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Authors: Turek-Herman, Joshua Ray
Advisors: Hyster, Todd K
Contributors: Chemistry Department
Keywords: Amines
Amino Acids
Directed Evolution
Peptides and Proteins
Subjects: Organic chemistry
Cellular biology
Issue Date: 2023
Publisher: Princeton, NJ : Princeton University
Abstract: Enzymes are remarkable catalysts able to facilitate chemical transformations with unparalleled selectivity. In addition to their selectivity, enzymes operation under mild reaction conditions and tunability by the tools of directed evolution have expanded their use from biologic systems to organic synthesis. Several classes of enzymes notable for their broad substrate scope and easy handling have become canon in the biocatalysis field. However, relatively few biocatalysts enable carbon–carbon bond formation, an important complexity-building disconnection in organic synthesis. Therefore, an expansion of reactions available to biocatalysts to generate enzymes competent at C–C bond formation is highly desired. The work described herein explores nonnatural reaction development from flavin-dependent ‘ene’-reductases (EREDs) and pyridoxal 5’-phosphate (PLP)-dependent threonine aldolases to create synthetically valuable carbon-carbon bonds. While their native mechanisms involve polar intermediates, both of these cofactors are known to undergo radical processes and therefore are hypothesized to be viable platforms for asymmetric radical chemistry. We imagined with the appropriately designed substrates we would enable C–C bond formation to generate motifs otherwise difficult to make such as sterically-crowded chiral amines and quaternary centers. Chapter 2 describes the evolution and application of EREDs evolved to catalyze the radical addition of an alpha-acyl radical into a ketoxime to form alpha-tertiary amines with high yields and enantioselectivities. This work builds upon previous reports from our lab that EREDs can form radicals from alpha-chloroamide radical precursors upon cyan light irradiation. Mechanistic investigations clarified the beneficial role of the mutants discovered during the directed evolution campaign. Exploration of the spectroscopic properties of the enzyme-templated charge transfer (CT) complex highlighted the role of an electron-rich pi-system for effective electron transfer. Chapter 3 further expands the utility of these evolved biocatalysts toward forming sterically related quaternary carbons. Owing to their sterically crowded nature, quaternary carbons are difficult to synthesize with few methods for their effective generation and even fewer reports of their asymmetric synthesis. Finally, chapter 4 describes our ongoing efforts to expand the reactivity of PLP-dependent threonine aldolases to enable C–C bond forming radical recombination from N-hydroxyphthalimide (NHPI) ester radical precursors. This enables formation of nonproteinogenic amino acids through a mechanistic regime unprecedented for PLP-dependent enzymes.
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Chemistry

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