Fluorescent Probes For H₂S And GSH Based On Benzoxadiazole（BD）derivatives:Design,Synthesis,and Applications

Glutathione（GSH）is the most abundant biothiol in cells（1-10 m M）,contributing to the maintenance of cellular redox homeostasis and defense against toxins;hydrogen sulfide（H₂S）,as an improtant gasotransmitter,is related to the regulation of angiogenesis,vasodilation,bioenergetic metabolism.Moreover,abnormal levels of GSH and H₂S are associated with tumors,which are considered as important biomarkers.Therefore,the development of chemical tools for specific detection to GSH and H₂S plays an important role in the early diagnosis and treatment of related cancers.Fluorescent probes have been widely used for the detection of biothiols due to their excellent properties such as high selectivity,sensitivity,rapid response,real-time visualization and tunable structure.But the current probes are mostly reacted with all biothiols and then distinguished each other by additional intermolecular reaction.Currently,few probes could detect GSH directly and the development of a new GSH universal receptor is in its infancy.Benzoxadiazole（BD）compounds have some excellent properties such as modifiability,small size,good aqueous solubility,so we chose BD compounds as the research modle to develop novel GSH-selective probes.Comparing the properties of BD derivatives modified with different substituents,we found that changing the nitro group of NBD,which has the strong ability of electron-withdrawing,to cyano and sulfonyl groups will lead to decreased reactivity but increased selectivity and stability.Therefore,the probes based on CBD arylether were designed and synthesized to selectively detect GSH for the first time.Probes exhibited excellent selectivity and appropriate dissociation constants to GSH and has been used to image endogenous GSH in He La cell.Besides,the comparison of reaction kinetics between probes based on CBD shows the tunable reactivity of CBD arylether.Based on NBD amine and CBD arylethers,fluorescent probe for simultaneous detection of H₂S and GSH was developed.Owning to the ICT-FRET double-quenching effect,the background fluorescence of probe was quite weak,and exhibit significant fluorescence enhancement（up to 500 fold）after it was dual-activated by H₂S and GSH.The probe exhibited excellent selectivity and biocompatibility,which was successful used for simultaneous detection of both H₂S and GSH in live cells.Importantly,the dual-reactive probe was successfully employed to reveal the biogenesis of both H₂S and GSH from L-Cys rather than from D-Cys,which may help understand the more H₂S generation from D-Cys than from L-Cys in live cells.