Novel Redox Reversible NIR / Ratiometric Fluorescent Probes

The redox state controls various biological processes including cellular proliferation,differentiation and apoptosis.The redox imbalance might indicate many diseases such as cancer,cardiovascular disease,hypertension,atherosclerosis and rheumatoid arthritis.Therefore,monitoring of intracellular redox state is of great significance for the study of related physiological and pathological mechanisms.In vivo,the dynamic balance between reactive oxygen species（ROS）and reduced glutathione（GSH）plays a key role in maintaining and regulating intracellular redox state.The relative level between ROS and GSH in cells can reflect cellular redox state.Ozone（O₃）as a strong gaseous oxidant has been widely used in daily life and industrial production.However,despite its powerful usage in different applications,O₃can increase human risk of asthma,bronchitis,heart disease,nerve poisoning and other diseases.Moreover,current studies demonstrated that O₃ has physiological functions in the immune system.Thence,accurate determination of O₃ is of great significance for preventing potential damage and studying the physiological function of O_3.Based on the specific recognition of fluorescent molecular probes,fluorescence imaging technology is able to achieve in-situ,real-time and dynamic visual detection of analytes in cells and organisms,thereby collecting the endogenous spatiotemporal information of the analyte.This technology has the advantages such as simple operation,good selectivity,high sensitivity,and the ability to observe multiple molecular events simultaneously,and has become the most widely used method of molecular imaging in biology and medicine.In this thesis,a series of redox reversible near-infrared（NIR）fluorescent probes were synthesized and applied to dynamic and reversible monitoring of cellular redox state,and a ratiometric specific fluorescent probe was developed for accurate determination of O₃ in in indoor air and living cells.The details are as follows:Three redox-reversible NIR fluorescent probes,Ge-pyronines（GePs）,were synthesized and used for dynamic and real-time imaging of cellular redox state.The results of spectroscopy,NMR,MS and theoretical calculation demostrated:Ge atom incorporation prolongs the absorption and emission of the traditional pyronine,and greatly increases the electrophilic reactivity of pyronine toward thiols.Based on the enhanced electrophilic affinity,GePs can undergo a rapid and reversible Michael addition with GSH to form a non-π-conjugated adduct GePs-GSH,resulting in fluorescence quenching.The generation of ROS（ClO-,ONOO-or HO·）promotes the decomposition of GePs-GSH intoπ-conjugated GePs,leading to fluorescence recovery.Therefore,GePs exhibit a reversible fast response to the GSH/ROS redox with high sensitivity and selectivity.GePs also show excellent photophysical properties including absorption and emission deep-red to NIR region（>600 nm）,high fluorescence quantum yield(Φ_fl=0.32-0.44)in aqueous media,suitable water solubility and good cell permeability.Our results indicated GePs can be applied for real-time,dynamic and continues monitoring of ROS stress and cellular self-regulation of ROS stress via GSH repair in living HeLa and HL-60 cells.Four GSH-reversible-reponsive long-wavelength fluorescent probes,Si-pyronies（SiPs）,were developed and used for dynamic and real-time fluorescence imaging of fluctuations in intracellular GSH homeostasis.The results of spectroscopy,NMR and MS analysis discolsed that the electrophilic reactivity of the traditional pyronine is greatly enhanced and effectively regulated by the replacement of the pyronine’s O atom with different Si atom functional groups.Based on the mechanism,SiPs can undergo a reversible and ultrafast Michael addition with GSH,leading to fluctuations in fluorescence intensity due to the interruption and restoration of theirπ-conjugation.Under physiological conditions,SiPs exhibit sensitive and ultrafast response（<5 s）to changes in GSH homeostasis.Additionally,SiPs also has long-wavelength absorption and emission,high fluorescence quantum yield(Φ_fl=0.32-0.44)in aqueous media,proper water solubility and membrane permeability.Cellular imaging showed that SiPs can be applied to real-time,dynamic and continuous imaging of GSH fluctuation in HeLa cells under external stimulation.Based on the ICT mechanism of the 4-hydroxy-1,8-naphthalimide fluorophore,a ratiometric and selective fluorescence probe for ozone（O₃）was synthesized by utilizing 1-butene as the recognition group.Because of the unique ozonation mechanism of terminal alkenyl groups,the probe exhibits a specific response to O₃ in various ions,ROS and reducing substances.After reaction with O₃,probe 1 shows a 76nm red shift of absorption spectra,accompanying a color change from colorless to yellow,that thereby can serve as a colorimetric indicator for O₃.Meanwhile,the fluorescence intensity ratio at 550 nm/465 nm shows a good linearity with the ozone concentration below 24.2μM,with a detection limit of 39 nM.The results show that probe 1 can be applied to the ratio determination of O₃ in indoor air and the dual-channel fluorescence imaging of O₃in living A549 cells.