Reversible Nitric Oxide Detention in Aqueous Solution

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DESCRIPTION (provided by applicant): Nitic oxide (NO) operates as a biological signalling agent, which regulates both beneficial and harmful biological processes, depending on a variety of not yet fully delineated factors. Elucidation of the physiological roles of NO would benefit substantially from a probe that can detect the molecule directly in living cells. Fluorescence-based methodologies offer one possible approach to satisfy these requirements, however, in general, biologically compatible systems of this type, which have been studied to date are irreversible in nature, making the spatiotemporal imaging of NO flux in live samples unfeasible. The proposed research is centered on the development of a molecular fluorescence-based sensors for nitric oxide (NO) that are capable of functioning reversibly under physiological conditions. Such systems are being designed for the bioimaging of NO in living biological samples. The sensing strategy we will employ to address this issue, will rely on the construction of copper(ll) dithiocarbamate and dithiolene complexes (NO receptors) tethered to electron donating BODIPY fluorescent dyes (reporters), which display "turn-on" emission upon NO binding to the copper(ll) site via a photoinduced electron-transfer (PET) abolition mechanism. Initial work will involve the construction of a diverse family of NO receptor complexes based on a physiologically compatible L-cysteic acid backbone. This array of copper(ll) complexes will be screened for reversible NO binding in buffered aqueous solutions that replicate biological conditions, using various physical techniques including UV-Vis, FT-IR and NMR spectroscopies. The copper(ll) complexes best suited for NO detection in-vivo will subsequently be incorporated into BODIPY-NO receptor conjugates, which will be nonemissive in the absence of NO. The reversible "turn-on" fluorescence response of these conjugates will be monitored under physiological conditions. The most efficacious NO-detection conjugates will be employed in the reversible bioimaging of both endogenous and artificially introduced NO for both macrophage and neuroblastoma cell types. The effect of various external stimuli on cellular NO flux will also be a point of interest as will the comparison of the spatiotemporal characteristics of NO flux in healthy and cancer cells. Finally, experiments using live hippocampal brain slices will be used to map NO release and track neural signaling and network formation upon introduction of external stimuli (i.e. olfactory response). This work is relevant to public health, as the bioimaging of NO in living samples will undoubtedly shed light on the molecule's role as a regulatory and pathogenic agent. In addition to being pertinent to neurological function, implications with respect to cardiovascular health and general oncology are also of extreme interest.