alpha-Thioglycoligase-based synthesis of O-aryl alpha-glycosides as chromogenic substrates for alpha-glycosidases

Abstract

alpha-Thioglycoligases are retaining alpha-glycosidase mutants, with modification of their general acid/base catalytic residue to an inactive amino acid residue, catalyzing the formation of S-glycosidic linkages using a sugar donor with an excellent leaving group and suitable sugar acceptors with a thiol group as the substrate. In this study, we describe the enzymatic synthesis of O-aryl alpha-glycosides catalyzed by alpha-thioglycoligases. An alpha-xylosidase mutant (Yicl-D482A) efficiently catalyzed the synthesis of O-aryl alpha-xylosides in near-quantitative yields (up to 99%) using 4-methylumbelliferone and nitrophenols. Synthesis did not occur with those acceptors having a nitro group at the ortho-position. The conversion yields of 3-nitrophenol markedly increased at pH 8.0, whereas those of other aryl compounds were nearly independent of pH, ranging from pH 6.0 to 8.0. The O-aryl alpha-xylosides were prepared on a preparative scale with yields of up to 96%. Upon employing the O-aryl alpha-xylosides as the substrate for the wild-type Yicl, Bronsted relationships of log k(cat) versus pK(a) and log (k(cat)/K-M) versus pK(a) both showed a linear monotonic dependence on the leaving group pK(a) with low beta(lg) values of 0.39 and 0.38, respectively. In addition, synthesis of O-aryl alpha-glucosides was successfully conducted by an alpha-glucosidase mutant (MalA-D416A) in the same fashion with high yields. Therefore, this strategy can be used for the synthesis of O-aryl alpha-glycosides using an acid/base mutant of retaining alpha-glycosidases that hydrolyze the glycosides. (c) 2012 Elsevier B.V. All rights reserved.