Preparation method of Semaglutide

Abstract

The invention discloses a preparation method of Semaglutide, which comprises the following steps: dividing the Semaglutide into six peptide fragments, respectively synthesizing side chains of five fragments (peptide fragments a, b, c, e and f) and a peptide fragment d by adopting a solid phase method, then completing the synthesis of the peptide fragment d by adopting a liquid phase method, finally preparing Semaglutide resin peptide by solid phase coupling of the six peptide fragments, and purifying crude peptide obtained after cracking of the resin peptide to obtain a Semaglutide finished product. According to the method, impurities are controllable, the reaction efficiency is greatly improved, the operation is simple and easy to control, and large-scale production is facilitated.

Description

Technical Field

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

[0002] Semaglutide, a GLP-1 analog developed by Novo Nordisk, was first launched globally in 2017. GLP-1 is a gastrointestinal hormone with incretin effects. Natural GLP-1 has a short half-life and is rapidly inactivated. GLP-1 analogs can extend the half-life and achieve better results. Semaglutide was initially mainly used for blood sugar control in adult patients with type 2 diabetes. In 2021, its weight loss indication was approved by the US FDA. On November 17, 2024, Semaglutide was launched in China. At the end of January 2025, the US FDA formally approved its use to reduce the risk of worsening kidney disease, renal failure, and death from cardiovascular disease in adult patients with type 2 diabetes and chronic kidney disease. As metabolic diseases such as diabetes and obesity have become major global public health issues, Semaglutide's outstanding blood sugar-lowering and weight-reducing effects have been highly sought after by the market, with a significant sales growth trend. Its structure is as follows:

[0003]

[0004] There are two main production strategies for Semaglutide: one is the bio-fermentation method (recombinant expression method). CN113278061B uses recombinant bacteria to produce fusion proteins, and then obtains LYS after enzyme cleavage and tag removal. 26 Arg 34 GLP-1 (10-37) peptide chain, the remaining peptide chain is synthesized by chemical coupling. This method requires complex genetic engineering steps, and the efficiency, specificity and subsequent chemical modification steps of enzyme cutting need to be carefully controlled. On the other hand, in the large-scale fermentation process, if there is a situation that is not conducive to the synthesis of the target product by microorganisms (pH changes, oxygen depletion, carbon dioxide accumulation), it will cause irreversible damage to the microorganisms, and ultimately affect the quality and yield of the target product, and the controllability is low; the other is a pure chemical synthesis method. CN112028986A adopts a method of coupling each amino acid in sequence; CN117624332A divides semaglutide into two long fragments for segmented synthesis. The long peptide chain is prone to long coupling time, generating missing peptides and racemic peptides, causing subsequent purification difficulties, and the degree of resin substitution is easily limited when coupling one by one, causing waste and increasing costs. Because semaglutide contains more hydrophobic amino acids in the sequence, unreasonable segment division during segmented synthesis will also cause low solubility of the peptide segments, and difficulty in coupling between resin and peptide chains. Therefore, it is necessary to provide a safe, efficient, low-cost method for preparing Semaglutide with controllable operating steps and quality. Summary of the Invention

[0005] The object of the present invention is to provide a new and efficient method for preparing Semaglutide, which can improve production efficiency, reduce production costs and purification difficulty, and is suitable for industrial production.

[0006] The present invention provides a new and efficient method for preparing Semaglutide. First, six peptide segments are prepared by a method combining solid phase and solid-liquid phases as follows:

[0007] (1) Peptide segment a: Boc-His(Trt) 1 -Aib 2 -Glu(otbu) 3 -Gly 4 -Thr(tbu) 5 -OH(1-5);

[0008] (2) Peptide segment b: Fmoc-Phe 6 -Thr(tbu) 7 -Ser(tbu) 8 -Asp(otbu) 9 -Val 10 -OH(6-10);

[0009] (3) Peptide segment c: Fmoc-Ser(tbu) 11 -Ser(tbu) 12 -Tyr(tbu) 13 -Leu 14 -Glu(otbu) 15 -Gly 16 -Gln(Trt) 17 -Ala 18 -Ala 19 -OH(11-19);

[0010] (4) Peptide segment d: Fmoc-Lys 20 (AEEA-AEEA-γ-Glu-Octadecanedioic)-OH;

[0011] (5) Peptide segment e: Fmoc-Glu(otbu) 21 -Phe 22 -Ile 23 -Ala 24 -Trp(Boc) 25 -Leu 26 -Val 27 -OH(21-27);

[0012] (6) Peptide f: Fmoc-Arg(Pbf) 28 -Gly 29 -Arg(Pbf) 30 -Gly 31 -OH(28 - 31);

[0013] Among them, the amino acid used for condensation at position 1 of the peptide a is Boc-His(Trt)-OH;

[0014] The peptide segments a, b, c, e, and f are synthesized by solid-phase synthesis;

[0015] The peptide segment d is synthesized by a solid-liquid combination method, and the amino acid used for condensation at position 20 is Fmoc-Lys-OH·HCl; among them, NH2-AEEA-AEEA-γ-Glu-Octadecanedioic-OH is the side chain of Semaglutide;

[0016] Then, the docking of each fragment is completed by the solid-phase method to synthesize the Semaglutide resin peptide. The resin peptide is then cleaved to obtain the crude peptide, and the obtained crude peptide is purified to obtain the Semaglutide finished product;

[0017] Furthermore, the preparation method of Semaglutide provided by the present invention includes the following steps:

[0018] (1) Using 2-CTC resin, the peptide segments a, b, c, e, and f are respectively synthesized by solid-phase synthesis;

[0019] (2) Using 2-CTC resin, the side chain NH2-AEEA-AEEA-γ-Glu-Octadecanedioic-OH is synthesized by solid-phase synthesis, and then the peptide segment d is synthesized by liquid-phase synthesis;

[0020] (3) Using 2-CTC resin, the peptide segments f, e, d, c, b, and a are successively coupled to obtain the Semaglutide resin peptide;

[0021] (4) The Semaglutide resin peptide obtained in step (3) is cleaved using a cleavage solution, methyl tert-butyl ether is added for crystallization, filtered, and the solid is collected to obtain the Semaglutide crude product;

[0022] (5) The Semaglutide crude product obtained in step (4) is subjected to reverse-phase preparative purification and freeze-dried to obtain the Semaglutide finished product; the structure of the Semaglutide resin peptide is:

[0023] Boc-His(Trt) 1 -Aib 2 -Glu(otbu) 3-Gly 4 -Thr(tbu) 5 -Phe 6 -Thr(tbu) 7 -Ser(tbu) 8 -Asp(otbu) 9 -Val 10 -Ser(tbu) 11 -Ser(tbu) 12 -Tyr(tbu) 13 -Leu 14 -Glu(otbu) 15 -Gly 16 -Gln(Trt) 17 -Ala 18 -Ala 19 -Lys 20 (AEEA - AEEA - γ - Glu - Octadecanedioic)-Glu(otbu) 21 -Phe 22 -Ile 23 -Ala 24 -Trp(Boc) 25 -Leu 26 -Val 27 -Arg(Pbf) 28 -Gly 29 -Arg(Pbf) 30 -Gly 31 -2CTC-Resin;

[0024] In the above preparation method,

[0025] The reaction solvent for coupling between each amino acid and peptide chain fragment in the above steps is one or more of DCM, DMF, 50% DMF / THF, 50% DMF / DCM, NMP, THF, DMSO, preferably DCM or 50% DMF / THF;

[0026] In step (1) of solid-phase synthesis, the selected resin is 2-CTC Resin, with a substitution degree of 0.8 - 1.6 mmol / g, preferably a substitution degree of 1.0 - 1.2 mmol / g. The dosage of Fmoc-protected amino acid is 1.5 - 2 times the total molar amount of the fed resin, and the coupling temperature of amino acids in the fragment is 30 - 35 °C;

[0027] In step (1), the cleavage solution for the resin peptide of each peptide segment is 30% TFE / DCM. Specifically, the volume of the cleavage solution is 7 - 8 times the weight of the resin peptide;

[0028] In step (2), the synthesis and cleavage of the side chain NH2-AEEA-AEEA-γ-Glu-Octadecanedioic-OH are the same as in step (1). Then, the obtained side chain is dissolved in THF, and HOSU and DIC are added for activation. After forming the active ester, Fmoc-Lys-OH·HCl and an aqueous sodium bicarbonate solution are added, and amidation is carried out in the THF solvent. Finally, the peptide segment d is obtained through column chromatography purification;

[0029] In steps (1), (2), and (3), the condensing agent used for coupling each amino acid or peptide chain fragment is one or more of HOBT / DIC, PyAOP / DIEA, PyOxim / DIEA, OXYMA / DIC, PyBOP / DIEA, HOAT / HATU / DIEA, and HATU / HOBT / DIEA. Among them, the preferred condensing agent in steps (1) and (2) is HOBT / DIC, and the preferred condensing agent in step (3) is HOAT / HATU / DIEA;

[0030] In step (3), the resin selected for solid-phase synthesis is 2-CTC Resin, with a substitution degree of 0.3 - 1.0 mmol / g, preferably 0.4 - 0.5 mmol / g. The dosage of each peptide segment is 1.5 - 3 times the total molar amount of the fed resin, and the amino acid coupling temperature in each peptide segment is 30 - 35 °C;

[0031] In step (4), the volume of the cleavage solution is 7 - 8 times the weight of the resin peptide. The formula of the cleavage solution is one of TFA:Tis:phenol:anisole = 85:5:5:5 or TFA:Tis:EDT:anisole:phenol:water = 87:5:2.5:2.5:2:1, preferably TFA:Tis:EDT:anisole:phenol:water = 87:5:2.5:2.5:2:1.

[0032] The present invention has achieved the following beneficial effects:

[0033] (1) By using segmented synthesis of peptide segments, the coupling conditions are mild, the reaction steric hindrance is reduced, impurities are controllable, parallel synthesis of segments is carried out, the efficiency is improved, and the reaction yield is increased by liquid-phase synthesis of the side chain, reducing the material cost;

[0034] (2) The peptide segment length is appropriate, which not only fully utilizes the advantages of solid-phase polypeptide synthesis, but also ensures the yield of the segmented synthesized peptide chain, and solves the solubility problem of fully protected peptide fragments;

[0035] (3) The combination of solid and liquid phases for peptide segments better controls and reduces the generation of finished product impurities, shortens the synthesis time, improves the resin yield, crude product purity and yield, and purification yield of Semaglutide. The operation is simple and easy to control, which is conducive to large-scale production. Specific embodiments

[0036] In order to enable those skilled in the art of the present technology to better understand the technical solution of the present invention, the present invention will be further elaborated below in conjunction with embodiments:

[0037] Example 1: Boc-His(Trt) 1 -Aib 2 -Glu(otbu) 3 -Gly 4 -Thr(tbu) 5 Preparation of -OH (peptide segment a)

[0038] (1) Resin swelling: Under nitrogen protection, weigh 100 g (substitution degree 1.10 mmol / g, specification 110 mmol) of 2-CTC Resin into a 2000 mL solid-phase synthesis bottle, add 1000 mL of anhydrous dichloromethane, mechanically stir at 25 - 30 °C for 1 h, filter by suction, complete resin swelling, and set aside for use.

[0039] (2) Preparation of Fmoc-Thr(tbu)-CTC Resin: Under nitrogen protection, weigh 0.22 mol of Fmoc-Thr(tbu)-OH and dissolve it in 600 mL of anhydrous dichloromethane. Add it to the resin in step 1, dropwise add 0.44 mol of DIEA under stirring, react for 4 h, filter by suction, add 800 mL * 3 DMF and wash 3 times, filter by suction. Add 0.22 mol of DIEA and methanol to 600 mL of 50% DMF / DCM, stir evenly, add it to the resin, and block for 30 min. After the blocking is completed, wash with 800 mL * 6 DMF 6 times to complete the preparation of Fmoc-Thr(tbu)-CTC Resin.

[0040] (3) Removal of Fmoc protecting group: Add 20% Pip / DMF to the solid-phase synthesis bottle to remove the Fmoc protecting group twice, 600 mL each time for 20 min. Take a small amount of resin and wash it three times with ethanol. Use the phenol / ninhydrin method to detect whether the Fmoc is removed. After confirming the removal, filter by suction, add 800 mL * 6 DMF and wash 6 times, and dry by suction for use.

[0041] (4) Peptide chain extension: Weigh 0.22 mol of Fmoc-Gly-OH and HOBT, dissolve them in 600 mL of 50% DMF / DCM, then dropwise add 0.22 mol of DIC, sonicate for 5 min, add it to the solid-phase synthesis bottle, react for 1 h. Take a small amount of resin and wash it three times with ethanol. Use the phenol / ninhydrin method to detect whether the reaction is completed. After confirming the completion of the reaction, filter by suction, add 800 mL * 6 DMF and wash 6 times, and dry by suction for use.

[0042] (5) Following the same method as in steps (3) and (4), continue to couple Fmoc-Glu(Otbu)-OH, Fmoc-Aib-OH, and Boc-His(Trt)-OH in the amino acid sequence of Boc-His(Trt)-Aib-Glu(Otbu)-Gly-Thr(tbu)-OH. After detecting the reaction by the phenol / ninhydrin method, perform suction filtration, add 800 mL * 6 DMF and wash 6 times, drain, add 800 mL MeOH and wash 4 times, and dry after suction filtration to obtain 177 g of Boc-His(Trt)-Aib-Glu(Otbu)-Gly-Thr(tbu)-OH resin peptide, with a yield of 85%.

[0043] (6) Cleavage of the resin peptide: Prepare the cleavage solution (trifluoroethanol:dichloromethane = 3:7), slowly add 177 g of the resin peptide with stirring. After addition, carry out cleavage at 25 - 30 °C for 1.5 h. Perform suction filtration, rotary evaporate the filtrate, and dry under vacuum to obtain 94 g of Boc-His(Trt)-Aib-Glu(Otbu)-Gly-Thr(tbu)-OH (peptide segment a), with a yield of 87%.

[0044] Example 2: Preparation of Fmoc-Phe 6 -Thr(tbu) 7 -Ser(tbu) 8 -Asp(otbu) 9 -Val 10 -OH (peptide segment b)

[0045] (1) Swelling of the resin: Under nitrogen protection, weigh 100 g (substitution degree 1.10 mmol / g, specification 110 mmol) of 2-CTC Resin into a 2000 mL solid-phase synthesis flask, add 1000 mL of anhydrous dichloromethane, mechanically stir at 25 - 30 °C for 1 h, perform suction filtration to complete the swelling of the resin for later use.

[0046] (2) Preparation of Fmoc-Val-CTC Resin: Under nitrogen protection, weigh 0.22 mol of Fmoc-Val-OH and dissolve it in 600 mL of anhydrous dichloromethane, add it to the resin, dropwise add 0.44 mol of DIEA with stirring, react for 4 h, perform suction filtration, add 800 mL * 3 DMF and wash 3 times, perform suction filtration, add 0.22 mol of DIEA and methanol to 600 mL of 50% DMF / DCM, stir evenly, add it to the resin, seal for 30 min, and wash 6 times with DMF after the sealing ends to complete the preparation of Fmoc-Val-2CTC Resin.

[0047] (3) Removal of the Fmoc protecting group: The same as step (3) of Example 1.

[0048] (4) Elongation of the peptide chain: Weigh 0.22 mol of Fmoc-Asp(Otbu)-OH and HOBT, dissolve them in 600 mL of 50% DMF / DCM, add 0.22 mol of DIC dropwise, sonicate for 5 min, add to the solid-phase synthesis flask, react for 1 h, take a small amount of resin, wash it three times with ethanol, and use the phenol / ninhydrin method to detect whether the reaction is completed. After confirming the completion of the reaction, filter by suction, add 800 mL of *6 DMF and wash 6 times, filter to dryness, and set aside.

[0049] (5) According to the amino acid sequence of Fmoc-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-OH, continue to couple Fmoc-Ser(tbu)-OH, Fmoc-Thr(tbu)-OH, and Fmoc-Phe-OH. After detecting the completion of the reaction by the phenol / ninhydrin method, filter by suction, add 800 mL of DMF and wash 6 times, filter to dryness, add 800 mL of MeOH and wash 4 times, filter by suction and dry to obtain 193 g of Fmoc-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-OH resin peptide, with a yield of 94%.

[0050] (6) Cleavage of the resin peptide: Prepare the cleavage solution (trifluoroethanol:dichloromethane = 3:7), slowly add 193 g of the resin peptide with stirring. After adding, carry out cleavage at 25 - 30 °C for 1.5 h. Filter by suction, rotary evaporate the filtrate, and dry under vacuum to obtain 94 g of Fmoc-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-OH (peptide segment b), with a yield of 90%.

[0051] Example 3: Preparation of Fmoc-Ser(tbu) 11 -Ser(tbu) 12 -Tyr(tbu) 13 -Leu 14 -Glu(otbu) 15 -Gly 16 -Gln(Trt) 17 -Ala 18 -Al a 19 -OH (peptide segment c)

[0052] (1) Swelling of the resin: Under nitrogen protection, weigh 100 g (substitution degree 1.10 mmol / g, specification 110 mmol) of 2-CTC Resin into a 2000 mL solid-phase synthesis flask, add 1000 mL of anhydrous dichloromethane, stir mechanically at 25 - 30 °C for 1 h, filter by suction to complete the swelling of the resin, and set aside.

[0053] (2) Preparation of Fmoc-Ala-CTC Resin: Under nitrogen protection, weigh 0.22 mol of Fmoc-Ala-OH and dissolve it in 600 mL of anhydrous dichloromethane. Add it to the resin, and dropwise add 0.44 mol of DIEA while stirring. React for 4 h, then filter by suction. Add 800 mL of DMF and wash 3 times, then filter by suction again. Add 0.22 mol of DIEA and methanol to 600 mL of 50% DMF / DCM, stir evenly, add it to the resin, and block for 30 min. After blocking, wash with DMF 6 times to complete the preparation of Fmoc-Ala-CTC Resin.

[0054] (3) Removal of Fmoc protecting group: The same as step (3) of Example 1.

[0055] (4) Peptide chain extension: Weigh 0.22 mol of Fmoc-Ala-OH and HOBT, dissolve them in 600 mL of 50% DMF / DCM, then dropwise add 0.22 mol of DIC, sonicate for 5 min, add it to the solid-phase synthesis bottle, and react for 1 h. Take a small amount of resin and wash it three times with ethanol. Use the phenol / ninhydrin method to detect whether the reaction is completed. After confirming that the reaction is completed, filter by suction, add 800 mL of DMF and wash 6 times, then dry. (5) According to the amino acid sequence of Fmoc-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(otbu)-Gly-Gln(Trt)-Ala-Ala-OH, continue to couple Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(otbu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tbu)-OH, Fmoc-Ser(tbu)-OH, Fmoc-Ser(tbu)-OH. After detecting the reaction with the phenol / ninhydrin method, filter by suction, add 800 mL of DMF and wash 6 times, then dry. Add 800 mL of MeOH and wash 4 times, filter by suction and dry to obtain 269 g of Fmoc-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(otbu)-Gly-Gln(Trt)-Ala-Ala-OH resin peptide, with a yield of 97%.

[0056] (6) Cleavage of resin peptide: Prepare the cleavage solution (trifluoroethanol:dichloromethane = 3:7), slowly add 269 g of resin peptide while stirring. After adding, cleave at 25 - 30 °C for 1.5 h. Filter by suction, rotary evaporate the filtrate, and dry in vacuum to obtain 167 g of Fmoc-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(otbu)-Gly-Gln(Trt)-Ala-Ala-OH (peptide segment c), with a yield of 94%.

[0057] Example 4: Preparation of NH2-AEEA-AEEA-γ-Glu-Octadecanedioic)-OH

[0058] (1) Resin swelling: Under nitrogen protection, weigh 100 g (substitution degree 1.10 mmol / g, specification 110 mmol) of 2-CTCResin into a 2000 mL solid phase synthesis bottle, add 1000 mL of anhydrous dichloromethane, stir mechanically at 25-30 °C for 1 h, filter and complete the resin swelling, and set aside.

[0059] (2) Preparation of Fmoc-AEEA-CTC Resin: Under nitrogen protection, weigh 0.22 mol Fmoc-AEEA-OH and dissolve it in 600 mL anhydrous dichloromethane, add it to the resin, add 0.44 mol DIEA dropwise with stirring, react for 4 h, filter, add 800 mL DMF to wash three times, filter, add 0.22 mol DIEA and methanol to 600 mL 50% DMF / DCM, stir evenly, add to the resin, block for 30 min, wash with DMF six times after blocking, and complete the preparation of Fmoc-AEEA-CTC Resin.

[0060] (3) Removal of Fmoc protecting group: Same as step (3) in Example 1.

[0061] (4) Peptide chain extension: Weigh 0.22 mol Fmoc-AEEA-OH, HOBT, dissolve in 600 mL 50% DMF / DCM, add 0.22 mol DIC, sonicate for 5 min, add to solid phase synthesis bottle, react for 1 h, take a small amount of resin and wash it three times with ethanol, check whether it is completed by phenol / ninhydrin method, after confirming that the reaction is completed, filter, add 800 mL DMF and wash 6 times, drain and set aside. (5) According to the amino acid sequence of AEEA-AEEA-γ-Glu-Octadecanedioic-OH, continue coupling Fmoc-Glu(Otbu)-OH, HOOC-C 16 H 32 -COOtBu, phenol / ninhydrin method detection after the reaction is completed, filter, add 800mL DMF to wash 6 times, dry, add 800mL MeOH to wash 4 times, filter and dry to obtain 173g of NH2-AEEA-AEEA-γ-Glu-Octadecanedioic-OH resin peptide, with a yield of 90%.

[0062] (6) Cleavage of resin peptide: Prepare cleavage solution (trifluoroethanol:dichloromethane = 3:7), slowly add 173 g of resin peptide with stirring. After addition, carry out cleavage at 25 - 30 °C for 1.5 h. Filter by suction, rotary evaporate the filtrate, and dry in vacuum to obtain 77 g of NH2-AEEA-AEEA-γ-Glu-Octadecanedioic-OH, with a yield of 83%.

[0063] Example 5: Fmoc-Lys 20 Preparation of (AEEA-AEEA-γ-Glu-Octadecanedioic)-OH (peptide segment d) (1) Take the tetrapeptide side chain obtained in Example 4, dissolve it in 5 vol of THF with stirring. After it is dissolved clearly, add 2 eq of HOSU and 2 eq of DIC, control the temperature at 35 - 40 °C, keep stirring for 10 min. After obvious solid precipitation, keep stirring for 2.0 h, filter by suction and set aside; (2) Add 2 eq of Fmoc-Lys-OH·HCl, 2.5 eq of 10% aqueous sodium bicarbonate solution and 2.5 vol of THF to the filtrate, stir until the system is completely dissolved clearly, control the temperature at 30 - 35 °C, and keep stirring for 2.0 h;

[0064] (3) After monitoring the reaction to completion by TLC plate (DCM:MeOH = 10:1, R f = 0.7), add hydrochloric acid dropwise to adjust the pH to 3 - 4, and concentrate under reduced pressure at 40 °C to remove THF;

[0065] (4) Add 10 vol of DCM for extraction, take the lower organic phase and concentrate it under reduced pressure at 40 °C to obtain the crude product;

[0066] (5) Dissolve with 6 vol of DCM, elute the target product with eluent 15% MeOH / DCM, collect the eluate and evaporate it to dryness under reduced pressure at 40 °C to obtain 92 g of Fmoc-Lys 20 (AEEA-AEEA-γ-Glu-Octadecanedioic)-OH (peptide segment d), with a yield of 85%.

[0067] Example 6: Fmoc-Glu(otbu) 21 -Phe 22 -Ile 23 -Ala 24 -Trp(Boc) 25 -Leu 26 -Val 27 Preparation of -OH (peptide segment e) (1) Swelling of resin: Under nitrogen protection, weigh 100 g (substitution degree 1.10 mmol / g, specification 110 mmol) of 2-CTC Resin into a 2000 mL solid-phase synthesis bottle, add 1000 mL of anhydrous dichloromethane, stir mechanically at 25 - 30 °C for 1 h, filter by suction to complete the resin swelling, and set aside.

[0068] (2) Preparation of Fmoc-Val-CTC Resin: Under nitrogen protection, weigh 0.22 mol of Fmoc-Val-OH and dissolve it in 600 mL of anhydrous dichloromethane. Add it to the resin, and dropwise add 0.44 mol of DIEA with stirring. React for 4 h, then filter by suction. Add 800 mL of DMF and wash 3 times, then filter by suction again. Add 0.22 mol of DIEA and methanol to 600 mL of 50% DMF / DCM, stir evenly, add it to the resin, and seal for 30 min. After sealing, wash with DMF 6 times to complete the preparation of Fmoc-Val-CTC Resin.

[0069] (3) Removal of Fmoc protecting group: Same as step (3) of Example 1.

[0070] (4) Elongation of peptide chain: Weigh 0.22 mol of Fmoc-Leu-OH and HOBT, dissolve them in 600 mL of 50% DMF / DCM, then dropwise add 0.22 mol of DIC, sonicate for 5 min, add it to the solid-phase synthesis bottle, and react for 1 h. Take a small amount of resin and wash it three times with ethanol. Use the phenol / ninhydrin method to detect whether the reaction is completed. After confirming the completion of the reaction, filter by suction, add 800 mL of DMF and wash 6 times, then dry. (5) According to the amino acid sequence of Fmoc-Glu(otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-OH, continue to couple Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, and Fmoc-Glu(otbu)-OH. After detecting the reaction with the phenol / ninhydrin method, filter by suction, add 800 mL of DMF and wash 6 times, then dry. Add 800 mL of MeOH and wash 4 times, filter by suction and dry to obtain 219 g of Fmoc-Glu(otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-OH resin peptide, with a yield of 92%. (6) Cleavage of resin peptide: Prepare the cleavage solution (trifluoroethanol:dichloromethane = 3:7), slowly add 219 g of resin peptide with stirring. After adding, carry out cleavage at 25 - 30 °C for 1.5 h. Filter by suction, rotary evaporate the filtrate, and dry it under vacuum to obtain 126 g of Fmoc-Glu(otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-OH (peptide segment e), with a yield of 91%.

[0071] Example 7: Preparation of Fmoc-Arg(Pbf) 28 -Gly 29 -Arg(Pbf) 30 -Gly 31 -OH (peptide segment f)

[0072] (1) Resin swelling: Under nitrogen protection, 100 g (substitution degree 1.10 mmol / g, specification 110 mmol) of 2-CTC Resin was weighed and placed in a 2000 mL solid-phase synthesis bottle. 1000 mL of anhydrous dichloromethane was added, and mechanical stirring was carried out at 25 - 30 °C for 1 h. Filtration was performed to complete the resin swelling and it was ready for use.

[0073] (2) Preparation of Fmoc-Gly-CTC Resin: Under nitrogen protection, 0.22 mol of Fmoc-Gly-OH was dissolved in 600 mL of anhydrous dichloromethane, added to the resin, and 0.44 mol of DIEA was added dropwise with stirring. The reaction was carried out for 4 h, and then filtration was performed. 800 mL of DMF was added for washing 3 times, followed by filtration. 0.22 mol of DIEA and methanol were added to 600 mL of 50% DMF / DCM, stirred evenly, and then added to the resin for blocking for 30 min. After the blocking was completed, it was washed 6 times with DMF to complete the preparation of Fmoc-Gly-CTC Resin.

[0074] (3) Removal of Fmoc protecting group: The same as in Example 1(3).

[0075] (4) Peptide chain extension: 0.22 mol of Fmoc-Arg(Pbf)-OH and HOBT were weighed, dissolved in 600 mL of 50% DMF / DCM, then 0.22 mol of DIC was added dropwise, and ultrasonic treatment was carried out for 5 min. It was added to the solid-phase synthesis bottle and the reaction was carried out for 1 h. A small amount of resin was taken and washed three times with ethanol. The phenol / ninhydrin method was used to detect whether the reaction was completed. After confirming that the reaction was completed, filtration was performed, 800 mL of DMF was added for washing 6 times, and it was dried and ready for use.

[0076] (5) According to the amino acid sequence of Fmoc-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH, Fmoc-Gly-OH and Fmoc-Arg(Pbf)-OH were continuously coupled. After detecting the reaction completion by the phenol / ninhydrin method, filtration was performed, 800 mL of DMF was added for washing 6 times, and it was dried. 600 mL of MeOH was added for washing 4 times, and after filtration and drying, 217 g of Fmoc-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH resin peptide was obtained, with a yield of 95%.

[0077] (6) Resin peptide cleavage: A cleavage solution (trifluoroethanol:dichloromethane = 3:7) was prepared, and 217 g of resin peptide was slowly added with stirring. After addition, cleavage was carried out at 25 - 30 °C for 1.5 h. Filtration was performed, the filtrate was rotary evaporated, and vacuum drying was carried out to obtain 116 g of Fmoc-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OH (peptide segment f), with a yield of 90%.

[0078] Example 8: Preparation of Semaglutide resin peptide 1

[0079] (1) Resin swelling: Under nitrogen protection, weigh 25 g (substitution degree 0.44 mmol / g) of 2-CTC Resin into a 500 mL solid-phase synthesis bottle, add 250 mL of anhydrous dichloromethane, mechanically stir at 25 - 30 °C for 1 h, filter by suction, complete the resin swelling, and set aside for use.

[0080] (2) Preparation of Fmoc-AA(28 - 31)-CTC Resin: Under nitrogen protection, weigh 22 mmol of the peptide segment f obtained in Example 7 and dissolve it in 150 mL of anhydrous dichloromethane. Add it to the resin, dropwise add 44 mmol of DIEA under stirring, react for 4 h, filter by suction, add 200 mL of DMF and wash 3 times, filter by suction. Add 22 mmol of DIEA and methanol to 150 mL of 50% DMF / DCM, stir evenly, add it to the resin, and block for 30 min. After the blocking is completed, wash with DMF 6 times to complete the preparation of Fmoc-AA(28 - 31)-2CTCResin.

[0081] (3) Removal of Fmoc protecting group: The same as step (3) of Example 1.

[0082] (4) Peptide chain elongation: Weigh 22 mmol of the peptide segment e and PyAOP obtained in Example 6, dissolve them in 150 mL of 50% DMF / DCM, dropwise add 22 mmol of DIEA, sonicate for 5 min, add it to the solid-phase synthesis bottle, react for 1.5 h. Take a small amount of resin and wash it three times with ethanol. Use the phenol / ninhydrin method to detect whether the reaction is completed. After confirming that the reaction is completed, filter by suction, add 200 mL of DMF and wash 6 times, filter dry, and set aside for use.

[0083] (4) Steps are the same as (3). In sequence, couple peptide segment d (Example 5), peptide segment c (Example 3), peptide segment b (Example 2), and peptide segment a (Example 1); use the phenol / ninhydrin method to detect that the resin does not change color; filter by suction, add 200 mL of DMF and wash 6 times; filter by suction, add 200 mL of MeOH and contract 4 times; filter by suction and dry to obtain 62 g of Semaglutide resin peptide, and the resin peptide yield is 68%.

[0084] Example 9: Preparation of Semaglutide resin peptide 2

[0085] (1) Resin swelling: Under nitrogen protection, weigh 25 g (substitution degree 0.44 mmol / g) of 2-CTC Resin into a 500 mL solid-phase synthesis bottle, add 250 mL of anhydrous dichloromethane, mechanically stir at 25 - 30 °C for 1 h, filter by suction, complete the resin swelling, and set aside for use.

[0086] (2) Preparation of Fmoc-AA(28-31)-CTC Resin: Under nitrogen protection, weigh 22 mmol of the peptide segment f obtained in Example 7 and dissolve it in 150 mL of anhydrous dichloromethane. Add it to the resin, and dropwise add 44 mmol of DIEA with stirring. React for 4 h, filter by suction, add 200 mL of DMF and wash 3 times, filter by suction again. Add 22 mmol of DIEA and methanol to 150 mL of 50% DMF / THF, stir evenly, add it to the resin, and block for 30 min. After blocking, wash with DMF 6 times to complete the preparation of Fmoc-AA(28-31)-2CTC Resin.

[0087] (3) Removal of Fmoc protecting group: The same as (3) in Example 1.

[0088] (4) Elongation of the peptide chain: Weigh 22 mmol of the peptide segment e obtained in Example 6, HOAT / HATU. After dissolving in 150 mL of 50% DMF / THF, dropwise add 22 mmol of DIEA, sonicate for 5 min, add it to the solid-phase synthesis bottle, and react for 1.5 h. Take a small amount of resin and wash it three times with ethanol. Use the phenol / ninhydrin method to detect whether the reaction is completed. After confirming that the reaction is completed, filter by suction, add 200 mL of DMF and wash 6 times, and dry it for later use.

[0089] (4) The steps are the same as (3). In (4), couple the peptide segments obtained in Example 5, Example 3, Example 2, and Example 1 in sequence. Use the phenol / ninhydrin method to detect that the resin does not change color; filter by suction, add 200 mL of DMF and wash 6 times; filter by suction, add 200 mL of MeOH and contract 4 times; after filtering by suction, dry to obtain 85 g of Semaglutide resin peptide, and the yield of the resin peptide is 93.3%.

[0090] Example 10: Preparation of Semaglutide crude product

[0091] (1) Prepare 320 mL of cleavage solution (TFA: Tis: phenol: anisole = 85:5:5:5), pre-cool it to 0 - 5 °C. With stirring, slowly add 40 g of the resin peptide obtained in Example 8. After adding, keep stirring at a constant temperature for 5 min, and then raise the temperature to 25 - 30 °C for a cleavage reaction for 3 h. (2) After the cleavage is completed, collect the filtrate and slowly add it to 7 times the volume of methyl tert-butyl ether (pre-cooled to 0 - 5 °C), stir for 10 min for crystallization, filter, collect the solid, and dry to obtain 14.7 g of Semaglutide crude product, with a yield of 65% and a purity of 65%;

[0092] Example 11: Preparation of Semaglutide crude product

[0093] (1) Prepare 320 mL of cleavage solution (TFA: Tis: EDT: anisole: phenol: water = 87:5:2.5:2.5:2:1), pre-cool it to 0 - 5 °C, and slowly add 40 g of the resin peptide obtained in Example 9 under stirring. After addition, stir at a constant temperature for 5 min, then raise the temperature to 25 - 30 °C and carry out the cleavage reaction for 3 h.

[0094] (2) After the cleavage is completed, collect the filtrate and slowly add it to 7 times the volume of methyl tert-butyl ether (pre-cooled to 0 - 5 °C), stir for crystal precipitation for 10 min, filter, collect the solid, and dry to obtain 19.2 g of semaglutide crude product, with a yield of 85% and a purity of 80%.

[0095] Example 12: Preparation of Semaglutide Finished Product

[0096] (1) Flush the column and equilibrate: First, flush the column with 40% mobile phase A for 15 min, then equilibrate with 5% mobile phase A for 10 min, with a flow rate of 80 mL / min. The column specification model is a silica gel C8 reversed-phase packing column, 10 μm, 50 mm * 250 mm; among them, mobile phase A is 10 mmol ammonium acetate + potassium dihydrogen phosphate, and mobile phase B is methanol.

[0097] (2) Sample treatment: Dissolve 6.0 g of the semaglutide crude product obtained in Example 10 in 0.1% TFA water, and filter it for use after dissolution.

[0098] (3) Load the sample and purify: The sample loading flow rate is 20 mL / min. After completion, perform gradient purification. The initial is 7% mobile phase B, which is increased to 25% within 40 min, held for 10 min, increased to 30% within 10 min, then isocratic for 20 min, increased to 50% within 0.1 min, and the isocratic process ends at 120 min. The detection wavelength is 220 nm.

[0099] (4) Post-treatment of the purified component: The collected target component is concentrated under reduced pressure and then loaded onto the column for enrichment, and rinsed with 80% acetonitrile until the collection ends. (5) Concentrate and freeze-dry: The enriched solution is concentrated under reduced pressure until no obvious liquid flows out, and then freeze-dried to obtain 3.3 g of semaglutide finished product, with a purity of 99.7% and a yield of 55.0%.

Claims

1. A method for preparing Semaglutide crude peptide, characterized in that, It includes the following steps: (1) Prepare polypeptide fragments a, b, c, e, and f by solid-phase synthesis; (2) After synthesizing the side chain NH2-AEEA-AEEA-γ-Glu-Octadecanedioic-OH by solid-phase synthesis, complete the synthesis of peptide segment d by liquid-phase method; (3) Couple the resin with peptide segment f by solid-phase synthesis, and then successively couple peptide segments e, d, c, b, and a to obtain Semaglutide resin peptide; (4) Cut the Semaglutide resin peptide obtained in step (3) with a cleavage solution to obtain crude Semaglutide peptide; Wherein, The polypeptide fragment a described above is Boc-His(Trt) 1 -Aib 2 -Glu(otbu) 3 -Gly 4 -Thr(tbu) 5 (1-5); The polypeptide fragment b is Fmoc-Phe 6 -Thr(tbu) 7 -Ser(tbu) 8 -Asp(otbu) 9 -Val 10 -OH(6-10); The polypeptide fragment c is Fmoc-Ser(tbu) 11 -Ser(tbu) 12 -Tyr(tbu) 13 -Leu 14 -Glu(otbu) 15 -Gly 16 -Gln(Trt) 17 -Ala 18 -Ala 19 -OH(11 - 19); The polypeptide fragment d described above is Fmoc-Lys 20 (AEEA-AEEA-γ-Glu-Octadecanedioic)-OH(20); The polypeptide fragment e is Fmoc-Glu(otbu) 21 -Phe 22 -Ile 23 -Ala 24 -Trp(Boc) 25 -Leu 26 -Val 27 -OH(21-27); The polypeptide fragment f described above is Fmoc-Arg(Pbf) 28 -Gly 29 -Arg(Pbf) 30 -Gly 31 -OH (28 - 31).

2. The preparation method according to claim 1, characterized in that, the resin selected in step (1) is 2-CTC resin, and the reaction solvent for coupling between each amino acid and peptide chain fragment is 50% DMF / THF, and the condensing agent is HOAT / HATU / DIEA.

3. The preparation method according to claim 1, wherein in the liquid-phase synthesis in step (2), Dissolve the obtained tetrapeptide side chain in THF, HOSU, and DIC for activation. After forming an active ester, add Fmoc-Lys-OH·HCL and an aqueous sodium bicarbonate solution, and carry out amidation in a THF solvent. Finally, obtain peptide segment d by column chromatography purification.

4. The preparation method according to claim 1, wherein the resin selected in step (3) is 2-CTC resin.

5. The preparation method according to claim 1, wherein the volume of the cleavage solution in step (4) is 7 to 8 times the weight of the resin peptide, and the formula of the cleavage solution is TFA:Tis:EDT:anisole:phenol:water = 87:5:2.5:2.5:2:

1.

6. The preparation method according to claim 1, wherein the crude Semaglutide peptide obtained in step (4) is subjected to preparative purification, enrichment, and lyophilization to obtain pure Semaglutide.

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