Development Of Fluorescent Probes For Hydrogen Sulfide And Cell Cytotoxicity

BackgroundThe past decade we has seen a boost of research interest in hydrogen sulfide?H₂S?,H₂S as an important signaling molecular with multiple,which is recognized as the third signaling gasotransmitter in the body as well as nitric oxide and carbon monoxide.Studies have shown that the concentration of H₂S in central nervous system is in the range of 50-160?M and the sulfide concentration in blood is in the range of 10-100?M,this reactive sulfide plays an important role in maintaining normal physiological activities.Beisdes,the abnormal level of H₂S in cells may cause many types of disorders including Down's syndrome,Alzheimer's disease,liver cirrhosis and diabetes.Therefore,for us to fully understand how H₂S acts on physiological and pathological mechanisms,it presents significant research value to develop efficient methods that can detect H₂S in living biological systems with high sensitive and selection.ObjectiveA series of fluorescent probes containing Schiff base and Coumarin derivative fluorobenzene derivatives to detection H₂S had been designed and synthesized.In order to select to high selectivity fluorescent molecular probes,the interaction of the probes in HEPES buffer solution with biologically important anions?HS^-,H₂PO₄^-,F^-,AcO^-,Cl^-,Br^-,I^-?were determined.The effect of fluorescent molecular probes on the stability of host-guest was investigated by substituting the substituent of fluorescent probes.Besides,we further explore the preliminary assessment of their cytotoxicity.Finally,we screened out the fluorescent molecular probes with high selectivity,high sensitively and low cytotoxicity which can provide scientific basis for H₂S detection in vivo.Methods1.Construction of fluorescent molecular probes:Based on theories of copper sulfide precipitation reaction and thiolysis 2,4-dinitrophenyl ether,a series of fluorescent probes containing Schiff base with copper and Coumarin derivative fluorobenzene derivatives had been designed and synthesized.The crystal structure was cultivated and obtained.The molecular structures of the complexes were characterized by ¹H NMR,HRMS,IR and elemental analysis.2.Detection of hydrogen sulfide in vitro:In order to select to high selectivity fluorescent molecular probes,the interaction of the probes in HEPES buffer solution with biologically important anions?HS^-,H₂PO₄^-,F^-,AcO^-,Cl^-,Br^-,I^-?was determined by UV-vis,Fluorescence experiments and based on the binding constants calculated using the method of non-linear least square calculation,which furthermore selected good selectivity and high sensitivity fluorescent probes for H₂S detection.Besides,we further analysized the possible interacted mechanism of host-guest,through the perspective of optics and HRMS titration experiment.3.The preliminary assessment of specific binding of fluorescent molecular probes with MCF-7 cell can provide scientific basis for H₂S detection in vivo.Results1.Eight Schiff base based copper complexes and four coumarin derivative fluorobenzene derivatives were successfully synthesized.All of molecular structures of the complexes were characterized by ¹H NMR,HRMS,IR and elemental analysis.2.Seven kinds of fluorescent molecular probes were selected by UV-vis absorption spectroscopy,fluorescence emission spectroscopy and HRMS titration for detecting hydrogen sulfide with good binding ability,good fluorescence response and high selectivity.3.The fluorescent probes were applied to MCF-7 cells and Hela cells for preliminary evaluation of cytotoxicity,and the results showed that the fluorescent molecular probes had less toxicity to cells.Conclusion1.We have synthesized a series of fluorescent molecular probes and a crystal structure of compound j was cultivated and obtained.2.Fluorescence molecular probes a,b,h,i with strong binding ability to hydrogen sulfide were selected by UV-vis,Fluorescence experiments and the detection line for hydrogen sulfide of the corresponding fluorescence molecular probes was calculated by UV-vis,Fluorescence titration data.The interference experiment also showed that the fluorescence molecular probes a,b and h had high selectivity to hydrogen sulfide detection.The colorimetric recognition of fluorescent molecular probe i indicated that compound i could be applied to"naked eye"recognition of hydrogen sulfide.3.The fluorescent molecular probes with fluorescent groups contaning schiff base and coumarin derivativesthat showed low cytotoxicity which could provide a theoretical basis for the design of fluorescent molecular probes in the future.