2-alpha-(N-dansyl-4-aminophenylthio)-N-acetyl-9-O-acetylneuraminic acid. A new specific and highly sensitive substrate in sialate-O-acetylesterase assay.

2-alpha-(N-Dansyl-4-aminophenylthio)-N-acetyl-9-O-acetylneuraminic acid (10) was prepared as a new specific and highly sensitively detectable sialate-9-O-acetyl-esterase substrate. It is built up from a sialidase-stable aminophenyl-alpha-thioketoside of N-acetylneuraminic acid. By labeling this thioketoside with dansyl chloride a fluorescent neuraminic acid derivative was prepared which allows determinations down to the picomol range. Regioselective acetylation with trimethylorthoacetate results in the corresponding 9-O-acetyl derivative. After incubation with esterase from bovine brain the hydrolysis products were separated on a HPLC column and fluorimetrically detected at 334 nm excitation and 564 nm emission. The Km value of 2.5 mM was in the range between the values of the completely unspecific methylumbelliferyl acetate and the less sensitively detectable N-acetyl-9-O-acetylneuraminic acid which have been used up to now as standard substrates.