Development Of Fluorescent Probes For The Specific Detection Of Hydroxyl Radicals And Their Application In Bioimaging

Hydroxyl radical（·OH）is the most oxidative reactive oxygen species（ROS）in biological systems.It is mainly generated by Fenton reaction and Haber-Weiss reaction,and can react with a variety of biomolecules including DNA,proteins,lipids and amino acids at a diffusion-controlled rate.The excessive production of·OH may cause cell damage,DNA/protein mutations,and various diseases.On the other hand,recent studies suggest that the production of ROS including·OH can be exploited as a therapeutic approach to treat clinical conditions such as cancer.Therefore,detecting and quantifying intracellular·OH is crucial for understanding its behavior and functions and further scrutinizing its therapeutic utilization.Because of its high sensitivity,noninvasiveness and extraordinary spatiotemporal resolution,fluorescent probes have become a powerful molecular tool for the detection of reactive oxygen species in biological systems.Combined with imaging technology,in situ,dynamic and visual monitoring of biological reactive oxygen species can be realized.However,endogenous·OH has the characteristics of short lifetime,small diffusion distance and low concentration in cells.Therefore,developing sensitive and selective fluorescent probes for detecting·OH in biological systems still faces great challenges.Based on the scavenging mechanism of the neuroprotective drug edaravone on·OH,this paper combines edaravone with fluorophores to construct fluorescent probes for specifically detecting·OH and simultaneously detecting of·OH and pH,respectively,and their properties and bioimaging applications were studied.The content of the thesis is mainly divided into three chapters:Chapter 1.Different kinds of fluorescencent probes for the selective detection of·OH were summarized based on their sensing mechansim.On the basis its current situation,the main research ideas of this thesis were proposed.Chapter 2.A rhodamine-based fluorescent probe for the specific detection of hydroxyl radicals with edaravone as a recognition moiety was proposed.Herein,inspired by the scavenging mechanism of a neuroprotective drug edaravone toward·OH,we developed a new·OH-specific fluorescent probe RH-EDA.RH-EDA is a hybrid of rhodamine and edaravone and exploits a·OH-specific 3-methyl-pyrazolone moiety to control its fluorescence behavior.RH-EDA itself is almost nonfluorescent in physiological conditions,which was attributed to the formation of a twisted intramolecular charge transfer（TICT）state upon photoexcitation and the acylation of its rhodamine nitrogen at the 3′position.However,upon a treatment with·OH,its edaravone subunit was converted to the corresponding 2-oxo-3-（phenylhydrazono）-butanoic acid（OPB）derivative（to afford RH-OPB）,the intramolecular TICT effect was inhibited and the 3′position nitrogen of rhodamine in the probe was deacylated,thus leading to a significant fluorescence increase at 579 nm.RH-EDA shows a high sensitivity and selectivity to·OH without interference from other ROS.RH-EDA has been utilized for imaging endogenous·OH production in living cells and zebrafishes under different stimuli.Moreover,RH-EDA allows a high-contrast discrimination of cancer cells from normal ones by monitoring their different·OH levels upon stimulation withβ-Lapachone（β-Lap）,an effective ROS-generating anticancer therapeutic agent.Chapter 3.Development of a coumarin-based fluorescent probe for the simultaneous detection of hydroxyl radicals and pH with edaravone as the recognition group.In this chapter,a fluorescent probe Cou-EDA for the simultaneous detection of·OH and pH was constructed using 7-diethylamino-coumarin as the fluorophore.Since the pK_a of the probe Cou-EDA was 5.97,its pyrazolone mainly exists in the form of enol anion under physiological pH conditions.After adding·OH to the probe solution,this group can specifically recognize·OH to generate Cou-OPB.Under neutral conditions,the imine moiety of Cou-OPB will be protonated to form the corresponding iminium,which will form intramolecular hydrogen bonds with the carbonyl group of 7-diethylaminocoumarin,resulting in the extension of its conjugated system,thus leading to the red-shift the emission maxima from 507 nm to 538 nm.Thus,the fluorescence signal of the detection system at 538 nm is proportional to the·OH concentration within a certain range.On the other hand,When the pH of the probe solution is decreased,the anionic form of the probe will be converted into the corresponding keto form,and the fluorescence signal of the detection system at 507 nm increases significantly.Thus,the probe Cou-EDA realizes the simultaneous detection of·OH and pH at yellow(λ_ex/λ_em=510/538 nm)and green emission bands(λ_ex/λ_em=450/507 nm),respectively.Preliminary experimental results show that Cou-EDA can be applied to the fluorescence imaging of exogenous·OH and pH in Hela cells.Furthermoe,Cou-EDA has been applided for monitoring the variation of·OH and pH value in cinnamaldehyde-induced cell apoptosis.