Coumarin-based Fluorescent Probes With Multiple Reaction Sites For Reactive Sulfur Species:Design,Synthesis And Application

Due to the development of optical imaging technology in recent years,the application of simple and sensitive fluorescent probes in the area of cell biology and biological detection has made great progress.Researchers have developed a series of florescent probes for the detection of ions,reactive molecules in living organisms.The biothiols and hydrogen sulfide are important biological reactive sulfur species in human physiology,participating in a series of physiological activities such as intracellular signaling and immune regulation.However,the abnormal level of these thiols is closely associated with Alzheimer’s disease,cardiovascular disease and other diseases.In this dissertation,the design method,sensing mechanism of fluorescent probes and research progress of fluorescent probes for biothils and hydrogen sulfide were briefly reviewed,on this basis,fluorescent probes with coumarin as the fluorophore were delevoped for the qualitative analysis and quantitative detection of biothiols and hydrogen sulfide.The details are listed as follow:(1)Fluorescent probes with coumarin as the fluorophore were developed for the detection of hydrogen sulfide,introducing chlorine atom,phenylthio and phenylselenium into 4-position of coumarin as the reaction site,and introducing dicyanovinyl into 3-position of coumarin as another reaction site.The spectral tests demonstrate that probe with phenylselenium as the leaving group has lower fluorescence quantum yield and faster response toward hydrogen sulfide.The probe PhSeCou has excellent sensing performance for the detection of hydrogen sulfide,with a short response time of 1 min and a detection limit of 8.8 nM,showing good selectivity.The sensing mechanism was analysed by separating,purifying and characterizing the sensing products.The cell imaging experiments show that probe PhSeCou can be applied to sense the exogenous and endogenous hydrogen sulfide successfully in living HepG2 cells.Thses results indicate that probe PhSeCou has potential application prospects,and provide a novel idea for the molecular design of fluorescent probes for the detection of hydrogen sulfide.(2)On the basis of previous work,fluorescent probe with coumarin as the fluorophore were developed for the detection of biothils,introducing phenylselenium into 4-position of coumarin as the reaction site,and introducing ethyl 2-cyanoacrylate into 3-position of coumarin as the second reaction site.Meanwhile,the corresponding fluorescent probes with chlorine atom and phenylthio as leaving group were synthesized.The spectral tests indicate that probe with phenylselenium as leaving group has faster response toward biothiols.The probe CouSePh exhibits briliiant sensing properties for the detection of biothiols with short response time(2 min for Cys,15 min for Hcy and 2 min for GSH),low LODs(3.0 nM for Cys,39.5 nM for Hcy and 9.7 nM for GSH)and discriminative detection of biothiols(Cys:λem=465 nm;Hcy:λem=542 nm;GSH:λem=533 nm).The sensing mechanism was analyzed in detail by tracking NMR spectrometry,mass spectrometry and separation and characterization of sensing products.Through dual-channel fluorescence imaging,probe CouSePh is capable of discriminatively sensing endogenous biothiols in living cells.(3)Fluorescent probes with coumarin as the fluorophore were developed for the detection of biothils,introducing chlorine atom,phenylthio and phenylselenium into 4-position of coumarin as the leaving group and first reaction site,and introducing nitroethenyl into 3-position of coumarin as the second reaction site.The spectral tests show that probe with phenylselenium as leaving group has faster response toward biothiols.In contrast to reported probes with nitroethenyl as Michael acceptor,the probe CouSeNO2 showes good sensing properties for the detection of biothiols,and is able to discriminatively detect Cys/Hcy and GSH.Finally,the probe CouSeNO2 was successfully applied to sense the endogenous and exogenous biothiols in living cells through dual-channel fluorescence imaging.