Synthesis Of Quinoline Bodipy Fluorescent Dye With Its Fluorescence Imaging In Vivo

Fluorescent probe was a functional detection tool that could convert physiological characteristics of living body into changes in optical signals.The switch of fluorescence or the change of fluorescent intensity could be used to detect the abnormal situation in the microenvironment of living organisms.Biothiols,as a very important biological molecule,played a very important role in the physiological metabolism of organisms.When some of thiols'content was abnormal,many diseases may follow soon.Therefore,the detection of biothiols was very important.Apart form the biothiols,viscosity of cell was also an important part of the biological microenvironment which could be used as indicator for apoptosis detection and carcinogenesis.Therefore,in this graduation thesis,the quinoline BODIPY structure was designed to variety of fluorescent probes,including a thiols detected probe based on hydrogen bond-induced dehydrogenation mechanism(Chapter 3),a viscosity probe with a unique RIR mechanism(Chapter 4)and a near-infrared fluorescent dyes with its'materialization applications(Chapter 5).Finally,as enhancing the detection of diseases,the treatment also need to strengthen.Therefore,two D-A-type triphenylamine BODIPY photosensitizers were synthesized for photodynamic therapy(Chapter 6).We hope to use this series of fluorescent dyes to detect micro-environmental abnormalities in the human body and provide a reliable alternative to photosensitizers for the development of photodynamic therapy.The design and construction strategy of these series of quinoline BODIPY dyes were described in second chapter.Firstly,the 8-hydroxyquinoline BODIPY dye was linked with 2,4-dinitrobenzenesulfonyl chloride at hydroxyl position to constructed the Q-BODIPY probe in the third chapter and modified by knoevenagel condensation at the 3.5-position with N-diethylmorpholine 3-furfural and bis-morphomorpholine benzaldehyde to synthesize a viscosity probe based on RIR mechanism(Lys-VBOP)and NIR fluorescent dye(M-BODIPY)in the fourth and fifth chapter respectively.This series of compounds synthesized in this paper have been characterized by hydrogen,carbon and high-resolution mass spectrometry,and both have good performance in their biological applications.In the third chapter,The 2,4-dinitrobenzenesulfonyl chloride and 8-hydroxyquinoline BODIPY dye was used to synthesize probe Q-BODIPY which could preferentially recognize cysteine(Cys)among these thiols.The 2,4-dinitrobenzenesulfonate was introduced as quench group and the fluorescence of quinoline BODIPY was quench through the PET effect.In the the recognition mechanism part,we proposed a bidirectional-inducted dehydrogenation mechanism,which based on the weak basicity of the nitrogen atom on the quinoline structure and the theoretical calculation results for the intermediate state of nucleophilic substitution.Under this special recognition mechanism,Q-BODIPY has a certain degree of preferential selectivity on Cys and allowed Q-BODIPY to quickly recognize Cys in 30s with its?_F change from 0.02 to 0.16.After the Q-BOIDPY probe reacted with Cys,its'molar extinction coefficient changed from 4.8×10⁴ M^-1 cm^-1 to 6.2×10⁴ M^-1 cm^-1and accompany with the color change from pink to yellow.Meanwhile,the concentration titration experiment of Q-BODIPY probe showed the detected limit for Cys was as low as 33 n M.Finally,we applied the probe to cell imaging and zebrafish imaging.The imaging results showed that the probe Q-BODIPY could quickly respond to changes of biothiols in Hep G2 and zebrafish sensitively.In the fourth chapter,a near-infrared BODIPY viscosity probe(Lys-VBOD)with lysosomal localization was synthesized.Along with the increase of the external environment viscosity,the?-bond between the indole structure and the BODIPY fluorophore,the single bond rotation between the quinoline and the BODIPY nucleus both were further limited.Thus,the fluorescence intensity of Lys-VBOD was continuously enhanced with 65 times maximum enhancement.The experimental results also showed that the change of fluorescence intensity has a good linear relationship with the viscosity value.Moreover,the fluorescence lifetime of Lys-VBOD was very promising in high viscosity solvent,which facilitated the fluorescence lifetime imaging in future application.In addition,the Lys-VBOD could work in an environment with a p H of 6 and the BSA titration experiment also proved that the presence of macromolecules and proteins does not affect the response of Lys-VBOD to viscosity.Furthermore,the cellular experiments showed that the fluorescence intensity of Lys-VBOD increased more than three times under the stimulation of dexamethasone.Therefore,Lys-VBOD was a very effective fluorescent probe that could be used to detect changes in lysosomal viscosity in living cells.In the fifth chapter,the novel D-A configuration M-BODIPY dye was composed by linking 8-hydroxyquino BODIPY with p-morpholine benzaldehyde styrene through knoevenagel condensation reaction.In DMSO solution,the maximum absorption wavelength of M-BODIPY was at 698 nm and its'molar extinction coefficient was5.9×10⁴ M^-1cm^-1,the maximum emission wavelength was at 770 nm and its'stokes shift was 72 nm.Besides that,this near-infrared fluorescent dye M-BODIPY also was prepared as silica nanoparticle(M-BODIPY/Si O₂)by forward microemulsion method.The maximum emission wavelength of M-BODIPY/Si O₂ was 726 nm in pure aqueous solution and the fluorescence quantum of M-BODIPY/Si O₂ was 0.33.In addtion,the HR-TEM morphology showed that the M-BODIPY/Si O₂ was spherical particle with uniform size and the DLS test indicated that the average particle size was 43 nm.The M-BODIPY contained a lysosomal localization-morpholine structure with an emission wavelength in the near-infrared region,which can be used for near-infrared localization imaging of intracellular lysosomes in future application.In the sixth chapter,two D-A type triphenylamine BODIPY photosensitizers(TPA-BOP and MA-BOP)were synthesized.Under the illumination of green monochromatic light,the two D-A type photosensitizers could generate the CT(charge transfer)states,thereby promoting the intersystem crossing from the S₁(singlet)state to the T₁(triplet)state.Then,the oxygen was transferd to singlet oxygen during the relaxation effect from T₁ state to LE(local excited)state.The TPA-BOP and MA-BOP photosensitizers have similar absorption peaks at 502 nm and their molar extinction coefficient were 7.43×10⁴ M^-1cm^-1 and 6.18×10⁴ M^-1cm^-1 respectively.Among free-heavy-atoms type photosensitizer,the TPA-BOP and MA-BOP both has a relatively high singlet oxygen yield at 19.28%and 20.12%.Besides that,the MA-BOP with higher singlet oxygen yield and better biological compatibility was chosen for further cell experiments.The dark toxic experiment showed MA-BOP had a low cell cytotoxicity at concentration of 2-12?M and the phototoxicity test indicated that,after ten minutes of irradiation,MA-BOP can eliminate more than 50%of A-549 cells with the concentration at 12?M.Furthermore,the intracellular photodynamic therapy showed that as the exposure time increased,more and more cells killed by singlet oxygen were stained red by EB.The results of intracellular PDT experiments prove that MA-BOP photosensitizer can be used as a potential material for photodynamic therapy and the triphenylamine BODIPY materials presented in this paper can provide a good candidate for the future synthesis of D-A type photosensitizer.