Functionalized boric acid affinity filler as well as preparation method and application thereof

Abstract

The invention discloses a functionalized boric acid affinity filler as well as a preparation method and application thereof, the functionalized boric acid affinity filler takes carboxyl modified porous polymer microspheres as a matrix, functional polypeptide micromolecular spacer arms are grafted on the surfaces, and biphenyl boric acid compounds are coupled at the tail ends of peptide chains. The functionalized boric acid affinity filler provided by the invention contains an auxiliary functional group, and can provide a secondary acting force to assist the combination of boric acid and cis-diol molecules, improve the loading capacity, maintain higher combining capacity at low pH, and separate cis-diol molecules with different structures; the preparation method of the functionalized boric acid affinity filler provided by the invention is simple and efficient, and has wide application prospects and good industrialization conditions.

Description

Technical Field

[0001] The invention relates to a boric acid affinity filler and a preparation method and application thereof, in particular to a boric acid affinity filler with auxiliary functional groups of multiple different functions and a preparation method and application thereof, belonging to the field of chromatography medium synthesis. Background Art

[0002] Boronic acid affinity filler is a material that can separate cis-diol compounds based on the specific binding of boric acid ligands to cis-diols. Cis-diol compounds include nucleosides, nucleic acids, carbohydrates, glycoproteins and enzymes, which are of great significance to the study of life activities of organisms and the detection and diagnosis of specific diseases. Taking glycoproteins as an example, protein glycosylation is a normal post-translational modification in organisms and is widely present in mammals. However, abnormal expression of glycosylation usually causes changes in protein structure and function, which in turn causes a variety of diseases such as Alzheimer's disease, breast cancer, diabetes and rheumatoid arthritis. Therefore, glycoproteins, as clinical markers for these diseases, require accurate detection methods to determine the occurrence and progression of related diseases.

[0003] In the method of separating and enriching cis-diol compounds such as glycoproteins, the method using boric acid affinity fillers has the advantages of high selectivity, simple steps, and low cost, but there are still some challenges. For example, the binding ability of traditional boric acid affinity materials at pH 8.0 is still weak, and the pH needs to be increased to enhance the binding force, but strong bases are likely to affect the stability of biological samples; and the existing boric acid functional groups with strong low pH binding ability are expensive and cannot meet the needs of industrial applications. For another example, the concentration of cis-diol compounds in biological samples is generally low, while the loading capacity of traditional boric acid affinity materials is limited and the capture efficiency is low. In addition, since boric acid affinity is indiscriminate for molecules containing cis-diol structures, traditional boric acid affinity materials cannot effectively separate cis-diol molecules of different structures.

[0004] Patent CN109225177A discloses a preparation method and application of a polyethyleneimine hyperbranched agarose-based boron affinity material, using polyethyleneimine as a boric acid affinity material with a spacer arm. This polymerized macromolecular spacer arm effectively increases the amount of boric acid that can be loaded; however, the introduction of polyethyleneimine will bring a large number of amino groups, and the strong anionic interaction will make the protein difficult to elute. Based on this, patent CN110711571A uses tris (2-aminoethyl) to replace polyethyleneimine and performs amino end-capping to reduce the impact of residual amino groups; however, this method is not only more expensive, but also limits the possibility of filler design. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a boric acid affinity filler containing an auxiliary functional group and a preparation method thereof, wherein the secondary force provided by the auxiliary functional group selectively promotes the binding of boric acid and cis-diol molecules.

[0006] Technical solution: The functionalized boric acid affinity filler of the present invention has the following general structural formula:

[0007] in: It is a porous polymer microsphere or a porous silica microsphere; R1 is an alkyl, carboxyl, amino, hydroxyl, hydrogen, phenyl, heteroaryl, thioether, amidine, amide or halogen functional group; R2 is an alkane group, a halogen or a carboxyl group; n is any integer from 1 to 50.

[0008] The functionalized boronic acid affinity filler is based on carboxyl-modified porous polymer microspheres or porous silica microspheres, with functional polypeptide small molecule spacer arms grafted on the surface, and phenylboronic acid compounds coupled at the end of the peptide chain. When the functionalized boronic acid affinity filler is used, the secondary force provided by the auxiliary functional groups R1 and R2 can assist the binding of boric acid and cis-diol molecules, increase the filler loading, maintain a high binding capacity at low pH, and different functional groups can also assist in the separation of cis-diol molecules of different structures.

[0009] Preferably, the porous polymer microspheres are agarose microspheres, dextran microspheres, chitosan microspheres, konjac microspheres, cellulose microspheres, polymethacrylate microspheres, polystyrene-divinylbenzene microspheres or polyvinyl alcohol microspheres.

[0010] The porous polymer microspheres or porous silica microspheres preferably have a diameter of 10-100 μm and a pore size of 50-1000 Å; further, the polymer-matrix Monomix microspheres or agarose-matrix Agarosix microspheres produced by Saifen are preferred.

[0011] The functionalized boric acid affinity filler of the present invention can be used to separate cis-diol molecules with different structures.

[0012] The method for preparing the functionalized boric acid affinity filler of the present invention comprises the following steps: Step 1: activating porous polymer microspheres or porous silica microspheres with a compound containing a carboxyl functional group to obtain a carboxylated porous material; Step 2: Using amino acid compounds as monomers, obtaining polypeptide polymers through amide condensation reaction; Step 3: Grafting the polypeptide polymer obtained in step 2 onto the surface of the carboxylated porous material obtained in step 1; Step 4: Add a boronic acid compound to the product obtained in step 3 for coupling to obtain a functionalized boronic acid affinity filler.

[0013] Furthermore, the coupling ligand density in step 1 is 10-200 μmol / mL resin. The reaction system of steps 1-4 is one or a mixture of water, dimethyl sulfoxide (DMSO), dioxane or N,N-dimethylformamide (DMF); the reaction is preferably carried out at 25-65°C, 10-24 h, and 50-300 rpm.

[0014] Preferably, in step 2, the polypeptide polymer has the following general structural formula:

[0015] in: R1 is an alkyl, carboxyl, amino, hydroxyl, hydrogen, phenyl, heteroaryl, thioether, amidine, amide or halogen functional group; n is any integer from 1 to 50.

[0016] The polypeptide polymer constitutes the spacer arm of the functionalized boric acid affinity filler, which can assist the boric acid in capturing the target cis-diol molecule; according to the different configurations of the polypeptide polymer, the glycoprotein captured by the boric acid filler is also different.

[0017] Preferably, in step 2, the amino acid compound is at least one of 20 common amino acids; the amount of the amino acid used is 0.1-10 times the mass of the porous polymer microspheres or porous silica microspheres.

[0018] The type and quantity of amino acids affect the configuration and properties of polypeptide polymers. For example, the boric acid filler with glycine as the spacer arm has low nonspecific binding and is suitable for capturing cis-diol molecules such as glycoproteins with strong affinity; phenylalanine has strong hydrophobicity and can give the boric acid filler a certain hydrophobic effect; lysine is a basic amino acid containing an amino group, with an isoelectric point of 9.74 and strong binding ability; the more basic amino acids a polypeptide polymer contains, the higher its isoelectric point and the stronger its binding force.

[0019] Preferably, in step 1, the compound containing a carboxyl functional group is oxalic acid, iodoacetic acid or malonic acid; and its amount is 0.1-5 times the mass of the porous polymer microspheres or porous silica microspheres.

[0020] Preferably, in steps 2-4, a condensing agent is used for the reaction, and the condensing agent is carbodiimide, onium salt or active ester; the amount of the condensing agent is 0.1-5 times the mass of the porous polymer microspheres or porous silica microspheres.

[0021] Further, the condensing agent is selected from dicyclohexylcarbodiimide (DCC), N,N-diisopropylcarbodiimide (DIC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and hydrochloride (EDCI), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid (TBTU), benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP) or tripyrrolidinophosphine hexafluorophosphate (PyAOP). The condensing agent is preferably 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

[0022] Preferably, in step 2-4, a catalyst and / or an organic base is used. The catalyst is 4-dimethylaminopyridine (DMAP), 4-pyrrolidinopyridine (4-PPY), N-hydroxysuccinimide (NHS), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt); the amount of the catalyst is 0.1-5 times the mass of the porous polymer microspheres or porous silica microspheres; the organic base is triethylamine (TEA), N,N-diisopropylethylamine (DIEA) or N-methylmorpholine (NMM); the amount of the organic base is 0.1-5 times the mass of the porous polymer microspheres or porous silica microspheres.

[0023] Furthermore, the catalyst is preferably N-hydroxysuccinimide. The organic base is preferably N,N-diisopropylethylamine.

[0024] Preferably, in step 4, the boronic acid compound is 3-aminophenylboronic acid, 4-aminophenylboronic acid, 4-aminophenylboronic acid hydrochloride, 3-aminophenylboronic acid hydrochloride, 3-aminophenylboronic acid hemisulfate, 3-amino-5-carboxyphenylboronic acid, (3-amino-4-chlorophenyl)boric acid, (2-amino-5-chlorophenyl)boric acid or 3-amino-4-methylphenylboric acid; the amount of the boronic acid compound is 0.1-5 times the mass of the porous polymer microspheres or porous silica microspheres.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The functional groups of the functionalized boronic acid affinity filler provided by the present invention can provide secondary forces to assist the binding of boric acid and cis-diol molecules, increase the loading capacity, maintain a high binding capacity at low pH, and separate cis-diol molecules of different structures; 2. The preparation method of the functionalized boronic acid affinity filler provided by the present invention is simple and efficient, and has broad application prospects and good industrialization conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The loading spectrum of the functionalized boronic acid affinity filler prepared in Example 1 of the present invention; Figure 2 This is a chromatogram of separation of glycated proteins in human serum albumin by the functionalized boric acid affinity filler prepared in Example 1 of the present invention; Figure 3 This is a chromatogram of separation of glycated proteins in human serum albumin by the functionalized boric acid affinity filler prepared in Example 2 of the present invention; Figure 4 This is a chromatogram of separation of glycated proteins in human serum albumin using the functionalized boric acid affinity filler prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0028] Example 1 A method for preparing a functionalized boric acid affinity filler In this embodiment, a functionalized boric acid affinity filler is obtained by the following method, including the following steps: (1) 5 g of polymethacrylate microspheres and 50 g of N, N-dimethylformamide (DMF) were added to the reaction, and 5 g of oxalic acid was added to the reaction. 1.82 g of hexadecyltrimethylammonium chloride was hydrothermally reacted at 65 °C for 10 h to obtain the carboxylated porous polymer material CPPM-1.

[0029] (2) 2.22 g of glycine, 9.5 g of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 7.65 g of N,N-diisopropylethylamine were added to 100 mL of dimethyl sulfoxide (DMSO) in sequence, activated at room temperature, and the reaction system was stirred for 2 h. Extraction was performed and the mixture was dried by rotary evaporation to obtain the polypeptide polymer M1. (3) CPPM-1 prepared in step (1), 4.59 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 4.03 g of 4-dimethylaminopyridine and 6.07 g of triethylamine were added to 50 mL of N, N-dimethylformamide (DMF) in sequence, activated at 35°C for 1 h, mixed evenly, and then added with polypeptide polymer M1. The reaction system was stirred for 4 h, then washed to neutrality, dried and set aside to obtain COOH-CPPM-1. (4) COOH-CPPM-1, 70 mL of water, 5 mL of 0.5 M sulfuric acid and 4.11 g of 3-aminophenylboronic acid were added in sequence, and the mixture was stirred at room temperature for 0.5 h. 6.9 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) was added and the system was maintained for 6 h. The functionalized boronic acid affinity filler was obtained by washing and drying.

[0030] Example 2 Preparation method of functionalized boric acid affinity filler This example obtains a functionalized boric acid affinity filler by the following method, which differs from Example 1 in that glycine in step (2) of Example 1 is replaced by phenylalanine. The specific steps are as follows: (1) 5 g of polymethacrylate microspheres and 50 g of N, N-dimethylformamide (DMF) were added with 5 g of oxalic acid and 1.82 g of hexadecyltrimethylammonium chloride, and the reaction was hydrothermally reacted at 65 °C for 10 h to obtain the carboxylated porous polymer material CPPM-2.

[0031] (2) 4.88 g of phenylalanine, 9.5 g of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 7.65 g of N,N-diisopropylethylamine were added to 100 mL of DMSO in sequence, activated at room temperature, and the reaction system was stirred for 2 h. The mixture was extracted and dried by rotary evaporation to obtain a polypeptide polymer M2. (3) CPPM-2 prepared in step (1), 4.59 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 4.03 g of 4-dimethylaminopyridine and 6.07 g of triethylamine were added to 50 mL of N, N-dimethylformamide (DMF) in sequence, activated at 35°C for 1 h, mixed evenly, and then added with polypeptide polymer M2. The reaction system was stirred for 4 h, then washed to neutrality, dried and set aside to obtain COOH-CPPM-2. (4) COOH-CPPM-2, 70 mL of water, 5 mL of 0.5 M sulfuric acid and 4.11 g of 3-aminophenylboronic acid were added in sequence, and the mixture was stirred at room temperature for 0.5 h. 6.9 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added and the system was maintained for 6 h. The functionalized boronic acid affinity filler was obtained by washing and drying.

[0032] Example 3 Preparation method of functionalized boric acid affinity filler This example obtains a functionalized boric acid affinity filler by the following method, which is different from Example 1 mainly in that glycine in step (2) of Example 1 is replaced by lysine. The specific steps are as follows: (1) 5 g of polymethacrylate microspheres and 50 g of water were added with 2.25 g of oxalic acid and 1.82 g of hexadecyltrimethylammonium chloride, and the reaction was carried out at 65 °C for 10 h to obtain the carboxylated porous polymer material CPPM-3.

[0033] (2) 4.66 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 3.68 g of 4-dimethylaminopyridine and 5.06 g of triethylamine were added to 50 mL of N, N-dimethylformamide (DMF) in sequence, and 3.65 g of lysine was added. The reaction system was stirred at room temperature for 4 h, extracted, and dried by rotary evaporation to obtain polypeptide polymer M3. (3) CPPM-3 prepared in step (1), 1.94 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 1.84 g of 4-dimethylaminopyridine and 2.53 g of triethylamine were added to 50 mL of N, N-dimethylformamide (DMF) in sequence, activated at room temperature for 1 h, mixed evenly, and then added with polypeptide polymer M3. The reaction system was stirred for 4 h, then washed to neutrality, dried and set aside to obtain COOH-CPPM-3. (4) COOH-CPPM-3, 70 mL of water, 5 mL of 0.5 M sulfuric acid and 2 g of 4-aminophenylboronic acid were added in sequence, and the mixture was stirred at room temperature for 0.5 h. 2.8 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added and the system was maintained for 6 h. The functionalized boronic acid affinity filler was obtained by washing and drying.

[0034] Example 4 Preparation method of functionalized boric acid affinity filler This example obtains a functionalized boric acid affinity filler by the following method, which is different from Example 1 mainly in that glycine in step (2) of Example 1 is replaced by asparagine. The specific steps are as follows: (1) 5 g of chitosan microspheres and 50 g of water were added with 5 g of malonic acid and 3.6 g of hexadecyltrimethylammonium chloride, and the reaction was carried out at 50 °C for 20 h to obtain the carboxylated porous polymer material CPPM-4.

[0035] (2) To 50 mL of N, N-dimethylformamide (DMF), 13.21 g of asparagine was added at room temperature, followed by 12.62 g of N, N-diisopropylcarbodiimide and 11.51 g of N-hydroxysuccinimide. The reaction system was stirred for 2 h, then washed to neutrality and dried by rotary evaporation to obtain polypeptide polymer M4. (3) CPPM-4 prepared in step (1), 10.32 g of dicyclohexylcarbodiimide, 11.12 g of 4-pyrrolidinopyridine and 5.06 g of triethylamine were added to 50 mL of N, N-dimethylformamide (DMF) in sequence, activated at room temperature for 1 h, mixed evenly, and then added with polypeptide polymer M4. The reaction system was stirred for 4 h, then washed to neutrality, dried and set aside to obtain COOH-CPPM-4; (4) COOH-CPPM-4, 3.73 g of N,N-diisopropylethylamine, 50 mL of 1,4-dioxane, and 2.5 g of 3-aminophenylboronic acid hydrochloride were mixed and stirred at room temperature for 0.5 h, 5.47 g of O-benzotriazole-tetramethyluronium hexafluorophosphate was added and the system was maintained for 2 h, and the functionalized boronic acid affinity filler was obtained by washing and drying.

[0036] Example 5 A method for preparing a functionalized boric acid affinity filler In this example, a functionalized boric acid affinity filler is obtained by the following method, which is different from Example 1 mainly in that the glycine in step (2) of Example 1 is replaced by proline. The specific steps are as follows: (1) 5 g of polystyrene-divinylbenzene microspheres and 50 g of water were added with 2.25 g of oxalic acid and 1.82 g of hexadecyltrimethylammonium chloride, and the reaction was carried out at 65 °C for 10 h to obtain the carboxylated porous polymer material CPPM-5.

[0037] (2) To 50 mL of N, N-dimethylformamide (DMF), 3.45 g of proline was added at room temperature, followed by 1.89 g of N, N-diisopropylcarbodiimide and 1.83 g of 4-dimethylaminopyridine. The reaction system was stirred for 2 h, then washed to neutrality and dried by rotary evaporation to obtain polypeptide polymer M5. (3) CPPM-5 prepared in step (1), 4.66 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 5.18 g of N-hydroxysuccinimide were added to 50 mL of water in sequence, activated at 40°C for 1 h, mixed evenly, and then added with polypeptide polymer M5. The reaction system was stirred for 4 h, then washed to neutrality, dried and set aside to obtain COOH-CPPM-5. (4) COOH-CPPM-5, 8.87 g of N,N-diisopropylethylamine, 70 mL of N,N-dimethylformamide (DMF), and 8 g of 3-amino-4-methylphenylboronic acid were mixed and stirred at room temperature for 0.5 h, and 11.02 g of O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate was added and the system was maintained for 4 h. The functionalized boronic acid affinity filler was obtained by washing and drying.

[0038] Example 6 Preparation method of functionalized boric acid affinity filler This example obtains a functionalized boric acid affinity filler by the following method, which is different from Example 1 mainly in that the glycine in step (2) of Example 1 is replaced by histidine. The specific steps are as follows: (1) 5 g agarose microspheres and 50 g water were added with 5 g iodoacetic acid to the reaction, and the reaction was carried out at 40 °C for 24 h to obtain the carboxylated porous polymer material CPPM-6.

[0039] (2) Add 3.88 g of histidine to 70 mL of DMF at room temperature, then add 4 g of benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate and 3.99 g of N,N-diisopropylethylamine, keep stirring the reaction system for 1 h, then wash to neutrality, and dry by rotary evaporation to obtain polypeptide polymer M6; (3) CPPM-6 prepared in step (1), 5.83 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and 4.32 g of N-hydroxysuccinimide were added to 50 mL of N, N-dimethylformamide (DMF) in sequence, activated at room temperature for 1 h, mixed evenly, and then added with polypeptide polymer M6. The reaction system was stirred for 4 h, then washed to neutrality, dried, and set aside to obtain COOH-CPPM-6. (4) COOH-CPPM-6, 5.82 g of N,N-diisopropylethylamine, 50 mL of 1,4-dioxane, and 2.5 g of 3-aminophenylboronic acid hydrochloride were mixed and stirred at room temperature for 0.5 h, 4.82 g of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate was added and the system was maintained for 2 h, and the functionalized boronic acid affinity filler was obtained by washing and drying.

[0040] Load test The functionalized boric acid affinity filler prepared in Example 1 was used to fill a chromatography column (6.6×30 mm) for a loading test. Test conditions: Mobile phase: A: 100 mM ammonium acetate, 200 mM sodium chloride (pH 9.0); B: 100 mM glycine (pH=3.0). Sample: horseradish peroxidase-Sigma-Aldrich (1 mg / mL). Flow rate: 0.2 mL / min. Detector wavelength: UV 280 nm. Column temperature: 25 ℃.

[0041] The loading profile is as follows Figure 1 As shown. According to the calculation based on 10% flow-through, the loading capacity of the functionalized boronic acid affinity filler obtained in Example 1 is 40.36 mg / mL, while the loading capacity of similar commercial fillers is generally 10-30 mg / mL. The loading capacity of the fillers obtained in Examples 2-6 was tested using the same method and conditions, and the results are shown in Table 1. The loading capacity test results show that the functionalized boronic acid affinity filler obtained by the preparation method of the functionalized boronic acid affinity filler provided by the present invention has a higher loading capacity, which is helpful for separating cis-diol molecules.

[0042] Table 1 Loading capacity of boric acid fillers for horseradish peroxidase in Examples 1-6 Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Loading capacity (mg / mL) 40.36 33.79 36.26 40.19 31.36 38.04 Purification test The functionalized boric acid affinity fillers prepared in Example 1, Example 2 and Example 3 were respectively filled into chromatographic columns (4.6×50 mm) and purified by HPLC. Test conditions: Sample: homemade human albumin original sample (100 mg / mL, wherein the content of glycated protein was 0-10%). Flow rate: 0.5 mL / min. Detector wavelength: UV 280 nm. Column temperature: 25 ℃. Injection volume: 100 μL.

[0043] The mobile phases used in Test Examples 1 and 2 were: A: 20 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 10 mM magnesium chloride (MgCl2), pH = 8.5; B: A + 100 mM sodium chloride (NaCl), pH = 8.5; C: A + 100 mM sorbitol, pH = 8.5. The elution conditions used were: 0-25 min 100% A, 25-40 min 100% B, 40-65 min 100% C.

[0044] The mobile phase used in Test Example 3: A: 20 mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 10 mM magnesium chloride (MgCl2), pH = 8.5; B: A + 100 mM sorbitol, pH = 8.5; C: B + 100 mM sodium chloride (NaCl), pH = 8.5. The elution conditions used: 0-25 min 100% A, 25-40 min 100% B, 40-65 min 100% C.

[0045] The glycoprotein content was quantified by CON-A column. The total protein content was quantified by SEC column. The standard curve was established by external standard method to calculate the content of glycosylated protein in protein. The recovery rate and sugar removal rate were calculated as follows: recovery rate = albumin mass in flow-through ÷ protein injection mass * 100%; sugar removal rate = (glycoprotein content before loading - glycoprotein content after loading) ÷ glycoprotein content before loading * 100%.

[0046] The purification test results of Example 1 are as follows Figure 2 As shown in Table 2. Unglycosylated proteins were separated by flow-through mode with a yield of more than 95% and a sugar removal rate of 94.00%, indicating that the functionalized boronic acid affinity filler prepared by the preparation method of Example 1 has a good separation effect.

[0047] The purification test results of Example 2 are as follows Figure 3As shown in Table 2. Unglycosylated proteins were separated by flow-through mode with a yield of 85.50% and a sugar removal rate of 89.00%. The reason why the yield and sugar removal performance of the functionalized boronic acid affinity filler prepared in Example 2 are lower than those in Example 1 is that the phenylalanine in the spacer arm enhances the hydrophobic effect of the filler, causing nonspecific adsorption and partial shielding of the boronic acid functional group, indicating that hydrophobic side chains may not be suitable for proteins in this system, and a suitable secondary ligand needs to be selected according to the sample system.

[0048] Table 2 The desugaring efficiency of boric acid fillers in Examples 1 and 2 on human serum albumin Glycoprotein content in protein before loading % Glycoprotein content in protein after loading Sugar removal rate% Yield % Example 1 5.00% 0.30% 94.00% 95.81% Example 2 5.00% 0.55% 89.00% 85.50% The purification test results of Example 3 are as follows Figure 4 It is shown that the secondary ion interaction of the functionalized boric acid affinity filler prepared in Example 3 can separate different glycoforms.

Claims

1. A functionalized boric acid affinity filler, characterized in that: It has the following general structural formula: in: It is a porous polymer microsphere or a porous silica microsphere; R1 is an alkyl, carboxyl, amino, hydroxyl, hydrogen, phenyl, heteroaryl, thioether, amidine, amide or halogen functional group; R2 is an alkane group, a halogen or a carboxyl group; n is any integer from 1 to 50.

2. The functionalized boric acid affinity filler according to claim 1, characterized in that: The porous polymer microspheres are agarose microspheres, dextran microspheres, chitosan microspheres, konjac microspheres, cellulose microspheres, polymethacrylate microspheres, polystyrene-divinylbenzene microspheres or polyvinyl alcohol microspheres.

3. Use of the functionalized boric acid affinity filler as claimed in claim 1 in separating cis-diol molecules with different structures.

4. A method for preparing a functionalized boric acid affinity filler as claimed in any one of claims 1 or 2, characterized in that: The following steps are involved: Step 1: activating porous polymer microspheres or porous silica microspheres with a compound containing a carboxyl functional group to obtain a carboxylated porous material; Step 2: Using amino acid compounds as monomers, a condensation reaction is performed to obtain polypeptide polymers; Step 3: Grafting the polypeptide polymer obtained in step 2 onto the surface of the carboxylated porous material obtained in step 1; Step 4: Add a boronic acid compound to the product obtained in step 3 for coupling to obtain a functionalized boronic acid affinity filler.

5. The preparation method according to claim 4, characterized in that: In step 2, the polypeptide polymer has the following general structural formula: in: R1 is an alkyl, carboxyl, amino, hydroxyl, hydrogen, phenyl, heteroaryl, thioether, amidine, amide or halogen functional group; n is any integer from 1 to 50.

6. The preparation method according to claim 4, characterized in that: In step 2, the amino acid compound is at least one of 20 common amino acids; and its usage is 0.1-10 times of the mass of the porous polymer microspheres or porous silica microspheres.

7. The preparation method according to claim 4, characterized in that: In step 1, the compound containing a carboxyl functional group is oxalic acid, iodoacetic acid or malonic acid; The dosage is 0.1-5 times the mass of the porous polymer microspheres or porous silica microspheres.

8. The preparation method according to claim 4, characterized in that: In step 2-4, the condensing agent used in the condensation reaction is one of carbodiimides, onium salts or active esters; and the amount used is 0.1-5 times the mass of the porous polymer microspheres or porous silica microspheres.

9. The preparation method according to claim 4, characterized in that: In step 2-4, a catalyst and / or an organic base is used; the catalyst is 4-dimethylaminopyridine, 4-pyrrolidinopyridine, N-hydroxysuccinimide, 1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole; and the amount thereof is 0.1-5 times the mass of the porous polymer microspheres or porous silica microspheres; the organic base is triethylamine, N,N-diisopropylethylamine or N-methylmorpholine; and the amount thereof is 0.1-5 times the mass of the porous polymer microspheres or porous silica microspheres.

10. The preparation method according to claim 4, characterized in that: In step 4, the boric acid compound is 3-aminophenylboronic acid, 4-aminophenylboronic acid, 4-aminophenylboronic acid hydrochloride, 3-aminophenylboronic acid hydrochloride, 3-aminophenylboronic acid hemisulfate, 3-amino-5-carboxyphenylboronic acid, (3-amino-4-chlorophenyl)boric acid, (2-amino-5-chlorophenyl)boric acid or 3-amino-4-methylphenylboric acid; and its amount is 0.1-5 times the mass of the porous polymer microspheres or porous silica microspheres.

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