Solid-phase preparation method of tierpotide

Abstract

The invention relates to the technical field of preparation methods of polypeptide drugs, and provides a solid-phase preparation method of tirpotide, which comprises the following steps: by taking amino resin as initial resin, sequentially coupling amino acid and small-fragment peptide according to an amino acid sequence of the tirpotide by using a solid-phase synthesis method to synthesize tirpotide resin; and cracking and purifying the tilpotide resin to obtain a pure tilpotide product. Wherein the small fragment peptide is 1-4 tetrapeptide fragment Boc-Tyr (tBu)-Aib-Glu (OtBu)-G1y-OH, 5-6 dipeptide fragment Fmoc-Thr (tBu)-Phe-OH, 7-8 dipeptide fragment Fmoc-Thr (tBu)-Ser (tBu)-OH, 10-11 dipeptide fragment Fmoc-Tyr (tBu)-Ser (tBu)-OH, 12-13 dipeptide fragment Fmoc-Ile-Aib-OH, 17-18 dipeptide fragment Fmoc-Lys (AEEA-AEA-gamma-Glu (alpha-OtBu)-Ecosanoic acid (mon-tBu), 20 side chain peptide Fmoc The coupling agent coupling the amino acid and the small fragment peptide is selected from the group consisting of Oxyma and DIC, TPTU and TMP or COMU and DIEA. The crude product of the tirpotide prepared by the method disclosed by the invention is high in yield and high in purity.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide drug preparation methods, and particularly relates to a preparation method of telpotide. Background Art

[0002] Tirzepatide is a dual agonist of the glucose-dependent insulinotropic polypeptide (GIP, also known as gastric inhibitory polypeptide) receptor and the glucagon-like peptide-1 (GLP-1) receptor. Both GIP and GLP-1 are hormones secreted by the intestine that can promote insulin secretion. Tirzepatide integrates two insulinotropic effects into a single molecule and represents a new class of drugs for the treatment of type 2 diabetes. Tirzepatide can improve β-cell function and increase insulin sensitivity, thus proving its efficacy.

[0003] The structure of tepoxetine is as follows: H-Tyr 1 -Aib 2 -G1u 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Ile 17 -Ala 18 -GIn 19 -Lys 20 (AEEA-AEEA-γ-Glu-Eicosanedioicacid)-Ala 21 -Phe 22 -Val 23 -GIn 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 3 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro38 -Ser 39 -NH 2 .

[0004] At present, the most effective chemical synthesis method for peptide drugs is solid phase synthesis, that is, starting from the carboxyl end of the peptide, after the first amino acid is connected to the resin, the amino acid amino protecting group is removed, and the next amino acid is coupled according to the peptide sequence, the amino protecting group is removed, and then coupled again, and the operation is repeated until the amino acid assembly is completed. The peptide can be cleaved from the resin and all the protecting groups are removed to obtain a crude peptide, which is then purified and freeze-dried to obtain the peptide API. In the existing solid phase synthesis of telpotide, due to the large number of telpotide amino acids and the large number of hydrophobic amino acids in the sequence, intramolecular hydrogen bonds are easily formed during the coupling process, resulting in severe β-folding and causing resin polycondensation. If step-by-step coupling is performed, the difficulty of amino acid coupling will increase and the efficiency of coupling will be reduced, resulting in each step of coupling after polycondensation being difficult and prone to producing a large number of missing peptides. If telpotide is synthesized by long-fragment peptides, the cost is high and the process is complicated, which is not conducive to industrial production.

[0005] Therefore, designing and developing a low-cost, simple synthetic process and high-quality preparation method of telpotide has important practical significance and industrial value. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a method for preparing telpotide with high yield and suitable for industrial production. The preparation method of the present invention can greatly reduce the number of coupling cycles and the use of solvents. In addition, in the precipitation process after the telpotide resin is cracked, centrifugation is not required, and a crude telpotide can be obtained by simple filtration. The yield and purity of the synthesized crude telpotide are high.

[0007] The present invention provides a method for preparing telpotide, wherein the amino acid sequence of the telpotide is H-Tyr 1 -Aib 2 -G1u 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Ile 17-Ala 18 -GIn 19 -Lys 20 (AEEA-AEEA-γ-Glu-Eicosanedioicacid)-Ala 21 -Phe 22 -Val 23 -GIn 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 3 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -NH 2 , the preparation method comprises: The amino resin is used as the starting resin, and the amino acids and small fragment peptides are sequentially coupled according to the amino acid sequence of the telpotide by solid phase synthesis to synthesize the telpotide resin. The telpotide resin is cleaved and purified to obtain a pure telpotide; wherein the small fragment peptide is a 1-4 tetrapeptide fragment Boc-Tyr(tBu)-Aib-Glu(OtBu)-G1y-OH, a 5-6 dipeptide fragment Fmoc-Thr(tBu)-Phe-OH, a 7-8 dipeptide fragment Fmoc-Thr(tBu)-Ser(tBu)-OH, 10~11 dipeptide fragment Fmoc-Tyr(tBu)-Ser(tBu)-OH, 12~13 dipeptide fragment Fmoc-Ile-Aib-OH, 17~18 dipeptide fragment Fmoc-Ile-Ala-OH, 20 side chain peptide Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-Eicosanedioic acid(mon-tBu))-OH, 28~30 tripeptide fragment Fmoc-Ala-Gly-G1y-OH, 32~34 tripeptide fragment Fmoc-Ser(tBu)-Ser(tBu)-Gly-OH and 36~38 tripeptide fragment Fmoc-Pro-Pro-Pro-OH; the coupling agent for coupling the amino acid and the small fragment peptide is selected from Oxyma and DIC, TPTU and TMP or COMU and DIEA; The order of coupling the amino acids and the small fragment peptide is as follows: .

[0008] Preferably, the coupling agents are: Oxyma and DIC for the amino acids with a coupling order of 1, 3, 5 and 7-13, TPTU and TMP for the small fragment peptides with a coupling order of 2, 4 and 6, and COMU and DIEA for the amino acids and small fragment peptides with a coupling order of 14-25.

[0009] Preferably, the preparation method of telpotide of the present invention comprises: (a) deprotecting the starting resin using a deprotecting agent; (b) adding the coupling agent to Fmoc-Ser(tBu)-OH to activate the amino acid; (c) adding the activated amino acid obtained in step (b) to the resin obtained in step (a) to perform a coupling reaction to obtain Fmoc-Ser(tBu)-resin; (d) repeating the steps of deprotection, amino acid activation and coupling reaction, and sequentially coupling the remaining amino acids and small fragment peptides to obtain telpotide resin; (e) adding the telpotide resin obtained in step (d) into a cleavage solution for cleavage, adding a precipitate solution into the cleavage solution for crystallization, filtering and drying to obtain a crude telpotide; (f) Purifying the crude telpotide obtained in step (e) by using a chromatography system, ultrafiltration, salt exchange and freeze drying to obtain pure telpotide.

[0010] Preferably, the starting resin is Rink Amide resin, Rink Amide-AM resin, Rink Amide-MBHA resin or Sieber resin, and the degree of substitution of the starting resin is 0.3-0.6 mmol / g.

[0011] Preferably, the deprotecting agent is a PIP / DMF solution, and the volume concentration of the PIP is 20% to 50%.

[0012] Preferably, the lysis solution is TFA / EDT / Tis / H2O / phenol lysis solution, and the TFA / EDT / Tis / H2O / phenol lysis solution is prepared by the following method: preparing a first solution with a volume ratio of TFA:EDT:Tis:H2O=90:5:2.5:2.5, and then adding phenol to the first solution, the mass of the phenol being 2% of the mass of the first solution.

[0013] Preferably, the precipitate is selected from MTBE and EA, MTBE and IPAc, or diethyl ether and EA, and the volume ratio of the two components in MTBE and EA, MTBE and IPAc, or diethyl ether and EA is 1:1.

[0014] Preferably, in the step (f), the crude telpotide obtained in the step (e) is purified twice using a chromatography system, the chromatography system using a reverse phase C18 chromatography column, and the purification comprises: (f-1): dissolving the crude telpotide obtained in step (e) in a 30% acetonitrile aqueous solution, filtering, and collecting the filtrate; (f-2): the filtrate is subjected to a first purification, wherein the mobile phase A is a phosphoric acid aqueous solution with a pH of 3, and the mobile phase B is acetonitrile, and more than 96% of the qualified fractions are collected; (f-3): The qualified fractions collected in step (f-2) are purified for the second time, wherein mobile phase A is sodium acetate solution with a pH of 8.5, and mobile phase B is acetonitrile, and fractions greater than 99.5% are collected.

[0015] Preferably, in the ultrafiltration salt exchange step of step (f), the pore size of the filter membrane is 1-2 nm and the pressure is set to 1.5 MPa.

[0016] Preferably, the solvent in the coupling reaction is at least one selected from DMF, DCM, DMAc and NMP.

[0017] The primary sequence of tepote in the present invention refers to the order of arrangement of amino acids in the polypeptide chain. The amino acid sequence of tepote in the present invention is specifically H-Tyr 1 -Aib 2 -G1u 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Ile 17 -Ala 18 -GIn 19 -Lys 20 (AEEA-AEEA-γ-Glu-Eicosanedioicacid)-Ala 21 -Phe 22 -Val 23 -GIn 24 -Trp 25 -Leu26 -Ile 27 -Ala 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 3 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -NH 2 The small peptide fragments of the present invention include dipeptide fragments, tripeptide fragments and tetrapeptide fragments, wherein the x~y N-peptide fragments represent small molecule peptides containing N amino acids numbered x, y and between x and y, for example, the 1~4 tetrapeptide fragments refer to tetrapeptide fragments containing Tyr numbered 1, Aib numbered 2, G1u numbered 3, Gly numbered 4 and a protecting group, and the small peptide fragments specifically include the 1~4 tetrapeptide fragments Boc-Tyr(tBu)-Aib-Glu(OtBu)-G1y-OH, the 5~6 dipeptide fragments Fmoc-Thr(tBu)-Phe -OH, 7~8 dipeptide fragment Fmoc-Thr(tBu)-Ser(tBu)-OH, 10~11 dipeptide fragment Fmoc-Tyr(tBu)-Ser(tBu)-OH, 12~13 dipeptide fragment Fmoc-Ile-Aib-OH, 17~18 dipeptide fragment Fmoc-Ile-Ala-OH, 20 side chain peptide Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-Eicosanedioic acid(mon-tBu))-OH, 28~30 tripeptide fragment Fmoc-Ala-Gly-G1y-OH, 32~34 tripeptide fragment Fmoc-Ser(tBu)-Ser(tBu)-Gly-OH and 36~38 tripeptide fragment Fmoc-Pro-Pro-Pro-OH.

[0018] Solid phase synthesis reaction is different from liquid phase synthesis reaction. Each step of solid phase synthesis reaction cannot be 100% coupled completely. The more times of coupling, the more impurities are generated, and the more the yield decreases. In the preparation method provided by the present invention, according to the amino acid sequence of telpotide, amino acids and small fragment peptides are coupled in sequence by solid phase synthesis method, and the number of coupling reactions in the solid phase synthesis method is greatly reduced by selecting specific small fragment peptides and specific coupling agents, and the efficiency of coupling reactions is improved, and the yield and purity of telpotide crude product are significantly improved. In addition, the preparation method of the present invention reduces the use of solvents, and in the precipitation process after the telpotide resin is cracked, there is no need for centrifugation, and the telpotide crude product can be obtained by simple filtration, which simplifies the production process, reduces production costs, and is more suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the HPLC spectrum of the crude telpotide of Example 4; Figure 2 is the HPLC spectrum of the crude telpotide of Example 5; Figure 3 is the HPLC spectrum of the crude telpotide of Example 6; Figure 4 is the HPLC spectrum of the pure telpotide of Example 7; Figure 5 is the mass spectrum of the pure telpotide of Example 7; Figure 6 is the HPLC spectrum of the pure telpotide of Example 8; Figure 7 is the HPLC spectrum of the pure telpotide of Example 9; Figure 8 This is the HPLC spectrum of the crude telpotide of Comparative Example 1. DETAILED DESCRIPTION

[0020] The technical scheme of the present invention is further described below in conjunction with the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention. The methods used in the present invention are conventional production methods unless otherwise specified; the raw materials used are conventional commercial products unless otherwise specified. In the following examples, the small fragment peptides of the present invention were purchased from Sichuan Tongsheng Biopharmaceutical Co., Ltd.

[0021] The English abbreviations and Chinese meanings of the reagents used in the present invention are shown in Table 1: Table 1 .

[0022] The present invention uses a certain number of small fragment peptides and a specific coupling agent, and compared with the use of individual amino acids for synthesis one by one, the coupling steps are significantly reduced, the use of raw materials such as solvents and the generation of impurities are reduced, the production process is simplified, the production cost is reduced, and the probability of serious β-folding and missing peptides is reduced, and the coupling efficiency is improved. Specifically, the preparation method of the present invention includes deprotecting the starting resin with a deprotecting agent; adding the coupling agent to Fmoc-Ser (tBu) -OH to activate the amino acid; adding the activated amino acid to the deprotected resin for coupling reaction to obtain Fmoc-Ser (tBu) -resin; repeating the steps of deprotection, amino acid activation and coupling reaction, sequentially coupling the remaining amino acids and small fragment peptides to obtain telpotide resin; adding the telpotide resin to a cleavage solution for cleavage, adding a precipitate to the cleaved solution for crystallization, filtering and drying to obtain a crude telpotide; purifying the crude telpotide using a chromatographic system, ultrafiltration, salt exchange and freeze drying to obtain a pure telpotide.

[0023] In a preferred embodiment of the present invention, the coupling agents used in each coupling step of the synthesis of telpotide are not exactly the same. The selection of coupling agents is related to the amino acid site. TPTU is suitable for the condensation of racemic fragments, and COMU has a good catalytic coupling effect and inhibits the occurrence of racemization. According to the difference between the carboxyl component and the amino component, the condensation reaction has difficulty and speed. For example, Val with a large steric hindrance and Pro with a secondary amine group are both amino acids that are difficult to condense. For this, enhanced conditions are required, such as the input of a high multiple excess of carboxyl components, a highly active condensing agent, and extending the reaction time or appropriately increasing the reaction temperature. In addition to the influence of the structure of the amino acids involved in the reaction, factors such as the length of the assembled peptide chain and the hydrophobicity of the peptide chain also have an important influence on the condensation reaction. The present invention improves the efficiency of the coupling reaction by selecting a specific coupling agent in each step of the coupling reaction, and can significantly improve the yield and purity of the crude telpotide. In a preferred embodiment, the solvent in the coupling reaction is at least one selected from DMF, DCM, DMAc, and NMP.

[0024] The amino acids, small fragment peptides, coupling agents and their amounts used in each synthesis step in the examples of the present invention are shown in the following Tables 2 and 3.

[0025] In a preferred embodiment of the present invention, the starting resin is Rink Amide resin, Rink Amide-AM resin, Rink Amide-MBHA resin or Sieber resin, and the degree of substitution of the starting resin is 0.3-0.6 mmol / g. In a preferred embodiment of the present invention, the deprotecting agent is PIP / DMF solution, wherein the volume concentration of PIP is 20%-50%.

[0026] In a preferred embodiment of the present invention, the lysate used in the present invention is a TFA / EDT / Tis / H2O / phenol lysate, and the lysate is prepared by the following method: a first solution having a volume ratio of TFA:EDT:Tis:H2O=90:5:2.5:2.5 is prepared, and then phenol is added to the first solution, and the mass of the phenol is 2% of the mass of the first solution. For example, 90 mL is prepared according to the volume ratio of TFA / EDT / Tis / H2O =90 / 5 / 2.5 / 2.5, and if the weighing is 100 grams, 2 grams of phenol are added. Phenol acts as an antioxidant to prevent Tyr and Trp from being oxidized.

[0027] In a preferred embodiment of the present invention, the precipitate is selected from MTBE and EA, MTBE and IPAc, or diethyl ether and EA, and the volume ratio of the two components in the MTBE and EA, MTBE and IPAc, and diethyl ether and EA is 1: 1. By using the above precipitate, the crude telportipin can be obtained directly by filtration without centrifugation, which simplifies the production steps and saves costs.

[0028] In a preferred embodiment of the present invention, the crude telportipeptide is purified twice using a chromatography system, the chromatography system using a reverse phase C18 chromatography column, and the purification comprises: First purification: dissolving the crude telpotide obtained in 30% acetonitrile aqueous solution, filtering, and collecting the filtrate; Second purification: the filtrate was subjected to the first purification, mobile phase A was a phosphoric acid aqueous solution with a pH of 3, mobile phase B was acetonitrile, and more than 96% of qualified fractions were collected; The collected qualified fractions were purified for the second time, with mobile phase A being sodium acetate solution with a pH of 8.5 and mobile phase B being acetonitrile, and fractions greater than 99.5% were collected.

[0029] In a preferred embodiment of the present invention, in the ultrafiltration salt exchange step, the filter membrane pore size used is 1-2 nm, and the pressure is set to 1.5 MPa.

[0030] By performing purification twice using a chromatography system and further purifying using ultrafiltration salt exchange, a high-purity pure product of telpotide can be obtained. Example 1: Preparation of Telbutide Resin

[0031] Step 1: Take 30g Rink Amide-AM resin (substitution degree 0.41mmol / g) and place it in a solid phase reactor, add DMF solution to swell for 30 minutes, filter, add 20% PIP / DMF (the volume ratio of PIP:DMF is 20%:80%) to deprotect for 10 minutes, filter, add 20% PIP / DMF to deprotect for 10 minutes again, filter, wash with DMF 6 times, and stir for 3 minutes each time.

[0032] Add 300 ml of DMF to another reaction bottle (hereinafter referred to as the "amino acid activation reaction bottle"), add 14.15 g of Fmoc-Ser(tBu)-OH and 6.3 g of Oxyma to the amino acid activation reaction bottle and stir to dissolve, slowly add 6.8 ml of DIC (Suzhou Haofan Biotechnology Co., Ltd.), stir for at least 5 minutes to activate the amino acid.

[0033] The activated amino acid solution was added to a solid phase reactor and reacted for 2 hours. A small amount of resin was taken for ninhydrin detection. The resin was colorless and was filtered and washed twice with DMF to obtain Fmoc-Ser(tBu)-amide resin.

[0034] Step 2: Add 20% PIP / DMF to the resin obtained in step 1 for deprotection for 10 minutes, filter, add 20% PIP / DMF again for deprotection for 10 minutes, filter, wash with DMF 6 times, and stir for 3 minutes each time. In the amino acid activation reaction bottle, add 300 ml of DCM / DMF = 1 / 1 mixed solvent, add 13.08g Fmoc-Pro-Pro-Pro-OH, add 7.31g TPTU (Suzhou Haofan Biotechnology Co., Ltd.) to dissolve and clarify, cool to below 0°C, drop 3.57 ml TMP (Annaiji (Shanghai) Pharmaceutical Chemistry Co., Ltd.), stir and activate below 0°C for at least 10 minutes, add to the solid phase reactor, and stir and react for 2 hours. Take a small amount of resin ninhydrin for detection, the resin is colorless, filter, and wash with DMF twice to obtain Fmoc-Pro-Pro-Pro-Ser(tBu)-amide resin.

[0035] The remaining amino acids and small fragment peptides were coupled in sequence, among which Fmoc-Ser(tBu)-Ser(tBu)-Gly-OH and Fmoc-Ala-Gly-G1y-OH were coupled using the amino acid coupling method of step 2, and the rest were coupled using the coupling method of step 1. The order of coupled amino acids and small fragment peptides, the coupling agents used and the amounts used are shown in Table 2 below: Table 2 .

[0036] Step 14: Amino Acid Activation: Add a mixed solution of DMAc / DMF / DCM = 1 / 1 / 1 into the reaction bottle, add 30.12g Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-Eicosanedioic acid (mon-tBu))-OH and 10.54g COMU (Suzhou Haofan Biotechnology Co., Ltd.) to dissolve, cool to below 0°C, drop 4.72ml DIEA (Annage (Shanghai) Pharmaceutical Chemistry Co., Ltd.) to activate for at least 5 minutes, add to the solid phase reactor for stirring reaction for 5 hours, and take samples to detect the end point of the reaction.

[0037] The above coupling operation was repeated to sequentially couple the remaining amino acids and small peptide fragments, wherein the coupling order of amino acids and small peptide fragments, coupling agents and their amounts are shown in Table 3. After all amino acids and small peptide fragments were coupled, methanol was added for shrinkage 3 times, 10 minutes each time, filtered, vacuum dried for 4 hours, and the target peptide resin was collected and weighed 130 g.

[0038] Table 3 . Example 2: Preparation of Telbutide Resin

[0039] Except for using Rink Amide-MBHA resin (substitution degree 0.47 mmol / g) as the starting resin, other steps were the same as Example 1 to obtain 138 g of the target peptide resin. Example 3: Preparation of Telpotide Resin

[0040] Except for using Sieber resin (substitution degree 0.45 mmol / g) as the starting resin, other steps were the same as in Example 1 to obtain 133 g of the target peptide resin. Example 4: Preparation of crude telpotide

[0041] The lysate was prepared in the following manner: a first solution with a volume ratio of TFA:EDT:Tis:H2O=90:5:2.5:2.5 was prepared, and then phenol was added to the first solution, the mass of the phenol being 2% of the mass of the first solution. The target peptide resin prepared in Example 1 was added to the lysate, the amount of the lysate was 10 times the weight of the target peptide resin prepared in Example 1, stirred for 3 hours, filtered, the filtrate was collected, cooled to below 0°C, and the filtrate was slowly added to a mixed system with a volume ratio of MTBE / EA=1 / 1 for precipitation, the amount of the mixed system was 60 times the weight of the target peptide resin prepared in Example 1, filtered, the filter cake was washed with MTBE, and the solid was dried in an oven for 4 hours to obtain 62g of crude telpotide, the crude yield was 105%, and the HPLC purity of the crude product was 74.00%, wherein the purity was the area ratio in the HPLC spectrum, and the yield was calculated as crude product mass / (resin substitution degree x resin feed amount x product molecular weight), and the product molecular weight was 4813.5. Chromatographic detection was performed using the following method: Liquid chromatograph: Waters 2695 Separations Module; Specifications: UV (ALLLANCE E2695 / ACQUITY Arc); Column: Kinetex 2.6μm C18 100Å 150x4.6mm; Column temperature: 30°C; Detection wavelength: 210nm; Injection volume: 10 μL; Flow rate: 0.7 mL / min; Mobile phase A: Weigh 18.4 g of ammonium dihydrogen phosphate, add 1600 g of ultrapure water to dissolve, adjust the pH to 3.6 with phosphoric acid, then add 150 g of acetonitrile and 200 g of ultrapure water, and mix well;

[0042] Mobile phase B: weigh 930 g acetonitrile, 310 g isopropanol, and 400 g ultrapure water and mix well;

[0043] . Example 5: Preparation of crude telportipeptide resin

[0044] The lysis solution was the same as that used in Example 4. The target peptide resin prepared in Example 2 was added to the lysis solution, and the amount of the lysis solution was 10 times the weight of the target peptide resin prepared in Example 2. The mixture was stirred for 3 hours, filtered, and the filtrate was collected. The temperature was lowered to below 0°C, and the filtrate was slowly added to a mixed system with a volume ratio of MTBE / IPAc=1 / 1 for precipitation. The amount of the mixed system was 60 times the weight of the target peptide resin prepared in Example 2. The mixture was filtered, the filter cake was washed with MTBE, and the solid was dried in an oven for 4 hours to obtain 69 g of a crude product of telportipeptide, with a crude product yield of 102% and a crude product HPLC purity of 75.74%. Example 6: Preparation of crude telportipeptide resin

[0045] The lysate was the same as that used in Example 4. The target peptide resin prepared in Example 3 was added to the above lysate, and the amount of the lysate was 10 times the weight of the target peptide resin prepared in Example 3. The mixture was stirred for 3 hours, filtered, and the filtrate was collected. The temperature was lowered to below 0°C, and the filtrate was slowly added to a mixed system with a volume ratio of ether / EA=1 / 1 for precipitation. The amount of the mixed system was 60 times the weight of the target peptide resin prepared in Example 3. The mixture was filtered, the filter cake was washed with ether, and the solid was dried in an oven for 4 hours to obtain 68 g of crude telportipeptide, with a crude yield of 105% and a crude HPLC purity of 73.95%. Example 7: Purification of crude tepote

[0046] The crude product of telportin obtained in Example 4 was dissolved in acetonitrile water with a volume concentration of 30% and filtered. Two injections were performed for purification using a liquid chromatography system. The high performance liquid chromatograph and its parameters used in the purification process are shown in Table 4 below: Table 4 .

[0047] For the first purification, mobile phase A was a phosphoric acid aqueous solution with a pH of 3, mobile phase B was acetonitrile, and the elution gradient was as shown in Table 5 below: Table 5 .

[0048] Qualified fractions with a concentration greater than 96% were collected and subjected to secondary purification.

[0049] The second purification mobile phase A was sodium acetate solution with a pH of 8.5, and the mobile phase B was acetonitrile. The elution gradient was shown in Table 6 below: Table 6 .

[0050] Collect the qualified fractions greater than 99.5%.

[0051] Afterwards, the qualified fractions were salt exchanged by a multifunctional membrane experimental device (Hangzhou Reina Membrane Engineering Co., Ltd., RNM-18G), where the pore size of the filter membrane was selected to be 1-2nm, the pressure was set to 1.5Mpa, and the concentrate was collected. The pure telpotide was obtained by freeze-drying. The total amount of pure telpotide was 38g, the yield was 64.2%, and the HPLC purity was 99.88%. The obtained pure telpotide was subjected to mass spectrometry and measured by liquid chromatography-mass spectrometry. The instruments and their parameters were as follows: Instrument name: Ultra-high performance liquid chromatography; Brand model: ACQUIIY UPLC H-CLASS PIUS; Supplier: Waters Technology (Shanghai) Co., Ltd. Column: 1.7μm PEPTIDE XB-C18 10C LC Column 150x2.1nm; Column temperature: 30°C; Injection volume: 10 μL; Flow rate: 0.4 mL / min; Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile; . Example 8 Purification of crude telpotide

[0052] The crude telpotide obtained in Example 5 was purified in the same manner as in Example 7 to obtain pure telpotide. The total amount of pure telpotide was 41 g, with a yield of 60.4% and an HPLC purity of 99.61%. Example 9 Purification of crude telpotide

[0053] The crude telpotide obtained in Example 6 was purified in the same manner as in Example 7 to obtain pure telpotide. The total amount of pure telpotide was 40 g, with a yield of 61.5% and an HPLC purity of 99.66%.

[0054] Comparative Example 1 Using 30 g of Rink Amide-AM resin (substitution degree 0.41 mmol / g) as the starting resin, single amino acids were sequentially coupled with the resin to prepare telpotide resin. Except for the amino acids and coupling agents used, the other steps were the same as in Example 1. The amino acids, coupling agents and their amounts used in the coupling process are shown in Table 7 below: Table 7

[0055] .

[0056] After drying, 126 g of the target peptide resin was obtained, which was cleaved in the same manner as in Example 4 to obtain 42 g of crude telpotide with a yield of 70.9% and an HPLC purity of 45.48%.

[0057] Comparative Example 2 Telpoitide was synthesized by a fragment method, wherein the fragments include fragment 1 (30-39AA), fragment 2 (14-29AA), and fragment 3 (1-13AA). The product peptide resin obtained by the reaction of fragment 2 and fragment 3 was cleaved and then continued to react with the cleavage product of fragment 1 by liquid phase synthesis to obtain an API with a protective group, and telpoitide was obtained after deprotection and precipitation. The preparation method of fragment 1 is to use 5.0g Sieber resin (substitution degree 0.81mmol / g) as the starting resin, sequentially couple amino acids to obtain a peptide resin, and the peptide resin is cleaved and dried to obtain 4.6g of fragment 1. The amino acids and coupling agents used and their amounts are shown in Table 8 below: Table 8 .

[0058] The preparation method of fragment 2 is to use 11.0g 2-CTC resin (substitution degree 0.74mmol / g) as the starting resin, sequentially couple amino acids to obtain peptide resin, and the peptide resin is cracked and dried to obtain 30.6g fragment 2. The amino acids, coupling agents and their amounts used are shown in Table 9 below: Table 9 .

[0059] The preparation method of fragment 3 is to use 12g 2-CTC resin (substitution degree 0.74mmol / g) as the starting resin, sequentially couple amino acids to obtain peptide resin, and the peptide resin is cracked and dried to obtain 4.1g fragment 3. The amino acids, coupling agents and their amounts used are shown in Table 10 below: Table 10 .

[0060] Solid phase synthesis of fragment 3 and fragment 2: The reaction effect is poor when Oxyma / DIC is selected as the coupling agent. COMU / DIC is used as the coupling agent. The reaction time is 4-6h, and fragment 2 requires 2 times the equivalent, which is more expensive.

[0061] It can be seen from the above embodiments and comparative examples that comparative example 1 uses a single amino acid as a material, has many coupling steps, takes a long time, and has low purity. However, when a large fragment peptide is used for coupling as in comparative example 2, there are problems such as poor reaction effect, long reaction time, high raw material consumption, and high cost. The preparation method of the present invention has fewer steps, simple operation, and improves the yield of the reaction. The yield of the crude product prepared is higher, and it is more suitable for industrial production.

[0062] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A solid phase preparation method of telpotide, wherein the amino acid sequence of telpotide is H-Tyr 1 -Aib 2 -G1u 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Tyr 10 -Ser 11 -Ile 12 -Aib 13 -Leu 14 -Asp 15 -Lys 16 -Ile 17 -Ala 18 -GIn 19 -Lys 20 (AEEA-AEEA-γ-Glu-Eicosanedioicacid)-Ala 21 -Phe 22 -Val 23 -GIn 24 -Trp 25 -Leu 26 -Ile 27 -Ala 28 -Gly 29 -Gly 30 -Pro 31 -Ser 32 -Ser 3 -Gly 34 -Ala 35 -Pro 36 -Pro 37 -Pro 38 -Ser 39 -NH 2 , characterized in that, The preparation method comprises: Using amino resin as the starting resin, sequentially coupling amino acids and small fragment peptides according to the amino acid sequence of the telpotide by solid phase synthesis to synthesize the telpotide resin, and the telpotide resin is cleaved and purified to obtain a pure telpotide; The small peptide fragments are 1-4 tetrapeptide fragments Boc-Tyr(tBu)-Aib-Glu(OtBu)-G1y-OH, 5-6 dipeptide fragments Fmoc-Thr(tBu)-Phe-OH, 7-8 dipeptide fragments Fmoc-Thr(tBu)-Ser(tBu)-OH, 10-11 dipeptide fragments Fmoc-Tyr(tBu)-Ser(tBu)-OH, 12-13 dipeptide fragments Fmoc-Ile-Aib-OH, 17-18 dipeptide fragments Fmoc-Ile-Ala-OH, 20 side chain peptides Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-Eicosanedioic acid(mon-tBu))-OH, 28~30 tripeptide fragment Fmoc-Ala-Gly-G1y-OH, 32~34 tripeptide fragment Fmoc-Ser(tBu)-Ser(tBu)-Gly-OH and 36~38 tripeptide fragment Fmoc-Pro-Pro-Pro-OH; The coupling agent for coupling the amino acid and the small fragment peptide is selected from Oxyma and DIC, TPTU and TMP or COMU and DIEA; The order of coupling the amino acids and the small fragment peptide is as follows: 。 2. The preparation method according to claim 1, characterized in that: The coupling agents are: Oxyma and DIC are used for the amino acids with a coupling order of 1, 3, 5 and 7-13, TPTU and TMP are used for the small fragment peptides with a coupling order of 2, 4 and 6, and COMU and DIEA are used for the amino acids and small fragment peptides with a coupling order of 14-25.

3. The preparation method according to claim 1, characterized in that: The preparation method comprises: (a) deprotecting the starting resin using a deprotecting agent; (b) adding the coupling agent to Fmoc-Ser(tBu)-OH to activate the amino acid; (c) adding the activated amino acid obtained in step (b) to the resin obtained in step (a) to perform a coupling reaction to obtain Fmoc-Ser(tBu)-resin; (d) repeating the steps of deprotection, amino acid activation and coupling reaction, and sequentially coupling the remaining amino acids and small fragment peptides to obtain telpotide resin; (e) adding the telpotide resin obtained in step (d) into a cleavage solution for cleavage, adding a precipitate solution into the cleavage solution for crystallization, filtering and drying to obtain a crude telpotide; (f) Purifying the crude telpotide obtained in step (e) by using a chromatography system, ultrafiltration, salt exchange and freeze drying to obtain pure telpotide.

4. The preparation method according to claim 1, characterized in that: The starting resin is Rink Amide resin, Rink Amide-AM resin, Rink Amide-MBHA resin or Sieber resin, and the degree of substitution of the starting resin is 0.3-0.6 mmol / g.

5. The preparation method according to claim 3, characterized in that: The deprotecting agent is a PIP / DMF solution, and the volume concentration of the PIP is 20% to 50%.

6. The preparation method according to claim 3, characterized in that: The lysis solution is TFA / EDT / Tis / H2O / phenol lysis solution, and the TFA / EDT / Tis / H2O / phenol lysis solution is prepared by the following method: preparing a first solution with a volume ratio of TFA:EDT:Tis:H2O=90:5:2.5:2.5, and then adding phenol to the first solution, wherein the mass of the phenol is 2% of the mass of the first solution.

7. The preparation method according to claim 3, characterized in that: The precipitate is selected from MTBE and EA, MTBE and IPAc, or diethyl ether and EA, and the volume ratio of the two components in the MTBE and EA, MTBE and IPAc, and diethyl ether and EA is 1:

1.

8. The preparation method according to claim 3, characterized in that: In the step (f), the crude telpotide obtained in the step (e) is purified twice using a chromatography system, wherein the chromatography system uses a reverse phase C18 chromatography column, and the purification comprises: (f-1): dissolving the crude telpotide obtained in step (e) in a 30% acetonitrile aqueous solution, filtering, and collecting the filtrate; (f-2): the filtrate is subjected to a first purification, wherein the mobile phase A is a phosphoric acid aqueous solution with a pH of 3, and the mobile phase B is acetonitrile, and more than 96% of the qualified fractions are collected; (f-3): The qualified fractions collected in step (f-2) are purified for the second time, wherein mobile phase A is sodium acetate solution with a pH of 8.5, and mobile phase B is acetonitrile, and fractions greater than 99.5% are collected.

9. The preparation method according to claim 3, characterized in that: In the ultrafiltration salt exchange step of step (f), the pore size of the filter membrane is 1-2 nm and the pressure is set to 1.5 MPa.

10. The preparation method according to claim 3, characterized in that: The solvent in the coupling reaction is at least one selected from DMF, DCM, DMAc, and NMP.

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