A synthetic method of semaglutide

Abstract

The invention discloses a method for synthesizing semaglutide, comprising synthesizing a first peptide fragment II, synthesizing a second peptide fragment IV, and one-pot isocyanate-mediated connection / de-Fmoc reaction of the first peptide fragment II and the second peptide fragment IV. The connection and de-Fmoc protection of the polypeptide fragments in the method of the invention are carried out in the same container, and no intermediate purification is required, and the connection efficiency is high. The first peptide fragment II and the second peptide fragment IV used are peptide fragments with unprotected side chains, which will not cause amino acid diastereomerization during the reaction, are easy to operate, and are rapidly converted, and have good solubility. They can be purified by high-performance liquid chromatography before coupling the polypeptide fragments. In the final liquid chromatography purification step, the impurities are not defective peptides lacking one or several amino acids, but uncondensed partial fragments, which greatly reduce the impurities generated by racemization, oxidation, and hydrolysis of the final product, reduce the difficulty of purification of the final finished product, and save costs.

Description

Technical Field

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

[0002] Diabetes is one of the top ten chronic diseases in the world. It is a metabolic disease characterized by chronic hyperglycemia. Most patients with diabetes are type 2 patients, accounting for more than 90%. According to statistics from the International Diabetes Federation (IDF), by the end of 2017, 425 million adults in the world had diabetes. It is estimated that by 2045, the number of patients worldwide will reach 629 million. The prevalence of diabetes in my country has exceeded the global average. In 2017, the number of people with diabetes reached 114 million, ranking first in the world. It is expected to further increase to about 150 million by 2045.

[0003] Semaglutide is a long-acting GLP-1 analogue for subcutaneous injection once a week developed by diabetes research and development giant Novo Nordisk. On December 5, 2017, the U.S. FDA approved the 0.5mg and 1mg prefilled injection pens (Ozempic) of Semaglutide for the treatment of adult patients with type 2 diabetes. On September 20, 2019, the FDA formally approved Novo Nordisk's application for the marketing of oral Semaglutide (Rybelsus), which is used in combination with diet and exercise to improve blood sugar control in patients with type 2 diabetes. In addition, on April 29 this year, the China National Drug Administration approved the marketing of Semaglutide in two prefilled injection pens of 0.5mg and 1mg in China. Structurally, Semaglutide is a GLP-1 (7-37) chain in which Ala at position 8 is replaced by Aib, Lys at position 34 is replaced by Arg, and Lys at position 26 is connected to an octadecanoic acid fatty chain. After PEG modification, it can not only bind tightly to albumin and cover up the DPP-4 enzyme hydrolysis site, but also reduce renal excretion, prolong the biological half-life, and achieve a long circulation effect.

[0004] The chemical name of semaglutide is N6,26-{18-[N-(17-carboxyheptadecanoyl)-L-γ-glutamyl]-10-oxo-3,6,12,15-tetraoxa-9,18-diazaocta decanoyl}-[8-(2-amino-2-propanoic acid),34-L-arginine]human glucagon-like peptide 1(7-37), and the molecular formula is C 187 H291 N 45 O 59 , molecular weight 4113.5775, sequence H-His-Aib-Glu-Gly-Thr 5 -Phe-Thr-Ser-Asp-Val 10 -Ser-Ser-Tyr-Leu-Glu 15 -Gly-Gln-Ala-Ala-Lys 20 (AEEA-AEEA-γ-Glu-Octadecanedioic Acid)-Glu-Phe-Ile-Ala-Trp 25 -Leu-Val-Arg-Gly-Arg 30 -Gly-OH. The structural formula is shown in Formula 1.

[0005]

[0006] Currently, the synthesis method of semaglutide reported mainly adopts solid phase stepwise coupling method (Fmoc-SPPS), such as patents: CN103848910B, CN106478806A, CN108359006A, CN106928343A, CN101133082A. However, the synthesis of semaglutide by conventional solid phase stepwise coupling method (Fmoc-SPPS) is very challenging, mainly reflected in the serious shrinkage of the resin during the coupling process, resulting in a large number of missing peptides, which not only significantly reduces the yield, but also increases the difficulty of purification of the final product, which greatly increases the research and development cost. In recent years, the synthesis of semaglutide by condensation of fully protected peptide fragments has developed rapidly, such as patents: CN106749613A, CN104356224A, CN108359006A, CN109456401A, CN109627317A. The basic idea of ​​this strategy is: first, several fully protected small fragments of polypeptides are obtained by solid phase synthesis, and then condensed in liquid phase or on a solid phase carrier to obtain a fully protected long peptide chain, and finally the side chain protecting group is cleaved to obtain semaglutide. However, since all polypeptide fragments are fully protected, poor solubility becomes the most difficult problem encountered using this method, and the length of each fragment should not be too long and the purity is not high. There are many impurities generated during the coupling process, and there are many impurities in the final crude semaglutide peptide, and purification requires a lot of manpower and material costs. Summary of the invention

[0007] In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide a method for synthesizing semaglutide. The specific technical scheme is as follows:

[0008] The present invention provides a method for synthesizing semaglutide, comprising the following steps:

[0009] Synthesis of the first peptide fragment II: (1) preparing Fmoc-Gly-Wang resin; (2) sequentially coupling amino acids with N-terminal Fmoc protection and side chain protection by solid phase synthesis to obtain a fully protected peptide fragment II solid phase resin; (3) removing the resin and the protecting group by cleavage and purifying to obtain the first peptide fragment II, wherein Lys is the pentapeptide fragment I;

[0010] The pentapeptide fragment I is Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH;

[0011] The first peptide fragment II is H-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Oct)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH;

[0012] Synthesis of the second peptide fragment IV: the second peptide fragment IV is Fmoc-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-COSH;

[0013] One-pot isonitrile-mediated coupling / de-Fmoc reaction: The first peptide fragment II and the second peptide fragment IV are coupled in the presence of a coupling agent. After the reaction, the Fmoc protection is removed and purified to obtain semaglutide.

[0014] In the above technical solution of the present invention, a liquid phase method is used to synthesize the pentapeptide fragment I, comprising the steps of:

[0015] 1) Oct(OtBu) and HOSu are coupled in the presence of a coupling agent to obtain Oct(OtBu)-OSu, and then Oct(OtBu)-OSu and H-Glu-OtBu react to obtain a dipeptide fragment Oct(OtBu)-Glu-OtBu;

[0016] 2) The dipeptide fragment Oct(OtBu)-Glu-OtBu and HOSu are coupled in the presence of a coupling agent to obtain Oct(OtBu)-Glu(OSu)-OtBu, and then Oct(OtBu)-Glu(OSu)-OtBu and AEEA-AEEA are reacted to obtain the tetrapeptide fragment Oct(OtBu)-Glu(AEEA-AEEA)-OtBu;

[0017] 3) The tetrapeptide fragment Oct(OtBu)-Glu(AEEA-AEEA)-OtBu and Fmoc-Lys-OH·HCl were coupled in the presence of a coupling agent to obtain the pentapeptide fragment I Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH.

[0018] Furthermore, the coupling agent in 1) and 2) is selected from DCC, DIC or EDC·HCl, preferably EDC·HCl;

[0019] 3) The coupling agent is selected from EDC·HCl+A, DIPEA+B, DIPEA+A+B or CDI, wherein A is HOBt or HOAt, and B is PyBOP, PyAOP, HATU, HBTU or TBTU.

[0020] In the above technical solution of the present invention, the preparation method of Fmoc-Gly-Wang resin is: in the presence of a coupling agent, the activated Wang resin is coupled with Fmoc-Gly-OH;

[0021] Preferably, the Wang resin substitution degree is 0.6 to 1.0 mmol / g, preferably 0.7 to 0.9 mmol / g;

[0022] Preferably, when preparing Fmoc-Gly-Wang resin, the coupling agent is DIC+A+DMAP, and A is HOBt or HOAt;

[0023] Preferably, in terms of molar ratio, DIC:A:DMAP=1.2:1.1:0.1;

[0024] Preferably, when preparing Fmoc-Gly-Wang resin, the solvent of the coupling agent is selected from one or more of NMP, THF, DCM, DMF and DMSO.

[0025] In the above technical solution of the present invention, the fully protected peptide fragment II solid phase resin is H-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin;

[0026] When synthesizing the fully protected peptide fragment II solid phase resin, the amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled: Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, 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(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH, Fmoc-Ala-OH, Fmoc-Ala-OH and Fmoc-Gln(Trt)-OH;

[0027] When synthesizing the fully protected peptide fragment II solid phase resin, the coupling agent used is DIC+A or DIPEA+A+B, wherein A is HOBt or HOAt, and B is PyBOP, PyAOP, HATU, HBTU or TBTU;

[0028] Preferably, in terms of molar ratio, DIC:A=1.2:1.1;

[0029] Preferably, in terms of molar ratio, DIPEA:A:B=1.5:1.1:1.0;

[0030] When synthesizing the fully protected peptide fragment II solid phase resin, the solvent of the coupling agent is selected from one or more of DMF, DCM, NMP, THF and DMSO;

[0031] The cleavage solution used in the synthesis of the first peptide fragment II (3) is a combination of TFA, H2O, PhOMe and PhSMe;

[0032] Preferably, the volume ratio of TFA, H2O, PhOMe and PhSMe is 90:5:4:1.

[0033] In the above technical scheme of the present invention, the steps of synthesizing the second peptide fragment IV include: (1) preparing Fmoc-Gly-2CTC resin; (2) sequentially coupling amino acids with N-terminal Fmoc protection and side chain protection through solid phase synthesis to obtain a fully protected peptide fragment III solid phase resin; (3) removing the resin by cleavage to obtain a fully protected peptide fragment III; (4) coupling the fully protected peptide fragment III with thiol to obtain a fully protected peptide fragment IV; (5) removing the protecting group by cleavage and purifying to obtain the second peptide fragment IV.

[0034] Furthermore, the preparation method of Fmoc-Gly-2CTC resin is as follows: in the presence of a coupling agent, the activated 2CTC resin is coupled with Fmoc-Gly-OH;

[0035] Preferably, the degree of substitution of the 2CTC resin is 0.4 to 1.0 mmol / g, preferably 0.6 to 0.8 mmol / g;

[0036] Preferably, when preparing Fmoc-Gly-2CTC resin, the solvent of the coupling agent is selected from one or more of DMF, DCM, NMP, THF and DMSO.

[0037] In the above technical solution of the present invention, the fully protected peptide fragment III solid phase resin is Fmoc-His(trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-2CTC resin;

[0038] When synthesizing the fully protected peptide fragment III solid phase resin, the amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled: 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, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Fmoc-His(trt)-OH;

[0039] When synthesizing the fully protected peptide fragment III solid phase resin, the coupling agent used is DIC+A or DIPEA+A+B, wherein A is HOBt or HOAt, and B is PyBOP, PyAOP, HATU, HBTU or TBTU;

[0040] Preferably, in terms of molar ratio, DIC:A=1.2:1.1, DIPEA:A:B=1.5:1.1:1.0;

[0041] Preferably, when synthesizing the fully protected peptide fragment III solid phase resin, the solvent of the coupling agent is selected from one or more of DMF, DCM, NMP, THF and DMSO;

[0042] The cleavage solution used in the synthesis of the second peptide fragment IV (3) is a mixture of TFE and DCM or a mixture of HFIP and DCM; preferably, it is a mixture of TFE and DCM, and more preferably, the volume ratio of TFE to DCM is 1:4.

[0043] In the above technical solution of the present invention, the thiol in the synthesis of the second peptide fragment IV (4) is 2,4,6-trimethoxybenzylthiol;

[0044] The coupling agent used in the synthesis of the second peptide fragment IV (4) is DIC+A or DIPEA+A+B, wherein A is HOBt or HOAt, and B is PyBOP, PyAOP, HATU, HBTU or TBTU;

[0045] Preferably, in terms of molar ratio, DIC:A=1.2:1.1, DIPEA:A:B=1.5:1.1:1.0

[0046] Preferably, the solvent of the coupling agent is selected from one or more of DMF, DCM, NMP, THF and DMSO;

[0047] The reagent used for cleavage in the synthesis of the second peptide fragment IV (5) is a combination of TFA, H2O, PhOMe and PhSMe, preferably, the volume ratio of TFA, H2O, PhOMe and PhSMe is 90:5:4:1.

[0048] In the above technical scheme of the present invention, in the one-pot isonitrile-mediated connection / de-Fmoc reaction, the coupling agent is tBuNC and HOBt, and the solvent of the coupling agent is selected from one or more of DMA, DMF, DCM, NMP, THF and DMSO;

[0049] The Fmoc removal reagent is selected from DBU, diethylamine or piperidine, preferably piperidine.

[0050] The beneficial effects of the present invention are:

[0051] 1. The present invention provides a one-pot isonitrile-mediated connection / de-Fmoc method for synthesizing semaglutide. The connection and de-Fmoc protection of polypeptide fragments in the method are carried out in the same container, and no intermediate purification is required. The connection efficiency is high and the reaction conditions are mild. The first peptide fragment II and the second peptide fragment IV used are peptide fragments with unprotected side chains. They will not cause amino acid isomerization during the reaction, are easy to operate, and are rapidly converted. They have good solubility and can be purified by high performance liquid chromatography before coupling the polypeptide fragments. In the final liquid chromatography purification step, the impurities are not defective peptides lacking one or several amino acids, but uncondensed partial fragments, which greatly reduce the impurities generated by racemization, oxidation, and hydrolysis of the final product, reduce the difficulty of purification of the final product, and save costs.

[0052] 2. During the solid phase synthesis of the first peptide fragment II of the present invention, Lys 20 The method for synthesizing the pentapeptide fragment I provided by the present invention adopts the pentapeptide fragment Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH, which is simple to operate and has a short synthesis path, and a high-purity pentapeptide fragment can be obtained in only five steps.

[0053] 3. The first peptide fragment II and the second peptide fragment IV used in the present invention can be synthesized simultaneously, which improves the synthesis efficiency of semaglutide, and the total yield of synthesizing semaglutide is more than 30%. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The synthetic route of the pentapeptide fragment I Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH;

[0055] Figure 2 This is the synthetic route of the first peptide fragment II;

[0056] Figure 3 This is the synthetic route of the second peptide fragment IV;

[0057] Figure 4 It is a liquid phase one-pot isonitrile-mediated ligation / Fmoc removal reaction;

[0058] Figure 5 It is the mass spectrum of pentapeptide fragment I Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH;

[0059] Figure 6 HPLC spectrum of pentapeptide fragment I Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH;

[0060] Figure 7 is the mass spectrum of semaglutide;

[0061] Figure 8 This is the HPLC spectrum of semaglutide. DETAILED DESCRIPTION

[0062] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0063] The abbreviations and their meanings used in the present invention are shown in the following table:

[0064]

[0065]

[0066] Example 1: Synthesis of Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH Pentapeptide Fragment I

[0067] The synthesis of pentapeptide fragment I comprises the following steps:

[0068] 1. Synthesis of Oct(OtBu)-Glu-OtBu dipeptide fragment

[0069] Weigh Oct(OtBu)-OH (92.7 g, 250 mmol) and HOSu (31.7 g, 275 mmol) and dissolve them in 1000 mL DMF, add EDC·HCl (71.9 g, 375 mmol), and react at room temperature for 16 hours. After the reaction is completed, slowly add 2000 mL of purified water to the reaction solution, precipitate a large amount of precipitate, filter, and then use purified water to beat and wash three times, drain to obtain Oct(OtBu)-OSu activated ester. Weigh H-Glu-OtBu (61.0 g, 300 mmol) and dissolve it in 400 mL of saturated NaHCO3 aqueous solution. Dissolve the obtained Oct(OtBu)-OSu activated ester with 1200 ml of THF and slowly add it to the reaction solution, and react at room temperature for 16 hours. After the reaction, most of the THF was removed by rotary evaporation, and then the pH of the reaction solution was adjusted to 3-4 with 15% citric acid, a large amount of oil was precipitated, and then extracted three times with EtOAc, the organic phases were combined, and then washed three times with brine, dried over anhydrous sodium sulfate overnight, filtered, and the solvent was removed by rotary evaporation. 132.1 g of Oct(OtBu)-Glu-OtBu dipeptide fragment was obtained by oil pump drying, and the yield was 95.0%.

[0070] 2. Synthesis of Oct(OtBu)-Glu(AEEA-AEEA)-OtBu tetrapeptide fragment

[0071] Oct(OtBu)-Glu-OtBu dipeptide fragment (132.1g, 238mmol) and HOSu (30.1g, 262mmol) were dissolved in 1000mL DMF, EDC·HCl (68.5g, 357mmol) was added, and the mixture was reacted at room temperature for 6 hours. After the reaction, 2000mL purified water was slowly added to the reaction solution to precipitate a large amount of precipitate, which was filtered and then washed three times with purified water and dried to obtain Oct(OtBu)-Glu(OSu)-OtBu activated ester. AEEA-AEEA (80.8g, 262mmol) was weighed and dissolved in 400mL saturated NaHCO3 aqueous solution. The obtained Oct(OtBu)-Glu(OSu)-OtBu activated ester was dissolved in 1200ml THF and slowly added to the reaction solution and reacted at room temperature for 16h. After the reaction, most of the THF was removed by rotary evaporation, and then the pH of the reaction solution was adjusted to 3-4 with 15% citric acid to precipitate a large amount of oil, which was then extracted three times with EtOAc. The organic phases were combined and then washed three times with brine, dried overnight with anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The product was dried by oil pump to obtain 176.8 g of Oct(OtBu)-Glu(AEEA-AEEA)-OtBu tetrapeptide fragment with a yield of 87.9%.

[0072] 3. Synthesis of Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH pentapeptide fragment I

[0073] Weigh Fmoc-Lys-OH·HCl (12.2 g, 30.0 mmol), dissolve it in 50 ml DMF, and cool it to 0-5°C for later use. Then weigh the Oct(OtBu)-Glu(AEEA-AEEA)-OtBu tetrapeptide fragment (16.9 g, 20 mmol) of step 1 in Example 1 above, dissolve it in 50 mL DMF, cool it to 0-5°C, add CDI (4.9 g, 30 mmol), continue stirring for 0.5 h, then drop the activation solution into the Fmoc-Lys-OH·HCl DMF solution, and continue the reaction for 0.5 h after the addition is complete. After the reaction, 400 ml of purified water was added to the reaction solution, and semi-preparative purification was carried out using a 100×250 mm reverse phase C18 column. The detection wavelength was 220 nm, the mobile phase was H2O (phase A) and MeOH (phase B) containing 0.1% TFA, the flow rate was 200 ml / min, the gradient was B%: 75%-95%-95%, and the target components were collected, concentrated, extracted, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 22.1 g of pure pentapeptide fragment I Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH, and the total yield was 92.5%. Figure 5 is the mass spectrum of pentapeptide fragment I, Figure 6 This is the HPLC spectrum of pentapeptide fragment I.

[0074] Example 2: Synthesis of Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH Pentapeptide Fragment I

[0075] The synthesis of pentapeptide fragment I comprises the following steps:

[0076] 1. Synthesis of Oct(OtBu)-Glu-OtBu dipeptide fragment, same as in Example 1;

[0077] 2. Synthesis of Oct(OtBu)-Glu(AEEA-AEEA)-OtBu tetrapeptide fragment, same as in Example 1;

[0078] 3. Synthesis of Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH pentapeptide fragment I

[0079] Weigh Fmoc-Lys-OH·HCl (12.2 g, 30.0 mmol), dissolve it in 50 ml DMF, and cool it to 0-5°C for use. Then weigh the Oct(OtBu)-Glu(AEEA-AEEA)-OtBu tetrapeptide fragment (16.9 g, 20 mmol) and PyBOP (10.4, 20 mmol) of step 1 in the above Example 1, dissolve them in 50 mL DMF, cool it to 0-5°C, and then drop DIPEA (6.6 ml, 40 mmol). After the addition is complete, continue stirring for 1 hour, then drop the activation solution into the Fmoc-Lys-OH·HCl DMF solution, and continue the reaction for 2 hours after the addition is complete. After the reaction, 400 ml of purified water was added to the reaction solution, and semi-preparative purification was carried out using a 100×250 mm reverse phase C18 column. The detection wavelength was 220 nm, the mobile phase was H2O (phase A) and MeOH (phase B) containing 0.1% TFA, the flow rate was 200 ml / min, the gradient was B%: 75%-95%-95%, and the target components were collected, concentrated, extracted, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 20.4 g of pure pentapeptide fragment I Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH, and the total yield was 85.4%.

[0080] Example 3: Synthesis of Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH Pentapeptide Fragment I

[0081] The synthesis of pentapeptide fragment I comprises the following steps:

[0082] 1. Synthesis of Oct(OtBu)-Glu-OtBu dipeptide fragment, same as in Example 1;

[0083] 2. Synthesis of Oct(OtBu)-Glu(AEEA-AEEA)-OtBu tetrapeptide fragment, same as in Example 1;

[0084] 3. Synthesis of Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH pentapeptide fragment I

[0085] Weigh Fmoc-Lys-OH·HCl (12.2 g, 30.0 mmol), dissolve it in 50 ml DMF, and cool it to 0-5°C for later use. Then weigh the Oct(OtBu)-Glu(AEEA-AEEA)-OtBu tetrapeptide fragment (16.9 g, 20 mmol) and HOAt (4.1, 30 mmol) in step 2 of Example 1, dissolve them in 50 mL DMF, cool it to 0-5°C, add EDC·HCl (3.8 g, 20 mmol), continue stirring for 2 h, then drop the activation solution into the Fmoc-Lys-OH·HCl DMF solution, and continue the reaction for 2 h after the addition is complete. After the reaction, 400 ml of purified water was added to the reaction solution, and semi-preparative purification was carried out using a 100×250 mm reverse phase C18 column. The detection wavelength was 220 nm, the mobile phase was H2O (phase A) and MeOH (phase B) containing 0.1% TFA, the flow rate was 200 ml / min, the gradient was B%: 75%-95%-95%, and the target components were collected, concentrated and extracted, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 20.8 g of pure pentapeptide fragment I Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH, with a yield of 87.0%.

[0086] Example 4: Synthesis of Semaglutide

[0087] 1. Synthesis of the first peptide fragment II

[0088] (1) Preparation of Fmoc-Gly-Wang resin with a substitution degree of 0.41 mmol

[0089] Weigh 48.2g of Wang resin with a substitution degree of 0.83mmol / g, add it to the reaction column, and swell it with DMF / DCM (1:1) for 30 minutes. Weigh Fmoc-Gly-OH (59.6g, 200mmol), HOBt (32.4g, 220mmol) and DMAP (2.4g, 2mmol), dissolve them in 150mL DMF, add DIC (40.6mL, 240mmol) under ice bath, activate it for 5 minutes and add it to the reaction column. After reacting for 2.5h, wash the resin with 150mL x 3DMF, add 70mL acetic anhydride and 60mL pyridine, and seal it for 2h. After the reaction is completed, wash the resin with 150mL x5 DMF, shrink it with 150mL x3 MeOH, and drain it to obtain Fmoc-Gly-Wang resin, and the substitution degree is measured to be 0.41mmol / g.

[0090] (2) Preparation of solid phase resin of fully protected peptide fragment II

[0091] Weigh 24.4 g of the Fmoc-Gly-Wang resin with a substitution degree of 0.41 mmol / g prepared in step (1) in a solid phase reaction column and swell with DMF / DCM (1:1) for 30 minutes. Deprotect with 80 mL x 2 DBLK for 5 min + 7 min, and wash the resin with 100 mL x 5 DMF. Weigh Fmoc-Arg(Pbf)-OH (32.4 g, 50.0 mmol) and HOBT (8.1 g, 55.0 mmol) and dissolve them with 60 mL DMF. Add DIC (10.2 mL, 60.0 mmol) to activate for 5 min under ice bath, then add the mixed solution to the reaction column and react at room temperature for 2 hours. After the reaction is completed, wash the resin with 80 mL x 3 DMF, add 80 mL DBLK x 2 for deprotection for 5 min + 7 min, and wash the resin with 80 mL x 5 DMF. Repeat the above coupling operation, and continue to couple Fmoc-Gly-OH, 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(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH, Fmoc-Ala-OH, Fmoc-Ala-OH and Fmoc-Gln(Trt)-OH in sequence according to the peptide sequence. After the reaction is completed, the resin is washed with 200mL x 5DMF, shrunk with 200mL x 3MeOH, and dried to obtain the fully protected peptide fragment II solid phase resin.

[0092] (3) Preparation of the first peptide fragment II

[0093] The resin obtained in step (2) was added to a 1L round-bottom flask, and 500mL of the pre-prepared lysis solution TFA / H2O / PhOMe / PhSMe (90:5:4:1, v / v) was added. The mixture was stirred magnetically at room temperature for 2 hours, and the resin was filtered under reduced pressure to collect the filtrate. The resin was washed with a small amount of TFA, and the filtrate was combined. The filtrate was slowly added to 5.0L of ice ether for precipitation, centrifuged, washed with 2.5L x 3 ice ether, and dried with nitrogen to obtain 25.4g of the crude peptide of the first peptide fragment II.

[0094] (4) Purification of the first peptide fragment II

[0095] Weigh 20.0 g of the crude product of the first peptide fragment II obtained in step (3), add 4 L of 0.6% ammonia water to dissolve it, and use a 100×250 mm reverse phase C18 column for semi-preparative purification. The detection wavelength is 220 nm, the mobile phase is water containing 0.1% TFA (phase A) and acetonitrile (phase B), the flow rate is 200 ml / min, the gradient is: B%: 32%-52%, 60 min, collect the target component, and lyophilize the above concentrate to obtain 10.7 g of the first peptide fragment II, with a yield of 56.3%.

[0096] 2. Synthesis of the second peptide fragment IV

[0097] (1) Preparation of Fmoc-Gly-2CTC Resin with a substitution degree of 0.36 mmol

[0098] Weigh 66.7g of 2CTC Resin with a substitution degree of 0.60mmol / g, add it to the reaction column, and swell it with DMF / DCM (1:1) for 30 minutes. Weigh Fmoc-Gly-OH (23.8g, 80mmol) and dissolve it in 200mL DMF and add it to the reaction column. Add DIPEA (20.8ml, 80mmol) and react at room temperature for 2h, then add 20mL MeOH and DIPEA (10.4ml, 40mmol) and continue to react for 0.5h. After the reaction is completed, wash the resin with 200mL x5 DMF, shrink it with 200mL x3 tertiary methyl ether, and drain it to obtain Fmoc-Gly-2CTC Resin resin, and the substitution degree is measured to be 0.36mmol / g.

[0099] (2) Preparation of fully protected peptide fragment III

[0100] Weigh 55.6 g of Fmoc-Gly-2CTC Resin with a substitution degree of 0.36 mmol / g prepared in step (1) in a solid phase reaction column and swell with DMF / DCM (1:1) for 30 minutes. Deprotect with 200 mL x 2 DBLK for 5 min + 7 min, and wash with 200 mL x 5 DMF. Weigh Fmoc-Glu(OtBu)-OH (42.5 g, 100.0 mmol) and HOBT (16.2 g, 110.0 mmol) and dissolve with 150 mL DMF. Add DIC (20.4 mL, 120.0 mmol) in an ice bath for activation for 5 min, then add the mixture to the reaction column and react at room temperature for 2 hours. After the reaction, the resin was washed with 200mL x 3DMF, 200mL DBLK x 2 was added for deprotection for 5min+7min, and the resin was washed with 200mL x 5DMF. The coupling was continued in sequence according to the peptide sequence: Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-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, Fmoc-Aib-OH and Fmoc-His(trt)-OH. After the reaction, the resin was washed with 400 mL x 5 DMF, and the resin was shrunk with 400 mL x 3 tert-methyl ether. After drying, 500 mL of pre-prepared lysis solution TFE / DCM (1:4, v / v) was added, and lysis was carried out at room temperature for 2 h. The lysis solution was filtered and collected. The resin was then washed twice with DCM, combined, and DCM was removed by rotary evaporation. 1000 mL of purified water was slowly added to produce a large amount of precipitate, which was filtered and washed twice with anhydrous ether, dried, and dried to obtain the fully protected peptide fragment III.

[0101] (3) Preparation of fully protected peptide fragment IV

[0102] The fully protected peptide fragment III obtained in step (2), 2,4,6-trimethoxybenzyl mercaptan (5.1 g, 24 mmol), HOBt (3.6 g, 26.4 mmol) and HBTU (9.1 g, 24 mmol) were weighed and dissolved in 400 mL of DMF, and DIPEA (5.9 mL, 36 mmol) was added dropwise under an ice bath. After the addition was completed, the mixture was returned to room temperature and the reaction was continued for 2 h. After the reaction was completed, 1000 mL of purified water was slowly added dropwise to the reaction solution to precipitate a large amount of precipitate, which was filtered and then washed three times with purified water and dried to obtain the fully protected peptide fragment IV.

[0103] (4) Preparation of the Second Peptide Fragment IV

[0104] The fully protected peptide fragment IV obtained in step (3) was added to a 1L round-bottom flask, and 400mL of the pre-prepared lysis solution TFA / H2O / PhOMe / PhSMe (90:5:4:1, v / v) was added, and magnetic stirring was performed at room temperature for 2 hours. After the reaction was completed, the lysis solution was slowly added to 4.0L of ice ether for precipitation, centrifuged, washed with 2.0L x 3 ice ether, and dried with nitrogen to obtain 41.6g of the second peptide fragment IV crude peptide.

[0105] (5) Purification of the second peptide fragment IV

[0106] Weigh 20 g of the crude product of the second peptide fragment IV obtained in step (4), add 4 L of distilled water to dissolve, and use a 100×250 mm reverse phase C18 column for semi-preparative purification, with a detection wavelength of 220 nm, a mobile phase of water containing 0.1% TFA (phase A) and acetonitrile (phase B), a flow rate of 200 ml / min, a gradient of B%: 22%-42%, 60 min, and collect the target component. Concentrate and freeze-dry to obtain 11.5 g of the second peptide fragment IV, with a yield of 61.5%.

[0107] 3. One-pot isonitrile-mediated ligation / Fmoc removal

[0108] The first peptide fragment II (2.4 g, 1.0 mmol), the second peptide fragment IV (2.3 g, 1.2 mmol) and HOBT (1.4 g, 10.0 mmol) were weighed and dissolved in 100 mL of DMA, tBuNC (0.34 mL, 3.0 mmol) was added, and the reaction was continued at room temperature for 48 h. After the reaction was completed by HPLC, 20 mL of piperidine was added and the reaction was continued at room temperature for 1 h. After the reaction was completed, 1 L of purified water was added, filtered, and semi-preparative purification was performed using a 50×250 mm reverse phase C18 column, the detection wavelength was 220 nm, the mobile phase was water containing 0.1% TFA (phase A) and acetonitrile (phase B), the flow rate was 70 ml / min, the gradient was: B%: 32%-52%, 60 min, and the target component was collected. Concentration and lyophilization were performed to obtain 2.4 g of semaglutide peptide with a yield of 58.4%. Figure 7 is the mass spectrum of semaglutide; Figure 8 This is the HPLC spectrum of semaglutide.

[0109] Example 5: Synthesis of Semaglutide

[0110] 1. Synthesis of the first peptide fragment II, same as in Example 4;

[0111] 2. Synthesis of the second peptide fragment IV, same as in Example 4;

[0112] 3. One-pot isonitrile-mediated ligation / Fmoc removal

[0113] Weigh the first peptide fragment II (2.4g, 1.0mmol), the second peptide fragment IV (2.3g, 1.2mmol) and HOBT (1.4g, 10.0mmol) and dissolve them in 100mL of DMF, add tBuNC (0.34mL, 3.0mmol), and react at room temperature for 48h. After the reaction is complete by HPLC, add 20mL of piperidine and continue to react at room temperature for 1h. After the reaction is completed, add 1L of purified water, filter, and use a 50×250mm reverse phase C18 column for semi-preparative purification. The detection wavelength is 220nm, the mobile phase is water (phase A) containing 0.1% TFA and acetonitrile (phase B), the flow rate is 70ml / min, the gradient is: B%: 32%-52%, 60min, and the target component is collected. Concentrate and freeze-dry to obtain 1.8g of semaglutide peptide with a yield of 43.8%.

[0114] Example 6: Synthesis of Semaglutide

[0115] 1. Synthesis of the first peptide fragment II, same as in Example 4;

[0116] 2. Synthesis of the second peptide fragment IV, same as in Example 4;

[0117] 3. One-pot isonitrile-mediated ligation / Fmoc removal

[0118] Weigh the first peptide fragment II (2.4g, 1.0mmol), the second peptide fragment IV (2.3g, 1.2mmol) and HOBT (1.4g, 10.0mmol) and dissolve them in 100mL of NMP, add tBuNC (0.34mL, 3.0mmol), and react at room temperature for 48h. After the reaction is complete by HPLC, add 20mL of piperidine and continue to react at room temperature for 1h. After the reaction is completed, add 1L of purified water, filter, and use a 50×250mm reverse phase C18 column for semi-preparative purification. The detection wavelength is 220nm, the mobile phase is water (phase A) containing 0.1% TFA and acetonitrile (phase B), the flow rate is 70ml / min, the gradient is: B%: 32%-52%, 60min, and the target component is collected. Concentrate and freeze-dry to obtain 2.1g of semaglutide peptide with a yield of 51.1%.

[0119] Example 7: Synthesis of Semaglutide

[0120] 1. Synthesis of the first peptide fragment II, same as in Example 4;

[0121] 2. Synthesis of the second peptide fragment IV, same as in Example 4;

[0122] 3. One-pot isonitrile-mediated ligation / Fmoc removal

[0123] Weigh the first peptide fragment II (2.4g, 1.0mmol), the second peptide fragment IV (2.3g, 1.2mmol) and HOBT (1.4g, 10.0mmol) and dissolve them in 100mL of DMA / NMP (1:1), add tBuNC (0.34mL, 3.0mmol), and react at room temperature for 48h. After the reaction is complete by HPLC, add 20mL of piperidine and continue to react at room temperature for 1h. After the reaction is completed, add 1L of purified water, filter, and use a 50×250mm reverse phase C18 column for semi-preparative purification. The detection wavelength is 220nm, the mobile phase is water (phase A) containing 0.1% TFA and acetonitrile (phase B), the flow rate is 70ml / min, the gradient is: B%: 32%-52%, 60min, and the target component is collected. Concentrate and freeze-dry to obtain 2.5g of semaglutide peptide with a yield of 60.8%.

[0124] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for synthesizing semaglutide, characterized in that: The steps include: Synthesis of the first peptide fragment II: (1) preparing Fmoc-Gly-Wang resin; (2) sequentially coupling amino acids with N-terminal Fmoc protection and side chain protection, wherein Lys is Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH, by solid phase synthesis to obtain a fully protected peptide fragment II solid phase resin; (3) removing the resin and the protecting group by cleavage and purifying to obtain the first peptide fragment II; The fully protected peptide fragment II solid phase resin is H-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Wang resin; The first peptide fragment II is H-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-Oct)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH; Synthesis of the second peptide fragment IV: (1) preparing Fmoc-Gly-2CTC resin; (2) sequentially coupling amino acids with N-terminal Fmoc protection and side chain protection by solid phase synthesis to obtain a fully protected peptide fragment III solid phase resin; (3) removing the resin by cleavage to obtain a fully protected peptide fragment III; (4) coupling the fully protected peptide fragment III with thiol to obtain a fully protected peptide fragment IV; (5) removing the protecting group by cleavage and purifying to obtain the second peptide fragment IV; The fully protected peptide fragment III solid phase resin is Fmoc-His(trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-2CTC resin; The second peptide fragment IV is Fmoc-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-COSH; One-pot isonitrile-mediated connection / de-Fmoc reaction: The first peptide fragment II and the second peptide fragment IV are coupled in the presence of a coupling agent, wherein the coupling agent is tBuNC and HOBt, and the solvent of the coupling agent is selected from one or more of DMA, DMF, and NMP. After the reaction, a de-Fmoc reagent is added, the Fmoc protection is removed, and purification is performed to obtain semaglutide.

2. The synthesis method according to claim 1, characterized in that Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Oct(OtBu))-OH is synthesized by a liquid phase method, comprising the steps of: 1) Oct(OtBu) and HOSu are coupled in the presence of a coupling agent to obtain Oct(OtBu)-OSu, and then Oct(OtBu)-OSu and H-Glu-OtBu react to obtain the dipeptide fragment Oct(OtBu)-Glu-OtBu; 2) The dipeptide fragment Oct(OtBu)-Glu-OtBu and HOSu are coupled in the presence of a coupling agent to obtain Oct(OtBu)-Glu(OSu)-OtBu, and then Oct(OtBu)-Glu(OSu)-OtBu and AEEA-AEEA are reacted to obtain Oct(OtBu)-Glu(AEEA-AEEA)-OtBu; 3) Oct(OtBu)-Glu(AEEA-AEEA)-OtBu and Fmoc-Lys-OH·HCl are coupled in the presence of a coupling agent to obtain Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)- Oct(OtBu))-OH.

3. The synthesis method according to claim 2, characterized in that The coupling agent in 1) and 2) is selected from DCC, DIC or EDC·HCl; 3) The coupling agent is selected from EDC·HCl + A, DIPEA + B, DIPEA + A + B or CDI, wherein A is HOBt or HOAt, and B is PyBOP, PyAOP, HATU, HBTU or TBTU.

4. The synthesis method according to claim 1, characterized in that The preparation method of Fmoc-Gly-Wang resin is as follows: in the presence of a coupling agent, the activated Wang resin is coupled with Fmoc-Gly-OH; The Wang resin substitution degree is 0.6 to 1.0 mmol / g; When preparing Fmoc-Gly-Wang resin, the coupling agent is DIC + A + DMAP, wherein A is HOBt or HOAt.

5. The synthesis method according to claim 4, characterized in that When preparing Fmoc-Gly-Wang resin, the molar ratio of DIC:A:DMAP is 1.2:1.1:0.

1.

6. The synthesis method according to claim 1, characterized in that When synthesizing the fully protected peptide fragment II solid phase resin, the amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled as Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, 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(AEEA-AEEA-γ-Glu(OtBu)- Oct(OtBu))-OH, Fmoc-Ala-OH, Fmoc-Ala-OH and Fmoc-Gln(Trt)-OH; When synthesizing the fully protected peptide fragment II solid phase resin, the coupling agent used is DIC + A or DIPEA + A + B, wherein A is HOBt or HOAt, and B is PyBOP, PyAOP, HATU, HBTU or TBTU; The cleavage solution used in the synthesis of the first peptide fragment II (3) is a combination of TFA, H2O, PhOMe and PhSMe.

7. The synthesis method according to claim 6, characterized in that When synthesizing the fully protected peptide fragment II solid phase resin, the molar ratio is DIC:A=1.2:1.1, DIPEA:A:B=1.5:1.1:1.

0.

8. The synthesis method according to claim 6, characterized in that In the synthesis of the first peptide fragment II (3), the volume ratio of TFA, H2O, PhOMe and PhSMe is 90:5:4:

1.

9. The synthesis method according to claim 1, characterized in that The preparation method of Fmoc-Gly-2CTC resin is as follows: in the presence of a coupling agent, the activated 2CTC resin is coupled with Fmoc-Gly-OH; The substitution degree of the 2CTC resin is 0.4 to 1.0 mmol / g.

10. The synthesis method according to claim 1, characterized in that When synthesizing the fully protected peptide fragment III solid phase resin, the amino acids with N-terminal Fmoc protection and side chain protection are sequentially coupled: 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, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Fmoc-His(trt)-OH; When synthesizing the fully protected peptide fragment III solid phase resin, the coupling agent used is DIC + A or DIPEA + A + B, wherein A is HOBt or HOAt, and B is PyBOP, PyAOP, HATU, HBTU or TBTU; The cleavage solution used in the synthesis of the second peptide fragment IV (3) is a mixture of TFE and DCM or a mixture of HFIP and DCM.

11. The synthesis method according to claim 10, characterized in that: When synthesizing the fully protected peptide fragment III solid phase resin, the molar ratio is DIC:A=1.2:1.1, DIPEA:A:B=1.5:1.1:1.

0.

12. The synthesis method according to claim 10, characterized in that: The cleavage solution used in the synthesis of the second peptide fragment IV (3) is a mixture of TFE and DCM, and the volume ratio of TFE to DCM is 1:

4.

13. The synthesis method according to claim 1, characterized in that: The thiol in the synthesis of the second peptide fragment IV (4) is 2,4,6-trimethoxybenzylthiol; The coupling agent used in the synthesis of the second peptide fragment IV (4) is DIC + A or DIPEA + A + B, wherein A is HOBt or HOAt, and B is PyBOP, PyAOP, HATU, HBTU or TBTU; The reagent used for cleavage in the synthesis of the second peptide fragment IV (5) is a combination of TFA, H2O, PhOMe and PhSMe.

14. The synthesis method according to claim 13, characterized in that: In the synthesis of the second peptide fragment IV (4), the molar ratios were DIC:A=1.2:1.1, DIPEA:A:B=1.5:1.1:1.

0.

15. The synthesis method according to claim 13, characterized in that: In the synthesis of the second peptide fragment IV (5), the volume ratio of TFA, H2O, PhOMe and PhSMe was 90:5:4:

1.

16. The synthesis method according to claim 1, characterized in that: In the one-pot isocyanate-mediated ligation / de-Fmoc reaction, the de-Fmoc reagent is selected from DBU, diethylamine or piperidine.

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