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Preparation method of plecanatide

A technology of plecanatide and plecanatide linear peptide, applied in the field of peptide synthesis, can solve the problems of affecting the coupling efficiency of plecanatide, high difficulty of linear peptide synthesis, low purity, etc., and is suitable for industrial production and operation. Simple and easy to reduce the effect of intermolecular disulfide bond formation

Pending Publication Date: 2021-01-08
杭州信海医药科技有限公司
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  • Claims
  • Application Information

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Problems solved by technology

However, since the sequence of plecanatide is easy to form resin polycondensation during the coupling process, the amino acids on the main chain are not modified, which affects the coupling efficiency of plecanatide, resulting in high difficulty in the synthesis of linear peptides, low purity, and low yield. low rate

Method used

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  • Preparation method of plecanatide
  • Preparation method of plecanatide
  • Preparation method of plecanatide

Examples

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preparation example Construction

[0037]The present invention provides a method for preparing pukanatide, which comprises the following steps:

[0038](1) Mix the starting resin, FMPB and the condensation reagent to modify to obtain a modified resin; the starting resin includes MBHA resin or AM resin;

[0039](2) Under a protective atmosphere, the modified resin, H-Leu-OtBu and furan solvents are mixed to carry out the aminoaldehyde condensation reaction, and the obtained Schiff base is mixed with a reducing agent to carry out a reduction reaction to obtain H-Leu -OtBu-FMPB MBHA / AM resin;

[0040](3) Connect Fmoc-Cys(Acm)-OH, R in sequence on the H-Leu-OtBu-FMPB MBHA / AM resin according to the sequence of the pukanatide backbone peptide1-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Cys(Acm)-OH, R2-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH and Fmoc-Asn(Trt)-OH, Obtain the fully protected pukanatide resin; the R1And R2Independen...

Embodiment 1

[0117](1) 10g of AM resin with a substitution degree of 1.0mmol / g, 200mL of 5v / v%DIEA / DCM swelling resin for 1h, DMF washed twice, suction filtered to remove the solution, add 3.33g FMPB (15mmol) into the reactor , Add DMF to dissolve, add 2.36mL DIC (15mmol) and 2.03g HoBt (15mmol) to mix, after modification for 1.5h, ninhydrin test becomes negative, wash with DMF 3 times, THF wash 2 times, suction filter to remove the solvent, A modified resin (13.56 g) was obtained.

[0118](2) Under the protection of nitrogen, mix the 1.36g modified resin obtained in step (1), 4.47g H-Leu-OtBu·HCl (20mmol), 200mL THF, add 2.3mL (40mmol) of acetic acid and mix evenly. Aldehyde condensation reaction for 30min, add 3mL of 13.3mol / L sodium cyanoborohydride / methanol solution and mix, reduce reaction for 16h, wash 3 times with DMF, wash 3 times with methanol, and vacuum dry to obtain H-Leu-OtBu FMPBAM resin (1.49 g, yield 96.3%).

[0119](3) Put 1.49g of the H-Leu-OtBu-FMPB AM resin (1mmol) obtained in step...

Embodiment 2

[0127](1) Put 6.30g of the H-Leu-OtBu-FMPBAM resin (1mmol) obtained in step (2) of Example 1 into 25mL DCM to swell for 30min, use 3mmol DIC and 3mmol HoBt as condensation reagents, and swell the swollen H-Leu- OtBu-FMPB AM resin and Fmoc-Cys(Acm)-OH are coupled to react at room temperature, and the end of the reaction is determined by the ninhydrin detection solution. The test result is negative, and then exists in 140mL 20v / v% piperidine in DMF solution The Fmoc protecting group is removed from the bottom, and the end point of the reaction is determined by the ninhydrin detection solution. The test result is positive. The Fmoc protecting group removal reaction is completed to obtain Cys(Acm)-Leu-OtBu-FMPB AM resin; repeat the above coupling reaction React with the removal of the Fmoc protecting group, and connect Fmoc-Hmb-Gly-OH and Fmoc-Thr(tBu)-OH to Cys(Acm)-Leu-OtBu-FMPB AM resin in sequence according to the sequence of the Pukana peptide backbone , Fmoc-Cys(Trt)-OH, Fmoc-Ala-...

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Abstract

The invention provides a preparation method of plecanatide, and relates to the technical field of polypeptide synthesis. AM resin or MBHA resin is modified through FMPB, H-Leu-OtBu serves as first amino acid to be coupled to the modified resin, amino acid at sites difficult to connect is modified and then is gradually coupled to the modified resin according to the sequence of plecanatide main chain peptide, so that the synthesis difficulty of plecanatide linear peptide is greatly reduced, and the problems of many impurities, low purity, low yield, amino acid racemization and the like in the existing synthesis are solved. The Cys at sites of 4 and 12 in the sequence are subjected to first oxidation bonding and then subjected to column chromatography coarse purification, so that the purity of the obtained plecanatide is improved; the purification liquid is diluted, and the Cys at the 7-site and the 15-site are subjected to the second oxidation bonding, such that the concentration of thepre-oxidized plecanatide line peptide can be reduced so as to reduce the intermolecular disulfide bond during the second oxidation bonding process, and improve the yield of plecanatide; and further, the operation is simple and convenient, and the method is suitable for industrial production.

Description

Technical field[0001]The invention relates to the technical field of polypeptide synthesis, in particular to a preparation method of pukanatide.Background technique[0002]Plecanatide was developed by Synergy Pharmaceuticals, USA. It is an analog of uroguanylin and contains a 16 amino acid cyclic polypeptide. Its sequence is: Asn1-Asp2-Glu3-Cys4-Glu5-Leu6-Cys7-Val8-Asn9-Val10-Ala11-Cys12-Thr13-Gly14-Cys15-Leu16(Two pairs of disulfide bonds: Cys4&Cys12And Cys7&Cys15), a guanylate cyclase receptor agonist with natriuretic excretion, can regulate acid-base ions in the gastrointestinal tract, induce fluid transport into the gastrointestinal tract, increase gastrointestinal motility and accelerate defecation.[0003]Chinese patent CN107383170A discloses a method for preparing pukanatide: (1) In the presence of an activator system, Fmoc-Leu-resin is obtained by coupling a resin solid phase carrier and Fmoc-Leu-OH; (2) through solid Phase synthesis method, according to the sequence of the main...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/08C07K1/06C07K1/04C07K1/20
CPCC07K7/08
Inventor 李雪豪纪东亮罗瑞昌
Owner 杭州信海医药科技有限公司
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