Design, Synthesis And Biological Applications Of Novel Fluorescent Probes Based On Cyanine Dye

Cancer is currently one of the deadliest diseases in the world,killing about 10 million people worldwide each year.The accurate diagnosis and effective treatment of cancer are still hot topics that need to be tackled urgently.Chemotherapy is still the priority choice of cancer treatment at present.Previous research in cancer therapeutics has demonstrated specifically targeting cancer cells and delivering cytotoxic drugs was indispensable to promote effective cancer treatment when generating detectable signals.Cells are the basic components of living organisms,responsible for maintaining and performing physiological functions.The dynamic changes of intracellular polarity,p H,viscosity,and bioactive substances in the cellular microenvironment determine the function of intracellular suborganelles.The dysfunction of suborganelles can induce physiological or pathological changes,leading to the occurrence of serious diseases such as cancer,stroke and Alzheimer’s disease.Different pathological process affects the state of cells,such as proliferation,ion transfer,apoptosis,self-repair,endocytosis,etc.Therefore,monitoring the dynamic changes of cell microenvironment has great potential in the study of disease physiological state,early cancer diagnosis and therapeutic effect evaluation.Due to the complexity of cell microenvironment,there is still a large space for accurate,real-time and in-situ monitoring of its changes.Fluorescent probes has caused widespread attention in recent years,benefit from their advantages of straight forward functioning,remarkable selectivity and remarkable sensitivity.With the rapid development of fluorescence detection methods and applications,imaging technology based on fluorescence probes has become an effective way to monitor cell microenvironment due to its non-invasive and real-time properties,and has been widely used in cell sensing,biomedicine and other fields.However,traditional fluorescent probes still have some limitations,such as poor water solubility,serious self-fluorescence interference,poor biocompatibility and low tissue penetration ability.Therefore,it remains challenging to further improve the probe to accurately quantify the fluctuations of various signals in living cells.（1）:In this study,we exploited an innovative fluorophore Cy496 based on light-initiated cleavage reaction.Cy496 contained the typical D-π-A structure,as well as a versatile building block for chemosensor construction through flexible side chains.Spectral experiments confirmed that the typical D-π-A structure endowed Cy496the ultra-sensitive fluorescence behavior toward surrounding environment.By regulation of lipophilicity and basicity through bis-site substitution,we synthesized a series of fluorescence probes by using different substituted groups（Cholesteryl chloroformate,palmitoyl chloride,n-Butyl iodide）as modified groups and screened a novel mitochondria-targeted ratiometric probe Cy1321.The experiments calculation data showed that the chemical modifications enhanced the values of Log P and p Kb in the molecules,which satisfied the criteria for mitochondria accumulation and avoided the lysosomal uptake effects.At the same time,the bis-cholesterol architecture was anticipated to be anchored on the inner membrane of mitochondrial and not affected by the changes of mitochondrial membrane potential（MMP）.In this study,we successfully demonstrated that Cy1321 can real-time evaluate the dynamic changes of mitochondrial micropolarity mediated by bis-cholesterol anchoring.Cy1321 has realized two-color quantification of polarity fluctuations on cisplatin-induced apoptosis through flow cytometry and real-time visualization,and also achieved the purpose of detecting mitochondria-related apoptosis at the level of tissues.It is envisioned that Cy1321 has sufficient capability as a promising and facile tool for evaluation of apoptosis and contributing to therapeutic drugs screening.（2）:In this study,two kinds of heptathylene matrix dyes（Cy796 and Cy758）were designed and synthesized by introducing p-chloromethylbenzene sulfonyl and furan rings as recognition groups and coupling groups,respectively.Cy796 and Cy758 molecules can efficiently produce singlet oxygen.Under the strong nucleophilic attack of H₂S,two probes Cy644 and Cy606 with near-infrared light absorption and blue shift emission were generated respectively,the production efficiency of singlet oxygen decreases and the quenching effect of fluorescence molecular oxidation mediated by singlet oxygen is avoided.Cy796 can produce singlet oxygen under 808 nm laser irradiation,which promotes the opening of furan rings and the binding of RNA around mitochondria,so as to be fixed around mitochondria,realizing the immobilization and continuous detection of tumor cells.In cell imaging experiments,both Cy796 and Cy758 successfully realized fluorescence imaging of H₂S in HCT116 cells.Compared with Cy758,the goal of detection and long-term imaging of H₂S was realized due to the coupling effect of furan rings in Cy796 on cytoplasmic RNA.In tumor imaging experiments,Cy796 has also successfully achieved the effect of long-term targeted tumor fluorescence imaging.Therefore,our study provides a feasible and ingenious strategy for imaging tumor markers over a long period of time.