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Method for preparing heparin derivatives by using biological enzyme to selectively modify heparin structure

A heparin derivative and selective technology, applied in the field of biomedicine, can solve the problems of reducing the content of 2-O-sulfate group and 6-O-sulfate group, and achieve a wide range of indications, great social value and economic value, Effects of Dosage and Toxicity Reduction

Inactive Publication Date: 2009-09-16
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Using biological enzymes to selectively modify the structure of heparin, while increasing the number of anticoagulant active centers, reduces the content of 2-O-sulfate groups and 6-O-sulfate groups that are not related to the activity. So far, there have been no related reports at home and abroad

Method used

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  • Method for preparing heparin derivatives by using biological enzyme to selectively modify heparin structure
  • Method for preparing heparin derivatives by using biological enzyme to selectively modify heparin structure
  • Method for preparing heparin derivatives by using biological enzyme to selectively modify heparin structure

Examples

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Effect test

example 1

[0020] Product I was synthesized using heparan sulfate 3-OH sulfotransferase. The usual sulfation reaction conditions are 1mg heparin in 20ml reaction liquid and shake (300rpm) at room temperature for 6h. The reaction solution contains 50mM Tris-HCl (pH7.2), 1% Triton X-100, 1% BSA, 1mM MgCl 2 , 1mM MnCl 2 , 1 mM PNPS, 40 μM PAP, 8 mg 3-OST-1 (or 3-OST-5) and 4 mg AST-IV. The reaction product was separated by DEAE resin, dialyzed and freeze-dried to obtain the synthetic product I. Use mixed heparin degrading enzymes to completely degrade heparin and synthetic product I into disaccharides, and use C 18 The composition and content of disaccharides were analyzed by reversed-phase column HPLC. The results are shown in figure 1 , indicating that the 3-O-sulfate disaccharide content of product I is about 3 times that of heparin, revealing that the number of anticoagulant active centers has increased by 3 times.

example 2

[0022] Product II was synthesized using heparan sulfate 2-O desulfatase. The usual desulfation reaction condition is that the product I is reacted in 20ml of reaction solution at room temperature with shaking (300rpm) for 6h. The reaction solution contains 50mM Tris-HCl (pH7.2), 1% Triton X-100, 1% BSA, 1mM MgCl 2 , 1mM MnCl 2 , 1mM CaCl 2 , 5mg 2-O-sulfatase. The reaction product was separated by DEAE resin, dialyzed and freeze-dried to obtain the synthetic product II. Heparin and synthetic product II were completely degraded into disaccharides with mixed heparin-degrading enzymes, and C 18 The composition and content of disaccharides were analyzed by reversed-phase column HPLC. The results are shown in figure 2 , indicating that the 2-O-sulfate disaccharide content of product II is about 20% of that of heparin.

example 3

[0024] Using heparan sulfate 6-O desulfatase, product III was synthesized. The usual desulfation reaction conditions are product II in 20ml of reaction solution at room temperature shaking (300rpm) reaction for 6h. The reaction solution contains 50mM Tris-HCl (pH7.2), 1% Triton X-100, 1% BSA, 1mM MgCl 2 , 1mM MnCl 2 , 1mM CaCl 2 , 5mg 6-O-sulfatase. The reaction product was separated by DEAE resin, dialyzed and freeze-dried to obtain the synthetic product III. Heparin and synthetic product III were completely degraded into disaccharides with mixed heparin-degrading enzymes, and C 18 The composition and content of disaccharides were analyzed by reversed-phase column HPLC. The results are shown in image 3 , indicating that the 6-O-sulfate disaccharide content of product III is about 20% of that of heparin.

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Abstract

The invention provides a method for selectively modifying a heparin structure by using biological enzyme, which can improve anticoagulation activity of heparin, reduce combination of heparin with blood protein such as platelet factors and the like, and reduce toxic and side effects. The invention belongs to the field of biological medicine. The antithrombus and anticoagulation activities of the heparin derivatives prepared by the method are 2 to 3 times higher than common heparin medicament, the 2-O-sulfate and 6-O-sulfate contents are about 20 percent of the common heparin, and the combination capability of the heparin derivatives with the platelet factors is about 20 percent of the common heparin. The novel heparin derivatives have high anticoagulation activity and low side effect.

Description

technical field [0001] The invention relates to a method for selectively modifying the structure of heparin with biological enzymes to prepare heparin derivatives, which belongs to the field of biomedicine. Background technique [0002] Heparin is a sulfated polysaccharide formed by the alternating copolymerization of highly sulfated uronic acid and glucosamine, which is mainly isolated from mammalian mast cells. The advantage of heparin is that it has high antithrombotic and anticoagulant activities at the same time, and can quickly produce anticoagulant effect after subcutaneous injection, the price is relatively cheap, and protamine can be used to neutralize the excessive use of heparin in the body[ 8]. However, the clinical use of heparin has great limitations. It is mainly manifested in the following aspects: (1) Heparin is isolated from animal viscera. Therefore, there are large differences in composition, molecular weight, and structure of heparin from different sou...

Claims

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Application Information

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IPC IPC(8): C08B37/10A61P7/02
Inventor 陈敬华金坚许正宏
Owner JIANGNAN UNIV
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