Vinyl compounds containing isatoic anhydride functional groups, copolymers thereof, preparation methods and applications thereof

Abstract

The present invention relates to a vinyl compound containing an isatoic anhydride functional group, a copolymer thereof, a preparation method and applications thereof. The vinyl compound containing an isatoic anhydride functional group has a structure shown in formula (1). The water-soluble copolymer containing isatoic anhydride of the present invention is prepared by copolymerizing a vinyl compound containing an isatoic anhydride functional group with a water-soluble vinyl monomer. The water-soluble copolymer can be completely dissolved in water and does not undergo self-assembly, thereby effectively exposing the isatoic anhydride functional group. It has a high reactivity with the amino side groups in proteins and can conjugate with proteins efficiently, and can be used for modifying and modifying proteins. Moreover, the reaction conditions for modifying proteins are mild, the reaction is rapid, the modified protein complex has high activity, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical fields of functional polymers and biomedicine, and particularly relates to a vinyl compound containing isatoic anhydride functional groups, its copolymer, and its preparation method and application. Background Art

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

[0003] Polymer modification is one of the important methods to solve the above problems, which can improve the functions of proteins in many aspects and expand the applications of proteins. However, the main problem in 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 available chemical modification types. Modifying and modifying polymers using the primary amine side groups of lysine units in proteins is the most commonly used method. Currently, the most used is to react N-hydroxysuccinimide (NHS) with the primary amine group to form an amide bond, but this reaction has problems such as slow reaction rate and easy hydrolysis of the NHS group, 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 isatoic anhydride functional groups and its copolymer. The isatoic anhydride functional group in it has a high reaction activity with primary amines, can conjugate with proteins containing amino side groups, and does not affect the biological activity of proteins, and can be used for the synthesis of polymer-protein composites.

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

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

[0008]

[0009] In the second aspect, the present invention provides a preparation method of the vinyl compound containing isatoic anhydride functional groups, including the following steps: reacting isatoic anhydride with 2-hydroxyethyl methacrylate under the action of triphenylphosphine and diisopropyl azodicarboxylate to obtain it.

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

[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] Among them, 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 to 5:95 to 99, preferably 1:99.

[0014] In a fourth aspect, the present invention provides 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, the aqueous vinyl monomer, and an initiator in an organic solvent, and reacting to obtain the product.

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

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

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

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

[0019] The present invention prepares a novel vinyl monomer containing isatoic anhydride by reacting isatoic anhydride with 2-hydroxyethyl methacrylate. This monomer can be copolymerized with other water-soluble vinyl monomers through free radical polymerization to prepare a polymer containing isatoic anhydride. The H on the N atom of the isatoic anhydride functional group has high reactivity after being substituted and can conjugate with a protein containing an amino side group 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 and will not self-assemble, thus effectively exposing the isatoic anhydride functional group. It has a high reactivity with the amino side groups in proteins and can efficiently conjugate with proteins, and can be used for modifying and modifying proteins. Moreover, the reaction conditions for modifying proteins are mild, the reaction is rapid, the modified protein complex has high activity, and it has broad application prospects. Description of the Drawings

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

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

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

[0024] Figure 4 SDS-PAGE image of the polymer-cytochrome C conjugate.

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

[0026] Figure 6 Catalytic activity UV-Vis spectrum of the polymer-BSA conjugate. Detailed Embodiments

[0027] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0028] The experimental methods without specific conditions noted in the following examples are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers. All kinds of commonly used chemical reagents used in the examples are commercially available products.

[0029] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field 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 used 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 other factors not described, unless the context clearly dictates otherwise. Further, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in" includes "in" and "on".

[0031] In some embodiments thereof, there is involved a vinyl compound containing an isatoic anhydride functional group, which has the structure shown in formula (1):

[0032]

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

[0034] In some embodiments thereof, there is involved a preparation method of the vinyl compound containing an isatoic anhydride functional group, which comprises the following steps: reacting isatoic anhydride and 2-hydroxyethyl methacrylate under the action of triphenylphosphine and diisopropyl azodicarboxylate to obtain the product.

[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 temperature of the reaction 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 to 1.5: 1 to 1.5: 1: 1 to 1.5, preferably 1.1 to 1.3: 1.1 to 1.3: 1: 1.1 to 1.3, more preferably 1.2: 1.2: 1: 1.2.

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

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

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

[0042] In some preferred embodiments, the molar ratio of the vinyl compound containing an isatoic anhydride functional group 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, and self-assembly will not occur, avoiding the hydrophobic isatoic anhydride functional group being wrapped in the core and reducing its reaction efficiency with the primary amino group of the protein.

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

[0044] In some of these embodiments, there is also involved a preparation method of the water-soluble copolymer containing isatoic anhydride, including the following steps: dissolving the vinyl compound containing an isatoic anhydride functional group, the aqueous vinyl monomer and an initiator in an organic solvent, and reacting to obtain the product.

[0045] In some preferred embodiments, the initiator is selected from one or more of azodiisobutyronitrile, azodiisovaleronitrile and benzoyl 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 an isatoic anhydride functional group and the aqueous 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 temperature of the reaction is 60°C-80°C, the reaction time is 6h-48h, preferably 8h-16h, and more preferably 12 hours.

[0049] In some embodiments of the present invention, there is also involved a polymer-protein conjugate, which is obtained by reacting the water-soluble copolymer containing isatoic anhydride described in the present invention with a protein.

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

[0051] Among them, the protein can be cytochrome C or bovine serum albumin, etc.

[0052] Some embodiments of the present invention also relate to a method for preparing the polymer-protein conjugate, which includes 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 it.

[0053] The water-soluble copolymer prepared by copolymerizing the vinyl monomer containing isatoic anhydride with other water-soluble vinyl monomers of the present invention can be completely dissolved in water and will not self-assemble, thereby effectively exposing the isatoic anhydride functional group. It has a high reaction activity with the amino side groups in proteins and can be efficiently conjugated with proteins, and can be used for modifying proteins. Moreover, the reaction conditions for modifying proteins are mild, the reaction is rapid, and the modified protein complex has high activity, having broad application prospects.

[0054] The following further elaborates on the present invention in conjunction with specific embodiments.

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

[0056] Example 1

[0057] (1) Synthesis of vinyl monomer 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 cool in an ice bath; under the ice bath condition, slowly dropwise add a solution of diisopropyl azodicarboxylate 29.8 g (147.5 mmol) in tetrahydrofuran (50 mL) to the obtained solution. After the addition is complete, raise the temperature to room temperature and react for 24 h; spin dry and separate by column chromatography to obtain 24.7 g of the vinyl monomer containing isatoic anhydride functional groups, with a yield of 73%.

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

[0060]

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

[0062] Dissolve 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 in 20 mL of N,N-dimethylformamide, react at 70 °C for 12 h, cool to room temperature, pour the reaction solution into ether for precipitation, and vacuum dry the obtained solid to obtain 16.5 g of a water-soluble copolymer containing isatoic anhydride functional groups, with a yield of 89%.

[0063] The obtained copolymer was analyzed by gel permeation chromatography, and the results are shown in Figure 2 , indicating that the polymer was successfully synthesized, and its number-average molecular weight was 13,500.

[0064] Prepare an aqueous solution of the obtained copolymer with a concentration of 10 mg / mL. The particle size could not be measured by dynamic light scattering, indicating complete dissolution.

[0065] Example 2

[0066] It 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] It 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] It 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 copolymer can self-assemble in water (1 mg / mL). As Figure 3 shown, self-assembly into spherical micelles with a particle size of ~21 nm was observed by atomic force microscopy. The formation of micelles is mainly because the monomer unit containing isatoic anhydride in the copolymer is hydrophobic. When the hydrophobic component exceeds the critical value, the polymer will spontaneously self-assemble in water, and isatoic anhydride is wrapped in the micelles and cannot contact with the primary amino group in the subsequent conjugation reaction to react.

[0072] Example 4 Complexation of the Water-Soluble Copolymer Containing Isatoic Anhydride Functional Groups with 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 purify the rest by dialysis and lyophilization to obtain the polymer-protein complex.

[0074] The obtained conjugation solution was analyzed for conjugation efficiency by 15% SDS-PAGE, and the results are as Figure 4 shown. At a low concentration and a reaction time of 3 h, its conjugation efficiency is as high as 92%, indicating that the conjugation reaction of the polymer and the protein prepared in the present invention is rapid and efficient.

[0075] Example 5 Complexation of Polymer-Bovine Serum Albumin (BSA) and Its Catalytic Activity

[0076] Dissolve bovine serum albumin BSA (1 mmol) in 10 mL of PBS buffer (pH = 7.4), add 10 mg of the water-soluble copolymer containing isatoic anhydride functional groups prepared in Example 1, react at room temperature for 6 hours, then quench the reaction with 50 μL of saturated glycine aqueous solution, and separate by preparative GPC to obtain the polymer-BSA conjugate.

[0077] The GPC spectra before and after conjugation are as Figure 5 shown. The molecular weight of the product after conjugation increased significantly, 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 characterization process is as follows:

[0079] Quickly mix 0.1 mL of PBS buffer of BSA or polymer-BSA conjugate (both at a concentration of 0.27 mM in terms of the concentration of BSA, pH = 8.0), 10 μL of acetonitrile solution of p-nitrophenyl acetate (10 mM), and 0.9 mL of PBS buffer (pH = 8.0), and react at 25 °C for 20 min. Measure the absorbance peak intensity at 405 nm with UV-Vis spectroscopy (the characteristic absorbance peak of the hydrolysis product p-nitrophenol), and the results are as Figure 6 shown. It can be found that the polymer-BSA conjugate still retains very high catalytic activity. By comparing the absorbance 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-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended 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 isatoic anhydride functional group according to claim 1, characterized in that, It includes the following steps: phthalic anhydride and 2-hydroxyethyl methacrylate react under the action of triphenylphosphine and diisopropyl azodicarboxylate to obtain the product.

3. The preparation method of the vinyl compound containing isatoic anhydride functional group according to claim 2, characterized in that, The reaction is carried out in an organic solvent; and / or The temperature of the reaction is 15°C to 40°C; and / or The time of the reaction is 12 h to 48 h; and / or The molar ratio of the phthalic anhydride, triphenylphosphine, 2-hydroxyethyl methacrylate and diisopropyl azodicarboxylate is 1 to 1.5: 1 to 1.5: 1: 1 to 1.

5.

4. The method for preparing a vinyl compound containing an isatoic anhydride functional group according to claim 3, characterized in that, The organic solvent is at least one of dioxane, tetrahydrofuran, N,N-dimethylformamide and dimethyl sulfoxide.

5. The preparation method of the vinyl compound containing isatoic anhydride functional group according to claim 3, characterized in that, The temperature of the reaction is 20°C to 30°C.

6. The preparation method of the vinyl compound containing isatoic anhydride functional group according to claim 3, characterized in that, The time of the reaction is 20 h to 30 h.

7. The method for preparing a vinyl compound containing an isatoic anhydride functional group according to claim 3, characterized in that, The molar ratio of the phthalic anhydride, triphenylphosphine, 2-hydroxyethyl methacrylate and diisopropyl azodicarboxylate is 1.1 to 1.3: 1.1 to 1.3: 1: 1.1 to 1.

3.

8. The method for preparing a vinyl compound containing an isatoic anhydride functional group according to claim 3, wherein The preparation method of the vinyl compound containing a phthalic anhydride functional group includes the following steps: dissolve phthalic anhydride, triphenylphosphine and 2-hydroxyethyl methacrylate in an organic solvent, cool in an ice bath, dropwise add an organic solvent solution of diisopropyl azodicarboxylate into the obtained solution, and raise the temperature to 15°C to 40°C for reaction for 12 h to 48 h to obtain the product.

9. A water-soluble copolymer containing isatoic anhydride, characterized in that, It is prepared by copolymerizing the vinyl compound containing a phthalic anhydride functional group described in claim 1 with a water-soluble vinyl monomer.

10. The water-soluble copolymer containing isatoic anhydride according to claim 9, 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 a phthalic anhydride functional group to the water-soluble vinyl monomer is 1 to 5: 95 to 99; and / or The number-average molecular weight of the water-soluble copolymer containing phthalic anhydride is 13000 to 14000.

11. The water-soluble copolymer containing isatoic anhydride according to claim 10, characterized in that, The molar ratio of the vinyl compound containing a phthalic anhydride functional group to the water-soluble vinyl monomer is 1:

99.

12. A method for preparing a water-soluble copolymer containing isatoic anhydride according to any one of claims 9-11, characterized in that, It includes the following steps: dissolve the vinyl compound containing a phthalic anhydride functional group, the aqueous vinyl monomer and the initiator in an organic solvent, and react to obtain the product.

13. The preparation method of the water-soluble copolymer containing isatoic anhydride according to claim 12, characterized in that, The initiator is selected from one or more of azodiisobutyronitrile, azodiisovaleronitrile 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 used to the initiator is 1: 0.001 to 0.03, and the total amount of monomers used is the total amount of the vinyl compound containing a phthalic anhydride functional group and the aqueous vinyl monomer; and / or The temperature of the reaction is 60°C to 80°C, and the time of the reaction is 6 h to 48 h.

14. The preparation method of the water-soluble copolymer containing isatoic anhydride according to claim 13, characterized in that, The molar ratio of the total amount of monomers used to the initiator is 1: 0.005 to 0.

015.

15. The preparation method of the water-soluble copolymer containing isatoic anhydride according to claim 13, characterized in that, The time of the reaction is 8 h - 16 h.

16. Use of the water-soluble copolymer containing phthalic anhydride described in any one of claims 9-11 in modifying proteins.

17. A polymer-protein conjugate, characterized in that, It is obtained by reacting the water-soluble copolymer containing phthalic anhydride described in any one of claims 9-11 with a protein.

18. The polymer-protein conjugate according to claim 17, wherein, The ratio of the water-soluble copolymer containing isatoic anhydride to the protein is 8 mg to 12 mg: 1 μmol.

19. The polymer-protein conjugate according to claim 17 or 18, characterized in that, The protein is cytochrome C or bovine serum albumin.

20. A method for preparing a polymer-protein conjugate according to any one of claims 17-19, characterized in that, It includes the following steps: adding the water-soluble copolymer containing isatoic anhydride functional groups into a buffer solution containing the protein, and reacting at a temperature of 15°C to 40°C for 2 hours to 6 hours to obtain the product.

Images