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Method for preparing linaclotide

A technology of linaclotide and crude peptide, which is applied in the field of linaclotide preparation, can solve the problems of low yield, high cost of raw materials, cumbersome synthesis steps, etc., and achieve high yield, avoid mismatch and reduce cost Effect

Inactive Publication Date: 2016-10-12
CS BIO SHANGHAI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to provide a method for quickly and simply mass-preparing linaclotide by using an automatic polypeptide synthesizer, which mainly solves the problems of cumbersome synthesis steps, high cost of raw materials, long synthesis cycle, low yield and impurities in the existing synthetic methods. Many, not suitable for technical problems such as industrial production

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  • Method for preparing linaclotide
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  • Method for preparing linaclotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Preparation of Linaclotide linear peptide protection resin

[0030] (1) Preparation of HMP-AM resin with substitution degree of 0.2~0.6 mmol / g

[0031] 445 g (200 mmol) of HMP-AM resin was prepared by the Fmoc protection method, and the degree of substitution measured with an ultraviolet absorbance photometer was 0.45 mmol / g.

[0032] (2) Preparation of Linaclotide linear peptide resin

[0033] Using an automatic peptide synthesizer, first react the Fmoc-Tyr(tBu)-resin from the previous step in a 20% mass percentage Pip / DMF solution for 20 minutes, remove the protective group Fmoc, and then automatically wash with DMF / DCM solution 2 times to prepare for the next amino acid link.

[0034] Use the program set up by the peptide synthesizer to couple amino acids one by one according to the peptide sequence. The specific reaction process is as follows: Fmoc-AA-OH (3 eq, 600 mmol) and HOBt (3 eq, 600 mmol) are dissolved in DMF (2.0 L), and then DIC (3 eq, 600 mmol) is adde...

Embodiment 2

[0035] Example 2: Preparation of Linaclotide Linear Peptide

[0036] Under the protection of nitrogen, linaclotide linear peptide resin is cleaved in a TFA / TIS / EDT / water volume ratio (94:2:2:2) solution. The specific operation is as follows: mix the linaclotide resin from the previous step with the TFA / TIS / EDT / water solution (5-10 mL / g), react for 4 hours, filter to remove the cracked resin, and then wash with TFA solution 2 all over. Combine the filtrate, evaporate the filtrate with a rotary evaporator, and then add cold ether (5-10 mL / g). The solid precipitates out. Filter and wash with cold ether 3 times to obtain a linear peptide of linaclotide, a white solid of 251.97 g, purity : 62%, yield: 50.9%.

Embodiment 3

[0037] Example 3: Preparation of linaclotide crude peptide

[0038] Linaclotide linear peptide (60.0 g) was dissolved in a mixed aqueous solution (25 L) of 1% by mass ammonia water and 30% by mass acetonitrile, and then added 2% by mass DMSO solution, During the reaction, the pH was adjusted to 9-10 with ammonia water, stirred at room temperature for 24 hours, and the progress of the reaction was monitored by LC-MS. Repeat the above experimental process to oxidize all linear peptides of linaclotide to linaclotide. 252.0 g of linaclotide was obtained, purity: 50.1%, yield: 41.1%.

[0039] Note: The monitoring and analysis conditions of LC-MS are as follows

[0040] Chromatographic conditions: Agilent 1290; C18 column (1.7µm, 150x3mm); detection wavelength: λ=214nm

[0041] Mobile phase A: 0.1% mass percent TFA aqueous solution;

[0042] Mobile phase B: 0.1% mass percent acetonitrile solution of TFA chromatography;

[0043] Gradient elution: mobile phase A: mobile phase B mass percentag...

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Abstract

The invention belongs to the technical field of biochemical polypeptide synthesis and relates to a method for preparing linaclotide. The method mainly solves the problem that the existing synthesis method has complicated steps of synthesis, high raw material cost, long synthesis period, low yield and more impurity and is unsuitable for industrial production. The method comprises that (1) Fmoc-Tyr(tBu)-OH and a carrier resin undergo a reaction to produce Fmoc-Tyr(tBu)-resin, (2) the Fmoc-Tyr(tBu)-resin and other amino acids with Fmoc protecting groups are coupled one by one through an activator so that linaclotide linear peptide resin is obtained, (3) the linaclotide resin is subjected to deprotection and cracking agent-based cracking to form linaclotide linear peptide, (4) three disulfide bonds of the linaclotide linear peptide is cyclized in an ammonium hydroxide / DMSO system to form linaclotide crude peptide, and (5) the linaclotide crude peptide is purified in an alkaline buffer solution and then is subjected to freeze-drying so that pure linaclotide is obtained.

Description

Technical field [0001] The invention belongs to the technical field of biochemical polypeptide synthesis, and specifically relates to a preparation method of linaclotide. Background technique [0002] Linaclotide was developed by Ironwood Corporation of the United States and was first approved for the market in the United States on December 17, 2012. It is a peptide drug for the treatment of adult chronic idiopathic constipation and constipation-type irritable bowel syndrome (IBS-C). , The product name is LINZESS ® . This product is a guanylate cyclase-C (GCC) agonist. The mechanism of action may be that linaclotide binds to the GC-C receptor in small intestinal epithelial cells, which activates GC-C, resulting in increased secretion of intestinal juice, and Accelerate the transport, but also reduce the sensitivity of the pain nerve in the small intestine. [0003] Linaclotide is a polypeptide composed of 14 amino acids and contains 3 pairs of disulfide bonds. The specific struct...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/20C07K1/06C07K1/04
CPCY02P20/55C07K7/08
Inventor 张恒维王为施学庚杨培朱国基张倩巢庆乔立超田晓博
Owner CS BIO SHANGHAI
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