Modified assay-procedure for guanosine diphosphate-l-fucose:2-acetamido-2-deoxy-β-d-glucopyranoside-(1→4)-α-l-fucosyltransferase with the aid of synthetic phenyl 2-acetamido-2-deoxy-4-O-α-l-fucopyranosyl-3-O-β-d-galactopyranosyl-β-d-glucopyranoside as a reference compound

Starting from phenyl 2-acetamido-2-deoxy-4,6-O-(p-methoxybenzylidene)-β-d-glucopyranoside (1), chemical syntheses were developed for phenyl 2-acetamido-2-deoxy-3-O-β-d-galactopyranosyl-β-d-glucopyranoside (4) and phenyl 2-acetamido-2-deoxy-4-O-α-l-fucopyranosyl-3-O-β-d-galactopyranosyl-β-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 (R_F 0.69) from synthetic disaccharide 4 (R_F 0.78), fucose (R_F 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→4)-α-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 α-(1→4)-l-fucosyltransferase activity; these include phosphorylase and α-l-fucosidase activities, and incorporation of α-l-[¹⁴C]-fucose into endogenous acceptors of enzyme preparations. Thus, the modified assay-procedure should facilitate determination of α-(1→4)-l-fucosyltransferase.