Action pattern of Sulfolobus O-α-glycoligase for synthesis of highly water soluble resveratrol 3,4′-α-diglucoside

This study presents the enzymatic synthesis of resveratrol-3,4′-O-α-diglucoside (RDG) using a hyperactive O-α-glycoligase (MalA-D416R/Q450S) and α-glucopyranosyl fluoride as the donor substrate. The transglycosylation rate for resveratrol by MalA-D416R/Q450S was maximized in 100 mM Tris-HCl (pH 9.5) containing 20 % DMSO at 45°C. Because the pK_a of the 4′-OH group of resveratrol is lower than that of the 3-OH group, the 4′-OH group is more nucleophilic at the alkaline pH, leading to a preference for glycosylation at the 4′-OH site rather than the 3-OH site. This preference makes resveratrol 3-O-α-glucoside (R3G) as the more efficient acceptor than resveratrol 4′-O-α-glucoside (R4′G), resulting in negligible production of resveratrol 3-O-α-glucoside (R3G) due to its complete consumption in the second transglycosylation reaction when using a 2:1 ratio of donor to acceptor substrates. From a preparative scale reaction, R4′G and RDG were isolated with yields of 41.2 % and 43.3 %, respectively. The water solubility of RDG exceeded 1.67 M, which represents more than a 9,800-fold improvement compared to resveratrol. In a hydrolysis experiment using intestinal α-glycosidase from rat, the α-glucosides of resveratrol (R4′G and RDG) were completely deglycosylated to the aglycone.