X-NeuNAc (5-Bromo-4-chloro-3-indolyl-alpha-D-N-acetylneuraminic acid sodium salt) is a new chromogenic assay substrate of neuraminidase activity in bacterial expression systems.

X-Neu5Ac was hydrolyzed by neuraminidase to release a halogenated product undergoing rapid aerobic oxidation to form the dark blue pigment. Preliminary kinetic studies indicated that X-Neu5Ac was a good and stable substrate for neuraminidase. In addition, X-Neu5Ac would also be hydrolyzed mutant enzymes

To visualize extracellular sialidase activity on the membrane surface in the rat brain, acute brain slices were incubated with X-NeuNAc at pH 7.3. After 1h, myelin-abundant regions showed intense fluorescence in the rat brain. Although the hippocampus showed weak fluorescence in the brain, mossy fiber terminals in the hippocampus showed relatively intense fluorescence. In addition, the fluorescence intensities caused by X-NeuNAc was correlated with the sialidase activity. Therefore, staining with X-NeuNAc was specific for sialidase and useful for quantitative analysis of sialidase activities [1].