The central theme of the principal investigator's overall research program is the design, chemical synthesis, characterization and collaborative application of new probes and reagents for the study of biological systems. Five subareas form the focus of this five year project. a) New Nitroxide-Based Molecular pH Indicators. The measurement of pH inside cells and vesicles is fundamental to the study of many biological processes. We propose to extend our series of Alpha-amino notroxides with pH dependent ESR hyperfine splitting constants aN to include those which respond optimally in the range pH 6-8. b) New Nitroxide Substituted Crown Ethers. Measurement of the intracellular activities of sodium and potassium ions is central to molecular studies of membrane ion transport processes in cell biology. We envisage a series of nitroxide spin labeled crown ethers and cryptands in which aN is sensitive to the activity of the metal ion in the physiologically interesting concentration range, constituting a new, highly sensitive ESR method for determination of ion activities. c) New Small Highly Electron Dense EM labels. High resolution EM biological studies require the development of smaller, highly electron dense labels capable of attachment to specific sites on a macromolecule. Organic functionalized heteropolytungstate ions hold high promise in this regard. The synthesis and application of several of these labels is proposed. d) A New ESR Probe for Singlet Oxygen. Single oxygen is a likely oxidant associated with the photodynamic effect in biological systems. We intend to develop anthracene nitroxides as sensitive ESR probes for this oxidant through endoperoxide formation with associated ESR spectral changes. e) New Nitroxide-Based NMR Whole Body Medical Imaging Agents. The use of NMR for whole body medical imaging is undergoing explosive development. Agents which enhance contrast between organs are needed. Nitroxides may be the answer if resistance to in situ reduction can be built into the molecules. This is our objective. The significance of this research project is to make available new molecules which have been designed to overcome some of the currently serious obstacles in biological and medical research.
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