Construction And Application Of Novel Pyridine/pyridinium Modified Bodipy Fluorescent Dyes

Mitochondria provide the cells with the energy they need,and changes in their quantity,structure,and distribution will affect the life activities of the cells and are closely related to various diseases.Hypochlorous acid(HOCl),as a kind of reative oxygen species(ROS),plays an important role in the innate immune system.It has bactericidal activity,helps the host resist the invasion of microorganisms,and can maintain the balance in the body,but over-produced hypochlorous acid may cause oxidative stress and cause various diseases.Therefore,visual tracking of the changes of mitochondria themselves and various active substances in organisms has very important biological significance for deepening understanding of the physiological and pathological rules.Although there are many types of fluorescent probe detection of hypochlorous acid but still exist many drawbacks,including water soluble,poor sensitivity,slow response and shorter wavelength(600 nm)and the orientation of the lack of mitochondrial or effect is not good,so the design with the performance of real-time detection of hypochlorous acid in the organelles of long wavelength fluorescence probe is still very difficult.According to the research progress of pyridine / pyridinium BODIPY fluorescent dyes,there are still bottleneck problems at this stage: the meso-position pyridinium BODIPY dye is usually a symmetric structure,which leads to limited dye structure modification;The synthetic routes of pyridine-subtituted dyes other than meso derivatives are complicated.They lack systematic study of the mitochondrial targeting ability of non-meso pyridinium BODIPY dyes.Therefore,we carried out a series of research work in Chapter 2: introduced pyridyl / pyridinium at 1-position,3-position,and 7-position,respectively,and instablled methoxyphenyl group at 3-position and/or 5-position to construct a series of new pyridyl / pyridinium BODIPY fluorescent dyes.We optimized the synthetic route of pyrrole and used a strong base Na H to prepare pyridyl azetidine propylene in situ.Subsequently,we successfully synthesized 8 pyridylpyrroles using one-pot method.Then we carried out the synthesis of pyridyl BODIPY dye,and found that only initial formylation on pyridylpyrrole could afford success condensation reaction with another molecule of pyrrole.We speculated that pyridyl is an electron-withdrawing group.When introduced into pyrrole,the density of the electron of ?-position of pyrrole will be reduced,and electrophilic substitution reaction is reluctant to occur.Therefore,we optimized the synthetic route of pyridyl BODIPY dyes and successfully prepared 10 pyridyl BODIPY fluorescent dyes.After that,some pyridyl BODIPY fluorescent dyes were quaternized,and 9 pyridinium BODIPY fluorescent dyes were successfully obtained.Then we explored the spectral properties of 10 pyridyl BODIPY fluorescent dyes and 9 pyridinium BODIPY fluorescent dyes,and conducted mitochondrial localization investigations on 3 of the pyridinium BODIPY fluorescent dyes.Through the study of their photophysical properties,we found that because as an electron-withdrawing group,pyridyl on BODIPY afforded the ICT effect to cause redshift of the absorption maximum and emission maximum.We also observed that the increased number of pyridine moiety on BODIPY further caused bathochromic shift of the absorption maximum and emission maximum.This is because the increase in the number of pyridyl groups strengthens the ICT effect in the BODIPY molecule,driving its maximum absorption wavelength and maximum emission wavelength further red shift.Then we found that the conformationally-restricted BODIPY dye with fixed cyclic structure to form a rigid planar structure can enhance the degree of conjugation of the BODIPY dye,which can extend the absorption maximum and emission maximum,enhance molar extinction coefficient and Fwhm.However,Stokes shift was decreased.In addition,we carried out pyridinium formation of ortho-pyridyl BODIPY dye A-1,meta-pyridyl BODIPY dye A-2 and para-pyridyl BODIPY dye A-3.We found that the corresponding absorption and emission wavelengths produced various degrees of redshift due to the ICT effect of electron-withdrawing pyridine substituent.The pyridinium species will make the dye wavelength redshift.In addition,the optical properties of the ortho-pyridinium salt BODIPY dye JA-1,the meta-pyridinium BODIPY dye JA-2 and the para-pyridinium BODIPY dye JA-3 are slightly different.The absorption and emission wavelengths for JA-2 are least red-shifted and the molar extinction coefficient increases,while the maximum absorption and emission wavelengths of JA-1 and JA-3 are red-shifted and the molar extinction coefficient decreases.We speculated that this is due to the difference in inductive effect of the nitrogen atom on the pyridinium group and the steric hindrance caused by methyl group.Finally,we conducted mitochondrial colocalization experiments.It was found that the pyridyl BODIPY dye may enable mitochondrial localization ability after quaternization.JA-1 and JA-2 had better mitochondrial localization ability and the co-localization coefficient was basically over 0.75.Aiming at the systematic regulation of pyridine / pyridinium BODIPY fluorescent dye structure and related research,we can construct mitochondrial positioning dyes with excellent performance.In Chapter 3,we chose the red wavelength BODIPY dye,and introduced N,N-dimethylthiocarbamate as the hypochlorous acid recognition group at the meso position of the dye through immolative linker to develop DM-BDP-OCl as probe of HOCl.Due to the PET effect,the probe DM-BDP-OCl has almost no fluorescence.When used for hypochlorous acid detection,the recognition group of the probe is oxidized to release phenolate.The phenolate further undergoes immolative reaction to generate DM-BDP with strong red fluorescence.Through systemic investigations,it is found that DM-BDP-OCl provides specific and sensitive detection of hypochlorous acid over other ROS / RNS.Upon treatment with hypochlorous acid,the fluorescence intensity increases rapidly by 112 times within 1 minute,and reaches relative saturation(170 times)after 10 minutes.Good linear relationship in the hypochlorous acid concentration range of 0–50 ?M could be obtained with a detection limit of 60 n M.These characteristics enable the probe DM-BDP-OCl to rapidly detect concentration fluctuation of exogenous and endogenous hypochlorous acid in He La cells and RAW 264.7 cells.The reaction mechanism and PET effect of the probe were verified by mass spectrometry and density functional theory(DFT)calculations,respectively.This work provides a reliable and convenient imaging detection method for exploring the physiological and pathological functions of hypochlorous acid.At the same time,it provides a theoretical basis for the further application of hypochlorous acid recognition group to the new pyridyl BODIPY dyes to develop a long-wavelength new fluorescent probe with mitochondrial targeting function.