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Method for synthesis of bivalirudin in solid-phase fragment approach

A technology of bivalirudin and synthesis ratio, applied in the field of solid-phase fragment method synthesis of peptides, can solve the problems of difficult large-scale production, difficult product purification, low product purity, etc., and achieves cost increase, less environmental pollution and high yield. rate effect

Active Publication Date: 2012-10-17
YANCHENG KAILI PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This scheme produces a large amount of waste liquid in large-scale production, and the product is relatively difficult to purify
The process of the above scheme is relatively cumbersome, and there are many impurities produced, and the product purity is relatively low, so it is not easy to carry out large-scale production

Method used

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  • Method for synthesis of bivalirudin in solid-phase fragment approach
  • Method for synthesis of bivalirudin in solid-phase fragment approach
  • Method for synthesis of bivalirudin in solid-phase fragment approach

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Preparation of 13 Peptide Fragment I-Wang Resin

[0027] Weigh 1g of Wang Resin with a substitution degree of 0.7mmol / g, add it to a solid-phase reaction column, add anhydrous 10ml DCM to swell for 30min, wash with anhydrous DMF 3 times (1min each time), and add 1.4mmol of Fmoc -Leu-OH and HOBt were dissolved in 5ml of anhydrous DMF, then 1.4mmol of DIC was added to activate at 0-5°C for 5min, then added to a solid-phase reaction column for 10min, then 0.2mmol of DMAP was added, and reacted at 30°C for 5h. After completion, use anhydrous DMF and CH respectively 3 Wash with OH, DCM, DMF 2, 1, 1, 2 times, each time not less than 1min, and the detection substitution is 0.65mmol / g. Use 0.1 mmol acetic anhydride and pyridine to block unreacted hydroxyl groups on the resin for 1 h to obtain Fmoc-Leu-Wang.

[0028] After removing the Fmoc protecting group on Fmoc-Leu-Wang with 10ml volume of 25% piperidine-DMF in turn (the first reaction was 5 minutes, the middle was washed ...

Embodiment 2

[0030] Preparation of 7-peptide Fragment II

[0031] Weigh 2g of 2-Chlorotrityl Chloride Resin with a degree of substitution of 0.7mmol / g, add it to a solid-phase reaction column, add anhydrous DCM to swell for 30min, wash with 10ml anhydrous DMF 3 times (1min each time), add 2.4 One mmol of Fmoc-Gly-OH and HOBt were dissolved in 5ml of anhydrous DMF, and then 1.4mmol of DIC was added to activate for 5min at 0-5°C, then added to the solid-phase reaction column for 10min, then 0.4mmol of DMAP was added, and the React at ℃ for 5 hours, wash twice with 10ml of anhydrous DMF, each time for 1min, and detect the substitution of 0.68mmol / g. Use 0.2mmol acetic anhydride and pyridine to block unreacted amino groups on the resin for 1h to obtain Fmoc-Gly-2-Chlorotrityl Chloride Resin. Use 20ml volume of 25% piperidine / DMF to remove the Fmoc protecting group on Fmoc-Leu-Wang for 2 times (the first reaction is 5min, the second reaction is 10min, the middle is washed once with 10ml DMF), ...

Embodiment 3

[0033] Synthesis of Bivalirudin by Solid Phase Fragmentation

[0034] Take a 1.5-fold excess of 7-peptide fragment II, a 2-fold excess of HOBt, HUBT, and anhydrous DMF dissolved in 0-5°C, add a 1.5-fold excess of DIEA to activate for 5 minutes, and add it to a solid-phase synthesis column. React at 30°C for 1-5h (the end point of the reaction is determined by the ninhydrin method), wash with anhydrous DMF for 3 times, dalirudin-Wang Resin (D-Phe-Pro-Arg(pbf)-Pro-Gly-Gly -Gly-Gly-Asn(Trt)-Gly-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(OtBu)-Leu- Wang Resin). After adding 15ml of 25% piperidine-DMF as the Fmoc deprotecting agent for 2 times (the first reaction is 5min, the second reaction is 10min, and the middle is washed once with 10ml DMF), sequentially wash with DMF, DCM, CH 3 Wash 2, 1, and 3 times with OH, each time not less than 2 min; add 15 ml of TFA / H with a volume ratio of 95:2.5:2.5 2 O / TIS was used as a reagent for cleavage and removal of s...

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Abstract

The invention provides a method for synthesis of bivalirudin in a solid-phase fragment approach. The method comprises: first employing a solid phase stepwise method to synthesize a 13-peptide fragment I-Wang Resin, and a 7-peptide fragment II with the terminal N protected by Fmoc, coupling the two fragments in the solid-phase fragment approach to synthesize bivalirudin -Wang Resin; and finally conducting deprotection, cracking, purification, and freezing drying so as to obtain pure bivalirudin. Compared with existing solid phase stepwise method or liquid phase fragment method, the method of the invention has the advantages of simple technological operation, high yield and purity, small environmental pollution, and easy large-scale production, etc.

Description

technical field [0001] The invention relates to a method for synthesizing polypeptides by a solid-phase fragment method, in particular to a method for synthesizing bivalirudin by a solid-phase fragment method. Background technique [0002] Bivalirudin is a direct, specific and repressible thrombin inhibitor that has been used clinically in recent years. Its effective anticoagulant component is a hirudin derivative fragment, which can directly inhibit thrombin by specifically Active and play an anticoagulant effect, the effect is reversible and short-lived. Early clinical studies have shown that anticoagulant therapy with bivalirudin is effective and has a lower incidence of bleeding events, and it is safer to use than traditional heparin anticoagulant therapy. A study conducted by Bittl of the Oakla Heart Institute in Florida showed that in patients with unstable angina and post-infarct angina undergoing coronary angioplasty, the effect of bivalirudin was better than that o...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K1/06C07K1/04
CPCY02P20/55
Inventor 姚程成杨毅跃康国伟蒙相锋
Owner YANCHENG KAILI PHARMA
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