Method for preparing Semaglutide through solid and liquid combination

A semaglutide and synthetic method technology, applied in the field of polypeptide drug preparation, can solve the problems of product purity reduction, easy production of missing peptides, low efficiency, etc.

Active Publication Date: 2018-05-22
润辉生物技术(威海)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Since semaglutide is a long peptide consisting of a main chain containing 31 amino acids and a long side chain, the above preparation method has the following disadvantages: the synthesis of semaglutide by amino acid condensation one by one has long synthesis cycle and high efficiency. Low, with the extension of the peptide chain, it is easy to produce missing peptides, and the purity of the product is reduced; at the same time, semaglutide contains 3 Gly, 4 Ala and 2 Arg residues. Due to the structural characteristics of Gly and Ala, it can be used in It is easy to repeat the coupling during the condensation process, resulting in [+Gly] and [+Ala] impurities; Arg is a difficult amino acid, the coupling efficiency is low during the condensation process, and it is easy to produce [-Arg] missing peptides, these impurities are compatible with the target peptide The properties are similar, which increases the difficulty of product separation and purification, resulting in low product yield

Method used

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  • Method for preparing Semaglutide through solid and liquid combination
  • Method for preparing Semaglutide through solid and liquid combination
  • Method for preparing Semaglutide through solid and liquid combination

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Preparation of Alloc-Lys(PEG-PEG-γ-Glu(OtBu)-Monobutyl octadecanate)-OH:

[0058] 1) Resin swelling: Take 100g of CTC resin with a substitution degree of 0.5mmol / g, add 700ml of DCM or DMF to swell the resin for 0.5h, drain the solvent, wash the resin twice with DMF, and drain the solvent.

[0059]2) Preparation of Alloc-Lys(Fmoc)-resin: a) Mix Alloc-Lys(Fmoc)-OH, DIEA and resin according to the molar mass ratio of 3:6:1, and react at 25°C for 2h , to obtain Alloc-Lys(Fmoc)-resin; b) add a mixed solution of MeOH, DMF and DIEA to the resin, react at 10-30° C. for 30 min, and block the resin.

[0060] 3) Removal of Fmoc protecting group: add 20% PIP-DMF solution at 25°C to remove Fmoc protection twice: remove Fmoc protection for the first time and remove Fmoc protection for the second time, then wash the resin with DMF When the pH reaches 7, the solvent is drained; the reaction time for the first removal of Fmoc protection is 5 minutes, and the reaction time for the seco...

Embodiment 2

[0066] Liquid phase synthesis of dipeptide, tripeptide and tetrapeptide fragments:

[0067] 1) Preparation of Fmoc-Arg(Pbf)-Gly-OH:

[0068] a) Synthesis of Fmoc-Arg(Pbf)-OSu: Dissolve 0.2mol Fmoc-Arg(Pbf)-OH and 0.24mol HOSu in 0.4L THF, put in ice-water bath; dissolve 0.24mol DCC in 0.2L THF, and Add dropwise to the mixed solution in the previous step, continue to react for 1 h after the dropwise addition, then raise the temperature to 25°C and continue to react for 3 h; filter the reaction solution, evaporate to dryness, add DCM to dissolve and filter, and evaporate to dryness; add ethyl acetate to dissolve The solid was dissolved and recrystallized to obtain Fmoc-Arg(Pbf)-OSu.

[0069] b) Synthesis of Fmoc-Arg(Pbf)-Gly-OH: 0.15mol H-Gly-OH and 0.15mol Na 2 CO 3 Dissolve in 0.2L 50% THF-H 2 O solution to obtain a mixed solution; 0.1 mol Fmoc-Arg(Pbf)-OSu prepared in step a) was dissolved in THF, and added dropwise to the mixed solution in the previous step, reacted over...

Embodiment 3

[0106] Preparation of semaglutide peptide resin:

[0107] 1) Resin swelling: Take 25g of CTC resin with a substitution degree of 0.4mmol / g, add 200ml of DCM or DMF to swell the resin for 0.5h, drain the solvent, wash the resin twice with DMF, and drain the solvent.

[0108] 2) Preparation of Fmoc-Arg(Pbf)-Gly-resin: a) Mix Fmoc-Arg(Pbf)-Gly-OH, DIEA and resin according to the molar mass ratio of 3:6:1, at 25°C Under the condition of reaction for 2h, Fmoc-Arg(Pbf)-Gly-resin is obtained; b) Add the mixed solution of MeOH, DMF and DIEA to the resin, react at 10-30°C for 30min, seal the resin, and then use The resin was washed twice with DMF and the solvent was drained.

[0109] 3) Removal of Fmoc protecting group: add 20% volume fraction of PIP-DMF solution to the resin, and perform two removal of Fmoc protection at 10-30°C: after the first removal of Fmoc protection and the second removal of Fmoc protection , and then wash the resin with DMF to pH 7; the first de-Fmoc protecti...

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Abstract

The invention relates to a method for preparing Semaglutide through solid and liquid combination, and solves the technical problems that in the process for synthesizing long-sequence polypeptide by the existing technology, the synthesis period is long; the purification difficulty is high; the yield is low. The method for preparing Semaglutide through solid and liquid combination provided by the invention is characterized in that firstly, Lys and resin are condensed in an Alloc-Lys(Fmoc)-OH form by adopting a solid phase synthesis method; Fmoc protecting groups on epsilon-NH2 are removed; sidechain connection is performed; cracking is performed to obtain Alloc-Lys(PEG-PEG-gamma-Glu(OtBu)-Monobutyl octadecanate)-OH; meanwhile, 10 dipeptide or tripeptide or tetrapeptide fragments are simultaneously synthesized by a liquid phase synthesis method; then, the condensation reaction of the synthesized peptide fragments and single amino acid is performed by using the resin as a carrier; the 15-step solid phase condensation reaction is reduced in the process; the generation of lacked peptide impurities is reduced; the product purity and the yield are improved; meanwhile, the generation of the impurities of [+Gly]-Semaglutide and [+Ala]-Semaglutide is effectively avoided; the purification difficulty is greatly reduced. The method is widely applied to the technical field of polypeptide medicine preparation.

Description

technical field [0001] The invention belongs to the technical field of polypeptide drug preparation, and in particular relates to a method for preparing semaglutide by solid-liquid combination. Background technique [0002] Glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted by human intestinal L cells, which can promote the secretion of insulin and inhibit the secretion of glucagon, and has the effect of reducing blood sugar concentration. For the treatment of type II diabetes. However, natural GLP-1 is unstable in vivo and is easily degraded by dipeptidyl peptidase-IV (DPP-IV). [0003] Semaglutide, whose English name is Semaglutide, is a new type of long-acting glucagon-like peptide-1 (GLP-1) analog developed and produced by Novo Nordisk, Denmark, for the treatment of type II diabetes. Semaglutide has the effects of lowering blood sugar, losing weight and protecting cardiovascular system, and was recommended by the FDA for approval on October 18, 2017. After ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C07K1/02C07K1/04C07K1/06C07K1/14C07K1/36
CPCY02P20/55C07K14/605
Inventor 姬胜利陈明鲁殷金岗刘超王广胜
Owner 润辉生物技术(威海)有限公司
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