Derivative synthesis of the transcriptional inhibitor α-amanitin and the translational inhibitor cycloheximide

Abstract

The following work focuses on my efforts towards the synthesis of the transcriptional inhibitor a-amanitin and the translational inhibitor cycloheximide as a means to exploit the potential of these natural products as probes for developing new biochemical methods. The first half of this thesis details my work to access derivatives of a-amanitin, a naturally occurring cytotoxin that is found in several species of poisonous mushrooms and demonstrates incredible selectivity and binding affinity to eukaryotic RNA Polymerase II (Pol II). We devised a modular synthesis of a click-compatible a-amanitin that would be amenable to late- stage derivatization. Our strategy was reliant on the linear assembly of amino acid building blocks - many of which were non-canonical - followed by the key Savige-Fontana macrocyclization to access the unique cysteine-tryptophan tryptathionine side chain linkage. This portion of my thesis details the synthesis of a solid-phase peptide synthesis-compatible bromopyrroloindoline, the enantioselective synthesis of (2S,3R,4R)-4,5-dihydroxy-isoleucine (DHIle), and finally the assembly of the bicyclic peptide framework to furnish the desired amanitin derivatives. The second half of this thesis outlines the work towards the synthesis and characterization of potent cycloheximide (CHX) analogues. Due to its ability to effectively freeze ribosomes along mRNA, CHX has long been employed as a biochemical tool to study protein synthesis, and more recently has gained the role as the de facto inhibitor used for ribosome profiling. The remaining chapters of this dissertation will outline our lab’s previous total synthesis of CHX and related analogues as well as our development of a semi-synthetic route to C13-amido CHX derivatives that is reliant on a diastereoselective C-H amination. This chapter is concluded with our mechanistic exploration of our most potent CHX analogue, using sequencing-based analysis and cryogenic electron microscopy to investigate the rationale of the increased activity of these synthetic derivatives.