Synthesis of aryl 3′-sulfo-β-lactosides as fluorogenic and chromogenic substrates for ceramide glycanases

We have previously reported that 4-methylumbelliferyl 6′-O-benzyl-β-lactoside (2) is a useful substrate for a fluorometric assay of ceramide glycanase (CGase) (L.-X. Wang, N.V. Pavlova, S.-C. Li, Y.-T. Li and Y.C. Lee, Glycoconjugate J., 13 (1996) 359–365). The introduction of a 6-O-benzyl group at the terminal Gal efficiently protected the substrate from its hydrolysis by exo-galactosidase, permitting the assay of CGase in crude biological materials. However, a drawback of this substrate is its low water-solubility and relatively high Km (at a mM level). Introduction of a sulfate group into 4-methylumbelliferyl β-lactoside (1) led to the formation of 4-methylumbelliferyl 3′-O-sulfo-β-lactoside (3), which was found to be a more effective substrate than 2. Moreover, the presence of a 3′-O-sulfate group not only increases the water solubility tremendously, but also protects the substrate from cleavage by exo-β-galactosidase as the 6′-O-benzyl group in 2 does. In addition to the fluorogenic substrate (3), two sulfated chromogenic substrates, N-tetradecanoyl-4-O-(3′-sulfo-β-lactosyl)-3-nitro-l-tyrosine methyl ester (9) and 2-N-(tetradecanoylamino)-4-nitrophenyl 3′-sulfo-β-lactoside (12), were synthesized and their suitability for a photometric assay of CGase was evaluated. Substrates 9 and 12, with a long fatty acid chain attached to the aglycon part, have a Km value close to that of the natural substrate GM1 (at a μM level).