NIR Fluorescent Probes Based On Acridinones:Synthesis, Characterization And Application

In recent years,fluorescence imaging using small-molecule probes has been an efficient method to visualize the spatial and temporal distribution of biomolecules in biological systems due to its high sensitivity,high spatial resolution,visualization and convenient operation.In vivo fluorescence imaging has been widely used in the fields of medicine,biology,clinical diagnosis and drug discovery.With the rapid development of in vivo fluorescence imaging,the requirements for fluorescent probes have also increased.However,the in vivo applications of most of probes were greatly limited due to the use of short-wavelength excitation lights?usually<600 nm?which lead to shallow tissue penetration depth,strong background auto-fluorescence and photodamge.An effective approach for overcoming this problem is to design near-infrared?NIR?fluorescent probes that are excited and emitted in the spectral window between wavelengths of 600-950 nm.For example,DDAO is a promising NIR fluorescent dye with several attractive properties including tunable excitation wavelength?600-650 nm?,moderate aqueous solubility,long emission wavelengths(l_em=660 nm)and relatively low pK_a?⁵?.So,two novel NIR fluorescent probes AB1 and AG were developed by modulating the ICT process within the dye DDAO.OBJECTIVE:Two new fluorescent probes were designed and synthesized using DDAO as the fluorescent nucleus.They were used to detect hydrogen peroxide and?-glucosidase,respectively.Vitro fluorescence properties of fluorescent probes and a test compound to treat responsive.Cellular experiments determine the toxicity of probes and their responsiveness to the compounds to be tested in cells.Finally,the fluorescence probes were tested for its responsiveness to the test compound in vivo imaging.METHODS:We synthesized two novel fluorescent probes AB1 and AG.The structural analysis,molecular weight and purity of the probe were determined by nuclear magnetic resonance spectroscopy,high resolution mass spectrometry,and high performance liquid chromatography,respectively.Selectivity,sensitivity and other properties of probes were evaluated by UV-Vis absorption spectroscopy,fluorescence emission spectroscopy.MTT assay determine the toxicity of probes,laser confocal imaging determines the responsivity of the probes to the compound to be tested in the Cell.The fluorescence probes were tested for its responsiveness to the test compound in vivo imaging.RESULTS:We designed and synthesized two novel fluorescent probes AB1 and AG.AB1 has a relatively strong orange fluorescence emission at 620 nm,the quantum yield of AB1 in ethanol was estimated to be 0.28,comparable to that of DDAO??_F=0.39?,AB1 can be used for ratiometric detection of H₂O₂.AB1 had very weak fluorescence at 660 nm.The addition of H₂O₂ to aqueous solution of AB1 led to a gradual fluorescence enhancement than AB1 at 660 nm after 1 h incubation,AB1 also can be used for detection of H₂O₂ by turn-on mode.Probe AB1 enables highly sensitive and reliable detection of H₂O₂ based on both fluorescent ratiometric and turn-on dual modes,the limit of detection?LOD?was calculated to be 0.42?M;probe AB1 also has high selectivity towards H₂O₂,allowing its applications in complex biological systems such as living cells.The low cytotoxicity of AB1 guarantees its uses in cells and animals.Results imply that the probe AB1 is able to image dynamic changes of endogenous H₂O₂ levels in living cells using the ratiometric fluorescent mode and can be used to detect various concentrations of H₂O₂ in mice.At the same time,AG had very weak fluorescence at 660 nm.The addition of?-glucosidase to aqueous solution of AG led to a gradual fluorescence enhancement than AG at 660nm after 1 h incubation,AG can be used for detection of?-glucosidase by turn-on mode.Results imply that the probe AG is able to image dynamic changes of endogenous?-glucosidase levels in living cells and can be used to detect?-glucosidase in mice.CONCLUSION:Two novel NIR fluorescent probes AB1 and AG were developed by modulating the ICT process within the dye DDAO.AB1 has great potential for sensitive and specific quantification of H₂O₂ levels in cells and animals.AG has great potential for sensitive and specific responsivity of?-glucosidase in cells and animals.Provides new research ideas for the development of late fluorescent probes.