Acceptor-induced modification of regioselectivity in CGTase-catalyzed glycosylations of p-nitrophenyl-glucopyranosides

Abstract

Cyclodextrin glycosyltransferases (CGTase) are reported to selectively catalyze α(1→4)-glycosyl transfer reactions besides showing low hydrolytic activity. Here, the effect of the anomeric configuration of the glycosyl acceptor on the regioselectivity of CGTase catalyzed glycosylations was investigated. For this purpose, the α and β anomers of p-nitrophenyl-d-glucopyranoside were used as glycosyl acceptors, Bacillus macerans and Thermoanaerobacter sp. CGTases were used as biocatalysts and β-cyclodextrin as the glycosyl donor. As expected, p-nitrophenyl-α-d-glucopyranosyl-(1→4)-O-α-d-glucopyranoside was produced when p-nitrophenyl-α-d-glucopyranoside was used as acceptor with B. macerans CGTase. Surprisingly, when p-nitrophenyl-β-d-glucopyranoside was used as glycosyl acceptor, besides the expected α(1→4)-glycosylation products both α(1→3)- and α(1→6)-transfer products were also obtained. This unexpected change in B. macerans CGTase regioselectivity leading to α(1→4)-, α(1→3)- and α(1→6)-glycosylation products was also observed for Thermoanaerobacter sp. CGTase with the β anomer. It is shown, applying time course analyses, that all isomers can be synthesized efficiently by adequate selection of enzyme and reaction conditions. In fact, when using Thermoanaerobacter sp. CGTase the yield of p-nitrophenyl-β-d-isomaltoside (the α(1→6)-transfer product) was the highest at long reaction time (19% yield). The previously unknown capacity of α(1→6)-glycosidic linkages formation by CGTases demonstrates an unexpected broader regioselectivity of CGTases in glycosyl-transfer reactions as well as an acceptor dependent transfer selectivity.