Thiogalactopyranosides are Resistant to Hydrolysis by α‐Galactosidases

Abstract

Fluorescently tagged glycosides containing terminal α(1→3) and α(1→4)‐linked thiogalactopyranosides have been prepared and tested for resistance to hydrolysis by α‐galactosidases. Eight fluorescent glycosides containing either galactose or 5‐thiogalactose as the terminal sugar were enzymatically synthesized using galactosyltransferases, with lactosyl glycosides as acceptors and UDP‐galactose or UDP‐5′‐thiogalactose, respectively, as donors. The glycosides were incubated with human α‐galactosidase A (CAZy family GH27, a retaining glycosidase), Bacteroides fragilis α‐1,3‐galactosidase (GH110, an inverting glycosidase), or homogenates of MCF‐7 human breast cancer cells or NG108‐15 rat glioma cells. Substrate hydrolysis was monitored by capillary electrophoresis with fluorescence detection. All compounds containing terminal O‐galactose were readily degraded. Their 5‐thiogalactose counterparts were resistant to hydrolysis by human α‐galactosidase A and the enzymes present in the cell extracts. B. fragilis α‐1,3‐galactosidase hydrolyzed both thio‐ and O‐galactoside substrates; however, the thiogalactosides were hydrolyzed at only 1–3 % of the rate of O‐galactosides. The hydrolytic resistance of 5‐thiogalactose was also confirmed by an in vivo study using cells in culture. The results suggest that 5‐thiogalactosides may be useful tools for the study of anabolic pathways in cell extracts or in single cells.