Solid material with surface glycopeptide molecule chemical modification, modification method and application

Abstract

The invention belongs to the technical field of material interface chemistry, and particularly relates to a surface glycopeptide molecule chemical modified solid material, a modification method and application, firstly, a commercial KH550 silane coupling agent is used for treating the surface of the material, so that an amino reaction site is introduced; grafting a polypeptide macromolecular chain with double bonds on a side chain on the surface of the material through amino-initiated N-carboxyl cyclic anhydride ring-opening polymerization reaction; and finally, preparing glycopeptide molecules on the surface of the material through water-phase terminal group activation of sugar molecules and sulfydryl-double bond click chemistry. By means of selective combination of glycopeptide to specific protein, rapid separation and purification of a protein mixed sample can be achieved. By utilizing the interaction of glycopeptide molecules with bacteria and cells and the antibacterial property of amino sugar, the material surface can be endowed with good antibacterial property and biocompatibility through surface glycopeptide modification, and a raw material is provided for the development of subsequent medical auxiliary materials and in-vivo implant materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of material interface chemistry, and in particular relates to a solid material with surface glycopeptide molecules chemically modified, a modification method and an application thereof. Background Art

[0002] There are a large number of glycosylation modifications on cell surface proteins. This ubiquitous glycopeptide structure, as an important antigen molecule, affects various cell activities. For example, the infection process of pathogenic microorganisms and the immune escape of tumor cells are all related to the specific recognition and interaction between sugars and proteins. By simulating the sugar cluster structure on the cell surface and fixing the glycopeptide molecules on the surface of the material by artificial chemical synthesis, new properties can be given to the material. For example, by fixing the glycopeptide molecules that specifically bind to the lectin protein on the surface of the microsphere, affinity chromatography fillers for protein separation and purification can be prepared. Based on the antibacterial activity of amino sugars, glycopeptide molecules containing chitosan oligosaccharide structures are fixed on the surface of fiber fabrics, which can give the material good antibacterial properties. At present, the interfacial chemical fixation methods of glycopeptide molecules include surface free radical polymerization, surface amino-carboxyl coupling, avidin-biotin coupling and other technologies. However, the existing methods have certain limitations in terms of universality. For example, surface-initiated free radical polymerization requires the synthesis of a chain transfer agent with a special molecular structure and fixation on the surface of the material. In addition, sugar monomers with double bond structures need to be synthesized through complex chemical processes, and these processes often require the use of a large amount of organic solvents. Therefore, developing a more general green aqueous glycopeptide interface immobilization method that does not require complex chemical synthesis is of great significance and has broad application value. Summary of the invention

[0003] The purpose of the present invention is to provide a simple and universal method for chemically fixing glycopeptides on material surfaces. First, the material surface is treated with a commercial KH550 silane coupling agent to introduce amino reaction sites; then, a polypeptide macromolecular chain containing double bonds in the side chain is grafted on the material surface through an amino-initiated N-carboxyl cyclic anhydride (NCA) ring-opening polymerization reaction; finally, glycopeptide molecules are prepared on the material surface through aqueous phase end group activation of sugar molecules and thiol-double bond click chemistry.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] A method for chemically modifying glycopeptide molecules on the surface of solid materials, the fixation technology roadmap is as follows:

[0006]

[0007] The modification method comprises the following steps:

[0008] S1. Ultrasonic dispersion of 10 g of solid material in 10 mL of ethanol solution, followed by sequential addition of 90 mL of acidic ethanol solution (generally acidic conditions help accelerate the hydrolysis of the silane coupling agent, and subsequent interfacial coupling reaction can only be carried out after hydrolysis, and acetic acid can be used here), 4 g of silane coupling agent KH-550, stirring at room temperature for 2 hours, taking out the solid material, washing it with distilled water 3 times, washing it with ethanol 3 times, and finally drying it in a vacuum oven at 110° C. for 1 hour to obtain a surface amino-functionalized material; wherein the volume ratio of ethanol / water in the acidic ethanol solution is 95 / 5, i.e., 95% of the acidic ethanol aqueous solution, and the pH value is 4 to 6;

[0009] S2. Take 1.0g of the surface amino functionalized material and 2.0g of the N-carboxyl-intracyclic anhydride derivative monomer of L-allylglycine, add them to a glass reaction bottle, replace the air in the reaction bottle with dry nitrogen three times, add 100mL of N,N-dimethylformamide with a water content of ≤30ppm under nitrogen atmosphere, stir and react at room temperature for 1 to 4 days, take out the solid material, wash it with tetrahydrofuran and ethanol in turn, and vacuum dry it at room temperature to obtain a solid material with a polypeptide macromolecular chain surface modified with a double bond in the side chain;

[0010] S3. Dissolve 1.0 mmol of sugar molecules in acetonitrile aqueous solution, cool the reaction solution to 0°C, add 1.7 mL of triethylamine, 1.0 g of sodium thiosulfate, and 0.7 g of 2-chloro-1,3-dimethylimidazoline chloride to the reaction solution respectively, maintain the reaction temperature at 0°C and stir for 2 hours, then add 0.25 g of sodium sulfide nonahydrate and continue stirring at room temperature for 1 hour, finally add 1.0 g of the solid material prepared in the second step reaction with the surface modified with a polypeptide macromolecular chain containing double bonds in the side chain, continue stirring at room temperature for 1 to 4 days, take out the solid material after the reaction is completed, wash it with water and ethanol 3 times respectively, and vacuum dry it to obtain the glycopeptide molecule surface modified material.

[0011] Furthermore, the preparation method of the N-carboxyl-intracyclic anhydride derivative monomer of L-allylglycine is as follows: 2.5 g L-allylglycine, 14 mL α-pinene, 2.6 g triphosgene, and 100 mL ultra-dry tetrahydrofuran are respectively added to a reaction bottle under nitrogen protection, the reaction system is heated to 50° C. for reaction for 3-5 hours, the reaction solution is vacuum concentrated to 30 mL, 100 mL of n-hexane is added to generate a precipitate, the white precipitate is collected by filtration, redissolved in 100 mL of ethyl acetate and filtered, vacuum concentrated to 20 mL, 100 mL of n-hexane is added to generate a precipitate, the precipitate is collected, and vacuum dried to obtain the NCA monomer.

[0012] Furthermore, the solid material in step S1 is one of a polymer material, an inorganic non-metallic material, and a metal material, and its morphology is a powder material, a fiber fabric material, a film material, or a sheet material.

[0013] Furthermore, in step S3, the sugar molecule is a monosaccharide or an oligosaccharide, the monosaccharide is one of glucose, galactose, mannose, fucose, glucosamine, and galactosamine, and the oligosaccharide is one of maltose, lactose, and chitosan oligosaccharide; and the volume concentration of the acetonitrile aqueous solution is 10% to 50%.

[0014] Another object of the present invention is to provide a solid material with surface glycopeptide molecules chemically modified, which is prepared by the above method.

[0015] Another object of the present invention is to provide an application of a solid material with chemically modified surface glycopeptide molecules, wherein the solid material is used in affinity chromatography fillers for protein separation and purification, especially when the solid material is magnetic microspheres and the sugar type is D-lactose, the magnetic microspheres modified with sugar chains have the ability to selectively separate concanavalin.

[0016] Another object of the present invention is to provide an application of a solid material with surface glycopeptide molecules chemically modified, wherein the solid material is used in medical auxiliary materials for wound infections caused by Pseudomonas aeruginosa, such as Band-Aids. In particular, when the solid material is a nylon fiber non-woven fabric and the sugar type is D-glucosamine, the nylon fiber non-woven fabric modified with sugar chains has a good antibacterial effect on Pseudomonas aeruginosa and Vibrio vulnificus that cause wound infections, with an antibacterial rate of 70% to 88%.

[0017] The advantages of the present invention are as follows: the present invention provides a simple and universal method for chemically fixing glycopeptides on the surface of materials. First, the surface of the material is treated with a commercial KH550 silane coupling agent to introduce amino reaction sites; then, a polypeptide macromolecular chain containing double bonds in the side chain is grafted onto the surface of the material through an amino-initiated N-carboxyl cyclic anhydride (NCA) ring-opening polymerization reaction; finally, glycopeptide molecules are prepared on the surface of the material through aqueous end group activation of sugar molecules and thiol-double bond click chemistry; the selective binding of glycopeptides to specific proteins can achieve rapid separation and purification of protein mixed samples. By utilizing the interaction between glycopeptide molecules and bacteria and cells and the antibacterial properties of amino sugars, surface glycopeptide modification can give the material surface good antibacterial properties and biocompatibility, providing raw materials for the subsequent development of medical auxiliary materials and in vivo implant materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a comparison chart of the X-ray photoelectron spectroscopy characterization of the surface of the sugar chain modified microspheres prepared in Application Example 1 of the present invention and the original microspheres.

[0019] Figure 2 This is a comparison chart of the selective separation effects of sugar chain modified microspheres prepared in Application Example 1 of the present invention on different proteins.

[0020] Figure 3This is a diagram of the antibacterial evaluation results of the non-woven fabric modified with glycopeptide molecules prepared in Application Example 2 of the present invention; wherein (A) is the original fabric; and (B) is the fabric modified with glycopeptide molecules.

[0021] Figure 4 This is the nuclear magnetic hydrogen spectrum of the NCA monomer prepared in Example 1 of the present invention. Among them, the three groups of peaks with chemical shifts in the range of 4.0-6.0ppm are characteristic peaks of double bond structures. NCA polymerization has two advantages: 1. It can simply and quickly prepare brush-like polymer chains with double bond structures in the side chains; 2. Because it is a polypeptide main chain structure derived from amino acids, compared with the common petroleum-based polymer structure, the polypeptide structure is an endogenous molecule, has better biocompatibility, and does not have the toxicity risk caused by in vivo and in vitro degradation fragments.

[0022] Figure 5 This is a cytotoxicity test diagram of nylon fiber fabric after surface modification with glycopeptide molecules in Application Example 2.

[0023] Figure 6 This is a graph showing the antibacterial evaluation results of the product prepared in Application Example 2 against Pseudomonas aeruginosa.

[0024] Figure 7 This is a graph showing the antibacterial evaluation results of the product prepared in Application Example 2 against Vibrio vulnificus. DETAILED DESCRIPTION

[0025] Example 1

[0026] A method for chemically modifying glycopeptide molecules on the surface of solid materials, the fixation technology roadmap is as follows:

[0027]

[0028] The modification method comprises the following steps:

[0029] S1. Ultrasonic dispersion of 10 g of solid material in 10 mL of ethanol solution, followed by sequential addition of 90 mL of acidic ethanol solution (generally acidic conditions help accelerate the hydrolysis of the silane coupling agent, and subsequent interfacial coupling reaction can only be carried out after hydrolysis, and acetic acid can be used here), 4 g of silane coupling agent KH-550, stirring at room temperature for 2 hours, taking out the solid material, washing it with distilled water 3 times, washing it with ethanol 3 times, and finally drying it in a vacuum oven at 110°C for 1 hour to obtain a surface amino-functionalized material; wherein the volume ratio of ethanol / water in the acidic ethanol solution is 95 / 5, and the pH value is 4-6;

[0030] S2. Take 1.0g of the surface amino functionalized material and 2.0g of the N-carboxyl-cyclic anhydride derivative monomer NCA of L-allylglycine, add them to a glass reaction bottle, replace the air in the reaction bottle with dry nitrogen three times, add 100mL of ultra-dry N,N-dimethylformamide (water content ≤30ppm) under nitrogen atmosphere, stir and react at room temperature for 1 to 4 days, take out the solid material, wash it with tetrahydrofuran and ethanol in turn, and vacuum dry it at room temperature to obtain a solid material with a polypeptide macromolecular chain surface modified with a double bond in the side chain;

[0031] S3. Dissolve 1.0 mmol of sugar molecules in acetonitrile aqueous solution, cool the reaction solution to 0°C, add 1.7 mL of triethylamine, 1.0 g of sodium thiosulfate, and 0.7 g of 2-chloro-1,3-dimethylimidazoline chloride to the reaction solution respectively, maintain the reaction temperature at 0°C and stir for 2 hours, then add 0.25 g of sodium sulfide nonahydrate and continue stirring at room temperature for 1 hour, finally add 1.0 g of the solid material prepared in the second step reaction with the surface modified with a polypeptide macromolecular chain containing double bonds in the side chain, continue stirring at room temperature for 1 to 4 days, take out the solid material after the reaction is completed, wash it with water and ethanol 3 times respectively, and vacuum dry it to obtain the glycopeptide molecule surface modified material.

[0032] Furthermore, the preparation method of the N-carboxyl-intracyclic anhydride derivative monomer of L-allylglycine is as follows: 2.5 g L-allylglycine, 14 mL α-pinene, 2.6 g triphosgene, and 100 mL ultra-dry tetrahydrofuran are respectively added to a reaction bottle under nitrogen protection, the reaction system is heated to 50° C. for reaction for 3-5 hours, the reaction solution is vacuum concentrated to 30 mL, 100 mL of n-hexane is added to generate a precipitate, the white precipitate is collected by filtration, redissolved in 100 mL of ethyl acetate and filtered, vacuum concentrated to 20 mL, 100 mL of n-hexane is added to generate a precipitate, the precipitate is collected, and vacuum dried to obtain the NCA monomer.

[0033] Furthermore, the solid material in step S1 is one of a polymer material, an inorganic non-metallic material, and a metal material, and its morphology is a powder material, a fiber fabric material, a film material, or a sheet material.

[0034] Furthermore, in step S3, the sugar molecule is a monosaccharide or an oligosaccharide, the monosaccharide is one of glucose, galactose, mannose, fucose, glucosamine, and galactosamine, and the oligosaccharide is one of maltose, lactose, and chitosan oligosaccharide; and the volume concentration of the acetonitrile aqueous solution is 10% to 50%.

[0035] Another object of the present invention is to provide a solid material with surface glycopeptide molecules chemically modified, which is prepared by the above method.

[0036] Application Example 1

[0037] The surface glycopeptide molecules were modified on the ferroferric oxide magnetic microspheres by the modification method of Example 1, and the selective separation effect of the ferroferric oxide microspheres on concanavalin A was tested. The ferroferric oxide microspheres were purchased from Xiamen Promag Biotechnology Co., Ltd., with a diameter of 0.4 microns. The sugar type used was D-lactose. Figure 1 X-ray photoelectron spectroscopy (XPS) was used to characterize the surface of the microspheres. After glycopeptide modification, characteristic peaks of the N element appeared on the surface of the microspheres, proving that the surface of the microspheres was successfully modified. The selective separation effect of the magnetic microspheres on different proteins in the solution was then tested. 10 mg of microspheres were respectively put into 0.1 mg / mL and 0.01 mg / mL protein solutions (1.0 mL), incubated for 5 minutes, and quickly magnetically separated using a magnetic stand. The supernatant was extracted for protein concentration determination. Here, a NanoDrop micro-spectrophotometer is used to test the protein concentration. Since the glycopeptide-modified magnetic beads can specifically adsorb the lectin protein in the solution, the protein concentration in the supernatant will be reduced through magnetic phase separation. The results are as follows Figure 2 As shown in the figure, after being treated with sugar chain-modified magnetic beads, the concentration of bovine serum albumin BSA in the solution did not change significantly, indicating that the magnetic beads had no adsorption effect on BSA. However, the content of concanavalin A lectin in the supernatant decreased significantly, proving that the sugar chain-modified magnetic microspheres had the ability to selectively separate the protein. This result also proved that the sugar chain was successfully modified on the surface of the magnetic microspheres.

[0038] Application Example 2

[0039] The surface of the non-woven fabric was modified with glycopeptide using the modification method of Example 1, and its antibacterial effect was tested. Among them, the nylon fiber non-woven fabric was produced by Changzhou Henglibao Nano New Materials Co., Ltd., and the sugar type used was D-glucosamine. The antibacterial evaluation of the nylon fiber non-woven fabric modified with glycopeptide molecules was carried out with reference to the national standard GB / T 20944.3-2008 "Evaluation of Antibacterial Properties of Textiles-Part 3 Oscillation Method". The results showed that the surface of the non-woven fabric was modified with glycopeptide, which had a good antibacterial effect on Pseudomonas aeruginosa and Vibrio vulnificus that caused wound infection, with an inhibition rate of 70% to 88%.

[0040] Performance Testing

[0041] 1. Biocompatibility testing

[0042] COS-7 mammalian cells were used to evaluate the cytotoxicity of glycopeptide-modified fiber materials. Among them, No. 1 was a PBS buffer control group, and Nos. 2-5 were glycopeptide-modified nylon fibers (the added concentrations were No. 2: 1 mg / mL, No. 3: 2 mg / mL, No. 4: 5 mg / mL, and No. 5: 10 mg / mL).

[0043] The test results show that with the increase in the amount of fiber material added, there is no obvious decrease in cell survival rate, indicating that the polyester fiber material modified with glycopeptide molecules has good biocompatibility.

[0044] 2. Antibacterial test experimental method:

[0045] 2.1 Take Pseudomonas aeruginosa strain (purchased from Beina Biotechnology, product number BNCC337005), streak it on an LB nutrient agar plate, and culture it at 37℃±1℃ for 24 hours. Then use an inoculation loop to pick a typical colony from the plate and inoculate it into 20mL of LB nutrient broth medium. Shake and culture it at 37℃±1℃ for 24 hours to prepare a bacterial suspension.

[0046] 2.2 Four-step dilution: take 1 mL of bacterial suspension + 9 mL of LB nutrient broth medium; then take 1 mL of the diluted bacterial suspension + 9 mL of LB nutrient broth medium; then take 1 mL of the diluted bacterial suspension + 9 mL of PBS; then take 5 mL of the diluted bacterial suspension + 45 mL of PBS to prepare the inoculum solution.

[0047] 2.3 Take 500μL and 50μL of inoculated bacterial solution and add them to a conical flask containing 70mL PBS, set up three experimental groups: blank control, positive control, and sample group, and set up two parallel controls for each group of experiments. The blank control group is 50μL of normal saline + 500μL of bacterial solution + 70mL PBS; the positive control group is 50μL of 20% sodium hypochlorite solution + 500μL of bacterial solution + 70mL PBS; the sample group is 5mg sample 1 + 500μL of bacterial solution + 70mL PBS.

[0048] 2.4 After sample preparation, shake for 30 minutes under appropriate culture conditions. After shaking, take 1 mL of the sample liquid and mix it with 15 mL of 50°C LB nutrient agar medium. Pour two plates into each conical flask. After the LB nutrient agar medium in the plate solidifies, culture it under appropriate culture conditions for 24 hours. After the culture is completed, count the colonies. The results are shown in Table 1.

Claims

1. A method for chemically modifying glycopeptide molecules on the surface of solid materials, characterized in that: The following steps are involved: S1. Ultrasonic dispersion of 10 g of solid material in 10 mL of ethanol solution, followed by the addition of 90 mL of acidic ethanol solution and 4 g of silane coupling agent KH-550, stirred at room temperature for 2 hours, the solid material was taken out and washed with distilled water 3 times and ethanol 3 times, and finally dried in a vacuum oven at 110°C for 1 hour to obtain a surface amino functionalized material; wherein the acidic ethanol solution is a 95% acidic ethanol aqueous solution with a pH value of 4-6; S2. Take 1.0 g of the surface amino functionalized material and 2.0 g of the N-carboxyl-intracyclic anhydride derivative monomer of L-allylglycine, add them to a glass reaction bottle, replace the air in the reaction bottle with dry nitrogen three times, add 100 mL of N, N-dimethylformamide with a water content of ≤30 ppm under a nitrogen atmosphere, stir and react at room temperature for 1 to 4 days, take out the solid material, wash it with tetrahydrofuran and ethanol in turn, and vacuum dry it at room temperature to obtain a solid material with a polypeptide macromolecular chain surface modified with a double bond in the side chain; S3. Dissolve 1.0 mmol of sugar molecules in acetonitrile aqueous solution, cool the reaction solution to 0℃, add 1.7 mL of triethylamine, 1.0 g of sodium thiosulfate, and 0.7 g of 2-chloro-1,3-dimethylimidazoline chloride to the reaction solution respectively, maintain the reaction temperature at 0℃ and stir for 2 hours, then add 0.25 g of sodium sulfide nonahydrate and continue stirring at room temperature for 1 hour, finally add 1.0 g of the solid material with the surface modified with the polypeptide macromolecular chain containing double bonds in the side chain prepared in the second step reaction to the reaction solution, continue stirring at room temperature for 1 to 4 days, take out the solid material after the reaction is completed, wash it with water and ethanol 3 times respectively, and vacuum dry it to obtain the glycopeptide molecule surface modified material.

2. The method for chemically modifying glycopeptide molecules on the surface of a solid material according to claim 1, characterized in that: The preparation method of the N-carboxyl-intracyclic anhydride derivative monomer of L-allylglycine is as follows: 2.5 g L-allylglycine, 14 mL α-pinene, 2.6 g triphosgene, and 100 mL ultra-dry tetrahydrofuran are respectively added to a reaction bottle under nitrogen protection, the reaction system is heated to 50° C. for reaction for 3-5 hours, the reaction solution is vacuum concentrated to 30 mL, 100 mL of n-hexane is added to generate a precipitate, the white precipitate is collected by filtration, the precipitate is redissolved in 100 mL ethyl acetate and filtered, vacuum concentrated to 20 mL, 100 mL of n-hexane is added to generate a precipitate, the precipitate is collected, and vacuum dried to obtain the NCA monomer.

3. The method for chemically modifying glycopeptide molecules on the surface of a solid material according to claim 1, characterized in that: The solid material in step S1 is one of a polymer material, an inorganic non-metallic material, and a metal material, and its morphology is a powder material, a fiber fabric material, a film material, or a sheet material.

4. The method for chemically modifying glycopeptide molecules on the surface of a solid material according to claim 1, characterized in that: In step S3, the sugar molecule is a monosaccharide or an oligosaccharide, the monosaccharide is one of glucose, galactose, mannose, fucose, glucosamine, and galactosamine, and the oligosaccharide is one of maltose, lactose, and chitosan oligosaccharide; and the volume concentration of the acetonitrile aqueous solution is 10% to 50%.

5. A solid material with surface glycopeptide molecules chemically modified, prepared by the method according to any one of claims 1 to 4.

6. The use of the solid material according to claim 5, characterized in that: The solid material is used in affinity chromatography filler for protein separation and purification.

7. The use of the solid material according to claim 5, characterized in that: The solid material is used in medical auxiliary materials for wound infection caused by Pseudomonas aeruginosa.

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