Polymer grafting by polysaccharide synthases

• US 6,444,447 B1
• Filed: 11/10/1999
• Issued: 09/03/2002
• Est. Priority Date: 11/11/1998
• Status: Expired due to Term

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: DeAngelis, Paul L.
• Primary Examiner(s): Nashed, Nashaat T.
• Application Number:
  US09/437,277
• Time in Patent Office: 1,028 Days
• Field of Search: 435/97, 435/72, 435/100, 435/101
• US Class Current: 435/97
• CPC Class Codes: A61K 31/728 Hyaluronic acid
• CPC Class Codes: A61K 31/737 Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate A61K31/727 takes precedence
• CPC Class Codes: A61K 47/36 Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
• CPC Class Codes: A61K 9/006 Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
• CPC Class Codes: A61L 24/08 Polysaccharides A61L24/043 takes precedence
• CPC Class Codes: A61L 29/085 Macromolecular materials
• CPC Class Codes: C08L 5/08 Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
• CPC Class Codes: C12N 9/1048 Glycosyltransferases (2.4)
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates
• CPC Class Codes: C12P 19/28 N-glycosides
• CPC Class Codes: Y10S 977/898 Enzymatic

Abstract

The present invention relates to methodology for polymer grafting by a polysaccharide synthase and, more particularly, polymer grafting using the hyaluronate synthase from Pasteurella multocida. The present invention also relates to coatings for biomaterials wherein the coatings provide protective properties to the biomaterial and/or act as a bioadhesive. Such coatings could be applied to electrical devices, sensors, catheters and any device which may be contemplated for use within a mammal. The present invention further relates to drug delivery matrices which are biocompatible and may comprise combinations of a biomaterial or a bioadhesive and a medicament or a medicament-containing liposome. The biomaterial and/or bioadhesive is a hyaluronic acid polymer produced by a hyaluronate synthase from Pasteurella multocida. The present invention also relates to the creation of chimeric molecules containing hyaluronic acid or hyaluronic acid-like chains attached to various compounds and especially carbohydrates or hydroxyl containing substances. The present invention also relates to a chondroitan synthase from Pasteurella multocida which is capable of producing polysaccharide polymers on an acceptor or primer molecule.

Claims

1. A method for elongating a hyaluronic acid polymer acceptor, comprising the steps of:

providing a hyaluronic acid polymer acceptor, wherein the hyaluronic acid polymer acceptor has at least two sugar units selected from the group consisting of GlcA and GlcNAc;

providing a hyaluronic acid synthase capable of elongating the hyaluronic acid polymer acceptor, wherein the hyaluronic acid synthase has an amino acid sequence as set forth in SEQ. ID NO;

1; and

providing UDP-GlcA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the hyaluronic acid polymer acceptor.

2. The method of claim 1, wherein the hyaluronic acid polymer acceptor is attached to a substrate.

3. The method of claim 2, wherein the substrate is a silica or silicon compound.

4. The method of claim 3, wherein the substrate is glass.

5. The method of claim 2, wherein the substrate is a polymer.

6. The method of claim 2, wherein the substrate is an organic compound.

7. A method for elongating a hyaluronic acid polymer acceptor, comprising the steps of:

providing a hyaluronic acid polymer acceptor, wherein the hyaluronic acid polymer acceptor has at least two sugar units selected from the group consisting of GlcA and GlcNAc;

providing a hyaluronic acid synthase capable of elongating the hyaluronic acid polymer acceptor, wherein the hyaluronic acid synthase is encoded by a nucleotide sequence as set forth in SEQ. ID NO;

2; and

providing UDP-GlcA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the hyaluronic acid polymer acceptor.

8. The method of claim 7, wherein the hyaluronic acid polymer acceptor is attached to a substrate.

9. The method of claim 8, wherein the substrate is a silica or silicon compound.

10. The method of claim 9, wherein the substrate is glass.

11. The method of claim 8, wherein the substrate is a polymer.

12. The method of claim 8, wherein the substrate is an organic compound.

13. A method for elongating a hyaluronic acid polymer acceptor, comprising the steps of:

providing a hyaluronic acid polymer acceptor, wherein the hyaluronic acid polymer acceptor has at least three sugar units selected from the group consisting of GlcA and GlcNAc;

providing a hyaluronic acid synthase capable of elongating the hyaluronic acid polymer acceptor, wherein the hyaluronic acid synthase has an amino acid sequence as set forth in SEQ. ID NO;

1; and

providing UDP-GlcA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the hyaluronic acid polymer acceptor.

14. The method of claim 13, wherein the hyaluronic acid polymer acceptor is attached to a substrate.

15. The method of claim 14, wherein the substrate is a silica or silicon compound.

16. The method of claim 15, wherein the substrate is glass.

17. The method of claim 14, wherein the substrate is a polymer.

18. The method of claim 14, wherein the substrate is an organic compound.

19. A method for elongating a hyaluronic acid polymer acceptor, comprising the steps of:

providing a hyaluronic acid polymer acceptor, wherein the hyaluronic acid polymer acceptor has at least three sugar units selected from the group consisting of GlcA and GlcNAc;

providing a hyaluronic acid synthase capable of elongating the hyaluronic acid polymer acceptor, wherein the hyaluronic acid synthase is encoded by a nucleotide sequence as set forth in SEQ. ID NO;

2; and

providing UDP-GlcA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the hyaluronic acid polymer acceptor.

20. The method of claim 19, wherein the hyaluronic acid polymer acceptor is attached to a substrate.

21. The method of claim 20, wherein the substrate is a silica or silicon compound.

22. The method of claim 21, wherein the substrate is glass.

23. The method of claim 20, wherein the substrate is a polymer.

24. The method of claim 20, wherein the substrate is an organic compound.

25. A method of making a glycosidic bond between a hyaluronic acid polymer acceptor and at least one of GlcA and GlcNAc, comprising the steps of:

providing a hyaluronic acid synthase capable of making a glycosidic bond between a hyaluronic acid polymer acceptor and at least one of GlcA and GlcNAc, wherein the hyaluronic acid polymer acceptor has at least two sugar units selected from the group consisting of GlcA and GlcNAc and further wherein the hyaluronic acid synthase has an amino acid sequence as set forth in SEQ. ID NO;

1; and

incubating the hyaluronic acid synthase with at least one of UDP-GlcA and UDP-GlcNAc in the presence of the hyaluronic acid polymer acceptor so as to form the glycosidic bond between the hyaluronic acid polymer acceptor and at least one of GlcA and GlcNAc.

26. The method of claim 25, wherein the hyaluronic acid polymer acceptor is attached to a substrate.

27. The method of claim 26, wherein the substrate is a silica or silicon compound.

28. The method of claim 27, wherein the substrate is glass.

29. The method of claim 26, wherein the substrate is a polymer.

30. The method of claim 26, wherein the substrate is an organic compound.

31. A method of making a glycosidic bond between a hyaluronic acid polymer acceptor and at least one of GlcA and GlcNAc, comprising the steps of:

providing a hyaluronic acid synthase capable of making a glycosidic bond between a hyaluronic acid polymer acceptor and at least one of GlcA and GlcNAc, wherein the hyaluronic acid polymer acceptor has at least two sugar units selected from the group consisting of GlcA and GlcNAc and further wherein the hyaluronic acid synthase is encoded by a nucleotide sequence as set forth in SEQ ID NO;

2; and

incubating the hyaluronic acid synthase with at least one of UDP-GlcA and UDP-GlcNAc in the presence of the hyaluronic acid polymer acceptor so as to form the glycosidic bond between the hyaluronic acid polymer acceptor and at least one of GlcA and GlcNAc.

32. The method of claim 31, wherein the hyaluronic acid polymer acceptor is attached to a substrate.

33. The method of claim 32, wherein the substrate is a silica or silicon compound.

34. The method of claim 33, wherein the substrate is glass.

35. The method of claim 32, wherein the substrate is a polymer.

36. The method of claim 32, wherein the substrate is an organic compound.

37. A method of making a glycosidic bond between a hyaluronic acid polymer acceptor and at least one of GlcA and GlcNAc, comprising the steps of:

providing a hyaluronic acid synthase capable of making a glycosidic bond between a hyaluronic acid polymer acceptor and at least one of GlcA and GlcNAc, wherein the hyaluronic acid polymer acceptor has at least three sugar units selected from the group consisting of GlcA and GlcNAc and further wherein the hyaluronic acid synthase has an amino acid sequence as set forth in SEQ. ID NO;

1; and

incubating the hyaluronic acid synthase with at least one of UDP-GlcA and UDP-GlcNAc inn the presence of the hyaluronic acid polymer acceptor so as to form the glycosidic bond between the hyaluronic acid polymer acceptor and at least one of GlcA and GlcNAc.

38. The method of claim 37, wherein the hyaluronic acid polymer acceptor is attached to a substrate.

39. The method of claim 38, wherein the substrate is a silica or silicon compound.

40. The method of claim 39, wherein the substrate is glass.

41. The method of claim 38, wherein the substrate is a polymer.

42. The method of claim 38, wherein the substrate is an organic compound.

43. A method of making a glycosidic bond between a hyaluronic acid polymer acceptor and at least one of GlcA and GlcNAc, comprising the steps of:

providing a hyaluronic acid synthase capable of making a glycosidic bond between a hyaluronic acid polymer acceptor and at least one of GlcA and GlcNAc, wherein the hyaluronic acid polymer acceptor has at least three sugar units selected from the group consisting of GlcA and GlcNAc and further wherein the hyaluronic acid synthase is encoded by a nucleotide sequence as set forth in SEQ. ID NO;

2; and

incubating the hyaluronic acid synthase with at least one of UDP-GlcA and UDP-GlcNAc in the presence of the hyaluronic acid polymer acceptor so as to form the glycosidic bond between the hyaluronic acid polymer acceptor and at least one of GlcA and GlcNAc.

44. The method of claim 43, wherein the hyaluronic acid polymer acceptor is attached to a substrate.

45. The method of claim 44, wherein the substrate is a silica or silicon compound.

46. The method of claim 45, wherein the substrate is glass.

47. The method of claim 44, wherein the substrate is a polymer.

48. The method of claim 44, wherein the substrate is an organic compound.