| Reactive oxygen species(ROS), mainly produced from the oxygen metabolism of organisms, are the sign of oxidative stress, and have been revealed to be closely related to aging, cancers, diabetes, neurodegenerative diseases, etc. However, in the normal state, ROS could also participate in the normal physiological process. For example, the burst out of reactive oxygen in the phagosome of neutrophils and macrophages can resist the invasion of pathogens.Hydrogen peroxide(H2O2) is one of the most important ROS, which plays an important role in physiological and pathological process. On the one hand, the high concentration of H2O2 could make cells apoptosis and necrosis, leading to oxidative stress and oxidative damage. On the other hand, H2O2 produced by the stimulus of growth factor, cell factor, neurotransmitter and so on, could be used as a second messenger molecule involved in cell growth, proliferation and differentiation.Compared to other ROS, the reactivity of H2O2 is mild, and it could participate in the signal transduction process through the reversible phosphatase oxidation and thioredoxin reaction.Peroxynitrite(ONOO-), another important ROS, is generated from the diffusion limited reaction between superoxide(O2.-) and nitric oxide(NO). As a strong oxidant and nucleophilic reagent, ONOO-could react with a variety of biological molecules,such as protein, enzyme transition metal centers, lipids and nucleic acid, eventually leading to cell death. As a result, ONOO-has been implicated in many diseases,including neurodegenerative disease(amyotrophic lateral sclerosis, Alzheimer's disease), chronic inflammation and autoimmune disease, ischemia reperfusion injury,and septic shock. At the same time, ONOO-produced in macrophages can kill invading pathogens such as Escherichia coli, Rhodococcus, G's cone insects, which plays an essential role in the immune process. In addition, ONOO-could also serve as a signal molecule involved in the signal transduction process.In this thesis, a new reactive moiety, ?-ketoamide, was designed and integrated into the cyanine or naphthalimide to obtain two small molecule fluorescent probes,Mito-NIRHP and TP-KA, which specificly responded to H2O2 and ONOO-,respectively. They have been used to detect H2O2 and ONOO- in biological systems.The details are as follows:(1) In this section, ?-ketoamide was first used as the responsive group to H2O2. A new fluorescent probe, Mito-NIRHP, characterized of good selectivity, high sensitivity,as well as mitochondria targeting, was obtained by integrating ?-ketoamide into the cyanine skeleton. By using Mito-NIRHP, the endogenous H2O2 in HepG2 cells and living mice were fluorescently imaged. In addition, Mito-NIRHP was utilized as an efficient molecular tool to prove that H2O2 was overgenerated during ischaemia-reperfusion injury(IRI) at both cell and organ level, corroborating the close relationship between H2O2 and this type of organism damage process. This work provides an efficient method for the in situ detection of H2O2 in living organism.(2) In this section, ?-ketoamide was incorporated into 1,8-naphthalimide to obtain a new probe TP-KA. Different from Mito-NIRHP, TP-KA specificly responded to ONOO- without the interference from H2O2, although they had the same reactive group. TP-KA is characterized of good selectivity, fast response, and high sensitivity,making it an ideal probe for intracellular ONOO-. In addition, taking advantage of the two photon emission property of TP-KA, endogenous ONOO- in living cells were visualized by TP-KA by a two-photon fluorescent microscope. |
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