Functionalized boronic acid affinity material, method of making and use thereof

Abstract

The application discloses a kind of functionalized boronic acid affinity material and its preparation method and application, the functionalized boronic acid affinity material is covered with metal organic framework structure on the outer layer of porous polymer microsphere, the metal organic framework is connected by coordination bond and consists of metal ion / cluster and polydentate organic ligand;The polydentate organic ligand is carboxylic acid organic ligand, part of carboxylic acid in it occurs condensation reaction with diamine compound, another part of carboxylic acid in carboxylic acid organic ligand is reacted with amino-functionalized ionic liquid, and finally with borate reaction is modified to be prepared.The functionalized boronic acid affinity material prepared by using the application has good separation effect on low concentration glycosylated protein.

Description

TECHNICAL FIELD

[0001] The present application belongs to a kind of material and its preparation method and application, specifically related to a kind of functional borate affinity material and its preparation method and application. BACKGROUND

[0002] The reaction of boronic acid with cis-diol compound dates back to 170 years ago, but the first application in the field of nucleic acid and carbohydrate separation appeared in 1970, Weith et al. used cellulose bonded phenylboronic acid affinity material, and then more and more boron affinity materials were developed, but few were used on a large scale. In recent years, with the development of the pharmaceutical industry, boronic acid affinity materials have received attention due to their unique ability to specifically bind glycoproteins. Boron affinity monolithic column and boron affinity imprinted polymer have become the main research focus and are mainly used for the separation of cis-diol biological samples and the separation and detection of glycoproteins, such as horseradish peroxidase (HRP), immunoglobulin G (IgG), ovalbumin (OVA), human serum albumin (HSA), and drug extraction. The principle of boronic acid affinity is that when the pH of the reaction system is alkaline, the boronic acid group is hydrolyzed and coordinates with the -OH in the environment, and the boron atom changes from sp2 hybridization to tetrahedral sp3 hybridization. Then it reacts with the adjacent dihydroxyl group in the cis-diol compound to form a stable five-membered or six-membered ring. Under acidic conditions, the cyclic boronic ester dissociates, the boron atom changes from sp3 hybridization to sp2 hybridization, and the cis-diol compound is released, achieving the purpose of selective separation and enrichment. This method is simple, efficient and has the advantage of selective recognition, but also has great challenges. Because the concentration of cis-diol in biological molecules is usually low, the traditional borate affinity material has only a very limited single-layer adsorption layer, and the adsorption site is small, which causes low separation and enrichment efficiency and difficulty in effective capture in actual application. Taking the glycosylated protein in recombinant human serum albumin as an example, the proportion of glycoprotein is not higher than 1%, and it is difficult to capture using traditional boronic acid affinity filler, making it more challenging to remove low-concentration glycosylated proteins. SUMMARY

[0003] The purpose of the present application is to provide a functional boronic acid affinity material for separating glycosylated proteins. Another purpose of the present application is to provide a preparation method of the functional boronic acid affinity material.

[0004] Technical solution: The functionalized boronic acid affinity material has a metal organic framework (MOF) structure on the outer layer of the porous polymer microsphere, the metal organic framework is composed of metal ions / clusters and polydentate organic ligands connected by coordination bonds, the polydentate organic ligand is a carboxylic acid organic ligand, part of the carboxylic acid in the carboxylic acid organic ligand is condensed with a diamine compound, another part of the carboxylic acid in the carboxylic acid organic ligand is reacted with an amino-functionalized ionic liquid, and finally modified with borate to obtain.

[0005] The preparation method of the functionalized boronic acid affinity material comprises the following steps:

[0006] (1) The porous polymer microspheres (PPM) are added to a mixed solution of carboxylic acid organic ligand and metal salt MX in water and ethanol, stirred at room temperature, and then moved into a pressure-resistant sealed container by adding sodium hydroxide aqueous solution dropwise, and hydrothermal treatment is carried out for 8-12 h, then washed repeatedly with water and ethanol and vacuum dried to obtain M-PPM; M represents metal ions in the metal salt MX, and X represents corresponding anions;

[0007] (2) The M-PPM microspheres of step (1) are mixed uniformly with N,N-dimethylformamide (DMF); condensing agent A, catalyst B and organic base C are mixed and added to the above solution, and the reaction is carried out at 0-45℃ for 0.5-3h; a diamine compound is added, stirred at room temperature, washed with hydrochloric acid aqueous solution until neutral, dried and used to obtain NH2-M-PPM;

[0008] (3) The amino-functionalized ionic liquid (AFILs) is dissolved in water to form an aqueous solution, then the NH2-M-PPM microspheres of step (2) are added to system 1, stirred for 2-6h; the borate, condensing agent A, catalyst B and organic base C are mixed to form system 2, system 2 is stirred at 40-50℃ for 20-40min, then added to system 1, and continue to stir at room temperature, washed and dried to obtain the functionalized boronic acid affinity material.

[0009] The preparation method of the functionalized boronic acid affinity material, the porous polymer microspheres in step (1) are selected from monodisperse polystyrene / divinylbenzene microspheres (PSDVB), polymethyl methacrylate microspheres (PMMA), polyglycidyl methacrylate microspheres (PGMA), agarose microspheres, polylactic acid-glycolic acid copolymer microspheres (PLGA), polylactic acid microspheres (PLA), dextran microspheres, chitosan microspheres, polyethyleneimine microspheres, polypropylene microspheres (PS), cellulose acetate microspheres (CAS), and the diameter is 10-100μm, the pore size is

[0010] The organic ligand is selected from one or more of 2,5-dithiophene carboxylic acid, fumaric acid (HFMA), terephthalic acid (HBDC), 4,4'-biphenyl dicarboxylic acid (BPDC), 2,6-naphthalene dicarboxylic acid (HNDC), 2,2'-bipyridine-5,5'-dicarboxylic acid, 1,3,5-benzenetricarboxylic acid, 55-trifluoromethyl-1,3-benzene dicarboxylic acid, and 4,4'-oxybisphthalic acid, and the amount of the organic ligand is 10-50 mmol;

[0011] The metal salt MX has M selected from one or more of nickel, copper, cobalt, zinc, zirconium, calcium, and magnesium, and X selected from one or more of halogen ions, acetylacetone, acetate ions, sulfate ions, and nitrate ions, and the stirring time is 0.5-6 h.

[0012] In the preparation method of the functionalized boronic acid affinity material, the mass ratio of the M-PPM microspheres to DMF in step (2) is 1:10-1:100, and the molar ratio of the condensing agent A to the catalyst B is 1:0.5-1:5. Preferably, the molar ratio of the condensing agent A to the catalyst B is 1:0.5-1:3.

[0013] In the preparation method of the functionalized boronic acid affinity material, the condensing agent A in steps (2) and (3) is a carbodiimide condensing agent selected from one or more of dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDCI), and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC).

[0014] The catalyst B is N-hydroxysuccinimide (NHS), 4-N,N-dimethylpyridine (DMAP), 1-hydroxybenzotriazole (HOBt), or 1-hydroxy-7-azobenzotriazole (HOAt).

[0015] The organic base C is selected from one or more of N-methylmorpholine, diisopropylethylamine (DIEA), triethylamine (TEA), sodium tert-butoxide, potassium tert-butoxide, lithium diisopropylamide (LDA), potassium bis(trimethylsilyl)amide (KHMDS), and imidazole, and the amount of the organic base C added in step (2) is 2-5 eq. of the condensing agent A.

[0016] In the preparation method of the functionalized boronic acid affinity material, the diamine compound in step (2) is selected from one or more of ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, and 1,6-hexanediamine, and the stirring time at room temperature is 4-48 h.

[0017] In the preparation method of the functionalized boric acid affinity material, the amino-functionalized ionic liquid (AFILs) in step (3) is selected from one or more of 1-aminopropyl-3-methylimidazolium tetrafluoroborate, 1-aminopropyl-3-methylimidazolium hexafluorophosphate, 1-aminopropyl-3-methylimidazolium bromide, 1-aminopropyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, 1-aminoethyl-3-methylimidazolium tetrafluoroborate, 1-aminoethyl-3-methylimidazolium bromide, and 1-aminoethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, and the mass ratio of the AFILs to the NH2-M-PMM microspheres is 1:20 to 1:100.

[0018] In the preparation method of the functionalized boric acid affinity material, the molar ratio of condensing agent A: catalyst B: organic base C in step (3) is 1:0.5-5:2-5, and the stirring time is continued at room temperature for 2-24 hours.

[0019] The method for preparing the functionalized boric acid affinity material, wherein in step (3), the borate is one or more of 3-carboxyphenylboronic acid, 4-carboxyphenylboronic acid, 3-acetamido-5-boronbenzoic acid, 3,5-dicarboxyphenylboronic acid, 3-carboxy-5-nitrophenylboronic acid, 3-ethoxycarbonylphenylboronic acid, 3-carboxyphenylboronic acid-1,3-propanediol ester, and 3-methoxy-4-carboxyphenylboronic acid; the amount of borate added is 3% to 50% of the mass ratio of the porous polymer microspheres in step (1).

[0020] The condensing agent A, catalyst B and organic base C in step (3) above can be different from those in step (2), but the present invention prefers to use the same ones.

[0021] The functionalized boric acid affinity material described above or prepared by the method described above is particularly suitable for the separation and purification of low-concentration glycosylated proteins.

[0022] The preparation process of functionalized boric acid affinity materials is as follows: Figure 1 As shown, porous polymer microspheres are represented as The outer circle of the microsphere represents a metal-organic framework composed of metal ions / clusters and multidentate organic ligands linked by coordination bonds. The molecular formula of the diamine compound is represented as follows: Amino-functionalized ionic liquids (AFILs) are represented as The final functionalized boric acid affinity material is shown below:

[0023]

[0024] The metal-organic framework of the boric acid material prepared in this invention is composed of metal ions / clusters and multidentate organic ligands connected by coordination bonds. Due to its controllable size, morphology, topology, and porosity, the material exhibits excellent orientation and structural confinement effects, thereby reducing non-specific interactions and enhancing its affinity. Simultaneously, the presence of ionic liquids can regulate the density of boric acid sites on the surface of the matrix spheres, enabling multiple binding in biomolecular interactions. The ligand structure possesses stronger binding properties, significantly enhancing the affinity of the high-extraction material and its ability to capture low concentrations of glycosylated proteins.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following advantages: (1) The present invention uses simple equipment and is easy to scale up production; the raw materials are readily available, the cost is low, the process is simple and has good repeatability, and it is convenient for industrial production;

[0026] (2) The functionalized boric acid affinity material prepared by the present invention has excellent capture ability for low concentration of glycosylated proteins and good separation effect. Attached Figure Description

[0027] Figure 1 Flowchart for the preparation of functionalized boric acid materials;

[0028] Figure 2 This is an HPLC chromatogram of human serum albumin separation using boric acid column in Example 1. Detailed Implementation

[0029] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0030] The polymethyl methacrylate microspheres were purchased from Suzhou Saifen Technology Co., Ltd.

[0031] The polyglycidyl methacrylate microspheres were purchased from Suzhou Saifen Technology Co., Ltd.

[0032] Monodisperse polystyrene / divinylbenzene microspheres were purchased from Suzhou Saifen Technology Co., Ltd.

[0033] The polylactic acid-glycolic acid copolymer microspheres were prepared in-house. For details on the preparation method, please refer to Pathak, S., Regmi, S., Shrestha, P., Choi, I., Doh, K., & Jeong, J. Small, 2019, 1901269.

[0034] The cellulose acetate microspheres were prepared in-house; for details of the preparation method, please refer to patent CN103724635A.

[0035] In addition, the microspheres used in the experiments of this application can also be commercially available microspheres.

[0036] Example 1

[0037] Step 1: Add 10g of polymethyl methacrylate (PMMA) microspheres (product name Monomix MC60-SEC-1000, specifications: particle size 60μm, pore size...) 3.44 g of 2,5-thiophene dicarboxylic acid and 10.258 g of nickel acetylacetonate were dissolved in 40 mL of ethanol-water mixture (V ethanol / V water = 1:1). After stirring at room temperature for 30 min, 20 mL of 1 mol / L sodium hydroxide solution was slowly added dropwise to the above system and stirred evenly. The mixture was then transferred to a reaction vessel and hydrothermally heated at 80 °C for 12 h. Afterward, the mixture was washed with water and ethanol in sequence and vacuum dried at 80 °C for 12 h to obtain Ni-PPM microspheres.

[0038] Step 2: Mix the Ni-PPM microspheres from Step 1 with 100 mL of LDM. Add 3.59 g of condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarboimide, 4.25 g of catalyst 1-hydroxybenzotriazole, and 4.08 g of triethylamine to the above solution and react at 25 °C for 2 h. Add 26 g of 1,6-hexanediamine, stir at room temperature, and wash with 0.1 mol / L hydrochloric acid water until neutral. Dry for later use to obtain NH2-Ni-PPM.

[0039] Step 3: Dissolve 0.5g of 1-aminopropyl-3-methylimidazolium tetrafluoroborate in 20mL of aqueous solution, then add NH2-Ni-PPM from Step 2 to form System 1 and stir for 4h. Mix 0.3g of 3-carboxyphenylboronic acid, 3.59g of condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarboimide, 4.25g of catalyst 1-hydroxybenzotriazole, and 4.08g of triethylamine in 40mL of LDM and stir at room temperature to form System 2. Add System 2 to System 1 while stirring at 45℃ for 30min, and keep stirring at room temperature for 4h. Wash the filler with water and ethanol sequentially, and vacuum dry at 45℃ for 12h to obtain the functionalized boric acid affinity material.

[0040] Example 2

[0041] Step 1: Take 10g of polyglycidyl methacrylate microspheres, with a diameter of 100μm and a pore size of... 7.27 g of 4,4'-biphenyldicarboxylic acid and 8.07 g of copper chloride were dissolved in 40 mL of ethanol-water mixture (V ethanol / V water = 1:1). After stirring at room temperature for 30 min, 100 mL of 1 mol / L sodium hydroxide solution was slowly added dropwise to the above system and stirred until homogeneous. The mixture was then transferred to a reaction vessel and hydrothermally heated at 80 °C for 12 h. Afterward, the mixture was washed sequentially with water and ethanol and vacuum dried at 80 °C for 12 h to obtain Cu-PPM microspheres.

[0042] Step 2: Mix the Cu-PPM microspheres from Step 1 with 100 mL of LDM. Add 3.59 g of condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarboimide, 6.91 g of catalyst N-hydroxysuccinimide, and 4.08 g of triethylamine to the above solution and react at 25 °C for 4 h. Add 16.58 g of 1,3-propanediamine, stir at room temperature, and wash with 0.1 mol / L hydrochloric acid water until neutral. Dry for later use to obtain NH2-Cu-PPM.

[0043] Step 3: Dissolve 0.1g of 1-aminopropyl-3-methylimidazolium hexafluorophosphate in 20mL of aqueous solution, then add NH2-Cu-PPM from Step 2 to form System 1 and stir for 4h. Mix 5g of 2,5-dicarboxyphenylboronic acid, 3.59g of condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarboimide, 6.91g of catalyst N-hydroxysuccinimide, and 4.08g of triethylamine in 40mL of water and stir at room temperature to form System 2. Add System 2 to System 1 while stirring at 45℃ for 30min, and keep stirring at room temperature for 4h. Wash the filler with water and ethanol sequentially, and vacuum dry at 45℃ for 12h to obtain the functionalized boric acid affinity material.

[0044] Example 3

[0045] Step 1: Take 10g of polylactic-co-glycolic acid copolymer microspheres (PLGA), with a diameter of 30μm and a pore size of... 2.11 g of 1,3,5-pyromellitic acid and 5.49 g of cobalt nitrate were dissolved in 40 mL of ethanol-water mixture (V ethanol / V water = 1:1). After stirring at room temperature for 30 min, 50 mL of 1 mol / L sodium hydroxide solution was slowly added dropwise to the above system and stirred until homogeneous. The mixture was then transferred to a reaction vessel and hydrothermally heated at 80 °C for 12 h. The mixture was then washed sequentially with water and ethanol and vacuum dried at 80 °C for 12 h to obtain Co-PPM microspheres.

[0046] Step 2: Mix the Co-PPM microspheres from Step 1 with 300 mL of LDM. Add 3.83 g of condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4.25 g of catalyst 1-hydroxybenzotriazole, and 5.18 g of diisopropylethylamine (DIEA) to the above solution and react at 25 °C for 2 h. Add 13.44 g of ethylenediamine, stir at room temperature, and wash with 0.1 mol / L hydrochloric acid water until neutral. Dry for later use to obtain NH2-Co-PPM.

[0047] Step 3: Dissolve 1g of 1-aminopropyl-3-methylimidazolium bromate in 20mL of aqueous solution, then add NH2-Co-PPM from Step 2 to form System 1 and stir for 4h. Mix 3g of 3-carboxyphenylboronic acid, 3.83g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 4.25g of 1-hydroxybenzotriazole and 7.77g of diisopropylethylamine (DIEA) in 40mL of System 2 obtained by stirring at room temperature. Add System 2 to System 1 while stirring at 45℃ for 30min, and keep stirring at room temperature for 6h. Wash the filler with water and ethanol sequentially, and vacuum dry at 45℃ for 12h to obtain the functionalized boric acid affinity material.

[0048] Example 4

[0049] Step 1: Take 10g of monodisperse polystyrene / divinylbenzene microspheres (PSDVB), with a diameter of 100μm and a pore size of... 8.31 g of terephthalic acid (HBDC) and 6.72 g of copper chloride were dissolved in 40 mL of ethanol-water mixture (V ethanol / V water = 1:1). After stirring at room temperature for 30 min, 20 mL of 1 mol / L sodium hydroxide solution was slowly added dropwise to the above system and stirred until homogeneous. The mixture was then transferred to a reaction vessel and hydrothermally heated at 80 °C for 12 h. Afterward, the mixture was washed with water and ethanol sequentially and vacuum dried at 80 °C for 12 h to obtain Cu-PPM microspheres.

[0050] Step 2: Mix the Cu-PPM microspheres from Step 1 with 200 mL of LDM. Add 4.13 g of the condensing agent dicyclohexylcarbodiimide, 1.22 g of 4-N,N-dimethylpyridine (DMAP), and 5.18 g of diisopropylethylamine (DIEA) to the above solution and react at 25 °C for 2 h. Add 20 g of 1,6-hexanediamine, stir at room temperature, and wash with 0.1 mol / L hydrochloric acid water until neutral. Dry the mixture to obtain NH2-Cu-PPM.

[0051] Step 3: Dissolve 0.5g of 1-aminoethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide salt in 20mL of aqueous solution, then add NH2-Cu-PPM from Step 2 to form System 1 and stir for 4h. Mix 3g of 3-acetamido-5-boronbenzoic acid, 4.13g of condensing agent dicyclohexylcarbodiimide, 1.22g of 4-N,N-dimethylpyridine and 5.18g of diisopropylethylamine (DIEA) in 40mL of water and stir at room temperature to form System 2. Add System 2 to System 1 while stirring at 45℃ for 30min, and keep stirring at room temperature for 8h. Wash the filler with water and ethanol sequentially, and vacuum dry at 45℃ for 12h to obtain the functionalized boric acid affinity material.

[0052] Example 5

[0053] Step 1: Take 10g of cellulose acetate microspheres (CAS), with a diameter of 60μm and a pore size of... 6.21 g of 4,4'-oxophthalic acid and 6.55 g of zirconium acetate were dissolved in 40 mL of ethanol-water mixture (V ethanol / V water = 1:1). After stirring at room temperature for 30 min, 100 mL of 1 mol / L sodium hydroxide solution was slowly added dropwise to the above system and stirred until homogeneous. The mixture was then transferred to a reaction vessel and hydrothermally heated at 80 °C for 12 h. The mixture was then washed sequentially with water and ethanol and vacuum dried at 80 °C for 12 h to obtain Zr-PPM microspheres.

[0054] Step 2: Mix the Zr-PPM microspheres from Step 1 with 100 mL of LDM. Add 2.53 g of diisopropylcarbodiimide as a condensing agent, 2.72 g of 1-hydroxy-7-azobenzotriazole as a catalyst, and 10.115 g of N-methylmorpholine to the above solution and react at 0 °C for 4 h. Add 16.58 g of 1,3-propanediamine, stir at room temperature, and wash with 0.1 mol / L hydrochloric acid water until neutral. Dry the mixture to obtain NH2-Zr-PPM.

[0055] Step 3: Dissolve 0.5g of 1-aminoethyl-3-methylimidazolium bromide in 20mL of aqueous solution, then add NH2-Zr-PPM from Step 2 to form System 1 and stir for 4h. Mix 2g of 3-carboxyphenylboronic acid-1,3-propanediol ester, 2.53g of diisopropylcarbodiimide as condensing agent, 2.72g of 1-hydroxy-7-azobenzotriazole as catalyst, and 10.115g of N-methylmorpholine in 40mL of water and stir at room temperature to form System 2. Add System 2 to System 1 while stirring at 45℃ for 30min, and keep stirring at room temperature for 2h. Wash the filler with water and ethanol sequentially, and vacuum dry at 45℃ for 12h to obtain the functionalized boric acid affinity material.

[0056] HPLC test

[0057] The functionalized boric acid affinity materials prepared in each example were packed into a 4605 stainless steel chromatographic column (4.6 × 50 mm).

[0058] Mobile phase: A: 20 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 200 mM sodium chloride (NaCl), 10 mM magnesium chloride (MgCl2), pH = 8.5;

[0059] B: A + 100mM sorbitol (pH = 8.5);

[0060] Elution conditions: 0-25 min 100% A, 25-50 min 100% B;

[0061] Flow rate: 0.5 mL / min;

[0062] Detector wavelength: UV 280nm;

[0063] Column temperature: room temperature;

[0064] Injection volume: 100 μL;

[0065] Sample: Original recombinant human serum albumin (200 mg / mL).

[0066] Figure 2 The image shows the separation and purification of recombinant human serum albumin using the functionalized boric acid affinity material prepared in Example 1. It can be seen that the glycosylated protein was separated by flow-through mode with a yield of 99.00% and a purity of 99.78%, indicating that it has a good separation effect.

[0067] The test samples in other embodiments were the same as in Example 1, with a recombinant human serum albumin concentration of 200 mg / mL and a glycated protein concentration of 2 mg / mL.

[0068] A standard curve was established using the external standard method, and the amount of protein in the flow-through solution (mg) was calculated.

[0069]

[0070]

[0071] The HPLC test results for each embodiment are shown in the table below.

[0072]

Claims

1. A functionalized boric acid affinity material, characterized in that, It is a porous polymer microsphere with a metal-organic framework structure on the outer layer. The metal-organic framework is composed of metal ions / clusters and multidentate organic ligands connected by coordination bonds. The multidentate organic ligands are carboxylic acid organic ligands. Some of the carboxylic acids undergo condensation reactions with diamine compounds, and another part of the carboxylic acid organic ligands reacts with amino-functionalized ionic liquids. Finally, it is modified with borate by reacting with borate.

2. A method for preparing a functionalized boric acid affinity material, characterized in that, Includes the following steps: (1) Porous polymer microspheres PPM were added to a mixture of water and ethanol containing carboxylic acid organic ligands and metal salt MX. The mixture was stirred at room temperature, and sodium hydroxide aqueous solution was added dropwise. The mixture was then transferred to a pressurized sealed container for hydrothermal reaction. After repeated washing and drying, M-PPM was obtained. In the metal salt MX, M represents a metal ion and X represents the corresponding anion. (2) After the M-PPM microspheres from step (1) are mixed evenly with N,N-dimethylformamide, condensing agent A, catalyst B and organic base C are mixed and added to the above solution for reaction; then diamine compounds are added, the reaction is stirred at room temperature, then washed until neutral, and dried for later use to obtain NH2-M-PPM; (3) Dissolve the amino-functionalized ionic liquid in water to prepare an aqueous solution, then add the NH2-M-PPM microspheres from step (2), stir and react to obtain system 1; mix borate, condensing agent A, catalyst B and organic base C to form system 2, then add system 2 to system 1, stir and react, wash and dry to obtain functionalized boric acid affinity material.

3. The method for preparing the functionalized boric acid affinity material according to claim 2, characterized in that, The porous polymer microspheres mentioned in step (1) are selected from monodisperse polystyrene / divinylbenzene microspheres, polymethyl methacrylate microspheres, polyglycidyl methacrylate microspheres, agarose microspheres, polylactic acid-glycolic acid copolymer microspheres, polylactic acid microspheres, dextran microspheres, chitosan microspheres, polyethyleneimine microspheres, polypropylene microspheres, and cellulose acetate microspheres, with a diameter of 10–100 μm and a pore size of [missing information]. The organic ligand is selected from one or more of 2,5-dithiophenecarboxylic acid, fumaric acid, terephthalic acid, 4,4'-biphenylcarboxylic acid, 2,6-naphthalenedicarboxylic acid, 2,2'-bipyridine-5,5'-dicarboxylic acid, 1,3,5-pyromellitic acid, 55-trifluoromethyl-1,3-dibenzoic acid, and 4,4'-oxobisphthalic acid, and the amount of the organic ligand is 10 to 50 mmol; In the metal salt MX, M is selected from one or more of nickel, copper, cobalt, zinc, zirconium, calcium, and magnesium, and X is selected from one or more of halide ions, acetylacetone, acetate ions, sulfate ions, and nitrate ions. The stirring time is 0.5 to 6 hours.

4. The method for preparing the functionalized boric acid affinity material according to claim 2, characterized in that, In step (2), the mass ratio of M-PPM microspheres to N,N-dimethylformamide is 1:10 to 1:100; the molar ratio of condensing agent A to catalyst B is 1:0.5 to 1:

5.

5. The method for preparing the functionalized boric acid affinity material according to claim 2, characterized in that, The condensing agent A mentioned in steps (2) and (3) is a carbodiimide condensing agent, selected from one or more of the following: dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; Catalyst B is N-hydroxysuccinimide, 4-N,N-dimethylpyridine, 1-hydroxybenzotriazole, or 1-hydroxy-7-azobenzotriazole; Organic base C is one or more of N-methylmorpholine, diisopropylethylamine, triethylamine, sodium tert-butoxide, potassium tert-butoxide, diisopropylaminolithium, bis(trimethylsilyl)aminopotassium, and imidazole; wherein, in step (2), the amount of organic base C added is 2 to 5 eq. of condensing agent A.

6. The method for preparing the functionalized boric acid affinity material according to claim 2, characterized in that, The diamine compound mentioned in step (2) is selected from one or more of ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, and 1,6-hexanediamine; the stirring time at room temperature is 4 to 48 hours.

7. The method for preparing the functionalized boric acid affinity material according to claim 2, characterized in that, The amino-functionalized ionic liquid in step (3) is selected from one or more of 1-aminopropyl-3-methylimidazolium tetrafluoroborate, 1-aminopropyl-3-methylimidazolium hexafluorophosphate, 1-aminopropyl-3-methylimidazolium bromide, 1-aminopropyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, 1-aminoethyl-3-methylimidazolium tetrafluoroborate, 1-aminoethyl-3-methylimidazolium bromide, and 1-aminoethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, and its mass ratio with NH2-M-PMM microspheres is 1:20 to 1:

100.

8. The method for preparing the functionalized boric acid affinity material according to claim 2, characterized in that, The molar ratio of condensing agent A: catalyst B: organic base C in step (3) is 1:0.5-5:2-5, and the stirring time is continued at room temperature for 2-24 hours.

9. The method for preparing the functionalized boric acid affinity material according to claim 2, characterized in that, In step (3), the borate is one or more of 3-carboxyphenylboronic acid, 4-carboxyphenylboronic acid, 3-acetamido-5-boronbenzoic acid, 3,5-dicarboxyphenylboronic acid, 3-carboxy-5-nitrophenylboronic acid, 3-ethoxycarbonylphenylboronic acid, 3-carboxyphenylboronic acid-1,3-propanediol ester, and 3-methoxy-4-carboxyphenylboronic acid; the amount of borate added is 3% to 50% of the mass ratio of the porous polymer microspheres in step (1).

10. The use of the functionalized boric acid affinity material according to claim 1 or the functionalized boric acid affinity material prepared by any one of claims 2-9 in the separation of glycated proteins.

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