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Method for synthesizing liraglutide

A technology for liraglutide and peptide resin, applied in the field of liraglutide synthesis, can solve the problems of wasting raw materials, increasing costs, affecting downstream purification, etc., so as to reduce the generation of D-Ala, reduce the generation of impurities, and improve the purity and yield effect

Inactive Publication Date: 2020-02-25
苏州天马医药集团天吉生物制药有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The currently reported synthesis methods of liraglutide mainly use solid-phase synthesis, which are mainly divided into conventional methods and fragment methods, all of which have certain defects: for example, Fmoc-Lys(Alloc)-OH is used in Chinese patent CN102286092B, The heavy metal palladium is used in the final removal, and it is difficult to remove the heavy metal in the follow-up, which affects the quality of the product; Chinese patent CN103980358B incorporates the modified Ly s into the main chain. Due to the large side chain group and low activity, the coupling is easy to be incomplete , and the conventional condensation system is used, which causes a certain degree of difficulty for the coupling of subsequent amino acids, and produces a large number of impurity peptides such as missing peptides and racemated peptides, which affects downstream purification; Chinese patent CN103864918B divides the peptide chain into three fragments Coupling, this method wastes raw materials, and requires quality control for intermediate fragments, the steps are cumbersome and increase the cost, the gain outweighs the gain

Method used

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  • Method for synthesizing liraglutide
  • Method for synthesizing liraglutide
  • Method for synthesizing liraglutide

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1: Liraglutide fully protected peptide synthesis

[0039] In this example, liraglutide fully protected peptide refers to: Boc-His (Trt) 7 -Ala 8 -Glu(OtBu) 9 -Gly 10 -T hr(tBu) 11 -Phe 12 -Thr(tBu) 13 -Ser(tBu) 14 -Asp(OtBu) 15 -Val 16 -Ser(tBu) 17 -Ser(tBu) 18 -Tyr(tBu) 19 -Leu 20 -Glu(OtBu) 21 -Gly 22 -Gln(Trt) 23 -Ala 24 -Ala 25 -Lys(Pal-Glu-OtBu) 26 -Glu(OtBu) 27 -Phe 28 -Ile 29 -Ala 30 -Trp(Boc) 31 -Leu 32 -Val 33 -Arg(Pbf)34 -Gly 35 -Arg(Pbf) 36 -Gly 37 -Wang Resin.

[0040] A. Add 100g (30mmol) of Fmoc-Gly-Wang Resin with a substitution degree of 0.3mmol / g into the reaction column, add 400mml of dichloromethane to swell for 30min, and drain it. Add 300ml of 20% piperidine / DMF solution, mix for 5min, then drain, add DMF 300ml, wash for 1min, then drain. Add 300 ml of 20% piperidine / DMF solution, mix for 15 minutes, and drain. Add 300ml of DMF to wash for 1min, then drain and repeat DMF washing 4 times. Take a small amount of...

Embodiment 2

[0045] Example 2: Preparation of crude liraglutide

[0046] Add 2500ml of lysate to the liraglutide fully protected peptide resin obtained in Example 1, the proportions are TFA: 90%-95%, m-cresol: 1%-5%, EDT: 1%-5%, Purified water: 1-5%, cleavage at room temperature for 2-3 hours, suction filtration, add the filtrate to 10 times the volume of pre-cooled methyl tert-butyl ether to precipitate, centrifuge, and then wash the precipitate with methyl tert-butyl ether for 3 times, vacuum-dried to obtain liraglutide crude peptide.

Embodiment 3

[0047] Example 3: Purification of Liraglutide Crude Peptide

[0048] The crude peptide was purified in two steps by reverse-phase high-performance liquid chromatography: the first step was purification, the mobile phase A was 20mmol / L sodium carbonate solution (pH was adjusted to 9 with phosphoric acid), and the B phase was: acetonitrile; the second step was purification For desalination of pure products, mobile phase A is purified water, and mobile phase B is ACN; the two-step purification is gradient elution, and the elution gradient: the first step is mobile phase B with 30% to 45%, nano-micro C18 packing ; The second step of purification and desalination is 10% to 50% of B phase, nano-micro C4 polymer filler, the elution time is 40min, and the ultraviolet detection wavelength is 220nm. The pure liraglutide was obtained by concentration and freeze-drying. The purity of liraglutide was tested to be 99.66%, the purity of the pure product was less than 0.15%, and the purifica...

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Abstract

The invention discloses a method for synthesizing liraglutide and belongs to the technical field of polypeptide synthesis. The method of the invention comprises the steps: with Fmoc-Gly-Wang Resin serving as a solid-phase synthesis starting support, performing activation coupling on 29 amino acids from positions 37 to 9 (sequence C end to N end) of liraglutide by means of a novel condensing agentPyBrop (bromotrispyrrolidinophosphoniumhexafluophosphate), wherein Fmoc-Lys(Pal-Glu-OtBu)-OH is used as a raw material in the position 26; Boc-His(Trt)-Osu and Fmoc-Ala-Osu are used to perform condensing in the positions 7 and 8 to obtain a liraglutide all-protected peptide, and cracking precipitation is then performed to obtain liraglutide. The method has the advantages that amino acid coupling efficiency is improved, racemic peptides and deleted peptide impurities generated in the synthesis process of liraglutide are greatly reduced, racemic impurities (D-His7 and D-Ala8) which are very similar to the product in property are particularly effectively inhibited or reduced, the purity of the crude product is improved, the purification difficulty is reduced, and industrial scaled productionis facilitated.

Description

technical field [0001] The invention relates to a method for synthesizing liraglutide, which belongs to the technical field of polypeptide synthesis. Background technique [0002] Liraglutide, English name Liraglutide, independently developed by Novo Nordisk, Denmark, is a long-acting glucagon-like peptide 1 (GLP-1) analogue for the treatment of type 2 diabetes, which belongs to the GLP-1 receptor Agonist, the first human glucagon-like polypeptide-1 (GLP-1) analog developed for the treatment of type II diabetes. The drug was approved for marketing by FDA on January 25, 2010, and was approved by SFDA for marketing in China on March 4, 2011. Liraglutide, as a new generation of incretin-based hypoglycemic drugs, not only has a long duration of action, but also fully retains multiple physiological activities of natural GLP-1, which can safely and effectively lower blood sugar and may prevent various cardiovascular risks. Factors have a protective effect, bringing new options f...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K1/08C07K1/04C07K1/20
CPCC07K14/605Y02P20/55
Inventor 王良友周黎郑春旭沈杰黄保胜陈维维宋亮张璐
Owner 苏州天马医药集团天吉生物制药有限公司
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