Preparation method of polyion liquid macromonomer with stable protein structure

Abstract

The invention discloses a preparation method of a polyion liquid macromonomer with a stable protein structure, and belongs to the technical field of high polymer material preparation and protein recognition, and a functional element, an inert element and a cross-linking element are polymerized by adopting an RAFT (reversible addition fragmentation chain transfer) polymerization method to form the polyion liquid macromonomer. The structural formula of the polyionic liquid macromonomer is p (PVIM-b-(VSPIM-co-VIMA-co-VIMCDs)), PVIM is 1-vinyl-3-styrene imidazole chloride ionic liquid, VSPIM is 1-vinyl-3-propane sulfonic acid imidazole chloride zwitterionic liquid, VIMA is 1-vinyl-3-acetamide imidazole chloride ionic liquid, and VSPIM is 1-vinyl-3-propane sulfonic acid imidazole chloride zwitterionic liquid. And the VIMCDs is a toluenesulfonated 1-vinyl-beta-cyclodextrin imidazole ionic liquid. The molecular formula of the ionic liquid is shown in the description. According to the preparation method disclosed by the invention, the preparation of the polyion liquid macromonomer which is designed by active / controllable free radical polymerization and has multiple anchoring protein effects is realized, and the problem of poor selective recognition caused by inaccurate imprinting and single functional monomer-protein interaction in the western blot process is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer material preparation and its application in protein recognition technology, and particularly relates to a preparation method of a polyionic liquid macromonomer for stabilizing protein structure. Background Art

[0002] By using functional monomers with multiple interaction modes to synergistically imprint proteins, it is also possible to better address the problems of the complex surface structure of proteins and the single interaction between proteins and monomers. In existing research, the metal coordination strategy has an enhancing effect on protein imprinting recognition. Designing and synthesizing multiple functional monomers for protein pre-fixation also improves the protein imprinting recognition performance. Aiming at the competition and interference of the imprinting medium on the interaction between the target protein and the functional monomer, Du et al. prepared a series of highly cross-linked raspberry balls functionalized with different ionic liquids on the surface and investigated their adsorption capacity for target molecules; the research results found that multiple interaction sites between proteins and functional groups can enhance the interaction between the two and thus improve the imprinting efficiency. For example, the hydrophobic inner cavity and hydrophilic outer wall structure of cyclodextrin enable it to include target molecules through host-guest interactions. Zhang et al. utilized the property of ionic liquid-β-cyclodextrin to provide multiple interactions and high affinity in an aqueous environment to prepare a novel ionic liquid-functional monomer-based antigenic determinant magnetic imprinted microsphere based on cyclodextrin and achieved effective recognition of cytochrome C.

[0003] Therefore, introducing design elements with multiple affinity effects on the target protein into the imprinting system to anchor proteins is an effective strategy to improve protein imprinting efficiency. For example, while not affecting the specific adsorption of the imprinting site to the target protein, introducing an "inert component", an amphoteric ion structure segment that resists protein adsorption, to increase the selectivity of the imprinted polymer for the target. The team of Zhang Qiuyu at Northwestern Polytechnical University designed and prepared a highly specific recognition oriented imprinting material for bovine serum albumin using the protein adsorption resistance property of the polymer chain segment of monomer 2-methacryloyloxyethyl phosphorylcholine (MPC). Therefore, the research and development of molecular structures that resist non-specific protein adsorption is also one of the key objectives to improve the directional imprinting and selective recognition performance of imprinted polymers.

[0004] It can be seen that one of the cores of precise protein recognition is to construct design elements that can undergo multiple affinity interactions such as electrostatic, hydrogen bond, π-π, and hydrophobic interactions with the target protein and resist non-specific protein adsorption to achieve multiple anchoring and directional imprinting. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of a polyionic liquid macromonomer for stabilizing protein structure, aiming to solve the problem of "poor selective recognition" caused by inaccurate imprinting and single interaction between functional monomer and protein during the protein imprinting process.

[0006] To achieve the above object, the present invention provides a method for preparing a polyionic liquid macromonomer for stabilizing protein structures, which includes a functional motif, an inert motif, and a crosslinking motif. The functional motif, the inert motif, and the crosslinking motif are polymerized by aqueous phase polymerization to form a polyionic liquid macromonomer.

[0007] Preferably, the functional motif is polymerized from 1-vinyl-3-acetamidoimidazolium chloride and tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomer. The inert motif is several 1-vinyl-3-propane sulfonate imidazolium chloride zwitterionic liquid monomers. The crosslinking motif is several 1-vinyl-3-styrene imidazolium chloride ionic liquid monomers. The structural formula of the polyionic liquid macromonomer is: p(PVIM -b-(VSPIM-co-VIMA -co-VIMCDs)) , where PVIM is 1-vinyl-3-styrene imidazolium chloride ionic liquid monomer, VSPIM is 1-vinyl-3-propane sulfonate imidazolium chloride zwitterionic liquid monomer, VIMA is 1-vinyl-3-acetamidoimidazolium chloride ionic liquid monomer, and VIMCDs is tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomer.

[0008] Preferably, the polymerizable imidazolium-based ionic liquid monomer is prepared by an alkylation substitution reaction of a functional monomer. The functional monomer reacts with any one of N-vinylimidazole and 4-vinylbenzyl chloride, 3-chloropropane sulfonic acid, chloroacetamide, and β-cyclodextrin to prepare 1-vinyl-3-styrene imidazolium chloride, 1-vinyl-3-propane sulfonate imidazolium chloride, 1-vinyl-3-acetamidoimidazolium chloride, and tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid respectively. For the preparation of 1-vinyl-3-styrene imidazolium chloride ionic liquid, N-vinylimidazole is first polymerized on 2-(benzylthiosulfanyl)acetic acid, and then 4-vinylbenzyl chloride is added through an alkylation reaction on the prepared precursor Precursor-3, so as to achieve the effect of polymerizing 1-vinyl-3-styrene imidazolium chloride ionic liquid on a macromolecular chain transfer agent.

[0009] Preferably, it includes the following steps: S1. Prepare polymerizable imidazolium-based ionic liquid monomers through an alkylation substitution reaction of a functional monomer, including 1-vinyl-3-propane sulfonate imidazolium chloride ionic liquid, 1-vinyl-3-acetamidoimidazolium chloride ionic liquid, and tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid; S2. Polymerize a chain transfer agent and vinylimidazole into a macromolecular chain transfer reagent by RAFT polymerization, and then prepare a precursor Precursor-1 of 1-vinyl-3-propane sulfonate imidazolium chloride zwitterionic liquid monomer on the macromolecular chain transfer reagent: p(VIM-b-VSPIM) ; S3. Using the precursor - 1 as a chain transfer agent, polymerize the 1 - vinyl - 3 - acetamidoimidazolium chloride ionic liquid monomer onto the precursor Precursor - 1 by RAFT polymerization to prepare precursor Precursor - 2: p(VIM-b-(VSPIM -co- VIMA)) ; S4. Using the precursor - 2 as a chain transfer agent, polymerize the tosylated 1 - vinyl - β - cyclodextrinimidazolium ionic liquid monomer onto the precursor Precursor - 1 by RAFT polymerization to prepare precursor Precursor - 3: p(VIM-b-(VSPIM -co-VIMA-co-VIMCDs)) ; S5. React the precursor Precursor - 3 with 4 - vinylbenzyl chloride and a very small amount of hydroquinone in dimethyl sulfoxide as a solvent to prepare the final product, the polyionic liquid macromonomer: p(PVIM -b-(VSPIM -co-VIMA -co-VIMCDs)); S6. Study the interaction between the synthesized polyionic liquid macromonomer and proteins through molecular dynamics simulation, and explore the effects of the polyionic liquid macromonomer on the secondary structure, hydrogen bonds, and energy of proteins.

[0010] Preferably, the preparation method of the 1 - vinyl - 3 - propane sulfonic acid imidazolium chloride zwitterionic liquid monomer in step S1 includes the following steps: Dissolve N - vinylimidazole in acetonitrile at a molar volume ratio of 2 mol:1 L at 50 °C. Under vigorous magnetic stirring, slowly drop 1,3 - propane sultone into the reactor using a constant - pressure funnel. The molar ratio of N - vinylimidazole to 1,3 - propane sultone is 2:3. Stir the mixture at 50 °C for 24 h to obtain a white precipitate. Further purify it by washing with acetone and perform freeze - drying by lyophilization to obtain the 1 - vinyl - 3 - propane sulfonic acid imidazolium chloride zwitterionic liquid monomer; The preparation method of the 1 - vinyl - 3 - acetamidoimidazolium chloride ionic liquid monomer in step S1 includes the following steps: Take N - vinylimidazole and chloroacetamide with a molar ratio of 33:31, dissolve and react them in a 50% ethanol solution for 24 h. After the reaction, extract with ethyl acetate, and lyophilize the extracted solution to obtain the 1 - vinyl - 3 - acetamidoimidazolium chloride ionic liquid monomer; The preparation method of the tosylated 1 - vinyl - β - cyclodextrinimidazole monomer in step S1 includes the following steps: Dissolve β-cyclodextrin in deionized water, and dropwise add an aqueous solution of sodium hydroxide and an acetonitrile solution of tosyl chloride thereto. After stirring at 23 °C and 800 rpm for 2 hours, remove the precipitate by suction filtration, and refrigerate the filtrate overnight at 4 °C. Recover the precipitate by suction filtration to obtain tosylated-β-cyclodextrin; Dissolve tosylated-β-cyclodextrin in a mixed solution of N-vinylimidazole and N,N-dimethylformamide, and react at 75 °C for 4 h under a N2 atmosphere; after cooling to ambient temperature, precipitate the crude product by using acetone, then filter, and dry overnight in a vacuum oven at 50 °C to obtain tosylated 1-vinyl-β-cyclodextrin imidazole ionic liquid.

[0011] Preferably, in step S3, precursor Precursor-1: p(VIM-b-VSPIM) The preparation specifically includes the following steps: S2-1: Using 1 mmol of 2-(benzylsulfanylthio)acetic acid as a chain transfer agent, N-vinylimidazole as a monomer, taking 40 ml of dimethyl sulfoxide as a solvent, and 0.4 mmol of azobisisobutyronitrile as an initiator, thermally initiate the reaction under a N2 atmosphere to obtain a macromolecular chain transfer reagent; S2-2: Add 1-vinyl-3-propane sulfonic acid imidazole chloride zwitterionic liquid monomer and azobisisobutyronitrile to the macromolecular chain transfer reagent to prepare precursor Precursor-1: p(VIM-b-VSPIM) ; The length of precursor Precursor-1 is adjusted by adjusting the ratio of the chain transfer agent, N-vinylimidazole, and 1-vinyl-3-propane sulfonic acid imidazole chloride zwitterionic liquid monomer.

[0012] Preferably, the preparation conditions of precursor Precursor-2 in step S3 are as follows: using precursor Precursor-1 as a chain transfer agent, dimethyl sulfoxide as a solvent, azobisisobutyronitrile as an initiator, and 1-vinyl-3-acetamide imidazole chloride ionic liquid as a monomer, and preparing at 70 °C under a N2 atmosphere.

[0013] Preferably, the preparation conditions of precursor Precursor-3 in step S4 are as follows: using precursor Precursor-2 as a chain transfer agent, dimethyl sulfoxide as a solvent, azobisisobutyronitrile as an initiator, and tosylated 1-vinyl-β-cyclodextrin imidazole ionic liquid as a monomer, and preparing at 70 °C under a N2 atmosphere.

[0014] Preferably, the preparation conditions of the polyionic liquid macromonomer in step S5 are as follows: using Precursor-3 as a chain transfer agent, dimethyl sulfoxide as a solvent, 4-vinylbenzyl chloride and a very small amount of hydroquinone for alkylation reaction, and preparing it at 60 °C under a N2 atmosphere.

[0015] Therefore, the present invention adopts the above-mentioned preparation method of a polyionic liquid macromonomer for stabilizing protein structure, and its beneficial effects are as follows: Aiming at the problem of "poor selective recognition" caused by inaccurate imprinting and single functional monomer-protein interaction during the protein blotting process, the present invention introduces an ionic liquid with protein structure stability into the imprinting system, and designs a polyionic liquid monomer with a multiple protein anchoring effect through living / controlled radical polymerization.

[0016] The technical solution of the present invention will be further described in detail below through the drawings and examples. Description of the Drawings

[0017] Figure 1 It is a flow chart of the preparation method of a polyionic liquid macromonomer for stabilizing protein structure according to the present invention; Figure 2 It is a structural schematic diagram of four polymerizable imidazolium-based ionic liquid monomers of the present invention, where (a) is 1-vinyl-3-styrylimidazolium chloride ionic liquid monomer, (b) is 1-vinyl-3-propane sulfonic acid imidazolium chloride zwitterionic liquid monomer, (c) is 1-vinyl-3-acetamide imidazolium chloride monomer, and (d) is toluenesulfonic acid 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomer; Figure 3 It is the conformations of the polymer and the protein at different simulation times; Figure 4 (a) is the change of the solvent accessible surface area (SASA) of the protein with the simulation time; (b) is the change of the number of hydrogen bonds between the protein and the small molecule with the simulation time; (c) is the interaction energy between the protein and the polymer during the whole simulation process; (d) is a schematic diagram of the change of the protein secondary structure; Detailed Embodiments

[0018] The technical solution of the present invention will be further described below through the drawings and examples.

[0019] Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meaning understood by those of ordinary skill in the field to which the present invention belongs.

[0020] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art. These other embodiments are also covered by the protection scope of the present invention.

[0021] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0022] The present invention discloses a preparation method of a polyionic liquid macromonomer for stabilizing protein structure, and its preparation method is as shown in the attached Figure 1 wherein A is a functional motif, Figure 1 B in the attached Figure 1 is an inert motif, Figure 1 and C in the attached

[0023] is a cross-linking motif. The functional motif, inert motif and cross-linking motif are polymerized by RAFT polymerization to form a polyionic liquid macromonomer. p(PVIM - b-(VSPIM -co-VIMA -co-VIMCDs)) .

[0024] Among them, PVIM is 1-vinyl-3-styrylimidazolium chloride ionic liquid monomer, VSPIM is 1-vinyl-3-propane sulfonate imidazolium chloride zwitterionic liquid monomer, VIMA is 1-vinyl-3-acetamidoimidazolium chloride ionic liquid monomer, and VIMCDs is p-toluenesulfonic acid modified 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomer. For the cytochrome C protein system, VIMCDs, VSPIM and PVIM have better recognition effects on this protein.

[0025] The polymerizable imidazolium-based ionic liquid monomer is prepared by an alkylation substitution reaction of a functional monomer: 1-vinyl-3-styrylimidazolium chloride is prepared by reacting N-vinylimidazole with vinylbenzyl chloride.

[0026] 1-vinyl-3-propane sulfonate imidazolium chloride is prepared by reacting N-vinylimidazole with 3-chloropropane sulfonic acid.

[0027] 1-vinyl-3-acetamidoimidazolium chloride is prepared by reacting N-vinylimidazole with chloroacetamide.

[0028] Prepare tosylated 1-vinyl-β-cyclodextrin imidazole ionic liquid by reacting N-vinylimidazole with β-cyclodextrin.

[0029] The structural diagrams of the above four functional monomers are shown in the attached Figure 2 specification. The above four functional monomers are A - D in the figure respectively.

[0030] It includes the following steps: S1. Prepare polymerizable imidazolium ionic liquid monomers from functional monomers through alkylation substitution reaction, including 1-vinyl-3-acetamidoimidazolium chloride ionic liquid monomer, tosylated 1-vinyl-β-cyclodextrin imidazole ionic liquid, and 1-vinyl-3-propane sulfonic acid imidazolium chloride zwitterionic liquid monomer.

[0031] S2. Polymerize a chain transfer agent and vinylimidazole into a macromolecular chain transfer reagent through RAFT polymerization, and then prepare precursor Precursor-1 of 1-vinyl-3-propane sulfonic acid imidazolium chloride zwitterionic liquid monomer on the macromolecular chain transfer reagent: p(VIM-b-VSPIM)

[0032] S3. Use precursor-1 as a chain transfer agent and polymerize 1-vinyl-3-acetamidoimidazolium chloride ionic liquid monomer onto precursor Precursor-1 through RAFT polymerization to prepare precursor Precursor-2: p(VIM-b-(VSPIM-co- VIMA))

[0033] S4. Use precursor-2 as a chain transfer agent and polymerize tosylated 1-vinyl-β-cyclodextrin imidazole ionic liquid monomer onto precursor Precursor-1 through RAFT polymerization to prepare precursor Precursor-3: p(VIM-b- (VSPIM-co-VIMA-co-VIMCDs))

[0034] S5. React precursor Precursor-3 with 4-vinylbenzyl chloride and a very small amount of hydroquinone in dimethyl sulfoxide as a solvent to prepare the final product, polyionic liquid macromonomer: p(PVIM -b-(VSPIM -co-VIMA -co-VIMCDs))。

[0035] S6. Study the interaction between the synthesized polyionic liquid macromonomer and protein through molecular dynamics simulation, and explore the effects of the polyionic liquid macromonomer on the secondary structure, hydrogen bonds, and energy of the protein.

[0036] In step S1, the synthesis of polymerizable imidazolium ionic liquid monomers from functional monomers includes the following steps: Preparation of 1-vinyl-3-styrylimidazolium chloride ionic liquid monomer: ​​​Take 4.7 g (50 mmol) of N-vinylimidazole and 6.5 g (43 mmol) of vinylbenzyl chloride, dissolve them in a mixed solution of 20 mL of water and 20 mL of ethyl acetate, and react for 24 h. After the reaction, extract with ethyl acetate, and freeze-dry the extraction solution to obtain 1-vinyl-3-styrylimidazolium chloride ionic liquid.

[0037] Preparation of 1-vinyl-3-acetamidoimidazolium chloride ionic liquid monomer: Take 3.1 g (33 mmol) of N-vinylimidazole and 2.9 g (31 mmol) of chloroacetamide, dissolve them in a mixed solution of 13 mL of ethanol and 13 mL of water, and react for 24 h. After the reaction, extract with ethyl acetate, and freeze-dry the extraction solution to obtain 1-vinyl-3-acetamidoimidazolium chloride ionic liquid.

[0038] Preparation of toluenesulfonated 1-vinyl-β-cyclodextrin imidazolium ionic liquid: Suspend 30 g of cyclodextrin in 250 mL of water, and add a solution of 3.29 g of sodium hydroxide in 10 mL of water. Dropwise add a 15 mL acetonitrile solution of 5.02 g of p-toluenesulfonyl chloride within 8 minutes. After stirring at 23 °C for 2 hours, remove the precipitate by suction filtration, and refrigerate the filtrate at 4 °C overnight. Recover the precipitate by suction filtration to obtain toluenesulfonated-β-cyclodextrin (6-TsO-β-CD).

[0039] Dissolve 6.45 g of 6-TsO-β-CD in a mixed solution of 1.8 mL of N-vinylimidazole and 15 mL of dimethylformamide, and react at 75 °C for 4 h under a N2 atmosphere. After cooling to ambient temperature, precipitate the crude product by using 70 mL of acetone, and then filter to obtain toluenesulfonated 1-vinyl-β-cyclodextrin imidazolium ionic liquid.

[0040] Preparation of 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer: Dissolve VIM (1.88 g, 0.02 mol) in 10 mL of acetonitrile at 50 °C. Under vigorous magnetic stirring, slowly drop 1,3-propanesultone (3.66 g, 0.03 mol) into the reactor using a constant-pressure funnel, stir the mixture at 50 °C for 24 h to obtain 4.53 g of a white precipitate, further purify it by washing with a small amount of acetone, and freeze-dry it by freeze-drying to obtain 1-vinyl-3-propanesulfonic acid imidazolium chloride; Precursor Precursor-1 in step S2: p(VIM-b-VSPIM) The preparation specifically includes the following steps: S2-1. Using 1 mmol of 2-(benzylsulfanylthio)acetic acid as a chain transfer agent, vinylimidazole as a monomer, taking 40 ml of dimethyl sulfoxide as a solvent, and 0.4 mmol of azobisisobutyronitrile as an initiator, a macromolecular chain transfer reagent was prepared by thermal initiation reaction under a N2 atmosphere; S2-2. Subsequently, 1-vinyl-3-propane sulfonic acid imidazole chloride zwitterionic liquid monomer and azobisisobutyronitrile were added to the macromolecular chain transfer reagent to prepare a precursor Precursor-1: p(VIM-b-VSPIM) ; The length of the precursor Precursor-1 was adjusted by regulating the ratio of the chain transfer agent, vinylimidazole, and 1-vinyl-3-propane sulfonic acid imidazole chloride zwitterionic liquid monomer.

[0041] The preparation conditions of the precursor Precursor-2 in step S3 were as follows: Using the precursor Precursor-1 as a chain transfer agent, dimethyl sulfoxide as a solvent, azobisisobutyronitrile as an initiator, and 1-vinyl-3-acetamidoimidazole chloride ionic liquid as a monomer, it was prepared at 70 °C under a N2 atmosphere.

[0042] The preparation conditions of the precursor Precursor-3 in step S4 were as follows: Using the precursor Precursor-2 as a chain transfer agent, dimethyl sulfoxide as a solvent, azobisisobutyronitrile as an initiator, and toluenesulfonic acid modified 1-vinyl-β-cyclodextrin imidazole ionic liquid as a monomer, it was prepared at 70 °C under a N2 atmosphere.

[0043] The preparation conditions of the polyionic liquid macromolecular monomer in step S5 were as follows: Using the precursor Precursor-3 as a chain transfer agent, dimethyl sulfoxide as a solvent, 4-vinylbenzyl chloride and a very small amount of hydroquinone were subjected to an alkylation reaction, and it was prepared at 60 °C under a N2 atmosphere.

[0044] Polyionic liquid macromolecular monomer p(PVIM -b-(VSPIM -co-VIMA -co-VIMCDs)) Preparation method: S1. Dissolve CA (32 mmol) in 50 mL of acetone, and a clear solution can be obtained by stirring at 45 °C for about 20 minutes. Then, under vigorous magnetic stirring, VIM (20 mmol) was added dropwise using a constant pressure funnel, and then reacted at 50 °C for 24 hours to prepare a polymerizable imidazolium-based ionic liquid monomer.

[0045] S2. Using reversible addition-fragmentation chain transfer living radical polymerization (RAFT polymerization), 2-(benzylsulfanylthio)acetic acid as the chain transfer agent (CTA), N-vinylimidazole as the monomer, dimethyl sulfoxide (DMSO) as the solvent, 2,2'-azobis(2-methylpropionitrile) (AIBN) as the initiator, a Macro-CTA was prepared by thermal initiation reaction under N2 atmosphere. Subsequently, VSPIM and AIBN were added to prepare the precursor 1 (Precursor-1: p(VIM-b-VSPIM)) ; By changing the ratio between Macro-CTA and VSPIM monomer, the p(VIM-b-VSPIM) length can be adjusted; By changing the monomer dosages of monomers VSPIM and VIM, the p(VIM-b-VSPIM) hydrophilicity and hydrophobicity can be regulated.

[0046] S3. Using the precursor Precursor-1 as the chain transfer agent, also adopting RAFT polymerization, DMSO as the solvent, AIBN as the initiator, and VIMA as the monomer, a precursor 2 ( Precursor-2: p(VIM- b-(VSPIM -co-VIMA)) .

[0047] S4. Using the precursor-2 as the chain transfer agent, adopting RAFT polymerization, DMSO as the solvent, AIBN as the initiator, and VIMCDs as the monomer, a precursor 3 (Precursor-3: p(VIM-b-(VSPIM - co-VIMA -co-VIMCDs)) ; S5. Reacting the precursor Precursor-3 with 4-vinylbenzyl chloride and a very small amount of hydroquinone in dimethyl sulfoxide as the solvent to prepare the final product polyionic liquid macromonomer: p(PVIM -b-(VSPIM -co-VIMA -co-VIMCDs)); S6. Studying the interaction between the synthesized polyionic liquid macromonomer and protein through molecular dynamics simulation, and exploring the effects of the polyionic liquid macromonomer on the secondary structure, hydrogen bonds, and energy of the protein.

[0048] Studying the interaction between the macromonomer synthesized from the monomer screened in S1 and protein through molecular dynamics simulation, and exploring the effects of the macromonomer on the secondary structure, hydrogen bonds, and energy of the protein. First, perform protein (PDB: 5ty3)-polymer molecular docking: Chem3D was used to preliminarily optimize the structure of the polymer. HDOCK was used to perform rigid docking on the protein and the optimized polymer. The molecular docking was carried out using the HDOCK online tool. The optimal conformation after docking is as Figure 3 shown, providing a reasonable initial structure for subsequent molecular dynamics simulation.

[0049] Molecular dynamics simulations were performed using Gromacs 2021 with the gromos force field, and the force field of the polymer was generated using ATB (http: / / atb.uq.edu.au / index.py). The complex formed by docking the protein and the polymer molecule was placed in a molecular dynamics simulation box of appropriate size, and then solvent water and sodium chloride were added to the simulation box to neutralize the charge of the simulation system. After constructing the simulation system, the simulation system was minimized in energy for 50,000 steps using the steep method with a force convergence criterion of 1000 kJ / mol / nm, and then NVT and NPT simulations were performed for 100 ps each for temperature control (298.15 K) and pressure control (1 atm). Subsequently, a 50 ns molecular dynamics simulation was carried out.

[0050] From the results of the molecular dynamics simulations, the conformations of the polymer and the protein at different simulation times are as shown Figure 3 below. From left to right are the conformations at 0, 10, 20, 30, 40, and 50 ns. It can be seen from the figure that the polymer tends to interact with the random coil regions of the protein.

[0051] The change in the solvent accessible surface area (SASA) of the protein with simulation time is as shown Figure 4 in (a). It can be seen from the figure that at the initial stage of the simulation, the solvent accessible surface area of the protein decreased slightly, but from the entire simulation time, the change in the SASA of the protein was not very obvious, indicating that the number of hydrogen bonds between the protein and the small molecule changed with the simulation time (the red line in the figure) as shown Figure 4 in (b). It can be seen from the figure that the change in the number of hydrogen bonds was relatively stable.

[0052] The interaction energy between the protein and the polymer during the entire simulation process is as shown Figure 4 in (c). The total interaction energy consists of the electrostatic interaction energy and the van der Waals interaction energy. It can be seen from the figure that the total interaction energy between the polymer and the protein was around -350 KJ / mol, and the contribution of the van der Waals interaction energy was greater than that of the electrostatic interaction, indicating that the addition of the monomer had an impact on the energy of the protein, but the impact was not significant.

[0053] A schematic diagram of the change in the secondary structure of the protein is as shown Figure 4 in (d). It can be seen from the figure that the α-helix structure (blue part), random coil (white part), and β-sheet (green part) of the protein were hardly affected and remained intact in shape, only the β-turn of the protein was affected. Generally speaking, the polyionic liquid macromolecular monomer had little impact on the structure of the protein.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements do not enable the modified technical solutions to depart from the spirit and scope of the technical solutions of the present invention.

Claims

1. A preparation method of a polyionic liquid macromonomer for stabilizing protein structures, characterized in that It includes functional units, inert units and crosslinking units. The functional units, the inert units and the crosslinking units are all polymerizable imidazolium ionic liquid monomers. The functional units, the inert units and the crosslinking units are polymerized into polyionic liquid macromonomers by RAFT polymerization.

2. The preparation method of a polyionic liquid macromonomer for stabilizing protein structure according to claim 1, characterized in that, The polymerizable imidazolium ionic liquid monomers are prepared by the alkylation substitution reaction between N-vinylimidazole and functional monomers. The functional monomers include any one of 4-vinylbenzyl chloride, 3-chloropropanesulfonic acid, chloroacetamide and β-cyclodextrin. 1-vinyl-3-styrylimidazolium chloride ionic liquid monomer, 1-vinyl-3-propane sulfonic acid imidazolium chloride zwitterionic liquid monomer, 1-vinyl-3-acetamide imidazolium chloride ionic liquid monomer, and toluenesulfonic acid modified 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomer are respectively prepared.

3. The preparation method of a polyionic liquid macromonomer for stabilizing protein structure according to claim 2, characterized in that The functional motif is formed by the polymerization of 1-vinyl-3-ethylacetamidoimidazolium chloride ionic liquid and 1-vinyl-β-cyclodextrin imidazolium tosylate ionic liquid monomer. The inert motif is several 1-vinyl-3-propane sulfonic acid imidazolium chloride zwitterionic liquid monomers, and the crosslinking motif is several 1-vinyl-3-styrene imidazolium chloride ionic liquid monomers. The structural formula of the polyionic liquid macromonomer is: p(PVIM-b-(VSPIM-co-VIMA-co-VIMCDs)) ; Among them, PVIM is 1-vinyl-3-styrylimidazolium chloride ionic liquid, VSPIM is 1-vinyl-3-propane sulfonic acid imidazolium chloride zwitterionic liquid , VIMA is 1-vinyl-3-acetamide imidazolium chloride ionic liquid , VIMCDs is toluenesulfonic acid modified 1-vinyl-β-cyclodextrin imidazolium ionic liquid.

4. The preparation method of a polyionic liquid macromonomer for stabilizing protein structure according to claim 2, wherein, It includes the following steps: S1. Prepare polymerizable imidazolium ionic liquid monomers by the alkylation substitution reaction between N-vinylimidazole and functional monomers, including 1-vinyl-3-propane sulfonic acid imidazolium chloride zwitterionic liquid monomer, 1-vinyl-3-acetamide imidazolium chloride ionic liquid monomer, and toluenesulfonic acid modified 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomer; S2. Polymerize a chain transfer agent and vinylimidazole into a macromolecular chain transfer reagent through RAFT polymerization, and then polymerize 1-vinyl-3-propane sulfonic acid imidazole chloride zwitterionic liquid monomer on the macromolecular chain transfer reagent to prepare a precursor Precursor-1: p(VIM-b-VSPIM) ; S3. Using the precursor-1 as a chain transfer agent, the 1-vinyl-3-ethylacetamidoimidazolium chloride ionic liquid monomer is polymerized onto the precursor Precursor-1 by RAFT polymerization to prepare the precursor Precursor-2: p(VIM-b-(VSPIM-co-VIMA)) ; S4. Using the precursor-2 as a chain transfer agent, polymerize the toluene-sulfonated 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomer onto the precursor Precursor-1 by RAFT polymerization to prepare the precursor Precursor-3: p(VIM-b-(VSPIM-co- VIMA-co-VIMCDs)) ; S5. React the precursor Precursor-3 with 1-vinyl-3-styrylimidazolium chloride ionic liquid monomer in dimethyl sulfoxide as the solvent to prepare the final product, the polyionic liquid macromonomer: p([PVIM]Cl-b-([VSPIM]Cl- co-[VIMA]Cl-co-[VIMCDs]OTs)); S6. Study the interaction between the synthesized polyionic liquid macromonomers and proteins by molecular dynamics simulation, and explore the effects of the polyionic liquid macromonomers on the secondary structure, hydrogen bonds and energy of proteins.

5. The preparation method of a polyionic liquid macromonomer for stabilizing protein structure according to claim 4, wherein, The preparation method of the 1-vinyl-3-propane sulfonic acid imidazolium chloride zwitterionic liquid monomer in the step S1 includes the following steps: Dissolve N-vinylimidazole in acetonitrile at a molar volume ratio of 2 mol:1 L at 50 °C. Under vigorous magnetic stirring, slowly drip 1,3-propanesultone into the reactor using a constant pressure funnel. The molar ratio of N-vinylimidazole to 1,3-propanesultone is 2:

3. Stir the mixture at 50 °C for 24 h to obtain a white precipitate. Purify it further by washing with acetone and perform freeze-drying by lyophilization to obtain the 1-vinyl-3-propane sulfonic acid imidazolium chloride zwitterionic liquid monomer; The preparation method of the 1-vinyl-3-acetamide imidazolium chloride ionic liquid monomer in the step S1 includes the following steps: Take N-vinylimidazole and chloroacetamide with a molar ratio of 33:31, dissolve and react them in a 50% ethanol solution for 24 h. After the reaction, extract with ethyl acetate and lyophilize the extracted solution to obtain the 1-vinyl-3-acetamide imidazolium chloride ionic liquid monomer; The preparation method of the toluenesulfonated 1-vinyl-β-cyclodextrin imidazole monomer in the step S1 includes the following steps: Dissolve β-cyclodextrin in deionized water, dropwise add an aqueous solution of sodium hydroxide and an acetonitrile solution of tosyl chloride thereto, stir at 23 °C and 800 rpm for 2 hours, remove the precipitate by suction filtration, refrigerate the filtrate overnight at 4 °C, and recover the precipitate by suction filtration to obtain toluenesulfonated-β-cyclodextrin; Dissolve toluenesulfonated-β-cyclodextrin in a mixed solution of N-vinylimidazole and N,N-dimethylformamide, react at 75 °C for 4 h under a N2 atmosphere; after cooling to ambient temperature, precipitate the crude product by using acetone, then filter, and dry overnight in a vacuum oven at 50 °C to obtain toluenesulfonated 1-vinyl-β-cyclodextrin imidazole ionic liquid; Preparation of 1-vinyl-3-styreneimidazolium chloride ionic liquid monomer: Take N-vinylimidazole and vinylbenzyl chloride with a molar ratio of 50:43, dissolve and react them in a mixed solution of water and ethyl acetate with a volume ratio of 1:1 for 24 h. After the reaction, extract with ethyl acetate, and lyophilize the extraction solution to obtain 1-vinyl-3-styreneimidazolium chloride ionic liquid.

6. The preparation method of a polyionic liquid macromonomer for stabilizing protein structure according to claim 4, characterized in that, Precursor Precursor-1 in step S2: p(VIM-b-VSPIM) The preparation specifically includes the following steps: S2-1: Using 1 mmol of 2-(benzylsulfanylthio)acetic acid as a chain transfer agent, taking N-vinylimidazole as a monomer, taking 40 ml of dimethyl sulfoxide as a solvent, and 0.4 mmol of azobisisobutyronitrile as an initiator, thermally initiate the reaction under a N2 atmosphere to obtain a macromolecular chain transfer agent; S2-2. Subsequently, a macromolecular chain transfer agent, 1-vinyl-3-propane sulfonic acid imidazole chloride zwitterionic liquid monomer, and azodiisobutyronitrile were added to prepare a precursor, Precursor-1: p(VIM-b-VSPIM) ; The length of the precursor Precursor-1 is adjusted by adjusting the ratio of the chain transfer agent, N-vinylimidazole, and 1-vinyl-3-propane sulfonic acid imidazolium chloride zwitterionic liquid monomer.

7. The preparation method of a polyionic liquid macromonomer for stabilizing protein structure according to claim 4, characterized in that The preparation conditions of the precursor Precursor-2 in the step S3 are as follows: using the precursor Precursor-1 as a chain transfer agent, using dimethyl sulfoxide as a solvent, azobisisobutyronitrile as an initiator, and using 1-vinyl-3-acetamidoimidazolium chloride ionic liquid monomer as a monomer, and preparing at 70 °C under a N2 atmosphere.

8. The preparation method of a polyionic liquid macromonomer for stabilizing protein structure according to claim 4, characterized in that, The preparation conditions of the precursor Precursor-3 in the step S4 are as follows: using the precursor Precursor-2 as a chain transfer agent, using dimethyl sulfoxide as a solvent, azobisisobutyronitrile as an initiator, and using toluenesulfonated 1-vinyl-β-cyclodextrin imidazole ionic liquid monomer as a monomer, and preparing at 70 °C under a N2 atmosphere.

9. The preparation method of a polyionic liquid macromonomer for stabilizing protein structure according to claim 8, characterized in that, The polymerization conditions of 1-vinyl-3-styreneimidazolium chloride ionic liquid on the precursor Precursor-3 are: polymerize N-vinylimidazole on 2-(benzylsulfanylthio)acetic acid, use the precursor Precursor-3 as a chain transfer agent, add 4-vinylbenzyl chloride and hydroquinone through an alkylation reaction on the precursor Precursor-3, and prepare and polymerize at 60 °C under a N2 atmosphere.

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