A process for the preparation of tirzepatide

Abstract

The application relates to the technical field of polypeptide drug synthesis, and discloses a preparation method of Tirzepatide, which comprises the following steps: preparing Tirzepatide peptide resin by a solid-phase polypeptide synthesis method, and obtaining Tirzepatide by cleaving the Tirzepatide peptide resin; wherein the method for introducing Pro is that a 2-6 peptide fragment containing Pro is used. The application can effectively reduce the generation of related inserted peptide or deleted peptide impurities, is beneficial to purification, has a higher yield, and is a Tirzepatide preparation method with wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of polypeptide drug synthesis technology, and in particular to a method for preparing Tirzepatide. Background Technology

[0002] Tirzepatide is a GIP-GLP1 dual receptor agonist that can improve pancreatic β-cell function and insulin sensitivity in patients with type 2 diabetes. It has a positive effect on glycemic control and weight loss, and tolerability improves with increasing dose. It can also improve biomarkers of non-alcoholic steatohepatitis (NASH) and gastrointestinal side effects. It is a blockbuster investigational product of Eli Lilly and Company, currently undergoing Phase III clinical trials, and is expected to be approved for market launch in 2022.

[0003] Tirzepatide has the following structure:

[0004] Tyr 1 -Aib-Glu-Gly-Thr 5 -Phe-Thr-Ser-Asp-Tyr 10 -Ser-Ile-Aib-Leu-Asp 15 -Lys-Ile-Ala-Gln-Lys 20 (AEEA-AEEA-γGlu-Eicosanedioic acid)-Ala-Phe-Val-Gln-Trp 25 -Leu-Ile-Ala-Gly-Gly 30 -Pro-Ser-Ser-Gly-Ala 35 -Pro-Pro-Pro-Ser-NH2.

[0005] Methods for preparing tirzepatide have been reported. Patent CN107207576A discloses a solid-phase preparation method for tirzepatide, which involves stepwise solid-phase synthesis of a 39-amino acid linear peptide, selective removal of the Lys side-chain protecting group Alloc, solid-phase coupling of side-chain modification groups, and cleavage to obtain the polypeptide product. This method suffers from numerous steps, a long cycle, many impurities, and difficult purification. Patent CN110903355A discloses another solid-phase preparation method for tirzepatide, also employing a stepwise coupling method. In this method, the insertion of amino acid 20 is selected from Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioicacid(mono-tBu))-OH, and the insertion of amino acids 1-4 is selected from the tetrapeptide fragment Boc-Tyr(tBu)-Aib-Glu(tBu-Gl). The γ-OH cleavage yields a polypeptide product. Although this method selects a partial fragment coupling method, the generation of related missing peptide impurities is still unavoidable. Patent WO2020159949 discloses a method for preparing Tirzepatide using a combination of solid and liquid phases. The method involves first synthesizing fragments of different lengths in the solid phase, then condensing the fragments in the liquid phase to obtain a fully protected peptide, and finally cleaving to obtain the polypeptide product. This method can solve some problems such as purification difficulties caused by the generation of related missing peptide impurities. However, the liquid phase reaction is not easy to control, the intermediates are not easy to purify, and it is not conducive to large-scale industrial production. Summary of the Invention

[0006] This invention addresses the problems of numerous impurities, difficult purification, and low yield in existing technologies by providing a novel method for synthesizing Tirzepatide. The method includes the following steps: preparing Tirzepatide peptide resin using a solid-phase polypeptide synthesis method, and cleaving the Tirzepatide peptide resin to obtain Tirzepatide; wherein the method for incorporating Pro is to use a 2-6 peptide fragment containing Pro.

[0007] Furthermore, during the synthesis of Tirzepatide using the above method, the 2-6 peptide fragments containing Pro are selected from: Pro-Ser-Ser-Gly-Ala-Pro, Ser-Ser-Gly-Ala-Pro-Pro, Ser-Gly-Ala-Pro-Pro-Pro, Ser-Ser-Gly-Ala-Pro, Ser-Gly-Ala-Pro-Pro, Gly-Ala-Pro-Pro-Pro, Ser-Gly-Ala-Pro, Gly-Ala-Pro-Pro, Ala-Pro-Pro-Pro, Gly-Ala-Pro, Ala-Pro-Pro-Pro, Pro-Pro-Pro, Pro-Pro, Ala-Pro.

[0008] Furthermore, during the synthesis of Tirzepatide using the above method, the 2-6 peptide fragments containing Pro are selected from: Gly-Gly-Pro-Ser-Ser-Gly, Ala-Gly-Gly-Pro-Ser-Ser, Ile-Ala-Gly-Gly-Pro-Ser, Leu-Ile-Ala-Gly-Gly-Pro, Pro-Ser-Ser-Gly-Ala, Gly-Pro-Ser-Ser-Gly, Gly-Gly-Pro-Ser-Ser, Ala-Gly-Gly-Pro-Ser, Ile-Ala-Gly-Gly-Pro, Pro-Ser-Ser-Gly, Gly-Pro-Ser-Ser, Gly-Gly-Pro-Ser, Ala-Gly-Gly-Pro, Pro-Ser-Ser-Gly, Gly-Pro-Ser, Gly-Gly-Pro-Ser, Gly-Pro, Pro 31 -Ser 32 .

[0009] In a preferred embodiment of the present invention, other dipeptide fragments or combinations thereof are used in the process of synthesizing Tirzepatide using the above method; wherein the dipeptide fragments are selected from Thr-Phe, Leu-Asp, Gly-Gly, and Ser-Ser.

[0010] In a preferred embodiment of the present invention, during the synthesis of Tirzepatide using the above method, the peptide fragment combination used for the incorporation of Pro is selected from: Pro 31 -Ser 32 and Pro-Pro; Gly-Pro-Ser and Pro-Pro; Gly-Gly-Pro-Ser and Pro-Pro; Gly-Gly-Pro-Ser-Ser and Pro-Pro; Pro 31 -Ser 32 And Pro-Pro-Pro; Gly-Gly-Pro-Ser and Pro-Pro-Pro; Gly-Gly-Pro-Ser and Ser-Gly-Ala-Pro-Pro-Pro.

[0011] In a preferred embodiment of the present invention, during the synthesis of Tirzepatide using the above method, a combination of the Pro-containing 2-6 peptide fragment and the dipeptide fragment is used, wherein the combination is selected from: Thr-Phe and Pro-Pro-Pro; Leu-Asp and Pro-Pro-Pro.

[0012] Furthermore, the prepared Tirzepatide was further purified and freeze-dried to obtain pure Tirzepatide.

[0013] In existing technologies for preparing tirzepatide, a stepwise coupling method is typically used to introduce protease (Pro). However, due to the structure and properties of Pro and the influence of the starting material synthesis process, stepwise coupling inevitably introduces related inserted and deleted peptide impurities. When multiple Pro are coupled consecutively, the content of these two impurities increases due to a cumulative effect. Since the properties of these two impurities are similar to the product, their increased content affects purification difficulty and yield. This invention uses Pro-containing 2-6 peptide fragments for solid-phase coupling, which can reduce the generation of Pro-related inserted or deleted peptide impurities, thus reducing purification difficulty and increasing yield. Furthermore, using Gly-Gly or Ser-Ser fragments can further reduce the generation of related inserted or deleted peptide impurities. The tirzepatide synthesis method provided by this invention can effectively reduce the generation of inserted or deleted peptide impurities, facilitating purification and improving yield. Detailed Implementation

[0014] The present invention will be further described in detail below through embodiments, which are intended to illustrate the invention and not limit it. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications also fall within the protection scope of the present invention.

[0015] The Chinese names corresponding to the English abbreviations involved in this invention are shown in Table 1:

[0016] Table 1 shows the Chinese names corresponding to the English abbreviations involved in this invention.

[0017]

[0018]

[0019] Unless otherwise specified, the explanations of relevant terms used in this invention shall adopt the conventional interpretations in the prior art.

[0020] Example 1: Preparation of Fmoc-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-OH

[0021] (1) Preparation of Fmoc-Pro-2-CTC resin

[0022] 1363.6 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 1.5 mol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Pro-OH (1012.11 g, 3.0 mol), DIEA (775.44 g, 6.0 mol), and 6 L of DMF were added, and the reaction was carried out for 6 h. The mixture was dried under vacuum, and methanol (96.12 g, 3.0 mol), DIEA (193.86 g, 1.5 mol), and 6 L of DMF were added. The reaction was carried out for 1 h, dried under vacuum, and washed three times each with DMF and DCM. The Fmoc-Pro-2-CTC resin was removed, dried, and its substitution degree was measured to be 0.66 mmol / g, with a mass of 1138.33 g.

[0023] (2) Preparation of Fmoc-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-OH

[0024] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1), add it to the solid-phase reactor, wash with DMF for swelling, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and the resin is positive by K test. Weigh 24.91 g (80 mmol) of Fmoc-Ala-OH and 12.97 g (96 mmol) of HOBt, dissolve them in 150 mL of DMF, add DIC (15.15 g, 120 mmol) under ice bath, and activate for 3 min. Add the activated solution to the solid-phase reactor, react for 2 h, the resin is negative by K test, dry under vacuum, and wash with DMF 4 times. The same steps were followed to continue coupling Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Pro-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 29.54 g of Fmoc-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-OH.

[0025] Example 2: Preparation of Fmoc-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-OH

[0026] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1) of Example 1, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and the resin is positive by K test. Weigh 26.99 g (80 mmol) of Fmoc-Pro-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash 4 times with DMF. The same steps were followed to continue coupling Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Ser(tBu)-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 30.22 g of Fmoc-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-OH.

[0027] Example 3: Preparation of Fmoc-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-OH

[0028] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1) of Example 1, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and the resin is positive by K test. Weigh 26.99 g (80 mmol) of Fmoc-Pro-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash 4 times with DMF. The same steps were followed to continue coupling Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, and Fmoc-Ser(tBu)-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 28.93 g of Fmoc-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-OH.

[0029] Example 4: Preparation of Fmoc-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-OH

[0030] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1) of Example 1, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and the resin is positive by K test. Weigh 24.91 g (80 mmol) of Fmoc-Ala-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash 4 times with DMF. The same steps were followed to continue coupling Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Ser(tBu)-OH. After the reaction was complete, the resin was washed three times with DMF and DCM respectively, and dried to obtain Fmoc-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 27.91 g of Fmoc-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-OH.

[0031] Example 5: Preparation of Fmoc-Ser(tBu)-Gly-Ala-Pro-Pro-OH

[0032] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1) of Example 1, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and the resin is positive by K test. Weigh 26.99 g (80 mmol) of Fmoc-Pro-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash 4 times with DMF. The same steps were followed to continue coupling Fmoc-Ala-OH, Fmoc-Gly-OH, and Fmoc-Ser(tBu)-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Ser(tBu)-Gly-Ala-Pro-Pro-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 24.77 g of Fmoc-Ser(tBu)-Gly-Ala-Pro-Pro-OH.

[0033] Example 6: Preparation of Fmoc-Gly-Ala-Pro-Pro-Pro-OH

[0034] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1) of Example 1, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and the resin is positive by K test. Weigh 26.99 g (80 mmol) of Fmoc-Pro-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash 4 times with DMF. The same steps were followed to continue coupling Fmoc-Pro-OH, Fmoc-Ala-OH, and Fmoc-Gly-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Gly-Ala-Pro-Pro-Pro-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 24.15 g of Fmoc-Gly-Ala-Pro-Pro-Pro-OH.

[0035] Example 7: Preparation of Fmoc-Ser(tBu)-Gly-Ala-Pro-OH

[0036] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1) of Example 1, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and the resin is positive by K test. Weigh 24.91 g (80 mmol) of Fmoc-Ala-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash 4 times with DMF. The same steps were followed to continue coupling Fmoc-Gly-OH and Fmoc-Ser(tBu)-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Ser(tBu)-Gly-Ala-Pro-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 22.47 g of Fmoc-Ser(tBu)-Gly-Ala-Pro-OH.

[0037] Example 8: Preparation of Fmoc-Gly-Ala-Pro-Pro-OH

[0038] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1) of Example 1, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and the resin is positive by K test. Weigh 26.99 g (80 mmol) of Fmoc-Pro-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash 4 times with DMF. The same steps were followed to continue coupling Fmoc-Ala-OH and Fmoc-Gly-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Gly-Ala-Pro-Pro-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 19.95 g of Fmoc-Gly-Ala-Pro-Pro-OH.

[0039] Example 9: Preparation of Fmoc-Ala-Pro-Pro-Pro-OH

[0040] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1) of Example 1, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and the resin is positive by K test. Weigh 26.99 g (80 mmol) of Fmoc-Pro-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash 4 times with DMF. The same steps were followed to continue coupling Fmoc-Pro-OH and Fmoc-Ala-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Ala-Pro-Pro-Pro-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 20.98 g of Fmoc-Ala-Pro-Pro-Pro-OH.

[0041] Example 10: Preparation of Fmoc-Gly-Ala-Pro-OH

[0042] 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1) of Example 1 was weighed and added to a solid-phase reactor. The resin was swollen and washed with DMF, deprotected twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), washed 6 times with DMF, and tested positive for K. 24.91 g (80 mmol) of Fmoc-Ala-OH and 12.97 g (96 mmol) of HOBt were weighed and dissolved in 150 mL of DMF. DIC (15.15 g, 120 mmol) was added under ice bath conditions and activated for 3 min. The activated solution was added to the solid-phase reactor and reacted for 2 h. The resin tested negative for K. The solution was dried and washed 4 times with DMF. The same steps were followed to continue coupling Fmoc-Gly-OH. After the reaction was complete, the resin was washed 3 times with DMF and 3 times with DCM, and dried to obtain Fmoc-Gly-Ala-Pro-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness, and then dried under vacuum to obtain 16.44 g of Fmoc-Gly-Ala-Pro-OH.

[0043] Example 11: Preparation of Fmoc-Ala-Pro-Pro-OH

[0044] 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1) of Example 1 was weighed and added to a solid-phase reactor. The resin was swollen and washed with DMF, deprotected twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), washed 6 times with DMF, and tested positive for K. 26.99 g (80 mmol) of Fmoc-Pro-OH and 12.97 g (96 mmol) of HOBt were weighed and dissolved in 150 mL of DMF. DIC (15.15 g, 120 mmol) was added under ice bath conditions and activated for 3 min. The activated solution was added to the solid-phase reactor and reacted for 2 h. The resin tested negative for K. The solution was dried and washed 4 times with DMF. The same steps were followed to continue coupling Fmoc-Ala-OH. After the reaction was complete, the resin was washed 3 times with DMF and 3 times with DCM, and dried to obtain Fmoc-Ala-Pro-Pro-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness, and then dried under vacuum to obtain 17.89 g of Fmoc-Ala-Pro-Pro-OH.

[0045] Example 12: Preparation of Fmoc-Pro-Pro-Pro-OH

[0046] 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1) of Example 1 was weighed and added to a solid-phase reactor. The resin was swollen and washed with DMF, deprotected twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), washed 6 times with DMF, and tested positive for K. 26.99 g (80 mmol) of Fmoc-Pro-OH and 12.97 g (96 mmol) of HOBt were weighed and dissolved in 150 mL of DMF. DIC (15.15 g, 120 mmol) was added under ice bath conditions and activated for 3 min. The activated solution was added to the solid-phase reactor and reacted for 2 h. The resin tested negative for K. The solution was dried and washed 4 times with DMF. The same steps were repeated to couple Fmoc-Pro-OH. After the reaction was complete, the resin was washed 3 times with DMF and 3 times with DCM, and dried to obtain Fmoc-Pro-Pro-Pro-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness, and then dried under vacuum to obtain 18.33 g of Fmoc-Pro-Pro-Pro-OH.

[0047] Example 13: Preparation of Fmoc-Pro-Pro-OH

[0048] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1) of Example 1, add it to a solid-phase reactor, wash with DMF for swelling, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and K test shows the resin is positive. Weigh Fmoc-Pro-OH (26.99 g, 80 mmol) and HOBt (12.97 g, 96 mmol) and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. K test shows the resin is negative. Dry the solution, wash with DMF and DCM 3 times each, and dry to obtain Fmoc-Pro-Pro-2-CTC resin. Add the resin to 1 L of 20% TFE / DCM (v / v) and react for 2 h. Filter, evaporate the filtrate to dryness, and vacuum dry to obtain 15.15g of Fmoc-Pro-Pro-OH.

[0049] Example 14: Preparation of Fmoc-Ala-Pro-OH

[0050] Weigh 60.61 g (0.66 mmol / g, 40 mmol) of Fmoc-Pro-2-CTC resin obtained in step (1) of Example 1, add it to a solid-phase reactor, wash with DMF for swelling, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and K test shows the resin is positive. Weigh 24.91 g (80 mmol) of Fmoc-Ala-OH and 12.97 g (96 mmol) of HOBt, dissolve them in 150 mL of DMF, add DIC (15.15 g, 120 mmol) under ice bath, and activate for 3 min. Add the activated solution to the solid-phase reactor, react for 2 h, K test shows the resin is negative, dry, wash 3 times with DMF and DCM respectively, and dry to obtain Fmoc-Ala-Pro-2-CTC resin. Add the resin to 1 L of 20% TFE / DCM (v / v) and react for 2 h. Filter, evaporate the filtrate to dryness, and vacuum dry to obtain 14.66 g of Fmoc-Ala-Pro-OH.

[0051] Example 15: Preparation of Fmoc-Pro-Ser(tBu)-OH

[0052] (1) Preparation of Fmoc-Ser(tBu)-2-CTC resin

[0053] 363.6 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 0.4 mol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (306.75 g, 0.8 mol), DIEA (1206.78 g, 1.6 mol), and 1.8 L of DMF were added, and the reaction was carried out for 6 h. The mixture was dried under vacuum, and methanol (9.61 g, 0.3 mol), DIEA (51.7 g, 0.4 mol), and 1.8 L of DMF were added. The reaction was carried out for 1 h, dried under vacuum, and washed three times each with DMF and DCM. The Fmoc-Ser(tBu)-2-CTC resin was removed, dried, and its substitution degree was measured to be 0.65 mmol / g, with a mass of 310.71 g.

[0054] (2) Preparation of Fmoc-Pro-Ser(tBu)-OH

[0055] Weigh 61.54 g (0.65 mmol / g, 40 mmol) of the Fmoc-Ser(tBu)-2-CTC resin obtained in step (1), add it to a solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and the resin is positive by K test. Weigh 26.99 g (80 mmol) of Fmoc-Pro-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash with DMF 4 times. Wash with DCM 3 times and dry to obtain Fmoc-Pro-Ser(tBu)-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness, and then dried under vacuum to obtain 16.79 g of Fmoc-Pro-Ser(tBu)-OH.

[0056] Example 16: Preparation of Fmoc-Gly-Pro-Ser(tBu)-OH

[0057] Weigh 61.54 g (0.65 mmol / g, 40 mmol) of Fmoc-Ser(tBu)-2-CTC resin obtained in step (1) of Example 15, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and the resin is positive by K test. Weigh 26.99 g (80 mmol) of Fmoc-Pro-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash 4 times with DMF. The same steps were followed to continue coupling Fmoc-Gly-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Gly-Pro-Ser(tBu)-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 20.07 g of Fmoc-Gly-Pro-Ser(tBu)-OH.

[0058] Example 17: Preparation of Fmoc-Gly-Gly-OH

[0059] (1) Preparation of Fmoc-Gly-2-CTC resin

[0060] 136.36 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 150 mmol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and washed three times. Fmoc-Gly-OH (89.19 g, 300 mmol), DIEA (77.55 g, 600 mmol), and 540 mL of DMF were added, and the reaction was carried out for 6 h. The mixture was dried under vacuum, and methanol (48.06 g, 1500 mmol), DIEA (12.92 g, 100 mmol), and 540 mL of DMF were added. The reaction was carried out for 1 h, dried under vacuum, and washed three times each with DMF and DCM. The Fmoc-Gly-2-CTC resin was then removed, dried, and its substitution degree was measured to be 0.67 mmol / g, with a mass of 113.69 g.

[0061] (2) Preparation of Fmoc-Gly-Gly-OH

[0062] Weigh 89.55 g (0.67 mmol / g, 60 mmol) of the Fmoc-Gly-2-CTC resin obtained in step (1), add it to the solid-phase reactor, wash with DMF for swelling, deprotect twice with 208% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and the resin is positive by K test. Weigh 51.06 g (120 mmol) of Fmoc-Gly-OH and 19.46 g (144 mmol) of HOBt and dissolve them in 250 mL of DMF. Add DIC (22.73 g, 180 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution, wash with DMF and DCM 3 times each, and dry to obtain Fmoc-Gly-Gly-2-CTC resin. The resin was added to 1000 mL of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness, and then dried under vacuum to obtain 18.96 g of Fmoc-Gly-Gly-OH.

[0063] Example 18: Preparation of Fmoc-Ser(tBu)-Ser(tBu)-OH

[0064] Weigh 50.72 g (0.69 mmol / g, 35 mmol) of Fmoc-Ser(tBu)-2-CTC resin obtained in step (1) of Example 15, add it to a solid-phase reactor, swell and wash with DMF, deprotect twice with 208% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and K test shows resin positive. Weigh 26.84 g (70 mmol) of Fmoc-Ser(tBu)-OH and 11.35 g (84 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (13.25 g, 105 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. K test shows resin negative. Dry the solution, wash 3 times with DMF and DCM respectively, and dry to obtain Fmoc-Ser(tBu)-Ser(tBu)-2-CTC resin. The resin was added to 1000 mL of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness, and then dried under vacuum to obtain 17.39 g of Fmoc-Ser(tBu)-Ser(tBu)-OH.

[0065] Example 19: Preparation of Fmoc-Gly-Gly-Pro-Ser(tBu)-OH

[0066] Weigh 61.54 g (0.65 mmol / g, 40 mmol) of Fmoc-Ser(tBu)-2-CTC resin obtained in step (1) of Example 15, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and the resin is positive by K test. Weigh 26.99 g (80 mmol) of Fmoc-Pro-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash 4 times with DMF. The same steps were followed to continue coupling Fmoc-Gly-OH and Fmoc-Gly-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Gly-Gly-Pro-Ser(tBu)-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 21.72 g of Fmoc-Gly-Gly-Pro-Ser(tBu)-OH.

[0067] Example 20: Preparation of Fmoc-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-OH

[0068] Weigh 61.54 g (0.65 mmol / g, 40 mmol) of Fmoc-Ser(tBu)-2-CTC resin obtained in step (1) of Example 15, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and the resin is positive by K test. Weigh 30.68 g (80 mmol) of Fmoc-Ser(tBu)-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash 4 times with DMF. The same steps were followed to continue coupling Fmoc-Pro-OH, Fmoc-Gly-OH, and Fmoc-Gly-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 26.58 g of Fmoc-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-OH.

[0069] Example 21: Preparation of Fmoc-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-OH

[0070] Weigh 61.54 g (0.65 mmol / g, 40 mmol) of Fmoc-Ser(tBu)-2-CTC resin obtained in step (1) of Example 15, add it to the solid-phase reactor, swell and wash with DMF, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash 6 times with DMF, and the resin is positive by K test. Weigh 32.68 g (80 mmol) of Fmoc-Ser(tBu)-OH and 12.97 g (96 mmol) of HOBt and dissolve them in 150 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution and wash 4 times with DMF. The same steps were followed to continue coupling Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, and Fmoc-Ala-OH. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Fmoc-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-2-CTC resin. The resin was added to 1 L of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 29.85 g of Fmoc-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-OH.

[0071] Example 22: Preparation of Fmoc-Thr(tBu)-Phe-OH

[0072] (1) Preparation of Fmoc-Phe-2-CTC resin

[0073] 136.36 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 150 mmol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and washed three times. Fmoc-Phe-OH (116.23 g, 300 mmol), DIEA (77.54 g, 600 mmol), and 750 mL of DMF were added, and the reaction was carried out for 6 h. The mixture was dried under vacuum, and methanol (48.06 g, 1500 mmol), DIEA (12.92 g, 100 mmol), and 750 mL of DMF were added. The reaction was carried out for 1 h, dried under vacuum, and washed three times each with DMF and DCM. The Fmoc-Phe-2-CTC resin was then removed, dried, and its substitution degree was measured to be 0.68 mmol / g, with a mass of 114.75 g.

[0074] (2) Preparation of Fmoc-Thr(tBu)-Phe-OH

[0075] Weigh 88.24 g (0.68 mmol / g, 60 mmol) of the Fmoc-Phe-2-CTC resin obtained in step (1), add it to the solid-phase reactor, wash with DMF for swelling, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and the resin is positive by K test. Weigh Fmoc-Thr(tBu)-OH (47.71 g, 120 mmol) and HOBt (19.46 g, 144 mmol) and dissolve them in 250 mL of DMF. Add DIC (22.73 g, 180 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution, wash with DMF and DCM 3 times each, and dry to obtain Fmoc-Thr(tBu)-Phe-2-CTC resin. The resin was added to 1000 mL of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness, and then dried under vacuum to obtain 28.41 g of Fmoc-Thr(tBu)-Phe-OH.

[0076] Example 23: Preparation of Fmoc-Leu-Asp(OtBu)-OH

[0077] (1) Preparation of Fmoc-Asp(OtBu)-2-CTC resin

[0078] 409 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 0.45 mol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and washed three times. Fmoc-Asp(OtBu)-OH (370 g, 0.9 mol), DIEA (232 g, 1.2 mol), and 2.4 L of DMF were added, and the reaction was carried out for 6 h. The mixture was then dried, and methanol (144 g, 4.5 mol), DIEA (39 g, 0.3 mol), and 2.4 L of DMF were added. The reaction was carried out for 1 h, dried, and washed three times with DMF and DCM respectively. The Fmoc-Asp(OtBu)-2-CTC resin was then removed, dried, and its substitution degree was measured to be 0.65 mmol / g, with a mass of 351 g.

[0079] (2) Preparation of Fmoc-Leu-Asp(OtBu)-OH

[0080] Weigh 92.31 g (0.65 mmol / g, 60 mmol) of the Fmoc-Asp(OtBu)-2-CTC resin obtained in step (1), add it to a solid-phase reactor, wash with DMF for swelling, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), wash with DMF 6 times, and the resin is positive by K test. Weigh 42.41 g (120 mmol) of Fmoc-Leu-OH and 19.46 g (144 mmol) and dissolve them in 200 mL of DMF. Add DIC (15.15 g, 120 mmol) under ice bath and activate for 3 min. Add the activated solution to the solid-phase reactor and react for 2 h. The resin is negative by K test. Dry the solution, wash with DMF and DCM 3 times each, and dry to obtain Fmoc-Leu-Asp(OtBu)-2-CTC resin. The resin was added to 1000 mL of 20% TFE / DCM (v / v) and reacted for 2 h. The mixture was filtered, the filtrate was evaporated to dryness, and then dried under vacuum to obtain 27.97 g of Fmoc-Leu-Asp(OtBu)-OH.

[0081] Example 24: Preparation of Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH

[0082] (1) Preparation of Fmoc-AEEA-2-CTC resin

[0083] 136.36 g of 2-CTC resin (substitution degree 1.1 mmol / g, scale 150 mmol) was weighed and added to a solid-phase reactor. The resin was swollen with DMF and washed three times. Fmoc-AEEA-OH (115.62 g, 300 mmol), DIEA (77.55 g, 600 mmol), and 700 mL of DMF were added, and the reaction was carried out for 6 h. The mixture was dried under vacuum, and methanol (48.06 g, 1500 mmol), DIEA (15.50 g, 150 mmol), and 700 mL of DMF were added. The reaction was carried out for 1 h, dried under vacuum, and washed three times each with DMF and DCM. The Fmoc-AEEA-2-CTC resin was then removed, dried, and its substitution degree was measured to be 0.67 mmol / g, with a mass of 118.47 g.

[0084] (2) Preparation of AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)

[0085] Weigh 89.55 g (0.67 mmol / g, 60 mmol) of Fmoc-AEEA-2-CTC resin from step (1), add it to a solid-phase reactor, wash with DMF to swell, deprotect twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), and wash 6 times with DMF. Weigh 46.25 g (120 mmol) of Fmoc-AEEA-OH and 19.46 g (144 mmol) of HOBt, dissolve them in 360 mL of DMF, add DIC (22.73 g, 180 mmol) under ice bath, and activate for 3 min. Add the activated solution to the solid-phase reactor, react for 2 h, dry under vacuum, and wash 6 times with DMF. The same steps were followed to continue coupling Fmoc-γGlu(α-OtBu)-OH and eicosanedioic acid monotert-butyl ester. After the reaction was complete, the resin was washed three times each with DMF and DCM, and dried to obtain Eicosaned(mon-tBu)-γGlu(α-OtBu)-AEEA-AEEA-2-CTC resin. The resin was added to 1700 mL of 20% TFE / DCM (v / v) and reacted for 2 h. After filtration, the filtrate was evaporated to dryness and then dried under vacuum to obtain 48.72 g of AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu).

[0086] (3) Preparation of Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH

[0087] Weigh 22.74 g (28 mmol) of AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu) and 5.42 g (30 mmol) of PFP-OH obtained in step (2), add 100 mL of dichloromethane, cool to 0 °C, and add EDCI (8.05 g, 42 mmol) in six batches with stirring. Continue the reaction for 15 min, then raise the temperature to 25 °C and react for 2 h. Monitor the reaction of the starting materials by TLC and HPLC to ensure complete reaction. Stop the reaction, wash the reaction solution with 70 mL of water, 70 mL of saturated sodium bicarbonate solution, and 70 mL of saturated brine, respectively. Dry the organic phase with anhydrous sodium sulfate, filter, and evaporate to dryness to obtain an oily viscous substance. Weigh 14.73 g (40 mmol) of Fmoc-Lys-OH and dissolve it in 100 mL of 10% sodium carbonate aqueous solution. Add 140 mL of tetrahydrofuran and, with stirring, slowly add 200 mL of the above oily viscous substance to the tetrahydrofuran solution using a constant pressure dropping funnel at 5 °C. After the addition is complete, raise the temperature to 25 °C and continue the reaction for 3 h. Monitor the reaction of the starting material by TLC and HPLC to ensure complete reaction. Stop the reaction, adjust the pH to 3-4 with 1 M hydrochloric acid solution, evaporate the tetrahydrofuran to dryness, extract with DCM in aqueous phase, dry and concentrate the organic phase to obtain crude Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosaned(mon-tBu)]-OH. Purify to obtain 19.6 g of pure product, yield 59%, purity 99.2%.

[0088] Example 25: Preparation of crude Tirzepatide peptide 1

[0089] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0090] Repeat the above steps, sequentially coupling the remaining amino acids or amino acid fragments according to the peptide sequence, wherein the amino acid fragments are Fmoc-Pro-Pro-OH prepared in Example 13 and Fmoc-Pro prepared in Example 15. 31 -Ser 32(tBu)-OH and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH prepared in Example 24 were used to obtain 38.11 g of Tirzepatide peptide resin.

[0091] The obtained Tirzepatide peptide resin was added to 380 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 12.08 g of crude Tirzepatide peptide with a purity of 73.9%.

[0092] Example 26: Preparation of crude Tirzepatide peptide 2

[0093] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0094] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the peptide sequence, wherein the amino acid fragments are Fmoc-Pro-Pro-OH prepared in Example 13, Fmoc-Gly-Pro-Ser(tBu)-OH prepared in Example 16, and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH prepared in Example 24, to obtain 38.54g of Tirzepatide peptide resin.

[0095] The obtained Tirzepatide peptide resin was added to 385 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 12.13 g of crude Tirzepatide peptide with a purity of 74.1%.

[0096] Example 27: Preparation of crude Tirzepatide peptide 3

[0097] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0098] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the peptide sequence, wherein the amino acid fragments are Fmoc-Pro-Pro-OH prepared in Example 13, Fmoc-Gly-Gly-Pro-Ser(tBu)-OH prepared in Example 19, and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH prepared in Example 24, to obtain 39.05g of Tirzepatide peptide resin.

[0099] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether and dried. The resulting white solid was 12.22 g of crude Tirzepatide peptide with a purity of 74.5%.

[0100] Example 28: Preparation of crude Tirzepatide peptide 4

[0101] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0102] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the peptide sequence, wherein the amino acid fragments are Fmoc-Pro-Pro-OH prepared in Example 13, Fmoc-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-OH prepared in Example 20, and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH prepared in Example 24, to obtain 38.77g of Tirzepatide peptide resin.

[0103] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 12.12 g of crude Tirzepatide peptide with a purity of 74.2%.

[0104] Example 29: Preparation of crude Tirzepatide peptide 5

[0105] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0106] Repeat the above steps, sequentially coupling the remaining amino acids or amino acid fragments according to the peptide sequence, wherein the amino acid fragments are Fmoc-Pro-Pro-Pro-OH prepared in Example 12 and Fmoc-Pro prepared in Example 15. 31 -Ser 32 (tBu)-OH and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH prepared in Example 24 were used to obtain 38.43 g of Tirzepatide peptide resin.

[0107] The obtained Tirzepatide peptide resin was added to 385 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 12.1 g of crude Tirzepatide peptide with a purity of 73.9%.

[0108] Example 30: Preparation of crude Tirzepatide peptide 6

[0109] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0110] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the peptide sequence, wherein the amino acid fragments are Fmoc-Pro-Pro-Pro-OH prepared in Example 12, Fmoc-Gly-Gly-Pro-Ser(tBu)-OH prepared in Example 19, and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH prepared in Example 24, to obtain 38.15g of Tirzepatide peptide resin.

[0111] The obtained Tirzepatide peptide resin was added to 380 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 12.18 g of crude Tirzepatide peptide with a purity of 74.4%.

[0112] Example 31: Preparation of crude Tirzepatide peptide 7

[0113] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0114] Repeat the above steps, and sequentially couple the remaining amino acids or amino acid fragments according to the peptide sequence, wherein the amino acid fragments are Fmoc-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-OH prepared in Example 3, Fmoc-Gly-Gly-Pro-Ser(tBu)-OH prepared in Example 19, and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioicacid(mon-tBu)]-OH prepared in Example 24, to obtain 38.79g of Tirzepatide peptide resin.

[0115] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 12.01 g of crude Tirzepatide peptide with a purity of 73.3%.

[0116] Example 32: Preparation of crude Tirzepatide peptide 8

[0117] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0118] Repeat the above steps, and sequentially couple the remaining amino acids or amino acid fragments according to the peptide sequence, wherein the amino acid fragments are Fmoc-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-OH prepared in Example 4, Fmoc-Gly-Gly-OH prepared in Example 17, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH prepared in Example 24, Fmoc-Leu-Asp(OtBu)-OH prepared in Example 23, and Fmoc-Thr(tBu)-Phe-OH prepared in Example 22, to obtain 39.05g of Tirzepatide peptide resin.

[0119] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 12.15 g of crude Tirzepatide peptide with a purity of 74.3%.

[0120] Example 33: Preparation of crude Tirzepatide peptide 9

[0121] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0122] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the peptide sequence, wherein the amino acid fragments are Fmoc-Pro-Pro-Pro-OH prepared in Example 12, Fmoc-Ser(tBu)-Ser(tBu)-OH prepared in Example 18, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH prepared in Example 24, and Fmoc-Thr(tBu)-Phe-OH prepared in Example 22, to obtain 39.26g of Tirzepatide peptide resin.

[0123] The obtained Tirzepatide peptide resin was added to 395 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 12.03 g of crude Tirzepatide peptide with a purity of 73.6%.

[0124] Example 34: Preparation of crude Tirzepatide peptide 10

[0125] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0126] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the peptide sequence, wherein the amino acid fragments are Fmoc-Pro-Pro-Pro-OH prepared in Example 12, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH prepared in Example 24, and Fmoc-Leu-Asp(OtBu)-OH prepared in Example 23, to obtain 39.14g of Tirzepatide peptide resin.

[0127] The obtained Tirzepatide peptide resin was added to 390 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 12.09 g of crude Tirzepatide peptide with a purity of 73.5%.

[0128] Example 35: Preparation of crude Tirzepatide peptide 11

[0129] 12.5 g of Rink Amide AM resin (substitution degree 0.40 mmol / g, 5 mmol) was weighed and added to a solid-phase reactor, swollen with DMF and washed three times. Fmoc-Ser(tBu)-OH (3.83 g, 10 mmol) and HOBt (1.62 g, 12 mmol) were weighed and dissolved in 60 mL of DMF. DIC (1.89 g, 15 mmol) was added under ice bath conditions, and activation was performed for 3 min. The activated solution was added to the solid-phase reactor, and the reaction was carried out for 2 h. The ninhydrin test result was negative. The solution was dried under vacuum and washed three times with DMF. Deprotection was performed twice with 20% piperidine / DMF solution (v / v) (5 min + 15 min), followed by six washes with DMF. The ninhydrin test result was positive.

[0130] Repeat the above steps, and couple the remaining amino acids or amino acid fragments sequentially according to the main chain peptide sequence. The amino acid fragments are Fmoc-Gly-Ala-Pro-Pro-OH prepared in Example 8 and Fmoc-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-OH prepared in Example 21. The 20th amino acid residue and the protecting group are Lys(Alloc). After removing Alloc from Pd(PPh3)4 / DMF, couple it with AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu) prepared in step (2) of Example 24 to obtain 38.34g of Tirzepatide peptide resin.

[0131] The obtained Tirzepatide peptide resin was added to 391.4 mL of cryolysis reagent (92.5% TFA / 2.5% TIS / 2.5% H2O / 2.5% MPa (v / v)) and reacted for 2 h. The mixture was filtered, the filtrate was concentrated, and 2 L of isopropyl ether was added, resulting in a white precipitate. The precipitate was washed three times with isopropyl ether, and the resulting white solid was 11.97 g of crude Tirzepatide peptide with a purity of 72.5%.

[0132] Example 36: Preparation of Tirzepatide peptides 1

[0133] The crude Tirzepatide peptide obtained in Example 25 was dissolved in an aqueous acetonitrile solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The purified Tirzepatide was then freeze-dried to obtain 7.49 g of refined Tirzepatide peptide with an HPLC purity of 99.3% and a yield of 31.2%.

[0134] Example 37: Preparation of Tirzepatide peptides 2

[0135] The crude Tirzepatide peptide obtained in Example 26 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The purified Tirzepatide was then freeze-dried to obtain 7.83 g of refined Tirzepatide peptide with an HPLC purity of 99.4% and a yield of 32.6%.

[0136] Example 38: Preparation of Tirzepatide peptides 3

[0137] The crude Tirzepatide peptide obtained in Example 27 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 8.02 g of refined Tirzepatide peptide with an HPLC purity of 99.6% and a yield of 33.4%.

[0138] Example 39: Preparation of Tirzepatide peptides 4

[0139] The crude Tirzepatide peptide obtained in Example 28 was dissolved in an aqueous acetonitrile solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then linearly eluted by HPLC using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The purified Tirzepatide was then freeze-dried to obtain 7.88 g of refined Tirzepatide peptide with an HPLC purity of 99.4% and a yield of 32.8%.

[0140] Example 40: Preparation of Tirzepatide peptides 5

[0141] The crude Tirzepatide peptide obtained in Example 29 was dissolved in an aqueous acetonitrile solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The purified Tirzepatide was then freeze-dried to obtain 7.42 g of refined Tirzepatide peptide with an HPLC purity of 99.2% and a yield of 30.9%.

[0142] Example 41: Preparation of Tirzepatide peptides 6

[0143] The crude Tirzepatide peptide obtained in Example 30 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 8.04 g of refined Tirzepatide peptide with an HPLC purity of 99.5% and a yield of 33.5%.

[0144] Example 42: Preparation of Tirzepatide peptides 7

[0145] The crude Tirzepatide peptide obtained in Example 31 was dissolved in an aqueous acetonitrile solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 7.08 g of refined Tirzepatide peptide with an HPLC purity of 99.1% and a yield of 29.5%.

[0146] Example 43: Preparation of Tirzepatide peptides 8

[0147] The crude Tirzepatide peptide obtained in Example 32 was dissolved in an aqueous acetonitrile solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 8.09 g of refined Tirzepatide peptide with an HPLC purity of 99.4% and a yield of 33.7%.

[0148] Example 44: Preparation of Tirzepatide peptides 9

[0149] The crude Tirzepatide peptide obtained in Example 33 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The purified Tirzepatide was then freeze-dried to obtain 7.25 g of refined Tirzepatide peptide with an HPLC purity of 99.1% and a yield of 30.2%.

[0150] Example 45: Preparation of Tirzepatide peptides 10

[0151] The crude Tirzepatide peptide obtained in Example 34 was dissolved in an acetonitrile aqueous solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The solution was then freeze-dried to obtain 7.32 g of refined Tirzepatide peptide with an HPLC purity of 99.1% and a yield of 30.5%.

[0152] Example 46: Preparation of Tirzepatide peptides 11

[0153] The crude Tirzepatide peptide obtained in Example 35 was dissolved in an aqueous acetonitrile solution. HPLC gradient elution was performed on the crude Tirzepatide peptide solution using octadecyl-bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and some acetonitrile was removed by rotary evaporation to obtain a primary purified Tirzepatide solution. The primary purified Tirzepatide solution was then subjected to linear HPLC elution using octadecyl-bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase. The Tirzepatide fraction was collected, and acetonitrile and most of the water were removed by rotary evaporation. The purified Tirzepatide was then freeze-dried to obtain 6.70 g of refined Tirzepatide peptide with an HPLC purity of 99.1% and a yield of 27.9%.

Claims

1. A method for preparing Tirzepatide, characterized in that, Mainly includes: using Tirzepatide peptide resin was prepared by solid-phase peptide synthesis, and Tirzepatide was obtained by cleavage of the Tirzepatide peptide resin; the peptide fragments used were: Fmoc-Pro-Pro-OH, Fmoc-Pro 31 -Ser 32 (tBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH; or Fmoc-Pro-Pro-OH, Fmoc-Gly-Pro-Ser(tBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH; or Fmoc-Pro-Pro-OH, Fmoc-Gly-Gly-Pro-Ser(tBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH; or or Fmoc-Pro-Pro-Pro-OH, Fmoc-Pro31-Ser32(tBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH; or Fmoc-Pro-Pro-Pro-OH, Fmoc-Gly-Gly-Pro-Ser(tBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH; or OR or or Fmoc-Pro-Pro-Pro-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH, Fmoc-Leu-Asp(OtBu)-OH; Fmoc-Gly-Ala-Pro-Pro-OH and Fmoc-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-OH have Lys(Alloc) as the 20th amino acid residue and the protecting group. After de-Allocing Pd(PPh3)4 / DMF, it is coupled with AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu).

2. The method for preparing Tirzepatide according to claim 1, characterized in that, The peptide fragments used are: Fmoc-Pro-Pro-OH, Fmoc-Pro 31 -Ser 32 (tBu)-OH, Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH.

3. The method for preparing Tirzepatide according to claim 1, characterized in that, The peptide fragments used are: Fmoc-Pro-Pro-OH, Fmoc-Gly-Pro-Ser(tBu)-OH, and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH.

4. The method for preparing Tirzepatide according to claim 1, characterized in that, The peptide fragments used are: Fmoc-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-OH, Fmoc-Gly-Gly-Pro-Ser(tBu)-OH, and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH.

5. The method for preparing Tirzepatide according to claim 1, characterized in that, The peptide fragments used are: Fmoc-Pro-Pro-OH, Fmoc-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-OH, and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH.

6. The method for preparing Tirzepatide according to claim 1, characterized in that, The peptide fragments used are: Fmoc-Pro-Pro-Pro-OH, Fmoc-Pro31-Ser32(tBu)-OH, and Fmoc-Lys[AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu)]-OH.

7. The method for preparing Tirzepatide according to claim 1, characterized in that, The obtained Tirzepatide was further purified and freeze-dried to obtain pure Tirzepatide.

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