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Preparation and purification method of heparin hexasaccharides containing N-unsubstituted glucosamine

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

Inactive Publication Date: 2014-04-16
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 method of heparin hexasaccharides containing N-unsubstituted glucosamine

Examples

Experimental program
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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-Gel P-10), the mobile phase is 0.2M NH 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 h. Then use SAX-HPLC, two-phase linear gradient elution under the conditions of 0-0.6M NaCl, 0.6-1.3M NaCl, separate and collect the highly sulfated heparin sodium hexasaccharide crude product. The specific parameters are as follows:

[0043] Chromatographic column: ProPac PA1 (9×250mm) Semi-Prep

[0044] Flow rate: 4ml / min

[0045] Mobile phase: A:pH3.5H ...

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-Gel P-10), the mobile phase is 0.2M NH 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 h. Then use SAX-HPLC, two-phase linear gradient elution under the conditions of 0-0.6M NaCl, 0.6-1.3M NaCl, separate and collect the highly sulfated heparin sodium hexasaccharide crude product. The specific parameters are as follows:

[0078] Chromatographic column: ProPac PA1 (9×250mm) Semi-Prep

[0079] Flow rate: 4ml / min

[0080] Mobile phase: A: pH3.5 H...

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Abstract

The invention relates to a preparation and purification method of heparin hexasaccharides containing (i)N(i)-unsubstituted glucosamine (GlcNH3+). The preparation and purification method comprises the following steps: using enzymolysis low molecular weight heparin as the raw material, separating and purifying the heparin sodium hexasaccharide by methods of gel chromatography and ion exchange HPLC; preparing heparin pyridine hexasaccharide by using cation exchange resin and pyridine neutralization process; finally preparing heparin hexasaccharides with different numbers of GlcNH3+ by the (i)N( / i)-sulfate removal method, and separating and purifying the heparin hexasaccharide by the SAX-HPLC method. The preparation and purification method can specifically prepare the heparin oligosaccharides containing different numbers and sequences of GlcNH3+, and provide important oligosaccharides libraries for researching the structure and function of the GlcNH3+ residues in the heparin sulfate and the relationship between the GlcNH3+ residues and 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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IPC IPC(8): C08B37/10
Inventor 魏峥梁群焘林江慧魏可镁
Owner FUZHOU UNIV
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