Production of fucosylated carbohydrates by enzymatic fucosylation synthesis of sugar nucleotides; and in situ regeneration of GDP-fucose
AssigneeThe Scripps Research Insitute
InventorsChi-Huey Wong, Yoshitaka Ichikawa, Gwo-Jenn Shen, Kun-Chin Liu
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Abstract
This invention contemplates improved methods of enzymatic production of carbohydrates especially fucosylated carbohydrates. Improved syntheses of glycosyl 1- or 2-phosphates using both chemical and enzymatic means are also contemplated. The phosphorylated glycosides are then used to produce sugar nucleotides that are in turn used as donor sugars for glycosylation of acceptor carbohydrates. Especially preferred herein is the use of a disclosed method for fucosylation.
Claims20
A method of producing a fucosylated carbohydrate in a single reaction mixture comprising the steps of:
- (a) using a fucosyltransferase to form an O-glycosidic bond between a nucleoside 5′-diphospho-fucose and an available hydroxyl group of a carbohydrate acceptor molecule to yield a fucosylated carbohydrate and a nucleoside 5′-diphosphate, and;
- (b) recycling in situ the nucleoside 5′-diphosphate to form the corresponding nucleoside 5′-diphospho-fucose.
A method for producing a fucosylated sialylated carbohydrate molecule through enzymatic formation of glycosidic linkages in a single reaction mixture comprising:
- (a) forming a first glycosidic linkage between an diphosphonucleoside-activated glycosyl donor and an available hydroxyl group of a carbohydrate acceptor molecule using a first glycosyltransferase;
- (b) forming a second glycosidic linkage between a monophosphonucleoside-activated sialyl donor and an available hydroxyl group of the carbohydrate acceptor molecule using a sialyltransferase;
- and
- (c) forming a third glycosidic linkage between a diphosphonucleoside-activated fucosyl donor and an available hydroxyl group of the carbohydrate acceptor molecule using a fucosyltransferase to yield a fucosylated carbohydrate acceptor and a nucleoside 5′-diphosphate, with in situ recycling of said nucleoside 5′-diphosphate to form more of said diphosphonucleoside-activated fucosyl donor, said glycosidic linkages of steps (a), (b) and (c) being formed in a single reaction mixture.
Dependencies are inferred from the claim text.