Novel Nbd-based Fluorescence Probes Fabricated For Discriminately Detecting Biothiols

Hydrogen sulfide(H2S),cysteine(Cys),homocysteine(Hcy)and glutathione(GSH)are important biothiols,which can maintain the cellular redox homeostasis,and involve in diverse physiological and pathological processes.Misregulation of these biomolecules is closely related to various diseases making them biomarkers of some diseases.Thus,it is of great physiological and medical value to differentiate these biothiols as well as investigate their physiological functions.Fluorescence-based method allows for the in-situ,real-time and visual detection of analytes.Although numerous fluorescent bithiol probes have been reported,few of them can be utilized to differentiate one or two biothiols as well as to expolore their inherent relationship.To this end,based on the strong nucleophilicity of biothiols,using nitrobenzofurazine(NBD)thioether/ether/amine as the reaction motif,a series of fluorescent biothiol probes were developed in this thesis.Dual-color NBD-S-rhodamine fluorescent probe 1,based on the fast thiolysis reaction of NBD thioether with thiol group,was designed and synthesized in this thesis.After reaction with GSH/H2S,probe 1 displayed red fluorescence,while treatment by Cys/Hcy trigerred both green and red emission,by which selective detection of the four biothiols and the Cys/Hcy was achieved.Furthermore,probe 1 exhibited good selectivity and high sensitivity,and was applied for specific detection of Cys/Hcy in human living fibrosarcoma cells in the presence of millimolar GSH.To simultaneously distinguish H2S and Cys/Hcy,a dual-reactive and dual-quenching fluorescent probe 3 was designed and synthesized,which was derived from the thiolysis reaction of H2S with NBD amine and the reactivity of Cys/Hcy with NBD ether.Very high selectivity and sensitivity of probe 3 torward H2S and Cys/Hcy in the corresponding blue and green channels were demonstrated.Probe 3 was successfully used for visualize Cys-induced endogenous H2S,which verificated the CBS enzymatic pathway for generating H2S from Cys.Due to the very similar chemical structure with Hcy,it has been challenging for specific detection of Cys.By combining the Michael addition reaction of acrylic ester with biothiols and the NBD thioether ammonolysis reaction,dynamic-identified probe 5 was ingeniously designed and synthesized.Owing to the difference in reactivity of the biothios,in living cells,probe 5 allowed for specific detection of Cys within 30 minutes and visualization of Hcy/GSH within 3 hours,through which simultaneously selective detection of Cys and Hcy/GSH was brought about.In summary,a series of fluorescent molecular probes for the specific detection of biothiols were developed in this thesis.Among them,probe 1 allowed for selective detection of four biothiols and Cys/Hcy through red and green channels respectively;while,in the blue and green channels,probe 3 showcased high specificity towards H2S and Cys/Hcy;and the final dynamic-identified probe 5 was used for specific detection of Cys and Hcy/GSH at different time points.Investigations in this thesis should expand the development and applications of NBD-based fluorescent probes,and provide a series of facile but effective fluorescent probe tools for the detection and research of biothiols.