New method for synthesizing texipatide fragment

Abstract

The invention relates to a novel method for synthesizing a tetradecapeptide fragment Boc-Tyr (tBu)-Aib-Glu (OtBu)-Gly-Thr (tBu)-Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-Tyr (tBu)-Ser (tBu)-Ile-Aib-Leu-OH of texipatide. The invention further relates to a preparation method of the tetradecapeptide fragment. According to the method, homogeneous coupling of a carrier or a peptide carrier and amino acid is achieved, full-protection splitting decomposition of a peptide chain can be completed in a splitting decomposition solution, and the full-protection tetradecapeptide fragment Boc-Tyr (tBu)-Aib-Glu (OtBu)-Gly-Thr (tBu)-Phe-Thr (tBu)-Ser (tBu)-Asp (OtBu)-Tyr (tBu)-Ser (tBu)-Ile-Aib-Leu-OH of the texipatide is obtained. Compared with the existing production technology, the production process can be effectively simplified, the production cost is reduced, the production efficiency is improved, the environmental pollution is reduced, and the method is green and environment-friendly.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and more specifically, relates to a novel synthesis method of a tesipatide fragment Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-OH. Background Art

[0002] Tirzepatide is a dual agonist of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors. It can activate two incretin receptors, GIP and GLP-1, which are involved in blood sugar control through a dual mechanism of action. It reduces food intake, body weight and fat content by reducing calorie intake and regulating appetite, and only requires one injection per week. After being approved for weight loss, it has become the first treatment method for weight loss that activates two incretin hormone receptors at the same time.

[0003] Since 2022, Eli Lilly's tesiparatide has been on the market as Mounjaro for the treatment of type 2 diabetes. In adult patients with type 2 diabetes, three doses of tesiparatide (subcutaneous injection, once a week) showed superiority in lowering blood sugar (A1C) and body weight compared with Novo Nordisk's injectable semaglutide fragment (Semaglutide, 1 mg, subcutaneous injection, once a week). In experimental studies, tesiparatide showed excellent effects in controlling blood sugar and reducing weight, both of which are important indicators of the health status of patients with type 2 diabetes. Compared with semaglutide, tesiparatide has better effects on lowering blood sugar and weight. Importantly, many patients treated with tesiparatide achieved a significant reduction in A1C without experiencing hypoglycemia (less than 54 mg / dL).

[0004] The peptide sequence of tesiparatide is as follows:

[0005] H-Tyr39-Aib38-Glu37-Gly36-Thr35-Phe34-Thr33-Ser32-Asp31-Tyr30-Ser29-Ile28-Aib27-Leu26-Asp25-Lys24-Ile23-Ala22-Gln21-{diacid-C20-ga mma-Glu-(AEEA)2-Lys}20-Ala19-Phe18-Val17-Gln16-Trp15-Leu14-Ile13-Ala12-Gly11-Gly10-Pro9-Ser8-Ser7-Gly6-Ala5-Pro4-Pro3-Pro2-Ser1-NH2

[0006] In 2021, Eli Lilly adopted a solid phase synthesis-liquid phase synthesis combined strategy to achieve kilogram-level GMP production of Tirzepatide. In this strategy, the tirzepatide fourteen peptide fragment Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-OH was synthesized using the traditional solid phase synthesis method. The coupling reaction is carried out in a heterogeneous phase. In order to ensure the complete reaction, excess amino acids and condensation reagents are often required to be added to the reaction system. At the same time, a huge amount of organic solvents for washing are required. The yield of solid phase synthesis is low and the cost is very high.

[0007] In order to solve the above problems, the present invention utilizes a novel non-classical solid phase carrier to synthesize the tetradecapeptide fragment Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-OH, and develops a new method for synthesizing the tetradecapeptide fragment which is green, environmentally friendly, process-reliable and effective. Compared with the existing technology, this project has the advantages of high efficiency, simplicity, low cost, good industrial applicability, etc., and has obvious process feasibility and technological advancement. Summary of the invention

[0008] The present invention provides a novel non-classical solid phase synthesis carrier, whose structural formula is selected from:

[0009]

[0010] The synthesis method is as follows:

[0011] In the presence of a base, the substrate is reacted with a halogenated alkane to obtain a benzaldehyde intermediate containing a long-chain alkyl ether fragment. In the presence of a reducing agent, a reduction reaction is performed to obtain a benzyl alcohol intermediate containing a long-chain alkyl ether fragment. The reaction formula is as follows:

[0012]

[0013] On the other hand, the present invention also provides a method for synthesizing the tetradecapeptide fragment Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-OH, and the reaction formula is as follows:

[0014]

[0015] The synthesis process includes the following steps:

[0016] A. Amino acid coupling reaction: In a reaction medium, the non-classical solid phase synthesis carrier of claim 1 and the Fmoc-amino acid are subjected to coupling reaction under the action of a condensation reagent, and a detection method is used to determine the reaction endpoint;

[0017] B. Reaction of removing Fmoc protecting group: adding deprotection reagent to the reaction solution in the previous step to carry out deprotection reaction to obtain the corresponding peptide carrier;

[0018] C. Repeat steps (A) and (B) until all amino acids in the polypeptide substance are connected to obtain a polypeptide substance carrier;

[0019] D. Peptide carrier cleavage reaction: adding the polypeptide carrier obtained in step (C) to a cleavage solution for cleavage at a reaction temperature of 20 to 40° C. After the cleavage is completed, adding the cleavage solution to a poor solvent, and obtaining a crude target peptide after sedimentation, centrifugation, washing, and drying;

[0020] E. The crude product is separated and purified to obtain the pure product.

[0021] Step A, amino acid coupling reaction: In a reaction medium, a novel non-classical solid phase synthesis carrier and Fmoc-amino acid are coupled under the action of a condensation reagent, and the reaction endpoint is determined by HPLC, TLC or NT detection method.

[0022] The coupling reaction medium of step A is a combination of one or more of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-diethylformamide, N-methylpyrrolidone or dimethyl sulfoxide, preferably a solvent or combination that can dissolve the coupling reaction system.

[0023] The condensation reagent in the above step A is any combination of DIC, DCC, EDCI, HOOBT, HOBT, HOAT, DIPEA, NMM, DMAP, HBTU, HATU, TBTU or PyBOP, preferably a combination of HOBT / EDCI and catalyst DMAP.

[0024] The feed ratio of the coupling reaction in the above step A is carrier: Fmoc-amino acid: HOBT: EDCI: DMAP = 1:1:1:0.1 to 1:3:3:0.3, preferably carrier: Fmoc-amino acid: HOBT: EDCI: DMAP = 1:1.2:1.2:1.2:0.1.

[0025] The reaction temperature in the above step A is 20-50°C, preferably 30-35°C.

[0026] The deprotection reagent in the above step B is a tetrahydrofuran solution of piperidine or diethylamine, and the preferred reaction solvent is a tetrahydrofuran solution of piperidine with a concentration of 20%.

[0027] The reaction temperature in the above step B is 20-50°C, preferably 30-35°C.

[0028] The poor solvent for the peptide carrier in the above steps B and C is one or any combination of water, methanol, ethanol, n-hexane, n-heptane, ethyl acetate or acetonitrile, preferably a combination of water and acetonitrile.

[0029] The reaction temperature of the above step D is 20-40°C, preferably 30-35°C.

[0030] The poor solvent in step D is one or any combination of n-hexane, n-heptane, methyl tert-butyl ether or diethyl ether, preferably n-hexane or methyl tert-butyl ether.

[0031] The most significant feature of this method is that in a benign solvent for the carrier or peptide carrier, homogeneous coupling of the carrier or peptide carrier with amino acids can be carried out; in a poor solvent, the carrier or peptide carrier can be washed and purified, and the fully protected cleavage of the peptide chain can be completed in the lysis solution to obtain a fully protected tetradecapeptide fragment of tesiparatide.

[0032] Compared with the existing technology, this method is easy to operate and has large-scale production capacity. Under the same production scale, the reactor volume can be reduced by 30%-50%, the amount of raw amino acid and condensation agent can be reduced by 50%-60%, and the amount of organic solvent used for reaction and washing can be reduced by 80%-90%, ultimately achieving a 50%-70% reduction in production costs.

[0033] The abbreviations used in the specification and claims of the present invention have the following meanings:

[0034]

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the nuclear magnetic spectrum of the synthetic carrier HZ-NST03 used in the present invention.

[0037] Figure 2 This is the HPLC spectrum of the purified tetradecapeptide fragment of Tetradecapeptide in Example 12 of the present invention. DETAILED DESCRIPTION

[0038] In order to further understand the present invention, the synthesis method provided by the present invention is described in detail below in conjunction with examples. It should be understood that these examples are only for further describing the features of the present invention in detail, and are not intended to limit the scope of the present invention or the scope of the claims of the present invention.

[0039] Example 1: Using HZ-NST03 as a synthetic carrier, the intermediate H-Leu-NST03 of the tetradecapeptide fragment of tesiparatide was synthesized. Carrier structure:

[0040]

[0041] Into a 250mL three-necked flask, add 100mL THF, 5.00g (5.47mmol; MW: 913.60) HZ-NST03, 3.87g (10.94mmol) Fmoc-Leu-OH and 0.13g (1.09mmol) DMAP, stir to dissolve, add 2.10g (10.94mmol) EDCI dropwise, heat in an oil bath to T = 30-35°C, start the reaction, and monitor the reaction endpoint by TLC.

[0042] Removal of Fmoc protecting group and post-treatment: Add Fmoc-Leu-HZ-NST03 to a 20% piperidine THF protecting solution, stir to dissolve, heat in an oil bath to T = 30-35°C, start the reaction, and monitor the reaction endpoint by TLC detection. After the deprotection reaction is completed, the reaction solution is slurried with 1000 ml of cold water, filtered, and the filter cake is rinsed with water and acetonitrile in turn, and dried to obtain 5.56 g of crude peptide carrier H-Leu-HZ-NST03 as a white solid with a yield of 96%.

[0043] Example 2: Synthesis of Tetradecapeptide Fragment Intermediate H-Aib-Leu-HZ-NST03.

[0044] In a 250mL three-necked flask, add 60mL THF, 5.56g (5.42mmol) H-Leu-HZ-NST03 crude product, heat in an oil bath to T = 30-35°C, stir to dissolve. In a reaction bottle, add 2.12g (6.50mmol) Fmoc-Aib-OH, 0.88g (6.50mmol) HOBT and 0.08g (0.65mmol) DMAP, stir to dissolve. In a reaction bottle, add 1.24g (6.50mmol) EDCI, start the reaction, and monitor the reaction endpoint by TLC detection.

[0045] Removal of Fmoc protecting group and post-treatment: Add Fmoc-Aib-Leu-HZ-NST03 to a 20% piperidine THF protecting solution, stir to dissolve, heat in an oil bath to T = 30-35°C, start the reaction, and monitor the reaction endpoint by TLC detection. After the deprotection reaction is completed, the reaction solution is slurried with 600 ml of cold water, filtered, and the filter cake is rinsed with water and acetonitrile in turn, and dried to obtain 5.96 g of crude peptide carrier H-Aib-Leu-HZ-NST03 as a white solid with a yield of 96%.

[0046] Example 3: Synthesis of Tetradecapeptide Fragment Intermediate H-Ile-Aib-Leu-HZ-NST03.

[0047] In a 250 mL three-necked flask, add 100 mL THF, 5.96 g (5.36 mmol) H-Aib-Leu-HZ-NST03 crude product, heat in an oil bath to T = 30-35 ° C, stir to dissolve. In a reaction bottle, add 2.27 g (6.43 mmol) Fmoc-Ile-OH, 0.87 g (6.43 mmol) HOBT and 0.08 g (0.64 mmol) DMAP, stir to dissolve. In a reaction bottle, add 1.23 g (6.43 mmol) EDCI, start the reaction, and monitor the reaction endpoint by TLC.

[0048] Removal of Fmoc protecting group and post-treatment: Add Fmoc-Ile-Aib-Leu-HZ-NST03 to a 20% piperidine THF protecting solution, stir to dissolve, heat in an oil bath to T = 30-35°C, start the reaction, and monitor the reaction endpoint by TLC detection. After the deprotection reaction is completed, the reaction solution is slurried with 600 ml of cold water, filtered, and the filter cake is rinsed with water and acetonitrile in turn, and dried to obtain 6.55 g of crude peptide carrier H-Ile-Aib-Leu-HZ-NST03 as a white solid with a yield of 96%.

[0049] Example 4: Synthesis of the tetradecapeptide fragment intermediate H-Ser(tBu)-Ile-Aib-Leu-HZ-NST03 of tesiparatide.

[0050] In a 250mL three-necked flask, add 60mL THF, 6.55g (5.35mmol) H-Ile-Aib-Leu-HZ-NST03 crude product, heat in an oil bath to T = 30-35°C, stir to dissolve. In a reaction bottle, add 2.46g (6.42mmol) Fmoc-Ser (tBu) -OH, 0.87g (6.42mmol) HOBT and 0.078 (0.64mmol) DMAP, stir to dissolve. In a reaction bottle, add 1.23g (6.42mmol) EDCI, start the reaction, and monitor the reaction endpoint by TLC detection.

[0051] Removal of Fmoc protecting group and post-treatment: Add Fmoc-Ser(tBu)-Ile-Aib-Leu-HZ-NST03 to a 20% piperidine THF protecting solution, stir to dissolve, heat in an oil bath to T = 30-35°C, start the reaction, and monitor the reaction endpoint by TLC detection. After the deprotection reaction is completed, the reaction solution is slurried with 600 ml of cold water, filtered, and the filter cake is rinsed with water and acetonitrile in turn, and dried to obtain 7.26 g of crude peptide carrier H-Ser(tBu)-Ile-Aib-Leu-HZ-NST03 as a white solid with a yield of 96%.

[0052] Example 5: Synthesis of the tetradecapeptide fragment intermediate H-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03 of tesiparatide.

[0053] In a 250mL three-necked flask, add 70mL THF, 7.26g (5.31mmol) H-Ser (tBu) -Ile-Aib-Leu-HZ-NST03 crude product, heat in an oil bath to T = 30-35°C, stir to dissolve. In a reaction bottle, add 2.93g (6.37mmol) Fmoc-Tyr (tBu) -OH, 0.86g (6.37mmol) HOBT and 0.078 (0.64mmol) DMAP, stir to dissolve. In a reaction bottle, add 1.22g (6.37mmol) EDCI, start the reaction, and monitor the reaction endpoint by TLC detection.

[0054] Removal of Fmoc protecting group and post-treatment: Add Fmoc-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03 to a 20% piperidine THF protecting solution, stir to dissolve, heat in an oil bath to T = 30-35°C, start the reaction, and monitor the reaction endpoint by TLC detection. After the deprotection reaction is completed, the reaction solution is slurried with 700 ml of cold water, filtered, and the filter cake is rinsed with water and acetonitrile in turn, and dried to obtain 8.33 g of crude peptide carrier H-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03 as a white solid with a yield of 97%.

[0055] Example 6: Synthesis of the tetradecapeptide fragment intermediate H-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03.

[0056] In a 250mL three-necked flask, add 70mL THF, 8.33g (5.25mmol) H-Tyr (tBu) -Ser (tBu) -Ile-Aib-Leu-HZ-NST03 crude product, heat in an oil bath to T = 30-35°C, stir to dissolve. In a reaction bottle, add 2.59g (6.30mmol) Fmoc-Asp (OtBu) -OH, 0.85g (6.30mmol) HOBT and 0.077 (0.63mmol) DMAP, stir to dissolve. In a reaction bottle, add 1.21g (6.30mmol) EDCI, start the reaction, and monitor the reaction endpoint by TLC detection.

[0057] Removal of Fmoc protecting group and post-treatment: Add Fmoc-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03 to a 20% piperidine THF protecting solution, stir to dissolve, heat in an oil bath to T = 30-35°C, start the reaction, and monitor the reaction endpoint by TLC detection. After the deprotection reaction is completed, the reaction solution is slurried with 800 ml of cold water, filtered, and the filter cake is rinsed with water and acetonitrile in turn, and dried to obtain 9.16 g of crude peptide carrier H-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03, which is a white solid with a yield of 97%.

[0058] Example 7: Synthesis of the tetradecapeptide fragment intermediate H-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03.

[0059] In a 250mL three-necked flask, add 90mL THF, 9.16g (5.21mmol) H-Asp (OtBu) -Tyr (tBu) -Ser (tBu) -Ile-Aib-Leu-HZ-NST03 crude product, heat in an oil bath to T = 30-35°C, stir to dissolve. In a reaction bottle, add 2.40g (6.25mmol) Fmoc-Ser (tBu) -OH, 0.84g (6.25mmol) HOBT and 0.077 (0.63mmol) DMAP, stir to dissolve. In a reaction bottle, add 1.20g (6.25mmol) EDCI, start the reaction, and monitor the reaction endpoint by TLC detection.

[0060] Removal of Fmoc protecting group and post-treatment: Add Fmoc-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03 to a 20% piperidine THF protecting solution, stir to dissolve, heat in an oil bath to T = 30-35°C, start the reaction, and monitor the reaction endpoint by TLC detection. After the deprotection reaction is completed, the reaction solution is slurried with 900 ml of cold water, filtered, and the filter cake is rinsed with water and acetonitrile in turn, and dried to obtain 9.76 g of crude peptide carrier H-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03, which is a white solid with a yield of 96%.

[0061] Example 8: Synthesis of the tetradecapeptide fragment intermediate H-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03.

[0062] In a 250mL three-necked flask, add 90mL THF, 9.76g (5.13mmol) H-Ser (tBu) -Asp (OtBu) -Tyr (tBu) -Ser (tBu) -Ile-Aib-Leu-HZ-NST03 crude product, heat in an oil bath to T = 30-35°C, stir to dissolve. In a reaction bottle, add 2.45g (6.16mmol) Fmoc-Thr (tBu) -OH, 0.83g (6.16mmol) HOBT and 0.075 (0.62mmol) DMAP, stir to dissolve. In a reaction bottle, add 1.18g (6.16mmol) EDCI, start the reaction, and monitor the reaction endpoint by TLC detection.

[0063] Removal of Fmoc protecting group and post-treatment: Fmoc-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03 was added to a 20% piperidine THF protecting solution, stirred to dissolve, heated in an oil bath to T = 30-35°C, and the reaction was started. The reaction end point was monitored by TLC detection. After the deprotection reaction was completed, the reaction solution was slurried with 900 ml of cold water, filtered, and the filter cake was rinsed with water and acetonitrile in turn, and dried to obtain 10.36 g of crude peptide carrier H-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03, which was a white solid with a yield of 97%.

[0064] Example 9: Synthesis of the tetradecapeptide fragment intermediate H-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03.

[0065] To a 250mL three-necked flask, add 100mL THF, 10.36g (5.03mmol) H-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Tyr (tBu) -Ser (tBu) -Ile-Aib-Leu-HZ-NST03 crude product, heat in an oil bath to T = 30-35°C, stir to dissolve. To a reaction bottle, add 2.34g (6.04mmol) Fmoc-Phe-OH, 0.82g (6.04mmol) HOBT and 0.073 (0.60mmol) DMAP, stir to dissolve. To a reaction bottle, add 1.16g (6.04mmol) EDCI, start the reaction, and monitor the reaction endpoint by TLC detection.

[0066] Removal of Fmoc protecting group and post-treatment: Add Fmoc-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03 to a 20% piperidine THF protecting solution, stir to dissolve, heat in an oil bath to T = 30-35°C, start the reaction, and monitor the reaction endpoint by TLC detection. After the deprotection reaction is completed, the reaction solution is slurried with 1000ml of cold water, filtered, and the filter cake is rinsed with water and acetonitrile in turn, and dried to obtain 10.90g of crude peptide carrier H-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03, which is a white solid with a yield of 98%.

[0067] Example 10: Synthesis of the tetradecapeptide fragment intermediate H-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03.

[0068] In a 250mL three-necked flask, add 100mL THF, 10.90g (4.94mmol) H-Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Tyr (tBu) -Ser (tBu) -Ile-Aib-Leu-HZ-NST03 crude product, heat in an oil bath to T = 30-35°C, stir to dissolve. In a reaction bottle, add 2.36g (5.93mmol) Fmoc-Thr (tBu) -OH, 0.80g (5.93mmol) HOBT and 0.072 (0.59mmol) DMAP, stir to dissolve. In a reaction bottle, add 1.14g (6.04mmol) EDCI, start the reaction, and monitor the reaction endpoint by TLC detection.

[0069] Removal of Fmoc protecting group and post-treatment: Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03 was added to a 20% piperidine THF protecting solution, stirred and dissolved, and heated in an oil bath to T = 30-35°C to start the reaction, and the reaction endpoint was monitored by TLC detection. After the deprotection reaction was completed, the reaction solution was slurried with 1000 ml of cold water, filtered, and the filter cake was rinsed with water and acetonitrile in turn, and dried to obtain 11.34 g of crude peptide carrier H-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03, which was a white solid with a yield of 97%.

[0070] Example 11: Synthesis of the tetradecapeptide fragment of tesiparatide Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03.

[0071] To a 250 mL three-necked flask, add 100 mL THF, 11.34 g (4.80 mmol) H-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Tyr (tBu) -Ser (tBu) -Ile-Aib-Leu-HZ-NST03 crude product, heat to T = 30-35 ° C in an oil bath, stir and dissolve. To a reaction bottle, add 3.83 g (5.76 mmol) Boc-Tyr (tBu) -Aib-Glu (OtBu) -Gly-OH, 0.78 g (5.76 mmol) HOBT and 0.071 (0.58 mmol) DMAP, stir and dissolve. To a reaction bottle, add 1.10 g (5.76 mmol) EDCI, start the reaction, and monitor the reaction endpoint by TLC.

[0072] Post-reaction treatment: After the coupling reaction is completed, the reaction solution is poured into 1000 mL of cold water, and a large amount of white solid is precipitated. The filter cake is washed with water and acetonitrile in turn, and dried to obtain 13.91 g of a crude peptide carrier fragment Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03, which is a white solid with a yield of 96%.

[0073] Example 12: Cleavage reaction of the peptide carrier of tesiparatide Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03:

[0074] Prepare lysis solution formic acid-TFE-DCM (10:10:80), add 140 mL lysis solution and 13.91 g (4.62 mmol) of tesiparatide Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-HZ-NST03 peptide carrier into a 500 mL lysis bottle, heat in an oil bath to T = 30-35°C, and lyse for 2-3 h. After the cleavage, the lysate was added to 1400 mL of n-hexane, precipitated, centrifuged, washed and dried to obtain 8.31 g of a crude product of tesiparatide Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-OH, with a crude product synthesis yield of 85% and a crude product HPLC purity.

[0075] Purification of crude tetradecapeptide fragment of tesiparatide:

[0076] 8.31 g of crude tetradecapeptide fragment Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-OH was purified by reverse preparative chromatography and concentrated to obtain 7.50 g of tetradecapeptide fragment as a white powder; the yield was 90%.

[0077] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A novel non-classical solid phase synthesis carrier, characterized in that: The structural formula of the carrier is selected from:

2. A method for synthesizing a tetradecapeptide fragment of tesiparatide Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-OH, characterized in that: The synthesis process includes the following steps: A. Amino acid coupling reaction: In a reaction medium, the non-classical solid phase synthesis carrier of claim 1 and the Fmoc-amino acid are subjected to coupling reaction under the action of a condensation reagent, and a detection method is used to determine the reaction endpoint; B. Reaction of removing Fmoc protecting group: adding deprotection reagent to the reaction solution in the previous step to carry out deprotection reaction to obtain the corresponding peptide carrier; C. Repeat steps (A) and (B) until all amino acids in the polypeptide substance are connected to obtain a polypeptide substance carrier; D. Peptide carrier cleavage reaction: adding the polypeptide carrier obtained in step (C) to a cleavage solution for cleavage at a reaction temperature of 20 to 40° C. After the cleavage is completed, adding the cleavage solution to a poor solvent, and obtaining a crude target peptide after sedimentation, centrifugation, washing, and drying; E. The crude product is separated and purified to obtain the pure product.

3. The synthesis method according to claim 2, characterized in that: The coupling reaction medium in step A is selected from one or any combination of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-diethylformamide, N-methylpyrrolidone or dimethyl sulfoxide.

4. The synthesis method according to claim 2, characterized in that: The condensation reagent in step A is selected from any combination of DIC, DCC, EDCI, HOOBT, HOBT, HOAT, DIPEA, NMM, DMAP, HBTU, HATU, TBTU or PyBOP.

5. The synthesis method according to claim 2, characterized in that: The feed ratio of the coupling reaction in step A is carrier: Fmoc-amino acid: condensation reagent: catalyst = 1:1:1:0.1 to 1:3:3:0.

3.

6. The synthesis method according to claim 2, characterized in that: The detection method for determining the reaction endpoint of the amino acid coupling reaction in step A is selected from HPLC, TLC or NT detection method.

7. The synthesis method according to claim 2, characterized in that: The deprotection reagent in step B is selected from a tetrahydrofuran solution of piperidine or diethylamine.

8. The synthesis method according to claim 2, characterized in that: The poor solvent for the peptide carrier in steps B and C is one or any combination of water, methanol, ethanol, n-hexane, n-heptane, ethyl acetate or acetonitrile.

9. The synthesis method according to claim 2, characterized in that: The reaction temperature in steps A, B and C is 20-50°C.

10. The synthesis method according to claim 2, characterized in that: The poor solvent in step D is one or any combination of n-hexane, n-heptane, methyl tert-butyl ether or diethyl ether.

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