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Method for preparing exenatide with natural coupling method

An exenatide and coupling technology, which is applied in the field of preparation of polypeptide drugs, can solve the problems of unfavorable large-scale production, strong hydrophobicity, long synthesis cycle, etc., and achieves advantages of large-scale production, easy post-processing, and short synthesis cycle. Effect

Inactive Publication Date: 2012-07-04
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since each fragment of this method is a fully protected peptide, there are problems such as strong hydrophobicity, poor solubility of the peptide chain, difficulty in fragment purification, and low coupling efficiency between fragments.
[0006] The existing synthetic method of exenatide has the disadvantages of cumbersome operation, long synthesis cycle, large waste liquid, unfavorable for environmental protection, high cost and unfavorable for large-scale production

Method used

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  • Method for preparing exenatide with natural coupling method
  • Method for preparing exenatide with natural coupling method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Preparation of fully protected peptide fragment 11 with Fmoc protection at the N-terminus

[0049] SEQ ID No.11, also known as peptide fragment 11 in the present invention, its molecular structure is His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-OH; fully protected peptide fragment 11 refers to The peptide fragment 11 protected by the protecting group tBu and OtBu has the same meaning as "Exenatide (1-10)" in the present invention.

[0050] A. Preparation of Fmoc-Leu-CTC resin

[0051] Weigh 20 g of 2-CTC resin with a substitution degree of 0.5 mmol / g, add it to a solid-phase reaction column, wash it twice with DMF, and swell the resin with DMF for 30 minutes; take 7.1 g of Fmoc-Leu-OH and dissolve it in DMF, ice Add 5.22mL DIEA to activate in a water bath; then add to the above-mentioned reaction column filled with resin, react for 2 hours; add 8mL of anhydrous methanol to block for 1 hour. Wash 3 times with DMF and 3 times with DCM, seal with anhydrous methanol...

Embodiment 2

[0056] Example 2: Preparation of Fmoc-Exenatide (1-10)-O-salicylaldehyde ester

[0057] Fmoc-Exenatide (1-10)-OH crude peptide (10.9g, 6mmol), salicylaldehyde (3.1ml, 30mmol) and DCC (2.5g, 12mmol) were dissolved in 400ml DCM, and reacted at room temperature for 2h. After the reaction was completed, the volume of the reaction solution was rotary evaporated to a volume ratio of <25%, and then added to 100 times the amount of anhydrous ether, centrifuged, washed with anhydrous ether, and vacuum-dried to obtain Fmoc-Exenatide (1-10)- O-salicylaldehyde ester 10.73g.

Embodiment 3

[0058] Embodiment 3: Remove Fmoc-exenatide (1-10)-O-salicylaldehyde ester protecting group

[0059] Fmoc-exenatide (1-10)-O-salicylaldehyde ester (9.6g, 5mmol) was placed in the reaction vessel, added in 200ml lysate (TFA:H 2 O:TIS volume ratio=95:2.5:2.5), stirred at room temperature for 2h. After the reaction, the reaction solution was poured into 2L of anhydrous ether, centrifuged, washed with anhydrous ether, and vacuum-dried to obtain the Fmoc-peptide fragment 11-O-salicylaldehyde ester, namely Fmoc-His-Gly-Glu-Gly -Thr-Phe-Thr-Ser-Asp-Leu-O-salicylaldehyde ester, product weight 6.85g.

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Abstract

The invention relates to a method for preparing exenatide. The method comprises the following steps of: (1) splitting exenatide into a plurality of peptide fragments, wherein peptide fragments of which the ends N are Thr or Ser and other peptide fragments needed by exenatide are obtained with a solid phase synthesis method; (2) forming an acetal structure by using the ends C of peptide fragments coupled to the ends N of the peptide fragments of which the ends N are Thr or Ser, and removing side chain protecting groups; (3) removing side chain protecting groups and section N protecting groups of the peptide fragments of which the ends N are Thr or Ser; (4) coupling the peptide fragments in the step (2) with the peptide fragments in the step (3) in a liquid phase to obtain exenatide of which the end N is protected; and (5) removing end N amine protecting groups and purifying to obtain exenatide. A process disclosed by the invention has the characteristics of simple reaction operation, intermediate controllability, short reaction period, high yield, low cost and the like, and has a considerable economic and practical value and a wide application prospect.

Description

technical field [0001] The invention relates to a preparation method of polypeptide medicine, in particular to a method for preparing exenatide by natural coupling method. Background technique [0002] Exenatide, the English name is exenatide, its molecular structure is His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala- Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Pro-Ser-NH 2 . [0003] Exenatide is the first incretin analog jointly developed by Eli Lilly and Amylin. It is an artificially synthesized polypeptide consisting of 39 amino acids, which is compatible with endogenous incretins such as glucagon Glucose-like peptide-1 (GLP-1) has a similar effect. It can promote glucose-dependent insulin secretion, restore first-phase insulin secretion, inhibit glucagon secretion, slow down the emptying of gastric contents, and improve pancreatic β-cells. functions etc. [0004] In the prior art US6924264, US7157555, and U...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/575C07K1/06
CPCC07K1/107C07K1/02C07K1/06C07K14/605C07K1/04C07K14/575Y02P20/55
Inventor 宓鹏程唐洋明刘建马亚平袁建成
Owner HYBIO PHARMA
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