Construction And Biological Application Of Coumarin-based Fluorescent Probes For Recognizing ClO~-

Hypochlorite ion（ClO^-）is an important reactive oxygen species（ROS）,which not only exists widely in the environment,but also plays an extremely important role in the organism.Appropriate concentration of ClO^-can kill a variety of pathogenic microorganisms in the organism,and play an essential role in the cellular immunity of humans and other animals.However,when ClO^-in the body is at an abnormal concentration level,it may cause multiple pathogens.Over the years,the emergence of fluorescent probes has provided a convenient and effective tool for the detection of various molecules and ions in organisms.Due to the stable photochemical properties and large stokes shift of coumarins,highly specific fluorescent probes can often be obtained by modifying the coumarin parent.In this paper,three coumarin-based fluorescent probes were constructed for the detection of ClO^-,and their biological applications were further studied.The main contents are as follows:1.A ratiometric fluorescent probe（Probe 1）was developed,which can be used to quickly identify ClO^-.In this work,Probe 1 itself had a relatively strong yellow fluorescence at 570 nm,and the addition of ClO^-would gradually weaken the fluorescence of the probe itself,and a regularly increased blue fluorescence emission appeared at 420 nm.In addition,the probe solution changed from yellow to colorless before and after the reaction,which proved that the probe can detect ClO^-by colorimetry.Studies have shown that the mechanism of the reaction was that Probe 1 was rapidly oxidized by ClO^-to an olefinic acid derivative,which led to the destruction of its conjugated system,which triggered a series of changes in spectral properties.We have verified this mechanism through ¹H NMR experiments and mass spectrometry,and confirmed it through theoretical calculations.In addition,the probe has been successfully applied to the detection of ClO^-in cells,zebrafish and Arabidopsis.2.A near-infrared fluorescent probe（Probe 2）that can specifically recognize ClO^-was designed and synthesized.In the DMF/PBS（v/v,1/1,p H=7.4）system,the probe had long emission wavelength（650 nm）and large Stokes shift（145 nm）.After adding ClO^-,the fluorescence intensity of the probe itself gradually weakened.Spectral experiments showed that in the concentration range of 0-73.7μM,Probe 2 can sensitively monitor ClO^-,with detection limit as low as 0.15μM,and illustrated a good linear relationship.In addition,the probe exhibited excellent selectivity and anti-interference ability in the detection of ClO^-.Finally,Probe 2 has been further applied to the fluorescence imaging of ClO^-in He La cells.3.A fluorescent probe（Probe 3）was constructed by combining coumarin and chalcone derivatives,which can dynamically monitor endogenous ClO^-in tumor animal models.The probe itself had a very weak fluorescence emission at 605 nm,and the reaction with ClO^-would greatly enhanced its fluorescence,and its detection limit was as low as 67 n M.We believed that the mechanism of the reaction was that the carbonyl group in the probe molecule was oxidized to hydroxyl by ClO^-,which led to a change in the conjugation structure of the probe,and finally caused an increase in fluorescence at 605 nm.Besides,the detection mechanism has been further confirmed by mass spectrometry and ¹³C NMR experiments.It was worth mentioning that due to its good fluorescence performance and extremely low detection limit,we not only used Probe 3 to detect exogenous ClO^-in cells,but also successfully applied it to real-time monitoring of the endogenous ClO^-in tumor animal models.