Design And Synthesis Of Novel Tetraphenylethene Derivatives For Protein Study

Fluorescence has become a widely-used technique in biosensors and bio-imaging, due to its advantages such as visual facility, real-time monitoring, in situ and non-invasive observation, and more importantly, being capable of analyzing biological events and complex structures on molecular level. Nowadays, a variety of fluorescent dyes have been incorporated into this thriving filed, and the fluorogens with aggregation-induced emission (AIE) property become promising fluorescent materials for detecting and imaging owing to their unique photophysical behaviours that they are non- or weakly emissive in solution but emit intensely upon aggregate formation or other processes involving the restriction of intramolecular motions. Therefore, the design and synthesis of novel AIE-active fluorescent probes and the exploration of their applications in bioassays and bioimaging has grown into a hot, important and frontier research area. Besides the AIE-activity, other requirements such as water solubility, red-to-infrared emission and cyto-compatibility must be matched for bioassay and bioimaging. Consequently, the available AIE-active probes are scarcely reported. Moreover, biomolecules are countless in the natural world but the fluorescent probes suitable for specific biomolecules are only a cantlet in human story. There is a practical demanding for novel fluorescent probes aiming at the detection, analysis and imaging. Considering that proteins are the most abundant in nature, most complex in molecular structure, most diversification in function, this thesis will focus on the design and synthesis of protein-targeted AIE-active fluorescent probes. Furthermore, the interactions between the AIE-active probes and representative proteins, and the tentative application of these probes in bioimaging will be explored.Amino acids are basic building blocks of proteins. A tetraphenylethene (TPE) derivative substituted with electron-acceptor 1,3-indandione (IND) group was designed and prepared for detecting alkaline amino acids. IND-TPE undergoes a hydrolysis reaction in basic aqueous solution, thus the red-orange emission can be quenched by OH- or other species that can induce the generation of sufficient OH-. Accordingly, IND-TPE has been used to discriminatively detect arginine and lysine from other amino acids, due to their basic nature. The experimental data are satisfactory. Moreover, the hydrolyzation product of IND-TPE is weakly emissive in the resultant mixture but becomes highly blue emissive after the illumination for a period by UV-light. Thus IND-TPE can be used as a dual-responsive fluorescent probe.Serum albumin is a typical kind of transport protein. The conformational dynamics of the protein determines its binding property towards various ligands. Through monitoring the fluorescent changes of two exogenous TPE derivatives bearing sodium sulfonate and pyridinium ion, respectively, the conformational behaviors of bovine serum albumin (BSA), in the native, unfolding and refolding states were probed into. As bound to the hydrophobic cavity of BSA, both of the probes displayed an enhanced emission. Their emission changes disclose useful information about the protein’s loading and releasing of charged species:(1) Two dyes were found to specifically bind to subdomain IIA of native BSA and each protein molecule binds a single dye molecule; (2) In addition to hydrophobic interaction, electrostatic interaction also played crucial role in BSA’s responding towards charged species; (3) An intermediate was mapped, in which the binding site of the two probes in subdomain ⅡA. was destroyed and a binding site suitable specifically for the cationic probe became available.Taking advantage of peptides with biological compatibility and high solubility in aqueous solutions, we constructed simple peptide-based fluorescent materials for cell imaging through covalently attaching peptides to tetraphenylethene core. Three kinds of peptides with different sequences and hydrophilic properties were selected. After dispersed in the aqueous media with a relative high concentration, aggregation of the probes occurred naturally, and they inherited high emission efficiency from tetraphenylethene in the aggregate state. Among them, the aggregation of the molecule decorated with the peptide that is rich in basic amino acid residues like arginine was found to be able to penetrate the cell membrane and specifically stain the cytoplasm of the HeLa cells with high contrast. Given that it is particularly demanding for in vivo cell tracing to shift the excitation and emission of the dyes to longer wavelengths, the tetraphenylethene core was modified with tricyanofuran. Togather with the peptide moiety, the whole molecule can be employed as a labeling agent of cytoplasm with red fluorescence.Inspired by some organic small protease inhibitors, which can react covalently with the catalytic active sites of certain proteases, tetraphenylethene derivatives containing protease inhibitor moiety were synthesized and used for detecting targeted proteases, like trypsin. The results indicated that the probes can covalently label the active site of the proteases through the interactions between inhibitor part of the probes and proteases. However, no distinct fluorescent response was observed. The structures of the probes will further be adjusted to make the tetraphnylethene moiety work.