Synthesis of exenatide through solid phase fragment method

A technology of exenatide and solid-phase synthesis, which can be applied in the direction of peptides, specific peptides, hormone peptides, etc., and can solve the problems of incomplete amino acid coupling reaction, long peptide, and long synthesis period.

Active Publication Date: 2013-10-02
HAINAN SHUANGCHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the relatively long peptide, this method has the characteristics of

Method used

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  • Synthesis of exenatide through solid phase fragment method
  • Synthesis of exenatide through solid phase fragment method
  • Synthesis of exenatide through solid phase fragment method

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preparation example Construction

[0041] Specifically, the exenatide solid-phase synthesis method provided by the invention comprises the following steps:

[0042] In the first step, peptide resin fragments (formula II and formula III) are prepared in solid phase respectively;

[0043] In the second step, the formula III is cut to obtain a polypeptide fragment with fully protected side chains (formula IV);

[0044] In the third step, the polypeptide fragment (formula IV) with fully protected side chains is coupled to the peptide resin (formula II), and Fmoc is removed to obtain the polypeptide resin (formula V);

[0045] The fourth step is to sequentially couple the remaining ten protected amino acids by solid-phase synthesis, and de-Fmoc to obtain Exenatide-Rink Amide Resin (Formula VI);

[0046] In the fifth step, the polypeptide on the polypeptide resin represented by formula VI is separated from the resin to obtain exenatide represented by formula I.

[0047]The synthesis of the peptide resin fragment sh...

Embodiment 1

[0079] Peptide resin H-[22-39]-Rink Amide AM resin with protective groups on the side chain

[0080] 1: Synthesis of Fmoc-Ser(tBu)-Rink Amide AM resin

[0081] (1) Fmoc-Rink Amide AM resin (manufactured by Tianjin Nankai Hecheng Technology Co., Ltd., with a substitution degree of 0.8mmol / g, 10g) was put into a solid-phase reaction column, washed twice with DMF, and swelled in DMF for 30 minutes.

[0082] (2) Drain the solution, and remove Fmoc twice with 20% piperidine in DMF at room temperature for 10 minutes and 20 minutes respectively.

[0083] (3) Drain the solution and wash the resin six times with DMF.

[0084] (4) Dissolve Fmoc-Ser(tBu)-OH (9.20g) and HOBt (4.86g) in DMF (30mL) and DCM (30mL), add DIPCDI (7.52ml), and pre-react in an ice bath for 10 minutes.

[0085] (5) Add the above-mentioned reaction solution into a solid-phase reactor, stir it mechanically, and react at room temperature for 3 hours. The ninhydrin test shows that the resin is colorless and transpar...

Embodiment 2

[0096] Peptide resin Fmoc-[11-21]-CTC resin with protective groups on the side chain

[0097] 1. Synthesis of Fmoc-Leu-CTC resin

[0098] Weigh 30 g of CTC resin with a substitution degree of 1.1 mmol / g, add it to a solid-phase reactor, and wash it twice with DMF. Weigh 17.49g of Fmoc-Leu-OH, add 200mL of DMF to dissolve, then add 20.20mL of DIPEA, stir for 5min, add to the solid phase reactor, and react for 2 hours. The reaction solution was drained and washed three times with DMF. Add a mixture of 60mL methanol, 180mL DMF and 15mL DIPEA, and block for 30min. Wash 3 times with DMF, twice with DCM, twice with methanol, and drain. 43.07 g of Fmoc-Leu-CTC resin was obtained, and the degree of substitution was 0.75 mmol / g after measurement.

[0099] 2. Synthesis of Fmoc-[11-21]-CTC resin

[0100] (1) Put 43.07g of Fmoc-Leu-CTC resin into the solid phase reactor, wash with DMF three times, and swell with DMF for 20 minutes

[0101] (2) Drain the solution, add 2% (g / mL) HOBt ...

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Abstract

The invention discloses a solid phase synthesis preparation method of exenatide represented by a formula I, wherein a Fmoc-resin and a deprotection agent are mixed to obtain a deprotection resin, a Fmoc-amino acid and a deprotection resin are subjected to condensation to obtain a Fmoc-amino acid-resin, and Fmoc protection removal and condensation of the polypeptides on the Fmoc-amino acid and the resin are repeated to carry out condensation of the amino acid and the polypeptide on the resin according to a C terminal-to-N-terminal sequence to form a polypeptide resin. The method is characterized by comprising the following steps: (1) respectively forming polypeptide resin fragments represented by a formula II and a formula III; (2) cutting the fragment represented by the formula III to obtain a polypeptide fragment with complete side chain protection and represented by a formula IV; (3) conjugating the polypeptide fragment with complete side chain protection and represented by the formula IV to the polypeptide resin fragment represented by the formula II, and removing Fmoc to obtain a polypeptide resin represented by a formula V; (4) sequentially conjugating the remaining 10 Fmoc-amino acids, and removing Fmoc to obtain an exenatide-Rink Amide resin represented by a formula VI; and (5) separating the polypeptide and the resin on the polypeptide resin represented by the formula VI to obtain the exenatide represented by the formula I.

Description

technical field [0001] The invention relates to the field of polypeptide solid-phase synthesis, in particular to the synthesis of exenatide by a solid-phase fragment method. Background technique [0002] Exenatide is a polypeptide composed of 39 amino acids, its sequence 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-Ser. The amino acid sequence of exenatide is 53% homologous to human glucagon-like peptide-1 (GLP-1), which has a high affinity with the GLP-1 receptor and also has a significant hypoglycemic effect. -1 has the same physiological function. Stimulates the body to produce insulin 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 subcutaneous injection for patients with type 2 diabetes whose blood sugar cannot be adequately controlled by metform...

Claims

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Application Information

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IPC IPC(8): C07K14/575C07K1/06C07K1/04
Inventor 谭松暖陈超陈绵绵吴四清袁剑琳张巍
Owner HAINAN SHUANGCHENG PHARMA
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