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Method for preparing linaclotide through solid-liquid combination

A technology for linaclotide and lotide, which is applied in the field of preparing linaclotide by solid-liquid combination, can solve the problems of low total yield, low purity of linear crude peptide, inability to solve the purity of linear crude peptide, etc. Conducive to the effect of synthesis scale and production cost reduction

Active Publication Date: 2020-08-07
汉肽生物医药集团有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the one-step oxidation method can convert linear peptides to target structures through different buffer oxidation systems, it still cannot solve the problem of low purity of linear crude peptides, resulting in low overall yields
[0008] In conclusion, in the process of preparing linaclotide, there is a technical problem that the purity of the linear crude peptide is low, resulting in a low overall yield

Method used

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  • Method for preparing linaclotide through solid-liquid combination

Examples

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Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Synthesis of Fmoc-Gly-Cys(tButhio)-OH

[0048] Accurately weigh 2.09Kg (10mol) of H-Cys(tButio)-OH into a 50L reaction kettle, add 12L of 10% sodium carbonate aqueous solution by mass percentage, and dissolve under stirring; after the solution is clear, add Fmoc-Gly- OSu 3.94kg (10mol) / 12L tetrahydrofuran solution, stirred and reacted, TLC monitored the end point; after concentration under reduced pressure, acid adjustment, ethyl acetate extraction, drying, and crystallization, the dipeptide monomer Fmoc-Gly-Ser with a purity of 99.1% was obtained (tBu)-OH 3.86kg, yield 76.2%.

Embodiment 2

[0049] Embodiment 2: the synthesis of Fmoc-Pro-Ala-OH

[0050] Accurately weigh 1.07kg (12mol) of alanine in a 50L reaction kettle, add 12L of 10% sodium carbonate aqueous solution by mass percentage, and dissolve under stirring; after the solution is clear, add Fmoc-Pro-OSu 4.34kg (1mol ) / 12L tetrahydrofuran solution, stirred and reacted, and the end point was monitored by TLC; after concentration under reduced pressure, acid adjustment, ethyl acetate extraction, drying, and crystallization, the dipeptide monomer Fmoc-Pro-Ala-OH with a purity of 99.2% was obtained 3.27kg, Yield 78.2%.

Embodiment 3

[0051] Example 3: Preparation of linear linaclotide resin

[0052] Weigh 75 g (50 mmol) of Fmoc-Tyr(tBu)-Wang resin with a degree of substitution of 0.67 mmol / g and place it in a peptide resin synthesis reactor, add 700 mL of DCM to swell for 2 h. After the swelling is completed, wash with DMF three times, 600 mL each time, and then add 600 mL of 20% by volume piperidine / DMF solution for deprotection twice, for 10 min and 10 min respectively. After the deprotection was completed, the resin was washed 6 times with DMF, 600 mL each time. Weigh 49g of Fmoc-Gly-Cys(tButhio)-OH, 13.5g of HOBt and dissolve in 300mL of DMF, add 17mL of DIC to activate, add the solution into the reactor, react for 2h, and monitor the reaction end point by Kaiser test. After the reaction, the resin was washed 5 times with DMF, and then the next protected amino acid was deprotected and coupled. Repeat the above steps, followed by Fmoc-Thr(tBu)-OH, Fmoc-Cys(tButio)-OH, Fmoc-Pro-Ala-OH, Fmoc-Asn(Trt)-OH...

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Abstract

The invention relates to the field of polypeptide synthesis, in particular to a method for preparing linaclotide through solid-liquid combination, thereby well avoiding the generation of impurity peptides, improving the purity of crude peptides and reducing the production cost. According to the preparation method disclosed by the invention, the linaclotide is synthesized by adopting dipeptide monomers Fmoc-Gly-Cys (tButhio)-OH and Fmoc-Pro-Ala-OH for the first time in a solid phase manner; meanwhile, a strategy of protecting cysteine sulfydryl by stable tButhio under a TFA cracking condition is introduced, so that the purity of the linear peptide can be effectively improved, the yield of a final product is improved, the amplification of a synthesis scale is facilitated, and the productioncost is reduced.

Description

technical field [0001] The invention relates to the field of polypeptide synthesis, in particular to a method for preparing linaclotide by solid-liquid combination. [0002] technical background [0003] Linaclotide (LINZESS), developed by Ironwood Pharmaceuticals of the United States, was first approved for marketing in the United States on December 17, 2012, as a peptide drug for the treatment of gastrointestinal diseases. In 2013, it was launched in Denmark, Listed in Finland, Germany, Norway, Sweden and the United Kingdom, it was approved to be listed in China in March 2019. This product is the only GC-C (guanylate cyclase-C) agonist drug approved by the FDA that can be used clinically in the intestinal tract, and it is also the first approved in Europe for the treatment of adults with moderate to severe IBS-C New prescription drugs for patients. Linaclotide capsules (LINACLOTIDE capsules) specification: 145MCG / capsule; 290MCG / capsule. [0004] Linaclotide is a polypep...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/04C07K1/06C07K1/02
CPCC07K7/08Y02P20/55
Inventor 马程云翟涛赵传海其他发明人请求不公开姓名
Owner 汉肽生物医药集团有限公司
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