Diversification of human milk oligosaccharides (HMOs) or precursors thereof

Patent metadata

Field	Value
Publication number	US-9382564-B2
Application number	US-201214117332-A
Country	US
Kind code	B2
Filing date	May 14, 2012
Priority date	May 13, 2011
Publication date	Jul 5, 2016
Grant date	Jul 5, 2016

Abstract

A method of diversification of human milk oligosaccharides (HMOs) or precursors thereof, compounds obtainable by the method, and uses and compositions involving such compounds. The method comprises a) providing at least one compound or a mixture of the compounds selected from the group consisting of: optionally sialylated and/or fucosylated lactose derivatives of general formula 2 and salts thereof; b) adding at least one enzyme comprising a transglycosidase activity to the at least one compound or a mixture of compounds provided according to step a); and c) incubating the mixture obtained according to step b).

First claim

The invention claimed is:

1. A method for preparation of two or more human milk oligosaccharides (HMOs) or derivatives or precursors thereof, the method comprising the steps of
a) providing at least one compound or a mixture of the compounds selected from the group consisting of:
optionally sialylated and/or fucosylated lactose derivatives of general formula 2 and salts thereof:

wherein
R is a group removable by hydrogenolysis,
R 1 independently of each other is fucosyl or H
R 4 independently of each other is sialyl or H,
provided that the compound of general formula 2 is not R-glycoside of lactose, if provided alone;
optionally sialylated and/or fucosylated lactose derivatives of general formula 4 and salts thereof:

wherein
R 1 independently of each other is fucosyl or H
R 4 independently of each other is sialyl or H,
provided that the compound of general formula 4 is not lactose, if provided alone;
lacto-N-tetraose (LNT):

lacto-N-tetraose (LNT) derivatives of the following formula:

wherein R is a group removable by hydrogenolysis;
lacto-N-neotetraose (LNnT):

lacto-N-neotetraose (LNnT) derivatives of the following formula:

wherein R is a group removable by hydrogenolysis;

b) adding at least one enzyme comprising a transglycosidase activity to the at least one compound or a mixture of compounds provided according to step a);
c) incubating the mixture obtained according to step b);
d) optionally repeating at least steps a) and c) or steps b) and c) with the mixture obtained according to step c);
e) optionally subjecting the mixture obtained after step c) or d) to a hydrogenolysis reaction;
wherein the incubation results in the production of two or more human milk oligosaccharides or derivatives or precursors thereof.

2. The method according to claim 1 , wherein the mixture of compounds comprises at least two-different compounds as defined according to step a).

3. The method according to claim 1 , wherein the at least one enzyme comprising transglycosidase activity is selected from two to ten different enzymes comprising transglycosidase activity.

4. The method according to claim 1 , wherein the compounds or enzymes are added simultaneously or sequentially.

5. The method according to claim 1 , wherein the at least one enzyme comprising transglycosidase activity is an enzyme comprising a trans-fucosidase, trans-sialidase, trans-lacto-N-biosidase and/or trans-N-acetyllactosaminidase activity.

6. The method according to claim 1 , wherein incubation leads to a mixture of human milk oligosaccharides and/or precursors thereof as defined according to
compounds of general formula 1 and salts thereof

wherein
R is a group removable by hydrogenolysis,
R 1 is fucosyl or H,
R 2 is selected from N-acetyl-lactosaminyl and lacto-N-biosyl groups, wherein the N-acetyl lactosaminyl group may carry a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; any N-acetyl-lactosaminyl and lacto-N-biosyl group can be substituted with one or more sialyl and/or fucosyl residue,
R 3 is H or N-acetyl-lactosaminyl group optionally substituted with a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; any N-acetyl-lactosaminyl and lacto-N-biosyl group can be substituted with one or more sialyl and/or fucosyl residue;
compounds of general formula 2 and salts thereof

wherein
R is a group removable by hydrogenolysis,
R 1 independently of each other is fucosyl or H
R 4 independently of each other is sialyl or H,
with the proviso that at least one R 1 or R 4 is not H;
compounds of general formula 3 and salts thereof

wherein
R 1 is fucosyl or H,
R 2 is selected from N-acetyl-lactosaminyl and lacto-N-biosyl groups, wherein the N-acetyl lactosaminyl group may carry a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; any N-acetyl-lactosaminyl and lacto-N-biosyl group can be substituted with one or more sialyl and/or fucosyl residue,
R 3 is H or N-acetyl-lactosaminyl group optionally substituted with a glycosyl residue comprising one or more N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups; any N-acetyl-lactosaminyl and lacto-N-biosyl group can be substituted with one or more sialyl and/or fucosyl residue; and/or
compounds of general formula 4 and salts thereof

wherein
R 1 independently of each other is fucosyl or H
R 4 independently of each other is sialyl or H
with the proviso that at least one R 1 or R 4 is not H.

7. The method according to claim 6 , wherein compounds of formulae 1 and 2 are further characterized by general formulae 1a, 1b or 2 or salts thereof

and compounds of formulae 3 and 4 are further characterized by general formulae 3a, 3b or 4 or salts thereof

wherein
R, R 1 and R 4 are as defined in claim 6 ,
R 2a is N-acetyl-lactosaminyl group optionally substituted with a glycosyl residue comprising one N-acetyl-lactosaminyl and/or one lacto-N-biosyl group; any N-acetyl-lactosaminyl and lacto-N-biosyl group can be substituted with one or more sialyl and/or fucosyl residue,
R 3a is H or N-acetyl-lactosaminyl group optionally substituted with a lacto-N-biosyl group; any N-acetyl-lactosaminyl and lacto-N-biosyl group can be substituted with one or more sialyl and/or fucosyl residue,
R 2b is lacto-N-biosyl group optionally substituted with sialyl and/or fucosyl residue,
R 3b is H or N-acetyl-lactosaminyl group optionally substituted with one or two N-acetyl-lactosaminyl and/or one lacto-N-biosyl group; any N-acetyl-lactosaminyl and lacto-N-biosyl group can be substituted with one or more sialyl and/or fucosyl residue.

8. The method according to claim 7 , wherein
the N-acetyl-lactosaminyl group in the glycosyl residue of R 2a in general formula 1a or 3a is attached to another N-acetyl-lactosaminyl group with 1-3 interglycosidic linkage, or
the lacto-N-biosyl group in t

Assignees

• Dekany Gyula
• Champion Elise
• Schroven Andreas
• Hederos Markus
• Glycom As

Inventors

• Dekany Gyula
• Champion Elise
• Schroven Andreas
• Hederos Markus

Classifications

• A61P37/02

  Immunomodulators · CPC title

• C12P19/14 Primary

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• C07H13/04

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

• C07H1/00

  Processes for the preparation of sugar derivatives · CPC title

• C07H5/04

  to nitrogen · CPC title