CN109265512B

CN109265512B - Preparation method of protein conjugate based on pyridine dicarbaldehyde

Info

Publication number: CN109265512B
Application number: CN201811113353.7A
Authority: CN
Inventors: 高卫平; 孙佳维; 赵文国; 刘欣宇
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2018-09-25
Filing date: 2018-09-25
Publication date: 2021-06-11
Anticipated expiration: 2038-09-25
Also published as: CN109265512A

Abstract

The invention provides a method for modifying the N terminal of a protein. The method comprises the following steps: contacting the protein to be modified with a compound shown as a formula (I) so as to obtain the protein subjected to N-terminal modification, wherein the second amino acid at the N terminal of the protein is not proline. The method has mild reaction conditions and high efficiency, can specifically modify the N terminal of the protein, is beneficial to the construction of an accurate protein conjugate, and the obtained product is stable to a certain degree.

Description

Preparation method of protein conjugate based on pyridine dicarbaldehyde

Technical Field

The invention relates to the field of biomedicine, in particular to a preparation method of a protein conjugate based on pyridine dicarbaldehyde.

Background

In recent decades, the rapid development of biology and chemical biology has greatly pushed the development of protein drugs, and more protein drugs are approved by the U.S. Food and Drug Administration (FDA) for the treatment of diseases, and at the same time, protein-conjugates have received increasing attention as a new class of biomacromolecule derivatives.

The protein-conjugate is a biological hybrid formed by coupling a protein with other functional components through covalent bonds or non-covalent bonds, can usually obtain the characteristics from new components while keeping the characteristics of the protein (such as precise structure and biological function), and the biological conjugates combining different characteristics can be widely applied to the biomedical fields of biocatalysis, molecular diagnosis, molecular imaging, biological materials, drug delivery, biomolecule delivery, tissue engineering, regenerative medicine, targeted therapy, biological therapy and the like.

The protein-conjugate is applied to the biological medicine through the multi-generation revolution, but the current protein-polymer coupling technology still has a series of key common scientific problems which need to be solved urgently, such as poor coupling site selectivity, low coupling efficiency, lack of a general platform technology and the like, and the wide application of the protein-conjugate in the biological medicine is severely restricted.

Disclosure of Invention

The present application is based on the discovery and recognition by the inventors of the following facts and problems:

the reaction condition of the pyridine carboxaldehyde derivative and the protein is mild, the efficiency is high, and the product is stable, but the pyridine carboxaldehyde derivative and the N-terminal amino group of the protein can be efficiently reacted, and simultaneously, other amino groups on the protein can be partially modified, so that the construction of an accurate protein conjugate is not facilitated. Based on the discovery of the above problems, the inventors have proposed a pyridine-dicarbaldehyde-based modification method, in which two aldehyde groups are structurally designed so that the amount of the two aldehyde groups can be reduced by half compared with other aldehyde-group modifiers, the aldehyde groups can be introduced into the protein after the reaction with the protein, and the excess hydroxylamine and hydrazine derivatives can compete for the destruction of the unstable imine bond formed by the side chain amino group of the protein amino acid residue and the pyridine-aldehyde-group reagent while coupling other functional components by the reaction of the hydroxylamine and hydrazine derivatives with the remaining aldehyde groups of the protein, thereby achieving site-specific modification while introducing new components (see fig. 1).

In the first aspect of the present invention, the present invention provides a method for modifying the N-terminus of a protein. According to an embodiment of the invention, the method comprises: contacting the protein to be modified with a compound shown as a formula (I) so as to obtain the protein subjected to N-terminal modification, wherein the second amino acid at the N terminal of the protein is not proline,

according to the embodiment of the invention, the compound shown in the formula (I) specifically performs nucleophilic addition reaction with amino at the N terminal of the protein to obtain an imine structure, and the imine structure is an unstable structure, so that secondary amino acid of an amide bond formed by a second amino acid at the N terminal of the protein (the second amino acid at the N terminal of the protein is not proline) and a first amino acid performs nucleophilic addition reaction with imine to generate a stable 5-membered ring structure, and further the protein with the modified N terminal is obtained. The method according to the present example enables specific modification of the N-terminus of a protein (only a small amount of free amino groups on the protein will be modified, and the resulting unstable imine structure will subsequently be unstable and will disappear as the reaction proceeds). According to the method disclosed by the embodiment of the invention, the aldehyde group is introduced into the N terminal of the protein, so that the construction of a precise protein conjugate is facilitated, the reaction condition is mild, the efficiency is high, and the obtained product is stable.

According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:

according to an embodiment of the invention, the protein is interferon, green fluorescent protein, myoglobin, rnase, bovine serum albumin, human serum albumin or glucose oxidase. It should be noted that the protein includes proteins, small peptides and antibodies related to pharmaceutical, agricultural, scientific and other industrial fields, and particularly includes therapeutic proteins such as insulin, monoclonal antibodies, blood factors, colony stimulating factors, growth hormones, interleukins, growth factors, therapeutic vaccines, calcitonin, Tumor Necrosis Factor (TNF), enzymes, and the like. Specific examples include, but are not limited to: glutaminase, arginase, arginine deaminase, adenosine deaminase ribonuclease, cytosine deaminase, trypsin, 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, somatostatin, growth hormone, somatostatin, calcitonin, parathyroid hormone, Colony Stimulating Factor (CSF), blood clotting factors, tumor necrosis factor, interferon, interleukins, gastrointestinal peptides, Vasoactive Intestinal Peptide (VIP), intestinal tryptic peptide (CCK), gastrin, secretin, erythropoietin, antidiuretic hormone, octreotide, pancreatic enzyme, superoxide dismutase, thyroid stimulating hormone releasing hormone (TRH), Thyroid stimulating hormone, Luteinizing Hormone Releasing Hormone (LHRH), tissue plasminogen activator, interleukin-1, interleukin-15, receptor antagonist (IL-1RA), glucagon-like peptide-1 (GLP-1), leptin, auxin, granulocyte colony stimulating factor (GM-CSF), interleukin-2 (IL-2), adenosine deaminase, uricase, asparaginase, human growth hormone; chorionic gonadotropin, heparin, atrial natriuretic peptide, hemoglobin, retroviral vectors, relaxin; cyclosporine, oxytocin, vaccines, monoclonal antibodies, single chain antibodies, ankyrin repeat proteins, affibodies and the like.

According to an embodiment of the invention, the protein is stable at 25 degrees celsius for 10-16 hours. The first N-terminal amino acid of the protein needs to react with pyridine-2, 6-diformaldehyde to generate an imine structure in a short time, then further performs an addition reaction with the second N-terminal amino acid, and then performs other reactions, so that the protein needs to be stably present within 10-16 hours.

According to a particular embodiment of the invention, said contact is carried out at 25 ℃ for 16 hours. According to the method provided by the embodiment of the invention, the reaction condition is mild, and the efficiency is high.

According to a particular embodiment of the invention, the molar ratio of the protein to be modified to the compound of formula (I) is 1: 200. The inventors found that the reaction efficiency was high and the obtained product was stable under the above molar ratio conditions.

According to an embodiment of the invention, the compound of formula (I) is pre-dissolved in a phosphate buffer at pH 7.4. According to a specific embodiment of the present invention, the concentration of the compound represented by formula (I) is 0.01-5mg/mL, preferably, the concentration of the compound represented by formula (I) is 4 mg/mL.

According to an embodiment of the present invention, the phosphate buffer solution with pH of 7.4 further comprises NaCl, ethylene diamine tetraacetic acid. The buffer can dissolve the compound shown in the formula (I) and the protein at the same time, so that the compound and the protein are prevented from reacting in an organic solvent, and the activity of the protein is not damaged.

According to a specific embodiment of the invention, the molar ratio of the compound shown in formula (I), phosphate, NaCl and EDTA is 3:5:15: 1. Furthermore, ethylenediaminetetraacetic acid can stabilize proteins to some extent.

In a second aspect of the invention, the invention features a method of making a protein-conjugate. According to an embodiment of the invention, the method comprises: 1) carrying out N-terminal modification treatment on the protein by using the method; 2) and (2) carrying out nucleophilic addition reaction on the product obtained in the step (1) and the molecule to be coupled, wherein the molecule to be coupled has a hydroxyl amine functional group or a hydrazine functional group. The product obtained in step 1) according to the example of the present invention has an aldehyde functional group and is capable of undergoing a nucleophilic addition reaction with a nucleophile. The compound containing hydroxylamine functional group or hydrazine functional group is nucleophilic reagent, and another end of said compound can be connected with other functional group, and can be coupled with other functional component by means of reaction of hydroxylamine, hydrazine derivative and aldehyde group on the protein, at the same time, the excess hydroxylamine and hydrazine derivative can be used for competitively destroying unstable imine bond of protein amino group and pyridine aldehyde group reagent, and the above-mentioned method can implement site-specific modification at the same time of introducing new component. According to the method provided by the embodiment of the invention, the specificity of the modification site is strong, the construction of the protein conjugate is accurate, the reaction efficiency is high, and various compounds containing other important functional groups can be introduced into the protein.

according to an embodiment of the invention, the molecule to be coupled has a hydroxylamine function and the nucleophilic addition reaction is carried out at a temperature of 4 to 37 ℃ for 2 to 36 hours. The method according to the specific embodiment of the invention has the advantages of mild reaction conditions, high yield and good specificity, and can obtain the protein-conjugate with a precise structure.

According to an embodiment of the invention, the molecule to be coupled has a hydroxylamine function and the nucleophilic addition reaction is carried out at a temperature of 25 ℃ for 16 hours. According to the method provided by the embodiment of the invention, the reaction condition is milder, the yield is higher, the specificity is better, and the protein-conjugate with a more accurate structure can be obtained.

According to an embodiment of the present invention, the product obtained in step 1) is pre-dissolved in a phosphate buffer solution with a pH of 5.5, and the phosphate buffer solution with a pH of 5.5 further comprises NaCl, wherein the buffer solution can simultaneously dissolve the product obtained in step 1) and the molecule to be coupled, so as to avoid contact with an organic solvent, and further avoid activity destruction of the protein and other active functional groups by the organic solvent.

According to an embodiment of the invention, the molar ratio of phosphate to NaCl of the product obtained in step 1) is 0.15:50: 150. According to the method of the embodiment of the present invention, the product obtained in step 1) is more sufficiently dissolved under the condition of the molar ratio.

According to an embodiment of the invention, the molecule to be coupled has a hydroxylamine function, the molecule to be coupled has a structure represented by formula (II),

wherein the A groups are independently atom transfer radical polymerization initiators, fluorescent molecules, chemotherapeutic drugs, polymers, small peptides, proteins, "click" chemically-related groups. According to specific embodiments of the present invention, atom transfer radical polymerization initiators include, but are not limited to, 2-bromobutyl derivatives and the like, fluorescent molecules including, but not limited to, fluorescein, rhodamine B, indocyanine green, and the like, chemotherapeutic drugs including, but not limited to, methotrexate, temozolomide, gemcitabine, dorzoledrine, and the like, polymers including, but not limited to, polyethylene glycol, poly-2-methacryloyloxyethyl phosphorylcholine (PMPC), and the like, small peptides including, but not limited to, F3, RGD, and the like, and "click" chemistry (i.e., click chemistry) related groups including, but not limited to, azide, alkynyl, maleimide, and the like.

According to an embodiment of the invention, the molecule to be coupled has a hydroxylamine function, the molecule to be coupled has a structure represented by formulae (1) to (7),

wherein each a, b, c, d, e, f is independently a positive integer from 1 to 10; n is 25 to 500, and the number average molecular weight of the compound represented by the formula (7) is 5000.

According to the embodiment of the invention, the molar ratio of the compound represented by the formula (1) to the compound represented by the formula (6) to the protein is (10-100): 1. The inventors found that the reaction efficiency was high and the obtained product was stable under the condition of the molar ratio of (10-100): 1.

According to the embodiment of the invention, the molar ratio of the compound of the formula (7) to the protein is (10-300): 1. The inventors found that the reaction efficiency was high and the obtained product was stable under the condition of the molar ratio of (10-300): 1.

According to the embodiment of the invention, the molar ratio of the compound of the formula (7) to the protein is (10-300): 1. The inventors found that at a molar ratio of 50:1, the reaction efficiency was higher and the resulting product was more stable.

According to an embodiment of the present invention, the molecule to be coupled has a structure represented by formula (8), and the surface of the protein does not have a free thiol group;

the product obtained by nucleophilic addition reaction of the compound represented by formula (8) and the product obtained in step (1) reacts with thiol, and further reacts with protein if there is free thiol on the surface of protein.

According to an embodiment of the invention, the molar ratio of the compound of formula (8) to the protein is (10-100):1, preferably 25: 1. The inventors found that the reaction efficiency was high and the obtained product was stable under the condition of the molar ratio of (10-100):1, and that the reaction efficiency was higher and the obtained product was more stable under the condition of the molar ratio of 25: 1.

According to a particular embodiment of the invention, the molecule to be coupled has a structure represented by formula (9),

according to an embodiment of the present invention, the molar ratio of the compound of formula (9) to the protein is (10-100):1, preferably 25: 1. The inventors found that the reaction efficiency was high and the obtained product was stable under the condition of the molar ratio of (10-100):1, and that the reaction efficiency was high and the obtained product was stable under the condition of the molar ratio of 25: 1.

According to the embodiment of the invention, the molecule to be coupled has the structure shown in formula (3), formula (6) and formula (9), and further comprises the step of carrying out in-situ atom transfer radical polymerization reaction on the nucleophilic addition reaction product and a monomer compound so as to obtain the protein-macromolecule conjugate. The structures shown in the formulas (3), (6) and (9) are common initiators of atom transfer radical polymerization, so that the molecules to be coupled shown in the formulas (3), (6) and (9) and the protein firstly undergo nucleophilic addition reaction to obtain a nucleophilic addition reaction product (namely a protein-initiator combination), and then undergo in-situ polymerization with a monomer compound.

According to an embodiment of the invention, the monomer compound comprises water-soluble methacrylate, acrylate, methacrylamide, acrylamide monomers. The water-soluble monomer compound and the nucleophilic addition reaction product containing the protein can generate in-situ polymerization reaction in the aqueous solution, so that the direct contact between the protein and the organic solvent is avoided, and the damage of the organic solvent to the biological activity of the protein is further avoided.

According to an embodiment of the present invention, the monomer compound includes oligoethylene glycol methyl ether methacrylate, 2-methacryloyloxyethyl phosphorylcholine, acrylamide, sugar derivative monomer.

According to an embodiment of the present invention, the molecule to be conjugated has a structure represented by formula (8), further comprising subjecting the nucleophilic addition reaction product to a linking reaction with a thiol-group-containing polymer, so as to obtain a protein-polymer conjugate.

It should be noted that the reaction of the nucleophilic addition reaction product with the thiol-group-containing polymer is a classical "click" chemical reaction, and thus the molar ratio of the two is not particularly limited as long as the "click" chemical reaction occurs. According to an embodiment of the present invention, the molar ratio of the nucleophilic addition reaction product to the thiol-group-containing polymer is 1 (1-300), preferably 1: 20. The inventors found that the reaction efficiency was high and the purification was simple under the condition of the molar ratio of 1 (1-300), and that the reaction efficiency was higher and the purification was simpler under the condition of the molar ratio of 1: 20.

According to an embodiment of the present invention, the thiol-group-containing polymer is a water-soluble thiol-group-containing polymer. The water-soluble sulfhydryl-containing polymer can react with the nucleophilic addition reaction product containing protein in aqueous solution, so as to avoid the contact of protein and organic solvent, and further avoid the damage of organic solvent to the biological activity of protein.

According to the embodiment of the invention, the water-soluble thiol-containing polymer is a water-soluble polymer of polymethacrylate, polyacrylate, polymethacrylate and polyacrylamide obtained by reversible addition-fragmentation chain transfer polymerization, atom transfer radical polymerization and polymerization; polymers with thiol groups produced by ring-opening polymerization, anionic polymerization, cationic polymerization, polyamino acids.

According to an embodiment of the present invention, the thiol-group-containing polymer is thiol-polyethylene glycol.

According to an embodiment of the invention, the molecule to be coupled has a hydrazine functional group, the molecule to be coupled has a structure represented by formula (III),

wherein the A groups are independently atom transfer radical polymerization initiators, fluorescent molecules, chemotherapeutic drugs, polymers, small peptides, proteins, "click" chemically-related groups. According to a specific embodiment of the present invention, the atom transfer radical polymerization initiator includes, but is not limited to, 2-bromobutyl derivatives. Fluorescent molecules include, but are not limited to, fluorescein, rhodamine B, indocyanine green, and the like, chemotherapeutic drugs include, but are not limited to, methotrexate, temozolomide, gemcitabine, polybrexpyrazine, and the like, polymers include, but are not limited to, polyethylene glycol, poly-2-methacryloyloxyethyl phosphorylcholine (PMPC), and the like, small peptides include, but are not limited to, F3, RGD, "click" chemically-related groups include, but are not limited to, azide, alkynyl, maleimide, and the like.

According to an embodiment of the invention, the molecule to be coupled has a structure represented by formula (10) to formula (12):

according to an embodiment of the present invention, the molecule to be coupled has a structure represented by formula (13), and the protein surface has no free thiol group:

the product obtained by nucleophilic addition reaction of the compound represented by formula (13) and the product obtained in step (1) reacts with thiol, and further reacts with protein if there is free thiol on the surface of protein.

According to an embodiment of the present invention, the molecule to be conjugated has a structure represented by formula (13), further comprising subjecting the nucleophilic addition reaction product to a linking reaction with a thiol-group-containing polymer, so as to obtain a protein-polymer conjugate.

It should be noted that the reaction of the nucleophilic addition reaction product with the thiol-group-containing polymer is a classical "click" chemical reaction, and thus the molar ratio of the two is not particularly limited as long as the "click" chemical reaction occurs. According to an embodiment of the present invention, the molar ratio of the nucleophilic addition reaction product to the thiol-group-containing polymer is 1 (5-200), preferably 1: 20. The inventors found that the reaction efficiency was high and the purification was simple under the condition of the molar ratio of 1 (5-200), and that the reaction efficiency was higher and the purification was simpler under the condition of the molar ratio of 1: 20.

According to an embodiment of the present invention, the water-soluble thiol-group-containing polymer includes water-soluble polymethacrylate, polyacrylate, polymethacrylamide, polyacrylamide polymers obtained by reversible addition-fragmentation chain transfer polymerization.

According to an embodiment of the present invention, the thiol-group-containing polymer includes thiol polyethylene glycol, polymethacryloxyethylphosphorylcholine, polyacrylamide.

According to the embodiment of the invention, the molecule to be coupled comprises a hydrazine functional group, and the obtained nucleophilic addition product is not only suitable for constructing a protein conjugate with a hydrazone bond connected with pH responsiveness, but also suitable for stabilizing the protein conjugate after reducing the hydrazone bond with sodium cyanoborohydride.

According to an embodiment of the invention, the molecule to be coupled has an amino function, the molecule to be coupled has a structure represented by formula (IV),

wherein the A groups are independently atom transfer radical polymerization initiators, amino acid derivatives, fluorescent molecules, chemotherapeutic drugs, polymers, small peptides, proteins, "click" chemically-related groups. According to a specific embodiment of the present invention, the atom transfer radical polymerization initiator includes, but is not limited to, 2-bromobutyl derivatives. Fluorescent molecules include, but are not limited to, fluorescein, rhodamine B, indocyanine green, and the like, chemotherapeutic drugs include, but are not limited to, methotrexate, temozolomide, gemcitabine, polybrexpyrazine, and the like, polymers include, but are not limited to, polyethylene glycol, oligomeric polyethylene glycol, poly-2-methacryloyloxyethyl phosphorylcholine (PMPC), and the like, small peptides include, but are not limited to, F3, RGD, "click" chemically-related groups include, but are not limited to, azide, alkynyl, maleimide, and the like.

According to an embodiment of the invention, the amino-functional reagent comprises an amino acid, a polypeptide derivative.

According to an embodiment of the invention, the amino-functional amino acid derivative is a cysteine derivative.

According to an embodiment of the invention, the molecule to be coupled has a structure represented by formula (14):

according to an embodiment of the present invention, the molar ratio of the compound of formula (14) to the protein is (10-100):1, preferably 25: 1. The inventors found that the reaction efficiency was high and the obtained product was stable under the condition of the molar ratio of (10-100):1, and that the reaction efficiency was high and the obtained product was stable under the condition of the molar ratio of 25: 1.

According to an embodiment of the present invention, the molecule to be coupled has a structure shown in formula (14), further comprising subjecting the nucleophilic addition reaction product to an in situ atom transfer radical polymerization reaction with a monomer compound, so as to obtain a protein-macromolecule conjugate.

In a third aspect of the invention, the invention features a protein-conjugate. According to an embodiment of the invention, the protein-conjugate is obtained according to the method described previously. The protein-conjugate according to the embodiment of the invention is stable and has accurate structure.

In a fourth aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, the pharmaceutical composition comprises the protein-conjugate as described above, and the molecule to be conjugated and/or the protein is a drug molecule. According to the specific embodiment of the present invention, the protein itself may be a pharmaceutical protein, and the molecule to be coupled may be a drug molecule connected with a hydroxylamine functional group or a hydrazine functional group, where the drug molecule includes, but is not limited to, an anti-tumor drug, an immunosuppressive drug, a receptor inhibitor, an agonist, and the like. After the two are coupled, protein coupled drug molecules or medicinal protein coupled high molecular polymers or medicinal protein coupled drug molecules can be obtained, and the three can realize in-vivo delivery of the drug molecules, combined treatment of protein drugs and combined small molecular drugs and the like.

In a fifth aspect of the invention, a fluorescence imaging method is presented. According to an embodiment of the invention, the protein-conjugate as described above is introduced into the microenvironment to be imaged, the molecule to be conjugated being a fluorescent molecule. It should be noted that the imaging microenvironment includes both ex vivo and in vivo cells and tissues.

In a sixth aspect of the invention, a method of fluorescent labeling is provided. According to an embodiment of the present invention, the method comprises 1) subjecting the protein to an N-terminal modification treatment using the method described above; 2) carrying out nucleophilic addition reaction on the product obtained in the step 1) and a fluorescent molecule, wherein the fluorescent molecule has a hydroxylamine functional group or a hydrazine functional group. The inventors have found that the method according to the embodiment of the present application can specifically bind a molecule having fluorescence to the N-terminus of a protein.

In a seventh aspect of the present invention, a method for detecting protein-protein interactions is provided. According to an embodiment of the invention, the method comprises: (1) labeling the at least two proteins to be detected separately using the method described above; (2) detecting the mixture of at least two proteins marked in the step (1) based on a fluorescence energy resonance transfer technology; (3) determining whether an interaction exists between the at least two proteins based on the detection result, wherein resonance transfer of fluorescence energy is indicative of the existence of the interaction between the at least two proteins. And if the fluorescence energy is subjected to resonance transfer, the two proteins are combined, and if the fluorescence energy is not subjected to resonance transfer, the two proteins are not combined. According to the method provided by the embodiment of the invention, whether the interaction between the proteins occurs can be conveniently, quickly and accurately known.

In another aspect of the invention, the invention provides a method for introducing aldehyde group into protein N-terminal specific modification. According to a specific embodiment of the present invention, the method comprises:

1) preparing a compound of formula (I): a50 ml round-bottom flask was charged with 10ml of 1, 4-dioxane, 0.42 g of 2, 6-pyridinedimethanol and 0.33 g of selenium dioxide were added under stirring, and the reaction was stirred at 65 ℃ for 24 hours. Suction filtering, removing insoluble solid, and rotary evaporating the reaction liquid to dryness. The product was purified by silica gel column chromatography. The product is a white solid, and the reaction chemical formula is as follows:

it is to be noted that the compound represented by the formula (I) can also be obtained commercially;

2) obtaining a protein: obtaining proteins by purchasing, fusion expression, extraction from organisms, purification and the like;

3) dissolving the compound shown in the formula (I) in a phosphate buffer solution, adding the phosphate buffer solution into the protein solution obtained in the step 2, reacting overnight, dialyzing for multiple times to remove unreacted pyridine dicarbaldehyde molecules to obtain protein with introduced aldehyde groups, and obtaining the protein with the modified N-terminal, which can be also called protein-aldehyde groups.

In yet another aspect of the invention, the invention features a method of constructing a protein-poly (oligo (ethylene glycol) methyl ether) (protein-POEGMA) conjugate. According to the specific embodiment of the invention, after an aldehyde group is introduced for the specific modification of the N terminal of the protein, an Atom Transfer Radical Polymerization (ATRP) initiator is introduced on the protein through the reaction of a hydroxylamine group in a compound containing a hydroxylamine functional group and the aldehyde group, the side reaction of the non-specific modification of the compound shown in the formula (I) is eliminated, and oligo-poly-ethylene glycol methyl ether (OEGMA) is polymerized and grown in situ on the protein, so that the protein-poly-oligo-ethylene glycol methyl ether (protein-POEGMA) conjugate is constructed, and the specific steps are as follows:

1) obtaining protein, specifically modifying and introducing aldehyde group at the N terminal of the protein, and placing the protein in phosphate buffer solution (PBS-5.5) with pH of 5.5; wherein, the protein can be obtained by purchasing, fusion expression, extraction from organisms, purification and the like; the protein includes interferon, green fluorescent protein, myoglobin, RNase, bovine serum albumin, human serum albumin, glucose oxidase, trichosanthin, etc.

2) Dissolving a hydroxylamine ATRP initiator ABM in phosphate buffer solution (PBS-5.5) with pH of 5.5, adding the solution into the protein aldehyde group solution prepared in the step 1 for reaction overnight, and dialyzing for multiple times by using the phosphate buffer solution to prepare a protein initiator (protein-bromine);

3) adding the protein initiator prepared in the step 2) into oligoethylene glycol monomethyl ether, introducing nitrogen to remove oxygen sufficiently, adding cuprous chloride, cupric chloride and 1,1,4,7,10, 10-hexamethyl triethylene tetramine (HMTETA) solution which are introduced with nitrogen to remove oxygen sufficiently, reacting for 4 hours, introducing air to terminate the reaction, dialyzing for many times to remove small molecules, and separating unreacted protein through an ion exchange column to obtain the protein-poly-oligoethylene glycol methyl ether (protein-POEGMA) conjugate.

The protein conjugate constructed according to the specific embodiment of the invention has good activity maintenance, high stability and high modification specificity. The pharmacokinetic parameters of the interferon-poly (oligo (ethylene glycol) methyl ether) conjugate constructed according to the specific embodiment of the invention are significantly better than that of interferon itself.

In yet another aspect of the invention, a method of constructing a protein-polymer conjugate is provided. According to a specific embodiment of the present invention, the method comprises the steps of:

1) obtaining protein, specifically modifying and introducing aldehyde group at the N terminal of the protein, and placing the protein in phosphate buffer solution (PBS-5.5) with pH of 5.5; wherein, the protein can be obtained by purchasing, fusion expression, extraction from organisms, purification and the like;

2) the preparation process of the hydroxylamine polyethylene glycol monomethyl ether is shown as follows, and the specific process is shown in the specific embodiment part;

3) and (3) dissolving the hydroxylamine polyethylene glycol monomethyl ether prepared in the step (2) in phosphate buffer solution (PBS-5.5) with pH of 5.5, adding the solution into the protein aldehyde group solution prepared in the step (1) for reaction overnight, concentrating the reaction solution, and removing unreacted hydroxylamine polyethylene glycol monomethyl ether and unreacted protein by using a molecular sieve.

In yet another aspect of the invention, a method of constructing a protein-polymer conjugate is provided. According to the specific embodiment of the invention, a molecule to be coupled (hydroxylamine-maleimide bifunctional molecule), for example, a compound shown in formula (8), is reacted with an aldehyde group, a protein-maleimide is constructed while eliminating a non-specific modification side reaction of the compound shown in formula (I), and then a thiol polymer is prepared by reversible addition-fragmentation chain transfer polymerization (RAFT), so that the thiol polymer and the protein-maleimide are subjected to a classical 'click' chemical reaction. According to a specific embodiment of the present invention, the preparation steps are as follows:

1) obtaining protein, specifically modifying and introducing aldehyde group at the N terminal of the protein, and placing the protein in phosphate buffer solution (PBS-5.5) with pH of 5.5;

2) preparing a thiol polymer, a specific process being shown in the detailed description section;

3) preparation of a hydroxylamine-maleimide bifunctional molecule (e.g., a compound of formula (8)) the preparation procedure is as follows, the specific procedure is shown in the detailed description,

4) dissolving the hydroxylamine-maleimide bifunctional molecule prepared in the step 3 in phosphate buffer solution (PBS-5.5) with pH of 5.5, adding the solution into the protein aldehyde group solution prepared in the step 1 for reaction overnight, and dialyzing for multiple times by using phosphate buffer solution with pH of 7.2 to prepare protein-maleimide;

5) and (3) dissolving the sulfhydryl polymer prepared in the step (2) in phosphate buffer solution (PBS-5.5) with the pH value of 7.2, adding the sulfhydryl polymer into the protein-maleimide solution prepared in the step (4) for reaction for 4 hours, concentrating the reaction solution, and removing the sulfhydryl polymer which does not participate in the reaction and unreacted protein by using a molecular sieve to prepare the protein-polymer conjugate.

In yet another aspect of the invention, a method of constructing a protein-polymer conjugate is provided. According to the specific embodiment of the present invention, through the reaction of a molecule to be coupled (hydrazine-maleimide bifunctional molecule), for example, a compound represented by formula (13), with a protein-aldehyde group, a hydrazone bond-linked protein-maleimide is constructed while the non-specific modification side reaction of dipicolinate is eliminated, and then a thiol polymer is prepared through reversible addition-fragmentation chain transfer polymerization (RAFT), so that the thiol polymer and the protein-maleimide undergo a classical "click" chemical reaction, thereby preparing a hydrazone bond-linked protein-polymer conjugate, which has pH responsiveness due to the presence of the hydrazone bond. According to a specific embodiment of the present invention, the preparation steps are as follows:

1) obtaining protein, specifically modifying and introducing aldehyde group at the N terminal of the protein, and placing the protein in phosphate buffer solution (PBS-5.5) with pH of 5.5, wherein the protein can be obtained by purchasing, fusion expression, extraction from organisms, purification and other modes;

3) dissolving hydrazine-maleimide bifunctional molecules (such as a compound shown as a formula (13)) in phosphate buffer solution (PBS-7.2) with pH of 7.2, adding aniline hydrochloride solution with pH of 7.2 into the protein aldehyde solution prepared in the step 1, reacting overnight, and dialyzing for multiple times by using phosphate buffer solution with pH of 7.2 to prepare protein-maleimide connected with hydrazone bonds;

4) and (3) dissolving the sulfhydryl polymer prepared in the step (2) in phosphate buffer solution (PBS-5.5) with the pH value of 7.2, adding the sulfhydryl polymer into the protein-maleimide solution prepared in the step (3) for reaction for 4 hours, concentrating the reaction solution, and removing the sulfhydryl polymer and the unreacted protein which do not participate in the reaction through a molecular sieve to prepare the hydrazone bond-linked protein-polymer conjugate.

According to an embodiment of the present invention, the hydrazone bond in the protein-polymer conjugate connected with the hydrazone bond is reduced by sodium cyanoborohydride, so that a stable protein-polymer conjugate can be prepared.

In yet another aspect of the present invention, the present invention provides a method of constructing a hydrazone linkage-linked protein-drug molecule conjugate. According to a specific embodiment of the present invention, the preparation steps are as follows:

2) preparing a hydrazine-drug molecule, a specific process being shown in the detailed description section;

3) and (3) dissolving the hydrazine-drug molecules prepared in the step (2) in phosphate buffer solution (PBS-7.2) with the pH value of 7.2, adding aniline hydrochloride solution with the pH value of 7.2 into the protein aldehyde group solution prepared in the step (1) to react overnight, and dialyzing for multiple times by using phosphate buffer solution with the pH value of 7.2 to prepare the hydrazone bond-connected protein-drug molecule conjugate.

According to the specific embodiment of the present invention, the hydrazone bond in the protein-drug molecule conjugate connected with the hydrazone bond is reduced by sodium cyanoborohydride, so that a stable protein-drug molecule conjugate can be prepared.

According to the specific embodiment of the invention, hydrazone-bond-linked human serum albumin-doxorubicin derivatives and human serum albumin-methotrexate conjugates, as well as hydrazone-bond-reduced human serum albumin-doxorubicin derivatives and human serum albumin-methotrexate conjugates were successfully prepared.

In yet another aspect of the present invention, a method of constructing a hydrazone linkage-linked protein-imaging molecule conjugate is presented. According to a specific embodiment of the present invention, the preparation steps are as follows:

2) preparing a hydrazine-imaging molecule, a specific process being shown in the detailed description section;

3) and (3) dissolving the hydrazine-imaging molecules prepared in the step (2) in phosphate buffer solution (PBS-7.2) with the pH value of 7.2, adding aniline hydrochloride solution with the pH value of 7.2, adding the aniline hydrochloride solution into the protein aldehyde solution prepared in the step (1) for reaction overnight, and dialyzing for multiple times by using phosphate buffer solution with the pH value of 7.2 to prepare the hydrazone bond-connected protein-imaging molecule conjugate.

According to the specific embodiment of the present invention, the hydrazone bond in the protein-drug molecule conjugate connected with the hydrazone bond is reduced by sodium cyanoborohydride, so that a stable protein-imaging molecule conjugate can be prepared. Interferon-fluorescein conjugates were successfully prepared according to the examples of the present invention.

The advantageous features of the present invention include, but are not limited to, the following:

1) the method for preparing the protein-conjugate related in the invention is a two-step reaction, wherein the reaction of hydroxylamine and hydrazine derivatives in the second step can eliminate the side reaction of non-N-terminal amino modification in the first step, which is favorable for determining the position of the modification site at the active center of the protein;

2) the method for preparing the protein-conjugate has mild reaction conditions and high conversion rate, the total conversion rate of the two-step reaction is more than 80%, and the modification conversion rate of partial protein can reach more than 90%.

3) The method for preparing the protein-conjugate has wide design space, and can be used for preparing various conjugates such as protein-imaging molecules, protein-drugs, protein-polymers, protein-protein/polypeptides, protein-nano materials and the like.

Drawings

FIG. 1 is an overall scheme for the preparation of protein-conjugates according to an embodiment of the invention;

FIG. 2 is an ESI mass spectrum of a prepared protein initiator according to an embodiment of the present invention

FIG. 3 is an enzymatic LC-MS spectrum of interferon-dipicolinate-ABM according to an embodiment of the present invention;

FIG. 4 is an SDS-PAGE gel of a portion of in situ growth protein-poly (oligo (ethylene glycol) methyl ether) conjugates according to an embodiment of the present invention;

FIG. 5 is a GPC chart of a portion of an in situ growth protein-poly (oligo (ethylene glycol) methyl ether) conjugate according to an embodiment of the present invention;

FIG. 6 is a graph showing activity tests of interferon, interferon-aldehyde group, interferon-initiator, interferon-poly (oligo (ethylene glycol) methyl ether, according to an embodiment of the present invention;

FIG. 7 is a pharmacokinetic profile of an interferon, in situ grown interferon-poly (oligo (ethylene glycol) methyl ether) conjugate, according to an embodiment of the present invention;

figure 8 is a human serum albumin-dolorubicin and dolorubicin cytotoxicity profile according to an embodiment of the invention;

FIG. 9 is an ESI mass spectrum of an interferon-fluorescein conjugate in accordance with an embodiment of the present invention;

FIG. 10 is a diagram of an interferon, an interferon aldehyde group, an interferon-aminoxy polyethylene glycol conjugate sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to an embodiment of the present invention;

FIG. 11 is a diagram of an interferon, an interferon aldehyde, an interferon-thiol polyethylene glycol conjugate sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Example 1 Synthesis of pyridine-2, 6-dicarboxaldehyde (PDA) (Compound represented by the formula (I))

A50 ml round bottom flask was charged with 10ml of 1, 4-dioxane, 0.42 g of 2, 6-pyridinedimethanol and 0.33 g of selenium dioxide under stirring, and stirred at 65 deg.CThe reaction was carried out for 24 hours. Suction filtering, removing insoluble solid, and rotary evaporating the reaction liquid to dryness. The product was purified by column chromatography on silica gel eluting with 20:1 volume ratio dichloromethane/methanol, R_f0.6. The product was a white solid, C₇H₅NO₂Yield was 0.36 g, 88.2%.¹HNMR(400MHz,CDCl₃):δ10.16(s,2H),8.06-8.19(m,3H)。ESI-mass m/z:136.2([M+H]⁺)。

Example 2

Pyridine-2, 6-dicarboxaldehyde and a hydroxylamine atom transfer radical polymerization initiator are used for modifying proteins in two steps, and oligomeric polyethylene glycol is polymerized in situ to construct a protein-poly (oligo (ethylene glycol) methyl ether) conjugate, wherein the proteins shown in the embodiment comprise: interferon, green fluorescent protein, myoglobin, RNase, bovine serum albumin, human serum albumin, glucose oxidase, trichosanthin, etc.

2.1 construction of the protein expression

2.11 construction and expression of Interferon

Using PCR techniques from

The IFN coding sequence is amplified in the T vector and inserted into the pET-25b (+) vector through Nde I/Eco RI enzyme cutting sites. The N-terminal of the IFN gene sequence is connected with Nde I enzyme cutting site and is connected with three glycins, the C-terminal is connected with a specific recognition sequence LPETG and a 6 XHis tag of SrtA, and then is connected with the enzyme cutting site of Eco RI, and the final nucleotide sequence is as follows:

GGCGGTGGGTGTGATCTGCCTCAGACTCATTCTCTGGGTAGTCGTCGTACGCTGATGCTGCTGGCTCAAATGCGCCGTATTAGCCTGTTTTCTTGCCTGAAAGATCGCCACGATTTTGGGTTTCCACAGGAAGAATTTGGCAACCAGTTCCAGAAAGCCGAAACAATTCCGGTACTGCACGAGATGATTCAACAAATCTTTAACCTGTTCAGCACCAAAGACTCTTCAGCTGCCTGGGATGAAACACTGCTGGACAAATTCTATACCGAGCTGTATCAGCAACTGAACGATCTGGAGGCATGTGTTATTCAGGGTGTTGGTGTGACTGAAACTCCTCTGATGAAAGAGGATAGCATTCTGGCAGTCCGTAAATATTTTCAGCGTATCACACTGTATCTGAAAGAGAAAAAATATAGCCCGTGTGCCTGGGAAGTTGTTCGTGCCGAAATCATGCGCAGCTTTAGTCTGTCTACCAACCTGCAAGAGAGCCTGCGTTCTAAAGAAGGATCCGGTGGCGGTGGCTCTCTGCCGGAAACCGGTGGCCACCATCATCATCATCAT(SEQ ID NO:1)。

the post-translational amino acid sequence is as follows:

GGGCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKEGSGGGGSLPETGGHHHHHH(SEQ ID NO:2)。

the primers used were designed as follows:

a forward primer: GGAATTCcatatgGGCGGTGGGTGTGATCTGCCTCAGACTCAT (SEQ ID NO: 3).

Reverse primer: CGgaattcTTATCAATGATGATGATGATG (SEQ ID NO: 4).

After verification by DNA sequencing, the constructed plasmid encoding G3-IFN-H6 was transformed into E.coli strain Rosetta-gami (DE3) pLysS competent cells and cultured overnight at 37 ℃ in ampicillin-resistant LB medium. The culture was transferred to 1L sterile ampicillin-resistant TB medium and the culture was continued until the OD600 of the bacterial suspension reached 0.5, induction was carried out by adding IPTG at a final concentration of 500. mu.M and overexpression was carried out overnight at 18 ℃. The cells were harvested by centrifugation, resuspended in 50mM Tris & HCl,150mM NaCl, pH7.4 buffer, sonicated in an ice water bath and centrifuged at 14,000 Xg for 10min to remove the pellet, and the supernatant was mixed with 2mL of 1% (w/v) Polyethyleneimine (PEI) and centrifuged again. The supernatant containing soluble proteins was purified by the AKTA Purifier 10 system by passing through a nickel affinity column. The column was equilibrated with 50mM Tris-HCl, 500mM NaCl, 10% glycerol, 25mM imidazole, pH7.4 buffer, and the eluted hetero-proteins were G3-IFN-H6 eluted with 50mM Tris-HCl, 500mM NaCl, 10% glycerol, 50mM imidazole, pH7.4 buffer, and finally further purified by HiPrep 26/10 desalting column, replaced with 50mM phosphate, 150mM NaCl, pH7.4 solution, stored at-80 ℃. The purification process and purity of IFN-LPETGGH6 were assessed by SDS-PAGE gel electrophoresis. The concentration of the protein was determined by NanoDrop 2000.

The Nanodrop 2000UV/Vis method was calibrated to a concentration of 6 mg/ml and a yield of 100 mg per liter of bacteria. Purifying, and freezing in solution.

2.2 construction and expression of Green fluorescent protein

The Green Fluorescent Protein (GFP) used in this study was a non-mutated, original protein, which was expressed clonally according to standard protocols. Samples were sent to GeneWiz for sequencing. The sequencing result is as follows:

MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLCYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYNVDHHHHHH(SEQ ID NO:5)。

the number of amino acids was 247, the molecular weight was 27978.44, and the isoelectric Point (PI) was 5.94 as calculated from the sequencing results. The GFP used in this example was the batch of purified GFP. After purification with AKTA protein purifier and DNA sequencing verification, the constructed GFP-encoding plasmid was transformed into competent cells of E.coli strain BL21 and cultured overnight at 37 ℃ in ampicillin-resistant LB medium. The culture was transferred to 1L sterile ampicillin-resistant TB medium and the culture was continued until OD600 of the bacterial suspension reached 0.5, induction was carried out by adding IPTG at a final concentration of 500. mu.M and overexpression was carried out overnight at 25 ℃. The cells were harvested by centrifugation, resuspended in 50mM Tris & HCl,150mM NaCl, pH7.4 buffer, sonicated in an ice water bath and centrifuged at 14,000 Xg for 10min to remove the pellet, and the supernatant was mixed with 2mL of 1% (w/v) Polyethyleneimine (PEI) and centrifuged again. The supernatant containing soluble proteins was purified by the AKTA Purifier 10 system by passing through a nickel affinity column. The column was equilibrated with 50mM Tris-HCl, 500mM NaCl, 10% glycerol, 25mM imidazole, pH7.4 buffer, and the eluted heteroproteins were eluted with 50mM Tris-HCl, 500mM NaCl, 10% glycerol, 50mM imidazole, pH7.4 buffer, and finally GFP was eluted with 50mM Tris-HCl, 500mM NaCl, 10% glycerol, 500mM imidazole, pH7.4 buffer, and further purified by HiPrep 26/10 desalting column, replaced with 50mM phosphate, 150mM NaCl, pH7.4 solution, and stored at-80 ℃. Purity was assessed by SDS-PAGE gel electrophoresis. After purification of the protein, the concentration was 13.5 mg/ml as calibrated by the Nanodrop 2000UV/Vis method, and the yield was 150 mg per liter of bacteria. Purifying, and freezing in solution.

2.3 preparation of N-terminal-specific pyridine-2, 6-dicarboxaldehyde (protein-aldehyde group) of protein

The reaction conditions of the various proteins with pyridine-2, 6-dicarbaldehyde were identical, as follows: mu.M of the protein solution was allowed to stand with 30mM of pyridine-2, 6-dicarbaldehyde (PDA) (4mg/mL) in 50mM phosphate, 150mM NaCl, 10mM EDTA, pH7.4 phosphate buffer at 25 ℃ for 16 hours. After the reaction was completed, the product (protein-CHO) to which the PDA molecule was ligated was obtained by conducting multiple dialysis with 50mM phosphate, 150mM NaCl, phosphate buffer solution (PBS-5.5) at pH 5.5.

In the examples of the present invention, interferon-aldehyde groups, green fluorescent protein-aldehyde groups, myoglobin-aldehyde groups, RNase-aldehyde groups, bovine serum albumin-aldehyde groups, and the like were prepared by the method.

2.4 preparation of protein-initiator

Protein aldehyde group (protein-CHO) connected with PDA molecule reacts with ABM to connect a small molecule ATRP initiator on protein to make the protein into a large molecule ATRP initiator.

The chemical structural formula of ABM is

150 μ M protein aldehyde (protein-CHO) solution in 50mM phosphate, 150mM NaCl, pH 5.5 phosphate buffer (PBS-5.5). 1.6mg of ABM & HCl was added to 1mL of the above solution, and the mixture was reacted at 25 ℃ for 16 hours. After completion of the reaction, dialysis (dialysis membrane type No. 131276, Spectrum, molecular weight cut-off 8-10kDa) was performed with PBS (150mM NaCl, pH 7.4) to obtain protein initiator (protein-Br).

The above reaction conditions were used for the reaction of all proteins with ABM in inventive example 1.

In the embodiment of the invention, the interferon initiator, the green fluorescent protein initiator, the myoglobin initiator, the RNase initiator and the bovine serum albumin initiator are prepared by the method.

In the embodiment of the invention, the specificity of the N-terminal modification of the protein is verified by Q-tof mass spectrum and LC-MS, and FIG. 2 is ESI of partial protein pyridine dicarbaldehyde and ABM two-step modificationThe mass spectrum chart of fig. 2 shows that the dipicolinate can be linked to three proteins (GFP, IFN, myoglobin Mb), and shows that most of the proteins are modified with one dipicolinate molecule, a small amount of protein peaks modified with two molecules of dipicolinate exist in the mass spectrum of the GFP protein, and after the second step of ABM modification, only the protein peaks modified with one molecule of ABM are visible in the protein spectrum, indicating that the second step of reaction can eliminate the by-products of the first step of reaction. In addition, LC-MS spectrogram obtained after modification of G3-IFN by dipicolinate and ABM and enzymolysis of Trypsin proves that the modification site is N-terminal of protein, FIG. 3 is a secondary mass spectrogram of N-terminal fragment generated by enzymolysis of G3-IFN two-step modified PDA and ABM, the N-terminal sequence is GGGCDLPQTHSLGSR, and the part with modified molecular weight increased is regarded as C₁₃H₁₃Br N₂O₃The second mass spectrum of the N-terminal fragment was matched with the theoretical results. FIGS. 2 and 3 demonstrate that the N-terminal-PDA and ABM two-step modification sites are unique and N-terminal to the protein. While other aldehyde protein modifiers modify the amino group at the N-terminus of the protein and also modify the amino group on the lysine side chain, see Jentoft N, Dearborn D.labeling of proteins by reduced reaction using sodium cyanohydrin [ J ]]Journal of Biological Chemistry,1979,254(11):4359-4365, whereas the modification method presented herein shows no non-specific modification and shows better site specificity.

In addition, in order to verify the cyclization process of the reaction of the pyridinedicarboxaldehyde and the N-terminal of the protein, a nuclear magnetic spectrum of a reaction product of the pyridinedicarboxaldehyde and three glycine derivatives is used, and it is verified that imine formed by the reaction of the pyridinedicarboxaldehyde and the protein or small peptide can be added with a second amino acid aminoamide to form an imidazolidinone five-membered ring. NMR (400MHz, CDCl)₃)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 preparation and purification of protein-Poly (oligo-ethylene glycol methyl Ether)

The protein initiator (protein-Br) was diluted to 50. mu.M with PBS, 1mL of the above solution was transferred to a 10mL reaction tube, 50. mu.L of OEGMA (number average molecular weight 500) was added, and high-purity nitrogen gas was introduced into the reaction tube and bubbled at a constant rate for 15 minutes. 370 mu L of 1mg/mL CuCl aqueous solution and 3.1 mu L of 1,1,4,7,10, 10-Hexamethyltriethylenetetramine (HMTETA) are added into a 10-mL ground test tube, the volume of the solution is made up to 1mL by deionized water, and the solution is transferred into protein-Br solution through a bidirectional solvent transfer needle after high-purity nitrogen gas is bubbled at a constant speed for 15 minutes. The reaction was stirred at 4 ℃ for 4 hours and then quenched by introducing air.

In the embodiment of the invention, the protein-pOEGMA in situ ATRP polymerization adopts the reaction conditions to prepare protein-pOEGMA conjugates such as interferon-pOEGMA, green fluorescent protein-pOEGMA, myoglobin-pOEGMA, RNase-pOEGMA, bovine serum albumin-pOEGMA and the like.

FIG. 4 is a SDS-PAGE gel of a portion of in situ grown protein-poly (oligo (ethylene glycol) methyl ether) conjugates of the present invention demonstrating the successful preparation and purification of these classes of protein-polymer conjugates.

FIG. 5 is a GPC chart of a portion of in situ grown protein-poly (oligo (ethylene glycol) methyl ether) conjugates of the present invention, which also demonstrates the successful preparation and purification of these protein-polymer conjugates, and also provides quantitative results on the molecular weight size and distribution of the conjugates.

2.6 Activity assay of proteins, protein conjugates

2.61 in vitro bioactivity assay of Interferon (IFN) and its modified products

The cytotoxicity of IFN, IFN-CHO, IFN-Br and IFN-POEGMA was determined by MTT method. Burkitt's B lymphoma cells (Daudi B) were selected for log-growth phase and 5000 cells (50. mu.l medium) were plated per well in 96-well plates, with 100. mu.l medium alone and wells without cells as background. After 5 hours of incubation at 37 deg.C, 50 microliters of serially diluted IFN, IFN-CHO, IFN-Br and IFN-POEGMA solutions were added, three wells for each concentration of drug were added for parallel experiments, and the wells to which saline was added were used as controls. After 96 hours of culture, 10 microliters of MTS reagent was added to each well, and after 3 hours of culture in a cell incubator, the 96-well plate was taken out, and the absorbance data at 495 nm was read on a microplate reader. Software Gr for acquired dataAphPad Prism 5.0 treatment, half maximal Inhibitory Concentration (IC)₅₀) The software was also used for fitting calculations and data results are given as mean ± standard deviation.

Half maximal Inhibitory Concentration (IC) of IFN, IFN-CHO, IFN-Br and IFN-POEGMA₅₀) Respectively as follows: 2.46 plus or minus 0.29pM, 2.64 plus or minus 0.23pM, 2.56 plus or minus 0.29pM, 4,19 plus or minus 0.16 pM. The specific map is shown in figure 6.

2.62 Activity assay of Myoglobin and modified products thereof

A guaiacol solution (20mM) and a hydrogen peroxide solution (19.4mM), and a myoglobin and modified product solution thereof (myoglobin concentration: 0.17mg/mL) were prepared in advance using 10mM of pH7.0 potassium phosphate buffer as a solvent. mu.L of guaiacol solution and 100. mu.L of hydrogen peroxide solution were mixed, 10. mu.L of myoglobin solution was added, absorption at 470nm was measured at different time points at 25 ℃ and recorded every 15 seconds for 5 minutes, and a mixed solution of 100. mu.L of guaiacol solution and 100. mu.L of hydrogen peroxide solution and 10. mu.L of Tris buffer was used as a blank. The data obtained (absorbance-time curve) were fitted linearly, the slope of the curve being proportional to the protein activity, and the relative activities of the other modified products were calculated, taking the original myoglobin activity as 100%.

The relative activities of MB, MB-CHO, MB-Br and MB-POEGMA were: 100 plus or minus 2%, 89.1 plus or minus 2.3%, 87.4 plus or minus 0.7% and 90.4 plus or minus 1%. Specific results are shown in table 1.

2.63 Activity assay of ribonuclease A (RNaseA) and modified products thereof

Taking Tris buffer solution with 50mM pH7.0 as solvent, ribonuclease A and modified products thereof are diluted to 0.15mg/mL of RNaseA concentration, and cytidine 2 '3' -cyclic monophosphate (CP) solution is prepared in advance with the concentration of 0.5 mg/mL. mu.L of RNaseA or the modified product solution was added to 190. mu.L of cytidine 2 '3' -cyclic monophosphate solution, and the absorbance at various time points of 290nm was measured at 25 ℃ for 5 minutes, and data was recorded every 15 seconds. mu.L of cytidine 2 ', 3' -cyclic monophosphate solution was added to 10. mu.L of Tris buffer as a blank. The data obtained (absorbance-time curve) were fitted linearly, the slope of the curve being proportional to the enzyme activity, and the relative enzyme activities of the other modified products were calculated with the enzyme activity of the original RNase A as 100%.

The relative activities of RNASEA, RNASEA-CHO, RNASEA-Br and RNASEA-POEGMA are respectively: 100 plus or minus 3.7 percent, 96.8 plus or minus 3.2 percent, 103.8 plus or minus 4.7 percent and 95.4 plus or minus 4.4 percent. Specific results are shown in table 1.

2.64 Activity assay of Green fluorescent protein and modified products thereof

The fluorescence intensity of GFP is directly proportional to the concentration of the solution over a range of concentrations, and therefore a standard curve method can be used to determine the fluorescence activity of GFP. To wells of a 96-well plate, each 2.5, 5, 10, 20. mu.l of GFP standard solution (1mg/mL) was added, 200. mu.l was made up with PBS, and wells to which 200. mu.l of PBS solution was added were used as a background. Measuring the fluorescence intensity of excitation wavelength 460nm and emission wavelength 525nm at 25 ℃ by using a microplate reader, subtracting the background, performing linear fitting by using microsoft Excel 2010 to obtain a standard curve, measuring the fluorescence of a sample to be measured while making the standard curve, calculating the relative fluorescence intensity of each step of reaction product containing GFP with the same equivalent concentration and an original GFP standard solution through the standard curve, and taking the fluorescence intensity of the original GFP standard solution as 100%, wherein the measurement result shows that the N-end site specificity modification of the PDA mediated GFP is carried out, and the fluorescence of each step of reaction product is not changed.

The relative activities of GFP, GFP-CHO, GFP-Br and GFP-POEGMA were: 100 plus or minus 10.5 percent, 97.1 plus or minus 9.1 percent, 98.4 plus or minus 10 percent and 99.6 plus or minus 11.2 percent. Specific results are shown in table 1.

Table 1:

activity of	Original protein (%)	Protein-aldehyde group (%)	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
					Myoglobin	100±2.0	89.1±2.3	87.4±0.7	90.4±1.0

2.7 pharmacokinetic testing of Interferon, Interferon conjugates

Female BALB/c nude mice (6 weeks old) were randomly divided into 5 groups (3 mice/group) and injected with IFN- α, and IFN-POEGMA conjugate at 1mg/kg body weight via tail vein. At appropriate time points (1,5,15,30min,1,3,6,24,48,72 and 96h), about 20 μ L of tail vein blood was collected (collection tubes were previously dried by soaking with sodium heparin), left at 4 ℃ for 30min and centrifuged at 4000 Xg for 15min, and plasma was collected and stored at-80 ℃. IFN concentrations were determined using an IFN-. alpha.2 ELISA kit, while background values were subtracted from the plasma of mice that were not injected with the drug. The data is analyzed and processed by DAS 3.0 software. The data show that t1/2 β (h) of the interferon-poly (oligo (ethylene glycol) methyl ether) conjugate and interferon in mice is 41.3 hours and 1.83 hours, respectively, and the blood clearance half-life of the interferon conjugate is 22.6 times that of interferon. The specific interferon, in situ grown interferon-poly (oligo (ethylene glycol) methyl ether) conjugate pharmacokinetic profile is shown in figure 7.

Example 3 preparative characterization of two classes of human serum albumin-drug molecule conjugates

3.1 preparation of human serum Albumin-methotrexate

3.11 preparation of human serum Albumin-aldehyde groups

A150. mu.M HSA protein solution was allowed to stand with 30mM Pyridine Dicarbaldehyde (PDA) (4mg/mL) in 50mM phosphate, 150mM NaCl, 10mM EDTA, pH7.4 phosphate buffer at 25 ℃ for 16 hours. After the reaction was completed, the product (HSA-CHO) to which PDA molecules were ligated was obtained by conducting multiple dialysis with 50mM phosphate, 150mM NaCl, phosphate buffer solution (PBS-5.5) at pH 5.5.

3.1.2 preparation of methotrexate-hydrazine derivatives

Dried Methotrexate (MTX) (400mg, 0.88mmol) was dissolved in anhydrous degassed DMF (6.0mL), EDC (338mg, 1.76mmol) was added to HOTU (240mg, 1.76mmol), and the reaction was stirred for 2 min. Hydrazine hydrate (44mg, 0.88mmol) was then added to the clear red solution and the reaction was stirred at ambient temperature in the dark for 36 h. Precipitating the reaction solution in 3/1 volume ratio diethyl ether/acetone for three times to obtain orange yellowA solid in a viscous color, which was placed in a vacuum oven overnight, yielded 139mg (37%) of the resulting methotrexate-hydrazine as an orange yellow solid.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 preparation of human serum Albumin-methotrexate conjugates

mu.M of the HSA-aldehyde group obtained in the above step was reacted with 500. mu.M Methotrexate (MTX) by allowing to stand in a buffer solution of 50mM phosphate, 150mM NaCl, pH7.4 while adding aniline hydrochloride for catalysis, and after 16 hours of the reaction, 20mM sodium cyanoborohydride (NaBH3CN) was added and allowed to stand at 4 ℃ for 16 hours, and then dialyzed against 50mM phosphate, 150mM NaCl, pH7.4 for a plurality of times to obtain a human serum albumin-methotrexate conjugate.

3.2 preparation and characterization of human serum Albumin-Doluropyracin

3.21 preparation of human serum Albumin-aldehyde groups

As described in 3.11

3.22 preparation of human serum Albumin-hydrazine

mu.M HSA-CHO obtained in the above step was allowed to react with 1mg/mL Adipic dihydrazide (adipoic dihydrazide) at 4 ℃ for 16 hours in 50mM phosphate, 150mM NaCl, pH 5.5 phosphate buffer, 20mM sodium cyanoborohydride (NaBH3CN) was added thereto and allowed to react at 4 ℃ for 16 hours, and after the completion of the reaction, dialysis was performed several times with 50mM phosphate, 150mM NaCl, pH7.4 buffer to obtain stable HSA-NHNH₂。

3.23 preparation of human serum Albumin-Doluropyran

The HSA-NHNH of 100 μ M obtained in the above step₂After allowing 500. mu.M Doxorubicin (DOX) to stand in a buffer solution of 50mM phosphate, 150mM NaCl, pH7.4 while adding aniline hydrochloride to catalyze the reaction, the reaction was subjected to dialysis several times with a buffer solution of 50mM phosphate, 150mM NaCl, pH7.4 after 16 hours to obtain HSA-DOX in which doxorubicin was linked to the amino terminus of HSA.

3.24 characterization of human serum Albumin-Doluropyracin Activity

The cytotoxicity of HSA-DOX and DOX was determined by MTT assay. Logarithmic growth phase 4T1 breast cancer cells (4T1) were selected and 5000 cells (50 microliters of medium) were plated per well in 96-well plates with only 100 microliters of medium added and wells without cells as background. After incubation at 37 ℃ for 5 hours, 50. mu.l of the serially diluted HSA-DOX and DOX solutions were added, three wells for each concentration of drug were used for parallel experiments, and the wells to which physiological saline was added were used as controls. After 96 hours of culture, 10 microliters of MTS reagent was added to each well, and after 3 hours of culture in a cell incubator, the 96-well plate was taken out, and the absorbance data at 495 nm was read on a microplate reader. The data obtained were processed with the software GraphPad Prism 5.0, half maximal Inhibitory Concentration (IC)₅₀) The software was also used to fit the calculations.

IC of DOX and HSA-DOX₅₀Respectively 0.435. mu.M and 5.07. mu.M.

The cytotoxicity curve of human serum albumin-doxofitabine is shown in figure 8.

Example 4 construction of Interferon-fluorescent molecule conjugates

4.1 preparation of Interferon-aldehyde groups

This step is the same as the interferon-aldehyde group preparation described in 2.2 of example 2.

4.2 preparation of fluorescein-hydrazine derivatives

Dried Fluorescein Isothiocyanate (FITC) (38.9mg, 0.1mmol) was dissolved in acetonitrile (6.0mL), potassium carbonate (27.6mg, 0.3mmol) was added and the reaction was stirred for 2 min. Hydrazine hydrate (5mg, 0.1mmol) was then added to the solution and the reaction was stirred at ambient temperature for 12h, protected from light. Filtering the reaction liquid to remove excessive potassium carbonate, selecting dry acetonitrile, dissolving orange solid in deionized water, adding hydrochloric acid into the solution until the pH value is 5 to generate a large amount of orange insoluble substances, centrifuging at 3000rpm for 10 minutes, removing supernatant, taking precipitate, and placing the precipitate in a vacuum oven overnight to generate 16mg (40%) of orange solid, namely the obtained fluorescein-hydrazine.

4.3 preparation of Interferon-fluorescein conjugates

And (3) standing and reacting the 100 mu M IFN-aldehyde group obtained in the step with 500 mu M fluorescein hydrazine in a buffer solution of 50mM phosphate, 150mM NaCl and pH7.4, adding aniline hydrochloride for catalysis, and dialyzing for multiple times by using the buffer solution of 50mM phosphate, 150mM NaCl and pH7.4 after reacting for 16 hours to obtain the hydrazone bond connected interferon-fluorescein conjugate.

To the interferon-fluorescein conjugate was added 20mM sodium cyanoborohydride (NaBH)₃CN) is left to stand at 4 ℃ for 16 hours, and after the reaction is finished, the stable interferon-fluorescein conjugate is obtained by multiple dialysis with 50mM phosphate, 150mM NaCl and a buffer solution with pH 7.4. The ESI mass spectrum of the interferon-fluorescein conjugate is shown in FIG. 9.

Example 5 preparation of interferon-polyethylene glycol conjugate by grafting of Interferon pyridine Dicarboxaldehyde conjugate with hydroxylamine tetraethylene glycol

5.1 preparation of hydroxylamine tetraethylene glycol

The route for the preparation of hydroxylamine tetraethylene glycol is shown below:

the specific operation steps are as follows:

5 g of polyethylene glycol (molecular weight 5000) is dissolved in 20 ml of acetonitrile, stirred, added with 0.52 g of triphenylphosphine and cooled in an ice bath. 0.66 g of carbon tetrabromide was dissolved in 22 ml of acetonitrile, and the solution was added dropwise at 0 ℃ for 3 hours. The ice bath was removed and the reaction temperature was slowly raised to room temperature over 3 hours. The temperature was raised to 40 ℃ and stirring was maintained for 18 hours. Acetonitrile was removed by rotary evaporation, 40 ml of water and 40 ml of n-hexane were added, and heating and refluxing were performed for 30 minutes. And cooling to obtain white precipitate. Filtering, and rotary evaporating to remove n-hexane. 200ml of dichloromethane were extracted 3 times and dried over anhydrous magnesium sulfate. The solvent is dried by spinning to obtain the polyethylene glycol bromine.

5 g of polyethylene glycol bromide and 0.27 g of N-Boc-hydroxylamine were dissolved in 20 ml of ethanol, and 0.8 g of potassium hydroxide was added to react at 60 ℃ for 2 hours. And (4) cooling, filtering, and removing the solvent by rotary evaporation to obtain a white solid residue. The residue was dissolved in 5ml dichloromethane and precipitated in 500ml cold ether in China, centrifuged and the precipitate dried under vacuum to yield a white solid, Boc-aminooxytetraethylene glycol.

And taking 3g of the obtained white solid, adding 10ml of 7M ethyl acetate hydrochloride solution, reacting at room temperature for 4 hours, performing rotary evaporation on ethyl acetate and hydrogen chloride, adding a small amount of dichloromethane for dissolution, precipitating in 200ml of glacial ethyl ether, filtering to obtain white fluffy powder, and drying the ethyl ether in a vacuum oven to obtain the final product, namely aminoxy polyethylene 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).

5.2 preparation of Interferon-aldehyde groups

This step is the same as the interferon-aldehyde group preparation method described in 2.3 in example 2.

5.3 preparation of Interferon-polyethylene glycol conjugates

150 μ M of an interferon aldehyde (protein-CHO) solution in 50mM phosphate, 150mM NaCl, pH 5.5 phosphate buffer (PBS-5.5). 30.5mg of aminoxy polyethylene glycol was added to 1mL of the above solution, and the reaction was carried out at 25 ℃ for 16 hours. FIG. 10 shows the electrophoresis of interferon, interferon aldehyde, and interferon-polyethylene glycol conjugate sodium dodecyl sulfate-polyacrylamide gel.

Example 6 preparation of interferon-Polymer conjugates with hydroxylamine-maleimide functionalized linker grafted thiol Polymer

6.1 preparation of mercapto polymers by reversible addition-fragmentation chain transfer polymerization (RAFT)

6.11 preparation of mercaptopolyoligoethylene glycol monomethyl ether (POEGMA-SH)

Dissolving oligoethylene glycol monomethyl ether methacrylate 128mg, a chain transfer reagent 4-cyano-4- (phenylthiocarbonylthio) pentanoic acid 0.838mg and an initiator azobisisobutyronitrile 0.1mg in 1ml DMF, removing oxygen through 5 times of freeze-thaw cycle, and placing the reaction solution in a 65 ℃ oil bath pan for reaction for 12 hours. After the reaction, unreacted small molecules were removed by dialysis several times through a semipermeable membrane having a molecular weight cut-off of 3000 in a 20% ethanol aqueous solution. The molecular weight and polydispersity of the polymer are calculated by PEG internal standard method through GPC, after dialysis, the polymer solution is freeze-dried, 60mg of poly-oligo-ethylene glycol monomethyl ether is taken and dissolved in 1ml of deionized water, 0.18mg of ethylenediamine is added into the solution under the protection of nitrogen atmosphere, and after overnight at room temperature, the ethylenediamine is dialyzed in water for multiple times to remove the ethylenediamine, thus obtaining the sulfhydryl poly-oligo-ethylene glycol monomethyl ether.

6.12 mercaptopoly-2-methacryloyloxyethyl phosphorylcholine (PMPC-SH)

130mg of methacryloyloxyethyl phosphorylcholine, 0.838mg of 4-cyano-4- (phenylthioformylthio) pentanoic acid as a chain transfer reagent and 0.1mg of azodiisobutyronitrile as an initiator are dissolved in 1ml of DMF, oxygen is removed through 5 times of freeze-thaw cycles, and the reaction solution is put into an oil bath kettle at 65 ℃ for reaction for 12 hours. After the reaction, unreacted small molecules were removed by dialysis several times through a semipermeable membrane having a molecular weight cut-off of 3000 in a 20% ethanol aqueous solution. The molecular weight and polydispersity of the polymer are calculated by PEG internal standard method through GPC, after dialysis, the polymer solution is freeze-dried, 60mg of polymethacryloyloxyethyl phosphorylcholine is taken, 0.18mg of ethylenediamine is added into the solution under the protection of nitrogen atmosphere, and after overnight at room temperature, the ethylenediamine is dialyzed in water for multiple times to remove the ethylenediamine, thus obtaining the sulfhydryl poly-oligo-ethylene glycol monomethyl ether.

6.2 preparation of hydroxylamine-maleimide functionalized linkers

The route of preparation of the hydroxylamine-maleimide functionalized linker is shown below:

the method comprises the following specific steps:

15.54 ml of tetraethylene glycol is dissolved in 200ml of acetonitrile, stirred, added with 44.3 g of triphenylphosphine and cooled in an ice bath. 56.0 g of carbon tetrabromide is dissolved in 220 ml of solution, and the solution is added dropwise at 0 ℃ for 3 hours. The ice bath was removed and the reaction temperature was slowly raised to room temperature over 3 hours. The temperature was raised to 40 ℃ and stirring was maintained for 18 hours. Acetonitrile was removed by rotary evaporation, 400 ml of water and 400 ml of n-hexane were added, and heating and refluxing were performed for 30 minutes. And cooling to obtain white precipitate. Filtering, and rotary evaporating to remove n-hexane. 200ml of dichloromethane were extracted 3 times and dried over anhydrous magnesium sulfate. Purification on silica gel (ether/ethyl acetate 3: 1). Product 22.83 g, 89% yield.¹H NMR(400MHz,CDCl₃):δ3.81(t，4H)，3.67(s，8H)，3.46(t，4H)。

16 g of 1, 11-dibromo-3, 6, 9-trioxaundecane and 14 g of N-Boc-hydroxylamine were dissolved in 100 ml of ethanol, 6.3 g of potassium hydroxide was added, and the reaction was carried out at 60 ℃ for 2 hours. After cooling, filtration was carried out, and the solvent was removed by rotary evaporation, and the residue was yellow oily. Silica gel column (EA/DCM/MeOH ═ 10:10: 1).¹H NMR(400MHz,CDCl₃):δ7.78(s，1H)，4.00-4.12(m，4H)，3.66-3.72(m，12H)，1.47(s，9H)。

Triphenylphosphine was added to super-dry tetrahydrofuran, and after stirring in ice bath for 15 minutes, DIAD was added, and further stirring for 15 minutes, after which the above-obtained yellow liquid was added, and after stirring for 15 minutes, a maleimide solid was directly added to the reaction liquid, after which the solid was gradually dissolved, the reaction was left to react overnight at room temperature, and then the reaction liquid was subjected to silica gel column purification (dichloromethane/ethyl acetate ═ 5: 3). So as to obtain a yellow oily substance,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).

taking 300mg of the obtained yellow oily substance, adding 10ml of 3M ethyl acetate hydrochloride solution, reacting at room temperature for 15 minutes, rotationally evaporating ethyl acetate and hydrogen chloride in an ice-water bath, adding a small amount of dichloromethane, dissolving in the ice-water bath, rotationally evaporating, precipitating in 200ml of glacial ethyl ether, centrifuging to obtain yellow viscous liquid, and drying the ethyl ether in a vacuum oven to obtain the final product of aminoxy polyethylene glycol maleimide (aminoxy-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 preparation of Interferon-pyridine Formaldehyde conjugate (Interferon-aldehyde group)

This step was the same as the interferon-aldehyde group preparation method described in 2.2 of example 2, with a yield of 95%.

6.4 preparation of Interferon-Maleimide conjugates

150 μ M protein aldehyde (IFN-CHO) solution in 50mM phosphate, 150mM NaCl, pH 5.5 phosphate buffer (PBS-5.5). The solution was added with 30 times molar ratio of hydroxylamine-maleimide functionalized linker and reacted at 25 ℃ for 16 hours. After completion of the reaction, dialysis (dialysis membrane type No. 131276, Spectrum, molecular weight cut-off 8-10kDa) was performed with PBS (150mM NaCl, pH 7.4) to obtain an interferon-maleimide conjugate. The yield thereof was found to be 80%.

6.5 preparation of Interferon-thiol Polymer conjugates

150 μ M of an interferon aldehyde (protein-CHO) solution in 50mM phosphate, 150mM NaCl, pH 745 in phosphate buffered saline (PBS-7.4). 1mL of the above solution was added to mg of a mercapto polymer (mercapto poly-oligo ethylene glycol monomethyl ether) and reacted at 25 ℃ for 8 hours. The yield of the interferon-thiol polymer conjugate was 35%, fig. 11.

Example 7 preparation of interferon-Polymer conjugates with hydrazine-maleimide functionalized linker grafted thiol Polymer

7.1 preparation of mercapto polymers by reversible addition-fragmentation chain transfer polymerization (RAFT)

This step is the same as the preparation of the thiol polymer described in 6.3 of example 6.

7.2 preparation of Interferon-pyridine dialdehyde conjugate (Interferon-aldehyde group)

The reaction conditions of the various proteins with pyridine dicarbaldehyde were identical, and the reaction conditions were as follows: mu.M of the protein solution was allowed to stand with 30mM of Pyridine Dicarbaldehyde (PDA) (4mg/mL) in 50mM phosphate, 150mM NaCl, 10mM EDTA, pH7.4 phosphate buffer at 25 ℃ for 16 hours. After the reaction was completed, the product (protein-CHO) to which the PDA molecule was ligated was obtained by multiple dialysis against 50mM phosphate, 150mM NaCl, phosphate buffer solution (PBS-5.5) pH 7.4. The yield thereof was found to be 95%.

7.3 preparation of Interferon-Maleimide conjugates

150 μ M protein aldehyde (IFN-CHO) solution in 50mM phosphate, 150mM NaCl, pH 5.5 phosphate buffer (PBS-5.5). The solution was added with 30 times molar ratio of hydroxylamine-maleimide functionalized linker and reacted at 25 ℃ for 16 hours. After completion of the reaction, the reaction mixture was dialyzed against PBS (150mM NaCl, pH 7.4). The dialyzed protein solution was added with 5-fold molar ratio of sodium cyanoborohydride, reacted at 4 ℃ for 10 hours, and dialyzed against PBS (150mM NaCl, pH 7.4) after the reaction was completed. (dialysis membrane type No. 131276, Spectrum, molecular weight cut-off 8-10kDa) to give an interferon-maleimide conjugate. The yield thereof was found to be 80%.

7.4 this step is the same as the preparation of the interferon-thiol polymer conjugate described in 6.5 of example 6

The yield of interferon-thiol polymer conjugate was 35%.

Example 8 preparation of bovine serum Albumin-Polymer conjugates from bovine serum Albumin by cysteine initiator derivatives

8.1 preparation of bovine serum Albumin-aldehyde groups

This procedure was the same as the protein-aldehyde group preparation described in 2.3 of example 2, with a yield of 95%.

8.2 preparation of bovine serum Albumin-initiator

150 μ M bovine serum albumin aldehyde (BSA-CHO) solution in 50mM phosphate, 150mM NaCl, pH7.4 phosphate buffer (PBS-7.4). 1.2mg of cysteine initiator of formula (14) was added to 1mL of the above solution, and the reaction was carried out at 25 ℃ for 16 hours. After completion of the reaction, the reaction mixture was dialyzed (dialysis membrane model No. 131276, Spectrum, molecular weight cut-off 8-10kDa) against PBS (150mM NaCl, pH 7.4) to obtain bovine serum albumin initiator (BSA-Br) in a yield of 75%.

8.3 preparation of bovine serum Albumin-Polymer conjugates

This procedure was identical to the procedure described for the preparation of the protein-polymer conjugates in 2.5 of example 2, with a yield of 80%.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> Qinghua university

<120> method for preparing 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> nucleotide sequence of interferon 2.11 in example 2

<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> amino acid sequence of interferon 2.11 in example 2

<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> Forward primer 2.11 in example 2

<400> 3

ggaattccat atgggcggtg ggtgtgatct gcctcagact cat 43

<210> 4

<211> 29

<212> DNA

<213> Artificial

<220>

<223> reverse primer in 2.11 of example 2

<400> 4

cggaattctt atcaatgatg atgatgatg 29

<210> 5

<211> 247

<212> PRT

<213> Artificial

<220>

<223> sequencing results of Green fluorescent protein used in 2.2 of example 2

<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

1. A method of preparing a protein-conjugate,

1) contacting the protein to be modified with a compound shown as a formula (I) so as to obtain the protein subjected to N-terminal modification, wherein the second amino acid at the N terminal of the protein is not proline,

2) carrying out nucleophilic addition reaction on the product obtained in the step 1) and the molecule to be coupled, wherein the molecule to be coupled has a hydroxylamine functional group, an amino functional group or a hydrazine functional group.

2. The method of claim 1, wherein the protein is interferon, green fluorescent protein, myoglobin, rnase, bovine serum albumin, human serum albumin, or glucose oxidase.

3. The method of claim 2, wherein the protein is stable at 25 degrees celsius for 10-16 hours.

4. The method of claim 1, wherein the contacting is at 25 ℃ for 16 hours.

5. The method according to claim 1, wherein the molar ratio of the protein to be modified to the compound of formula (I) is 1: 200.

6. The method according to claim 1, wherein the compound of formula (I) is pre-dissolved in a phosphate buffer solution having a pH of 7.4, the phosphate buffer solution having a pH of 7.4 further comprising NaCl, ethylene diamine tetraacetic acid.

7. The method according to claim 1, characterized in that the molecule to be coupled has a hydroxylamine function and the nucleophilic addition reaction is carried out at a temperature of 4-37 ℃.

8. The method of claim 7, wherein the nucleophilic addition reaction is performed at a temperature of 25 ℃.

9. The method of claim 7, wherein the nucleophilic addition reaction is carried out at a temperature of 4-37 ℃ for 16 hours.

10. The process according to claim 1, characterized in that the product obtained in step 1) is pre-dissolved in a phosphate buffer solution at pH 5.5, said phosphate buffer solution at pH 5.5 further comprising NaCl.

11. The method according to claim 10, wherein the molar ratio of the phosphate to the NaCl of the product obtained in step 1) is 0.15:50: 150.

12. The method according to claim 1, characterized in that the molecule to be coupled has a hydroxylamine function, the molecule to be coupled having a structure of formula (II),

wherein, the A group is independently atom transfer free radical polymerization initiator, fluorescent molecule, chemotherapy drug, polymer, small peptide, protein, click chemistry related group.

13. The method according to claim 1, wherein the molecule to be coupled has a structure represented by one of formulae (1) to (7),

14. The method according to claim 13, wherein the molar ratio of the compound of formula (1) to the compound of formula (6) to the protein is (10-100): 1.

15. The method according to claim 13, wherein the molar ratio of the compound of formula (7) to the protein is (10-300): 1.

16. the method of claim 15, wherein the molar ratio of the compound of formula (7) to the protein is 50: 1.

17. The method according to claim 1, wherein the molecule to be coupled has a structure represented by formula (8), and the surface of the protein has no free thiol group;

18. the method of claim 17, wherein the molar ratio of the compound of formula (8) to the protein is (10-100): 1.

19. The method of claim 18, wherein the molar ratio of the compound of formula (8) to the protein is 25: 1.

20. The method according to claim 1, wherein the molecule to be coupled has a structure represented by formula (9);

21. the method of claim 20, wherein the molar ratio of the compound of formula (9) to the protein is (10-100): 1.

22. The method according to claim 13, wherein the molecule to be coupled has a structure represented by formula (3), formula (6) or formula (9), and further comprising subjecting the nucleophilic addition reaction product to an in situ atom transfer radical polymerization reaction with a monomer compound to obtain a protein-polymer conjugate.

23. The method of claim 22, wherein the monomer compound comprises water-soluble methacrylate, acrylate, methacrylamide, acrylamide monomers.

24. The method of claim 23, wherein the monomer compound comprises oligoethylene glycol methyl ether methacrylate, 2-methacryloyloxyethyl phosphorylcholine, acrylamide, a sugar monomer.

25. The method of claim 17, wherein the molecule to be conjugated has the structure of formula (8), and further comprising subjecting the nucleophilic addition reaction product to a linking reaction with a thiol-group-containing polymer to obtain a protein-polymer conjugate.

26. The method of claim 25, wherein the molar ratio of the nucleophilic addition reaction product to the thiol-group-containing polymer is 1 (1-300).

27. The method of claim 26, wherein the molar ratio of the nucleophilic addition reaction product to the thiol-group-containing polymer is 1: 20.

28. The method of claim 25, wherein the thiol-group-containing polymer is a water-soluble thiol-group-containing polymer.

29. The method of claim 28, wherein the water-soluble thiol-group-containing polymer is a water-soluble polymethacrylate, polyacrylate, polymethacrylamide, polyacrylamide polymer obtained by reversible addition-fragmentation chain transfer polymerization, atom transfer radical polymerization, polymerization; polymers with thiol groups produced by ring-opening polymerization, anionic polymerization, cationic polymerization, polyamino acids.

30. The method of claim 25, wherein the thiol-group-containing polymer is a thiol-polyethylene glycol.

31. The method according to claim 1, wherein the molecule to be coupled has a hydrazine functional group, the molecule to be coupled has a structure represented by formula (III),

wherein A is independently atom transfer radical polymerization initiator, fluorescent molecule, chemotherapeutic drug, polymer, small peptide, protein, click chemistry related group.

32. The method according to claim 1, wherein the molecule to be coupled has a structure represented by formula (10) to formula (12):

33. the method according to claim 1, wherein the molecule to be coupled has a structure represented by formula (13), and the protein has no free thiol group:

34. the method of claim 33, wherein the molecule to be conjugated has the structure of formula (13), and further comprising subjecting the nucleophilic addition reaction product to a linking reaction with a thiol-group-containing polymer to obtain the protein-polymer conjugate.

35. The method of claim 34, wherein the molar ratio of the nucleophilic addition reaction product to the thiol-group-containing polymer is from 1: (5-200).

36. The method of claim 35, wherein the molar ratio of the nucleophilic addition reaction product to the thiol-group-containing polymer is 1: 20.

37. The method of claim 34, wherein the thiol-group-containing polymer is a water-soluble thiol-group-containing polymer.

38. The method of claim 37, wherein the water-soluble thiol-group-containing polymer comprises water-soluble polymethacrylate, polyacrylate, polymethacrylate, polyacrylamide polymers obtained by reversible addition-fragmentation chain transfer polymerization.

39. The method of claim 28, wherein the thiol-group-containing polymer comprises thiol-polyethylene glycol, polymethacryloxyethylphosphorylcholine, polyacrylamide.

40. The method according to claim 1, wherein the molecule to be coupled has an amino function and the molecule to be coupled has a structure of formula (IV),

41. The method of claim 40, wherein the amino-functional reagent comprises an amino acid derivative, a polypeptide derivative.

42. The method of claim 41, wherein the amino acid derivative is a cysteine derivative.

43. The method according to claim 1, wherein the molecule to be coupled has a structure represented by formula (14):

44. the method of claim 43, wherein the molar ratio of the compound of formula (14) to the protein is (10-100): 1.

45. The method of claim 44, wherein the molar ratio of the compound of formula (14) to the protein is 25: 1.

46. The method according to claim 43, wherein the molecule to be coupled has the structure shown in formula (14), further comprising subjecting the nucleophilic addition reaction product to an in situ atom transfer radical polymerization reaction with a monomer compound to obtain the protein-macromolecule conjugate.

47. A protein-conjugate obtained according to the method of claim 1.

48. Use of the protein-conjugate according to claim 47, wherein the molecule and/or protein to be conjugated is a drug molecule, for the preparation of a medicament for the treatment of tumors.

49. A pharmaceutical composition comprising the protein-conjugate of claim 1, wherein the molecule and/or protein to be conjugated is a drug molecule.

50. A fluorescence imaging method for non-diagnostic or therapeutic purposes, characterized in that a protein-conjugate according to claim 1 is introduced into the microenvironment to be imaged, the molecule to be conjugated being a fluorescent molecule.

51. A method of fluorescent labeling for non-diagnostic or therapeutic purposes,

1) subjecting a protein to an N-terminal modification treatment by the method according to claim 1;

2) carrying out nucleophilic addition reaction on the product obtained in the step 1) and a fluorescent molecule, wherein the fluorescent molecule has a hydroxylamine functional group, an amino functional group or a hydrazine functional group.

52. A method for detecting protein-protein interactions for non-diagnostic purposes, characterized in that,

(1) labeling the at least two proteins to be detected separately using the method of claim 51;

(2) detecting the mixture of at least two proteins marked in the step (1) based on a fluorescence energy resonance transfer technology;

(3) determining whether there is an interaction between the at least two proteins based on the detection result,

wherein resonance transfer of fluorescence energy is indicative of the presence of an interaction between at least two proteins.