One-pot multienzyme synthesis of Lewis x and sialyl Lewis x antigens.

L-Fucose has been found abundantly in human milk oligosaccharides, bacterial lipopolysaccharides, glycolipids, and many N- and O-linked glycans produced by mammalian cells. Fucose-containing carbohydrates have important biological functions. Alterations in the expression of fucosylated oligosaccharides have been observed in several pathological processes such as cancer and atherosclerosis. Chemical formation of fucosidic bonds is challenging due to its acid lability. Enzymatic construction of fucosidic bonds by fucosyltransferases is highly efficient and selective but requires the expensive sugar nucleotide donor guanosine 5'- diphosphate-L-fucose (GDP-Fuc). Here, we describe a protocol for applying a one-pot three-enzyme system in synthesizing structurally defined fucose-containing oligosaccharides from free L-fucose. In this system, GDP-Fuc is generated from L-fucose, adenosine 5'-triphosphate (ATP), and guanosine 5'-triphosphate (GTP) by a bifunctional L-fucokinase/GDP-fucose pyrophosphorylase (FKP). An inorganic pyrophosphatase (PpA) is used to degrade the by-product pyrophosphate (PPi) to drive the reaction towards the formation of GDP-Fuc. In situ generated GDP-Fuc is then used by a suitable fucosyltransferase for the formation of fucosides. The three-enzyme reactions are carried out in one pot without the need for high cost sugar nucleotide or isolation of intermediates. The time for the synthesis is 4-24 hours. Purification and characterization of products can be completed in 2-3 days.