Designing Fluorescent Probes For Sensing Hydrogen Sulfide And Peroxynitrite In Mitochondria

Intracellular redox-active species,including reactive oxygen species?ROS?,reactive nitrogen species?RNS?,reactive sulfur species?RSS?and so on,have a significant role in regulating intracellular redox homeostasis and maintaining cellular function.Mitochondria are not only the powerhouses of cells,but also the apoptotic signaling hubs,and work significantly depending on their redox state.Therefore,developing a sensitive and selective method for the detection of redox-active species in mitochondria is conducive to explore their biological functions.Compared with traditional analytical techniques,fluorescent imaging has been recognized as one of the most efficient tools for monitoring target bio-analytes in complicated biological systems due to its high sensitivity and selectivity,easiness of manipulating,high imaging resolution,real-time imaging and nondestructive detection.Herein we focus on developing fluorescent probes for monitoring two mitochondrial redox-active species,hydrogen sulfide?H₂S?and peroxynitrite?ONOO^-?.The specific research contents are summarized as follows:?1?H₂S is recognized as a critical gaseous signaling molecule involved in multiple physiological and pathological processes.Using a piperazidine-bridged styrylpyridinium as the push-pull fluorophore and a 7-nitro-1,2,3-benzoxadiazole moiety as the response group,herein we developed a mitochondria-targeted fluorescent probe MNOP-H₂S,which exhibited remarkable fluorescence turn-on?>130 fold?,large Stokes shift?190 nm?,excellent sensitivity and selectivity.Importantly,the probe was characterized by an ultralow detection limit?29 nM?,thereby being successfully applied to monitor mitochondrial basal H₂S levels in various types of cells and zebrafish,to distinguish cancer cells from normal cells,and to reveal that the endogenously produced H₂S in mitochondria is a key mediator for the endothelial cell migration stimulated by vascular endothelial growth factor.?2?ONOO^-as an important ROS/RNS,has strong oxidative and nucleophilic properties.Exceptionally high levels of intracellular ONOO^-contribute to a plethora of pathogenic effects,for instance,acute inflammation,neurodegenerative and cardio-vascular diseases,and even cancer.Using salicyladazine as the fluorescent reporter,boronates as the response group and triphenylphosphonium as the mitochondria-targeted group,we developed a fluorescent probe TBSA for mitochondrial ONOO^-with the following main features:a)high selectivity and sensitivity;b)aggregation-induced emission?AIE?and excited-state intramolecular proton transfer?ESIPT?properties;c)capable of monitoring the exogenous and endogenous production of ONOO^-in HepG2cells and zebrafish.Theranostic agents are dual function systems that offer both therapeutic promise and potential for concurrent diagnosis.They are particularly attractive in the context of personalized cancer therapy,as well as in high precision cancer imaging.Compared with normal cells,cancer cells display mitochondrial dysfunction and altered redox homeostasis,such as increased levels of ROS and glutathione?GSH?,to maintain their malignant phenotype.The biochemical differences have opended a window for developing mitochondria-targeted pro-oxidative anticancer theranostic prodrugs.?3?It was previously reported that conjugation of a salicyladazine fluorophore with the mitochondria-targeting TPP generated a theranostic agent with the AIE characteristics and the ability to kill cancer cells through promoting ROS generation?Angew.Chem.Int.Ed.2014,53,14225?.Accordingly,we designed a GSH-activated pro-oxidative anticancer theranostic prodrug Mito-TSSA using 2,4-dinitrobenzenesulfonate to mask the two phenolic hydroxyl groups on the salicyladazine fluorophore.We reasoned that it can exploit increased levels of GSH in cancer cells to in situ release free phenolic hydroxyl groups,thereby allowing a turn-on fluorescent for its release,targeting mitochondria and therapeutic efficacy via the AIE and ESIPT mechanisms,and kill selectively cancer cells due to its strong ability to aggregate in mitochondria,generating ROS and interfering energy metabolism.Currently,we have completed the synthesis of Mito-TSSA,whose activity experiments are in progress.