Highly sensitive detection of cysteine over glutathione and homo-cysteine: New insight into the Michael addition of mercapto group to maleimide

A fluorescence "off on" probe CMP for thiols was designed with coumarin as the fluorophore and maleimide as the receptor. The fluorescence of the coumarin was quenched through photoinduced electron transfer (PET) from the fluorophore to maleimide group. The Michael addition of the mercapto group toward maleimide formed a thioether with relatively weak fluorescence. The intramolecular nucleophilic substitution of amino group in cysteine (Cys) to alkylthio produced a much stronger fluorescent amino adduct, which was supported by UPLC-MS and NMR titration. The above sensing mechanism ensured CMP a highly sensitive probe toward Cys over GSH and Hcy. The fluorescence intensity at 495 nm was linear with Cys concentration over the range of 0-10 mu M with a detection limit of 14 nM and a rapid response time of 20 min. High selectivity and good competition of the probe toward thiols over other biologically relevant species enabled us to monitor mercaptocontaining proteins as well as fluorescence imaging Cys in living cells.