Method of covalent coupling

• US 6,653,457 B1
• Filed: 06/27/2002
• Issued: 11/25/2003
• Est. Priority Date: 11/23/1999
• Status: Expired due to Term

• Current Assignee: Medicarb AB
• Original Assignee: Medicarb AB
• Inventors: Gouda, Ibrahim, Larm, Olle
• Primary Examiner(s): Barts, Samuel
• Assistant Examiner(s): Henry, Michael C.
• Application Number:
  US10/130,312
• Time in Patent Office: 516 Days
• Field of Search: 536/21, 536/124, 536/55.1, 536/55.3, 536/123.1, 525/54.2
• US Class Current: 536/20
• CPC Class Codes: A61L 33/08 Polysaccharides
• CPC Class Codes: C08B 37/0021 Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
• CPC Class Codes: C08B 37/0069 Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
• CPC Class Codes: C08B 37/0075 Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
• CPC Class Codes: C08L 5/02 Dextran; Derivatives thereof
• CPC Class Codes: C08L 5/10 Heparin; Derivatives thereof

Abstract

A method of preparing conjugates by means of covalent coupling of a polysaccharide selected from heparin, heparin derivatives, at least partially deacerylated dermatan sulphate and dextran sulphate and a solid substance containing primary amino groups, said method comprising the following steps: a) reduction of the saccharide so that its terminal monosaccharide unit is converted to an alditol; b) periodate oxidation of the alditol formed in step a) to the formation of a terminal aldehyde group under cleavage of the monosaccharide unit between two vicinal groups selected from hydroxyl and amino groups; and c) reductive coupling of the alditol via the aldehyde group to the amino group of the solid substrate.

Claims

1. A method of preparing conjugates by means of covalent coupling of a polysaccharide selected from heparin, heparin derivatives, at least partially deacetylated dermatan sulphate and dextran sulphate and a solid substance containing primary amino groups, comprising the steps:

a) reduction of the saccharide so that its terminal monosaccharide unit is converted to an alditol;

b) periodate oxidation of the alditol formed in step a) to the formation of a terminal aldehyde group under cleavage of the monosaccharide unit between two vicinal groups selected from hydroxyl and amino groups; and

c) reductive coupling of said alditol via the aldehyde group to the amino group of the solid substrate.

2. A method according to claim 1, characterized in that the reduction in step a) is carried out with a reducing agent selected from borohydrides, catalytic reducing agents, and Raney nickel.

3. A method according to claim 2, wherein the reduction in step 2) is carried out with a borohydride.

4. A method according to claim 1, wherein the oxidation in step b) is carried out with periodic acid or a sodium or potassium salt thereof.

5. A process according to claim 1, wherein the reductive coupling in step c) is carried out with a cyanoborohydride.

6. A method according to claim 1, wherein the reduction in step a) is carried out with sodium borohydride;

the oxidation in step b) is carried out with sodium periodate; and

the reductive coupling in step c) is carried out with sodium cyanoborohydride.

7. A method according to claim 1, wherein the saccharide is a polysaccharide which is biologically active.

8. A method according to claim 7, wherein the polysaccharide is heparin or heparan sulphate.

9. A method according to claim 1, wherein the substrate is selected from polymeric objects aminated on the surface thereof.

10. A method according to claim 1, wherein the substrate is provided with a coating of polyethylenimine or chitosan.

11. Conjugate made by the method according to claim 1.

12. A method according to claim 2, wherein the oxidation in step b) is carried out with periodic acid or a sodium or potassium salt thereof.

13. A method according to claim 3, wherein the oxidation in step b) is carried out with periodic acid or a sodium or potassium salt thereof.

14. A process according to claim 2, wherein the reductive coupling in step c) is carried out with a cyanoborohydride.

15. A process according to claim 3, wherein the reductive coupling in step c) is carried out with a cyanoborohydride.

16. A process according to claim 4, wherein the reductive coupling in step c) is carried out with a cyanoborohydride.

17. A method according to claim 2, wherein the saccharide is a polysaccharide which is biologically active.

18. A method according to claim 2, wherein the substrate is selected from polymeric objects aminated on the surface thereof.

19. A method according to claim 2, wherein the substrate is provided with a coating of polyethylenimine or chitosan.

20. Conjugate made by the method according to claim 2.

21. A method according to claim 3, wherein the borohydride is sodium borohydride or potassium borohydride.