Enzymatic modification of glycopeptides

• US 20090292110A1
• Filed: 07/25/2005
• Published: 11/26/2009
• Est. Priority Date: 07/23/2004
• Status: Abandoned Application

• Current Assignee: Ratiopharm GmbH (Teva Pharmaceutical Industries Limited)
• Original Assignee: Ratiopharm GmbH (Teva Pharmaceutical Industries Limited)
• Inventors: Defrees, Shawn
• Application Number:
  US11/658,218
• Publication Number:
  US 20090292110A1
• Time in Patent Office: Days
• US Class Current: 530/345
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins

Abstract

The present invention provides glycoconjugates that are formed through the enzymatically-mediated coupling of a glycosyl moiety, e.g., on a peptide or lipid, and a modifying group that includes an acyl group. The conjugates include the modifying group tethered to the glycosyl moiety through a linking moiety that includes an acyl residue. Also provided are methods for preparing the conjugates of the invention

Claims

1. A peptide conjugate comprising the glycosyl moiety:

2. The conjugate according to claim 1 wherein

R¹ is a member selected from:

3. The conjugate according to claim 2 wherein said glycosyl moiety is a sialic acid.

4. The conjugate according to claim 1 wherein said modifying group is a member selected from poly(ether), poly(sialic acid), and poly(amino acid).

5. The conjugate according to claim 2 wherein R¹⁰ has the formula:

6. The conjugate according to claim 5, wherein R¹¹ is an acyl moiety, such that said acyl moiety, taken together with the atoms to which it is covalently attached, comprises a moiety selected from an ester, amide and urethane.

7. The conjugate according to claim 1 having the structure:

8. The conjugate according to claim 1 having the structure:

9. The conjugate according to claim 8 in which a member selected from R³ and R⁴ is N-acetyl.

10. The conjugate according to claim 8 wherein R⁶ is a member selected from Gal, GalNAc, Man, GlcNAc and Glu.

11. A method of preparing a conjugate according to claim 1, said method comprising:

(a) contacting a peptide comprising a glycosyl residue with

(i) an acylating agent comprising an activated acyl moiety; and

(ii) an enzyme for which said acylating agent is a substrate, under conditions appropriate to acylate said glycosyl residue, thereby preparing said conjugate.

12. The method according to claim 11, wherein said glycosyl residue is acylated by said acylating agent at a site that is a member selected from an OH, NH₂ and SH.

13. The method according to claim 11, wherein said enzyme is a member selected from a lipase, a protease, an esterase, an acylase and an acyltransferase.

14. The method according to claim 11, wherein said enzyme has an amino acid sequence that is a wild-type sequence for said enzyme.

15. The method according to claim 11, wherein said enzyme is a mutated enzyme

wherein said mutated enzyme has a mutated amino acid sequence.

16. The method according to claim 15, wherein said mutated enzyme has an acylation activity that is enhanced relative to a corresponding wild-type enzyme.

17. The method according to claim 15;

wherein said mutated amino acid sequence comprises a mutation wherein an amino acid residue, implicated in hydrolysis of a member selected from an amide and an ester, is replaced by an amino acid residue that is not implicated in said hydrolysis.