Dicyanoisophorone-based Fluorescent Probes For The Detection Of Cysteine And Their Bioimaging Application

BackgroundCysteine(Cys),a common amino acid in organism,is involved in many physiological processes,including protein synthesis,maintenance of redox homeostasis,and post-translational modification of proteins.In addition,abnormal levels of Cys can induce a series of diseases such as liver injury,cancer and Alzheimer's disease.Therefore,the development of Cys-specific methods can be conducive to studying the production,distribution,metabolism,and mechanism of Cys in complex biological systems as well as the diagnosis of Cys-related diseases.Organic small molecule fluorescent probes have been widely used for the detection of Cys in organisms due to their low cost,simple operation,temporal-spatial resolution,non-invasiveness and real-time monitoring.However,most of the fluorescent probes for detecting Cys limited their practical applications in biological imaging,because of short emission wavelengths,small Stokes shifts,and poor water solubility,which could lead to shallow tissue-penetration depth,auto-fluorescence interference,photobleaching or photodamage.Therefore,it is particularly urgent to construct the Cys-specific fluorescent probes with long emission wavelength,large Stokes shift and good water solubility.PurposeTo selectively detect Cys in biological system,a series of fluorescent probes based on dicyanoisophorone derivatives were designed and synthesied,which have long emission wavelengths and large Stokes shifts.Methods1.Synthesis and characterization:The probes were prepared by chemical methods such as dehydration condensation and substitution.Their structures were characterized by ~1H NMR,¹³C NMR and HRMS.2.Spectral measurement:The absorption/emission spectrum,limit of detection(LOD),response rate,selectivity,anti-interference ability and p H stability were investigated by UV-visible spectrophotometer and fluorescence spectrophotometer.3.Cytotoxicity evaluation:The cytotoxicity of probes was determined by MTT assay.4.Fluorescence imaging application:Fluorescence imaging of Cys in cells and organisms was realized by using fluorescence inversion microscope and spectra multiapplication imager.Results1.Three Cys-specific fluorescent probes based on dicyanoisophorone derivatives(DCI-Cys,TMN-NCS,DCI-C-Cys)were successfully prepared.2.DCI-Cys had the advantages of long emission wavelength(626 nm),large Stokes shift(185 nm),and rapid response(20 min).DCI-Cys could not only be applied for the fluorescence imaging of endogenous/exogenous Cys in cells as well as zebrafish,but also successfully monitored the fluctuations of intracellular Cys levels during the redox process induced by Hg²⁺and resveratrol.3.TMN-NCS had the advantages of long emission wavelength(660 nm),large Stokes shift(210 nm),high sensitivity(LOD:120 n M),and low cytotoxicity.TMN-NCS was successfully applied to the fluorescence imaging of Cys in cells and mice.4.DCI-C-Cys had the advantages of long emission wavelength(702 nm),large Stokes shift(240 nm),low cytotoxicity,good water solubility and high sensitivity(LOD:93 n M).In addition,DCI-C-Cys was successfully used to detect endogenous/exogenous Cys in cells and zebrafish.Meanwhile,the imaging results showed that balsam pear polysaccharides could induce the upregulation of intracellular Cys levels.Conclusion1.Three Cys-specific fluorescent probes,which exhibited long emission wavelengths and large Stokes shifts,were successfully prepared and applied to the fluorescence imaging of Cys in cells and organisms.2.The development of three probes realized the visualization of intracellular Cys concentration fluctuations.The utilization of probe DCI-Cys and DCI-C-Cys confirmed the ability of mercury ions to induce down-regulation of intracellular Cys levels,while the treatment with resveratrol and balsam pear polysaccharides could up-regulate intracellular Cys levels.