Preparation method of liraglutide

A technology of liraglutide and liraglutide, which is applied in the field of preparation of liraglutide, can solve the problems of low amino acid access rate and reduced yield, and achieve the reduction of purification difficulty, production cost and preparation cost low effect

Active Publication Date: 2017-08-18
SICHUAN JISHENG BIOPHARM CO LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] The object of the present invention is to: aim at the problems of multiple couplings of amino acids caused by amino folding in the synthesis process and low access rate to further reduce yield and product purity, the present invention provides a high-yield, low-cost, reaction The preparation method of liraglutide with mild conditions is conducive to the realization of industrialization

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  • Preparation method of liraglutide
  • Preparation method of liraglutide
  • Preparation method of liraglutide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Preparation of Liraglutide Peptide Backbone Resin:

[0045] Using Fmoc-Gly-Wang resin as the initial carrier, the liraglutide peptide backbone resin was prepared by de-Fmoc protection and coupling reaction, and the amino acids were sequentially coupled. Gly was used as the first coupled amino acid, and His was used as the first coupling amino acid. The 31 coupled amino acids are sorted, and the coupled amino acid fragments are:

[0046]Boc-His(trt)-Ala-Glu(otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(otbu)-Y(tbu)-Tyr(tbu)-Leu- Glu(otbu)-Gly-Gln(trt)-X(alloc)-Glu(otbu)-Phe-Ile-Ala-Trp(boc)-Leu-Val-Arg(pbf)-Gly-Arg(pbf)-Gly -Wang Resin,

[0047] Where X is Ala-Ala-Lys, Y is Val-Ser-Ser;

[0048] When X is inserted, the corresponding protected amino acid is Fmoc-Ala-Ala-Lys(alloc)-OH;

[0049] When Y is inserted, the corresponding protected amino acid is Fmoc-Val-Ser(tbu)-Ser(tbu)-OH;

[0050] When His is inserted, the corresponding protected amino acid is Boc-His(trt...

Embodiment 2

[0078] On the basis of Example 1, the crude product of liraglutide was prepared:

[0079] Take liraglutide peptide resin, add the lysate with a volume ratio of TFA:EDT:TIS:water=90:4:2:4, the amount of lysate is 5mL / g resin, and stir evenly. Stir the reaction at room temperature for 3 hours, filter the reaction mixture with a sand core funnel, collect the filtrate, wash the resin three times with a small amount of TFA, combine the filtrate and concentrate under reduced pressure, add anhydrous ether to precipitate, then wash the precipitate three times with anhydrous ether, pump The dried off-white powder is crude liraglutide.

Embodiment 3

[0081] On the basis of Example 2, the purification process of crude liraglutide:

[0082] The crude liraglutide was dissolved in 20% acetonitrile aqueous solution with ammonia water to adjust the pH value to 10, filtered through a 0.45 μm mixed microporous membrane, and purified for later use;

[0083] High performance liquid chromatography is used for purification. The purified chromatographic column is octadecyl bonded silica gel with a diameter of 10cm and a filler of 10um. The mobile phase is dilute ammonia solution and acetonitrile solution respectively, the flow rate is 120ml / min, the sample loading is 5-10g, and the detection wavelength of the chromatograph is 230nm.

[0084] After multiple purifications, the qualified main peak was collected, analyzed in liquid phase to detect its purity, and concentrated under reduced pressure to obtain a dilute ammonia solution of liraglutide, which was freeze-dried to obtain 18.8 g of liraglutide, with a total yield of 10.1%.

[0...

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Abstract

The invention discloses a preparation method of liraglutide. The preparation method comprises the following steps: using a deprotection reagent Fmoc-Gly-wang resin for deprotection, and removing Fmoc protective groups; coupling three protective amino acids at the 12th-14th sites by protective peptide fragments X; coupling three protective amino acids at 20-22 sites by protective peptide fragments Y; coupling protective amino acid at the 31th site by a Boc-His(trt)-OH protective form; removing alloc protection of lysine side chains for main-chain peptide resin of the liraglutide, coupling the side chains one by one and thus obtaining peptide resin of the liraglutide; cracking the peptide resin of the liraglutide in cracking liquid and thus obtaining a liraglutide crude product; purifying the liraglutide crude product and thus obtaining a liraglutide fine product. The preparation method disclosed by the invention has the advantages that segment coupling is carried out on amino sites which are easiest for folding in the process of synthesizing the main chain of the liraglutide, so that the problems of multiple times of coupling and low access rate for the amino acids due to folding in the synthesis process are avoided, the production cycle of the liraglutide is shortened, and the synthetic yield of the liraglutide is increased.

Description

technical field [0001] The present invention relates to the synthesis of polypeptides, in particular to a preparation method of liraglutide. Background technique [0002] At present, with the improvement of living standards, the number of people suffering from diabetes is gradually increasing, and liraglutide, a human glucagon-like peptide-1 (GLP-1) analogue, is used for the treatment of diabetes. The structure of liraglutide is: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Na- PAL-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. Therefore, there is a great demand for liraglutide in the market. There are many synthetic methods available. The method of genetic engineering is difficult and technically difficult; during the synthesis process, the most easily foldable amino sites (20-22 and 12-14) in the main chain synthesis process are likely to cause amino folding, resulting in by-products. The product and other impurities affect th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C07K1/16C07K1/08C07K1/06C07K1/04
CPCC07K14/605Y02P20/55
Inventor 龙春艳易文铖杨玲玲倪红飞田安全
Owner SICHUAN JISHENG BIOPHARM CO LTD
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