Design And Synthesis Of Novel Fluorescent Probes For Biothiolsin Living Cell Imaging

Low-weight bio-molecules, including amino acids, small peptides, monosaccharides, hormone and cholesterol, play roles as crucial as the organelles in cell. Amino acids are the basic units of various proteins. Monosaccharides are the main source of energy in cells. Level of cholesterol is now an important parameter for the physical condition. Therefore, the detection of the intracellular levels and distributions of various low-weight molecules are significant for biology and pharmacology. However, unlike the detection of the organelles, to visualize small intracellular molecules using optical microscopy or even the electron microscope is impossible. Consequently, many kinds of methods, such as high performance liquid chromatography, chromatography-mass spectrometry and capillary electrophoresis methods plasmon resonance based on nanoparticles, have been developed for the detection of these molecules. Among these methods, optical measurements based on fluorescent probes exhibit many advantages, including convinence performance, low damage to intact cells and permission for visualization in situ, and thus has been widely studied and developed.Molecular functions are usually tightly related to molecular structures, and the roles of small intracellular molecules are thought to be determined by their structures. Molecules with thiol group often exhibits high nucleophilic reaction activities, high ability to bind the metal ions and are easily oxidated to disulfide bond, and thus have distinct intracellular roles. Cysteine (Cys) is a basic amido acid for the synthesis of proteins, and was proved to be relative to the formation of the three dimension structures of proteins. Moreover, Cys could strongly bind to metal ions and balance their intracellular concentration, which protects the cell from damages caused by high concentration of heavy ions. According to many studies, abnormal levels of intracellular Cys are closely related to Parkinson’s disease, Alzheimer’s disease, adverse pregnancy outcomes and many other diseases. Intracellular redox glutathione (GSH) have a balance with its oxidation state (GSSG). GSH could be oxidated to GSSH by cellular oxidizing subjects, which limits the concentration of cellular oxides and prevents the stacking of these compounds. Intracellular concentration of homocysteine (Hcy) is rather low, only5-15μM. However, it was an independent factor for cardiovascular disease, Alzheimer’s disease and many other diseases. Particularly, Hey has attracted more and more attentions and the level of Hey was considered as one of important parameters for disease. Therefore, determination of cellular levels and distributions of various bio-thiols is important for diagnosis, medicine development and keeping health.As stated above, the strong nucleophilic reaction activity of thiols is the base of their distinctive intracellular roles. Simultaneously, this reaction activity also offers the methods for their detection:linking a thiol reactive functional group to a fluorophore could generate a fluorescent probe, which could react with thiols, give fluorescence responses and enable determination of intracellular thiols in situ. When this functional group reacts with specific thiols, this probe could have more specific selectivity. Up to now, many kinds of probes have been developed for the determination of GSH, Cys, Hey or total thiols. Cys and Hey were first detected by an aldehyde group by Strongin and co-workers. After that, many probes with an aldehyde as the receptor group have been synthesized and reported. Cys exhibits relatively stronger reactivity than other thiols, and thus the probes selectively detecting Cys have been developed. For example, Jung and co-workers have employed chalcone as probes for successfully detecting Cys over Hey and GSH. Our group has also reported two fluorescent probes containing aldehyde groups, AM1and CA1, which enables selective detection of Cys. As Hey has a weaker reactivity than Cys, fluorescent probes that could discriminate Hey remain rare so far. Zhang et al. and Strongin et al. have reported fluorescent probes for specific detection of Hey under equimolar measurements. It was worthwhile to notice that the intracellular concentrations of various thiols differ a lot. Cellular GSH has a rather higher concentration of1～10mM, while intracellular concentrations of Cys and Hey were30-200μM and5-15μM, respectively. Accordingly, large interferences from high concentration of GSH is bound to be considered for the detection of cellular Cys and Hey, which raises new challenge for the design of probes.In this work, we have designed and synthesized9probes:CBl and HOTA with olefin aldehyde group; HMCA, BMCA and NMCA with aldehyde group; HMCN with the nitroolefin group; HOVC, HODVC and HOBVC with the vinyl cyanide group.CBl is a fluorescent probe which exhibits3-(4-pyridinvinyl)-9-ethylcarbazole as the fluorophore, and olefin aldehyde as the receptor group. Particularly, a probe CBl molecule could react with two Cys molecules in two steps while could only react with one GSH molecule. CBl itself was weakly fluorescent in the mixture of ethanol and buffer solution, and its fluorescence could be largely enhanced after reaction with one thiol molecule. However, the reaction rates of CB1with one molecule of various thiols differ a lot:the reaction of CB1with one molecule of Cys could be completed in about40min, while the reaction was completed within24h of Hey and7days of GSH. Meanwhile, the product of CBl-Cys and CBl-Hcy could react with one more molecule of Cys and Hey, which could quench the fluorescence of the system. Fortunately, the second step reaction of Cys could be completed in24h, and this reaction of Hey could be completed in7days, which enables CBl a fluorescent probe for discriminative detection of Cys, Hey and GSH at three time windows. Only the system of CBl-Cys exhibits strong fluorescence at40min, while only the system of CBl-Hcy was strongly fluorescent at24h. After7days, the fluorescence of CBl-Cys and CBl-Hcy was quenched, and the system of CB1-GSH was emissive. Considering high selectivity and good two-photon excited fluorescent properties of CB1, the cell imaging experiments with CBl under wide-field microscopy and two-photon microscopy were successfully performed.HOTA is a fluorescent probe which exhibits4-phenolvinyltriphenylamine as the fluorophore, and olefin aldehyde as the receptor unit. Probe HOTA has the same receptor unit as CB1, which could selectively react with Cys in a short response time. The selectivity is super-high and Hcy and GSH could hardly bring interferences. In particular, the concentration of intracellular GSH (1～10mM) is much higher than Cys (30～200μM), and probe HOTA could discriminate Cys over such high concentration of GSH. Consequently, cell imaging experiments were successfully performed with this probe. To testify the source of cellular emission, the inhibitor of GSH synthetase, BSO, has been used to prevent cellular synthesis of GSH before capturing cell images, and then these images were compared with the cells directly stained with HOTA. The experiment results showed that these two kinds of cells possess similar fluorescent intensity, which proved that cellular fluorescence was from the reaction of Cys and the probe.HMCA, BMCA and NMCA were probes with an aldehyde as the receptor group. Due to PET process caused by the aldehyde group, these three probes were weakly fluorescent. The experiment results demonstrated that these probes could react with Cys and Hcy, which bring obvious fluorescent enhancement. Unlike other reported probes with aldehyde, these three probes have stronger interaction with Hcy. With equimolar measurements, HMCA, BMCA and NMCA were Hcy probes. According to the molecular structure of three kinds of thiols, the steric effect should be a key parameter for the selectivity. Therefore, we have designed three molecules HOVC, HODVC and HOBVC, to study the steric effects. HOVC has vinyl cyanide as a receptor unit, HODVC has an unsaturated malononitrile as a receptor unit and HOBVC bearing an unsaturated benzyl cyanide as a receptor unit. The three probes have rather different steric effects, and this study would provide inspiration for molecule design of probes for thiol detection.