SYNTHESIS OF BI-FLUORESCENCE-LABELED LACTOSIDE - A SUBSTRATE FOR CONTINUAL ASSAY OF CERAMIDE GLYCANASE

A bi-fluorescence labeled derivative suitable for analysis of ceramide glycanase activity was constructed from 4-pentenyl lactoside. Selecti ve modification of the galactosyl residue was attained by formation of 4',6'-naphthylmethylidene derivative, which was followed by regiosele ctive reductive ring opening. The aglycon was extended by Michael addi tion of 2-aminoethanetiol, and dansylated at the terminal amino group. Excitation of the naphthyl group results in emission from the dansyl group, while the emission from the naphthyl group is quenched by the d ansyl group. Upon digestion with ceramide glycanase, the energy transf er is severed and a decrease in the dansyl emission concommitant with an increase in the naphthyl emission was observed. This substrate was successfully used to analyze ceramide glycanase activity.