A kind of synthetic method of semaglutide

Abstract

The present invention provides a method for synthesizing semaglutide. It includes coupling amino acids or peptides from the C-terminus to the N-terminus in sequence according to the amino acid sequence of semaglutide by a solid phase synthesis method to obtain a fully protected semaglutide resin, and semaglutide is obtained by cracking and purification; the peptide includes a dipeptide S20-S21 formed by the 20-21 amino acids, and the S20-S21 is R1-Lys(Fmoc)-Glu(OtBu)-OH, wherein R1 is Dde or ivDde, Fmoc is a side chain protecting group, and R1 is a main chain protecting group. The total yield of the crude semaglutide produced by the present invention reaches 57.07%, and the HPLC purity reaches 77.71%, which reduces the production of impurities, and the synthesis and purification process is simple, the selected materials are cheap and easy to obtain, the comprehensive production cost is low, and it is more conducive to industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide synthesis, and in particular to a method for synthesizing semaglutide. Background Art

[0002] GLP-1 (Glucagon-like peptide-1), also known as glucagon-like peptide-1, was first isolated by scientists from intestinal extracts in the 1970s. GLP-1 works in a glucose-dependent manner, has excellent and mild hypoglycemic effects, and is not prone to hypoglycemia. In addition, GLP-1 protects pancreatic β cells, promotes proliferation, inhibits apoptosis, inhibits the synthesis and release of glucagon, delays gastric emptying, suppresses appetite, helps lose weight, and improves large and small vascular lesions and neuropathy in chronic complications of diabetes. However, GLP-1 is unstable and easily degraded, and is easily degraded by DPP-4 (dipeptidyl peptidase-4). In order to increase the "endurance" of GLP-1, the molecular structure of GLP-1 was artificially modified, resulting in GLP-1 analogs. Compared with GLP-1, GLP-1 analogs have a longer half-life, stronger biological activity and lower immunogenicity, better efficacy and safety, and patients use the drug less frequently and have higher compliance.

[0003] Semaglutide (formerly temporarily known as semaglutide; English name: Semaglutide) is a long-acting GLP-1 analog developed by Novo Nordisk. It has a half-life of up to 7 days, is suitable for once-weekly injection, and has a stable blood concentration. It can effectively lower glycated hemoglobin and reduce body weight. In addition to developing it as a subcutaneous injection drug, Novo Nordisk is also developing an oral version of semaglutide, which is taken orally once a day.

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

[0005] H-His 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Val 10 -Ser 11 -Ser 12 -Tyr 13 -Leu 14 -Glu 15 -Gly 16 -Gln 17 -Ala 18 -Ala 19 -Lys20 (AEEA-AEEA-γ-Glu-OctadecanedioicAcid)-Glu 21 -Phe 22 -Ile 23 -Ala 24 -Trp 25 -Leu 26 -Val 27 -Arg28-Gly 29 -Arg 30 -Gly 31 -OH.

[0006] Molecular formula: C 187 H 291 N 45 O 59 ,Exact Mass:4111.1154,Molecular Weight:4113.6410,Structural formula is as follows:

[0007]

[0008] From the peptide sequence, the main chain has 31 amino acids, the 2nd amino acid is a non-natural α-aminoisobutyric acid (Aib), and the 20th amino acid lysine in the peptide sequence is connected to two AEEA, γ-glutamic acid and octadecanedioic acid fatty chains to form a side chain.

[0009] In December 2017, the U.S. Food and Drug Administration (FDA) approved the marketing of semaglutide injection (Ozempic) for blood sugar control in adult patients with type 2 diabetes; in September 2019, semaglutide oral preparation (Rybelsus) was approved by the FDA for use in combination with diet and exercise to improve blood sugar control in patients with type 2 diabetes; in June 2021, the FDA further approved semaglutide subcutaneous injection (Wegovy) for chronic weight management in obese or overweight adults. In April 2021, it was approved by the National Medical Products Administration (NMPA) for the treatment of adult type 2 diabetes and to reduce the risk of adverse cardiovascular events in patients with T2DM and cardiovascular disease. On December 3, 2021, it was included in the National Basic Medical Insurance, Work-related Injury Insurance and Maternity Insurance Drug Catalog (2021). In 2020, semaglutide injection has been launched in 43 countries around the world, with sales in the first half of the year continuing to grow by 151%, or approximately US$1.5 billion. It is estimated that by 2024, global sales of semaglutide injection will reach US$5.28 billion, and semaglutide oral preparations will reach US$3.23 billion, with annual sales exceeding US$10 billion in the future. Semaglutide has an extremely broad market prospect.

[0010] At present, the chemical solid phase synthesis method of semaglutide reported at home and abroad generally uses CTC Resin (2-triphenylchlororesin) (CN106928343 A) and Wang Resin (Wang Resin) (CN112250755 A) as solid phase reaction carriers in production, but both resins have shortcomings. When CTC Resin is used as a solid phase reaction carrier, the linker can be detached and the polypeptide fragment can be removed under weak acid conditions; when Wang Resin is used as a solid phase reaction carrier, it is sequentially coupled to Fmoc-Arg(Pbf)-Gly-Wang Resin, and after deprotection, Arg 30 The α-amino group of the residue is in a free state, and it may perform intramolecular cyclization on the benzyl ester bond at the junction of the resin and the peptide chain, generating a six-membered ring diketopiperazine derivative that is released from the carrier and causes the reaction to terminate (DKPS). This reaction is very rapid, which will greatly reduce the yield, increase production costs, and reduce economic benefits.

[0011] According to the coupling sequence of amino acids, the solid phase chemical synthesis of semaglutide is generally divided into two synthetic strategies. One is to gradually couple the main chain and then remove the Lys 20 Side chain protecting groups, and then gradually coupled to the side chains (CN113402598 A; CN11212590B; CN101133082 B); the second is to gradually couple to the main chain Lys 20 After that, the side chain is preferentially coupled, and then the coupling of the main chain is completed (CN112028986 A; CN 113880935 B). In order to obtain better synthesis effects, the above two synthesis strategies are generally combined with special peptide segments, special amino acid protection strategies, O-isoacyl dipeptides and other technologies. CN112028986 A uses Fmoc-Gly-Wang resin as a solid phase carrier. In October 2022, CDE issued the "Technical Guidelines for Pharmaceutical Research of Chemically Synthesized Polypeptide Drugs (Draft for Comments)", which stated that due to the lack of effective characterization methods and targeted quality control methods, resins connected to the C-terminal amino acid are not recommended as starting materials; and there are still a certain amount of impurities in the reaction product, and the purity needs to be further improved.

[0012] CN113444164B discloses a method for synthesizing semaglutide, and CN111217901 A discloses a method for synthesizing semaglutide. The above two methods for preparing semaglutide are simple and can obtain a crude semaglutide product with high purity; however, the tBuOC-C 16 H 32-CONH-γ-Glu(α-OtBu)-AEEA-AEEA-OSu or Fmoc-Lys[AEEA-AEEA-γ-Glu(α-OtBu)-Octadecanedioic Acid-18(tert-butoxy)]-OH has a high price, which greatly increases the production cost. Summary of the invention

[0013] In view of the deficiencies in the prior art, the present invention provides a method for synthesizing semaglutide, which solves the problems of low semaglutide synthesis yield, high cost and impurities in the prior art.

[0014] On the one hand, the present invention provides a method for synthesizing semaglutide. According to the amino acid sequence of semaglutide, amino acids or peptide segments are sequentially coupled from the C-terminus to the N-terminus by a solid phase synthesis method to obtain a fully protected semaglutide resin, and semaglutide is obtained by cleavage and purification; the peptide segment includes a dipeptide S20-S21 formed by amino acids at positions 20-21, and the S20-S21 is R1-Lys(Fmoc)-Glu(OtBu)-OH, wherein R1 is Dde or ivDde, Fmoc is a side chain protecting group, and R2 is a main chain protecting group.

[0015] Furthermore, the peptide segment also includes a dipeptide S1-S2 formed by amino acids at positions 1-2, a dipeptide S3-S4 formed by amino acids at positions 3-4, a dipeptide S15-S16 formed by amino acids at positions 15-16, and a dipeptide S29-S30 formed by amino acids at positions 29-30, wherein S1-S2 is R2-His(Trt)-Aib-OH, and R2 is Fmoc or Boc; S3-S4 or S15-S16 is Fmoc-Glu(OtBu)-Gly-OH; and S29-S30 is Fmoc-Gly-Arg(Pbf)-OH.

[0016] Further, the following steps are included:

[0017] S1: Using Wang resin as a solid phase synthesis carrier, Fmoc-Gly-OH, Fmoc-Gly-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH.H2O, Fmoc-Ile-OH, Fmoc-Phe-OH and R1-Lys(Fmoc)-Glu(OtBu)-OH were coupled in sequence to obtain R1-Lys(Fmoc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin;

[0018] S2: Remove the side chain protecting group Fmoc in S20-S21, and couple Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu and HOOC-C in sequence 16 H 32 -COOtBu, get R1-Lys[AEEA-AEEA-γ-Glu(α-OtBu)-Octadecanedioic

[0019] Acid-18(tert-butoxy)]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin;

[0020] S3: Remove the main chain protecting group R1 in S20-S21, and couple Fmoc-Ala-OH.H2O, Fmoc-Ala-OH.H2O, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Va l-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-Gly-OH and R2-His(Trt)-Aib-OH to obtain semaglutide fully protected resin;

[0021] S4: The semaglutide fully protected resin is subjected to cleavage, sedimentation, centrifugation and drying to obtain semaglutide.

[0022] Furthermore, in step S1, the degree of substitution of the Wang resin is 0.1-1.0 mmol / g, preferably 0.1-0.4 mmol / g.

[0023] Furthermore, in step S2, the deprotection agent of Fmoc is a solution of piperidine in N,N-dimethylformamide, with a concentration of 20% to 50%, preferably 20%.

[0024] Furthermore, in step S3, the deprotecting agent of R1 is a solution of hydrazine hydrate in N,N-dimethylformamide, with a concentration of 1% to 5%, preferably 2% to 3%.

[0025] Furthermore, steps S1-S3 also include one or more of the following features:

[0026] (1) The condensation reagent used in the coupling is any one or more of A, B, C, and D,

[0027] Wherein, A is selected from any one of DIC, DCC, and EDCI, B is selected from any one of HOOBT, HOBT, and BOAT, C is selected from any one of DIPEA and NMM, and D is selected from any one of DMAP, HBTU, HATU, TBTU, and PyBOP;

[0028] (2) The coupling reaction medium is one or more of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-diethylformamide, N-methylpyrrolidone and dimethyl sulfoxide, preferably N,N-dimethylformamide;

[0029] (3) The coupling and deprotection temperature is 25-45°C, preferably 30-40°C.

[0030] Furthermore, in terms of mass ratio, Wang resin: amino acid or peptide: condensation reagent = 1:2:2 to 1:4:4, preferably Wang resin: amino acid or peptide: condensation reagent = 1:3:3.

[0031] Furthermore, in step S4, the cleavage solution used for the cleavage is a mixture of trifluoroacetic acid, triisopropylsilane, 1,2-ethanedithiol, phenol and water, preferably, by mass ratio, trifluoroacetic acid: 1,2-ethanedithiol: triisopropylsilane: phenol: water = 87.5:5:2.5:2.5:2.5.

[0032] Furthermore, in step S4, the sedimentation liquid used for the sedimentation is one or more of n-hexane, diethyl ether and methyl tert-butyl ether, preferably methyl tert-butyl ether.

[0033] In one embodiment of the present invention, the preparation method comprises the following steps: (1) using Wang Resin as a solid phase synthesis carrier to synthesize Fmoc-Gly-Wang Resin starting resin. (2) according to the sequence of semaglutide, sequentially coupling amino acids or peptides to Lys 20 , where the S29-S30 fragment is Fmoc-Gly-Arg(Pbf)-OH; the S20-S21 fragment is R1-Lys(Fmoc)-Glu(OtBu)-OH, and R1 is Dde or ivDde. (3) Removal of Lys 20 ε-amino protecting group, and then the amino acids or compounds on the side chains are coupled. (4) Removal of Lys 20 α-amino protecting group, and then the amino acids or compounds on the main chain are coupled in sequence, wherein the S3-S4 and S15-S16 fragments are Fmoc-Glu(OtBu)-Gly-OH; the S1-S2 fragment is R2-His(Trt)-Aib-OH, and R2 is Fmoc or Boc. (5) The fully protected semaglutide peptide resin is cleaved and precipitated by a cleavage solution to obtain a crude semaglutide peptide.

[0034] The fully protected resin sequence of semaglutide in the present invention is:

[0035] H-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tB u)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys[AEEA-AEEA-γ-Glu(α-OtBu)-Octadecanedioic Acid-18(tert-butoxy)]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin.

[0036] or

[0037] Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-V al-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-GlyEE-Glyn A-γ-Glu(α-OtBu)-Octadecanedioic Acid-18(tert-butoxy)]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin。

[0038] She also lives in:

[0039] H-His 1 -Aib 2 -Glu 3 -Gly 4 -Thr 5 -Phe 6 -Thr 7 -Ser 8 -Asp 9 -Val 10 -Ser 11 -Ser 12 -Tyr 13 -Able 14 -Glu 15 -G ly 16 -Gln 17 -Going to 18 -Going to 19 -Lys 20 (AEEA-AEEA-γ-Glu-Octadecanedioic Acid)-Glu 21 -Phe 22 -Ile 23 -Going to 24 -Trp 25 -Able 26 -Val 27 -Arg 28 -Gly 29 -Arg 30 -Gly 31 -OH。

[0040] The technical principle of the present invention is as follows: in the production of semaglutide, the inventors found that H-Arg(Pbf)-Gly-WangResin has a more significant tendency to undergo diketopiperazine cyclization side reaction; further, the inventors also found that Dde-Lys(Fmoc)-OH is more difficult to couple than ordinary amino acids, resulting in more impurities HOOC-C in the crude semaglutide product. 16 H 32 -CONH-γ-Glu-AEEA-AEEA-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. Therefore, the inventors tried to form the dipeptide R1-Lys(Fmoc)-Glu(OtBu)-OH with the amino acids at positions 20 and 21, and at the same time, the dipeptide R2-His(Trt)-Aib-OH with the amino acids at positions 1-2, the dipeptide Fmoc-Gly-Arg(Pbf)-OH with the amino acids at positions 29-30, and the dipeptide Fmoc-Glu(OtBu)-Gly-OH with the amino acids at positions 3-4 and 15-16. The results showed that: not only the HOOC-C 16 H 32 -CONH-γ-Glu-AEEA-AEEA-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH and other impurities are reduced, thereby improving the purity of the product and increasing the yield of the product.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] (1) The product of the present invention has high yield and high purity. The total yield of crude semaglutide is 57.07%, and the HPLC purity of crude semaglutide is 77.71%.

[0043] (2) During the peptide chain elongation process, Lys 20 -Glu 21 Choosing to connect in the form of S20-S21 fragment Dde-Lys(Fmoc)-Glu(OtBu)-OH or ivDde-Lys(Fmoc)-Glu(OtBu)-OH can solve the problem of Dde-Lys(Fmoc)-OH or ivDde-Lys(Fmoc)-OH coupling difficulty and improve the synthesis yield; and when connected with Lys 20 Finally, the strategy of coupling the side chain first and then the main chain was adopted. The access of the side chain can solve the problem of difficult residue coupling and low yield caused by the β-folding of the semaglutide sequence itself; further, the impurity HOOC-C 16 H 32-Generation of CONH-γ-Glu-AEEA-AEEA-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-O H.

[0044] (3) The present invention uses Wang resin as a solid phase synthesis carrier, compared with CTC Resin (2-triphenylchlororesin), which can effectively avoid the shedding of linker under weak acid conditions and the removal of polypeptide fragments, thereby effectively improving the synthesis yield.

[0045] (4) During the peptide chain elongation process, Gly 29 -Arg 30 Selecting to insert the S29-S30 fragment in the form of Fmoc-Gly-Arg(Pbf)-OH can significantly inhibit the occurrence of diketopiperazine cyclization side reactions and improve the synthesis yield.

[0046] (5) During the peptide chain elongation process, Glu 3 -Gly 4 and Glu 15 -Gly 16 Selecting to access in the form of S3-S4 and S15-S16 fragments Fmoc-Glu(OtBu)-Gly-OH can reduce side reactions, reduce the formation of Gly-added or Gly-deficient impurities, improve product purity, and be more conducive to the purification of crude products.

[0047] (6) During the peptide chain elongation process, His 1 -Aib 2 Choosing to access in the form of S1-S2 fragment Fmoc-His(Trt)-Aib-OH or Boc-His(Trt)-Aib-OH can reduce the formation of His-deficient impurities and inhibit the risk of amino acid His racemization, thereby improving the purity of the product and facilitating the purification of the crude product. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a synthetic route diagram of semaglutide in the present invention;

[0049] Figure 2 is the HPLC spectrum of the crude semaglutide in Example 6 of the present invention;

[0050] Figure 3 is the HPLC spectrum of pure semaglutide in Example 6 of the present invention;

[0051] Figure 4 This is the LC-MS spectrum of pure semaglutide in Example 6 of the present invention. DETAILED DESCRIPTION

[0052] The technical scheme of the present invention is further described below in conjunction with the accompanying drawings and examples. The names and English abbreviations of the reagents and instruments used in this application are shown in Table 1.

[0053] Table 1 Reagents and instruments used

[0054]

[0055]

[0056]

[0057] Example 1 Synthesis of semaglutide starting resin Fmoc-Gly-Wang Resin

[0058] First amino acid loading: In a 1000 ml reaction flask, add 27.1 g Wang Resin (substitution degree 0.37 mmol / g, 10 mmol) and 200 ml DMF, stir evenly, heat in a water bath, and set the temperature to T = 35 ± 3 ° C; In a 250 ml activation bottle, add 8.9 g Fmoc-Gly-OH (30 mmol), 4.2 g HOBt (30 mmol) and 100 ml DMF, stir to dissolve, and control the temperature to T in an ice water bath. 内 =5±5°C, continue to add 4.7ml DIC (30mmol), maintain the temperature, and activate for 5-10min. After activation, add the activation solution to the reaction flask, add 0.4g DMAP (3mmol), and stir to react. After 5h of reaction, stop the reaction, transfer the reaction solution to a suitable solid phase reactor, filter, and wash the peptide resin 4 times with 300ml DMF, and drain each time.

[0059] Peptide resin capping: Measure 252 ml DMF, 30 ml acetic anhydride and 18 ml DIPEA (300 ml in total), mix well, add to the solid phase reactor, open the N2 valve, start the reaction, and the reaction temperature is T 内 =35±3°C, react for 0.5h. After the reaction is completed, filter and wash the peptide resin 4 times with 300ml DMF, each time draining to obtain Fmoc-Gly-Wang Resin. The synthetic route of semaglutide is as follows Figure 1 shown.

[0060] Example 2 Semaglutide side chain extension reaction

[0061] Deprotection of the N-terminal amino group of the peptide resin: Add 300 ml of deprotection solution (20% PIP in DMF) to the solid phase reactor, open the N2 valve, and start the reaction. The reaction temperature is T 内=35±3℃, react for 0.5h. After the reaction, filter and wash the peptide resin with 300ml DMF for 6 times, each time draining. Take a sample for NT test, the test result is positive.

[0062] Coupling amino acids or peptides: Pre-activation: Weigh amino acids or peptides (30 mmol) and HOBt (30 mmol), add them to an activation bottle, add 300 ml DMF, stir to dissolve. Control the temperature in an ice water bath T 内 =5±5℃, continue to add DIC (30mmol), maintain the temperature, and activate for 5-10min. Coupling: After activation, add the activation solution to the solid phase reactor, open the N2 valve, and start the reaction. After 2h of reaction, take a sample for NT detection. After the NT test is negative, stop the reaction, filter, and wash the peptide resin with 300ml DMF 4 times, each time draining. The extended reaction of semaglutide peptide resin gives:

[0063] Dde-Lys(Fmoc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin. The coupling order and amount of each amino acid or peptide are shown in Table 2.

[0064] Table 2 Coupling order and dosage of each amino acid or peptide

[0065] Coupling order Amino acid or peptide name MW. Mole / mmol Mass / g 1 Fmoc-Gly-Arg(Pbf)-OH 705.82 30 21.2 2 Fmoc-Arg(Pbf)-OH 648.77 30 19.5 3 Fmoc-Val-OH 339.39 30 10.2 4 Fmoc-Leu-OH 353.41 30 10.6 5 Fmoc-Trp(Boc)-OH 526.58 30 15.8 6 <![CDATA[Fmoc-Ala-OH H2O]]> 329.33 30 9.8 7 Fmoc-Ile-OH 353.41 30 10.6 8 Fmoc-Phe-OH 387.43 30 11.6 9 Dde-Lys(Fmoc)-Glu(OtBu)-OH 717.86 30 21.5

[0066] Example 3 Semaglutide side chain extension reaction

[0067] Deprotection of the N-terminal amino group of the peptide resin: Add 300 ml of deprotection solution (20% PIP in DMF) to the solid phase reactor, open the N2 valve, and start the reaction. The reaction temperature is T 内 =35±3℃, react for 0.5h. After the reaction, filter and wash the peptide resin with 300ml DMF for 6 times, each time draining. Take a sample for NT test, the test result is positive.

[0068] Coupling amino acids or peptides: Pre-activation: Weigh amino acids or peptides (30 mmol) and HOBt (30 mmol), add them to an activation bottle, add 300 ml DMF, stir to dissolve. Control the temperature in an ice water bath T 内 =5±5℃, continue to add DIC (30mmol), maintain the temperature, and activate for 5-10min. Coupling: After activation, add the activation solution to the solid phase reactor, open the N2 valve, and start the reaction. After 2h of reaction, take a sample for NT detection. When the NT test is negative, stop the reaction, filter, and wash the peptide resin 4 times with 300ml DMF, and drain each time.

[0069] Dde-Lys[AEEA-AEEA-γ-Glu(α-OtBu)-Octadecanedioic Acid-18(tert-butoxy)]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin was synthesized by side chain extension reaction of semaglutide. The coupling order and amount of each amino acid or peptide are shown in Table 3.

[0070] Table 3 Coupling order and dosage of each amino acid or peptide

[0071] Coupling order Amino acid or peptide name MW. Mole / mmol Mass / g 1 Fmoc-AEEA-OH 385.41 30 11.6 2 Fmoc-AEEA-OH 385.41 30 11.6 3 Fmoc-Glu-OtBu 425.47 30 12.8 4 <![CDATA[HOOC-C 16 H 32 -COOtBu]]> 370.57 30 11.1

[0072] Example 4 Semaglutide Lys 20 Deprotection of amino acids on the main chain

[0073] Semaglutide Lys 20 Deprotection of amino acid backbone amino groups: Add 300 ml of deprotection solution (2% N2H4·H2O in DMF) to the solid phase reactor, open the N2 valve, start the reaction, and the reaction temperature T 内 =35±3℃, react for 0.5h. After the reaction, filter and wash the peptide resin 6 times with 300ml DMF, each time draining. Take a sample for NT test, the test result is positive. The resin sequence synthesized is:

[0074] H-Lys[AEEA-AEEA-γ-Glu(α-OtBu)-Octadecanedioic Acid-18(tert-butoxy)]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin.

[0075] Example 5: Main chain extension reaction of semaglutide

[0076] Deprotection of the N-terminal amino group of the peptide resin: Add 300 ml of deprotection solution (20% PIP in DMF) to the solid phase reactor, open the N2 valve, and start the reaction. The reaction temperature is T 内 =35±3℃, react for 0.5h. After the reaction, filter and wash the peptide resin with 300ml DMF for 6 times, each time draining. Take a sample for NT test, the test result is positive.

[0077] Coupling amino acids or peptides: Pre-activation: Weigh amino acids or peptides (30 mmol) and HOBt (30 mmol), add them to the activation bottle, add 300 ml DMF, stir to dissolve. Control the temperature in an ice water bath T 内 =5±5℃, continue to add DIC (30mmol), maintain the temperature, and activate for 5-10min. Coupling: After activation, add the activation solution to the solid phase reactor, open the N2 valve, and start the reaction. After 2h of reaction, take a sample for NT detection. After the NT test is negative, stop the reaction, filter, and wash the peptide resin with 300ml DMF 4 times, each time draining. Obtain 60.35g of peptide resin with a yield of 71.8%. The sequence of the synthetic peptide resin is Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-S er(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys[AEEA-AEEA-γ-Glu(α-OtBu)-Octadecanedioic Acid-18(tert-butoxy)]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin. The coupling order and amount of each amino acid or peptide segment are shown in Table 4.

[0078] Table 4 Coupling order and dosage of each amino acid or peptide

[0079]

[0080]

[0081] Example 6 Cleavage to obtain crude semaglutide

[0082] Take 87.5 ml TFA, 5 ml EDT, 2.5 ml TIS, 2.5 ml PhOH and 2.5 ml purified water in sequence, add them to the lysis bottle, mix well, and precool to T 内 = 0-10°C. Under stirring, 10 g of semaglutide fully protected resin was slowly added to the cleavage bottle to start cleavage. Reaction temperature T 内 =35±3℃, react for 2-3h. After the cleavage is complete, filter and rinse the filter cake with 10ml TFA for 2 times, each time draining. Combine the filtrates and slowly add them into 1000ml cold MTBE under stirring, and a white solid will precipitate.

[0083] Centrifuge, remove the supernatant, and continue to use 500 ml of cold MTBE to beat, wash, and centrifuge the solid to complete the washing. Wash three times in total to obtain 3.9 g of crude semaglutide, with a total yield of 57.07% and a crude HPLC purity of 77.71%. The HPLC detection spectrum of crude semaglutide is as follows Figure 2 The HPLC detection spectrum of pure semaglutide is shown in Figure 3 The LC-MS detection spectrum of pure semaglutide is shown in Figure 4 shown.

[0084] Comparative Example 1

[0085] 1. Synthesize semaglutide starting resin Fmoc-Gly-Wang Resin.

[0086] According to the method of Example 1, Fmoc-Gly-Wang Resin was synthesized with a reaction scale of 4 mmol to obtain 11.2 g of the starting peptide resin.

[0087] 2. Sequential coupling of amino acids to complete the semaglutide fragment-peptide resin extension

[0088] According to the method of Example 2, 5.5 g of the starting peptide resin Fmoc-Gly-Wang Resin of step 1 was used to synthesize 7.2 g of semaglutide fragment fully protected peptide resin H-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin; after cleavage, 798 mg of crude peptide H-Arg-Gly-Arg-Gly-OH was obtained, with a crude yield of 89.76%.

[0089] The coupling sequence and dosage of each amino acid or peptide segment are shown in Table 5.

[0090] Table 5 Coupling order and dosage of each amino acid or peptide

[0091] Coupling order Amino acid or peptide name MW. Mole / mmol Mass / g 1 Fmoc-Arg(Pbf)-OH 648.77 6 3.9 2 Fmoc-Gly--OH 297.31 6 1.8 3 Fmoc-Arg(Pbf)-OH 648.77 6 3.9

[0092] 3. Use the S29-S30 fragment to complete the semaglutide fragment-peptide resin extension

[0093] According to the method of Example 2, 5.5 g of the starting peptide resin Fmoc-Gly-Wang Resin of step 1 was used to synthesize 7.7 g of semaglutide fragment fully protected peptide resin H-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin, and 903 mg of crude peptide H-Arg-Gly-Arg-Gly-OH was obtained after cleavage, with a crude yield of 101.57%.

[0094] The coupling order and dosage of each amino acid or peptide segment are shown in Table 6.

[0095] Table 6 Coupling order and dosage of each amino acid or peptide

[0096] Coupling order Amino acid or peptide name MW. Mole / mmol Mass / g 1 Fmoc-Gly-Arg(Pbf)-OH 705.82 6 4.2 2 Fmoc-Arg(Pbf)-OH 648.77 6 3.9

[0097] It can be seen that through the comparison of step 2 (sequential coupling of amino acids to complete the semaglutide fragment-peptide resin extension) and step 3 (using the S29-S30 fragment to complete the semaglutide fragment-peptide resin extension), it is proved that the use of the S29-S30 fragment Fmoc-Gly-Arg(Pbf)-OH can effectively inhibit the DKPS side reaction and improve the synthesis yield.

[0098] Comparative Example 2

[0099] Semaglutide was synthesized according to the methods of Examples 1 to 6, wherein the amino acid residue Lys 20 and Glu 21 The crude semaglutide obtained had an HPLC purity of 58.85%. Compared with the crude semaglutide obtained in Example 6, a major impurity with an HPLC purity of 23.33% (RT = 21.98 min, RRT = 1.22) was added. The structure of the impurity was identified as:

[0100] HOOC-C 16 H 32 -CONH-γ-Glu-AEEA-AEEA-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0101] Comparative Example 3

[0102] Semaglutide was synthesized according to the methods of Examples 1 to 6, wherein the amino acid residue His 1 and Aib 2 The crude semaglutide obtained had an HPLC purity of 69.57%. Compared with the crude semaglutide obtained in Example 6, a major impurity with an HPLC purity of 10.13% (RT = 17.59 min, RRT = 0.98) was added. The structure of the impurity was identified as:

[0103] H-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Octadecanedioic)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0104] Comparative Example 4

[0105] Semaglutide was synthesized according to the methods of Examples 1 to 6, wherein the amino acid residue Glu 3 Glu 15 , Gly 4 and Gly 16 The crude semaglutide was connected in the form of Fmoc-Glu(OtBu)-OH and Fmoc-Gly-OH. The HPLC purity of the crude semaglutide was 72.53%. The LC-MS spectrum showed that several new impurities with added Gly and lacked Gly were generated, and these impurities added a lot of difficulties to the subsequent purification process.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for synthesizing semaglutide, characterized in that: According to the amino acid sequence of semaglutide, amino acids or peptides are sequentially coupled from C-terminus to N-terminus by solid phase synthesis method to obtain semaglutide fully protected resin, and semaglutide is obtained by cleavage and purification; the peptide includes dipeptide S20-S21 formed by amino acids at positions 20-21, and S20-S21 is R1-Lys(Fmoc)-Glu(OtBu)-OH, Wherein, R1 is Dde or ivDde, Fmoc is a side chain protecting group, and R2 is a main chain protecting group; The peptide segment also includes dipeptide S1-S2 formed by amino acids at positions 1-2, dipeptide S3-S4 formed by amino acids at positions 3-4, dipeptide S15-S16 formed by amino acids at positions 15-16, and dipeptide S29-S30 formed by amino acids at positions 29-30. Among them, S1-S2 is R2-His(Trt)-Aib-OH, R2 is Fmoc or Boc; S3-S4 or S15-S16 is Fmoc-Glu(OtBu)-Gly-OH; S29-S30 is Fmoc-Gly-Arg(Pbf)-OH; The following steps are involved: S1: Using Wang resin as a solid phase synthesis carrier, Fmoc-Gly-OH, Fmoc-Gly-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH.H2O, Fmoc-Ile-OH, Fmoc-Phe-OH and R1-Lys(Fmoc)-Glu(OtBu)-OH were coupled in sequence to obtain R1-Lys(Fmoc)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin; S2: Remove the side chain protecting group Fmoc in S20-S21, and couple Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu and HOOC-C in sequence 16 H 32 -COOtBu, get R1-Lys[AEEA-AEEA-γ-Glu(α-OtBu)-Octadecanedioic Acid-18(tert-butoxy)]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang Resin; S3: Remove the main chain protecting group R1 in S20-S21, and couple Fmoc-Ala-OH.H2O, Fmoc-Ala-OH.H2O, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Va l-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-Gly-OH and R2-His(Trt)-Aib-OH to obtain semaglutide fully protected resin; S4: The semaglutide fully protected resin is subjected to cleavage, sedimentation, centrifugation and drying to obtain semaglutide.

2. The method for synthesizing semaglutide according to claim 1, characterized in that: In step S1, the degree of substitution of the Wang resin is 0.1-1.0 mmol / g.

3. A method for synthesizing semaglutide according to claim 2, characterized in that: The degree of substitution of the Wang resin is preferably 0.1-0.4 mmol / g.

4. The method for synthesizing semaglutide according to claim 1, characterized in that: In step S2, the deprotection agent of Fmoc is a solution of piperidine in N,N-dimethylformamide with a concentration of 20% to 50%.

5. A method for synthesizing semaglutide according to claim 4, characterized in that: The concentration of the piperidine N,N-dimethylformamide solution is preferably 20%.

6. The method for synthesizing semaglutide according to claim 1, characterized in that: In step S3, the deprotecting agent of R1 is a solution of hydrazine hydrate in N,N-dimethylformamide with a concentration of 1% to 5%.

7. The method for synthesizing semaglutide according to claim 6, characterized in that: The concentration of the hydrazine hydrate N,N-dimethylformamide solution is preferably 2% to 3%.

8. The method for synthesizing semaglutide according to claim 1, characterized in that: Steps S1-S3 may also include one or more of the following features: (1) The condensation reagent used in the coupling is any one or more of A, B, C, and D, Wherein, A is selected from any one of DIC, DCC, and EDCI, B is selected from any one of HOOBT, HOBT, and BOAT, C is selected from any one of DIPEA and NMM, and D is selected from any one of DMAP, HBTU, HATU, TBTU, and PyBOP; (2) The coupling reaction medium is one or more of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-diethylformamide, N-methylpyrrolidone and dimethyl sulfoxide; (3) The coupling and deprotection temperature is 25-45°C.

9. The method for synthesizing semaglutide according to claim 8, characterized in that: The coupling reaction medium is preferably N,N-dimethylformamide.

10. The method for synthesizing semaglutide according to claim 8, characterized in that: The coupling and deprotection temperature is preferably 30-40°C.

11. The method for synthesizing semaglutide according to claim 8, characterized in that: By mass ratio, Wang resin: amino acid or peptide: condensation reagent = 1:2:2~1:4:

4.

12. The method for synthesizing semaglutide according to claim 11, characterized in that: The preferred ratio of Wang resin: amino acid or peptide: condensation reagent is 1:3:

3.

13. The method for synthesizing semaglutide according to claim 1, characterized in that: In step S4, the cleavage solution used for the cleavage is a mixture of trifluoroacetic acid, triisopropylsilane, 1,2-ethanedithiol, phenol and water.

14. The method for synthesizing semaglutide according to claim 13, characterized in that: Preferably, by mass ratio, trifluoroacetic acid:1,2-ethanedithiol:triisopropylsilane:phenol:water=87.5:5:2.5:2.5:2.

5.

15. The method for synthesizing semaglutide according to claim 1, characterized in that: In step S4, the sedimentation liquid used for the sedimentation is one or more of n-hexane, diethyl ether and methyl tert-butyl ether.

16. The method for synthesizing semaglutide according to claim 15, characterized in that: The sedimentation liquid is preferably methyl tert-butyl ether.

Images