Chemical probes for the study of mitochondrial oxidative stress.
PhD thesis, University of Glasgow.
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The implication of oxidative stress in ageing, and various disease states, has resulted in great interest in understanding the processes associated with it. Mitochondria are the major source of reactive oxygen species (ROS) via the electron transport chain. ROS may be free radicals, or molecules that rapidly form free radicals. These free radicals can undergo various rapid reactions with cellular components resulting in cell damage, and eventually cell death. Therefore, there is much interest in studying ROS production particularly from mitochondria.
In this thesis three novel classes of chemical probes for the study of oxidative stress are presented.
Firstly, progress was made towards a novel mitochondria-targeted hydroxylamine iii based on the 1,1,3,3-tetraethylisoindoline (TEIO) scaffold. This hydroxylamine iii should undergo reaction with radicals to form a stable nitroxide which gives a simple, persistent, quantifiable EPR (electron paramagnetic resonance) signal. Phosphonium salt i was prepared and EPR spectroscopy studies in a trial oxidation reaction indicated the formation of the late stage nitroxide precursor ii.
Three new pyridinium salt nitrone spin traps iv-vi, designed to accumulate in mitochondria, were synthesised using the Zincke reaction as the key pyridinium salt forming reaction. The compatibility of nitrones with the Zincke reaction was demonstrated for the first time. The capability of spin traps iv-vi to trap methyl radicals was demonstrated using EPR spectroscopy.
Two novel selective uncoupling molecules (SUMs), DNP-SUM vii and FCCP-SUM viii, were designed to react with H2O2 and release a mitochondrial uncoupler. These were synthesised and their reaction with H2O2 was examined using UV spectrophotometry. DNP-SUM vii reacted slowly with a second order rate constant of 0.20 M-1s-1 and was deemed unsuitable for further investigation. However, the reaction of H2O2 and FCCP-SUM viii displayed good pseudo-first order kinetics when FCCP-SUM viii was used in excess and a second order rate constant of 64 M-1s-1 for the release of the mitochondrial uncoupler FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) in its ionised form. New analogues of FCCP-SUM viii are now in development to enable targeting to mitochondria.
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