Development of New Imaging Agents for the Study of Cellular Copper Dynamics

Copper is an essential element in biological systems. Its participation as a catalytic and structural cofactor in enzymes that function in biological processes such as energy generation, oxygen transport, cellular metabolism and signal transduction renders it vital for the life of eukaryotic organisms. However, if misregulated, it can cause cellular oxidative damage through the production of highly reactive oxygen species. Consequently, it is not surprising that organisms have developed intricate cellular machineries to tightly regulate the trafficking of copper. The fascinating balance between the functional and pathological roles of copper has significantly motivated the study of the processes involved in the proper management of this metal in the cell by inspection of the total metal content in biological samples and its distribution into static and labile cellular pools. Traditional bulk techniques, in combination with biochemical and genetic methods, have been used probe the static copper pool and provide a general understanding of how cells handle copper ions, allowing for the identification of major proteins and ligands involved in copper homeostasis. Alternatively, molecular imaging provides a complementary, versatile approach that can be used to monitor dynamic fluxes of the labile copper pools in real time with spatial and temporal resolution. This dissertation describes the design, synthesis and characterization of new copper-responsive fluorescent sensors in order to expand the available toolbox for molecular imaging of copper dynamics. Application of a novel carbo-rhodol scaffold allowed for the development of the Copper CarboRhodol (CCR) and Copper CarboFluor (CCF) probes, that in combination with additional rhodol platforms extended the available color palette of copper-selective probes. Additionally, the design of unique metal binding motifs with altered ligand architectures, binding geometries and coordination units resulted in the synthesis of new copper-responsive fluorophores. Alteration of the coordination unit and inclusion of a tripodal ligand topology afforded a family of xanthene-based sensors with an assortment of fluorescent responses to copper and ability to sense fluctuations in endogenous, labile copper pools. Finally, development of a modified donor set that increases the number of available donors in the ligand framework was investigated as an alternative strategy to modulate the binding properties of the resulting copper sensors