Synthesis of nucleotide-activated disaccharides with β-galactosidase from Bacillus circulans and α-galactosidase from Bifidobacterium adolescentis

Abstract

The enzymatic synthesis of nucleotide-activated disaccharides by glycosidase-catalyzed transglycosylation reactions was investigated. High product yields were obtained when the kinetically driven syntheses were performed in frozen aqueous solutions at −5 °C. β-Galactosidase from Bacillus circulans catalyzed the transfer of d-galactose from lactose onto the nucleotide sugar UDP-Gal stereo- and regioselectively, forming the nucleotide disaccharide Gal(β1–4)Gal(α1-UDP. α-Galactosidase from Bifidobacterium adolescentis accepted UDP-Gal and UDP-Glc only when the reaction was performed at −5 °C with melibiose as glycoside donor. Enzyme and donor concentrations were optimized for the preparative synthesis yielding Gal(α1–3)Glc(α1-UDP and Gal(α1–3)Gal(α1-UDP as analyzed by MALDI-TOF mass spectrometry and 1D and 2D 1H and 13C NMR spectroscopy. The reported products are invaluable tools for studies on their possible biochemical function as donor substrates or inhibitors of Leloir glycosyltransferases.