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A kind of preparation method of linaclotide

A technology of linaclotide and peptidyl peptide, which is applied in the field of linaclotide preparation, can solve the problems of increased production cost, unfavorable large-scale production, cumbersome operation steps, etc.

Active Publication Date: 2021-12-14
浙江肽昇生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Patent CN 103626849A discloses a preparation method of linaclotide. After the crude linaclotide line peptide is prepared by this method, 3 pairs of disulfide bonds are formed by stepwise oxidation, and the operation steps are cumbersome; A large amount of hydrazine hydrate, iodine and acetic acid are used in the process of disulfide bond, which increases the production cost and obviously reduces the economic benefit of the process
Patent CN 102875655A uses the GSH / GSSH system as the oxidant, which is expensive and unfavorable for large-scale production; and the final linaclotide product has many impurities and low purity

Method used

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  • A kind of preparation method of linaclotide
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  • A kind of preparation method of linaclotide

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[0028] The present invention provides a kind of preparation method of linaclotide, comprising the following steps:

[0029] Provide crude linaclotide line peptide;

[0030] dissolving the crude linaclotide line peptide in an ammonium chloride solution, and adjusting the pH value of the obtained system to 7.5-9.5 with an alkaline reagent to obtain a linaclotide line peptide solution;

[0031] Mixing the linaclotide linear peptide solution with an aqueous hydrogen peroxide solution, continuously feeding air into the resulting reaction solution for oxidation reaction, to obtain an oxidation product system;

[0032] The oxidation product system is freeze-dried to obtain linaclotide.

Embodiment 1

[0043] Preparation of crude linaclotide line peptide, comprising the following steps:

[0044] Take Fmoc-Tyr(tBu)-Wang Resin 140.12g (100mmol) with a degree of substitution of 0.70mmol / g; wherein, Fmoc is 9-fluorenylmethoxycarbonyl, Wang Resin is 4-benzyloxybenzyl alcohol resin, and tBu is tertiary Butyl), added in the solid-phase reaction column, after swelling the resin with N,N-dimethylformamide (DMF) for 60min, vacuum filtration, adding 3 times the mixture of piperidine and DMF of the resin bed volume (the The volume fraction of piperidine in the mixture is 20%), stirred for 30min to remove Fmoc protection, and washed 5 times with DMF after vacuum filtration;

[0045] Dissolve 176.3g (300mmol) Fmoc-Cys(Trt)-OH and 40.5g (300mmol) 1-hydroxybenzotriazole (HOBt) in DMF and cool at -10°C for 20min, add 47.3mL (300mmol) N ,N-diisopropylcarbodiimide (DIC) and continue to activate for 30min; put the activated reaction solution into the solid-phase reaction column, and carry out ...

Embodiment 2

[0057] Weigh 1g of linaclotide line peptide crude product (purity is 84.37%), ultrasonically dissolve in 4L ammonium chloride solution with a concentration of 0.05mol / L, adjust the pH value to 8.0 with ammonia water with a concentration of 0.5wt%, and then add 900 μL of hydrogen peroxide with a concentration of 10wt% was continuously fed into the reaction solution obtained by magnetic stirring at room temperature (25° C.) to carry out the oxidation reaction for 4 hours, wherein the air was specifically fed into the reaction through a corrosion-resistant hose In the liquid, the air pressure passed into the flexible pipe is 0.01MPa (after deducting the atmospheric pressure), and the diameter of the flexible pipe is 8mm; after the oxidation reaction finishes, the purity of the resulting product system is 72.92% (HPLC figure is shown image 3 As shown, the unit of the abscissa is min, and the unit of the ordinate is AU; the data information corresponding to the chromatographic peak...

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Abstract

The invention provides a preparation method of linaclotide, which belongs to the technical field of pharmaceutical synthesis. The method provided by the present invention comprises the following steps: providing a crude linaclotide line peptide; dissolving the crude linaclotide line peptide in an ammonium chloride solution, and adjusting the pH value of the obtained system to 7.5~ 9.5, to obtain a linaclotide line peptide solution; mix the linaclotide line peptide solution with an aqueous hydrogen peroxide solution, and continuously feed air into the obtained reaction solution for oxidation reaction to obtain an oxidation product system; The product system was freeze-dried to obtain linaclotide. The present invention uses hydrogen peroxide aqueous solution and air as the oxidizing agent to generate linaclotide through one-step oxidation reaction, which is simple in operation and low in cost, and is suitable for large-scale production; meanwhile, the present invention carries out the oxidation reaction in the ammonium chloride solution, and controls it properly. The pH value is conducive to ensuring the smooth progress of the oxidation reaction, and the product purity and yield are high.

Description

technical field [0001] The invention relates to the technical field of pharmaceutical synthesis, in particular to a preparation method of linaclotide. Background technique [0002] Linaclotide (linaclotide) is the first guanylate cyclase agonist (GCCA) drug so far. In August 2012, the US FDA approved linaclotide for the treatment of adults with chronic idiopathic constipation and constipation-type constipation. Irritable bowel syndrome (IBS-C). Linaclotide is composed of 14 amino acids, including 6 cysteine ​​residues connected by 3 pairs of disulfide bonds, and its structural sequence is as follows: [0003] H-Cys1-Cys2-Glu3-Tyr4-Cys5-Cys6-Asn7-Pro8-Ala9-Cys10-Thr11-Gly12-Cys13-Tyr14-OH (3 pairs of disulfide bonds: 1-6, 2-10 and 5-13 ). [0004] Patent CN 103626849A discloses a preparation method of linaclotide. After the crude linaclotide line peptide is prepared by this method, 3 pairs of disulfide bonds are formed by stepwise oxidation, and the operation steps are cum...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K1/14
CPCC07K7/08
Inventor 俞智杰纪东亮罗瑞昌
Owner 浙江肽昇生物医药有限公司
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