Chemical synthesis method of hirudin with tyrosine sulfation modification

A chemical synthesis, hirudin technology, applied in chemical instruments and methods, organic chemistry, peptide preparation methods, etc., to avoid side reactions and improve synthesis yield

Active Publication Date: 2022-07-12
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Despite the importance of these modifications, obtaining purified O-glycosylated and Tyr-sulfated proteins is extremely challenging

Method used

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  • Chemical synthesis method of hirudin with tyrosine sulfation modification
  • Chemical synthesis method of hirudin with tyrosine sulfation modification
  • Chemical synthesis method of hirudin with tyrosine sulfation modification

Examples

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Embodiment 1

[0113] Example 1: One-pot synthesis method of ligation and refolding of hirudin HIRV1 with tyrosine sulfate modification:

[0114] (1) Fmoc-Lys(Boc)-NHNH 2 Preparation of resin: Take 1 g of 2-Chlorotryl Chloride resin (referred to as 2-Cl resin) in a peptide synthesis tube, with a loading of 0.3-0.5 mmol / g, add 15 mL of DMF to swollen twice at room temperature, 30 min each time, drain, Add 15 mL of 5% hydrazine hydrate / DMF to the resin, shake for 30 min at room temperature, wash twice with DMF, add 15 mL of 5% hydrazine hydrate / DMF for the second time, shake for 30 min at room temperature, and wash twice with DMF Then, 15 mL of 5% hydrazine hydrate / DMF was added for the third time, the reaction was shaken for 30 min at room temperature, washed twice with DMF, DCM, and DMF in turn, and the solvent was drained to obtain a resin with a hydrazide end at the C-terminus (referred to as hydrazide resin). ), add 15 mL of 5% MeOH / DMF to the resin, shake the reaction for 10 min at room...

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Abstract

The invention provides a chemical synthesis method of hirudin with tyrosine sulfation modification. The preparation method comprises the following steps: firstly synthesizing a C-terminal hydrazide fragment 1 and a fragment 2 with sulfated modified tyrosine, then utilizing a natural chemical ligation reaction and an accompanying neopentyl removal reaction to obtain a full-length linear polypeptide, finally removing impurities in a system, dropwise adding the full-length linear polypeptide into a refolding reaction system, and carrying out oxidation and refolding reactions to generate three pairs of disulfide bonds, so as to obtain the high-purity peptide. A target product is obtained. The post-translational modification of the sulfated tyrosine is introduced to the polypeptide resin through a solid-phase polypeptide synthesis method, so that the sulfated tyrosine stably exists on the solid-phase resin, side reaction is avoided, and separable purification is realized; natural chemical connection is combined with automatic removal of neopentyl in an aqueous solution, so that the reaction time is shortened; natural chemical connection and refolding reaction of linear polypeptide are combined into one-pot reaction without separation and purification, so that the synthesis yield is greatly improved, and the method has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of solid-phase synthesis and preparation of polypeptide sulfated modification, in particular to a chemical synthesis method of hirudin with tyrosine sulfated modification. Background technique [0002] Hirudin is a protein found in leech saliva. The amino acid sequence of hirudin contains about 65 amino acid residues, its amino terminus (N terminus) contains six cysteines, which form a spherical compact stable structure through three pairs of disulfide bonds, while its exposed carboxyl terminus ( C-terminal) sequence has homology in amino acid sequence with fibrinogen in animals. Thrombin in animals can specifically recognize and cut fibrinogen, so that fibrinogen with better water solubility can form water-insoluble fibrin monomer, which can block blood vessels and prevent excessive bleeding through the above mechanism. effect. "Hirudin and thrombin" have a stronger interaction than "fibrinogen and throm...

Claims

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Application Information

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IPC IPC(8): C07K14/815C07K1/04C07K1/107C07K1/06C07K1/34A61K38/58A61P7/04
CPCC07K14/815A61P7/02A61K38/00Y02P20/55
Inventor 何春茂杨烨
Owner SOUTH CHINA UNIV OF TECH
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