Isatoic anhydride functional group-containing vinyl compound, copolymer thereof, preparation method and application thereof

Abstract

The invention relates to a vinyl compound containing an isatoic anhydride functional group, a copolymer thereof, and a preparation method and application of the vinyl compound. The vinyl compound containing the isatoic anhydride functional group has a structure as shown in a formula (1). The water-soluble copolymer containing isatoic anhydride is prepared by copolymerizing a vinyl compound containing an isatoic anhydride functional group and a water-soluble vinyl monomer. The water-soluble copolymer can be completely dissolved in water and does not generate self-assembly, so that an isatoic anhydride functional group can be effectively exposed, and the water-soluble copolymer has very high reaction activity with an amino side group in protein, can be efficiently conjugated with the protein and can be used for modifying the protein. And the reaction conditions for modifying the protein are mild, the reaction is rapid, and the modified protein compound is high in activity and has a wide application prospect. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of functional polymers and biomedicine, and specifically relates to a vinyl compound containing an isatoic anhydride functional group, a copolymer thereof, and a preparation method and application thereof. Background Art

[0002] As an important class of biomacromolecules, proteins have a wide range of applications in chemical and medical fields such as biocatalysis, drug delivery, and molecular imaging due to their special three-dimensional spatial structure and efficient catalytic activity. However, their application is limited due to their low stability, strong immunogenicity, and poor solubility in organic solvents.

[0003] Polymer modification is one of the important methods to solve the above problems. It can improve the function of proteins in many aspects and expand the application of proteins. However, the main problem of modifying proteins with polymers is how to retain the original biological activity of proteins while ensuring the efficiency and stability of modification.

[0004] Currently, there are not many types of chemical modifications that can be used. The most commonly used method is to use the primary amine side groups of lysine units in proteins to modify polymers. The most commonly used method is to use N-hydroxysuccinimide (NHS) to react with primary amine groups to form amide bonds, but this reaction has problems such as slow reaction rate and easy hydrolysis of NHS groups, which has obvious limitations in the preparation of polymer-protein composites. Summary of the invention

[0005] Based on this, the present invention provides a vinyl compound containing an isatoic anhydride functional group and a copolymer thereof, wherein the isatoic anhydride functional group has a high reactivity with primary amines, can be conjugated with proteins containing amino side groups, and does not affect the biological activity of the protein, and can be used for the synthesis of polymer-protein composite materials.

[0006] The present invention includes the following technical solutions.

[0007] In a first aspect, the present invention provides a vinyl compound containing an isatoic anhydride functional group, having a structure shown in formula (1):

[0008]

[0009] In a second aspect, the present invention provides a method for preparing the vinyl compound containing isatoic anhydride functional group, comprising the following steps: isatoic anhydride and 2-hydroxyethyl methacrylate react under the action of triphenylphosphine and diisopropyl azodicarboxylate to obtain.

[0010] Preferably, the preparation method of the vinyl compound containing isatoic anhydride functional group comprises the following steps: dissolving isatoic anhydride, triphenylphosphine and 2-hydroxyethyl methacrylate in an organic solvent, placing in an ice bath, dropping an organic solvent solution of diisopropyl azodicarboxylate into the obtained solution, heating to 15°C to 40°C and reacting for 12h to 48h to obtain the vinyl compound.

[0011] In a third aspect, the present invention provides a water-soluble copolymer containing isatoic anhydride, which is prepared by copolymerizing the vinyl compound containing isatoic anhydride functional group with a water-soluble vinyl monomer.

[0012] The water-soluble vinyl monomer can be selected from one or more of acrylamide, N,N-dimethylacrylamide, polyethylene glycol methacrylate and polyethylene glycol acrylate.

[0013] Preferably, the molar ratio of the vinyl compound containing isatoic anhydride functional group to the water-soluble vinyl monomer is 1-5:95-99, preferably 1:99.

[0014] In a fourth aspect, the present invention provides a method for preparing the water-soluble copolymer containing isatoic anhydride, comprising the steps of: dissolving the vinyl compound containing isatoic anhydride functional group, an aqueous vinyl monomer and an initiator in an organic solvent, and reacting to obtain the water-soluble copolymer.

[0015] In a fifth aspect, the present invention provides the use of the water-soluble copolymer containing isatoic anhydride in modifying proteins.

[0016] In a sixth aspect, the present invention provides a polymer-protein conjugate obtained by reacting the water-soluble copolymer containing isatoic anhydride described in the present invention with protein.

[0017] In a seventh aspect, the present invention provides a method for preparing the polymer-protein conjugate, comprising the following steps: adding the water-soluble copolymer containing isatoic anhydride functional groups to a buffer solution containing protein, reacting at a temperature of 15°C to 40°C for 2 hours to 6 hours, to obtain

[0018] The present invention has the following beneficial effects:

[0019] The invention prepares a novel vinyl monomer containing isocyanate by reacting isocyanate with hydroxyethyl methacrylate. The monomer can be used to prepare a polymer containing isocyanate by free radical polymerization with other water-soluble vinyl monomers. The polymer has high reactivity with primary amine after H on the N atom of the isocyanate functional group is replaced, and can be conjugated with proteins containing amino side groups for protein modification.

[0020] The water-soluble copolymer prepared by copolymerizing a vinyl monomer containing isatoic anhydride with other water-soluble vinyl monomers can be completely dissolved in water without self-assembly, thereby effectively exposing the isatoic anhydride functional group, having high reactivity with the amino side group in the protein, being able to be efficiently conjugated with the protein, and being used to modify the protein. Moreover, the reaction conditions for modifying the protein are mild, the reaction is rapid, the modified protein complex is highly active, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A vinyl monomer containing an isatoic anhydride functional group 1 H NMR spectrum.

[0022] Figure 2 This is the GPC spectrum of the water-soluble copolymer containing isatoic anhydride functional groups prepared in Example 1.

[0023] Figure 3 This is the AFM image of the copolymer self-assembled micelles prepared in Comparative Example 1.

[0024] Figure 4 It is the SDS-PAGE picture of polymer-cytochrome C conjugate.

[0025] Figure 5 This is the GPC spectrum of polymer-BSA conjugate.

[0026] Figure 6 The UV-Vis spectrum of the catalytic activity of the polymer-BSA conjugate. DETAILED DESCRIPTION

[0027] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0028] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0029] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0030] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

[0031] Some of the embodiments involve a vinyl compound containing an isatoic anhydride functional group, which has a structure shown in formula (1):

[0032]

[0033] The vinyl compound containing isatoic anhydride functional groups of the present invention has high reactivity with primary amines and can be conjugated with proteins containing amino side groups, and thus can be used to modify proteins.

[0034] Some of the embodiments involve a method for preparing a vinyl compound containing an isatoic anhydride functional group, comprising the following steps: isatoic anhydride and 2-hydroxyethyl methacrylate react under the action of triphenylphosphine and diisopropyl azodicarboxylate to obtain.

[0035] In some preferred embodiments, the reaction is carried out in an organic solvent, and the organic solvent is preferably at least one of dioxane, tetrahydrofuran, N,N-dimethylformamide and dimethyl sulfoxide, more preferably tetrahydrofuran.

[0036] In some preferred embodiments, the reaction temperature is 15°C to 40°C, preferably 20°C to 30°C.

[0037] In some preferred embodiments, the reaction time is 12 h to 48 h, preferably 20 h to 30 h.

[0038] In some preferred embodiments, the molar ratio of isatoic anhydride, triphenylphosphine, 2-hydroxyethyl methacrylate and diisopropyl azodicarboxylate is 1-1.5:1-1.5:1:1-1.5, preferably 1.1-1.3:1.1-1.3:1:1.1-1.3, and more preferably 1.2:1.2:1:1.2.

[0039] In some preferred embodiments, the preparation method of the vinyl compound containing isatoic anhydride functional group comprises the following steps: dissolving isatoic anhydride, triphenylphosphine and 2-hydroxyethyl methacrylate in an organic solvent, placing in an ice bath, dropping an organic solvent solution of diisopropyl azodicarboxylate into the obtained solution, heating to 15°C to 40°C and reacting for 12h to 48h to obtain the vinyl compound.

[0040] Some of the embodiments also involve a water-soluble copolymer containing isatoic anhydride, which is prepared by copolymerizing the vinyl compound containing isatoic anhydride functional group described in the present invention with a water-soluble vinyl monomer.

[0041] The water-soluble vinyl monomer is preferably one or more selected from acrylamide, N,N-dimethylacrylamide, polyethylene glycol methacrylate and polyethylene glycol acrylate.

[0042] In some preferred embodiments, the molar ratio of the vinyl compound containing isatoic anhydride functional groups to the water-soluble vinyl monomer is 1-5:95-99, preferably 1:99. The water-soluble copolymer prepared under this ratio can be completely dissolved in water without self-assembly, thus avoiding the hydrophobic isatoic anhydride functional groups from being wrapped in the core to reduce the reaction efficiency with the primary amine groups of the protein.

[0043] In some embodiments, the water-soluble copolymer containing isatoic anhydride has a number average molecular weight of 13,000 to 14,000.

[0044] Some of the embodiments also involve a method for preparing the water-soluble copolymer containing isatoic anhydride, which comprises the following steps: dissolving the vinyl compound containing isatoic anhydride functional group, water-based vinyl monomer and initiator in an organic solvent, and reacting to obtain the water-soluble copolymer.

[0045] In some preferred embodiments, the initiator is selected from one or more of azobisisoheptanonitrile, azobisisobutyronitrile and dibenzoyl peroxide.

[0046] In some preferred embodiments, the molar ratio of the total amount of monomers to the initiator is 1:0.001-0.03, preferably 1:0.005-0.015, and the total amount of monomers refers to the total amount of the vinyl compound containing isatoic anhydride functional group and the water-based vinyl monomer.

[0047] In some preferred embodiments, the organic solvent is selected from one or more of dioxane, tetrahydrofuran, N,N-dimethylformamide and dimethyl sulfoxide.

[0048] In some preferred embodiments, the reaction temperature is 60° C. to 80° C., and the reaction time is 6 h to 48 h, preferably 8 h to 16 h, and more preferably 12 hours.

[0049] Some embodiments of the present invention also relate to a polymer-protein conjugate, which is obtained by reacting the water-soluble copolymer containing isatoic anhydride of the present invention with protein.

[0050] In some preferred embodiments, the ratio of the water-soluble copolymer containing isatoic anhydride to protein is 8 mg to 12 mg: 1 μmol.

[0051] Wherein, the protein can be cytochrome C or bovine serum albumin.

[0052] Some embodiments of the present invention also involve a method for preparing the polymer-protein conjugate, comprising the following steps: adding the water-soluble copolymer containing isatoic anhydride functional groups of the present invention to a buffer solution containing protein, and reacting at a temperature of 15° C. to 40° C. for 2 hours to 6 hours to obtain

[0053] The water-soluble copolymer prepared by copolymerizing a vinyl monomer containing isatoic anhydride with other water-soluble vinyl monomers can be completely dissolved in water without self-assembly, thereby effectively exposing the isatoic anhydride functional group, having high reactivity with the amino side group in the protein, being able to be efficiently conjugated with the protein, and being used to modify the protein. Moreover, the reaction conditions for modifying the protein are mild, the reaction is rapid, the modified protein complex is highly active, and has broad application prospects.

[0054] The present invention is further described in detail below with reference to specific embodiments.

[0055] In the following examples, the room temperature refers to a room temperature of 20°C-30°C unless otherwise specified.

[0056] Example 1

[0057] (1) Synthesis of vinyl monomers containing isatoic anhydride functional groups

[0058] Dissolve 24.1 g (147.7 mmol) of isatoic anhydride, 38.7 g (147.7 mmol) of triphenylphosphine and 16.0 g (122.9 mmol) of 2-hydroxyethyl methacrylate in 100 mL of tetrahydrofuran and place in an ice bath; slowly dropwise add a tetrahydrofuran (50 mL) solution containing 29.8 g (147.5 mmol) of diisopropyl azodicarboxylate to the resulting solution under ice bath conditions, raise the temperature to room temperature after the addition is complete, and react for 24 hours; spin dry, separate by column chromatography, and obtain 24.7 g of a vinyl monomer containing an isatoic anhydride functional group, with a yield of 73%.

[0059] The product structure was characterized by H NMR spectrum. Figure 1 , indicating that the monomer has been synthesized, and its structural formula is:

[0060]

[0061] (2) Synthesis of water-soluble copolymers containing isatoic anhydride functional groups

[0062] 0.5 g (1.8 mmol) of the monomer obtained in step (1), 17.67 g (178.2 mmol) of N,N-dimethylacrylamide and 0.3 g (1.8 mmol) of azobisisobutyronitrile were dissolved in 20 mL of N,N-dimethylformamide, reacted at 70° C. for 12 h, cooled to room temperature, poured the reaction solution into ether for precipitation, and the obtained solid was vacuum dried to obtain 16.5 g of a water-soluble copolymer containing isocyanate functional groups, with a yield of 89%.

[0063] The obtained copolymer was analyzed by gel permeation chromatography. Figure 2 , indicating that the polymer was successfully synthesized and its number average molecular weight was 13500.

[0064] The obtained copolymer was added with water to prepare a 10 mg / mL aqueous solution. The particle size could not be measured by dynamic light scattering, indicating complete dissolution.

[0065] Example 2

[0066] The method is basically the same as Example 1, except that the water-soluble monomer used in step (2) is polyethylene glycol methacrylate (the molecular weight of the polyethylene glycol chain is 400).

[0067] Example 3

[0068] The method is basically the same as Example 1, except that the molar ratio of the vinyl monomer containing isatoic anhydride to dimethylacrylamide used in step (2) is 5:95.

[0069] Comparative Example 1

[0070] The method is basically the same as Example 1, except that the molar ratio of the vinyl monomer containing isatoic anhydride to dimethylacrylamide used in step (2) is 7:93.

[0071] The obtained copolymers were able to self-assemble in water (1 mg / mL), as Figure 3 As shown, atomic force microscopy observations revealed that the micelles were self-assembled into spherical micelles with a particle size of ~21 nm. The formation of micelles is mainly due to the hydrophobicity of the monomer units containing isatoic anhydride in the copolymer. When the hydrophobic component exceeds the critical value, the polymer will spontaneously self-assemble in water, and isatoic anhydride is encapsulated in the micelles and cannot contact and react with the primary amine group in the subsequent conjugation reaction.

[0072] Example 4 Composite of water-soluble copolymer containing isatoic anhydride functional groups and cytochrome C

[0073] Take 10 mL of PBS buffer solution containing cytochrome C (pH = 7.4, cytochrome C concentration 100 μmol / L), add 10 mg of the water-soluble copolymer containing isatoic anhydride functional groups prepared in Example 1, react at room temperature for a period of time, then add 50 μL of saturated glycine aqueous solution to quench the reaction, take a small amount for SDS-PAGE analysis, and dialysis and freeze-drying to purify the rest to obtain a polymer-protein complex.

[0074] The obtained conjugation solution was analyzed for conjugation efficiency by 15% SDS-PAGE. Figure 4 As shown, the conjugation efficiency is as high as 92% at low concentration and 3 h reaction time, indicating that the conjugation reaction between the polymer prepared by the present invention and protein is fast and efficient.

[0075] Example 5 Polymer-bovine serum albumin (BSA) complex and its catalytic activity

[0076] Bovine serum albumin BSA (1 mmol) was dissolved in 10 mL PBS buffer (pH = 7.4), and 10 mg of the water-soluble copolymer containing isocyanate functional groups prepared in Example 1 was added. The mixture was reacted at room temperature for 6 hours, and then the reaction was quenched with 50 μL saturated glycine aqueous solution. The mixture was separated by preparative GPC to obtain a polymer-BSA conjugate.

[0077] GPC spectra before and after conjugation are as follows Figure 5 As shown, the molecular weight of the product increased significantly after conjugation, indicating that the polymer-BSA conjugate was successfully prepared.

[0078] The polymer-BSA conjugate can be used to catalyze ester hydrolysis, and its catalytic performance is characterized as follows:

[0079] 0.1 mL of BSA or polymer-BSA conjugate in PBS buffer (both 0.27 mM in terms of BSA concentration, pH = 8.0), 10 μL of acetonitrile solution of p-nitrophenyl acetate (10 mM), and 0.9 mL of PBS buffer (pH = 8.0) were quickly mixed and reacted at 25°C for 20 min. The absorption peak intensity at 405 nm (characteristic absorption peak of the hydrolysis product p-nitrophenol) was measured by UV-Vis spectroscopy. The results are shown in FIG. Figure 6 As shown, it can be found that the polymer-BSA conjugate still retains a very high catalytic activity. By comparing the absorption intensity of the BSA system at 405 nm, it can be found that its catalytic activity is about 95% of that before conjugation.

[0080] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A vinyl compound containing an isatoic anhydride functional group, characterized in that: It has the structure shown in formula (1):

2. A method for preparing a vinyl compound containing an isatoic anhydride functional group according to claim 1, characterized in that: The method comprises the following steps: isatoic anhydride and 2-hydroxyethyl methacrylate react under the action of triphenylphosphine and diisopropyl azodicarboxylate to obtain the product.

3. The method for preparing a vinyl compound containing an isatoic anhydride functional group according to claim 2, characterized in that: The reaction is carried out in an organic solvent, and the organic solvent is preferably at least one of dioxane, tetrahydrofuran, N,N-dimethylformamide and dimethyl sulfoxide; and / or, The reaction temperature is 15°C to 40°C, preferably 20°C to 30°C; and / or, The reaction time is 12h to 48h, preferably 20h to 30h; and / or, The molar ratio of isatoic anhydride, triphenylphosphine, 2-hydroxyethyl methacrylate and diisopropyl azodicarboxylate is 1-1.5:1-1.5:1:1-1.5, preferably 1.1-1.3:1.1-1.3:1:1.1-1.3; Preferably, the preparation method of the vinyl compound containing isatoic anhydride functional group comprises the following steps: dissolving isatoic anhydride, triphenylphosphine and 2-hydroxyethyl methacrylate in an organic solvent, placing in an ice bath, dropping an organic solvent solution of diisopropyl azodicarboxylate into the obtained solution, heating to 15°C to 40°C and reacting for 12h to 48h to obtain the vinyl compound.

4. A water-soluble copolymer containing isatoic anhydride, characterized in that: The product is prepared by copolymerizing the vinyl compound containing isatoic anhydride functional group as claimed in claim 1 with a water-soluble vinyl monomer.

5. The water-soluble copolymer containing isatoic anhydride according to claim 4, characterized in that: The water-soluble vinyl monomer is selected from one or more of acrylamide, N,N-dimethylacrylamide, polyethylene glycol methacrylate and polyethylene glycol acrylate; and / or, The molar ratio of the vinyl compound containing isatoic anhydride functional group to the water-soluble vinyl monomer is 1-5:95-99, preferably 1:99; and / or, The number average molecular weight of the water-soluble copolymer containing isatoic anhydride is 13,000 to 14,000.

6. A method for preparing a water-soluble copolymer containing isatoic anhydride according to claim 4 or 5, characterized in that: The method comprises the following steps: dissolving the vinyl compound containing isatoic anhydride functional group, water-based vinyl monomer and initiator in an organic solvent, and reacting to obtain the product.

7. The method for preparing a water-soluble copolymer containing isatoic anhydride according to claim 6, characterized in that: The initiator is selected from one or more of azobisisoheptanonitrile, azobisisobutyronitrile and dibenzoyl peroxide; and / or, The organic solvent is selected from one or more of dioxane, tetrahydrofuran, N,N-dimethylformamide and dimethyl sulfoxide; and / or, The molar ratio of the total amount of monomers to the initiator is 1:0.001-0.03, preferably 1:0.005-0.015, and the total amount of monomers is the total amount of the vinyl compound containing isatoic anhydride functional group and the water-based vinyl monomer; and / or, The reaction temperature is 60° C. to 80° C., and the reaction time is 6 h to 48 h, preferably 8 h to 16 h.

8. Use of the water-soluble copolymer containing isatoic anhydride according to claim 4 or 5 in modifying proteins.

9. A polymer-protein conjugate, characterized in that Obtained by reacting the water-soluble copolymer containing isatoic anhydride according to claim 4 or 5 with protein; Preferably, the ratio of the water-soluble copolymer containing isatoic anhydride to the protein is 8 mg to 12 mg: 1 μmol; Preferably, the protein is cytochrome C or bovine serum albumin.

10. A method for preparing a polymer-protein conjugate according to claim 9, characterized in that: The method comprises the following steps: adding the water-soluble copolymer containing isatoic anhydride functional groups into a buffer solution containing protein, and reacting at a temperature of 15 to 40° C. for 2 to 6 hours to obtain the product.

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