Design And Synthesis New Types Regional Location Of Fluorescence Probes For Intracellular Radicals And Their Application In Vivo

Cell is the basic unit of life about biological morphology, structure and biological activities. A large number of minor or trace amounts of reactive species (small molecules and ions), such as a variety of reactive oxygen species (mainly superoxide anion radical O₂^-·, hydrogen peroxide H2O2, hydroxyl radical·OH, lipid hydroperoxide ROOH, lipid free radicals ROO·, peroxide nitrosyl ONOO-, nitric oxide NO, etc.), various metal ions (such as iron ion Fe²⁺ or Fe³⁺, zinc ion Zn²⁺, copper ions Cu²⁺, etc.), the intracellular activity species through a variety of biochemical reactions involved in the regulation of apoptosis, variation and differentiation, signal transduction and other physiological functions and phenomena. Proper amount of these active species are essential to organisms, but in some special environments (such as oxidative stress), the bio-active molecules will cause damage to organisms. Because the cell morphology of different organelles and different functions in the same cell organelles in different species exist in the biological activity of the type and concentration are not the same. Such as mitochondria is the main existing sites of O₂^-·and other reactive oxygen species (ROS), the cytoplasm and plasma membrane have a lot of Fe²⁺ or Fe³⁺, acidic organelles such as lysosomes of H+ concentrations were higher, prominent vesicles in the nerve cells there is a higher concentration of Zn²⁺. If the different organelles within the existing functionality of the active species can be timely and accurate regional localization test, then either a more detailed understanding of reactive species production and role of the mechanism of biological function, but also further sub-cellular levels of the different organelles function, allowing the generation of these active species, the existence of mechanism of action much more refined and profound.To date, several methods for O₂^-·detection include electron spin resonance spectroscopy, chemiluminescence, fluorescence and chromatography, etc., in which fluorescence as high sensitivity, good selectivity, but simple, for the activity determination of the species. The fluorescence and laser scanning confocal microscopy, to effectively implement the ROS in living cells and tissues such as functional active species "real-time, visual, quantitative," the high sensitivity detection, not only for in situ real-time dynamic monitoring, with fast experiment, easy sample handling, comprehensive data, etc., but also displays a single organelles within cells and fluorescence intensity changes will increase the single cell level and subcellular level detection. Thus, the development of high selective and sensitive fluorescent probe molecules, the dynamic active species in situ visualization of image analysis within a given region in living cells of plants and animals is currently a major focus and the difficulty one of life science research fields.In this paper, based on the changes in spectrum characters of the fluorescent probes reacting with ROS or bio-active molecules, we detected these specific compounds. The designed fluorescent probes were also applied to confocal imaging of several kinds of cells, which showed the localization of living cells. Mainly carried out in the following two aspects of dissertation:First, we designed and synthesized a new near-infrared (NIR)fluorescence probe for detecting O₂^-·. The structure of the probe consists of two parts: one is with carboxylic Cy7 as the NIR fluorophore; the other is benzothiazolinone as the superoxide anion response group. It was synthesized conveniently by only two steps. Structure of the Cy-DBZ was characterized by ¹H NMR and ¹³C NMR, and fluorescence properties of the reaction between Cy-DBZ and O₂^-·were optimized in chemical system. It showed that the probe possessed potent selectivity to O₂^-·and could respond to changes of O₂^-·concentrations. In addition, the experimental results showed that the probe successful targeted mitochondria, and specific responsed to the changes of O₂^-·concentrations.Second, we designed and synthesized two mitochondrial-targeted NIR fluorescent probes for detecting O₂^-·in living cells. Experimental results showed that the probes can be detected O₂^-·with selectivity in the near infrared region, and the probes successful realized mitochondrial CPC Focus fluorescence imaging analysis through introduction of phosphonium head groups and lysine groups.