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Method for preparing linaclotide

A technology of linaclotide and a synthesis method, which is applied in the field of peptide medicine synthesis, can solve the problems of many impurities, high cost, unfavorable large-scale production and the like

Inactive Publication Date: 2014-11-26
ADLAI NORTYE BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the first method, the purity of the linear peptide is only 65%, and the yield is low, which is not conducive to large-scale production. The second and third methods use a variety of side chain removal and oxidation reagents, which will inevitably lead to increased impurities, which is not conducive to obtaining high-quality peptides. Pure, high-yield products are not conducive to process scale-up
[0008] Chinese patent CN 102875655A uses the Mmt protecting group to protect the cysteine ​​side chain, and synthesizes the linear crude peptide of linaclotide by one-by-one coupling, and finally uses the GSH / GSSH oxidation system to oxidize to obtain linaclotide. The linear peptide obtained by this method The purity is not high and there are relatively many impurities, which is not conducive to large-scale production
[0009] In summary, the existing solid-phase synthesis process of linaclotide is not suitable for industrial production due to long synthesis period, high cost, low yield and many impurities.

Method used

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  • Method for preparing linaclotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment one: the synthesis of the Fmoc-Cys(Trt)-CTC resin that the degree of substitution is 0.10mmol / g

[0086] Weigh 50.00g of 2-CTC resin with a substitution degree of 0.40mmol / g, add it to the solid phase reaction column, add it to the solid phase reaction column, wash once with DMF, swell the resin with DMF for 30 minutes, and take 58.57g Fmoc-Cys(Trt)-OH (100mmol) was dissolved in DMF, activated by adding 17ml DIEA (100mmol) in an ice-water bath, then added to the above-mentioned reaction column equipped with resin, reacted for 2 hours, added 500ml of anhydrous methanol to seal for 1 hour . Wash 3 times with DMF and 3 times with DCM, block with anhydrous methanol for 30 minutes, shrink and dry with methanol to obtain Fmoc-Cys(Trt)-CTC resin, the detection degree of substitution is 0.10mmol / g.

Embodiment 2

[0087] Embodiment two: the degree of substitution is the synthesis of Fmoc-Cys(Trt)-CTC resin of 0.90mmol / g

[0088] Weigh 133.33g of 2-CTC resin with a degree of substitution of 1.50mmol / g, add it to the solid phase reaction column, add it to the solid phase reaction column, wash once with DMF, swell the resin with DMF for 30 minutes, and take 585.70g Fmoc-Cys(Trt)-OH (1000mmol) was dissolved in DMF, activated by adding 165ml DIEA (1000mmol) in an ice-water bath, then added to the above-mentioned reaction column equipped with resin, reacted for 2 hours, added 2000ml of anhydrous methanol to block for 1 hour . Wash 3 times with DMF and 3 times with DCM, block with anhydrous methanol for 30 minutes, shrink and dry with methanol to obtain Fmoc-Cys(Trt)-CTC resin, the detection degree of substitution is 0.90mmol / g.

Embodiment 3

[0089] Embodiment three: the degree of substitution is the synthesis of Fmoc-Cys(Trt)-CTC resin of 0.50mmol / g

[0090] Weigh 200.00g of 2-CTC resin with a degree of substitution of 1.00mmol / g, add it to the solid phase reaction column, add it to the solid phase reaction column, wash once with DMF, swell the resin with DMF for 30 minutes, and take 585.70g Fmoc-Cys(Trt)-OH (1000mmol) was dissolved in DMF, activated by adding 165ml DIEA (1000mmol) in an ice-water bath, then added to the above-mentioned reaction column equipped with resin, reacted for 2 hours, added 2000ml of anhydrous methanol to block for 1 hour . Wash 3 times with DMF and 3 times with DCM, block with anhydrous methanol for 30 minutes, shrink and dry with methanol to obtain Fmoc-Cys(Trt)-CTC resin, the detection degree of substitution is 0.50mmol / g.

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Abstract

The invention relates to a method for preparing linaclotide. The method comprises the following steps of A, synthesizing a pentapeptide fragment I of Fmoc-CysCysGluTyrCys-SBzl by a solid-liquid phase method, B, a nonapeptide fragment resin II of H-CysAsn(Trt)ProAlaCys(Trt)Thr(tBu)GlyCys(Trt)Tyr(tBu)-Wang resin by the solid-liquid phase method, C, adding the pentapeptide fragment I into the nonapeptide fragment resin II, carrying out thioester exchange and S-to-N acyl transfer to obtain a novel tetrakaideca-peptide fragment resin, removing a Fmoc protective group, and carrying out pyrolysis to obtain high-purity linaclotide linear peptide, and D, carrying out oxidation by a GSH / GSSH oxidation system to obtain linaclotide. Through thioester exchange and S-to-N acyl transfer, the method for preparing linaclotide is realized and has the advantages of mild synthesis conditions, high product purity, high yield and large-scale production feasibility.

Description

technical field [0001] The invention relates to the field of polypeptide medicine synthesis, in particular to a method for preparing linaclotide. Background technique [0002] Linaclotide, English name: Linaclotide, structural formula is as follows: [0003] [0004] The peptide sequence is: [0005] [0006] Linaclotide is a polypeptide containing 14 amino acids and is the world's first guanylate cyclase-C agonist, which can bind and activate guanylate cyclase C on the luminal surface of intestinal epithelial cells receptor, resulting in increased intracellular and extracellular cyclic guanylate. The net effect is increased secretion of chlorine and bicarbonate into the intestinal lumen, which in turn leads to increased fluid secretion and accelerated stool passage. It is used in the treatment of constipation-predominant irritable bowel syndrome and in patients with chronic idiopathic constipation. On August 30, 2012 and November 26, 2012, it was approved for mark...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/06C07K1/04
CPCY02P20/55
Inventor 朱明月周亮路杨杨东晖
Owner ADLAI NORTYE BIOPHARMA CO LTD
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