Glycoconjugate synthesis

Patent metadata

Field	Value
Publication number	US-9816122-B2
Application number	US-201314430930-A
Country	US
Kind code	B2
Filing date	Sep 25, 2013
Priority date	Sep 25, 2012
Publication date	Nov 14, 2017
Grant date	Nov 14, 2017

Abstract

The invention relates to a method for producing a glycoconjugate comprising an oligosaccharide part covalently linked to a non-sugar moiety selected from the group consisting of amino acids, peptides, proteins, lipids, longer alkyl groups, polyethylene glycols, α,β-unsaturated amido group and polyvinyl alcohols, using a genetically modified cell.

First claim

The invention claimed is:

1. A method for producing a glycoconjugate comprising an oligosaccharide part covalently linked to a polyethylene glycol, the method comprising:
(i) providing a genetically modified cell comprising a gene encoding a glycosyl transferase enzyme that is able to transfer the glycosyl residue of an activated sugar nucleotide to an internalized acceptor and a gene encoding the E. coli lactose permease,
(ii) culturing said genetically modified cell in the presence of an exogenous acceptor according to formula 7

wherein n is an integer from 2 to 6,
inducing
(a) internalization of the exogenous acceptor by the genetically modified cell, and
(b) formation of said glycoconjugate from said internalized acceptor molecule by a glycosyl transfer mediated by said glycosyl transferase enzyme expressed by said cell, and then

(iii) collecting said glycoconjugate from the fermentation broth.

2. The method according claim 1 , wherein said genetically modified cell provided in step (i) further comprises a set of genes encoding enzymes responsible for the synthesis of said activated sugar nucleotide by a de novo pathway, and wherein said culturing step (ii) further induces producing said activated sugar nucleotide by a de novo pathway.

3. The method according to claim 1 , wherein said glycosyl transferase enzyme is selected from the group consisting of N-acetyl-glucosaminyl transferases, galactosyl transferases, N-acetyl-galactosaminyl transferases, glucuronosyl transferases, sialyl transferases and fucosyl transferases.

4. The method according to claim 3 , wherein said glycosyl transferase enzyme is α-1,2-fucosyl transferase.

5. The method according to claim 3 , wherein said glycosyl transferase enzyme is selected from the group consisting of β-1,3-N-acetyl-glucosaminyl transferase, β-1,6-N-acetyl-glucosaminyl transferase, β-1,3-galactosyl transferase, β-1,4-galactosyl transferase, α-2,3-sialyl transferase, α-2,6-sialyl transferase, α-1,2-fucosyl transferase, α-1,3-fucosyl transferase and α-1,4-fucosyl transferase.

6. The method according to claim 1 , wherein the genetically modified cell is a bacterium or yeast.

7. The method according to claim 1 , wherein the genetically modified cell is E. coli of LacZ − , LacY + genotype, the exogenous acceptor is a lactose covalently linked to a polyethylene glycol, the glycosyl transferase is a fucosyl transferase, and/or a sialyl transferase, and/or an N-acetylglucosaminyl transferase and/or a galactosyl transferase, and the glycoconjugate produced by the method is a fucosylated and/or sialylated and/or N-acetylglucosaminylated and/or galactosylated lactose covalently linked to a polyethylene glycol.

8. The method according to claim 7 , wherein the exogenous acceptor consists of lactose covalently linked to said polyethylene glycol and the glycoconjugate is a fucosylated lactose covalently linked to polyethylene glycol.

9. The method according to claim 7 , wherein the oligosaccharide part of the glycoconjugate is a human milk oligosaccharide.

10. The method according to claim 9 , wherein the human milk oligosaccharide is selected from the group consisting of 2′-fucosyllactose, 3-fucosyllactose, difucosyllactose, 3′-sialyllactose, 6′-sialyllactose, 3′-sialyl-3-fucosyllactose, lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT).

11. The method according to claim 8 , wherein the glycoconjugate is a compound of formula 7a

wherein Y is a human milk oligosaccharide glycosyl residue, and n is an integer from 2-6.

12. The method according to claim 11 , wherein n is an integer of from 2 to 4.

13. The method according to claim 11 , wherein the compound of formula 7a is of formula 8d

wherein R 1 is fucosyl or H, R 2 is fucosyl or H, n is an integer from 2 to 6, provided that at least one of the R 1 and R 2 groups is fucosyl.

14. The method according to claim 13 , wherein n is an integer of from 2 to 4.

Assignees

• Glycom As

Inventors

• Peltier-Pain Pauline
• Dekany Gyula
• Pain Christophe
• Chassagne Pierre
• Fierfort Nicolas
• Molnàr-Gàbor Dóra

Classifications

• C07H15/26

  Acyclic or carbocyclic radicals, substituted by hetero rings · CPC title

• C12P19/44 Primary

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

• C07H15/04

  attached to an oxygen atom of the saccharide radical · CPC title

• C07H13/04

  having the esterifying carboxyl radicals attached to acyclic carbon atoms · CPC title

• C07H15/08

  Polyoxyalkylene derivatives · CPC title