A method for preparing a polyionic liquid macromonomer stabilizing protein structure

Abstract

The application discloses a preparation method of polyionic liquid macromonomer for stabilizing protein structure, belongs to the technical field of polymer material preparation and protein recognition technology, and is prepared by polymerization addition of a functional unit, an inert unit and a crosslinking unit through a RAFT polymerization method. p(PVIM-b-(VSPIM-b- co-VIMA-co-VIMCDs)) Wherein, 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 VIMCDs is toluenesulfonated 1-vinyl-β-cyclodextrin imidazole ionic liquid. Through the preparation method, the application realizes preparation of polyionic liquid macromonomer with multiple anchoring protein effects through active / controllable free radical polymerization design, and solves the problems of inaccurate imprinting in the protein imprinting process and "poor selective recognition" caused by single function monomer-protein interaction.

Description

Technical Field

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

[0002] By using functional monomers with multiple modes of action to synergistically imprint proteins, it is also possible to better address the challenges of complex protein surface structures and single protein-monomer interactions. Previous studies have shown that metal coordination strategies have enhanced protein imprint recognition, and the design and synthesis of multifunctional monomers for protein pre-immobilization have also improved protein imprint recognition performance. To address the competition and interference of imprinting media on the interaction between target proteins and functional monomers, Du et al. prepared a series of highly cross-linked raspberry spheres functionalized with different ionic liquids and examined their adsorption capacity for target molecules. The results showed that multiple interaction sites between proteins and functional groups can enhance the interaction between the two and thus improve imprinting efficiency. For example, the structural characteristics of cyclodextrin, with its hydrophobic inner cavity and hydrophilic outer wall, enable it to encapsulate target molecules through host-guest interactions. Zhang et al., taking advantage of the multiple interactions and high affinity provided by the ionic liquid β-cyclodextrin in an aqueous environment, prepared a novel cyclodextrin-based ionic liquid functional monomer antigen-determinant magnetic imprinting microspheres and achieved effective recognition of cytochrome C.

[0003] Therefore, introducing motif anchor proteins with multiple affinities for target proteins into the imprinting system is an effective strategy to improve the efficiency of protein imprinting. For example, without affecting the specific adsorption of the target protein to the imprinting site, the "inert component" zwitterionic structural segments that are resistant to protein adsorption are introduced to increase the selectivity of the imprinted polymer for the target. Zhang Qiuyu's team at Northwestern Polytechnical University used the anti-protein adsorption properties of the monomer 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer segment to design and prepare a directional imprinting material that can specifically recognize bovine serum albumin. Therefore, the research and development of molecular structures that are resistant to nonspecific protein adsorption is also one of the key goals to improve the directional imprinting and selective recognition performance of imprinted polymers.

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

[0005] The purpose of the present invention is to provide a method for preparing a polyionic liquid macromolecular monomer that stabilizes protein structure, aiming to solve the problem of "poor selectivity and recognition" caused by inaccurate imprinting and single functional monomer-protein interaction during protein imprinting.

[0006] To achieve the above objectives, the present invention provides a method for preparing a polyionic liquid macromolecular monomer for stabilizing protein structure, which comprises a functional unit, an inert unit and a cross-linking unit. The functional unit, the inert unit and the cross-linking unit are polymerized and added into a polyionic liquid macromolecular monomer by an aqueous phase polymerization method.

[0007] Preferably, the functional unit is formed by polymerization of 1-vinyl-3-acetamido imidazolium chloride and tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomers, the inert unit is a plurality of 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomers, the cross-linking unit is a plurality of 1-vinyl-3-styrene imidazolium chloride ionic liquid monomers, and the structural formula of the polyionic liquid macromonomer is: p(PVIM -b-(VSPIM-co-VIMA -co-VIMCDs)) ,

[0008] Among them, PVIM is 1-vinyl-3-styrene imidazolium chloride ionic liquid monomer, VSPIM is 1-vinyl-3-propanesulfonic acid 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.

[0009] Preferably, the polymerizable imidazolium-based ionic liquid monomer is prepared by an alkylation substitution reaction of a functional monomer, wherein the functional monomer is N-vinylimidazole reacted with any one of 4-vinylbenzyl chloride, 1,3-propanesulfonic acid imidazole chloride, chloroacetamide and β-cyclodextrin to prepare 1-vinyl-3-styrene imidazole chloride, 1-vinyl-3-propanesulfonic acid imidazole chloride, 1-vinyl-3-acetamide imidazole chloride, and toluenesulfonated 1-vinyl-β-cyclodextrin imidazole ionic liquid, wherein the preparation of 1-vinyl-3-styrene imidazole chloride ionic liquid is firstly carried out by polymerizing N-vinylimidazole on 2-(phenylmethylsulfonylthio)acetic acid, and then adding 4-vinylbenzyl chloride to the prepared precursor Precursor-3 by an alkylation reaction, thereby achieving the effect of polymerizing 1-vinyl-3-styrene imidazole chloride ionic liquid on a macromolecular chain transfer agent.

[0010] Preferably, the method comprises the following steps:

[0011] S1. Prepare a polymerizable imidazolium-based ionic liquid monomer by an alkylation substitution reaction of a functional monomer, including 1-vinyl-3-propanesulfonic acid imidazolium chloride ionic liquid, 1-vinyl-3-acetamide imidazolium chloride ionic liquid, and toluenesulfonated 1-vinyl-β-cyclodextrin imidazolium ionic liquid;

[0012] S2. The chain transfer agent and vinyl imidazole are polymerized into a macromolecular chain transfer reagent by RAFT polymerization. Then, 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer is added to the macromolecular chain transfer reagent to prepare Precursor-1: p(VIM-b-VSPIM) ;

[0013] S3, using Precursor-1 as a chain transfer agent, RAFT polymerization was used to polymerize 1-vinyl-3-acetamidoimidazolium chloride ion liquid monomer on Precursor-1 to prepare Precursor-2: p(VIM-b-(VSPIM -co- VIMA)) ;

[0014] S4. Using Precursor-2 as a chain transfer agent, RAFT polymerization was used to polymerize the tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomer on Precursor-1 to prepare Precursor-3: p(VIM-b-(VSPIM -co-VIMA-co-VIMCDs)) ;

[0015] S5. The precursor Precursor-3 is reacted with 4-vinylbenzyl chloride and a very small amount of hydroquinone in dimethyl sulfoxide as a solvent to prepare the final product, a polyionic liquid macromonomer: p(PVIM -b-(VSPIM -co-VIMA -co-VIMCDs));

[0016] S6. Through molecular dynamics simulation, the interaction between the synthesized polyionic liquid macromolecular monomer and protein is studied to explore the effect of polyionic liquid macromolecular monomer on the secondary structure, hydrogen bonding and energy of protein.

[0017] Preferably, the preparation method of the 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer in step S1 comprises the following steps:

[0018] N-vinylimidazole was dissolved in acetonitrile at a molar volume ratio of 2 mol:1 L at 50 °C. 1,3-propane sultone was slowly added dropwise into the reactor using a constant pressure funnel under vigorous magnetic stirring. The molar ratio of N-vinylimidazole to 1,3-propane sultone was 2:3. The mixture was stirred at 50 °C for 24 h to obtain a white precipitate, which was further purified by washing with acetone and freeze-dried to obtain 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer.

[0019] The preparation method of the 1-vinyl-3-acetamidoimidazole chloride ion liquid monomer in step S1 comprises the following steps:

[0020] N-vinylimidazole and chloroacetamide at a molar ratio of 33:31 were dissolved in a 50% ethanol solution and reacted for 24 hours. After the reaction, the mixture was extracted with ethyl acetate, and the extracted solution was freeze-dried to obtain a 1-vinyl-3-acetamidoimidazole chloride ion liquid monomer.

[0021] The method for preparing the tosylated 1-vinyl-β-cyclodextrin imidazole monomer in step S1 comprises the following steps:

[0022] β-cyclodextrin was dissolved in deionized water, and an aqueous solution of sodium hydroxide and an acetonitrile solution of tosyl chloride were added dropwise thereto. After stirring at 23°C and 800 rpm for 2 hours, the precipitate was removed by suction filtration, and the filtrate was refrigerated at 4°C overnight. The precipitated tosylated-β-cyclodextrin was recovered by suction filtration.

[0023] Tosylated β-cyclodextrin was dissolved in a mixed solution of N-vinylimidazole and N,N-dimethylformamide, and reacted at 75°C for 4 hours under a N2 atmosphere; after cooling to ambient temperature, the crude product was precipitated by using acetone, then filtered, and dried in a vacuum oven at 50°C overnight to obtain tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid.

[0024] Preferably, in step S3, the precursor Precursor-1: p(VIM-b-VSPIM) The preparation specifically comprises the following steps:

[0025] S2-1. A macromolecular chain transfer reagent was prepared by thermally initiating a reaction under N2 atmosphere using 1 mmol of 2-(phenylmethylsulfonylthio)acetic acid as a chain transfer agent, N-vinylimidazole as a monomer, 40 ml of dimethyl sulfoxide as a solvent, and 0.4 mmol of azobisisobutyronitrile as an initiator.

[0026] S2-2, add 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer and azobisisobutyronitrile to the macromolecular chain transfer reagent to prepare Precursor-1: p(VIM-b-VSPIM) ;

[0027] The length of the precursor Precursor-1 was adjusted by adjusting the ratio of chain transfer agent, N-vinylimidazole and 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer.

[0028] Preferably, the preparation conditions of the precursor Precursor-2 in step S3 are 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 ion liquid as a monomer, and preparing it at 70° C. under N2 atmosphere.

[0029] Preferably, the preparation conditions of the precursor Precursor-3 in step S4 are as follows: using the precursor Precursor-2 as a chain transfer agent, dimethyl sulfoxide as a solvent, azobisisobutyronitrile as an initiator, and tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid as a monomer, and preparing it at 70° C. under N2 atmosphere.

[0030] 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 N2 atmosphere.

[0031] Therefore, the present invention adopts the above-mentioned method for preparing a polyionic liquid macromolecular monomer with a stable protein structure, and its beneficial effects are:

[0032] To address the problem of "poor selectivity" caused by inaccurate imprinting and single functional monomer-protein interaction during protein imprinting, the present invention introduces ionic liquids with protein structure stability into the imprinting system, and designs polyionic liquid monomers with multiple protein anchoring effects through active / controllable free radical polymerization.

[0033] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a flow chart of a method for preparing a polyionic liquid macromolecular monomer for stabilizing protein structure according to the present invention;

[0035] Figure 2 Schematic diagrams of the structures of four polymerizable imidazolium-based ionic liquid monomers of the present invention, wherein (a) is a 1-vinyl-3-styrene imidazolium chloride ionic liquid monomer, (b) is a 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer, (c) is a 1-vinyl-3-acetamido imidazolium chloride monomer, and (d) is a tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomer;

[0036] Figure 3 are the conformations of polymers and proteins at different simulation moments;

[0037] Figure 4 (a) The change of the solvent accessible surface area (SASA) of the protein with simulation time; (b) The change of the number of hydrogen bonds between the protein and small molecules with simulation time; (c) The interaction energy between the protein and the polymer during the entire simulation process; (d) Schematic diagram of the change of protein secondary structure; DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0039] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0040] Furthermore, it should be understood that although this specification describes the embodiments, not every embodiment includes only one independent technical solution. This description is for clarity only. Those skilled in the art should consider the specification as a whole. The technical solutions in the various embodiments may also be appropriately combined to form other embodiments that are understandable to those skilled in the art. These other embodiments are also encompassed within the scope of protection of the present invention.

[0041] The present invention is further described below with reference to the accompanying drawings and specific embodiments.

[0042] The present invention discloses a method for preparing a polyionic liquid macromolecular monomer that stabilizes protein structure. Figure 1 As shown, attached Figure 1 A is a functional primitive, Figure 1 B is an inert primitive, Figure 1 C is a cross-linking unit. The functional unit, the inert unit and the cross-linking unit are polymerized and added by RAFT polymerization to form a polyionic liquid macromolecular monomer.

[0043] The functional unit is formed by polymerization of 1-vinyl-3-acetamido imidazolium chloride and tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomers, the inert unit is a plurality of 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomers, the cross-linking unit is a plurality of ionic liquid monomers, and the structural formula of the polyionic liquid macromolecular monomer is: p(PVIM - b-(VSPIM -co-VIMA -co-VIMCDs)) .

[0044] PVIM stands for 1-vinyl-3-phenylimidazolium chloride, VSPIM stands for 1-vinyl-3-propanesulfonic acid imidazolium chloride, VIMA stands for 1-vinyl-3-acetamidoimidazolium chloride, and VIMCDs stands for tosylated 1-vinyl-β-cyclodextrin imidazolium. Specifically, VIMCDs, VSPIM, and PVIM demonstrate superior performance in recognizing cytochrome C.

[0045] The polymerizable imidazolyl ionic liquid monomer is prepared by alkylation substitution reaction of functional monomer:

[0046] 1-Vinyl-3-styrene imidazole chloride was prepared by the reaction of N-vinylimidazole and vinylbenzyl chloride.

[0047] 1-Vinyl-3-propanesulfonic acid imidazolium chloride was prepared by reacting N-vinylimidazole with 1,3-propanesultone.

[0048] 1-vinyl-3-acetamidoimidazole chloride was prepared by the reaction of N-vinylimidazole with chloroacetamide.

[0049] Tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid was prepared by reacting N-vinylimidazole with β-cyclodextrin.

[0050] The structural diagrams of the above four functional monomers are shown in the attached manual. Figure 2 As shown, the above four functional monomers are AD in the figure.

[0051] The following steps are involved:

[0052] S1. Prepare a polymerizable imidazolyl ionic liquid monomer through an alkylation substitution reaction of a functional monomer, including a 1-vinyl-3-acetamido imidazolium chloride ionic liquid monomer, a toluenesulfonated 1-vinyl-β-cyclodextrin imidazolium ionic liquid, and a 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer.

[0053] S2. The chain transfer agent and vinyl imidazole are polymerized into a macromolecular chain transfer reagent by RAFT polymerization. Then, 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer is added to the macromolecular chain transfer reagent to prepare Precursor-1: p(VIM-b-VSPIM) .

[0054] S3, using Precursor-1 as a chain transfer agent, RAFT polymerization was used to polymerize 1-vinyl-3-acetamidoimidazolium chloride ion liquid monomer on Precursor-1 to prepare Precursor-2: p(VIM-b-(VSPIM-co- VIMA)) .

[0055] S4. Using Precursor-2 as a chain transfer agent, RAFT polymerization was used to polymerize the tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomer on Precursor-1 to prepare Precursor-3: p(VIM-b- (VSPIM-co-VIMA-co-VIMCDs)) .

[0056] S5. The precursor Precursor-3 is reacted with 4-vinylbenzyl chloride and a very small amount of hydroquinone in dimethyl sulfoxide as a solvent to prepare the final product, a polyionic liquid macromonomer: p(PVIM -b-(VSPIM -co-VIMA -co-VIMCDs)).

[0057] S6. Through molecular dynamics simulation, the interaction between the synthesized polyionic liquid macromolecular monomer and protein is studied to explore the effect of polyionic liquid macromolecular monomer on the secondary structure, hydrogen bonding and energy of protein.

[0058] In step S1, the synthesis of polymerizable imidazolyl ionic liquid monomers from functional monomers includes the following steps:

[0059] Preparation of 1-vinyl-3-styrene imidazolium chloride ionic liquid monomer:

[0060] Take 4.7 g (50 mmol) of N-vinylimidazole and 6.5 g (43 mmol) of vinylbenzyl chloride and dissolve them in a mixed solution of 20 mL of water and 20 mL of ethyl acetate to react for 24 hours. After the reaction, use ethyl acetate to extract, and the extract solution is freeze-dried to obtain 1-vinyl-3-styrylimidazole chloride ion liquid.

[0061] Preparation of 1-vinyl-3-acetamidoimidazolium chloride ionic liquid monomer:

[0062] 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 hours. After the reaction, extract with ethyl acetate, and freeze-dry the extracted solution to obtain 1-vinyl-3-acetamidoimidazole chloride ion liquid.

[0063] Preparation of tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid:

[0064] 30 g of cyclodextrin was suspended in 250 mL of water, and a solution of 3.29 g of sodium hydroxide in 10 mL of water was added. A solution of 5.02 g of p-toluenesulfonyl chloride in 15 mL of acetonitrile was added dropwise over 8 minutes. After stirring at 23°C for 2 hours, the precipitate was removed by suction filtration, and the filtrate was refrigerated at 4°C overnight. The precipitate was recovered by suction filtration to obtain tosylated β-cyclodextrin (6-TsO-β-CD).

[0065] 6.45 g of 6-TsO-β-CD was dissolved in a mixed solution of 1.8 mL of N-vinylimidazole and 15 mL of dimethylformamide and reacted at 75° C. for 4 h under a N2 atmosphere. After cooling to ambient temperature, the crude product was precipitated with 70 mL of acetone and then filtered to obtain the tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid.

[0066] Preparation of 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer:

[0067] VIM (1.88 g, 0.02 mol) was dissolved in 10 mL of acetonitrile at 50 °C. Under vigorous magnetic stirring, 1,3-propane sultone (3.66 g, 0.03 mol) was slowly added dropwise to the reactor using a constant pressure funnel. The mixture was stirred at 50 °C for 24 h to obtain 4.53 g of a white precipitate, which was further purified by washing with a small amount of acetone and freeze-dried to obtain 1-vinyl-3-propanesulfonic acid imidazolium chloride.

[0068] Precursor-1 in step S2: p(VIM-b-VSPIM) The preparation specifically comprises the following steps:

[0069] S2-1. A macromolecular chain transfer reagent was prepared by thermally initiating a reaction under N2 atmosphere using 1 mmol of 2-(phenylmethylsulfonylthio)acetic acid as a chain transfer agent, vinylimidazole as a monomer, 40 ml of dimethyl sulfoxide as a solvent, and 0.4 mmol of azobisisobutyronitrile as an initiator.

[0070] S2-2, followed by the addition of 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer and azobisisobutyronitrile to the macromolecular chain transfer reagent to prepare Precursor-1: p(VIM-b-VSPIM) ;

[0071] The length of the precursor Precursor-1 was adjusted by adjusting the ratio of chain transfer agent, vinyl imidazole and 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer.

[0072] The preparation conditions of the precursor Precursor-2 in step S3 are 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 ion liquid as a monomer, it is prepared at 70°C under N2 atmosphere.

[0073] The preparation conditions of the precursor Precursor-3 in step S4 are as follows: using the precursor Precursor-2 as a chain transfer agent, dimethyl sulfoxide as a solvent, azobisisobutyronitrile as an initiator, and tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid as a monomer, it is prepared at 70°C under N2 atmosphere.

[0074] 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 N2 atmosphere.

[0075] Polyionic liquid macromonomer p(PVIM -b-(VSPIM -co-VIMA -co-VIMCDs)) Preparation method:

[0076] S1. Dissolve CA (32 mmol) in 50 mL of acetone and stir at 45°C for approximately 20 minutes to obtain a clear solution. Then, add VIM (20 mmol) dropwise using a constant pressure funnel under vigorous magnetic stirring. The mixture is reacted at 50°C for 24 hours to prepare a polymerizable imidazolyl ionic liquid monomer.

[0077] S2, using reversible addition-fragmentation chain transfer controlled radical polymerization (RAFT polymerization), 2-(phenylmethylsulfonylthio)acetic acid as a chain transfer agent (CTA), N-vinylimidazole as a monomer, dimethyl sulfoxide (DMSO) as a solvent, azobisisobutyronitrile (AIBN) as an initiator, thermal initiation reaction under N2 atmosphere to prepare Macro-CTA, followed by the addition of VSPIM and AIBN to prepare Precursor-1 (Precursor-1: p(VIM-b-VSPIM)) ; Adjustable by changing the ratio between Macro-CTA and VSPIM monomers p(VIM-b-VSPIM) The length can be controlled by changing the monomer dosage of VSPIM and VIM p(VIM-b-VSPIM) hydrophilicity.

[0078] S3, using the precursor Precursor-1 as a chain transfer agent, the same RAFT polymerization method was used, DMSO as a solvent, AIBN as an initiator, and VIMA as a monomer, and thermal initiation was performed under N2 atmosphere to prepare the precursor 2 ( Precursor-2: p(VIM- b-(VSPIM-co-VIMA)) .

[0079] S4, using precursor-2 as a chain transfer agent, RAFT polymerization method, DMSO as solvent, AIBN as initiator, VIMCDs as monomers, thermal initiation under N2 atmosphere to prepare precursor 3 (Precursor-3: p(VIM-b-(VSPIM - co-VIMA -co-VIMCDs) ;

[0080] S5. The precursor Precursor-3 is reacted with 4-vinylbenzyl chloride and a very small amount of hydroquinone in dimethyl sulfoxide as a solvent to prepare the final product, a polyionic liquid macromonomer: p(PVIM -b-(VSPIM -co-VIMA -co-VIMCDs));

[0081] S6. Through molecular dynamics simulation, the interaction between the synthesized polyionic liquid macromolecular monomer and protein is studied to explore the effect of polyionic liquid macromolecular monomer on the secondary structure, hydrogen bonding and energy of protein.

[0082] The interaction between the macromonomers synthesized from the monomers screened by S1 and proteins was studied through molecular dynamics simulations to explore the effects of macromonomers on the secondary structure, hydrogen bonding, and energy of proteins. First, protein (PDB: 5ty3)-polymer molecular docking was performed: Chem3D was used to preliminarily optimize the structure of the polymer. HDOCK was used to perform rigid docking between the protein and the optimized polymer. Molecular docking was performed using the HDOCK online tool. The optimal conformation after docking is shown below. Figure 3 As shown, it provides a reasonable initial structure for subsequent molecular dynamics simulations.

[0083] Molecular dynamics simulations were performed using Gromacs 2021, using the gromos force field and the polymer force field generated using ATB (http: / / atb.uq.edu.au / index.py). The complex formed after docking the protein and polymer molecules was placed in an appropriately sized molecular dynamics simulation box. Water and sodium chloride were then added to the box to neutralize the charge in the simulation system. After the simulation system was constructed, the steep method was used to minimize the system for 50,000 steps with a force convergence criterion of 1000 kJ / mol / nm. NVT and NPT simulations were then performed sequentially, each with 100 ps of temperature control (298.15 K) and pressure control (1 atm). This was followed by a 50 ns molecular dynamics simulation.

[0084] From the molecular dynamics simulation results, we can see that the conformations of polymers and proteins at different simulation times are shown in the attached figure. Figure 3 From left to right, they are the conformations of 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 part of the protein.

[0085] The solvent accessible surface area (SASA) of the protein changes with simulation time as shown in the attached figure. Figure 4 As shown in (a). As can be seen from the figure, at the beginning of the simulation, the solvent accessible surface area of ​​the protein decreases slightly, but from the perspective of the entire simulation time, the SASA of the protein does not change very significantly, indicating that the number of hydrogen bonds between the protein and the small molecule changes with the simulation time (red line in the figure) as shown in the attached figure. Figure 4 As shown in (b), the number of hydrogen bonds changes relatively steadily.

[0086] The interaction energy between protein and polymer during the whole simulation is shown in the attached figure. Figure 4(c) shows the total interaction energy, which is composed of electrostatic and van der Waals interactions. As shown in the figure, the total interaction energy between the polymer and the protein is around -350 kJ / mol, and the contribution of van der Waals interactions is greater than that of electrostatic interactions, indicating that the addition of monomers has an impact on the protein's energetics, but the effect is not significant.

[0087] The schematic diagram of the changes in protein secondary structure is shown in the attached Figure 4 As shown in (d), it can be seen in the figure that the α-helix structure (blue part) of the protein, the random curl (white part) and the β-fold (green part) of the protein are almost unaffected, and the shape remains intact. Only the protein β-turn is affected. In general, the polyionic liquid macromolecular monomer has little effect on the structure of the protein.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a polyionic liquid macromolecular monomer for stabilizing protein structure, characterized in that: The invention comprises a functional unit, an inert unit and a cross-linking unit, wherein the functional unit, the inert unit and the cross-linking unit are all polymerizable imidazolium-based ionic liquid monomers, and the functional unit, the inert unit and the cross-linking unit are polymerized and added into a polyionic liquid macromolecular monomer by RAFT polymerization; The functional unit is formed by polymerization of 1-vinyl-3-acetamido imidazolium chloride ionic liquid and tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomers, the inert unit is a plurality of 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomers, the cross-linking unit is a plurality of 1-vinyl-3-styrene imidazolium chloride ionic liquid monomers, and the structural formula of the polyionic liquid macromolecular monomer is: p(PVIM-b-(VSPIM-co-VIMA-co-VIMCDs)) ; in, PVIM It is 1-vinyl-3-styrene imidazolium chloride ion liquid, VSPIM 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid VIMA 1-vinyl-3-acetamidoimidazole chloride ionic liquid , VIMCDs It is a tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid; The following steps are involved: S1. Prepare a polymerizable imidazolyl ionic liquid monomer by an alkylation substitution reaction between N-vinylimidazole and a functional monomer, wherein the functional monomer includes any one of 4-vinylbenzyl chloride, 1,3-propanesultone, chloroacetamide and β-cyclodextrin, and the polymerizable imidazolyl ionic liquid monomers prepared respectively are 1-vinyl-3-styrene imidazolium chloride ionic liquid monomer, 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer, 1-vinyl-3-acetamide imidazolium chloride ionic liquid monomer, and toluenesulfonated 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomer; S2, polymerizing the chain transfer agent 2-(phenylmethylsulfonylthio)acetic acid and N-vinylimidazole by RAFT polymerization to form a macromolecular chain transfer reagent, and then polymerizing 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer on the macromolecular chain transfer reagent to prepare Precursor-1; S3, using Precursor-1 as a chain transfer agent, RAFT polymerization was used to polymerize 1-vinyl-3-acetamidoimidazolium chloride ion liquid monomer on Precursor-1 to prepare Precursor-2; S4, using Precursor-2 as a chain transfer agent, to polymerize the tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid monomer on Precursor-1 by RAFT polymerization to prepare Precursor-3; S5, reacting the precursor Precursor-3 with 1-vinyl-3-styrene imidazolium chloride ion liquid monomer in dimethyl sulfoxide as a solvent to prepare the final product polyionic liquid macromonomer; S6. Through molecular dynamics simulation, the interaction between the synthesized polyionic liquid macromolecular monomer and protein is studied to explore the effect of polyionic liquid macromolecular monomer on the secondary structure, hydrogen bonding and energy of protein.

2. The method for preparing a polyionic liquid macromolecular monomer for stabilizing protein structure according to claim 1, characterized in that: The preparation method of the 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer in step S1 comprises the following steps: N-vinylimidazole was dissolved in acetonitrile at a molar volume ratio of 2 mol:1 L at 50 °C. 1,3-propane sultone was slowly added dropwise into the reactor using a constant pressure funnel under vigorous magnetic stirring. The molar ratio of N-vinylimidazole to 1,3-propane sultone was 2:

3. The mixture was stirred at 50 °C for 24 h to obtain a white precipitate, which was further purified by washing with acetone and freeze-dried to obtain 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer. The preparation method of the 1-vinyl-3-acetamidoimidazole chloride ion liquid monomer in step S1 comprises the following steps: N-vinylimidazole and chloroacetamide at a molar ratio of 33:31 were dissolved in a 50% ethanol solution and reacted for 24 hours. After the reaction, the mixture was extracted with ethyl acetate, and the extracted solution was freeze-dried to obtain a 1-vinyl-3-acetamidoimidazole chloride ion liquid monomer; The method for preparing the tosylated 1-vinyl-β-cyclodextrin imidazole monomer in step S1 comprises the following steps: β-cyclodextrin was dissolved in deionized water, and an aqueous solution of sodium hydroxide and an acetonitrile solution of tosyl chloride were added dropwise thereto. After stirring at 23°C and 800 rpm for 2 hours, the precipitate was removed by suction filtration, and the filtrate was refrigerated at 4°C overnight. The precipitated tosylated-β-cyclodextrin was recovered by suction filtration. Tosylated β-cyclodextrin was dissolved in a mixed solution of N-vinylimidazole and N,N-dimethylformamide, and the mixture was reacted at 75°C for 4 hours under a N2 atmosphere; after cooling to ambient temperature, the crude product was precipitated with acetone, filtered, and dried in a vacuum oven at 50°C overnight to obtain tosylated 1-vinyl-β-cyclodextrin imidazolium ionic liquid; Preparation of 1-vinyl-3-styrene imidazolium chloride ionic liquid monomer: N-vinylimidazole and 4-vinylbenzyl chloride in a molar ratio of 50:43 were dissolved in a mixed solution of water and ethyl acetate in a volume ratio of 1:1 and reacted for 24 hours. After the reaction, ethyl acetate was used for extraction, and the extract solution was freeze-dried to obtain 1-vinyl-3-styrylimidazole chloride ion liquid.

3. The method for preparing a polyionic liquid macromolecular monomer for stabilizing protein structure according to claim 1, characterized in that: The preparation of the precursor Precursor-1 in step S2 specifically includes the following steps: S2-1. A macromolecular chain transfer reagent was prepared by thermally initiating a reaction under N2 atmosphere using 1 mmol of 2-(phenylmethylsulfonylthio)acetic acid as a chain transfer agent, N-vinylimidazole as a monomer, 40 ml of dimethyl sulfoxide as a solvent, and 0.4 mmol of azobisisobutyronitrile as an initiator. S2-2, followed by the addition of 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer and azobisisobutyronitrile to a macromolecular chain transfer reagent to prepare Precursor-1; The length of the precursor Precursor-1 was adjusted by adjusting the ratio of chain transfer agent, N-vinylimidazole and 1-vinyl-3-propanesulfonic acid imidazolium chloride zwitterionic liquid monomer.

4. The method for preparing a polyionic liquid macromolecular monomer for stabilizing protein structure according to claim 1, characterized in that: The preparation conditions of the precursor Precursor-2 in step S3 are 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 ion liquid monomer as a monomer, and preparing it at 70°C under N2 atmosphere.

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

6. The method for preparing a polyionic liquid macromolecular monomer for stabilizing protein structure according to claim 5, characterized in that: The conditions for the polymerization of 1-vinyl-3-styrene imidazolium chloride ionic liquid on the precursor Precursor-3 are as follows: N-vinylimidazole is polymerized on 2-(phenylmethylsulfonylthio)acetic acid, the precursor Precursor-3 is used as a chain transfer agent, 4-vinylbenzyl chloride and hydroquinone are added to the precursor Precursor-3 through an alkylation reaction, and the polymerization is prepared at 60°C under a N2 atmosphere.

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