The Studies On Photo-regulation Of Bio-molecule Activity By Caging Group With Exact Spatiotemporal Resolution

Photo-labile caging compound offer an ideal method for regulating cellular or tissue functions with a high spatiotemporal resolution and therefore, have become increasingly important in the Chemcal Biology research. In this dissertation, we firstly reviewed the research history and recent development of caging compound and its application in biosystem. Nevertheless, the existing photo-caging groups are still limited by various unfavorable properties including poor water solubility, slowly photolysis sensitivity, and lower caging and uncaging efficiency. Current research is focus on improve the physical and chemical properity of caging group which based on coumarin and quinoline group, and exert its further applications in precisely probing biological systems.We firstly synthesized a series of quinoline-based photo-removable caging groups by changing the position of the hydroxyl and bromine group or incorporate an aromatic heterocycle into the chromophore. Among these quinoline-based derivatives, 7-hydroxy-8-pyridin-2-yl quinoline exhibit dramatically better water solubility (nearly 10 folds),1.5 fold photolysis sensitivity and much lower self-fluorescence than BHQ, which should contribute to further applications for photo-regulation of various bioactive molecules under physiological conditions. Furthermore, we have demonstrated in the present study that the replacement of a halogen group by a pyridine-like heterocycle may provide a general strategy to improve the existing photo-caging groups.Coumarin and quinoline-based caging groups were further attempted to control Thrombin aptamer and Green fluorescence protein (GFP) activity with exact spatiotemporal resolution in living system. Through multiple-caging strategy, compounds could bind with target nucleic acid conviently. The test result indicated that the specific affinity of aptamer to thrombin was successfully blocked by caging with both 8-bromo-7-hydroxyquinoline chromophore (BHQ) and 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc) and the effect was partially restored with subsequent transitory period illumination. Compared with Bhc caging, BHQ exhibited bout 40% percent higher restoration (uncaging) efficiency and substantially improved photolysis sensitivity. Moreover, BHQ was successfully used in cellular system to light-control GFP activity with high spatial and temporal resolution.Additional, zinc(â…¡) probe FluoZin-3AM was used to distinguish different stages of apoptotic cell according to trace intracellular zinc(â…¡) fluorescence flux. FluoZin-3AM was found cell permeable to detect the intracellular zinc(â…¡) fluorescence enhancement within transitory period with low cytotoxicity. Confocol and cytomety test demonstrated that FluoZin-3AM was selectively and strongly stained apoptotic cells only at early and middle stages, which was attributed to significantly increased free zinc(â…¡) flux during these stages. Compared with Annexin-V-FITC and PI, zinc(â…¡) probe FluoZin-3AM was found to be a suitable probe acting alone to distinguish the stage of cell apoptosis.