Preparation method for linaclotide

A technology of linaclotide and crude peptide, applied in the field of preparation of linaclotide, can solve the problems of low purity, high price and low yield of the final linear peptide, avoid heavy metal toxicity, reduce synthesis cost, and reduce production cost effect

Active Publication Date: 2014-12-24
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In the method adopted by Miriam et al., the peptide resin uses a chlorine resin. Due to the acid sensitivity of the chlorine resin, the peptide will fall off during the coupling process, resulting in a low yield of linear peptides.
In addition, a strategy in this method is that all cysteines are protected by Trt. Due to the large Trt group, it will affect the coupling effect, resulting in low purity and low yield of the final linear peptide.
At t...

Method used

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  • Preparation method for linaclotide
  • Preparation method for linaclotide
  • Preparation method for linaclotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the preparation of the Fmoc-Tyr(tBu)-Wang Resin that the degree of substitution is 0.5mmol / g

[0037] Weigh 120g (120mmol) of Wang resin with a substitution degree of 1.0mmol / g, add it to a solid-phase reaction column, wash it twice with DMF, and swell the resin with DMF for 30 minutes, take 110.3g (240mmol) of Fmoc-Tyr(tBu) -OH, 38.9g (288mmol) HOBt, 36.3g (288mmol) DIC, 2.93g (24mmol) DMAP were dissolved in a mixed solution of DCM and DMF with a volume ratio of 1:1, added to a solid-phase reaction column, and reacted at room temperature for 2h. After the reaction, wash 4 times with DMF and 2 times with DCM. Then add 189.8g (2400mmol) pyridine and 245.04g (2400mmol) acetic anhydride mixture to seal the resin for 6h. After washing with DMF for 4 times and DCM for 2 times, methanol was shrunk and dried to obtain Fmoc-Tyr(tBu)-Wang Resin with a detection substitution degree of 0.498mmol / g.

Embodiment 2

[0038] Embodiment 2: the preparation of the Fmoc-Tyr(tBu)-Wang Resin that the degree of substitution is 0.7mmol / g

[0039] Weigh 120g (120mmol) of Wang resin with a substitution degree of 1.0mmol / g, add it to a solid-phase reaction column, wash it twice with DMF, and swell the resin with DMF for 30 minutes, take 165.4g (360mmol) of Fmoc-Tyr(tBu) -OH, 58.4g (432mmol) HOBt, 54.5g (432mmol) DIC, 4.39g (36mmol) DMAP were dissolved in a mixed solution of DCM and DMF with a volume ratio of 1:1, added to a solid-phase reaction column, and reacted at room temperature for 2h. After the reaction, wash 4 times with DMF and 2 times with DCM. Then add 189.8g (2400mmol) pyridine and 245.04g (2400mmol) acetic anhydride mixture to seal the resin for 6h. After washing with DMF for 4 times and DCM for 2 times, methanol was shrunk and dried to obtain Fmoc-Tyr(tBu)-Wang Resin with a detection substitution degree of 0.705mmol / g.

Embodiment 3

[0040] Embodiment 3: the preparation of the Fmoc-Tyr(tBu)-Wang Resin that the degree of substitution is 1.0mmol / g

[0041] Weigh 100g (120mmol) of Wang resin with a substitution degree of 1.2mmol / g, add it to a solid-phase reaction column, wash it twice with DMF, and swell the resin with DMF for 30 minutes, take 165.4g (360mmol) of Fmoc-Tyr(tBu) -OH, 58.4g (432mmol) HOBt, 54.5g (432mmol) DIC, 4.39g (36mmol) DMAP were dissolved in a mixed solution of DCM and DMF with a volume ratio of 1:1, added to a solid-phase reaction column, and reacted at room temperature for 2h. After the reaction, wash 4 times with DMF and 2 times with DCM. Then add 189.8g (2400mmol) pyridine and 245.04g (2400mmol) acetic anhydride mixture to seal the resin for 6h. After washing with DMF for 4 times and DCM for 2 times, methanol was shrunk and dried to obtain Fmoc-Tyr(tBu)-Wang Resin with a detection substitution degree of 0.989mmol / g.

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Abstract

Belonging to the technical field of pharmaceutical synthesis, the invention discloses a preparation method for linaclotide. During synthesis of linear peptides, the carrier resin adopts Wang Resin, which has stable nature relative to chlorine resin and makes peptide difficult to fall off. Meanwhile, during synthesis of linear peptides, five of six cysteines in a peptide sequence adopt relatively cheap Fmoc-Cys(Trt)-OH as the reaction raw material, thus greatly reducing the synthesis cost and being beneficial to industrial production. In addition, during formation of three disulfide bonds, the one-step oxidation method is employed to oxidize crude peptide directly so as to form correctly paired three disulfide bonds. The method has the advantages of simple and practical operation, high yield and low cost, and is in favor of industrial production. The linaclotide prepared by the method provided by the invention can have a linear crude peptide weight yield up to 103.9%, purity up to 82.9%, refined peptide purity stabilized at 99.12-99.30%, and a total yield up to 32.15%.

Description

technical field [0001] The invention relates to the technical field of drug synthesis, in particular to a preparation method of linaclotide. Background technique [0002] Linaclotide, the English name is Linaclotide; the trade name is Linzess; the Chinese common name is Linaclotide. It is a peptide drug for the treatment of chronic idiopathic constipation and irritable bowel syndrome with constipation (IBS-C) in adults. The specific peptide sequence is shown in the following formula: [0003] [0004] There are few domestic and foreign reports on the preparation method of linaclotide. At present, there are mainly the synthesis method described in Chinese patent CN102875655A and the synthesis method attempted by Miriam et al. (“Optimized Fmo c Solid-Phase Synthesis of the Cysteine-Rich Peptide Linaclotide”, Peptide Science, 2011, Volume96, Issue1, pages69-80) . [0005] Chinese patent CN102875655A describes the synthesis method of linaclotide, which uses Mmt as the side...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/06C07K1/04
CPCY02P20/55
Inventor 朱日成宓鹏程马亚平袁建成
Owner HYBIO PHARMA
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