Fluorescent detection of biothiols with maleimide-based probes: Effect of the spacer on the selectivity

Three naphthalimide-based fluorescent probes (NHA, NHB and NHC) with maleimide attached at different sites were designed and synthesized for the detection of thiols. The inherently weak fluorescence of NHA is caused by both photoinduced electron transfer (PET) and intramolecular charge transfer (ICT), while those of NHB and NHC could be ascribed to PET mechanism. The spacer between maleimide and the fluorophore plays an important role in the fluorescent response toward thiols: the probe with a spacer of a single bond (NHA) exhibited significant fluorescence response toward cysteine (Cys) rather than glutathione (GSH) or homocysteine (Hcy); whereas that with a connecting arm of four single bonds (NHC) displayed evidently fluorescent enhancement to the above three thiols. NMR-titration, HPLC and DFT calculation were employed to investigate the sensing mechanism: the spectral responses of the probes toward Cys underwent a two-step process of mercapto Michael addition followed by intramolecular transfer cyclization amidation, and the latter affected the electron effect of the receptor significantly; however, only thiol-addition occurred between the probes and GSH to produce thioethers. The electron push-pull effect of Cys addition product was influenced by the spacer, which led to different selectivity of the probes.