Fluorescent Detection of Biothiols with Maleimide-based Probes: Effect of the Spacer on the Selectivity

•Three naphthalimide-based fluorescent probes for thiols with maleimide attached at different sites were synthesized.•The probe NHA with the shortest spacer exhibited high selectivity toward Cys over other thiols.•The photophysical responses of NHA toward Cys could be ascribed to both PET and ICT.•A two-step process of mercapto Michael addition followed by intramolecular transfer cyclization amidation was proposed.