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Preparation method of polypeptide solid-phase synthesis bivalirudin crude product

A peptide solid-phase synthesis, bifaludin technology, applied in the field of peptide solid-phase synthesis, can solve the problems of cumbersome operation, high cost, limited product purity, etc.

Active Publication Date: 2009-10-14
HAINAN SHUANGCHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] US20070093423A discloses a method for the solid-phase synthesis of polypeptide bivaludin, which uses a solid-liquid phase synthesis method, the operation is cumbersome, and the cutting agent used contains acidic substances, ethanedithiol and other substances
The cost of this method is high, the impurities produced are many, and the product purity is limited

Method used

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  • Preparation method of polypeptide solid-phase synthesis bivalirudin crude product

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preparation example Construction

[0047] In an example of the present invention, the preparation method of polypeptide solid-phase synthesis bivalirudin of the present invention comprises the following steps:

[0048] In the first step, Fmoc-leucine (Fmoc-Leu) is bonded to the resin. The resin used can be well known in the art, preferably Wang resin, more preferably the substitution rate of Wang resin is 1.0-1.4 mmol / g.

[0049] The second step is to use the deprotecting agent of the present invention to remove the Fmoc group, that is, to wash the Fmoc-Leu-resin obtained in the first step with the deprotecting agent of the present invention to remove the Fmoc group.

[0050] The third step is to condense the Fmoc-amino acid and the amino acid on the resin. In a preferred example of the present invention, the amino acid is condensed one by one from the C-terminal to the N-terminal according to the polypeptide shown in formula I until it is bonded to the resin. There is a polypeptide as shown in formula I.

[...

Embodiment 1

[0072] Preparation of Bifaludine 1

[0073] step one:

[0074] Fmoc-leucine bonding step: 2.0 equivalent mol Fmoc-leucine, activated by 2,6-dichlorobenzoyl chloride and pyridine and Wang resin (substitution rate 1.2-1.4mmol / g) in N,N-di reaction in methylformamide solution.

[0075] Step two:

[0076] Fmoc removal procedure: Fmoc group was removed with 15% piperidine / 5% DBU in DMF for 30 minutes. Then wash the resin 1 time with DMF; wash the resin 3 times with methanol; wash the resin 3 times with DMF.

[0077] Fmoc-amino acid (the last amino acid condensation of Boc-D-Phe-OH) condensation step: 2.0-3.0 times of resin equivalent of Fmoc-amino acid, 1.5 times of resin equivalent of HOBt dissolved in 3mL / g resin amount of DMF and added to the resin , and then add 3.0 times of resin equivalent DIC. After reacting for 90 minutes, monitor with ninhydrin chromogenic method (Kaiser), control the temperature to 10°C, and then dilute the above solution to a volume of 4mL / g resin wi...

Embodiment 2

[0087] Preparation of Bifaludin 2

[0088] Fmoc-leucine bonding step: 3.0 equivalent mol Fmoc-leucine is activated by 2,6-dichlorobenzoyl chloride and pyridine and Wang resin (substitution rate 1.0-1.2mmol / g) in N,N-dimethyl reaction in the formamide solution. Unreacted groups on the resin were blocked with benzoyl chloride / triethylamine.

[0089] Step two:

[0090]Fmoc removal step: 10% piperidine / 7% DBU / 3% HOOBT DMF solution of 5 times the volume of the resin bed was used for 30 minutes to remove the Fmoc group. Then wash the resin with 5 times the resin bed volume of DMF for 1 time; wash the resin with 5 times the resin bed volume of methanol for 3 times; wash the resin with 5 times the resin bed volume of DMF for 3 times.

[0091] Fmoc-amino acid (the last amino acid condensation of Boc-D-Phe-OH) condensation step: 1.5-2.0 times of resin equivalent of Fmoc-amino acid, 3.0 times of resin equivalent of HOBt dissolved in 5mL / g resin amount of DMF and added to the resin , ...

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Abstract

The invention discloses a preparation method of polypeptide solid-phase synthesis bivalirudin crude product. The preparation method comprises the following steps of: (1) mixing Fmoc-amino-acid resin or Fmoc-polypeptide resin and an unprotecting agent and removing Fmoc protecting group; (2) in the existence of a condensing agent, leading amino acid with Fmoc or Boc and amino acid or polypeptide onthe resin to carry out condensation; (3) repeating the step (1) and step (2) and obtaining polypeptide resin shown as formula II; and (4) in the existence of a cutting agent, leading the polypeptide and the resin on the polypeptide resin to be separated, and obtaining bivalirudin shown as formula I. The unprotecting agent contains 3 to 20 percent of piperidine and 0.5 to 10 percent of bicyclic amidine by total volume.

Description

technical field [0001] The invention relates to the field of polypeptide solid-phase synthesis, in particular to a solid-phase synthesis method for a crude product of Bivalirudin. Background technique [0002] It was R. Bruce Merrifield who made a breakthrough in the technology of peptide synthesis. He designed a peptide synthesis pathway and named it the solid-phase synthesis pathway. First, an amino acid whose amino group is protected by a blocking group is covalently linked to a solid-phase support; under the action of a deprotecting agent, the amino-protecting group is removed, so that the first amino acid is connected to the solid-phase support. Then the carboxyl group of the second amino acid whose amino group is blocked is activated by a reagent, and the second amino acid whose carboxyl group is activated by DCC reacts with the amino group of the first amino acid that has been attached to the solid-phase carrier to form a peptide bond, so that on the solid-phase carri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/06C07K1/04A61P7/02
CPCC07K14/815Y02P20/55A61P7/02
Inventor 白俊才张若平刘亚东张国庆
Owner HAINAN SHUANGCHENG PHARMA
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