Methods for modifying proteins in an open environment and protein modifiers

Abstract

This invention discloses a method for modifying proteins in an open environment and the protein modifiers thereof. The method includes constructing an open reaction system, reacting the open reaction system at a first temperature, heating it to a second temperature, allowing it to stand, and then directly centrifuging to obtain a supernatant containing the protein modifier. The open reaction system comprises a monomer of a polymer modifier, a protein containing an initiating group, and a deoxygenation system. The deoxygenation system comprises a catalytic deoxygenation polymer, a substrate, and a product scavenger. The method of this invention can be carried out in an open environment, eliminating the need for complex deoxygenation steps. Primary purification of the protein-polymer conjugate and the reaction system can be achieved simply by changing the temperature. It exhibits broad monomer compatibility, protein adaptability, and excellent scalability, suitable for scale-up from 10 μL to 100 L.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and materials science, specifically to methods for modifying proteins in an open environment and protein modifiers. The methods of this invention are particularly applicable to long-acting protein therapeutics, antibody drugs, novel vaccines, and in vitro diagnostic reagents. Background Technology

[0002] Protein-polymer conjugates are a new generation of semi-synthetic biomolecules, showing broad application prospects in protein therapy, drug delivery, novel vaccines, tissue engineering scaffolds, in vitro diagnostic reagents, and biocatalysis. Currently, common synthetic strategies mainly include "grafting to" and "grafting from" methods, with "grafting from" gaining increasing popularity due to its higher coupling efficiency, wider molecular weight range, and simpler purification process.

[0003] In recent years, the rise of controlled radical polymerization (CRP) technology has significantly promoted the application of "grafting" methods. Among them, atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer polymerization (RAFT) are widely used due to their site selectivity and mild reaction conditions. However, a key limitation of these methods lies in their dependence on a strict deoxygenation environment, especially in the synthesis of protein-containing polymers, where oxygen significantly interferes with the radical polymerization reaction, reducing reaction efficiency and the quality of the final product. Although some oxygen-tolerant polymerization methods have been developed, such as systems utilizing enzyme-assisted deoxygenation, these methods usually still require complex subsequent purification steps, increasing operational complexity and production costs.

[0004] Furthermore, existing technologies exhibit significant limitations in adaptability for both small-scale and large-scale production. For small-scale synthesis, the complex deoxygenation steps of existing systems are unfavorable for the rapid preparation of high-cost therapeutic proteins. In large-scale production, closed systems place high demands on equipment, making high-throughput or continuous operation difficult. Simultaneously, limitations in monomer selection and protein compatibility of existing methods prevent them from meeting diverse functional requirements. Therefore, developing a simple, adaptable, and efficient deoxygenation general synthesis strategy that can be achieved in an open environment has become a pressing issue in the field.

[0005] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] To address at least some of the technical problems in the prior art, the present invention provides a method for modifying proteins in an open environment and the resulting protein modifiers. Specifically, the present invention includes the following.

[0007] A first aspect of the present invention provides a method for modifying proteins in an open environment, comprising the following steps:

[0008] (1) Construct an open reaction system comprising a monomer of a polymer modifier, a protein containing an initiating group, and a deoxygenation system, wherein the deoxygenation system comprises a catalytic deoxygenation polymer, a substrate, and a product scavenger;

[0009] (2) Allow the open reaction system to react at the first temperature; and

[0010] (3) After heating to the second temperature and letting it stand, the supernatant containing the protein modifier is directly separated.

[0011] In some embodiments, according to the method for modifying proteins in an open environment according to the first aspect of the present invention, the polymer modifier is a water-soluble polymer or a hydrophilic polymer, or a polymer that forms dispersed particles in an aqueous solution.

[0012] In some embodiments, according to the method for modifying proteins in an open environment according to the first aspect of the present invention, the catalytic deoxygenating polymer is in a dissolved or dispersed state and has catalytic activity at a first temperature, and the catalytic deoxygenating polymer is in an aggregated or precipitated state at a second temperature.

[0013] In some embodiments, according to the method for modifying proteins in an open environment according to the first aspect of the present invention, the catalytic deoxypolymer has the activity of at least one enzyme selected from glucose oxidase, lipoxygenase, cholesterol oxidase, L-amino acid oxidase, ethanol oxidase and polyphenol oxidase.

[0014] In some embodiments, according to the method for modifying proteins in an open environment according to the first aspect of the present invention, the first temperature is 0-40°C, the second temperature is 20-50°C, and the second temperature is higher than the first temperature.

[0015] In some embodiments, according to the method for modifying proteins in an open environment according to the first aspect of the present invention, the open reaction system further comprises at least one selected from the group consisting of free radical stabilizers, reducing agents, and copper salts and their ligand compounds.

[0016] In some embodiments, the method for modifying proteins in an open environment according to the first aspect of the present invention further includes step (4) of further purifying the supernatant to obtain a protein modifier.

[0017] A second aspect of the present invention provides a method for modifying proteins in an open environment, comprising the following steps:

[0018] (1) Construct an open reaction system comprising a monomer of a polymer modifier, a first modified protein containing an initiating group, and a deoxygenation system, wherein the deoxygenation system comprises a catalytic deoxygenation polymer, a substrate, and a product scavenger;

[0019] (2) Allow the open reaction system to react at the first temperature; and

[0020] (3) After heating to the second temperature and letting it stand, centrifuge directly to obtain the supernatant containing the second modified protein.

[0021] In some embodiments, according to the method for modifying a protein in an open environment according to the first aspect of the present invention, the first modified protein is a modified protein obtained by the method described in the first aspect.

[0022] In a third aspect, the present invention provides a protein modifier obtained by the method described in the first or second aspect.

[0023] The method of this invention can serve as a general strategy for the efficient synthesis and purification of protein-polymer conjugates, without requiring complex deoxygenation steps in an open environment, and possesses broad monomer compatibility, protein adaptability, and excellent potential for large-scale production. In some embodiments, the invention achieves primary purification of the protein-polymer conjugate and reaction system by centrifugation at, for example, above 23°C, followed by removal of unreacted monomers and low-molecular-weight byproducts using a desalting column. For specific proteins, ion-exchange chromatography can be used for fine fractionation of the products. These approaches combine temperature-induced phase separation with optional chromatographic purification, enabling product purification in an open environment through a two- or three-step combined process, and are adaptable to various reaction system sizes, such as scale extensions from 10 μL to 100 L, including 50 μL, 100 μL, 500 μL, 1 L, 10 L, 50 L, and 100 L. Attached Figure Description

[0024] Figure 1 MALDI-TOF mass spectra of GOX and GOX-Br in Example 1.

[0025] Figure 2 SDS-PAGE images of GOX, GOX-Br, and GOX-PDEGMA in Example 1. Lane 1 is GOX, lane 2 is GOX-Br, lane 3 is GOX-PDEGMA, and lane 4 is the pre-stained protein marker.

[0026] Figure 3 The GOX-PDEGMA prepared in Example 1 1 H NMR.

[0027] Figure 4 The GOX-PDEGMA prepared in Example 1 was cut out and subjected to GPC detection.

[0028] Figure 5 The particle size results of GOX and GOX-PDEGMA in Example 1. The left side shows the particle size distribution of GOX, and the right side shows the particle size distribution of GOX-PDEGMA.

[0029] Figure 6 Dynamic light scattering (DLS) experiments on the GOX-PDEGMA prepared in Example 1 showed that it underwent a low critical solution temperature (LCST) phase transition in the range of 21-23 °C.

[0030] Figure 7 Enzyme activity assays of GOX and GOX-PDEGMA at 4°C and 37°C in Example 1.

[0031] Figure 8 Thermal stability of GOX and GOX-PDEGMA in the examples. Compared with GOX, the thermal stability of GOX-PDEGMA is significantly improved.

[0032] Figure 9 The circular dichroism (CD) spectra of GOX, GOX-Br, and GOX-PDEGMA in Example 1 confirmed that the secondary structure of GOX was not affected by the initiator and polymer conjugation.

[0033] Figure 10 (a) GPC curves of HSA-POEGMA during the reaction process. (b) Evolution of molecular weight (Mw) with conversion. (c) Semi-logarithmic kinetics of copper wire insertion and removal during OEGMA polymerization. (d) GPC curves of HSA and its polymer conjugates. (e) TEM image of HSA-PHPMA. (f) SDS-PAGE of HSA (lane 2), HSA-Br (lane 3), HSA-POEGMA (lane 4), and HSA-POEGMA-PHPMA (lane 5). (g) CD curves of HSA, HSA-Br, and their conjugates. (h) Normalized esterase-like activities of HSA, HSA-Br, and their conjugates.

[0034] Figure 11 After three rounds of temperature-induced precipitation and redissolution treatment, GOX-PDEGMA retained more than 50% of its enzyme activity.

[0035] Figure 12The SDS-PAGE electrophoresis analysis results of HSA (lane 2), HSA-Br (lane 3), and HSA-POEGMA synthesized using different cycles of GOX-PDEGMA (cycles 1 to 3 correspond to lanes 4 to 6 respectively) further confirmed the continued success of the polymerization reaction in these cycles.

[0036] Figure 13 To verify the universality of the polymerization method for other therapeutic proteins, recombinant human interferon-α (IFN) was used as a representative protein for testing. IFN is a cytokine widely used in antiviral and antitumor therapy. (a) Schematic diagram of IFN-POEGMA preparation. (b) SDS-PAGE electrophoresis analysis of IFN-α (lane 2), IFN-Br (lane 3), and purified IFN-POEGMA (lane 4) showed that IFN-POEGMA was successfully synthesized and purified. (c) In vitro antitumor proliferation activity of IFN and IFN-POEGMA showed that the activity of IFN-POEGMA was 61.1% of that of IFN, indicating that this polymerization method has a significant advantage in maintaining the therapeutic activity of the protein.

[0037] Figure 14 To further verify the applicability of the polymerization method to therapeutic antibodies, Herceptin was used as a representative antibody for testing. Herceptin is a widely used targeted antibody drug for the treatment of breast cancer. (a) A schematic diagram of the preparation of Herceptin-POEGMA is provided. (b) SDS-PAGE electrophoresis analysis of Herceptin (lane 2), Herceptin-Br (lane 3), and purified Herceptin-POEGMA (lane 4) showed that Herceptin-POEGMA was successfully synthesized and purified. (c) Comparison of fluorescence intensity histograms of the control group, Herceptin group, and Herceptin-POEGMA group. (d) Quantitative analysis of fluorescence intensity of the Herceptin group and Herceptin-POEGMA group showed that the biological activity of Herceptin-POEGMA was 86.4% of that of Herceptin, indicating that this polymerization method has a significant advantage in maintaining the activity of antibody drugs.

[0038] Figure 15(a) Comparison photos of the GOX-PDEGMA solution before and after centrifugation at 10,000 rpm for 10 minutes at room temperature. (b) Comparison of relative enzyme activities between the GOX-PDEGMA solution before centrifugation (+GOX-PDEGMA) and the supernatant after centrifugation (-GOX-PDEGMA). Based on the direct proportionality between the enzyme activity and mass of GOX-PDEGMA under experimental conditions, the removal efficiency of the temperature-induced phase change combined with centrifugation was evaluated by measuring the enzyme activity of GOX-PDEGMA. The enzyme activity of GOX-PDEGMA before centrifugation was defined as 100%, and the residual enzyme activity in the supernatant after centrifugation was only 0.77%. Therefore, the centrifugation method achieves a removal efficiency of over 99% for GOX-PDEGMA.

[0039] Figure 16 SDS-PAGE analysis was performed on HSA (lane 2), HSA-Br (lane 3), and unpurified HSA-POEGMA prepared with different concentrations of GOX-PDEGMA (lanes 4-15, corresponding to 1 μM, 500 nM, 100 nM, 20 nM, 4 nM, 400 pM, 40 pM, 4 pM, 400 fM, 40 fM, 4 fM, and 0 fM, respectively). The results showed that when the GOX-PDEGMA concentration was in the range of 100 nM to 1 μM, obvious high molecular weight bands were observed on the SDS-PAGE gel, while weak residual bands appeared around 66 kDa. These residual bands slightly increased with decreasing GOX-PDEGMA concentration, indicating good monomer conversion and initiation efficiency at higher GOX-PDEGMA concentrations, thus enabling successful polymerization. In contrast, when the GOX-PDEGMA concentration was between 4 pM and 20 nM, the monomer conversion and the initiation efficiency of the HSA-Br macromolecular initiator decreased significantly. At GOX-PDEGMA concentrations as low as ≤400 fM, the initiation efficiency and monomer conversion were extremely low or almost negligible. These results indicate that a GOX-PDEGMA concentration of at least 100 nM is required to achieve acceptable polymerization conversion. To ensure optimal reaction results, a 1 μM concentration was consistently used thereafter. Furthermore, the control group (lane 15), lacking GOX-PDEGMA, showed almost no polymerization reaction, further confirming the significant inhibitory effect of oxygen on the polymerization process. Detailed Implementation

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0043] In this article, the term "open environment" refers to a reaction process that is not limited to a pressure-controlled environment, or a reaction system that is in a state where it can freely diffuse with the outside world, especially an oxygen-containing environment, such as air. It is usually contrasted with a "sealed environment" such as a sealed reactor.

[0044] In this article, the term "protein modification" refers to the process of establishing chemical or non-chemical bonds between a protein and a modifying agent to form a complex.

[0045] In this article, the term "modifier" refers to any molecule, including macromolecules, particularly polymer macromolecules, that is used to establish a chemical or non-chemical binding with a protein to alter its properties, enhance its performance, or add new functions.

[0046] In this document, the term "protein" refers to polymers of amino acid residues and their variants, as well as synthetic and naturally occurring analogs. This includes amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids (such as chemical analogs of the corresponding naturally occurring amino acids), and naturally occurring amino acid polymers and their naturally occurring chemical derivatives. Such derivatives include, for example, post-translational modifications and degradation products, including pyroglutamyl, isoaspartic, proteolytic, phosphorylated, glycosylated, oxidized, isomerized, and deamination variants. Exemplarily, proteins in this document include pharmaceutical proteins, including but not limited to peptides, enzymes, long-acting protein therapeutics, antibodies, antigens, vaccines, and in vitro diagnostic reagents.

[0047] In this article, the term "protein modifier" refers to a protein-polymer conjugate obtained by reacting a protein with a modifier.

[0048] [First Modification Method]

[0049] A first aspect of the present invention provides a method for modifying proteins in an open environment, which is a method for modifying proteins containing initiating groups, specifically including but not limited to the following steps:

[0050] (1) Construct an open reaction system comprising a monomer of a polymer modifier, a protein containing an initiating group and a deoxygenation system, and optionally further comprising other components, such as free radical stabilizers, reducing agents, copper salts and their ligand compounds.

[0051] (2) Allow the open reaction system to react at the first temperature; and

[0052] (3) After heating to the second temperature and letting it stand, centrifuge directly to obtain the supernatant containing the protein modifier.

[0053] Those skilled in the art should understand that the numbers (1), (2), etc., are only for distinguishing different steps and do not indicate the order of the steps. As long as the purpose of the present invention can be achieved, the order of the above steps is not particularly limited. In addition, two or more of the above steps can be combined and performed simultaneously. For example, steps (1) and (2) can be performed simultaneously, that is, the two steps can be performed at the same time, or steps (1) and (2) can be combined into one step.

[0054] Step (1):

[0055] Step (1) of the present invention is a step of constructing an open reaction system, wherein the open reaction system comprises a monomer of a polymer modifier, a protein containing an initiating group and a deoxygenation system, and optionally further comprises other components, such as a free radical stabilizer, a reducing agent copper salt and its ligand compound.

[0056] In this invention, the monomer of the polymer modifier (sometimes simply referred to herein as "monomer" or "the monomer of this invention") refers to the structural unit used to polymerize and obtain the polymer modifier. For example, it is a monomer containing a vinyl double bond structure capable of forming a polymer chain through free radical polymerization or controlled free radical polymerization, such as (meth)acrylates or (meth)acrylamides. Preferably, it is hydrophilic or hydrophobic, suitable for polymerization reactions in the corresponding environment. Preferably, the monomer contains a variety of functional side chain groups, such as hydroxyl, phosphate, quaternary ammonium salt, and amino groups, thereby endowing the polymer with diverse physicochemical properties and good biocompatibility. Preferably, by regulating the monomer structure, the polymer's stimuli responsiveness, stability, and good interaction with proteins can be achieved. Examples of monomers include, but are not limited to, OEGMA, HEMAP, MPC, DMAEMA, HPMA, methyl methacrylate (MMA), hydroxyethyl methacrylate (HEMA), SBMA (sulfobetaine methacrylate), CBMA (carboxybetaine methacrylate), acrylic acid (AAc), NIPAM (N-isopropylacrylamide), GMA (glycidyl methacrylate), propyl 3-sulfonate methacrylate (SPMA), isooctyl methacrylate (EHMA), and VIM (vinylimidazole). The open reaction system of this invention may contain multiple monomers simultaneously, i.e., it may be one or a combination of the above monomers.

[0057] In this invention, the monomers of the polymer modifier are polymerized to form the polymer modifier, which binds to proteins, particularly covalently. The polymer modifier generally possesses physical properties different from those of the catalytic deoxygenating polymer; preferably, it is not thermally responsive, or although it is thermally responsive, the LCST of the polymer modifier differs from that of the catalytic deoxygenating polymer, for example, by more than 5°C, preferably more than 10°C, more preferably more than 15°C, such as more than 20°C. Typically, the polymer modifier is a water-soluble polymer or a hydrophilic polymer, or a polymer that forms dispersible particles in an aqueous solution. Examples of dispersible particles include, but are not limited to, micelles, nanoparticles, and self-assemblies.

[0058] In this invention, a protein containing an initiating group refers to a protein that has been activated or modified to contain an initiating group, thereby enabling it to react with monomers. The initiating group is not limited, and includes initiating groups used in ATRP reactions, as well as initiating groups used in RAFT, NMP, iniferter, and ROMP reactions. The initiating groups of this invention are not limited to specific structures, and examples include, but are not limited to: bromine, chlorine, and iodine groups for atom transfer radical polymerization (ATRP), and some sulfonate groups can also be used as ATRP initiators; chain transfer agents with thiocarbonyl structures for reversible addition-fragmentation chain transfer polymerization (RAFT), including dithiobenzoate, trithiocarbonate, dithiocarbamate, and xanthate; stable nitroxide radical groups for nitroxide-mediated polymerization (NMP), such as TEMPO and its derivatives (non-nitroso groups); iniferter groups (such as diphenyl dithiocarbamate) and conventional photoinitiators for photoinduced radical polymerization; and metal catalytic initiating groups for cyclic olefin ring-opening polymerization (ROMP), such as ruthenium-based Grubbs catalysts and molybdenum / tungsten-based Schrock catalysts.

[0059] In the open reaction system of the present invention, the molar ratio of the monomer of the polymer modifier to the protein containing the initiating group is generally 100-100000:1, preferably 500-50000:1, more preferably 800-10000:1, for example, 1000:1, 1500:1, 2000:1, 2500:1, 3000:1, 3500:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, etc.

[0060] In this invention, a deoxygenation system refers to a system used to react with oxygen in an open environment to consume or remove oxygen, which typically includes a catalytic deoxygenation polymer, a substrate, and a product scavenger. In this invention, a polymer modifier refers to a molecule used to modify a protein that is a polymer; specifically, polymers include molecules obtained from monomers through polymerization reactions.

[0061] In this invention, the catalytic deoxygenating polymer refers to a polymer with catalytic activity that converts oxygen into other substances, thereby removing or eliminating oxygen. It typically exists in two different states: dissolved or dispersed, and aggregated or precipitated. The state can be altered by changing conditions such as temperature or pH. For example, it may be in a dissolved or dispersed state at a first temperature, and in an aggregated or precipitated state at a second temperature.Catalytic deoxygenation polymers may comprise a catalytic moiety and a thermally responsive moiety. The catalytic moiety can be an enzyme, such as glucose oxidase, lipoxygenase, cholesterol oxidase, L-amino acid oxidase, D-amino acid oxidase, lactate oxidase, alcohol oxidase, polyphenol oxidase, xanthine oxidase, tyrosinase, ceruloplasmin (Laccase), aldehyde oxidase, amine oxidase (including copper-dependent types), uricase oxidase, galactose oxidase, L-glutamate oxidase, glycolate oxidase, thiol oxidase, tryptophan 2,3-dioxygenase, methylamine oxidase, aromatic compound dioxygenases (such as benzene dioxygenase), polyamine oxidase, etc. The thermally responsive moiety includes polymers... Polymers, especially those with a low critical solution temperature (LCST), i.e., polymers that change from a soluble state to an insoluble state at temperatures above the LCST, include, but are not limited to, poly(N-isopropylacrylamide) (PNIPAM), poly(N-vinylcaprolactam) (PVCL), poly(N,N-diethylacrylamide) (PDEAM), poly(N-ethylacrylamide) (PEAM), poly(N,N-dimethylacrylamide) (PDMAM), poly(N-n-propylacrylamide) (PnNPAM), poly(N-cyclopropylacrylamide) (PCPAM), and poly(2-ethoxyethyl acrylate) (PEOE). A) Poly(2-ethoxyethoxyethyl acrylate) (PDEGMA), Poly(2-propoxyethyl acrylate) (PPOEGA), Poly(2-isopropoxyethyl acrylate) (PiPGEA), Poly(2-methoxyethyl acrylate) (PMEO2MA), Poly(2-(2-methoxyethoxy)ethyl acrylate) (PMDEGA), Poly(2-(diethylamino)ethyl methacrylate) (PDEAEMA), Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), Poly(N-vinylcaprolactam) (PVCL), Poly(methyl vinyl ether) (PMVE) and their derivatives. Copolymers such as methyl vinyl ether-maleic anhydride copolymer (PMVE-MA), poly(2-methoxyethoxyphosphazene) (PMOEOP), hydroxybutyl chitosan (HBC), some methyl cellulose (MC) derivatives, poly(sulfobetaine methacrylate) (PSBMA) and other zwitterionic polymers, as well as block or graft copolymers such as poly(N-isopropylacrylamide)-b-polyethylene glycol (PNIPAM-b-PEG), poly(lactic acid-co-glycolic acid)-g-poly(N-vinylcaprolactam) (PLGA-g-PVCL), polyoxyethylene-polyoxypropylene block copolymer (Pluronic), etc.

[0062] In some embodiments, the concentration of the catalytic deoxygenating polymer of the present invention in the open reaction system is generally 0.01-10 μM, preferably 0.05-5 μM, more preferably 0.08-3 μM, and most preferably 0.1 μM-1 μM. Examples include 0.1 μM, 0.2 μM, 0.5 μM, 0.8 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, and 4 μM. When the concentration is too low, the polymerization reaction tends to be inhibited. For example, when the catalytic component in the catalytic deoxygenating polymer is glucose oxidase, if its concentration is below 100 nM, the polymerization reaction tends to be inhibited. However, when its concentration is above 1 μM, the reaction efficiency is high.

[0063] In this invention, the substrate refers to the consumable component required for the catalytic deoxygenated polymer to react with oxygen, and reducing sugars can be used, including but not limited to mannose, glucose, fructose, etc.

[0064] In some embodiments, the concentration of the substrate (e.g., glucose) in the reaction system is generally 0.001-10 M, preferably 0.01-5 M, more preferably 0.05-1 M, for example, 0.08 M, 0.09 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, etc. If the concentration is too low, deoxygenation tends to be incomplete, or even significantly reduced by oxygen interference.

[0065] In this invention, the product scavenger refers to a substance used to remove the products of the reaction between catalytically deoxygenated polymers and oxygen. Examples include, but are not limited to, pyruvate or its salts, thiosulfate or its salts, and acetylacetonate or its salts. Here, the salt is generally a monovalent or divalent salt, and examples include, but are not limited to, sodium salts and potassium salts. From the perspective of system stability, this invention preferably uses pyruvate or its salts, such as sodium pyruvate.

[0066] In some embodiments, the concentration of the product scavenger (such as sodium pyruvate) in the reaction system is 0.01-5 M, preferably 0.05-2 M, for example, 0.08 M, 0.09 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 1 M, 1.5 M, etc. If the concentration of the product scavenger is too low, harmful products generated in the system, such as hydrogen peroxide, tend not to be removed, and their oxidative byproducts damage proteins, ultimately affecting the quality and bioactivity of the product.

[0067] In this invention, the open reaction system may optionally further comprise a free radical stabilizer, examples of which include, but are not limited to, halide salts, such as fluorides, bromides, or iodides. Combinations of the above two salts may be used in this invention. Examples of salts are not limited, including monovalent salts such as sodium and / or potassium salts. For example, sodium chloride or sodium iodide.

[0068] In some embodiments, the concentration of the free radical stabilizer of the present invention in the open reaction system is generally 0.01-5 M, preferably 0.05-1 M, for example, 0.08 M, 0.09 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, etc. If the concentration is too low, it tends to cause the polymerization reaction to lose free radical stability, the molecular weight distribution to broaden, and the polymerization efficiency to decrease.

[0069] In this invention, the open reaction system may optionally further include a reducing agent. Examples include copper, iron, silver, and other metals, particularly copper-containing reducing agents such as Cu(I) salts (e.g., CuCl or CuBr), and especially copper metals such as copper wire. Without a reducing agent, a stable source of Cu(I) cannot be provided, resulting in a significant reduction in catalytic efficiency and polymerization rate, or even complete cessation of polymerization.

[0070] In this invention, the open reaction system may optionally further comprise a copper salt and its ligand compound. The copper salt includes monovalent and / or divalent copper salts, such as CuBr2. Examples of ligand compounds are not limited to Me6TREN or TREN.

[0071] Step (2):

[0072] Step (2) of the present invention is a polymerization reaction step, which includes reacting the open reaction system at a first temperature.

[0073] In this invention, polymerization reaction generally refers to controlled radical polymerization (CRP), which includes, but is not limited to, atom transfer radical polymerization (ATRP), reversible addition-fracture transfer polymerization (RAFT), nitride-mediated polymerization (NMP), iniferter polymerization, iodine transfer polymerization (ITP), organotellurium-mediated polymerization (TERP), ROMP polymerization, and ROP polymerization.

[0074] In this invention, the first temperature is typically the temperature during a mild reaction, such as room temperature, or a temperature range of 0-40°C, preferably 5-35°C, more preferably a range of 10-30°C, such as 15°C, 20°C, 25°C, 30°C, etc.

[0075] The polymerization reaction conditions of the present invention are not particularly limited. Generally, the reaction is carried out at a first temperature, such as room temperature or below, for example, 4°C. The reaction can be carried out under static or stirring conditions, and there is no limitation on this. The polymerization reaction is not limited, and generally lasts from 10 minutes to 10 hours, preferably from 30 minutes to 8 hours, more preferably from 1 hour to 6 hours, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, etc.

[0076] In some embodiments, the reaction rate is controlled by inserting and removing a copper wire during the polymerization process. For example, after the reactants are added to a centrifuge tube, the copper wire is added and reacted for 1-20 minutes, then the copper wire is removed for 1-20 minutes, and then the copper wire is reinserted, and this process is repeated until the total reaction time reaches 50-200 minutes, for example, 120 minutes. Preferably, samples are taken every 1-30 minutes.

[0077] Step (3):

[0078] Step (3) of this invention is a separation step, which includes heating to a second temperature and allowing it to stand, followed by direct centrifugation to obtain a supernatant containing the protein modifier. It should be noted that after heating and allowing it to stand, separation can be performed directly without any other operations, such as centrifugation, thereby greatly simplifying the separation or purification steps.

[0079] In this invention, the first temperature and the second temperature refer to different temperatures or temperature ranges for the purpose of distinction only, and do not imply that the temperature is high or low. However, in some embodiments herein, the first temperature is lower than the second temperature.

[0080] In this document, the range of the second temperature can be, for example, 20-50°C, preferably 25-45°C, more preferably 30-40°C, for example, 35°C. It should be noted that the second temperature can be freely chosen within the above range, as long as the second temperature is higher than the first temperature. In some embodiments, the first temperature in this document needs to be lower than the phase transition temperature (LCST) of the catalytic deoxygenated polymer, while the second temperature needs to be higher than the LCST.

[0081] Other steps:

[0082] Those skilled in the art should also understand that other steps or operations may be included before or after steps (1)-(3) above, or between any of these steps, for example, to further optimize and / or improve the method described in this invention.

[0083] By way of example, after steps (1) to (3) above, the present invention further includes a step of further purifying the obtained supernatant. These purification steps are not particularly limited and can be any purification means known in the art, including but not limited to filtration, chromatography, centrifugation, etc., for example, a combination of chromatographic purification.

[0084] [Second Modification Method]

[0085] A second aspect of the present invention provides a first method for modifying proteins in an open environment, which is a method for further modifying a modified protein, specifically including but not limited to the following steps:

[0086] (1) Construct an open reaction system comprising a monomer of a polymer modifier, a first modified protein containing an initiating group, and a deoxygenation system, wherein the deoxygenation system comprises a catalytic deoxygenation polymer, a substrate, and a product scavenger;

[0087] (2) Allow the open reaction system to react at the first temperature; and

[0088] (3) After heating to the second temperature and letting it stand, centrifuge directly to obtain the supernatant containing the second modified protein.

[0089] Except for replacing the protein containing the initiating group in the first modification method with the first modified protein containing the initiating group, all other steps or conditions are the same as in the first modification method.

[0090] In this invention, the first modified protein refers to a protein that has been modified. The first modified protein can be a protein-polymer obtained by the first modification method, or any known modified protein or any modified protein obtained by a known method.

[0091] [Protein Modifiers]

[0092] A third aspect of the invention provides a protein modifier obtained by the first or second modification method of the invention.

[0093] In some embodiments, the number of monomers in the polymer of the protein modifier of the present invention is not limited, for example, it can be 50-50000, preferably 80-30000, more preferably 100-20000, such as 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 8000, 10000, 12000, 14000, 16000, etc.

[0094] Example 1

[0095] 1. Synthesis of GOX-PDEGMA:

[0096] 80 mg of lyophilized GOX powder was dissolved in 10 mL of PBS buffer (10 mM, NaCl 150 mM, pH 8.0), and 250 μL of 200 mM NHS-TEG-Br (dissolved in DMF) was added. The reaction was allowed to proceed statically at 4 °C for 12 hours without stirring. GOX-Br was obtained after purification by a desalting column. Subsequently, 8 mg of GOX-Br and 37.6 μL of DEGMA were dissolved in 1 mL of PBS solution. After deoxygenation by bubbling under nitrogen for 30 minutes, a deoxygenation catalyst solution consisting of 0.2 mg CuBr, 0.8 mg CuBr2, and 3.13 mg HMTETA (deoxygenated under nitrogen) was added. The Schlenk tube was sealed, and the reaction was terminated by exposure to air after 2 hours at room temperature. The product was purified by temperature-induced phase separation and column chromatography to obtain GOX-PDEGMA.

[0097] 2. Synthesis of HSA-Br:

[0098] 132 mg of lyophilized HSA powder was dissolved in 20 mL of Tris-HCl buffer (50 mM, NaCl 150 mM, pH 7.4), and 400 μL of 50 mM DBMP (dissolved in DMF) was added. The reaction was carried out at room temperature for 2 hours. HSA-Br was obtained by purification using a desalting column.

[0099] 3. Synthesis of HSA-POEGMA:

[0100] Add 30 μL of 2 M glucose solution, 30 μL of 1 M sodium bromide solution, 30 μL of 1 M sodium pyruvate solution, 1.8 mg of HSA-Br, and 14 μL of OEGMA (molar ratio of OEGMA to HSA-Br is 1000) to a centrifuge tube or 96-well plate. Then add 0.1 mg of CuBr2, 0.4 μL of tris(2-dimethylaminoethyl)amine (Me6TREN), and 100 μL of 3 μM GOX-PDEGMA solution, adjusting the total reaction volume to 300 μL. Soak 10 mg of copper wire (0.2 mm in diameter, purity >99.999%) in 37% concentrated hydrochloric acid for 15 minutes, then transfer the copper wire to the reaction tube after rinsing. Incubate the reaction system at 4 °C for 2 hours without stirring. After the reaction was completed, the copper wire was removed and allowed to stand at room temperature for a period of time. Then, it was centrifuged at 10,000 rpm for 10 minutes to remove GOX-PDEGMA. Finally, HSA-PDEGMA was obtained by purification by desalting column and anion exchange chromatography.

[0101] Example 2

[0102] This embodiment describes the synthesis of HSA-PHEMAP. Except for the following steps, the rest of the steps are the same as in Example 1.

[0103] Add 30 μL of 2 M glucose solution, 30 μL of 1 M sodium bromide solution, 30 μL of 1 M sodium pyruvate solution, 1.8 mg HSA-Br, and 90 μL of 100 mM HEMAP (HEMAP to HSA-Br molar ratio of 300) to a centrifuge tube or 96-well plate. Then add 0.1 mg CuBr2, 0.4 μL of tris(2-dimethylaminoethyl)amine (Me6TREN), and 100 μL of 3 μM GOX-PDEGMA solution, adjusting the total reaction volume to 300 μL. Soak 10 mg of copper wire (0.2 mm diameter, purity >99.999%) in 37% concentrated hydrochloric acid for 15 minutes, rinse, and transfer to the reaction tube. Incubate the reaction system at 4 °C for 2 hours without stirring. After the reaction was completed, the copper wire was removed and allowed to stand at room temperature for a period of time. Then, it was centrifuged at 10,000 rpm for 10 minutes to remove GOX-PDEGMA. Finally, HSA-PHEMAP was obtained by desalting column and anion exchange chromatography.

[0104] Example 3

[0105] This embodiment describes the synthesis of HSA-PMPC. Except for the following steps, the rest of the steps are the same as in Example 1.

[0106] Add 30 μL of 2 M glucose solution, 30 μL of 1 M sodium bromide solution, 30 μL of 1 M sodium pyruvate solution, 1.8 mg of HSA-Br, and 60 μL of 1 M MPC (MPC to HSA-Br molar ratio of 2000) to a centrifuge tube or 96-well plate. Then add 0.1 mg of CuBr2, 0.4 μL of tris(2-dimethylaminoethyl)amine (Me6TREN), and 100 μL of 3 μM GOX-PDEGMA solution, adjusting the total reaction volume to 300 μL. Soak 10 mg of copper wire (0.2 mm diameter, purity >99.999%) in 37% concentrated hydrochloric acid for 15 minutes, rinse, and transfer to the reaction tube. Incubate the reaction system at 4 °C for 2 hours without stirring. After the reaction was complete, the copper wire was removed and allowed to stand at room temperature for a period of time. Then, it was centrifuged at 10,000 rpm for 10 minutes to remove GOX-PDEGMA. Finally, HSA-PMPC was obtained by desalting column and anion exchange chromatography.

[0107] Example 4

[0108] This embodiment describes the synthesis of HSA-PDMAEMA. Except for the following steps, the rest of the steps are the same as in Example 1.

[0109] Add 30 μL of 2 M glucose solution, 30 μL of 1 M sodium bromide solution, 30 μL of 1 M sodium pyruvate solution, 1.8 mg of HSA-Br, and 9.4 μL of DMAEMA (DMAEMA to HSA-Br molar ratio of 2000) to a centrifuge tube or 96-well plate. Then add 0.1 mg of CuBr2, 0.4 μL of tris(2-dimethylaminoethyl)amine (Me6TREN), and 100 μL of 3 μM GOX-PDEGMA solution, adjusting the total reaction volume to 300 μL. Soak 10 mg of copper wire (0.2 mm diameter, purity >99.999%) in 37% concentrated hydrochloric acid for 15 minutes, rinse, and transfer to the reaction tube. Incubate the reaction system at 4 °C for 2 hours without stirring. After the reaction was completed, the copper wire was removed and allowed to stand at room temperature for a period of time. Then, it was centrifuged at 10,000 rpm for 10 minutes to remove GOX-PDEGMA. Finally, HSA-PDMAEMA was obtained by purification by desalting column and anion exchange chromatography.

[0110] Example 5

[0111] This embodiment describes the synthesis of HSA-PHPMA. Except for the following steps, the remaining steps are the same as in Example 1.

[0112] Add 30 μL of 2 M glucose solution, 30 μL of 1 M sodium bromide solution, 30 μL of 1 M sodium pyruvate solution, 1.8 mg HSA-Br, and 8.6 μL HPMA (HPMA to HSA-Br molar ratio of 1500) to a centrifuge tube or 96-well plate. Then add 0.1 mg CuBr2, 0.4 μL tris(2-dimethylaminoethyl)amine (Me6TREN), and 100 μL of 3 μM GOX-PDEGMA solution, adjusting the total reaction volume to 300 μL. Soak 10 mg of copper wire (0.2 mm diameter, purity >99.999%) in 37% concentrated hydrochloric acid for 15 minutes, rinse, and transfer to the reaction tube. Incubate the reaction system at 4 °C for 2 hours without stirring. After the reaction, remove the copper wire, allow it to stand at room temperature for a period of time, then centrifuge at 10,000 rpm for 10 minutes to remove GOX-PDEGMA. Purify the HSA-PDEGMA by size exclusion chromatography (SEC).

[0113] Example 6

[0114] This embodiment describes the synthesis of HSA-POEGMA-PHPMA. Except for the following steps, the remaining steps are the same as in Example 2.

[0115] After the synthesis and purification of HSA-POEGMA, in-situ ATRP-initiated HPMA polymerization was performed. In a centrifuge tube or 96-well plate, 30 μL of 2 M glucose solution, 30 μL of 1 M sodium bromide solution, 30 μL of 1 M sodium pyruvate solution, 100 µM HSA-POEGMA, and 8.6 μL of HPMA (HPMA to HSA-POEGMA molar ratio of 1500) were added, followed by 0.1 mg CuBr2, 0.4 μL of tris(2-dimethylaminoethyl)amine (Me6TREN), and 100 μL of 3 μM GOX-PDEGMA solution, adjusting the total reaction volume to 300 μL. 10 mg of copper wire (0.2 mm diameter, purity >99.999%) was soaked in 37% concentrated hydrochloric acid for 15 minutes, rinsed, and then transferred to the reaction tube. The reaction system was incubated at 4 °C for 2 hours without stirring. After the reaction was completed, the copper wire was removed and allowed to stand at room temperature for a period of time. Then, it was centrifuged at 10,000 rpm for 10 minutes to remove GOX-PDEGMA. HSA-PDEGMA-PHPMA was obtained by size exclusion chromatography (SEC).

[0116] Example 7

[0117] This example illustrates polymerization reactions of varying volumes. In the reaction systems of Examples 1-6, such as the HSA-POEGMA reaction system, the concentration of reactants was kept constant, and the reaction volume was increased to 100 mL or decreased to 10 µL for polymerization. All other conditions remained unchanged.

[0118] Example 8

[0119] 1. Preparation and polymerization reaction of IFN-Br:

[0120] The reaction mixture containing 100 mM IFN-LPETGGH6, 50 mM Sortase A (SrtA), and 2 mM AEBBM (buffer: 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, pH 7.4) was incubated overnight at room temperature without stirring. The mixture was then purified using an anion exchange column (HiTrap Capto Q, Cytiva). The equilibration buffer was 20 mM Tris-HCl, 1 M NaCl, pH 7.4, and the eluent was the same buffer system. A gradient elution was performed on the reaction mixture, and the fraction showing the IFN-Br peak was collected. The solution was then replaced with a desalting column containing 10 mM PBS (pH 7.4) and 10% glycerol, and concentrated by ultrafiltration (Amicon ultra 4 mL, 3K MWCO, Millipore) for subsequent use.

[0121] 2. Recovery of GOX-PDEGMA from Example 1:

[0122] In the preparation of HSA-POEGMA, 30 µL of 2 M glucose, 30 µL of 1 M sodium bromide, 30 µL of 1 M sodium pyruvate, 1.8 mg HSA-Br, 14 µL of OEGMA (OEGMA to HSA-Br molar ratio of 1000), 0.1 mg CuBr2, 0.4 µL of Me6TREN, and 100 µL of 3 µM GOX-PDEGMA were added to a centrifuge tube, bringing the reaction volume to 300 µL. Copper wire (10 mg, diameter 0.2 mm, purity >99.999%) was pretreated with 37% concentrated hydrochloric acid for 15 minutes, rinsed thoroughly, and added to the reaction tube. The system was incubated at 4 °C for 2 hours. After the reaction, the copper wire was removed, and the mixture was allowed to stand at room temperature for a period of time before centrifugation at 10,000 rpm for 10 minutes to remove GOX-PDEGMA. The recovered GOX-PDEGMA was washed several times with PBS and stored for later use.

[0123] 3. Synthesis of IFN-POEGMA:

[0124] In the polymerization reaction, 1 µL of 2 M glucose, 1 µL of 1 M sodium bromide, 1 µL of 1 M sodium pyruvate, 100 µM IFN-Br, 0.5 µL of OEGMA (OEGMA to IFN-Br molar ratio of 1000), 0.003 mg of CuBr2, 0.013 µL of Me6TREN, and 3.3 µL of 3 µM GOX-PDEGMA were added to a centrifuge tube, for a total reaction volume of 10 µL. Copper wire (0.3 mg, 0.2 mm diameter, purity >99.999%) was pretreated with 37% concentrated hydrochloric acid for 15 minutes, rinsed thoroughly, and transferred to the above reaction vessel. The reaction system was allowed to stand at 4 °C for 2 hours without stirring.

[0125] Example 9

[0126] 1. Preparation and polymerization of Herceptin-Br:

[0127] Herceptin was dissolved in 1 mL of Tris-HCl buffer (20 mM, 150 mM NaCl, 5 mM EDTA, pH 7.4) to a final concentration of 4 mg / mL. 15 µL of 10 mM TCEP (dissolved in water) was added to the mixture, and the mixture was incubated at 37 °C for 2 hours. Then, 15 µL of 100 mM DBEB was added, and the reaction was carried out overnight at 37 °C. Herceptin-Br was purified by desalting column chromatography and concentrated using ultrafiltration (Amicon ultra 4 mL, 10K MWCO, Millipore) for subsequent use.

[0128] 2. Same as step 2 in Example 8.

[0129] 3. Synthesis of Herceptin-OEGMA:

[0130] In the polymerization reaction system, 1 µL of 2 M glucose, 1 µL of 1 M sodium bromide, 1 µL of 1 M sodium pyruvate, 100 µM Herceptin-Br, 0.5 µL of OEGMA (OEGMA to Herceptin-Br molar ratio of 1000), 0.003 mg of CuBr2, 0.013 µL of Me6TREN, and 3.3 µL of 3 µM GOX-PDEGMA were added, bringing the final volume to 10 µL. 0.3 mg of copper wire (0.2 mm diameter, purity >99.999%) was soaked in 37% concentrated hydrochloric acid for 15 minutes, rinsed thoroughly, and then added to the reaction system. The reaction was allowed to proceed at 4 °C for 2 hours without stirring.

[0131] Example 10

[0132] This embodiment evaluates the oxygen-deoxidizing capacity-induced polymerization reaction of different concentrations of GOX-PDEGMA: Following the aforementioned preparation method for HSA-PDEGMA, polymerization reactions were conducted using 12 different concentration gradients of GOX-PDEGMA (1 µM, 500 nM, 100 nM, 20 nM, 4 nM, 400 pM, 40 pM, 4 pM, 400 fM, 40 fM, 4 fM, and 0 fM). The reaction products were analyzed by SDS-PAGE to evaluate their polymerization effect. Furthermore, higher concentrations of GOX-PDEGMA were also tested, demonstrating that the effects of this invention can be achieved even at 100 µM.

[0133] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A method for modifying proteins in an open environment, characterized in that, Includes the following steps: (1) Construct an open reaction system comprising a monomer of a polymer modifier, a protein containing an initiating group, a deoxygenation system, a stabilizer, a reducing agent, and a divalent copper salt and its ligand, wherein the ligand includes Me6TREN or TREN, wherein the deoxygenation system comprises a catalytic deoxygenation polymer, a substrate, and a product scavenger, wherein the protein containing the initiating group is a protein containing a bromine group, the stabilizer is sodium bromide, the reducing agent is copper metal, the substrate is glucose, and the product scavenger is sodium pyruvate; (2) The open reaction system is subjected to controlled free radical polymerization at a first temperature to obtain a reaction solution containing polymer-modified proteins and catalytically deoxygenated polymers; and (3) After heating the reaction solution to the second temperature and letting it stand, the reaction solution is directly centrifuged to obtain the supernatant containing the protein modifier without any other operation; The polymer modifier is a water-soluble polymer or a hydrophilic polymer, or a polymer that forms dispersed particles in an aqueous solution, and the polymer modifier is not thermally responsive or, when it is thermally responsive, its LCST differs from that of the catalytic deoxygenating polymer by more than 5°C. The catalytic deoxygenating polymer is in a dissolved or dispersed state and has catalytic activity at a first temperature, and in an aggregated or precipitated state at a second temperature. The first temperature is lower than the LCST of the catalytic deoxygenating polymer, while the second temperature is higher than the LCST of the catalytic deoxygenating polymer. The catalytic deoxygenating polymer comprises a catalytic portion and a thermally responsive polymer. The catalytic deoxygenating polymer is GOX-PDEGMA and its LCST is 21-23°C. In GOX-PDEGMA, GOX and PDEGMA have a conjugated relationship.

2. The method for modifying proteins in an open environment according to claim 1, characterized in that, The process further includes step (4), which involves further purifying the supernatant to obtain a protein modifier.

3. A method for modifying proteins in an open environment, characterized in that, Includes the following steps: (1) Construct an open reaction system comprising a monomer of a polymer modifier, a first modified protein containing an initiating group, a deoxygenation system, a stabilizer, a reducing agent, and a divalent copper salt and its ligand, wherein the ligand includes Me6TREN or TREN, wherein the deoxygenation system comprises a catalytic deoxygenation polymer, a substrate, and a product scavenger, wherein the protein containing the initiating group is a protein containing a bromine group, the stabilizer is sodium bromide, the reducing agent is copper metal, the substrate is glucose, and the product scavenger is sodium pyruvate; (2) The open reaction system is subjected to controlled free radical polymerization at a first temperature to obtain a reaction solution containing polymer-modified proteins and catalytically deoxygenated polymers; and (3) After heating the reaction solution to the second temperature and letting it stand, the reaction solution is directly centrifuged to obtain the supernatant containing the second modified protein without any other operation. The polymer modifier is a water-soluble polymer or a hydrophilic polymer, or a polymer that forms dispersed particles in an aqueous solution, and the polymer modifier is not thermally responsive or, when it is thermally responsive, its LCST differs from that of the catalytic deoxygenating polymer by more than 5°C. The catalytic deoxygenating polymer is in a dissolved or dispersed state and has catalytic activity at a first temperature, and in an aggregated or precipitated state at a second temperature. The first temperature is lower than the LCST of the catalytic deoxygenating polymer, while the second temperature is higher than the LCST of the catalytic deoxygenating polymer. The second temperature is higher than the first temperature. The catalytic deoxygenating polymer is GOX-PDEGMA and its LCST is 21-23°C. In GOX-PDEGMA, GOX and PDEGMA have a conjugated relationship.

4. The method for modifying proteins in an open environment according to claim 3, characterized in that, The first modified protein is a modified protein obtained by the method of claim 1 or 2.

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