Diversification of human milk oligosaccharides (HMOs) or precursors thereof

Patent metadata

Field	Value
Publication number	US-9963729-B2
Application number	US-201615161887-A
Country	US
Kind code	B2
Filing date	May 23, 2016
Priority date	May 13, 2011
Publication date	May 8, 2018
Grant date	May 8, 2018

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 one or more human milk oligosaccharides (HMOs) or derivatives or precursors thereof, the method comprising the steps of
a) providing at least two compounds or a mixture of the compounds selected from the group consisting of:

compounds of general formula 5, wherein general formula 5 is:

wherein
R′ independently is fucosyl or H, with the proviso that at least one R′ is fucosyl, and
R* is a group removable by hydrogenolysis or H;

optionally sialylated lactose derivatives of general formula 6 and salts thereof, wherein general formula 6 is:

wherein
R* is a group removable by hydrogenolysis or H, and
R″ independently of each other is sialyl or H;

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):

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

wherein R is a group removable by hydrogenolysis;

wherein at least one compound is a compound of general formula 5;
b) adding at least one enzyme comprising a trans-fucosidase activity to the mixture of the at least two compounds provided according to step a);
c) incubating the mixture of at least two compounds with the at least one enzyme obtained according to step b);
d) optionally:
I. adding to the mixture obtained according to step c), at least one compound or a mixture of compounds selected from the group consisting of:
i. 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;
ii. 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;
iii. lacto-N-tetraose (LNT):

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

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

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

wherein R is a group removable by hydrogenolysis; and
incubating the mixture obtained;
II. adding at least one enzyme comprising a transglycosidase activity to the mixture obtained according to step c) or step d)-I.), wherein the transglycosidase activity is selected from the group consisting of trans-fucosidase, trans-sialidase, trans-lacto-N-biosidase and trans-N-acetyllactosaminidase activity, and incubating the mixture obtained; or III. adding at least one compound or mixture of compounds selected from the group defined in step I and at least one enzyme comprising a transglycosidase activity as defined in step II to the mixture obtained according to step c), and incubating the mixture obtained, wherein

the incubation according to step (c) or step (d), if present, leads to a mixture of human milk oligosaccharides (HMOs) or derivatives 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 or one or more lacto-N-biosyl groups; any N-acetyl-lactosaminyl or lacto-N-biosyl group can be substituted with one or more sialyl 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 or one or more lacto-N-biosyl groups; any N-acetyl-lactosaminyl or lacto-N-biosyl group can be substituted with one or more sialyl 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 or one or more lacto-N-biosyl groups; any N-acetyl-lactosaminyl or lacto-N-biosyl group can be substituted with one or more sialyl 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 or one or more lacto-N-biosyl groups; any N-acetyl-lactosaminyl or lacto-N-biosyl group can be substituted with one or more sialyl or fucosyl residue;
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; or
a mixture of compounds selected from the group consisting of general formulas 1, 2, 3, and 4,

wherein the mixture of human milk oligosaccharides or derivatives or precursors thereof comprises at least one compound selected from the group consisting of compounds of general formula 1, general formula 2, general formula 3, and general formula 4 that bears at least one fucosyl group; and
e) optionally subjecting the mixture obtained after step c) or step d) to a hydrogenolysis reaction.

2. The method according to claim 1 , wherein step a) comprises providing 2′-fucosyllactose or 3-fucosyllactose, and one further compound selected from the group consisting of 2′-fucosyllactose, 3-fucosyllactose, 3′-sialyllactose, 6′-sialyllactose, LNT and LNnT.

Assignees

• Glycom As

Inventors

• Dekany Gyula
• Champion Elise
• Hederos Markus Jondelius

Classifications

• A61P37/02

  Immunomodulators · 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

• C12P19/12

  Disaccharides · CPC title