PmST3 enzyme for chemoenzymatic synthesis of alpha-2-3-sialosides

Patent metadata

Field	Value
Publication number	US-9783838-B2
Application number	US-201214356376-A
Country	US
Kind code	B2
Filing date	Nov 7, 2012
Priority date	Nov 7, 2011
Publication date	Oct 10, 2017
Grant date	Oct 10, 2017

Abstract

The present invention provides novel methods for preparing glycosylated molecules such as oligosaccharides, glycolipids, and glycoproteins/peptides. Novel sialyltransferases are also disclosed. The method includes forming a reaction mixture containing an acceptor molecule, a donor substrate having a sugar moiety and a nucleotide, and a sialyltransferase selected from PmST3 (SEQ ID NO:7) and certain variants thereof. The reaction mixture is formed under conditions sufficient to transfer the sugar moiety from the donor substrate to the acceptor molecule, thereby forming the glycosylated molecule. In some embodiments, the acceptor molecule is selected from a natural product, an oligosaccharide, a glycoprotein, and a glycolipid. In some embodiments, the donor substrate is formed via conversion of a suitable hexosamine derivative to a cytidine 5′-monophosphate(CMP)-sialic acid in a one-pot reaction mixture containing asialic acid aldolase and a CMP-sialic acid synthetase.

First claim

What is claimed is:

1. A method of preparing a glycosylated molecule, the method comprising:
a) forming a reaction mixture comprising an acceptor molecule, a donor substrate, and the polypeptide according to SEQ ID NO:7, wherein the polypeptide lacks the C-terminal 35 amino acid residues of SEQ ID NO:7;
wherein the acceptor molecule is selected from the group consisting of an oligosaccharide, a polysaccharide, a glycopeptide, a glycoprotein, and a glycolipid;
wherein the donor substrate is selected from the group consisting of a cytidine 5′-monophosphate-sialic acid (CMP-sialic acid), an O-acetyl CMP-sialic acid, and an N-acetyl CMP-sialic acid; and
wherein the reaction mixture is formed under conditions sufficient to transfer the sialic acid from the donor substrate to the acceptor molecule, thereby forming the glycosylated molecule.

2. The method of claim 1 , wherein the acceptor molecule comprises a galactoside moiety.

3. The method of claim 2 , wherein the galactoside moiety is selected from the group consisting of a β1-4 linked galactoside moiety and a β1-3 linked galactoside moiety.

4. The method of claim 1 , wherein the acceptor molecule comprises a lactoside (LacβOR) or an N-acetyl lactosaminide (LacNAcβOR), wherein R represents the remainder of the acceptor molecule.

5. The method of claim 1 , wherein the acceptor molecule comprises a lacto-N-bioside moiety or a galacto-N-bioside moiety.

6. The method of claim 1 , wherein the CMP-sialic acid is selected from the group consisting of cytidine 5′-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac), cytidine 5′-monophosphate N-glycolylneuraminic acid (CMP-Neu5Gc), an O-acetyl derivative thereof, or an N-acetyl derivative thereof.

7. The method of claim 6 , further comprising:
b) forming a reaction mixture comprising a CMP-sialic acid synthetase; cytidine triphosphate; and Neu5Ac, Neu5Gc, an O-acetyl derivative thereof, or an N-acetyl derivative thereof;
wherein the reaction mixture is formed under conditions suitable to form the CMP-Neu5Ac, the CMP-Neu5Gc, the O-acetyl derivative thereof, or the N-acetyl derivative thereof.

8. The method of claim 7 , wherein steps a) and b) are performed in one pot.

9. The method of claim 7 , further comprising:
c) forming a reaction mixture comprising a sialic acid aldolase; pyruvic acid or thereof; and N-acetylmannosamine N-glycolylmannosamine, an O-acetyl derivative thereof, or an N-acetyl derivative thereof;
wherein the reaction mixture is formed under conditions sufficient to form the Neu5Ac, the Neu5Gc, the O-acetyl derivative thereof, or the N-acetyl derivative thereof.

10. The method of claim 9 , wherein steps a), b), and c) are performed in one pot.

11. The method of claim 1 , wherein the glycosylated molecule comprises an α2-3-linked sialic acid residue.

12. The method of claim 1 , wherein the reaction mixture is formed in vitro.

13. The method of claim 1 , wherein the polypeptide further comprises a tag sequence to facilitate purification.

14. The method of claim 13 , wherein the polypeptide further comprising the tag sequence to facilitate purification has the amino acid sequence according to SEQ ID NO: 11.

15. A method of preparing a glycosylated molecule, the method comprising:
forming a reaction mixture comprising an acceptor molecule comprising a galactoside moiety; a donor substrate comprising a cytidine 5′-monophosphate-sialic acid (CMP-sialic acid); and the polypeptide according to SEQ ID NO:11;
wherein the reaction mixture is formed under conditions sufficient to transfer the sialic acid from the donor substrate to the galactoside moiety, thereby forming the glycosylated molecule.

16. The method of claim 15 , wherein the acceptor molecule is selected from the group consisting of an oligosaccharide, a glycopeptide, a glycoprotein, and a glycolipid.

17. The method of claim 15 , wherein the glycosylated molecule comprises an α2-3-linked sialic acid residue.

Assignees

• Univ California

Inventors

• Chen Xi
• Thon Vireak
• Yu Hai

Classifications

• C12P19/44

  Preparation of O-glycosides, e.g. glucosides {(polysaccharides and not substituted disaccharides C12P19/04, C12P19/12)} · CPC title

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• C12P21/005 Primary

  Glycopeptides, glycoproteins · CPC title

• C12N9/1081

  transferring other glycosyl groups (2.4.99) · CPC title

• C12P19/18

  produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins · CPC title