Polymer grafting by polysaccharide synthases

• US 7,575,904 B2
• Filed: 07/11/2005
• Issued: 08/18/2009
• Est. Priority Date: 11/11/1998
• Status: Expired due to Fees

• 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.
• Assistant Examiner(s): MEAH, MOHAMMAD Y
• Application Number:
  US11/178,560
• Publication Number:
  US 20060116348A1
• Time in Patent Office: 1,499 Days
• Field of Search: None
• 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 polysaccharide to produce a polysaccharide polymer on a substrate, comprising the steps of:

providing a substrate having an immobilized polysaccharide primer thereon to provide a primed substrate, wherein the primed substrate comprises at least two sugar units selected from the group consisting of GIcUA, GIcNAc, GIc, GaINAc, GIcN, and GaIN;

combining the primed substrate within a reaction medium with an isolated, enzymatically active hyaluronan synthase having an empty acceptor site, wherein the isolated, enzymatically active hyaluronan synthase is a single protein that is a dual-action catalyst that utilizes UDP-GIcA and UDP-GIcNAc to synthesize hyaluronic acid (HA) on a substrate, wherein the reaction medium contains at least one sugar precursor selected from the group consisting of UDP-GIcA and UDP-GIcNAc, and wherein the enzymatically active hyaluronan synthase has an amino acid sequence that is at least one of;

(A) an amino acid sequence that is at least 90% identical to SEQ ID NO;

1;

(B) an amino acid sequence encoded by a nucleic acid segment that is at least 90% identical to SEQ ID NO;

2; and

(C) an amino acid sequence selected from the group consisting of SEQ ID NOS;

7, 8, 11 and 13-17; and

reacting the isolated, enzymatically active hyaluronan synthase with the primed substrate to produce a polysaccharide polymer coating on the primed substrate.

2. The method of claim 1 wherein, in the step of providing a substrate having an immobilized polysaccharide primer thereon to provide a primed substrate, the polysaccharide primer is hyaluronic acid.

3. The method of claim 1 wherein, in the step of providing a substrate having an immobilized polysaccharide polymer thereon to provide a primed substrate, the polysaccharide primer is chondroitin.

4. The method of claim 2, wherein the polysaccharide primer comprises at least two sugar units.

5. The method of claim 3, wherein the polysaccharide primer comprises at least five sugar units.

6. The method of claim 1 wherein, in the step of reacting the isolated, enzymatically active hyaluronan synthase with the primed substrate to produce a polysaccharide polymer coating on the primed substrate, the polysaccharide polymer comprises at least 20 sugar units.

7. The method of claim 1 wherein, in the step of reacting the isolated, enzymatically active hyaluronan synthase with the primed substrate to produce a polysaccharide polymer coating on the primed substrate, the polysaccharide polymer comprises at least 100 sugar units.

8. The method of claim 1 wherein, in the step of reacting the isolated, enzymatically active hyaluronan synthase with the primed substrate to produce a polysaccharide polymer coating on the primed substrate, the polysaccharide polymer comprises at least 400 sugar units.

9. The method of claim 1 wherein, in the step of providing a substrate, the substrate comprises an organic substrate.

10. The method of claim 1 wherein, in the step of providing a substrate, the substrate comprises an inorganic substrate.

11. The method of claim 1 wherein, in the step of providing a substrate, the substrate comprises at least one of an electronic and a metallic substrate.

12. The method of claim 1 wherein, in the step of providing a substrate, the substrate comprises a polymer.

13. The method of claim 1 wherein, in the step of providing a substrate, the substrate comprises a polyacrylamide surface.

14. The method of claim 1 wherein, in the step of providing a substrate, the substrate comprises a silica or silicon compound.