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Method for preparing linaclotide

A linaclotide and a pair technology, applied in the field of preparation of linaclotide, can solve the problems of complex processing process, few raw material sources and high cost, achieve simple process operation, avoid disulfide bond mismatch isomer impurities , the effect of reducing the difficulty of purification

Inactive Publication Date: 2015-11-04
JINAN KANGHE MEDICAL TECH
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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 the linear peptide to the target structure as much as possible through the buffer system, it still cannot avoid the generation of disulfide bond mismatch isomer impurities, and the yield is low
[0007] Patent CN103626849A discloses the use of the same solution system to form three pairs of disulfide bonds step by step. Although this method can avoid the production of disulfide bond mismatch isomer impurities, the same system reacts, the processing process is complicated, and the source of raw materials is less , the cost is relatively high

Method used

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  • Method for preparing linaclotide
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  • Method for preparing linaclotide

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1: Preparation of Fmoc-Cys(Mmt)-CTC resin

[0043] Accurately weigh 320 g of CTC resin (1.0 mmol / g) and place it in a peptide resin synthesis reactor, add 3 L of DCM to swell for 1 h. After swelling, wash 3 times with DMF, 3 L each time. Weigh 394g of Fmoc-Cys(Mmt)-OH and 87g of HOBt and dissolve them in 2L of DMF, add 110mL of DIC to activate, add the solution into the reactor, and react for 4h. After the reaction was completed, the resin was washed three times with DMF, and then a pre-prepared capping reagent (2.5L DCM, 0.3L methanol, 0.2L DIEA) was added for capping reaction for 1h. After the capping is completed, wash 4 times with DMF, 2 times with DCM, and 3 times with methanol, 3 L each time, and then vacuum-dry the resin to obtain 507 g of Fmoc-Cys(Mmt)-CTC resin. Take a small amount of resin and measure Fmoc-Cys The substitution degree of (Mmt)-CTC resin is 0.61mmol / g. The calculated synthesis scale was 309 mmol.

Embodiment 2

[0044] Embodiment 2: the preparation of fragment I peptide resin

[0045] Weigh 493g (300mmol) of the Fmoc-Cys(Mmt)-CTC resin with a substitution degree of 0.61mmol / g in Example 1 and place it in a peptide resin synthesis reactor, add 4L DCM to swell for 2h. After the swelling is completed, wash with DMF three times, 4 L each time, and then add 4 L of 20% 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, 4 L each time. Weigh 352g of Fmoc-Cys(Trt)-OH and 81g of HOBt and dissolve them in 2L of DMF, add 103mL 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 above-mentioned steps, carry out the coupling of Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Cys...

Embodiment 3

[0046] Example 3: Preparation of Fragment I Linear Peptide

[0047] Weigh 700 g (200 mmol) of the Fragment I peptide resin obtained in Example 2 and place it in a 10 L reaction bottle, add 7 L of cleavage reagent TFA-TIS-DCM (5-5-90), and react for 5 minutes at room temperature. After the reaction was completed, the lysate was filtered into a suction filter flask. The above cleavage reaction was repeated 3 times, and the combined filtrates were mixed with the same volume of 0.1mol / L NaHCO 3 Wash the filtrate with aqueous solution until the pH of the solution is 7-8, concentrate by rotary evaporation at 25°C, collect the separated precipitate by filtration, and dry in vacuo to obtain 261 g of Fragment I linear peptide, with a purity of 82.3% and a yield of 95%.

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Abstract

The invention relates to the field of polypeptide synthesis, and in particular to a method for preparing linaclotide. The method adopts a fragment method to perform synthesis of peptide chains of linaclotide, can be used for forming three pairs of disulfide bonds by three steps in a completely selective manner, and specifically comprises the following steps: (a) synthesizing linear peptide of a segment I; (b) forming the first pair of disulfide bonds to obtain oxidized peptide of the segment I; (c) synthesizing peptide resin of a segment II; (d) synthesizing linaclotide crude peptide containing a pair of disulfide bonds; (e) synthesizing the second pair of the disulfide bonds; and (f) synthesizing the third pair of the disulfide bonds. The method adopts a process for forming the three pairs of the disulfide bonds by three steps in a completely selective manner to prepare linaclotide, isomer impurities with mismatched disulfide bonds can be avoided, the difficulty of a purification process is reduced, and meanwhile, the segment method reduces the difficulty for forming the first pair of the disulfide bonds, so that the purity and yield of the finally obtained crude peptide are relatively high, the operation process is simple and convenient, and the method is suitable for large-scale production.

Description

technical field [0001] The invention relates to the field of polypeptide synthesis, in particular to a method for preparing linaclotide. technical background [0002] 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, and it was launched in Denmark in 2013. , Finland, Germany, Norway, Sweden and the United Kingdom and other countries, and has not yet been declared for import into China (but international multi-center clinical phase III trials have been conducted in China). 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:...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/08C07K1/06C07K1/04
CPCY02P20/55
Inventor 张颖王德龙王仁友李同金石鑫磊
Owner JINAN KANGHE MEDICAL TECH
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