described herein Cover polypeptide (including (but not limited to) the polypeptide including alpha-non-natural amino acid, the polypeptide including alpha-non-natural amino acid antibody, The combination collocation object of polypeptide including alpha-non-natural amino acid) can partial purification or substantially purifying for homogeneity.Therefore, retouch herein The polypeptide stated can be recycled and purified by any one of well known numerous methods in fields, these methods include (but not It is limited to) ammonium sulfate or ethanol precipitation, acid or alkali carries take, column chromatography, affine column chromatography, anion or cation exchange color Spectrum, phosphocellulose chromatography, hydrophobic interaction chromatograph, hydroxyapatite chromatography, agglutinin chromatography, gel electrophoresis and its is any Combination.When preparing the maturation protein appropriately folded, Protein refolding steps can be used if necessary.Needing the final of high-purity High performance liquid chromatography (HPLC), affinity chromatography or other appropriate methodologies can be used in purification step.In one embodiment, will be directed to Alpha-non-natural amino acid (or polypeptide including alpha-non-natural amino acid) prepare antibody be used as (include, but are not limited to) include one or The purified reagent of the purifying based on compatibility of the polypeptide of more than one alpha-non-natural amino acid.It partial purification or is purified on demand After homogeneity, polypeptide i.e. be optionally used for a variety of effectiveness, it includes (but not limited to) as calibrating component, therapeutic agent, prophylactic, Diagnosticum, investigational agent and/or as Antibody preparation immunogene.

In addition to other dated herein bibliography, known a variety of purifying/protein folding method in fields, It includes those methods proposed in (but not limited to) documents below：R.Scopes,Protein Purification, Springer-Verlag,N.Y.(1982)；Deutscher,Methods in Enzymologv volumes 182:Guide to Protein Purification,Academic Press,Inc.N.Y.(1990)；Sandana(1997)Bioseparation of Proteins.Academic Press,Inc.；Bollag et al. (1996)Protein Methods.Second edition Wiley- Liss,NY；Walker(1996)The Protein Protocols HandbookHumana Press,NJ；Harris and Angal(1990)Protein Purification Applications:A Practical Approach IRL Press at Oxford,Oxford,England；Harris and AngalProtein Purification Methods:A Practical ApproachIRL Press at Oxford,Oxford,England；Scopes(1993)Protein Purification:Principles and Practice the 3rd editionSpringer Verlag,NY；Janson and Ryden (1998)Protein Purification:Principles.High Resolution Methods and Applications. second editionWiley-VCH,NY；And Walker (1998)Protein Protocols on CD-ROM Humana Press,NJ；And references cited therein.

Preparation includes the polypeptide of at least one alpha-non-natural amino acid in eukaryotic host cell or non-eukaryotic host cell One advantage is that usual polypeptide can be folded with its native conformation.However, the method and composition being described herein is some In embodiment, after synthesis, expression and/or purifying, polypeptide can have the conformation of the required conformation different from related polypeptide. In the one side of method and composition described herein, optionally make expressed protein denaturation and subsequent renaturation.This can Choosing denaturation and renaturation are realized using known method in fields, and wherein these methods are including (but not limited to) by companion's egg (chaperonin) is added in polypeptide of interest and polypeptide is dissolved in chaotropic agent (including (but not limited to) hydrochloric acid in vain Guanidine) in, and utilize protein disulphideisomerase.

In general, it is sometimes necessary to be denatured and reduce expressed polypeptide and these polypeptide refoldings is then made to be preferred Conformation.For example, the refolding can be by being added to of interest turn over by guanidine, urea, DTT, DTE and/or chaperone It translates in product to realize.The known reduction of those skilled in the art, denaturation and recombinant protein matter method (referring to, more than Bibliography and Debinski et al. (1993)J.Biol.Chem..268:14065-14070；Kreitman and Pastan (1993)Bioconjug.Chem..4:581-585；And Buchner, et al., (1992)Anal.Biochem..205:263- 270).Debinski et al. (for example) describes the denaturation and reduction of inclusion body protein in guanidine-DTE.Protein can contain (bag Contain, but be not limited to) refolding in the potential buffer solution of oxidized form of glutathione and L-arginine.Refolding reagent is flowable Or otherwise move to be contacted with one or more kinds of polypeptide or other expression products, or vice versa.

In the case where protokaryon prepares non-natural amino acid polypeptides, the polypeptide that so prepares may false folding and therefore Lack bioactivity or with the bioactivity reduced.The bioactivity of protein can be recovered by " refolding ".Implement one In example, by using (for example) one or more kinds of chaotropic agent (including (but not limited to) urea and/or guanidine) and can Reducing agent (including (but not limited to) dithiothreitol (DTT), the DTT or 2 mercapto ethanol, 2-ME) dissolving of Reduction of Disulfide is (wherein more Peptide is also to be insoluble), unfolding and reducing polypeptide chain make the polypeptide refolding of false folding.In the chaotropic agent of intermediate concentration Under, oxidant (including (but not limited to) oxygen, cystine or cystamine) is then added, allows to re-form disulfide bond.Unfolding or The polypeptide of false folding can be used known standard method refolding, such as U.S. Patent No. 4 in fields, 511, No. 502, Those methods described in 4th, 511, No. 503 and the 4th, 512, No. 922, each of these documents is by reference In being fully incorporated herein.Polypeptide also can altogether fold to form heterodimer or heteromultimeric together with other protein.It is rolling over again After folded or common folding, optionally polypeptide is further purified.

Multiple technologies can also be used to realize in the purifying of non-natural amino acid polypeptides, are retouched herein it includes (but not limited to) Those technologies stated, for example, hydrophobic interaction chromatograph, size exclusion chromatography, ion-exchange chromatography, reversed-phase high performance liquid chromatography, Affinity chromatography and its similar techniques or any combination thereof.Other purifying can also include drying or precipitate purified protein Step.

After purification, non-natural amino acid polypeptides can be exchanged in different buffer solutions and/or by fields Any one of a variety of methods (including (but not limited to) diafiltration and dialysis) known concentration.It can make in the form of single protein purification The hGH experience aggregation of offer and precipitation.In certain embodiments, purified non-natural amino acid polypeptides can be at least 90% pure (as by reversed-phase high performance liquid chromatography (RP-HPLC) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) institute Measurement).In some other embodiments, purified non-natural amino acid polypeptides can be that at least 95% is pure or at least 98% is pure, Or at least 99% or higher purity.No matter the exact numerical values recited of the purity of non-natural amino acid polypeptides, non-natural amino acid polypeptides are equal It is sufficiently pure for use as pharmaceutical product or for being processed further, it includes (but not limited to) and water-soluble polymer (such as PEG) Engagement.

In certain embodiments, non-natural amino acid polypeptides molecule there is no other active components or protein (except Excipient, supporting agent and stabilizer, seralbumin with and the like) in the case of can be used as therapeutic agent, and in some realities It applies in example, non-natural amino acid polypeptides molecule can be compound with another polypeptide or polymer.

2.The purifying of non-natural amino acid polypeptides

General purification processThe technology disclosed in this part can be applied to non-natural amino acid polypeptides described herein General purifying.

Can to cell lysates extract, culture medium, inclusion body, the periplasmic space of host cell, host cell it is thin Kytoplasm or other substances including required polypeptide or by including (but not limited to) affinity chromatography, ion-exchange chromatography, it is hydrophobic mutually Action chromatography, gel filtration chromatography, high performance liquid chromatography (" HPLC "), reversed-phase HPLC (" RP-HPLC "), expanded bed adsorption or its Any combinations and/or any separating step repeated and carry out a variety of points with any mixtures of polypeptides that any appropriate order generates From any one of step.

It can bought on the market for performing the equipment of technology described herein and other necessary materials.Pump, fraction are received Storage, monitor, logger and whole system be obtained from (for example) Applied Biosystems (Foster City, CA), Bio-Rad Laboratories, Inc. (Hercules, CA) and Amersham Biosciences, Inc. (Piscataway,NJ).Including (but not limited to) exchange matrix material, medium and buffer solution chromatographic material also available from this A little companies.

Using the professional equipment such as pumped can more quickly realize balance in column chromatography method described herein and Other steps are such as washed and eluted.It is commercially available pump including (but not limited to)Pump P-50, peristaltic pump P-1, pump P- 901 and pump P-903 (Amersham Biosciences, Piscataway, NJ).

The example of fraction collector include RediFrac fraction collectors, FRAC-100 and FRAC-200 fraction collectors with AndFraction collector (Amersham Biosciences, Piscataway, NJ).Also can use mixer with Form pH value and linear concentration gradient.Commercial mixer includes gradient mixer GM-1 and tandem mixer (Amersham Biosciences,Piscataway,NJ)。

Any commercially available monitor can be used to monitor chromatographic process.These monitors can be used for collecting such as UV, fluorescence, pH value And the information of conductivity.The example of detector include monitor UV-1,S II, monitor UV-M II, prison Control device UV-900, monitor UPC-900, monitor pH/C-900 and conductivity monitor (Amersham Biosciences, Piscataway,NJ).In fact, comprising from the various of Amersham BiosciencesSystem The whole system of (Piscataway, NJ) can bought on the market.

In one embodiment of the method and composition being described herein, for example, polypeptide can make by first in urea The purified polypeptide denaturation of gained, is then diluted in the TRIS buffer solutions containing reducing agent (such as DTT) for being suitble to pH value In reduce and be denatured.In another embodiment, polypeptide is in the urea in about 2M to the concentration range between about 9M Denaturation, is then diluted in the TRIS buffer solutions under the pH value in the range of about 5.0 to about 8.0.This reality can then be cultivated Apply the refolding mixture of example.In one embodiment, refolding mixture cultivate at room temperature 4 to 24 it is small when.Reduction and denaturation Mixtures of polypeptides then can be through further isolated or purified.

As set forth herein, the pH value of the first mixtures of polypeptides can be adjusted first, then perform any later separation step Suddenly.In addition, known technology in fields can be used to concentrate the first mixtures of polypeptides or its any subsequent mixtures.This Outside, the elution buffer including the first mixtures of polypeptides or its any subsequent mixtures can be used those skilled in the art ripe The exchange of skills known is the buffer solution suitable for next separating step.

Ion-exchange chromatographyThe technology disclosed in this part can be applied to non-natural amino acid polypeptides described herein Ion chromatography.

In one embodiment, and as other optional steps, ion-exchange chromatography can be carried out to the first mixtures of polypeptides. Usually referring to ION EXCHANGE CHROMATOGRAPHY:PRINCIPLES AND METHODS (catalog number (Cat.No.) 18-1114-21, Amersham Biosciences(Piscataway,NJ)).Commercially available ion exchange column includes AndTubing string (Amersham Biosciences, Piscataway, NJ).These tubing strings are handed over using strong anion Change agent, such as QFast Flow、QHigh Performance and QXL；Strong cation exchanger, such as SPHigh Performance、SPFast Flow and SPXL；Weak anion exchanger, such as DEAEFast Flow；And weak cation exchanger, such as CM Fast Flow (Amersham Biosciences,Piscataway,NJ).The polypeptide in any stage for being in purification process can be carried out it is cloudy from Son or cation exchange column chromatography are to separate substantially purified polypeptide.Can be used any suitable cation exchange matrix into Row cation-exchange chromatography step.Cation exchange matrix is including (but not limited to) fibrous, porous, non-porous, microgranular, bead Or cross-linked cationic exchange matrix material.These cation exchange matrix materials are including (but not limited to) cellulose, agarose, Portugal Glycan, polyacrylate, polyethylene, polystyrene, silica, the compound of polyethers or any aforementioned substances.It is adsorbed in polypeptide After in cation-exchanger matrix, substantially purified polypeptide can be by making matrix with having enough high ph-values or ion strong The Buffer fluid contacts of degree are eluted so that polypeptide is cemented out from matrix.For the high pH of substantially purified polypeptide Suitable buffer solution in value elution is including (but not limited to) citrate, phosphate, formates, acetate, HEPES, Yi Jinong Spend the MES buffer solutions in the range of at least about 5mM at least about 100mM.

Reverse-phase chromatographyThe technology disclosed in this part can be applied to the reverse phase of non-natural amino acid polypeptides described herein Chromatography.

It can be suitble to scheme according to known to those skilled in the art to carry out RP-HPLC with protein purification.Referring to (for example) Pearson et al., ANAL BIOCHEM. (1982) 124:217-230(1982)；Rivier et al., J.CHROM. (1983)268:112-119；Kunitani et al., J.CHROM. (1986) 359:391-402.RP-HPLC can be carried out to polypeptide To separate substantially purified polypeptide.In this respect, can be used has different lengths (including (but not limited to) at least about C₃Extremely At least about C₃₀, at least about C₃To at least about C₂₀Or at least about C₃To at least about C₁₈) silica with alkyl functional base of resin spreads out Raw resin.Alternatively, polymer resin can be used.For example, TosoHaas Amberchrome CG1000sd trees can be used Fat is styrenic polymer resins.Cyano or polymer resin with a variety of long alkyl chains can also be used.Furthermore, it is possible to The solvent washing RP-HPLC tubing strings of such as ethyl alcohol.Contain ion-pairing agent and organic modifiers (such as methanol, isopropanol, tetrahydrochysene Furans, acetonitrile or ethyl alcohol) suitable elution buffer can be used for polypeptide is eluted out from RP-HPLC tubing strings.The most frequently used ion To reagent including (but not limited to) acetic acid, formic acid, perchloric acid, phosphoric acid, trifluoroacetic acid, hyptafluorobutyric acid, triethylamine, tetramethyl-ammonium, Tetrabutylammonium, acetic acid triethyl ammonium.One or more kinds of gradients or isocratic condition can be used to be eluted, wherein gradient strip Part preferably reduces disengaging time and reduces peak width.Another method is directed to use with two kinds of ladders with different solvents concentration range Degree.The example of suitable elution buffer used herein can be including (but not limited to) ammonium acetate and acetonitrile solution.

Hydrophobic interaction chromatograph purification techniqueThe technology disclosed in this part can be applied to non-natural described herein The hydrophobic interaction chromatograph purifying of amino acid polypeptide.

Hydrophobic interaction chromatograph (HIC) can be performed to polypeptide.Usually referring to HYDROPHOBIC INTERACTION CHROMATOGRAPHY HANDBOOK:PRINCIPLES AND METHODS (catalog number (Cat.No.) 18-1020-90, Amersham Biosciences (Piscataway, NJ)), it is to be incorporated herein by reference.It is suitble to HIC matrix that can include (but not It is limited to) matrix that substitutes through alkyl or aryl, the matrix such as substituted through butyl, hexyl, octyl group or phenyl, the matrix includes Agarose, Sepharose, Ago-Gel, cellulose, silica, glucan, polystyrene, polymethacrylates matrix, And the resin of mixed form, the polymethyl substituted it includes (but not limited to) polyethylenimine resins or through butyl or phenyl Acid esters matrix.The commercial source of hydrophobic interaction column chromatography including (but not limited to)With AndTubing string (Amersham Biosciences, Piscataway, NJ).In short, before loading, HIC pipes Standard buffer solution known to one of ordinary skill in the art can be used (such as acetic acid/sodium chloride solution or to contain sulfuric acid for column The HEPES of ammonium) it balances.Ammonium sulfate can be used as loading the buffer solution of HIC tubing strings.After polypeptide is loaded, can then it make Tubing string is washed with standard buffer solution and condition to remove unwanted substance, but polypeptide is retained on HIC tubing strings.About 3 can be used To about 10 tubing string volumes standard buffer solution (wherein, such as containing EDTA and ammonium sulfate concentrations lower than level pad HEPES buffer solution or acetic acid/sodium chloride buffer) elution polypeptide.It is linear using continuously decreasing for (for example) potassium phosphate gradient Salt gradient can also be used for elution peptide molecule.Then can elutriant (for example) be concentrated by filtering (such as diafiltration or ultrafiltration). Using diafiltration to remove to elute the salt of polypeptide.

Other purification techniquesThe technology disclosed in this part can be applied to non-natural amino acid polypeptides described herein Other purification techniques.

First mixtures of polypeptides or its any subsequent mixtures can be carried out using (for example) gel filtration (GEL FILTRATION:PRINCIPLES AND METHODS (catalog number (Cat.No.) 18-1022-18, Amersham Biosciences, Piscataway, NJ), be in being hereby incorporated by reference in their entirety), hydroxylapatite chromatography (be suitble to matrix include (but Be not limited to) HA-Ultrogel, High Resolution (Calbiochem), CHT ceramic hydroxyapatites (BioRad), Bio- Gel HTP hydroxyapatites (BioRad)), HPLC, expanded bed adsorption, ultrafiltration, diafiltration, desivac and its similar approach it is another One separating step is to remove any excess salt and be suitable for next separating step or even allocate the buffer solution of final drug products To replace buffer solution.Various technologies (it includes (but not limited to) those described herein technologies) can be used to monitor herein The yield of the polypeptide comprising substantially purified polypeptide under each step of description.It is last that these technologies can also be used for assessment The yield of substantial purified polypeptide after separating step.For example, can be used several with a variety of long alkyl chains Reverse-phase HPLC tubing string any one (such as cyano RP-HPLC, C₁₈RP-HPLC) and cation exchange HPLC and Gel filtration HPLC monitors the yield of polypeptide.

Standard technique (such as SDS-PAGE) can be used or by using Western blot (Western blot) and ELISA calibratings measure polypeptide to measure purity.For example, polyclonal antibody can be directed to from negative control yeast fermentation and sun from Son, which exchanges, recycles separated protein generation.These antibody can also be used for the presence of detection pollution host cell proteins matter.

In certain embodiments, the polypeptide after each purification step can be in the initial substance of each purification step Polypeptide at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least About 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least About 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least About 98%, at least about 99%, at least about 99.9% or at least about 99.99%.

RP-HPLC material Vydac C4 (Vydac) are made of silica gel particle, and surface carries C₄- alkyl chain.Polypeptide and egg The separation of white matter impurity is the difference according to the intensity of hydrophobic interaction.It is washed with the acetonitrile gradient in dilute trifluoroacetic acid It carries.Preparation HPLC is carried out using stainless-steel tubing pillar (the Vydac C4 silica gel for rising to 3.2 liters filled with 2.8).Pass through addition three Hydroxyapatite Ultrogel eluates are acidified and are loaded on Vydac C4 tubing strings by fluoroacetic acid.For washing and eluting, make For the acetonitrile gradient in dilute trifluoroacetic acid.It is neutralized immediately by fraction collection and with phosphate buffer.It collects in IPC limits Polypeptide fraction.

DEAE Sepharose (Pharmacia) materials are by the diethyl that is covalently bound on the surface of Sepharose beads Base amino-ethyl (DEAE) group forms.The combination of polypeptide and DEAE groups is mediated by ionic interaction.Acetonitrile and trifluoro second Acid without being detained passes through tubing string.In these substances after wash-off, by with the acetate buffer of low ph value wash tubing string come Remove trace impurity.Tubing string is then washed and to be eluted with the buffer solution of increased ionic strength with neutral phosphate buffer liquid Polypeptide.Tubing string is loaded with DEAE Sepharose fast flow.Regulation pipe column volume is coagulated with ensuring that polypeptide is loaded in every milliliter In the range of glue 3-10mg polypeptides.With water and level pad (sodium phosphate/potassium phosphate) washing tubing string.Load HPLC eluates Collect fraction and with equilibration buffer solution tubing string.Then with lavation buffer solution (sodium acetate buffer) wash tubing string, then with Equilibration buffer solution.Then, with elution buffer (sodium chloride, sodium phosphate/potassium phosphate) polypeptide eluted out from tubing string and According to main elution overview with single fraction collection.The eluate of DEAE Sepharose tubing strings is adjusted to specified conductivity. Gained drug substance is sterile filtered in Teflon bottles and is stored at -70 DEG C.

Can be used the assessment of a variety of methods and procedures include the yield of the polypeptide of one or more alpha-non-natural amino acids with Purity, it includes (but not limited to) Bradford calibratings, SDS-PAGE, silver staining SDS-PAGE, coomassie brilliant blue staining SDS- PAGE (coomassie stained SDS-PAGE), mass spectrum (including (but not limited to) MALDI-TOF) and fields The known other methods for being used to characterize protein of technical staff.

Other methods are including (but not limited to) the endotoxic step of removal.Endotoxin is positioned at Gram-negative host cells Lipopolysaccharides (LPS) on the outer membrane of (such as, Escherichia coli).The method of endotoxin content is reduced including (but not limited to) using silicon The purification technique of stone carrier, glass powder or hydroxyapatite, reverse-phase chromatography, affinity chromatography, size exclusion chromatography, anion are handed over Colour changing spectrum, hydrophobic interaction chromatograph, the combination of these methods and its similar approach.It may need modification or other methods To remove the pollutant for such as migrating albumen altogether from polypeptide of interest.In the known measurement of one of ordinary skill in the art The method of content of toxins and it includes (but not limited to) horseshoe crab ameboid cell lysate (Limulus Amebocyte Lysate, LAL) examine and determine.

Other methods and program including (but not limited to) the SDS-PAGE coupled with protein staining method, immune turn stain Method, substance assistant laser desorpted/ionization-mass spectrometry (MALDI-MS), liquid chromatography/mass spectrometry, isoelectric focusing, analytic type anion are handed over It changes, chromatofocusing and circular dichroism spectra.

In certain embodiments, comprising the model in Formulas I-XVIII, XXX-XXXIV (A and B) and XXXX-XXXXIII Formulas I-XVIII, the XXX-XXXIV (A and B) of any minor or specific compound in farmland and the amino acid of XXXX-XXXXIII It can be incorporated to by biosynthesis in polypeptide, and then generate non-natural amino acid polypeptides.In other embodiments, these amino acid are It is incorporated at specific site in polypeptide.In other embodiments, these amino acid are to be incorporated to using translation system in polypeptide. In other embodiment, these translation systems include：(i) polynucleotide of coded polypeptide, wherein polynucleotide include corresponding to simultaneously Entering the selection codon of the predetermined site of above-mentioned amino acid and (ii) includes the tRNA of amino acid, and wherein tRNA is close to selecting Numeral has specificity.In the other embodiment of these translation systems, polynucleotide is the mRNA generated in translation system. In the other embodiment of these translation systems, translation system includes plasmid or the bacteriophage including polynucleotide.It is turned at these In the other embodiment for translating system, translation system includes the genomic DNA comprising polynucleotide.In other of these translation systems In embodiment, polynucleotide stable integration enters in genomic DNA.In the other embodiment of these translation systems, translation system Including having the tRNA of specificity to selection codon, wherein the selection codon is selected from close by amber codon, ochre Numeral, opal codon, unique codon, rare codon, unnatural codons, five base codons and four bases are close The molecular group of code.In the other embodiment of these translation systems, tRNA is inhibition tRNA.In its of these translation systems In his embodiment, translation system includes will be by the aminoacylated tRNA on above-mentioned amino acid.In other of these translation systems In embodiment, translation system includes the amino acyl synthetase for having specificity to tRNA.In other implementations of these translation systems In example, translation system includes orthogonal tRNA and orthogonal aminoacyl tRNA synzyme.In the other embodiment of these translation systems, Polypeptide is synthesized by ribosomes, and in other embodiments, translation system is to include being selected from by bacterial cell, archeabacterial cell And the in vivo translation system of the cell of the group of eukaryocyte composition.In other embodiments, cell is thin for Escherichia coli Born of the same parents, yeast cells, cell, mammalian cell, plant cell or the insect cell of species from pseudomonad.It is turned at these In the other embodiment for translating system, translation system is to include the cell from bacterial cell, archeabacterial cell or eukaryocyte to carry Take the in vitro translation system of object.In other embodiments, cell extract is from Bacillus coli cells, from pseudomonad Species cell, yeast cells, mammalian cell, plant cell or insect cell.In other embodiments, at least one Dividing polypeptide is synthesized by solid phase or solution phase peptide synthetic method or its combination, and in other embodiments, further comprise Polypeptide is connected on another polypeptide.In other embodiments, comprising in Formulas I-XVIII, XXX-XXXIV (A and B) and Formulas I-XVIII, the XXX-XXXIV (A and B) of any minor or specific compound in the scope of XXXX-XXXXIII and The amino acid of XXXX-XXXXIII can be incorporated to by biosynthesis in polypeptide, wherein polypeptide with selected from being made of following object The homologous protein of the treatment albumen matter of group：α -1 antitrypsins, angiostatin, anti-hemolytic factor, antibody, load Lipoprotein, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, C-X-C chemotactic factor (CF)s, T39765, NAP- 2nd, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit coordinations Body, cell factor, CC chemotactic factor (CF)s, monocyte chemoattractant protein-1, monocyte chemoattractant protein -2, monocyte chemotactic egg In vain -3, -1 α of monocyte inflammatory protein, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligands, collagen, colony stimulating factor (CSF), Complement factor 5a, complement inhibitor, complement receptor 1, cell factor, epithelium neutrophilic granulocyte activation peptide -78, MIP-16, MCP- 1st, epidermal growth factor (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin(EPO) (EPO), exfoliative toxin,or exfoliatin, the factor IX, factor Ⅴ II, Factor IX, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four Helix bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, promoting sexual gland hormone, growth factor, growth factor receptors, Grf, Hedgelog protein, hemochrome, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), the white egg of human serum In vain, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 Receptors, insdri, insulin-like growth factor (IGF), IGF-I, IGF- II, interferon (IFN), IFN-α, IFN-β, IFN-γ, interleukins (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL- 6th, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactotransferrin, leukaemia Inhibiting factor, luciferase, neural element, neutrophil inhibitory factor (nif) (NIF), oncostatin M, osteogenic protein, oncoprotein, The outer poison of paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, multi-effector, albumin A, Protein G, pth, pyrogenicity Plain A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxain, feritin, SCF, atom synthetic proteins, Soluble complement receptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, Growth hormone, streptokinase, super antigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroids swash It is plain receptor, superoxide dismutase, toxic-shock syndrome toxin, thymosin α1, histiotype plasminogen activation factor, swollen Knurl growth factor (TGF), tumor necrosis factor, tumor necrosis factor α, tumor necrosis factor β, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, P53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, testosterone receptor, aldosterone receptor, LDL by Body and cortisone.

B. in vivo posttranslational modification

By generating the polypeptide of interest at least one alpha-non-natural amino acid in eukaryocyte, these polypeptides can It is modified after eukaryotic translation being included.In certain embodiments, protein includes at least one alpha-non-natural amino acid and at least one The posttranslational modification that kind is in vivo carried out by eukaryocyte, wherein posttranslational modification are not carried out by prokaryotic cell.Citing comes Say, posttranslational modification including (but not limited to) acetylation, acylation, lipid-modified, palmitoylation, palmitate addition, phosphorylation, Glycolipids key modification, glycosylation and its similar to mechanism.On the one hand, posttranslational modification includes bonded by GlcNAc- asparagines By oligosaccharides (including (but not limited to) (GlcNAc-Man)₂- Man-GlcNAc-GlcNAc)) it is connected on asparagine.Referring to table 1, list some examples (other residues also may be present, do not show) of the bonded oligosaccharides of N- of eukaryotic protein.On the other hand, Posttranslational modification is included through GalNAc- serines or GalNAc- threonines are bonded or GlcNAc- serines or GlcNAc- Soviet Unions Propylhomoserin is bonded to be connected to oligosaccharides on serine or threonine (including (but not limited to) Gal-GalNAc, Gal-GlcNAc etc.).

Table 1：Pass through the example of the oligosaccharides of the bonded connections of GlcNAc

In another aspect, posttranslational modification includes predecessor (including (but not limited to) calcitonin predecessor, drop blood calcium Plain gene related peptide predecessor, Pre Pro PTH, preproinsulin, proinsulin, pre-pro-opiomelanocortin, preceding Ah's casting skin Element with and the like) proteolysis processing, be assembled into multi-subunit protein matter or macromolecular assembly, translate into cell Another site (including (but not limited to) organelle, such as endoplasmic reticulum, golgiosome (golgi apparatus), core, lyase Body, peroxisome, mitochondria, chloroplaset, vacuole etc. pass through secretory pathway).In certain embodiments, protein bag Include secretion or positioning sequence, epitope label, FLAG labels, polyhistidyl tags, GST fusions or its analog.

One advantage of alpha-non-natural amino acid is that it has other chemical parts that can be used for adding other molecules.These Modification can in vivo or in vitro carry out in eukaryocyte or non-eukaryocyte.Therefore, in certain embodiments, after translation Modification is carried out by alpha-non-natural amino acid.For example, posttranslational modification can be carried out by nucleophilic-electrophilic reaction.At present For the selective modification of protein most of translations be related to nucleophilic and electrophilic reaction collocation object between form covalent bond, It includes the reactions of (but not limited to) α-halogen ketone and histidine or cysteine side chain.Selectivity in the case of these is by egg The number of nucleophilic residues and accessibility determine in white matter.It is described herein or is prepared using method described herein In polypeptide, other more selectively reactions can be used, the in vitro and in vivo non-natural ketone it includes (but not limited to) The reaction of base amino acid and hydrazides or aminooxy compound.Referring to (for example) Cornish et al., (1996)Am.Chem.Soc.118:8150-8151；Mahal et al., (1997)Science,276:1125-1128；Wang et al., (2001)Science292:498-500；Chin et al., (2002)Am.Chem.Soc.124:9026-9027；Chin et al., (2002)Proc.Natl.Acad.Sci..99:11020-11024；Wang et al., (2003)Proc.Natl.Acad.Set 100:56-61；Zhang et al., (2003)Biochemistry.42:6735-6746；And Chin et al., (2003)Science300:964-967.This allows that (it includes fluorogen, crosslinking agent, sugar derivatives and cytotoxicities with many reagents Molecule) the substantially any protein of selected marker.Referring also to entitled " Glycoprotein filed in 16 days January in 2003 The U.S. Patent Application No. of synthesis " 10/686,944, is to be incorporated herein by reference.It is repaiied after translation Decorations can also connect (Staudinger ligation) (including (but not limited to) azido amino acid is passed through) by Staudinger (including (but not limited to) with triaryl phosphine reagent) carries out.Referring to (for example) Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligtation,PNAS 99(1):19-24。

IX. the alternate systems of non-natural amino acid polypeptides are used to prepare

Using it is several strategy in non-recombinant hosts cell, mutagenesis host cell or cell free system by non-natural ammonia Base acid is introduced into protein.The alternate systems disclosed in this part can be applied to prepare alpha-non-natural amino acid described herein Polypeptide.For example, (such as Lys, the Cys and Tyr) derivatization of the amino acid with reactive side chain is made so that lysine changes For N²- acetyl-lysine.Chemical synthesis also provides the straightforward procedure for being incorporated to alpha-non-natural amino acid.With the enzymatic of peptide fragment Connect the latest developments connected with native chemical, it is possible to manufacture larger protein.Referring to (for example) P.E.Dawson and S.B.H.Kent,Annu.Rev.Biochem,69:923(2000).The connection of chemical peptide connects that be described in the U.S. special with native chemical Profit the 6,184,344th, U.S. Patent Publication case the 2004/0138412nd, U.S. Patent Publication case the 2003/0208046th Number, be in being hereby incorporated by reference in their entirety in WO 02/098902 and WO 03/042235.It wherein will be through required The inhibition tRNA of alpha-non-natural amino acid chemical acylation is added in the in vitro extract that can support Protein synthesis General in vitro biological synthesis method has been used for more than 100 kinds alpha-non-natural amino acid locus specificities being incorporated to substantially any big In small multiple proteins.Referring to (for example) V.W.Cornish, D.Mendel and P.G.Schultz,Angew.Chem.Int.Ed.Engl.1995,34:621-633(1995)；C.J.Noren,S.J.Anthony-Cahill, M.C.Griffith,P.G.Schultz,A general method for site-specific incorporation of unnatural amino acids into proteins,Science244182-188(1989)；And J.D.Bain, C.G.Glabe,T.A.Dix,A.R.Chamberlin,E.S.Diala,Biosynthetic site-specific incorporation of a unnatural amino acid into a polypeptide,J.Am.Chem.Soc.1118013-8014(1989).A variety of functional groups have been introduced into protein to study protein stabilization Property, protein folding, enzyme mechanism and signal transduction.

The in vivo method that is incorporated to referred to as selection pressure is developed to develop the hybridization of wild type synzyme.Referring to (for example) N.Budisa, C.Minks, S.Alefelder, W.Wenger, F.M.Dong, L.Moroder and R.Huber,FASEB J..13:41-51(1999).Lack the nutrition being wherein cut off to the relevant metabolic pathway of the specific natural amino acid of cell supply Swaged strain growth is in the minimal medium of the natural amino acid containing Finite Concentration, while the transcription of target gene is pressed down System.When the fixed growth stage starts, natural amino acid is exhausted and is replaced with non-natural amino acid analogs.Induction restructuring egg White expression is so that the protein accumulation containing non-natural analogs.For example, using this strategy, by adjacent the third ammonia of fluorobenzene Acid, fluorophenylalanine and P-fluoropnenylalanine are incorporated in protein, and displaying can be easy to two differentiated in UV spectrum Distinctive shoulder.Referring to (for example) C.Minks, R.Huber, L.Moroder and N.Budisa,Anal.Biochem.,284:29- 34(2000)；Using trifluoro methionine the methionine in bacteriophage T4 Lysozyme is substituted to pass through¹⁹F NMR study it With the interaction of chitosan oligosaccharide ligand, referring to (for example) H.Duewel, E.Daub, V.Robinson and J.F.Honek,Biochemistry,36:3404-3416(1997)；And trifluoro leucine is incorporated to instead of leucine, cause leucine zipper protein White thermal stability and chemical stability increase.Referring to (for example) Y.Tang, G.Ghirlanda, W.A.Petka, T.Nakajima, W.F.DeGrado and D.A.Tirrell, Angew.Chem.Int.Ed.Engl..40(8):1494-1496 (2001).In addition, selenomethionine and tellurium methionine are incorporated in various recombinant proteins in favor of in parsing X-ray crystallography Phase.Referring to (for example) W.A.Hendrickson, J.R.Horton and D.M.Lemaster,EMBO J.,9(5):1665- 1672(1990)；J.O.Boles, K.Lewinski, M.Kunkle, J.D.Odom, B.Dunlap, L.Lebioda and M.Hatada,Nat.Struct.Biol,1:283-284(1994)；N.Budisa,B.Steipe,P.Demange, C.Eckerskorn, J.Kellermann and R.Huber,Eur.J.Biochem.,230:788-796(1995)；And N.Budisa, W.Karnbrock, S.Steinbacher, A.Humm, L.Prade, T.Neuefeind, L.Moroder and R.Huber,J.Mol.Biol.270:616-623(1997).Methionine analogs with alkene or alkynes functional group are also effectively Ground is incorporated to, and allows to carry out other modifications of protein by chemical means.Referring to (for example) J.C.M.vanHest and D.A.Tirrell,FEBS Lett..428:68-70(1998)；J.C.M.van Hest, K.L.Kiick and D.A.Tirrell,J.Am.Chem.Soc.122:1282-1288(2000)；And K.L.Kiick and D.A.Tirrell, Tetrahedron, 56: 9487-9493(2000)；U.S. Patent No. 6,586,207；U.S. Patent Publication case 2002/0042097 is with reference During mode is fully incorporated herein.

The success of this method depends on identifying non-natural amino acid analogs by aminoacyl-tRNA synthetase, leads to It often needs highly selective to ensure the fidelity of protein translation.An approach for expanding the scope of the method is to loosen aminoacyl The substrate specificity of base-tRNA synzyme, is realized in the case of finite population.Only for example, in Escherichia coli benzene Ala is replaced by Gly in alanyl-tRNA synzyme (PheRS)²⁹⁴Increase the size of Binding Capacity bag, and cause tRNAPhe It is acylated by fenclonine (p-Cl-Phe).Referring to, M.Ibba, P.Kast and H.Hennecke,Biochemistry.33: 7107-7112(1994).Coli strain containing this mutant PheRS allows to be incorporated to fenclonine or to bromobenzene third Propylhomoserin replaces phenylalanine.Referring to (for example) M.Ibba and H.Hennecke,FEBS Lett..364:272-275(1995)；With And N.Sharma, R.Furter, P.Kast and D.A.Tirrell,FEBS Lett..467:37-40(2000).Similarly, connect Diazonium is allowed in point mutation Phe130Ser displayings at the amino acid binding site of nearly Escherichia coli tyrosyl--tRNA synzyme Tyrosine is more effectively incorporated to than tyrosine.Referring to, F.Hamano-Takaku, T.Iwama, S.Saito-Yano, K.Takaku, Y.Monden, M.Kitabatake, D.Soil and S.Nishimura, LBiol. Chem..275(51): 40324-40328(2000)。

Alpha-non-natural amino acid is in vivo incorporated to another strategy in protein as synthesis of the modification with check and correction mechanism Enzyme.These synzyme cannot distinguish between and the amino acid of homologous natural amino acid is therefore similar on activatable structural.This mistake is a Other site is corrected, and mispairing amino acid is deacylated to keep the fidelity of protein translation from tRNA.If synthesis The check and correction loss of activity of enzyme, then the analogue of mistake activation can avoid editting function and be merged in.Recently with figured silk fabrics ammonia Acyl group-tRNA synzyme (ValRS) confirms the method.Referring to, V.Doring, H.D.Mootz, L.A.Nangle, T.L.Hendrickson, V.de Crecy-Lagard, P.Schimmel and P.Marliere,Science.292:501-504 (2001).ValRS can use Cys, Thr or the wrong aminoacylated tRNAVal of aminobutyric acid salt (Abu)；Then hydrolyzed by edit field These non-homologous amino acids.After random mutagenesis escherichia coli chromosome, select that there is mutation in the editing sites of ValRS Mutant Escherichia coli bacterial strain.This editor's deficiency ValRS mistake loads tRNAVal with Cys.Because Abu spatially classes Like Cys (in the Abu-SH group warps-CH of Cys₃Displacement), so when this mutant Escherichia coli bacterial strain is raw in the presence of Abu When long, Abu is also incorporated in protein by mutant ValRS.Mass spectral analysis is illustrated in each valine position of native protein The valine at place about 24% is replaced by Abu.

Synthesis in solid state and semisynthesis also allow many protein containing new amino acid of synthesis.For example, join See following discloses case and references cited therein, the publication is as follows：Crick,F.J.C.,Barrett, L.Brenner,S.Watts-Tobin,R.General nature of the genetic code for proteins.Nature,192(4809):1227-1232(1961)；Hofmann,K.,Bohn,H.Studies on polypeptides.XXXVI.The effect of pyrazole-imidazole replacements on the S- protein activating potency of an S-peptide fragment,J,Am Chem,88(24):5914- 5919(1966)；Kaiser,E.T.Synthetic approaches to biologically active peptides and proteins including enyzmes,Acc Chem Res.22(2):47-54(1989)；Nakatsuka,T., Sasaki,T.,Kaiser,E.T.Peptide segment coupling catalyzed by the semisynthetic enzyme thiosubtilisin,J Am Chem Soc,109,3808-3810(1987)；Schnolzer,M.,Kent,S B H.Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease,Science,256,221-225(1992)；Chaiken, I.M.Semisynthetic peptides and proteins,CRC Crit Rev Biochemu255-301(1981)； Offord,R.E.Protein engineering by chemical meansProtein Eng.,1(3):151-157 (1987)；And Jackson, D.Y., Burnier, J., Quan, C, Stanley, M., Tom, J., Wells, J.A.A Designed Peptide Ligase for Total Synthesis of Ribonuclease A with Unnatural Catalytic Residues,Science,266,243-247(1994)。

Chemical modification is had been used for a variety of non-natural sides for including co-factor, spin labeling and oligonucleotides in vitro Chain is introduced into protein.Referring to (for example) Corey, D.R., Schultz, P.G.Generation of a hybrid sequence-specific single-stranded deoxyribonuclease,Science,238,1401-1403 (1987)；Kaiser,E.T.,Lawrence D.S.,Rokita,S.E.The chemical modification of enzymatic specificity,Ann.Rev Biochem,54,565-595(1985)；Kaiser,E.T.,Lawrence, D.S.Chemical mutation of enyzme active sites,Science,226,505-511(1984)；Neet, K.E.,Nanci A,Koshland,D.E.Properties of thiol-subtilisin,J Biol.Chem,243(24): 6392-6401(1968)；Polgar,L.B.,M.L.A new enzyme containing a synthetically formed active site.Thiol-subtilisin.J.Am Chem Soc,88(13):3153-3154(1966)；And Pollack,S.J.,Nakayama,G.Schultz,P.G.Introduction of nucleophiles and spectroscopic probes into antibody combining sites,Science,1(242):1038-1040 (1988)。

Alternatively, it has been used for visiting several biophysics using the biological synthesis method of the aminoacyl-tRNA through chemical modification Pin is incorporated in the protein in vitro synthesized.Referring to following discloses case and references cited therein：Brunner,J.New Photolabeling and crosslinking methods,Annu.Rev Biochem,483-514(1993)；And Krieg,U.C.,Walter,P.,Hohnson,A.E.Photocrosslinking of the signal sequence of nascent preprolactin of the 54-kilodalton polypeptide of the signal recognition particle,Proc.Natl.Acad.Sci.83,8604-8608(1986)。

In the past, it has therefore proved that can be in vitro by the stylized albumen of the gene through being mutated with amber nonsense needed for containing Alpha-non-natural amino acid locus specificity is incorporated to albumen by addition through the aminoacylated inhibition tRNA of chemistry in matter synthetic reaction In matter.Using these methods, it can be used and taken for specific amino acids for the bacterial strain of nutritional deficiency type with tight structure homologue For numerous common 20 kinds of amino acid, for example, substituting phenylalanine with fluorophenylalanine.Referring to (for example) Noren, C.J., Anthony-Cahill,Griffith,M.C.,Schultz,P.G.A general method for site-specific incorporation of unnatural amino acids into proteins,Science.244:182-188 (1989)；M.W.Nowak et al.,Science268:439-42(1995)；Bain,J.D.,Glabe,C.G.,Dix,T.A., Chamberlin,A.R.,Diala,E.S.Biosynthetic site-specific Incoiporation of a non- natural amino acid into a polypeptide,J.Am Chem Soc.111:8013-8014(1989)； N.Budisa et al.,FASEB J.13:41-51(1999)；Ellman,J.A.,Mendel,D.,Anthony-Cahill,S., Noren,C.J.,Schultz,P.G.Biosynthetic method for introducing unnatural ammo acids site-specifically into proteins.Methods in Enz.,202,301-336(1992)；And Mendel, D., Cornish, V.W. and Schultz, P.G.Site-Directed Mutagenesis with an Expanded Genetic Code,Annu Rev Biophys.Biomol Struct.24,435-62(1995)。

For example, identification terminator codon UAG is prepared and through the aminoacylated inhibition tRNA of alpha-non-natural amino acid chemistry. It is formed in using conventional rite-directed mutagenesis at the site of interest in protein gene and introduces terminator codon TAG.Referring to (for example) Sayers,J.R.,Schmidt,W.Eckstein,F.5',3'Exonuclease in phosphorothioate-based oligonucleotide-directed mutagenesis,Nucleic Acids Res.16(3):791-802(1988).When Acylated will inhibit tRNA in vitro transcribe with mutant gene/translation system in when combining, alpha-non-natural amino acid response UAG is close Numeral is incorporated to obtain the protein for containing that amino acid in specified location.Use [³H]-Phe experiment and use α-hydroxyl The experiment of base acid confirms that only required amino acid is incorporated in the position specified by UAG codons and this amino acid is not in protein In any other site at be incorporated to.Referring to (for example) Noren et al., it is same as above；Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432；And Ellman, J.A., Mendel, D., Schultz, P.G.Site- specific incorporation of novel backbone structures into proteins,Science, 255,197-200(1992)。

Microinjection also has been used for alpha-non-natural amino acid being incorporated in protein.Referring to (for example) M.W.Nowak, P.C.Kearney,J.R.Sampson,M.E.Saks,C.G.Labarca,S.K.Silverman,W.G.Zhong, J.Thorson, J.N.Abelson, N.Davidson, P.G.Schultz, D.A.Dougherty and H.A.Lester,Science,268:439-442(1995)；And D.A.Dougherty,Curr.Opin.Chem.Biol,4:645(2000)。 Two kinds of RNA substances that co-injection in vitro manufactures into xenopus leavis oocytes：Have at amino acid position of interest The mRNA of the encoding target protein of UAG terminator codons and inhibit through the aminoacylated amber of required alpha-non-natural amino acid tRNA.The translating mechanism of subsequent egg mother cell is inserted into alpha-non-natural amino acid in the position specified by UAG.The method is allowed completely The in vivo structure-function research of memebrane protein, the memebrane protein are generally free from vitro expression system effect.Example include (but Be not limited to) by Fluorescent amino acid be incorporated in tachykinin neurokinin-2 receptors with by fluorescence resonance energy transfer come measure away from From, referring to (for example) G.Turcatti, K.Nemeth, M.D.Edgerton, U.Meseth, F.Talabot, M.Peitsch, J.Knowles, H.Vogel and A.Chollet, IBiol.Chem.,271(33):19991-19998(1996)；It is incorporated to biology Elementization amino acid is to differentiate the surface exposed residue in ion channel, referring to (for example) J.P.Gallivan, H.A.Lester and D.A.Dougherty,Chem.Biol.,4(10):739-749(1997)；Cover Tyrosine Analogues using cage with monitor in real time from Conformation change in subchannel, referring to (for example) J.C.Miller, S.K.Silverman, P.M.England, D.A.Dougherty and H.A.Lester, Neuron, 20:619-624(1998)；And use change ion channel skeleton α hydroxy-amino-acids are to detect its door control mechanism.Referring to (for example) P.M.England, Y.Zhang, D.A.Dougherty and H.A.Lester,Cell,96:89-98(1999)；And T.Lu, A.Y.Ting, J.Mainland, L.Y.Jan, P.G.Schultz and J.Yang,Nat.Neurosci.,4(3):239-246(2001)。

The ability in vivo alpha-non-natural amino acid being directly incorporated into protein provide mutein high yield, Technology is simple, may study in cell or in living organism the possibility of mutein and in therapeutic treatment The advantages of using these muteins.It will be with the non-of all size, acidity, nucleophilicity, hydrophobicity and other properties The ability that natural amino acid is included in protein can greatly extend us rationally and the systematically structure of operon protein Ability, to detect protein function and generate new protein or organism with novel property.

During P-fluoropnenylalanine is incorporated to attempting locus specificity, yeast amber is inhibited into tRNAPheCUA/ Phenylalanyl-tRNA synzyme is to being used in p-F-Phe resistances, Phe auxotrophic E. coli bacterial strains.Referring to (example As) R.Furter,Protein Sci.,7:419-426(1998)。

It is also possible to the expression of polynucleotide needed for being obtained using acellular (in vitro) translation system.In these systems In, the DNA (combining in vitro transcription and translation) as the mRNA (in vitro translating) of template or as template can be included, it is living External synthesis is instructed by ribosomes.Sizable effort has been applied to develop cell-free protein expression system.Referring to (for example) Kim, D.-M. and J.R.Swartz, Biotechnology and Bio engineering, 74 (4):309-316(2001)； Kim, D.-M. and J.R.Swartz, Biotechnology Letters, 22,1537-1542, (2000)；Kim, D.-M. and J.R.Swartz,Biotechnology Progress,16,385-390,(2000)；Kim, D.-M. and J.R.Swartz, Biotechnology and Bioengineering,66(3):180-188,(1999)；And Patnaik, R. and J.R.Swartz,Biotechniques 24(5):862-868,(1998)；U.S. Patent No. 6,337,191；United States Patent (USP) Publication the 2002/0081660th；WO 00/55353；WO 90/05785 is to be hereby incorporated by reference in their entirety In.Another method that can be applied to the expression for the polypeptide for including alpha-non-natural amino acid is merged including (but not limited to) mRNA- peptides Technology.Referring to (for example) R.Roberts and J.Szostak, Proc.Natl Acad.Sci. (USA) 9412297-12302 (1997)；A.FranKel et al., Chemistry＆Biology 10,1043-1050 (2003).In this approach, it is connected to MRNA templates on puromycin are translated as peptide on ribosomes.If modify one or more kinds of tRNA molecules, then Alpha-non-natural amino acid also may be incorporated into peptide.After last mRNA codons are read, puromycin captures the C- ends of peptide.Such as Fruit finds that gained mRNA- peptides binding element has interesting property in vitro examining and determine, then its identity can be easy to according to mRNA Sequence discloses.By this method, can screening including one or more alpha-non-natural amino acids polypeptide storehouse to differentiate with institute Need the polypeptide of property.Recently, report the translation of the in vitro ribosomes with purified components and allow synthesis through alpha-non-natural amino acid Substituted peptide.Referring to (for example) A.Forster et al., Proc.Natl Acad.Sci. (USA) 100 (11):6353-6357 (2003)。

X. the posttranslational modification of the non-natural amino acid constituents of polypeptide

For convenience, the posttranslational modification one of the non-natural amino acid constituents of the polypeptide described in this part (XA to XJ) As property and/or described with particular instance.However, it is repaiied after the translation of the non-natural amino acid constituents of polypeptide described in this part It adorns and the general description or particular instance that are provided in this part is provided, but the non-natural ammonia of the polypeptide described in this part The posttranslational modification of base acid constituents is equally fully adapted in Formulas I-XVIII, XXX-XXXIV (A and B) and XXXX- All compounds in the scope of XXXXIII, it includes described in description herein book, claim and the schema Formulas I-XVIII, XXX-XXXIV (A and B) and any minor or specific compound in the scope of XXXX-XXXXIII.

Method, composition, technology and strategy have been developed so that site is specifically between the in vivo translation phase of protein It is incorporated to alpha-non-natural amino acid.There is the non-natural of the pendant chemical orthogonal with those side chains of naturally occurring amino acid by being incorporated to Amino acid, this technology make it possible the site-specific derivatization of recombinant protein.Therefore, method described herein, The major advantage of composition, technology and strategy is nowadays the protein of derivatization can be prepared as determining homologues.However, this Method, composition, reaction mixture, technology and the strategy of described in the text are not limited to what is formed by vivo protein translation technology Non-natural amino acid polypeptides, but comprising the non-natural amino acid polypeptides formed by any technology, the technology is included and (only illustrated For) marking protein connection, chemical synthesis, based on ribozyme technology (referring to (for example) herein entitled " alternative system The part of expression in system ").

The ability wide spread that alpha-non-natural amino acid is incorporated in recombinant protein can be realized for derivatization after translation Chemistry, wherein this derivatization occur in vivo or in vitro.More particularly, the shape on the non-natural amino acid moieties of polypeptide Into several advantages of protein derived offer of oxime key.First, naturally occurring amino acid is not generally formed oxime key and therefore through setting Meter (can assume of course that non-natural amino to form the reagent of oxime key with the reaction of the non-natural amino acid constituents locus specificity of polypeptide Acid has been designed to form oxime key with corresponding reagent), therefore the mixing of the derived protein with using prior art processes generation Object is on the contrary, the ability of site selectivity derivatization albumen matter provides single homogeneous product.Second, oxime adduct is under biotic factor To be stable, it is effective candidate for the treatment of use to imply the protein as derived from exchanging oxime.3rd, the stabilization of gained oxime key Property can have been formed on the property (that is, functional group and/or structure) of alpha-non-natural amino acid thereon to manipulate according to oxime key.Therefore, As shown in example 16, the pH stability of the oxime key of alpha-non-natural amino acid can from less than 1 it is small when be changed to far more than 1 week. Therefore, in some embodiments, the oxime key of non-natural amino acid polypeptides is having less than 1 half life of decomposition when small, in other realities Apply in example is less than 1 day, in other embodiments to be less than 2 days, in other embodiments to be less than 1 week and in other embodiment In be more than 1 week.In other embodiments, gained oxime was in appropriate stable under acidic conditions at least two weeks, in other embodiments, Gained oxime was in appropriate stable under acidic conditions at least 5 days.In other embodiments, non-natural amino acid polypeptides between about 2 with Stablize at least 1 day under pH value between about 8；In other embodiments, stablize at least 1 day under the pH value of about 2 to about 6；At it In his embodiment, stablize at least 1 day under the pH value of about 2 to about 4.In other embodiments, using it is described herein strategy, Method, composition and technology, those skilled in the art should be able to synthesize with adjust to needed for skilled craftsman (for example, with In therapeutical uses (such as sustained release) or diagnostic uses or industrial use or military use) half life of decomposition non-natural The oxime key of amino acid polypeptide.

Above-mentioned non-natural amino acid polypeptides are suitable for (include, but are not limited to) novel therapeutic agents, diagnosticum, catalyzing enzyme, work Industry enzyme, binding protein (including (but not limited to) antibody and antibody fragment) and (include, but are not limited to) protein structure and work( The research of energy.Referring to (for example) Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function,Current Opinion in Chemical Biology,4:645-652.Above-mentioned non-day Other purposes of right amino acid polypeptide include (only for example) purposes based on calibrating, cosmetic use, phytobiology and use On the way, environmental application, energy production purposes and/or military use.It is further repaiied however, above-mentioned non-natural amino acid polypeptides can be undergone It is decorated with and is incorporated to new or modified functional group, it includes the therapeutic efficiencies for manipulating polypeptide；Improve the security overview of polypeptide；It adjusts Pharmacokinetics, pharmacology and/or the drug effect of polypeptide are saved (for example, increase is water-soluble, biological usability；Increase serum half-life； Increase treatment half-life period；Adjust immunogenicity；Adjust bioactivity；Or extend circulation time)；Other functions are provided to polypeptide Base；Label, mark or detectable signal are incorporated into polypeptide；The separating property of easyization polypeptide；And any group of foregoing modification It closes.

It is the method for the separating property of easyization polypeptide in certain embodiments, using including at least one non-natural The homologous organisms synthesis non-natural amino acid polypeptides of amino acid, the alpha-non-natural amino acid is selected from by containing oxime non-natural amino The group of acid, alpha-non-natural amino acid containing carbonyl and the composition of alpha-non-natural amino acid containing azanol.In other embodiments, these non-days Right amino acid is incorporated to by biosynthesis in polypeptide as described herein.In other or alternate embodiment, these non-days Right amino acid polypeptide includes at least one amino acid selected from Formulas I-XVIII, XXX-XXXIV (A and B) or XXXX-XXXXIII Alpha-non-natural amino acid.

Method described herein, composition, strategy and technology are not limited to the specific type, species or family of polypeptide.It is real On border, substantially any polypeptide can include at least one alpha-non-natural amino acid described herein.Only for example, polypeptide can be with choosing Freely the treatment albumen matter of the group of following object composition is homologous：α -1 antitrypsins, angiostatin, anti-hemolysis because Son, antibody, apolipoprotein, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, C-X-C chemotactic factor (CF)s, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, drop blood calcium Element, c-kit ligands, cell factor, CC chemotactic factor (CF)s, monocyte chemoattractant protein-1, monocyte chemoattractant protein -2, monokaryon Cell chemotaxis protein-3, -1 α of monocyte inflammatory protein, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligands, collagen, colony thorn Swash the factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cell factor, epithelium neutrophilic granulocyte activation peptide -78, MIP-16, MCP-1, epidermal growth factor (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin(EPO) (EPO), epidermis stripping Detoxification element, factors IX, factor Ⅴ II, Factor IX, factor X, fibroblast growth factor (FGF), fibrinogen, fiber Connect albumen, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, promoting sexual gland hormone, growth factor, life Growth factor receptor body, grf, Hedgelog protein, hemochrome, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), Human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 Receptors, insdri, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-α, IFN-β, IFN-γ, interleukins (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), breast Siderophillin, LIF ELISA, luciferase, neural element, neutrophil inhibitory factor (nif) (NIF), oncostatin M, into Bone protein, oncoprotein, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, multi-effector, albumin A, Protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxain, feritin, SCF, atom synthesis Albumen, Soluble complement receptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, growth are adjusted Save element, Somat, growth hormone, streptokinase, super antigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic-shock syndrome toxin, thymosin α1, tissue Type plasminogen activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor α, tumor necrosis factor β, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, Mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, testosterone receptor, Aldosterone receptor, ldl receptor and cortisone.Non-natural amino acid polypeptides also can be any more with growth hormone supergene family Peptide member is homologous.

Comprising other functional groups is made to be incorporated into the non-natural amino acid constituents of polypeptide, these functional groups include for these modifications (but not limited to)：Mark；Dyestuff；Polymer；Water-soluble polymer；The derivative of polyethylene glycol；Photocrosslinking agent；Cytotoxicity Close object；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Resin；Second protein or polypeptide or polypeptide Like object；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbohydrate；Polynucleotide；DNA；RNA；Antisense Polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial；Nano-particle；Spin labeling；Fluorogen contains The part of metal；Radioactive segment；Novel functional group；With other molecule covalents or the group of noncovalent interaction；Light cage is covered Part；Can actinic radiation excitation part；Ligand；It can photoisomerization part；Biotin；Biotin analog；It is combined with weight The part of atom；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active Agent；Aminothio acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups； Electron dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；Small point Son；Inhibition ribonucleic acid, radioactive nucleotides；Neutron capture agent；The derivative of biotin；Quantum dot；Nanometer transfers element；It puts Penetrating property transfers element；It is abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, anti- Hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecular, intend epitope, receptor, reverse micelle with And any combination thereof.

In addition, non-natural amino acid polypeptides contain the part that can be changed into other functional groups, such as, only for example, Carbonyl, dicarbapentaborane or azanol.Figure 63 A illustrate that non-natural amino acid polypeptides are changed into the alpha-non-natural amino acid containing carbonyl or dicarbapentaborane The chemical transformation of polypeptide, and Figure 63 B illustrate that non-natural amino acid polypeptides are changed into the change of the non-natural amino acid polypeptides containing azanol Learn transformation.Non-natural amino acid polypeptides of the gained containing carbonyl or dicarbapentaborane and the non-natural amino acid polypeptides containing azanol can be used for Or it is incorporated to and is used to prepare, purify, characterizing and using alpha-non-natural amino acid described herein, non-natural amino acid polypeptides and through repairing In any one of method, composition, technology and strategy of the non-natural amino acid polypeptides of decorations.Chemical part is changed into other officials The chemical transformation of energy base (such as, only for example, carbonyl, dicarbapentaborane or azanol) can be used known to those skilled in the art Technology and material realize, it is all such as (e.g.) in March, A_DVANCED O_RGANIC C_HEMISTRY5th edition, (Wiley 2001)；With And Carey and Sundberg, A_DVANCED O_RGANIC C_HEMISTRY4th edition, described in A volumes and B volumes (Plenum 2000,2001), (all documents are to be fully incorporated by reference).

Therefore, only for example, the non-natural amino acid polypeptides containing any one of following amino acid can be used herein Described in method and composition further modify：

Wherein：

J is

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R " is each independently H, alkyl, substituted alkyl or protecting group or when there are more than one R " during group, two A R " optionally forms Heterocyclylalkyl；

R₁For H, amino protecting group, resin；And

R₂For OH, ester protecting group, resin；

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R₁For H, amino protecting group, resin；And

R₂For OH, ester protecting group, resin；

Or R₅For L-X, wherein X is selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymeric Object；The derivative of polyethylene glycol；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reaction Property compound；Resin；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor； Aliphatic acid；Carbohydrate；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, ring paste Essence, biomaterial；Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With it The group of his molecule covalent or noncovalent interaction；Light cage covers part；Can actinic radiation excitation part, ligand, can light Isomerized part；Biotin；Biotin analog；It is combined with the part of heavy atom；The group chemically cracked；It can photodestruciton Group；Extended side chain；The bonded sugar of carbon；Redox active agent；Aminothio acid；Toxin part；Through isotope marks Part；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electron dense group；Magnetic group；It is inserted into group；Color development Group；Energy transfer agent；Bioactivator；Detectable label；Small molecule；Inhibition ribonucleic acid, radioactive nucleotides；Neutron is captureed Obtain agent；The derivative of biotin；Quantum dot；Nanometer transfers element；Radioactivity transfers element；Abzyme, activation composite activating agent, disease Poison, adjuvant, anaphylactogen, angiostatin, antihormones, antioxidant, aptamer, guide RNA, saponin, are worn aglycone Shuttle carrier, macromolecular intend epitope, receptor, reverse micelle and any combination thereof；And L is optional, and be when it is present Connexon selected from the group being made of following group：Alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,- O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)_k- (wherein k is 1st, 2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted Asia Alkyl)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-(alkylidene is substituted alkylidene)- O-N=CR'- ,-(alkylidene is substituted alkylidene)-C (O) NR'- (alkylidene is substituted alkylidene)-,-(alkylidene or It is substituted alkylidene)-S (O)_k- (alkylidene is substituted alkylidene)-S- ,-(alkylidene is substituted alkylidene)-S-S- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, Wherein R' is each independently H, alkyl or substituted alkyl；

Wherein：

K is-NR₆R₇Or-N=CR₆R₇；

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R₁For H, amino protecting group, resin；And

R₂For OH, ester protecting group, resin；

X is selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymer；Polyethylene glycol Derivative；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Tree Fat；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbon water Compound；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial； Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other molecule covalents or The group of noncovalent interaction；Light cage covers part；Can actinic radiation excitation part；Ligand；It can photoisomerization part；It is raw Object element；Biotin analog；It is combined with the part of heavy atom；The group chemically cracked；Can photodestruciton group；Extended side Chain；The bonded sugar of carbon；Redox active agent；Aminothio acid；Toxin part；Part through isotope marks；Biophysics Probe；Phosphorescence groups；Chemiluminescent groups；Electron dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent； Bioactivator；Detectable label；Small molecule；Inhibition ribonucleic acid, radioactive nucleotides；Neutron capture agent；Biotin Derivative；Quantum dot；Nanometer transfers element；Radioactivity transfers element；Abzyme, activation composite activating agent, virus, adjuvant, glucosides are matched somebody with somebody Base, anaphylactogen, angiostatin, antihormones, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecular, Intend epitope, receptor, reverse micelle and any combination thereof；And L is optional, and be selected from by following base when it is present The connexon of the group of group's composition：Alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O- ,-O- (alkylidene or Be substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_kIt is (sub- Alkyl is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylene Base is substituted alkylidene)-,-N (R')-,-NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')- (alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S) N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N =,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl；

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

X₁For C, S or S (O)；And L is alkylidene, is substituted alkylidene, N (R') (alkylidene) or N (R') and (is substituted Asia Alkyl), wherein R' is each independently H, alkyl or substituted alkyl；Or

Wherein：

M is-C (R₃)-,

The one side of method and composition described herein be have comprising at least one it is at least one (comprising (but not It is limited to) at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, Or at least ten or ten or more) composition of the polypeptide of the alpha-non-natural amino acid of posttranslational modification.Modification after translated Alpha-non-natural amino acid may be the same or different, including (but not limited to) including 1,2,3,4,5,6,7,8,9,10,11,12,13, 14th, may be present in the polypeptide of the alpha-non-natural amino acid of 15,16,17,18,19,20 or 20 or more different translated rear modifications 1st, the different loci of 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 20 or more.The opposing party Face, the specific amino acids that composition includes (but all or less than) at least one wherein present in polypeptide are with modifying after translated The polypeptide of alpha-non-natural amino acid substitution.For the given polypeptide of the alpha-non-natural amino acid with more than one translated rear modification For, the alpha-non-natural amino acid of translated rear modification, which may be the same or different, (include, but are not limited to, polypeptide can include two or two It is a it is above it is different types of it is translated after modification alpha-non-natural amino acid or can include two it is identical it is translated after modification it is non- Natural amino acid).It is translated for the given polypeptide of the alpha-non-natural amino acid with more than two translated rear modifications The alpha-non-natural amino acid modified afterwards can it is identical, different or for multiple identical types it is translated after modification alpha-non-natural amino acids with The combination of the alpha-non-natural amino acid of at least one different translated rear modification.

A. it is used for the method for posttranslational modification non-natural amino acid polypeptides：Alpha-non-natural amino acid containing carbonyl and the examination containing azanol The reaction of agent

The side chain of naturally occurring amino acid lacks height electrophilicity site.Therefore, it is incorporated to containing electrophilic reagent The alpha-non-natural amino acid (including the carbonyl or the amino acid of dicarbapentaborane (only for example) containing such as ketone or aldehyde) of side chain causes By the site-specific derivatization of carbonyl or the nucleophilic attack of dicarbapentaborane, this side chain is possibly realized.Attack nucleopilic reagent wherein In the case of for azanol, protein derived from oxime can be generated.The method for derivatization and/or further modified, which can be used in, to be derived The polypeptide purified before changing step or after derivatization step carries out.In addition, the side for derivatization and/or further modified Method can be used in synthetic polymer, polysaccharide or the polynucleotide purified before or after these modifications and carry out.In addition, derivatization step Can be acid to occurring under the conditions of alkalescence in appropriateness, the condition including (for example) under the pH value between about 2-8 or between Under pH value between about 4-8.

According to the reaction of the polypeptide containing carbonyl or dicarbapentaborane and the molecule substituted through azanol come the method for derivatization polypeptide With clear advantage.First, azanol in the pH value range of 2-8 (and in other embodiments in the pH value range of 4-8) The condensation with the compound containing carbonyl or dicarbapentaborane is undergone to generate oxime adduct.Under these conditions, naturally occurring amino The side chain of acid is anergy.Second, this selective chemical makes it possible site-specific derivatization recombinant protein： Nowadays derivative protein can be prepared as definite homologues.3rd, realize azanol described herein with it is described herein The required temperate condition of reaction of the polypeptide containing carbonyl or dicarbapentaborane usually will not irreversibly destroy the three-level of polypeptide Structure (certainly, unless the purpose wherein reacted is to destroy this tertiary structure).Finally, although hydroxyamine groups amino seems by big Enterobacteria is metabolized, but the condensation of azanol and the molecule containing carbonyl or dicarbapentaborane generates the oxime adduction stablized under biotic factor Object.

Only for example, following alpha-non-natural amino acid is to can be used for further modifying the non-day containing carbonyl or dicarbapentaborane The reagent described herein containing azanol of right amino acid polypeptide has the class of the amino acid containing carbonyl or dicarbapentaborane of reactivity Type：

Wherein：

J is

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R₁For H, amino protecting group, resin；And

R₂For OH, ester protecting group, resin；

Or-J-R groups are collectively formed including at least one carbonyl (comprising dicarbapentaborane), through carbonyl is protected (to include through protection Dicarbapentaborane) or masked carbonyl (include masked dicarbapentaborane) monocyclic or bicyclic cycloalkyl or Heterocyclylalkyl.

In certain embodiments, formula (I) compound has reactivity under appropriate acid condition with azanol in aqueous solution. In certain embodiments, the acid condition is pH 2 to 8.

Only for example, it is object defined above, formula (I) compound includes the compound having following structure：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

X₁For C, S or S (O)；And L is a key, alkylidene, is substituted alkylidene, N (R') (alkylidene) or N (R') (through taking For alkylidene), wherein R' is each independently H, alkyl or substituted alkyl.

Only in further example, it is object defined above, formula (I) compound includes the chemical combination of the structure with formula (XXXX) Object：

Wherein：

M is-C (R₃)-,

Actually there is no restriction for the type of polypeptide including these alpha-non-natural amino acids for containing carbonyl or dicarbapentaborane, as long as Alpha-non-natural amino acid containing carbonyl or dicarbapentaborane be located on polypeptide so as to azanol reagent can with carbonyl or dicarbapentaborane reaction and not The modified alpha-non-natural amino acid of gained for the tertiary structure for destroying polypeptide is generated (certainly, if unless this destruction is reaction Purpose).

Only for example, the reagent containing azanol is to the non-natural ammonia described herein containing carbonyl or dicarbapentaborane below Base acid has reactivity and available for the examination containing azanol of further non-natural amino acid polypeptides of the modification containing carbonyl or dicarbapentaborane The type of agent：

Wherein：

X is each independently H, alkyl, substituted alkyl, alkenyl, is substituted alkenyl, alkynyl, is substituted alkynyl, alcoxyl Base, be substituted alkoxy, alkyl alkoxy, substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, Aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,- (alkylidene is substituted alkylidene)-ON (R ")₂,-(alkylidene is substituted alkylidene)-C (O) SR " ,-(alkylidene or through taking For alkylidene)-S-S- (aryl or substituted aryl) ,-C (O) R " ,-C (O)₂R " or-C (O) N (R ")₂, the respective independences of wherein R " Ground is hydrogen, alkyl, substituted alkyl, alkenyl, is substituted alkenyl, alkoxy, is substituted alkoxy, is aryl, substituted aryl, miscellaneous Aryl, alkaryl are substituted alkaryl, aralkyl or are substituted aralkyl；

Or X is each independently selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymer； The derivative of polyethylene glycol；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactivity Compound；Resin；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Fat Fat acid；Carbohydrate；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, Biomaterial；Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other points The group that son covalently or non-covalently interacts；Light cage covers part；Can actinic radiation excitation part；Ligand；It can light isomery Change part；Biotin；Biotin analog；It is combined with the part of heavy atom；The group chemically cracked；Can photodestruciton base Group；Extended side chain；The bonded sugar of carbon；Redox active agent；Aminothio acid；Toxin part；Portion through isotope marks Point；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electron dense group；Magnetic group；It is inserted into group；Chromophore； Energy transfer agent；Bioactivator；Detectable label；Small molecule；Inhibition ribonucleic acid, radioactive nucleotides；Neutron absorption Agent；The derivative of biotin；Quantum dot；Nanometer transfers element；Radioactivity transfers element；Abzyme, activation composite activating agent, virus, Adjuvant, aglycone, anaphylactogen, angiostatin, antihormones, antioxidant, aptamer, guide RNA, saponin, shuttle Carrier, macromolecular intend epitope, receptor, reverse micelle and any combination thereof；

L is each independently selected from the group being made of following group：Alkylidene is substituted alkylidene, alkenylene, through taking For alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or through taking For alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-, (alkylidene is substituted-NR'- Alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-(alkylidene is substituted alkylidene) NR'C (O) O- (alkylidene is substituted alkylidene)-,-O-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-N (R') C (O) O- (alkylidene is substituted alkylidene)-,-S (O)_kN(R')-、-N(R')C(O)N (R')-,-N (R') C (O) N (R')-(alkylidene is substituted alkylidene)-,-N (R') C (S) N (R')-,-N (R') S (O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and- C(R')₂-N(R')-N(R')-；

L₁To be optional, and it is-C (R') when it is present_p- NR'-C (O) O- (alkylidene is substituted alkylidene)-, wherein p For 0,1 or 2；R' is each independently H, alkyl or substituted alkyl；

W is-N (R₈)₂, wherein R₈It is each independently H or amino protecting group；And n is 1 to 3；

Its restrictive condition is L-L₁- W provide jointly it is at least one can with alpha-non-natural amino acid or " through modification or without repairing Decorations " the azanol base of carbonyl (including dicarbapentaborane) reaction on non-natural amino acid polypeptides.

In some embodiments of formula (XIX) compound, X be include alkyl, substituted alkyl, alkenyl, be substituted alkenyl, Alkynyl, be substituted alkynyl, alkoxy, be substituted alkoxy, alkyl alkoxy, substituted alkyl alkoxy, polyoxyalkylene, It is substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, is substituted alkaryl, virtue Alkyl or the polymer for being substituted aralkyl.In some embodiments of formula (XIX) compound, X is to include polyoxyalkylene Or it is substituted the polymer of polyoxyalkylene.In some embodiments of formula (XIX) compound, X be include-[(alkylidene or It is substituted alkylidene)-O- (hydrogen, alkyl or substituted alkyl)]_xThe polymer of (wherein x is 20-10,000).Change in formula (XIX) In some embodiments for closing object, X is the m-PEG with the molecular weight in the range of 2KDa to 40KDa.In formula (XIX) chemical combination In some embodiments of object, X is selected from by peptide, protein, enzyme, antibody, drug, dyestuff, lipid, nucleosides, oligonucleotides, thin The bioactivator for the group that born of the same parents, virus, liposome, particle and micella form.In some embodiments of formula (XIX) compound In, X be selected from by antibiotic, fungicide, antivirotic, antiphlogistic, antitumor agent, cardiovascular agents, antianxiety agent, hormone, The drug of the group of growth factor and steroid agent composition.In some embodiments of formula (XIX) compound, X is selected from by peppery The enzyme for the group that root peroxidase, alkaline phosphatase, beta galactosidase and glucose oxidase form.At formula (XIX) In some embodiments of compound, X is selected from by fluorescence part, phosphorescent moieties, chemiluminescent moiety, chelating moiety, electronics cause The group of compact part point, magnetic part, insertion portion, radioactive segment, chromophoric moiety and energy transfer part composition can Detection mark.In some embodiments of formula (XIX) compound, L is selected from by-N (R') CO-, (alkylidene is substituted alkylene Base)-,-CON (R')-(alkylidene is substituted alkylidene)-,-N (R') C (O) N (R')-(alkylidene is substituted alkylene Base)-,-O-CON (R')-(alkylidene is substituted alkylidene)-,-O- (alkylidene is substituted alkylidene)-,-C (O) N (R')-and-N (R') C (O) O- (alkylidene is substituted alkylidene)-composition group.

In some embodiments of formula (XIX) compound, for the compound of the structure with formula (XX)：

In some embodiments of formula (XX) compound, to be selected from the compound for the group being made of following object：

Wherein in other embodiments, these m-PEG or PEG group have the molecule in the range of 5kDa to 30kDa Amount.

In some embodiments of formula (XIX) compound, for the compound of the structure with formula (XXI)：

In some embodiments of formula (XXI) compound, to be selected from the compound for the group being made of following object：

In some embodiments of formula (XIX) compound, for the compound of the structure with formula (XXII)：

In some embodiments of formula (XXII) compound, L is-(alkylidene is substituted alkylidene)-N (R') C (O) O- (alkylidene is substituted alkylidene)-.In some embodiments of formula (XXII) compound, for the structure with formula (XXIII) Compound：

Wherein in the other embodiment of formula (XXII) compound, these m-PEG groups have the model in 5kDa to 30kDa Enclose interior molecular weight.

In some embodiments of formula (XIX) compound, for the compound of the structure with formula (XXIV)：

In some embodiments of formula (XXIV) compound, L is-(alkylidene is substituted alkylidene)-N (R') C (O) O- (alkylidene is substituted alkylidene)-or-N (R') C (O) O- (alkylidene is substituted alkylidene)-.In formula (XXIV) compound Some embodiments in, for the compound of the structure with formula (XXV)：

Wherein in the other embodiment of formula (XXIV) compound, these m-PEG groups have the model in 5kDa to 30kDa Enclose interior molecular weight.

In some embodiments of formula (XIX) compound, for the compound of the structure with formula (XXVI)：

Wherein R₁₀It is each independently H or amino protecting group.

In some embodiments of formula (XXVI) compound, polyoxyalkylene PEG.In its of formula (XXVI) compound In his embodiment, PEG group has the molecular weight in the range of 5kDa to 30kDa.In another reality of formula (XXVI) compound It applies in example as the compound corresponding to following formula：

For azanol to be coupled to three illustrative embodiments of the method on the alpha-non-natural amino acid containing carbonyl on polypeptide In shown in Figure 7.In these illustrative embodiments, it is more that reagent derived from azanol is added to the alpha-non-natural amino acid containing carbonyl In the buffer solution (pH 2-8) of peptide.Reaction carries out at most day of multiple hours at ambient temperature.To accelerate to engage, addition is such as Those additives of presentation in Fig. 8；These compounds are also referred herein as accelerating agent.In certain embodiments, accelerating agent Or additive being capable of base catalysis.Non-natural amino acid polypeptides containing oxime obtained by being purified as HPLC, FPLC or size exclusion chromatography.

In one embodiment, multiple connexon chemistry can be with the non-natural amino acid polypeptides position through carbonyl or dicarbapentaborane substitution Point specifically reacts.In one embodiment, connection submethod described herein is utilized at least one connexon end Connexon (mono-functional, difunctionality or multi-functional) containing azanol functional group.Connexon derived from azanol through ketone with taking The condensation of the protein in generation, which generates, stablizes oxime key.Bifunctional and/or multifunctional connexon is (for example, with one or more The azanol of other bonded chemistry) allow to connect different molecular (for example, other protein, polymer or small molecule) locus specificity It is connected on non-natural amino acid polypeptides, and simple function connexon (substituting on all ends through azanol) promotes alpha-non-natural amino acid The locus specificity dimerization or oligomerization of polypeptide.By the way that this is connected substrategy and in vivo translation technology described herein It is combined so that the three-dimensional structure that the protein through chemical refining is described in detail is possibly realized.

It is to include Formulas I-XVIII, XXX-XXXIV (A and B) or XXXX-XXXXIII for derivatization in certain embodiments Amino acid (it includes any minors in Formulas I-XVIII, XXX-XXXIV (A and B) or the scope of XXXX-XXXXIII Or specific compound) polypeptide method, wherein the described method includes make to include at least one Formulas I-XVIII, XXX-XXXIV (A And B) or the polypeptide of amino acid of XXXX-XXXXIII contacted with the reagent of formula (XIX).In certain embodiments, polypeptide with formula (XIX) it is purified before or after reagent contact.It is to include at least one containing corresponding to formula (XI) in other embodiments The gained of oxime amino acid derives polypeptide, and in other embodiments, these derive polypeptide under appropriate acid condition in aqueous solution Middle stable at least one moon.In other embodiments, these derive polypeptides in appropriate stable under acidic conditions at least 2 weeks.At other In embodiment, these derive polypeptides in appropriate stable under acidic conditions at least 5 days.In other embodiments, the condition is pH 2 to 8.In certain embodiments, the tertiary structure of derivative polypeptide is kept.In other embodiments, these derivatizations of polypeptide into One step is connected to including that will derive polypeptide on another polypeptide.In other embodiments, these derivative polypeptides with selected from by with Under each object composition group treatment albumen matter it is homologous：It is α -1 antitrypsins, angiostatin, anti-hemolytic factor, anti- Body, apolipoprotein, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, C-X-C chemotactic factor (CF)s, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit Ligand, cell factor, CC chemotactic factor (CF)s, monocyte chemoattractant protein-1, monocyte chemoattractant protein -2, monocyte chemotactic Protein-3, -1 α of monocyte inflammatory protein, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligands, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cell factor, epithelium neutrophilic granulocyte activation peptide -78, MIP- 16th, MCP-1, epidermal growth factor (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin(EPO) (EPO), exfoliation poison Element, factors IX, factor Ⅴ II, Factor IX, factor X, fibroblast growth factor (FGF), fibrinogen, fiber connection Albumen, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, promoting sexual gland hormone, growth factor, growth because Sub- receptor, grf, Hedgelog protein, hemochrome, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), the mankind Seralbumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 Receptors, insdri, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-α, IFN-β, IFN-γ, interleukins (IL), IL-1, IL-2, IL-3, IL- 4th, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), newborn iron transfer egg In vain, LIF ELISA, luciferase, neural element, neutrophil inhibitory factor (nif) (NIF), oncostatin M, osteogenic protein, cancer Gene outcome, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, multi-effector, albumin A, Protein G, pth, Pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxain, feritin, SCF, atom synthetic proteins, solubility Complement receptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, growth swash Plain inhibin, growth hormone, streptokinase, super antigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic-shock syndrome toxin, thymosin α1, tissue plasminogen Former activation factor, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor α, tumor necrosis factor β, tumour are bad Necrosis factor receptor (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, Raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, testosterone receptor, aldosterone by Body, ldl receptor and cortisone.

In certain embodiments to generate the method for polypeptide dimer, wherein the described method includes：

(i) the first polypeptide and (ii) for including the amino acid of formula (I) with the reagent derivatization of formula (XXVI) make step (i) the second protein contacts of gained derived protein and the amino acid including formula (I), so formed include the first polypeptide and The dimer of second polypeptide.In other embodiments to generate the method for polypeptide dimer, wherein the first polypeptide and the second polypeptide Amino acid including corresponding to formula (II).In certain embodiments, polypeptide be before being contacted with the reagent of formula (XXVI) or it By purifying.In other embodiments, the gained derived protein of step (i) includes at least one corresponding to formula (XXVIII) Amino acid containing oxime：

B. it is used for the method for posttranslational modification non-natural amino acid polypeptides：Make alpha-non-natural amino acid containing oxime with containing carbonyl Reagent reacting

Had based on protein containing oxime and the protein derived method of the exchange reaction of the molecule substituted through carbonyl or dicarbapentaborane There is clear advantage.First, research instruction the oxime adduct based on amino acid by with the compound than being used to generate original oxime The balance experience oxime of the more reactive compound containing carbonyl or dicarbapentaborane exchanges.This exchange reaction is happened at the pH of 4-8 In the range of value：Under the described conditions, the side chain of naturally occurring amino acid does not have reactivity.Therefore, be used to prepare suitable for containing oxime The conventional method of the molecule substituted through carbonyl or dicarbapentaborane of albumen qualitative response can provide to obtain derived from a variety of locus specificities The approach of protein.Herein in vivo in the case of translation technology, those points commonly used in derivatization albumen matter are used to prepare The form substituted through carbonyl or dicarbapentaborane of son (comprising (only for example) hydrophilic polymer, such as polyethylene glycol) it is general Method is valuable and can provide to obtain the approach of non-natural amino acid polypeptides derived from a variety of locus specificities.Second, this Selective chemical makes it possible the site-specific derivatization of recombinant protein：Nowadays derived protein can be definite homologous Product.3rd, realize that the required temperate condition of exchange reaction described herein usually will not irreversibly destroy polypeptide Tertiary structure (certainly, unless the purpose wherein reacted is this tertiary structure of destruction).Finally, exchange reaction is generated in biotic factor The new oxime adduct of lower stabilization.

Only for example, following alpha-non-natural amino acid is the sheet with containing the oxime non-natural amino acid polypeptides new available for generation The reagent containing carbonyl or dicarbapentaborane of described in the text has the type of the amino acid containing oxime of reactivity：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

Or R₅For L-X, wherein X is selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymeric Object；The derivative of polyethylene glycol；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reaction Property compound；Resin；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor； Aliphatic acid；Carbohydrate；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, ring paste Essence, biomaterial；Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With it The group of his molecule covalent or noncovalent interaction；Light cage covers part；Can actinic radiation excitation part；Ligand；It can light Isomerized part；Biotin；Biotin analog；It is combined with the part of heavy atom；The group chemically cracked；It can photodestruciton Group；Extended side chain；The bonded sugar of carbon；Redox active agent；Aminothio acid；Toxin part；Through isotope marks Part；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electron dense group；Magnetic group；It is inserted into group；Color development Group；Energy transfer agent；Bioactivator；Detectable label；Small molecule；Inhibition ribonucleic acid, radioactive nucleotides；Neutron is captureed Obtain agent；The derivative of biotin；Quantum dot；Nanometer transfers element；Radioactivity transfers element；Abzyme, activation composite activating agent, disease Poison, adjuvant, anaphylactogen, angiostatin, antihormones, antioxidant, aptamer, guide RNA, saponin, are worn aglycone Shuttle carrier, macromolecular intend epitope, receptor, reverse micelle and any combination thereof；And L is optional, and be when it is present Connexon selected from the group being made of following group：Alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,- O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)_k- (wherein k is 1st, 2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted Asia Alkyl)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-(alkylidene is substituted alkylidene)- O-N=CR'- ,-(alkylidene is substituted alkylidene)-C (O) NR'- (alkylidene is substituted alkylidene)-,-(alkylidene or It is substituted alkylidene)-S (O)_k- (alkylidene is substituted alkylidene)-S- ,-(alkylidene is substituted alkylidene)-S-S- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, Wherein R' is each independently H, alkyl or substituted alkyl.

Only in further example, following alpha-non-natural amino acid is also and containing the oxime alpha-non-natural amino acid new available for generation The reagent described herein containing carbonyl or dicarbapentaborane of polypeptide has the type of the amino acid containing oxime of reactivity：

Wherein：

X is selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymer；Polyethylene glycol Derivative；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Tree Fat；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbon water Compound；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial； Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other molecule covalents or The group of noncovalent interaction；Light cage covers part；Can actinic radiation excitation part；Ligand；It can photoisomerization part；It is raw Object element；Biotin analog；It is combined with the part of heavy atom；The group chemically cracked；Can photodestruciton group；Extended side Chain；The bonded sugar of carbon；Redox active agent；Aminothio acid；Toxin part；Part through isotope marks；Biophysics Probe；Phosphorescence groups；Chemiluminescent groups；Electron dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent； Bioactivator；Detectable label；Small molecule；Inhibition ribonucleic acid, radioactive nucleotides；Neutron capture agent；Biotin Derivative；Quantum dot；Nanometer transfers element；Radioactivity transfers element；Abzyme, activation composite activating agent, virus, adjuvant, glucosides are matched somebody with somebody Base, anaphylactogen, angiostatin, antihormones, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecular, Intend epitope, receptor, reverse micelle and any combination thereof；And L is optional, and be selected from by following base when it is present The connexon of the group of group's composition：Alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O- ,-O- (alkylidene or Be substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_kIt is (sub- Alkyl is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylene Base is substituted alkylidene)-,-N (R')-,-NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')- (alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S) N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N =,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl.

Only for example, the reagent below containing carbonyl or dicarbapentaborane is to have with alpha-non-natural amino acid containing oxime described herein Have reactivity and available for realize exchange reaction with formed new oxime key and therefore modify the non-natural amino acid polypeptides containing oxime containing carbonyl The type of the reagent of base or dicarbapentaborane：

Wherein：

W is-C (=O) R₉, wherein R₉For H or OR'；And n is 1 to 3；

Or wherein L-L₁- W is collectively formed including at least one carbonyl (comprising dicarbapentaborane), through carbonyl is protected (to include through protecting Protect dicarbapentaborane) or masked carbonyl (include masked dicarbapentaborane) monocyclic or bicyclic cycloalkyl or Heterocyclylalkyl；

Its restrictive condition is L-L₁- W provides at least one can undergo with alpha-non-natural amino acid or " through modification or not jointly It is modified " carbonyl (including dicarbapentaborane) of oxime exchange reaction occurs for the oximido on non-natural amino acid polypeptides.

It is used to implement two of the method for the oxime exchange reaction between the amino acid containing oxime on polypeptide and the reagent containing carbonyl Illustrative embodiment is in shown in Figure 9.In these illustrative embodiments, the reagent containing carbonyl is added to the ammonia of non-natural containing oxime In the buffer solution (pH 2-8) of base acid polypeptide.Reaction carries out at most day of multiple hours at ambient temperature.Pass through HPLC, FPLC Or size exclusion chromatography come purify through modify non-natural amino acid polypeptides containing oxime.

In one embodiment, multiple connexon chemistry can locus specificity with the non-natural amino acid polypeptides that substitute through oxime Reaction.In one embodiment, connection submethod described herein utilize at least one connexon end containing carbonyl or The connexon (simple function, difunctionality or multifunctional) of dicarbapentaborane functional group.Connexon through oxime with taking derived from carbonyl or dicarbapentaborane The condensation of the non-natural amino acid polypeptides in generation generates new stabilization oxime key.Bifunctional and/or multifunctional connexon is (for example, have The carbonyl or dicarbapentaborane of one or more other bonded chemistry) allow different molecular (for example, other protein, polymerization Object or small molecule) locus specificity is connected on non-natural amino acid polypeptides, and simple function connexon is (through carbonyl on all ends Base or dicarbapentaborane substitution) promote non-natural amino acid polypeptides locus specificity dimerization or oligomerization.By by this connexon It is tactful combined in vivo translation technology described herein so that the three-dimensional knot of the protein through chemical refining is described in detail It is configured to possibility.

C. it is used for the method for posttranslational modification non-natural amino acid polypeptides：Make the alpha-non-natural amino acid containing azanol with containing carbonyl Reagent reacting

Above-mentioned posttranslational modification technology and composition can also be used for the reagent reacting containing carbonyl or dicarbapentaborane generating warp The alpha-non-natural amino acid containing azanol of the non-natural amino acid polypeptides containing oxime of modification.

Had based on the protein containing azanol and the protein derived method of the reaction of the molecule substituted through carbonyl or dicarbapentaborane There is clear advantage.First, azanol undergone in the pH value range of 4-8 with the condensation of the compound containing carbonyl or dicarbapentaborane with Generate oxime adduct.Under the described conditions, the side chain of naturally occurring amino acid does not have reactivity.Second, this selective chemical makes The site-specific derivatization for obtaining recombinant protein is possibly realized：Nowadays derived protein can be prepared as determining homologues.The Three, realize that the reaction of the reagent described herein containing carbonyl or dicarbapentaborane and the polypeptide described herein containing azanol is required Temperate condition usually will not irreversibly destroy polypeptide tertiary structure (certainly, unless the purpose wherein reacted for destroy this three Level structure).Finally, although azanol base amino acid seems by Metabolism of E. coli, the reagent containing carbonyl or dicarbapentaborane is with containing hydroxyl The condensation of the amino acid of amine generates the oxime adduct stablized under biotic factor.

Only for example, following alpha-non-natural amino acid be with it is more available for further alpha-non-natural amino acid of the modification containing azanol The reagent described herein containing carbonyl or dicarbapentaborane of peptide has the type of the amino acid containing azanol of reactivity：

Wherein：

K is-NR₆R₇Or-N=CR₆R₇；

In some embodiments of formula (XIV) compound, K NH₂。

Including these, actually there is no restriction for the type of the polypeptide of the alpha-non-natural amino acid containing azanol, as long as containing the non-of azanol Natural amino acid is located on polypeptide destroys polypeptide so that the reagent containing carbonyl or dicarbapentaborane can be reacted with azanol base and do not generated The modified alpha-non-natural amino acid of gained (certainly, if unless this destruction is the purpose of reaction) of tertiary structure.

Only for example, the reagent below containing carbonyl or dicarbapentaborane is and the non-natural amino described herein containing azanol Acid has reactivity and available for the reagent containing carbonyl or dicarbapentaborane of further non-natural amino acid polypeptides of the modification containing azanol Type：

Wherein：

W is-J-R, and wherein J is

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；R " is each independently H, alkyl, is substituted Alkyl or protecting group or " during group, two R " optionally form Heterocyclylalkyl when there are more than one R；And n is 1 to 3；

Its restrictive condition is L-L₁- W provide jointly it is at least one can with alpha-non-natural amino acid or " through modification or without repairing Decorations " carbonyl (including dicarbapentaborane) of azanol base reaction on non-natural amino acid polypeptides.

It is the compound of the structure with formula (XXI) in some embodiments of formula (XIX) compound：

For the reagent for containing carbonyl to be coupled to the illustrative of the method on the alpha-non-natural amino acid containing azanol on polypeptide Embodiment is in shown in Figure 10.In this illustrative embodiment, the reagent through carbonyl derivatization is added to the non-day containing azanol In the buffer solution (pH 2-8) of right amino acid polypeptide.Reaction carries out at most day of multiple hours at ambient temperature.To accelerate to connect It closes, adds those additives of presentation in such as Fig. 8.It is non-containing oxime obtained by being purified as HPLC, FPLC or size exclusion chromatography Natural amino acid polypeptide.

In one embodiment, multiple connexon chemistry can locus specificity it is more with the alpha-non-natural amino acid that substitutes through azanol Peptide reacts.In one embodiment, connection submethod described herein is utilized contains carbonyl at least one connexon end Or the connexon (simple function, difunctionality or multifunctional) of dicarbapentaborane functional group.Through connexon derived from carbonyl or dicarbapentaborane and warp The condensation of the protein of azanol substitution, which generates, stablizes oxime key.Bifunctional and/or multifunctional connexon (for example, tool there are one or one The carbonyl or dicarbapentaborane of other a above bonded chemistry) allow different molecular (for example, other protein, polymer or small point Son) locus specificity is connected on non-natural amino acid polypeptides, and simple function connexon is (through carbonyl or two carbonyls on all ends Base substitute) promote non-natural amino acid polypeptides locus specificity dimerization or oligomerization.By by this connection substrategy and originally Described in the text in vivo translation technology combination so that be described in detail the protein through chemical refining three-dimensional structure become can Energy.

It is that (it includes in Formula XIV-XVI's for amino acid of the derivatization including Formula XIV-XVI in certain embodiments Any minor or specific compound in scope) polypeptide method, wherein the described method includes make to include at least one formula The polypeptide of the amino acid of XIV-XVI is contacted with the reagent of formula (XIX).In certain embodiments, polypeptide is in the examination with formula (XIX) It is purified before or after agent contact.It is to include at least one amino acid containing oxime corresponding to formula (XXIX) in other embodiments Gained derive polypeptide,

Wherein：

A is optional, and when it is present for low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted it is low Grade alkenylene, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, Sub- aralkyl is substituted sub- aralkyl；

B is optional, and is the connexon selected from the group being made of following group when it is present：Low-grade alkylidene, Be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,- S- ,-S- (alkylidene is substituted alkylidene)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylene Base)-,-C (O)-,-NS (O)₂-、-OS(O)₂- ,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylene Base is substituted alkylidene)-,-N (R')-,-NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')- (alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S) N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N =,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl；

L is the connexon independently selected from the group being made of following group：Alkylidene is substituted alkylidene, sub- alkene Base, be substituted alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-, (alkylidene is substituted alkylene by-S- ,-S- Base)-,-S (O)_k- (wherein k is 1,2 or 3) ,-S (O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene Or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidene or Be substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-(alkylidene is substituted Alkylidene) NR'C (O) O- (alkylidene is substituted alkylidene)-,-O-CON (R')-(alkylidene is substituted alkylidene)-,- CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-N (R') C (O) O- (alkylidene is substituted alkylidene)-,-S (O)_kN(R')-、-N(R')C(O)N (R')-,-N (R') C (O) N (R')-(alkylidene is substituted alkylidene)-,-N (R') C (S) N (R')-,-N (R') S (O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and- C(R')₂- N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl；

R₂To be optional, and it is OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide when it is present；

R₃With R₄It is each independently H, halogen, low alkyl group or is substituted low alkyl group；And

X independently is detectable label, bioactivator or polymer.

In other embodiments, these derive polypeptide and stablize at least one moon in aqueous solution under appropriate acid condition. In other embodiment, these derive polypeptides in appropriate stable under acidic conditions at least 2 weeks.In other embodiments, these derive Polypeptide was in appropriate stable under acidic conditions at least 5 days.In other embodiments, the condition is pH 2 to 8.In some embodiments In, the tertiary structure of the derivative polypeptide of holding.In other embodiments, these derivatizations of polypeptide further comprise derivative polypeptide It is connected on another polypeptide.In other embodiments, these derivative polypeptides are with being selected from the group being made of following object Treatment albumen matter is homologous：α -1 antitrypsins, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoenzyme egg In vain, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, C-X-C chemotactic factor (CF)s, T39765, NAP-2, ENA-78, gro-a, Gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligands, cell factor, CC Chemotactic factor (CF), monocyte chemoattractant protein-1, monocyte chemoattractant protein -2, monocyte chemoattractant protein-3, monocyte are scorching - 1 α of property albumen, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligands, collagen, colony stimulating factor (CSF), complement factor 5a, complement Inhibitor, complement receptor 1, cell factor, epithelium neutrophilic granulocyte activation peptide -78, MIP-16, MCP-1, epidermal growth factor (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin(EPO) (EPO), exfoliative toxin,or exfoliatin, factors IX, factor Ⅴ II, the factor VIII, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helix bundle albumen, G- CSF, glp-1, GM-CSF, glucocerebrosidase, promoting sexual gland hormone, growth factor, growth factor receptors, grf, Hedgelog protein, Hemochrome, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptors, LFA-1, LFA-1 Receptors, insdri, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-α, IFN-β, IFN-γ, interleukins (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactotransferrin, leukaemia inhibit because Son, luciferase, neural element, neutrophil inhibitory factor (nif) (NIF), oncostatin M, osteogenic protein, oncoprotein, The outer poison of paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, multi-effector, albumin A, Protein G, pth, pyrogenicity Plain A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxain, feritin, SCF, atom synthetic proteins, Soluble complement receptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, Growth hormone, streptokinase, super antigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroids swash It is plain receptor, superoxide dismutase, toxic-shock syndrome toxin, thymosin α1, histiotype plasminogen activation factor, swollen Knurl growth factor (TGF), tumor necrosis factor, tumor necrosis factor α, tumor necrosis factor β, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, P53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, testosterone receptor, aldosterone receptor, LDL by Body and cortisone.

D. the example of functional group is added：The macromolecule polyalcohol being coupled on non-natural amino acid polypeptides

Composition described herein, method, technology and strategy can be used to realize to non-natural ammonia described herein The various modifications of base acid polypeptide.These modifications, which include, makes other functional groups be incorporated into the non-natural amino acid constituents of polypeptide, this A little functional groups including (but not limited to)：Mark；Dyestuff；Polymer；Water-soluble polymer；The derivative of polyethylene glycol；Photo-crosslinking Agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Resin；Second protein or Polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbohydrate；Polynucleotide； DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial；Nano-particle；Spin mark Note；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other molecule covalents or noncovalent interaction Group；Light cage covers part；Can actinic radiation excitation part；Ligand；It can photoisomerization part；Biotin；Biotin is similar Object；It is combined with the part of heavy atom；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon； Redox active agent；Aminothio acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups； Chemiluminescent groups；Electron dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；It can examine Mark is remembered；Small molecule；Inhibition ribonucleic acid, radioactive nucleotides；Neutron capture agent；The derivative of biotin；Quantum dot；It receives Rice transfers element；Radioactivity transfers element；Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, blood vessel life Into inhibin, antihormones, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecular, intend epitope, by Body, reverse micelle and any combination thereof.As the illustrative, unrestricted of composition described herein, method, technology and strategy Property example, be described below to concentrate on macromolecule polyalcohol be added on non-natural amino acid polypeptides, while should be appreciated that in addition Composition, method, technology and the strategy of description can also be used for (be subject to if necessary suitably modified, and those skilled in the art Disclosure herein can be used to carry out) other functional groups are added, it includes (but not limited to) those functions listed above Base.

A variety of macromolecule polyalcohols and other molecules can be coupled on non-natural amino acid polypeptides described herein with Adjust the biological property of non-natural amino acid polypeptides (or corresponding natural amino acid polypeptide) and/or to non-natural amino acid polypeptides (or corresponding natural amino acid polypeptide) provides new biological property.These macromolecule polyalcohols can by alpha-non-natural amino acid or Any sense substituent of alpha-non-natural amino acid is coupled to added to any substituent group on alpha-non-natural amino acid or functional group On non-natural amino acid polypeptides.

Water-soluble polymer can be coupled to the polypeptide described in being incorporated herein (natural or synthetic), polynucleotide, polysaccharide or On alpha-non-natural amino acid in synthetic polymer.Water-soluble polymer can pass through the alpha-non-natural amino acid being incorporated in polypeptide or non-day Any functional group of right amino acid or substituent group are coupled added to any functional group on alpha-non-natural amino acid or substituent group. In some cases, non-natural amino acid polypeptides described herein are coupled to water-soluble polymer including one or more On alpha-non-natural amino acid and one or more bonded naturally occurring amino acid on water-soluble polymer.Hydrophily Covalent linkage of the polymer with bioactive molecule represents to increase bioactive molecule (comprising protein, peptide and especially hydrophobic Molecule) water solubility (such as in physiological environment), biological usability, increase serum half-life, increase treatment half-life period, adjust Immunogenicity adjusts bioactivity or extends a kind of method of circulation time.Other key characters of these hydrophilic polymers Include biocompatibility, nontoxicity and non-immunogenicity.For the therapeutical uses of final product preparation, preferred polymers should be Pharmaceutically acceptable.

The example of hydrophilic polymer including (but not limited to)：Poly alkyl ether and its alkoxy end-capped analog (for example, Polyoxyethylene glycol, polyoxyethylene glycol/polyoxypropylene glycol and its methoxy or ethoxy capped analogs, especially polyoxy second two Alcohol, the latter are also referred to as polyethylene glycol or PEG)；Polyvinylpyrrolidone；Polyvinylalkylethers；Polyoxazoline, Ju Wan Ji Evil Oxazoline and poly- Qiang Wan oxazolins；Polyacrylamide, poly- alkyl acrylamide and poly- alkylol acrylamides are (for example, poly- Hydroxypropylmethacrylamide and its derivative)；Poly- hydroxyalkyl acrylates；Poly sialic acid with and the like；It is hydrophilic Property peptide sequence；Polysaccharide and its derivative, it includes glucan and glucan derivative, for example, Sensor Chip CM 5, sulfuric acid Portugal Glycan, aminoglucan；Cellulose and its derivative, for example, carboxymethyl cellulose, hydroxy alkyl cellulose；Chitin and Its derivative, for example, chitosan, succinyl group chitosan, carboxymethyl chitin, carboxymethyl chitosan；Hyaluronic acid and its Derivative；Starch；Alginates；Chondroitin sulfate；Albumin；Amylopectin and carboxymethyl amylopectin；Polyaminoacid and Its derivative, for example, polyglutamic acid, polylysine, poly-aspartate, poly-asparagine；Copolymer-maleic anhydride, such as：Benzene Ethylene maleic acid anhydride copolymer, divinyl ethylether copolymer-maleic anhydride；Polyvinyl alcohol；Its copolymer；Its ternary polymerization Object；Its mixture；And the derivative of aforementioned substances.Water-soluble polymer can be any structure type, it includes (but it is unlimited In) linear, forked or branch.In some embodiments, the water-soluble polymer main chain with 2 to about 300 ends is especially suitable With.Multifunctional polymer derivant including (but not limited to) tool there are two end linear polymer, wherein each end with can Identical or different functional group's bond.In some embodiments, aqueous-based polymers include polyalkylene glycol moiety.The molecule of polymer Amount can be in broad range, and it includes scope of the (but not limited to) between about 100Da and about 100,000Da or higher.It is poly- Close object molecular weight can between about 100Da and about 100,000Da, it includes (but not limited to) 100,000Da, 95,000Da, 90,000Da、85,000Da、80,000Da、75,000Da、70,000Da、65,000Da、60,000Da、55,000Da、50, 000Da、45,000Da、40,000Da、35,000Da、30,000Da、25,000Da、20,000Da、15,000Da、10, 000Da、9,000Da、8,000Da、7,000Da、6,000Da、5,000Da、4,000Da、3,000Da、2,000Da、1, 000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In some embodiments, The molecular weight of polymer is between about 100Da and 50,000Da.In some embodiments, the molecular weight of polymer is in about 100Da Between 40,000Da.In some embodiments, the molecular weight of polymer is between about 1,000Da and 40,000Da.At some In embodiment, the molecular weight of polymer is between about 5,000Da and 40,000Da.In some embodiments, the molecule of polymer Amount is between about 10,000Da and 40,000Da.In some embodiments, peg molecule is branched polymers.Branch PEG Molecular weight can be between about 1,000Da and about 100,000Da, it includes (but not limited to) 100,000Da, 95,000Da, 90, 000Da、85,000Da、80,000Da、75,000Da、70,000Da、65,000Da、60,000Da、55,000Da、50, 000Da、45,000Da、40,000Da、35,000Da、30,000Da、25,000Da、20,000Da、15,000Da、10, 000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da and 1, 000Da.In some embodiments, the molecular weight of branch PEG is between about 1,000Da and 50,000Da.In some embodiments, The molecular weight of branch PEG is between about 1,000Da and 40,000Da.In some embodiments, the molecular weight of branch PEG is about 5, Between 000Da and 40,000Da.In some embodiments, the molecular weight of branch PEG is between about 5,000Da and 20,000Da. It will be understood by one of ordinary skill in the art that the aforementioned list on substantial water-soluble main chain is detailed absolutely not and only illustrative , and it is expected that all polymeric materials with above-mentioned quality suitable for method and composition described herein.

As described above, an example of hydrophilic polymer is polyethylene glycol (being abbreviated as PEG), drug is widely used in In, on prostheses and implants and wherein biocompatibility, nontoxicity and non-immunogenicity have in the other application of importance. Polymer described herein:Polypeptide embodiment will use PEG as illustrative hydrophilic polymer, wherein it should be appreciated that other are close Waterborne polymeric can be similarly used in these embodiments.

PEG be well known water-soluble polymer, can buy on the market or can according to well known method in fields by The ring-opening polymerisation of ethylene glycol prepares (Sandler and Karo, Polymer Synthesis, Academic Press, New York, volume 3, the 138-161 pages).PEG normally transparents, it is colourless, tasteless, water-soluble in, to thermostabilization, to many chemistry Agent inertia is not hydrolyzed or gone bad, and usually nontoxic.Polyethylene glycol is thought for bio-compatible, and PEG can be with group living in other words It knits or organism coexists, without damaging.More particularly, PEG is essentially non-immunogenic, and PEG exists in other words It is not intended to generate immune response in vivo.When be connected to have the function of in vivo some need molecule (such as bioactivator) When upper, PEG is intended to shelter medicament and can reduce or eliminate any immune response so that organism can tolerate the presence of medicament. PEG binding elements are intended to not generate substantive immune response or cause condensation or other unwanted effects.

It widely uses term " PEG " and covers any peg molecule (modification without considering size or on PEG ends), And when it is bonded on non-natural amino acid polypeptides when can be expressed from the next：

XO-(CH₂CH₂O)_n-CH₂CH₂-Y

Wherein n is that 2 to 10,000 and X is H or end modified, and it includes (but not limited to) C_1-4Alkyl, protecting group or end Functional group.Term PEG is including (but not limited to) its any type of polyethylene glycol, and it includes bifunctional PEG, multi-arm PEG, derivatives PEG, forked PEG, branch PEG (wherein each chain have about 1kDa to about 100kDa, about 1kDa to about 50kDa or about 1kDa extremely The molecular weight of about 20kDa), pendency PEG (PEG i.e. with one or more functional groups for overhanging main polymer chain or Related polymer) or wherein with degradable bonded PEG.In one embodiment, wherein n is that the PEG of about 20 to about 2000 is fitted For in method and composition described herein.In some embodiments, aqueous-based polymers include polyalkylene glycol moiety.PEG The molecular weight of polymer can in broad range, it includes (but not limited to) between about 100Da and about 100,000Da or higher it Between scope.The molecular weight of PEG polymer can between about 100Da and about 100,000Da, it includes (but not limited to) 100, 000Da、95,000Da、90,000Da、85,000Da、80,000Da、75,000Da、70,000Da、65,000Da、60, 000Da、55,000Da、50,000Da、45,000Da、40,000Da、35,000Da、30,000Da、25,000Da、20, 000Da、15,000Da、10,000Da、9,000Da、8,000Da、7,000Da、6,000Da、5,000Da、4,000Da、3, 000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In some embodiments, the molecular weight of polymer is between about 100Da and 50,000Da.In some embodiments, gather The molecular weight of object is closed between about 100Da and 40,000Da.In some embodiments, the molecular weight of polymer is about 1,000Da Between 40,000Da.In some embodiments, the molecular weight of polymer is between about 5,000Da and 40,000Da.At some In embodiment, the molecular weight of polymer is between about 10,000Da and 40,000Da.In some embodiments, peg molecule For branched polymers.The molecular weight of branch PEG can be between about 1,000Da and about 100,000Da, and it includes (but not limited to) 100,000Da、95,000Da、90,000Da、85,000Da、80,000Da、75,000Da、70,000Da、65,000Da、60, 000Da、55,000Da、50,000Da、45,000Da、40,000Da、35,000Da、30,000Da、25,000Da、20, 000Da、15,000Da、10,000Da、9,000Da、8,000Da、7,000Da、6,000Da、5,000Da、4,000Da、3, 000Da, 2,000Da and 1,000Da.In some embodiments, the molecular weight of branch PEG is in about 1,000Da and 50,000Da Between.In some embodiments, the molecular weight of branch PEG is between about 1,000Da and 40,000Da.In some embodiments, The molecular weight of branch PEG is between about 5,000Da and 40,000Da.In some embodiments, the molecular weight of branch PEG is about 5, Between 000Da and 20,000Da.Broad range of PEG molecules are described in and (include, but are not limited to) Shearwater It is to be incorporated herein by reference in Polymers, Inc. catalogue, Nektar Therapeutics catalogues.

In document the particular instance of terminal functional group including (but not limited to) carbonic acid N- succinimide esters (referring to (for example) U.S. Patent No. 5,281, No. 698, the 5th, 468, No. 478), amine (referring to (for example) Buckmann et al., Makromol.Chem.182:1379(1981)；Zalipsky et al., Eur.Polym.J.19:1177 (1983)), hydrazides (ginseng See (for example) Andresz et al., Makromol.Chem.179:301 (1978)), succinimidyl propionate and butyric acid amber Imide ester is (referring to (for example) Olson et al., in Poly (ethylene glycol) Chemistry＆Biological The 170-181 pages in Applications, Harris and Zalipsky are compiled, ACS, Washington, D.C., and 1997；Referring also to U.S. Patent No. 5,672,662), succinimidyl succinate is (referring to (for example) Abuchowski et al., Cancer Biochem.Biophys.7:175 (1984) and Joppich et al., Makromol.Chem.180:1381 (1979)), amber Acid imide base ester (referring to (for example) U.S. Patent No. 4,670,417), carbonic acid benzotriazole are (referring to (for example) U.S. Patent No. 5,650, No. 234), glycidol ether is (referring to (for example) Pitha et al., Eur.J Biochem.94:11(1979)；Elling Et al., Biotech.Appl.Biochem.13:354 (1991)), Epoxide carbonyl imidazoles (referring to (for example) Beauchamp et al., Anal.Biochem.131:25(1983)；Tondelli et al., J.Controlled Release 1:251 (1985)), carbonic acid P-nitrophenyl ester is (referring to (for example) Veronese et al., Appl.Biochem.Biotech., 11:141(1985)；And Sartore et al., Appl.Biochem.Biotech., 27:45 (1991)), aldehyde (referring to (for example) Harris et al., J.Polym.Sci.Chem.Ed.22:341(1984)；U.S. Patent No. 5,824,784；U.S. Patent No. 5,252,714 Number), maleimide is (referring to (for example) Goodson et al., Bio/Technology8:343(1990)；Romani et al., Chemistry of Peptides and Proteins 2:In 29 (1984)；And Kogan, Synthetic Comm.22: 2417 (1992)), adjacent pyridyl disulfide is (referring to (for example) Woghiren et al., Bioconj.Chem.4:314 (1993)), propenyl is (referring to (for example) Sawhney et al., Macromolecules, 26:581 (1993)), vinyl sulfone (ginseng See (for example) U.S. Patent No. 5,900,461).All above-mentioned bibliography and patent are to be fully incorporated this by reference Wen Zhong.

In some cases, PEG is terminated at one end with hydroxyl or methoxyl group, i.e. X is H or CH₃(" methoxyl group PEG "). Alternatively, PEG can be terminated with reactive group, and then form double functional copolymer.Typical reaction group, which can include, to be commonly used in It ((is included including (but not limited to) maleimide base group, activated carbonate with functional group present in 20 kinds of common amino acids (but not limited to) p-nitrophenyl ester), Acibenzolar (including (but not limited to) n-hydroxysuccinimide, p-nitrophenyl ester) and Aldehyde) and to 20 kinds of common amino acid inertia but with the function of complementary functional group's specific reaction present in alpha-non-natural amino acid Those reactive groups of base (including (but not limited to) oximido, carbonyl or dicarbapentaborane and azanol base).

It should be noted that it should directly or indirectly be connected by alpha-non-natural amino acid by the other end of the Y PEG represented in above formula Onto polypeptide (synthesis is natural), polynucleotide, polysaccharide or synthetic polymer.When Y is azanol base, then the PEG containing azanol Reagent can with react to be formed containing the alpha-non-natural amino acid of carbonyl or dicarbapentaborane in polypeptide it is bonded on polypeptide by oxime key PEG group.When Y be carbonyl or dicarbapentaborane when, then the PEG reagents containing carbonyl or dicarbapentaborane can in polypeptide containing the non-of azanol Natural amino acid reacts to be formed through the bonded PEG group on polypeptide of oxime key.When Y is carbonyl or dicarbapentaborane, then contain There are the PEG reagents of carbonyl or dicarbapentaborane that can also react to be formed through new oxime key key with the alpha-non-natural amino acid containing oxime in polypeptide The PEG group being coupled on polypeptide.The example of appropriate reaction condition, purification process and reagent throughout this specification and subsidiary schema into Row description.For example, non-natural amino acid polypeptides of Fig. 7 presentations containing carbonyl are formed bonded with the PEG reagent reactings containing azanol Three examples of the non-natural amino acid polypeptides containing oxime on to PEG group.In addition, alpha-non-natural amino acid of Fig. 9 presentations containing oxime Polypeptide forms the two of the bonded new non-natural amino acid polypeptides containing oxime in PEG group with the PEG reagent reactings containing carbonyl A example.And non-natural amino acid polypeptides of Figure 10 presentations containing azanol and the PEG reagent reactings containing carbonyl formed it is bonded to PEG group One example of the non-natural amino acid polypeptides containing oxime in group.

Only for example and it is not intended as trying the PEG that can be used for composition described herein, method, technology and strategy The limitation of the type or species of agent, Figure 11 presentations can react bonded to PEG to be formed with the non-natural amino acid polypeptides containing carbonyl Other illustrative examples of the PEG reagents containing azanol of the non-natural amino acid polypeptides containing oxime on group and can with containing oxime Non-natural amino acid polypeptides or non-natural amino acid polypeptides containing azanol react to be formed and bonded contain to new in PEG group The example of the PEG reagents containing carbonyl of oxime non-natural amino acid polypeptides.Figure 12 presentations be used to being formed PEG reagents containing azanol or Four explanations of the synthetic method of the masked form through forms of protection or the PEG reagents containing azanol of the PEG reagents containing azanol Property example.Figure 13 presentations are used to be formed through the bonded PEG reagents containing azanol of amide or through the bonded PEG containing azanol of amide The synthetic method of the masked form through forms of protection or through the bonded PEG reagents containing azanol of amide of reagent it is illustrative Example.Figure 14 and Figure 15 presentations are used to be formed through the bonded PEG reagents containing azanol of carbamate or through amino-formate bond The masked shape through forms of protection or through the bonded PEG reagents containing azanol of carbamate of the PEG reagents containing azanol of connection The illustrative example of the synthetic method of formula.Figure 16 presentations are used to form PEG reagents simply containing azanol or simply containing azanol The illustrative example of the synthetic method of the masked form of the PEG reagents through forms of protection or simply containing azanol of PEG reagents. In addition, Figure 17 presentations have the illustrative example of the reagent containing azanol of the branch of multiple bonded PEG groups, and further exhibition Show that this kind of multiple PEG branches reagent containing azanol and the non-natural amino acid polypeptides containing carbonyl are formed with bonded multiple The reaction of the non-natural amino acid polypeptides containing oxime of PEG branched groups.

When needing different molecular being connected on each end of polymer, Heterobifunctional derivative is also to be especially suitable 's.For example, ω-N- amino-N- azidos PEG can allow by with activation electrophilic group (such as aldehyde, ketone, Acibenzolar, Activated carbonate etc.) molecule be connected on an end of PEG and the molecule with sub- acetyl group be connected to the another of PEG On a end.

In some embodiments, strong nucleopilic reagent (including (but not limited to) azanol) can with present in alpha-non-natural amino acid Aldehyde radical or ketone group reaction are to form oxime, it can be by handling further to reduce with appropriate reducing agent in some cases.Alternatively, Strong nucleopilic reagent can be incorporated in polypeptide by alpha-non-natural amino acid and is used for and ketone group present in water-soluble polymer or aldehyde radical Preferential reaction.In general, at least one end of PEG molecules can be used for reacting with alpha-non-natural amino acid.

Therefore, in some embodiments, the polypeptide including alpha-non-natural amino acid is bonded by the side chain of alpha-non-natural amino acid Onto water-soluble polymer (such as polyethylene glycol (PEG)).Alpha-non-natural amino acid method and composition described herein provides For in the highly effective method of PEG derivatives selectively modifying proteins, including by alpha-non-natural amino acid (comprising (but not It is limited to) containing 20 kinds of natural those amino acid for being incorporated to the functional group being not present in amino acid or substituent group) selectively it is incorporated to egg In white matter codon is selected to respond and then with those amino acid of suitable reactivity PEG Derivatives Modifieds.A variety of knownization Method is suitable for alpha-non-natural amino acid method and composition described herein water-soluble polymer is incorporated to protein.

Main polymer chain can be linear or branch.Branched polymers main chain is usually known in fields.In general, Branched polymers have center branch core part and multiple linear polymer chains being connected on the branch core of center.PEG is to divide Branch form uses, can be by the way that ethylene oxide is added to various polyalcohols (such as glycerine, glycerine oligomer, Ji Wusi Alcohol and D-sorbite) on prepare.Center branch part point can also be derived from several amino acid, such as lysine.The poly- second of branch Glycol can be expressed as R (- PEG-OH) with common version_m, wherein R is derived from core part, such as glycerine, glycerine oligomer Or pentaerythrite, and m represents the number of arm.Multi-arm PEG molecules (such as U.S. Patent No. 5,932,462；5,643,575th Number；No. 5,229,490；No. 4,289,872；U.S. patent application case 2003/0143596；WO 96/21469；And WO Those PEG molecules described in 93/21259, each of these documents are in being hereby incorporated by reference in their entirety) also may be used As main polymer chain.

Branch PEG is alternatively by PEG (- YCHZ₂)_nThe form of the forked PEG represented, wherein Y is bonded group and Z is logical It crosses and determines that the atomic link of length is bonded to the activated terminus group on CH.Another branched forms dangle PEG along PEG main chains rather than The end of PEG chains has the reactive group of such as carboxyl.

In addition to these forms of PEG, the polymer in main chain with weak bond or degradable linkage can be also prepared.Citing comes It says, the PEG in the polymer backbone with ester bond, experience hydrolysis can be prepared.As shown in this article, this is hydrolyzed so that polymer It is cracked into the segment of lower molecular weight：

-PEG-CO₂-PEG-+H₂O→PEG-CO₂H+HO-PEG-。

It will be understood by one of ordinary skill in the art that term polyethylene glycol or PEG are represented or comprising known in fields Form of ownership, it includes (but not limited to) those forms disclosed herein.The molecular weight of polymer can in broad range, Including (but not limited to) the scope between about 100Da and about 100,000Da or higher.The molecular weight of polymer can be about Between 100Da and about 100,000Da, it includes (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da、75,000Da、70,000Da、65,000Da、60,000Da、55,000Da、50,000Da、45,000Da、40, 000Da、35,000Da、30,000Da、25,000Da、20,000Da、15,000Da、10,000Da、9,000Da、8,000Da、 7,000Da、6,000Da、5,000Da、4,000Da、3,000Da、2,000Da、1,000Da、900Da、800Da、700Da、 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In some embodiments, the molecular weight of polymer about 100Da with Between 50,000Da.In some embodiments, the molecular weight of polymer is between about 100Da and 40,000Da.In some implementations In example, the molecular weight of polymer is between about 1,000Da and 40,000Da.In some embodiments, the molecular weight of polymer exists Between about 5,000Da and 40,000Da.In some embodiments, the molecular weight of polymer about 10,000Da and 40,000Da it Between.

To maximize total molecule of the required property of PEG, PEG polymer or the polymer being connected on bioactive molecule Amount and hydration status are sufficiently high usually be connected relevant favorable characteristics to assign with PEG polymer, it is such as water-soluble with follow Ring half-life period increases, the bioactivity without adversely affecting parent molecule.

Method and composition described herein can be used for the polymer for preparing substantial homogeneous:The system of protein conjugates Agent." substantial homogeneous ", which means, as used herein observes polymer:Protein conjugates molecular proportion gross protein Half bigger.Polymer:Protein conjugates have bioactivity and " substantial homogeneous " PEG of the present invention provided herein Change polypeptide formulations are the advantages of enough homogeneous are to show homogeneous preparation (for example, being easy to clinical practice pharmacokinetics between batch In prediction) those preparations.

Also may be selected to prepare polymer:The mixture of protein conjugates molecule, and it is provided herein the advantages of be can Selection includes single polymer in the mixture:The ratio of protein conjugates.Therefore, if necessary, various protein can be prepared Mixture with the polymer moieties (that is, dimer part, trimer fractions, tetramer part etc.) of various numbers being connected And the single polymer prepared by these binding elements and using method described herein:Protein conjugates combine, and make mixing Object has single polymer of predetermined ratio:Protein conjugates.

The ratio regular meeting of peg molecule and protein molecule changes with its concentration in the reactive mixture.Generally For, best ratio (being there is minimum excessive unreacted protein or polymer for reaction efficiency) can be by selected It the molecular weight of polyethylene glycol and can be determined on available with the number of reactive group.On molecular weight, usual polymer Molecular weight it is higher, the number that may be connected to polymer molecule on protein is fewer.Similarly, when optimizing these parameters, It should be taken into account the branch of polymer.In general, molecular weight is higher (or branch is more), polymer:Protein rate is higher.

As used herein, and hydrophilic polymer ought be covered:During polypeptides/proteins binding element, " treatment is effective for term Amount " is further referred to the increased amount of benefit needed for patient's offer.The amount can change with Different Individual and should depend on In many factors, it includes the overall physical state of patient and the potential causes of disease of disease, illness or the symptom to be treated.This hair The therapeutically effective amount of bright composition can be easy to by those skilled in the art using can disclose obtain material and program Lai It determines.

It is adjustable it is described herein be bonded to it is water-soluble on " through modification or unmodified " non-natural amino acid polypeptides Property polymer number (that is, PEGylated or glycosylated degree) with provide change (including (but not limited to) it is increased or reduce ) pharmacology, pharmacokinetics or pharmacodynamic properties, such as vivo half-life.In some embodiments, the half-life period ratio of polypeptide Unmodified polypeptide increase at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, twice, five Again, 10 times, 50 times or at least about 100 times.

In one embodiment, passed through including the polypeptide containing carbonyl or the alpha-non-natural amino acid of dicarbapentaborane and contain end hydroxylamine Part PEG Derivatives Modifieds, be directly bonded on PEG main chains.

In some embodiments, azanol end PEG derivatives have following structure：

RO-(CH₂CH₂O)_n-O-(CH₂)_m-O-NH₂

Wherein R is simple alkyl (methyl, ethyl, propyl etc.), and m is 2-10 and n is 100-1,000 (that is, average molecular weight Between 5kDa-40kDa).The molecular weight of polymer can be in broad range, it includes (but not limited to) between about 100Da With the scope between about 100,000Da or higher.The molecular weight of polymer can wrap between about 100Da and about 100,000Da 100,000Da containing (but not limited to), 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da、60,000Da、55,000Da、50,000Da、45,000Da、40,000Da、35,000Da、30,000Da、25, 000Da、20,000Da、15,000Da、10,000Da、9,000Da、8,000Da、7,000Da、6,000Da、5,000Da、4, 000Da、3,000Da、2,000Da、1,000Da、900Da、800Da、700Da、600Da、500Da、400Da、300Da、200Da And 100Da.In some embodiments, the molecular weight of polymer is between about 100Da and 50,000Da.In some embodiments, The molecular weight of polymer is between about 100Da and 40,000Da.In some embodiments, the molecular weight of polymer is about 1, Between 000Da and 40,000Da.In some embodiments, the molecular weight of polymer is between about 5,000Da and 40,000Da. In some embodiments, the molecular weight of polymer is between about 10,000Da and 40,000Da.

In another embodiment, including the polypeptide containing carbonyl or the amino acid of dicarbapentaborane through containing end hydroxylamine Part PEG Derivatives Modifieds are bonded to by amido bond on PEG main chains.

In some embodiments, azanol end PEG derivatives have following structure：

RO-(CH₂CH₂O)_n-O-(CH₂)₂-NH-C(O)(CH₂)_m-O-NH₂

In another embodiment, including the polypeptide containing carbonyl or the amino acid of dicarbapentaborane through containing end hydroxylamine Part Each chain of PEG Derivatives Modifieds, wherein branch PEG has the MW in the range of 10kDa-40kDa, and in other embodiment In, in the range of 5kDa-20kDa.The molecular weight of branched polymers can be in broad range, it includes (but not limited to) between about Scope between 100Da and about 100,000Da or higher.The molecular weight of polymer can about 100Da and about 100,000Da it Between, it includes (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70, 000Da、65,000Da、60,000Da、55,000Da、50,000Da、45,000Da、40,000Da、35,000Da、30, 000Da、25,000Da、20,000Da、15,000Da、10,000Da、9,000Da、8,000Da、7,000Da、6,000Da、5, 000Da、4,000Da、3,000Da、2,000Da、1,000Da、900Da、800Da、700Da、600Da、500Da、400Da、 300Da, 200Da and 100Da.In some embodiments, the molecular weight of polymer is between about 100Da and 50,000Da.One In a little embodiments, the molecular weight of polymer is between about 100Da and 40,000Da.In some embodiments, the molecule of polymer Amount is between about 1,000Da and 40,000Da.In some embodiments, the molecular weight of polymer is in about 5,000Da and 40, Between 000Da.In some embodiments, the molecular weight of polymer is between about 10,000Da and 40,000Da.

In another embodiment, the polypeptide including alpha-non-natural amino acid derives through at least one PEG with apparatus derivatorius Object is modified.In some embodiments, the PEG derivatives containing azanol base have following structure：

[RO-(CH₂CH₂O)_n-O-(CH₂)₂-C(O)-NH-CH₂-CH₂]₂CH-X-(CH₂)_m-O-NH₂

Wherein R is simple alkyl (methyl, ethyl, propyl etc.), and X optionally for NH, O, S, C (O) or is not present, m 2- 10 and n is 100-1,000.The molecular weight of polymer can be between about 1,000Da and about 100,000Da, and it includes (but unlimited In) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da、55,000Da、50,000Da、45,000Da、40,000Da、35,000Da、30,000Da、25,000Da、20, 000Da、15,000Da、10,000Da、9,000Da、8,000Da、7,000Da、6,000Da、5,000Da、4,000Da、3, 000Da, 2,000Da and 1,000Da.In some embodiments, the molecular weight of branch PEG about 1,000Da and 50,000Da it Between.In some embodiments, the molecular weight of branch PEG is between about 1,000Da and 40,000Da.In some embodiments, prop up The molecular weight of chain PEG is between about 5,000Da and 40,000Da.In some embodiments, the molecular weight of branch PEG is about 5, Between 000Da and 20,000Da.

It can obtain on the functionalization of PEG and several general introductions of engagement and monograph.Referring to (for example) Harris, Macromol.Chem.Phys.C25:325-373(1985)；Scouten,Methods in Enzymology 135:30-65 (1987)；Wong et al., Enzyme Microb.Technol.14:866-874(1992)；Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9:249-304(1992)；Zalipsky, Bioconjugate Chem.6:150-165(1995)。

For activated polymer method also seen in WO 94/17039, U.S. Patent No. 5,324,844, WO 94/ 18247th, WO 94/04193, U.S. Patent No. 5,219,564, U.S. Patent No. 5,122,614, WO 90/13540, U.S. In state's patent the 5th, 281,698 and more WO 93/15189, and for the side of the engagement between activated polymer and enzyme Method is including (but not limited to) blood coagulation factor VIII (WO 94/15625), hemoglobin (WO 94/09027), the oxygen carrier molecule (U.S. Patent the 4th, 412,989), ribalgilase and superoxide dismutase (Veronese et al., App.Biochem.Biotech.11:141-152 (1985)), all these documents are to be hereby incorporated by reference in their entirety In.

If necessary, the PEGylated non-natural amino acid polypeptides described herein obtained from hydrophobic chromatography can by a kind of or Program known to more than one those skilled in the art is further purified, these programs are including (but not limited to) affine color Spectrum；Anion or cation-exchange chromatography (using (include, but are not limited to) DEAE SEPHAROSE)；Silica chromatography；Reverse phase HPLC；Gel filtration (uses (include, but are not limited to) SEPHADEX G-75)；Hydrophobic interaction chromatograph；Size exclusion color Spectrum；Immobilized metal ion afinity chromatography；Ultrafiltration/diafiltration；Ethanol precipitation；Ammonium sulfate precipitation；Chromatofocusing；Displcement chromatography；Electrophoretic procedures (include The isoelectric focusing of (but not limited to) preparative), differential dissolubility (including (but not limited to) ammonium sulfate precipitation) or extraction.Led to by GPC Apparent molecular weight (Preneta AZ, PROTEIN PURIFICATION can be estimated compared with globular preteins reference substance by crossing METHODS, A PRACTICAL APPROACH (Harris and Angal are compiled) IRL Press 1989,293-306).Non-natural ammonia Base acid polypeptide:The purity of PEG binding elements can pass through proteolytic degradation (being cracked including (but not limited to) trypsase), then matter Spectrum analysis is assessed.Pepinsky RB. et al., J.Pharmacol.＆Exp.Ther.297 (3):1059-66(2001).

Be bonded to water-soluble polymer on the alpha-non-natural amino acid of polypeptide described herein can without stint pass through into One step is derivative or substitutes.

E. enhance to sero-abluminous compatibility

Various molecules can also merge to adjust the half-life period in serum with non-natural amino acid polypeptides described herein. In some embodiments, non-natural amino acid polypeptides are bonded or melt with " through modification or unmodified " described herein for molecule It closes to enhance to endogenous sero-abluminous compatibility in animal.

For example, in some cases, polypeptide is carried out to merge with the restructuring of albumin combination sequence.Illustrative albumin Binding sequence including (but not limited to) from streptococcus protein G albumin combination domain (referring to (for example) Makrides et al., J.Pharmacol Exp.Ther.277(l):534-542 (1996) and Sjolander et al., J, Immunol.Methods 201:115-123 (1997)) or albumin binding peptide, it is all such as (e.g.) Dennis et al., J.Biol.Chem.277 (38): Those albumin binding peptides described in 35035-35043 (2002).

In other embodiments, " through modification or unmodified " non-natural amino acid polypeptides described herein are through fat Acylating acid.In some cases, aliphatic acid promotes and sero-abluminous combination.Referring to (for example) Kurtzhals et al., Biochem.J.312:725-731(1995)。

In other embodiments, it is described herein " through modification or it is unmodified " non-natural amino acid polypeptides directly with Seralbumin (including (but not limited to) human serum albumin) merges.It will be understood by one of ordinary skill in the art that it is a variety of its His molecule can also be bonded to as described herein through modification or unmodified non-natural amino acid polypeptides on adjust and blood The combination of pure albumen or other serum components.

F. the glycosylation of non-natural amino acid polypeptides described herein

Method and composition described herein includes and is combined with the one or more non-natural with saccharide residue The polypeptide of amino acid.Saccharide residue can be natural (including (but not limited to) N- acetyl glucosamines) or non-natural (comprising (but not It is limited to) 3- fluorine galactolipin).The sugar can be bonded by N or bonded O glycosidic bond (including (but not limited to) N- acetyl group galas Sugar-Serine) or non-natural key be bonded to non-natural (including (but not limited to) oxime or corresponding C is bonded or S is bonded glucosides) On amino acid.

Sugared (including (but not limited to) glycosyl) partly can in vivo or in vitro be added on non-natural amino acid polypeptides. In some embodiments, including the polypeptide containing carbonyl or the alpha-non-natural amino acid of dicarbapentaborane through being repaiied with the sugar of amino oxygroup derivatization It is decorated with the corresponding glycosylated polypeptides generated through oxime key connection.It is once connected on alpha-non-natural amino acid, sugar can be by with glycosyl Transferase and other enzymatic treatments further refine to generate the oligosaccharides being attached on non-natural amino acid polypeptides.Referring to (for example) H.Liu et al., J.Am.Chem.Soc.125:1702-1703(2003).

G. bonded group use and the application comprising polypeptide dimer and polymer

In addition to directly adding functional group to non-natural amino acid polypeptides, the non-natural amino acid moieties of polypeptide can be first Through multifunctional (for example, difunctionality, trifunctional, tetrafunctional) connexon molecular modification, through further modification after.Namely It says, at least one end of multifunctional connexon molecule and at least one alpha-non-natural amino acid reaction in polypeptide and multifunctional company At least one other end for connecing son can be used for further functionalization.If all ends of multifunctional connexon are identical, then (depending on stoichiometric condition) can form the homopolymer of non-natural amino acid polypeptides.If the end of multifunctional connexon With different chemical reactivities, then at least one end of multifunctional connection subbase group can be with non-natural amino acid polypeptides knot It closes and other ends can then be reacted from different functional groups, these functional groups include (only for example)：Mark；Dyestuff；It is poly- Close object；Water-soluble polymer；The derivative of polyethylene glycol；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Light Affinity marker；Reactive compounds；Resin；Second protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal Chelating agent；Co-factor；Aliphatic acid；Carbohydrate；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble branch Shaped polymer, cyclodextrin, biomaterial；Nano-particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Newly Clever functional group；With other molecule covalents or the group of noncovalent interaction；Light cage covers part；Can actinic radiation excitation portion Point；Ligand；It can photoisomerization part；Biotin；Biotin analog；It is combined with the part of heavy atom；Chemically crack Group；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active agent；Aminothio acid；Toxin part； Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electron dense group；Magnetic group； It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；Small molecule；Inhibition ribonucleic acid, radioactivity Nucleotide；Neutron capture agent；The derivative of biotin；Quantum dot；Nanometer transfers element；Radioactivity transfers element；Abzyme, activation are multiple Close activator, virus, adjuvant, aglycone, anaphylactogen, angiostatin, antihormones, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecular, plan epitope, receptor, reverse micelle and any combination thereof.

Multifunctional connection subbase group has following universal architecture：

Wherein：

X is each independently NH₂,-C (=O) R₉,-SR' or-J-R, wherein R₉It is for H or OR', wherein J

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；R " is each independently H, alkyl, is substituted Alkyl or protecting group or " during group, two R " optionally form Heterocyclylalkyl when there are more than one R；

R' is each independently H, alkyl or substituted alkyl；

L₁To be optional, and it is-C (R') when it is present_p- NR'-C (O) O- (alkylidene is substituted alkylidene)-, wherein p For 0,1 or 2；W is NH₂,-C (=O) R₉,-SR' or-J-R；And n is 1 to 3；

Its restrictive condition is X and L-L₁- W provides at least one of following group independently jointly：(a) can With carbonyl (including dicarbapentaborane) reaction on alpha-non-natural amino acid or " through modification or unmodified " non-natural amino acid polypeptides Azanol base；It (b) can be with the azanol on alpha-non-natural amino acid or " through modification or unmodified " non-natural amino acid polypeptides The carbonyl (including dicarbapentaborane) of base reaction；(c) can undergo with alpha-non-natural amino acid or " through modification or it is unmodified " it is non- The carbonyl (including dicarbapentaborane) of exchange reaction occurs for the oximido on natural amino acid polypeptide.

(there are two same ends for wherein described connexon tool, i.e. azanol for the bifunctional homotype connexon of Figure 18 presentations synthesis Base) illustrative, non-limiting examples.This connexon can be used to form the non-natural amino acid polypeptides containing carbonyl or dicarbapentaborane Homodimer is to form two oxime keys.Alternatively, if an end of this connexon is through protection, then this part is through protection Connexon can be used for making the azanol end without protection and the non-natural amino acid polypeptides knot containing carbonyl or dicarbapentaborane by oxime key It closes, leaves another end through protection after deprotection for other bonded reactions.Alternatively, to the stoichiometry of reagent Carefully operation can provide similar result (heterodimer), can be similarly by one although wherein required heterodimer can be obtained The result of a little homodimeric body pollutions.

The wherein each connexon of Figure 19 presentations has terminal-reactive group (that is, azanol base, the oxime of more than one type Base and thioester substrate) two kinds of multifunctional special-shaped connexons illustrative, non-limiting examples.It is discussed using throughout this specification The chemistry based on oxime, this connexon can be used to form the heterodimer of non-natural amino acid polypeptides.

PEG group is connected to alpha-non-natural amino acid by Figure 20 presentations in multi-step synthesis using special-shaped bifunctional linker Schematically illustrating property, non-limiting examples on polypeptide.In a first step, described in such illustrative drawings, containing carbonyl The non-natural amino acid polypeptides of base and the bifunctional linker containing azanol react that form modified alpha-non-natural amino acid containing oxime more Peptide.However, still retain can be with the reagent with appropriate reaction (in the drawings by the object of matched shape by bifunctional linker Illustrate) react to be formed the functional group of the modified functionalization non-natural amino acid polypeptides containing oxime (herein by visible object Illustrate).In this certain illustrative schema, function turns to PEG group, but can also include any one of foregoing functional group or In the case of trifunctional or tetrafunctional connexon, the functional group comprising more than one types or polytype identical function Base.Figure 21 presentations are used for the connection subbase for four types being bonded to the non-natural amino acid polypeptides for containing azanol in PEG group The illustrative example of group.As previously mentioned, PEG functional groups are only illustrative purpose and provide.Therefore, connexon described herein Group provides another method that non-natural amino acid polypeptides are further modified in a manner of site selectivity.

Method and composition described herein also provides polypeptides in combination, such as homodimer, heterodimer, homotype Polymer or heteromultimer (that is, tripolymer, tetramer etc.).Only for example, it is described below and focuses on GH supergene families Member, however, the method, technology and composition described in this part can be applied to providing in dimer and multimeric forms Other substantially any polypeptides of benefit, it includes (only for example)：α -1 antitrypsins, angiostatin, anti-hemolysis The factor, antibody, apolipoprotein, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, C-X-C chemotactic factor (CF)s, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, drop blood calcium Element, c-kit ligands, cell factor, CC chemotactic factor (CF)s, monocyte chemoattractant protein-1, monocyte chemoattractant protein -2, monokaryon Cell chemotaxis protein-3, -1 α of monocyte inflammatory protein, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligands, collagen, colony thorn Swash the factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cell factor, epithelium neutrophilic granulocyte activation peptide -78, MIP-16, MCP-1, epidermal growth factor (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin(EPO) (EPO), epidermis stripping Detoxification element, factors IX, factor Ⅴ II, Factor IX, factor X, fibroblast growth factor (FGF), fibrinogen, fiber Connect albumen, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, promoting sexual gland hormone, growth factor, life Growth factor receptor body, grf, Hedgelog protein, hemochrome, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), Human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 Receptors, insdri, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-α, IFN-β, IFN-γ, interleukins (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), breast Siderophillin, LIF ELISA, luciferase, neural element, neutrophil inhibitory factor (nif) (NIF), oncostatin M, into Bone protein, oncoprotein, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, multi-effector, albumin A, Protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxain, feritin, SCF, atom synthesis Albumen, Soluble complement receptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, growth are adjusted Save element, Somat, growth hormone, streptokinase, super antigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic-shock syndrome toxin, thymosin α1, tissue Type Plasminogen Activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor α, tumor necrosis factor β, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, Mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, testosterone receptor, Aldosterone receptor, ldl receptor and cortisone.

Therefore, cover in method described herein, technology and composition and be bonded directly to polypeptide backbone or pass through connection Son is bound to any other polypeptide or its change of another GH supergene families member or its variant or non-GH supergene families member The GH supergene family member polypeptides containing one or more alpha-non-natural amino acids on body.Due to itself and single phase score Son amount increase, so GH supergene family member dimers or polymer binding element can show novel or needs properties, bag It is different pharmacology, pharmacokinetics, pharmacodynamics, adjusted containing (but not limited to) compared with monomer GH supergene family members Treatment half-life period or adjusted plasma half-life.In some embodiments, GH supergene families member described herein Dimer can adjust the dimerization of GH supergene family member receptors.In other embodiments, GH supergenes described herein Family member's dimer or polymer can serve as GH supergene family member receptors antagonist, agonist or conditioning agent.

In some embodiments, through directly bonded, it includes (but not limited to) to pass through GH supergene families member polypeptide Asn-Lys amido bonds or Cys-Cys disulfide bond.In some embodiments, bonded GH supergene families member polypeptide and/or key The non-GH supergene families member of connection can include the different alpha-non-natural amino acids for promoting dimerization, and it includes (but not limited to) the One GH supergene families member and/or bonded non-GH supergene families member, including with including the non-natural amino containing azanol The polypeptide and the polypeptide of the alpha-non-natural amino acid containing ketone of the 2nd GH supergene families member polypeptide engagement of acid are by forming phase Oxime is answered to react.

Alternatively, two kinds of GH supergene families member polypeptides and/or bonded non-GH supergene families member are to pass through connection Son connection.Any special-shaped bifunctional linker or homotype bifunctional linker can be used to connect two kinds of GH supergene family members, And/or bonded non-GH supergene families member, polypeptide, there can be identical or different primary sequence.In some cases, use Can be in the connexon for being bound together GH supergene families member and/or bonded non-GH supergene families member, polypeptide Bifunctional PEG reagent.

In some embodiments, method and composition described herein provides the water solubility difunctionality with dumbbell structure Connexon, the structure include：A) azide in at least first end of main polymer chain, alkynes, hydrazine, hydrazides, azanol Or the part containing carbonyl or dicarbapentaborane；And b) at least one the second functional group in the second end of main polymer chain. Second functional group can be identical or different with the first functional group.In some embodiments, the second functional group is not anti-with the first functional group It should.In some embodiments, method and composition offer described herein includes the arm of at least one branched molecule structure Water soluble compound.For example, branched molecule structure can be dendroid.

In some embodiments, method and composition offer described herein passes through the water solubility with having following structure Activated polymer reacts the polymer for including one or more GH supergene family members to be formed：

R-(CH₂CH₂O)_n-O-(CH₂)_m-X

Wherein n is about 5 to 3,000, m 2-10, and X can be azide, alkynes, hydrazine, hydrazides, amino oxygroup, azanol, acetyl Base or the part containing carbonyl or dicarbapentaborane, and R be end-capping group, functional group or can be identical or different with X leaving group. R can be the functional group (for example) selected from the group being made of following group：Hydroxyl, through protect hydroxyl, alkoxy, N- hydroxyl ambers Amber vinegar imines base ester, 1- benzotriazole base ester, carbonic acid N- hydroxysuccinimidyl vinegar imines base ester, carbonic acid 1- benzotriazole base ester, acetal, Aldehyde, aldehydrol, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, amino oxygroup, through protecting amine, acyl Hydrazine, through protect hydrazides, through protect mercaptan, carboxylic acid, through protect carboxylic acid, isocyanates, isothiocyanates, maleimide, ethylene Base sulfone, dithiopyridines, vinylpyridine, iodoacetamide, epoxides, dialdehyde, diketone, methanesulfonates, tosylate, with And tresylate, alkene and ketone.

Figure 22 presentations form non-natural amino acid polypeptides described herein using connection subbase group described herein Illustrative, the non-limiting examples of homodimer.In this illustrative example, the non-natural amino acid polypeptides containing carbonyl are made There are two can use azanol base with tool under conditions of bonded homodimer non-natural amino acid polypeptides containing oxime are suitably formed Bifunctional linker's radical reaction.The method of presentation is not limited to bifunctional linker's coupling containing azanol containing carbonyl in schema Base non-natural amino acid polypeptides.Non-natural amino acid polypeptides can further contain can be with the multifunctional connection subbase group containing carbonyl Experience exchange reaction is more to form the oximido of the homopolymer connected by the structure for containing multiple oximidos or alpha-non-natural amino acid Peptide further can form the knot by containing multiple oximidos containing that can undergo reaction with the multifunctional connection subbase group containing carbonyl The azanol base of the homopolymer of structure connection.Certainly, homopolymer can be that homodimer, homotrimer or homotype four are poly- Body.

Figure 23 presentations connect illustrative, the nonrestrictive reality of the heterodimer of subbase group formation polypeptide using special-shaped function Example, at least one member wherein in heterodimer regard feelings for non-natural amino acid polypeptides described herein and other members Condition is non-natural amino acid polypeptides as described herein, other kinds of non-natural amino acid polypeptides or naturally occurring amino Sour polypeptide.In this schema in the example of presentation, connection subbase group contains two identical azanol bases, by controlling chemistry meter Amount, temperature and the other parameter of reaction, connexon and the primary product of the non-natural amino acid polypeptides reaction containing carbonyl are connection Modified non-natural amino acid polypeptides containing oxime to the connexon with available azanol base.This latter group can further with Another kind reacts to form double officials of the non-natural amino acid polypeptides containing oxime containing the non-natural amino acid polypeptides of carbonyl or dicarbapentaborane It can heterodimer.Certainly, the functional group on connexon need not be identical, and it is not necessarily azanol base.Using throughout this specification The chemistry of detailed description, those skilled in the art can design wherein at least one functional group and can be formed with non-natural amino acid polypeptides The connexon of oximido；Other functional groups on connexon can utilize other known chemistry, and it includes known in organic chemistry filed The chemistry based on nucleopilic reagent/electrophilic reagent.

H. the example of functional group is added：The separating property of easyization polypeptide

Naturally occurring or non-natural amino acid polypeptides may be because of the dissolubility or binding characteristic including (but not limited to) polypeptide Many reasons be difficult to separate from sample.It for example, may be from during the polypeptide for therapeutical uses is prepared This polypeptide is separated in the recombination system of polypeptide excessively to produce through Engineering Design.However, dissolubility or combination due to polypeptide Feature, the purity for reaching required degree are usually proved to be difficulty.Method described herein, composition, technology and strategy carry For the solution of this situation.

Using method described herein, composition, technology and strategy, those skilled in the art can prepare with it is required The non-natural amino acid polypeptides containing oxime of homologous peptide, wherein the non-natural amino acid polypeptides containing oxime have the separation spy of improvement Sign.In one embodiment, homologous non-natural amino acid polypeptide makes to prepare by biosynthesis.In another or Additional examples of composition, Alpha-non-natural amino acid by its structure be incorporated herein described in one of alpha-non-natural amino acid in.In another or additional implementation In example, alpha-non-natural amino acid is to be incorporated in end or middle position and be further incorporated to for locus specificity.

In one embodiment, such as the alpha-non-natural amino acid as obtained by prepared by biosynthesis has had required improvement point From feature.In other or Additional examples of composition, alpha-non-natural amino acid includes the oxime key of the group with providing improvement separation characteristic. In other or Additional examples of composition, alpha-non-natural amino acid is more to form modified alpha-non-natural amino acid containing oxime through further modification Peptide, wherein the modification provides the oxime key of the group with providing improvement separation characteristic.In some embodiments, this group is direct It is bonded on alpha-non-natural amino acid, and in other embodiments, this group is bonded to non-natural amino by connecting subbase group On acid.In certain embodiments, this group is connected to by single chemical reaction on alpha-non-natural amino acid, in other embodiment In, it is necessary to which this group is connected on alpha-non-natural amino acid by series of chemical.It is preferred that assign improvement separation characteristic Group site under the reaction condition utilized is specifically bonded on the alpha-non-natural amino acid in non-natural amino acid polypeptides, And it is not bonded on naturally occurring amino acid.

In other or Additional examples of composition, gained non-natural amino acid polypeptides and GH supergene family members are homologous, however, Method, technology and composition described in this part can be applied to from improvement separation characteristic can to benefit substantially any other are more Peptide, the polypeptide include (only for example)：α -1 antitrypsins, angiostatin, anti-hemolytic factor, antibody, load fat Albumen, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, C-X-C chemotactic factor (CF)s, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit coordinations Body, cell factor, CC chemotactic factor (CF)s, monocyte chemoattractant protein-1, monocyte chemoattractant protein -2, monocyte chemotactic egg In vain -3, -1 α of monocyte inflammatory protein, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligands, collagen, colony stimulating factor (CSF), Complement factor 5a, complement inhibitor, complement receptor 1, cell factor, epithelium neutrophilic granulocyte activation peptide -78, MIP-16, MCP- 1st, epidermal growth factor (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin(EPO) (EPO), exfoliative toxin,or exfoliatin, the factor IX, factor Ⅴ II, Factor IX, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four Helix bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, promoting sexual gland hormone, growth factor, growth factor receptors, Grf, Hedgelog protein, hemochrome, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), the white egg of human serum In vain, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 Receptors, insdri, insulin-like growth factor (IGF), IGF-I, IGF- II, interferon (IFN), IFN-α, IFN-β, IFN-γ, interleukins (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL- 6th, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactotransferrin, leukaemia Inhibiting factor, luciferase, neural element, neutrophil inhibitory factor (nif) (NIF), oncostatin M, osteogenic protein, oncoprotein, The outer poison of paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, multi-effector, albumin A, Protein G, pth, pyrogenicity Plain A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxain, feritin, SCF, atom synthetic proteins, Soluble complement receptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, Growth hormone, streptokinase, super antigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroids swash Plain receptor, superoxide dismutase, toxic-shock syndrome toxin, thymosin α1, tissue type plasminogen activator, tumour Growth factor (TGF), tumor necrosis factor, tumor necrosis factor α, tumor necrosis factor β, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, P53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, testosterone receptor, aldosterone receptor, LDL by Body and cortisone.

In other or Additional examples of composition, the water solubility of the group improvement polypeptide of improvement separation characteristic is assigned；In other realities It applies in example, the binding property of the group improvement polypeptide；In other embodiments, the group provides new associativity to polypeptide Matter (includes (only for example) biotin group or biotin conjugated group).The water solubility of the group improvement polypeptide wherein Embodiment in, group is selected from water-soluble polymer described herein, and it includes (only for example) described herein Any one of PEG polymeric groups.

I. the example of functional group is added：Detect the presence of polypeptide

Naturally occurring or non-natural amino acid polypeptides can be because of bag in sample (including in vivo sample and in vitro sample) Containing (but not limited to) shortage can be easy to polypeptide with reference to reagent or mark many reasons and be difficult to detect.It is described herein Method, composition, technology and strategy provide the solution of this situation.

Using method described herein, composition, technology and strategy, those skilled in the art can prepare with it is required The non-natural amino acid polypeptides containing oxime of homologous peptide, wherein the non-natural amino acid polypeptides containing oxime allow detection sample in vivo Polypeptide in sheet and in vitro sample.In one embodiment, homologous non-natural amino acid polypeptide is prepared by biosynthesis. In another or Additional examples of composition, alpha-non-natural amino acid by its structure be incorporated herein described in one of alpha-non-natural amino acid In.In another or Additional examples of composition, alpha-non-natural amino acid is to be incorporated in end or middle position and is further site spy The opposite sex is incorporated to.

In one embodiment, such as the non-natural amino acid polypeptides as obtained by prepared by biosynthesis have had required inspection Survey feature.In other or Additional examples of composition, non-natural amino acid polypeptides include at least one be selected from by the non-natural ammonia containing oxime The alpha-non-natural amino acid of the group of base acid, the alpha-non-natural amino acid containing carbonyl and the composition of the alpha-non-natural amino acid containing azanol. In other embodiment, these alpha-non-natural amino acids are incorporated to by biosynthesis in polypeptide as described herein.At other or replace For in embodiment, non-natural amino acid polypeptides include at least one selected from Formulas I-XVIII, XXX-XXXIV (A and B) or XXXX- The alpha-non-natural amino acid of the amino acid of XXXXIII.In other or Additional examples of composition, alpha-non-natural amino acid is included with providing improvement Detect the oxime key of the group of feature.In other or Additional examples of composition, alpha-non-natural amino acid is formed through further modifying through repairing The non-natural amino acid polypeptides containing oxime of decorations, wherein the modification provides the oxime key of the group with providing improved detection feature. In some embodiments, this group direct key is coupled on alpha-non-natural amino acid, and in other embodiments, this group passes through company It is bonded on alpha-non-natural amino acid to connect subbase group.In certain embodiments, this group is connected to non-by single chemical reaction On natural amino acid, in other embodiments, it is necessary to which this group is connected to alpha-non-natural amino acid by series of chemical On.It is preferred that the group for assigning improved detection feature is specifically bonded to non-natural amino in site under the reaction condition utilized On alpha-non-natural amino acid in sour polypeptide, and not it is bonded on naturally occurring amino acid.

In other or Additional examples of composition, gained non-natural amino acid polypeptides and GH supergene family members are homologous, however, Method, technology and composition described in this part can be applied to sample in vivo and in vitro in sample need to detect several Any other polypeptide, the polypeptide include (only for example)：α -1 antitrypsins, angiostatin, anti-hemolysis because Son, antibody, apolipoprotein, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, C-X-C chemotactic factor (CF)s, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, drop blood calcium Element, c-kit ligands, cell factor, CC chemotactic factor (CF)s, monocyte chemoattractant protein-1, monocyte chemoattractant protein -2, monokaryon Cell chemotaxis protein-3, -1 α of monocyte inflammatory protein, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligands, collagen, colony thorn Swash the factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cell factor, epithelium neutrophilic granulocyte activation peptide -78, MIP-16, MCP-1, epidermal growth factor (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin(EPO) (EPO), epidermis stripping Detoxification element, factors IX, factor Ⅴ II, Factor IX, factor X, fibroblast growth factor (FGF), fibrinogen, fiber Connect albumen, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, promoting sexual gland hormone, growth factor, life Growth factor receptor body, grf, Hedgelog protein, hemochrome, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), Human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 Receptors, insdri, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-α, IFN-β, IFN-γ, interleukins (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), breast Siderophillin, LIF ELISA, luciferase, neural element, neutrophil inhibitory factor (nif) (NIF), oncostatin M, into Bone protein, oncoprotein, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, multi-effector, albumin A, Protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxain, feritin, SCF, atom synthesis Albumen, Soluble complement receptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, growth are adjusted Save element, Somat, growth hormone, streptokinase, super antigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic-shock syndrome toxin, thymosin α1, tissue Type Plasminogen Activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor α, tumor necrosis factor β, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, Mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, testosterone receptor, Aldosterone receptor, ldl receptor and cortisone.

In other or Additional examples of composition, the group for assigning improved detection feature is selected from the group being made of following object Group：Mark；Dyestuff；Affinity marker；Photoaffinity marks；Spin labeling；Fluorogen；Radioactive segment；It is combined with heavy atom Part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electron dense group；Magnetic Property group；Chromophore；Energy transfer agent；Detectable label and any combination thereof.

J. the example of functional group is added：Improve the therapeutic properties of polypeptide

Naturally occurring or non-natural amino acid polypeptides should be able to provide certain to the patient with particular condition, disease or symptom One treatment benefit.This treatment benefit should depend on many factors, and it includes (only for example)：The security overview of polypeptide, with And pharmacokinetics, pharmacology and/or the pharmacodynamics of polypeptide are (for example, water solubility, biological usability, serum half-life, treatment half It declines phase, immunogenicity, bioactivity or circulation time).In addition, it can be favourable to provide other functional groups to polypeptide, such as connect The cytotoxic compound or drug that connect may need to be connected to form homopolymer described herein with other polypeptides And hetero multimer.It is preferred that these modifications do not destroy the activity and/or tertiary structure of original polypeptide.Side described herein Method, composition, technology and strategy provide the solution of these problems.

Using method described herein, composition, technology and strategy, those skilled in the art can prepare with it is required The non-natural amino acid polypeptides containing oxime of homologous peptide, wherein the non-natural amino acid polypeptides containing oxime have the treatment spy of improvement Sign.In one embodiment, homologous non-natural amino acid polypeptide is prepared by biosynthesis.In another or Additional examples of composition, Alpha-non-natural amino acid by its structure be incorporated herein described in one of alpha-non-natural amino acid in.In another or additional implementation In example, alpha-non-natural amino acid is to be incorporated in end or middle position and be further incorporated to for locus specificity.

In one embodiment, such as there is the alpha-non-natural amino acid as obtained by prepared by biosynthesis required improvement to control Treat feature.In other or Additional examples of composition, alpha-non-natural amino acid includes the oxime key of the group with providing improved treatment feature. In other or Additional examples of composition, alpha-non-natural amino acid is more to form modified alpha-non-natural amino acid containing oxime through further modification Peptide, wherein the modification provides the oxime key of the group with providing improved treatment feature.In some embodiments, this group is direct It is bonded on alpha-non-natural amino acid, and in other embodiments, this group is bonded to non-natural amino by connecting subbase group On acid.In certain embodiments, this group is connected to by single chemical reaction on alpha-non-natural amino acid, in other embodiment In, it is necessary to which this group is connected on alpha-non-natural amino acid by series of chemical.It is preferred that assign improvement separation characteristic Group site under the reaction condition utilized is specifically bonded on the alpha-non-natural amino acid in non-natural amino acid polypeptides, And it is not bonded on naturally occurring amino acid.

In other or Additional examples of composition, gained non-natural amino acid polypeptides and GH supergene family members are homologous, however, Method, technology and composition described in this part can be applied to from the treatment feature of improvement can to benefit substantially any other are more Peptide, it is described comprising (only for example)：α -1 antitrypsins, angiostatin, anti-hemolytic factor, antibody, load fat egg In vain, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, C-X-C chemotactic factor (CF)s, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit coordinations Body, cell factor, CC chemotactic factor (CF)s, monocyte chemoattractant protein-1, monocyte chemoattractant protein -2, monocyte chemotactic egg In vain -3, -1 α of monocyte inflammatory protein, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligands, collagen, colony stimulating factor (CSF), Complement factor 5a, complement inhibitor, complement receptor 1, cell factor, epithelium neutrophilic granulocyte activation peptide -78, MIP-16, MCP- 1st, epidermal growth factor (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin(EPO) (EPO), exfoliative toxin,or exfoliatin, the factor IX, factor Ⅴ II, Factor IX, factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four Helix bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, promoting sexual gland hormone, growth factor, growth factor receptors, Grf, Hedgelog protein, hemochrome, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), the white egg of human serum In vain, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 Receptors, insdri, insulin-like growth factor (IGF), IGF-I, IGF- II, interferon (IFN), IFN-α, IFN-β, IFN-γ, interleukins (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL- 6th, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactotransferrin, leukaemia Inhibiting factor, luciferase, neural element, neutrophil inhibitory factor (nif) (NIF), oncostatin M, osteogenic protein, oncoprotein, The outer poison of paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, multi-effector, albumin A, Protein G, pth, pyrogenicity Plain A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxain, feritin, SCF, atom synthetic proteins, Soluble complement receptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, Growth hormone, streptokinase, super antigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroids swash Plain receptor, superoxide dismutase, toxic-shock syndrome toxin, thymosin α1, tissue type plasminogen activator, tumour Growth factor (TGF), tumor necrosis factor, tumor necrosis factor α, tumor necrosis factor β, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, P53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, testosterone receptor, aldosterone receptor, LDL by Body and cortisone.

In other or Additional examples of composition, the water solubility of the group improvement polypeptide of improved treatment feature is assigned；In other realities It applies in example, the binding property of the group improvement polypeptide；In other embodiments, the group provides new associativity to polypeptide Matter (includes (only for example) biotin group or biotin conjugated group).The water solubility of the group improvement polypeptide wherein Embodiment in, group is selected from water-soluble polymer described herein, and it includes (only for example) PEG polymer matrixes Group.In other or Additional examples of composition, the group is cytotoxic compound, and in other embodiments, the group is Drug.In other embodiments, bonded drug or cytotoxic compound can crack to incite somebody to action from non-natural amino acid polypeptides Drug or cytotoxic compound are delivered to required treatment position.In other embodiments, the group is the second polypeptide, is wrapped The non-natural amino acid polypeptides containing oxime containing (for example), further including (for example) has and the first non-natural amino The polypeptide of the identical amino acid structure of sour polypeptide.

In other or Additional examples of composition, the non-natural amino acid polypeptides containing oxime are the modified non-natural amino containing oxime Sour polypeptide.In other or Additional examples of composition, compared with homologous naturally occurring amino acid polypeptide, the alpha-non-natural amino acid containing oxime is more Peptide increases the biological usability of polypeptide.In other or Additional examples of composition, compared with homologous naturally occurring amino acid polypeptide, containing oxime Non-natural amino acid polypeptides increase polypeptide security overview.In other or Additional examples of composition, naturally deposited compared with homologous In amino acid polypeptide, the non-natural amino acid polypeptides containing oxime increase the water solubility of polypeptide.In other or Additional examples of composition, relatively In homologous naturally occurring amino acid polypeptide, the non-natural amino acid polypeptides containing oxime increase the treatment half-life period of polypeptide.Other or In Additional examples of composition, compared with homologous naturally occurring amino acid polypeptide, the non-natural amino acid polypeptides containing oxime increase the blood of polypeptide Clear half-life period.In other or Additional examples of composition, compared with homologous naturally occurring amino acid polypeptide, the alpha-non-natural amino acid containing oxime The circulation time of polypeptide protracting polypeptide.In other or Additional examples of composition, compared with homologous naturally occurring amino acid polypeptide, containing oxime Non-natural amino acid polypeptides adjust polypeptide activity.In other or Additional examples of composition, compared with homologous naturally occurring amino Sour polypeptide, the non-natural amino acid polypeptides containing oxime adjust the immunogenicity of polypeptide.

XI. the therapeutical uses through modified polypeptide

For convenience, " through modification or unmodified " non-native polypeptide described in this part it is general and/or with Particular instance describes.However, " through modification or unmodified " non-native polypeptide described in this part should not be limited only to this The general description or particular instance provided in part, but " through modification or unmodified " non-natural described in this part is more Peptide be equally fully adapted to it is all including at least one in Formulas I-XVIII, XXX-XXXIV (A and B) and XXXX-XXXXIII Scope in amino acid " through modification or it is unmodified " non-native polypeptide, it includes in description herein book, power It is any in Formulas I-XVIII, XXX-XXXIV (A and B) and the scope of XXXX-XXXXIII described in profit requirement and schema Minor or specific compound.

" through modification or unmodified " non-natural amino acid polypeptides described herein are (comprising its homopolymer and different Type polymer) there are multiple use, it includes (but not limited to)：Therapeutical uses, diagnostic uses, the purposes based on calibrating, industry Purposes, cosmetic use, phytobiology purposes, environmental application, energy production purposes and/or military use.As unrestricted Property explanation, " through modification or unmodified " following therapeutical uses of non-natural amino acid polypeptides are provided.

" through modification or unmodified " non-natural amino acid polypeptides described herein are suitable for treatment various diseases, disease Shape or disease.Administration " through modification or unmodified " non-natural amino acid polypeptides product described herein generates in the mankind Any activity confirmed by commercially available polypeptide formulations.The average magnitude of " through modification or unmodified " non-natural amino acid polypeptides product It can change and should especially be changed according to the suggestion and prescription of qualified physicians." through modification or unmodified " alpha-non-natural amino acid The exact amount of polypeptide should be selected preferentially according to following factor：The exact type for the symptom such as treated, the patient that is treated Other compositions in symptom and composition.The amount to be given can be easy to by those skilled in the art according to use " warp Modification is unmodified " therapies of non-natural amino acid polypeptides determines.

A. dispensing and medical composition

" through modification or unmodified " non-natural amino acid polypeptides described herein are suitable for treatment various diseases.It throws It is generated with " through modification or unmodified " non-natural amino acid polypeptides product described herein in the mankind by commercially available polypeptide Any activity that preparation confirms." through modification or unmodified " average magnitude of non-natural amino acid polypeptides product can change and especially It should change according to the suggestion and prescription of qualified physicians." through modification or it is unmodified " non-natural amino acid polypeptides it is definite Amount should be selected preferentially according to following factor：The exact type for the symptom treated, the symptom of the patient treated and composition In other compositions.The amount to be given can be easy to by those skilled in the art according to use " through modification or it is unmodified " therapies of non-natural amino acid polypeptides determines.

As described herein through modification or unmodified non-natural amino acid polypeptides (including (but not limited to) synthesis Enzyme, protein including one or more alpha-non-natural amino acids etc.) optionally for therapeutical uses, it includes (but it is unlimited In) combine with suitable pharmaceutical carrier.These compositions (for example) including therapeutically effective amount it is as described herein through modification or Unmodified non-natural amino acid polypeptides and pharmaceutically acceptable supporting agent or excipient.The supporting agent or excipient Including (but not limited to) brine, buffered saline, dextrose, water, glycerine, ethyl alcohol and/or its combination.It is allocated to adapt to throw Medicine pattern.In general, the method for administration protein is known in fields and it can be applied to as described herein Dispensing through modification or unmodified non-natural amino acid polypeptides.

According to well known method in fields, including it is one or more kinds of as described herein through modification or without The therapeutic combination of the non-natural amino acid polypeptides of modification optionally in it is one or more kinds of diseased it is appropriate in vitro and/or It in vivo tests to determine effect, tissue metabolism and estimation dosage in animal model.Specifically, dosage just begins by non-natural Amino acid is with respect to natural amino acid homologue (including (but not limited to) will be through modifying with comprising one or more non-natural ammonia The polypeptide of base acid is compared with natural amino acid polypeptide) activity, stability or other measurement is suitble to (that is, to be examined and determine in correlation In) measure.

Dispensing is by being commonly used in introducing molecule with any one of approach for finally being contacted with blood or histocyte Come carry out.As described herein through modification or unmodified non-natural amino acid polypeptides be optionally with it is a kind of or it is a kind of with Upper pharmaceutically acceptable supporting agent administration in any suitable manner together.It can obtain and be passed through as described herein to patient's administration Modification or the appropriate methodology of unmodified non-natural amino acid polypeptides, and although can be used more than one approach administration specific Composition, but particular approach usually provides than another way and more directly and more effectively acts on or react.

Particular composition of the pharmaceutically acceptable supporting agent part by institute's administration and the spy by being used for administration composition The method of determining determines.Accordingly, there exist the suitable formulas of a variety of medical compositions described herein.

Non-natural amino acid polypeptides described herein and composition including these polypeptides can by be suitable for protein or Any conventional route of peptide carrys out administration, it includes the parenteral administration of (but not limited to), such as injects, it includes (but not limited to) skins Lower injection or intravenous injection or any other form of injection or infusion.Polypeptide medical composition is (comprising described herein Various non-natural amino acid polypeptides) can by numerous approach come administration, it includes (but not limited to) is oral, in intravenous, peritonaeum, flesh Meat is interior, percutaneous, subcutaneous, local, sublingual or rectal.Including as described herein through modification or unmodified non-natural The composition of amino acid polypeptide also can be by liposome come administration.These dosing ways and proper formulation are usually fields Known to technical staff.Non-natural amino acid polypeptides described herein also can be individually or with other suitable components (comprising (but not It is limited to) pharmaceutical carrier) it is applied in combination.

That combines individually or with other suitable components is as described herein through modification or unmodified non-natural amino Sour polypeptide may be made as can be by sucking the aerosol formulations (that is, it " can be atomized ") come administration.Aerosol formulations, which can be inserted, to be added Pressure acceptable propellant in, such as dicholorodifluoromethane, propane, nitrogen with and the like.

Suitable for parenteral dispensing (such as, by intra-articular (intra-articular), intravenous, intramuscular, intracutaneous, peritonaeum And subcutaneous route) formula include aqueous and non-aqueous isotonic aseptic injectable solution, can containing antioxidant, buffer, Bacteriostatic agent and the solute for making formula isotonic with the blood of predetermined receptor；And aqueous and non-aqueous sterile suspensions, it can wrap Suspending agent-containing, solubilizer, thickener, stabilizer and preservative.The formula of packaged nucleic acid may be present in unit dose or In multiple dose sealing container (such as ampoule and bottle).

Parenteral dispensing and Intravenous administration are preferred medication administration method.Specifically, natural amino acid homologue is had been used for The dosing way of therapeutic agent (is situated between including (but not limited to) commonly used in EPO, IFN, GH, G-CSF, GM-CSF, IFN, leucocyte Those dosing ways of element, antibody and/or any other medicine delivering protein) and the formula that uses at present provide for such as Preferred dosing way and formula through modification or unmodified non-natural amino acid polypeptides described herein.

In the case of the composition and method that are described herein, the dosage in administration to patient is enough at any time in patient Middle generation advantageous treatment reaction.Dosage is by the effect of special formulation and used through modification or unmodified non-natural amino Activity, stability or the serum half-life of sour polypeptide and the symptom of patient and the weight or surface area of the patient to be treated To determine.Dosage size also depositing by the adjoint any harmful side effect of the administration special formulation in particular patient or its analog It is determined in, property and degree.

It is determining treating or preventing disease (including (but not limited to) cancer, genetic disease, diabetes, AIDS or its class Like disease) when be intended to administration formula effective quantity when, doctor's assessments blood plasma level, formula toxicity, the progress of disease and/ Or the generation of (if related) anti-non-natural amino acid polypeptides antibody.

Dosage in the patient of administration to (for example) 70 kilograms is usually being equivalent to the work suitable for compositions related change In the range of the dosage of property or the presently used treatment albumen matter of serum half-life.Medical formulation described herein can be by any (it includes antibody dispensing, vaccine dispensing, cytotoxic agent, natural amino acid polypeptide, nucleic acid, ucleotides for known conventional therapy Like object, biological response modifiers with and the like dispensing) carry out supplementary therapy situation.

For dispensing, medical formulation described herein is with by the LD-50 or ED-50 of associated formula and/or right Under various concentration (during it includes (but not limited to) when the quality and general health applied to patient) through modification or without The rate that the observation of any side effect of the non-natural amino acid polypeptides of modification is definite carrys out administration.Dispensing can by single-dose or Divided doses are realized.

If there is fever, feels cold or courbature in the patient of experience formula infusion, then him/her is made to receive suitable dosage Aspirin (aspirin), brufen (ibuprofen), acetaminophen (acetaminophen) or other pain/fever control Drug.Make experience infusion reaction (such as fever, courbature and feel cold) patient future with aspirin, acetaminophen or Medication in 30 minutes in advance before (include, but are not limited to) diphenhydramine (diphenhydramine) infusion.For to antipyretic and Antihistaminic without react rapidly it is more serious feel cold and courbature, use meperidine (Meperidine).Visual response it is serious Property is slowed or shut off neutropenia.

As described herein through modification or unmodified non-natural amino acid polypeptides can directly administration to mammal Person under inspection.It is offerd medicine by any one of approach for being commonly used in polypeptide being introduced into person under inspection.Warp as described herein Modification or unmodified non-natural amino acid polypeptides, which include, is suitable for oral, per rectum, part, sucking (comprising (but unlimited In) pass through aerosol), oral cavity (including (but not limited to) sublingual), Via vagina, parenteral (including (but not limited to) subcutaneous, muscle In interior, intracutaneous, intra-articular, pleura, in peritonaeum, big intracerebral, intra-arterial or intravenous), it is local (that is, skin and mucous membrane surface, Include tracheae surface) and administered transdermal those polypeptides, although in the case that it is any give most suitable approach should be depending on being controlled Depending on the property and seriousness of the symptom for the treatment of.Dispensing can be topically or systemically.Formula may be present in unit dose or multi-agent It measures in sealing container (such as ampoule and bottle).As described herein through modification or unmodified non-natural amino acid polypeptides It can be prepared in the unit dosage injectable form of pharmaceutically acceptable supporting agent (including (but not limited to) solution, suspension Or lotion) mixture in.It as described herein also can be by continuous through modification or unmodified non-natural amino acid polypeptides Infusion (minipump for using (include, but are not limited to) such as osmotic pumps), single bolus or slow release storage tank formula are matched somebody with somebody Side carrys out administration.

Include aqueous and non-aqueous solution, isotonic aseptic injectable solution suitable for the formula of dispensing, can contain antioxidant, Buffer, bacteriostatic agent and the solute for making formula isotonic；And aqueous and non-aqueous sterile suspensions, can include suspending agent, Solubilizer, thickener, stabilizer and preservative.Solution and suspension can be by aseptic powdery, particle and previous description species Tablet prepare.

It freezes as the technology of usually used presentation protein, is used for removing water from protein formulation of interest. (Freeze-drying) or lyophilized (lyophilization) is freeze-dried as first by be intended to dry material freeze and then By distilling to remove the process of ice or chilled solvent in vacuum environment.Excipient may be included in in advance lyophilized formula with Increase the stability of stability and/or improvement lyophilized products in storage in freeze-drying process.Pikal,M.Biopharm.3(9) 26-30 (1990) and Arakawa et al., Pharm.Res.8 (3):285-291(1991).

The spray drying of drug is also known to those skilled in the art.For example, referring to Broadhead, J. Et al., " The Spray Drying of Pharmaceuticals, " Drug Dev.Ind.Pharm, 18 (11 and 12), 1169-1206(1992).In addition to small-molecule drug, a variety of biomaterials are spray-dried and these materials include：Enzyme, blood Clearly, microorganism and yeast.The technology to be applicable in is spray-dried, because liquid pharmaceutical formulation can be changed into essence by it with one-step method Carefully, dustless or cohesion powder.Basic fundamental includes following four step：A) material solution is atomized into spraying；B) spraying-sky Gas contacts；C) spray drying is made；And d) dried product is separated with dry air.U.S. Patent No. 6,235,710 It is red thin come Prepare restructuring by being spray-dried with No. 6,001,800 (it is in being hereby incorporated by reference in their entirety) description Born of the same parents generate element.

Medical composition described herein may include pharmaceutically acceptable supporting agent, excipient or stabilizer.Medicine Acceptable supporting agent part is determined by the particular composition of institute's administration and the ad hoc approach for administration composition on.Cause This, there are a variety of medical compositions described herein through modification or unmodified non-natural amino acid polypeptides (optionally Include pharmaceutically acceptable supporting agent, excipient or stabilizer) suitable formula, (referring to (for example) Remington:The Science and Practice of Pharmacy, the 19th edition (Easton, Pa.:Mack Publishing Company, 1995)；Hoover,John E.,Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton,Pennsylvania 1975；Liberman, H.A. and Lachman, L. are compiled, Pharmaceutical Dosage Forms,Marcel Decker,New York,N.Y.,1980；And Pharmaceutical Dosage Forms and Drug Delivery Systems, the 17th edition (Lippincott Williams＆Wilkins, 1999)).Suitable supporting agent includes Buffer, containing succinate, phosphate, borate, HEPES, citrate, imidazoles, acetate, bicarbonate and its His organic acid；Antioxidant, it includes (but not limited to) ascorbic acid；Low molecular weight polypeptide, it includes (but not limited to) to be less than The those polypeptides of about 10 residues；Protein, it includes (but not limited to) seralbumin, gelatin or immunoglobulins；It is hydrophilic Property polymer, it includes (but not limited to) polyvinylpyrrolidones；Amino acid, it includes (but not limited to) glycine, glutamy Amine, asparagine, arginine, histidine or histidine derivative, methionine, glutamic acid or lysine；Monose, disaccharides and Other carbohydrate, it includes (but not limited to) trehalose, sucrose, glucose, mannose or dextrin；Chelating agent, it includes (but not limited to) EDTA；Bivalent metal ion, it includes (but not limited to) zinc, cobalt or copper；Sugar alcohol, it is sweet it includes (but not limited to) Reveal sugar alcohol or D-sorbite；Salt-forming counterion, it includes (but not limited to) sodium；And/or nonionic surfactant, it includes (but not limited to) Tween^TM(including (but not limited to) 20 (polysorbate of Tween 80 (polysorbate80) and Tween 20))、Pluronics^TMAnd other general Lip river niacins (pluronic acid) are (including (but not limited to) general Lip river niacin F68 (Pa Luoshamu 188 (poloxamer 188))) or PEG.Surfactant is suitble to include and is such as (but not limited to) based on polyoxygenated Ethylene-polypropylene oxide-polyethylene glycol oxide (that is, (PEO-PPO-PEO)) or polypropylene oxide-polyethylene glycol oxide-polyoxygenated third The polyethers of alkene (that is, (PPO-PEO-PPO)) or its combination.PEO-PPO-PEO and PPO-PEO-PPO is with brand name PluronicsTM, R-PluronicsTM, TetronicsTM and R-TetronicsTM (BASF Wyandotte Corp., Wyandotte, Mich.) it is commercially available and be further described in U.S. Patent No. 4,820,352 (its be by reference all It is incorporated herein) in.Other ethylene/polypropylene block polymers can be suitable surfactant.Surfactant or surface The combination of activating agent can be used for resisting one or more kinds of stress (including (but not limited to) the stress from stirring) to stablize PEGylated non-natural amino acid polypeptides.Some above-mentioned surfactants can be referred to " swelling agent ".Some are also known as " tension Conditioning agent ".

It (includes through modification or unmodified non-natural amino acid polypeptides as described herein and is bonded to water-soluble polymeric Those non-natural amino acid polypeptides on object (such as PEG)) also can be by sustained release system or as sustained release system portion Divide and carry out administration.Sustained-release composition includes (include, but are not limited to), and for formed article, (it includes (but not limited to) films or micro- Capsule) form semipermeable polymer matrices.Sustained-release matrix includes biocompatible materials, such as poly- (2- ethoxys Methacrylate) (Langer et al., J.Biomed.Mater.Res., 15:167-277(1981)；Langer, Chem.Tech.,12:98-105 (1982)), ethylene vinyl acetate (Langer et al., as before) or poly- D- (-) -3- hydroxyls Butyric acid (EP 133,988), polyactide (polylactic acid) (U.S. Patent No. 3,773,919；EP 58,481), polyglycolide (second The polymer of alkyd), polylactide-co-glycolide (copolymer of lactic acid and glycolic), polyanhydride, Pidolidone and γ-ethyl- The copolymer (U.Sidman et al., Biopolymers, 22,547-556 (1983)) of Pidolidone ester, poly- (original) ester, polypeptide, Hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, aliphatic acid, phosphatide, polysaccharide, nucleic acid, polyaminoacid, amino acid are (such as Phenylalanine, tyrosine, isoleucine), polynucleotide, polyvinyl propylene, polyvinylpyrrolidone and poly- silica.Continue It discharges composition and also includes liposomal encapsulated compound.Containing compound liposome is by method preparation known per se： DE 3,218,121；Epstein et al., Proc.Natl.Acad.Sci.U.S.A., 82:3688-3692(1985)；Hwang etc. People, Proc.Natl Acad.Sci.U.S.A., 77:4030-4034(1980)；EP 52,322；EP 36,676；EP 88, 046；EP 143,949；EP 142,641；Japanese patent application case 83-118008；U.S. Patent No. 4,485,045 and the 4th, No. 544,545；And EP 102,324.

Liposomal encapsulated polypeptide can be prepared by the method described in documents below：For example, DE 3,218,121； Epstein et al., Proc.Natl Acad.Sci.U.S.A., 82:3688-3692(1985)；Hwang et al., Proc.Natl Acad.Sci.U.S.A.,77:4030-4034(1980)；EP 52,322；EP 36,676；EP 88,046；EP 143,949； EP 142,641；Japanese patent application case 83-118008；U.S. Patent No. No. 4,485,045 and No. 4,544,545；And EP 102,324.The composition and size of liposome are well known or can be by those skilled in the art rule of thumb easily It determines.Some examples of liposome are described in (for example) Park JW et al., Proc.Natl.Acad.Sci.USA 92:1327- 1331(1995)；Lasic D and Papahadjopoulos D (eds.):MEDICAL A_PPLICATIONS _OF L_IPOSOMES(1998)； Drummond DC et al., Liposomal drug delivery systems for cancer therapy, in Teicher B (eds.):In CANCER DRUG DISCOVERY AND DEVELOPMENT (2002)；Park JW et al., Clin.Cancer Res.8:1172-1181(2002)；Nielsen UB et al., Biochim.Biophys.Acta 1591 (1-3):109-118 (2002)；Mamot C et al., Cancer Res.63:3154-3161(2003).

The dosage of administration to patient should be enough to exist at any time in the case of the composition, formula and the method that are described herein Beneficial reaction is generated in person under inspection.It is in general, as described herein through modification or unmodified per the parenteral administration of dosage Non-natural amino acid polypeptides total medical effective quantity in daily about 0.01 μ g of per kilogram patient's weight to about 100 μ g or about In the range of 0.05mg to about 1mg, although it should be through treated judgement.Administration frequency also is subjected to treatment and judges, and than through criticizing Mutatis mutandis frequency higher or frequency in the commercial product of the mankind is lower.In general, polymer:Polypeptide binding element is (comprising (only citing comes Say) PEGylated polypeptide as described herein) it can be by any one of above-mentioned dosing way come administration.

Example

Example 1

The synthesis of the amino acid containing carbonyl of presentation in this Examples detail Fig. 4.Alpha-non-natural amino acid containing carbonyl be as It is prepared described in Fig. 4.

Example 2

The synthesis through protecting the amino acid containing azanol of presentation in this Examples detail Fig. 5 a.Through protecting the non-day containing azanol Right amino acid is prepared as described in Fig. 5 a.

Example 3

The synthesis of the amino acid containing azanol of presentation in this Examples detail Fig. 5 b.Alpha-non-natural amino acid containing azanol be as It is prepared described in Fig. 5 b.

Example 4

The synthesis of the amino acid containing azanol of presentation in this Examples detail Fig. 5 c.Alpha-non-natural amino acid containing azanol be as It is prepared described in Fig. 5 c.

Example 5

The synthesis of the amino acid containing oxime of presentation in this Examples detail Fig. 5 d.Alpha-non-natural amino acid containing oxime is such as Fig. 5 d Described in prepare.

Example 6

The synthesis of the amino acid containing oxime of presentation in this Examples detail Fig. 6 a.Alpha-non-natural amino acid containing oxime is such as Fig. 6 a Described in prepare.

Example 7

The synthesis of the amino acid containing oxime of presentation in this Examples detail Fig. 6 b.Alpha-non-natural amino acid containing oxime is such as Fig. 6 b Described in prepare.

Example 8

The synthesis of the amino acid containing oxime of presentation in this Examples detail Fig. 6 c.Alpha-non-natural amino acid containing oxime is such as Fig. 6 c Described in prepare.

Example 9

The synthesis of the amino acid containing carbonyl of presentation in this Examples detail Figure 24.

Synthesis

Ether (60mL) is added into NaOH solution (40mL, 25 volume %) at 0 DEG C.Blast shield object is placed in reaction Before flask.N- nitroso-N-methylureas (6.0g, 57.9mmol) are added into gained mixture through 3 minutes points 3 parts.At 0 DEG C Under be stirred to react 10 minutes.Then ether layer is made to be separated with sodium hydroxide layer.Through 5 minutes by organic layer portionwise (about 6 times addition) It is added in solution of the N-Boc-4- hydroxymethyls phenylalanine (7.5g, 25.4mmol) in anhydrous THF (20mL) up to Beginning substance is completely disappeared and (monitored by TLC).Then 5 drop glacial acetic acid of addition is with stopped reaction.It is removed by rotary evaporation organic molten After agent, ethyl acetate is added.By organic layer with NaHCO₃Saturated solution, H₂O and brine wash successively, through anhydrous MgSO after₄ Drying filters and concentrates to generate white powdered product (5.9g, 75%).

Synthesis

To alcohol (6.0g, 19.4mmol) and pyridine (12mL, 150mmol) in CH at 0 DEG C₂Cl₂In (400mL) through stirring It mixes addition Dai Si-Martin in solution and crosses iodine alkane (Dess-Martin periodinane) (14.2g, 33.4mmol).At room temperature The mixture was stirred overnight.It will then react with Na₂S₂O₃-NaHCO₃Saturated aqueous solution (1:1,300mL) stop and use CH₂Cl₂ Extraction.Organic layer is combined and with H₂O and salt water washing, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.It is logical Cross flash chromatography (silica, 1:100-1:1 hexane:EtOAc) purifying residue obtain white solid-like aldehyde product (5.48g, 92%).

Synthesis

Acetic acid hydrazides (1.7g, 20mmol) are added into solution of the aldehyde (3.07g, 10mmol) in EtOH (40mL).It will Reaction mixture is stirred at room temperature 30 minutes and concentrates.H is added into residue₂O (200mL), then adds CH₂Cl₂.It will Organic layer is separated and concentrated in a vacuum.By flash chromatography (silica, 3:7-1:9 hexanes:EtOAc) purifying residue is in The product (3.29g, 90%) of white solid.

Synthesis

At 0 DEG C LiOH is added into solution of the above-mentioned methyl esters (3.29g, 9.1mmol) in dioxane (10mL) (10mL, 1N).Mixture is stirred at the same temperature 1 it is small when and then by add citric acid (5g) stopped reaction and with H₂O dilutes.Mixture is extracted with EtOAc.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Dry, mistake It filters and concentrates to obtain white solid (3.05g, 96%).

Synthesis

To above-mentioned sour (3.02g, 8.6mmol) in CH at 0 DEG C₂Cl₂Trifluoroacetic acid is added in solution in (20mL) (20mL).Reaction mixture is stirred at 0 DEG C 2 it is small when and concentration.MeOH (1mL) is added into residue, then adds HCl (in 2.0mL, 4N, Yu dioxane).Then addition ether (200mL) with precipitate in yellow solid product (2.07g, 83%).

Example 10

The synthesis of the amino acid containing dicarbapentaborane of presentation in this Examples detail Figure 25.

Synthesis

Added at 0 DEG C into agitated solution of the amine (10g, 34mmol) in DMF (70mL) pyruvic acid (5mL, 72mmol), 1- (3- dimethylaminopropyls) -3- ethyl-carbodiimide hydrochlorides (EDC, 20g, 104mmol), 1- hydroxy benzenes And triazole hydrate (HOBt, 85g, 71mmol) and N, N- diisopropyl ethyl amine (DIEA, 35mL, 200mmol).It will mixing Object be stirred at room temperature 6 it is small when and then with aqueous citric acid solution (5%, 500mL) stop and with EtOAc (500mL) extract.It will Organic layer is with H₂O and brine wash successively, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.Pass through flash chromatography (silica, 3:1-1:1 hexane:EtOAc) purifying residue obtains the product (4.78g, 40%) in solid-like.

Synthesis

At 0 DEG C LiOH is added into solution of the above-mentioned methyl esters (2.96g, 8.1mmol) in dioxane (10mL) (10mL, 1N).Mixture is stirred at the same temperature 3 it is small when.Then will reaction with aqueous citric acid solution (5%) stop and with EtOAc dilutes.Organic layer is separated and with H₂O and brine wash successively, through anhydrous Na after₂SO₄Dry, filtering and concentrate with Obtain the product (2.87g, 100%) in yellow solid.

Synthesis

To above-mentioned sour (2.05g, 5.9mmol) in CH at 0 DEG C₂Cl₂Trifluoroacetic acid is added in solution in (10mL) (10mL).Stir the mixture for 2 it is small when and concentrate in a vacuum.HCl (in 1mL, 4N, Yu dioxane) is added into residue, Then ether (400mL) is added.Collect the sediment (1.38g, 82%) of white solid-like.

Example 11

The synthesis of the amino acid containing dicarbapentaborane of presentation in this Examples detail Figure 26.

Synthesis

At 90 DEG C to 4'- methyl phenyl ketones (20g, 122mmol) and N-bromosuccinimide (NBS, 23g, 130mmol) 2,2'- azobis isobutyronitriles (AIBN, 0.6g, 3.6mmol) are added in the solution in benzene (300mL).By gained Solution is heated to being refluxed overnight.Then the reaction is cooled to room temperatures.By brown solution with H₂O and brine wash successively, are passed through after Anhydrous Na₂SO₄Drying is filtered and concentrated in a vacuum.Residue is crystallized from hexane to obtain the production in pale-yellow solid Object (27g, 87%).

Synthesis

Acetamido the third two is added into solution of the EtONa (14.5g, 203mmol) in EtOH (400mL) at 0 DEG C Diethyl phthalate (39g, 180mmol) then adds solution of the above-mentioned bromide (27g, 119mmol) in EtOH (100mL).It will Gained mixture be heated to reflux last 1 it is small when and stopped with citric acid (30g) and use H₂O (300mL) dilutes.It moves in a vacuum After most of solvent, with EtOAc extracted residues.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄ Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 10:1-3:1 hexane:EtOAc) purifying residue is in The product (37g, 88%) of yellow solid.

Synthesis

At 0 DEG C Br is added into solution of the ketone (5g, 13.8mmol) in ether (100mL)₂(0.8mL, 15.6mmol).Be stirred at room temperature mixture 3 it is small when and then with NaHCO₃Saturated aqueous solution stops.With Et₂O extraction mixing Object.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Dry, filtering and concentrate in a vacuum be in The product (5.4g, 88%) of yellow solid, need not be further purified and can be directly used in next step.

Synthesis

To α-bromoketone (5.4g, 12.2mmol) and Na₂CO₃Add in the solution of (2.0g, 18.9mmol) in DMSO (20mL) Add KI (2.1g, 13.2mmol).When 90 DEG C stirring mixture 28 is small under nitrogen atmosphere.It will then react with H₂O stop and with EtOAc dilutes.Organic layer is separated and with H₂O and brine wash successively, through anhydrous Na after₂SO₄Drying is filtered and in vacuum Middle concentration.By flash chromatography (silica, 6:1-1:10 hexanes:EtOAc residue) is purified to obtain the product in solid-like (1.12g, 24%).

Synthesis

By diketone (1.12g, 3.0mmol), in dense HCl, (solution in 10mL) He dioxane (10mL) is heated to flowing back Night.After removing solvent in a vacuum, MeOH (3mL) is added to dissolve residue.Then addition ether (300mL) is in shallow to precipitate The product (302mg, 42%) of yellow solid.

Example 12

The synthesis of the amino acid containing dicarbapentaborane of presentation in this Examples detail Figure 27.

Synthesis

To C at 0 DEG C₃H₇MgCl (2M, 50mmol) is made an addition in the solution in ether (25mL) in ether (50mL) Benzaldehyde (5mL, 42.5mmol).Acquired solution is stirred at 0 DEG C 30 minutes.It will then react with NH₄Cl saturated solutions stop And it is diluted with ether.Organic layer is separated and with H₂O and brine wash successively, through anhydrous Na after₂SO₄Drying is filtered and true Aerial concentration can be directly used in next reaction to obtain crude product (7.2g), without purifying.

Synthesis

To above-mentioned alcohol (7.2g, 43.9mmol) and pyridine (7mL, 86.7mmol) in CH at 0 DEG C₂Cl₂In (300mL) Dai Si-Martin is added in solution and crosses iodine alkane (19.2g, 45.3mmol).Gained mixture is stirred overnight and with Na₂S₂O₃Saturation Aqueous solution and NaHCO₃Saturated aqueous solution (1:1) stop.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄ Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 8:1-4:1 hexane:EtOAc) purifying residue is in Product (the 6.28g, for two steps for 91%) of colorless oil.

Synthesis

To above-mentioned ketone (4.43g, 27.3mmol) and N-bromosuccinimide (NBS, 5.5g, 30.9mmol) at 90 DEG C 2,2'- azobis isobutyronitriles (AIBN, 0.2g, 1.2mmol) are added in solution in benzene (150mL).Acquired solution is heated To being refluxed overnight and be then cooled to room temperature.By brown solution with H₂O and brine wash successively, through anhydrous Na after₂SO₄Dry, It filters and concentrates in a vacuum.Residue is crystallized from hexane to obtain the product (6.21g, 95%) of white solid-like.

Synthesis

Acetamido the third two is added into solution of the EtONa (2.5g, 34.9mmol) in EtOH (200mL) at 0 DEG C It is molten in EtOH (100mL) then to add above-mentioned bromide (6.2g, 25.8mmol) for diethyl phthalate (6.7g, 30.9mmol) Liquid.By gained mixture be heated to reflux last 1 it is small when and then with citric acid (9g) stop and with H₂O dilutes.Remove big portion After dividing solvent, with EtOAc extracted residues.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Dry, mistake It filters and concentrates in a vacuum.By flash chromatography (silica, 4:1-2:1 hexane:EtOAc residue) is purified to obtain in light yellow The product (8.92g, 92%) of solid-like.

Synthesis

Br is added into solution of the above-mentioned ketone (1.4g, 3.71mmol) in HOAc (50mL)₂(0.7mL, 13.6mmol). Mixture is stirred at room temperature overnight and then with NaHCO₃Saturated aqueous solution stops.With Et₂O extracts mixture.By organic layer With H₂O and brine wash successively, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 5:1-3:2 hexanes:EtOAc residue) is purified to obtain the product (1.23g, 73%) in yellow solid.

Synthesis

To α-bromoketone (1.12g, 2.46mmol) and Na₂CO₃In the solution of (0.4g, 3.77mmol) in DMSO (30mL) Add KI (0.45g, 13.2mmol).Mixture is stirred overnight at 90 DEG C and then with citric acid (2g) and H₂O(200mL) Stop.Mixture is extracted with EtOAc.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Dry, filtering and It concentrates in a vacuum.By flash chromatography (silica, 6:1-1:10 hexanes:EtOAc) purifying residue obtains α-hydroxyl in oily Base ketone (0.62g, 64%).

To above-mentioned alcohol (0.62g, 1.58mmol) and pyridine (0.5mL, 6.19mmol) in CH at 0 DEG C₂In Cl (100mL) Solution in addition Dai Si-Martin cross iodine alkane (0.9g, 2.12mmol).Gained mixture is stirred overnight and then with Na₂S₂O₃ Saturated aqueous solution and NaHCO₃Saturated aqueous solution (1:1) stop.By organic layer with H₂O and brine wash successively, through anhydrous after Na₂SO₄Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 9:1-3:2 hexanes:EtOAc) purify residue with Obtain the product (287mg, for two steps for 30%) in yellow oily.

Synthesis

By above-mentioned diketone (272mg, 0.7mmol), in dense HCl, (mixture in 10mL) He dioxane (10mL) is heated to It is refluxed overnight.After removing solvent in a vacuum, MeOH (1mL) is added to dissolve residue.Then addition ether (200mL) is with heavy The product (162mg, 81%) to form sediment in yellow solid.

Example 13

The synthesis of the amino acid containing dicarbapentaborane of presentation in this Examples detail Figure 28.These compounds are such as institute in Figure 28 Show to synthesize.

Example 14

This Examples detail is through clone of the modified polypeptide in Escherichia coli and expression.Using including orthogonal tRNA (O- TRNA) and the introduced translation system expression of orthogonal aminoacyl tRNA synzyme (O-RS) contains the more of alpha-non-natural amino acid Peptide.O-RS is preferentially with the aminoacylated O-tRNA of alpha-non-natural amino acid.Next, alpha-non-natural amino acid is inserted into polypeptide by translation system In, encode selection codon to respond.The poly-nuclear glycosides of amino acid and O-tRNA and O-RS suitable for being incorporated to alpha-non-natural amino acid Acid sequence is described in the United States Patent (USP) of entitled " In vivo Incorporation of Unnatural Amino Acids " Application case the 10/126,927th and entitled " Methods and Compositions for the Production of The U.S. Patent Application No. 10/126,931 of Orthogonal tRNA-Aminoacyl tRNA Synthetase Pairs " In, these documents are to be incorporated herein by reference.Following O-RS and O-tRNA sequences can also be used：

To contain through modifier and orthogonal aminoacyl tRNA synzyme/tRNA to (having to required alpha-non-natural amino acid Specificity) plasmid conversion Escherichia coli allow alpha-non-natural amino acid locus specificity being incorporated in polypeptide.At 37 DEG C containing There are the inverted Escherichia coli grown in the culture medium of the specific alpha-non-natural amino acid between 0.01mM-100mM with height The fidelity and efficiency of degree express modified polypeptide.The polypeptide containing alpha-non-natural amino acid marked through His is by large intestine bar Bacterium host cell is generated with inclusion body or aggregate form.These aggregations are dissolved and passed through in 6M guanidine hydrochlorides under Denaturing Affinity purification.In 50mM TRIS-HCl (pH 8.0), 40 μM of CuSO₄With 2% (w/v) N- sarcosyls (Sarkosyl) stayed overnight at 4 DEG C by progress refolding of dialysing.Then by the substance with 20mM TRIS-HCl (pH 8.0)、100mM NaCl、2mM CaCl₂Dialysis then removes His- labels.Referring to Boissel et al., J.Biol.Chem., (1993)268:15983-93.It is in the art for known to and black by SDS-PAGE, west for the method for purified polypeptide Point analysis or electron spray ionisation ion trap mass spectrometry and its similar approach confirm.

Example 15：Test alpha-non-natural amino acid

This example provide four tests that some illustrative alpha-non-natural amino acids are carried out as a result, to help to predict it The property being incorporated in non-natural amino acid polypeptides.

Example 16：Test alpha-non-natural amino acid

This example provide to the pH stability that some illustrative alpha-non-natural amino acids carry out test as a result, to contribute to Predict that it is incorporated to the property in non-natural amino acid polypeptides.

Example 17

The synthesis of the amino acid containing dicarbapentaborane of presentation in this Examples detail Figure 29.

Synthesis

To amino acid pAF (10g, 41.1mmol) in H₂O- dioxanes (300mL, 1:1) NaHCO is added in the solution in₃ (12g, 142.9mmol) and Boc₂O (12g, 55.0mmol).By mixture be stirred at room temperature 7 it is small when and then with citric acid Stop.Mixture is extracted with EtOAc.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Dry, filtering and It concentrates to obtain the N-Boc-pAF (13.7g, quantitative) of white solid-like.

Synthesis

Ether (60mL) is added into NaOH (40mL, 25 volume %) at 0 DEG C.Blast shield object is placed in reaction flask Front.N- nitroso-N-methylureas (6.0g, 57.9mmol) are added into gained mixture through 3 minutes points 3 parts.It is stirred at 0 DEG C Mix reaction 10 minutes.Then ether layer is made to be separated with sodium hydroxide layer.Through 5 minutes, by organic layer, (about 6 additions) was added portionwise Into solution of the N-Boc-pAF (5.0g, 16.2mmol) in anhydrous THF (20mL) until initial substance is completely disappeared (by TLC Monitoring).Then 5 drop glacial acetic acid of addition is with stopped reaction.After removing organic solvent by rotary evaporation, ethyl acetate is added. By organic layer with NaHCO₃Saturated solution, H₂O and brine wash successively, through anhydrous MgSO after₄Drying filters and concentrates to produce Raw white powder (4.1g, 80%).

Synthesis

The pAF (3.82g, 11.9mmol) through protection is slowly added into t-BuOK (60mL, 1.0M, in THF) in new Solution in the methyl propionate (20mL, 208mmol) of distillation.Gained mixture is stirred at room temperature 30 minutes and molten with citric acid Liquid (10%, 300mL) stops.Mixture is extracted with EtOAc.By organic layer with H₂O and salt water washing, through anhydrous Na after₂SO₄ Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 4:1-1:1 hexane:EtOAc residue) is purified to obtain The product (3.89g, 87%) of white solid-like.

Synthesis

Added at 0 DEG C into solution of the above-mentioned methyl esters (1.12g, 2.97mmol) in dioxane (4mL) LiOH (4mL, 1N).Mixture is stirred at 0 DEG C 3 it is small when and stopped with aqueous citric acid solution (5%, 200mL) and diluted with EtOAc.To have Machine layer separates and with H₂O and brine wash successively, through anhydrous Na after₂SO₄Drying filters and concentrates to obtain white solid (1.02g, 94%).

Synthesis

To above-mentioned sour (1.0g, 2.75mmol) in CH at 0 DEG C₂Cl₂Trifluoroacetic acid is added in solution in (10mL) (10mL).Mixture is stirred at 0 DEG C 2 it is small when and then concentration.MeOH (1mL) is added into residue, then adds HCl (in 1.5mL, 4N, Yu dioxane).Then addition ether (200mL) with precipitate the product of white solid-like (701mg, 96%).

Example 18

The synthesis of the amino acid containing dicarbapentaborane of presentation in this Examples detail Figure 30.

Synthesis

The pAF (1.09g, 3.4mmol) through protection is slowly added into t-BuOK (15mL, 1.0M, in THF) in difluoro Solution in methyl acetate (6mL, 68.7mmol).By gained mixture be stirred at room temperature 30 minutes and with citric acid (5g, 25.4mmol) stop and with H₂O dilutes.Mixture is extracted with EtOAc.By organic layer with H₂O and brine wash successively, are passed through after Anhydrous Na₂SO₄Dry, filtering and concentration.By flash chromatography (silica, 20:1-3:2 hexanes:EtOAc residue) is purified to obtain To the product (1.27g, 94%) in brown solid shape.

Synthesis

At 0 DEG C LiOH is added into solution of the above-mentioned methyl esters (1.26g, 3.17mmol) in dioxane (30mL) (30mL, 1N).Mixture is stirred at 0 DEG C 0.5 it is small when and stopped and with H with citric acid (10g, 51mmol)₂O dilutes.With EtOAc extracts mixture.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Dry, filtering and concentration.It is logical Cross flash chromatography (silica, 100:1-10:1CH₂Cl₂:MeOH, 0.5%HOAc) purifying residue with obtain brown oil (1.19g, 98%).

Synthesis

To above-mentioned sour (1.19g, 3.1mmol) in CH at 0 DEG C₂Cl₂Trifluoroacetic acid is added in solution in (15mL) (15mL).Stir the mixture for 0.5 it is small when and concentration.Into residue add MeOH (2mL), then add HCl (2mL, 4N, In Yu dioxanes).Ether (200mL) is then added to precipitate the product of white solid-like (0.82mg, 82%).

Example 19

The synthesis of the PEG reagents containing azanol of presentation in this Examples detail Figure 12 a.PEG reagents containing azanol are such as figure It is prepared described in 12a.

Example 20

The synthesis of the PEG reagents containing azanol of presentation in this Examples detail Figure 12 b.PEG reagents containing azanol are such as figure It is prepared described in 12b.

Example 21

The synthesis of the PEG reagents containing azanol of presentation in this Examples detail Figure 12 c.PEG reagents containing azanol are such as figure It is prepared described in 12c.

Example 22

The synthesis of the PEG reagents containing azanol of presentation in this Examples detail Figure 12 d.PEG reagents containing azanol are such as figure It is prepared described in 12d.

Example 23

The synthesis of the PEG reagents containing azanol of presentation in this Examples detail Figure 13.PEG reagents containing azanol are such as Figure 13 Described in prepare.

Example 24

The synthesis of the PEG reagents containing azanol of presentation in this Examples detail Figure 14.PEG reagents containing azanol are such as Figure 14 Described in prepare.

Example 25

The synthesis of the PEG reagents containing azanol of presentation in this Examples detail Figure 15.PEG reagents containing azanol are such as Figure 15 Described in prepare.

Example 26

The synthesis of the PEG reagents containing azanol of presentation in this Examples detail Figure 16 a.PEG reagents containing azanol are such as figure It is prepared described in 16a.

Example 27

The synthesis of the PEG reagents containing azanol of presentation in this Examples detail Figure 16 b.PEG reagents containing azanol are such as figure It is prepared described in 16b.

Example 28

The synthesis of the connexon reagent containing azanol of presentation in this Examples detail Figure 18.Connexon reagent containing azanol is Preparation as shown in Figure 18.

Example 29

The synthesis of double (4- (bromomethyl) phenyl) disulphanes (1) of the 1,2- of presentation in this Examples detail Figure 36.In nitrogen Added under pressure into the round-bottomed flask through oven drying with stirring rod p-methylphenyl disulphide (5.0g, 20.3mmol), N-bromosuccinimide (8.6g, 48.4mmol) and 60mL anhydrous benzenes.Solution is heated to 95 DEG C.With portion Add azobis isobutyronitrile (.106g .64mmol).Will reaction reflux 16 it is small when.Solvent is removed and by brown by rotary evaporation Solid is dissolved in 100mL ethyl acetate.With saturated aqueous solution of sodium bicarbonate (2 × 50mL), deionized water (1 × 50mL) and salt Water (1 × 50mL) washing reaction mixture successively.Organic layer is separated and through anhydrous magnesium sulfate is dry, filtering and dense under reduced pressure Contracting.By using Biotage Inc.HORIZON^TMThe silica chromatogram purification crude product of chromatographic system, concentrate appropriate fraction and After removing trace solvent (vacuum pump), obtain white solid-like double (4- (bromomethyl) phenyl) disulphanes of 1,2- (2.1g, 25%).It obtains¹H NMR spectras data and mass spectrum.Reaction is repeated to obtain the product of (2.0g, 23%).

Example 30

Double (4- diethyl -2- acetamidomalonic acids ester) phenyl disulphanes of the 1,2- of presentation in this Examples detail Figure 36 (2) synthesis.Acetamidomalonic acid is added into the round-bottomed flask through oven drying with stirring rod under nitrogen pressure Diethylester (6.48g, 30mmol) and the anhydrous EtOH of 50mL.Ethoxyquin sodium (2.6g, 38mmol) is added into solution with portion.It will Reaction is cooled to 0 DEG C.Double (4- (bromomethyl) phenyl) disulphanes (4.1g, 10.1mmol) of 1,2- are dissolved in 20mL 1: It is added in 1EtOH/THF and by charging hopper through 1 process when small in cold soln.It removes ice bath and is stirred at room temperature anti- Answer 6 it is small when.Solvent is removed by rotary evaporation and red solid is dissolved in 100mL ethyl acetate.With 5% citric acid solution (2 × 50mL), deionized water (1 × 50mL) and brine (1 × 50mL) washing reaction mixture successively.Organic layer is separated and passed through Anhydrous magnesium sulfate is dry, filters and is concentrated under reduced pressure.By using the silica of Biotage Inc.HORIZONTM chromatographic systems Chromatogram purification crude product after concentrating appropriate fraction and removing trace solvent (vacuum pump), obtains 1,2- in yellow solid Double (4- diethyl -2- acetamidomalonic acids ester) phenyl disulphanes (5.0g, 73%).It obtains¹H NMR spectras data, HPLC Trace and mass spectrum.

Example 31

Double (the synthesis of 4- (2- amino -3- propionic acid) phenyl disulphanes (3) of the 1,2- of presentation in this Examples detail Figure 36. Double (4- diethyl -2- the acetamides of 1,2- are added into the round-bottomed flask through oven drying with stirring rod under nitrogen pressure Propylmalonic acid ester group) phenyl disulphanes (1.0g, 1.4mmol), HCl (8mL, 12M) and 8mL Isosorbide-5-Nitraes-dioxane.It stirs under reflux Mix reaction 16 it is small when.Solvent is removed by rotary evaporation and vacuum pump to obtain double (4- (the 2- ammonia of the thick 1,2- of transparent oily Base -3- propionic acid) phenyl disulphanes (0.75g, 135%).It obtains¹H NMR spectras data and mass spectrum.

Example 32

Double (4- (2- amino -3- propionic acid) benzene of bis- tertbutyloxycarbonyl -1,2- of the N of presentation in this Examples detail Figure 36, N'- The synthesis of base disulphanes (4).To double (4- (2- amino -3- propionic acid) phenyl disulphanes of 1,2- in dry round-bottomed flask - dioxane of addition 5mL Isosorbide-5-Nitraes, 5mL deionized waters, di-tert-butyl dicarbonate (.65g, 3.0mmol) in (0.75g, 1.9mmol) With sodium acid carbonate (0.98g, 12mmol).Be stirred at room temperature reaction 16 it is small when.Solvent and will be transparent is removed by rotary evaporation Oil is dissolved in 100mL ethyl acetate.With 5% citric acid solution (5mL × 2), deionized water (50mL) and brine (50mL) according to Secondary washing reaction mixture.By organic layer separate and through anhydrous magnesium sulfate it is dry, filter and be concentrated under reduced pressure.By using Biotage Inc.HORIZON^TMThe silica chromatogram purification crude product of chromatographic system is concentrating appropriate fraction and is removing trace solvent After (vacuum pump), the N of white solid-like, double (4- (2- amino -3- propionic acid) phenyl two of bis- tertbutyloxycarbonyls -1,2- of N'- are obtained (0.5g is 44% from crude product, through 2 steps for 52%) to sulfane.It obtains¹H NMR spectras data, HPLC traces and matter Spectrum.

Example 33

The conjunction of N- tertbutyloxycarbonyl -2- amino -3- (4- mercaptophenyls) propionic acid (5) of presentation in this Examples detail Figure 36 Into.N, bis- tertbutyloxycarbonyl -1,2- of N'- are added into the round-bottomed flask through oven drying with stirring rod under nitrogen pressure Double (4- (2- amino -3- propionic acid) phenyl disulphanes (0.5g, 0.84mmol), normal-butyl phosphine (0.6mL, 2.44mmol) and 15mL Anhydrous THF.Be stirred at room temperature reaction 2 it is small when.Solvent is removed by rotary evaporation and clean oil is dissolved in 50mL acetic acid second In ester.With 5% citric acid solution (2 × 25mL), deionized water (25mL) and brine (25mL) successively washing reaction mixture.It will Organic layer separate and through anhydrous magnesium sulfate it is dry, filter and be concentrated under reduced pressure.By using Biotage Inc.HORIZON^TM The silica chromatogram purification crude product of chromatographic system after concentrating appropriate fraction and removing trace solvent (vacuum pump), is obtained in white N- tertbutyloxycarbonyl -2- amino -3- (4- mercaptophenyls) propionic acid (0.5g, 100%) of color solid-like.It obtains¹H NMR spectra numbers According to, HPLC traces and mass spectrum.

Example 34

The synthesis of 2- amino -3- (4- mercaptophenyls) the propionate hydrochlorates (6) of presentation in this Examples detail Figure 36.To tool There is addition N- tertbutyloxycarbonyl -2- amino -3- (4- mercaptophenyls) propionic acid in the round-bottomed flask through oven drying of stirring rod (.5g, 1.6mmol), 10mL anhydrous methylene chlorides and 3mL trifluoroacetic acids.Be stirred at room temperature reaction 2 it is small when.It is steamed by rotating Hair removes solvent and adds 4.0M hydrogen chloride of the 1mL in 1,4- dioxanes.Simple turn round-bottomed flask, then adds 100mL Anhydrous ether is to precipitate 2- amino -3- (4- mercaptophenyls) propionate hydrochlorate (0.39g, 100%) of white solid-like.It obtains ¹H NMR spectras data, HPLC traces and mass spectrum.

Example 35

2- (4- (2- oxo rosickyite base) benzyl) -2- acetamidomalonic acid diethyls of presentation in this Examples detail Figure 37 The synthesis of ester (7).Under nitrogen pressure into the round-bottomed flask through oven drying with stirring rod add (2) (1.1g, 1.6mmol)、n-Bu₃P (1.2mL, 4.8mmol) and the anhydrous THF of 25mL (25mL).Be stirred at room temperature reaction 2 it is small when.To anti- It should middle addition chlroacetone (0.16mL, 2.0mmol) and NaHCO₃(0.98g, 12mmol).Be stirred at room temperature reaction 2 it is small when.It is logical Rotary evaporation is crossed to remove solvent and white solid is dissolved in 100mL ethyl acetate.With saturated aqueous solution of sodium bicarbonate (50mL × 2), deionized water (50mL) and brine (50mL) washing reaction mixture successively.Organic layer is separated and through anhydrous magnesium sulfate Drying is filtered and is concentrated under reduced pressure.By using Biotage Inc.HORIZON^TMThe silica chromatogram purification of chromatographic system is thick Product after concentrating appropriate fraction and removing trace solvent (vacuum pump), obtains 2- (4- (the 2- oxos third of white solid-like Sulfenyl) benzyl) -2- diethyl acetamidos (0.62g, 98%).It obtains¹H NMR spectras data, HPLC traces and Mass spectrum.

Example 36

The synthesis of 3- (4- (2- oxo rosickyite base) phenyl) -2- alanines (8) of presentation in this Examples detail Figure 37. 7 (0.62g, 1.5mmol), 10mL1,4- are added into the round-bottomed flask through oven drying with stirring rod under nitrogen pressure Dioxane and 10mL 12M HCl.Reaction is made to flow back and is stirred overnight.Solvent is removed by rotary evaporation to generate 3- (4- (2- Oxo rosickyite base) phenyl) -2- alanines (0.40g, 99% crude product).

Example 37

N- tertbutyloxycarbonyls -3- (4- (2- oxo rosickyite base) phenyl) -2- amino of presentation in this Examples detail Figure 37 The synthesis of propionic acid (9).Under nitrogen pressure in the round-bottomed flask through oven drying with stirring rod 8 (0.35g, Addition di-tert-butyl dicarbonate (0.63g, 3.0mmol), sodium acid carbonate (0.98g, 12mmol), 8mL Isosorbide-5-Nitraes-two in 1.3mmol) Oxane and 8mL deionized waters.Be stirred at room temperature reaction 16 it is small when.Solvent is removed by rotary evaporation and is dissolved in clean oil In 100mL ethyl acetate.It is washed successively with 5% citric acid solution (50mL × 2), deionized water (50mL) and brine (50mL) anti- Answer mixture.By organic layer separate and through anhydrous magnesium sulfate it is dry, filter and be concentrated under reduced pressure.By using Biotage Inc.HORIZON^TMThe silica chromatogram purification crude product of chromatographic system is concentrating appropriate fraction and is removing trace solvent (vacuum pump) Afterwards, obtain white solid-like N- tertbutyloxycarbonyls -3- (4- (2- oxo rosickyite base) phenyl) -2- alanines (0.30g, From crude product for 66%).It obtains¹H NMR spectras data, HPLC traces and mass spectrum.

Example 38

N- tertbutyloxycarbonyls-the 3- (4- (2- oxopropyls sulfinyl) phenyl) of presentation in this Examples detail Figure 37- The synthesis of 2- alanines (10).9 are added into the round-bottomed flask through oven drying with stirring rod under nitrogen pressure The 30%v/v hydrogen peroxide of (150mg .4mmol), 8mL glacial acetic acids and 2mL in water.Be stirred at room temperature reaction 2 it is small when.It is logical Rotary evaporation is crossed to remove solvent and clean oil is dissolved in 50mL ethyl acetate.With 5% citric acid solution (25mL × 2), go Ionized water (25mL) and brine (25mL) washing reaction mixture successively.Organic layer is separated and through anhydrous magnesium sulfate drying, mistake It filters and is concentrated under reduced pressure.By using Biotage Inc.HORIZON^TMThe silica chromatogram purification crude product of chromatographic system, After concentrating appropriate fraction and removing trace solvent (vacuum pump), N- tertbutyloxycarbonyls -3- (4- (2- oxopropyl sulfenyls are obtained Base) phenyl) -2- alanines (0.13g, 86% crude product).Obtain HPLC traces and mass spectrum.

Example 39

3- (4- (2- oxopropyls sulfinyl) phenyl) -2- alanines (11) of presentation in this Examples detail Figure 37 Synthesis.N- tertbutyloxycarbonyls -3- (4- (2- oxopropyls are added into the round-bottomed flask through oven drying with stirring rod Sulfinyl) phenyl) -2- alanines 10 (0.13g, 0.35mmol), 10mL anhydrous methylene chlorides and 3mL trifluoroacetic acids. Be stirred to react at room temperature 2 it is small when.Solvent is removed by rotary evaporation and adds 4.0M hydrogen chloride of the 1mL in 1,4- dioxanes. Simple turn round-bottomed flask, then adding 100mL anhydrous ethers, ((2- oxopropyls are sub- by 4- to precipitate the 3- of white solid-like Sulfonyl) phenyl) -2- alanines (0.072g, from crude product for 74%).It obtains¹H NMR spectras data, HPLC traces with And mass spectrum.

Example 40

N- tertbutyloxycarbonyls -3- (4- (2- oxopropyls sulfonyl) phenyl) -2- of presentation in this Examples detail Figure 37 The synthesis of alanine (12).9 are added into the round-bottomed flask through oven drying with stirring rod under nitrogen pressure The 30%v/v hydrogen peroxide of (150mg, 0.4mmol), 8mL glacial acetic acids and 2mL in water.Be stirred at room temperature reaction 24 it is small when. Solvent is removed by rotary evaporation and clean oil is dissolved in 50mL ethyl acetate.With 5% citric acid solution (25mL × 2), Deionized water (25mL) and brine (25mL) washing reaction mixture successively.By organic layer separate and through anhydrous magnesium sulfate it is dry, It filters and is concentrated under reduced pressure.By using Biotage Inc.HORIZON^TMThe silica chromatogram purification crude product of chromatographic system, After concentrating appropriate fraction and removing trace solvent (vacuum pump), N- tertbutyloxycarbonyls -3- (4- (2- oxopropyl sulphonyl is obtained Base) phenyl) -2- alanines (12) (0.13g, 86% crude product).Obtain HPLC traces and mass spectrum.

Example 41

3- (4- (2- oxopropyls sulfonyl) phenyl) -2- alanines (13) of presentation in this Examples detail Figure 37 Synthesis.N- tertbutyloxycarbonyls -3- (4- (2- oxopropyl sulphurs are added into the round-bottomed flask through oven drying with stirring rod Acyl group) phenyl) -2- alanines 12 (0.13g, 0.35mmol), 10mL anhydrous methylene chlorides and 3mL trifluoroacetic acids.In room temperature Under be stirred to react 2 it is small when.Solvent is removed by rotary evaporation and adds 4.0M hydrogen chloride of the 1mL in 1,4- dioxanes.Simply Turn round-bottomed flask then adds 100mL anhydrous ethers to precipitate the 3- of white solid-like (4- (2- oxopropyls sulfonyl) Phenyl) -2- alanines (0.067g, from crude product for 65%).It obtains¹H NMR spectras data, HPLC traces and mass spectrum.

Example 42

The conjunction of 3- (4- (2- oxo rosickyite base) phenyl) -2- alanines (14) of presentation in this Examples detail Figure 37 Into.N- tertbutyloxycarbonyls -3- (4- (2- oxo rosickyite base) benzene is added into the round-bottomed flask through oven drying with stirring rod Base) -2- alanines 9 (0.10g .28mmol), 10mL anhydrous methylene chlorides and 3mL trifluoroacetic acids.Reaction is stirred at room temperature 2 it is small when.Solvent is removed by rotary evaporation and adds 4.0M hydrogen chloride of the 1mL in 1,4- dioxanes.Simple turn round bottom is burnt Bottle then adds 100mL anhydrous ethers to precipitate the 3- of white solid-like (4- (2- oxo rosickyite base) phenyl) -2- aminopropans Acid (0.062g, from crude product for 85%).It obtains¹H NMR spectras data, HPLC traces and mass spectrum.

Example 43

N- tertbutyloxycarbonyls -3- (4- (2- oxocyclopentyls sulfenyl) phenyl) -2- of presentation in this Examples detail Figure 38 The synthesis of alanine (15).5 are added into the round-bottomed flask through oven drying with stirring rod under nitrogen pressure (0.15g, 0.76mmol), 2- chlorine cyclopentanone (0.12mL, 1.25mmol), sodium acid carbonate (0.98g, 12mmol), 15mL are anhydrous THF.Be stirred at room temperature reaction 16 it is small when.Solvent is removed by rotary evaporation and white solid is dissolved in 100mL acetic acid second In ester.It is mixed with saturated aqueous solution of sodium bicarbonate (50mL × 2), deionized water (50mL) and brine (50mL) successively washing reaction Object.By organic layer separate and through anhydrous magnesium sulfate it is dry, filter and be concentrated under reduced pressure.By using Biotage Inc.HORIZON^TMThe silica chromatogram purification crude product of chromatographic system is concentrating appropriate fraction and is removing trace solvent (vacuum pump) Afterwards, N- tertbutyloxycarbonyls -3- (4- (2- oxocyclopentyls sulfenyl) phenyl) -2- alanines of white solid-like are obtained (0.15g, 51%).Obtain HPLC traces and mass spectrum.

Example 44

3- (4- (2- oxocyclopentyls sulfenyl) phenyl) -2- alanines (16) of presentation in this Examples detail Figure 38 Synthesis.N- tertbutyloxycarbonyls -3- (4- (2- oxocyclopentyls are added into the round-bottomed flask through oven drying with stirring rod Sulfenyl) phenyl) -2- alanines 15 (0.15g, 0.39mmol), 10mL anhydrous methylene chlorides and 3mL trifluoroacetic acids.In room temperature Under be stirred to react 2 it is small when.Solvent is removed by rotary evaporation and adds 4.0M hydrogen chloride of the 1mL in 1,4- dioxanes.Simply Turn round-bottomed flask then adds 100mL anhydrous ethers to precipitate the 3- of white solid-like (4- (2- oxocyclopentyls sulfenyl) Phenyl) -2- alanines (0.108g, 100%).It obtains¹H NMR spectras data, HPLC traces and mass spectrum.

Example 45

N- tertbutyloxycarbonyls -3- (4- (2- oxos butylthio) phenyl) -2- amino of presentation in this Examples detail Figure 38 The synthesis of propionic acid (18).Under nitrogen pressure into the round-bottomed flask through oven drying with stirring rod addition 5 (0.15g, 0.76mmol), the bromo- 2- butanone (0.12mL, 1.25mmol) of 1-, sodium acid carbonate (0.98g, 12mmol), the anhydrous THF of 15mL. Be stirred to react at room temperature 16 it is small when.Solvent is removed by rotary evaporation and white solid is dissolved in 100mL ethyl acetate.With Saturated aqueous solution of sodium bicarbonate (50mL × 2), deionized water (50mL) and brine (50mL) washing reaction mixture successively.To have Machine layer separate and through anhydrous magnesium sulfate it is dry, filter and be concentrated under reduced pressure.By using Biotage Inc.HORIZON^TMColor The silica chromatogram purification crude product of spectra system after concentrating appropriate fraction and removing trace solvent (vacuum pump), obtains white N- tertbutyloxycarbonyls -3- (4- (2- oxos butylthio) phenyl) -2- alanines (0.15g, 51%) of solid-like.Obtain HPLC Trace and mass spectrum.

Example 46

The synthesis of the compound 19-22 of presentation in this Examples detail Figure 38.Compound 19-22 be use be similar on Method described in compound 10-14 synthesizes.

Example 47

The conjunction of 3- (4- (2- oxos butylthio) phenyl) -2- alanines (23) of presentation in this Examples detail Figure 38 Into.N- tertbutyloxycarbonyls -3- (4- (2- oxos butylthio) benzene is added into the round-bottomed flask through oven drying with stirring rod Base) -2- alanines 18 (0.15g .56mmol), 10mL anhydrous methylene chlorides and 3mL trifluoroacetic acids.It is stirred at room temperature anti- Answer 2 it is small when.Solvent is removed by rotary evaporation and adds 4.0M hydrogen chloride of the 1mL in 1,4- dioxanes.Simple turn round bottom Flask then adds 100mL anhydrous ethers to precipitate the 3- of white solid-like (4- (2- oxos butylthio) phenyl) -2- amino Propionic acid (0.149g, 100%).It obtains¹H NMR spectras data, HPLC traces and mass spectrum.

Example 48

The synthesis of the compound 24-27 of presentation in this Examples detail Figure 39.Compound 24-27 be use be similar on Method described in compound 10-14 synthesizes.

Example 49

The synthesis of the amino acid containing dicarbapentaborane of presentation in this Examples detail Figure 31.

Synthesis

The pAF (1.0g, 3.1mmol) through protection is slowly added into t-BuOK (15mL, 1.0M, in THF) in trifluoro Solution in methyl acetate (5mL, 50mmol).By reaction mixture be stirred at room temperature 30 minutes and with citric acid (5g, 25.4mmol) stop and diluted with EtOAc.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Dry, mistake Filter and concentration.By flash chromatography (silica, 20:1-3:2 hexanes:EtOAc residue) is purified to obtain in brown solid shape Product (1.07g, 83%).

Synthesis

Added at 0 DEG C into solution of the above-mentioned methyl esters (1.0g, 2.4mmol) in dioxane (30mL) LiOH (30mL, 1N).Mixture is stirred at 0 DEG C 0.5 it is small when and stopped and with H with citric acid (10g, 51mmol)₂O dilutes.Extracted with EtOAc Take mixture.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Dry, filtering and concentration.By quick Chromatography (silica, 100:1-10:1CH₂Cl₂:MeOH, 0.5%HOAc) residue is purified to obtain brown oil (0.87g, 98%).

Synthesis

To above-mentioned sour (1.0g, 2.5mmol) in CH at 0 DEG C₂Cl₂Trifluoroacetic acid is added in solution in (15mL) (15mL).By gained mixture stirring 0.5 it is small when and concentrate in a vacuum.MeOH (2mL) is added into residue, is then added HCl (in 2mL, 4N, Yu dioxane).Then addition ether (200mL) with precipitate the product of white solid-like (0.56g, 75%).

Example 50

The synthesis of the amino acid containing dicarbapentaborane of presentation in this Examples detail Figure 32.

Synthesis

The pAF (1.0g, 3.1mmol) through protection is slowly added into t-BuOK (15mL, 1.0M, in THF) in five fluorine Solution in methyl propionate (8mL, 62mmol).By gained mixture be stirred at room temperature 1 it is small when and with citric acid (5g, 25.4mmol) stop and with H₂O (100mL) dilutes.After removing most of solvent, with EtOAc extracted residues.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Dry, filtering and concentration.By flash chromatography (silica, 20:1-3:2 oneself Alkane:EtOAc residue) is purified to obtain the product (1.1g, 76%) in brown solid shape.

Synthesis

Added at 0 DEG C into solution of the above-mentioned methyl esters (1.0g, 2.1mmol) in dioxane (30mL) LiOH (30mL, 1N).At 0 DEG C by gained mixture stirring 0.5 it is small when and with citric acid (10g, 51mmol) and H₂O stops.Extracted with EtOAc Take mixture.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Dry, filtering and concentration.By quick Chromatography (silica, 100:1-10:1CH₂Cl₂:MeOH, 0.5%HOAc) residue is purified to obtain the product in yellow solid (0.8g, 84%).

Synthesis

To above-mentioned sour (0.7g, 2.5mmol) in CH at 0 DEG C₂Cl₂Trifluoroacetic acid is added in solution in (15mL) (15mL).Mixture is stirred at the same temperature 0.5 it is small when and concentration.MeOH (2mL) is added into residue, is then added HCl (in 2mL, 4N, Yu dioxane).Then addition ether (200mL) with precipitate the product of white solid-like (0.62g, 70%).

Example 51

The synthesis of the amino acid containing dicarbapentaborane of presentation in this Examples detail Figure 33.

Synthesis

To alcohol 1 (2.35g, 7.6mmol) and pyridine (1.5mL, 18.6mmol) in CH at 0 DEG C₂Cl₂Warp in (150mL) Dai Si-Martin is added in agitating solution and crosses iodine alkane (3.5g, 8.3mmol).By mixture be stirred at room temperature overnight and with Na₂S₂O₃-NaHCO₃Saturated aqueous solution (1:1,100mL) stop and with CH₂Cl₂Dilution.Organic layer is separated and with H₂O and brine Washing, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 20:1-3:1 hexane: EtOAc residue) is purified to obtain the aldehyde 2 of white solid-like (2.15g, 92%).ESI-MS,m/z:292(M⁺-OH), 232,204,175,131,115(100)。

Synthesis

At 0 DEG C positive fourth is added into agitated solution of the diisopropylamine (0.33mL, 2.33mmol) in THF (60mL) Base lithium (1.46mL, 2.34mmol).20 minutes are stirred the mixture at 0 DEG C and is cooled to -78 DEG C and then adds cyclopentanone. It is stirred the mixture at -78 DEG C after twenty minutes, addition aldehyde 2 (0.5g, 1.63mmol) is in THF (20mL is washed with 20mL) Solution.At -78 DEG C by gained mixture stirring 1.0 it is small when and with NH₄Cl saturated aqueous solutions stop.Remove most of solvent Afterwards, with EtOAc extracted residues.By organic layer with H₂O and salt water washing, through anhydrous Na after₂SO₄Drying is filtered and in vacuum Middle concentration.By flash chromatography (silica, 10:1-1:1 hexane:EtOAc) purifying residue obtains 3 in colorless oil (482mg, 80%).

Synthesis

To alcohol 3 (0.44g, 1.13mmol) and pyridine (0.6mL, 7.44mmol) in CH at 0 DEG C₂Cl₂In (150mL) Dai Si-Martin is added in agitated solution and crosses iodine alkane (0.6g, 1.41mmol).Mixture is stirred at room temperature overnight.It will be anti- It should be with Na₂S₂O₃-NaHCO₃Saturated aqueous solution (1:1,100mL) stop and with CH₂Cl₂Extraction.Organic layer is combined and with H₂O and Salt water washing, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 20:1-2:1 oneself Alkane:EtOAc) purifying residue obtains the diketone 4 (342mg, 78%) in colorless oil.ESI-MS,m/z:412(M⁺+Na), 356,312,230,212,184,146(100)。

Synthesis

At 0 DEG C to methyl esters 4 (in the solution in 330mg, 0.85mmol) Yu dioxanes (4mL) add LiOH (4mL, 1N).Gained mixture is stirred 30 minutes at 0 DEG C and is stopped with aqueous citric acid solution (5%, 100mL).It is extracted with EtOAc Mixture.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Drying, filtering and concentration are solid to obtain white Body (310mg, 97%).ESI-MS,m/z:342,330(M⁺-COOH),298,230,185,119(100)。

Synthesis

To 5 (310mg, 0.83mmol) of acid in CH at 0 DEG C₂Cl₂Trifluoroacetic acid (4mL) is added in solution in (4mL). Mixture at 0 DEG C is stirred 30 minutes and is concentrated in a vacuum.MeOH (1mL) is added into residue, then adds HCl (in 1mL, 4N, Yu dioxane).Ether (100mL) is then added to precipitate the product of white solid-like (241mg, 94%). ESI-MS,m/z:298(M⁺+Na),276(M⁺+1),230(M⁺-COOH),184,119(100)。

Example 52

The synthesis of the amino acid containing dicarbapentaborane of presentation in this Examples detail Figure 34.

Synthesis

To alcohol (6.0g, 19.4mmol) and pyridine (12mL, 150mmol) in CH at 0 DEG C₂Cl₂In (400mL) through stirring It mixes addition Dai Si-Martin in solution and crosses iodine alkane (14.2g, 33.4mmol).Mixture is stirred at room temperature overnight.Will reaction with Na₂S₂O₃-NaHCO₃Saturated aqueous solution (1:1,300mL) stop and with CH₂Cl₂Extraction.Organic layer is combined and with H₂O and brine Washing, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 1:100-1:1 hexane: EtOAc) purifying residue obtains the aldehyde (5.48g, 92%) of white solid-like.

Synthesis

H is made an addition into solution of the above-mentioned aldehyde (3.41g, 11.1mmol) in acetone (70mL)₂KMnO in O (10mL)₄ (2.5g, 15.8mmol).Gained mixture is stirred at room temperature overnight.After removing most of solvent, residue is dissolved in It is extracted in aqueous citric acid solution (5%, 300mL) and with EtOAc.Organic layer is combined and with H₂O and salt water washing, through nothing after Water Na₂SO₄Drying filters and concentrates to obtain the product (2.83g, 79%) of white solid-like in a vacuum, need not be into one Step purifying can be directly used in next step.

Synthesis

At 0 DEG C 1- amino -2- propyl alcohol is added into above-mentioned sour (2.83g, 8.76mmol) solution in DMF (60mL) (1.4mL, 17.9mmol), 1- (3- dimethylaminopropyls) -3- ethyl-carbodiimide hydrochlorides (EDC, 4.1g, 21.4mmol), I-hydroxybenzotriazole hydrate (HOBt, 2.2g, 18.5mmol) and N, N- diisopropyl ethyl amine (DIEA, 9mL, 51.6mmol).Mixture is stirred at room temperature overnight and then with aqueous citric acid solution (5%, 200mL) stop and with EtOAc is extracted.Organic layer is combined and with H₂O and salt water washing, through anhydrous Na after₂SO₄It dries, filter and is dense in a vacuum Contracting.By flash chromatography (silica, 10:1-1:1 hexane:EtOAc) purifying residue obtains the product of white foam-like (2.45g, 74%).

Synthesis

To above-mentioned alcohol (2.44g, 6.4mmol) and pyridine (4mL, 49.6mmol) in CH at 0 DEG C₂Cl₂In (100mL) Dai Si-Martin is added in agitated solution and crosses iodine alkane (4.1g, 9.7mmol).Mixture is stirred at room temperature overnight.It will reaction With Na₂S₂O₃-NaHCO₃Saturated aqueous solution (1:1,300mL) stop and with CH₂Cl₂Extraction.By organic layer with H₂O and salt washing It washs, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 1:1-1:3 hexanes: EtOAc) purifying residue obtains the product (1.84g, 76%) in yellow solid.

Synthesis

At 0 DEG C LiOH is added into solution of the above-mentioned methyl esters (1.72g, 4.6mmol) in dioxane (10mL) (10mL, 1N).Mixture is stirred at the same temperature 3 it is small when and with aqueous citric acid solution (5%) stop.It is extracted with EtOAc Mixture.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Drying filters and concentrates in a vacuum to obtain To the product (1.7g) in solid-like, can be directly used for without purifying in next step.

To above-mentioned sour (1.7g, 4.7mmol) in CH at 0 DEG C₂Cl₂Trifluoroacetic acid is added in solution in (15mL) (15mL).Mixture is stirred at the same temperature 2 it is small when and concentrate in a vacuum.Into residue add HCl (1.5mL, In 4N, Yu dioxane), then add ether (400mL).Collect the precipitated product (1.52g, for 2 steps of white solid-like 90%) rapid is.

Example 53

The synthesis of the amino acid containing hydrazides of presentation in this Examples detail Figure 44.

Synthesis

Into solution of the aldehyde (410mg, 1.34mmol) in EtOH (15mL) add formic hydrazide (170mg, 2.83mmol).By reaction mixture be stirred at room temperature 1 it is small when.After removing most of solvent, with CH₂Cl₂Extracted residues.It will Organic layer is combined and concentrated in a vacuum.By flash chromatography (silica, 1:6-1:1 hexane:EtOAc) purifying residue obtains white Color solid (390mg, 83%).

Synthesis

Added at 0 DEG C into solution of the above-mentioned methyl esters (349mg, 1mmol) in dioxane (7mL) LiOH (7mL, 1N).Mixture is stirred at the same temperature 10 minutes and stopped and with H with citric acid (2.5g)₂O is extracted.It is extracted with EtOAc Mixture.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Drying, filtering and concentration are solid to obtain white Body (290mg, 87%).

Synthesis

To above-mentioned sour (290mg, 0.87mmol) in CH at 0 DEG C₂Cl₂Trifluoroacetic acid is added in solution in (20mL) (20mL).It stirs the mixture for 20 minutes and concentrates.Into residue add MeOH (1mL), then add HCl (1.0mL, 4N, In Yu dioxanes).Ether (200mL) is then added to precipitate the product (195mg, 83%) in pale-yellow solid.

Example 54

The synthesis of the amino acid containing hydrazides of presentation in this Examples detail Figure 45.

Synthesis

It is molten in DMF (100mL) to N- tertbutyloxycarbonyl -4- hydroxymethyls phenylalanines (11.73g, 39.8mmol) Alanine methyl ester hydrochloride (9.0g, 64.5mmol), 1- (3- dimethylaminopropyls) 3- ethylcarbodiimines are added in liquid Hydrochloride (EDC, 15.4g, 80.3mmol), N, N- diisopropyl ethyl amines (DIEA, 30mL, 172mmol) and 1- hydroxy benzos Triazole hydrate (HOBt, 8.4g, 70.6mmol).Reaction mixture is stirred at room temperature overnight and is then diluted with EtOAc. Organic layer is separated and with H₂O, citric acid (5%), H₂O、NaHCO₃、H₂O and brine wash successively, through anhydrous Na after₂SO₄It is dry It is dry, filtering and concentrate with obtain white solid-like through protect dipeptides (13.74g, 91%).

Synthesis

To above-mentioned through protecting dipeptides (10.33g, 27.2mmol) in CH at 0 DEG C₂Cl₂It is added in solution in (300mL) Pyridine (8mL, 99.1mmol) and Dai Si-Martin cross iodine alkane (14g, 33.0mmol).Reaction mixture is stirred overnight and then With NaHCO₃/Na₂S₂O₃Saturated aqueous solution (1:1) stop.By organic layer with H₂O, citric acid, H₂O and brine wash successively, then Through anhydrous Na₂SO₄Dry, filtering and concentration.By flash chromatography (silica, 9:1-1:1 hexane:EtOAc residue) is purified to obtain To the product (10.12g, 98%) of white solid-like.

Synthesis

Into solution of the dipeptides aldehyde (10.1g, 26.7mmol) in EtOH (200mL) add acetic acid hydrazides (3.7g, 45mmol).Reaction mixture is stirred at room temperature 30 minutes and concentrated.H is added into residue₂O (1L) and CH₂Cl₂ (500mL).Organic layer is separated and concentrates to obtain white solid (11.21g, 97%).

Synthesis

At 0 DEG C LiOH is added into solution of the above-mentioned methyl esters (11.1g, 25.6mmol) in dioxane (50mL) (50mL, 1N).Mixture is stirred at the same temperature 30 minutes and then stopped and with H with citric acid (20g)₂O(200mL) Dilution.Mixture is extracted with EtOAc.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Dry, filtering and It concentrates to obtain white solid (9.52g, 88%).

Synthesis

To above-mentioned sour (9.5g, 22.6mmol) in CH at 0 DEG C₂Cl₂Trifluoroacetic acid is added in solution in (50mL) (50mL).Mixture is stirred at 0 DEG C 1 it is small when and concentrate in a vacuum.HCl (7mL, 4N, Yu bis- Evil are added into residue In alkane), then add ether (500mL).Collect the sediment (7.25g, 90%) of white solid-like.

Example 55

The synthesis of the amino acid containing oxime of presentation in this Examples detail Figure 46 A.

Synthesis

To aldehyde (3.0g) in MeOH/H⁺In solution in add 2 equivalent hydroxylamine hydrochlorides.By reaction mixture at room temperature Stir 2 it is small when and concentration.H is added into residue₂O (200mL), then adds CH₂Cl₂.By organic layer separation and in a vacuum Concentration.By flash chromatography (silica, 3:7-1:9 hexanes:EtOAc product (96%) of the residue generation in solid-like) is purified.

Synthesis

LiOH (10mL, 1N) is added into solution of the above-mentioned methyl esters (3.0g) in dioxane (10mL) at 0 DEG C.It will be mixed Close object stir at the same temperature 3 it is small when and then by add citric acid (5g) stop and with H₂O dilutes.It is extracted with EtOAc Mixture.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Drying filters and concentrates to obtain solid. Then, at 0 DEG C to gained acid in CH₂Cl₂Trifluoroacetic acid (20mL) is added in solution in (20mL).It is at 0 DEG C that reaction is mixed Close object stirring 1 it is small when and concentration.MeOH (1mL) is added into residue, then adds HCl (in 2.0mL, 4N, Yu dioxane). Ether (200mL) is then added to precipitate the product (87%) in solid-like.

Example 56

The synthesis of the amino acid containing oxime of presentation in this Examples detail Figure 46 B.

Synthesis

To aldehyde (3.0g) in MeOH/H⁺In solution in add 2 equivalent methoxy amine hydrochlorates.By reaction mixture in room When the lower stirring 2 of temperature is small and concentration.H is added into residue₂O (200mL), then adds CH₂Cl₂.Organic layer is separated and true Aerial concentration.By flash chromatography (silica, 3:7-1:9 hexanes:EtOAc product of the residue generation in solid-like) is purified (93%).

Synthesis

LiOH (10mL, 1N) is added into solution of the above-mentioned methyl esters (3.0g) in dioxane (10mL) at 0 DEG C.It will be mixed Close object stir at the same temperature 3 it is small when and then by add citric acid (5g) stop and with H₂O dilutes.It is extracted with EtOAc Mixture.By organic layer with H₂O and brine wash successively, through anhydrous Na after₂SO₄Drying filters and concentrates to obtain solid. Then, at 0 DEG C to gained acid in CH₂Cl₂Trifluoroacetic acid (20mL) is added in solution in (20mL).By reaction mixture 0 At DEG C stirring 1 it is small when and concentration.MeOH (1mL) is added into residue, then adds HCl (in 2.0mL, 4N, Yu dioxane). Ether (200mL) is then added to precipitate the product (89%) in solid-like.

Example 57

The synthesis of the amino acid containing hydrazine of presentation in this Examples detail Figure 46 C.

Synthesis

To aldehyde (3.0g) in MeOH/H⁺In solution in add 2 equivalent methyl hydrazines.Reaction mixture is stirred at room temperature 2 it is small when and concentration.H is added into residue₂O (200mL), then adds CH₂Cl₂.By organic layer separation and it is dense in a vacuum Contracting.By flash chromatography (silica, 3:7-1:9 hexanes:EtOAc product (93%) of the residue generation in solid-like) is purified.

Synthesis

Example 58

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 48 A.

Synthesis

To mPEG (30K)-OH (1.0g, 0.033mmol) in anhydrous CH₂Cl₂P-nitrophenyl is added in solution in (10mL) Phenol chloro-formate (60mg, 0.28mmol).By mixture be stirred at room temperature 15 it is small when.Add ether (200mL).By sediment Filtering is washed with ether and is dried in a vacuum to obtain white powdered product (1.0g, 100%).

Synthesis

N- hydroxyls are added into solution of the 3- hydroxyethylcarbamates tert-butyl ester (1.75g, 10mmol) in THF (60mL) Phthalimide (3.2g, 20mmol), triphenylphosphine (2.0g, 15mmol).Reaction mixture is stirred at room temperature 10 Minute and be then cooled to 0 DEG C.Through 1 it is small when by syringe be added dropwise diisopropyl azodiformate (DIAD, 2.0mL, 10.5mmol).It removes ice bath and the mixture was stirred overnight and concentrates.White solid is dissolved in ethyl acetate (100mL). By reaction mixture with saturated aqueous solution of sodium bicarbonate (100mL), H₂O (100mL) and brine (100mL) wash successively, then Through anhydrous MgSO₄Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 100:1-10:1 hexane:EtOAc it is) pure Change crude product to obtain the title compound of white solid-like (2.6g, 81%).

Synthesis

To the connexon (2.0g, 9.1mmol) protected through tertbutyloxycarbonyl in CH₂Cl₂Three are added in solution in (5mL) Fluoroacetic acid (5mL).By gained mixture be stirred at room temperature 1 it is small when and concentration.HCl (4N, Yu dioxanes are added into residue In, 1.5mL), then add Et₂O(150mL).Sediment is filtered, is washed with ether and is dried in a vacuum to obtain in white The amine connexon (1.1g, 85%) of color solid-like.

Synthesis

To mPEG (30K) p-nitrophenol carbonic esters (1.0g, 0.033mmol) and amine connexon (53mg, 0.21mmol) In DMF-CH₂Cl₂(10mL, 1:2) diisopropyl ethyl amine (50 μ L, 0.28mmol) and DMAP are added in the mixture in (5mg, 0.041mmol).When mixture 15 obtained by being stirred at room temperature is small.Add ether (200mL).Sediment is filtered, with Ether washs and is dried in a vacuum to obtain white powdered product (0.83g, 83%).

Synthesis

Hydrazine is added into solution of the mPEG phthalimides (30K, 0.8g, 0.0266mmol) in MeOH (5mL) (8.5 μ L, 0.27mmol).When mixture 1.0 obtained by stirring is small at 45 DEG C.After reaction is cooled to room temperature, CH is added₂Cl₂ (150mL) and with HCl/water solution (0.1N, 100mL) wash solution.With CH₂Cl₂(150mL) aqueous layer extracted.Organic layer is combined And with H₂O (100mL) is washed, through anhydrous Na after₂SO₄Dry, filtering and concentration.Residue is dissolved in CH₂Cl₂In (5mL). Ether (200mL) is added to precipitate white powdered hydroxylamine product (0.72g, 90%).

Example 59

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 48 B.

Synthesis

To mPEG (30K)-OH (1.0g, 0.033mmol) in anhydrous CH₂Cl₂P-nitrophenyl is added in solution in (10mL) Phenol chloro-formate (60mg, 0.28mmol).Be stirred at room temperature mixture 15 it is small when.Add ether (200mL).By sediment mistake Filter is washed with ether and is dried in a vacuum to obtain white powdered product (1.0g, 100%).

Synthesis

N- hydroxyls are added into solution of the 2- hydroxyethylcarbamates tert-butyl ester (2.8mL, 18mmol) in THF (60mL) Phthalimide (5.8g, 36mmol), triphenylphosphine (3.6g, 27mmol).Reaction mixture is stirred at room temperature 10 Minute and be then cooled to 0 DEG C.Through 1 it is small when by syringe be added dropwise diisopropyl azodiformate (DIAD, 3.6mL, 19mmol).It removes ice bath and the mixture was stirred overnight and concentrates.White solid is dissolved in ethyl acetate (100mL).It will Reaction mixture is with saturated aqueous solution of sodium bicarbonate (2 × 50mL), H₂O (50mL) and brine (50mL) wash successively, are passed through after Anhydrous MgSO₄Drying is filtered and concentrated in a vacuum.By using Biotage Inc.HORIZON^TMThe quick color of chromatographic system Purification of crude product is composed to obtain the title compound (12g, 206%) containing impurity of white solid-like.

Synthesis

To the thick connexon (12g) through tertbutyloxycarbonyl protection in CH₂Cl₂Trifluoroacetic acid is added in solution in (5mL) (5mL).By gained mixture be stirred at room temperature 1 it is small when and concentration.Into residue add HCl (in 4N, Yu dioxane, 1.5mL), Et is then added₂O(150mL).Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white Amine connexon (the 3.0g, for two steps for 68%) of solid-like.

Synthesis

To mPEG (30K) p-nitrophenol carbonic esters (1.0g, 0.033mmol) and amine connexon (50mg, 0.21mmol) In DMF-CH₂Cl₂(10mL, 1:2) diisopropyl ethyl amine (50 μ L, 0.28mmol) and 4- dimethyl are added in the mixture in Aminopyridine (4mg, 0.033mmol).When mixture 15 obtained by being stirred at room temperature is small.Add ether (200mL).By sediment Filtering is washed with ether and is dried in a vacuum to obtain white powdered product (0.81g, 81%).

Synthesis

Hydrazine is added into solution of mPEG (30K) phthalimide (0.8g, 0.0266mmol) in MeOH (5mL) (8.5 μ L, 0.27mmol).When mixture 1.0 obtained by stirring is small at 45 DEG C.After reaction is cooled to room temperature, CH is added₂Cl₂ (150mL) and with HCl/water solution (0.1N, 100mL) wash solution.With CH₂Cl₂(150mL) aqueous layer extracted.Organic layer is combined And with H₂O (100mL) is washed, through anhydrous Na after₂SO₄Dry, filtering and concentration.Residue is dissolved in CH₂Cl₂In (5mL). Ether (200mL) is added to precipitate white powdered hydroxylamine product (0.68g, 85%).

Example 60

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 49 A.

Synthesis

Solution at 0 DEG C to N- (3- bromopropyls) phthalimides (4.0g, 15.0mmol) in DMF (50mL) Middle addition K₂CO₃(10g, 73mmol) and the N- hydroxy carbamic acids tert-butyl ester (2.5g, 18.8mmol).Reaction is stirred at room temperature When mixture 3 is small.By mixture with H₂O is diluted (200mL) and is extracted with EtOAc (200mL).By organic layer with H₂O and salt washing It washs, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 20:1-3:1 hexane: EtOAc) purifying residue obtains the product (3.5g, 72%) in colorless oil.

Synthesis

Hydrazine is added into solution of the above-mentioned phthalimide (500mg, 1.6mmol) in EtOH (10mL) (0.25mL, 8.0mmol).When mixture 3 obtained by being stirred at room temperature is small.After removing sediment, filtrate is concentrated.Residue is stayed In high vacuum overnight.By flash chromatography (silica, 3:1-1:1EtOAc:MeOH) purifying residue obtains white solid-like Amine connexon (252mg, 85%).

Synthesis

To mPEG (30K)-OH (1.0g, 0.033mmol) in anhydrous CH₂Cl₂P-nitrophenyl is added in solution in (10mL) Phenol chloro-formate (60mg, 0.28mmol).Be stirred at room temperature mixture 15 it is small when.Add ether (200mL).By sediment mistake Filter is washed with ether and is dried in a vacuum to obtain the product (1.0g, 100%) of white solid-like.

Synthesis

To above-mentioned activation mPEG (30K) (3.0g, 0.1mmol) in anhydrous CH₂Cl₂Addition two is different in solution in (30mL) Ethylamine (88 μ L, 0.5mmol) and amine connexon (76mg, 0.4mmol).It is small that gained mixture 15 is stirred at room temperature When.Ether (700mL) is added into reaction mixture.Sediment is filtered, is washed with ether and is dried in a vacuum to obtain White powder (2.8g, 93%).

Synthesis

To the mPEG (30K) (2.0g, 0.067mmol) protected through tertbutyloxycarbonyl in anhydrous CH at 0 DEG C₂Cl₂(10mL) In solution in add trifluoroacetic acid (10mL).When mixture 5 obtained by being stirred at room temperature is small.Ether (500mL) is added to anti- It answers in mixture.Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white powder (1.8g, 90%).

Example 61

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 49 B.

Synthesis

It is added at 0 DEG C into solution of the N- hydroxy carbamic acids tert-butyl ester (5.0g, 37.6mmol) in DMF (30mL) K₂CO₃(12g, 87.6mmol) and N- (2- bromoethyls) phthalimide (10.0g, 39.7mmol).It is stirred at room temperature anti- When answering mixture 3 small.By mixture with H₂O is diluted (200mL) and is extracted with EtOAc (200mL).By organic layer with H₂O and brine Washing, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 20:1-1:1 hexane: EtOAc) purifying residue obtains the product (5.2g, 55%) of white solid-like.

Synthesis

Hydrazine is added into solution of the above-mentioned phthalimide (500mg, 1.6mmol) in EtOH (10mL) (0.25mL, 8.0mmol).When mixture 3 obtained by being stirred at room temperature is small.After removing sediment, filtrate is concentrated.By residue It stays in high vacuum overnight.By flash chromatography (silica, 3:1-1:1EtOAc:MeOH) purifying residue obtains white solid The amine connexon (301mg, 86%) of shape.

Synthesis

To above-mentioned activation mPEG (30K) (1.0g, 0.033mmol) in anhydrous CH₂Cl₂Two are added in solution in (10mL) Diisopropylethylamine (58 μ L, 0.33mmol), 4-dimethylaminopyridine (4mg, 0.033mmol) and above-mentioned amine connexon (64mg, 0.31mmol).When mixture 15 obtained by being stirred at room temperature is small.Ether (200mL) is added into reaction mixture.It will Sediment filtering is washed with ether and is dried in a vacuum to obtain white powder (0.85g, 85%).

Synthesis

To the mPEG (30K) (0.85g, 0.028mmol) protected through tertbutyloxycarbonyl in anhydrous CH at 0 DEG C₂Cl₂ Trifluoroacetic acid (10mL) is added in solution in (10mL).When mixture 5 obtained by being stirred at room temperature is small.Add ether (200mL) is into reaction mixture.Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white powder (0.68g, 80%).

Example 62

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 50 A.

Synthesis

Solution at 0 DEG C to single tertbutyloxycarbonyl phthalimide (2.1g, 6.9mmol) in pyridine (50mL) Middle addition Boc₂O (3.3g, 15.1mmol).Gained mixture is heated to 60 DEG C overnight.It, will be residual after removing solvent in a vacuum Excess is diluted with EtOAc (200mL) and washed with citric acid (5%, 200mL), water (200mL) and brine (200mL), is passed through after Anhydrous Na₂SO₄Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 20:1-3:1 hexane:EtOAc) purify residual Excess obtains the product (2.37g, 85%) in yellow oily.

Synthesis

It is molten in MeOH (15mL) to two tertbutyloxycarbonyl phthalimides (1.21g, 2.98mmol) at 0 DEG C The ammonia (15mL, 7N, 105mmol) in MeOH is made an addition in liquid.Gained mixture is stirred at room temperature to stay overnight.Filter out sediment and Filtrate is concentrated in a vacuum.By flash chromatography (silica, 10:1-6:4EtOAc:MeOH) purifying residue obtains white solid The amine connexon (0.61g, 74%) of body shape.

Synthesis

To mPEG (30K)-OH (1.0g, 0.033mmol) in anhydrous CH₂Cl₂P-nitrophenyl is added in solution in (10mL) Phenol chloro-formate (60mg, 0.28mmol).Be stirred at room temperature mixture 15 it is small when.Ether (200mL) is added to precipitate mPEG (30K) product.Product is filtered, washs and is dried in a vacuum (1.0g, 100%) with ether.

Synthesis

To above-mentioned activation mPEG (30K) (1.0g, 0.033mmol) in anhydrous CH₂Cl₂Two are added in solution in (10mL) Diisopropylethylamine (58 μ L, 0.33mmol), 4-dimethylaminopyridine (5mg, 0.041mmol) and above-mentioned two tertbutyloxycarbonyl Amine connexon (90mg, 0.33mmol).When mixture 15 obtained by being stirred at room temperature is small.It is mixed to reaction to add ether (200mL) It closes in object.Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white powder (0.82g, 82%).

Synthesis

To the mPEG (30K) (0.82g, 0.027mmol) protected through tertbutyloxycarbonyl in anhydrous CH at 0 DEG C₂Cl₂(8mL) In solution in add trifluoroacetic acid (8mL).When mixture 5 obtained by being stirred at room temperature is small.Ether (200mL) is added to reaction In mixture.Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white powder (0.57g, 70%).

Example 63

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 50 B.

Synthesis

It is added at 0 DEG C into solution of single tertbutyloxycarbonyl phthalimide (1.5g, 4.7mmol) in pyridine Boc₂O (2.2g, 10.0mmol).Gained mixture is heated to 60 DEG C overnight.In a vacuum remove solvent after, by residue with EtOAc (200mL) is diluted and washed with citric acid (5%, 200mL), water (200mL) and brine (200mL), through anhydrous after Na₂SO₄Drying is filtered and concentrated in a vacuum.By flash chromatography (silica, 20:1-3:1 hexane:EtOAc residue) is purified Obtain the product (1.6g, 81%) in oily.

Synthesis

Solution at 0 DEG C to two tertbutyloxycarbonyl phthalimides (1.5g, 3.6mmol) in MeOH (15mL) In make an addition to the ammonia (15mL, 7N, 105mmol) in MeOH.Gained mixture is stirred at room temperature to stay overnight.Filter out sediment and Filtrate is concentrated in vacuum.By flash chromatography (silica, 10:1-6:4EtOAc:MeOH) purifying residue obtains white solid The amine connexon (0.85g, 82%) of shape.

Synthesis

To above-mentioned activation mPEG (30K) (1.0g, 0.033mmol) in anhydrous CH₂Cl₂Two are added in solution in (10mL) Diisopropylethylamine (58 μ L, 0.33mmol), 4-dimethylaminopyridine (5mg, 0.041mmol) and above-mentioned two tertbutyloxycarbonyl Amine connexon (100mg, 0.34mmol).When mixture 15 obtained by being stirred at room temperature is small.It is mixed to reaction to add ether (200mL) It closes in object.Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white powder (0.89g, 89%).

Synthesis

To the mPEG (30K) (0.89g, 0.030mmol) protected through tertbutyloxycarbonyl in anhydrous CH at 0 DEG C₂Cl₂(8mL) In solution in add trifluoroacetic acid (8mL).When mixture 5 obtained by being stirred at room temperature is small.Ether (200mL) is added to reaction In mixture.Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white powder (0.65g, 73%).

Example 64

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 51 A.

Synthesis

To mPEG (30K) propionic aldehyde (0.5g, 0.0166mmol) and amine connexon (73mg, 0.25mmol) in MeOH (10mL) In mixture in add NaCNBH₃(20mg, 0.30mmol).When mixture 48 obtained by being stirred at room temperature is small.Remove big portion After dividing solvent, ether (200mL) is added.Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white powder (0.43g, 86%).

Synthesis

To the mPEG (30K) (0.42g, 0.014mmol) protected through two tertbutyloxycarbonyls in anhydrous CH at 0 DEG C₂Cl₂ Trifluoroacetic acid (4mL) is added in solution in (4mL).When mixture 8 obtained by being stirred at room temperature is small.Add ether (100mL) Into reaction mixture.Sediment is filtered, is washed with ether and be dried in a vacuum to obtain white powder (0.35g, 83%).

Example 65

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 51 B.

Synthesis

To mPEG (30K) (6.0g, 0.2mmol) and pyridine (0.1mL, 1.2mmol) in CH at 0 DEG C₂Cl₂In (60mL) Agitated solution in addition Dai Si-Martin cross iodine alkane (0.2g, 0.47mmol).Mixture is stirred at room temperature to stay overnight.Then It will react with Na₂S₂O₃-NaHCO₃Saturated aqueous solution (1:1,100mL) stop and with CH₂Cl₂(500mL × 2) extract.It will be organic Layer combination and Yi Shui and salt water washing, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.Residue is dissolved in CH₂Cl₂In (50mL).Ether (1L) is added into solution.Sediment is filtered, washs and be dried in a vacuum to obtain with ether To white powder (4.9g, 82%).

Synthesis

To mPEG (30K) acetaldehyde (0.5g, 0.0166mmol) and amine connexon (73mg, 0.25mmol) in MeOH (10mL) In mixture in add NaCNBH₃(20mg, 0.30mmol).When mixture 48 obtained by being stirred at room temperature is small.Remove big portion After dividing solvent, ether (200mL) is added.Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white powder (0.43g, 85%).

Synthesis

Example 66

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 52 A.

Synthesis

To mPEG (30K)-NH₂Boc-Ser-OH is added in the solution of (6.0g, 0.2mmol) in DMF (60mL) (205mg, 1.0mmol), 1- ethyls -3- (3- dimethylaminopropyls) carbodiimide hydrochloride (EDC, 190mg, 1.0mmol) and N, N'- diisopropyl ethyl amine (0.17mL, 1.0mmol).Be stirred at room temperature mixture 10 it is small when and with EtOAc (500mL) dilutes.By gained mixture with NaHCO₃Saturated aqueous solution (300mL), H₂O (300mL) and brine (300mL) It washs successively, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.Residue is dissolved in CH₂Cl₂In (50mL). Add ether (700mL).Sediment is filtered, is washed with ether and be dried in a vacuum to obtain white powder (5.1g, 82%).

Synthesis

To above-mentioned mPEG (30K) (3.0g, 0.1mmol) in CH at 0 DEG C₂Cl₂Trifluoro second is added in solution in (15mL) Sour (15mL).Be stirred at room temperature gained mixture 3 it is small when and concentration.CH is added into residue₂Cl₂(5mL), is then added HCl (in 4N, Yu dioxane, 2mL).Ether (400mL) is added to precipitate the dihydroxylamine of white solid-like (2.6g, 85%).

Synthesis

To above-mentioned mPEG (30K) (2.0g, 0.067mmol) in H₂O-CH₃CN(1:It is added in solution in 1,20mL) NaIO₄(15mg, 0.07mmol).By mixture be stirred at room temperature 4.0 it is small when and with CH₂Cl₂(500mL) dilutes.Gained is mixed It closes object to wash with water (100mL) and brine (100mL), through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.It will be residual Excess is dissolved in CH₂Cl₂In (50mL).Add ether (700mL).Sediment is filtered, washs and is dried in a vacuum with ether To obtain white powder (1.8g, 90%).

Synthesis

To mPEG (30K) acetaldehyde (0.5g, 0.0166mmol) and amine connexon (73mg, 0.25mmol) in MeOH (10mL) In mixture in add NaCNBH₃(20mg, 0.30mmol).When mixture 48 obtained by being stirred at room temperature is small.Remove big portion After dividing solvent, ether (200mL) is added.Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white powder (0.43g, 86%).

Synthesis

Example 67

The synthesis of the mPEG4- azanols of presentation in this Examples detail Figure 52 B.

Synthesis

To mPEG propionic aldehyde (30K, 0.5g, 0.0166mmol) and amine connexon (70mg, 0.25mmol) in MeOH (10mL) In mixture in add NaCNBH₃(20mg, 0.30mmol).When mixture 48 obtained by being stirred at room temperature is small.Remove big portion After dividing solvent, ether (200mL) is added.Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white powder (0.40g, 80%).

Synthesis

To the mPEG (30K) (0.40g, 0.013mmol) protected through two tertbutyloxycarbonyls in anhydrous CH at 0 DEG C₂Cl₂ Trifluoroacetic acid (4mL) is added in solution in (4mL).When mixture 8 obtained by being stirred at room temperature is small.Add ether (100mL) Into reaction mixture.Sediment is filtered, is washed with ether and be dried in a vacuum to obtain white powder (0.35g, 87%).

Example 68

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 53 A.

Synthesis

To mPEG (30K) (6.0g, 0.2mmol) and pyridine (0.1mL, 1.2mmol) in CH at 0 DEG C₂Cl₂In (60mL) Agitated solution in addition Dai Si-Martin cross iodine alkane (0.2g, 0.47mmol).Mixture is stirred at room temperature to stay overnight.It will be anti- It should be with Na₂S₂O₃-NaHCO₃Saturated aqueous solution (1:1,100mL) stop and with CH₂Cl₂(500mL × 2) extract.By organic layer group It closes and with H₂O and salt water washing, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.Residue is dissolved in CH₂Cl₂In (50mL).Ether (1L) is added into solution.Sediment is filtered, washs and be dried in a vacuum to obtain with ether To white powder (4.9g, 82%).

Synthesis

To mPEG (30K) acetaldehyde (0.5g, 0.0166mmol) and amine connexon (70mg, 0.25mmol) in MeOH (10mL) In mixture in add NaCNBH₃(20mg, 0.30mmol).When mixture 48 obtained by being stirred at room temperature is small.Remove big portion After dividing solvent, ether (200mL) is added.Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white powder (0.40g, 80%).

Synthesis

Example 69

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 53 B.

Synthesis

To mPEG (30K)-NH₂Boc-Ser-OH is added in the solution of (6.0g, 0.2mmol) in DMF (60mL) (205mg, 1.0mmol), 1- ethyls -3- (3- dimethylaminopropyls) carbodiimide hydrochloride (EDC, 190mg, 1.0mmol) and N, N'- diisopropyl ethyl amine (0.17mL, 1.0mmol).By mixture be stirred at room temperature 10 it is small when and with EtOAc (500mL) dilutes.By gained mixture with NaHCO₃Saturated aqueous solution (300mL), H₂O (300mL) and brine (300mL) It washs successively, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.Residue is dissolved in CH₂Cl₂In (50mL). Add ether (700mL).Sediment is filtered, is washed with ether and be dried in a vacuum to obtain white powder (5.1g, 82%).

Synthesis

To above-mentioned mPEG (30K) (3.0g, 0.1mmol) in CH at 0 DEG C₂Cl₂Trifluoro second is added in solution in (15mL) Sour (15mL).By gained mixture be stirred at room temperature 3 it is small when and concentration.CH is added into residue₂Cl₂(5mL), then adds Add HCl (in 4N, Yu dioxane, 2mL).Addition ether (400mL) with precipitate the dihydroxylamine of white solid-like (2.6g, 85%).

Synthesis

To above-mentioned mPEG (30K) (2.0g, 0.067mmol) in H₂O-CH₃CN(1:1,20mL) added in the solution in NaIO₄(15mg, 0.07mmol).By mixture be stirred at room temperature 4.0 it is small when and with CH₂Cl₂(500mL) dilutes.Gained is mixed Object is closed with H₂O (100mL) and brine (100mL) washing, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.It will be residual Excess is dissolved in CH₂Cl₂In (50mL).Add ether (700mL).Sediment is filtered, washs and is dried in a vacuum with ether To obtain white powder (1.8g, 90%).

Synthesis

Example 70

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 54 A.

Synthesis

To mPEG (30K) propionic aldehyde (0.5g, 0.0166mmol) and amine connexon (40mg, 0.16mmol) in MeOH (10mL) In mixture in add NaCNBH₃(12mg, 0.17mmol).When mixture 60 obtained by being stirred at room temperature is small.Remove big portion After dividing solvent, residue is dissolved in CH₂Cl₂It is washed in (200mL) and with citric acid (5%, 100mL).By organic layer through anhydrous Na₂SO₄Drying filters and concentrates to obtain white solid (0.42g, 84%) in a vacuum.

Synthesis

It is added into mixture of mPEG (30K) phthalimide (0.4g, 0.013mmol) in MeOH (4mL) H₂NNH₂(4.2 μ L, 0.13mmol).Mixture is stirred at 45 DEG C 1.0 it is small when and concentration.Residue is dissolved in CH₂Cl₂ It is washed in (100mL) and with HCl (0.1N, 100mL).With CH₂Cl₂(100mL) aqueous layer extracted.Organic layer is combined and with H₂O is washed It washs, through anhydrous Na after₂SO₄Dry, filtering and concentration.Residue is dissolved in CH₂Cl₂In (5mL).Add ether (200mL) To precipitate white powdered hydroxylamine product (0.34g, 85%).

Example 71

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 54 B.

Synthesis

To mPEG (30K) (6.0g, 0.2mmol) and pyridine (0.1mL, 1.2mmol) in CH at 0 DEG C₂Cl₂In (60mL) Agitated solution in addition Dai Si-Martin cross iodine alkane (0.2g, 0.47mmol).Mixture is stirred at room temperature to stay overnight.Then It will react with Na₂S₂O₃-NaHCO₃Saturated aqueous solution (1:1,100mL) stop and with CH₂Cl₂(500mL × 2) extract.It will be organic Layer combines and with H₂O and salt water washing, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.Residue is dissolved in CH₂Cl₂In (50mL).Ether (1L) is added into solution.Sediment is filtered, washs and be dried in a vacuum to obtain with ether To white powder (4.9g, 82%).

Synthesis

To mPEG (30K) acetaldehyde (0.5g, 0.0166mmol) and amine connexon (40mg, 0.16mmol) in MeOH (10mL) In mixture in add NaCNBH₃(12mg, 0.17mmol).When mixture 60 obtained by being stirred at room temperature is small.Remove big portion After dividing solvent, residue is dissolved in CH₂Cl₂It is washed in (200mL) and with citric acid (5%, 100mL).By organic layer through anhydrous Na₂SO₄Drying filters and concentrates to obtain white solid (0.42g, 84%) in a vacuum.

Synthesis

Example 72

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 55.

Synthesis

To mPEG (30K)-NH₂Boc-Ser-OH is added in the solution of (6.0g, 0.2mmol) in DMF (60mL) (205mg, 1.0mmol), 1- ethyls -3- (3- dimethylaminopropyls) carbodiimides (EDC, 190mg, 1.0mmol) and N, N'- diisopropyl ethyl amines (0.17mL, 1.0mmol).By mixture be stirred at room temperature 10 it is small when and with EtOAc (500mL) Dilution.By gained mixture with NaHCO₃Saturated aqueous solution (300mL), H₂O (300mL) and brine (300mL) wash successively, with By anhydrous Na₂SO₄Drying is filtered and concentrated in a vacuum.Residue is dissolved in CH₂Cl₂In (50mL).Add ether (700mL).Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white powder (5.1g, 82%).

Synthesis

Example 73

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 56 A.

Synthesis

To mPEG (30K) propionic aldehyde (0.5g, 0.0166mmol) and amine connexon (40mg, 0.16mmol) in MeOH (10mL) In mixture in add NaCNBH₃(12mg, 0.17mmol).When mixture 60 obtained by being stirred at room temperature is small.Remove big portion After dividing solvent, residue is dissolved in CH₂Cl₂It is washed in (200mL) and with citric acid (5%, 100mL).By organic layer through anhydrous Na₂SO₄Drying filters and concentrates to obtain white solid (0.41g, 82%) in a vacuum.

Synthesis

It is added into mixture of mPEG (30K) phthalimide (0.4g, 0.013mmol) in MeOH (4mL) H₂NNH₂(4.2 μ L, 0.13mmol).Mixture is stirred at 45 DEG C 1.0 it is small when and concentration.Residue is dissolved in CH₂Cl₂ It is washed in (100mL) and with HCl (0.1N, 100mL).With CH₂Cl₂(100mL) aqueous layer extracted.Organic layer is combined and with H₂O is washed It washs, through anhydrous Na after₂SO₄Dry, filtering and concentration.Residue is dissolved in CH₂Cl₂In (5mL).Add ether (200mL) To precipitate white powdered hydroxylamine product (0.34g, 83%).

Example 74

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 56 B.

Synthesis

To mPEG (30K) acetaldehyde (0.5g, 0.0166mmol) and amine connexon (40mg, 0.16mmol) in MeOH (10mL) In mixture in add NaCNBH₃(12mg, 0.17mmol).When mixture 60 obtained by being stirred at room temperature is small.Remove big portion After dividing solvent, residue is dissolved in CH₂Cl₂It is washed in (200mL) and with citric acid (5%, 100mL).By organic layer through anhydrous Na₂SO₄Drying filters and concentrates to obtain white solid (0.41g, 82%) in a vacuum.

Synthesis

Example 75

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 57.

Synthesis

Example 76

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 58 A.

The synthesis of mPEG (5K)-OMs

To mPEG (5K)-OH (1.0g, 0.2mmol) in CH₂Cl₂In solution in (40mL) add triethylamine (110 μ L, 0.79mmol) and MsCl (50 μ L, 0.64mmol).By mixture be stirred at room temperature 10 it is small when and concentration.Crude product (1.0g) It can be directly used for without purifying in next step.

mPEG(5K)-O-NHBocSynthesis

To thick mPEG (5K)-OMs (1.0g, 0.2mmol) in CH₂Cl₂Tert-butyl-n-hydroxyl is added in solution in (10mL) Aminocarbamic acid ester (0.3g, 2.2mmol) and triethylamine (0.4mL, 2.9mmol).Gained mixture is stirred 10 at 45 DEG C Hour and be cooled to room temperature.Add ether (200mL).Sediment is filtered, is washed and is dried in a vacuum to obtain white The product (0.42,42%) of solid-like.

mPEG(5K)-O-NH₃ ⁺Cl^- Synthesis

DEG C to mPEG (5K)-ONHBoc (0.2g, 0.04mmol) in CH₂Cl₂Trifluoro is added in solution in (3mL) Acetic acid (7mL).By gained mixture be stirred at room temperature 1 it is small when and concentration.CH is added into residue₂Cl₂(5mL), then adds Add HCl (in 4N, Yu dioxane, 2mL).Ether (300mL) is added to precipitate the PEG dihydroxy amine derivatives of white solid-like (170mg, 85%).

Example 77

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 58 B.

mPEG(30K)-OTfSynthesis

To mPEG (30K)-OH (3.0g, 0.1mmol) in CH₂Cl₂2,6- lutidines is added in solution in (30mL) (60 μ L, 0.5mmol) and Tf₂O (65 μ L, 0.4mmol).Be stirred at room temperature mixture 10 it is small when.Add ether (400mL) extremely In mixture.Sediment is filtered, is washed with ether and is dried in a vacuum to obtain white powder (2.7g, 90%).

mPEG(30K)-O-NHBocSynthesis

To mPEG (30K)-OTf (2.5g, 0.083mmol) in CH₂Cl₂N- hydroxyl aminos are added in solution in (25mL) T-butyl formate (110mg, 0.84mmol) and diisopropyl ethyl amine (0.2mL, 1,1mmol).Mixture is stirred at room temperature Overnight.Ether (200mL) is added to precipitate white powdered product (2.2g, 88%).

mPEG(30K)-O-NH₃ ⁺Cl^- Synthesis

To above-mentioned mPEG (30K)-ONHBoc (2.0g, 0.067mmol) in CH at 0 DEG C₂Cl₂In solution in (15mL) Add trifluoroacetic acid (15mL).By gained mixture be stirred at room temperature 3 it is small when and concentration.CH is added into residue₂Cl₂ (5mL) then adds HCl (in 4N, Yu dioxane, 2mL).Ether (300mL) is added to precipitate the PEG bis- of white solid-like Hydroxylamine derivative (1.72g, 86%).

Example 78

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 59 A.

Synthesis

To 2- (2- hydroxyl-oxethyls) phthalimides (0.5g, 2.4mmol) in CH₂Cl₂In solution in (20mL) Add phosgene (20% (in toluene), 8.0mL, 15.0mmol).By reaction mixture be stirred at room temperature 10 it is small when and true Aerial concentration.Residue (0.45g, 70%) can be directly used for next reaction without purifying.

Synthesis

To mPEG (30K)-NH₂(3g, 0.1mmol) and chloro-formate connexon (0.27g, 1.0mmol) are in CH₂Cl₂ Diisopropyl ethyl amine (0.2mL, 1.1mmol) is added in mixture in (30mL).Gained mixture is stirred at room temperature 15 it is small when.Ether (500mL) is added into mixture.Sediment is filtered, is washed and is dried in a vacuum to obtain white The product (2.7g, 90%) of solid-like.

Synthesis

Ammonia is added into solution of mPEG (30K) phthalimide (2.1g, 0.07mmol) in MeOH (15mL) (7N, in methanol, 15mL).By gained mixture be stirred at room temperature 15 it is small when and concentration.CH is added into residue₂Cl₂ (5mL) then adds HCl (in 4N, Yu dioxane, 1mL).Ether (300mL) is added to precipitate the product of white solid-like (2.4g, 89%).

Example 79

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 59 B.

Synthesis

To (t-butoxycarbonyl-amino oxygroup) acetic acid (3.0g, 16mmol) in CH₂Cl₂N is added in solution in (80mL), N'- Diisopropylcarbodiimides (DIC, 1.3mL, 8mmol).By mixture be stirred at room temperature 1 it is small when and it is dense in a vacuum Contracting.Crude product (4.9g, 84%), which need not be further purified, can be directly used in next step.

Synthesis

MPEG (5K)-NH is added into solution of the acid anhydride (7.3g, 20mmol) in DMF (20mL)₂(20g, 4mmol).It will Mixture be stirred at room temperature 10 it is small when and with H₂O dilutes (200mL).With CH₂Cl₂(500mL) extracts mixture.By organic layer With H₂O and brine (100mL) washing, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.It is added into residue CH₂Cl₂(10mL) then adds ether (500mL).Sediment is filtered, with ether wash and be dried in a vacuum be in The product (19.8g, 99%) of white powder.

Synthesis

To the mPEG (5K) (1.0g, 0.2mmol) protected through tertbutyloxycarbonyl in CH₂Cl₂It is added in solution in (5mL) Trifluoroacetic acid (5mL).By gained mixture be stirred at room temperature 1 it is small when and concentration.CH is added into residue₂Cl₂(2mL), connects Addition HCl (in 4N, Yu dioxane, 0.1mL) and ether (40mL).Sediment is filtered, with ether wash and in a vacuum It dries to obtain product (0.75g, 75%).

Example 80

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 60 A.

Synthesis

To mPEG (30K)-OH (4.5g, 0.15mmol) in CH₂Cl₂In solution in (50mL) add phosgene (20%, in In toluene, 1.6mL, 3.0mmol).By reaction mixture be stirred at room temperature 10 it is small when and concentrate in a vacuum.Residue (4.2g, 93%) can be directly used in next reaction without purifying.

Synthesis

To above-mentioned activation mPEG (30K) II (4.2g, 0.14mmol) and amine connexon VIII (67mg, 0.28mmol) in DMF-CH₂Cl₂(10mL, 1:2) diisopropyl ethyl amine (75 μ L, 0.42mmol) is added in the mixture in.It is stirred at room temperature After when gained mixture 15 is small, ether (200mL) is added.Sediment is filtered, washs and be dried in a vacuum to obtain with ether To white powdered product (3.8g, 90%).

Synthesis

It is added into solution of mPEG (30K) the phthalimide III (3.5g, 0.12mmol) in MeOH (15mL) Ammonia (7N, in methanol, 15mL).By gained mixture be stirred at room temperature 15 it is small when and concentration.CH is added into residue₂Cl₂ (5mL) then adds HCl (in 4N, Yu dioxane, 1mL).Ether (300mL) is added to precipitate the product of white solid-like (3.0g, 86%).

Synthesis

N- hydroxyls are added into solution of the 2- hydroxyethylcarbamates tert-butyl ester (2.8mL, 18mmol) in THF (60mL) Phthalimide (5.8g, 36mmol), triphenylphosphine (3.6g, 27mmol).Reaction mixture is stirred at room temperature 10 Minute and be then cooled to 0 DEG C.Through 1 it is small when by syringe be added dropwise diisopropyl azodiformate (DIAD, 3.6mL, 19mmol).It removes ice bath and the mixture was stirred overnight and concentrates.White solid is dissolved in ethyl acetate (100mL).It will Reaction mixture is washed successively with saturated aqueous solution of sodium bicarbonate (2 × 50mL), deionized water (50mL) and brine (50mL), with By anhydrous MgSO₄Drying is filtered and concentrated in a vacuum.By using Biotage Inc.HORIZON^TMChromatographic system it is fast Fast chromatogram purification crude product is to obtain the title compound (12g, 206%) containing impurity of white solid-like.

Synthesis

Example 81

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 60 B.

Synthesis

To mPEG (5K)-OH (10g, 2.0mmol), Ph at 0 DEG C₃P (790mg, 3.0mmol) and N- hydroxyl O-phthalics Acid imide (0.49g, 3.0mmol) is in CH₂Cl₂-THF(2:3,45mL) diisopropyl azodiformate is added in the solution in (DIAD, 409 μ L, 2.0mmol).By mixture be stirred at room temperature 15 it is small when after, addition ether (1L) into mixture.It will be heavy Starch filtering is washed with ether and is dried in a vacuum to obtain white powdered mPEG (5K)-phthalimide (9.8g, 98%).

mPEG(5K)-O-NH₂ Synthesis

Into solution of mPEG (5K) phthalimide (3g, 0.6mmol) in MeOH (25mL) add ammonia (7N, In methanol, 25mL).By gained mixture be stirred at room temperature 15 it is small when and concentration.CH is added into residue₂Cl₂(5mL), Then add HCl (in 4N, Yu dioxane, 1mL).Addition ether (300mL) with precipitate the azanol of white solid-like (2.5g, 83%).

Synthesis

To mPEG (30K)-OH (6g, 0.2mmol), Ph at 0 DEG C₃P (80mg, 0.3mmol) and N- hydroxyl O-phthalics Acid imide (49mg, 0.3mmol) is in CH₂Cl₂-THF(2:Diisopropyl azodiformate is added in solution in 3,45mL) (DIAD, 41 μ L, 0.2mmol).Be stirred at room temperature mixture 15 it is small when.Ether (200mL) is added into mixture.It will precipitation Object is washed with ether and is dried in a vacuum to obtain white powdered mPEG (30K)-phthalimide product (5.8g, 96%).

mPEG(30K)-O-NH₂ Synthesis

Into solution of mPEG (30K) phthalimide (3g, 0.1mmol) in MeOH (20mL) add ammonia (7N, In methanol, 20mL).By gained mixture be stirred at room temperature 15 it is small when and concentration.CH is added into residue₂Cl₂(5mL), Then add HCl (in 4N, Yu dioxane, 1mL).Addition ether (300mL) with precipitate the mPEG of white solid-like (30K)- ONH₂(2.6g, 87%).

Example 82

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 61 A.

Synthesis

Into 2- (2- (2- amino ethoxies) ethyoxyl) solution of ethamine (5.0g, 33.8mmol) in DMF (100mL) Add (t-butoxycarbonyl-amino oxygroup) acetic acid (14.2g, 74.2mmol), 1- ethyls -3- (3- dimethylaminopropyls) carbonizations Diimmonium salt hydrochlorate (EDC, 28.5g, 0.15mol) and N, N'- diisopropyl ethyl amine (26mL, 0.15mol).Mixture is existed It is diluted when stirring 10 is small at room temperature and with EtOAc (500mL).By gained mixture with NaHCO₃Saturated aqueous solution (300mL), H₂O (300mL) and brine (300mL) wash successively, through anhydrous Na after₂SO₄Drying is filtered and concentrated in a vacuum.Crude product (14.2g, 85%) can be directly used in next reaction without purifying.

Synthesis

To above-mentioned two tertbutyloxycarbonyls connexon (3.0g, 6.1mmol) in CH at 0 DEG C₂Cl₂In solution in (15mL) Add trifluoroacetic acid (15mL).By gained mixture be stirred at room temperature 3 it is small when and concentration.CH is added into residue₂Cl₂ (5mL) then adds HCl (in 4N, Yu dioxane, 2mL).Ether (400mL) is added to precipitate the dihydroxy of white solid-like Amine (1.47g, 82%).

Example 83

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 61 B.

Synthesis

At 0 DEG C Ph is added into solution of three (ethylene glycol) (1.5g, the 10mmol) in THF (100mL)₃P (8.0g, 30mmol) and n-Hydroxyphthalimide (4.9g, 30mmol).Azoformic acid diisopropyl is slowly added into mixture Ester (DIAD, 4.08mL, 20mmol).Gained mixture is stirred at 0 DEG C 4 it is small when and be stirred at room temperature 2 days.Add ether (25mL) is into reaction mixture.Sediment with ether is washed and is dried in a vacuum to obtain white two powdered neighbours Phthalimide product (3.72g, 82%).

Synthesis

Ammonia (7N, in first is added into solution of two phthalimides (2.2g, 5.0mmol) in MeOH (20mL) In alcohol, 20mL).By gained mixture be stirred at room temperature 15 it is small when and concentration.CH is added into residue₂Cl₂(5mL), then Add HCl (in 4N, Yu dioxane, 1mL).Ether (300mL) is added to precipitate the three of white solid-like (ethylene glycol) dihydroxies Amine connexon (1.1g, 87%).

Example 84

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 61 C.

Synthesis

At 0 DEG C Ph is added into solution of four (ethylene glycol) (1.94g, the 10mmol) in THF (100mL)₃P (8.0g, 30mmol) and n-Hydroxyphthalimide (4.9g, 30mmol).Azoformic acid diisopropyl is slowly added into mixture Ester (DIAD, 4.08mL, 20mmol).Gained mixture is stirred at 0 DEG C 4 it is small when and be stirred at room temperature 2 days.Add ether (25mL) is into reaction mixture.Sediment with ether is washed and is dried in a vacuum to obtain white two powdered neighbours Phthalimide product (3.58g, 74%).

Synthesis

Ammonia (7N, in first is added into solution of two phthalimides (2.42g, 5.0mmol) in MeOH (20mL) In alcohol, 20mL).By gained mixture be stirred at room temperature 15 it is small when and concentration.CH is added into residue₂Cl₂(5mL), then Add HCl (in 4N, Yu dioxane, 1mL).Ether (300mL) is added to precipitate the four of white solid-like (ethylene glycol) dihydroxies Amine connexon (1.27g, 85%).

Example 85

The synthesis of the mPEG- azanols of presentation in this Examples detail Figure 62 A.

Synthesis

At 0 DEG C Ph is added into solution of six (ethylene glycol) (2.82g, the 10mmol) in THF (100mL)₃P (8.0g, 30mmol) and n-Hydroxyphthalimide (4.9g, 30mmol).Azoformic acid diisopropyl is slowly added into mixture Ester (DIAD, 4.08mL, 20mmol).Gained mixture is stirred at 0 DEG C 4 it is small when and be stirred at room temperature 2 days.Add ether (25mL) is into reaction mixture.Sediment with ether is washed and is dried in a vacuum to obtain white two powdered neighbours Phthalimide product (4.40g, 77%).

Synthesis

Ammonia (7N, in first is added into solution of two phthalimides (2.86g, 5.0mmol) in MeOH (20mL) In alcohol, 20mL).By gained mixture be stirred at room temperature 15 it is small when and concentration.CH is added into residue₂Cl₂(5mL), then Add HCl (in 4N, Yu dioxane, 1mL).Ether (300mL) is added to precipitate the six of white solid-like (ethylene glycol) dihydroxies Amine connexon (1.68g, 87%).

Example 86

The synthesis of the mPEG compounds of presentation in this Examples detail Figure 62 B.

Synthesis

To mPEG-OH (30K, 3.0g, 0.1mmol) in anhydrous CH₂Cl₂Surpalite (63 μ are added in solution in (30mL) L, 0.5mmol).Mixture is stirred at room temperature overnight.Ether (700mL) is added to precipitate mPEG.Product is filtered, with second Ether is washed and is dried in a vacuum (3.0g, 100%).

Following instance description measurement and more modified therapeutic activity non-natural amino acid polypeptides in vitro and live body Interior activity and the method active in vitro and in vivo of therapeutic activity natural amino acid polypeptide.

Example 87：Cell combination is examined and determine

It is being not present or there are various concentration (volumes at 0 DEG C：10 μ l) un-marked GH, hGH or GM-CSF feelings Under condition and¹²⁵By cell (3 × l0 in the presence of I-GH (about 100,000cpm or 1ng)⁶) training in PBS/1%BSA (100 μ l) Support (two parts) 90 minutes (total volumes：120μl).Then cell is suspended and 200 μ l in 350 μ l plastic cement centrifuge tubes are ice-cold FCS higher slices and centrifugation (1000g；1 minute).Centrifugal-block is collected by cutting off the bottom of pipe and with gamma counter (Packard) centrifugal-block and supernatant are counted respectively.

It is subtracted with the total binding (dual multiple average value) in the case of there is no competitor there are 100 times of excess not The form of combination (cpm) (non-specific binding) in the case of labeled GH specifically binds (cpm) to measure.To being made Each measurement non-specific binding of cell type.Experiment is using identical¹²⁵I-GH preparations are carried out in the number of days separated And it should show internal consistency.¹²⁵I-GH confirms and generates the combination of the cell of GH receptors.With reference to dosage-dependent manner by Inhibit to un-marked natural GH or hGH, but inhibit from GM-CSF or other negative controls.HGH competitions are natural¹²⁵I-GH The ability that (being similar to natural GH) combines implies that receptor equally well identifies two kinds of forms.

Example 88：The in vivo research of PEGylated hGH

By PEG-hGH, unmodified hGH and buffer solution administration mouse or rat.As a result should show notable by weight Increase instruction compared with unmodified hGH, PEGylated hGH of the invention have outstanding active and extended half-life period.

Example 89：Engagement hGH and the measurement for not engaging hGH and the vivo half-life of its variant

All zooperies are in the up-to-standard equipment of AAALAC and with by Saint Louis University (St.Louis University management of laboratory animal and the use committee (Institutional Animal Care and Use) Committee) scheme of accreditation carries out.Indoors with the cycling of 12 light/dark when small by the indivedual stable breedings of rat in cage.Make Animal can be arbitrarily close to qualified Purina rodent chows 5001 and water.For the rat to hypophysectomize, drinking water volume Contain 5% glucose outside.

Example 90：Pharmacokinetic study

The quality of each PEGylated mutant hGH is assessed by examining and determine three times before zoopery is entered.By using non- MES SDS electrophoretic buffers (Invitrogen, Carlsbad, CA) electrophoresis 4%-12% acrylamides under reductive condition The purity of NuPAGE Bis-Tris gel detections PEG-hGH.Gel is dyed with Coomassie brilliant blue (Coomassie blue). It is scanned according to density measurement, the purity of PEG-hGH bands is more than 95%.By using from Charles River The KTA of Laboratories (Wilmington, MA)²The dynamics LAL calibratings of kit are interior in every PEG-hGH to test Content of toxins, and it is less than 5EU per dosage.With the bioactivity of IM-9pSTAT5 biological standardizations assessment PEG-hGH, and confirm EC₅₀ Value is less than 15nM.

In obtained from the male Sprague-Dawley rat of Charles River Laboratories (261g-425g) Be compared to each other the growth hormone compound through PEG modifications pharmacokinetic property and itself and non-PEGylated growth hormone are carried out Compare.Conduit is installed in arteria carotis by operation and is used for blood collection.After conduit is successfully installed, before administration will Animal is distributed into treatment group (every group three only to six).With the administered volume of 0.41-0.55ml/kg to animal subcutaneous administration 1mg/ The compound of kg.Blood sample is gathered by inlying catheter in Each point in time and is made it into the micro-centrifuge tube through EDTA coatings. Blood plasma is collected after centrifugation, and is stored at -80 DEG C until analysis.Using from BioSource International The antibody sandwich growth hormone of (Camarillo, CA) or Diagnostic Systems Laboratories (Webster, TX) ELISA kit measures compound concentration.Concentration is calculated using the standard corresponding to the analog given.Use model program WinNonlin (Pharsight, edition 4 .1) estimates pharmacokinetic parameter.It is accumulated using with linear upper/logarithm lower trapezoid The non-compartment model analysis (Noncompartmental analysis) divided, and homogeneous weighting concentration data.

After the single SC administration in rat, interval obtains plasma concentration at regular times.To rat (every group of n= 3-6) give the single bolus dosages of per kilogram protein 1mg.HGH wild-type proteins (WHO hGH), His marks hGH is more Peptide (his-hGH) is included at each of six different positions and the covalent bonded alpha-non-natural amino acids pair of 30kDa PEG The His of acetyl-phenylalanine marks hGH polypeptides compared with WHO hGH and (his)-hGH.In specific time interval Take plasma sample and according to the calibrating injection compound therein.Following table shows the medicine of the various hGH polypeptides of single dose administration Object dynamic parameter value.Concentration vs. time curve is assessed by non-compartment model analysis (Pharsight, edition 4 .1).Display It is worth for average value (+/- standard deviation).Cmax：Maximum concentration；Latter stage_t1/2：Terminal half-life；AUC_0->inf：It is extrapolated to infinity Concentration time curve under area；MRT：Mean residence time；Cl/f：Apparent total plasma clearance；Vz/f：In latter stage rank Apparent volume of distribution during section.Compared with compareing hGH, observe that 30KPEG-pAF92 (his) hGH Xun Huans are obviously prolonged, blood Clear half-life period increases and biological usability increases.

Table：The drug of the 1mg/kg bolus of subcutaneous administration single dose moves in normal male Sprague-Dawley rats Mechanics parameter value.

Example 91：Pharmacodynamic study

The male Sprague-Dawley rat to hypophysectomize is to be obtained from Charles River Laboratories.In 3- During 4 week old hypophysis is removed by performing the operation.In the period that animal is made to shake down 3 weeks, weight is monitored during this period.It will start to grind It is treatment group that animal through the period weight gain 0g-8g of 7 days before studying carefully, which is put into random combine,.To rat administration bolus agent Amount or daily subcutaneous administration.In entire research, daily and successively to rat weight, anesthesia, bloodletting and administration (as appropriate).Make Blood is gathered from socket of the eye sinus and insert in the micro-centrifuge tube through EDTA coatings with heparinised capillary.Pass through centrifugal separation plasma and storage In the presence of at -80 DEG C until analysis.Draw the curve at average (+/- S.D.) Plasma concentrations versus time interval.

Peptide IGF-1 is the member of the family of somatomedin or insulin-like growth factor.IGF-1 mediating growth hormones Many effects for promoting growth.Use the competitive binding enzyme immunoassays reagent of the rat/mouse IGF-1 standards for offer Box (Diagnosic Systems Laboratories) measures IGF-1 concentration.Cut off the hypophysis of rat.To rat (every group of n =5-7) subcutaneous administration single dose or daily dosage.Weigh, anaesthetize to animal successively daily, bloodletting and administration (as appropriate). It obtains placebo treatment, wild type hGH (hGH), His mark hGH ((his) hGH) and is included at position 35 and 92 and 30kDa The weight result of the covalent bonded hGH polypeptides to acetyl-phenylalanine of PEG.It observes for 30KPEG-pAF35 (his) Weight gain of the hGH compounds at the 9th day (p statistically different from 30KPEG-pAF92 (his) hGH compounds< 0.0005), the difference is that observing the weight gain of bigger.In including through PEGylated non-natural for administration single dose After the hGH polypeptides of coded amino acid, by using double tails distribution, not paired, equal variance t- measurements determinations to circulating The effect for starching IGF-1 levels has significant difference.

Example 92：The security of PEGylated hGH and/or the human clinical trial of effect including non-naturally encoded amino acids.

PurposeTo compare the PEGylated recombinant human hGH and one kind including non-naturally encoded amino acids through subcutaneous administration Or more than one commercially available hGH products are (including (but not limited to) Humatrope^TM(Eli Lilly&Co.)、Nutropin^TM (Genentech)、Norditropin^TM(Novo-Nordisk)、Genotropin^TM(Pfizer) and Saizen/Serostim^TM (Serono)) security and pharmacokinetics.

Patient18 health that the age is in the range of -40 years old 20 years old and weight is between 60kg-90kg are recruited in research Volunteer.Person under inspection should not have clinically significantly abnormal hematology or serum chemistry and negative urine toxicity screening, HIV Screening and Hepatitis B Surface antigen experiment value.It should not have any following sign：Hypertension；Any primary hematologic disease history； Great hepatopathy, nephrosis, angiocardiopathy, gastrointestinal disease, urogenital disease, metabolic disease, neuropathy medical history；Anaemia is insane Epilepsy medical history；To the known sensibility of the product of bacterium or mammal source, PEG or human serum albumin；Containing caffeine The habituation of beverage and Heavy User；Any other clinical test is participated in 30 days into research or through transfusing blood or offering Blood；HGH had been contacted in 3 months into research；It is diseased in 7 days into research；And 14 days in entrance research It is interior that there is significantly exception to physical examination before research or clinical laboratory assessments.All persons under inspection are assessed with security and timing is adopted Collection gathers object for the whole blood of pharmacokinetic analysis.Agree to owned through institutional ethics committee approval and patient Research.

Research and designIts should be stage i in healthy male volunteers, single center, open-label, it is random, two weeks Phase crossing research.18 persons under inspection are randomly assigned into one group in two treatment sequence groups (every group of 9 persons under inspection).It uses The PEGylated hGH including non-naturally encoded amino acids of equal dose and selected commercial product are in upper leg portion with quickly subcutaneous note It penetrates form and is administered alone period administration GH through two.The dosage and frequency of administration commercial product are according to saying in mark of parcels It is bright.As required, using commercial product other dosage, administration frequency or other parameter can by comprising other person under inspection's groups and It is added in research.Each medicine-feeding period was separated by the cleaning phase of 14 days.For each of medicine-feeding period twice, person under inspection Before administration at least 12 it is small when and administration after 72 it is small when be limited at research center, but without so between medicine-feeding period. If there is other dosage, frequency or other parameter, then can also add additional person under inspection's group to test PEGylated hGH.It is approved It can be used for for a variety of GH formulas of human use in this research.Humatrope^TM(Eli Lilly&Co.)、Nutropin^TM (Genentech)、Norditropin^TM(Novo-Nordisk)、Genotropin^TM(Pfizer) and Saizen/Serostim^TM (Serono) it is to be approved for the commercially available GH products of human use.The experimental formula of hGH is to include non-naturally encoded amino acids PEGylated hGH.

Blood samplingPass through direct venipuncture continuous blood sampling before and after administration hGH.About 30 minutes before administration, 20 minutes and 10 minutes (3 baseline samples) and upon administration about following time：30 minutes and 1 it is small when, 2 it is small when, 5 it is small when, it is 8 small When, 12 it is small when, 15 it is small when, 18 it is small when, 24 it is small when, 30 it is small when, 36 it is small when, 48 it is small when, it is 60 small and when 72 is small is used for Measure the venous samples (5mL) of Serum GH concentration.Each serum sample is divided into two aliquots.All serum samples are stored up In the presence of at -20 DEG C.Serum sample transports on dry ice.On day 1 before initial administration at once, the administration of the 4th day morning, the 16th day Before at once and the 19th day morning carry out fasting clinical trial test (hematology, serum chemistry and urine test).

Bioanalytical methodUsing ELISA kit program (Diagnostic Systems Laboratory [DSL], Webster TX) measure Serum GH concentration.

Security testBefore (the 1st day and the 16th day) is administered each time and each time after administration 6 it is small when, it is 24 small When, 48 it is small when and 72 it is small when immediate record vital sign.Security test be incidence based on adverse events and type and The variation from baseline is tested in clinical trial.In addition, in assessment vital sign measurement (including blood pressure) and Physical examination results With the variation before research.

Data analysisIt is subtracted by each by being worth after being administered by equalizing 30 minutes before administration, 20 minutes and 10 points After the horizontal average baselining GH concentration measured of the GH of three samples gathered during clock is on baseline GH concentration corrections administration before administration Serum concentration.If Serum GH concentration is less than the quantitative level of calibrating before administration, then it is not included in the calculating of average value In.Pharmacokinetic parameter is measured from the serum concentration data on baseline GH concentration corrections.Use the BIOAVL of recent release Software in 8600 computer systems of Digital Equipment Corporation VAX in passing through the method independent of model To calculate pharmacokinetic parameter.Measure following pharmacokinetic parameter：Peak serum-concentration (C_max)；Reach peak serum-concentration Time (t_max)；By using that linear trapezoidal rule calculates from time zero blood sampling time (AUC to the end_0-72) concentration- Area (AUC) under time graph；And terminal elimination half-life (the t calculated by elimination rate constant_1/2).In Log-Linear Elimination rate constant is estimated by the linear regression at consecutive numbers strong point in the terminal linear region of concentration time curve.For Each treatment calculates the mean standard deviation (SD) of pharmacokinetic parameter and the coefficient of variation (CV).Calculating parameter average value Ratio (keeps formula/non-holding formula).

Safety resultsThe incidence distribution of adverse events is identical in treatment group.It is surveyed with clinical trial before baseline or research It tries or blood pressure is compared without clinically significant changes, and Physical examination results and the vital sign measurement nothing compared with before research are aobvious Write variation.The security overview of Liang Ge treatment groups should be apparently similar.

Pharmacokinetics resultsUnder measured each time point, the one or more of single dose will be received Commercially available hGH products are (including (but not limited to) Humatrope^TM(Eli Lilly&Co.)、Nutropin^TM(Genentech)、 Norditropin^TM(Novo-Nordisk)、Genotropin^TM(Pfizer) and Saizen/Serostim^TM(Serono)) after All 18 persons under inspection average serum GH Concentration-times overview (not on the amendment of baseline GH contents) with including non-natural compile The PEGylated hGH of code amino acid is compared.All persons under inspection should be dense with baseline GH before the administration in normal physiologic range Degree.By measuring pharmacokinetic parameter on the sera data of average baselining GH concentration corrections before administration and measuring C_maxAnd t_max。 Selected clinical comparative (Humatrope^TM(Eli Lilly&Co.)、Nutropin^TM(Genentech)、Norditropin^TM (Novo-Nordisk)、Genotropin^TM(Pfizer)、Saizen/Serostim^TM(Serono)) average t_maxThan including The t of the PEGylated hGH of non-naturally encoded amino acids_maxIt is significantly shorter.With the end of the PEGylated hGH including non-naturally encoded amino acids End half-life period is compared, and the Terminal half-life value of the commercially available hGH products of test is significantly shorter.

Although the research of the present invention is carried out in healthy male person under inspection, it is anticipated that (such as suffering from other PATIENT POPULATIONs There are male or female patient, children's patients with renal failure, the patient in self stored Procedure or predetermined of cancer or chronic renal failure The patient of elective surgery) in can have similar Absorption Characteristics and security overview.

On the whole, the PEGylated hGH including non-naturally encoded amino acids of the single dose through subcutaneous administration should be safety And healthy male person under inspection it good is received.According to the opposite incidence of adverse events, the hGH of commercial form and including non- Clinical trial value, vital sign and the Physical examination results and security overview of the PEGylated hGH of natural coded amino acid answer phase When.PEGylated hGH including non-naturally encoded amino acids potentially provides patient and healthcare provider big clinical effect With.

Example 93：The water-soluble of PEGylated hGH and non-PEGylated hGH is compared

HGH wild-type proteins (WHO hGH), His are obtained by measuring the amount for the polypeptide out of the ordinary for being dissolvable in water 100 μ L water Mark hGH polypeptides (his-hGH) are included at position 92 with the covalent bonded alpha-non-natural amino acids of 30kDa PEG to acetyl The water solubility of the His mark hGH polypeptides of base-phenylalanine.The amount of PEGylated hGH is more than the amount of WHO hGH and hGH, proves non- The PEGylated increase of natural amino acid polypeptide is water-soluble.

Example 94：The in vivo research of modified therapeutic activity non-natural amino acid polypeptides

Prostate cancer xenograft is implanted into mouse, is then classified as two groups.One group daily with through repairing The therapeutic activity non-natural amino acid polypeptides of decorations are treated and another group is used therapeutic activity natural amino acid polypeptide therapeutic daily.Daily Tumor size is measured, and such as by the reduction for the tumor size of group treated with modified therapeutic activity non-natural amino acid polypeptides Indicated, compared with therapeutic activity natural amino acid polypeptide, modified therapeutic activity non-natural amino acid polypeptides have improvement Therapeutic efficiency.

Example 95：The in vivo research of modified therapeutic activity non-natural amino acid polypeptides

It will be appreciated that example described herein and embodiment be only illustrative purpose and its various modification or variation should can be by Those skilled in the art proposes and in the spirit and scope of present application and in the scope of appended claims. All publication cited herein, patents and patent applicationss case are hereby incorporated by reference in their entirety for all purposes In.

Sequence table

<110> AMBRX, INC.

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Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Gly

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

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Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Thr Tyr Tyr

145 150 155 160

Tyr Leu Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

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Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

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Arg Leu

305

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Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Gly

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Thr Ser His

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Tyr Leu Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

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Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

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Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

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Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

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Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

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Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

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Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

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Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

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Arg Leu

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Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

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Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ala Ala Ile

20 25 30

Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln Ile

35 40 45

Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile Leu

50 55 60

Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp Glu

65 70 75 80

Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met Gly

85 90 95

Leu Lys Ala Lys Tyr Val Tyr Gly Ser Pro Phe Gln Leu Asp Lys Asp

100 105 110

Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys Arg

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Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro Lys

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Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Ala Ile Tyr Leu

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Ala Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile His Met

165 170 175

Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His Asn Pro

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Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser Lys Gly

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Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala Lys Ile

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Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro Ile Met

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Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys Arg Pro

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Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu Leu Glu

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Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys Asn Ala

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Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys Arg Leu

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Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

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Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Pro Phe Gln Leu Asp Lys

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Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

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Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Ile Pro Tyr

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Leu Pro Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile His

165 170 175

Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His Asn

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Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser Lys

195 200 205

Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala Lys

210 215 220

Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro Ile

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Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys Arg

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Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu Leu

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Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys Asn

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Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys Arg

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Leu

305

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Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

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Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

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Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

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Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Lys Phe Gln Leu Asp Lys

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Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

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Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

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Leu Ala Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile His

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Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser Lys

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Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala Lys

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Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro Ile

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Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys Arg

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Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu Leu

260 265 270

Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys Asn

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Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys Arg

290 295 300

Leu

305

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Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Asn Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Leu His

145 150 155 160

Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210> 10

<211> 306

<212> PRT

<213> Methanocaldococcus jannaschii

<400> 10

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Leu His

145 150 155 160

Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210> 11

<211> 306

<212> PRT

<213> Methanocaldococcus jannaschii

<400> 11

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Val His

145 150 155 160

Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210> 12

<211> 306

<212> PRT

<213> Methanocaldococcus jannaschii

<400> 12

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Ser His

145 150 155 160

Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210> 13

<211> 306

<212> PRT

<213> Methanocaldococcus jannaschii

<400> 13

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gly Cys His

145 150 155 160

Tyr Arg Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210> 14

<211> 306

<212> PRT

<213> Methanocaldococcus jannaschii

<400> 14

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gly Thr His

145 150 155 160

Tyr Arg Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210> 15

<211> 306

<212> PRT

<213> Methanocaldococcus jannaschii

<400> 15

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gly Gly His

145 150 155 160

Tyr Leu Gly Val Asp Val Ile Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210> 16

<211> 306

<212> PRT

<213> Methanocaldococcus jannaschii

<400> 16

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Arg Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Val Ile His

145 150 155 160

Tyr Asp Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

<210> 17

<211> 306

<212> PRT

<213> Methanocaldococcus jannaschii

<400> 17

Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser

1 5 10 15

Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Gly

20 25 30

Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln

35 40 45

Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile

50 55 60

Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp

65 70 75 80

Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met

85 90 95

Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp Lys

100 105 110

Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys

115 120 125

Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro

130 135 140

Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Thr Tyr Tyr

145 150 155 160

Tyr Leu Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile

165 170 175

His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His

180 185 190

Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser

195 200 205

Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala

210 215 220

Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro

225 230 235 240

Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys

245 250 255

Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu

260 265 270

Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys

275 280 285

Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys

290 295 300

Arg Leu

305

Claims

1. a kind of compound or its salt, including formula (I)：

Wherein：

A is optional, and when it is present for low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted it is rudimentary Cycloalkylidene, lower alkenylene are substituted lower alkenylene, alkynylene, rudimentary sub- miscellaneous alkyl, are substituted sub- miscellaneous alkyl, are rudimentary Sub- Heterocyclylalkyl, be substituted rudimentary sub- Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, Alkarylene is substituted alkarylene, sub- aralkyl or is substituted sub- aralkyl；

B is optional, and is the connexon selected from the group being made of following group when it is present：Low-grade alkylidene, through taking For low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, rudimentary sub- miscellaneous alkyl, be substituted rudimentary sub- miscellaneous alkyl ,- O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-, wherein k be 1,2 or 3- S(O)_k-、-S(O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted Asia Alkyl)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-S (O)_kN(R')-、-N(R')C(O)N (R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, wherein R' be each independently H, Alkyl or substituted alkyl；

J is

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R " is each independently H, alkyl, substituted alkyl or protecting group or when there are more than one R " during group, two R " Optionally form Heterocyclylalkyl；

R₃With R₄It is each independently H, halogen, low alkyl group or is substituted low alkyl group or R₃With R₄Or two R₃Group regards feelings Condition forms cycloalkyl or Heterocyclylalkyl；

Or bicyclic or three annular cycloalkyl or Heterocyclylalkyl including at least one following group is collectively formed in-A-B-J-R groups： Carbonyl, it includes dicarbapentaborane；Through protecting carbonyl, it includes through protecting dicarbapentaborane；Or masked carbonyl, it includes masked two carbonyls Base；

Or monocyclic or bicyclic cycloalkyl or Heterocyclylalkyl including at least one following group is collectively formed in-J-R groups：Carbonyl Base, it includes dicarbapentaborane；It is at least one through protect carbonyl, it includes through protect dicarbapentaborane；At least one masked carbonyl, bag Containing masked dicarbapentaborane；Or its combination；

Its restrictive condition is when A is phenylene and R₃During respectively H, B exists；And when A is-(CH₂)₄- and R₃During respectively H, B is not For-NHC (O) (CH₂CH₂)-；And when A and B are not present and R₃During respectively H, R is not methyl；

Or its active metabolite or pharmaceutically acceptable prodrug or solvate.

2. compound according to claim 1, wherein A are the low-grade alkylidene that is substituted or is unsubstituted or without taking Generation or the arlydene for being selected from the group being made of phenylene, sub-pyridyl group, sub- pyrimidine radicals or sub- thienyl being substituted.

3. compound according to claim 1 corresponds to formula (XXXIII)：

Wherein X₁For C, S or S (O)；And L is alkylidene, is substituted alkylidene, N (R') (alkylidene) or N (R') and (is substituted alkylene Base).

4. compound according to claim 3 corresponds to formula (XXXIII-A)：

5. compound according to claim 3 corresponds to formula (XXXIII-B)：

6. compound according to claim 1 corresponds to formula (XXXIV)：

Wherein X₁For C, S or S (O)；And n is 0,1,2,3,4 or 5；And every CR⁸R⁹R on group⁸With R⁹Group is independently Selected from the group or any R being made of H, alkoxy, alkylamine, halogen, alkyl, aryl⁸And R⁹=O or cycloalkanes can be collectively formed Base or any and adjacent R⁸Cycloalkyl can be collectively formed in group.

7. compound according to claim 6 corresponds to formula (XXXIV-A)：

8. compound according to claim 6 corresponds to formula (XXXIV-B)：

9. compound according to claim 1 corresponds to formula (XXXV)：

10. compound according to claim 9 corresponds to formula (XXXV-A)：

11. compound according to claim 9 corresponds to formula (XXXV-B)：

12. compound according to claim 1 corresponds to formula (XXXX)：

Wherein:

M is-C (R₃)-,

Wherein (a) instruction is bonded with A groups bond and (b) instruction with carbonyl out of the ordinary；And

T₃For a key, C (R) (R), O or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl.

13. compound according to claim 12 corresponds to formula (XXXXIII)：

14. compound according to claim 13 is to be selected from：

15. compound according to claim 1 corresponds to formula (III)：

Wherein R_aIt is each independently selected from the group being made of following group：H, halogen, alkyl, substituted alkyl ,-N (R')₂、 Wherein k is 1,2 or 3-C (O)_kR'、-C(O)N(R')₂,-OR' and-S (O)_kR', wherein R' are each independently H, alkyl Or substituted alkyl.

16. compound according to claim 15 is selected from the group being made of following object：

17. compound according to claim 1 corresponds to formula (VI)：

18. compound according to claim 17 is selected from the group being made of following object：

19. compound according to claim 1 corresponds to formula (IX)：

20. compound according to claim 19 is selected from the group being made of following object：

21. compound according to claim 1 the, wherein-A-B-J-R groups are collectively formed including at least one carbonyl Base, bicyclic or three annular cycloalkyl or Heterocyclylalkyl through protecting carbonyl or masked carbonyl.

22. compound according to claim 21 is selected from the group being made of following object：

23. compound according to claim 1 the, wherein-J-R groups are collectively formed including at least one carbonyl, warp Protect the monocyclic or bicyclic cycloalkyl or Heterocyclylalkyl of carbonyl or masked carbonyl.

24. compound according to claim 23, has following structure：

25. a kind of polypeptide, and have at least one compound according to claim 1.

26. a kind of compound or its salt, including formula (XI)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R₅For H, alkyl, substituted alkyl, alkenyl, be substituted alkenyl, alkynyl, be substituted alkynyl, alkoxy, be substituted alkoxy, Alkyl alkoxy, substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, substituted aryl, Heteroaryl, substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene is substituted Alkylidene)-ON (R ")₂,-(alkylidene is substituted alkylidene)-C (O) SR " ,-(alkylidene is substituted alkylidene)-S-S- (aryl or substituted aryl) ,-C (O) R " ,-C (O)₂R " or-C (O) N (R ")₂, wherein R " is each independently hydrogen, alkyl, warp Substitution alkyl, alkenyl are substituted alkenyl, alkoxy, are substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, warp Substituted alkaryl, aralkyl are substituted aralkyl；

Or R₅For L-X, wherein

X is selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymer；The derivative of polyethylene glycol Object；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Resin；The Two protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbon hydrate Object；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial；Nanometer Particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other molecule covalents or non-common The group of valency interaction；Light cage covers part；It can photoisomerization part；Biotin；Biotin analog；It is combined with heavy atom Part；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active agent；Ammonia Base thio-acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electronics Dense group；

Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；And above-mentioned substance Any combinations；And

L is optional, and is the connexon selected from the group being made of following group when it is present：Alkylidene is substituted Asia Alkyl, alkenylene, be substituted alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidenes or through taking For alkylidene)-, wherein k be 1,2 or 3-S (O)_k-、-S(O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)- (alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,

- N (R')-,-NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted Alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-, (alkylidene is substituted-N (R') CO- Alkylidene)-,-N (R') C (O) O- ,-(alkylidene is substituted alkylidene)-O-N=CR'- ,-(alkylidene is substituted alkylene Base)-C (O) NR'- (alkylidene is substituted alkylidene)-,-(alkylidene is substituted alkylidene)-S (O)_k- (alkylidene or warp Substituted alkylene)-S- ,-(alkylidene is substituted alkylidene)-S-S- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N (R')C(S)N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,

- C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂- N (R')-N (R')-, wherein R' is each independently H, alkane Base or substituted alkyl；

Its restrictive condition is in the absence of A and B, and R is not methyl；

Or its active metabolite or pharmaceutically acceptable prodrug or solvate.

27. compound according to claim 26, wherein A is phenylenes or are substituted phenylene.

28. compound according to claim 26 corresponds to formula (XII)：

29. a kind of polypeptide, and have at least one compound according to claim 26.

30. compound according to claim 26, wherein X are selected from by peptide, protein, enzyme, antibody, drug, dyestuff, fat The bioactivator for the group that matter, nucleosides, oligonucleotides, cell, virus, liposome, particle and micella form.

31. compound according to claim 30, wherein X are selected from by antibiotic, fungicide, antivirotic, anti-inflammatory The drug for the group that agent, antitumor agent, cardiovascular agents, antianxiety agent, hormone, growth factor and steroid agent form.

32. compound according to claim 30, wherein X are selected from by horseradish peroxidase, alkaline phosphatase, β-half The enzyme of the group of lactoside enzyme and glucose oxidase composition.

33. compound according to claim 26, wherein X are selected from by fluorescence part, phosphorescent moieties, chemiluminescence portion Point, chelating moiety, electron dense part, magnetic part, insertion portion, radioactive segment, chromophoric moiety and energy transfer The detectable label of the group of part composition.

A kind of 34. method for the polypeptide for preparing the amino acid including at least one structure with formula (I)：

The described method includes the amino acid of formula (I) is incorporated in the terminal position or interior location in polypeptide, wherein：

B is optional, and is the connexon selected from the group being made of following group when it is present：Low-grade alkylidene, through taking For low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, rudimentary sub- miscellaneous alkyl, be substituted rudimentary sub- miscellaneous alkyl ,- O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidene is substituted alkylidene)-, wherein k be 1,2 or 3- S(O)_k-、-S(O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,

- CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,

-N(R')C(O)O-、-S(O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and- C(R')₂- N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl；

J is

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

Or monocyclic or bicyclic cycloalkyl or Heterocyclylalkyl including at least one following group is collectively formed in-J-R groups：Carbonyl Base, it includes dicarbapentaborane；Through protecting carbonyl, it includes through protecting dicarbapentaborane；Or masked carbonyl, it includes masked two carbonyls Base；

Its restrictive condition is when A is phenylene and R₃During respectively H, B exists；And when A is-(CH₂)₄- and R₃During respectively H, B is not For-NHC (O) (CH₂CH₂)-；And when A and B are not present and R₃During respectively H, R is not methyl.

35. according to the method for claim 34, wherein the amino acid is to be incorporated to using translation system in the polypeptide In specific site, the translation system includes：

(i) polynucleotide of coding said polypeptide, wherein the polynucleotide, which includes corresponding to, is incorporated to the formula (I) amino acid Preassign site selection codon and

(ii) tRNA of the amino acid is included, wherein the tRNA has specificity to the selection codon.

36. according to the method for claim 35, wherein the translation system includes aminoacylated arrive on formula (I) amino acid tRNA。

37. according to the method for claim 36, wherein the translation system be include being selected from it is thin by bacterial cell, archeobacteria The in vivo translation system of the cell of the group of born of the same parents and eukaryocyte composition.

38. according to the method for claim 36, wherein the amino acid has the structure corresponding to formula (III)：

39. according to the method for claim 38, wherein the amino acid is selected from the group being made of following object：

40. according to the method for claim 37, wherein the amino acid has the structure corresponding to formula (VI)：

41. according to the method for claim 40, wherein the amino acid is selected from the group being made of following object：

42. according to the method for claim 37, wherein the amino acid has the structure corresponding to formula (IX)：

43. according to the method for claim 42, wherein the amino acid is selected from the group being made of following object：

44. according to the method for claim 37, wherein-A-B-J-R the groups are collectively formed including at least one following The bicyclic or three annular cycloalkyl or Heterocyclylalkyl of group：Carbonyl, it includes dicarbapentaborane；Through protecting carbonyl, it includes through protection Dicarbapentaborane；Or masked carbonyl, it includes masked dicarbapentaborane.

45. according to the method for claim 44, wherein the amino acid is selected from the group being made of following object：

46. according to the method for claim 37, wherein-J-R the groups are collectively formed including at least one following group Monocyclic or bicyclic cycloalkyl or Heterocyclylalkyl：Carbonyl, it includes dicarbapentaborane；Through protecting carbonyl, it includes through protecting two carbonyls Base；Or masked carbonyl, it includes masked dicarbapentaborane.

47. according to the method for claim 46, wherein the amino acid is：

48. according to the method for claim 37, wherein the formula (I) amino acid has the structure of formula (XXX)：

49. according to the method for claim 37, wherein the formula (I) amino acid has the structure of formula (XXXIII)：

50. according to the method for claim 37, correspond to formula (XXXX)：

Wherein：

M is-C (R₃)-,

51. according to the method for claim 50, correspond to formula (XXXXIII)：

52. one kind includes the method for the polypeptide of formula (I) amino acid for derivatization, the described method includes make the polypeptide and formula (XIX) reagent contact, wherein formula (I) corresponds to：

Wherein：

A is optional, and is when it is present low-grade alkylidene, is substituted low-grade alkylidene, lower alkenylene, is substituted rudimentary Asia Alkenyl, arlydene are substituted arlydene, inferior heteroaryl, are substituted inferior heteroaryl, alkarylene, are substituted alkarylene, sub- virtue Alkyl is substituted sub- aralkyl；

B is optional, and is the connexon selected from the group being made of following group when it is present：Low-grade alkylidene, through taking For low-grade alkylidene, lower alkenylene, be substituted lower alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,- S- (alkylidene is substituted alkylidene)-, wherein k be 1,2 or 3-S (O)_k-、-S(O)_k(alkylidene is substituted alkylene Base)-,-C (O)-,-C (O)-(alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylene Base)-,-N (R')-,-NR'- (alkylidene is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene or through taking For alkylidene)-,-CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidenes or through taking For alkylidene)-,-N (R') C (O) O- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R') S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- And-C (R')₂- N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl；

J is

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R' is each independently H, alkyl or substituted alkyl；

Wherein formula (XIX) corresponds to：

Wherein：

X is each independently detectable label, bioactivator or polymer；

L is respectively the connexon independently selected from the group being made of following group：Alkylidene is substituted alkylidene, sub- alkene Base, be substituted alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-, (alkylidene is substituted alkylene by-S- ,-S- Base)-, wherein k be 1,2 or 3-S (O)_k-、-S(O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene Or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- (alkylidene or Be substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-(alkylidene is substituted Alkylidene) NR'C (O) O- (alkylidene is substituted alkylidene)-,-O-CON (R')-(alkylidene is substituted alkylidene)-,- CSN (R')-,-CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-N (R') C (O) O- (alkylidene is substituted alkylidene)-,-S (O)_kN(R')-、-N(R')C(O)N (R')-,-N (R') C (O) N (R')-(alkylidene is substituted alkylidene)-,-N (R') C (S) N (R')-,-N (R') S (O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and- C(R')₂- N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl；

L₁To be optional, and it is-C (R') when it is present_p- NR'-C (O) O- (alkylidene is substituted alkylidene)-, wherein p for 0, 1 or 2；

W is-ON (R₁)₂Or-C (=O) R₂, wherein R₁It is each independently H or amino protecting group, and R₂For H or OR'；And

N is 1 to 3.

53. method according to claim 52, wherein the amino acid corresponds to formula (II)：

54. method according to claim 52, wherein the reagent corresponds to formula (XXVII)：

55. method according to claim 52, wherein the derivative polypeptide includes at least one structure with formula (XI) Amino acid containing oxime：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

R₅For H, alkyl, substituted alkyl, alkenyl, be substituted alkenyl, alkynyl, be substituted alkynyl, alkoxy, be substituted alkoxy, Alkyl alkoxy, substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, substituted aryl, Heteroaryl, substituted heteroaryl, are substituted alkaryl, aralkyl, are substituted aralkyl ,-C (O) R " ,-C (O) alkaryl₂R" Or-C (O) N (R ")₂, wherein R " is each independently hydrogen, alkyl, substituted alkyl, alkenyl, is substituted alkenyl, alkoxy, warp Substituted alkoxy, aryl, heteroaryl, alkaryl, are substituted alkaryl, aralkyl or are substituted aralkyl substituted aryl；Or R₅For L-X, wherein

X is detectable label, bioactivator or polymer；And

L is optional, and is the connexon selected from the group being made of following group when it is present：Alkylidene is substituted Asia Alkyl, alkenylene, be substituted alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidenes or through taking For alkylidene)-, wherein k be 1,2 or 3-S (O)_k-、-S(O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)- (alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- it is (sub- Alkyl is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,- CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O-、-S(O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)_kN(R')-、-N(R')-N =,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂- N=N- and-C (R')₂-N(R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl；

Its restrictive condition is in the absence of A and B, and R is not methyl.

56. method according to claim 52, wherein make the polypeptide in aqueous solution under appropriate acid condition with institute State the reagent contact of formula (XIX).

57. method according to claim 56, wherein the condition is pH 2 to 8.

58. method according to claim 52, wherein making examination of the polypeptide with the formula (XIX) in the presence of accelerating agent Agent contacts, and the accelerating agent is selected from the group being made of following object：

59. method according to claim 52, wherein the formula (I) amino acid has the structure of formula (XXX)：

60. method according to claim 52, wherein the formula (I) amino acid has the structure of formula (XXXIII)：

61. method according to claim 52 corresponds to formula (XXXX)：

Wherein：

M is-C (R₃)-,

62. method according to claim 61 corresponds to formula (XXXXIII)：

63. a kind of method for treating illness, symptom or disease, the described method includes administration therapeutically effective amount including at least one The non-natural amino acid polypeptides of alpha-non-natural amino acid containing oxime, wherein the alpha-non-natural amino acid containing oxime is to use translation system It is incorporated in the specific site in the polypeptide, the translation system includes：

(i) polynucleotide of coding said polypeptide, wherein the polynucleotide, which includes corresponding to, is incorporated to the amino acid containing oxime Preassign site selection codon and

(ii) tRNA of the amino acid containing oxime is included, wherein the tRNA has specificity to the selection codon.

64. method according to claim 63, wherein the translation system is the organism for including being selected from following group The in vivo translation system of cell：It is prokaryotes, eucaryote, mammal, Escherichia coli (Escherichia coli), true Bacterium, pseudomonad kind (species of Pseudomonas), yeast, archeobacteria, eubacteria, plant, insect and primary life Object.

65. method according to claim 63, wherein at least one alpha-non-natural amino acid is amino acid containing oxime and institute Stating the alpha-non-natural amino acid containing oxime has the structure for corresponding to formula (XI)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

Or R₅For L-X, wherein

X is selected from the group being made of following object：Mark；Dyestuff；Polymer；Water-soluble polymer；The derivative of polyethylene glycol Object；Photocrosslinking agent；Cytotoxic compound；Drug；Affinity marker；Photoaffinity marks；Reactive compounds；Resin；The Two protein or polypeptide or polypeptide analog；Antibody or antibody fragment；Metal-chelator；Co-factor；Aliphatic acid；Carbon hydrate Object；Polynucleotide；DNA；RNA；Antisense polynucleotide；Carbohydrate, water-soluble dendritic, cyclodextrin, biomaterial；Nanometer Particle；Spin labeling；Fluorogen, the part containing metal；Radioactive segment；Novel functional group；With other molecule covalents or non-common The group of valency interaction；Light cage covers part；It can photoisomerization part；Biotin；Biotin analog；It is combined with heavy atom Part；The group chemically cracked；Can photodestruciton group；Extended side chain；The bonded sugar of carbon；Redox active agent；Ammonia Base thio-acid；Toxin part；Part through isotope marks；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electronics Dense group；Magnetic group；It is inserted into group；Chromophore；Energy transfer agent；Bioactivator；Detectable label；And above-mentioned object Any combinations of matter；And

L is optional, and is the connexon selected from the group being made of following group when it is present：Alkylidene is substituted Asia Alkyl, alkenylene, be substituted alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidenes or through taking For alkylidene)-, wherein k be 1,2 or 3-S (O)_k-、-S(O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)- (alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- it is (sub- Alkyl is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,- CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-(alkylidene is substituted alkylidene)-O-N=CR'- ,-(alkylidene is substituted alkylidene)-C (O) NR'- (alkylidenes Or be substituted alkylidene)-,-(alkylidene is substituted alkylidene)-S (O)_k- (alkylidene is substituted alkylidene)-S- ,-(sub- Alkyl is substituted alkylidene)-S-S- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N (R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂-N =N- and-C (R')₂- N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl；

Its restrictive condition is in the absence of A and B, and R is not methyl.

66. method according to claim 65, wherein the amino acid containing oxime has the structure corresponding to formula (XII)：

67. method according to claim 65, wherein the amino acid containing oxime has the structure corresponding to formula (XIII)：

68. method according to claim 65, wherein X are water-soluble polymer.

69. method according to claim 65, wherein X are the derivative of polyethylene glycol.

70. method according to claim 65, wherein X are cytotoxic compound.

71. method according to claim 65, wherein X are drug.

72. method according to claim 65, wherein X are the second polypeptide.

73. the method according to claim 72, wherein second polypeptide is the non-natural amino acid polypeptides containing oxime.

74. the method according to claim 72, wherein second polypeptide have with it is according to claim 62 non- The identical amino acid structure of natural amino acid polypeptide.

75. method according to claim 63 further comprises pharmaceutically acceptable supporting agent.

76. method according to claim 63, wherein the non-natural amino acid polypeptides containing oxime contain oxime to be modified Non-natural amino acid polypeptides.

77. it is a kind of for detecting the existing method of polypeptide in patient, it is a effective amount of including at least one the described method includes administration The homologous non-natural amino acid polypeptide of a alpha-non-natural amino acid containing oxime, wherein the amino acid containing oxime is to use translation system simultaneously Enter in the specific site in the polypeptide, the translation system includes：

78. the method according to claim 77, wherein the translation system is the organism for including being selected from following group The in vivo translation system of cell：Prokaryotes, eucaryote, mammal, Escherichia coli, pseudomonad kind, fungi, ferment Mother, archeobacteria, eubacteria, plant, insect and protist.

79. the method according to claim 77, wherein the non-natural amino acid polypeptides containing oxime include at least one tool There is the amino acid containing oxime of the structure of formula (XI)：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

Or R₅For L-X, wherein

L is optional, and is the connexon selected from the group being made of following group when it is present：Alkylidene is substituted Asia Alkyl, alkenylene, be substituted alkenylene ,-O- ,-O- (alkylidene is substituted alkylidene)-,-S- ,-S- (alkylidenes or through taking For alkylidene)-, wherein k be 1,2 or 3-S (O)_k-、-S(O)_k(alkylidene is substituted alkylidene)-,-C (O)-,-C (O)- (alkylidene is substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene is substituted alkylidene)-,-N (R')-,-NR'- it is (sub- Alkyl is substituted alkylidene)-,-C (O) N (R')-,-CON (R')-(alkylidene is substituted alkylidene)-,-CSN (R')-,- CSN (R')-(alkylidene is substituted alkylidene)-,-N (R') CO- (alkylidene is substituted alkylidene)-,-N (R') C (O) O- ,-(alkylidene is substituted alkylidene)-O-N=CR'- ,-(alkylidene is substituted alkylidene)-C (O) NR'- (alkylidenes Or be substituted alkylidene)-,-(alkylidene is substituted alkylidene)-S (O) k- (alkylidene is substituted alkylidene)-S- ,-it is (sub- Alkyl is substituted alkylidene)-S-S- ,-S (O)_kN(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-；-N (R')S(O)_kN (R')-,-N (R')-N=,-C (R')=N- ,-C (R')=N-N (R')-,-C (R')=N-N=,-C (R')₂-N =N- and-C (R')₂- N (R')-N (R')-, wherein R' is each independently H, alkyl or substituted alkyl；

Its restrictive condition is in the absence of A and B, and R is not methyl.

80. the method according to claim 79, wherein the amino acid containing oxime has the structure corresponding to formula (XII)：

81. the method according to claim 79, wherein the amino acid containing oxime has the structure corresponding to formula (XIII)：

82. the method according to claim 79, wherein X are selected from the group being made of following object：Mark；Dyestuff；Parent With property mark；Photoaffinity marks；Spin labeling；Fluorogen；Radioactive segment；It is combined with the part of heavy atom；Through isotope The part of mark；Biophysics probe；Phosphorescence groups；Chemiluminescent groups；Electron dense group；Magnetic group；Chromophore；Energy Measure transfer agent；Detectable label；And any combinations of above-mentioned substance.

83. the method according to claim 79, wherein the biosynthesis non-natural amino acid polypeptides containing oxime are through modification Biosynthesis non-natural amino acid polypeptides containing oxime.

84. a kind of method of the separating property of easyization polypeptide, using including at least one alpha-non-natural amino acid containing oxime Homologous non-natural amino acid polypeptide, wherein the amino acid containing oxime is the specific site being incorporated to using translation system in the polypeptide In, the translation system includes：

85. according to the method for claim 84, wherein the translation system is the cell for including being selected from the organism of following group In vivo translation system：Prokaryotes, eucaryote, mammal, Escherichia coli, fungi, pseudomonad kind, yeast, Gu Bacterium, eubacteria, plant, insect and protist.

86. according to the method for claim 84, wherein the non-natural amino acid polypeptides containing oxime have formula including at least one (XI) amino acid containing oxime of structure：

Wherein：

R is H, alkyl, substituted alkyl, cycloalkyl or is substituted cycloalkyl；

Or R₅For L-X, wherein

Its restrictive condition is in the absence of A and B, and R is not methyl.

87. the method according to claim 84, wherein the amino acid containing oxime has the structure corresponding to formula (XII)：

88. the method according to claim 87, wherein the amino acid containing oxime has the structure corresponding to formula (XIII)：

89. the method according to claim 87, wherein X are water-soluble polymer.

90. the method according to claim 87, wherein X are the derivative of polyethylene glycol.

91. the method according to claim 79, wherein the non-natural amino acid polypeptides containing oxime contain oxime to be modified Non-natural amino acid polypeptides.