Synthesis of Fluorescent Analogues of FluoBar1 for Studying Protein Expression Rates in Living Cells

Abstract: L-type voltage gated calcium channels are membrane bound ion channels that regulate the intracellular concentration of calcium ions as a response to changes in the membrane potential. They exist in four isoforms and recent studies have brought extra attention to the Cav1.2 isoform as it has been shown to be critical for the maturation of oligodendrocyte progenitor cells in the brain. These cells are involved in neural myelination and if they do not mature properly, it can lead to abnormal neural myelination. This is a condition that is strongly correlated with neurodegenerative diseases such as Multiple Sclerosis and has been associated with psychiatric diseases such as schizophrenia. The number of tools for studying these proteins are few, and therefore this thesis aimed to expand the toolbox by developing an analogue (FluoBar2) to an existing probe (FluoBar1). The analogous probe could resolve some of the issues currently associated with labeling using FluoBar1 as well as open up the possibility of conducting pulse-chase experiments when used with FluoBar1. The short absorption and emission wavelengths of FluoBar1 causes large amounts of autofluorescence in cellular imaging, making the images hard to interpret. In FluoBar2, the Pacific Blue fluorophore used in FluoBar1 is exchanged for 5-carboxyfluorescein, causing the absorption and emission to be redshifted which theoretically should decrease the cellular autofluoresence. The project begun with the synthesis of FluoBar1 which was isolated in a 0.19% overall yield. During this, the synthetic route towards the linker used in FluoBar1 and FluoBar2 was developed as well, increasing the yield with respect to the most expensive reagent from 41.5% to 61%. An attempt at synthesizing FluoBar2 was made and its presence was supported by 1H NMR as well as UPLC-MS, but it was not fully isolated and characterized.