Preparation method of semeglutide with high fragment utilization rate

Abstract

The invention relates to the field of drug synthesis, and discloses a preparation method of semeglutide with a high fragment utilization rate, which comprises the following steps: taking amino acids from the first site to the sixteenth site of a semeglutide sequence as a fragment I, and taking amino acids from the 17th site to the 29th site of the semeglutide sequence as a fragment II; the 30th to 31st amino acids of the sequence of the semeglutide are used as a fragment III; a semeglutide product is prepared from the first fragment, the second fragment and the third fragment through a solid-liquid combination method, three fragments with C-terminal amino acids being Gly are preferably selected in the method, racemization impurities cannot be generated during condensation of the fragments, the synthesized fragments are high in purity and good in solubility, polypeptide fragment condensation reaction is easy to carry out, and the method is suitable for industrial production. The input amount of amino acid is only 1.5-2 times when the fragment is synthesized, the synthesized fragment does not need to be purified, the input amount of a carboxyl terminal fragment is only 0.95-1.05 times that of the amino acid fragment when the fragment is condensed, the fragment utilization rate is high, the cost is low, the unutilized fragment can be extracted and separated, and the post-treatment is simple.

Description

A method for preparing smegglutinin with high fragment utilization Technical Field

[0001] This invention relates to the field of drug synthesis, and in particular to a method for preparing smegglutinin with high fragment utilization. Background Technology

[0002] Smegglutide is a novel, long-acting glucagon-like peptide-1 receptor agonist (GLP-1RA) with a structure similar to natural human glucagon-like peptide-1 (GLP-1), exhibiting up to 94% amino acid sequence homology. It is used for glycemic control in adults with type 2 diabetes. Smegglutide acts through the GLP-1 receptor, selectively binding to and activating it to stimulate insulin secretion and reduce glucagon secretion, thereby lowering blood glucose. Compared to traditional GLP-1RAs, its half-life is extended to 7 days. It can be used for long-term weight management in adult patients based on dietary control and increased physical activity. Its molecular formula is: C 187 H 291 N 45 O 59 ;The amino acid sequence is: H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0003] There are several methods for synthesizing semaglutide, including solid-phase synthesis, liquid-phase synthesis, and a combined solid-liquid synthesis method. For example, CN106928343A discloses a solid-phase synthesis method for preparing semaglutide using the Fmoc strategy to couple amino acids one by one. Another example is CN110041219B, which discloses a liquid-phase synthesis method for the side chain of semaglutide. Yet another example is CN109311961B, which discloses a combined solid-liquid synthesis method for preparing semaglutide.

[0004] The existing methods for preparing semaglutide have the following main problems: (1) The solid-phase synthesis method involves long coupling cycles of amino acids one by one, large solvent usage, high cost, and is not conducive to scale-up production. Moreover, during gradual coupling, the resin shrinks severely, and the reaction is prone to incomplete reaction, resulting in missing peptides. There are many hydrophobic amino acids, making condensation difficult and time-consuming, resulting in more impurities and making purification difficult. (2) The solid-liquid combination method has two cases: 1. Liquid-phase synthesis uses small fragments as starting materials for solid-phase synthesis. In this case, the amount of fragment input is 2 to 5 times, resulting in high fragment utilization and low cost. The yield of purified semaglutide is 30 to 40%, which is low. 2. Solid-phase synthesis of fragments, followed by liquid-phase condensation. In this case, the solubility of the fragment and the selection of the C-terminal amino acid of the fragment are key factors. Otherwise, racemic impurities are easily generated or it is difficult for fragments to condense. Solving the above problems is of great significance for achieving cost reduction and high yield of semaglutide. Summary of the Invention

[0005] The purpose of this invention is to synthesize semaglutide by selecting three highly soluble fragments with C-terminal amino acids of Gly, which significantly improves fragment utilization, purity, and yield of semaglutide products. The purity of semaglutide products can reach 99.8%, and the yield can reach 62%.

[0006] The specific technical solution of the present invention is as follows: a method for preparing smegglutinin with high fragment utilization rate, comprising the following steps: (1) solid-phase synthesis of fragment one and fragment two, liquid-phase synthesis of fragment three, condensation of fragment two and fragment three and removal of amino protecting groups to form fragment four; then condensation of fragment four with fragment one to form fully protected smegglutinin. The amino acid sequence of fragment one is the 1st to 16th amino acid of the smegglutinin sequence; the amino acid sequence of fragment two is the 17th to 29th amino acid of the smegglutinin sequence; the amino acid sequence of fragment three is the 30th to 31st amino acid of the smegglutinin sequence; (2) removal of the protecting group of the fully protected smegglutinin to form crude peptide; (3) purification of crude peptide to form smegglutinin product.

[0007] The amino acid sequence of the side-chain protected fragment 1 is: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser[Psi(Me,Me)Pro]-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH.

[0008] The amino acid sequence of fragment two protected by the side chain is: Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu(OtBu)-Octadecanedioic(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-OH.

[0009] The amino acid sequence of fragment three is: NH2-Arg(Pbf)-Gly-OH.

[0010] The amino acid sequence of fragment four, which is protected by the side chain, is: NH2-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu(OtBu)-Octadecanedioic(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu.

[0011] The amino acid sequence of the fully protected smegglutinin is: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser[Psi(Me,Me)Pro]-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu(OtBu)-Octadecanedioic(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-OtBu.

[0012] As a preferred embodiment, the conditions for solid-phase synthesis include: the support is an acid-sensitive resin, preferably a 2-chloro-triphenylmethyl chloride resin.

[0013] The coupling agent is one of DIC / Oxyma with a molar ratio of 1:1, DIC / HOBt with a molar ratio of 1:1, and HATU / HOAt / DIEA with a molar ratio of 1:1:2; the coupling agent is preferably DIC / Oxyma, and the molar ratio of the amino acid to be coupled to DIC is 1:1.

[0014] The amino deprotecting agent is one of a 20% (v / v) DMF solution of piperidine and a 3% (v / v) DBU DMF solution; the amino deprotecting agent is preferably a 20% (v / v) DMF solution of piperidine; the pyrolysis agent is one of a 0.5-1% (v / v) DCM solution of TFA and a 20% (v / v) DCM solution of HFIP; the pyrolysis agent is preferably a 0.5-1% (v / v) DCM solution of TFA.

[0015] Preferably, the conditions for liquid-phase synthesis include: the condensation reagent is one of EDCI / HOBT / DIEA and HATU / HOAt / DIEA; and the amino deprotection reagent is Pd / C / H2.

[0016] Preferably, the condensation conditions include: the coupling agent being one of the following: HBTU / HOBt / DIEA with a molar ratio of 1:1:2, HATU / HOAt / DIEA with a molar ratio of 1:1:2, EDCI / HOBt with a molar ratio of 1:1, EDCI / HOBt / DIEA with a molar ratio of 1:1:2, and PyBOP / HOBt / DIEA with a molar ratio of 1:1:2; and the solvent for the coupling reaction being one or more of DMF, DMAc, NMP, and DMSO.

[0017] Preferably, the conditions for removing the amino protecting group in step (2) include: the deamination agent is one of 5~10 eq. of piperidine, 5~10 eq. of TEA and 5~10 eq. of DEA; and the reaction solvent is one of DMF, DMAc and NMP.

[0018] Preferably, the protecting group of the fully protected smegglutinin is removed using a lysis buffer with a TFA / TIS / H2O mixed solution volume ratio of 90~100:2~3:2~3, a TFA / phenol / H2O / TIS mixed solution volume ratio of 80~90:4~8:4~8:4~8:1~3, and a TFA / EDT / TIS / H2O mixed solution volume ratio of 90~95:2~3:2~3:2~3. Preferably, the TFA / TIS / H2O mixed solution volume ratio is 95:2.5:2.5, a TFA / phenol / H2O / TIS mixed solution volume ratio of 88:5:5:5:2, or a TFA / EDT / TIS / H2O mixed solution volume ratio of 92.5:2.5:2.5:2.5.

[0019] As a preferred method, purification is performed using reversed-phase high-performance liquid chromatography.

[0020] Preferably, the chromatographic column for reversed-phase high-performance liquid chromatography is a C18 column.

[0021] Preferably, the mobile phase for reversed-phase high-performance liquid chromatography is a 0.25% aqueous acetic acid solution and an acetonitrile solution.

[0022] Compared with existing technologies, this application has the following technical advantages: This method uses liquid-phase synthesis to synthesize polypeptide fragment three, and solid-phase synthesis to synthesize polypeptide fragments one and two. The amount of amino acids added during synthesis is 1.5 to 2 times the amount required, resulting in low material costs. The synthesized polypeptide fragments one and two have high purity and can proceed with subsequent reactions without purification. Polypeptide fragments can be synthesized simultaneously, and the synthesis cycle is short. This method uses polypeptide fragments two and three to condense into polypeptide fragment four, and then uses polypeptide fragments one and four to condense into fully protected smegglutinin. During the condensation process, the amount of carboxyl-terminal fragment added is only 0.95 to 1.05 times the amount of polypeptide fragments, resulting in low cost, high raw material utilization, and the removal of unreacted fragments through extraction, simplifying post-processing. The crude smegglutinin obtained by this method does not contain defective peptides lacking one or more amino acids. Purification is simple, and the purity of the purified smegglutinin product can reach 99.8%, with a yield of 62%. Attached Figure Description

[0023] Figure 1 shows the chromatogram of smegglutinin prepared in the example. Detailed Implementation

[0024] The present invention will be further described below with reference to embodiments.

[0025] To better understand the content of this invention, further explanation is provided below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of this invention.

[0026] The abbreviations for the materials used in this invention are shown in Table 1; Table 1: Abbreviations for the materials used in this invention Example 1: (Preparation of Fmoc-Gly-2-chloro-triphenylmethyl resin) (1) Weigh 46.3g of CTC resin (content is 1.08 mmol / g) and place it in solid phase reactor R1. Then add 460mL of DCM to solid phase reactor R1 for swelling for 1 h. After swelling, remove the DCM to prepare swollen resin.

[0027] (2) Weigh 22.3 g of Fmoc-Gly-OH and place it into the jacketed reactor R2. Add 370 mL of DCM and 38.8 g of DIEA to the jacketed reactor R2 to dissolve the amino acids in the solvent. While dissolving the amino acids, control the temperature of the solid-phase reactor R1 to 10±5 ℃. After the amino acids in the jacketed reactor R2 have dissolved and become clear, slowly add the amino acids to the solid-phase reactor R1. Use 93 mL of DCM to clean the jacketed reactor R2 and add the cleaning solution to the solid-phase reactor R1. Control the temperature of the solid-phase reactor R1 at 25±5 ℃ for 2 h. After the reaction is completed, add 47 mL of MeOH to the solid-phase reactor R1 and carry out the end-capping reaction for 0.5 h under stirring and at a temperature of 25±5 ℃. After the reaction was completed, the reaction solution was dried under vacuum. The resin was washed with DMF, DCM and MeOH. After washing, it was dried under vacuum to constant weight to prepare Fmoc-Gly-2-chloro-triphenylmethyl resin. The loading of Fmoc-Gly-2-chloro-triphenylmethyl resin was determined by ultraviolet spectrophotometry to be 0.72 mmol / g.

[0028] Example 2: (Solid-phase synthesis of polypeptide fragment 1) Solid-phase synthesis was performed according to the amino acid sequence of polypeptide fragment 1. The amino acid sequence of polypeptide fragment 1 is: Boc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser[Psi(Me,Me)Pro]-Tyr(tBu)-Leu-Glu(OtBu)-Gly-OH; (1) 27.8 g of Fmoc-Gly-2-chloro-triphenylmethyl resin was weighed and treated with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection. Fmoc byproducts and residual piperidine were washed with DMF and ninhydrin was used to detect whether the color development was clean.

[0029] (2) Weigh Fmoc-Glu(OtBu)-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is complete, dry the resin and wash it with DMF 6 times.

[0030] (3) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0031] (4) Weigh Fmoc-Leu-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is complete, dry the resin and wash it with DMF 6 times.

[0032] (5) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0033] (6) Weigh Fmoc-Tyr(tBu)-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to prepare an activation solution. Add the activation solution to the solid-phase reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid-phase reactor R1. Control the temperature of the solid-phase reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is complete, dry the resin and wash it with DMF 6 times.

[0034] (7) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0035] (8) Weigh Fmoc-Ser(tBu)-Ser[Psi(Me,Me)Pro]-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC dropwise to activate the amino acids for 10~15 min to prepare an activation solution. Add the activation solution to the solid-phase reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid-phase reactor R1. Control the temperature of the solid-phase reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0036] (9) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0037] (10) Weigh Fmoc-Val-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0038] (11) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0039] (12) Weigh Fmoc-Asp(OtBu)-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0040] (13) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0041] (14) Weigh Fmoc-Ser(tBu)-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is complete, dry the resin and wash it with DMF 6 times.

[0042] (15) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0043] (16) Weigh Fmoc-Thr(tBu)-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0044] (17) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0045] (18) Weigh Fmoc-Phe-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0046] (19) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0047] (20) Weigh Fmoc-Thr(tBu)-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0048] (21) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0049] (22) Weigh Boc-His(Trt)-Aib-Glu(OtBu)-Gly-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC dropwise to activate the amino acids for 10~15 min to prepare an activation solution. Add the activation solution to the solid-phase reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid-phase reactor R1. Control the temperature of the solid-phase reactor at 25±5℃ and react for 3h. Use a mixture of DMF, DCM and MeOH to wash the resin and vacuum dry to constant weight to prepare peptide resin one.

[0050] (23) Controlling the temperature of the pyrolysis vessel at 10±5 ℃, add 1% TFA / DCM lysis buffer to pyrolysis vessel R4, then add peptide resin to the pyrolysis vessel in batches, control the temperature of pyrolysis vessel R4 at 25±5 ℃ and react for 1 h. After the reaction is complete, filter and collect filtrate one. Then add 1% TFA / DCM lysis buffer to pyrolysis vessel R5, and add the filter cake from pyrolysis vessel R4 to pyrolysis vessel R5, controlling the temperature of pyrolysis vessel R5 at 25±5 ℃. After the reaction was completed, the mixture was filtered and filtrate 2 was collected. The filter cake was then washed twice with DCM and filtrate 3 was collected. Filtrate 1, filtrate 2 and filtrate 3 were added to reactor R6 and the temperature of reactor R6 was controlled at 10±5℃. Pyridine was added to neutralize the reaction solution to pH 7. (24) The lysis solution in the reaction solution of step (23) was evaporated to obtain the polypeptide. The polypeptide was then dissolved with EA solution and the organic phase was washed twice with saturated brine. The organic phase was collected and dried with anhydrous sodium sulfate. The mixture was evaporated and then slurried with MTBE to precipitate a white solid. The solid was filtered and washed twice with MTBE. The filtered solid polypeptide was placed in a 40℃ oven and dried for 24 hours to obtain polypeptide fragment 1. The yield of polypeptide fragment 1 was 90% and the purity was 96%.

[0051] Example 3: (Solid-phase synthesis of peptide fragment 2) Solid-phase synthesis was performed according to the amino acid sequence of peptide fragment 2. The amino acid sequence of peptide fragment 2 is: Fmoc-Gln(Trt)-Ala-Ala-Lys(AEEA-AEEA-γ-Glu(OtBu)-Octadecanedioic(OtBu))-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-Gly-OH; (1) 27.8 g of Fmoc-Gly-2-chloro-triphenylmethyl resin was weighed and treated with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection. Fmoc byproducts and residual piperidine were washed with DMF and ninhydrin was used to detect whether the washing was clean.

[0052] (2) Weigh Fmoc-Arg(Pbf)-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0053] (3) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0054] (4) Weigh Fmoc-Val-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is complete, dry the resin and wash it with DMF 6 times.

[0055] (5) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0056] (6) Weigh Fmoc-Leu-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is complete, dry the resin and wash it with DMF 6 times.

[0057] (7) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0058] (8) Weigh Fmoc-Trp(Boc)-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to prepare an activation solution. Add the activation solution to the solid-phase reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid-phase reactor R1. Control the temperature of the solid-phase reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0059] (9) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0060] (10) Weigh Fmoc-Ala-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0061] (11) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0062] (12) Weigh Fmoc-Ile-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0063] (13) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0064] (14) Weigh Fmoc-Phe-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is complete, dry the resin and wash it with DMF 6 times.

[0065] (15) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0066] (16) Weigh Fmoc-Glu(OtBu)-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid-phase reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid-phase reactor R1. Control the temperature of the solid-phase reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0067] (17) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0068] (18) Weigh Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Octadecanedioic(OtBu))-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC dropwise to activate the amino acids for 10~15 min to prepare an activation solution. Add the activation solution to the solid-phase reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid-phase reactor R1. Control the temperature of the solid-phase reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0069] (19) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0070] (20) Weigh Fmoc-Ala-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0071] (21) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0072] (22) Weigh Fmoc-Ala-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC to activate the amino acids for 10~15 min to make an activation solution. Add the activation solution to the solid reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid reactor R1. Control the temperature of the solid reactor at 25±5℃ and react for 3h. Take a small amount of resin for ninhydrin detection. After the reaction is completed, dry the resin and wash it with DMF 6 times.

[0073] (23) Treat the resin with an amino deprotection reagent (20% volume fraction of piperidine / DMF) to remove amino protection, wash with DMF to remove Fmoc byproducts and residual piperidine, and use ninhydrin to detect whether the washing is clean.

[0074] (24) Weigh Fmoc-Gln(Trt)-OH and Oxyma and place them in a clean jacketed reactor R2. Add DMF to dissolve the amino acids. Control the temperature of the jacketed reactor R2 at 10±5℃ and add DIC dropwise to activate the amino acids for 10~15 min to prepare an activation solution. Add the activation solution to the solid-phase reactor R1. Use DMF to wash the jacketed reactor R2 and inject the washing solution into the solid-phase reactor R1. Control the temperature of the solid-phase reactor at 25±5℃ and react for 3h. Use a mixture of DMF, DCM and MeOH to wash the resin and vacuum dry to constant weight to prepare peptide resin II.

[0075] (25) Controlling the temperature of the pyrolysis vessel at 10±5 ℃, add 1% TFA / DCM lysis buffer to pyrolysis vessel R4, then add peptide resin to the pyrolysis vessel in batches, control the temperature of pyrolysis vessel R4 at 25±5 ℃ and react for 1 h. After the reaction is complete, filter and collect filtrate one. Then add 1% TFA / DCM lysis buffer to pyrolysis vessel R5, and add the filter cake from pyrolysis vessel R4 to pyrolysis vessel R5, controlling the temperature of pyrolysis vessel R5 at 25±5 ℃. After the reaction was completed, the mixture was filtered and filtrate 2 was collected. The filter cake was then washed twice with DCM and filtrate 3 was collected. Filtrate 1, filtrate 2 and filtrate 3 were added to reactor R6 and the temperature of reactor R6 was controlled at 10±5℃. Pyridine was added to neutralize the reaction solution to pH 7. (26) The lysis solution in the reaction solution of step (25) was evaporated to obtain the polypeptide. The polypeptide was then dissolved with EA solution and the organic phase was washed twice with saturated brine. The organic phase was collected and dried with anhydrous sodium sulfate. The mixture was evaporated and then slurried with MTBE to precipitate a white solid. The solid was filtered and washed twice with MTBE. The filtered solid polypeptide was placed in a 40℃ oven and dried for 24 hours to obtain polypeptide fragment 2. The yield of polypeptide fragment 2 was 93% and the purity was 95%.

[0076] Example 4: (Liquid-phase synthesis of polypeptide fragment 3) Liquid-phase synthesis was carried out according to the amino acid sequence of polypeptide fragment 3. The amino acid sequence of polypeptide fragment 2 is: NH2-Arg(Pbf)-Gly-OH; (1) Z-Arg(Pbf)-OH and H-Gly-OtBu.HCl were added to a jacketed bottle containing DMF, and then HOBT was added to make a mixture. The mixture was cooled to 0~5 ℃ and then EDCI and DIEA were added and stirred at 0~5 ℃ for 16 h. After the reaction was completed, MTBE and 5% H3PO4 aqueous solution with a volume ratio of 5:15 were added and the mixture was allowed to stand for layering. The aqueous phase was back-extracted twice with 3 volumes of MTBE and the organic phase was combined. The organic phase was washed twice with 5% phosphoric acid aqueous solution, 5% potassium bicarbonate aqueous solution and saturated sodium chloride. The organic phase was desolventized to obtain Z-Arg(Pbf)-Gly-OtBu.

[0077] (2) Z-Arg(Pbf)-Gly-OtBu was dissolved using MTBE, and then DMAc and 0.1X of Pd / C were added. After nitrogen purging, the nitrogen was replaced with hydrogen, and then the reaction was carried out and filtered. A potassium bicarbonate solution with 3 times the amount of DMAc was added and allowed to stand for separation. The aqueous phase was back-extracted with ethyl acetate. The organic phases were combined and washed once with potassium bicarbonate and twice with sodium chloride. The organic phase was desolvated to obtain polypeptide fragment 3. The purity of polypeptide fragment 3 was 99.8% and the yield was 95%.

[0078] Example 5: (Preparation of Smegglutinin) (1) Liquid-phase condensation of peptide fragment 2 and peptide fragment 3 into peptide fragment 4: Weigh 1.0 eq. of peptide fragment 2 and 1.1 eq. of peptide fragment 3 and add them to a jacketed reactor. Add 20 volumes of DMF / NMP (mass ratio 1:1) and stir until the solution is clear. Then cool down to 0~5 ℃. Subsequently, add 1.5 eq. of Cl-HOBT, 1.5 eq. of HBTU and 2.5 eq. of DIEA in sequence for reaction. After the reaction is completed, continue to the next step. Cool down to 0~5 ℃ and add 5.0 eq. of piperidine and stir for 3 h. Add 10% citric acid dropwise and stir for 20 min after the addition is complete. Filter and then slurry once each with 10% citric acid, 5% sodium bicarbonate solution, water and MTBE. Filter and dry the solid to obtain peptide fragment 4.

[0079] (2) Liquid-phase synthesis of fully protected smegglutinin from peptide fragments 4 and 1: NMP / DMF (mass ratio 1:1) and 1.0 eq. of peptide fragment 1 were added under stirring until the solution was clear. The temperature was lowered to 0-5℃, and then 1.2 eq. of HATU and 2.2 eq. of DIEA were added. 1.1 eq. of peptide fragment 4 were added in batches and reacted at 0-5℃ for 2h. After the reaction was confirmed to be complete, the reaction solution was squeezed into 10% citric acid, filtered, and the filter cake was successively slurried with 10% citric acid, 5% sodium bicarbonate and water. After filtration and drying, the fully protected smegglutinin was obtained with a yield of 85%.

[0080] (3) Cleavage of fully protected semaglutide to obtain crude peptide: The fully protected semaglutide was added to a trifluoroacetic acid / phenol / water / triisopropylsilane solution (volume ratio of 88:5:5:2), cooled to 0 °C, and stirred until the solid dissolved. Then the solution was heated to room temperature and stirred for 3 hours. The solution was added to 0 °C MTBE precipitate, washed, and the solid was dried in an oven to obtain crude semaglutide peptide with a yield of 98.5%.

[0081] (4) Purification of crude semaglutide by HPLC: HPLC conditions included: Waters C18 250×19, 5u, 130A column; flow rate of 8 mL / min; UV detection wavelength of 220 nm; mobile phase A was acetonitrile; mobile phase B was 0.25% acetic acid / water; conditions: 10-30% mobile phase A, 10 min; 30-50% mobile phase A, 40 min. 100 mg of crude semaglutide was purified by RP-HPLC to obtain 62 mg of semaglutide with a purity of 99.8% and a yield of 62%.

[0082] In the above-described method for preparing semaglutide, the amount of amino acids used in the synthesis of polypeptide fragments one, two, and three is only 1.5 to 2 times that of the polypeptide fragments. This results in low material consumption, low cost, and high purity and yield of the obtained polypeptide fragments, eliminating the need for further purification before subsequent operations. In the synthesis of semaglutide using the aforementioned polypeptide fragments, the amount of the carboxyl-terminal fragment is 0.95 to 1.05 times that of the polypeptide fragments, leading to high raw material utilization and significantly reduced costs. The yield of the crude peptide can reach 98%. Purification of 100 mg of crude semaglutide yields 62 mg of semaglutide product with a purity of 99.8% and a yield of 62%, demonstrating a significant increase in yield.

[0083] Comparative Example: Comparative Example 1 screened peptide sequences with Gly as the starting amino acid in the C-terminal amino acid of the smegglutinin sequence, and the following peptide fragments were selected: Peptide fragment 1 has amino acid sequences of amino acids 1-4 of the smegglutinin sequence; Peptide fragment 2 has amino acid sequences of amino acids 1-16 of the smegglutinin sequence; Peptide fragment 3 has amino acid sequences of amino acids 17-29 of the smegglutinin sequence; Peptide fragment 4 has amino acid sequences of amino acids 30-31 of the smegglutinin sequence; Peptide fragment 5 has amino acid sequences of amino acids 1-29 of the smegglutinin sequence; Peptide fragment 6 has amino acid sequences of amino acids 1-31 of the smegglutinin sequence; Peptide fragment 7 has amino acid sequences of amino acids 5-16 of the smegglutinin sequence; Peptide fragment 8 has amino acid sequences of amino acids 1-5 of the smegglutinin sequence.

[0084] The solubility of the above peptide fragments was tested. The results showed that the solubility of peptide fragment 1 was 1 g / mL (DMF dissolved), peptide fragment 2 was 1 g / 20 mL (DMF / NMP dissolved), peptide fragment 3 was 1 g / 20 mL (DMF / NMP dissolved), peptide fragment 4 was 1 g / 1 mL (DMF dissolved), peptide fragment 5 was 1 g / 80 mL (DMF / NMP dissolved), peptide fragment 6 was 1 g / 100 mL (DMSO dissolved), peptide fragment 7 was 1 g / 10 mL (DMF / NMP dissolved), and peptide fragment 8 was 1 g / 5 mL. (DMF dissolution) Analysis of the above results revealed that the solubility of polypeptide sequences with Gly as the starting amino acid at the C-terminus was significantly higher than that of polypeptide sequences with non-Gly starting amino acids at the C-terminus. Furthermore, the solubility of polypeptide sequences with Gly as the starting amino acid at the C-terminus was also related to the amino acid sequence length. When the amino acid sequence length exceeded 16 positions, the solubility of the polypeptide fragment decreased significantly. Further analysis showed that when the amino acid sequence length of the polypeptide fragment exceeded 16 positions, coiling and folding occurred, significantly reducing the solubility of the polypeptide. The solubility of the polypeptide fragment has a significant impact on the polypeptide condensation efficiency and the amount of raw materials used. Higher polypeptide fragment solubility results in higher polypeptide condensation efficiency and less raw material required for synthesis. In this invention, the amount of carboxyl-terminal fragment used in the polypeptide fragment condensation process was only 0.95 to 1.05 times the amount of polypeptide fragment used, resulting in high raw material utilization.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing smegglutinin with high fragment utilization, characterized in that, Includes the following steps: (1) Solid-phase synthesis of fragment 1 and fragment 2, liquid-phase synthesis of fragment 3, condensation of fragment 2 and fragment 3 and removal of amino protecting groups to form fragment 4; then condensation of fragment 4 with fragment 1 to form fully protected smegglutinin. The amino acid sequence of fragment 1 is the 1st to 16th amino acid of the smegglutinin sequence; the amino acid sequence of fragment 2 is the 17th to 29th amino acid of the smegglutinin sequence; the amino acid sequence of fragment 3 is the 30th to 31st amino acid of the smegglutinin sequence; (2) Removal of the protecting group of the fully protected smegglutinin to form crude peptide; (3) Purification of crude peptide to form smegglutinin product.

2. The method according to claim 1, characterized in that, The conditions for liquid-phase synthesis include: the condensation reagent is one of EDCI / HOBT / DIEA and HATU / HOAt / DIEA; the amino deprotection reagent is Pd / C / H2.

3. The method according to claim 1, characterized in that, The condensation conditions include: the coupling agent is one of HBTU / HOBt / DIEA, HATU / HOAt / DIEA, EDCI / HOBt, EDCI / HOBt / DIEA and PyBOP / HOBt / DIEA; the solvent for the coupling reaction is one or more of DMF, DMAc, NMP and DMSO.

4. The method according to claim 1, characterized in that, The lysis buffer was used to remove the protecting group of the fully protected smegglutinin. The lysis buffer was one of the following: a TFA / TIS / H2O mixed solution, a TFA / phenol / H2O / TIS mixed solution, or a TFA / EDT / TIS / H2O mixed solution.

5. The method according to claim 4, characterized in that TF The volume ratio of the A / TIS / H2O mixed solution is 90~100:2~3:2~3.

6. The method according to claim 4, characterized in that, The volume ratio of the TFA / phenol / H2O / TIS mixed solution is 80~90:4~8:4~8:4~8:1~3.

7. The method according to claim 4, characterized in that, The volume ratio of the TFA / EDT / TIS / H2O mixed solution is 90~95:2~3:2~3:2~3.

8. The method according to claim 1, characterized in that, Purification was performed using reversed-phase high-performance liquid chromatography.

9. The method according to claim 8, characterized in that, The chromatographic column used in the reversed-phase high-performance liquid chromatography is a C18 column.

10. The method according to claim 8 or 9, characterized in that, The mobile phase for reversed-phase high-performance liquid chromatography is an aqueous solution of acetic acid and an acetonitrile solution.

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