First and facile enzymatic synthesis of β-fucosyl-containing disaccharide nucleosides through β-galactosidase-catalyzed regioselective glycosylation

β-Galactosidase from bovine liver was purified to homogeneity. Its molecular mass was estimated to be 54 kDa by SDS-PAGE, 60 kDa by gel permeation chromatography, and 57 kDa by matrix-assisted laser desorption ionization – time of flight tandem mass spectrum. This enzyme displayed the highest catalytic efficiency with p-nitrophenyl β-d-galactopyranoside (Vmax/Km value, 0.0173 min−1) as the substrate, lower with p-nitrophenyl β-d-fucopyranoside (0.0156 min−1) and the lowest with p-nitrophenyl β-d-glucopyranoside (0.0126 min−1). With the enzymatic fucosylation of floxuridine as a model reaction, four key reaction conditions were optimized. Under the optimum conditions, the enzymatic synthesis of a group of β-fucosyl-containing disaccharide nucleosides using the purified β-galactosidase was conducted. The desirable 5′-O-β-d-fucosyl derivatives of pyrimidine nucleosides were obtained with 44–60% yields. Besides, the 5′-regioselectivities decreased markedly with increasing bulk of 5-substituents present in the base moiety of nucleosides. In addition, the enzyme could accept acyclic nucleoside analogs as the substrates and catalyze the enzymatic fucosylation of these nucleosides, furnishing the glycosylated products with the yields of 32–36%.

► First enzymatic synthesis of β-fucosyl-containing disaccharide nucleosides.
► A broad substrate spectrum of bovine liver β-galactosidase.
► Decrease of 5′-regioselectivity with increasing bulk of 5-group of nucleoside.
► Satisfactory yields of the desired 5′-O-β-fucosyl nucleosides.