Development Of Fluorescent Probes For Hypochlorous Acid Based On A Novel Strategy For The Synthesis Of 1,2,4-Triazolopyridine And Their Biological Applications

Reactive oxygen species(ROS)are a kind of oxidative substance closely related to oxygen metabolism in biosystem.As a highly active ROS,endogenous hypochlorous acid(HOCl)plays a crucial role in normal biochemical processes and abnormal pathological processes related to oxidative stress.Therefore,it is necessary to establish a detection method to in situ and in real time monitor the subtle changes of endogenous HOCl without interference of other ROS.The effective approach to solve the above problems is to develop new fluorescent probes with high sensitivity,high selectivity and high spatiotemporal resolution.In this paper,we discovered that HOCl can specifically induce 2-pyridylhydrazones to produce1,2,4-triazolo[4,3-?]pyridines.Based on the above-mentioned findings,we report here a novel synthetic method for 1,2,4-triazolo[4,3-?]pyridine and in turn develop new fluorescent probes with high sensitivity,high selectivity and good biocompatibility.The specific contents are as follows:1.We designed and synthesized six 2-pyridylhydrazones derivatives N1-N6.Next,the1,2,4-triazolo[4,3-?]pyridines NT1-NT6 were synthesized by the reaction of N1-N6 with HOCl.Among these constructed substrates,N6 as a new Rhodol derivative containing2-pyridylhydrazone is non-fluorescent because of the isomerization quenching of C=N bond.N6 reacts with HOCl to generate rigid NT6,leading to recovery of fluorescence.Therefore,N6 can be a fluorescence probe for HOCl based on the novel strategy of triazolopyridine formation.The experiment results show that N6 has high selectivity and sensitivity to HOCl with a detection limit as low as 5.3 n M.Moreover,N6 was successfully applied to image exogenous and endogenous HOCl in living cells and zebrafish.2.Mitochondrial HOCl is closely related to the redox balance in mitochondria.Therefore,it is critically important for the detection and prevention of HOCl-related diseases to detect the mitochondrial HOCl in situ and in real time.Based on the novel strategy for the synthesis of1,2,4-triazolo[4,3-?]pyridine,we developed a novel fluorescence probe NM1 to target mitochondria to in situ and in real time monitor mitochondrial HOCl by appending the mitochondria-targeted group triphenylphosphine cation into Rhodol molecular skeleton containing 2-pyridylhydrazone.NM1 can detect HOCl in a few seconds in vitro,with a low detection limit as 2.2 n M.In the colocalization experiment,the fluorescence of NM1 almost completely overlaps with the fluorescence of Mito Tracker Green,with the Pearson's correlation coefficient as 0.94.The results of the colocalization experiment show that NM1 has good ability of mitochondrial localization.Moreover,NM1 was successfully applied to image endogenous HOCl stimulated by lipopolysaccharide(LPS)in living Hep G2 cells and zebrafish.