Design, Synthesis And Application Of Two-photon And Near-infrared Superoxide Anion Fluorescent Probes

Fluorescence analysis has the advantages of simple operation,high sensitivity,and in situ detection of biomolecules and biological processes.It has been widely used in testing and imaging of biological activity of small molecules?such as hydrogen peroxide,peroxynitrite anion,cysteine,homocysteine,etc.?,biological macromolecules?such as alkaline phosphatase,nitroreductase,Amyloid-?Plaques,etc.?,metal ions?copper ion,mercury ion,iron ion,etc.?and biological microenvironment?such as viscosity,polarity etc.?.Two-photon and near-infrared fluorescence analysis has the advantages of near-infrared excitation,low background fluorescence,small photo damage to biological samples,and strong tissue penetration.It has broad application prospects in the fields of cell,tissue and in vivo imaging analysis.Drug-induced acute kidney injury?AKI?is a kidney injury caused by the drug itself or its metabolites,and has become one of the most common causes of drug development failure and drug withdrawal.In addition,it is difficult to achieve early detection of acute kidney injury by conventional means,and the early detection of AKI is still facing challenges.Superoxide anion(O₂^·-)is a reactive oxygen species,and studies have shown that changes in its concentration in kidney tissue are closely related to the occurrence of drug-induced acute kidney injury.Therefore,the development of non-invasive detection of superoxide anion is an effective method to evaluate and predict drug-induced acute kidney injury.Two novel mitochondrial-targeted two-photon or near-infrared O₂^·-probes have been developed to detect O₂^·-sensitively and selectively at the cellular,tissue and in vivo levels,which have been successfully applied to visualize the changes of O ₂^·-concentration during drug-induced acute kidney injury in this paper.In addition,it is the first time to demonstrate that L-carnitine?LC?can alleviate drug-induced nephrotoxicity with fluorescent probes.The details are as follows:Firstly,it is still a great challenge to detect superox ide anions with high specificity,because some bioactive species in organisms have similar properties with superoxide anions.In response to the above problems,we first modified various superoxide anion recognition groups on the hydroxyl group of hydroxy-naphthalimide to construct a series of fluorescent probes.Fluorescence analysis was used to study the response properties of different recognition groups to superoxide anion?such as selectivity and stability?,and the superoxide anion recognition group?trifluoromethyl sulfonate?with the best comprehensive performance was selected.Secondly,based on the above work,we constructed a novel two-photon mitochondrial targeting O₂^·-probe Naph-O₂^·-,using hydroxy-naphthalimide as two-photon chromophore,triphenyl phosphorus cation as mitochondrial targeting group,and trifluoromethanesulfonate as O₂^·-recognition site.The fluorescence signal of the free probe is weak.When it reacts with O ₂^·-,Naph-OH with enhanced ICT effect will be formed,which can enhance the fluorescence signal significantly and achieve the purpose of"Turn-On"detection of O₂^·-.Two-photon probe Naph-O₂^·-has been successfully applied to detect superoxide anions in cisplatin-induced renal cell and tissue damage models and to repair cispl atin-induced acute renal injury by LC at cell level,with its advantages of high sensitivity?0.39?M?,high stability and high selectivity.Thirdly,in order to investigate the changes of superoxide anion concentration during drug-induced acute kidney injury in vivo,we further developed a highly selective and sensitive near-infrared mitochondrial targeted superoxide anion fluorescence probe NIR-O₂^·-.The probe NIR-O₂^·-exhibited higher sensitivity?0.24?M?and lower fluorescence background interference than the two-photon probe Naph-O₂^·-.The in vivo imaging system of mice showed that the probe NIR-O₂^·-could detect the concentration of superoxide anion in cisplatin-induced renal injury and its repair process with high sensitivity.It also indicated that cisplatin-induced acute renal injury was closely related to the dose and stimulation time of cisplatin.The development of this near infrared probe is expected to provide an intuitive and effective new method for nephrotoxicity screening in the development of new drugs.