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Structural Elucidation of Metabolites via Electron Diffraction at the Interface of Synthetic Chemistry and Biology

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This dissertation describes the application and development of electron crystallography in the realm of small molecules and, in particular, natural products (NPs). Natural products serve as a major inspiration for drug discovery, but the rate of discovering novel NPs is hindered by limitations in existing characterization techniques. This dissertation highlights microcrystal electron diffraction (MicroED) as a powerful approach to overcome these limitations and structurally characterize NPs and other small molecules. Overall, this work demonstrates the general applicability of MicroED in natural product and small molecule characterization. Moreover, using MicroED as a platform, an efficient workflow for NP identification is developed, with the goal of accelerating the rate of novel natural product discovery as well as drug discovery. Chapter One is a brief overview of the current state of research on using electron diffraction to elucidate structures of natural products. This chapter serves as both a prelude to and a summary of the remaining chapters, and will be referenced throughout this dissertation. Brief history of electron diffraction and background of MicroED is provided, focusing on the scope of MicroED in small molecule regime. Furthermore, the application of MicroED in natural products and currently remaining challenges are highlighted, drawing on our experiences as well as others’. Chapters Two through Five describe the applications of MicroED in NP characterization. Chapter Two highlights the structural elucidation of a glucosyluric metabolite in C. elegans, whose MicroED structure was used to complement the NMR data that was ambiguous due to poor line shapes. Chapter Three showcases the structure determination of a biosynthetic intermediate to a fungal metabolite (–)-sambutoxin, whose relative stereochemistry was validated using MicroED. Chapter Four discusses the synergy between MicroED and genome mining by rediscovering the biosynthetic pathway to fischerin and revising its relative stereochemistry that was incorrectly assigned using computational studies. In this chapter, a novel compound is isolated and characterized using MicroED, demonstrating the first example of a novel, non-peptidyl NP structurally elucidated via MicroED. Additionally, this study highlights MicroED’s impressive sensitivity by detecting structural information from only 3 ng of material. Chapter Five reports the structural revision of the lomaiviticins enabled by MicroED analysis. The lomaiviticins are highly cytotoxic bacterial metabolites that have been extensively studied for decades, with multiple total synthesis efforts that failed. This structural revision, supported by high-field NMR as well as DFT calculations, enables scientists to embark on future synthetic efforts and studies on mechanisms of action with more confidence. Chapter Six highlights the development of a MicroED platform to enable highthroughput screening for crystalline NPs. Initial studies demonstrate MicroED’s ability to obtain crystal structures in complex mixtures of natural products. These preliminary experiments are used to envision a workflow coupling liquid chromatography and MicroED, which is further developed into a high-throughput strategy. Using this platform, NPs produced in insufficient amounts for other analytical techniques can be detected and characterized, accelerating the rate of NP discovery. Chapter Seven describes the expansion of the scope of MicroED further in the small molecule regime by solving structures of reactive organometallic species and synthetic reaction products. These examples showcase the ability of MicroED in obtaining crystal structures that were previously unattainable, and demonstrate the structural integrity of MicroED structures when compared with structures obtained by X-ray crystallography. Notably, we discuss the utility of room temperature MicroED experiments, which provide a facile and quick method of screening for diffraction and become an integral part of the workflow in structural elucidation of small molecules.

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This item is under embargo until June 9, 2024.