Facile Synthesis Of Dihydroquinolines As New Scaffolds For Fluores-cence Sensing Of Hydroxyl Radical

ObjectiveA series of new dihydroquinoline fluorescent probes were constructed through simple,mild and efficient chemical synthesis methods.By exploring their photophysical properties the most suitable fluorescent probe was screened out,which was used to detect ·OH specifically and sensitively.In addition,the probe can realize the fluorescence visual detection of ·OH in living cells and study the physiological activities of flavonoids against oxygen free radicals.Method1.Organic synthesis of small molecule probes:3-formyl-1-methylquinoline salt and different aromatic acetyl compounds reacted in acetonitrile solution catalyzed by 1methylpiperazine,and the reaction was monitored by thin layer chromatography(TLC).After the reaction was terminated,the product was extracted,concentrated,and purified by silica gel column chromatography.2.Characterization and identification of comounds:All intermediates and final products were structurally characterized by nuclear magnetic resonance spectroscopy NMR and high-resolution mass spectrometry HRMS.If neccesary,the single crystal X-ray diffraction analysis technique was used for further confirmation.3.The green preparation method of dihydroquinoline probes:In mixed water phase system H2O/EtOH(1:1,v/v),3-forml-1-methylquinoline salt and different aromatic acetyl compounds reacted under the alkaline condition provided by NaOH.The reaction process was detected by thin layer chromatography TLC.After the reaction was terminated,the product is extracted,concentrated,and purified by silica gel column chromatography.4.The optimization of the green preparation of fluorescent probes:2-acetnaphthalene 1a and 3-formyl-1-methylquinoline salt 2 were choosen as template substrates to optimize the water phase chemical reaction conditions through a single factor experiment.The influencing factors include catalyst type,catalyst equivalent,reaction temperature and reaction time,etc.The separation yield of dihydroquinoline compound 3a was used as an evaluation index.5.Research on the response mechanism and photophysical properties of the probes:the color and fluorescence changes of the solution which constained probe and ·OH were observed and recorded.The structure characterization data such as spectroscopy and highresolution mass spectrometry were infered the response mechanism.Then the Hirshfeld charge of all atoms and the electrophilicity auxiliary of 3a were calcalated by density functional theory(DFT)for verification mechanism.Ultraviolet-visible light(UV-vis)photometer,Fluorescence photometer and multi-functional microplate reader were used to detect and record the absorption spectrum,fluorescence spectrum stokes shift,fluorescence quantum yield and other photophysical properties of oxidation products.And the effect of substituents on the oxidation products were studied by DFT.Based on the physical properties,the fluorescent probe with excellent photophysical properties were screened out.6.The dihydroquinoline probe 3c was used to detect the content of ·OH in the Fe2+/H2O2 Fenton reaction system,and the effect of 3c on other active analytes ROS/RNS(HOCl,ONOO-,O2·-,NO2-,1O2,ROO·,TBHP,H2O2)and some metal ions(Fe2+,Fe3+)were investigated.7.The burst of ·OH in living cells was observed by 3c under the laser confocal fluorescence microscope.8.The ·OH clearance rate of flavonoids were calculated by the multifunctional microplate reader and the fluorescence intensity changes of the probe 3c.Results1.21 kinds of reduced dihydroquinoline fluorescent probes 3a-3u were successfully obtained by the addition reaction,which were characterized by 1H-NMR,13C-NMR and HRMS,and their structures were correct.2.It was proved that many aromatic acetyl compounds could react with 3-formyl-1methylquinoline salt respectively under the alkaline aqueous reaction conditions provided by NaOH;2-acetylnaphthalene 1a and 3-formyl-1-methylquinoline salt 2 were used as template substrates to obtain the most optimized reaction condition,which was as follows:2 eq NaOH was used as catalyst and the reactants reacted for 5 hours at room temperature to obtain the target product 3a,with a yield as high as 90%.3.The response ability of 3a-3u to ·OH was different.the photophysical properties of oxidation products 3a-pr?3u-pr were closely related to substituent effect.The electron donor group caused obvious fluorescence red shift,while the electron acceptor group caused fluorescence blue shift.3c-pr has excellent spectral performance and the highest fluorescence relative quantum yield(?=0.57).4.Based on two hydrogen abstraction reactions mediated by ·OH,the reduced dihdroquinoline compounds were oxidized into corresponding fluorescent products.5.·OH could be detected by probe 3c quantitatively with specificity and sensitivity,and the response time was about 8 min.The fluorescence of 3c was enhanced by 400 times with 100 ?M·OH.The reaction was not interfered by other bioactive analytes,and fluorescence imaging at lysosome could be realized.Conclusion21 kinds of dihydroquinoline compounds can be obtained by a green,mild,efficient,and simple preparation method.Their photophysical properties are adjustable and can be used as fluorescent probes to identify ·OH.Among them,compound 3c has good practical application value.It can not only performed specific quantitative analysis on the ·OH,but also realized the flurescence imaging of ·OH in living cells and was applied on the antioxidant properties of flavonoids.The work of this article proved that dihydroquinoline fluorescent probes have great potential in the identification and detection of ·OH,which was first reported by our group.