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Solid-phase synthesis method for liraglutide

A technology of liraglutide and solid-phase synthesis, which is applied to the preparation method of peptides, chemical instruments and methods, peptides, etc., can solve the problems of many fragments, large resin consumption, and high cost.

Active Publication Date: 2014-06-18
哈尔滨吉象隆生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The domestically reported patented method for solid-phase synthesis of liraglutide (CN102875665A) uses 5 fragments to synthesize liraglutide. Although this method can shorten the synthesis cycle and improve efficiency, there are many fragments and the resin consumption Large, high cost, and the 20th lysine uses Fmoc-Lys(Alloc)-OH, and selective removal of Alloc requires a heavy metal catalyst Pd(Ph3P)4, which generally needs to be reacted under nitrogen protection, and industrial production will restricted

Method used

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  • Solid-phase synthesis method for liraglutide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: Synthesis of Fragment 1

[0057] Weigh 10g of Wang resin with a substitution degree of 1.08mmol / g, add it to a solid-phase reaction column, and swell the resin with DMF for 30 minutes, weigh 3.21g Fmoc-Gly-OH, 1.46gHOBt and 0.13gDMAP and dissolve them with DMF, add 1.84 mL of DIC was activated for 5 minutes, and added to the above-mentioned reaction column filled with resin. After 3 hours of reaction, 20 mL of pyridine and 20 mL of acetic anhydride were added to block for 8 hours. Washed 6 times with DMF and 2 times with methanol, the detection substitution degree was 0.35mmol / g.

[0058] Use 25% piperidine / DMF to remove FMOC in FMOC-Gly-King resin, 10 minutes each time, twice in total, and wash with DMF 6 times after deprotection. , weigh

[0059] Fmoc-Arg(Pbf)-OH6.81g, HOBt1.42g, dissolved in 40ml of DMF, added DIC1.63ml to activate for 5 minutes, added to the reaction column for 2 hours to obtain

[0060] Fmoc-Arg(Pbf)-Gly-Wang resin. Repeat the ste...

Embodiment 2

[0062] Embodiment 2: the synthesis of fragment 2

[0063] Weigh 20g of CTC resin with a substitution degree of 1.0mmol / g, add it to a solid-phase reaction column, swell the resin with DMF for 30 minutes, dissolve 12.44g of Fmoc-Ala-OH with DMF, add 26.4mL of DIEA to activate, add the above In the reaction column equipped with the resin, after reacting for 2 hours, 20 mL of anhydrous methanol was added to block for 1 hour, and washed 6 times with DMF to obtain Fmoc-Ala-CTC.

[0064] Use 25% piperidine / DMF to remove FMOC in Fmoc-Ala-CTC, 10 minutes each time, twice in total, and wash 6 times with DMF after deprotection. , Weighed Fmoc-Ala-OH18.68g, HOBt8.1g, dissolved in 80ml DMF, added DIC9.3ml to activate for 5 minutes, added to the reaction column and reacted for 2 hours to obtain Fmoc-Ala-Ala-CTC. Repeat the above steps of de-FMOC and amino acid coupling, followed by Fmoc-Gln(trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(Otbu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tbu)- OH, Fmoc-Ser(tbu)-OH, Fmoc-Se...

Embodiment 3

[0067] Embodiment 3: the synthesis of fragment 2

[0068]Weigh 20g of CTC resin with a substitution degree of 1.0mmol / g, add it to a solid-phase reaction column, swell the resin with DMF for 30 minutes, dissolve 12.44g of Fmoc-Ala-OH with DMF, add 26.4mL of DIEA to activate, add the above In the reaction column equipped with the resin, after reacting for 2 hours, 20 mL of anhydrous methanol was added to block for 1 hour, and washed 6 times with DMF to obtain Fmoc-Ala-CTC.

[0069] Use 25% piperidine / DMF to remove FMOC in Fmoc-Ala-CTC, 10 minutes each time, twice in total, and wash 6 times with DMF after deprotection. , Weighed Fmoc-Ala-OH18.68g, TBTU19.26g, dissolved in 80ml DMF, added DIEA19.8ml to activate for 5 minutes, added to the reaction column and reacted for 2 hours to obtain Fmoc-Ala-Ala-CTC. Repeat the above steps of de-FMOC and amino acid coupling, followed by Fmoc-Gln(trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(Otbu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tbu)- OH, Fmoc-Ser(tbu)-OH, Fmoc...

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Abstract

The invention discloses a solid-phase synthesis method for liraglutide. The solid-phase synthesis method for liraglutide comprises the following steps: 1) firstly synthesizing first to tenth amino acid segments 3, eleventh to nineteenth amino acid segments 2, and twentieth to thirty-first amino acid segments 1, wherein the twentieth lysine adopts Fmoc-Lys(Mtt)-OH, and the first histidine adopts Boc-His(Trt)-OH; 2) synthesizing pal-Glu(OH)-Otbu by adopting a liquid phase synthesis method; 3) selectively removing the Mtt protecting group on the twentieth lysine by use of 5% TFA (Trifluoroacetic Acid), connecting pal-Glu(OH)-Otbu with the side chain of the twentieth lysine to obtain a segment 4; 4) sequentially connecting the segments 2, the segments 3 and the segments 4 to obtain peptide resin completely protected by liraglutide; 5) cracking, purifying, and lyophilizing to obtain the liraglutide product.

Description

technical field [0001] The invention relates to a method for synthesizing a polypeptide, in particular to a method for solid-phase synthesis of liraglutide. Background technique [0002] Liraglutide is a human glucagon-like peptide-1 (GLP-1) analogue developed by Novo Nordisk, consisting of 31 amino acid residues, and its molecular structure is as follows: [0003] H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-ε-(N-α -Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH [0004] Liraglutide is a GLP-1 analog developed by Novo Nordisk. The drug was approved for marketing in the United States on January 25, 2010. The dosage form is subcutaneous injection, and only one subcutaneous injection per day can exert a good hypoglycemic effect. [0005] Liraglutide is a synthetic drug based on the natural GLP-1(7-37) sequence. It has 95% homology with GLP-1 and retains the biological activity of GLP-1 (7-37). Its molecular st...

Claims

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Application Information

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IPC IPC(8): C07K14/605C07K1/20C07K1/06C07K1/04
CPCY02P20/55C07K14/605
Inventor 冷国庆余荣熹王艳王浩迟帅刘宇帅田辉张琪李迎新肖明辉李喜全赵民喜苏宏健
Owner 哈尔滨吉象隆生物技术有限公司
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