Development And Application Of Coumarin-semicyanine Dyes With High Fluorescence Quantum Yield

Fluorescence technology has many advantages for biomedical research,including non-invasiveness,high spatial and temporal resolution,and real-time imaging.It has been widely used in various fields of biomedical research,particularly in the fluorescence diagnosis of cancer,which has greatly promoted the progress of biomedical research.Additionally,as photosensitizers for photodynamic therapy（PDT）usually come from fluorescent dyes,fluorescence technology has also greatly promoted the development of PDT.In fact,fluorescent dyes are the material basis of fluorescence imaging and PDT.The photophysical properties of fluorescent dyes,such as absorption/emission wavelength,molar absorption coefficient,fluorescence quantum yield,etc.,largely determine their application effect.However,although fluorescence microscopy equipment has developed rapidly in recent years,the development of fluorescent dyes has been relatively slow.Currently,the most commonly used fluorescent dyes are still mainly concentrated in several traditional dye families,such as cyanines,rhodamines,and boron-dipyrromethenes（BODIPYs）.Although the fluorescence quantum yield of these dyes has been improved by means such as conformational restriction and suppression of twisted intramolecular charge transfer（TICT）,these dyes usually have a small Stokes shift,which will suffer from the interference of excitation light scattering and spontaneous fluorescence of biological molecules in practical applications.Therefore,developing fluorescent dyes with a large Stokes shift and bright fluorescence is currently the focus of fluorescence imaging and PDT fields,but it is also a challenging task.Coumarin-semi-cyanines（CHCs）are hybrid molecules of coumarin dyes and cyanine dyes,with a largeπ-conjugated system.They are characterized by a large Stokes shift and long emission wavelength（over 650 nm）and have been widely used to prepare fluorescent probes and applied in biological imaging.However,reported CHCs typically have a low fluorescence quantum yield,which reduces the spatial and temporal resolution of related fluorescent probes.We speculate that the low fluorescence quantum yield of CHCs may be due to non-radiative transitions caused by C=C bond photoisomerization.Therefore,suppressing C=C bond photoisomerization should be an effective strategy to improve their fluorescence quantum yield,while retaining the advantages of large Stokes shift of traditional dyes.In this study,starting from molecular structure design,we designed and synthesized a new class of conformationally restricted cyclic coumarin-semi-cyanines（RCHCs）by introducing a bicyclic structure into the molecular skeleton of traditional CHCs to suppress C=C bond photoisomerization.The results show that RCHCs not only inherit the advantages of large Stokes shift and long-wavelength absorption/emission of traditional CHCs but also exhibit significantly improved fluorescence quantum yield.Based on the RCHCs platform,we further developed fluorescent diagnostic reagents for tumors and photosensitizers for PDT and explored their applications in live-cell fluorescence imaging,fluorescence diagnosis of cancer cells,and tumor PDT at the cellular and in vivo levels.The main research contents of this study are summarized as follows:In Chapter 2,a series of cyclized flavone-semiquinone（RCHCs）dyes and their corresponding linear CHCs were designed and synthesized by introducing a bicyclic structure into the molecular structure of traditional CHCs dye,and by restricting the rotation of their C=C double bonds.The results indicated that the RCHCs with conformational constraints inherited the inherent large Stokes shift characteristics of traditional CHCs dye and showed a significant improvement in their fluorescence quantum yield compared to CHCs.Moreover,cellular imaging experiments showed that RCHCs exhibited not only significantly higher intracellular fluorescence brightness than CHC1,but also good targeting ability for lysosomes（RCHC1）and mitochondria（RCHC2）.Due to their large Stokes shift,this class of dyes showed a significantly improved spatiotemporal resolution in cellular imaging.These dyes have been successfully applied to the study of mitochondrial autophagy.In Chapter 3,a conformationally restricted,carboxyl-functionalized flavone-semiquinone dye,RCHC1-COOH,was synthesized by introducing a carboxyl functional group into the indole unit of traditional CHCs dye and further cyclizing the structure.Cellular imaging experiments showed that this fluorescent dye could specifically target the cell membrane.Further co-localization imaging studies indicated that the dye was mainly enriched in lipid rafts,which are microdomains of the cell membrane that are rich in lipids and cholesterol.Given that tumor cells express a higher level of lipid rafts than normal cells,this dye has been successfully used to distinguish between cancer cells and normal cells,as well as between cancer tissues and normal tissues,with a contrast far greater than the clinical threshold of 2.0.In Chapter 4,a photosensitizer,RCHC2-I,with photodynamic therapeutic effect was designed and synthesized by introducing an iodine atom into the indole ring of a conformationally restricted flavone-semiquinone dye.The iodine atom could induce a heavy-atom effect,accelerating the intersystem crossing（ISC）rate from the singlet excited state(S₁)to the triplet excited state（T₁）and enhancing the photodynamic therapeutic effect of the photosensitizer.Further studies showed that RCHC2-I could form J-aggregates under physiological conditions,reducing the energy gap between the S₁ and T₁ states and further accelerating the ISC from the S₁ to T₁ state,thus greatly improving the photodynamic therapeutic effect of the photosensitizer.The photodynamic therapeutic effect of the photosensitizer was confirmed in cellular and tumor mouse models:after incubating cancer cells with the photosensitizer and irradiating them for 15 minutes,almost all the cancer cells died after 24 hours;after injecting the photosensitizer into the tumor of a mouse and irradiating it for 30 minutes,the tumor volume decreased day by day and almost disappeared after 10 days.