Preparation and Purification of Heparin Hexasaccharide Containing n-Non-Substituted Glucosamine

A technology of glucosamine and heparin pyridine hexasaccharide, which is applied in the field of preparation and purification of natural products, and can solve problems such as high prices

Inactive Publication Date: 2016-03-30
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, the methods for preparing heparin oligosaccharides are many steps and expensive
There are few reports on direct chemical modification of the backbone structure of oligosaccharide fragments obtained after enzymatic hydrolysis of heparan sulfate

Method used

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  • Preparation and Purification of Heparin Hexasaccharide Containing n-Non-Substituted Glucosamine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] To prepare GlcNH containing 3 + Heparin hexasaccharide as an example:

[0041] 1 Preparation and purification of heparin sodium hexasaccharide

[0042] Heparinase Ⅰ, Ⅱ, Ⅲ Enzymolysis of low molecular weight heparin (3-8kD) at 37°C for 48h (commercially available) enzymatic hydrolysis of low molecular weight heparin through gel chromatography column (Bio-GelP-10), the mobile phase is 0.2MNH 4 HCO 3 , at UV 232nm, measure the absorbance of the eluate. Collect the elution peak of heparin sodium hexasaccharide mixture, and then remove NH in 55°C water bath for 24h×3 4 HCO 3 , Freeze-dried at -80°C for 5 hours. Then SAX-HPLC was used to elute with a two-phase linear gradient under the conditions of 0-0.6M NaCl and 0.6-1.3M NaCl, and the highly sulfated crude heparin sodium hexasaccharide was separated and collected. The specific parameters are as follows:

[0043] Chromatographic column: ProPacPA1 (9×250mm) Semi-Prep

[0044] Flow rate: 4ml / min

[0045] Mobile p...

Embodiment 2

[0075] To prepare GlcNH containing 3 + Heparin hexasaccharide as an example:

[0076] 1 Preparation and purification of heparin sodium hexasaccharide

[0077] Heparinase Ⅰ, Ⅱ, Ⅲ Enzymolysis of low molecular weight heparin (3-8kD) at 37°C for 48h (commercially available) enzymatic hydrolysis of low molecular weight heparin through gel chromatography column (Bio-GelP-10), the mobile phase is 0.2MNH 4 HCO 3 , at UV 232nm, measure the absorbance of the eluate. Collect the elution peak of heparin sodium hexasaccharide mixture, and then remove NH in 55°C water bath for 24h×3 4 HCO 3 , Freeze-dried at -80°C for 5 hours. Then SAX-HPLC was used to elute with a two-phase linear gradient under the conditions of 0-0.6M NaCl and 0.6-1.3M NaCl, and the highly sulfated crude heparin sodium hexasaccharide was separated and collected. The specific parameters are as follows:

[0078] Chromatographic column: ProPacPA1 (9×250mm) Semi-Prep

[0079] Flow rate: 4ml / min

[0080] Mobile...

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Abstract

The present invention relates to a N - Preparation and purification of unsubstituted glucosamine (GlcNH3+) hexasaccharide. The method uses enzymatic hydrolysis of low molecular weight heparin as a raw material, and separates and purifies heparin sodium hexasaccharide through gel chromatography and ion exchange high performance liquid chromatography; uses cation exchange resin and pyridine neutralization method to prepare heparin hexasaccharide; finally uses take off N - Prepare heparin hexasaccharides with different GlcNH3+ numbers by sulfation method, and use SAX-HPLC and other methods to separate and purify. The method can specifically prepare a series of heparin oligosaccharides containing different GlcNH3+ numbers and sequences, and provide an important sugar library for the research on the structure and function of GlcNH3+ residues in heparan sulfate and its relationship with diseases.

Description

technical field [0001] The present invention relates to the preparation of GlcNH-containing 3 + The method for heparin oligosaccharide and its purification belong to the field of preparation and purification of natural products. Background technique [0002] There are 12 representative disaccharides in heparan sulfate, including GlcNAc, GlcNSO 3 The disaccharide of the residue is the main form, while the positively charged GlcNH 3 + The disaccharide content of the residue is minimal and its presence varies depending on the cell and tissue of origin. Although GlcNH 3 + The content of heparan sulfate in heparan sulfate is very small, but it is closely related to the invasion of viruses, the occurrence of inflammation, brain tissue damage and other diseases, and plays an important role in biology and pathophysiology in organisms. GlcNH 3 + The research on the structure and function of carbohydrate compounds is of great significance in the treatment of diseases and the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08B37/10
Inventor 魏峥梁群焘林江慧魏可镁
Owner FUZHOU UNIV
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