Synthesis And Fluorescent Imaging Of Organelle-targetable Probes

Fluorescence imaging technology based on small-molecule fluorescent probe(SMFP)has been widely applied in biology,medicine,diagnosis and etc.However,the technology depends strictly on the nature of SMFP,such as brightness,photostablity,selectivity,sensitivity,biocompatibility,and etc.In recent years,much efforts has been made to improve the chemical and optical performance of SMFP,for example,extending the absorption and emission wavelength to far red or near infrared(NIR)region,grafting an analyte-specific trigger on to a suitable fluorophore to impove selectivity and sensitivity and introducing amphiphilic group to enhance water-solubility and liposolubility.Based on these improvements,a great number of SMFPs has been employed for the detection of biologically relevant molecules,ions,enzyme,and etc.Among them,HClO/ClO~-and Cu2+ attract much attention due to its important roles in maintaining the normal physiological function of cell.Up to date,many physiological and pathological processes in cells or organelles involving in ClO~-or Cu2+have not been not fully understood owing to the limitions of SMFPs and complex biological environment.Therefore,it is significant to tracing ClO~-or Cu2+ relevant events in cells or organelles by novel SMFPs with organelle-targeting ability.To solve some key problems in the design and sensing processes of fluorescent probes for Cu2+or ClO~-,an ongoing program was carried out to design and synthesize of long-wavelength emission dyes,fluorescent probe models and organelle-targeting fluorescent probes for cell imaging applications.As such,this paper is foucs on the mainly contents from following aspects:The Rhodol and phenothiazine fused hybrid NIR fluorescent dye PtzRhlMe has been easily synethsized,which was further developed into mitochondria-targetable NIR fluorescent probe PtzRhlMe-Mito.Based on a built-in reaction site,PtzRhlMe and PtzRhlMe-Mito responded fastly to ClO~-in PBS-Et OH solution through fluorescence ratio changes with high selectivity and high sensitivity.Further,PtzRhlMe and PtzRhlMe-Mito can be used in ratiometric imaging of exogenous ClO~-in living cell and tracing the endogenous ClO~-in stimulated cells.More importantly,the application in mitochondrial labeling and ratiometric tracing ClOof PtzRhlMe-Mito provided a model for constructing ClO~-fluorescent probes with mitochondria-targetability.As such,the rhodamine and phenothiazine fused hybrid NIR fluorescent dye Ptz RhEt has been prepared based on SNArHreaction,which was further developed into mitochondria-targeting NIR fluorescent probe PtzRhEt-Mito.Based on the same sensing mechanism,PtzRhEt and PtzRhEt-Mito responded fastlyto ClO~-in PBS-CH3CN(EtOH)solution through fluorescence ratio changes with high selectivity and high sensitivity.Further,Ptz RhEt can be employed in ratiometric imaging of exogenous and endogenous ClO~-in living cells,while PtzRhEt-Mito can be used in mitochondrial labeling and ratiometric imaging of ClO~-.The results not only provide useful tools for understanding the subcellular roles of ClO~-in physiological and pathological processes,but also present a new method for the development of fluorescent dyes with long-wavelength emission.The rhodamine and naphthocoumarin fused hybrid fluorescent dye NCR with long-wavelength emission has been prepared and further developed into Cu2+-selective fluorescent probe model NCR-Cu.Based on Cu2+-induced rhodamine spirolactam ring-opening and hydrolysis processes,NCR-Cu showed selective“turn-on” fluorescence response to Cu2+ in HEPES-CH3 CN soltution and can be used in imaging of Cu2+in living cell,which provided a model for constructing Cu2+fluorescent probe.Based on the above mentioned model for Cu2+ fluorescent probe,the rhodamine and phenoxazine fused hybrid NIR fluorescent probe PhxRh-Golgi has been prepared based on SNAr H reaction.PhxRh-Golgi exhibited selective“turn-on” fluorescence response towards Cu2+in HEPES-CH3 CN soltution.Cell imaging experiments indicated that the probe can be used in tracing Cu2+ in living cell.Subcellular co-localization experiments showed PhxRh-Golgi exhibits golgi-targetability in the process of tracing Cu2+.The results revealed that excessive Cu2+could exisit in Golgi apparatus of cells.The fluorescence regulation of Fluoran dye was implemented by chemical modification with electron withdrawing group to block the intermolecular PET process.Based on this strategy,two novel fluorescent probes ABAF-Cu and AF-ClO were designed and synthesized.ABAF-Cu exhibited selective “turn-on”fluorescence response towards Cu2+ in HEPES-CH3 CN soltution and can be used in tracing Cu2+ in living cell.While AF-ClO showed selective “turn-on” fluorescence response towards ClO~-in PBS-Et OH soltution and can be utilized in tracing ClO~-in living cell.The results provide new avenue for applying Fluoran dyes to fluorescent probe.