Fluorescent Probes Based On Dual-reactive And Dual-quenching Strategy:Design,Synthesis And Applications In H₂S Chemical Biology

Hydrogen sulfide(H2S)is an important intracellular gasotransmitter in a variety of tissues and organs,playing vital roles in numerous physiological and pathological progresses.Human NAD(P)H:quinone acceptor oxidoreductase ?(hNQO1,EC 1.6.99.2),a protein with multiple protective roles,promotes the 2-electron reductions of quinones.H2S as well as hNQO1 are both important reductive cancerous biomarkers.While hypochlorous acid(HClO)is a vital cellular reactive oxygen species(ROS),which protects cells against microbial invasion.All these endogenous reactive molecules are participants in cellular redox regulation,so monitoring of levels of these molecules,investigations on the crosstalk of H2S and hNQO1 under oxidative stress as well as studies on the metabolism of endogenous HCIO under reductive stress induced by H2S will benefit the improvement of H2S chemical biology and the cellular redox regulation.Fluorescence-based methods are widely used in the detection of biological reactive molecules due to the facile manipulation,good biocompatibility,in-situ and real-time visualization of analytes.However,the probes based on single-reactive and single-quenching effect do not always display ideal properties.To polish up the probe,in this thesis,we propose a dual-reactive and dual-quenching strategy.We aim to improve the selectivity and stability of probes through different chemical properties or reactions of the analytes,that is daul-reactive effect.While the dual-quenching effect is using the suppression of fluorescence from the two acceptors to decrease the background fluorescence,so as to enhance the emission response,sensitivity and the signal to noise in bioimaging.Based on the nucleophilicity of the sulfhydryl group,in this thesis,we first developed a dual-reactive and dual-quenching thiol probe 1.The selectivity and competitive selectivity of 1 was was remarkably improved by the dual-reactive strategy;and the background fluorescence of 1 was heavily quenched through dual-quenching effect,as a result,the fluorescence response was significantly increased(>400 fold).These results indicated the feasibility of this strategy.We popularized this strategy in the construction of H2S probes.Based on the high nucleophilicity of H2S,we developed the first FRET-ICT dual-quenching H2S probe 4.Owning to the dual-quenching effect,4 showed extremely weak background fluorescence.After reaction with H2S,a significant fluorescent off-on response up to 2000 fold was observed for 4,which was the largest fluorescence enhancement among H2S probes in the existing literatures.Cellular imaging results indicated that 4 displayed good biocompatibility and could be used for detection of exogenous and endogenous H2S in living cells.GSH/Cys,however,showed weak interference to the detection of H2S with 4.To address this problem,dual-reactive strategy was introduced,based on the redox activity and high nucleophilicity of H2S,we further developed dual-reactive and dual-quenching H2S probe 7.This dual-reactive probe not only displayed higher selectivity,sensitivity and stability but also showed a multiplication relationship with the single-reactive probes in terms of selectivity and fluorescence enhancement.Probe 7 was successfully utilized for fast and selective detection of H2S in HEK293 cells.These data demonstrated the universality of the dual-reactive and dual-quenching strategy.We further accessed the simultaneous detection of reductive cancer biomarkers H2S and hNQO1 using this strategy.We first developed near-infrared hNQO1 probes 10 and 11,corforming that the trimethyl-lock containing quinone propionic acid(Q3PA)moiety was a suitable receptor for hNQO1.Based on this,H2S-hNQO1 dual-reactive and dual-quenching probes 12 and 13 were rationally constructed by installing two chemoselective triggering groups that responded to H2S and hNQO1,respectively,into one fluorophore.Probe 12 provided a small turn-on fluorescence response toward H2S but a much larger response(200 fold)to both H2S and hNQO1 in tandem.By contrast,probe 13 was activated only in the presence of both H2S and hNQO1 with a large fluorescence turn-on(>400 fold),high sensitivity,excellent selectivity as well as good biocompatibility.Bioimaging results indicated that probe 13 could differentiate HT29 and HepG2 cancer cells from HCT116,FHC and HeLa cells.Expanded investigations using 13 revealed a synergistic antioxidant effect between H2S and hNQO1 under conditions of cellular oxidative stress.In addition,to explore the metabolism of endogenous HClO under reductive stress induced by H2S,we developed a new HClO probe 14 employing the fast oxidation of p-aminophenyl ether moiety by HClO,which displayed fast response(t1/2<30 s)and large off-on fluorescence increase(1046 fold)toward 1 eq.HClO with very high selectivity and sensitivity(the detection limit was as low as 0.65 nM),and could be applied for detection of endogenous HClO in living RAW264.7 and MCF-7 cells.Importantly,using the probe-based tool,we observed the H2S-induced HClO biogenesis in living cells for the first time,indicating an up-regulated level of endogenous HClO under reductive stress.In this thesis,we detected the intracellular H2S selectively and sensitively by dual-reactive and dual-quenching H2S fluorescence probes.Using the H2S-hNQ01 dual-reactive and dual-quenching probes,we differentiated some cancer cells and revealed a synergistic antioxidant effect between H2S and hNQOl under conditions of cellular oxidative stress.We also got a first insight into the generation of endogenous HC10 under reductive stress induced by H2S based on probe 14.Investigations in this thesis provided a new strategy for designing fluorescent probes,enriched the understanding of intracellular redox regulation,and promoted the explorations on H2S chemical biology.