RESUMO
A synthetic three-fluorophore system with two enzymatically cleavable linkers has been developed for the simultaneous detection of two proteases in a mixture. The probe was designed to afford single excitation/triple emission ratiometric detection through a fluorescence change during the cleavage of a peptide linker. The developed assays were verified for trypsin and chymotrypsin as the model enzymes.
Assuntos
Aminocumarinas/química , Quimotripsina/análise , Fluoresceínas/química , Corantes Fluorescentes/química , Rodaminas/química , Tripsina/análise , Aminocumarinas/síntese química , Aminocumarinas/efeitos da radiação , Estabilidade de Medicamentos , Ensaios Enzimáticos , Fluoresceínas/síntese química , Fluoresceínas/efeitos da radiação , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/efeitos da radiação , Hidrólise , Lisina/análogos & derivados , Lisina/química , Fenilalanina/análogos & derivados , Fenilalanina/química , Rodaminas/síntese química , Rodaminas/efeitos da radiaçãoRESUMO
Vicinal dithiol-containing proteins (VDPs) play a significant role in maintaining the cellular redox homeostasis and are implicated in many diseases. To provide new chemical tools for VDPs imaging, we report here a ratiometric fluorescent probe CAsH2 for VDPs using 7-diethylaminiocoumarin as the fluorescent reporter and cyclic 1,3,2-dithiarsenolane as the specific ligand. CAsH2 shows peculiar dual fluorescence emission from the excited intramolecular charge transfer (ICT) and twisted intramolecular charge transfer (TICT) states in aqueous media. However, upon selective binding of protein vicinal dithiols to the trivalent arsenical of CAsH2, the probe was brought from the polar water media into the hydrophobic protein domain, causing the excited state ICT to TICT conversion to be restricted; as a result, an increase from the ICT emission band and a decrease from the TICT emission band were observed simultaneously. The designed probe shows high selectivity toward VDPs over other proteins and biological thiols. Preliminary experiments show that CAsH2 can be used for the ratiometric imaging of endogenous VDPs in living cells. So far as we know, this is a rare example of the ratiometric fluorescent probe designed via modulating the ICT-TICT conversion process, which provides a new way to construct various protein-specific ratiometric fluorescent probes.
