Solid-liquid combined synthesis method of semeglutide

Abstract

The invention provides a solid-liquid combined synthesis method of semeglutide, which belongs to the technical field of semeglutide synthesis, and specifically comprises the following steps: segmenting a main chain of the semeglutide into three shorter polypeptide fragments, namely a fragment 4, a fragment 5 and a fragment 2 at sites 7-14, 15-22 and 23-37, and respectively synthesizing at the same time; performing solid-phase assembly on the fragment 4 and the fragment 5 to form 7-22 peptide fragments, namely a fragment 1, and after the fragment 1 is cut off from the resin, forming NHS ester at a C end; after the fragment 2 is assembled, selectively removing a protecting group A11oc on the Lys26, then connecting with a side chain for fatty acid treatment to form a fragment 3, and cutting off the fragment 3 from the resin; and connecting the NHS ester of the fragment 1 with the fragment 3 in a liquid phase reaction system. The crude semeglutide prepared by the synthetic method is relatively high in yield and purity. The refined semeglutide of which the purity is more than 99.3% and the content of single impurity is less than 0.1% can be obtained at high yield through a conventional purification means.

Description

Technical Field

[0001] The present invention relates to the technical field of semaglutide synthesis, and particularly to a solid-liquid combination synthesis method of semaglutide. Background Art

[0002] Semaglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist, mainly used for blood glucose control in patients with type 2 diabetes and can also reduce cardiovascular risk. It stimulates insulin secretion, suppresses appetite, and delays gastric emptying by mimicking the action of the GLP-1 hormone. Semaglutide is available in two dosage forms: an injection once a week and an oral tablet once a day.

[0003] The polypeptide sequence of semaglutide is as follows:

[0004] H-His 7 -Aib 8 -Glu 9 -Gly 10 -Thr 11 -Phe 12 -Thr 13 -Ser 14 -Asp 15 -Val 16 -Ser 17 -Ser 18 -Tyr 19 -Leu 20 -Glu 21 -Gly 22 -Gln 23 -Ala 24 -Ala 25 -Lys 26 (AEEA-AEEA-γ-Glu-OctadecanedioicAcid)-Glu 27 -Phe 28 -Ile 29 -Ala 30 -Trp 31 -Leu 32 -Val 33 -Arg 34 -Gly 35 -Arg 36 -Gly 37 -OH.

[0005] Patent WO2009083549A1 prepares semaglutide by a recombinant-biofermentation tandem chemical synthesis process. The process uses Fmoc-His-Aib-OSu and Lys 26The main chain 29-peptide with the side chain already connected reacts to obtain Fmoc-semaglutide, and then piperidine is used to remove Fmoc to obtain the crude semaglutide. However, the product produced by this method has 12-17% of disubstituted impurities (33-peptide), namely His-Aib-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. Due to partial removal of the Fmoc of the main chain, disubstituted impurities are formed.

[0006] CN116120427A discloses a synthesis method of semaglutide that couples amino acids or peptide segments sequentially from the C-terminus to the N-terminus. The total yield of the crude semaglutide synthesized is only 57.07%. The purity of the crude semaglutide by HPLC is only 77.71%.

[0007] The synthesis method of semaglutide disclosed in CN103848910A couples Gly with the resin to obtain Gly-resin, and through step-by-step coupling of amino acids or amino acid derivatives, after obtaining the main chain peptide segment, the protecting group of Lys is removed and then the side chain is connected. Due to the presence of more impurities, the purification yield of the final product is only 54.05%, and the total yield is 14.92%. 26

[0008] Currently, the most effective chemical synthesis method for polypeptide drugs is the solid-phase synthesis method. It is very challenging to use the conventional solid-phase synthesis method for semaglutide, which is mainly reflected in that there are a large number of hydrophobic amino acids on the main chain, making the hydrogen bonds between peptide chains stable, which can cause serious folding; the interaction force between peptide chains is enhanced, causing resin shrinkage, increasing the difficulty of amino acid coupling; reducing the coupling reaction activity and efficiency, resulting in a decrease in the reaction yield and an increase in cost, and there is a great difficulty in the industrial chemical preparation of semaglutide. Summary of the Invention

[0009] The purpose of the present invention is to provide a solid-liquid combined synthesis method of semaglutide. The crude peptide of semaglutide prepared by this synthesis method has relatively high yield and purity. Through conventional purification means, semaglutide fine peptide with a purity of more than 99.3% and a single impurity of less than 0.1% can be obtained with a high yield.

[0010] To solve the above technical problems, the technical solution adopted by the present invention is:

[0011] A solid-liquid combined synthesis method of semaglutide specifically includes the following steps: ​

[0012] The main chain of semaglutide is divided into three shorter polypeptide fragments at positions 7 - 14, 15 - 22, and 23 - 37, namely fragment 4, fragment 5, and fragment 2, which are synthesized simultaneously respectively.

[0013] Fragment 4 and fragment 5 are solid-phase assembled into a 7 - 22 peptide segment, namely fragment 1. After fragment 1 is cleaved from the resin, an NHS ester is formed at the C-terminus.

[0014] After fragment 2 is assembled, the protecting group A11oc on Lys 26 is selectively removed, and then it is connected to the side chain for fatty acylation to form fragment 3, and fragment 3 is cleaved from the resin.

[0015] In a liquid-phase reaction system, the NHS ester of fragment 1 is connected to fragment 3. After separation and purification, the protecting groups are removed to obtain a crude product, and then the semaglutide finished product is obtained through separation and purification.

[0016] Furthermore, the amino acid sequence of fragment 1 is:

[0017] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Osu.

[0018] Furthermore, the amino acid sequence of the said fragment 2 is:

[0019] Fmoc-Gln(Trt)-Ala-Ala-Lys(alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

[0020] Furthermore, the amino acid sequence of the said fragment 3 is:

[0021] Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

[0022] Furthermore, the amino acid sequence of the said fragment 4 is:

[0023] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH。

[0024] Furthermore, the 5-amino acid sequence of the fragment is as follows:

[0025] Fmoc-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH。

[0026] Furthermore, the solid-phase synthesis uses Wang resin as the solid-phase carrier, with a substitution degree of 0.1 - 0.6 mmol / g.

[0027] Furthermore, fragment 2 and fragment 5 are coupled with Fmoc-Gly-OH to prepare Fmoc-Gly-Wang resin; fragment 4 is coupled with Fmoc-Ser(tBu)-OH to prepare Fmoc-Ser(tBu)-Wang, and then the protected amino acids are sequentially coupled and connected according to the sequence to obtain the target peptide fragments 2, 4, and 5 respectively.

[0028] Furthermore, in the coupling reaction, after removing the Fmoc protection, the corresponding Fmoc-protected amino acid is added for reaction at a feeding ratio of 2 - 5 times, and the condensing agent is one of DIC / HOBT, DIC / HOAT, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, HATU / HOAT / DIPEA.

[0029] Furthermore, when removing the protecting group, the deprotecting reagent is a 20% piperidine DMF solution.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] In the present invention, the main chain is divided into 3 shorter polypeptide fragments at positions 7 - 14, 15 - 22, and 23 - 37 and synthesized simultaneously. The 7 - 14 peptide fragment (fragment 4) and the 15 - 22 peptide fragment (fragment 5) are assembled into a 7 - 22 peptide fragment (fragment 1). On the other hand, after completing the assembly of the 23 - 37 peptide fragment (fragment 2), the protecting group A11oc on Lys 26 is selectively removed. Then, the side chain of Lys 26 is fatty acylated to form fragment 3. Then, the 7 - 22 peptide fragment and the 23 - 37 peptide fragment are connected in a liquid-phase reaction system. After separation and purification, the protecting group is removed to obtain a crude product. Then, through separation and purification, the finished product is obtained. This method can not only make full use of the characteristics of high efficiency and easy purification of the solid-phase synthesis method, but also combine His 7 with Lys 26Fragmentation of the segment with a large number of hydrophobic amino acids into smaller parts can avoid the formation of severe β-sheets and resin polycondensation. On the other hand, when connecting the 7-22 peptide segment and the 23-37 peptide segment using a liquid-phase reaction system, the molar ratio of the 7-22 peptide segment to the 23-37 peptide segment is close to 1:1 (between 1:0.6 - 1.4), which has significant economic advantages compared to the traditional solid-phase synthesis method (usually requiring 1:3 - 8). The crude peptide of semaglutide prepared by the present invention has both high yield and purity. Through conventional purification means, semaglutide fine peptide with a purity of over 99.3% and a single impurity less than 0.1% can be obtained with a high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0033] Figure 1 It is a schematic diagram of the synthesis method flow of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] In the following text, only some exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the embodiments of the present invention. Therefore, the drawings and the description are considered to be exemplary in nature rather than restrictive.

[0035] The following will describe the embodiments of the present invention in detail with reference to the drawings.

[0036] Since His 7 and Lys 26 The presence of a large number of hydrophobic amino acids between them is a decisive factor affecting the solid-phase synthesis yield of the main chain of semaglutide. Therefore, in the present invention, the main chain of semaglutide is divided into 3 shorter polypeptide fragments at positions 7-14, 15-22, and 23-37 and synthesized simultaneously.

[0037] Assemble the 7-14 peptide segment (fragment 4) and the 15-22 peptide segment (fragment 5) into the 7-22 peptide segment (fragment 1).

[0038] After fragment 1 is cleaved from the resin, an NHS ester is formed at the C-terminus, that is:

[0039] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Osu。

[0040] On the other hand, after the 23-37 peptide segment (fragment 2) is assembled and completed, the protecting group A11oc on Lys is selectively removed. 26 After that, it is linked to the side chain (fatty acylation) to form fragment 3. After fragment 3 is excised from the resin, fragment 3 is obtained:

[0041] Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic Acid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH;

[0042] After that, in the liquid-phase reaction system, the 7-22 peptide segment (fragment 1): Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Osu and the 23-37 peptide segment:

[0043] Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic Acid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH are linked. After separation and purification, the protecting groups are removed to obtain a crude product. After separation and purification, the finished product is obtained.

[0044] The above method can not only make full use of the characteristics of high efficiency and easy purification of solid-phase synthesis method, but also combine His 7 with Lys 26Fragmentation of the fragment with a large number of hydrophobic amino acids into smaller parts can avoid the formation of severe β-sheets and resin polycondensation. On the other hand, when connecting the 7-22 peptide segment and the 23-37 peptide segment using a liquid-phase reaction system, the molar ratio of the 7-22 peptide segment to the 23-37 peptide segment is close to 1:1 (between 1:0.6 and 1.4), showing significant economic advantages compared to the traditional solid-phase synthesis method (usually 1:3 - 8).

[0045] The synthesis method process of solid-liquid combination of semaglutide is as Figure 1 shown.

[0046] The present invention proposes to solve the above problems through a fragment synthesis method. That is, the main chain is decomposed into 2 key fragments:

[0047] Fragment 1:

[0048] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH;

[0049] Fragment 2:

[0050] Fmoc-Gln(Trt)-Ala-Ala-Lys(alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH, where the alloc protecting group will be selectively removed and formed after connecting to the side chain;

[0051] Fragment 3:

[0052] Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

[0053] Among them, Fragment 1 can be further decomposed into 2 sub-fragments:

[0054] Fragment 4:

[0055] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH;

[0056] Fragment 5:

[0057] Fmoc-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH。

[0058] To facilitate further understanding of the present invention by those skilled in the art, the present invention will be further elaborated below in conjunction with specific implementation steps and cases.

[0059] a. Using Wang resin solid-phase carrier, fragment 2 and fragment 5 are coupled with Fmoc-Gly-OH to prepare Fmoc-Gly-Wang resin; fragment 4 is coupled with Fmoc-Ser(tBu)-OH to prepare Fmoc-Ser(tBu)-Wang. Then, the protected amino acids are sequentially coupled and connected in sequence to obtain the target peptide fragments 2, fragment 4 and fragment 5 respectively.

[0060] b. Fragment 4 is cut off from the resin, and then it is connected to fragment 5 that is connected to the resin and has its Fmoc protection removed:

[0061] (Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Wang) to form fragment 1 connected to the resin:

[0062] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Wang.

[0063] After that, fragment 1 is cut off from the resin to form fragment 1:

[0064] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH.

[0065] c. React the C-terminus of fragment 1 with an activating group such as NHS. To form fragment 1 with an activated C-terminus:

[0066] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu。

[0067] d. Deprotect the protecting group Alloc on the resin-attached fragment 2:

[0068] (Fmoc-Gln(Trt)-Ala-Ala-Lys(alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang) to form Fmoc-Gln(Trt)-Ala-Ala-Lys-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH, and link it to the side chain to obtain the resin-attached fragment 3:

[0069] Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang.

[0070] e. Selectively deprotect the N-terminus of the resin-attached fragment 3 and cleave it from the resin to obtain:

[0071] Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

[0072] f. React the C-terminus-activated fragment 1 with the peptide prepared in Example e in the liquid phase to obtain:

[0073] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH-Gln(Trt)-Ala-Ala

[0074] -Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-OH。

[0075] g. The peptide segment prepared in Example f is purified, deprotected and then purified again to obtain the finished product of semaglutide.

[0076] In step a, the solid-phase carrier is Wang resin with a substitution degree of 0.1 - 0.6 mmol / g. After the amino acid is linked to the resin, after removing the Fmoc protection, the corresponding Fmoc-protected amino acid is added for coupling reaction at a feeding ratio of 2 - 5 times. Each coupling reaction is a solid-phase peptide coupling reaction in the presence of a condensing agent. The end point of each coupling reaction is detected with Kaiser reagent. After the reaction is completed, the Fmoc is removed with a deprotecting reagent, and then coupled with the next Fmoc-protected amino acid; the operation is repeated until the fully protected fragment 2, fragment 4 and fragment 5 are synthesized respectively. The deprotecting reagent is preferably a 20% piperidine in DMF solution (volume ratio); the condensing agent used in the coupling reaction is one of the following combinations: DIC / HOBT, DIC / HOAT, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, HATU / HOAT / DIPEA.

[0077] In step b, the method for cleaving the peptide chain from the resin is: reacting the peptide chain linked to the resin with a mixture of TFA, triisopropylsilane (TIPS) and water, and the preferred ratio is 90% TFA, 5% TIPS and 5% water. The mixture is stirred at room temperature for 2 - 3 hours, a large amount of ether (5 - 50 times the mass of the resin, preferably 15 times) is added to precipitate the crude peptide, and then centrifuged and washed with ether 2 - 4 times to obtain it.

[0078] In step c, the C-terminal exposed peptide segment is dissolved in a dry organic solvent (such as dichloromethane or DMF). NHS is added to the solution, and then a coupling reagent such as DCC or EDC is added. The reaction is stirred at room temperature or slightly higher temperature (usually about 10 - 60 °C, preferably 25 °C) for 0.5 - 20 hours to form the NHS ester intermediate.

[0079] In step d, for the removal of the protecting group A11oc on Lys 26 The method is to use Pd(PPh3)4 in an amount of 0.1 - 0.4 times the synthetic scale and phenylsilane (or morpholine) in an amount of 10 - 30 times the synthetic scale to remove it under solid-phase conditions for 10 - 90 minutes.

[0080] In steps b, c, d, and f, the condensing agent used in the coupling reaction is one of the following combinations: DIC / HOBT, DIC / HOAT, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, HATU / HOAT / DIPEA.

[0081] In step e, the cleavage reagent is a TFA solution containing a scavenger with a volume ratio of 1 - 5%, and the scavenger is one or several of anisole, benzyl mercaptan, ethylene dithiol, mercaptoethanol, phenol, water, and TIS.

[0082] The preferred ratio of the cleavage reagent is: TFA / benzyl mercaptan / water / TIS = 90 / 2.5 / 5.0 / 2.5.

[0083] The abbreviations used in the specification and claims have the following meanings:

[0084]

[0085] The specific preparation examples are as follows:

[0086] Example 1: Synthesis of Fmoc-Gly-Wang;

[0087] Place 500.0 g of Wang resin support (sub = 0.42 mmol / g) in a synthesis column, wash it twice with 2400 mL of DMF, add 2400 mL of DCM to swell for 30 min; after filtering off the DCM by suction, add a mixed DCM solution of Fmoc-Gly-OH / DIC / HOBT [weigh 282.4 g (400 mmol) of Fmoc-Gly-OH and 64.8 g (480 mmol) of HOBT and place them in an amino acid activation bottle, add 2000 mL of a mixed solution of DMF and DCM with a volume ratio of 1:1 and stir to dissolve, add 76.4 mL (480 mmol) of DIC at 0 °C, activate for 5 minutes, add 4.8 g (4 mmol) of DMAP after reacting for 10 min; react for 2 h, filter off the reaction solution, wash it twice with 2400 mL of DMF, add 2400 mL of capping reagent (480 mL of acetic anhydride and 408 mL of pyridine dissolved in 1512 mL of DMF) and react for 2 h, filter off the reaction solution by suction, wash it twice with DMF, DCM, and methanol respectively, and obtain 609.3 g of Fmoc-Gly-Wang after vacuum drying: Take a sample to measure the substitution degree as 0.26 mmol / g.

[0088] Example 2: Synthesis of Fmoc-Ser(tBu)-Wang;

[0089] Place 500.0 g of Wang resin support (sub = 0.42 mmol / g) in a synthesis column, wash it twice with 2400 mL of DMF, add 2400 mL of DCM to swell for 30 min; after filtering off the DCM by suction, add a mixed DCM solution of Fmoc-Ser(tBu)-OH / DIC / HOBT [weigh 312.6 g (400 mmol) of Fmoc-Ser(tBu)-OH and 64.8 g (480 mmol) of HOBT and place them in an amino acid activation bottle, add 2000 mL of a mixed solution of DMF and DCM with a volume ratio of 1:1 and stir to dissolve, add 76.4 mL (480 mmol) of DIC at 0 °C, activate for 5 minutes, add 4.8 g (4 mmol) of DMAP after reacting for 10 min; react for 2 h, filter off the reaction solution, wash it twice with 2400 mL of DMF, add 2400 mL of capping reagent (480 mL of acetic anhydride and 408 mL of pyridine dissolved in 1512 mL of DMF) and react for 2 h, filter off the reaction solution by suction, wash it twice with DMF, DCM, and methanol respectively, and obtain 682.3 g of Fmoc-Ser(tBu)-Wang after vacuum drying: Take a sample to measure the substitution degree as 0.30 mmol / g.

[0090] Example 3: Preparation of peptide chain fragment 2 resin;

[0091] Accurately weigh 69.23 g (synthesis scale: 18 mmol) of Fmoc-Gly-Wang resin with a substitution degree of 0.26 mmol / g in Example 1 and place it in a synthesis column. Add 1000 ml of DCM to swell for 30 min. After suction filtration to remove DCM, wash twice with 800 ml of DMF. Add 1000 ml of 20% piperidine / DMP solution for deprotection twice, reacting for 10 min and 10 min respectively. Then wash twice with 800 ml of DMF, DCM, and DMF respectively. Add 500 ml of a DMF solution containing 25.41 g (36 mmol) of Fmoc-Arg(Pbf)-OH, 5.35 g (39.6 mmol) of HOBT, and 6.3 ml (39.6 mmol) of DIC, stir the reaction under nitrogen bubbling for 2 h. The end point of the reaction is determined by the Kaiser reagent test result. After the reaction reaches the end point, suction off the reaction solution and wash twice with 800 ml of DMF, DCM, and DMF respectively. Subsequently, perform deprotection again. Repeat such cyclic operations, and couple with protected amino acids one by one according to the sequence of peptide chain fragment 2. The protected amino acids connected in sequence are: Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH. Obtain the peptide chain fragment 2 resin with side chains protected: Fmoc-Gln(Trt)-Ala-Ala-Lys(alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang.

[0092] Example 4: Preparation of peptide chain fragment 4 resin;

[0093] Accurately weigh 75.26 g (synthesis scale 18 mmol) of Fmoc-Ser(tBu)-Wang resin with a substitution degree of 0.30 mmol / g in Example 1 and place it in a synthesis column. Add 1000 ml of DCM to swell for 30 min. After filtering off the DCM by suction, wash it twice with 800 ml of DMF. Add 1000 ml of 20% piperidine / DMP solution to deprotect twice, reacting for 10 min and 10 min respectively. Then wash it twice with 800 ml of DMF, DCM, and DMF respectively. Add 500 ml of a DMF solution containing 14.61 g (36 mmol) of Fmoc-Thr(Trt)-OH, 5.35 g (39.6 mmol) of HOBT, and 6.3 ml (39.6 mmol) of DIC. Stir the reaction under a nitrogen atmosphere for 2 h. The end point of the reaction is determined by the Kaiser reagent test result. After the reaction reaches the end point, suck out the reaction solution and wash it twice with 800 ml of DMF, DCM, and DMF respectively. Subsequently, deprotect again. Repeat the above cyclic operation. According to the sequence of peptide chain fragment 2, couple with protected amino acids one by one. The protected amino acids connected in sequence are: Fmoc-Thr(Trt)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Fmoc-His(Trt)-OH. Obtain peptide chain fragment 4 resin: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Wang.

[0094] Example 5: Preparation of peptide chain fragment 5 resin;

[0095] Accurately weigh 69.23 g (synthesis scale 18 mmol) of Fmoc-Gly-Wang resin with a substitution degree of 0.26 mmol / g in Example 1 and place it in a synthesis column. Add 1000 ml of DCM to swell for 30 min. After filtering off the DCM, wash with 800 ml of DMF twice. Add 1000 ml of 20% piperidine / DMP solution for deprotection twice, reacting for 10 min and 10 min respectively. Then wash with 800 ml of DMF, DCM, and DMF twice respectively. Add 500 ml of a DMF solution containing 19.42 g (36 mmol) of Fmoc-Glu(OtBu)-OH, 5.35 g (39.6 mmol) of HOBT, and 6.3 ml (39.6 mmol) of DIC, and stir the reaction under nitrogen for 2 h. The end point of the reaction is based on the detection result of Kaiser reagent. After the reaction reaches the end point, draw off the reaction solution and wash with 800 ml of DMF, DCM, and DMF twice respectively. Subsequently, deprotect again. Repeat the above cyclic operation, and couple with protected amino acids one by one according to the sequence of peptide chain fragment 2. The protected amino acids connected in sequence are: Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH. Obtain peptide chain fragment 5 resin: Fmoc-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Wang.

[0096] Example 6: Method for cleaving the fully protected peptide chain fragment 4 from the resin;

[0097] Put the peptide chain fragment 4 connected to the resin into a mixed solvent of DCM and hexafluoroisopropanol (HFIP). The ratio of the mixed solvent of DCM and hexafluoroisopropanol (HFIP) is 1 - 50:1. The preferred ratio is 5:1. After stirring the mixture at room temperature for 2 - 3 h, filter to remove the resin. After the filtrate is dried by suction, peptide chain fragment 4 is obtained:

[0098] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH. After purification, it is used in Example 7.

[0099] Example 7: Connect peptide chain fragment 4 and fragment 5 resin;

[0100] Take the peptide chain fragment 5 resin prepared in Example 5:

[0101] Fmoc-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Wang 110g was deprotected twice by adding 1000 ml of 20% piperidine / DMP solution, with reaction times of 10 min and 10 min respectively. Then it was washed twice with 800 ml of DMF, DCM, and DMF respectively, and the peptide chain fragment 4 prepared in Example 6 was added.

[0102] 500 ml of a DMF solution containing Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH, 5.35 g (39.6 mmol) of HOBT, and 6.3 ml (39.6 mmol) of DIC was stirred under a nitrogen atmosphere for 2 h. The end point of the reaction was determined by the Kaiser reagent. After the reaction reached the end point, the reaction solution was removed by suction, and it was washed twice with 800 ml of DMF, DCM, and DMF respectively. The peptide chain fragment 1 attached to the resin was obtained.

[0103] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Wang.

[0104] Example 8: Method for cleaving the fully protected peptide chain fragment 1 from the resin;

[0105] The peptide chain fragment 1 attached to the resin prepared in Example 7 was put into a mixed solvent of DCM and hexafluoroisopropanol (HFIP). The ratio of DCM to hexafluoroisopropanol (HFIP) in the mixed solvent was 1 - 50:1. The preferred ratio was 5:1. After the mixture was stirred at room temperature for 2 - 3 h, the resin was removed by filtration. After the filtrate was dried by suction, the peptide chain fragment 1 was obtained.

[0106] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH. After purification, it was used in Example 9.

[0107] Example 9: Activation of the C-terminus of the peptide chain fragment 1;

[0108] Dissolve 50 g of the peptide chain fragment 1 prepared in Example 8 in 200 mL of dry dichloromethane. Add 6.2 g of NHS to the solution, and then add 12.6 g of EDC. Stir and react at 25 °C for 5 hours to form a peptide chain with an activated C-terminus:

[0109] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OSu.

[0110] Example 10: Lys of peptide chain fragment 5 26 Selective deprotection of the protecting group on the side chain;

[0111] Add 1000 ml of DCM to the peptide chain fragment 2 resin prepared in Example 3 and wash it twice, then add 1000 ml of DCM, slowly add 27 ml of phenylsilane while stirring the resin evenly, after reacting for 3 min, add 5.64 g of Pd(PPh3)4, react at room temperature for 50 min, drain the reaction solution, and treat the above resin with a hydrazine hydrate / DMF (1 / 15) mixed solution 3 times. Wash it 6 times with 1000 ml of DCM: Take a sample for Kaiser reagent detection, and the resin shows blue. Thus, Lys is obtained 26 Peptide chain fragment 5 with side chain deprotected and attached to the resin:

[0112] Fmoc-Gln(Trt)-Ala-Ala-Lys 26 -Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang.

[0113] Example 11: Lys of peptide chain fragment 5 26 Fatty acylation of the side chain;

[0114] Weigh 86.96 g of tBuO-Ste-Glu(AEEA-AEEA-OH)-OtBu and 8.02 g (59.4 mol) of HOBT into a dissolution flask, add 500 ml of DMF to dissolve, after complete dissolution, place it in an ice-water bath and let it stand for 10 min, add 9.2 ml (59.4 mmol) of DIC and mix evenly, then activate it in an ice-water bath for 10 min; Add the activated solution to the Fmoc-Gln(Trt)-Ala-Ala-Lys obtained in Example 8 26-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin, uniform stirring reaction: Kaiser reagent was used to detect the reaction progress. After the resin was detected colorless, the coupling reaction was terminated. After the peptide resin was prepared, it was washed twice with 1000 ml of DMF, DCM, and methanol respectively, and then dried in vacuum to obtain the peptide resin:

[0115] Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang, weighing 262.6 g.

[0116] Example 12: The N-terminus of peptide chain fragment 5 was selectively deprotected and cleaved from the resin;

[0117] The resin linked with the peptide chain prepared in Example 11 was taken and deprotected twice with 500 ml of 20% piperidine / DMP solution for 10 min and 10 min respectively. Then it was washed twice with 400 ml of DMF, DCM, and DMF respectively. After that, a mixed solvent of DCM and hexafluoroisopropanol (HFIP) was added. The ratio of DCM to hexafluoroisopropanol (HFIP) in the mixed solvent was 1 - 50:1. The preferred ratio was 5:1. After the mixture was stirred at room temperature for 2 - 3 hours, the resin was filtered off. After the filtrate was dried by suction, the peptide chain fragment was obtained:

[0118] Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH. After purification, it was used in Example 13.

[0119] Example 13: Assembly of the semaglutide peptide chain;

[0120] Weigh the peptide chain fragment prepared in Example 12:

[0121] Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH and the C-terminally activated peptide chain prepared in Example 9:

[0122] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Osu was added to 500 ml of DMF, and nitrogen was bubbled and stirred at room temperature for 2 hours. The solvent was removed under reduced pressure, and after purification, a peptide chain fragment was obtained:

[0123] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

[0124] Example 14: Deprotection of semaglutide

[0125] The semaglutide peptide chain obtained in Example 13 was added to 1600 ml of frozen cleavage solution (volume ratio of TFA / thioanisole / TIS / 120 = 92.5 / 2.5 / 2.5 / 2.5), and the reaction was stirred at room temperature for 4 h: After the cleavage reaction was completed, the resin was filtered, and the resin was washed twice with 100 ml of TFA. The filtrate and the washing solution were combined, concentrated by rotary evaporation to 1000 ml, poured into 10 L of frozen methyl tert-butyl ether, and a white precipitate was precipitated; after standing for 30 min, it was filtered, washed 6 times with methyl tert-butyl ether, and vacuum dried to obtain 67.8 g of crude semaglutide, with a crude peptide yield of 96.8% and a purity of 93.8%.

[0126] Example 15: Purification of semaglutide;

[0127] 50.0 g of the crude peptide prepared in Example 12 was dissolved in 200 ml of acetic acid. After complete dissolution, it was diluted with water to 4000 ml. The solution was filtered through a 0.45-μm filter membrane for standby. Purification method: a C18 preparative column with a diameter of 150 mm, the mobile phase was a 0.1% acetic acid / water - 0.1% acetic acid / acetonitrile system, the sample loading amount was 25 g / time, the flow rate was 300 ml / min, and gradient elution was performed; pre-peak and post-peak cyclic injection was carried out to obtain a refined peptide solution with qualified central control analysis purity. After desalting, it was freeze-dried to obtain 31.4 g of refined peptide, with a total yield, a purity of over 99.5%, and each single impurity being less than 0.1%.

[0128] The yield and purity of the semaglutide crude peptide prepared by this synthesis method are both relatively high. Through conventional purification means, semaglutide refined peptide with a purity of over 99.5% and each single impurity being less than 0.1% can be obtained with a high yield.

[0129] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0130] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. It should be noted that any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A solid-liquid combined synthesis method of semaglutide, characterized in that: The specific steps include: The semaglutide main chain is divided into three shorter polypeptide fragments at positions 7-14, 15-22, and 23-37, namely fragment 4, fragment 5, and fragment 2, which are synthesized simultaneously; Fragment 4 and fragment 5 were solid-phase assembled into a 7-22 peptide fragment, i.e., fragment 1. After fragment 1 was cleaved from the resin, an NHS ester was formed at the C-terminus; After assembling fragment 2, Lys 26 The protecting group A11oc on the resin is then connected to the side chain for fatty acidization to form fragment 3, which is then removed from the resin; The NHS ester of fragment 1 and fragment 3 are connected in a liquid phase reaction system, and the protecting group is removed after separation and purification to obtain a crude product, which is then separated and purified to obtain a finished semaglutide product.

2. The solid-liquid combined synthesis method of semaglutide according to claim 1, characterized in that: The solid phase synthesis adopts Wang resin solid phase carrier, and the substitution degree is 0.1-0.6mmol / g.

3. The solid-liquid combined synthesis method of semaglutide according to claim 7, characterized in that: Fragment 2 and fragment 5 were coupled with Fmoc-G1y-OH to prepare Fmoc-Gly-Wang resin; Fmoc-Ser(tBu)-OH was coupled with fragment 4 to prepare Fmoc-Ser(tBu)-Wang, and then the protected amino acids were coupled in sequence to obtain the target peptide fragments 2, 4 and 5, respectively.

4. The solid-liquid combined synthesis method of semaglutide according to claim 7, characterized in that: In the coupling reaction, after removing the Fmoc protection, the corresponding Fmoc protected amino acid is added at a feed ratio of 2-5 times to react, and the condensing agent is one of DIC / HOBT, DIC / HOAT, TBTU / HOBT / DIPEA, HBTU / HOBT / DIPEA, and HATU / HOAT / DIPEA.

5. A solid-liquid combined synthesis method of semaglutide according to any one of claims 1 to 4, characterized in that: The deprotection reagent for removing the protecting group is a 20% DMF solution of piperidine.

6. The solid-liquid combined synthesis method of semaglutide according to claim 1, characterized in that: The amino acid sequence of fragment 1 is: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-As p(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Osu.

7. The solid-liquid combined synthesis method of semaglutide according to claim 1, characterized in that: The amino acid sequence of fragment 2 is: Fmoc-Gln(Trt)-Ala-Ala-Lys(alloc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

8. The solid-liquid combined synthesis method of semaglutide according to claim 1, characterized in that: The amino acid sequence of fragment 3 is: Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-OctadecanedioicAcid-OtBu)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH.

9. The solid-liquid combined synthesis method of semaglutide according to claim 1, characterized in that: The amino acid sequence of fragment 4 is: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(Trt)-Ser(tBu)-OH.

10. The solid-liquid combined synthesis method of semaglutide according to claim 1, characterized in that: The amino acid sequence of fragment 5 is: Fmoc-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH。

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