WO2023004686A1 - 一种非天然氨基酸及其应用、包含其的重组蛋白以及重组蛋白偶联物 - Google Patents

一种非天然氨基酸及其应用、包含其的重组蛋白以及重组蛋白偶联物 Download PDF

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WO2023004686A1
WO2023004686A1 PCT/CN2021/109276 CN2021109276W WO2023004686A1 WO 2023004686 A1 WO2023004686 A1 WO 2023004686A1 CN 2021109276 W CN2021109276 W CN 2021109276W WO 2023004686 A1 WO2023004686 A1 WO 2023004686A1
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amino acid
group
unnatural amino
recombinant protein
add
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PCT/CN2021/109276
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English (en)
French (fr)
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杨金纬
叶诚浩
夏钢
霍鹏超
丁文
陈龙飞
焦琳
黄浩
衡新
宫丽颖
祝静静
应跃斌
梁学军
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浙江新码生物医药有限公司
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Priority to PCT/CN2021/109276 priority Critical patent/WO2023004686A1/zh
Priority to KR1020247002441A priority patent/KR20240024235A/ko
Priority to CA3222899A priority patent/CA3222899A1/en
Priority to AU2021458069A priority patent/AU2021458069A1/en
Publication of WO2023004686A1 publication Critical patent/WO2023004686A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH] (Somatotropin)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C235/18—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the singly-bound oxygen atoms further bound to a carbon atom of a six-membered aromatic ring, e.g. phenoxyacetamides
    • C07C235/20—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the singly-bound oxygen atoms further bound to a carbon atom of a six-membered aromatic ring, e.g. phenoxyacetamides having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/72—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms
    • C07C235/76—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
    • C07C235/78—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of an unsaturated carbon skeleton the carbon skeleton containing rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/12—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/40—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of six-membered aromatic rings
    • C07C271/42—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of six-membered aromatic rings with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/54—Esters of carbamic acids having oxygen atoms of carbamate groups bound to carbon atoms of six-membered aromatic rings with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormones [GH] (Somatotropin)

Definitions

  • the invention relates to the field of biopharmaceuticals, in particular to a non-natural amino acid, a recombinant protein comprising the non-natural amino acid and a conjugate formed by the recombinant protein.
  • unnatural amino acids containing special groups By introducing unnatural amino acids containing special groups into proteins, a variety of scientific research and product development applications can be realized, for example, introducing photosensitive unnatural amino acids into proteins, or special labeling of unnatural amino acids to facilitate the study of protein interactions interactions; another example, the use of unnatural amino acids for directional modification of enzymes to improve enzyme activity, enzyme stability, or facilitate efficient enzyme immobilization; another example, the use of unnatural amino acid insertions that cannot be achieved in conventional hosts It can prepare safe live bacteria or live virus vaccines.
  • codon extension technology to introduce unnatural amino acids into proteins is the site-specific modification of proteins, changing the function, stability, half-life and other characteristics of proteins, and can be used for the development of innovative biopharmaceuticals.
  • the azide structure (-N 3 ) at the end of Lys-azido can be combined with an alkyne-containing structure (such as BCN, ie ) modified carrier drugs (such as PEG, etc.) to obtain conjugates (for example, Chinese patent CN 103153927B), which have high specific selectivity.
  • an alkyne-containing structure such as BCN, ie
  • modified carrier drugs such as PEG, etc.
  • conjugates for example, Chinese patent CN 103153927B
  • this coupling method and chemical modification method need to introduce a relatively expensive alkyne structure, and an acceptable drug-antibody coupling ratio can only be obtained when the equivalent is used, which increases the corresponding production cost and process.
  • the process is also more complex and the process conditions are harsh.
  • an object of the present invention is to provide an unnatural amino acid.
  • Another object of the present invention is to provide the use of said unnatural amino acid.
  • Another object of the present invention is to provide a recombinant protein and a recombinant protein conjugate.
  • the unnatural amino acid provided by the present invention is a compound having the structure shown in formula (I) or its enantiomer,
  • X and Z each independently represent a substituted or unsubstituted C0 ⁇ C20 linear or branched chain alkylene group, wherein one or more -CH 2 - can be optionally replaced by -O-, -S-, One or more of -NH-, -C(O)-, -S(O)-, Y represents -C(O)-, -S(O)- or -CH 2 -, and A represents substitution or Unsubstituted C6 ⁇ C20 aryl;
  • the substituents may be selected from hydroxyl, mercapto, halogen, nitro, cyano, alkyl, alkenyl, alkynyl, alkoxy, acyl, One or more of amide group, carboxyl group, ester group, amino group, sulfonyl group, sulfinyl group, cycloalkyl group, heterocyclic group, aryl group, heteroaryl group.
  • Example 11 the inventors of the present invention found that, in addition to the high cost and complex process, the azide structure (-N 3 ) at the end of Lys-azido is easily reduced to an amino structure (-N 3 ) when inserted into a protein. NH 2 ), thereby losing the activity of the coupling, so the reduction reaction reduces the yield in the process of preparation.
  • the non-natural amino acid provided by the present invention introduces a carbonyl group as an active reactive group at its end, which not only has a novel structure and is easy to prepare, but also has mild coupling conditions and low production costs, and is not prone to structural changes when inserted into protein sequences, resulting in loss of reactivity ;
  • the non-natural amino acid provided by the present invention also includes an aryl group connected to the terminal carbonyl group, the introduction of the aryl group can further enhance the stability of the resulting conjugate, and the conjugate is not easy to occur even at a lower pH condition break down.
  • the non-natural amino acid provided by the present invention also contains an alkylene group with a certain chain length, the compound has better flexibility, and it is easier to form various conjugates.
  • C0 ⁇ Cn includes C0 ⁇ C1, C0 ⁇ C2, ... C0 ⁇ Cn, when it means C0, it means that the group does not exist, and the C atoms at both ends of it are directly connected to form key.
  • the "C0-C6” group means that the part has 0-6 carbon atoms, that is, the group does not exist, contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 Carbon atoms, 5 carbon atoms or 6 carbon atoms;
  • the "C6-C10” group means that the part has 6-10 carbon atoms, that is, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms atom, 9 carbon atoms or 10 carbon atoms.
  • aryl refers to a carbocyclic aromatic system containing one or two rings, wherein the rings can be connected together in a fused manner.
  • Aryl includes monocyclic or bicyclic aryl groups, such as phenyl, naphthyl, tetrahydronaphthyl aromatic groups. Preferred aryl groups are C6-C10 aryl groups, more preferred aryl groups are phenyl and naphthyl, most preferably phenyl.
  • the substituents can be selected from hydroxyl, mercapto, halogen, nitro, cyano, C1 ⁇ C6 alkyl, C1 ⁇ C6 alkoxy, acyl, amido, carboxyl , one or more of ester group, amino group, sulfonyl group, sulfinyl group, C3-C8 cycloalkyl group, C3-C8 heterocyclic group, C6-C20 aryl group, and C4-C10 heteroaryl group.
  • the X and Z can each independently represent a C0 ⁇ C10 straight chain or branched chain alkylene group, wherein one or more -CH 2 - can be optionally replaced by One or more of -O-, -S-, -NH-; in some more preferred embodiments of the present invention, the X and Z can each independently represent a C0 ⁇ C6 linear alkylene group, One or more of -CH 2 - can be optionally replaced with one or more of -O-, -S-, -NH-; in some more preferred embodiments according to the present invention, the X An alkylene group that is not C0 at the same time as Z, that is, the X and Z groups cannot be absent at the same time.
  • the A may represent a substituted or unsubstituted C6-C10 aryl group, more preferably, the A may represent a substituted or unsubstituted phenyl or naphthyl group.
  • the unnatural amino acid may be a compound having the structure shown in formula (I-1),
  • said X, Z and A are each independently as defined in any one of the above technical solutions.
  • the unnatural amino acid may be a compound having the structure shown in formula (I-2),
  • R1 and R2 each independently represent hydrogen , hydroxyl, mercapto, halogen, nitro, cyano, C1 -C6 alkyl, C1-C6 alkoxy, acyl, amido, carboxyl, ester, amino, sulfonyl , sulfinyl, C3-C8 cycloalkyl, C3-C8 heterocyclyl, C6-C20 aryl or C4-C10 heteroaryl.
  • the unnatural amino acid is as shown in formula (I-3), formula (I-4), formula (I-5) or formula (I-6) structural compounds,
  • X' represents a straight-chain alkylene group of C0-C6, more preferably a straight-chain alkylene group of C0-C4, wherein one or more -CH 2 - can be optionally replaced by -O- and/or -NH-;
  • the R 1 and R 2 are each independently defined in any one of the above technical solutions.
  • the unnatural amino acids provided by the present invention include optically pure enantiomers and racemates.
  • the unnatural amino acid provided by the present invention is a compound with one of the following structures:
  • the present invention also provides the application of the non-natural amino acid described in any one of the above technical solutions in the preparation of recombinant protein or recombinant protein conjugate.
  • the recombinant protein can be obtained by inserting the non-natural amino acid described in any one of the above technical solutions into any type of protein commonly seen in the art at any position and in any number.
  • the recombinant protein conjugate can be a conjugate obtained after any recombinant protein obtained is coupled with a common coupling moiety in the art, wherein the coupling moiety can include but is not limited to a polymer (for example, polyethylene glycol with any molecular weight) Alcohol), protein, polypeptide or one or more of small molecule drugs.
  • the recombinant protein may be recombinant human growth hormone
  • the recombinant protein conjugate may be recombinant human growth hormone-polyethylene glycol conjugate.
  • the present invention also provides a recombinant protein, wherein at least one position in the amino acid sequence of the recombinant protein is the non-natural amino acid described in any one of the above technical solutions.
  • the recombinant protein may have a structure as shown in formula (II),
  • D represents the residue of the non-natural amino acid as described in any one of the above technical solutions except the amino carboxylic acid part, and P 1 and P 2 respectively represent the amino and carboxyl groups of the non-natural amino acid in the amino acid Links in the sequence.
  • the recombinant protein provided by the present invention can be prepared by common preparation methods in the art, including the cloning and expression of recombinant protein containing unnatural amino acids by using gene codon extension technology.
  • the recombinant protein provided by the present invention can be a recombinant protein obtained by inserting any unnatural amino acid described in any one of the above technical solutions into any type of protein commonly seen in the art at any position and in any number, such as recombinant human growth hormone .
  • the present invention also provides a recombinant protein conjugate, wherein the coupling between the end group carbonyl of the non-natural amino acid in the recombinant protein described in any one of the above technical solutions and the "NH 2 -O-" end group Formed partly through the formation of oxime bonds.
  • the recombinant protein conjugate may have a structure as shown in formula (III),
  • D' represents the residue of the recombinant protein as described in any one of the above technical schemes except for the carbonyl part of the terminal group of the unnatural amino acid, and D" represents the coupling part without the terminal group of "NH 2 -O-" residues.
  • the conjugation moiety may include one or more of polymers (for example, polyethylene glycol with any molecular weight), proteins, polypeptides or small molecule drugs.
  • polymers for example, polyethylene glycol with any molecular weight
  • proteins for example, polyethylene glycol with any molecular weight
  • polypeptides for example, polypeptides or small molecule drugs.
  • Different types of coupling moieties can be coupled to the recombinant protein alone, or different types of coupling moieties can be linked first and then coupled to the recombinant protein.
  • the recombinant protein may be recombinant human growth hormone, and the coupling moiety containing the "NH 2 -O-" end group may be a "NH 2 -O-" end group based polyethylene glycol.
  • the polyethylene glycol containing "NH 2 -O-" terminal group has the following structural formula:
  • the molecular weight of polyethylene glycol containing "NH 2 -O-" end group can be 10-100KD, including but not limited to about 10KD, 20KD, 30KD, 40KD, 50KD, 60KD, 70KD, 80KD, 90KD, 100KD, etc. Molecular weight values or molecular weight intervals in any combination.
  • the molecular weight of polyethylene glycol containing "NH 2 -O-" end group may be 20-50KD.
  • the amino acid sequence of the recombinant human growth hormone is shown in SEQ ID NO: 1; more preferably, it corresponds to the 107th in the amino acid sequence SEQ ID NO: 1
  • the present invention also provides the preparation of the recombinant protein conjugate described in any one of the above technical solutions for the treatment of growth and development disorders caused by insufficient secretion of endogenous growth hormone, Turner Use in medicines for growth disorders caused by syndromes or growth hormone deficiency in adults.
  • the unnatural amino acid provided by the present invention introduces a terminal carbonyl group and an aryl group connected to it in its structure, which is easier to prepare than the current terminal azido unnatural amino acid (for example, Lys-azido) , better security, less inactivation when inserted into the protein, higher binding rate with the coupling part, and higher stability of the resulting conjugate.
  • the current terminal azido unnatural amino acid for example, Lys-azido
  • the non-natural amino acid provided by the present invention can have the properties of an amino acid itself, thereby expanding the potential types of amino acids and making it applicable to many fields as amino acid derivatives, especially in Preparation of recombinant protein or recombinant protein conjugates.
  • the unnatural amino acids provided by the present invention can be successfully identified and inserted into proteins in prokaryotic expression systems and eukaryotic expression systems, thereby producing proteins containing unnatural amino acids at specific sites, which can form proteins with different physical and chemical properties , Biochemically active recombinant proteins, expanding the types and potential applications of proteins.
  • the non-natural amino acid of the present invention has higher expression efficiency in protein and has better practicability.
  • the terminal carbonyl active group contained in it can conveniently form protein conjugates (or conjugates), such as polyethylene glycol conjugates , polyethylene glycol-active drug conjugates, etc.
  • protein conjugates can have a variety of improved biological activities (such as anti-tumor activity) due to performance modification.
  • the present invention also provides a novel protein conjugate platform, which can realize the combination of various proteins and various coupling moieties through the novel unnatural amino acids contained in the protein and the coupling moieties connected thereto.
  • Fig. 1 is a map of recombinant human growth hormone expression plasmid pET21-rhGH107 in Example 8.
  • Fig. 2 is the SDS-PAGE electrophoresis diagram of the fermentation product obtained by adding different kinds of unnatural amino acids obtained in Example 8, wherein each swimming lane is respectively represented as follows: swimming lane 1: protein molecular weight marker; swimming lane 2: wild type recombinant human growth hormone; swimming lane 3 : the expression product of recombinant human growth hormone fed with NBOK; swimming lane 4: the expression product of recombinant human growth hormone fed with NPAK; lane 5: the expression product of recombinant human growth hormone fed with NBPK; lane 6: the expression product of recombinant human growth hormone fed with NBGK; swimming lane 7: the expression product of recombinant human growth hormone fed with NPOK; lane 8: the expression product of recombinant human growth hormone fed with NPOK-2; lane 9: the expression product of recombinant human growth hormone fed with NBGK-2; lane 10: without adding any non- Recombinant Human Growth Hormone Expression
  • Fig. 3 is the SDS-PAGE electrophoresis image of the recombinant human growth hormone containing unnatural amino acid and PEG coupling product in Example 8, wherein each swimming lane is respectively represented as follows: swimming lane 1: molecular weight marker; swimming lane 2: wild type recombinant human growth Hormone; lane 3: recombinant human growth hormone containing NBOK and PEG conjugated product; lane 4: recombinant human growth hormone containing NPAK and PEG conjugated product; lane 5: recombinant human growth hormone containing NBPK and PEG conjugated product; Lane 6: NBGK-containing recombinant human growth hormone and PEG conjugated product; swimming lane 7: NPOK-containing recombinant human growth hormone and PEG-conjugated product; swimming lane 8: NPOK-2-containing recombinant human growth hormone and PEG-conjugated product.
  • Fig. 4 is the SDS-PAGE electrophoresis image of the purified PEG-coupled recombinant human growth hormone in Example 8, wherein each swimming lane is respectively represented as follows: swimming lane 1: molecular weight marker; swimming lane 2: recombinant human growth hormone standard; swimming lane 3: Conjugate product of recombinant human growth hormone containing NPOK and PEG; Lane 4: Conjugate product of recombinant human growth hormone containing NBOK and PEG.
  • Figure 5A- Figure 5G are the cell activity curves in Example 8, wherein, Figure 5A is the cell activity curve of the rhGH standard product of the Chinese Academy of Sciences; The cell activity curve; Fig. 5C is Jin Sai Zeng Figure 5D is the cell activity curve of rhGH (NPOK); Figure 5E is the cell activity curve of PEG-rhGH (NPOK); Figure 5F is the cell activity curve of rhGH (NBOK); Figure 5G is Cell activity curve of PEG-rhGH (NBOK).
  • FIG. 6 is a fluorescence microscopic image of CHO cells transiently expressing inserted unnatural amino acids in Example 9.
  • FIG. 6 is a fluorescence microscopic image of CHO cells transiently expressing inserted unnatural amino acids in Example 9.
  • Figure 7 is a map of the expression plasmid pCDNA3.1-Trastuzumab-UAG142 in Example 10.
  • FIG. 9 is a diagram showing the inhibitory effects of NBOK-containing trastuzumab and DM1-modified NBOK-containing trastuzumab in Example 10 on BT-474 cells.
  • Figure 10A and Figure 10B are the mass spectra of rhGH whose 140th position is mutated to Lys-azido and rhGH whose 140th position is mutated to NBOK in Example 11, respectively.
  • Figure 11A and Figure 11B are the SDS-PAGE electrophoresis images of the coupling process of rhGH with Lys-azido mutation at position 140 and rhGH with NBOK mutation at position 140 in Example 11, respectively, with 30KD PEG.
  • Lane 1 Molecular Weight Marker
  • Lane 2 Wild-type recombinant human growth hormone
  • Lane 3 rhGH-Lys-azido-140
  • Lane 4 rhGH-Lys-azido-140: 30K
  • BCN- PEG is the product of 1:15 (molar ratio, the same below) coupling reaction for 72 hours
  • lane 5 rhGH-Lys-azido-140:30K
  • BCN-PEG is the product of 1:25 coupling reaction for 72 hours
  • Expressed as follows: Lane 1: Molecular Weight Marker; Lane 2: rhGH-NBOK-140; Lane 3: rhGH-NBOK-140: 30K BCN-PEG is the product of 1:15 coupling reaction for 6 hours
  • Lane 4: rhGH-NBOK-140 : 30K BCN-PEG is the product of 1:15 coupling reaction for 9 hours
  • lane 5: rhGH-NBOK-140: 30K BCN-PEG
  • reagents or raw materials used in the examples of the present invention are all commercially available products unless otherwise specified.
  • the preparation process includes the following steps:
  • NPAK structural formula
  • the preparation process includes the following steps:
  • NBPK The structural formula of NBPK is as follows:
  • the preparation process includes the following steps:
  • NPOK The structural formula of NPOK is as follows:
  • the preparation process includes the following steps:
  • NBGK The structural formula of NBGK is as follows:
  • the preparation process includes the following steps:
  • NPOK-2 The structural formula of NPOK-2 is as follows:
  • the preparation process includes the following steps:
  • NBGK-2 The structural formula of NBGK-2 is as follows:
  • the preparation process includes the following steps:
  • NPOK, NPAK, NBOK, NBPK, NBGK, NPOK-2, and NBGK-2 prepared in Examples 1-7 were used to prepare recombinant human growth hormone in a prokaryotic expression system, and to prepare PEG site-directed conjugates.
  • the helper plasmid pSupAR-MbPylRS was purchased from Addgene (product number #91705), a plasmid depository organization. This plasmid can express tRNA and tRNA synthetase that specifically recognize pyrrolysine-derived unnatural amino acids in Escherichia coli. After adding 37.5mg/L chlorine The helper plasmid was extracted after cultured in shake flask cultured with mycin in LB medium.
  • amino acid sequence (SEQ ID NO:1) of Homo sapiens growth hormone is as follows:
  • the gene sequence (SEQ ID NO:2) of recombinant human growth hormone is as follows:
  • pET21-rhGH107 can be used to express recombinant human growth hormone in which the 107th amino acid codon is replaced by an amber codon, and contains 6 histidine purification tags at the C-terminal of the protein.
  • the map of recombinant human growth hormone expression plasmid pET21-rhGH107 is shown in FIG. 1 .
  • each bacterial solution was centrifuged at 10000rpm for 5min, and the precipitate was taken, and NTA Buffer (20mM Tris-HCl pH7.9, 0.5M NaCl, 10% glycerol) and PMSF with a final concentration of 1mM were added to 1/20 of the cell growth volume.
  • Ultrasonic homogenate to disrupt the cells centrifuge at 10000rpm for 20min, take the supernatant, use Ni-NTA chromatography column to absorb the target protein with His tag, and then use the eluent NTA Buffer (20mM Tris-HCl pH7.9, 0.5M NaCl, 10% glycerol, 250mM imidazole) to obtain a sample of the target protein (ie, a recombinant protein inserted with an unnatural amino acid) with a purity of about 90%.
  • NTA Buffer 20mM Tris-HCl pH7.9, 0.5M NaCl, 10% glycerol, 250mM imidazole
  • Each sample was placed in a 10K nitrocellulose dialysis bag, dialyzed in PBS buffer solution of pH 7.0 (each 1ml protein solution corresponds to 200ml dialysate) overnight, and the medium was changed once, and the dialyzed protein solution was collected, SDS- Protein concentration was detected by PAGE.
  • the coupling reaction operation is as follows: Before the coupling reaction, adjust the target protein obtained above to 0.5 mg/ml with pH 7.0 PBS buffer According to 1:15 (molar ratio, recombinant protein: aminooxy PEG), add 30KD aminooxy PEG solid (sourced from Beijing Jiankai Technology Co., Ltd.), fully shake and dissolve to obtain a clear and transparent solution, and then seal the reaction solution. The reaction was shaken in a constant temperature shaker (25° C., 100 rpm). After 48 hours, SDS-PAGE was used to analyze the coupling situation, as shown in Figure 3.
  • the coupled PEG-rhGH was separated and purified by Butyl HP hydrophobic chromatography column (loading buffer: 50mM NaH 2 PO 4 ⁇ 2H 2 O, 0.8M (NH 4 ) 2 SO 4 , pH8.5; elution buffer : 20mM Tris-HCl, pH8.5, 40-fold column volume gradient elution), to obtain electrophoresis pure grade (>95%) pure PEG-rhGH.
  • loading buffer 50mM NaH 2 PO 4 ⁇ 2H 2 O, 0.8M (NH 4 ) 2 SO 4 , pH8.5
  • elution buffer 20mM Tris-HCl, pH8.5, 40-fold column volume gradient elution
  • PEG-rhGH containing the non-natural amino acid of the present invention also reached electrophoresis pure grade after purification.
  • HEK293-GHR cells see Chinese patent: 2020112649827 for the source of the cell line
  • HEK293-GHR cells were treated with 0.1 mg/mL hygromycin B (Sangon Bioengineering (Shanghai) Co., Ltd., catalog number A600230-0001), 0.75 mg/mL G418 (Sangon Bioengineering (Shanghai) Co., Ltd., Cat. No. A600958-0005), 10% fetal bovine serum (Gibco, Cat. No. 10099141) and DMEM (Gibco, Cat.
  • HEK293-GHR cells were inserted into each well of a black 96-well cell culture plate (Corning, Cat. No. 3904), 90 ⁇ L/well, and starved for 16 hours at 37°C and 5% carbon dioxide.
  • the rhGH standard (purchased from the China National Institutes for Food and Drug Control, referred to as "the Institute"), rhGH containing NPOK (ie, rhGH (NPOK)), PEG-rhGH with PEG-modified NPOK (ie, PEG-rhGH (NPOK) )), PEG-rhGH containing NBOK (i.e., rhGH(NBOK)), PEG-rhGH with PEG-modified NBOK (i.e., PEG-rhGH(NBOK)), commercially available rhGH drug Sai Zeng (Changchun Jinsai Pharmaceutical Co., Ltd.), commercially available PEG-rhGH drug Jinsai Zeng (Changchun Jinsai Pharmaceutical Co., Ltd.) Samples were serially diluted to a total of 9 concentrations, and incubated at 37° C.
  • the cell activity of other PEG-rhGH containing the unnatural amino acid of the present invention is also reduced to 25%-50% of the standard product after coupling, and the change rule of the biological activity is also consistent with the marketed PEG-rhGH.
  • the recombinant GFP protein was prepared in a eukaryotic expression system using the unnatural amino acids NPOK, NPAK, NBOK, NBPK, NBGK, NPOK-2 and NBGK-2 prepared in Examples 1-7.
  • helper plasmid pCMV-MbPylRS was purchased from addgene (product number #91706), which encodes aminoacyl-tRNA synthetase and corresponding tRNA (recognizing amber codon) in mammalian cells that specifically recognize pyrrolysine-derived unnatural amino acids UAG).
  • the expression vector expressing the wild-type green fluorescent protein (its gene coding sequence shown in SEQ ID NO: 3) containing the purification tag is subjected to point mutation to obtain the expression plasmid in which the 40th amino acid codon in the reading frame is mutated into an amber codon pEGFP40, its complete sequence is shown in SEQ ID NO:4.
  • the gene sequence (SEQ ID NO:3) of the wild-type green fluorescent protein containing the purification tag is as follows:
  • the gene sequence (SEQ ID NO:4) of expression plasmid pEGFP40 is as follows:
  • Chinese hamster ovary cells CHO-K1 (product number #CCL-61-ATC) were purchased from the American Type Culture Collection (ATCC), and cultured in RPMI1640 medium containing 10% fetal bovine serum. Extract the auxiliary plasmid pCMV-MbPylRS and the green fluorescent protein expression plasmid pEGFP40 in the above steps, use lipo2000 transfection reagent (invitrogen company, product number #12566014), and perform transient transfection according to the instructions, (the cells were inoculated at 50,000 cells/well Inoculated into 24-well plates, 8 wells in total, transfected after 24 hours of inoculation and culture, 500ng plasmid per well) and added NPOK (final concentration: 1mM) to the first well while transfecting, and added to the second well Add NPAK (final concentration: 1mM), add NBOK (final concentration: 1mM) to the 3rd well, add NBPK (final concentration: 1mM) to
  • NPOK NPAK
  • NBOK NBPK
  • NBGK NPOK-2
  • NBGK-2 NBGK-2
  • helper plasmid pCMV-MbPylRS was purchased from addgene (product number #91706), which encodes aminoacyl-tRNA synthetase and corresponding tRNA (recognizing amber codon) in mammalian cells that specifically recognize pyrrolysine-derived unnatural amino acids UAG).
  • the heavy and light chain DNAs encoding trastuzumab were synthesized by whole gene synthesis (the corresponding amino acid sequences are SEQ ID NO:5 and SEQ ID NO:6), and subcloned into the eukaryotic expression vector pCDNA3. 1+, and then perform point mutation on the obtained expression vector to obtain the expression plasmid pCDNA3.1-Trastuzumab-UAG142 in which the 142nd amino acid codon in the heavy chain reading frame is mutated into an amber codon, its map is shown in Figure 7, and its complete sequence See SEQ ID NO:7.
  • the heavy chain amino acid sequence (SEQ ID NO:5) of trastuzumab is as follows:
  • the light chain amino acid sequence (SEQ ID NO:6) of trastuzumab is as follows:
  • the gene sequence (SEQ ID NO: 7) of the expression plasmid pCDNA3.1-Trastuzumab-UAG142 is as follows:
  • HEK293 cells Suspended and acclimatized HEK293 cells were inoculated into Wayne293 TM medium (Quacell Biotechnology, product number A21501) at a density of 0.3 ⁇ 10 5 /mL in a 1 L shake flask with a volume of 240 mL, at 120 rpm, 5% CO 2 , 80% Shake culture under humidity conditions, transfect when the cell density reaches about 1 ⁇ 10 6 /mL, extract the helper plasmid pCMV-MbPylRS and expression plasmid pCDNA3.1-Trastuzumab-UAG142 described in steps (1) and (2), And remove endotoxin for later use.
  • Each shaker flask uses 120 ⁇ g of expression plasmid and helper plasmid for transfection. Put the plasmid into a centrifuge tube, dilute it to 7.2 mL with 1 ⁇ PBS buffer, and add PEI transfection in another centrifuge tube.
  • Dyeing reagent polyethyleneimine, Polyethyleneimine 720 ⁇ g, then add 1 ⁇ PBS buffer to dilute to 7.2mL, after mixing, let the two centrifuge tubes stand for 5 minutes, and then gently mix the liquid in the two centrifuge tubes After standing still for 10 minutes, slowly add a total of 14.4mL to the 240mL cell suspension in a 1L shaker flask, and shake gently constantly during the dropping process, shaking under the conditions of 120rpm, 5% CO 2 and 80% humidity After culturing for 4 hours, unnatural amino acid with a final concentration of 1 mM was added, followed by shaking culture at 120 rpm, 5% CO 2 , and 80% humidity for 5 days, and the cell culture supernatant was harvested for antibody purification.
  • the elution buffer is 100mmol/L glycine, 200mmol/L acetate, pH3.5, to obtain the purified trastuzumab inserted with unnatural amino acid .
  • the synthetic route is as follows (taking NBOK as an example):
  • the toxin containing ammoxy terminal group (DM1-PEG-ONH2) was combined with the purified trastuzumab inserted with unnatural amino acid (wherein the direction of R 1 to R 2 is from the N-terminus of the amino acid sequence to C-terminal direction) for site-directed coupling to obtain monoclonal antibody-toxin conjugates.
  • the specific operation of the coupling process is as follows: the purified trastuzumab inserted into the unnatural amino acid and DM1-PEG-ONH2 were mixed and dissolved at a molar ratio of 1:15, and then the pH was adjusted to 4.0 with 10M acetic acid. Shake on a shaker (25° C., 200 rpm), take a sample after 48 hours, and detect the reaction of trastuzumab by HPLC (HIC-HPLC) based on the principle of hydrophobic chromatography.
  • HPLC HPLC
  • the coupled monoclonal antibody-toxin conjugate was exchanged through a 50kDa ultrafiltration centrifuge tube to remove unreacted toxin raw materials, and the replacement buffer was 20mM histidine buffer (pH6.5).
  • HIC-HPLC analysis conditions are as follows:
  • Mobile phase B (75 mM K 2 HPO 4 , 25% isopropanol, pH 7.2 ⁇ 0.2).
  • the BT-474 cell line (ATCC, product number HTB-20) was used to detect the inhibitory effect of the sample on cell proliferation.
  • BT-474 cells were cultured in ATCC Hybri-Care Medium (ATCC, product number 46-X) medium containing 10% fetal bovine serum (Gibco, product number 10099141) at 37°C and 5% carbon dioxide until sufficient Inoculate 1.5 ⁇ 10 4 BT-474 cells per well (including vehicle control wells) in a 96-well cell culture plate (Corning, Cat. No. 3599), and add cell-free medium to the blank control wells, 80 ⁇ L/well.
  • ATCC ATCC Hybri-Care Medium
  • Gibco 10% fetal bovine serum
  • the NBOK-containing trastuzumab and DM1-modified NBOK-containing trastuzumab were serially diluted from 17 ⁇ g/mL to 0.13 ⁇ g/mL, with 8 concentrations in total, and 2 replicate wells for each dilution. Transfer the diluted sample to the culture plate inoculated with BT-474 cells, 10 ⁇ L per well, add 10 ⁇ L of medium to the vehicle control well and the blank control well, and incubate at 37°C and 5% carbon dioxide.
  • the conjugated monoclonal antibody-toxin conjugate was exchanged through a 50kDa ultrafiltration centrifuge tube (Millipore, catalog number UFC905024#) to remove unreacted toxin raw materials.
  • the replacement buffer was 20 mM histidine buffer (pH 6.0).
  • the protein solution was divided into three parts, adjusted to pH 4.0, pH 6.0, and pH 8.0 with 1M citric acid solution or 1M Tris solution, placed in a water bath at 25°C, and sampled at 24h, 48h, 72h, 96h, and 120h, respectively, and HIC -HPLC detection (detection condition is the same as above), the results are shown in Table 2.
  • an OmpA signal peptide is added to the N-terminus of rhGH on the expression vector Coding sequence, in combination with the helper plasmid pSupAR-MbPylRS in Example 8, construct the OrigamiB (DE3) expression strain of rhGH whose K140 codon is mutated into an amber codon (TAG), by adding Lys-azido during fermentation, express the 140th Natural rhGH mutated to Lys-azido at position 140, numbered rhGH-Lys-azido-140; by adding NBOK during fermentation, expressing natural rhGH mutated to NBOK at position 140, numbered rhGH-NBOK-140, and referring to the above References were purified by corresponding purification means.
  • RhGH-Lys-azido-140 and rhGH-NBOK-140 were used by liquid chromatography-mass spectrometry (high resolution mass spectrometer: XevoG2-XS Q-Tof, Waters Company; ultra-high performance liquid chromatography: UPLC (Acquity UPLC I-Class), Waters Company) to analyze the complete molecular weight.
  • 30KD BCN-PEG (self-produced with reference to Chinese patent CN112279906A) is coupled with rhGH-Lys-azido-140 through Click reaction (wherein the direction of R 1 to R 2 is from the N-terminus of the amino acid sequence to the C end direction).
  • Use pH 7.0 PBS buffer to adjust the target protein obtained above to 0.5 mg/ml, and put the BCN-PEG solid into rhGH-Lys- Azido solution, fully shaken to dissolve to obtain a clear and transparent solution, then seal the reaction solution, and shake the reaction in a constant temperature shaker (25°C, 70rpm). At intervals, samples were taken to detect the reaction results by SDS-PAGE, and the reaction stopped after 72 hours, and the conversion rate was about 50%-70%. The results of the reaction are shown in Figure 11A.

Abstract

提供一种非天然氨基酸，为具有如式(Ⅰ)所示结构的化合物或其对映异构体。还提供了所述非天然氨基酸的应用。进一步地，还提供了一种包含所述非天然氨基酸的重组蛋白以及由所述重组蛋白制得的蛋白偶联物。所述非天然氨基酸制备简便，安全性好，***蛋白时不易失活，与偶联部分的结合率高，所得偶联物的稳定性也更高，所述非天然氨基酸可应用至众多领域，尤其是在制备重组蛋白或重组蛋白偶联物中。

Description

一种非天然氨基酸及其应用、包含其的重组蛋白以及重组蛋白偶联物 技术领域
本发明涉及生物制药领域，具体涉及一种非天然氨基酸、包含所述非天然氨基酸的重组蛋白以及所述重组蛋白形成的偶联物。
背景技术
通过向蛋白质中引入含有特殊基团的非天然氨基酸，可以实现多种科学研究与产品开发应用，例如，向蛋白质中引入光敏非天然氨基酸，或者对非天然氨基酸进行特殊标记，便于研究蛋白质之间的相互作用；又例如，利用非天然氨基酸进行酶的定向改造，以提高酶活、酶的稳定性，或者便于进行酶的高效固定化；再例如，利用在常规宿主中不可实现非天然氨基酸***的特点，可制备安全的活菌或活病毒疫苗。使用密码子扩展技术向蛋白质中引入非天然氨基酸的一项重要应用在于对蛋白质进行定点修饰，改变蛋白质的功能、稳定性、半衰期等特征，并可用于创新型生物药的开发。目前已有一系列成果，如PEG定点偶联重组人生长激素制备长效重组蛋白、小分子毒素定点偶联单抗开发抗体偶联药物等。由此可见，非天然氨基酸具有非常重要的作用和非常广泛的应用领域。
现有技术(例如CN 102838671B、CN 106146663A、J.Am.Chem.Soc.2009,131,8720等)公开了一种非天然氨基酸Lys-azido，结构式如下所示：
Figure PCTCN2021109276-appb-000001
Lys-azido末端的叠氮结构(-N 3)能与含炔烃结构(如BCN，即
Figure PCTCN2021109276-appb-000002
)修饰的载体药物(如PEG等)进行化学连接得到偶联物(例如，中国专利CN 103153927B)，具有很高的特异选择性。但是，该种偶联方法和化学修饰方法需引入成本较高的炔烃类结构，且使用当量较大时才可获得可接受的药物抗体偶联比率，由此增加了相应的生产成本，工艺过程也较为复杂，工艺条件苛刻。
因此，需要开发一种结构新颖、制备简便、成本低廉的非天然氨基酸，以扩大氨基酸的种类以及应用。
发明内容
为克服现有技术中存在的上述不足，本发明的一个目的是提供一种非天然氨基酸。
本发明的另一个目的是提供所述非天然氨基酸的应用。
本发明的还一个目的是提供一种重组蛋白以及一种重组蛋白偶联物。
本发明提供的非天然氨基酸为具有如式(Ⅰ)所示结构的化合物或其对映异构体，
Figure PCTCN2021109276-appb-000003
其中，X和Z各自独立地表示取代或未取代的C0～C20直链或支链亚烷基，其中的一个或多个-CH 2-可任选地替换为-O-、-S-、-NH-、-C(O)-、-S(O)-中的一个或多个，Y表示-C(O)-、-S(O)-或-CH 2-，以及A表示取代或未取代的C6～C20芳基；
当所述X、Z和A各自独立地表示取代的基团时，取代基可以选自羟基、巯基、卤素、硝基、氰基、烷基、烯基、炔基、烷氧基、酰基、酰胺基、羧基、酯基、氨基、磺酰基、亚磺酰基、环烷基、杂环基、芳基、杂芳基中的一种或多种。
如实施例11所示，本发明的发明人发现，除了成本昂贵、工艺复杂之外，Lys-azido末端的叠氮结构(-N 3)在***到蛋白中时容易被还原为氨基结构(-NH 2)，从而失去偶联的活性，因此该还原反应降低了工艺制备过程中的收率。
Figure PCTCN2021109276-appb-000004
本发明提供的非天然氨基酸在其末端引入了羰基作为活性反应基团，不仅结构新颖、制备简便，而且偶联条件温和，生产成本较低，***蛋白序列时不易发生结构变化而导致反应活性丧失；本发明提供的非天然氨基酸中还包含与端基羰基相连的芳基，芳基的引入可进一步增强所得偶联物的稳定性，偶联物即使在较低的pH条件下也不容易发生分解。此外，本发明提供的非天然氨基酸中还包含有一定链长的亚烷基，化合物的柔性较好，更容易形成各种偶联物。
本发明提供的非天然氨基酸中，“C0～Cn”包括C0～C1、C0～C2、……C0～Cn，当表示C0时，意为该基团不存在，其两端的C原子直接连接成键。举例而言，所述“C0～C6”基团是指该部分中具有0～6个碳原子，即基团不存在、包含1个碳原子、2个碳原子、3个碳原子、4个碳原子、5个碳原子或6个碳原子；所述“C6～C10”基团是指该部分中具有6～10个碳原子，即包含6个碳原子、7个碳原子、8个碳原子、9个碳原子或10个碳原子。
本发明提供的非天然氨基酸中，“芳基”是指含有一个或者两个环的碳环芳香***，其中所述环可以以稠合的方式连接在一起。“芳基”包括单环或双环的芳基，比如苯基、萘基、 四氢萘基的芳香基团。优选芳基为C6～C10芳基，更优选芳基为苯基和萘基，最优选为苯基。
在根据本发明的一些优选的实施方式中，所述取代基可以选自羟基、巯基、卤素、硝基、氰基、C1～C6烷基、C1～C6烷氧基、酰基、酰胺基、羧基、酯基、氨基、磺酰基、亚磺酰基、C3～C8环烷基、C3～C8杂环基、C6～C20芳基、C4～C10杂芳基中的一种或多种。
在根据本发明的一些优选的实施方式中，所述X和Z可以各自独立地表示C0～C10直链或支链亚烷基，其中的一个或多个-CH 2-可任选地替换为-O-、-S-、-NH-中的一个或多个；在根据本发明的一些更优选的实施方式中，所述X和Z可以各自独立地表示C0～C6直链亚烷基，其中的一个或多个-CH 2-可任选地替换为-O-、-S-、-NH-中的一个或多个；在根据本发明的一些更优选的实施方式中，所述X和Z不同时为C0的亚烷基，即X和Z基团不能同时不存在。
在根据本发明的一些优选的实施方式中，所述A可以表示取代或未取代的C6～C10芳基，更优选地，所述A可以表示取代或未取代的苯基或萘基。
在根据本发明的一些优选的实施方式中，所述非天然氨基酸可以为具有如式(Ⅰ-1)所示结构的化合物，
Figure PCTCN2021109276-appb-000005
其中，所述X、Z和A各自独立地如上述技术方案任一项所定义。
在根据本发明的一些优选的实施方式中，所述非天然氨基酸可以为具有如式(Ⅰ-2)所示结构的化合物，
Figure PCTCN2021109276-appb-000006
其中，所述X如上述技术方案任一项所定义；
R 1和R 2各自独立地表示氢、羟基、巯基、卤素、硝基、氰基、C1～C6烷基、C1～C6烷氧基、酰基、酰胺基、羧基、酯基、氨基、磺酰基、亚磺酰基、C3～C8环烷基、C3～C8杂环基、C6～C20芳基或C4～C10杂芳基。
在根据本发明的一些较优选的实施方式中，所述非天然氨基酸为具有如式(Ⅰ-3)、式 (Ⅰ-4)、式(Ⅰ-5)或式(Ⅰ-6)所示结构的化合物，
Figure PCTCN2021109276-appb-000007
其中，X’表示C0～C6的直链亚烷基，更优选表示C0～C4的直链亚烷基，其中的一个或多个-CH 2-可任选地替换为-O-和/或-NH-；
所述R 1和R 2各自独立地如上述技术方案任一项所定义。
本发明提供的非天然氨基酸包括光学纯的对映异构体和外消旋体。
在根据本发明的一些更优选的实施方式中，本发明提供的非天然氨基酸为具有以下结构之一的化合物：
Figure PCTCN2021109276-appb-000008
Figure PCTCN2021109276-appb-000009
本发明还提供了上述技术方案任一项所述的非天然氨基酸在制备重组蛋白或重组蛋白偶联物中的应用。
本发明所述的应用中，重组蛋白可以是如上述技术方案任一项所述的非天然氨基酸以任意位点、任意数量***至本领域常见的任意种类的蛋白之中而得到重组蛋白。重组蛋白偶联物可以是所得的任意重组蛋白与本领域常见的偶联部分偶联后所得的偶联物，其中，偶联部分可以包括但不限于聚合物(例如，任意分子量的聚乙二醇)、蛋白、多肽或小分子药物中的一种或多种。
在根据本发明的一些优选的实施方式中，重组蛋白可以为重组人生长激素，重组蛋白 偶联物可以为重组人生长激素-聚乙二醇偶联物。
本发明还提供了一种重组蛋白，其中，所述重组蛋白的氨基酸序列中的至少一个位点上为上述技术方案任一项所述的非天然氨基酸。
进一步地，所述重组蛋白可以具有如式(Ⅱ)所示的结构，
Figure PCTCN2021109276-appb-000010
式(Ⅱ)中，D表示如上述技术方案任一项所述的非天然氨基酸除去氨基羧酸部分的残基，P 1、P 2分别表示所述非天然氨基酸的氨基和羧基在所述氨基酸序列中的连接部分。
本发明提供的重组蛋白可以使用本领域常见的制备方法制备得到，包括使用基因密码子扩展技术实现含非天然氨基酸的重组蛋白的克隆与表达。
本发明提供的重组蛋白可以是如上述技术方案任一项所述的非天然氨基酸以任意位点、任意数量***至本领域常见的任意种类的蛋白之中而得到重组蛋白，例如重组人生长激素。
本发明还提供了一种重组蛋白偶联物，其中，通过上述技术方案任一项所述的重组蛋白中的非天然氨基酸的端基羰基与含有“NH 2-O-”端基的偶联部分形成肟键而形成。
进一步地，所述重组蛋白偶联物可以具有如式(Ⅲ)所示的结构，
Figure PCTCN2021109276-appb-000011
式(Ⅲ)中，D’表示如上述技术方案任一项所述的重组蛋白除去非天然氨基酸的端基羰基部分的残基，D”表示偶联部分除去“NH 2-O-”端基的残基。
本发明提供的重组蛋白偶联物中，偶联部分可以包括聚合物(例如，任意分子量的聚乙二醇)、蛋白、多肽或小分子药物中的一种或多种。不同种类的偶联部分可以单独与重组蛋白偶联，也可以不同种类的偶联部分先形成连接后再与重组蛋白偶联。
在根据本发明的一些优选的实施方式中，所述重组蛋白可以为重组人生长激素，所述含有“NH 2-O-”端基的偶联部分可以为含有“NH 2-O-”端基的聚乙二醇。
在根据本发明的一些更优选的实施方式中，含有“NH 2-O-”端基的聚乙二醇具有如下结构式：
Figure PCTCN2021109276-appb-000012
其中，含有“NH 2-O-”端基的聚乙二醇的分子量可以为10～100KD，包括但不限于约10KD、20KD、30KD、40KD、50KD、60KD、70KD、80KD、90KD、100KD等分子量值或任意组合的分子量区间。优选地，含有“NH 2-O-”端基的聚乙二醇的分子量可以为20～50KD。
在根据本发明的一些更优选的实施方式中，所述重组人生长激素的氨基酸序列如SEQ ID NO:1所示；进一步优选地，对应于所述氨基酸序列SEQ ID NO:1中的第107位点上如上述技术方案任一项所述的非天然氨基酸。
当重组蛋白为重组人生长激素时，本发明还提供了上述技术方案任一项所述的重组蛋白偶联物在制备用于治疗内源性生长激素分泌不足所引起的生长发育障碍、特纳综合征引起的生长发育障碍或成人生长激素缺乏症的药物中的用途。
本发明提供的技术方案具有以下优点：
(1)本发明提供的非天然氨基酸在其结构中引入了端基羰基以及与之相连的芳基，相对于目前的端基叠氮基非天然氨基酸(例如，Lys-azido)，制备更简便，安全性更好，***蛋白时不易失活，与偶联部分的结合率更高，所得偶联物的稳定性也更高。
(2)本发明提供的非天然氨基酸作为一种氨基酸衍生物，本身可具备氨基酸的性质，由此扩大了氨基酸的潜在种类，使其可作为氨基酸衍生物应用至众多领域之中，尤其是在制备重组蛋白或重组蛋白偶联物中。
(3)本发明提供的非天然氨基酸可以在原核表达***和真核表达***中被成功识别并***至蛋白中，从而产生特定位点含有非天然氨基酸的蛋白，由此可形成具有不同理化性质、生化活性的重组蛋白，扩大了蛋白的种类和潜在的应用范围。此外，本发明的非天然氨基酸在蛋白中具有较高的表达效率，实用性更好。
(4)本发明提供的重组蛋白由于含有本发明的非天然氨基酸，其所含的末端羰基活性基团可方便地形成蛋白偶联物(或缀合物)，例如聚乙二醇偶联物、聚乙二醇-活性药物偶联物等。这些蛋白偶联物由于经过了性能改造，因而可具有多种改进的生物活性(例如抗肿瘤活性)。
(5)本发明还提供了一种新型的蛋白偶联物平台，通过蛋白质所含的新型非天然氨基酸以及所连接的偶联部分，可实现多种蛋白、多种偶联部分的结合。
附图说明
图1为实施例8中重组人生长激素表达质粒pET21-rhGH107的图谱。
图2为实施例8所得的添加不同种类非天然氨基酸获得的发酵产物SDS-PAGE电泳图，其中各泳道分别表示如下：泳道1：蛋白分子量Marker；泳道2：野生型重组人生长激素；泳道3：投料NBOK的重组人生长激素表达产物；泳道4：投料NPAK的重组人生长激素表达产物；泳道5：投料NBPK的重组人生长激素表达产物；泳道6：投料NBGK 的重组人生长激素表达产物；泳道7：投料NPOK的重组人生长激素表达产物；泳道8：投料NPOK-2的重组人生长激素表达产物；泳道9：投料NBGK-2的重组人生长激素表达产物；泳道10：不添加任何非天然氨基酸的重组人生长激素表达产物。
图3为实施例8中的含非天然氨基酸的重组人生长激素与PEG偶联产物的SDS-PAGE电泳图，其中各泳道分别表示如下：泳道1：分子量Marker；泳道2：野生型重组人生长激素；泳道3：含NBOK的重组人生长激素与PEG偶联产物；泳道4：含NPAK的重组人生长激素与PEG偶联产物；泳道5：含NBPK的重组人生长激素与PEG偶联产物；泳道6：含NBGK的重组人生长激素与PEG偶联产物；泳道7：含NPOK的重组人生长激素与PEG偶联产物；泳道8：含NPOK-2的重组人生长激素与PEG偶联产物。
图4为实施例8中经纯化的PEG偶联重组人生长激素的SDS-PAGE电泳图，其中各泳道分别表示如下：泳道1：分子量Marker；泳道2：重组人生长激素标准品；泳道3：含NPOK的重组人生长激素与PEG偶联产物；泳道4：含NBOK的重组人生长激素与PEG偶联产物。
图5A-图5G为实施例8中的细胞活性曲线图，其中，图5A为中检院rhGH标准品的细胞活性曲线图；图5B为赛增
Figure PCTCN2021109276-appb-000013
的细胞活性曲线图；图5C为金赛增
Figure PCTCN2021109276-appb-000014
的细胞活性曲线图；图5D为rhGH(NPOK)的细胞活性曲线图；图5E为PEG-rhGH(NPOK)的细胞活性曲线图；图5F为rhGH(NBOK)的细胞活性曲线图；图5G为PEG-rhGH(NBOK)的细胞活性曲线图。
图6为实施例9中CHO细胞瞬时表达***非天然氨基酸的荧光显微图像。
图7为实施例10中表达质粒pCDNA3.1-Trastuzumab-UAG142的图谱。
图8A-图8C分别为实施例10中含NBPK曲妥珠单抗、含NBOK曲妥珠单抗以及含NPOK-2曲妥珠单抗与毒素偶联后的HIC-HPLC谱图。
图9为实施例10中含NBOK的曲妥珠单抗和DM1修饰的含NBOK曲妥珠单抗分别对BT-474细胞的抑制效果图。
图10A、图10B分别为实施例11中第140位突变为Lys-azido的rhGH和第140位突变为NBOK的rhGH的质谱图。
图11A、图11B分别为实施例11中第140位突变为Lys-azido的rhGH和第140位突变为NBOK的rhGH分别与30KD PEG偶联过程的SDS-PAGE电泳图。其中图11A各泳道分别表示如下：泳道1：分子量Marker；泳道2：野生型重组人生长激素；泳道3：rhGH-Lys-azido-140；泳道4：rhGH-Lys-azido-140：30K BCN-PEG为1:15(摩尔比，下同)偶联反应72h的产物；泳道5：rhGH-Lys-azido-140：30K BCN-PEG为1:25偶联反应72h的产物；图11B各泳道分别表示如下：泳道1：分子量Marker；泳道2：rhGH-NBOK-140；泳道3：rhGH-NBOK-140：30K BCN-PEG为1:15偶联反应6h的产物；泳道4：rhGH-NBOK-140：30K BCN-PEG为1:15偶联反应9h的产物；泳道5：rhGH-NBOK-140： 30K BCN-PEG为1:15偶联反应12h的产物；泳道6：rhGH-NBOK-140：30K BCN-PEG为1:15偶联反应24h的产物；泳道7：rhGH-NBOK-140：30K BCN-PEG为1:15偶联反应48h的产物。
具体实施方式
以下结合具体实施例对本发明的技术方案做进一步详细说明。
本发明的实施例中所使用的试剂或原料如无特别说明均为商购产品。
实施例1非天然氨基酸NBOK的制备
NBOK的结构式如下所示：
Figure PCTCN2021109276-appb-000015
反应过程如下图所示：
Figure PCTCN2021109276-appb-000016
制备过程包括以下步骤：
a)在反应烧瓶中，加入对甲基苯乙酮(4.0mL,30.0mmol)，加入溶剂DCE(50.0mL)，加入NBS(6.41g,36.0mmol)和BPO(0.05g,0.3mmol)，混合物在80℃下回流24小时后，容器放入冰水中冷却，析出固体，过滤除去固体，用饱和Na 2CO 3洗涤3次，用DCM萃取3次，合并有机相，加入无水硫酸钠干燥，过滤，减压浓缩后得到粗产物1-1(5.46g,收率85％)，无需纯化直接用于下一步。
b)在反应烧瓶中，加入产物1-1(2.73g,12.80mmol)，加入溶剂二氧六环(40mL)和水(40mL)，再加入碳酸钙(7.68g,76.8mmol)，混合物在105℃回流24小时，冷却至室温，过滤除去固体，用DCM萃取3次，合并有机相，减压浓缩，经柱层析(洗脱剂：PE:EA＝3:1)纯化后得到产物1-2(1.80g,收率94％)。
c)在两口反应烧瓶中，加入对硝基苯基氯甲酸酯(2.90g，14.4mmol)，加入溶剂DCM(10.0mL)，降温至0℃，加入产物1-2(1.80g,12.0mmol)和吡啶(1.2mL，14.4mmol)，室温搅拌18小时后，反应液中加入饱和碳酸钠溶液(10mL)，用DCM(50mL)萃取3次，合并有机相，用水洗涤2次，无水硫酸钠干燥，过滤，减压浓缩，经柱层析(洗脱剂：PE:EA＝5:1) 纯化得到产物1-3(3.14g,收率83％)。
d)在反应烧瓶中，加入产物1-3(1.26g,4.0mmol)和Fmoc-Lys-OH盐酸盐(1.40g,3.33mmol)，加入溶剂二氧六环(15mL)和水(5mL)，再加入三乙胺(1.2mL,8.3mmol)，混合物在室温下反应24小时后，加入适量1M HCl溶液，用DCM萃取，减压浓缩，得到粗产物1-4，直接用于下一步。
e)在反应烧瓶中，将产物1-4(1.10g,0.19mmol)溶于DCM(10mL)中，加入二乙胺(5.0mL)，在室温下反应6小时，产物析出，过滤后用DCM打浆3次，即得目标产物NBOK(1-5,817mg,两步收率63％)。
1H-NMR(400MHz,重水)δ8.04(d,J＝8.4Hz,2H),7.55(d,J＝8.0Hz,2H),5.21(s,2H),3.74(t,J＝6.0Hz,1H),3.17(t,J＝6.4Hz,2H),2.70(s,3H),1.95–1.83(m,2H),1.62–1.52(m,2H),1.47–1.35(m,2H).
实施例2非天然氨基酸NPAK的制备
NPAK的结构式如下所示：
Figure PCTCN2021109276-appb-000017
反应过程如下图所示：
Figure PCTCN2021109276-appb-000018
制备过程包括以下步骤：
a)在反应烧瓶中，加入对氯苯乙酮(1.00g,6.47mmol)，氮气氛围下，加入丙二酸二乙酯(6.84g,47.70mmol)、KHCO 3(0.97g,9.70mmol)和K 2CO 3(1.34g,9.70mmol)，然后加入Pd(dba) 2(0.019g,0.030mmol)和P(t-Bu) 3HBF 4(0.021g,0.071mmol)，加入完毕后置换氮气保护，升温至160℃反应40h。TLC检测反应完全后，反应液中加入水(30mL)，用EA萃取3次，合并有机相，用水洗涤2次，无水硫酸钠干燥，过滤，在0-5℃减压浓缩，得到无色透明液体，柱层析(洗脱剂：PE:EA＝10:1)纯化得到产物2-1(0.80g,收率60％)。
b)在反应烧瓶中，加入LiOH(0.30g,11.64mmol)和水(5.0mL)，加入乙醇(10mL)，加入产物2-1(0.80g,3.88mmol)，室温搅拌2h后，TLC检测反应完全，反应液中加入2M  HCl溶液调节pH＝1～2，用EA萃取3次，合并有机相，用水洗涤2次，无水硫酸钠干燥，过滤，减压浓缩，得到产物2-2(0.5g,收率72％)。
c)在反应烧瓶中，加入产物2-2(0.20g,1.12mmol)，依次加入N-羟基琥珀酰亚胺(NHS,0.19g,1.68mmol)、DIPEA(0.07g，0.56mmol)、DCM(2.0mL)。降温至0～5℃，加入DCC(0.23g,1.12mmol)和DCM(2.0mL)的溶液，保温反应2h。升至室温搅拌过夜。TLC检测反应完全后，过滤，用DCM洗涤，母液减压浓缩，柱层析(洗脱剂：PE:EA＝5:1)纯化得到产物2-3(0.19g,收率62％)。
d)在反应烧瓶中，加入产物2-3(0.10g,0.36mmol)，依次加入三乙胺(0.04g,0.36mmol)、Fmoc-Lys-OH盐酸盐(0.13g,0.36mmol)、二氧六环(2.0mL)和水(2.0mL)，室温下搅拌反应18h。TLC检测反应完全后，减压浓缩，用EA萃取3次，无水硫酸钠干燥，减压浓缩，柱层析(洗脱剂：DCM:MeOH＝15:1)纯化得到油状液体产物2-4(0.03g,收率61％)。
e)在反应烧瓶中，加入产物2-4(0.08g,0.15mmol)、DCM(1.0mL)和哌啶(0.04g，0.47mmol)，在室温下搅拌3h。TLC检测反应完全后，减压浓缩，用石油醚(5mL)打浆1h，过滤，滤饼再用石油醚(5mL)打浆1h，过滤，滤饼用乙醇重复打浆4次除去残余的哌啶，最终得到类白色固体2-5(0.02g,收率43％)。
1H-NMR(400MHz,重水)δ7.85(d,J＝8.2Hz,2H),7.33(d,J＝8.2Hz,2H),3.94(t,J＝6.3Hz,1H),3.56(s,2H),3.12(t,J＝6.8Hz,2H),2.54(s,3H),1.80–1.70(m,2H),1.54–1.45(m,2H),1.40-1.224(m,2H).
实施例3非天然氨基酸NBPK的制备
NBPK的结构式如下所示：
Figure PCTCN2021109276-appb-000019
反应过程如下图所示：
Figure PCTCN2021109276-appb-000020
制备过程包括以下步骤：
a)在反应烧瓶中，加入对甲基苯乙酮(4.0mL,30.0mmol)，加入溶剂DCE(50.0mL)，加入NBS(6.41g,36.0mmol)和BPO(0.05g,0.3mmol)，混合物在80℃下回流24小时。TLC检测反应完全后，容器放入冰水中冷却，析出固体，过滤除去固体，用饱和Na 2CO 3洗涤3次，用DCM萃取3次，合并有机相，加入无水硫酸钠干燥，过滤，减压浓缩后得到粗产物3-1(5.46g,收率85％)，无需纯化直接用于下一步。
b)在反应烧瓶中，加入NaH(0.58g,14.64mmol,60％)，加入干燥的溶剂THF(20mL)，冰浴冷却下，缓慢加入乙二醇(6.7mL,122.0mmol)，在室温条件下搅拌1小时。然后加入产物3-1(2.60g,12.2mmol)，70℃加热回流48小时至反应完全。冰浴冷却下，缓慢滴入饱和NH 4Cl淬灭NaH，用水洗涤，EtOAc萃取3次，合并有机相，加入无水硫酸钠干燥，过滤，减压浓缩后，经柱层析(洗脱剂：PE:EA＝2:1)纯化得到产物3-2(1.39g,收率59％)。
c)在反应烧瓶中，加入产物3-2(1.39g,7.2mmol)，加入溶剂DCM(10mL)，冰浴冷却下，加入对硝基苯基氯甲酸酯(1.74g,8.64mmol)和吡啶(0.7mL,8.64mmol)，室温搅拌18小时。TLC检测反应完全后，加入水洗涤，EtOAc萃取3次，合并有机相，加入无水硫酸钠干燥，过滤，减压浓缩后，经柱层析(洗脱剂：PE:EA＝3:1)纯化得到产物3-3(2.27g,收率88％)。
d)在反应烧瓶中，加入产物3-3(2.27g,6.32mmol)，加入溶剂二氧六环(16mL)和水(4mL)，加入Fmoc-Lys-OH盐酸盐(2.13g,5.27mmol)，再加入三乙胺(1.85mL,13.2mmol)，在室温下搅拌18小时至反应完全。加入适量1M HCl调pH至2左右，用乙酸乙酯萃取，合并有机相，加入无水硫酸钠干燥，过滤，减压浓缩，得到粗产物3-4，直接用于下一步。
e)在反应烧瓶中，将上一步的产物3-4溶于DCM(10mL)，加入二乙胺(5mL)，在室温下反应6小时。TLC检测反应完全后，减压浓缩，经柱层析(洗脱剂：DCM:MeOH:H 2O＝40:10:1)纯化得到产物白色固体(3-5,0.95g,两步收率49％)。
1H-NMR(400MHz,重水)δ8.04(d,J＝8.0Hz,2H),7.56(d,J＝8.0Hz,2H),4.72(s,2H),4.25(s,2H),3.81(s,2H),3.74(t,J＝6.0Hz,1H),3.16–3.08(m,2H),2.70(s,3H),1.97–1.79(m,2H),1.60–1.48(m,2H),1.48–1.35(m,2H).
实施例4非天然氨基酸NPOK的制备
NPOK的结构式如下所示：
Figure PCTCN2021109276-appb-000021
反应过程如下图所示：
Figure PCTCN2021109276-appb-000022
制备过程包括以下步骤：
a)在反应烧瓶中，加入三光气(BTC,2.18g,7.35mmol)，加入溶剂THF(10.0mL)，在冰浴冷却下，加入对羟基苯乙酮(2.0g,14.7mmol)和吡啶(1.5mL,17.64mmol)，混合物在室温下反应24小时。TLC检测反应完全后，加入适量水，用EtOAc萃取3次，合并有机相。加入无水硫酸钠干燥，过滤，减压浓缩后得到粗产物4-1(1.20g)，直接用于下一步。
b)在反应烧瓶中，加入Boc-赖氨酸(1.1g,5.0mmol)，加入溶剂DCM(10.0mL)，加入产物4-1(1.20g)和三乙胺(2mL,15mmol)。在室温下搅拌24小时，TLC检测反应完全后，加入适量1M HCl调pH至弱酸性，用DCM萃取3次，合并有机相。加入无水硫酸钠干燥，过滤，减压浓缩后，经柱层析(洗脱剂：DCM:MeOH＝5:1)得到产物4-2(1.70g,收率84％)。
c)在反应烧瓶中，加入产物4-2(1.70g,4.2mmol)，加入溶剂DCM(5mL)，加入三氟乙酸(5mL)，混合物在室温下反应1小时。TLC检测反应完全后，直接减压浓缩，经柱层析(洗脱剂：DCM:MeOH:H 2O＝40:10:1)后得到产物4-3(1.19g,收率92％)。
1H-NMR(400MHz,重水)δ8.09(d,J＝8.6Hz,2H),7.31(d,J＝8.6Hz,2H),3.78(t,J＝6.0Hz,1H),3.27(t,J＝6.8Hz,2H),2.70(s,3H),1.98–1.87(m,2H),1.72–1.60(m,2H),1.55–1.43(m,2H).
实施例5非天然氨基酸NBGK的制备
NBGK的结构式如下所示：
Figure PCTCN2021109276-appb-000023
反应过程如下图所示：
Figure PCTCN2021109276-appb-000024
制备过程包括以下步骤：
a)在反应烧瓶中，加入对甲基苯乙酮(8.0mL,60.0mmol)，加入溶剂DCE(80.0mL)，加入NBS(12.82g,72.0mmol)和BPO(145mg,0.6mmol)，混合物在90℃下回流24小时。TLC检测反应完全后，容器放入冰水中冷却，析出固体，过滤除去固体，用饱和Na 2CO 3洗涤3次，用DCM萃取3次，合并有机相。加入无水硫酸钠干燥，过滤，减压浓缩后得到粗产物5-1(11.12g,收率87％)，无需纯化直接用于下一步。
b)在反应烧瓶中，将
Figure PCTCN2021109276-appb-000025
MS(14g)和LiOH(1.45g,34.54mmol)用DMF(70mL)溶解，在室温下搅拌20min后，加入甘氨酸甲酯盐酸盐(2.0g,15.7mmol)，再搅拌45min后，加入产物5-1(4.0g,18.8mmol)，室温搅拌18小时。TLC监测反应完全后，过滤除去固体，用EA洗滤饼，滤液用水洗涤两次。无水硫酸钠干燥，减压浓缩后得粗产物5-2，直接用于下一步。
c)在反应烧瓶中，将上一步产物5-2溶于二氧六环(20mL)，缓慢滴加1M NaOH，反应2小时后，TLC监测到水解反应完成得到产物5-3。加入20mL饱和NaHCO 3后，缓慢加入溶于二氧六环(10mL)的Fmoc-OSu，室温搅拌过夜，TLC监测反应完全后，用1M HCl调至弱酸性，用EA萃取，无水硫酸钠干燥，减压浓缩，经柱层析(洗脱剂：DCM:MeOH＝10:1)得到产物5-4(3.60g,收率89％)。
d)在反应烧瓶中，加入产物5-3(3.60g,8.0mmol)、NBS(1.10g,9.6mmol)、EDCI(1.85g,9.6mmol)，加入溶剂DCM(50mL)，混合物在室温下反应18小时。TLC监测反应完全后，用水洗涤3次，无水硫酸钠干燥，减压浓缩后得到产物5-5(3.20g,收率75％)。
e)在反应烧瓶中，加入产物5-5(3.20g,6.0mmol)，加入溶剂二氧六环(40mL)和水(10 mL)，加入Fmoc-Lys-OH盐酸盐(3.0g,7.2mmol)，再加入三乙胺(2.0mL,15.0mmol)，在室温下搅拌18小时至反应完全。加入适量1M HCl调pH至2左右，用EA萃取，合并有机相，加入无水硫酸钠干燥，过滤，减压浓缩，经柱层析(洗脱剂：DCM:MeOH:AcOH＝20:1:0.5)得到产物5-5(3.50g,收率75％)。
f)在反应烧瓶中，将产物5-5溶于DCM(20mL)，加入二乙胺(20mL)，在室温下反应6小时。TLC监测反应完全后，减压浓缩，经柱层析(洗脱剂：DCM:MeOH:H 2O＝30:10:1)得到终产物白色粉末5-7(0.55g,收率37％)。
1H-NMR(400MHz,重水)δ7.98(d,J＝8.2Hz,2H),7.50(d,J＝8.2Hz,2H),3.84(s,2H),3.71(s,1H),3.31(s,2H),3.17(t,J＝6.9Hz,2H),2.67(s,3H),1.97–1.73(m,2H),1.58–1.45(m,2H),1.44-1.27(m,2H).
实施例6非天然氨基酸NPOK-2的制备
NPOK-2的结构式如下所示：
Figure PCTCN2021109276-appb-000026
反应过程如下图所示：
Figure PCTCN2021109276-appb-000027
制备过程包括以下步骤：
a)在反应烧瓶中，加入对乙酰基苯酚(2.05g,15.0mmol)和溴乙酸(2.50g,18.0mmol)，再加入NaOH(1.20g,30mmol)的水溶液(6mL)。混合物在100℃下回流24小时至反应完全。反应冷却至室温，加1M盐酸调至酸性，析出固体，过滤得到白色粗产物6-1(3.32g,收率113％)，无需纯化直接用于下一步。
b)在反应烧瓶中，将上一步粗产物6-1(3.32g,17.0mmol)溶于DCM(50mL)中，加入NHS(2.35g,20.4mmol)和EDCI(3.90g,20.4mmol)。混合物在常温下搅拌18小时至反应完全。用DCM萃取,合并有机相，加入无水硫酸钠干燥，过滤，减压浓缩。再经柱层析(洗脱剂：DCM:MeOH:AcOH＝20:1:0.5)纯化，得到产物6-2(1.67g,收率38％)。
c)在反应烧瓶中，加入产物6-2(1.67g,5.7mmol)，加入溶剂二氧六环(20mL)和水(50mL)，加入Fmoc-Lys-OH盐酸盐(1.9g,4.8mmol)，再加入三乙胺(1.7mL,12.0mmol)，在 室温下搅拌18小时至反应完全，加入适量1M HCl调pH至2左右，用EA萃取，合并有机相，加入无水硫酸钠干燥，过滤，减压浓缩。将所得粗产物6-3直接溶于DCM(10mL)，再加入二乙胺(5mL)。混合物在常温下搅拌18小时至反应完全。减压浓缩，经柱层析(洗脱剂：DCM:MeOH:H 2O＝40:10:1)纯化得到终产物6-4(709mg,两步收率39％)。
1H-NMR(400MHz,重水)δ7.91(d,J＝8.8Hz,2H),6.98(d,J＝8.8Hz,2H),4.60(s,2H),3.59(t,J＝6.4Hz,1H),3.19(t,J＝6.8Hz,2H),2.52(s,3H),1.87–1.65(m,2H),1.56–1.40(m,2H),1.35–1.15(m,2H).
实施例7非天然氨基酸NBGK-2的制备
NBGK-2的结构式如下所示：
Figure PCTCN2021109276-appb-000028
反应过程如下图所示：
Figure PCTCN2021109276-appb-000029
制备过程包括以下步骤：
a)在反应烧瓶中，加入溴乙酸(2.10g,15.0mmol)和NaOH(0.80g,20mmol)的水溶液(10mL)，搅拌10分钟。然后加入对乙酰基苯胺(1.40g,10.0mmol)，混合物在100℃下回流18小时至反应完全。反应冷却至室温，过滤，用水洗涤，得到白色粗产物7-1(1.30g,收率67％)，无需纯化直接用于下一步。
b)在反应烧瓶中，加入产物7-1(1.30g,6.7mmol)和NaHCO 3(1.70g,20.1mmol)的水溶液(20mL)。再加入Fmoc-OSu(2.80g,8.1mmol)和DMF(20mL)。混合物在60℃下搅拌18小时至反应完全。冷却至室温，用EA萃取。保留水相用1M盐酸调pH至2左右，再用EA萃取，得有机相。加入无水硫酸钠干燥，过滤，减压浓缩。得产物7-2，无需纯化直 接用于下一步。
c)在反应烧瓶中，将上一步产物7-2(约6.7mmol)，NHS(0.90g,8.0mmol)和EDCI(1.50g,8.0mmol)溶解于DMF(50mL)。反应混合物在室温下搅拌24小时至反应完全。加水，用DCM萃取，得有机相。加入无水硫酸钠干燥，过滤，减压浓缩。得到产物7-3，无需纯化直接用于下一步。
d)在反应烧瓶中，加入上一步产物7-3(约6.7mmol)，Fmoc-Lys-OH盐酸盐(2.30g,5.6mmol)，以及三乙胺(2.0mL,14.0mmol)。反应混合物在室温下搅拌3小时至反应完全。然后用1M盐酸调pH至2左右，用EA萃取。加入无水硫酸钠干燥，过滤，减压浓缩。得到产物7-4，无需纯化直接用于下一步。
e)在反应烧瓶中，加入上一步产物7-4。加入溶剂DCM(20mL)和二乙胺(10mL)。反应混合物在室温下搅拌12小时至反应完全。先减压浓缩，再加入乙腈(50mL)重新溶解，再减压浓缩，反复3次操作除去多余二乙胺。加入DCM打浆两次，得到最终产物7-5(1.65g，总收率51％)。
1H-NMR(400MHz,重水)δ7.71(d,J＝8.8Hz,2H),6.51(d,J＝8.8Hz,2H),3.80(s,2H),3.53(t,J＝6.8Hz,1H),3.09(t,J＝6.8Hz,2H),2.39(s,3H),1.73–1.60(m,2H),1.42–1.33(m,2H),1.25–1.14(m,2H).
实施例8
使用实施例1-7制备的NPOK、NPAK、NBOK、NBPK、NBGK、NPOK-2、NBGK-2在原核表达***中制备重组人生长激素，并制备PEG定点偶联物。
(1)辅助质粒的获得
辅助质粒pSupAR-MbPylRS购自质粒保藏组织Addgene(货号#91705)，该质粒可在大肠杆菌中表达特异识别吡咯赖氨酸衍生非天然氨基酸的tRNA和tRNA合成酶，经添加有37.5mg/L氯霉素的LB培养基进行摇瓶培养后提取得到辅助质粒。
(2)阅读框内部含终止密码子的重组人生长激素表达质粒构建
从美国国家生物信息中心数据库中获得智人生长激素(氨基酸序列如SEQ ID NO:1所示)的编码基因的mRNA序列，在其翻译的蛋白质C端添加6个组氨酸组成的纯化标签，同时将SEQ ID NO:1第107位氨基酸的密码子改成琥珀密码子(TAG)，再通过全基因合成的方式合成该完整的DNA序列得到重组人生长激素的基因序列(SEQ ID NO:2)。
智人生长激素的氨基酸序列(SEQ ID NO:1)如下：
Figure PCTCN2021109276-appb-000030
重组人生长激素的基因序列(SEQ ID NO:2)如下：
Figure PCTCN2021109276-appb-000031
通过一步亚克隆，将重组人生长激素的基因序列(SEQ ID NO:2)克隆至pET21a(Novagen，货号#69740-3)的Nde I和Xho I两个酶切位点之间，得到表达质粒pET21-rhGH107，经测序验证与预期序列一致。pET21-rhGH107可以用于表达第107位氨基酸密码子被替换为琥珀密码子的重组人生长激素，且在该蛋白的C端包含6个组氨酸纯化标签。重组人生长激素表达质粒pET21-rhGH107的图谱如图1所示。
(3)目的表达菌株的获得
将上述辅助质粒pSupAR-MbPylRS和表达质粒pET21-rhGH107共转化至大肠杆菌OrigamiB(DE3)(Novagen，货号#70911-3)感受态细胞，并使用含100mg/L氨苄青霉素和37.5mg/L氯霉素的LB培养基筛选获得双抗性菌株，即为重组人生长激素表达菌株。
(4)***非天然氨基酸的重组蛋白质表达
将筛选后的重组人生长激素表达菌株接种至8份2×YT培养基(酵母提取物16g/L，胰蛋白胨10g/L，NaCl 5g/L，含100mg/L氨苄青霉素和37.5mg/L氯霉素)中，37℃培养至菌液OD600为2.0±0.2，向8份菌液中分别添加IPTG(终浓度为1mM)和***糖(终浓度为0.2％)，向第1份菌液中添加NBOK(终浓度为1mM)、向第2份菌液中添加NPAK(终浓度为1mM)、向第3份菌液中添加NBPK(终浓度为1mM)、向第4份菌液中添加NBGK(终浓度为1mM)、向第5份菌液中添加NPOK(终浓度为1mM)、向第6份菌液中添加NPOK-2(终浓度为1mM)，向第7份菌液中添加NBGK-2(终浓度为1mM)，第8份菌液不添加非天然氨基酸，作为阴性对照。37℃培养诱导表达5-6小时后取各培养液1ml，10000rpm离心1min，用PBS重悬至OD600为10，分别取各菌悬液进行SDS-PAGE电泳，各个菌株的SDS-PAGE电泳图见图2。图2结果表明表达菌株在分别添加了7种非天然氨基酸的情况下可以表达目的蛋白。
(5)***非天然氨基酸的重组蛋白质的纯化
上述诱导表达后的各菌液10000rpm离心5min，取沉淀，加入1/20细胞生长体积的NTA Buffer(20mM Tris-HCl pH7.9，0.5M NaCl，10％甘油)和终浓度为1mM的PMSF，超声波匀浆破碎细胞，10000rpm离心20min，取上清，使用Ni-NTA层析柱吸附带有His 标签的目的蛋白，再用洗脱液NTA Buffer(20mM Tris-HCl pH7.9，0.5M NaCl，10％甘油，250mM咪唑)洗脱，得到纯度约90％的目的蛋白(即，***非天然氨基酸的重组蛋白质)样品。将各样品置于10K硝酸纤维素透析袋，置于pH7.0的PBS缓冲液中透析(每1ml蛋白溶液对应200ml透析液)过夜，并换液1次，收集透析后的蛋白溶液，SDS-PAGE检测蛋白浓度。
(6)***非天然氨基酸的重组蛋白质与PEG偶联反应
Figure PCTCN2021109276-appb-000032
如上述合成路线所示(其中R 1到R 2的方向为氨基酸序列的N末端到C末端方向)。
以30KD氨氧基PEG肟化反应偶联***非天然氨基酸的重组蛋白质为例，偶联反应操作如下：在偶联反应前，用pH7.0PBS缓冲液调节上述获得的目的蛋白至0.5mg/ml，按照1：15(摩尔比，重组蛋白：氨氧基PEG)加入30KD氨氧基PEG固体(来源北京键凯科技有限公司)，充分摇晃溶解，得到澄清透明溶液，之后将反应液密封，在恒温摇床(25℃，100rpm)中摇晃反应。48h后使用SDS-PAGE分析偶联情况，见图3。图3结果显示6种目标蛋白均实现了30KD PEG偶联，进一步表明了上述7种非天然氨基酸***了目标蛋白中。***非天然氨基酸的重组蛋白质与PEG偶联产物简写为“PEG-rhGH”。
(7)PEG-rhGH的纯化
偶联后的PEG-rhGH经Butyl HP疏水层析柱分离纯化(上样缓冲液：50mM NaH 2PO 4˙2H 2O，0.8M(NH 4) 2SO 4，pH8.5；洗脱缓冲液：20mM Tris-HCl，pH8.5，40倍柱体积梯度洗脱)，获得电泳纯级(>95％)纯品PEG-rhGH。以含有NPOK和含有NBOK的PEG-rhGH为例，电泳图见图4。
其他的含有本发明的非天然氨基酸的PEG-rhGH在纯化后也达到了电泳纯级。
(8)PEG-rhGH的活性分析
具体过程如下：HEK293-GHR细胞(细胞株来源见中国专利：2020112649827)用含 0.1mg/mL潮霉素B(生工生物工程(上海)股份有限公司，货号A600230-0001)、0.75mg/mL的G418(生工生物工程(上海)股份有限公司，货号A600958-0005)、10％胎牛血清(Gibco，货号10099141)的DMEM(Gibco，货号11995040)培养基，于37℃、5％二氧化碳条件下培养至足够量，在黑色96孔细胞培养板(Corning，货号3904)中每孔接入9×10 4个HEK293-GHR细胞，90μL/孔，37℃、5％二氧化碳条件下饥饿培养16h。将rhGH标准品(购自中国食品药品检定研究院，简称“中检院”)、含NPOK的rhGH(即，rhGH(NPOK))、PEG修饰NPOK的PEG-rhGH(即，PEG-rhGH(NPOK))、含NBOK的PEG-rhGH(即，rhGH(NBOK))、PEG修饰NBOK的PEG-rhGH(即，PEG-rhGH(NBOK))、市售的rhGH药品赛增
Figure PCTCN2021109276-appb-000033
(长春金赛药业有限责任公司)、市售的PEG-rhGH药品金赛增
Figure PCTCN2021109276-appb-000034
(长春金赛药业有限责任公司)样品进行梯度稀释，共9个浓度，于37℃、5％二氧化碳条件下培养4h。每孔加入50μL ONE-Glo TM(Promega，货号E6120)检测试剂，酶标仪读取化学发光值。根据样品浓度和酶标仪的读值拟合曲线，计算EC50。结果见图5A-图5G和表1。结果表明偶联前各位点细胞活性与中检院rhGH标准品相当。偶联后细胞活性降低，约为标准品的25％～50％，其生物学活性变化规律与上市的PEG-rhGH一致。
其他的含有本发明的非天然氨基酸的PEG-rhGH在偶联之后细胞活性也降低为标准品的25％～50％，生物学活性变化规律也与上市的PEG-rhGH一致。
表1
Figure PCTCN2021109276-appb-000035
实施例9
使用实施例1-7制备的非天然氨基酸NPOK、NPAK、NBOK、NBPK、NBGK、NPOK-2和NBGK-2在真核表达***中制备重组GFP蛋白。
(1)辅助质粒的获得
辅助质粒pCMV-MbPylRS购自质粒保藏组织addgene(货号#91706)，该质粒编码哺乳动物细胞中特异性识别吡咯赖氨酸衍生非天然氨基酸的氨酰tRNA合成酶及对应的tRNA(识别琥珀密码子UAG)。
(2)基因阅读框内部含有琥珀密码子的绿色荧光蛋白表达载体的构建
将表达含有纯化标签的野生型绿色荧光蛋白(其基因编码序列如SEQ ID NO:3所示)的表达载体进行点突变，获取阅读框第40位氨基酸密码子被突变为琥珀密码子的表达质粒pEGFP40，其完整序列见SEQ ID NO:4。
含有纯化标签的野生型绿色荧光蛋白的基因序列(SEQ ID NO:3)如下：
Figure PCTCN2021109276-appb-000036
表达质粒pEGFP40的基因序列(SEQ ID NO:4)如下：
Figure PCTCN2021109276-appb-000037
Figure PCTCN2021109276-appb-000038
Figure PCTCN2021109276-appb-000039
Figure PCTCN2021109276-appb-000040
(3)***非天然氨基酸的重组GFP蛋白表达
从美国模式培养物集存库(ATCC)购买中国仓鼠卵巢细胞CHO-K1(货号#CCL-61-ATC)，并使用含10％胎牛血清的RPMI1640培养基进行贴壁培养。提取上述步骤的辅助质粒pCMV-MbPylRS与绿色荧光蛋白表达质粒pEGFP40，使用lipo2000转染试剂(invitrogen公司，货号#12566014)，根据说明书进行瞬时转染，(将细胞以50000个细胞/孔的接种量接种到24孔板中，共8个孔，在接种培养24h后进行转染，每孔500ng质粒)并在转染的同时向第1孔中添加NPOK(终浓度为1mM)、向第2孔中添加NPAK(终浓度为1mM)、向第3孔中添加NBOK(终浓度为1mM)、向第4孔中添加NBPK(终浓度为1mM)、向第5孔中添加NBGK(终浓度为1mM)，向第6孔中添加NPOK-2(终浓度为1mM)，向第7孔中添加NBGK-2(终浓度为1mM)，第8孔不添加非天然氨基酸，作为阴性对照。在二氧化碳培养箱中静置培养48h后在荧光显微镜下观察细胞，结果显示第1孔～第7孔存在明显的绿色荧光(见图6)，证明非天然氨基酸能够***到绿色荧光蛋白中，得到完整的绿色荧光蛋白，且不影响荧光蛋白的功能。
实施例10
使用实施例1-7制备的NPOK、NPAK、NBOK、NBPK、NBGK、NPOK-2、NBGK-2在真核表达***中表达含非天然氨基酸的抗HER2单抗，并制备与毒素的偶联物。
(1)辅助质粒的获得
辅助质粒pCMV-MbPylRS购自质粒保藏组织addgene(货号#91706)，该质粒编码哺乳动物细胞中特异性识别吡咯赖氨酸衍生非天然氨基酸的氨酰tRNA合成酶及对应的 tRNA(识别琥珀密码子UAG)。
(2)基因阅读框内部含有琥珀密码子的抗HER2抗体(曲妥珠单抗)表达载体的构建
通过全基因合成的方式合成编码曲妥珠单抗的重链和轻链DNA(对应的氨基酸序列分别为SEQ ID NO:5和SEQ ID NO:6)，并亚克隆至真核表达载体pCDNA3.1+，再将得到的表达载体进行点突变，获取重链阅读框第142位氨基酸密码子被突变为琥珀密码子的表达质粒pCDNA3.1-Trastuzumab-UAG142，其图谱见图7，其完整序列见SEQ ID NO:7。
曲妥珠单抗的重链氨基酸序列(SEQ ID NO:5)如下：
Figure PCTCN2021109276-appb-000041
曲妥珠单抗的轻链氨基酸序列(SEQ ID NO:6)如下：
Figure PCTCN2021109276-appb-000042
表达质粒pCDNA3.1-Trastuzumab-UAG142的基因序列(SEQ ID NO:7)如下：
Figure PCTCN2021109276-appb-000043
Figure PCTCN2021109276-appb-000044
Figure PCTCN2021109276-appb-000045
Figure PCTCN2021109276-appb-000046
Figure PCTCN2021109276-appb-000047
(3)***非天然氨基酸
使用悬浮驯化的HEK293细胞，以0.3×10 5/mL密度接种至Wayne293 TM培养基(Quacell Biotechnology，货号A21501)，使用1L的摇瓶，装液量240mL，在120rpm、5％CO 2、80％湿度的条件下震荡培养，当细胞密度达到约1×10 6/mL时进行转染，提取步骤(1)和(2)描述的辅助质粒pCMV-MbPylRS与表达质粒pCDNA3.1-Trastuzumab-UAG142，并去除内毒素备用，每个摇瓶转染时使用表达质粒和辅助质粒各120μg，将质粒加入离心管中，用1×PBS缓冲液稀释至7.2mL，再在另一个离心管中加入PEI转染试剂(聚乙烯亚胺，Polyethyleneimine)720μg，再加入1×PBS缓冲液稀释至7.2mL，混匀后将两个离心管各静置5分钟，再将两个离心管中的液体轻混匀后静置10分钟，缓慢滴加共14.4mL至1L摇瓶的240mL的细胞悬液中，并在滴加过程中不断轻轻摇匀，120rpm、5％CO 2和80％湿度的条件下震荡培养4h后添加终浓度为1mM的非天然氨基酸，再继续120rpm、5％CO 2、80％湿度的条件下震荡培养5天，收获细胞培养上清用于纯化抗体。
(4)纯化
采用HiTrap Protein A，1ml预装柱纯化细胞培养上清，洗脱缓冲液为100mmol/L甘氨酸，200mmol/L醋酸盐，pH3.5，得到纯化后的***非天然氨基酸的曲妥珠单抗。
(5)偶联
合成路线如下所示(以NBOK为例)：
Figure PCTCN2021109276-appb-000048
通过肟化反应将含有氨氧末端基团的毒素(DM1-PEG-ONH2)与纯化得到的***非天然氨基酸的曲妥珠单抗(其中R 1到R 2的方向为氨基酸序列的N末端到C末端方向)进行定点偶联，得到单抗-毒素偶联物。
DM1-PEG-ONH2制备过程如下：
Figure PCTCN2021109276-appb-000049
偶联过程的具体操作如下：将纯化得到的***非天然氨基酸的曲妥珠单抗与DM1-PEG-ONH2以1:15的摩尔比进行混合溶清，然后采用10M醋酸调节pH至4.0，在摇床上摇晃震荡(25℃，200rpm)，48h取样，采用疏水层析原理的HPLC(HIC-HPLC)检测曲妥珠单抗的反应情况。结果显示93.0％的含NBPK曲妥珠单抗被毒素修饰，93.0％的含NBOK曲妥珠单抗被毒素修饰，87.4％的含NPOK-2曲妥珠单抗被毒素修饰，如图8A-8C所示。曲妥珠单抗偶联比例＝100％-剩余未偶联原料的比例。
偶联后的单抗-毒素偶联物通过50kDa超滤离心管进行换液，去除未反应的毒素原料，置换缓冲液为20mM组氨酸缓冲液(pH6.5)。
HIC-HPLC分析条件如下：
流动相A(2M硫酸铵，75mM K 2HPO 4，pH 7.2±0.2)；
流动相B(75mM K 2HPO 4，25％异丙醇，pH 7.2±0.2)。
Figure PCTCN2021109276-appb-000050
(6)活性分析
采用BT-474细胞株(ATCC，货号HTB-20)，检测样品对细胞增殖抑制作用。
具体过程如下：BT-474细胞用含10％胎牛血清(Gibco，货号10099141)的ATCC Hybri-Care Medium(ATCC，货号46-X)培养基，于37℃、5％二氧化碳条件下培养至足够量，在96孔细胞培养板(Corning，货号3599)中每孔接种1.5×10 4个BT-474细胞(包括溶媒对照孔)，空白对照孔中加入不含细胞的培养基，80μL/孔。将含NBOK的曲妥珠单抗和DM1修饰的含NBOK曲妥珠单抗从17μg/mL梯度稀释至0.13μg/mL，共8个浓度，每一稀释度2个复孔。将稀释好的样品转移至已接种BT-474细胞的培养板内，每孔10μL，在溶媒对照孔和空白对照孔中加入10μL培养基，于37℃、5％二氧化碳条件下培养。第5天每孔加入10μL CCK-8(碧云天，货号C0043)检测试剂，于37℃、5％二氧化碳条件下显色6h，酶标仪于450nm波长下读取光密度值。用下列公式来计算检测样品的抑制率(Growth inhibition)：抑制率(％)＝(OD化合物–OD空白对照)/(OD溶媒对照–OD空白对照)×100％。在Excel中计算不同浓度化合物的抑制率，然后用GraphPad Prism7软件作抑制曲线图和计算IC50。结果见图9。该结果显示DM1修饰的含NBOK曲妥珠单抗相比含NBOK曲妥珠单抗，IC50低约3倍，肿瘤杀伤效果得到显著提升。
(7)DM1修饰的含非天然氨基酸的曲妥珠单抗的稳定性考察
偶联后的单抗-毒素偶联物通过50kDa超滤离心管(Millipore，货号UFC905024#)进行换液，去除未反应的毒素原料。置换缓冲液为20mM组氨酸缓冲液(pH6.0)。蛋白溶液分成三份，分别用1M柠檬酸溶液或1M Tris溶液调节pH4.0、pH6.0、pH8.0，置于25℃水浴中，分别于24h、48h、72h、96h和120h取样，HIC-HPLC检测(检测条件同上)，结果见表2。
表2 DM1修饰的含NBOK的曲妥珠单抗在25℃、不同pH下的稳定性
Figure PCTCN2021109276-appb-000051
表2的结果表明，本发明提供的DM1修饰的含非天然氨基酸的曲妥珠单抗中，所形成的肟键在低pH下仍相对较稳定。
实施例11 Lys-azido的活性基团还原现象
(1)高分辨质谱分析产物分子量
参考文献(宋礼华等，生物学杂志，16(1)：6-8,1999)的分泌型生长激素表达方法，根据文献中提供的信号肽序列，在表达载体上rhGH的N端添加OmpA信号肽编码序列，结合实施例8中的辅助质粒pSupAR-MbPylRS，构建K140密码子突变为琥珀密码子(TAG)的rhGH的OrigamiB(DE3)表达菌株，通过在发酵过程中添加Lys-azido，表达第140位突变为Lys-azido的天然rhGH，编号为rhGH-Lys-azido-140；通过在发酵过程中添加NBOK，表达第140位突变为NBOK的天然rhGH，编号为rhGH-NBOK-140，并参考上述参考文献相应纯化手段提纯。将rhGH-Lys-azido-140和rhGH-NBOK-140通过液质联用(高分辨质谱仪：XevoG2-XS Q-Tof，Waters公司；超高效液相色谱：UPLC(Acquity UPLC I-Class)，Waters公司)分析完整分子量。
如图10A所示，rhGH-Lys-azido-140样品中出现了比理论分子量(22265Da)小26Da的组分，该组分推断为由Lys-azido末端的叠氮结构(-N 3)被还原为(-NH 2)的产物，说明目前常用的非天然氨基酸Lys-azido在***蛋白时叠氮基不稳定，容易被还原形成还原产物，因此会失去偶联活性。如图10B所示，rhGH-NBOK-140样品未出现比例较高的杂质，表明NBOK相比Lys-azido，制备获得的重组蛋白产物更加稳定。
(2)偶联效率比较
Figure PCTCN2021109276-appb-000052
如上述反应路线所示，通过Click反应将30KD BCN-PEG(参照中国专利CN112279906A自制)与rhGH-Lys-azido-140定点偶联(其中R 1到R 2的方向为氨基酸序列的N末端到C末端方向)。用pH7.0PBS缓冲液调节上述获得的目的蛋白至0.5mg/ml，分别按照1：15和1：25(摩尔比，重组蛋白：30KD BCN-PEG)，将BCN-PEG固体投入rhGH-Lys-azido溶液中，充分摇晃溶解，得到澄清透明的溶液，之后将反应液密封，在恒温摇床(25℃，70rpm)中摇晃反应。每隔一段时间，取样用SDS-PAGE检测反应结果，72h后反应停止，转化率约50％-70％。反应结果见图11A。
参考实施例8，通过肟化反应将30KD氨氧基PEG与rhGH-NBOK-140定点偶联。每隔一段时间，取样用SDS-PAGE检测反应结果，48h后反应停止，转化率约90％。反应结果见图11B。
结合高分辨质谱分子量检测结果可以判断Lys-azido的还原导致产物无法与BCN-PEG偶联，由此降低了偶联率。而包含本发明的非天然氨基酸的重组蛋白与PEG进行偶联时，转化率明显高于包含Lys-azido的重组蛋白，而且反应时间也明显缩短，因此显著提高了反应效率。
除非特别限定，本发明所用术语均为本领域技术人员通常理解的含义。
本发明所描述的实施方式仅出于示例性目的，并非用以限制本发明的保护范围，本领域技术人员可在本发明的范围内作出各种其他替换、改变和改进，因而，本发明不限于上述实施方式，而仅由权利要求限定。

Claims (10)

  1. 一种非天然氨基酸，其特征在于，为具有如式(Ⅰ)所示结构的化合物或其对映异构体，
    Figure PCTCN2021109276-appb-100001
    其中，X和Z各自独立地表示取代或未取代的C0～C20直链或支链亚烷基，其中的一个或多个-CH 2-可任选地替换为-O-、-S-、-NH-、-C(O)-、-S(O)-中的一个或多个，Y表示-C(O)-、-S(O)-或-CH 2-，以及A表示取代或未取代的C6～C20芳基；
    当所述X、Z和A各自独立地表示取代的基团时，取代基选自羟基、巯基、卤素、硝基、氰基、烷基、烯基、炔基、烷氧基、酰基、酰胺基、羧基、酯基、氨基、磺酰基、亚磺酰基、环烷基、杂环基、芳基、杂芳基中的一种或多种。
  2. 根据权利要求1所述的非天然氨基酸，其特征在于，所述取代基选自羟基、巯基、卤素、硝基、氰基、C1～C6烷基、C1～C6烷氧基、酰基、酰胺基、羧基、酯基、氨基、磺酰基、亚磺酰基、C3～C8环烷基、C3～C8杂环基、C6～C20芳基、C4～C10杂芳基中的一种或多种；
    优选地，所述X和Z各自独立地表示C0～C10直链或支链亚烷基，优选表示C0～C6直链亚烷基，其中的一个或多个-CH 2-可任选地替换为-O-、-S-、-NH-中的一个或多个；更优选地，所述X和Z不同时为C0的亚烷基；和/或
    所述A表示取代或未取代的C6～C10芳基，更优选地，所述A表示取代或未取代的苯基或萘基。
  3. 根据权利要求1或2所述的非天然氨基酸，其特征在于，所述非天然氨基酸为具有如式(Ⅰ-1)所示结构的化合物，
    Figure PCTCN2021109276-appb-100002
    其中，所述X、Z和A各自独立地如权利要求1或2所定义；
    优选地，所述非天然氨基酸为具有如式(Ⅰ-2)所示结构的化合物，
    Figure PCTCN2021109276-appb-100003
    其中，所述X如权利要求1或2所定义；
    R 1和R 2各自独立地表示氢、羟基、巯基、卤素、硝基、氰基、C1～C6烷基、C1～C6烷氧基、酰基、酰胺基、羧基、酯基、氨基、磺酰基、亚磺酰基、C3～C8环烷基、C3～C8杂环基、C6～C20芳基或C4～C10杂芳基。
  4. 根据权利要求3所述的非天然氨基酸，其特征在于，所述非天然氨基酸为具有如式(Ⅰ-3)、式(Ⅰ-4)、式(Ⅰ-5)或式(Ⅰ-6)所示结构的化合物，
    Figure PCTCN2021109276-appb-100004
    其中，X’表示C0～C6的直链亚烷基，优选表示C0～C4的直链亚烷基，其中的一个或多个-CH 2-可任选地替换为-O-和/或-NH-；
    所述R 1和R 2各自独立地如权利要求3所定义。
  5. 根据权利要求1-4任一项所述的非天然氨基酸，其特征在于，所述非天然氨基酸为具有以下结构之一的化合物：
    Figure PCTCN2021109276-appb-100005
    Figure PCTCN2021109276-appb-100006
  6. 权利要求1-5任一项所述的非天然氨基酸在制备重组蛋白或重组蛋白偶联物中的应用；优选地，所述重组蛋白为重组人生长激素；或者，优选地，所述重组蛋白偶联物为重组人生长激素-聚乙二醇偶联物。
  7. 一种重组蛋白，其特征在于，所述重组蛋白的氨基酸序列中的至少一个位点上为权利要求1-5任一项所述的非天然氨基酸。
  8. 一种重组蛋白偶联物，其特征在于，通过权利要求7所述的重组蛋白中的非天然氨基酸的端基羰基与含有“NH 2-O-”端基的偶联部分形成肟键而形成。
  9. 根据权利要求8所述的重组蛋白偶联物，其特征在于，所述重组蛋白为重组人生长激素，所述含有“NH 2-O-”端基的偶联部分为含有“NH 2-O-”端基的聚乙二醇，优选分子量为10～100KD的聚乙二醇；更优选地，所述重组人生长激素的氨基酸序列如SEQ ID NO:1所示；进一步优选地，对应于所述氨基酸序列SEQ ID NO:1中的第107位点上包含权利要求1-5任一项所述的非天然氨基酸。
  10. 权利要求9所述的重组蛋白偶联物在制备用于治疗内源性生长激素分泌不足所引起的生长发育障碍、特纳综合征引起的生长发育障碍或成人生长激素缺乏症的药物中的用途。
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