Linaclotide synthesis method

A technology of linaclotide and protecting group, which is applied in the field of synthesizing linaclotide, can solve the problems of process amplification, improvement of the purity of linear crude peptide, and increase of impurities, so as to achieve the improvement of crude product purity and yield, and increase the purity of linear crude peptide , the effect of improving the purity

Active Publication Date: 2013-01-16
HYBIO PHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, methods 2 and 3 use a variety of side chain removal and oxidation reagents, and each step will inevitably bring about an increase in impurities, so it is not conducive to obtaining high-purity, high-yield products, and is also not conducive to the enlargement of the process.
[0005] Although method 1 has simple steps, only one protecting group is used to protect the cysteine ​​side chain sulfhydryl group, but the choice of cysteine ​​side chain protecting group is one of the key factors affecting the synthesis of linaclotide. Method 1 uses The Trt protecting group is not conducive to the improvement of the purity of the linear crude peptide of linaclotide, resul

Method used

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  • Linaclotide synthesis method

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Experimental program
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Effect test

Embodiment 1

[0036] Embodiment 1: Preparation of Fmoc-Tyr(tBu)-Wang Resin

[0037] Weigh 300g 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, add 206.8g of Fmoc-Tyr(tBu)-OH (450mmol), 72.9g HOBt (540mmol) and 5.50g DMAP (45mmol) were dissolved in DMF / DCM=1:1 (V / V) mixed solution, and 84.4mL DIPCDI (540mmol) was added under ice-water bath to activate for 5min, then the above-mentioned resin containing In the reaction column, after 2 hours of reaction. A mixture of 567.2ml acetic anhydride (6mol) and 482.2ml pyridine (6mol) was added to block for 12h. Wash with DMF for 3 times, DCM for 3 times, shrink and dry with methanol to obtain Fmoc-Tyr(tBu)-Wang Resin, the detection degree of substitution is 0.408mmol / g.

Embodiment 2

[0038] Embodiment 2: Preparation of Fmoc-Tyr(tBu)-Wang Resin

[0039] Weigh 300g 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, add 413.6g of Fmoc-Tyr(tBu)-OH (450mmol), 145.8g HOBt (540mmol) and 11.0g DMAP (45mmol) were dissolved in DMF / DCM=1:1 (V / V) mixture, and 168.8mL DIPCDI (1080mmol) was added under ice-water bath to activate for 5min, then the above-mentioned resin containing In the reaction column, after 2 hours of reaction. A mixture of 567.2ml acetic anhydride (6mol) and 482.2ml pyridine (6mol) was added to block for 12h. Wash with DMF for 3 times, DCM for 3 times, shrink and dry with methanol to obtain Fmoc-Tyr(tBu)-Wang Resin, the detection degree of substitution is 0.612mmol / g.

Embodiment 3

[0040] Embodiment 3: Preparation of Fmoc-Tyr(tBu)-Wang Resin

[0041] Weigh 300g of Wang Resin with a degree of substitution 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, add 344.6g of Fmoc-Tyr(tBu)-OH (750mmol) , 121.6g HOBt (900mmol), 9.2g DMAP (75mmol) were dissolved in DMF / DCM=1:1 (V / V) mixed solution, and 140.7mL DIPCDI (900mmol) was added under ice-water bath to activate for 5min, then the above-mentioned resin containing In the reaction column, after 2 hours of reaction. A mixture of 567.2ml acetic anhydride (6mol) and 482.2ml pyridine (6mol) was added to block for 12h. Wash with DMF for 3 times, DCM for 3 times, shrink and dry with methanol to obtain Fmoc-Tyr(tBu)-Wang Resin, the detection degree of substitution is 0.489mmol / g.

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Abstract

The invention relates to the field of pharmaceutical synthesis and discloses a linaclotide synthesis method. The linaclotide synthesis method includes: performing solid-phase synthesis to obtain linaclotide resin with an N terminal, a Thr side chain, a Cys side chain, an Asn side chain, a Tyr side chain and a Glu side chain of an amino acid sequence shown in SEQ ID NO:1 coupled with protecting groups and with a C terminal coupled with a resin solid-phase carrier, cracking to remove the protecting groups and the resin solid-phase carrier prior to carrying out oxidizing reaction by the aid of a GSH (glutathione)/GSSH (oxidized glutathione) oxidization system to obtain a crude linaclotide product, and purifying the crude linaclotide product so that linaclotide is obtained. By the method, an Mmt protecting group is used for protecting a cysteine side chain, crude linear linaclotide peptide is synthesized by a one-by-one coupling mode, and the linaclotide is obtained by oxidization by the aid of the GSH /GSSH oxidization system. Compared with existing methods, the linaclotide synthesis method has the advantages that purity of the crude linear peptide is improved, the oxidization step can be performed without purification, and purity and yield of the crude linaclotide product are remarkably improved.

Description

technical field [0001] The invention relates to the field of pharmaceutical synthesis, in particular to a method for synthesizing linaclotide. Background technique [0002] Linaclotide is a new type of GC-C (intestinal cell uridylate cyclase C) receptor agonist. The compound is a polypeptide composed of 14 amino acids, which is obtained by solid-phase synthesis technology. Linaclotide activates GC-C receptors on the apical surface of intestinal epithelial cells, resulting in increased intracellular and extracellular cyclic guanylate. Its 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, for the treatment of adults with slow transit constipation and irritable bowel syndrome with constipation (IBS-C), structural sequence for: [0003] NH 2 -Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-COOH (3 pairs of disulfide bonds are 1-6, 2-10, 5-13). [000...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/06C07K1/04
CPCY02P20/55
Inventor 宓鹏程潘俊锋马亚平袁建成
Owner HYBIO PHARMA
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