Synthesis method of tilpotide

Abstract

The invention provides a synthesis method of Tirzeptide. According to the method disclosed by the invention, the use of complex starting materials is avoided. According to the present invention, the used special materials comprise Fmoc-Lys (AEEA-AEEA-gamma-Glu-Eicosanoic acid)-OH, Fmoc-Ile-Aib-OH, and Boc-Tyr-Aib-OH, and the used special materials comprise the following raw materials, by using the simple materials, the condensation efficiency can be ensured, the fragment quality can be effectively controlled, and finally, the final product can be obtained at a relatively high content yield.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and specifically relates to a method for synthesizing Tirzepatide. Background Art

[0002] Tirzepatide is a GLP-1 and GIP dual-target agonist developed by Eli Lilly and Company. It was approved by the FDA on May 13, 2022 and launched in the United States.

[0003] Its structure (peptide sequence):

[0004] NH2-Tyr-Aib-Glu-Gly-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Asn-Lys(AEEA-AE EA-γ-Glu-eicosanedioic acid)-Ala-Phe-Val-Asn-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2

[0005] Among them, AEEA is a special amino acid: 2-(2-(2-aminoethoxy)ethoxy)acetic acid.

[0006] Specifically, it is shown in the following formula:

[0007]

[0008] The existing synthesis processes of Tirzepatide are generally divided into two categories:

[0009] The first category is the liquid-phase splicing method: In the patent with the application number CN113330024A by Eli Lilly and Company, the liquid-phase splicing method is used for the synthesis of Tirzepatide. The four fully protected fragments for liquid-phase splicing are fragment (1-14), fragment (15-21), fragment (22-29), and fragment (30-39). Fragment (22-29) and fragment (30-39) are processed by liquid-phase splicing and using a nanofiltration device to obtain fragment (22-39); then fragment (22-39) is subjected to liquid-phase condensation and nanofiltration treatment with fragment (15-21) to obtain fragment (15-39); finally, fragment (15-39) is liquid-phase condensed with fragment (1-14), and then deprotected to obtain the crude peptide. In this process, it is difficult to control the quality of the four fully protected fragments, especially for the identification of racemic impurities. In addition, the purification of intermediates in the liquid-phase reaction is not easy to carry out.

[0010] The second category is the Fmoc solid-phase synthesis method. There are relatively many reports on this type of synthesis method. There is the standard Fmoc solid-phase synthesis in sequence, and the side chain of Lys is deprotected and then the side chain is continued to be coupled and synthesized. There is also fragment condensation.

[0011] 1) Patent CN113330024A: The synthesis of tezepelumab was carried out using the standard Fmoc solid-phase synthesis strategy, in which the Lys side chain at the 20th position was protected by ivDde. Hydrazine hydrate was used to selectively remove Lys at the 20th position, and then (AEEA)2-γ-Glu-C 20 diacid was connected, and finally the crude peptide was obtained by cleavage and deprotection. The limitation of this method is that the coupling time is too long. The condensation time for conventional amino acids is generally 4-8 hours, and for special amino acids, the condensation time is even up to 18 hours.

[0012] 2) Patents CN112592387B and CN114736271A use pseudo-proline dipeptides containing serine or glycine protected by Dmb as special materials, and stepwise coupling can effectively inhibit the shrinkage problem during the synthesis of peptide resin. However, after the condensation of glycine containing Dmb, dipeptide defects at this site and the next site will occur.

[0013] 3) Patents CN115651075B and CN110903355A use the fragment (1-4) as an overall unit, aiming to solve the coupling difficulty problem of the first and second amino acids. However, the fragment (1-4) requires additional solid-phase or liquid-phase synthesis, and the cost is beneficial for large-scale production.

[0014] 4) Patents CN116120403A and CN115991742A use large fragments such as (1-16), (17-26), (27-39) or (1-14), (22-29) as splicing units. The disadvantage of this type of method is that the large fragments require additional synthesis steps, and the dosage of fragment peptides is large. In addition to the cost problem, there are also quality control problems with fully protected large fragments.

[0015] 5) Patents CN115181173A, CN115181174A and CN115160429A use 2-6 peptides or 5-7 peptides containing special sites, which can partially solve the defect problem. However, its disadvantages are also quite obvious. First, the dosage of fragment peptides is large, and second, there are cost and quality control problems of fragment peptides. Summary of the Invention

[0016] To solve the problems of low synthesis efficiency, low purity, difficult access to raw materials, and high purification difficulty of tirzepatide in the prior art, the present invention provides a method for synthesizing tirzepatide by a solid-phase stepwise coupling strategy. The method of the present invention avoids the use of complex starting materials. The special materials used are Fmoc-Lys(AEEA-AEEA-γ-Glu-eicosanedioicacid)-OH, Fmoc-Ile-Aib-OH, and Boc-Tyr-Aib-OH. The use of these simple materials can not only ensure the condensation efficiency but also effectively control the quality of its fragments, and finally the final product can be obtained with a high content yield.

[0017] The synthetic route of the present invention is as follows:

[0018]

[0019] Specifically, the present invention provides a solid-phase synthesis method of tirzepatide, and the solid-phase synthesis invention comprises the following steps:

[0020] S1) Using amide resin as a solid-phase carrier and the Fmoc solid-phase synthesis strategy, successively coupling Fmoc-protected amino acids or special materials to obtain a fully protected tirzepatide peptide resin;

[0021] S2) The synthesized fully protected tirzepatide peptide resin is subjected to cleavage, sedimentation, filtration, and drying to obtain tirzepatide;

[0022] The Fmoc-protected amino acids or special materials are coupled in sequence as follows: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosanedioic acid(mon-tBu))-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Boc-Tyr-Aib-OH.

[0023] Further, the method of the Fmoc solid-phase synthesis strategy in step S1) includes:

[0024] S11) Remove the Fmoc protecting group;

[0025] S12) Couple the Fmoc-protected amino acid or special material;

[0026] Among them, in S11), 20% piperidine / DMF is used as the Fmoc deprotection reagent; 20% piperidine / DMF is a mixed solution with a volume ratio of piperidine to DMF of 1:4.

[0027] The method of coupling the Fmoc-protected amino acid or special material in S12) is carried out under the action of an amino acid or special material coupling agent.

[0028] Further, the deprotection method in S11) is to add an Fmoc deprotection reagent for deprotection, with a deprotection time of 5 to 10 minutes. After washing with DMF, the Fmoc deprotection reagent is added again for deprotection for 5 to 10 minutes and washed with DMF.

[0029] Further, the coupling agent for coupling the protected amino acid is a combination of DIC and A or a combination of DIPEA, A, and B, where A is Oxyma, HOBt, or HOAt, and B is one of PyBOP, PyAOP, HATU, HBTU, TBTU; preferably a combination of DIC and HOBt.

[0030] Further, the molar amount of the coupling agent when coupling the protected amino acid is 1 - 1.5 times the molar amount of the amino acid.

[0031] Further, the coupling agent for coupling special materials Fmoc-Lys(AEEA - AEEA - γ - Glu(α - OtBu)-eicosanedioic acid(mon - tBu)) - OH, Fmoc - Ile - Aib - OH, and Boc - Tyr(tBu) - Aib - OH is a combination of DIC, HOBt, and DIPEA.

[0032] Further, the molar ratio of the special material, DIC, HOBt, and DIPEA when coupling the special material is 1:1 - 1.5:1 - 1.5:0.1 - 0.5, preferably 1:1.2:1.2:0.1.

[0033] Further, the amide resin in S1) is Rink amide resin, Rink amide AM resin, Rinkamide MBHA resin, Sieber resin. Among them, Rink amide MBHA resin and Sieber resin are preferred.

[0034] Further, the substitution degree of the amide resin in S1) is 0.2 - 0.8 mmol / g, and preferably 0.4 - 0.6 mmol / g.

[0035] Further, the reaction temperature in S1) is room temperature or 20 - 35°C.

[0036] Further, the cleavage in S2) uses a mixed solution of TFA, TIPS, EDT, and H2O. Preferably, the volume ratio of TFA, TIPS, EDT, and H2O is 91:3:3:3.

[0037] Further, in S2), the sedimentation method is to add an excessive amount of methyl tert-butyl ether to the pyrolysis reaction system for sedimentation. Preferably, the added methyl tert-butyl ether is 5-10 times the volume of the pyrolysis reaction system.

[0038]

[0039]

[0040] Beneficial effects

[0041] 1) The synthesis method of Tirzepatide of the present invention has simple synthesis steps and easily available starting materials. By stepwise coupling, the use of large peptide fragments is avoided, and the quality of the starting materials is easy to control.

[0042] 2) For special materials that are difficult to couple, small special fragments are used for synthesis, and at the same time, a small amount of DIPEA is added, which can significantly improve the coupling efficiency.

[0043] 3) The synthesis method of Tirzepatide of the present invention can finally synthesize Tirzepatide with a high content yield, while maintaining a high purity, with obvious cost advantages, which is conducive to large-scale synthesis. Description of the drawings

[0044] Figure 1 is the synthesis route of the present invention.

[0045] Figure 2 is the HPLC result of the crude peptide of tirzepatide synthesized in Example 1.

[0046] Figure 3 is the HPLC result of the crude peptide of tirzepatide synthesized in Comparative Example 1. Detailed implementation manners

[0047] In order to make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific implementation manners of the present invention is provided, but it should not be construed as a limitation on the scope of the present invention that can be implemented.

[0048] The following is combined with Figure 1 for illustration. The present invention provides a solid-phase synthesis method of tirzepatide, which includes the following steps:

[0049] S1) Using amide resin as the solid-phase carrier and Fmoc solid-phase synthesis strategy, successively coupling Fmoc-protected amino acids or special materials to obtain the fully protected tirzepatide peptide resin;

[0050] The Fmoc-protected amino acids or special materials are coupled in sequence as follows: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosanedioic acid(mon-tBu))-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Boc-Tyr-Aib-OH.

[0051] The method of the Fmoc solid-phase synthesis strategy in step S1) includes:

[0052] S11) Removal of the Fmoc protecting group: 20% piperidine / DMF is used as the Fmoc deprotection reagent; 20% piperidine / DMF is a mixed solution with a volume ratio of piperidine to DMF of 1:4; the deprotection method is to add the Fmoc deprotection reagent for deprotection, the deprotection time is 5 - 10 minutes, after washing with DMF, add the Fmoc deprotection reagent again for deprotection for 5 - 10 minutes and wash with DMF.

[0053] S12) Coupling of the Fmoc-protected amino acids or special materials; the coupling agent for the protected amino acids is a composition of DIC and HOBt. The dosages of DIC and HOBt are both 1.2 times the molar amount of the protected amino acid.

[0054] The coupling agents for coupling the special materials Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosanedioic acid(mon-tBu))-OH, Fmoc-Ile-Aib-OH, and Boc-Tyr(tBu)-Aib-OH are a combination of DIC, HOBt, and DIPEA. The molar ratio of the special materials, DIC, HOBt, and DIPEA during the coupling of the special materials is 1:1.2:1.2:0.1.

[0055] S2) The fully protected tirzepatide peptide resin synthesized is subjected to cleavage, sedimentation, filtration, and drying to obtain tirzepatide; the cleavage uses a mixed solution of TFA, TIPS, EDT, and H2O, and the volume ratio of TFA, TIPS, EDT, and H2O is 91:3:3:3. The sedimentation method in S2) is to add methyl tert-butyl ether with a volume 8 times that of the cleavage reaction system for sedimentation; the filtration method is filtration with a filter cloth.

[0056] Furthermore, it may further include the step of further purifying the synthesized tirzepatide.

[0057] Example 1

[0058] Select 23.3 g of Rink amide MBHA Resin amino acid resin with a substitution degree of 0.43 mmol / g, add it to the reaction column, swell it with DMF for 30 minutes, drain it, add 20% piperidine / DMF solution to remove Fmoc twice (10 minutes + 10 minutes). After removal, wash it with DMF 5 times. Weigh Fmoc-Ser(tBu)-OH (11.5 g, 30 mmol), HOBt (4.86 g, 36 mmol), add them to the reaction column, dissolve them with DMF, then add DIC (5.63 mL, 36 mmol), and react at room temperature for 1 hour. Ninhydrin detection shows that the reaction is complete, filter by suction, and wash it with an appropriate amount of DMF 5 times. Continue to repeat the above operations for the coupling of other amino acids and special materials. The reaction time for conventional amino acids is 1 h. Among them, when coupling the special materials Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosanedioic acid(mon-tBu))-OH, Fmoc-Ile-Aib-OH, and Boc-Tyr-Aib-OH, 0.2 eq. of DIPEA needs to be added to promote the reaction. The coupling times are 3 hours, 3 hours, and 2 hours respectively. After the coupling is completed, add methyl tert-butyl ether to shrink the resin, and finally obtain 91.5 g of peptide resin:

[0059] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosane dioic acid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink amide MBHA resin。

[0060] The specific coupling conditions are as follows:

[0061]

[0062]

[0063]

[0064] Cleavage step: The cleavage reagent used in this technology is TFA / TIDPS / EDT / H2O = 91 / 3 / 3 / 3, and the volume of the cleavage reagent is 8 times the weight of the resin. Cleavage is carried out at room temperature for 2 hours. The resin is removed by filtration. Methyl tert-butyl ether is added to the cleavage solution. The addition amount of methyl tert-butyl ether is 8 times (V / V) of the cleavage solution. Sedimentation, filtration, and drying of the filter cake are carried out, and finally 48.0 g of the crude peptide product is obtained, with a yield of 99.8%, a purity of 81.73%, and a content of 77.4%.( Figure 2 )

[0065] Comparative Example 1

[0066] Select 23.3 g of Rink amide MBHA Resin amino acid resin with a substitution degree of 0.43 mmol / g, add it to the reaction column, swell with DMF for 30 minutes, drain, add 20% piperidine / DMF solution to remove Fmoc twice (10 minutes + 10 minutes). After removal, wash with DMF 5 times. Weigh Fmoc-Ser(tBu)-OH (11.5 g, 30 mmol), HOBt (4.86 g, 36 mmol), add them to the reaction column, dissolve with DMF, then add DIC (5.63 mL, 36 mmol), react at room temperature for 1 hour. Ninhydrin test shows that the reaction is complete. Filter by suction and wash with an appropriate amount of DMF 5 times. Continue to repeat the above operations for other amino acid couplings. After the coupling is completed, add methyl tert-butyl ether to contract the resin, and finally obtain 72 g of peptide resin:

[0067] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(t Bu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosane dioic acid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink amide MBHA resin。

[0068] The specific coupling conditions are as follows:

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] Cleavage step: The cleavage reagent used in this technology is TFA / TIDPS / EDT / H2O = 91 / 3 / 3 / 3, and the volume of the cleavage reagent is 8 times the weight of the resin. Cleavage is carried out at room temperature for 2 hours. The resin is removed by filtration. Methyl tert-butyl ether is added to the cleavage solution. The addition amount of methyl tert-butyl ether is 8 times that of the cleavage solution (V / V). Sedimentation, filtration, and drying of the filter cake are carried out, and finally 29.0 g of the crude peptide product is obtained, with a yield of 60.3%, a purity of 47.68%, and a content of 32.0%.( Figure 3 )

[0076] By comparing Example 1 with Comparative Example 1, it can be seen that the present invention uses special material fragments to improve the synthesis efficiency. The peptide resin synthesis is shortened from 43 steps to 37 steps, the yield is increased from 60 to 99.8, and the purity is nearly doubled. The present invention achieves a significant improvement in synthesis efficiency and purity only by adjusting the synthesis sequence and using special materials.

Claims

1. A solid-phase synthesis method of tirzepatide, characterized in that, The solid-phase synthesis invention comprises the following steps: S1) Using amide resin as the solid-phase carrier and adopting the Fmoc solid-phase synthesis strategy, successively coupling Fmoc-protected amino acids or special materials to obtain the fully protected telotristat peptide resin; S2) The synthesized fully protected telotristat peptide resin is subjected to cleavage, sedimentation, filtration, and drying to obtain telotristat; The successive coupling of Fmoc-protected amino acids or special materials is as follows: successively coupling Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(AEEA-AEEA-γ-Glu(α-OtBu)-eicosanedioic acid(mon-tBu))-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Boc-Tyr-Aib-OH.

2. The solid-phase synthesis method according to claim 1, characterized in that, The method of the Fmoc solid-phase synthesis strategy in step S1) includes: S11) Removing the Fmoc protecting group; S12) Coupling Fmoc-protected amino acids or special materials; Among them, in S11), 20% piperidine / DMF is used as the Fmoc deprotection reagent; 20% piperidine / DMF is a mixed solution with a volume ratio of piperidine to DMF of 1:4; The method of coupling Fmoc-protected amino acids or special materials in S12) is to carry out the coupling under the action of an amino acid or special material coupling agent.

3. The solid-phase synthesis method according to claim 2, characterized in that, Furthermore, the deprotection method in S11) is to add the Fmoc deprotection reagent for deprotection, the deprotection time is 5 to 10 minutes, after washing with DMF, add the Fmoc deprotection reagent again for deprotection for 5 to 10 minutes and wash with DMF.

4. The solid-phase synthesis method according to claim 2, characterized in that, The coupling agents for coupling protected amino acids are a combination of DIC and A or a combination of DIPEA, A and B, where A is Oxyma, HOBt or HOAt, and B is one of PyBOP, PyAOP, HATU, HBTU, TBTU; preferably a composition of DIC and HOBt.

5. The solid-phase synthesis method according to claim 4, characterized in that, When coupling protected amino acids, the molar amount of the coupling agent is 1 - 1.5 times the molar amount of the amino acid.

6. The solid-phase synthesis method according to claim 2, characterized in that, The coupling agents for coupling special materials Fmoc-Lys(AEEA - AEEA - γ - Glu(α - OtBu)-eicosanedioic acid(mon - tBu))-OH, Fmoc-Ile-Aib-OH and Boc-Tyr(tBu)-Aib-OH are a combination of DIC, HOBt and DIPEA.

7. The solid-phase synthesis method according to claim 6, characterized in that, When coupling special materials, the molar ratio of the special material, DIC, HOBt and DIPEA is 1:1 - 1.5:1 - 1.5:0.1 - 0.

5. Preferably it is 1:1.2:1.2:0.

1.

8. The solid-phase synthesis method according to claim 1, characterized in that, In S1), the amide resin is Rink amide resin, Rink amide AM resin, Rink amide MBHA resin, Sieber resin. Among them, Rink amide MBHA resin and Sieber resin are preferred; Preferably, the substitution degree of the amide resin in S1) is 0.2 - 0.8 mmol / g.

9. The solid-phase synthesis method according to claim 1, characterized in that, In S2), cleavage is carried out using a mixed solution of TFA, TIPS, EDT and H2O. Preferably, the volume ratio of TFA, TIPS, EDT and H2O is 91:3:3:

3.

10. The solid-phase synthesis method according to claim 1, characterized in that, In S2), the sedimentation method is to add an excessive amount of methyl tert-butyl ether to the cleavage reaction system for sedimentation; Preferably, the added methyl tert-butyl ether is 5 - 10 times the volume of the cleavage reaction system.

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