Development and Characterization of Non-heme Iron Biocatalysts for Complex Molecule Synthesis
Doyon, Tyler
2020
Abstract
Nature has evolved myriad biocatalytic tools for selective synthesis. The three-dimensional architecture of an enzyme active site enables the direct construction of new bonds with exquisite site-, chemo-, and stereo-selectivity. Seeking to take advantage of these characteristics, researchers have leveraged biocatalysts for the rapid synthesis of natural products and complex molecules, developing sustainable methods to address long-standing challenges in synthetic chemistry. In recent years, this approach has been expanded to combine chemo- and biocatalytic methods in a single vessel, enabling transformations of increasing complexity to occur in a streamlined process. This thesis describes the development of novel, one-pot chemoenzymatic methods for the synthesis of complex molecules and natural products. Specifically, this work leveraged non-heme iron (NHI) alpha-ketoglutarate-dependent enzymes to access reactive ortho-quinone methide (o-QM) and radical intermediates for the construction of chroman and tropolone natural products. These studies provide a platform for the development of NHI enzymes as scalable and selective catalysts for the synthesis of complex molecules. The described research involved the development NHI enzymes CitB and ClaD to perform selective benzylic C–H hydroxylation of ortho-phenolic compounds. This biocatalytic method offered numerous advantages over small molecule oxidants, which often exhibit poor site- and chemo-selectivity for benzylic hydroxylation reactions. In comparison, CitB and ClaD provided strict control over the site of oxidation, avoiding the need for blocking or protecting groups to achieve selective catalysis. The substrate scope of this transformation was evaluated for these biocatalysts and a scalable reaction platform was developed for this transformation, demonstrating the ability of NHI enzymes to serve as sustainable and selective catalysts for benzylic C–H hydroxylation. The products of this selective oxidation were fully characterized and were shown to serve as reactive precursors for the formation of o-QMs in a one-pot process. Compared to traditional synthetic approaches to one-pot o-QM generation, a biocatalytic route offers the advantage of selective oxidation, leading to controlled generation of the reactive o-QM intermediate. These intermediates were elaborated in one-pot, modular chemoenzymatic fashion through 1,4-addition and [4+2] cycloaddition reactions demonstrating the synthetic utility of this approach for synthesizing complex scaffolds. Overall, this biocatalytic reaction platform offered an improved selectivity profile over traditional oxidative approaches to o-QM synthesis, enabling facile one-pot benzylic oxidation and functionalization in a scalable reaction format. A second focus of this work involved the chemoenzymatic synthesis of 7-membered aromatic compounds known as tropolones. Efficient synthetic access to this structurally-diverse class of metabolites represents a significant challenge to the development of novel tropolone pharmaceuticals. To address this hurdle, NHI enzymes XenC and TropC were leveraged for their native ring expansion function to develop an efficient one-pot, two-enzyme reaction for the synthesis of substituted tropolones. Reactions with XenC were leveraged in the efficient chemoenzymatic synthesis of a variety of non-natural tropolones including the deoxygenated form of tropolone natural product epolone B. The reactivity of a putative Diels-Alderase was also explored in this reaction, providing a potentially selective route to the synthesis of bioactive tropolone natural products. Furthermore, this work explored the mechanism of the native ring expansion reaction performed by NHI enzyme TropC and provided evidence of a radical-based reaction through structural characterization of the enzyme, as well as mutagenesis and computational analysis. These observations led to a revised proposal for fungal tropolone biosynthesis and provided critical insight into an understudied NHI-catalyzed transformation.Subjects
biocatalysis chemoenzymatic natural product synthesis non-heme iron enzyme
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