Polysaccharide conjugates of biomolecules

• US 20030027788A1
• Filed: 07/02/2002
• Published: 02/06/2003
• Est. Priority Date: 08/08/1997
• Status: Active Grant

• Current Assignee: Siemens Healthcare Diagnostics Products GmbH (Siemens AG)
• Original Assignee: Harshvardhan Mehta, Rajendra Singh
• Inventors: Singh, Rajendra, Mehta, Harshvardhan
• Granted Patent: US 6,949,524 B2
• Time in Patent Office: Days
• US Class Current: 514/54
• CPC Class Codes: A01N 43/16 with oxygen as the ring hetero atom
• 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: C08H 1/00 Macromolecular products derived from proteins food proteins A23; glue, gelatine C09H
• CPC Class Codes: G01N 2333/988 Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase
• CPC Class Codes: G01N 33/564 for pre-existing immune complex or autoimmune disease , i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Abstract

Compounds that are modified polysaccharides having pendant aldehyde functionalities are disclosed. Each of the aldehyde functionalities is attached through a linker to a position corresponding to a hydrogen atom of a different hydroxyl group of unmodified polysaccharide. Also disclosed is a method for introducing an amine-reactive functionality into a dextran. The method comprises (a) reacting the dextran with an alkylating agent having a functionality that reacts with an hydroxyl group of the dextran thereby forming an alkylated dextran wherein the alkylating agent has an olefin group and (b) treating the alkylated dextran to convert the olefin group to an amine-reactive functionality. A polysaccharide can be conjugated to a biomolecule by carrying out the above method and reacting the amine-reactive functionality with an amine functionality on the biomolecule to produce polysaccharide conjugated to the biomolecule.

Claims

1. A polymer comprisng a sequence of repeating monosaccharide units, each of which is independently selected from monosaccharide units of the formula:

2. The polymer of claim 1 having from about 50 to 50,000 repeating monosaccharide units.

3. The polymer of claim 1 wherein Q is C(Z)═

D wherein D is O and Z is H.

4. The polymer of claim 1 wherein Q is C(Z)═

D wherein D is CR¹R² and Z is H.

5. The polymer of claim 1 wherein Q is C(Z)═

D wherein D is O and Z is C(Y)═

O wherein Y is OH.

6. The polymer of claim 1 wherein at least two of the A'"'"'s are QL in at least one of said monosaccharide units for every 20 monosaccharides units of said polymer.

7. A compound produced by reaction of the polymer of claim 3 with a polypeptide.

8. A compound produced by the reductive amination of the polymer of claim 1 wherein D is O with a polypeptide.

9. The compound of claim 8 wherein said polypeptide is selected from the group consisting of avidin, streptavidin, protein A, protein G and antibodies.

45. The compound according to claim 7 wherein the polypeptide is a biomolecule.

46. The compound according to claim 7 wherein the polypeptide is a receptor.

47. The compound according to claim 7 wherein the polypeptide is a binding pair member.

48. The compound according to claim 7 wherein the polypeptide is an antibody.

10. A compound comprising:

11. The compound of claim 10 wherein Q¹ is C(Z)═

D wherein D is O and Z is H.

12. The compound of claim 10 wherein Q¹ is C(Z)═

D wherein D is CR¹R² and Z is H.

13. The compound of claim 10 wherein Q¹ is C(Z)═

D wherein D is O and Z is C(Y)═

O wherein Y is OH.

14. The compound of claim 10 wherein at least two of the A¹'"'"'s are Q¹L¹ in at least one of said monosaccharide units for every 20 monosaccharides units of said compound.

15. A compound produced by reaction of the compound of claim 11 with a polypeptide.

16. A compound produced by the reductive amination of the compound of claim 10 wherein D is O and Z is H with a polypeptide.

17. The compound of claim 16 wherein said polypeptide is selected from the group consisting of avidin, streptavidin, protein A, protein G and antibodies.

18. A compound that is a modified polysaccharide having pendant aldehyde functionalities wherein each of said aldehyde functionalities is attached through a linker to a position corresponding to a hydrogen atom of a hydroxyl group of unmodified polysaccharide.

19. The compound of claim 18 having two or more of said pendant aldehyde functionalities.

20. The compound of claim 18 wherein there is at least one aldehyde functionality for every 20 monosaccharides of said modified polysaccharide.

21. The compound of claim 18 wherein said polysaccharide is dextran.

22. A method for introducing an amine-reactive functionality into a dextran, said method comprising:

(a) reacting said dextran with an alkylating agent having a functionality that reacts with an hydroxyl group of said dextran thereby forming an alkylated dextran wherein said alkylating agent has an olefin group and

(b) treating said alkylated dextran to convert said olefin group to an amine-reactive functionality.

23. The method of claim 22 wherein step (b) comprises ozonolysis of said alkylated dextran.

24. The method of claim 22 wherein said alkylating agent comprises the structure:

L³(Z¹)C═

CR⁵R⁶

wherein L³ is a group comprising a functionality reactive with a hydroxyl group of said dextran, R⁵ and R⁶ are independently H, alkyl, aryl or may be taken together with each other or with L³ to form a ring and Z¹ is H or C(W)═

O wherein W is R⁷, OR⁷, or NR⁷R⁸ wherein R⁷ and R⁸ are independently H, alkyl or aryl.

25. The method of claim 24 wherein L³ comprises an epoxide or an alkyl radical comprising a leaving group.

26. The method of claim 24 wherein L³ is an alkyl radical comprising a leaving group and said leaving group is selected from the group consisting of bromide, chloride, iodide, aryl or alkyl sulfonates and sulfates.

27. The method of claim 26 wherein L³ is CH₂OCH₂(C₂H₃O) wherein (C₂H₃O) is an oxiranyl radical.

28. The method of claim 26 wherein L³ is CH₂Br.

29. The method of claim 22 wherein said amine-reactive functionality is an aldehyde.

30. The method of claim 29 wherein said olefin is subjected to ozonolysis to yield said aldehyde functionality.

31. The method of claim 22 wherein said amine-reactive functionality is an alpha-keto carboxylic acid functionality.

32. A method for conjugating a polysaccharide to a biomolecule, said method comprising:

(a) reacting said polysaccharide with an alkylating agent having a functionality that reacts with a hydroxy group of said polysaccharide thereby forming an alkylated polysaccharide wherein said alkylating agent has an olefin group,

(b) treating said alkylated polysaccharide to convert said olefin group to an amine-reactive functionality and

(c) reacting said amine-reactive functionality with an amine functionality on said biomolecule to produce polysaccharide conjugated to said biomolecule.

33. The method of claim 32 wherein step (b) comprises ozonolysis of said alkylated polysaccharide.

34. The method of claim 32 wherein said polysaccharide is dextran.

35. The method of claim 32 wherein said alkylating agent comprises the structure:

L³(Z¹)C═

CR⁵R⁶

wherein L³ is a group reactive with a hydroxyl group of said dextran, R⁵ and R⁶ are independently H, alkyl, aryl or may be taken together with each other or with L³to form a ring and Z¹ is H or C(W)═

O wherein W is R⁷, OR⁷, or NR⁷R⁸ wherein R⁷ and R⁸ are independently H, alkyl or aryl.

36. The method of claim 35 wherein L³ comprises an epoxide or an alkyl radical comprising a leaving group.

37. The method of claim 35 wherein L³ is an alkyl radical comprising a leaving group and said leaving group is selected from the group consisting of bromide, chloride, iodide, aryl or alkyl sulfonates and sulfates.

38. The method of claim 35 wherein L³ is CH₂OCH₂(C₂H₃O) wherein (C₂H₃O) is an oxiranyl radical.

39. The method of claim 35 wherein L³ is CH₂Br.

40. The method of claim 34 wherein said amine-reactive functionality is an aldehyde.

41. The method of claim 40 wherein said olefin is subjected to ozonolysis to yield said aldehyde functionality.

42. The method of claim 38 wherein said amine-reactive functionality is an alpha-keto carboxylic acid functionality.

43. The method of claim 34 wherein said biomolecule is a polypeptide.

44. The method of claim 43 wherein said polypeptide is selected from the group consisting of avidin, streptavidin, protein A, protein G and antibodies.