SYNTHESIS OF ARYL 3'-SULFO-BETA-LACTOSIDES AS FLUOROGENIC AND CHROMOGENIC SUBSTRATES FOR CERAMIDE GLYCANASES

We have previously reported that 4-methylumbelliferyl 6'-O-benzyl-beta -lactoside (2) is a useful substrate for a fluorometric assay of ceram ide glycanase (CGase) (L.-X. Wang, N.V. Pavlova, S.-C. Li, Y.-T. Li an d Y.C. Lee, Glycoconjugate J., 13 (1996) 359-365). The introduction of a 6-O-benzyl group at the terminal Gal efficiently protected the subs trate from its hydrolysis by exo-galactosidase, permitting the assay o f CGase in crude biological materials. However, a drawback of this sub strate is its low water-solubility and relatively high Km (at a mM lev el). Introduction of a sulfate group into 4-methylumbelliferyl beta-la ctoside (1) led to the formation of 4-methylumbelliferyl 3'-O-sulfo-be ta-lactoside (3), which was found to be a more effective substrate tha n 2. Moreover, the presence of a 3'-O-sulfate group not only increases the water solubility tremendously, but also protects the substrate fr om cleavage by exo-beta-galactosidase as the 6'-O-benzyl group in 2 do es. In addition to the fluorogenic substrate (3), two sulfated chromog enic substrates, yl-4-O-(3'-sulfo-beta-lactosyl)-3-nitro-L-tyrosine me thyl ester (9) and 2-N-(tetradecanoylamino)-4-nitrophenyl 3'-sulfo-bet a-lactoside (12), were synthesized and their suitability for a photome tric assay of CGase was evaluated. Substrates 9 and 12, with a long fa tty acid chain attached to the aglycon part, have a Km value close to that of the natural substrate GM1 (at a mu M level). (C) 1998 Elsevier Science Ltd. All rights reserved.