Synthesis Of Novel Hydrogen Ion-sensitive Fluorescent Probes And Their Application In Cell Imaging

Objective:Benzimidazole and its derivatives were connected with a fluorescent group through C=C bond to form a series of new hydrogen ion sensitive fluorescent probe with benzimidazole as the hydrogen ion response part,fluorescent group as the signal emission part and ethylene bridge as the connection part.Two types of probes were designed and synthesized.The first type is probe C2(4-(2-(1H-benzo[d]imidazol-2-yl)vinyl)-N,N-dimethylaniline),which is based on benzimidazole bridged dimethylamine.The second type is a series of probes C4(4-(2-(5-halo-1H-benzo[d]imidazol-2-yl)vinyl)-N,N-diphenylaniline),which are divided into C4-F,C4-Cl,and C4-Br,C4-I,based on halogenated benzimidazole bridged triphenylamine.Study the properties of the two types of probes such as UV absorption and fluorescence spectroscopy,fluorescence stability,reversibility,selectivity,biocompatibility,lysosome targeting,etc.,and make preliminary explorations of their application in cell imaging.Methods:1.To synthesize the above two types of probes through a one-step condensation reaction and to purify the products.2.The probes structure was basically characterized by means of NMR,mass spectrometry,etc.The optical indications and response performance of the synthesized probes were detected by measuring the absorption spectrum and the fluorescence spectrum.According to application needs,calculate the fluorescence quantum yield and fluorescence lifetime of the probes.Analyze the probes'hydrogen ion response and fluorescence mechanism by means of acid-base titration and proton nuclear magnetic resonance spectroscopy.3.The light stability of the probes was studied by monitoring the fluorescence intensity of the probes solution for a long time.The reversibility of the probes response to hydrogen ion was studied by repeated acid-base titration in the probe solution.The selectivity of the probes to H⁺was analyzed by adding appropriate amount of interfering ions to the probes solutions with different pH values.4.We use the MTT method to analyze the damage of the probes to cell in preparation for subsequent cell research.5.By analyzing the Pearson's co-localization coefficient of the synthesized probes and the commercial lysosome targeting probe in the cell fluorescence image,the ability of the probes to aggregate in the lysosome was studied,and its targeting mechanism was discussed.6.The response of the probes to intracellular hydrogen ion was observed by laser confocal microscope.Cells were stimulated with chloroquine,NAC and H₂O₂ to study the ability of the probes to monitor the spontaneous pH changes of cells.The cells were only treated with Hanks'balanced salt solution to induce autophagy,and the ability of the probe to monitor autophagy was studied.Results:1.Two types and five new probes were synthesized,and the success of the synthesis was proved by means of analytical chemistry.2.Probe C2 has double absorption and double emission fluorescence characteristics,and exhibits ratio-based fluorescence properties.Its luminescence mechanism is:after protonation of nitrogen atom on benzimidazole group C=N,diphenylamine transfers electrons to benzimidazole through ethylene bridge,causing the change of fluorescence wavelength.The luminescence mechanism of the series of probes C4 is similar to that of probe C2,but they generally have strong single emission fluorescence signal.Among them,C4-F and C4-Cl have relatively stable fluorescence quantum yields of 0.26 and0.22,respectively.The series of probes C4 all have two fluorescence lifetimes,one long and one short,and all belong to the static quenching fluorescence type.3.The probe C2 has good optical stability,reversibility and selectivity,and the series of probes C4 have good optical stability,reversibility and selectivity.4.The results showed that the cytotoxicity of probe C2 was weak and the biocompatibility was good.Among the series of probes C4,the cytotoxicity of C4-F and C4-Cl was relatively weak,and C4-Br and C4-I were safe to cells in a lower concentration range.5.Both types of probes have good lysosomal targeting ability.The Pearson's colocalization coefficient of probe C2 was 0.9099,with imidazole and dimethylamino groups as targeting groups.The Pearson's colocalization coefficients of the series of probes C4 were all greater than 0.89,the highest or even more than 0.94,and the targeting groups are imidazole groups and halogen atoms coupled to the benzene ring.6.The two types of probes performed well in cell pH quantitative ability,cell identification ability and monitoring ability of cell physiological changes.Probe C2 showed good cell autophagy monitoring ability.Conclusion:1.Probe C2 can assist in monitoring the occurrence and development of autophagy.It is a good probe for cell imaging and has great potential for tissue or in vivo imaging.2.The series of probes C4 can target cell lysosomes well,and monitor the pH value of cell lysosomes in real time.It can also respond quickly to changes in the pH value of cells due to stress.It is a kind of hydrogen ion sensitive fluorescent probe with good performance and certain application potential.