Construction And Performance Study Of "off-on" Fluorescent Dyes Based On Coumarin/Indanedione

Bioactive species such as reactive oxygen species,reactive sulfur species reactive carbonyl species and enzymes play crucial roles for participating the metabolism of intracellular materials,regulating the corresponding biological functions,and maintaining the homeostasis of the living cells.In particular,the study of the existence of Cl O~-and ONOO~-active molecules in physiology has become a hot topic in recent years.In recent years,fluorescent probe labeling imaging technology is an important means to detect and monitor living cells and bioactive substances in vivo due to its characteristics of high sensitivity,high spatial and temporal resolution,little light damage to living cells and low background fluorescence interference.Due to the short life of active substances and high reactivity,high requirements are put forward for the stability and real-time performance of probes,and the application of probes in fluorescence imaging is limited to a certain extent.Therefore,it is of great significance to develop fluorescent probes for detecting reactive oxygen species and reactive nitrogen species.The main contents from following aspects:(1)Phenothiazine was used as the recognition site,coumarin was used as the fluorophore group to formπ-π*conjugate through aldehyde methyl condensation reaction.The oxidation of phenothiazine by hypochlorous acid was used to generate sulfoxide structure,which inhibited the quenching effect of PET and triggered the"opening"of fluorescence signal.A novel enone-tethered fluorescence probe C-Ptz with a single recognition site for HOCl was designed and synthesized.Spectroscopic studies showed that probe C-Ptz had excellent fluorescence properties,such as high selectivity(fluorescence enhanced 42 times),high sensitivity(detection limit 12.6n M),fast response and large Stoker shift(97 nm).The fluorescence imaging of probe C-Ptz on exogenous hypochlorous acid in He La cells and endogenous hypochlorous acid in RAW 264.7 live cells was systematically studied in cell imaging experiments.In addition,the ability of probe C-Ptz to quantitatively detect Cl O~-in complex water samples was also explored.The recovery rate of hypochlorous acid was between 98.3-102.6%.These results indicate that the probe C-Ptz has high accuracy in the quantitative analysis of hypochlorous acid in complex water samples.(2)A new dye of IDBp with push-pull electron system was synthesized through the condensation of aldehyde methyl.The fluorescence intensity of the dye in glycerol with high viscosity system was enhanced significantly 38 times compared with that in aqueous solution system.Experimental cell imaging studies showed that dye IDBp could achieve fluorescence imaging of cell viscosity microenvironment induced by different drug stimuli in He La cells.The dye also tracks changes in the cellular microenvironment during iron death and can target lysosomes.Based on the excellent spectral properties of dye IDBp,we introduced diphenylphosphonic acid as a reaction site to recognize ONOO~-,and designed and synthesized near infrared ratio fluorescent probe IDPp,accompanied by a visible color change of solution(from orange to light purple).