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UBC Theses and Dissertations

Synthesis and evaluation of probes of RNA polymerase II : adaptation of the bicyclic scaffold of the octapeptide amanitin Dietrich, David John


This thesis covers the synthesis and evaluation of bicyclic octapeptides as intracellular probes of RNAP polymerase II. In Chapter 2, the synthetic methodology used to achieve the rigid bicyclic octapeptide and the introduction of modifications to gain spatio-temporal control and visualization is presented. This includes the synthesis of a nitrorveratryl protected derivative of hydroxyproline, and a diethylaminocoumarin labeled asparagine residue. These amino acids were incorporated into amatoxins through solid-phase peptide synthesis. The synthesis and properties of these amatoxins, including their photolability and fluorescent properties is discussed. Chapter 3 describes the initial evaluation of the amatoxin probes in various eukaryotic cell lines. The cytotoxicity of α-amanitin in a variety of cell lines was investigated, where Chinese hamster ovary cells proved to be most sensitive to the toxin. These cytotoxic effects were observed at ~1 μM, which is much higher than the reported binding constant (~ 3 nM). The cytotoxicity was slow, requiring 60-72 hours to achieve 100% cell death. This diminished activity was attributed to cell uptake. Cell permeabilizing agents digitonin and saponin were applied to improve α-amanitin uptake and toxicity. All synthetic amatoxins were shown to have minimal cytotoxic effects. Confocal microscopy demonstrated no cell uptake of a fluorescent amatoxin unless the cells were treated with detergent, pointing to critical issues of cell permeability. The optimization of amatoxin synthesis is presented in Chapter 4. It is shown that up to 20% water can inhibit epimerization during macrolactamization of amatoxins. A convergent synthetic approach to the bicyclic octapeptide was also developed. This allowed for the incorporation of a variety of amino acids at position three. This new synthetic approach was applied to the synthesis of tryptathionine-containing analogs of the opioid receptor agonist enkephalin. The attempted synthesis of the unnatural amino acid (2S),(3R),(4R)-dihydroxyisoleucine is described in Chapter 5. This was achieved through the use of a diastereoselective [3,3] sigmatropic rearrangement followed by Sharpless asymmetric dihydroxylation (AD). Surprising selectivity was noted during the AD reaction using phthalazine-based ligands, but this selectivity was reversed using pyrimidine-based ligands.

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