CN109265512A - The preparation method of protein conjugate based on pyridine dicarbaldehyde - Google Patents
The preparation method of protein conjugate based on pyridine dicarbaldehyde Download PDFInfo
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- CN109265512A CN109265512A CN201811113353.7A CN201811113353A CN109265512A CN 109265512 A CN109265512 A CN 109265512A CN 201811113353 A CN201811113353 A CN 201811113353A CN 109265512 A CN109265512 A CN 109265512A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- 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
- A61K47/62—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 a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
- G01N21/6404—Atomic fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
Abstract
The invention proposes a kind of methods for modifying protein N-terminal.This method comprises: protein to be finished is contacted with compound shown in formula (I), to obtain the protein by N-terminal modification, wherein second amino acid of the protein N-terminal is not proline.This method reaction condition is mild, high-efficient, and the special sex modification protein N-terminal of energy, conducive to the building of accurate protein conjugate, obtained product is stable to a certain degree.
Description
Technical field
The present invention relates to biomedicine fields, in particular it relates to the protein conjugate based on pyridine dicarbaldehyde
Preparation method.
Background technique
In recent ten years, biology and chemicobiological fast development have greatly pushed the research and development of pharmaceutical grade protein,
More and more pharmaceutical grade proteins are used for the treatment of disease, at the same time, egg by U.S. Food and Drug Administration (FDA) approval
White matter-conjugate is received more and more attention as a kind of new bio macromolecular derivatives.
Protein-conjugate refers to that protein is formed by covalent bond or non-covalent bond and the coupling of other functional ized components
A kind of biological hybrid also tend to obtain while retaining protein characteristic (such as accurate structure and biological function)
From the characteristic of new component, these have gathered the bioconjugate of different characteristics, and especially protein-polymer conjugate can
Be widely used in biocatalysis, molecular diagnosis, molecular image, biomaterial, drug delivery, biomolecule delivery, organizational project,
The biomedicine fields such as regenerative medicine, targeted therapy, biological therapy.
Change of the protein-conjugate through excessive generation, is applied in biological medicine, but current protein-height
Molecule coupling labeled technology still has a series of problem in science of crucial general character urgently to be solved, such as conjugation sites poor selectivity, idol
Join low efficiency and lack general-purpose platform technology etc., seriously constrain extensive use of the protein-conjugate in biological medicine.
Summary of the invention
The application is to be made based on inventor to the discovery of following facts and problem and understanding:
The reaction condition of pyridine carboxaldehyde derivative and protein is mild, high-efficient, and product is stablized, but and protein N-terminal
While amino highly effective reaction, other amino on protein can be partially modified, accurate protein conjugate is unfavorable for
Building.Discovery based on the above issues, inventors herein proposes the method for modifying based on pyridine dicarbaldehyde, and the structure of two aldehyde radicals is set
Meter and can introduce aldehyde after albumen qualitative response so that its dosage compared with other aldehyde group modified dose can reduce half on protein
Base, by azanol, hydrazine derivate with protein residue aldehyde radical while reacting to be coupled other functional components, excessive hydroxyl
Amine, hydrazine derivate can compete the unstable imines for destroying that gal4 amino acid residue side chains amino and pyridine aldehyde radical reagent are formed
Key can also accomplish that locus specificity modifies (see Fig. 1) while accomplishing to introduce new component by this method.
In first invention of the invention, the invention proposes a kind of methods for modifying protein N-terminal.It is according to the present invention
Embodiment, the above method includes: to contact protein to be finished with compound shown in formula (I), to obtain by N-terminal
The protein of modification, wherein second amino acid of the protein N-terminal is not proline,
Nucleophilic occurs first according to the amino of compound specificity shown in the formula (I) of inventive embodiments and protein N-terminal
Addition reaction, obtains an imine structure, and imine structure is a unstable structure, thus second of protein N-terminal
Amino acid (second amino acid of protein N-terminal is not proline) and first amino acid are formed by the parahelium of amido bond
Nucleophilic addition can occur with imines, generate a 5 relatively stable ring structures, and then obtain the egg after N-terminal modification
White matter.According to the method for the embodiment of the present invention can special sex modification protein the end N-, (can only modify on protein on a small quantity
Remaining free amino, formation are that unstable imine structure is subsequent unstable, can be disappeared with the progress of reaction).
Aldehyde radical is introduced in the N-terminal of protein according to the method for the embodiment of the present invention, conducive to the building of accurate protein conjugate, and root
Mild, high-efficient according to the method reaction condition of the embodiment of the present invention, obtained product is stablized.
According to an embodiment of the invention, the above method can further include at least one following additional technical feature:
According to an embodiment of the invention, the protein is interferon, green fluorescent protein, myoglobins, RNA enzyme, ox
Seralbumin, human serum albumins or glucose oxidase.It should be noted that the protein includes medicine, agricultural, section
It grinds and the relevant albumen of other industrial circles, small peptide and antibody, particularly including human cytokines such as insulin, monoclonal is anti-
Body, blood factor, colony stimulating factor, growth hormone, interleukin, growth factor, therapeutic vaccine, calcitonin, neoplasm necrosis
The factor (TNF) and enzyme etc..Specific example includes, but is not limited to: glutamic acid enzyme, arginase, arginine deaminase, adenosine
Deaminase ribalgilase, cytosine deaminase, trypsase, chymotrypsin, papain, epidermal growth factor
(EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), platelet-derived
Growth factor (PDGF), bone morphogenetic protein (BMP), fibroblast growth factor, growth hormone release inhibiting hormone, growth hormone, growth
Somatostatin, calcitonin, parathyroid hormone, colony stimulating factor (CSF), coagulation factor, neoplasm necrosis factor, interference
Element, interleukins, gastrointestinal peptide, vasoactive intestinal peptide (VIP), cholecystokinin (CCK), gastrin, secretin, rush are red thin
Born of the same parents generate element, antidiuretic hormone, Octreotide, pancreas enzyme, superoxide dismutase, thyrotropin-releasing hormone (TRH) (TRH), promote
It is thyroid hormone, luteinizing principle, luteinising hormone-releasing hormo (LHRH), tissue-type plasminogen activator, white thin
Born of the same parents' interleukin -1, interleukin-15, receptor antagonist (IL-1RA), glucagon-like-peptide-1 (GLP-1), leptin, growth
Element, G-GSF (GM-CSF), interleukin 2 (IL-2), adenosine deaminase, uricase, asparagus fern acyl
Amine enzyme, human growth hormone (HGH)；Human chorionic gonadtropin, heparin, atrial natriuretic peptide, hemoglobin, retroviral vector, relaxation
Peptide；Cyclosporin, oxytocins, vaccine, monoclonal antibody, single-chain antibody, ankyrin repeat protein, affine body etc..
According to an embodiment of the invention, stablizing in the protein is 10-16 hours lower at 25 degrees Celsius.Protein N-terminal
First amino acid the short time need with pyridine -2,6- diformazan aldehyde reaction generate imine structure, later further with N-
Second amino acid in end occur addition reaction and it is subsequent carry out other reactions again, thus protein was needed at 10-16 hours
Inside it is stabilized.
According to a particular embodiment of the invention, the contact is carried out 16 hours under conditions of 25 DEG C.According to the present invention
Embodiment method, reaction condition is mild, high-efficient.
According to a particular embodiment of the invention, the molar ratio of the protein to be finished and compound shown in formula (I) is 1:
200.Inventors have found that reaction efficiency is high under conditions of above-mentioned molar ratio, resulting product is stablized.
According to an embodiment of the invention, compound shown in formula (I) is previously dissolved in the phosphate buffer that pH is 7.4.Root
According to specific embodiments of the present invention, the concentration of compound shown in the formula (I) is 0.01-5mg/mL, it is preferable that the formula (I)
The concentration of shown compound is 4mg/mL.
It according to a particular embodiment of the invention, further comprise NaCl in the phosphate buffer solution that the pH is 7.4,
Ethylenediamine tetra-acetic acid.Above-mentioned buffer can dissolve compound and protein shown in formula (I) simultaneously, avoid the two in organic solvent
Middle reaction, and then the activity of protein will not be destroyed.
According to a particular embodiment of the invention, compound, phosphate, NaCl shown in the formula (I), ethylenediamine tetra-acetic acid
Molar ratio is 3:5:15:1.In turn, ethylenediamine tetra-acetic acid can stable protein to a certain extent.
In the second aspect of the present invention, protein-conjugate method is prepared the invention proposes a kind of.According to the present invention
Embodiment, which comprises 1) using method noted earlier to protein carry out N-terminal modification processing；2) by step (1)
Product obtained carries out nucleophilic addition with to coupling molecule, described to have azanol functional group or hydrazine function to coupling molecule
Group.Step 1) product obtained according to an embodiment of the present invention has aldehyde functions, nucleophilic can occur with nucleopilic reagent and add
At reaction.Compound containing azanol functional group or hydrazine functional group is nucleopilic reagent, these compound other ends can be connected with it
His functional group, and then other functional components of energy coupling are reacted by azanol, hydrazine derivate and the aldehyde radical on protein, meanwhile,
Excessive azanol, hydrazine derivate can compete the unstable imine linkage for destroying protein amino and pyridine aldehyde radical reagent, Jin Ershang
Stating method can also accomplish that locus specificity is modified while accomplishing to introduce new component.According to the method for the embodiment of the present invention, it repairs
It adorns that locus specificity is strong, the building of protein conjugate is accurate, reaction efficiency is high, can be introduced on protein and a variety of contain other
The compound of important functional group.
According to an embodiment of the invention, the above method can further include at least one following additional technical feature:
According to an embodiment of the invention, it is described to coupling molecule have azanol functional group, the nucleophilic addition be
Temperature carries out 2-36 hours under conditions of being 4-37 DEG C.Method according to a particular embodiment of the invention, reaction condition is mild, receives
Rate is high, and specificity is good, can obtain protein-conjugate of accurate structure.
According to an embodiment of the invention, it is described to coupling molecule have azanol functional group, the nucleophilic addition be
Temperature carries out 16 hours under conditions of being 25 DEG C.Method according to a particular embodiment of the invention, reaction condition milder, yield
Higher, specificity is more preferable, can obtain protein-conjugate of more accurate structure.
According to an embodiment of the invention, the step 1) product obtained is previously dissolved in the phosphate-buffered that pH is 5.5
In solution, the phosphate buffer solution that the pH is 5.5 further comprises the above-mentioned buffer solution energy of NaCl dissolving step 1 simultaneously)
Product obtained and to coupling molecule, avoids the contact with organic solvent, and then avoid protein and rest activity
Functional group's activity is destroyed by organic solvent.
According to an embodiment of the invention, the molar ratio of the step 1) product obtained, phosphate and NaCl are
0.15:50:150.The method of specific embodiment according to the present invention, step 1) product obtained are molten under conditions of molar ratio
Solution is more abundant.
According to an embodiment of the invention, described have azanol functional group to coupling molecule, it is described that there is formula to coupling molecule
(II) structure shown in,
Wherein, A group independently be atom transfer radical polymerization initiator, fluorescent molecule, chemotherapeutics, polymer,
Small peptide, protein, the related group of " click " chemistry.According to a particular embodiment of the invention, atom transfer radical polymerization causes
Agent includes but is not limited to 2- brombutyl derivative etc., and fluorescent molecule includes but is not limited to fluorescein, rhodamine B, indocyanine green etc.,
Chemotherapeutics includes but is not limited to methotrexate (MTX), Temozolomide, gemcitabine, how soft pyrrole magnitude, and polymer includes but is not limited to
Polyethylene glycol, polyethylene glycol, poly- 2- methacryloxyethyl Phosphorylcholine (PMPC) etc., small peptide include but is not limited to F3,
RGD etc., the related group of " click " chemistry (i.e. click chemistry) includes but is not limited to nitrine, alkynyl, maleimide etc..
According to an embodiment of the invention, described have azanol functional group to coupling molecule, it is described that there is formula to coupling molecule
(1) structure shown in~(7),
Wherein, each a, b, c, d, e, f are each independently positive integer described in 1-10；N is 25-500, formula (7) shownization
The number-average molecular weight for closing object is 5000.
According to an embodiment of the invention, the molar ratio of compound described in the formula (1)~formula (6) and protein is (10-
100):1.Inventors have found that at (10-100): under conditions of 1 molar ratio, reaction efficiency is high, and resulting product is stablized.
According to an embodiment of the invention, the molar ratio of compound described in the formula (7) and protein is (10-300): 1.
Inventors have found that at (10-300): under conditions of 1 molar ratio, reaction efficiency is high, and resulting product is stablized.
According to an embodiment of the invention, the molar ratio of compound described in the formula (7) and protein is (10-300): 1.
Inventors have found that reaction efficiency is higher under conditions of the molar ratio of 50:1, resulting product is more stable.
According to an embodiment of the invention, described have structure shown in formula (8) to coupling molecule, the surface of the protein is not
With free sulfhydryl groups；
The product that compound shown in formula (8) and step (1) obtain carries out the product that nucleophilic addition obtains, and can occur with sulfydryl
Reaction, can further react with protein if having free sulfydryl if protein surface.
According to an embodiment of the invention, the molar ratio of compound described in the formula (8) and the protein is (10-
100): 1, preferably 25:1.Inventors have found that at (10-100): under conditions of 1 molar ratio, reaction efficiency is high, resulting product
Stablize, under conditions of 25:1 molar ratio, reaction efficiency is higher, and resulting product is more stable.
It is according to a particular embodiment of the invention, described that there is structure shown in formula (9) to coupling molecule,
According to an embodiment of the invention, the molar ratio of compound described in the formula (9) and protein is (10-100): 1,
Preferably 25:1.Inventors have found that at (10-100): under conditions of 1 molar ratio, reaction efficiency is high, and resulting product is stablized,
Under conditions of 25:1 molar ratio, reaction efficiency is high, and resulting product is stablized.
According to an embodiment of the invention, described have formula (3), formula (6), structure shown in formula (9) to coupling molecule, into one
Step includes that nucleophilic addition product and monomeric compound are carried out atom transition free radical polymerization reaction in situ, to obtain egg
White matter-macromolecule conjugate.Formula (3), formula (6), structure is common atom transition free radical polymerization reaction shown in formula (9)
Initiator, thus nucleophilic addition first occurs to coupling molecule and protein shown in formula (3), formula (6), formula (9), obtain parent
Core addition reaction product (i.e. protein-initiator combination), and then home position polymerization reaction occurs with monomeric compound.
According to an embodiment of the invention, the monomeric compound includes water-soluble methyl acrylic ester, acrylate
Class, methacryl amine, acrylamide monomers.Water-soluble monomeric compound can be anti-with the nucleophilic addition containing protein
It answers product that home position polymerization reaction occurs in aqueous solution, avoids protein and contacted with the direct of organic solvent, and then avoid
Destruction of the organic solvent to proteins biological activity.
According to an embodiment of the invention, the monomeric compound includes oligomeric ethylene glycol methyl ether methacrylate, 2 one first
Base acrylyl oxy-ethyl Phosphorylcholine, acrylamide, sugar derivatives monomer.
According to an embodiment of the invention, described have structure shown in formula (8) to coupling molecule, it further comprise by nucleophilic
Addition reaction product is attached with polymer containing sulfydryl to react, to obtain protein-polymer conjugate.
It should be noted that nucleophilic addition product and polymer reaction containing sulfydryl are that classical " click " chemistry is anti-
It answers, thus the two molar ratio is not particularly limited, and occurs as long as meeting " click " chemical reaction.According to the present invention
Embodiment, the molar ratio of the nucleophilic addition product and the polymer containing sulfydryl is 1:(1-300), preferably 1:
20.Inventors have found that in 1:(1-300) molar ratio under conditions of, reaction efficiency is high, and purify it is simple, in mole of 1:20
Than under conditions of, reaction efficiency is higher, and purifies simpler.
According to an embodiment of the invention, the polymer containing sulfydryl is water-soluble polymer containing sulfydryl.It is water-soluble to contain mercapto
Based polyalcohol can react in aqueous solution with the nucleophilic addition product containing protein, avoid protein with it is organic molten
The contact of agent, and then avoid destruction of the organic solvent to proteins biological activity.
According to an embodiment of the invention, the water solubility polymer containing sulfydryl is poly- by reversible addion-fragmentation chain transfer
Water-soluble polymethacrylate, the polyacrylate, poly- methyl that conjunction, atom transfer radical polymerization, polymerization obtain
Acrylic amide, polyacrylamide polymer；The band sulfydryl generated by ring-opening polymerisation, anionic polymerisation, cationic polymerization
Polymer, polyaminoacid.
According to an embodiment of the invention, the polymer containing sulfydryl is mercapto-polyglycol.
According to an embodiment of the invention, described have hydrazine functional group to coupling molecule, it is described that there is formula to coupling molecule
(III) structure shown in,
Wherein, A group independently be atom transfer radical polymerization initiator, fluorescent molecule, chemotherapeutics, polymer,
Small peptide, protein, the related group of " click " chemistry.According to a particular embodiment of the invention, atom transfer radical polymerization causes
Agent includes but is not limited to 2- brombutyl derivative.Fluorescent molecule includes but is not limited to fluorescein, rhodamine B, indocyanine green etc., is changed
Treating drug includes but is not limited to methotrexate (MTX), Temozolomide, gemcitabine, how soft pyrrole magnitude, and polymer includes but is not limited to poly-
Ethylene glycol, polyethylene glycol, poly- 2- methacryloxyethyl Phosphorylcholine (PMPC) etc., small peptide include but is not limited to F3,
RGD, the related group of " click " chemistry includes but is not limited to nitrine, alkynyl, maleimide etc..
According to an embodiment of the invention, described have structure shown in formula (10)~formula (12) to coupling molecule:
According to an embodiment of the invention, described have structure shown in formula (13) to coupling molecule, the protein surface does not have
There are free sulfhydryl groups:
The product that compound shown in formula (13) and step (1) obtain carries out the product that nucleophilic addition obtains, and can send out with sulfydryl
Raw reaction, can further react with protein if having free sulfydryl if protein surface.
It further comprise adding nucleophilic according to an embodiment of the invention, there is structure shown in formula (13) to coupling molecule
It is attached and reacts with polymer containing sulfydryl at reaction product, to obtain protein-polymer conjugate.
It should be noted that nucleophilic addition product and polymer reaction containing sulfydryl are that classical " click " chemistry is anti-
It answers, thus the two molar ratio is not particularly limited, and occurs as long as meeting " click " chemical reaction.According to the present invention
Embodiment, the molar ratio of the nucleophilic addition product and the polymer containing sulfydryl is 1:(5-200), preferably 1:
20.Inventors have found that in 1:(5-200) molar ratio under conditions of, reaction efficiency is high, and purify it is simple, in mole of 1:20
Than under conditions of, reaction efficiency is higher, and purifies simpler.
According to an embodiment of the invention, the polymer containing sulfydryl is water-soluble polymer containing sulfydryl.It is water-soluble to contain mercapto
Based polyalcohol can react in aqueous solution with the nucleophilic addition product containing protein, avoid protein with it is organic molten
The contact of agent, and then avoid destruction of the organic solvent to proteins biological activity.
According to an embodiment of the invention, the water solubility polymer containing sulfydryl includes passing through reversible addion-fragmentation chain transfer
It polymerize the water-soluble polymethacrylate obtained, polyacrylate, polymethacrylamide class, polyacrylamide
Polymer.
According to an embodiment of the invention, the polymer containing sulfydryl includes mercapto-polyglycol, polymethyl acyl-oxygen second
Base Phosphorylcholine, polyacrylamide.
It to coupling molecule include hydrazine functional group according to an embodiment of the invention, described, obtained nucleophilic addition product is not
It is only applicable to building hydrazone bond connection pH responsiveness protein conjugate, the stabilization after applying also for sodium cyanoborohydride reduction hydrazone bond
Protein conjugate.
According to an embodiment of the invention, described have amido functional group to coupling molecule, it is described that there is formula to coupling molecule
(IV) structure shown in,
Wherein, A group independently is atom transfer radical polymerization initiator, amino acid derivativges, fluorescent molecule, chemotherapy
Drug, polymer, small peptide, protein, the related group of " click " chemistry.According to a particular embodiment of the invention, atom transfer from
It include but is not limited to 2- brombutyl derivative by base polymerization initiator.Fluorescent molecule include but is not limited to fluorescein, rhodamine B,
Indocyanine green etc., chemotherapeutics include but is not limited to methotrexate (MTX), Temozolomide, gemcitabine, how soft pyrrole magnitude, polymer packet
Include but be not limited to polyethylene glycol, oligomerization polyethylene glycol, poly- 2- methacryloxyethyl Phosphorylcholine (PMPC) etc., small peptide packet
F3, RGD are included but are not limited to, the related group of " click " chemistry includes but is not limited to nitrine, alkynyl, maleimide etc..
According to an embodiment of the invention, the amido functional group reagent includes amino acid, polypeptide derivative.
According to an embodiment of the invention, the amido functional group amino acid derivativges are cysteine derivative.
According to an embodiment of the invention, described have structure shown in formula (14) to coupling molecule:
According to an embodiment of the invention, the molar ratio of compound described in the formula (14) and protein is (10-100):
1, preferably 25:1.Inventors have found that at (10-100): under conditions of 1 molar ratio, reaction efficiency is high, and resulting product is stablized,
Under conditions of 25:1 molar ratio, reaction efficiency is high, and resulting product is stablized.
According to an embodiment of the invention, described have structure shown in formula (14) to coupling molecule, further comprising will be close
Core addition reaction product and monomeric compound carry out atom transition free radical polymerization reaction in situ, to obtain protein-high score
Sub- conjugate.
In the third aspect of the present invention, the invention proposes a kind of protein-conjugates.According to an embodiment of the invention,
Protein-the conjugate is to be obtained according to mentioned-above method.Protein-conjugate according to an embodiment of the present invention is steady
Calmly, structure is accurate.
In the fourth aspect of the present invention, the invention proposes a kind of pharmaceutical compositions.According to an embodiment of the invention, described
Pharmaceutical composition includes mentioned-above protein-conjugate, and described to coupling molecule and/or protein is drug molecule.Root
According to specific embodiments of the present invention, above-mentioned protein itself can be pharmaceutical protein, meanwhile, molecule to be coupled is also possible to medicine
Object molecule is formed by connecting with azanol functional group or hydrazine functional group, and drug molecule includes but is not limited to anti-tumor drug, immunosupress
Drug, acceptor inhibitor, agonist etc..After the two coupling, protein coupling drug molecule or medical protein coupling can be obtained
High molecular polymer or medical protein coupling drug molecule, the above-mentioned three kinds internal deliverings for being able to achieve drug molecule and egg
The combination therapy etc. of white matter drug and chemical combination small-molecule drug.
In the fifth aspect of the invention, the invention proposes a kind of fluorescence imaging methods.According to an embodiment of the invention, will
Mentioned-above protein-conjugate imports in microenvironment to be imaged, and described to coupling molecule is fluorescent molecule.It needs to illustrate
It is that it also includes the cell and tissue of living body that the imaging microenvironment, which had both included in vitro cell and tissue,.
In the sixth aspect of the present invention, the invention proposes a kind of fluorescence labeling methods.According to an embodiment of the invention, institute
Stating method includes 1) carrying out N-terminal modification processing to protein using method noted earlier；2) by step 1) product obtained
Nucleophilic addition is carried out with fluorescent molecule, the fluorescent molecule has azanol functional group or hydrazine functional group.Inventors have found that root
According to the method for the embodiment of the present application, can will be specifically bound with fluorescent molecule specificity and protein N-temiinal.
In the seventh aspect of the present invention, the invention proposes a kind of detection protein-protein interaction methods.According to this hair
Bright embodiment, which comprises (1) utilize mentioned-above method, mark at least two protein to be detected respectively；
(2) fluorescence resonance energy transfer technology, the mixture of at least two protein after detecting step (1) label are based on；(3) it is based on
Testing result determines between at least two protein with the presence or absence of interaction, wherein resonance transfer occurs for fluorescent energy
It is the instruction that there is interaction between at least two protein.Resonance transfer occurs for fluorescent energy, then shows described two eggs
White matter, which exists, to be combined, and resonance transfer does not occur for fluorescent energy, then showing described two protein, there is no combine.According to the present invention
The method of embodiment, energy is convenient, fast, accurately knows whether interact between protein.
In another aspect of this invention, the invention proposes the sides that a kind of special sex modification of protein N-terminal introduces aldehyde radical
Method.According to a particular embodiment of the invention, which comprises
1) it prepares compound shown in formula (I): 10 milliliters of Isosorbide-5-Nitrae-dioxane being added in 50 milliliters of round-bottomed flask,
0.42 gram of 2,6- pyridine dimethanol and 0.33 gram of selenium dioxide are added under stirring condition, is stirred to react at 65 DEG C 24 hours.It takes out
Filter, removes insoluble solids, and reaction solution is rotated to dry.Product silica gel chromatography.Product is white solid, reactionization
Learn equation are as follows:
It should be noted that formula (I) compound represented can also pass through commercially available acquisition；
2) it obtains protein: being extracted by purchase, amalgamation and expression, from organism, the modes such as purification obtain protein；
3) it takes compound shown in formula (I) to be dissolved in phosphate buffer, is added in the protein solution of step 2 acquisition,
Overnight, repeatedly dialysis removes unreacted pyridine dicarbaldehyde molecule for reaction, obtains the protein for introducing aldehyde radical, obtains N-terminal
Protein after modification, alternatively referred to as protein-aldehyde radical.
In still another aspect of the invention, the invention proposes a kind of poly- oligomeric ethylene glycol methyl ether (albumen of building protein-
Matter-POEGMA) conjugate method.According to a particular embodiment of the invention, the method is the special sex modification of protein N-terminal
After introducing aldehyde radical, by reacting for hydroxyamine groups in the compound containing azanol functional group and aldehyde radical, introduced on protein
Atom transfer radical polymerization polymerization (ATRP) initiator and the side reaction for eliminating the modification of compound non-specificity shown in formula (I),
And in-situ polymerization growth oligomeric ethylene glycol methyl ether (OEGMA) on protein, the poly- oligomeric ethylene glycol methyl ether of protein-is constructed according to this
(protein-POEGMA) conjugate, the specific steps are as follows:
1) it obtains protein and introduces aldehyde radical in the special sex modification of protein N-terminal, protein is placed in the phosphorus of pH 5.5
In phthalate buffer (PBS-5.5)；Wherein it is possible to be extracted by purchase, amalgamation and expression, from organism, the modes such as purification obtain
Take protein；Protein includes interferon, green fluorescent protein, myoglobins, RNA enzyme, bovine serum albumin(BSA), human seralbumin egg
White, glucose oxidase, trichosanthin etc..
2) it takes azanol ATRP initiator A BM to be dissolved in the phosphate buffer (PBS-5.5) of pH 5.5, is added to step 1
In in protein aldehyde radical solution obtained reaction overnight, repeatedly dialysed using phosphate buffer, protein initiator (egg be made
White matter-bromine)；
3) after being passed through the abundant deoxygenation of nitrogen after taking in step 2) protein initiator obtained that oligomeric ethylene glycol methyl ether is added,
The stannous chloride, copper chloride, 1,1,4,7,10,10- hexamethyl trien for being equally passed through the abundant deoxygenation of nitrogen is added
(HMTETA) solution, reaction 4 hours after be passed through air terminate reaction, repeatedly dialysis remove small molecule, by ion exchange column come
Unreacted protein is separated, the poly- oligomeric ethylene glycol methyl ether of protein-(protein-POEGMA) conjugate is obtained.
The protein conjugate activity for constructing generation according to a particular embodiment of the invention keeps good, and stability is high, repairs
Decorations specificity is high.In the medicine generation of the poly- oligomeric ethylene glycol methyl ether conjugate of the interferon-constructed according to a particular embodiment of the invention, is dynamic
Mechanics parameter is significantly better than interferon itself.
In another aspect of the invention, the invention proposes a kind of methods for constructing protein-polymer conjugate.According to
Specific embodiments of the present invention, described method includes following steps:
1) it obtains protein and introduces aldehyde radical in the special sex modification of protein N-terminal, protein is placed in the phosphorus of pH 5.5
In phthalate buffer (PBS-5.5)；Wherein it is possible to be extracted by purchase, amalgamation and expression, from organism, the modes such as purification obtain
Take protein；
2) azanol poly glycol monomethyl ether is prepared, preparation flow is as follows, and detailed process is in specific embodiment part
It shows；
3) azanol poly glycol monomethyl ether prepared by step 2 is taken to be dissolved in the phosphate buffer (PBS-5.5) of pH 5.5,
Reaction in protein aldehyde radical solution obtained is added in step 1 overnight, to remove after reaction solution is concentrated by molecular sieve and do not join
With the azanol poly glycol monomethyl ether and unreacted protein reacted.
In another aspect of the invention, the invention proposes a kind of methods for constructing protein-polymer conjugate.According to
Specific embodiments of the present invention, by coupling molecule (the Bifunctionalized molecule of azanol-maleimide) such as formula (8) shownization
Conjunction object is reacted with aldehyde radical, while eliminating the modification side reaction of compound non-specificity shown in formula (I), constructs protein-horse
Carry out acid imide, then sulfydryl polymer is prepared by reversible addion-fragmentation chain transfer polymerization (RAFT), allows sulfydryl polymer and albumen
Classical " click " chemical reaction occurs for matter-maleimide.According to a particular embodiment of the invention, the following institute of preparation step
Show:
1) it obtains protein and introduces aldehyde radical in the special sex modification of protein N-terminal, protein is placed in the phosphorus of pH 5.5
In phthalate buffer (PBS-5.5)；
2) sulfydryl polymer is prepared, detailed process is shown in specific embodiment part；
3) the Bifunctionalized molecule of azanol-maleimide (such as compound shown in formula (8)) is prepared, preparation flow is as follows,
Detailed process specific embodiment part show,
4) the Bifunctionalized molecule of azanol-maleimide prepared in step 3 is taken to be dissolved in the phosphate buffer of pH 5.5
(PBS-5.5) in, it is added in step 1 that reaction overnight, uses 7.2 phosphate-buffered of pH in protein aldehyde radical solution obtained
Liquid is repeatedly dialysed, and protein-maleimide is made；
5) it takes sulfydryl polymer prepared by step 2 to be dissolved in the phosphate buffer (PBS-5.5) of pH 7.2, is added to step
It reacts 4 hours in protein-maleimide amine aqueous solution obtained in rapid 4, is not joined after reaction solution is concentrated by molecular sieve removing
With the sulfydryl polymer and unreacted protein reacted, protein-polymer conjugate is made.
In another aspect of the invention, the invention proposes a kind of methods for constructing protein-polymer conjugate.According to
Specific embodiments of the present invention, by coupling molecule (the Bifunctionalized molecule of hydrazine-maleimide), such as formula (13) shownization
Object is closed to react with protein-aldehyde radical, while eliminating the modification side reaction of pyridine dicarbaldehyde non-specificity, building hydrazone bond connection
Protein-maleimide, then sulfydryl polymer is prepared by reversible addion-fragmentation chain transfer polymerization (RAFT), allows sulfydryl
Polymer and protein-maleimide occur classical " click " chemical reaction, the protein-polymer of hydrazone bond connection is made
Conjugate, since there are the conjugates to have pH responsiveness for hydrazone bond.According to a particular embodiment of the invention, preparation step is as follows
It is shown:
1) it obtains protein and introduces aldehyde radical in the special sex modification of protein N-terminal, protein is placed in the phosphorus of pH 5.5
In phthalate buffer (PBS-5.5), wherein can be obtained by purchase, amalgamation and expression, from modes such as organism extraction, purifications
Take protein；
2) sulfydryl polymer is prepared, detailed process is shown in specific embodiment part；
3) the Bifunctionalized molecule of hydrazine-maleimide (such as compound shown in formula (13)) is taken to be dissolved in the phosphate of pH 7.2
In buffer (PBS-7.2), it is added in 7.2 aniline hydrochloride solution of pH and is then added to protein aldehyde radical solution obtained in step 1
Middle reaction overnight, is repeatedly dialysed using 7.2 phosphate buffer of pH, and protein-maleimide of hydrazone bond connection is made；
4) it takes sulfydryl polymer prepared by step 2 to be dissolved in the phosphate buffer (PBS-5.5) of pH 7.2, is added to step
It reacts 4 hours in protein-maleimide amine aqueous solution obtained in rapid 3, is not joined after reaction solution is concentrated by molecular sieve removing
With the sulfydryl polymer and unreacted protein reacted, the protein-polymer conjugate of hydrazone bond connection is made.
According to a particular embodiment of the invention, the hydrazone bond in the protein-polymer conjugate of above-mentioned hydrazone bond connection passes through
Sodium cyanoborohydride reduction, can prepare stable protein-polymer conjugate.
In another aspect of the invention, the invention proposes a kind of protein-drug molecule coupling labeleds of building hydrazone bond connection
The method of object.According to a particular embodiment of the invention, preparation step is as follows:
1) it obtains protein and introduces aldehyde radical in the special sex modification of protein N-terminal, protein is placed in the phosphorus of pH 5.5
In phthalate buffer (PBS-5.5)；Wherein it is possible to be extracted by purchase, amalgamation and expression, from organism, the modes such as purification obtain
Take protein；
2) hydrazine-drug molecule is prepared, detailed process is shown in specific embodiment part；
3) it takes the hydrazine-drug molecule prepared in step 2 to be dissolved in the phosphate buffer (PBS-7.2) of pH 7.2, is added
It is then added in step 1 that reaction overnight, uses pH 7.2 in protein aldehyde radical solution obtained in 7.2 aniline hydrochloride solution of pH
Phosphate buffer is repeatedly dialysed, and the protein-drug molecule conjugate of hydrazone bond connection is made.
According to a particular embodiment of the invention, the hydrazone bond in the protein-drug molecule conjugate of above-mentioned hydrazone bond connection is logical
Sodium cyanoborohydride reduction is crossed, stable protein-drug molecule conjugate can be prepared.
According to a particular embodiment of the invention, be successfully prepared hydrazone bond connection human serum albumins-doxorubicin derivative,
Human serum albumins-doxorubicin derivative, human serum after human serum albumins-methotrexate (MTX) conjugate and hydrazone bond reduction
Albumin-methotrexate (MTX) conjugate.
In another aspect of the invention, the invention proposes a kind of protein of building hydrazone bond connection-imaging molecule couplings
The method of object.According to a particular embodiment of the invention, preparation step is as follows:
1) it obtains protein and introduces aldehyde radical in the special sex modification of protein N-terminal, protein is placed in the phosphorus of pH 5.5
In phthalate buffer (PBS-5.5)；Wherein it is possible to be extracted by purchase, amalgamation and expression, from organism, the modes such as purification obtain
Take protein；
2) hydrazine-imaging molecule is prepared, detailed process is shown in specific embodiment part；
3) it takes the hydrazine-imaging molecule prepared in step 2 to be dissolved in the phosphate buffer (PBS-7.2) of pH 7.2, is added
It is then added in step 1 that reaction overnight, uses pH 7.2 in protein aldehyde radical solution obtained in 7.2 aniline hydrochloride solution of pH
Phosphate buffer is repeatedly dialysed, and protein-imaging molecule conjugate of hydrazone bond connection is made.
According to a particular embodiment of the invention, the hydrazone bond in the protein-drug molecule conjugate of above-mentioned hydrazone bond connection is logical
Sodium cyanoborohydride reduction is crossed, stable protein-imaging molecule conjugate can be prepared.According to an embodiment of the invention, success
It is prepared for interferon-fluorescein conjugates.
Advantageous feature of the invention includes but is not limited to the following aspects:
1) protein-conjugate method for preparing involved in the present invention is two-step reaction, wherein second step azanol and hydrazine
The reaction of derivative can eliminate the amido modified side reaction of the non-N-terminal of the first step, be conducive to clear decorating site in protein
The position in activated centre；
2) protein-conjugate method is prepared involved in the present invention, reaction condition is mild, high conversion rate, and two steps are anti-
Answer total conversion 80% or more, the modification conversion ratio of part albumen is up to 90% or more.
3) protein-conjugate method for preparing involved in the present invention has wide design space, can be used for preparing
All kinds of protein-imaging molecule, protein-drug, protein-polymer, protein-protein/polypeptide, protein-are received
The conjugates such as rice material.
Detailed description of the invention
Fig. 1 is according to an embodiment of the present invention to prepare the whole route map of protein-conjugate；
Fig. 2 is the ESI mass spectrogram according to an embodiment of the present invention for preparing protein initiator
Fig. 3 is interferon according to an embodiment of the present invention-pyridine dicarbaldehyde-ABM enzymatic hydrolysis LC-MS spectrogram；
Fig. 4 is growth in situ protein in part according to an embodiment of the present invention-poly- oligomeric ethylene glycol methyl ether conjugate
SDS-PAGE glue figure；
Fig. 5 is growth in situ protein in part according to an embodiment of the present invention-poly- oligomeric ethylene glycol methyl ether conjugate GPC
Spectrogram；
Fig. 6 is interferon according to an embodiment of the present invention, interferon-aldehyde radical, interferon-initiator, the poly- oligomerization of interferon-
The active testing figure of ethylene glycol monomethyl ether；
Fig. 7 is the poly- oligomeric ethylene glycol methyl ether conjugate of interferon-of interferon according to an embodiment of the present invention, growth in situ
Pharmacokinetics spectrogram；
Fig. 8 is the how soft pyrrole star of human serum albumins-according to an embodiment of the present invention and how soft pyrrole astrocyte toxicity profile；
Fig. 9 is the ESI mass spectrogram of interferon-fluorescein conjugates according to an embodiment of the present invention；
Figure 10 is interferon according to an embodiment of the present invention, interferon aldehyde radical, interferon-aminooxy group polyethylene glycol conjugation object
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis figure；
Figure 11 is interferon according to an embodiment of the present invention, interferon aldehyde radical, interferon-mercapto-polyglycol conjugate ten
Sodium dialkyl sulfate-polyacrylamide gel electrophoresis figure.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings.Below with reference to
The embodiment of attached drawing description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.
The synthesis of 1 pyridine -2,6- dicarbaldehyde (PDA) of embodiment (compound shown in formula (I))
10 milliliters of Isosorbide-5-Nitrae-dioxane is added in 50 milliliters of round-bottomed flask, 0.42 gram 2,6- is added under agitation
Pyridine dimethanol and 0.33 gram of selenium dioxide, are stirred to react 24 hours at 65 DEG C.It filters, insoluble solids is removed, by reaction solution
It rotates to dry.Product silica gel chromatography, eluent are the methylene chloride/methanol of volume ratio 20:1, Rf=0.6.Product
For white solid, C7H5NO2, yield is 0.36 gram, yield 88.2%.1HNMR(400MHz,CDCl3):δ10.16(s,2H),
8.06-8.19(m,3H)。ESI-mass m/z:136.2([M+H]+)。
Embodiment 2
Protein is modified with pyridine -2,6- dicarbaldehyde and two step of hydroxylamination atom transfer radical polymerization initiator, and former
Position polymerization oligomerization polyethylene glycol constructs the poly- oligomeric ethylene glycol methyl ether conjugate of protein-, and protein shown in the present embodiment has:
Interferon, green fluorescent protein, myoglobins, RNA enzyme, bovine serum albumin(BSA), human serum albumins, glucose oxidase, day
Pollen protein etc..
The building of 2.1 protein is expressed
The construction and expression of 2.11 interferon
Using round pcr, fromIFN coded sequence is expanded in carrier T, passes through Nde I/Eco RI restriction enzyme site
It is inserted into pET-25b (+) carrier.The end N- of IFN gene order is connected with glycine there are three the connections of Nde I restriction enzyme site,
The end C- is connected with unique identification sequence LPETG and 6 × His label of SrtA, reconnects the restriction enzyme site of Eco RI, finally
Nucleotide sequence are as follows:
GGCGGTGGGTGTGATCTGCCTCAGACTCATTCTCTGGGTAGTCGTCGTACGCTGATGCTGCTGGCTCA
AATGCGCCGTATTAGCCTGTTTTCTTGCCTGAAAGATCGCCACGATTTTGGGTTTCCACAGGAAGAATTTGGCAAC
CAGTTCCAGAAAGCCGAAACAATTCCGGTACTGCACGAGATGATTCAACAAATCTTTAACCTGTTCAGCACCAAAG
ACTCTTCAGCTGCCTGGGATGAAACACTGCTGGACAAATTCTATACCGAGCTGTATCAGCAACTGAACGATCTGGA
GGCATGTGTTATTCAGGGTGTTGGTGTGACTGAAACTCCTCTGATGAAAGAGGATAGCATTCTGGCAGTCCGTAAA
TATTTTCAGCGTATCACACTGTATCTGAAAGAGAAAAAATATAGCCCGTGTGCCTGGGAAGTTGTTCGTGCCGAAA
TCATGCGCAGCTTTAGTCTGTCTACCAACCTGCAAGAGAGCCTGCGTTCTAAAGAAGGATCCGGTGGCGGTGGCTC
TCTGCCGGAAACCGGTGGCCACCATCATCATCATCAT(SEQ ID NO:1)。
Amino acid sequence is as follows after it is translated:
GGGCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFN
LFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWE
VVRAEIMRSFSLSTNLQESLRSKEGSGGGGSLPETGGHHHHHH(SEQ ID NO:2)。
The design of primers used is as follows:
Forward primer: GGAATTCcatatgGGCGGTGGGTGTGATCTGCCTCAGACTCAT (SEQ ID NO:3).
Reverse primer: CGgaattcTTATCAATGATGATGATGATG (SEQ ID NO:4).
After verifying by DNA sequencing, the plasmid of the coding G3-IFN-H6 of building is transformed into coli strain
In Rosetta-gami (DE3) pLysS competent cell, and 37 DEG C of overnight incubations in ammonia benzyl resistance LB culture medium.It will culture
Object, which is forwarded in the sterile ammonia benzyl resistance TB culture medium of 1L, to be continued to cultivate, and when bacterial suspension OD600 reaches 0.5, final concentration is added
It is induced for 500 μM of IPTG, overnight 18 DEG C of overexpressions.Cell is harvested by centrifugation, with 50mM TrisHCl, 150mM NaCl, pH
7.4 buffers are resuspended, sonicated cells in ice-water bath, and with 14,000 × g is centrifuged 10min and removes precipitating, is added in supernatant
2mL 1% (w/v) polyethyleneimine (PEI) is centrifuged again after mixing.Supernatant containing soluble protein is affine by nickel
Chromatographic column is purified by 10 system of AKTA Purifier.50mM TrisHCl, 500mM are used when balance columns
NaCl, 10% glycerol, 25mM imidazoles, 7.4 buffer of pH, elution foreign protein use 50mM TrisHCl, 500mM NaCl,
10% glycerol, 50mM imidazoles, pH7.4 buffer finally use 50mM TrisHCl, 500mM NaCl, 10% glycerol, 500mM
Imidazoles, the G3-IFN-H6 of 7.4 buffer of pH elution are simultaneously further purified by 26/10 desalting column of HiPrep, and 50mM is replaced into
Phosphate in 7.4 solution of 150mM NaCl, pH, is stored in -80 DEG C.IFN- is assessed by PAGE gel electrophoresis
LPETGGH6 purification process and purity.The concentration of albumen is determined by NanoDrop 2000.
It is 6 mg/mls that Nanodrop 2000UV/Vis method, which demarcates its concentration, and the yield of every liter of bacterium is 100 milligrams.It mentions
Freezen protective in the solution after pure.
The building of 2.2 green fluorescent proteins is expressed
The green fluorescent protein (GFP) used in this research is unmanifest original protein, according to standard practice instructions gram
Grand expression.Sample Song Jinwei intelligence company (GeneWiz) sequencing.Sequencing result are as follows:
MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLCYG
VQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY
NYNSHNVYIMADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMV
LLEFVTAAGITHGMDELYNVDHHHHHH(SEQ ID NO:5)。
It is 247 according to the amino acid number that sequencing result calculates, molecular weight 27978.44, isoelectric point (PI) is 5.94.
GFP used in this example is the GFP of this batch purification.Purified with AKTA protein purification instrument, is verified by DNA sequencing
Afterwards, the plasmid of the coding GFP of building is transformed into coli strain BL21 competent cell, and is cultivated in ammonia benzyl resistance LB
37 DEG C of overnight incubations in base.Culture is forwarded in the sterile ammonia benzyl resistance TB culture medium of 1L and continues to cultivate, bacterial suspension
When OD600 reaches 0.5, final concentration of 500 μM of IPTG induction is added, overnight 25 DEG C of overexpressions.Cell is harvested by centrifugation, uses 50mM
7.4 buffer of TrisHCl, 150mM NaCl, pH is resuspended, sonicated cells in ice-water bath, and with 14,000 × g is centrifuged
10min removes precipitating, is added after 2mL 1% (w/v) polyethyleneimine (PEI) is mixed in supernatant and is centrifuged again.It will contain solvable
Property albumen supernatant pass through nickel affinity chromatography column, purified by 10 system of AKTA Purifier.It is used when balance columns
50mM TrisHCl, 500mM NaCl, 10% glycerol, 25mM imidazoles, 7.4 buffer of pH, elution foreign protein use 50mM
TrisHCl, 500mM NaCl, 10% glycerol, 50mM imidazoles, pH7.4 buffer finally use 50mM TrisHCl, 500mM
NaCl, 10% glycerol, 500mM imidazoles, 7.4 buffer of pH elute GFP, and further pure by 26/10 desalting column of HiPrep
Change, be replaced into 50mM phosphate, in 7.4 solution of 150mM NaCl, pH, is stored in -80 DEG C.Purity passes through PAGE gel
Electrophoresis assessment.Demarcating its concentration with Nanodrop 2000UV/Vis method after protein purification is 13.5 mg/mls, every liter of bacterium
Yield be 150 milligrams.Freezen protective in the solution after purification.
The special sex modification pyridine -2,6- dicarbaldehyde of 2.3 protein N-terminals (protein-aldehyde radical) preparation
Various protein and the reaction condition of pyridine -2,6- dicarbaldehyde are consistent, and reaction condition is as follows: 150 μM of protein
Pyridine -2,6- dicarbaldehyde (PDA) (4mg/mL) of solution and 30mM are in 50mM phosphate, 150mM NaCl, 10mM EDTA, pH
It is reacted 16 hours in 7.4 phosphate buffer in 25 DEG C of standings.50mM phosphate, 150mM NaCl, pH are used after reaction
Repeatedly dialysis obtains the product (protein-CHO) for being connected to PDA molecule to 5.5 phosphate buffer (PBS-5.5).
Interferon-aldehyde radical, green fluorescent protein-aldehyde radical, myoglobins-aldehyde are prepared for using this method in the embodiment of the present invention
Base, RNA enzyme-aldehyde radical, bovine serum albumin(BSA)-aldehyde radical etc..
The preparation of 2.4 protein-initiator
The protein aldehyde radical (protein-CHO) for being connected to PDA molecule is reacted with ABM can connect one on protein
Protein is made into macromolecular ATRP initiator by a small molecule ATRP initiator.
ABM chemical structural formula is
150 μM protein aldehyde radical (protein-CHO) solution, solvent are 50mM phosphate, 150mM NaCl, pH 5.5
Phosphate buffer (PBS-5.5).Take above-mentioned solution 1mL that 1.6mg ABMHCl is added, 25 DEG C are reacted 16 hours.Reaction knot
Shu Houyong PBS (150mM NaCl, pH 7.4) dialysis (dialysis membrane model 131276, Spectrum, molecular cut off 8-
10kDa), protein initiator (protein-Br) is obtained.
All proteins are all made of above-mentioned reaction condition with reacting for ABM in present invention implementation 1.
Interferon initiator, green fluorescent protein initiator, myoglobins are prepared for using this method in the embodiment of the present invention
Initiator, RNA enzyme initiator, bovine serum albumin(BSA) initiator.
The specificity of protein N-terminal modification, Fig. 2 are verified in the embodiment of the present invention by Q-tof mass spectrum and LC-MS
ESI mass spectrogram is modified for the partially protein pyridine dicarbaldehyde, two step of ABM of the present embodiment, can prove pyridine diformazan from Fig. 2
Aldehyde may be coupled on three kinds of protein (GFP, IFN, myoglobins Mb), be shown one on most of protein modification in mass spectrogram
A pyridine dicarbaldehyde molecule has in the mass spectrogram of GFP protein the protein peak for having modified two molecule pyridine dicarbaldehydes on a small quantity to deposit
, second step ABM modification after, in proteomic image figure only it is visible modification one molecule ABM protein peak, show that second step is anti-
It should can eliminate the by-product of first step reaction.Furthermore G3-IFN is after pyridine dicarbaldehyde and ABM modification, by Trypsin
It is protein N-terminal that LC-MS spectrogram after enzymatic hydrolysis, which demonstrates decorating site, and Fig. 3 is to digest after two step of G3-IFN modifies PDA, ABM
The second order ms figure of the N-terminal segment of generation, N- terminal sequence are GGGCDLPQTHSLGSR, and decorating molecule amount increases part and is considered as
C13H13Br N2O3, the second order ms of N-terminal segment and notional result are coincide.Fig. 2 and Fig. 3 demonstrates two step of N-terminal-PDA and ABM
Decorating site is unique and is protein N terminal.And other aldehyde radical protein modifiers are while modifying protein N-terminal amino,
The amino on lysine side-chain can be also modified, reference can be made to Jentoft N, Dearborn D G.Labeling of proteins
by reductive methylation using sodium cyanoborohydride[J].Journal of
Biological Chemistry, 1979,254 (11): 4359-4365, and the method for modifying that the present invention is shown then be not present it is non-
Special sex modification shows better locus specificity.
In addition, using pyridine dicarbaldehyde and three to verify the annulation process that pyridine dicarbaldehyde is reacted with protein N terminal
The nuclear magnetic spectrum of glycine derivative reaction product demonstrates pyridine dicarbaldehyde and reacts the imines meeting to be formed with protein or small peptide
With second amino amino amide addition, an imidazolidinone five-membered ring is formed.NMR(400MHz,CDCl3) δ=9.94 (s,
1H), 8.11-7.79 (m, 3H), 5.69 (s, 1H), 4.17-4.12 (m, 2H), 3.49 (d, 2H), 3.39-3.35 (m, 2H),
3.12-3.09(m,2H),1.19(s,3H).
2.5 protein-poly- oligomeric ethylene glycol methyl ether preparation and purifying
Protein initiator (protein-Br) is diluted to 50 μM with PBS, above-mentioned solution 1mL is taken to be transferred to 10mL reaction
Guan Zhong is added OEGMA (number-average molecular weight 500) 50 μ L, high pure nitrogen will be passed through in reaction tube and is at the uniform velocity bubbled 15 minutes.In 10 millis
Rise the CuCl aqueous solution 370 μ L that 1mg/mL is added in rub oral examination tube, 1, Isosorbide-5-Nitrae, 7,10,10- hexamethyl triens
(HMTETA) above-mentioned liquor capacity is supplied 1mL with deionized water by 3.1 μ L, and high pure nitrogen will be above-mentioned after being at the uniform velocity bubbled 15 minutes
Solution shifts syringe needle by two-way solvent and is transferred in protein-Br solution.Air is passed through after being stirred to react 4 hours at 4 DEG C to quench
It goes out reaction.
The ATRP polymerization in situ of protein-pOEGMA is all made of above-mentioned reaction condition in the embodiment of the present invention, is prepared for doing
Disturb element-pOEGMA, green fluorescent protein-pOEGMA, myoglobins-pOEGMA, RNA enzyme-pOEGMA, bovine serum albumin(BSA)-
Protein-pOEGMA the conjugate such as pOEGMA.
Fig. 4 is part growth in situ protein-poly- oligomeric ethylene glycol methyl ether conjugate SDS- of the embodiment of the present invention
PAGE glue figure, the glue figure demonstrate the successful preparation and purification of this few proteinoid-polymer conjugates.
Fig. 5 is that part growth in situ protein-poly- oligomeric ethylene glycol methyl ether conjugate GPC of the embodiment of the present invention is composed
Figure, has been also demonstrated that the successful preparation and purification of this few proteinoid-polymer conjugates, while also to the molecular weight of conjugate
Size and distribution are made that quantitative result.
The active testing of 2.6 protein, protein conjugate
The measurement of the Bioactivity of 2.61 interferon (IFN) and its modified outcome
The cytotoxicity of IFN, IFN-CHO, IFN-Br and IFN-POEGMA are measured with mtt assay.Select logarithm growth
Burkitt ' the s B lymphoma cell (Daudi B) of phase, each hole is implanted into (the 50 microlitres of cultures of 5000 cells in 96 orifice plates
Base), only to add 100 microlitres of culture mediums, not celliferous hole is as background.After 37 DEG C are cultivated 5 hours, it is dilute that 50 microlitres of gradients are added
IFN, IFN-CHO, IFN-Br and IFN-POEGMA solution released, the drug of each concentration are added three holes and do parallel laboratory test, add
Enter the hole of physiological saline as control.10 microlitres of MTS reagents are added in each hole after culture 96 hours, in cell incubator culture 3
96 orifice plates are taken out after hour, the absorption data under 495 nanometers are read on microwell plate plate reader.The data software of acquirement
The processing of GraphPad Prism 5.0, half maximum suppression concentration (IC50) also with the software the Fitting Calculation, data result is with average
The form of value ± standard deviation provides.
Half maximum suppression concentration (the IC of IFN, IFN-CHO, IFN-Br and IFN-POEGMA50) it is respectively as follows: 2.46 ±
0.29pM,2.64±0.23pM,2.56±0.29pM,4,19±0.16pM.Specific map is shown in attached drawing 6.
The determination of activity of 2.62 myoglobins and its modified outcome
Using the kaliumphosphate buffer of 10mMpH7.0 as solvent, it is pre-configured with guaiacol solution (20mM) and peroxidating
Hydrogen solution (19.4mM), myoglobins and its modified outcome solution (myoglobin concentration 0.17mg/mL).100 μ L guaiaci lignum
Phenol solution and the mixing of 100 μ L hydrogenperoxide steam generators, are added 10 μ L myoglobin solutions, different time points exist when measuring 25 DEG C
The absorption of 470nm, record is primary within every 15 seconds, and minute is 5 minutes, with 100 μ L guaiacol solution and 100 μ L hydrogen peroxide
The mixed solution of the Tris buffer of solution mixing and 10 μ L is as blank control.To the data obtained (absorbance-time change
Curve) linear fit is carried out, the slope of curve is directly proportional to protein active, with original myoglobins activity for 100%, calculates other
The relative activity of modified outcome.
The relative activity of MB, MB-CHO, MB-Br and MB-POEGMA are respectively as follows: 100 ± 2%, 89.1 ± 2.3%, 87.4
± 0.7%, 90.4 ± 1%.It specifically the results are shown in Table 1.
The determination of activity of 2.63 ribonuclease As (RNaseA) and its modified outcome
Using the Tris buffer of 50mMpH7.0 as solvent, ribonuclease A and its modified outcome are diluted to RNaseA
Concentration is 0.15mg/mL, prepares 2 ' 3 '-ring of cytidine phosplate (CP) solution, concentration 0.5mg/mL in advance.By 10 μ L's
RNaseA or modified outcome solution are added in cytidine 2 ' 3 '-ring phosplate solution of 190 μ L, measure it in 290nm difference
The absorbance at time point, measuring temperature are 25 DEG C, and minute is 5 minutes, data of every 15 seconds records.The cytidine of 190 μ L
The Tris buffer of 10 μ L is added as blank control in 2 ', 3 '-ring phosplate solution.By the data obtained, (absorbance-time becomes
Change curve) linear fit is carried out, the slope of curve is directly proportional to enzymatic activity, with the enzymatic activity of original RNase A for 100%, calculates
The relative activity of other modified outcomes.
The relative activity of RNASEA, RNASEA-CHO, RNASEA-Br and RNASEA-POEGMA is respectively as follows: 100 ±
3.7%, 96.8 ± 3.2%, 103.8 ± 4.7%, 95.4 ± 4.4%.It specifically the results are shown in Table 1.
The determination of activity of 2.64 green fluorescent proteins and its modified outcome
Within the scope of a certain concentration, the fluorescence intensity and solution concentration of GFP is directly proportional, therefore measures the fluorescence of GFP
Activity can use calibration curve method.Each 2.5 are added in the hole of 96 orifice plates, 5,10,20 microlitres of GFP standard solution (1mg/
ML), 200 microlitres are supplied with PBS, the hole of 200 microlitres of PBS solutions is added as background.It is measured at 25 DEG C with microwell plate plate reader
Excitation wavelength 460nm, the fluorescence intensity of launch wavelength 525nm carry out line with microsoft Excel 2010 after background correction
Property fitting, obtain standard curve, while doing standard curve, measure the fluorescence of sample to be tested, and by standard curve, calculate
The relative intensity of fluorescence of each step reaction product containing identical centinormal 1 GFP and original GFP standard solution, with original
The fluorescence intensity of GFP standard solution is as 100%, and measurement result shows the N-terminal site-specific sex modification for the GFP that PDA is mediated, respectively
The fluorescence of step reaction product does not change.
The relative activity of GFP, GFP-CHO, GFP-Br and GFP-POEGMA is respectively as follows: 100 ± 10.5%, 97.1 ±
9.1%, 98.4 ± 10%, 99.6 ± 11.2%.It specifically the results are shown in Table 1.
Table 1:
| Activity | Original protein (%) | Protein-aldehyde radical (%) | Protein initiator (%) | Protein-POEGMA (%) |
| Green fluorescent protein | 100±10.5 | 97.0±9.1 | 98.4±10.0 | 99.6±11.2 |
| Glucose oxidase | 100±12.4 | 99.0±1.6 | 101.9±1.0 | 90.8±2.8 |
| Interferon | 100±5.6 | 60.0±6.0 | 62.2±10.9 | 56.3±10.3 |
| RNA enzyme | 100±3.7 | 96.8±3.2 | 103.8±4.7 | 95.4±4.4 |
| Myoglobins | 100±2.0 | 89.1±2.3 | 87.4±0.7 | 90.4±1.0 |
The pharmacokinetics test of 2.7 interferon, interferon conjugate
Female BAl BIc/c nude mice (6 week old) is randomly divided into 5 groups (3/group), and passes through tail vein injection 1mg/kg weight
IFN-α and IFN-POEGMA conjugate.In suitable time point (1,5,15,30min, 1,3,6,24,48,72 and 96h), tail
About 20 μ L of venous blood collection (collection tube impregnates drying with heparin sodium in advance), is centrifuged at 4000 × g after standing 30min at 4 DEG C
15min collects blood plasma and is stored in -80 DEG C.Using the concentration of IFN-α 2ELISA kit measurement IFN, while deducting and not injecting
The background value of the mice plasma of drug.Data are analyzed and are handled by 3.0 software of DAS.Data show that interferon-is poly- few
Methoxypolyethylene glycol conjugate and interferon are respectively 41.3 hours and 1.83 hours, interferon in the intracorporal t1/2 β (h) of mouse
The blood removing half-life period of conjugate is 22.6 times of interferon.The poly- oligomerization second of interferon-of specific interferon, growth in situ
The pharmacokinetics spectrogram of glycol methyl ether conjugate is shown in attached drawing 7.
3 liang of class human serum albumins-drug molecule conjugate preparation characterizations of embodiment
The preparation of 3.1 human serum albumins-methotrexate (MTX)
The preparation of 3.11 human serum albumins-aldehyde radical
The pyridine dicarbaldehyde (PDA) (4mg/mL) of 150 μM of HSA protein solution and 30mM in 50mM phosphate,
It is reacted 16 hours in the phosphate buffer of 150mMNaCl, 10mM EDTA, pH 7.4 in 25 DEG C of standings.It uses after reaction
50mM phosphate, repeatedly dialysis obtains being connected to PDA molecule the phosphate buffer (PBS-5.5) of 150mM NaCl, pH 5.5
Product (HSA-CHO).
3.1.2 methotrexate (MTX)-hydrazine derivate preparation
Dry methotrexate (MTX) (MTX) (400mg, 0.88mmol) is dissolved in the DMF (6.0mL) of no water degasification, is added
HOTU (240mg, 1.76mmol) is added in EDC (338mg, 1.76mmol), and reactant is stirred 2 minutes.Then by hydrazine hydrate
(44mg, 0.88mmol) is added in clear red solution, and reaction is protected from light lower stirring 36h in environment temperature.It will reaction
Liquid precipitates three times in ether/acetone that volume ratio is 3/1, generates orange-yellow thick solid, places it in vacuum drying oven
Overnight, generating 139mg (37%) orange/yellow solid is gained methotrexate (MTX)-hydrazine.1H NMR(400MHz,(CD3)2SO) δ=
12.12 (s, 1H), 10.12-9.96 (m, 1H), 8.59 (d, 1H, J=1.6Hz), 8.27-8.19 (m, 1H), 7.80-7.73 (m,
3H), 6.83 (d, 2H, J=9.2Hz), 6.74 (s, 2H), 6.09-5.99 (m, 1H), 4.80 (s, 2H), 4.34 (s, 1H), 3.22
(s,1H),2.09-1.70(m,4H).
3.1.3 human serum albumins-methotrexate (MTX) conjugate preparation
100 μM of HSA- aldehyde radical that upper step obtains and 500 μM of methotrexate (MTX)s (MTX) are in 50mM phosphate, 150mM NaCl,
Reaction is stood in the buffer of pH7.4, while aniline hydrochloride catalysis is added, and after reaction 16 hours, the cyano boron hydrogen of 20mM is added
Change sodium (NaBH3CN) in 4 DEG C standing reaction response 16 hours, the buffer with 50mM phosphate, 150mM NaCl, pH 7.4 is more
Secondary dialysis obtains human serum albumins-methotrexate (MTX) conjugate.
3.2 human serum albumins-how soft pyrrole star preparation and characterization
The preparation of 3.21 human serum albumins-aldehyde radical
With described in 3.11
The preparation of 3.22 human serum albumins-hydrazine
150 μM of HSA-CHO and the 1mg/mL adipic dihydrazide (Adipic dihydrazide) that upper step obtains exists
50mM phosphate is added 20mM's in the phosphate buffer of 150mM NaCl, pH 5.5 after 4 DEG C of standings are reacted 16 hours
Sodium cyanoborohydride (NaBH3CN) reacts 16 hours in 4 DEG C of standings, uses 50mM phosphate, 150mM NaCl, pH after reaction
7.4 buffer repeatedly dialyses and obtains stable HSA-NHNH2。
The preparation of 3.23 human serum albumins-how soft pyrrole star
100 μM of the HSA-NHNH that upper step obtains2With 500 μM of adriamycins (DOX) in 50mM phosphate, 150mM NaCl,
Reaction is stood in the buffer of pH7.4, while aniline hydrochloride catalysis is added, and uses 50mM phosphate, 150mM after reaction 16 hours
The buffer of NaCl, pH7.4, which are repeatedly dialysed, obtains the HSA-DOX for the aminoterminal that adriamycin is connected to HSA.
3.24 human serum albumins-how soft pyrrole star activity characterization
The cytotoxicity of HSA-DOX and DOX is measured with mtt assay.Select the 4T1 breast cancer cell of logarithm growth stage
(4T1), each hole is implanted into 5000 cells (50 microlitres of culture mediums) in 96 orifice plates, only to add 100 microlitres of culture mediums, without thin
The hole of born of the same parents is as background.After 37 DEG C are cultivated 5 hours, HSA-DOX the and DOX solution of 50 microlitres of gradient dilutions, each concentration is added
Drug three holes be added do parallel laboratory test, the hole of physiological saline is added as control.Each hole is added 10 after culture 96 hours
Microlitre MTS reagent takes out 96 orifice plates after cell incubator culture 3 hours, reads under 495 nanometers on microwell plate plate reader
Absorption data.The data of acquirement are handled with software GraphPad Prism 5.0, half maximum suppression concentration (IC50) also use and be somebody's turn to do
Software the Fitting Calculation.
The IC of DOX and HSA-DOX50It is 0.435 μM and 5.07 μM respectively.
The how soft pyrrole astrocyte toxicity profile of human serum albumins-is shown in attached drawing 8.
The building of 4 interferon of embodiment-fluorescent molecule conjugate
The preparation of 4.1 interferon-aldehyde radical
The step is identical as interferon-aldehyde radical preparation method described in embodiment 2 2.2.
The preparation of 4.2 fluoresceins-hydrazine derivate
Dry fluorescein isothiocynate (FITC) (38.9mg, 0.1mmol) is dissolved in (6.0mL) acetonitrile, carbon is added
Sour potassium (27.6mg, 0.3mmol) simultaneously stirs reactant 2 minutes.Then hydrazine hydrate (5mg, 0.1mmol) is added to solution
In, and reaction is protected from light lower stirring 12h in environment temperature.Reaction solution is filtered to remove excessive potassium carbonate, it, will after selecting dry acetonitrile
Orange/yellow solid is dissolved in deionized water, and it is 5 that hydrochloric acid is added into solution to pH, generates a large amount of orange-yellow insoluble matters, 3000rpm
Centrifugation after ten minutes, discards supernatant, and precipitating is taken to place it in vacuum drying oven overnight, generates 16mg (40%) orange/yellow solid and is
Gained fluorescein-hydrazine.
The preparation of 4.3 interferon-fluorescein conjugates
100 μM of the IFN- aldehyde radical and 500 μM of fluorescein hydrazines that upper step obtains, in 50mM phosphate, 150mM NaCl, pH
Reaction is stood in 7.4 buffer, while aniline hydrochloride catalysis is added, and uses 50mM phosphate, 150mM after reaction 16 hours
The buffer of NaCl, pH 7.4, which is repeatedly dialysed, obtains interferon-fluorescein conjugates of hydrazone bond connection.
Sodium cyanoborohydride (the NaBH of 20mM is added into interferon-fluorescein conjugates3CN) in 4 DEG C of standing reactions 16
Hour, 50mM phosphate is used after reaction, and the buffer of 150mM NaCl, pH 7.4, which is repeatedly dialysed, obtains stable interference
Element-fluorescein conjugates.Interferon-fluorescein conjugates ESI mass spectrogram is shown in attached drawing 9.
5 interferon pyridine dicarbaldehyde conjugate of embodiment grafting azanol tetraethylene glycol prepares interferon-polyethylene glycol conjugation
Object
The preparation of 5.1 azanol tetraethylene glycols
The preparation route of azanol tetraethylene glycol is as follows:
Itself specific steps are as follows:
5 grams of polyethylene glycol (molecular weight 5000) are dissolved in 20 milliliters of acetonitriles, and 0.52 gram of triphenylphosphine, ice bath is added in stirring
Cooling.0.66 gram of carbon tetrabromide is dissolved in 22 milliliters of acetonitriles, is added dropwise at 0 DEG C, is added dropwise within 3 hours.Ice bath is removed, reaction temperature exists
Room temperature is to slowly warm up in 3 hours.It is warming up to 40 DEG C and is kept stirring 18 hours.Revolving remove acetonitrile, be added 40 milliliters of water and
40 milliliters of n-hexanes are heated to reflux 30 minutes., there is white precipitate in cooling.Filtering, revolving remove n-hexane.200 milliliters of dichloros
Methane extracts 3 times, and anhydrous magnesium sulfate is dry.Polyethylene glycol bromine is obtained after being spin-dried for solvent.
5 grams of polyethylene glycol bromines and 0.27 gram of N-Boc- azanol are dissolved in 20 milliliters of ethyl alcohol, are added 0.8 gram of potassium hydroxide, and 60 DEG C
Reaction 2 hours.It is filtered after cooling, revolving removes solvent, and residue is white solid.5ml methylene chloride is dissolved the residue in, in
500ml cold ether China precipitating, centrifuging and taking precipitating vacuum drying, obtains white solid, Boc- aminooxy group tetraethylene glycol.
Above-mentioned gained white solid 3g is taken, the Hydrochloride/ethyl acetate 10ml of 7M is added, is reacted 4 hours at room temperature
Afterwards, rotary evaporation ethyl acetate and hydrogen chloride are added a small amount of methylene chloride dissolution, precipitate in 200ml ice ether, filtering obtains
Fluffy white powder dries ether in vacuum drying oven, obtains final product aminooxy group poly glycol monomethyl ether.
NMR(400MHz,CDCl3) δ=10.57 (s, 1H), 4,20 (t, 2H, J=4.8Hz), 3.80 (t, 4H, J=
4.8Hz), 3.69-3.51 (m, 460H), 3.44 (t, 4H, J=4.8Hz), 3.36 (s, 3H)
The preparation of 5.2 interferon-aldehyde radical
The step is identical as interferon-aldehyde radical preparation method described in embodiment 2 2.3.
The preparation of 5.3 interferon-polyethylene glycol conjugation object
150 μM interferon aldehyde radical (protein-CHO) solution, solvent are 50mM phosphate, 150mM NaCl, pH 5.5
Phosphate buffer (PBS-5.5).Take above-mentioned solution 1mL that 30.5mg aminooxy group polyethylene glycol is added, 25 DEG C are reacted 16 hours.
Attached drawing 10 is interferon, interferon aldehyde radical, interferon-polyethylene glycol conjugation object sodium dodecyl sulfate-polypropylene acrylamide gel
Electrophoretogram.
6 interferon of example prepares interferon-by the maleimide-functionalised connexon grafting sulfydryl polymer of azanol-and gathers
Close object conjugate
6.1 prepare sulfydryl polymer by reversible addion-fragmentation chain transfer polymerization (RAFT)
The preparation of the poly- oligomeric ethylene glycol monomethyl ether (POEGMA-SH) of 6.11 sulfydryls
Take oligomeric ethylene glycol monomethyl ether metacrylic acid ester 128mg, chain transfer agents 4- cyano -4- (phenyl formyl
Sulfenyl) valeric acid 0.838mg, initiator azodiisobutyronitrile 0.1mg is dissolved in 1mlDMF, and deoxidation is removed by 5 Frozen-thawed cycleds
Gas is placed reaction liquid into 65 DEG C of oil bath pans and is reacted 12 hours.After reaction, pass through retention point in 20% ethanol water
The semi-permeable membrane that son amount is 3000, which is repeatedly dialysed, removes unreacted small molecule.High molecular molecular weight and polydispersity are used by GPC
PEG internal standard method calculates, and after the completion of dialysis, Polymer Solution is lyophilized, and the gained poly- oligomeric ethylene glycol monomethyl ether of 60mg is taken to be dissolved in 1ml
In ionized water, under nitrogen atmosphere protection, ethylenediamine 0.18mg is added into solution, repeatedly dialyses and removes in water after ambient temperature overnight
Remove the ethylenediamine i.e. gained poly- oligomeric ethylene glycol monomethyl ether of sulfydryl.
The poly- 2- methacryloxyethyl Phosphorylcholine (PMPC-SH) of 6.12 sulfydryls
Take methacryloxyethyl Phosphorylcholine 130mg, chain transfer agents 4- cyano -4- (phenyl formyl sulphur
Base) valeric acid 0.838mg, initiator azodiisobutyronitrile 0.1mg is dissolved in 1ml DMF, and deoxidation is removed by 5 Frozen-thawed cycleds
Gas is placed reaction liquid into 65 DEG C of oil bath pans and is reacted 12 hours.After reaction, pass through retention point in 20% ethanol water
The semi-permeable membrane that son amount is 3000, which is repeatedly dialysed, removes unreacted small molecule.High molecular molecular weight and polydispersity are used by GPC
PEG internal standard method calculates, and after the completion of dialysis, Polymer Solution is lyophilized, and takes gained 60mg polymethyl trimethylammonium phosphinylidyne gallbladder
Ethylenediamine 0.18mg is added into solution, repeatedly dialyses in water after ambient temperature overnight and removes second two under nitrogen atmosphere protection for alkali
Amine is the gained poly- oligomeric ethylene glycol monomethyl ether of sulfydryl.
The preparation of 6.2 azanols-maleimide-functionalised connexon
The preparation route of the maleimide-functionalised connexon of azanol-is as follows:
The specific steps are that:
15.54 milliliters of tetraethylene glycols are dissolved in 200 milliliters of acetonitriles, stirring, and 44.3 grams of triphenylphosphines, ice bath cooling is added.
56.0 grams of carbon tetrabromides have been dissolved in 220 milliliters, are added dropwise at 0 DEG C, are added dropwise within 3 hours.Ice bath is removed, reaction temperature is small 3
When interior be to slowly warm up to room temperature.It is warming up to 40 DEG C and is kept stirring 18 hours.Revolving removes acetonitrile, and 400 milliliters of water and 400 are added
Milliliter n-hexane, is heated to reflux 30 minutes., there is white precipitate in cooling.Filtering, revolving remove n-hexane.200 milliliters of methylene chloride
Extraction 3 times, anhydrous magnesium sulfate are dry.Silica gel column purification (ether/ethyl acetate=3:1).22.83 grams of product, yield 89%.1H
NMR(400MHz,CDCl3): δ 3.81 (t, 4H), 3.67 (s, 8H), 3.46 (t, 4H).
16 grams of 1,11- bis- bromo- 3,6,9- trioxaundecanes and 14 grams of N-Boc- azanols are dissolved in 100 milliliters of ethyl alcohol, are added
6.3 grams of potassium hydroxide, 60 DEG C are reacted 2 hours.It is filtered after cooling, revolving removes solvent, and residue is yellow oily.Silicagel column
(EA/DCM/MeOH=10:10:1).1H NMR(400MHz,CDCl3): δ 7.78 (s, 1H), 4.00-4.12 (m, 4H), 3.66-
3.72 (m, 12H), 1.47 (s, 9H).
Triphenylphosphine is added in super dry tetrahydrofuran, after being stirred 15 minutes in ice bath, DIAD is added, is again stirring for 15 points
Clock is subsequently to added into above-mentioned gained yellow liquid and maleimide solid is added directly into reaction liquid after stirring 15 minutes,
Hereafter solid gradually dissolves, and reaction is placed in ambient temperature overnight reaction, back spin dry reaction liquid silica gel column purification (methylene chloride/acetic acid
Ethyl ester=5:3).Yellow oily substance is obtained,1HNMR(400MHz,CDCl3): δ=11.04 (s, 2H), 6.78 (s, 2H), 4.47
(t, 2H, J=4.0Hz), 3.95 (t, 2H, J=2.4Hz), 3.90 (t, 2H, J=4.8Hz), 3.79-3.77 (m, 2H), 3.71
(t, 2H, J=5.2Hz), 3.67-3.64 (m, 6H)
Gained yellow oily substance 300mg is taken, the Hydrochloride/ethyl acetate 10ml of 3M is added, reacts 15 points at room temperature
Zhong Hou, rotary evaporation ethyl acetate and hydrogen chloride in ice-water bath are added a small amount of methylene chloride and are dissolved in rotate in ice-water bath and steams
Hair, is precipitated in 200ml ice ether, and centrifugation obtains yellow viscous liquid, and ether is dried in vacuum drying oven, obtains final product ammonia
Oxygroup polyethylene glycol maleimide (aminooxy group-maleimide linker).1H NMR(400MHz,CDCl3): δ=7.62
(s,1H),6.73(s,2H),4.05-4.03(m,2H),3.77-3.62(m,14H).
6.3 prepare interferon-pyridine dicarbaldehyde conjugate (interferon-aldehyde radical)
The step is identical as interferon-aldehyde radical preparation method described in embodiment 2 2.2, yield 95%.
6.4 prepare interferon-maleimide conjugate
150 μM protein aldehyde radical (IFN-CHO) solution, solvent are 50mM phosphate, the phosphorus of 150mM NaCl, pH 5.5
Phthalate buffer (PBS-5.5).Above-mentioned solution is taken to be added the maleimide-functionalised connexon of azanol-of 30 times of molar ratios, 25
DEG C reaction 16 hours.Dialysed after reaction with PBS (150mM NaCl, pH 7.4) (dialysis membrane model 131276,
Spectrum, molecular cut off 8-10kDa), obtain interferon-maleimide conjugate.Yield is 80%.
6.5 prepare interferon-sulfydryl polymer conjugates
150 μM interferon aldehyde radical (protein-CHO) solution, solvent are 50mM phosphate, 150mM NaCl, pH 745
Phosphate buffer (PBS-7.4).Take above-mentioned solution 1mL that mg sulfydryl polymer (the poly- oligomeric ethylene glycol list first of sulfydryl is added
Ether), 25 DEG C are reacted 8 hours.Interferon-sulfydryl polymer conjugates yield is 35%, attached drawing 11.
7 interferon of embodiment prepares interferon-by the maleimide-functionalised connexon grafting sulfydryl polymer of hydrazine-and gathers
Close object conjugate
7.1 prepare sulfydryl polymer by reversible addion-fragmentation chain transfer polymerization (RAFT)
The step is identical as sulfydryl method for producing polymer described in 6.3 in embodiment 6.
7.2 prepare interferon-pyridine dicarbaldehyde conjugate (interferon-aldehyde radical)
Various protein are consistent with the reaction condition of pyridine dicarbaldehyde, and reaction condition is as follows: 150 μM of protein solution with
The pyridine dicarbaldehyde (PDA) (4mg/mL) of 30mM is in 50mM phosphate, the phosphate of 150mM NaCl, 10mM EDTA, pH 7.4
It is reacted 16 hours in buffer in 25 DEG C of standings.50mM phosphate, the phosphate of 150mM NaCl, pH 7.4 are used after reaction
Repeatedly dialysis obtains the product (protein-CHO) for being connected to PDA molecule to buffer (PBS-5.5).Yield is 95%.
7.3 prepare interferon-maleimide conjugate
150 μM protein aldehyde radical (IFN-CHO) solution, solvent are 50mM phosphate, the phosphorus of 150mM NaCl, pH 5.5
Phthalate buffer (PBS-5.5).Above-mentioned solution is taken to be added the maleimide-functionalised connexon of azanol-of 30 times of molar ratios, 25
DEG C reaction 16 hours.It is dialysed after reaction with PBS (150mM NaCl, pH 7.4).Protein solution after taking dialysis is added
The sodium cyanoborohydride of 5 times of molar ratios is dialysed with PBS (150mM NaCl, pH 7.4) after reaction in 4 DEG C of reaction 10h.
(dialysis membrane model 131276, Spectrum, molecular cut off 8-10kDa) obtains interferon-maleimide conjugate.It receives
Rate is 80%.
7.4 steps are identical as interferon-sulfydryl polymer conjugates preparation method described in embodiment 6 6.5
Interferon-sulfydryl polymer conjugates yield is 35%.
8 bovine serum albumin(BSA) of embodiment prepares bovine serum albumin(BSA)-polymer idol by cysteine initiator derivative
Join object
The preparation of 8.1 bovine serum albumin(BSA)s-aldehyde radical
The step is identical as protein-aldehyde radical preparation method described in embodiment 2 2.3, yield 95%.
The preparation of 8.2 bovine serum albumin(BSA)s-initiator
150 μM bovine serum albumin(BSA) aldehyde radical (BSA-CHO) solution, solvent are 50mM phosphate, 150mM NaCl, pH
7.4 phosphate buffer (PBS-7.4).Take above-mentioned solution 1mL that 1.2mg formula (14) cysteine initiator is added, 25 DEG C anti-
It answers 16 hours.With PBS (150mM NaCl, pH 7.4) dialysis, (dialysis membrane model 131276, Spectrum is cut after reaction
Stay molecular weight 8-10kDa), obtain bovine serum albumin(BSA) initiator (BSA-Br), yield 75%.
The preparation of 8.3 bovine serum albumin(BSA)s-polymer conjugates
The step is identical as protein-polymer conjugate preparation method described in embodiment 2 2.5, yield 80%.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
SEQUENCE LISTING
<110>Tsinghua University
<120>preparation method of the protein conjugate based on pyridine dicarbaldehyde
<130> PIDC3181312
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 561
<212> DNA
<213> Artificial
<220>
<223>in embodiment 2 2.11 interferon nucleotide sequence
<400> 1
ggcggtgggt gtgatctgcc tcagactcat tctctgggta gtcgtcgtac gctgatgctg 60
ctggctcaaa tgcgccgtat tagcctgttt tcttgcctga aagatcgcca cgattttggg 120
tttccacagg aagaatttgg caaccagttc cagaaagccg aaacaattcc ggtactgcac 180
gagatgattc aacaaatctt taacctgttc agcaccaaag actcttcagc tgcctgggat 240
gaaacactgc tggacaaatt ctataccgag ctgtatcagc aactgaacga tctggaggca 300
tgtgttattc agggtgttgg tgtgactgaa actcctctga tgaaagagga tagcattctg 360
gcagtccgta aatattttca gcgtatcaca ctgtatctga aagagaaaaa atatagcccg 420
tgtgcctggg aagttgttcg tgccgaaatc atgcgcagct ttagtctgtc taccaacctg 480
caagagagcc tgcgttctaa agaaggatcc ggtggcggtg gctctctgcc ggaaaccggt 540
ggccaccatc atcatcatca t 561
<210> 2
<211> 187
<212> PRT
<213> Artificial
<220>
<223>in embodiment 2 2.11 interferon amino acid sequence
<400> 2
Gly Gly Gly Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg
1 5 10 15
Thr Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys
20 25 30
Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn
35 40 45
Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln
50 55 60
Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
65 70 75 80
Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn
85 90 95
Asp Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro
100 105 110
Leu Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
115 120 125
Ile Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu
130 135 140
Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu
145 150 155 160
Gln Glu Ser Leu Arg Ser Lys Glu Gly Ser Gly Gly Gly Gly Ser Leu
165 170 175
Pro Glu Thr Gly Gly His His His His His His
180 185
<210> 3
<211> 43
<212> DNA
<213> Artificial
<220>
<223>2.11 in embodiment 2 in forward primer
<400> 3
ggaattccat atgggcggtg ggtgtgatct gcctcagact cat 43
<210> 4
<211> 29
<212> DNA
<213> Artificial
<220>
<223>2.11 in embodiment 2 in reverse primer
<400> 4
cggaattctt atcaatgatg atgatgatg 29
<210> 5
<211> 247
<212> PRT
<213> Artificial
<220>
<223>2.2 in embodiment 2 used in green fluorescent protein sequencing result
<400> 5
Met Ala Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Cys Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Arg His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Val Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Asn Val
225 230 235 240
Asp His His His His His His
245
Claims (10)
1. a kind of method for modifying protein N-terminal characterized by comprising by chemical combination shown in protein to be finished and formula (I)
Object is contacted, to obtain the protein by N-terminal modification, wherein second amino of the protein N-terminal
Acid is not proline,
2. the method according to claim 1, wherein the protein is that interferon, green fluorescent protein, flesh are red
Albumen, RNA enzyme, bovine serum albumin(BSA), human serum albumins or glucose oxidase；
Preferably, the protein it is 10-16 hours lower at 25 degrees Celsius in stablize；
Optionally, the contact is carried out 16 hours under conditions of 25 DEG C；
Optionally, the molar ratio of the protein to be finished and compound shown in formula (I) is 1:200；
Optionally, compound shown in the formula (I) is previously dissolved in the phosphate buffer solution that pH is 7.4；
It optionally, further comprise NaCl, ethylenediamine tetra-acetic acid in the phosphate buffer solution that the pH is 7.4.
3. a kind of prepare protein-conjugate method, which is characterized in that
1) N-terminal modification processing is carried out to protein using any one of claim 1~2 the method；
2) step 1) product obtained is subjected to nucleophilic addition with to coupling molecule, it is described that there is azanol to coupling molecule
Functional group or hydrazine functional group.
4. according to the method described in claim 3, it is characterized in that, described have azanol functional group, the parent to coupling molecule
Core addition reaction be temperature be 4-37 DEG C, preferably 25 DEG C under conditions of carry out 2-36 hours, preferably 16 hours；
Optionally, the step 1) product obtained is previously dissolved in the phosphate buffer solution that pH is 5.5, and the pH is
5.5 phosphate buffer solution further comprises NaCl；
Optionally, the molar ratio of the step 1) product obtained, phosphate and NaCl are 0.15:50:150；
Optionally, described that there is azanol functional group to coupling molecule, it is described that there is structure shown in formula (II) to coupling molecule,
Wherein, A group independently be atom transfer radical polymerization initiator, fluorescent molecule, chemotherapeutics, polymer, small peptide,
Protein, click chemistry correlation group；
It is optionally, described that there is structure shown in formula (1)~(7) to coupling molecule,
Wherein, each a, b, c, d, e, f are each independently positive integer described in 1-10；N is 25-500, compound shown in formula (7)
Number-average molecular weight be 5000；
Optionally, the molar ratio of compound and protein described in the formula (1)~formula (6) is (10-100): 1；
Optionally, the molar ratio of compound and protein described in the formula (7) is (10-300): 1, preferably 50:1；
Optionally, described to have structure shown in formula (8) to coupling molecule, the surface of the protein does not have free sulfhydryl groups；
Optionally, the molar ratio of compound described in the formula (8) and the protein is (10-100): 1, preferably 25:1；
Optionally, described that there is structure shown in formula (9) to coupling molecule；
Optionally, the molar ratio of compound and protein described in the formula (9) is (10-100): 1；
Optionally, described that there is formula (3), formula (6), structure shown in formula (9) to coupling molecule, it further comprise by nucleophilic addition
Reaction product and monomeric compound carry out atom transition free radical polymerization reaction in situ, to obtain protein-macromolecule coupling
Object；
Optionally, the monomeric compound includes water-soluble methyl acrylic ester, esters of acrylic acid, Methacrylamide
Class, acrylamide monomers；
Preferably, the monomeric compound includes oligomeric ethylene glycol methyl ether methacrylate, 2- methylacryoyloxyethyl phosphorus
Phatidylcholine, acrylamide, sugar monomer；
Optionally, it is described to coupling molecule have formula (8) shown in structure, further comprise by nucleophilic addition product with contain
Sulfydryl polymer is attached reaction, to obtain protein-polymer conjugate；
Optionally, the molar ratio of the nucleophilic addition product and the polymer containing sulfydryl is 1:(1-300), preferably 1:
20；
Optionally, the polymer containing sulfydryl is water-soluble polymer containing sulfydryl；
Optionally, the water-soluble polymer containing sulfydryl is by reversible addion-fragmentation chain transfer polymerization, atom transferred free radical
Water-soluble polymethacrylate, the polyacrylate, polymethacrylamide class, polypropylene that polymerization, polymerization obtain
Acylamide polymer；The polymer with sulfydryl, the polyaminoacid generated by ring-opening polymerisation, anionic polymerisation, cationic polymerization；
Optionally, the polymer containing sulfydryl is mercapto-polyglycol.
5. according to the method described in claim 3, it is characterized in that, it is described to coupling molecule have hydrazine functional group, it is described to idol
Joining molecule has structure shown in formula (III),
Wherein, A independently is atom transfer radical polymerization initiator, fluorescent molecule, chemotherapeutics, polymer, small peptide, albumen
Matter, click chemistry correlation group；
Optionally, described that there is structure shown in formula (10)~formula (12) to coupling molecule:
Optionally, described to have structure shown in formula (13) to coupling molecule, the protein surface does not have free sulfhydryl groups:
Optionally, there is structure shown in formula (13) to coupling molecule, further comprises by nucleophilic addition product and containing mercapto
Based polyalcohol is attached reaction, to obtain protein-polymer conjugate；
Optionally, the molar ratio of the nucleophilic addition product and the polymer containing sulfydryl is 1:(5-200), preferably 1:
20；
Optionally, the polymer containing sulfydryl is water-soluble polymer containing sulfydryl；
Optionally, the water-soluble polymer containing sulfydryl includes the water solubility obtained by reversible addion-fragmentation chain transfer polymerization
Polymethacrylate, polyacrylate, polymethacrylamide class, polyacrylamide polymer；
Optionally, the polymer containing sulfydryl includes mercapto-polyglycol, polymethyl acyloxyethyl Phosphorylcholine, polypropylene
Amide；
Optionally, described that there is amido functional group to coupling molecule, it is described that there is structure shown in formula (IV) to coupling molecule,
Wherein, A independently is atom transfer radical polymerization initiator, fluorescent molecule, chemotherapeutics, polymer, small peptide, albumen
Matter, click chemistry correlation group；
Optionally, the amido functional group reagent includes amino acid, polypeptide derivative；
Preferably, the amido functional group amino acid derivativges are cysteine derivative；
Optionally, described that there is structure shown in formula (14) to coupling molecule:
Optionally, the molar ratio of compound and protein described in the formula (14) is (10-100): 1, preferably 25:1；
Optionally, it is described to coupling molecule have formula (14) shown in structure, further comprise by nucleophilic addition product with
Monomeric compound carries out atom transition free radical polymerization reaction in situ, to obtain protein-macromolecule conjugate.
6. a kind of protein-conjugate, which is characterized in that the protein-conjugate is according to any one of claim 3~5
What the method obtained.
7. a kind of pharmaceutical composition, which is characterized in that described wait be coupled point including protein-conjugate as claimed in claim 6
Son and/or protein are drug molecule.
8. a kind of fluorescence imaging method, which is characterized in that import protein as claimed in claim 6-conjugate to be imaged micro-
In environment, it is described to coupling molecule be fluorescent molecule.
9. a kind of fluorescence labeling method, which is characterized in that
1) N-terminal modification processing is carried out to protein using any one of claim 1~2 the method；
2) step 1) product obtained and fluorescent molecule are subjected to nucleophilic addition, the fluorescent molecule has azanol function
Group or hydrazine functional group.
10. a kind of method for detecting protein-protein interaction, which is characterized in that
(1) method as claimed in claim 9 is utilized, marks at least two protein to be detected respectively；
(2) fluorescence resonance energy transfer technology, the mixture of at least two protein after detecting step (1) label are based on；
(3) it is based on testing result, determines and whether there is interaction between at least two protein,
Wherein, it is the instruction that there is interaction between at least two protein that resonance transfer, which occurs, for fluorescent energy.
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