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CN-121974996-A - Method for synthesizing semaglutin and semaglutin side chain fragment

CN121974996ACN 121974996 ACN121974996 ACN 121974996ACN-121974996-A

Abstract

The invention belongs to the field of polypeptide medicaments, and provides a semaglutin and a synthesis method of a semaglutin side chain fragment. The synthesis method of the semaglutin comprises the steps of taking resin as a solid-phase carrier, coupling amino acid or polypeptide fragments from the C end to the N end in sequence through a solid-phase synthesis method according to the primary sequence of the semaglutin, firstly synthesizing a first polypeptide resin of Ala 24 , removing Mmt or Mtt protecting groups on Lys 26 in the first polypeptide resin, connecting side chain fragments ivDde-AEEa-aEEA to obtain a second polypeptide resin, continuing synthesizing a semaglutin main chain to His 7 to obtain a third polypeptide resin, sequentially connecting side chain residues Fmoc-Glu-OtBu and Boc-C16-COOH after removing the ivDde, and cracking to obtain the semaglutin. The synthesis method of the invention has high efficiency, and greatly improves the yield and purity of the semaglutin.

Inventors

  • CHEN CHENGYUN
  • HE ZHIQUAN
  • PAN DELIN
  • WANG RUIRUI
  • FAN LINLIN

Assignees

  • 福建基诺厚普生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260128

Claims (13)

  1. 1. A method for synthesizing semaglutin, wherein the amino acid sequence of semaglutin is H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala 24- Ala-Lys 26 (AEEa-aEEA-G1u(α-Octa))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH,, the method comprising: Step 1, taking resin as a solid-phase carrier, and sequentially coupling amino acid or polypeptide fragments from a C end to an N end according to a primary sequence of the semaglutin by a solid-phase synthesis method to obtain a first polypeptide resin Fmoc-Ala 24 -Ala-Lys 26 (R1)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly- resin; Step 2, deprotecting the first polypeptide resin prepared in the step 1, removing the R1 protecting group, and connecting a first side chain segment ivDde-AEEA-AEEA to obtain a second polypeptide resin Fmoc-Ala 24 -Ala-Lys 26 (AEEa-aEEA-ivDde)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly- resin; step 3, coupling the second polypeptide resin prepared in the step 2 with the rest amino acid or polypeptide fragments in sequence by a solid phase synthesis method to obtain a third polypeptide resin Boc-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-Gln(Trt)-Ala 24 -Ala-Lys 26 (AEEa-aEEA-ivDde)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly- resin; Step 4, removing the ivDde protecting group on the third polypeptide resin prepared in the step 3, and sequentially connecting a second side chain segment Fmoc-Glu-OtBu and Boc-C16-COOH to obtain fully-protected semaglutin resin Boc-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-Gln(Trt)-Ala 24 -Ala-Lys 26 [(AEEa-aEEA-G1u(α-Octa(OtBu))]-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly-; Step 5, cracking the fully-protected semaglutin resin prepared in the step 4 to obtain semaglutin; wherein, R1 is Mmt or Mtt.
  2. 2. The synthetic method of claim 1 wherein the resin is wang resin.
  3. 3. The synthetic method of claim 2 wherein the Wang resin has a degree of substitution of 0.56 mmol/g.
  4. 4. The method according to claim 1, wherein the reagent for removing the R1 protecting group is DCM/TFA/TIS solution, and the volume ratio of DCM/TFA/TIS solution is DCM: TFA: TIS=95:4:1.
  5. 5. The synthetic method according to claim 1, wherein the reagent for removing the ivDde protecting group is a hydroxylamine hydrochloride/NMP/DIEA solution with a mass concentration of 10%, wherein the mass ratio of hydroxylamine hydrochloride, NMP and DIEA is 1.8:13.326:3.821.
  6. 6. The synthetic method according to claim 1, wherein the cleavage solution for cleaving the fully protected semaglutinin resin is a TFA/TIS/water/phenol solution, and the TFA/TIS/water/phenol solution has a mass ratio of TFA/TIS/water/phenol of water/phenol=7.60:0.21:0.14:0.14.
  7. 7. The synthetic method according to any one of claims 1 to 6, wherein the amino acid or polypeptide fragments used in the preparation method are Fmoc-Gly-OH、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(R1)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Glu(OtBu)-Gly-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Thr(tBu)-Phe-OH、Fmoc-Glu(OtBu)-Gly-OH and Boc-His (Trt) -Aib-OH.
  8. 8. The synthetic method of claim 7 wherein the coupling agent for coupling Fmoc-Lys (R1) -OH is PyOxim/DIEA.
  9. 9. The synthetic method of claim 7 wherein, in the preparation method, coupling is performed in the following order: 。
  10. 10. The method according to claim 9, wherein the molar ratio of the resin, the amino acid or the polypeptide fragment to the coupling agent is 1:2-3:2-3.
  11. 11. A synthesis method of a side chain fragment ivDde-AEEA-AEEA of semaglutin is characterized in that the ivDde-OH and AEEA-AEEA are reacted in ethanol as shown in a reaction formula I: 。
  12. 12. The method of synthesis according to claim 11, wherein the method of synthesis comprises: And respectively adding the ivDde-OH and the AEEA-AEEA into EtOH, heating and refluxing, wherein the reaction temperature is not lower than 78 ℃, and the reaction time is 8 hours.
  13. 13. The synthetic method of claim 11 or 12 wherein the molar ratio of ivDde-OH to AEEA-AEEA is 1:1.

Description

Method for synthesizing semaglutin and semaglutin side chain fragment Technical Field The invention belongs to the technical field of polypeptide medicaments, and particularly relates to a semaglutin and synthesis of semaglutin side chain fragments. Technical Field Semaglutin (Semaglutide) is a long-acting glucagon-like peptide-1 (GLP-1) analogue which, by activating the GLP-1 receptor, promotes glucose-dependent insulin secretion, inhibits glucagon secretion, delays gastric emptying, thereby reducing blood glucose. After the semaglutin is combined with GLP-1 receptor, the expression of insulin gene and the synthesis and secretion of insulin are promoted by activating the signal transmission path in cells. At the same time, it can suppress appetite and reduce food intake, thereby reducing weight. The semaglutin is mainly used for treating type 2 diabetes, can be used alone or in combination with other hypoglycemic drugs, and can be used as an initial therapeutic drug for patients with poor blood sugar control through diet control and exercise therapy. The active differentiation theory of the semaglutin is N epsilon 26 [ (S) - (22, 40-dicarboxylic acid-10,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazatetramantane-1-acyl) ] [ Aib 8,Arg34 ] GLP-1- (7-37) peptide, the molecular formula is C 187H291N45O59, the molecular weight is 4113.58g/mol, and the chemical structure is shown as the following formula 1. The preparation method of the semaglutin reported at present is mainly solid phase total synthesis (SPPS). The advantage of solid phase total synthesis is mainly represented by the fact that the original reactants and products are all connected to a solid phase carrier, so that all reactions can be carried out in one reaction vessel, and automation operation is facilitated. The solid phase total synthesis method is mainly divided into 2 strategies, wherein the first strategy is to directly connect Lys containing a side chain as a fragment to a main chain of the semaglutin to complete target peptide resin, namely, connecting the side chain to Lys 26-ε-NH2 or adopting a material of Lys 26 -epsilon-side chain, and gradually coupling amino acids to prepare the semaglutin. The process route is simple, but the complete side chain is connected due to the fact that the Lys 26 is coupled, so that space obstruction of rear end coupling is large, synthesis efficiency is low, impurities are increased, and yield is low. And secondly, disassembling the side chain, and respectively and gradually completing the coupling of the main chain and the side chain of the semaglutin, namely removing the protecting group on Lys 26-ε-NH2 after the coupling of the main chain peptide of the semaglutin is gradually completed in a solid phase, and then coupling the side chain fragment to finally obtain the target peptide resin. The process avoids the space obstruction caused by introducing side chains in the solid phase synthesis process, but the sequence of the semaglutin is longer and has more hydrophobic amino acids, and folding, resin shrinkage and reaction time prolongation are easy to form in the solid phase synthesis process, so that more racemized peptide impurities, missing peptides or inserted peptide impurities which are close to the properties of products are generated, the purification difficulty of the semaglutin is increased, and the product yield is reduced. Therefore, there is an urgent need in the art to provide a synthesis method of semaglutin, so as to solve the technical barrier of side chain grafting in the existing semaglutin synthesis process. Disclosure of Invention In order to solve the defects in the prior art, the invention provides a synthesis method of the semaglutin, which has high synthesis efficiency and greatly improves the yield and purity of the semaglutin. The invention provides a synthesis method of semaglutin, which is characterized in that the amino acid sequence of semaglutin is H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala24-Ala-Lys26(AEEa-aEEA-G1u(α-Octa))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH,, and the preparation method comprises the following steps: Step 1, taking resin as a solid-phase carrier, and sequentially coupling amino acid or polypeptide fragments from a C end to an N end according to a primary sequence of the semaglutin by a solid-phase synthesis method to obtain a first polypeptide resin Fmoc-Ala24-Ala-Lys26(R1)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly- resin; step 2, deprotecting the first polypeptide resin prepared in the step 1, removing the R1 protecting group, and connecting a side chain segment ivDde-AEEA-AEEA to obtain a second polypeptide resin Fmoc-Ala24-Ala-Lys26(AEEa-aEEA-ivDde)-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly- resin; step 3, coupling the second polypeptide resin prepared in the step 2 with the rest amino acid or polypeptide fragments in sequence by a solid phase synthesis method t