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Preparation method of liraglutide

A technology of liraglutide and peptide fragments, applied in the field of medicine, can solve the problems of severe resin shrinkage, reduced yield, difficult purification, etc., and achieve the effects of low cost, reduced generation of missing peptides, and easy purification

Active Publication Date: 2017-05-24
哈药集团股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since there are 15 hydrophobic amino acids in the liraglutide sequence, the resin shrinks more severely during the coupling process, and missing peptides are easily produced, resulting in a lower yield
At the same time, there are many impurities in the crude peptide that are similar in nature to the product, making purification very difficult

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] This example provides a preparation method of liraglutide, which is prepared according to the following steps:

[0037] 1. Preparation of peptide fragments

[0038] 1.1 Preparation of peptide fragment 1

[0039] Weigh 300 g (0.36 mol) of resin with a substitution degree of 1.2 mmol / g, add it to a polypeptide synthesis reactor, add 2 LDCM to swell the resin for 30 minutes, and wash once with DCM. Weigh 119.3g Fmoc-Asp(Otbu)-OH (0.29mol) and 69.8g DIEA (0.54mol) and dissolve them in DCM, add them to the above-mentioned reactor, react for 1.5 hours, drain them, and wash them once with DCM. Add a solution of DCM and methanol at a volume ratio of 10:1, and stir to block for 30 minutes. Drain the solvent, wash with DCM 3 times, shrink the resin 3 times with methanol, and vacuum-dry to obtain Fmoc-Asp(Otbu)-2-chloro-trityl resin. After testing, the resin substitution degree is 0.45mmol / g.

[0040] Weigh 360 g of Fmoc-Asp(Otbu)-2-chloro-trityl resin, remove the Fmoc protecti...

Embodiment 2

[0074] The difference with Example 1 is: the degree of substitution of Fmoc-Asp(Otbu)-2-chloro-trityl resin in step 1 is 0.32mmol / g; the degree of substitution of Fmoc-Leu-2-chloro-trityl resin The degree of substitution is 0.31mmol / g; the degree of substitution of Fmoc-Ile-2-chloro-trityl resin is 0.35mmol / g; the degree of substitution of Fmoc-Gly-2-chloro-trityl resin is 0.31mmol / g; the combination of condensing agent and activator in step 1 is DIEA / HATU, and the combination of condensing agent and activator in step 2 is DIEA / TBTU.

[0075] Step 4 was vacuum-dried to obtain 92.8 g of liraglutide crude peptide with a purity of 72.8% and a crude peptide yield of 74.5%.

[0076] After purification in step 4, 33.8 g of pure liraglutide was finally obtained, with a purity of 99.5%, a purification yield of 49.8%, and a total yield of 37.1%.

Embodiment 3

[0078] The difference with Example 2 is: the degree of substitution of Fmoc-Asp(Otbu)-2-chloro-trityl resin in step 1 is 0.78mmol / g; the degree of substitution of Fmoc-Leu-2-chloro-trityl resin The degree of substitution is 0.72mmol / g; the degree of substitution of Fmoc-Ile-2-chloro-trityl resin is 0.75mmol / g; the degree of substitution of Fmoc-Gly-2-chloro-trityl resin is 0.78mmol / g; the combination of condensing agent and activator in step 1 is DIC / HOBt.

[0079] Step 4 was vacuum-dried to obtain 118.5 g of liraglutide crude peptide with a purity of 57.6% and a crude peptide yield of 75.2%.

[0080] After purification in step 4, 32.5 g of pure liraglutide was finally obtained, with a purity of 99.3%, a purification yield of 47.3%, and a total yield of 35.6%.

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Abstract

The invention discloses a preparation method of liraglutide, and belongs to the technical field of medicines. The method comprises the following steps of (1) carrying out solid-phase synthesis to form four peptide segments of the liraglutide, wherein the peptide segment 1 is the (1-9)th amino acids in a liraglutide main chain sequence, the peptide segment 2 is the (10-14)th amino acids in the liraglutide main chain sequence, the peptide segment 3 is the (15-23)th amino acids in the liraglutide main chain sequence and the peptide segment 4 is the (24-31)th amino acids in the liraglutide main chain sequence; (2) stepwise coupling various peptide segments to obtain an all-protected liraglutide main chain peptide; (3) removing a protecting group of the 20th lysine and completing connection of side chains to obtain all-protected liraglutide peptide resin; and (4) carrying out cracking, purification and freeze-drying to obtain the liraglutide. The preparation method is high in production efficiency and stable in process, and a product is high in purity, high in yield and suitable for industrial production.

Description

technical field [0001] The invention relates to a preparation method of liraglutide, belonging to the technical field of medicine. Background technique [0002] Liraglutide, developed by Novo Nordisk, is an analogue of human glucagon-like polypeptide-1 (GLP-1), which is 97% homologous to human natural GLP-1 and has GLP-1 Receptor agonism. It has multiple functions such as lowering blood sugar, reducing body weight, promoting islet cell regeneration and protecting cardiovascular system, and can be used in combination with other antidiabetic drugs to treat type II diabetes. After 10 years of research and development, liraglutide was approved for marketing in the European Union and the United States in July 2009 and January 2010, and was launched in China in March 2011. Based on the unique fatty acid chain modification technology platform developed by Novo Nordisk, by replacing the 34th lysine of the natural GLP-1 molecule with arginine, and adding a 16-carbon fatty acid side...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K1/20C07K1/06C07K1/04
CPCC07K14/605Y02P20/55
Inventor 袁淑杰李郑武齐岩刘佳吉丁辉曹翊婕户巧芬王立东高晶张珊珊葛京城刘磊曲学伟赵华南黄炎
Owner 哈药集团股份有限公司
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