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Solid-phase preparation method for buserelin

A solid-phase preparation and solid-phase synthesis technology, which is applied in the preparation methods of peptides, chemical instruments and methods, organic chemistry, etc., can solve the problems of low application value, unfavorable industrial production, complicated operation, etc., and achieves easy post-processing, The effect of less environmental pollution and simple reaction operation

Inactive Publication Date: 2011-01-05
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, Buserelin (Buserelin) and its analogs have high medicinal value and broad market prospects, but the existing preparation method is mainly a liquid phase synthesis process, which is complicated to operate, is unfavorable for industrial production, and has low application value. High, so a more excellent production process is required

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Preparation of Fmoc-Pro-HMPB-MBHA resin

[0032] 11.1g of HMPB-MBHA resin (Resin) with a substitution degree of 0.9mmol / g was added to the solid-phase reaction column, and after adding DCM to swell the resin for 30 minutes, 7.76g of Fmoc-Pro-OH, 8.30g of HATU, 2.98g of HOAt It was dissolved in DMF under ice bath, added to the above resin and reacted for 10 minutes, then added 2.4ml of TMP, and reacted at room temperature for 45 minutes. After washing with DMF for 3 times, washing with DCM for 3 times, and shrinking with methanol for 3 minutes, 5 minutes and 8 minutes respectively, the Fmoc-Pro-HMPB-MBHA resin was obtained by shrinking, and the detected substitution degree was 0.6mmol / g.

[0033] The route is as follows:

[0034]

Embodiment 2

[0035] Embodiment 2: Preparation of Buserelin-HMPB-MBHA resin

[0036] Weigh 10mmol Fmoc-Pro-HMPB-MBHA resin into the reactor, swell with DCM for 0.5 hours, then use 20% DBLK to remove the Fmoc protection twice for 10 minutes and 5 minutes respectively, and connect Fmoc-Arg (HCl )-OH. Dissolve 12.9g Fmoc-Arg(HCl)-OH, 4.9g HOBt, and 6.1ml DIC in DCM (a small amount of DMF can be added to aid dissolution), activate in an ice-water bath for 7 minutes, add to a solid-phase reactor, and react at room temperature for 1- 2 hours. The end point of the reaction was determined by the ninhydrin method. Repeat the above steps to sequentially complete the connection of the remaining amino acids to obtain Buserelin-HMPB-MBHA resin (Buserelin-HMPB-MBHA resin). Wherein the amount of raw materials: amino acid 3.0mmol, DIC 6.1ml, HOBt 4.9g.

Embodiment 3

[0037] Example 3: Preparation of pGlu-His(Trt)-Trp-Ser(Trt)-Tyr-D-Ser(tBu)-Leu-Arg(HCl)-Pro-OH

[0038] 1. Add 23.6g of Buserelin-HMPB-MBHA Resin into a 100ml round bottom flask.

[0039] 2. Prepare 200ml of lysis reagent, including 2ml of trifluoroacetic acid and 198ml of DCM, and pre-cool in the refrigerator for 30 minutes.

[0040] 3. Pour the cleavage reagent into the resin, ice bath while stirring, and blow nitrogen. After reacting for 30 minutes, the ice bath was removed, and the reaction was continued for 2 hours at room temperature. Filter the resin and collect the filtrate. The resin was washed with a small amount of DCM, and the filtrates were combined. The filtrate was slowly added to 800ml of glacial ether, and a white precipitate appeared. Centrifuge at 3000 rpm, wash with glacial ether for 5 times, and dry under reduced pressure to obtain 18.3 g of crude peptide, HPLC purity > 90%.

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Abstract

The invention provides a solid-phase preparation method for buserelin. The method comprises the following steps of: 1) preparing a Fmoc-Pro-HMPB-MBHA resin with degree of substitution of between 0.15 and 0.80mmol / g from Fmoc-Pro-OH and a HMPB-MBHA resin with degree of substitution of between 0.2 and 0.9mmol / g by a solid-phase synthesis method; 2) gradually coupling remaining protected amino acid of the Fmoc-Pro-HMPB-MBHA resin according to a peptide sequence to obtain buserelin-HMPB-MBHA resin; 3) cracking the buserelin-HMPB-MBHA resin to obtain fully-protected peptide; and 4) performing ethyl amination, deprotection and purification on the obtained fully-protected peptide to obtain buserelin. The invention aims to provide a buserelin solid-phase synthesis method which has the advantages of high yield, low cost, mild reaction conditions, small environmental pollution and contribution to realizing industrialization.

Description

【Technical field】 [0001] The present invention relates to a method for synthesizing polypeptides, in particular to a method for solid-phase synthesis of buserelin. 【Background technique】 [0002] In the past 10 years, peptide drugs have become a hot research topic in the international pharmaceutical community, and have become the target of many manufacturers competing for development. According to a well-known international medical technology consulting company, the total output value of raw materials of peptide drugs in the world is estimated to be about 400 million US dollars. According to statistics, there are about 35 kinds of polypeptide drugs in the international market, among which the best-selling products include: buserelin, leuprolide, gorelin, luteinizing hormone antagonists and Roche's new polypeptide drug Fuzeon, etc. There are also several new active peptide drugs against SARS virus and HIV virus that will soon be approved by the US FDA. Not only American com...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/23C07K1/14C07K1/06C07K1/04
Inventor 陈大来李红玲马亚平袁建成
Owner HYBIO PHARMA
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