Molecular Imaging Approaches to Understanding the Roles of Copper in Biology.

摘要

Cells exert precise control over their cellular copper pools through a sophisticated array of uptake, trafficking, and storage mechanisms that effectively maintain a low concentration of thermodynamically free copper ions while maintaining excellent kinetic lability of cellular copper stores. In higher eukaryotes and humans, and particularly in specialized cell types associated with brain, heart, intestine, and liver tissue, the molecular specifics of how kinetically labile copper pools are regulated at the subcellular level and the consequences of copper misregulation in aging and disease remain insufficiently understood. Biochemical and genetic studies have established a broad understanding of how cells acquire, maintain, redirect and release copper ions, while also identifying key proteins involved in these activities. The precise role of the copper ion, however, is more difficult to determine, owing mainly to a dearth of methods for directly following the fate of cellular copper stores. This dissertation describes the design, synthesis, and characterization a several new Cu(I)-responsive fluorophores. Through a targeted synthetic survey and comprehensive electrochemical study, the properties of our previously reported fluorophore, Coppersensor-1, were improved to yield a compound (Coppernsensor-3) that exhibits the largest fluorescent response to Cu(I) to date. Along with X-ray fluorescence microscopy, CS3 was used to investigate disruptions in copper homeostasis in a cell model for Menkes disease. The following report describes the synthesis, characterization and applications of Ratio-Coppersensor-1 (RCS1), the first ratiometric fluorophore for live-cell imaging. This compound was used to investigate the effect of ascorbate on rat brain and human kidney cells, and proved able to be able to detect increases in endogenous labile Cu(I) that occurs upon ascorbate treatment. A slight alteration to the ligand of RCS1 gave RCS2, which has similar spectroscopic properties to RCS1. Single-molecule X-ray crystallography and VT-NMR studies provide molecular dynamic details of RCS2 coordination to Cu(I).