X-Neu5Ac: A novel substrate for chromogenic assay of neuraminidase activity in bacterial expression systems

A chromogenic substrate 1, 5-bromo-4-chloroindol-3-yl 5-acetamido-3,5-dideoxy-α-d-glycero-d-galacto-2-nonulopyranosidoni c acid (X-Neu5Ac), has been synthesized to facilitate the screening of bacterial colonies or plaques for the detection of either natural or mutant neuraminidase activity. Substrate 1 was hydrolyzed by neuraminidase isolated from Clostridium perfringens to release a halogenated indol-3-ol 2 that undergoes rapid aerobic oxidation to form the dark blue pigment, 5,5′-dibromo-4,-4′-dichloroindigo 3. Preliminary kinetic studies indicate that this compound is a good substrate (K_m 0.89 × 10^-3 M) for neuraminidase and is quite stable under identical conditions in the absence of enzyme. These results suggest that X-Neu5Ac 1 can be useful to screen for bacterially-encoded enzyme production directly on agar plates.