Design Of BODIPY-based Fluorescent Probes With Large Stokes Shifts And Its Application In Analysis

The development of fluorescence analysis is not only inseparable from the development of instruments,but also closely related to the design and synthesis of fluorescent probes.Small organic molecule fluorescent probes have advantages of high sensitivity,good selectivity and low toxicity,and can combine with fluorescence spectrophotometer,high performance liquid chromatography(HPLC),capillary electrophoresis(CE),fluorescence microscope to achieve detection of ions,small molecules and biomacromolecules,such as DNA,proteins and polysaccharides.Till now,fluorescent probes have been widely used in analytical chemistry,biomedicine,food science,environmental science and other fields.BODIPY-based probes have excellent properties including high molar absorption coefficient,high fluorescence quantum yield,good light stability,and insensitivity to pH.However,the Stokes shift of BODIPY is always as small as 5-15 nm.The small Stokes shift leads to a large overlap of the absorption and emission spectra,which cause self-absorption and excitation scattered light interference,and then greatly reduce the signal-to-noise ratio and accuracy of the detection.We have summarized the existing methods for improving the Stokes shift of BODIPY probes,and proposed an effective strategy to extend the Stokes shift of BODIPY probes by introducing substituents at the 3,5-position phenyl.According to such strategy,five BODIPY-based fluorescence probes with large Stokes shifts were designed:4,4-difluoro-8-phenyl-(4-oxy-aceticacid-N-hydroxysuccinimidylester)-3,5-bis(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indancene(MOOAPB-OSu)for aminocompounds,4,4-difluoro-8-(3,4-diaminophenyl)-3,5-bis(2,4-dimethoxyphenyl)-4-bora-3a,4a-diaza-s-indancene(DMOPB)fornitricoxide(NO),4,4-difluoro-8-{4-(2,4-dinitrophenoxy)phenyl}-3,5-bis(2,4-dimethoxyphenyl)-4-bora-3a,4a-diaza-s-indancene(DMOEPB)and4,4-difluoro-8-(4-nitrophenyl)-3,5-bis(2,4-dimethoxyphenyl)-4-bora-3a,4a-diaza-s-ind ancene(DMONPB)forhydrogensulfide(H₂S)and4,4-difluoro-8-(3,4-diaminophenyl)-3,5-bis(4-methoxyphenyl)-2-sodium sulfonate-4-bora-3a,4a-diaza-s-indancene(MOSPB)with improved water-solubility for NO.In this thesis,we successfully synthesized the above five kinds of fluorescent probes and carried out the following works:(1)By introducing methoxyphenyl group into the 3,5 position of BODIPY and taking N-hydroxysuccinimide ester as reactive group,a novel amino compounds derivative reagent MOOAPB-OSu with long wavelength and large Stokes shift was synthesized.The structure and fluorescence properties were characterized and researched.The maximum excitation and emission wavelengths are 573 nm and 615nm,and the Stokes shift is 42 nm.The fluorescence quantum yield of MOOAPB-OSu is 0.57,and the fluorescence quantum yield of MOOAPB-methylamine by MOOAPB-OSu reacting with methylamine is 0.59.Firstly,a new method for the separation and determination of eleven aliphatic amines by HPLC-fluorescence detection(HPLC-FD)was developed using MOOAPB-OSu as derivatization reagent.The derivatization conditions were as follows:a certain amount of mixed amines and 25 folds of MOOAPB-OSu was derivatized in CH₃CN:borate buffer solution(120 m M,pH 7.2)=1:1(V/V)at 25? for 4 min.Under optimized separation conditions,eleven aliphatic amines were separated within 19 min.The established method has been successfully applied to separate and detect aliphatic amines in lake water,beer and red wine.The spiked recoveries were 91.0%-108.8%and RSDs were 0.7%-5.0%.Secondly,a new method for the separation of glutamic acid,glutamine and?-aminobutyric acid by HPLC-FD was also established with MOOAPB-OSu as the derivatization reagent.The derivatization conditions were as follows:a certain amount of three amino acids and 12 equvi.MOOAPB-OSu were reacted in CH₃CN:borate buffer(40 m M,pH 8.4)=1:1(V/V)at 25? for 10 min.Three amino acid derivatives achieved baseline separation within 14 min under optimized separation conditions,and the method has been successfully applied for the separation and determination of glutamic acid,glutamine and?-aminobutyric acid in vegetables.The spiked recoveries were from 95.5%to 108.9%,and the RSDs were from 0.7%to 7.6%.The method for separation of fluorescent detection of amino compounds by high performance liquid chromatography based on MOOAPB-OSu has the advantages of simple operation,rapid sensitivity,good selectivity,which can be used for separation and detection of amino compounds in various complex samples.(2)We propose a simple strategy to introduce more methoxy groups on phenyl to further extend the Stokes shift of BODIPY based on quantum chemical calculations,and a"turn-on"NO fluorescent probe DMOBP has been designed and synthesized.Its structure and fluorescence properties were characterized and studied.The fluorescence quantum yield of DMOPB is only 0.002,while the fluorescence quantum yield of the triazole derivative DMOPB-T formed by DMOPB reacting with NO is 0.32.The excitation and emission wavelengths are 574 nm and 622 nm,respectively,and the Stokes shift is 48 nm.Under physiological conditions,DMOPB can react with NO within 2 min,and the fluorescence intensity has a good linear relationship with the concentration of NO from 0.5?M to 4?M.The limit of detection(LOD)is 1 n M.In the MTT assay,cell viability rates of 10?M and 50?M DMOPB were 83.3%and 77.9%,which can meet the requirements of cell imaging.In terms of analytical method development,firstly,a fluorescence imaging analysis method for NO in cells and tissues was developed.The long wavelength emission of DMOPB and the large Stokes shift can avoid the interference of background fluorescence in cells and tissues,and also reduce self-absorption and the interference of scattered light,thus improving the sensitivity of the probe.The method can be used for imaging endogenous and exogenous NO in Hela cells and HUVEC cells,and can also image NO in liver tissue and onion epidermal tissue.The method can observe the changes of NO concentration in cells and tissues simply and sensitively,and provides a visual means for studying the relationship between disease and NO.Secondly,a new method for the determination of NO in tomato,cucumber and onion by HPLC-FD with DMOPB as derivatization reagent was established.A certain amount of NO and 3 equiv.DMOPB were reacted in a medium containing CH₃CN:phosphate buffer solution(25 m M,pH 6.5)=70:30(V/V)at 30? for 9 min.The recoveries ranged from 96.4%to 100.8%,and the RSDs were from 0.8%to 6.6%.The method has the advantages of simple and efficient,good selectivity and high sensitivity.Considering the lack of methods to increase the Stokes shift of BODIPY-based fluorescent probes,our proposed strategy of simply extending the Stokes shift by introducing substituents has great potential.(3)In order to verify the universality of the strategy for synthesizing BODIPY-based probes with large Stokes shift proposed in the second section,two H₂S fluorescent probes DMOEPB and DMONPB have been designed and synthetized by using the same fluorophore of DMOPB.Their reactive groups were m-dinitrophenyl ether and nitro group,respectively.Their structures and fluorescence properties were characterized and studied.Their Stokes shifts are 49 nm and 51 nm,respectively.For DMOEPB,since the electron withdrawing group nitro is separated from the fluorophore by two benzene rings which are not conjugated to the fluorophore,thus the fluorescence of the probe is not effectively quenched by the photoinduced electron transfer mechanism(PET)with fluorescence quantum yield of 0.16.After m-dinitrophenyl ether is removed,the change of fluorescence intensity and wavelength are not obvious,which cannot be applied to the biological imaging of H₂S.For DMONPB,it has almost no fluorescence,and the fluorescence quantum yield is 0.007,while DMOAPB is generated after reacting with H₂S,which emit at 627 nm and has a fluorescence quantum yield of 0.13.DMONPB can react completely with100 equiv.H₂S in PIPES(50 m M,pH 7.4)buffer solution containing 50%DMSO at37? for 40 min.An excellent linear relationship was observed in the range of H₂S concentrations from 20?M to 800?M and the detection limit was 1.3?M.DMONPB shows excellent properties such as good photostability,insensitive to pH,low cytotoxicity,specificity for H₂S,and has been used in cell and tissue imaging.This work validated the feasibility of the strategy of synthesizing probes with large Stokes shift,and also provides new ideas and experience for designing the same type of probes.(4)A novel BODIPY-based probe MOSPB with large Stokes shift and water solubility was designed and synthesized by introducing p-methoxyphenyl group at the3,5 position and sodium sulfonate at the 2 position of BODIPY core.The structure and fluorescence properties were characterized and studied.The saturation concentration of MOSPB in pure water is higher than 0.61 g/L and the Stokes shift is54 nm.In aqueous solution,the fluorescence quantum yield of MOSPB is 0.004,while the fluorescence quantum yield of MOSPB-T formed by MOSPB reacted with NO is 0.17.MOSPB and NO can react completely at 35? for 12 min in PBS,which is not interfered by other coexisting ions and free radicals.There is a linear relationship between the fluorescence intensity and the concentration of NO in the range of 0.5?M to 5?M with a detection limit of 5 n M(S/N=3).The established method has been used to detect NO in immature and fully mature bananas and kiwifruit,and found that the levels of NO in immature bananas and kiwifruit were 5.3nmol/g and 32.4 nmol/g,respectively,while the full maturity were 2.6 nmol/g and13.9 nmol/g.The range of recoveries is from 92.4%to 103.4%,and the range of RSDs is from 1.2%to 6.7%.The derivatization process of the method can carried out without organic solvent and complicated pretreatment.It can be directly used for the determination of NO in biological samples,and has important significance for studying the action mechanism of NO in plants and the preservation and storage of fruits.