| Superoxide anion radical(O2·-)is an important anion radical in the biological and medical fields,etc.O2·-is a toxic by-product through partial reduction of oxygen during aerobic metabolism.In addition,O2·-is also a primary component of reactive oxygen species.The reactive activity of O2·-is poor,but it can transform into other highly active substances in the presence of other chemicals species.Therefore,O2·-can play crucial roles in causing adverse effects of DNA damage,polysaccharide depolymerization,lipid peroxidation,mammalian cell death and enzyme inactivation and so on.In view of the above important role of O2·-,it is vital to make use of imaging technology to study and analyze O2·-level in biological research,exploration of disease mechanisms,early clinical diagnosis,and intervention therapy.Bioluminescence imaging technology is a sensitive,reliable,convenient and non-invasive method for optical imaging based on enzymatic reactions.Due to no external excitation,bioluminescence imaging avoids the high background signal from tissue autofluorescence.Therefore,this method is particularly suitable for the study of organisms.The firefly luciferase-luciferin pair is one kind of a classical bioluminescence system.In the presence of adenosine triphosphate,oxygen,and magnesium ions,the firefly luciferase catalyzes the decarboxylation of the luciferin or aminoluciferin substrate,emitting visible photons.Based on the modification of D-luciferin molecules,herein my thesis focuses on the developing one new technology for bioluminescence imaging detection of O2·-in cells and animals,including the following two parts:In first section,a novel bioluminescent probe phosphinate-luciferin for specifically recognizing O2·-was designed and synthesized.The probe was constructed by the modification of diphenylphosphonyl chloride on the 6’hydroxyl of D-luciferin molecule.Two experimental routes were screened and the optimized synthesis route was found to be that 2-cyano-6-hydroxybenzothiazole first reacted with diphenylphosphinl chloride,and then followed by cysteine monohydrate for spontaneous cyclization.In addition,we used high performance liquid chromatography,nuclear magnetic resonance and mass spectrometry methods to verify the purity of the product and characterize its structure.In second section,the detection ability of phosphinate-luciferin for O2·-in cells and in animals are studied.Phosphinate group would quench the bioluminescence of D-luciferin due to its electron-withdrawing effect,however,free luciferin can be released upon treatment with O2·-,leading to a turn-on bioluminescence.In vitro,the probe exhibited high sensitivity for capturing O2·-at the nanomole level and high selectivity against other ROS.For endogenous O2·-analysis in Huh7 cells,the probe generated an obvious enhancement by using phorbol-12-myristate-13-acetate and polystyrene particles as O2·-stimulator.The experimental results demonstrated that phosphinate-luciferin was membrane-permeable and has low toxicity.Furthermore,bioluminescence imaging of the mice has confirmed that the probe was successfully employed for the detection of endogenous O2·-with fast response in minutes in nude mice. |
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