| Peroxynitrite(ONOO~-),a reaction product from nitric oxide with superoxide radical via diffusion-controlled rate,is one of the most important reactive oxygen species(ROS)in life system.It harbors high oxidative,nitrosative and nucleophilic activity,and thus works as either a defense agent against invasion of pathogens or a signaling molecule for regulating various physiological processes via selective nitration of tyrosine residues.However,its excessive production also causes oxidative damage of DNA and protein,which is closely related to the development of various diseases,such as cardiovascular diseases,neurodegenerative diseases,inflammatory diseases and diabetes.In addition,ONOO~-produced is one of the most important reasons of hepatotoxicity in the process of drug metabolism.Therefore,real-time detection of ONOO~-level in cells is essential for exploring its pathophysiological roles.Viscosity is an important parameter of intracellular microenvironment,which influences interaction of biological macromolecules,signal transduction and diffusion of metabolites.Abnormal viscosity levels are closely related to a plethora of pathological conditions including diabetes,atherosclerosis alzheimer’s disease and so on.Therefore,it is extremely important to accurately quantify change in intracellular viscosity.Fluorescent probes are recognized as ideal molecular tools for exploring biological events due to their high sensitivity,easy operation,non-invasiveness and real-time detection in vivo.Coumarin fluorophores have received widespread attention due to their good photostability,large stokes shift and high quantum yield.Herein we engineered two coumarin-based fluorescent probes for monitoring ONOO~-and viscosity in living cells.The main results are shown as follows:(1)The fluorescent probe COU-Mito for ONOO~-was designed by bridging the coumarin skeleton with a cyanopyridylethylene unit.The probe can effectively identify ONOO~-via oxidative cleavage of the carbon-carbon double bond by ONOO~-,and exhibits a turn-on fluorescence response,excellent selectivity,high sensitivity and mitochondrial-targeting.The probe was successfully applied for monitoring endogenous and exogenous ONOO~-generation in human liver cancer cells(Hep G2)and endogenous ONOO~-generation zebrafish,and evaluating on the acetaminophen-induced hepatotoxicity.(2)The dual-response fluorescent probe COU-NB for simultaneously monitoring ONOO~-and viscosity was designed by bridging the coumarin skeleton and a cyanopyridylethylene unit and introducing an additional phenylborate group.The oxidative cleavage of carbon-carbon double bonds in the probe by ONOO~-results in a turn-on fluorescence response at 498 nm,while blocking rotation of the carbon-carbon single bond between the double bond and the pyridyl group by increasing viscosity allows a turn-on fluorescence response at 650 nm,due to inhibition againt a twisted intramolecular charge transfer effect(TICT).Therefore,the probe is capable of simultaneously monitoring ONOO~-and viscosity by virtue of its two sets of distinct signals,green for ONOO~-and red for viscosity.With the aid of the probe,the production of endogenous and exogenous ONOO~-in Hep G2 cells and the change in intracellular viscosity induced by nystatin,as well as the acetaminophen-induced hepatotoxicity,were visualized.Natural products are starting points for drug discovery.Vitamin C widely exists in vegetables and fruits,and is also an extremely cheap drug.It has been identified as a prooxidant to induce death of cancer cells selectively via ROS generation at pharmacological doses(Proc.Natl.Acad.Sci.USA 2008,105,11105;2007,104,8749;2005,102,13604).Additionally,at pharmacological doses,it has been suggested to kill KRAS and BRAF mutant colorectal cancer cells selectively by ROS-driven inactivation of the glycolytic glyceraldehyde 3-phosphate dehydrogenase(Science2015,350,1391).However,the concentrations of vitamin C used in the above studies are very high reaching millimolar levels.Accordingly,we designed a vitamin C prodrug to improve its stability,ROS-generative efficiency,selective anticancer activity.(3)The glutathione(GSH)-activated vitamin C prodrug(G-PVC)were designed by masking its 4-position enol hydroxyl group using a 2,4-dinitrobenzenesulfonyl group.We reasoned that it is capable of exploiting increased levels of GSH in cancer cells to in situ release vitamin C through nucleophilic aromatic substitution followed by 1,6-elimination;in the presence of intracellular copper and iron ions,the free vitamin C is oxidized to DHA followed by its regeneration at the expense of GSH,thereby constructing an efficient redox cycle to generate ROS and kill cancer cells selectively.The synthesis of G-PVC was explored using vitamin C as the starting material according to a convergent strategy,which is currently underway. |
