Method for preparing exenatide

A technology of exenatide and peptide resin, applied in the field of peptide solid-phase synthesis, can solve the problems of difficult stability, many control items, and many by-products, etc.

Active Publication Date: 2011-09-07
SHANGHAI AMBIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Liquid-phase synthesis of Exenatide has complex steps, GMP production has many control items, it is not easy to be stable, and t...

Method used

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  • Method for preparing exenatide

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preparation example Construction

[0031] In one example of the present invention, the preparation method of solid-phase synthesis of exenatide by the polypeptide of the present invention comprises the following steps:

[0032] In the first step, AM polystyrene resin, Fmoc-Rink-Amide-Linker, TBTU, HOBt, and NMM are mixed to obtain Fmoc-Rink amide AM resin, whose degree of substitution is 0.4-1.6mmol / g;

[0033] In the second step, the deprotecting agent of the present invention is mixed with the Fmoc-Rink amide AM resin to remove the Fmoc group to obtain the Rink amide AM resin;

[0034] The third step is to condense Fmoc-Ser(tBu)-OH and Rink amide AM resin to obtain Fmoc-Ser(tBu)-Rink amide AM resin;

[0035] The fourth step, using the deprotecting agent of the present invention to remove the Fmoc group;

[0036] In the fifth step, the above-mentioned peptide bond formation steps are repeated to make the peptide chain grow from the C-terminus to the N-terminus until Fmoc-His(Trt)-Gly-Glu(OtBu)-Gly-Thr(tBu)-Ph...

Embodiment 1

[0061] Load Fmoc-Rink Amide Linker

[0062] Use 24.0g of AM polystyrene resin (substitution degree 0.6-0.9mmole / g) and 1.0 equivalent of Fmoc-Rink-Amide-Linker, 1.425 equivalent of TBTU, 1.5 equivalent of HOBT and 3 equivalents of NMM to react with stirring for 3 hours, and the reaction on the resin is not complete The amino group was capped with Ac2O / Pyridine / DMF (v / v / v), and finally the obtained resin was 34g, and the degree of substitution was 0.58mmole / g.

[0063] Deprotection

[0064] 8%piperidine / 1.5DBU / 5%HOBt / DMF (v / v / w / v) was used to deprotect twice consecutively, and the time was 10min and 20min respectively. Wash with DMF and methanol, respectively. After exhaustion, Kaiser test was used to evaluate the removal of Fmoc.

[0065] amino acid condensation

[0066] Add Fmoc-AA-OH / HOBt (1.0 equivalent / 1.0 equivalent) to the reactor, 1.0 equivalent relative to Fmoc-Rinkamide AM resin) / DMF solution, then add DIC (1.5 equivalent relative to Fmoc-Rinkamide AM resin), stir...

Embodiment 2

[0077] Load Fmoc-Rink Amide Linker

[0078] Use 24.0g of AM polystyrene resin (substitution degree 0.8-1.0mmole / g) and 1.5 equivalents of Fmoc-Rink-Amide-Linker, 1.425 equivalents of TBTU, 1.5 equivalents of HOBT and 3 equivalents of NMM to stir for 3 hours, and the reaction on the resin is not complete The amino group was capped with Ac2O / Pyridine / DMF, and finally the obtained resin was 34g, and the degree of substitution was 0.60mmole / g.

[0079] Deprotection

[0080] Use 6%piperidine / 1.3DBU / 2%HOBt / DMF (v / v / w / v) for two consecutive deprotection times for 10min and 20min respectively. Wash with DMF and methanol, respectively. After exhaustion, Kaiser test was used to evaluate the removal of Fmoc.

[0081] amino acid condensation

[0082] Add Fmoc-AA-OH / HOBt (1.5 equivalents / 1.5 equivalents) to the reactor, 1.5 equivalents relative to Fmoc-Rinkamide AM resin) / DMF solution, then add DIC (2.0 equivalents relative to Fmoc-Rinkamide AM resin), stir After 45 minutes, DIC (2.0 ...

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Abstract

The invention discloses a method for preparing polypeptide solid-phase synthesized exenatide. The method comprises the following steps of: (1) mixing Fmoc-Rink amide AM resin with a protection removing agent to obtain Rink amide AM resin; (2) condensing Fmoc-Ser(tBu)-OH and the Rink amide AM resin to obtain Fmoc-Ser(tBu)-Rink amide AM resin; (3) repeating the Fmoc protection removal in the step (1) and condensation of amino acid and polypeptide on the resin in the step (2) according to a solid-phase synthesis method and condensing the amino acids from C end to N end and the polypeptide on the resin in the sequence of Ser to His to form the polypeptide resin shown as the formula II; and separating the polypeptide and resin on the polypeptide resin shown as the formula II to obtain the exenatide shown as the formula I.

Description

technical field [0001] The invention relates to the field of polypeptide solid-phase synthesis, in particular to a method for solid-phase synthesis of exenatide. Background technique [0002] Exenatide, a polypeptide composed of 39 amino acids, is the first incretin drug. Incretins are a new type of treatment for type 2 diabetes that mimic the anti-diabetic or glucose-lowering responses of the body's natural gut hormones, which include stimulating insulin production in the body when blood sugar rises , inhibits the secretion of glucagon after meals, slows down the rate of blood uptake of nutrients and reduces food intake. Exenatide is a new type II diabetes drug. Subcutaneous injection, 2 times a day, for patients with type II diabetes whose blood sugar cannot be adequately controlled by metformin, sulfonylureas or the combination of metformin and sulfonylureas. [0003] Currently, the synthesis methods of exenatide include solid-phase method and solid-liquid phase collec...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/06C07K1/04
Inventor 白俊才张国庆张若平
Owner SHANGHAI AMBIOPHARM
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