Chemoenzymatic synthesis of heparin and heparan sulfate analogs

Patent metadata

Field	Value
Publication number	US-10160986-B2
Application number	US-201615017365-A
Country	US
Kind code	B2
Filing date	Feb 5, 2016
Priority date	Jul 21, 2011
Publication date	Dec 25, 2018
Grant date	Dec 25, 2018

Abstract

The present invention provides a one-pot multi-enzyme method for preparing UDP-sugars from simple sugar starting materials. The invention also provides a one-pot multi-enzyme method for preparing oligosaccharides from simple sugar starting materials.

First claim

What is claimed is:

1. A method of synthesizing a UDP-sugar, the method comprising forming a reaction mixture comprising: i) a first sugar, ii) a glucosamine uridylyltransferase (GlmU) or a promiscuous UDP-sugar pyrophosphorylase (USP), wherein the GlmU comprises the amino acid sequence set forth in SEQ ID NO: 46 and the USP comprises the amino acid sequence set forth in SEQ ID NO: 47, and iii) a kinase or a dehydrogenase, under conditions sufficient to form the UDP-sugar.

2. The method of claim 1 , wherein the first sugar is selected from the group consisting of substituted or unsubstituted glucose (Glc), substituted or unsubstituted glucose-1-phosphate (Glc-1-P), substituted or unsubstituted glucuronic acid (GlcA), substituted or unsubstituted glucuronic acid-1-phosphate (GlcA-1-P), substituted or unsubstituted iduronic acid (IdoA), substituted or unsubstituted iduronic acid-1-phosphate (IdoA-1-P), substituted or unsubstituted N-acetylglucosamine (GlcNAc), substituted or unsubstituted N-acetylglucosamine-1-phosphate (GlcNAc-1-P), substituted or unsubstituted glucosamine (GlcNH 2 ), substituted or unsubstituted glucosamine-1-phosphate (GlcNH 2 1-P), substituted or unsubstituted galactose (Gal), substituted or unsubstituted galactose-1-phosphate (Gal-1-P), substituted or unsubstituted galacturonic acid (GalA), substituted or unsubstituted galacturonic acid-1-phosphate (GalA-1-P), substituted or unsubstituted N-acetylgalactosamine (GalNAc), substituted or unsubstituted N-acetylgalactosamine-1-phosphate (GalNAc-1-P), substituted or unsubstituted galactosamine (GalNH 2 ), substituted or unsubstituted galactosamine-1-phosphate (GalNH 2 1-P), substituted or unsubstituted mannose (Man), substituted or unsubstituted mannose-1-phosphate (Man-1-P), and substituted or unsubstituted N-acetylmannosamine (ManNAc), substituted or unsubstituted N-acetylmannosamine-1-phosphate (ManNAc-1-P), substituted or unsubstituted mannosamine (ManNH 2 ), and substituted or unsubstituted mannosamine-1-phosphate (ManNH 2 1-P).

3. The method of claim 1 , wherein the kinase is selected from the group consisting of an N-acetylhexosamine 1-kinase (NahK), a galactokinase (GalK), and a glucuronokinase (GlcAK).

4. The method of claim 1 , wherein the dehydrogenase is UDP-glucose dehydrogenase (Ugd).

5. The method of claim 1 , wherein the UDP-sugar is selected from the group consisting of substituted or unsubstituted UDP-Glc, substituted or unsubstituted UDP-GlcA, substituted or unsubstituted UDP-IdoA, substituted or unsubstituted UDP-GalA, substituted or unsubstituted UDP-GlcNAc, substituted or unsubstituted UDP-GlcNH 2 , substituted or unsubstituted UDP-Gal, substituted or unsubstituted UDP-GalNAc, substituted or unsubstituted UDP-GalNH 2 , substituted or unsubstituted UDP-Man, and substituted or unsubstituted UDP-ManNAc, and substituted or unsubstituted UDP-ManNH 2.

6. The method of claim 1 , wherein the reaction mixture further comprises a pyrophosphatase.

7. The method of claim 1 , wherein the GlmU comprises the amino acid sequence set forth in SEQ ID NO: 8 and the USP comprises the amino acid sequence set forth in SEQ ID NO: 10.

8. The method of claim 1 , wherein the reaction mixture comprises: i) the first sugar, ii) the GlmU comprising the amino acid sequence set forth in SEQ ID NO: 46, and iii) the kinase or the dehydrogenase.

9. The method of claim 1 , wherein the reaction mixture comprises: i) the first sugar, ii) the USP comprising the amino acid sequence set forth in SEQ ID NO: 47, and iii) the kinase or the dehydrogenase.

10. A method of preparing an oligosaccharide, the method comprising:
forming a first reaction mixture comprising: i) a first sugar, ii) an acceptor sugar, iii) a glycosyltransferase, iv) a glucosamine uridylyltransferase (GlmU) or a promiscuous UDP-sugar pyrophosphorylase (USP), wherein the GlmU comprises the amino acid sequence set forth in SEQ ID NO: 46 and the USP comprises the amino acid sequence set forth in SEQ ID NO: 47, and iv) a kinase or a dehydrogenase,
wherein the first sugar is selected from the group consisting of a substituted or unsubstituted N-acetylglucosamine (GlcNAc), a substituted or unsubstituted glucosamine (GlcNH 2 ), a substituted or unsubstituted glucuronic acid (GlcA), a substituted or unsubstituted galacturonic acid (GalA), a substituted or unsubstituted iduronic acid (IdoA), and a substituted or unsubstituted glucose-1-phosphate (Glc-1-P),
wherein the acceptor sugar comprises at least one member selected from the group consisting of a substituted or unsubstituted N-acetylglucosamine (GlcNAc), a substituted or unsubstituted glucosamine (GlcNH 2 ), a substituted or unsubstituted glucuronic acid (GlcA), and a substituted or unsubstituted iduronic acid (IdoA),
under conditions sufficient to convert the first sugar to a UDP-sugar, and sufficient to couple the sugar in the UDP-sugar to the acceptor sugar, such that:
when the first sugar is substituted or unsubstituted GlcNAc or GlcNH 2 , the sugar in the UDP-sugar is coupled to substituted or unsubstituted GlcA or substituted or unsubstituted IdoA of the acceptor sugar, and
when the first sugar is substituted or unsubstituted Glc-1-P, substituted or unsubstituted GlcA, or substituted or unsubstituted IdoA, the sugar in the UDP-sugar is coupled to substituted or unsubstituted GlcNH 2 or substituted or unsubstituted GlcNAc of the acceptor sugar,
thereby preparing the oligosaccharide.

11. The method of claim 10 , wherein the acceptor sugar is selected from the group consisting of a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide, a hexasaccharide, a heptasaccharide, an octasaccharide, and a nonasaccharide.

12. The method of claim 10 , wherein the glycosyltransferase is selected from the group consisting of PmHS1, PmHS2, PmCS, PmHAS, NmLgtA, NmLgtB, KfiC, and KfiA.

13. The method of claim 10 , wherein the kinase is selected from the group consisting of an N-acetylhexosamine 1-kinase (NahK), a galactokinase (GalK), and a glucuronokinase (GlcAK).

14. The method of claim 10 , wherein the dehydrogenase is UDP-glucose dehydrogenase (Ugd).

15. The method of claim 10 , wherein the UDP-sugar is selected from the group consisting of substituted or unsubstituted UDP-GlcNAc, substituted or unsubstituted UDP-GlcNH 2 , substituted or unsubstituted UDP-Glc, substituted or unsubstituted UDP-GlcA, substituted or unsubstituted UDP-GalA, and substituted or unsubstituted UDP-IdoA.

16. The method of claim 10 , wherein the reaction mixture further comprises a pyrophosphatase.

17. The method of claim 10 , wherein the oligosaccharide is selected from the group consisting of:
GlcNAc-GlcA,
GlcA-GlcNAc-GlcA,
GlcNAc-GlcA-GlcNAc-GlcA,
GlcA-GlcNAc-GlcA-GlcNAc-GlcA,
GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA,
GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA,
GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA,
GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA,
GlcA-GlcNAc,
GlcNAc-GlcA-GlcNAc,
GlcA-GlcNAc-GlcA-GlcNAc,
GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc,
GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc,
GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc,
GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc,
GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc, and
GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc.

18. The method of claim 10 , wherein the GlmU comprises the amino acid sequence set forth in SEQ ID NO: 8 and the USP comprises the amino acid sequence set forth in SEQ ID NO: 10.

Assignees

• Univ California

Inventors

• Chen Xi
• Yu Hai
• Li Yanhong
• Chen Yi
• Qu Jingyao
• Muthana Musleh M
• Lau Kam
• Bode Lars

Classifications

• C12P19/18

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

• C12P19/04

  Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds · CPC title

• C12P19/305 Primary

  Pyrimidine nucleotides · CPC title

• C12P19/12

  Disaccharides · CPC title

• C12P19/00

  Preparation of compounds containing saccharide radicals (ketoaldonic acids C12P7/58) · CPC title