Method for generating human milk oligosaccharides (HMOs) or precursors thereof

A method for making a mixture of human milk oligosaccharides (HMOs) or derivatives or precursors thereof, the method comprising the steps of

• a) providing at least one donor selected from the group consisting of compounds of any of formulae 5 to 10
• and salts thereof,
• wherein X is selected from the group consisting of azide, fluoro, optionally substituted phenoxy, optionally substituted pyridinyloxy, group A, group B, group C and group D
• wherein Ra is independently H or alkyl, or two vicinal Ra groups represent a=C(Rb)2 group, wherein Rb is independently H or alkyl, Rc is independently selected from the group consisting of alkoxy, amino, alkylamino and dialkylamino, Rd is selected from the group consisting of H, alkyl and —C(═O)Re, wherein Re is OH, alkoxy, amino, alkylamino, dialkylamino, hydrazino, alkylhydrazino, dialkylhydrazino or trialkylhydrazino;
• b) providing at least one acceptor selected from the group of compounds consisting of:
• lactose
• lactose derivatives of the following formula:
• wherein R is a group removable by hydrogenolysis,
• LNT:
• LNT derivatives of the formula:
• wherein R is a group removable by hydrogenolysis, or salts thereof
• LNnT:
• LNnT derivatives of the formula:
• wherein R is a group removable by hydrogenolysis, or salts thereof
• c) providing at least one enzyme comprising a transglycosidase activity and/or a glycosynthase activity;
• d) preparing a mixture of the at least one donor, at least one acceptor and at least one enzyme provided in steps a), b) and c);
• e) incubating the mixture prepared according to step d);
• in the case where a single HMO or derivative or precursor thereof is made as a result of step e), repeating at least one of the steps a) to c), then repeating step d) and step e);
• g) optionally:
• repeating at least one of the steps a) to c), then repeating step d) and step e) with the mixture obtained according to step e) or f);
• h) optionally subjecting the mixture obtained as a result of step e), f) or g) to a hydrogenolysis reaction.