A bi-fluorescence-labeled substrate for ceramide glycanase based on fluorescence energy transfer

An alkyl lactoside containing two different fluorescence probes as an energy donor and an energy acceptor was synthesized as a substrate for ceramide glycanase. n-Pentenyl β-lactoside was converted into its 4′,6′-O-(2-naphthylmethylidene) derivative with subsequent benzoylation of all remaining OH groups. The fully protected lactoside was treated with borane-trimethylamine complex and aluminum chloride in tetrahydrofuran [P.J. Garegg, Pure Appl. Chem., 56 (1984) 845–858] for selective opening of the 4′,6′-acetal group to give the 6′-O-(2-naphthylmethyl) derivative in high yield. After O-debenzoylation, the ω-alkenyl group at the reducing end was extended by Michael addition with HS(CH2)2NH2 · HCl to provide an amino group at the terminal position. The amino group was then dansylated to give the target lactoside, which has two different fluorescent probes at each end. Excitation at 290 nm (of the 2-naphthyl group) of the bi-fluorescence-labeled lactoside showed emissions at 335 nm (2-naphthyl) and at 540 nm (dansyl). The distance between the naphthyl group and the dansyl group was estimated to be 12 Å by the Förster relationship. Digestion of this lactoside with American leech (Macrobdella decora) ceramide glycanase [B. Zhou et al., J. Biol. Chem., 264 (1989) 12,272–12,277] resulted in an increase in the naphthyl emission with a concomitant decrease in the dansyl emission. These changes can be used for continuous monitoring of the ceramide glycanase activity.