Enzymatic Synthesis of a Fluorogenic Reporter Substrate and the Development of a High-Throughput Assay for Fucosyltransferase VIII Provide a Toolkit to Probe and Inhibit Core Fucosylation

Abstract

We report a straightforward enzymatic synthesis of the 4-methylumbelliferyl glycoside of a complex-type oligosaccharide substrate for core fucosylation. We demonstrate the use of this synthetic glycoconjugate in a newly developed enzyme assay to probe the activity and inhibition of fucosyltransferase VIII, which catalyzes the core fucosylation of N-glycans on eukaryotic glycoproteins. In this fucosyltransferase assay, we use the fluorogenic probe and a specific glycosidase in a sequentially coupled enzyme reaction to distinguish an unmodified 4-methylumbelliferyl oligosaccharide probe from a fucosylated probe. Our findings show that this strategy is very sensitive and specific in its detection of enzyme activity and can even be used for analyzing impure tissue lysate samples.

An Economical High‐Throughput “FP‐Tag” Assay for Screening Glycosyltransferase Inhibitors**

“…Our "FP-Tag" assay format could be easily transferred to other glycosyltransferases (GTs) of interest, such as galactosyl, [16] fucosyl, [17] and sialy [18] transferases, as only minimally modified sugar nucleotide analogs are needed. It is well-known that GT inhibitors are difficult to find, thus sometimes screening a large compound set is needed to get suitable hits.…”