Functional Activity In The Intracellular Species (free Radicals, Metal Ions) Design And Synthesis Of Novel Fluorescent Probe And Its Visualization Imaging Analysis

There exist a large number of minim or trace bio-active substances such as reactive oxygen species and metal ions in organisms. These substances have great effect on life activities and can cause various bio-changes due to their special functions in vivo. On one hand, bio-active molecules are essential to organisms, but on the other hand, they can cause damage in some special conditions like stress. To deeply study the functions of these bio-active substances, we need to achieve their accurately detection. However, bio-active molecules are diverse and complex in organisms. The devolopment of molecular fluorescent probe with high selectivity and sensitivity is one of important hotspots life science studies to vasulize reactive species in living cells of animal and plant.Among the Reactive Species, one of ROS (Reactive Oxygen Species) lipid hydroperoxides (ROOH) was extensively investigated. ROOH appear at an early stage and then degrade rapidly to other products including alkanes and carbonyl compounds. In order to study lipid peroxidation process in detail, it is important to detect ROOH in site. The hydroxyl radical, one of the strongest oxidants known, is thought to be generated in the course of biology metabolism within cells and tissues where it can attack proteins, lipids, and DNA, thus initiating secondary radical reactions that can produce irreparable cellular damage initiating secondary radical reactions that can produce irreparable cellular damage. Currently, it is desired to develop a effective method for detecting lipid hydroperoxides and hydroxyl radicals in biological system, which is not only desired in life science studies, but also the hotspot and difficulties in chemical studies. Besides, iron is an essential element of life. Studies showed that intracellular chelating iron was a crucial pathogenic factor of cell damages. To further study the biological function of ferrous ion (Fe2+), novel detecting method with high selectivity and sensitivity need to be developed.To fleetly detect lipid hydroperoxides, hydroxyl radicals and ferrous ion in plants and animals with high selectivity and high sensitivity, we carried out three aspects of investigation:First, A NIR (Near-infrared) fluorescent probe TCP ( tricarbocyanine diphenylphosphine) including a non-conjugated"pre-tricarbocyanine"was designed and synthesized for visualizing lipid hydroperoxides (ROOH) in living cells. The excitation and emission spectra of tricarbocyanine in NIR region can effectively avoid background fluorescence interference in biological systems. The probe exhibited rapid fluorescence response to ROOH and high selectivity for ROOH over other ROS (reactive oxygen species) and some biological compounds,and the limit of detection was 38 pM. In addition, the probe was stable, and less cytotoxic, results supplied the ideal probe and analytical method detecting ROOH in other cells of animals and plants.Second, based on the mechanism of intramolecular electron exchange interaction, we designed and synthesized a novel fluorescent probe TEMPO-BDP for detecting hydroxyl radicals (?OH) in vivo. The detection was achieved because ?OH could rapidly react with DMSO to produce ?CH3 which then combined with probes and induced the formation of strongly fluoresced product. The probe can rapidly detect ?OH with good selectivity and high sensitivity in simulated physiological condition. Besides, it is stable to light in kinetic assay. The low toxicity probe TEMPO-BDP was also succeeded in appling to detecting and"visualizing"?OH of live RAW264.7 macrophages in both stimulated (PMA) and unstimulated conditions. In addition, TEMPO-BDP was used to detect the content of ?OH in thorax lavage fluids of various rats including normal ones, rats with bleomycin-induced pulmonary fibrosis and cured rats, which demonstrated the practicability of the proposed method in ainmals and plants'samples and cells.Third, we proposed a new method to detect chelating iron in both chemical system and cell plasm. A novel fluorescent probe with two thiophene combined with BODIPY was designed and synthesized to detect Fe2+. The strongly fluorescent probe could chelate with Fe2+, resulting in the fluorescence quenching and the colour change from blue to green. And there was a good proportion relationship between Fe²⁺ concentration and the extent of fluorescence quenching. The results demonstrated that the probe has high selectivity to Fe²⁺, and could be expected to be used as labile iron pool in cell plasm of plants and apply to detecing Fe²⁺ in complex organisms.