Modified assay-procedure for guanosine diphosphate-L-fucose: 2-acetamido-2-deoxy-beta-D-glucopyranoside-(1 leads to 4)-alpha-L-fucosyltransferase with the aid of synthetic phenyl 2-acetamido-2-deoxy-4-O-alpha-L-fucopyranosyl-3-O-beta-D-galactopy-ranosyl -beta-D-glucopyranoside as a reference compound

Starting from phenyl 2-acetamido-2-deoxy-4,6-O-(p-methoxybenzylidene)-beta-D-glucopyranoside (1), chemical syntheses were developed for phenyl 2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-beta-D-glucopyranoside (4) and phenyl 2-acetamido-2-deoxy-4-O-alpha-L-fucopyranosyl-3-O-beta-D-galactopyranosyl -beta-D-glucopyranoside (8). Thin-layer chromatography in the solvent system 6:4:1:5 (v/v) 2-propanol-ethyl acetate-ammonium hydroxide-water clearly separated the synthetic trisaccharide 8 (RF 0.69) from synthetic disaccharide 4 (RF 0.78), fucose (RF 0.56), and GDP-fucose (which remained at the origin). Based upon this observation, a modified method for the determination of GDP-L-fucose: N-acetylglucosaminide-(1 leads to 4)-alpha-L-fucosyltransferase was developed that employed the synthetic disaccharide 4 as an acceptor, and compound 8 as an authentic reference-compound. This modified assay-procedure can simultaneously monitor possible competing reactions which may interfere with determination of alpha-(1 leads to 4)-L-fucosyltransferase activity; these include phosphorylase and alpha-L-fucosidase activities, and incorporation of alpha-L-14C]-fucose into endogenous acceptors of enzyme preparations. Thus, the modified assay-procedure should facilitate determination of alpha-(1 leads to 4)-L-fucosyltransferase.