Preparation method for solid-phase synthesis of MAPS branch polypeptide

Abstract

The invention discloses a preparation method for solid-phase synthesis of MAPS branched polypeptide, and mainly solves the technical problem that pure MAPS branched polypeptide products with high purity and accurate target molecular weight products cannot be obtained in the prior art. Comprising the following steps: 1) dissolving solid-phase synthesized MAPS branch polypeptide with pure water, and filtering; 2) taking a dipotassium phosphate aqueous solution as a phase A of a preparation system, and taking pure acetonitrile as a phase B; (3) enabling the filtrate to pass through a macroporous-mesoporous combined titanium gel monolithic column with a two-stage pore structure; (4) enabling the one-step preparation liquid to pass through a mesoporous structure titanium gel monolithic column respectively; (5) enabling the secondary preparation liquid to pass through a microporous structure titanium gel monolithic column respectively; the method comprises the following steps of 1, preparing a peptide solution, 2, preparing a TFA salt-containing MAPS branched polypeptide, 3, preparing a TFA salt-containing MAPS branched polypeptide, 4, preparing a TFA salt-containing MAPS branched polypeptide, 6, carrying out time-of-flight mass spectrometry qualitative detection on the peptide solution to obtain a target product with a correct molecular weight, 7, passing the correct peptide solution through a macroporous structure titanium gel monolithic column with a TFA aqueous solution-acetonitrile system as a mobile phase, and 8, freezing and drying the collected peptide solution to obtain a TFA salt-containing MAPS branched polypeptide finished product.

Description

Technical Field

[0001] This invention belongs to the technical field of liquid-phase preparation and separation using a titanium gel monolithic column combined with time-of-flight mass spectrometry (MALID-TOF), and in particular relates to a method for preparing MAPS branched peptides that can be synthesized in the solid phase. Background Technology

[0002] MAPS branched peptides are multiple-antigen peptides, a type of branched polypeptide. They are formed by linking linear peptide chains to a polylysine core at its C-terminus, creating a branched structure that increases the overall molecular size. MAPS peptides are branched synthetic peptides with α- or ε- groups of lysine residues forming the backbone and multiple copies of the peptide antigen forming the outer layer. Depending on the number of lysine residues, polyantigen peptides with different numbers of side chains can be synthesized using solid-phase synthesis methods, such as the two-branched peptide MAPS2, the four-branched peptide MAPS4, and the eight-branched peptide MAPS8.

[0003] MAPS-branched peptides are widely used in drug and vaccine research. Due to their good affinity, high molecular weight, and large spatial structure, MAPS-branched peptides can be directly used as antigens for subsequent drug research without needing to be coupled to carrier proteins. Furthermore, MAPS-branched peptides can be modified through chelation to enhance the targeting properties of drug peptide antigens, which is of great significance for drug and vaccine scientific research.

[0004] MAPS-branched peptides synthesized via solid-phase chromatography exhibit increasing defects due to steric hindrance from intermolecular molecules, with the gradual addition of amino acids. In liquid chromatography analysis, MAPS-branched peptides, even with conventional columns using silica-bonded (C18, C8, C4) or polymer-bonded (polystyrene, methacrylic, etc.) stationary phases, still suffer from peak diffusion, peak extension, low response, peak collapse, and poor peak shape, even with increased column pore size. No matter how the linear gradient is altered, the peptide peaks cannot be separated. Furthermore, MAPS-branched peptides are highly hydrophilic and generally weakly basic; even changing the mobile phase to a weakly basic system cannot improve the resolution of these peptides in conventional liquid chromatography columns. For these peptides, it is impossible to obtain target products with the correct molecular weight or prepare high-purity products, significantly increasing the difficulties for subsequent drug research.

[0005] Currently, among MAPS-branched peptides, a few short-chain bibranched peptides can still be purified to low purity using conventional liquid chromatography columns. However, for their corresponding tetra-, octagonal, or larger-chained peptides, or bibranched peptides with long sequences, due to their large molecular weight, numerous defects, and severe steric hindrance, it is virtually impossible to obtain high-purity products, or even accurately identify the target product, using the aforementioned liquid chromatography columns. Summary of the Invention

[0006] The purpose of this invention is to provide a method suitable for solid-phase synthesis of MAPS branched peptides, which mainly solves the technical problem that existing methods cannot obtain high-purity MAPS branched peptides with precise target molecular weights.

[0007] The concept of this invention is as follows: This invention introduces a titanium-gel monolithic column within an organic-inorganic hybrid monolithic column. By utilizing the titanium-gel monolithic column formed with different particle sizes and pore sizes, combined with a weakly basic system, the various chromatographic peaks of the branched peptides are progressively separated from macropores to mesopores and micropores. Target qualitative detection is performed using more precise time-of-flight mass spectrometry (MALID-TOF) to obtain the target product with the correct molecular weight. Then, utilizing the high flowability of the macroporous titanium-gel monolithic column, buffer salts are removed again through liquid chromatography, and the peptide is converted into a peptide solution containing TFA salts. Finally, quantitative detection is performed by liquid HPLC to obtain high-purity MAPS branched peptides suitable for scientific research.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: A method for preparing MAPS-branched peptides by solid-phase synthesis includes the following steps: 1) First, dissolve the MAPS branched peptide synthesized in solid phase in pure water, filter it, and set the filtrate aside for later use; 2) Prepare a dipotassium hydrogen phosphate aqueous solution as the mobile phase A and a pure acetonitrile solution as the mobile phase B; 3) The filtrate is prepared by liquid chromatography, first passing it through a monolithic titanium gel column with a macroporous-mesoporous dual-pore structure, setting a linear gradient, collecting the separated chromatographic peaks, and numbering the collected chromatographic peaks sequentially. 4) The chromatographic peaks collected in the first step are prepared in a second step and passed through a mesoporous titanium gel monolithic column according to their numbers. The separated chromatographic peaks are collected and numbered sequentially. 5) The chromatographic peaks collected in the two-step preparation were prepared three times and passed through a microporous titanium gel monolithic column according to their numbers. The separated chromatographic peaks were collected and numbered in three levels. 6) The preparation solutions after the three preparations were subjected to time-of-flight mass spectrometry (MALID-TOF) for qualitative analysis to determine the target product with the correct molecular weight; 7) Pass the correct target preparation solution through a macroporous titanium gel monolithic column. Mobile phase conditions: TFA aqueous solution as phase A, pure acetonitrile as phase B; 8) Freeze and dry the collected peptide solution to obtain a white powdery product containing TFA salt; 9) The MAPS branched peptide product was quantitatively analyzed by liquid chromatography-HPLC and the content exceeded 95%.

[0009] A more preferred option is that the mobile phase system A in step 2) is an aqueous solution of dipotassium hydrogen phosphate with a pH of 7.5-8.0.

[0010] Titanium Glue Monolithic Column: Distributor: Beijing Yinglaike Technology Development Co., Ltd., Brand: ZirChrom, Model: ZirChrom Titania Sachtopore-RP; Divided into the following four types: Bipolar Pore Structure Titanium Glue Monolithic Column, Mesoporous Structure Titanium Glue Monolithic Column, Microporous Structure Titanium Glue Monolithic Column, Macroporous Structure Titanium Glue Monolithic Column.

[0011] A more preferred option is that the integral titanium glue column with a dual-pore structure in step 3) has a diameter of 3cm, a column length of 25cm, a macropore particle size of 50nm, a mesopore particle size of 20nm, and a pore size of 300A.

[0012] A more preferred option is that the integral titanium glue column with mesoporous structure in step 4) has a diameter of 3cm, a column length of 20cm, a pore particle size of 10nm, and a pore size of 200A.

[0013] A more preferred option is that the microporous titanium glue monolithic column in step 5) has a diameter of 2cm, a column length of 15cm, a pore particle size of 2nm, and a pore diameter of 120A.

[0014] A more preferred option is that: in step 7), the macroporous titanium glue monolithic column has a diameter of 2cm, a column length of 20cm, a pore particle size of 80nm, and a pore size of 300Å; phase A is a TFA aqueous solution with a mass percentage concentration of 0.12%.

[0015] The beneficial effects of this invention are as follows: This invention proposes a method for preparing MAPS branched peptides suitable for solid-phase synthesis. It uses a monolithic column composed of a hybrid organic and inorganic polymer as the liquid-phase preparation column, which effectively improves the problem of MAPS branched peptides failing to aggregate and diffuse, and thus failing to form chromatographic peaks, in conventional chromatographic columns. Titanium colloid monolithic columns packed with titanium dioxide of different particle sizes and pore structures are used, progressively increasing the resolution of MAPS branched peptides within the chromatographic column from macroporous titanium dioxide monolithic columns with larger particle sizes and pore sizes to mesoporous and microporous titanium dioxide monolithic columns. A weakly alkaline solution is configured as the mobile phase preparation system, which not only stabilizes the spatial stability of the MAPS branched peptides but also enhances the separation effect due to the alkalinity of the MAPS branched peptides. After multiple steps of liquid-phase preparation, the chromatographic absorption peaks of the MAPS branched peptides are finally separated. Then, the more precise time-of-flight mass spectrometry (MALID-TOF) technology is used for qualitative detection to confirm the correctness of each separated chromatographic peak, thus obtaining the target product. Finally, the peptide is passed through a monolithic titanium dioxide column with a macroporous structure. Utilizing the high adsorption and permeability of macroporous titanium dioxide, it is converted into TFA salt form in the liquid phase, ultimately yielding a high-purity MAPS branched peptide product containing TFA salt. This invention addresses the technical deficiencies in existing liquid-phase preparation methods for MAPS branched peptides. Furthermore, it allows for customization and easy availability of monolithic titanium dioxide columns of different types and structures, providing both high-purity, targeted peptide products and a novel method for the liquid-phase preparation of larger branched MAPS peptides. Attached Figure Description

[0016] Figure 1 This is a MALID-TOF detection image of a short peptide with a two-branched structure.

[0017] Figure 2 This is an HPLC chromatogram of a short peptide structured bibranched polypeptide.

[0018] Figure 3 This is a MALID-TOF detection image of a short peptide with a four-branched structure.

[0019] Figure 4 This is an HPLC chromatogram of a short peptide structure with four branches.

[0020] Figure 5 This is a MALID-TOF detection image of an eight-branched peptide with a short peptide structure.

[0021] Figure 6 This is an HPLC chromatogram of an eight-branched polypeptide with a short peptide structure.

[0022] Figure 7 This is a MALID-TOF detection image of a long peptide with a two-branched structure.

[0023] Figure 8 This is an HPLC chromatogram of a bibranched polypeptide with a long peptide structure. Detailed Implementation

[0024] Using the bibranched, tetrabranched, and octagonal peptides of the short peptide structure HDYKEDG and the bibranched peptide of the long peptide structure NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR as examples, the coded sequence listing of the peptide sequences is shown in the table below: Example 1

[0025] Taking (HDYKEDG) 2K bibranched peptide (manufacturer: Jier Biochemical (Shanghai) Co., Ltd., production batch: GLS021402500001) as an example, the specific implementation steps are as follows: 1. Solid-phase synthesis: The crude (HDYKEDG)2K bibranched polypeptide was first synthesized using the Fmoc solid-phase direct method. ([Yang Xuesen, Wang Huaqiao, Yuan Qunfang, Xie Yao, Yao Zhibin, Ye Xiaozhou, Wu Jiming, Zou Aiguo. Synthesis of Aβ by Fmoc solid-phase direct method]) 1-15 Peptide vaccines and their immunogenicity [J]. Journal of Sun Yat-sen University (Medical Sciences). 2006, (02): 121-125.]), with a molecular weight of 1128.39.

[0026] 2. Sample preparation: The crude (HDYKEDG)2K bibranched peptide synthesized by solid phase synthesis was directly dissolved in pure water by sonication (the concentration of the solution was about 50 mg / ml), filtered through a 0.45 μm aqueous phase filter, and the filtrate was reserved for later use.

[0027] 3. Preparation system configuration: Weigh 136.1g of dipotassium hydrogen phosphate and dissolve it in 5L of pure water. Stir thoroughly until completely dissolved. Adjust the pH to 7.5 with 0.2mol / L sodium hydroxide. This solution is the mobile phase A for the one-step, two-step, and three-step preparations. Pure acetonitrile is the B phase.

[0028] 4. First step: Liquid phase preparation Preparation conditions: Monolithic column: Bipolar porous titanium dioxide monolithic column with a macropore-mesopore combination, column diameter and length: 3×25cm, macropore particle size: 50nm, mesopore particle size: 20nm, pore size: 300Å. Flow rate: 30ml / min. Gradient: Mobile phase B mass percentage concentration: 8-65%, gradient treatment time: 60min. Injection volume: 500mg; Preparation process: After rinsing the chromatographic column with acetonitrile with a mass percentage concentration of 90% or higher, the sample solution is loaded. Linear gradient elution is performed. According to the separation of the chromatographic peaks in the whole column, the peptide solutions are collected in separate bottles according to the elution time, and marked with ①②③…… for later use.

[0029] 5. Second step: Liquid phase preparation Preparation conditions: Monolithic column: Mesoporous titanium dioxide monolithic column, column diameter and length: 3*20cm, particle size 10nm, pore size 200Å. Flow rate: 30ml / min. Gradient: Mobile phase B mass percentage concentration: 10-50%. Gradient treatment time: 40min. Injection volume: One-step preparation concentrate; Preparation process: The concentrated solution after the first step of liquid phase preparation is eluted linearly according to the labeling order. According to the separation, the peptide solution is collected in separate bottles and labeled as ①-1, ①-2, ①-3...②-1, ②-2, ②-3...③-1, ③-2... for later use.

[0030] 6. Third step: Liquid phase preparation Preparation conditions: Monolithic column: Microporous titanium dioxide monolithic column, column diameter and length: 2*15cm, particle size 2nm, pore size 120Å. Flow rate: 20ml / min. Gradient: Mass percentage concentration of mobile phase B: 12-45%. Gradient treatment time: 30min.

[0031] The injection volume is the concentrate prepared in the two steps; Preparation process: The concentrated solution prepared in the second step of liquid phase was eluted linearly according to the labeling order. The peptide solutions were collected in separate bottles according to the separation results and labeled as ①-1-1, ①-1-2……①-2-1, ①-2-2……①-3-1……②-1-1……②-2-1……③-1-1…….

[0032] 7. Qualitative detection: The prepared solutions of each chromatographic peak collected after the three-step preparation were analyzed by time-of-flight mass spectrometry (MALID-TOF) to determine the correct target product, with a molecular weight of 1128.39. Figure 1 ).

[0033] 8. Fourth step: Liquid phase preparation: Preparation conditions: Monolithic column: Macroporous titanium dioxide monolithic column, column diameter and length: 2*20cm, particle size 80nm, pore size 300Å. Mobile phase system: 0.12% TFA aqueous solution (mass percentage) as phase A, pure acetonitrile as phase B. Flow rate: 25ml / min. Gradient: Mass percentage concentration of mobile phase B: 8-50%. Gradient treatment time: 25min.

[0034] 9. Post-processing: The MAPS branched peptide liquid, which has been replaced with TFA salt, is freeze-dried and vacuum-dried to finally obtain a white powdered peptide product.

[0035] 10. Quantitative detection: The final product peptides are analyzed by liquid chromatography-HPLC, and the purity is greater than 95.0%. Figure 2 ). Example 2

[0036] Taking (HDYKEDG) 4K2K four-branched peptide (manufacturer: Jier Biochemical (Shanghai) Co., Ltd., production batch: GLS021402500002) as an example, the specific implementation steps are as follows: 1. Solid-phase synthesis: The crude (HDYKEDG) 4K2K four-branched polypeptide was first synthesized using the Fmoc solid-phase direct method, with a molecular weight of 5215.14. (Refer to step 1 of Example 1) 2. Sample preparation: The crude (HDYKEDG)4K2K four-branched peptide synthesized by solid phase was directly dissolved in deionized water by sonication (dissolution concentration of about 40 mg / ml), filtered through a 0.45 μm aqueous phase filter, and the filtrate was reserved for later use.

[0037] 3. Preparation system configuration: Weigh 136.5g of dipotassium hydrogen phosphate and dissolve it in 5L of pure water. Stir thoroughly until completely dissolved. Adjust the pH to 7.8 with 0.2mol / L sodium hydroxide. This solution is the mobile phase A for the one-step, two-step, and three-step preparations. Pure acetonitrile is the B phase.

[0038] 4. First step: Liquid phase preparation Preparation conditions: Monolithic column: Bipolar porous titanium dioxide monolithic column with a macropore-mesopore combination, column diameter and length: 3×25cm, macropore particle size: 50nm, mesopore particle size: 20nm, pore size: 300Å. Flow rate: 30ml / min. Gradient: Mobile phase B mass percentage concentration: 8-70%, gradient treatment time: 60min. Injection volume: 400mg; Preparation process: After rinsing the chromatographic column with acetonitrile with a mass percentage concentration of 90% or higher, the sample solution is loaded. Linear gradient elution is performed. According to the separation of the chromatographic peaks in the whole column, the peptide solutions are collected in separate bottles according to the elution time, and marked with ①②③…… for later use.

[0039] 5. Second step: Liquid phase preparation Preparation conditions: Monolithic column: Mesoporous titanium dioxide monolithic column, column diameter and length: 3*20cm, particle size 10nm, pore size 200Å. Flow rate: 30ml / min. Gradient: Mobile phase B mass percentage concentration: 9-60%. Gradient treatment time: 40min. Injection volume: One-step concentrate. Preparation process: Collect all peptide solutions from the first step of liquid phase preparation, and continue linear gradient elution according to the labeling order. Collect peptide solutions in separate bottles according to the separation results, and label them as ①-1, ①-2, ①-3... ②-1, ②-2, ②-3... ③-1, ③-2... for later use.

[0040] 6. Third step: Liquid phase preparation Preparation conditions: Monolithic column: Microporous titanium dioxide monolithic column, column diameter and length: 2*15cm, particle size 2nm, pore size 120Å. Flow rate: 20ml / min. Gradient: Mass percentage concentration of mobile phase B: 10-55%. Gradient treatment time: 30min.

[0041] The injection volume is the concentrate prepared in the two steps; Preparation process: Collect all peptide solutions from the second step of liquid phase preparation, and continue linear gradient elution according to the labeling order. Collect peptide solutions in separate bottles according to the separation status and label them as ①-1-1, ①-1-2……①-2-1, ①-2-2……①-3-1……②-1-1……②-2-1……③-1-1…….

[0042] 7. Qualitative Detection: The prepared solutions of each chromatographic peak collected after the three-step preparation were analyzed by time-of-flight mass spectrometry (MALID-TOF) to determine the correct target product, with a molecular weight of 5215.14. Figure 3 ).

[0043] 8. Fourth step: Liquid phase preparation: Preparation conditions: Monolithic column: Macroporous titanium dioxide monolithic column, column diameter and length: 2*20cm, particle size 80nm, pore size 300Å. Mobile phase system: 0.12% TFA aqueous solution (mass percentage) as phase A, pure acetonitrile as phase B. Flow rate: 25ml / min. Gradient: Mass percentage concentration of mobile phase B: 9-55%. Gradient treatment time: 25min.

[0044] 9. Post-processing: The MAPS branched peptide liquid, which has been replaced with TFA salt, is freeze-dried and vacuum-dried to finally obtain a white powdered peptide product.

[0045] 10. Quantitative detection: The final product peptides are analyzed by liquid chromatography-HPLC, and the purity is greater than 95.0%. Figure 4 ). Example 3

[0046] Taking (HDYKEDG) 8K4K2K eight-branched peptide (manufacturer: Jier Biochemical (Shanghai) Co., Ltd., production batch: GLS021402500003) as an example, the specific implementation steps are as follows: 1. Solid-phase synthesis: The crude (HDYKEDG) 8K4K2K eight-branched polypeptide was first synthesized using the Fmoc solid-phase direct method, with a molecular weight of 11973.74. (Refer to step 1 of Example 1) 2. Sample preparation: The crude (HDYKEDG)8K4K2K eight-branched peptide synthesized by solid phase synthesis was directly dissolved in deionized water by sonication (dissolution concentration was about 30 mg / ml), filtered through a 0.45 μm aqueous phase filter, and the filtrate was reserved for later use.

[0047] 3. Preparation system configuration: Weigh 136.9g of dipotassium hydrogen phosphate and dissolve it in 5L of pure water. Stir thoroughly until completely dissolved. Adjust the pH to 8.0 with 0.2mol / L sodium hydroxide. This solution is the mobile phase A for the one-step, two-step, and three-step preparations. Pure acetonitrile is the B phase.

[0048] 4. First step: Liquid phase preparation Preparation conditions: Monolithic column: Bipolar porous titanium dioxide monolithic column with a macropore-mesopore combination, column diameter and length: 3×25cm, macropore particle size: 50nm, mesopore particle size: 20nm, pore size: 300Å. Flow rate: 30ml / min. Gradient: Mobile phase B mass percentage concentration: 8-75%, gradient treatment time: 60min. Injection volume: 300mg; Preparation process: After rinsing the chromatographic column with acetonitrile with a mass percentage concentration of 90% or higher, the sample solution is loaded. Linear gradient elution is performed. According to the separation of the chromatographic peaks in the whole column, the peptide solutions are collected in separate bottles according to the elution time, and marked with ①②③…… for later use.

[0049] 5. Second step: Liquid phase preparation Preparation conditions: Monolithic column: Mesoporous titanium dioxide monolithic column, column diameter and length: 3*20cm, particle size 10nm, pore size 200Å. Flow rate: 30ml / min. Gradient: Mobile phase B mass percentage concentration: 9-65%. Gradient treatment time: 40min. Injection volume: One-step preparation concentrate; Preparation process: Collect all peptide solutions from the first step of liquid phase preparation, and continue linear gradient elution according to the labeling order. Collect peptide solutions in separate bottles according to the separation results, and label them as ①-1, ①-2, ①-3... ②-1, ②-2, ②-3... ③-1, ③-2... for later use.

[0050] 6. Third step: Liquid phase preparation Preparation conditions: Monolithic column: Microporous titanium dioxide monolithic column, column diameter and length: 2*15cm, particle size 2nm, pore size 120Å. Flow rate: 20ml / min. Gradient: Mass percentage concentration of mobile phase B: 9-55%. Gradient treatment time: 30min.

[0051] The injection volume is the concentrate prepared in the two steps; Preparation process: Collect all peptide solutions from the second step of liquid phase preparation, and continue linear gradient elution according to the labeling order. Collect peptide solutions in separate bottles according to the separation status and label them as ①-1-1, ①-1-2……①-2-1, ①-2-2……①-3-1……②-1-1……②-2-1……③-1-1…….

[0052] 7. Qualitative detection: The prepared solutions of each chromatographic peak collected after the three-step preparation were analyzed by time-of-flight mass spectrometry (MALID-TOF) to determine the correct target product, with a molecular weight of 11973.74. Figure 5 ).

[0053] 8. Fourth step: Liquid phase preparation: Preparation conditions: Monolithic column: Macroporous titanium dioxide monolithic column, column diameter and length: 2*20cm, particle size 80nm, pore size 300Å. Mobile phase system: 0.12% TFA aqueous solution (mass percentage) as phase A, pure acetonitrile as phase B. Flow rate: 25ml / min. Gradient: Mass percentage concentration of mobile phase B: 8-55%. Gradient treatment time: 25min.

[0054] 9. Post-processing: The MAPS branched peptide liquid, which has been replaced with TFA salt, is freeze-dried and vacuum-dried to finally obtain a white powdered peptide product.

[0055] 10. Quantitative detection: The final product peptides are analyzed by liquid chromatography-HPLC, and the purity is greater than 95.0%. Figure 6 ). Example 4

[0056] Taking (NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR)2K bibranched peptide (manufacturer: Jier Biochemical (Shanghai) Co., Ltd., production batch: GLS 021402500004) as an example, the specific implementation steps are as follows: 1. Solid-phase synthesis: The crude product of the 2K bibranched polypeptide (NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR) was first synthesized using the Fmoc solid-phase direct method, with a molecular weight of 12554.11. (Refer to step 1 of Example 1) 2. Sample preparation: The crude (NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR)2K bibranched peptide synthesized in solid phase was directly dissolved in deionized water by sonication (dissolution concentration was about 20 mg / ml), filtered through a 0.45 μm aqueous phase filter, and the filtrate was reserved for later use.

[0057] 3. Preparation system configuration: Weigh 136.7g of dipotassium hydrogen phosphate and dissolve it in 5L of pure water. Stir thoroughly until completely dissolved. Adjust the pH to 7.9 with 0.2mol / L sodium hydroxide. This solution is the mobile phase A for the one-step, two-step, and three-step preparations. Pure acetonitrile is the B phase.

[0058] 4. First step: Liquid phase preparation Preparation conditions: Monolithic column: Bipolar porous titanium dioxide monolithic column with a macropore-mesopore combination, column diameter and length: 3×25cm, macropore particle size: 50nm, mesopore particle size: 20nm, pore size: 300Å. Flow rate: 30ml / min. Gradient: Mobile phase B mass percentage concentration: 10-70%, gradient treatment time: 60min. Injection volume: 200mg; Preparation process: After rinsing the chromatographic column with acetonitrile with a mass percentage concentration of 90% or higher, the sample solution is loaded. Linear gradient elution is performed. According to the separation of the chromatographic peaks in the whole column, the peptide solutions are collected in separate bottles according to the elution time, and marked with ①②③…… for later use.

[0059] 5. Second step: Liquid phase preparation Preparation conditions: Monolithic column: Mesoporous titanium dioxide monolithic column, column diameter and length: 3*20cm, particle size 10nm, pore size 200Å. Flow rate: 30ml / min. Gradient: Mobile phase B mass percentage concentration: 12-60%. Gradient treatment time: 40min. Injection volume: One-step preparation concentrate; Preparation process: Collect all peptide solutions from the first step of liquid phase preparation, and continue linear gradient elution according to the labeling order. Collect peptide solutions in separate bottles according to the separation results, and label them as ①-1, ①-2, ①-3... ②-1, ②-2, ②-3... ③-1, ③-2... for later use.

[0060] 6. Third step: Liquid phase preparation Preparation conditions: Monolithic column: Microporous titanium dioxide monolithic column, column diameter and length: 2*15cm, particle size 2nm, pore size 120Å. Flow rate: 20ml / min. Gradient: Mass percentage concentration of mobile phase B: 12-55%. Gradient treatment time: 30min.

[0061] The injection volume is the concentrate prepared in the two steps; Preparation process: Collect all peptide solutions from the second step of liquid phase preparation, and continue linear gradient elution according to the labeling order. Collect peptide solutions in separate bottles according to the separation status and label them as ①-1-1, ①-1-2……①-2-1, ①-2-2……①-3-1……②-1-1……②-2-1……③-1-1…….

[0062] 7. Qualitative Detection: The prepared solutions of each chromatographic peak collected after the three-step preparation were analyzed by time-of-flight mass spectrometry (MALID-TOF) to determine the correct target product, with a molecular weight of 12554.11. Figure 7 ).

[0063] 8. Fourth step: Liquid phase preparation: Preparation conditions: Monolithic column: Macroporous titanium dioxide monolithic column, column diameter and length: 2*20cm, particle size 80nm, pore size 300Å. Mobile phase system: 0.12% TFA aqueous solution (mass percentage) as phase A, pure acetonitrile as phase B. Flow rate: 25ml / min. Gradient: Mass percentage concentration of mobile phase B: 10-55%. Gradient treatment time: 25min.

[0064] 9. Post-processing: The MAPS branched peptide liquid, which has been replaced with TFA salt, is freeze-dried and vacuum-dried to finally obtain a white powdered peptide product.

[0065] 10. Quantitative detection: The final product peptides are analyzed by liquid chromatography-HPLC, and the purity is greater than 95.0%. Figure 8 ).

Claims

1. A method for preparing MAPS-branched polypeptides by solid-phase synthesis, characterized in that, Includes the following steps: 1) First, dissolve the MAPS branched peptide synthesized in solid phase in deionized water and filter it to obtain the filtrate for later use; 2) Prepare a dipotassium hydrogen phosphate aqueous solution as the mobile phase A and a pure acetonitrile solution as the mobile phase B; 3) The filtrate is prepared by liquid chromatography, first passing it through a monolithic titanium gel column with a bi-level pore structure of macroporous-mesoporous combination, setting a linear gradient, and collecting the separated chromatographic peaks; 4) The chromatographic peaks collected in the first step are prepared a second time and passed through a mesoporous titanium gel monolithic column to collect the separated chromatographic peaks. 5) Pass the chromatographic peaks collected in the two-step preparation through a microporous titanium gel monolithic column to collect the separated chromatographic peaks; 6) The preparation solutions after the three preparations were subjected to time-of-flight mass spectrometry (MALID-TOF) for qualitative analysis to determine the target product with the correct molecular weight; 7) Pass the correct target preparation solution through a macroporous titanium gel monolithic column. Mobile phase conditions: TFA aqueous solution as phase A, pure acetonitrile as phase B; 8) Freeze and dry the collected peptide solution to obtain a white powdery product containing TFA salt.

2. The method for preparing MAPS branched peptides by solid-phase synthesis according to claim 1, characterized in that, The mobile phase A is an aqueous solution of dipotassium hydrogen phosphate with a pH of 7.5-8.

0.

3. The method for preparing MAPS branched polypeptides by solid-phase synthesis according to claim 1, characterized in that, Step 3) describes a bi-level porous titanium glue monolithic column with a macropore-mesopore combination. Model: 3cm in diameter, 25cm in length, macropore particle size 50nm, mesopore particle size 20nm, pore size 300A.

4. The method for preparing MAPS branched polypeptides by solid-phase synthesis according to claim 1, characterized in that, Step 4) The mesoporous titanium glue monolithic column has the following specifications: diameter 3cm, column length 20cm, mesoporous particle size 10nm, and pore size 200A.

5. The method for preparing MAPS branched peptides by solid-phase synthesis according to claim 1, characterized in that, Step 5) The microporous titanium glue monolithic column has the following specifications: diameter 2cm, column length 15cm, micropore particle size 2nm, and pore size 120A.

6. The method for preparing MAPS branched peptides by solid-phase synthesis according to claim 1, characterized in that, Step 7) describes a large-pore structure titanium glue monolithic column with a diameter of 2cm, a column length of 20cm, a large pore particle size of 80nm, and a pore size of 300A.

7. The method for preparing MAPS branched polypeptides by solid-phase synthesis according to claim 1, characterized in that, Step 7) The A phase mentioned is a TFA aqueous solution with a mass percentage concentration of 0.12%.

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