Year: 2022
Type: dissertation
Abstract: The precision and efficiency offered by enzyme catalysis has propelled the incorporation of biocatalytic strategies into synthetic campaigns in academic and industrial spheres. Nevertheless, biocatalytic methods remain prohibitively underdeveloped compared to the vast repertoire of their small molecule-based counterparts, including for the formation of carbon–carbon (C–C) bonds. Oxidative coupling reactions offer a direct and complexity-generating approach to C–C bond formation; however, controlling the selectivity of these reactions remains an outstanding challenge in organic synthesis. In contrast, Nature has evolved a wealth of cytochrome P450 enzymes that catalyze selective oxidative C–C coupling reactions in the biosynthesis of biaryl natural products. Inspired by this catalyst-controlled selectivity, the work presented in this dissertation describes a platform for biocatalytic biaryl bond formation using P450 catalysts. Using a yeast-based platform for whole-cell biocatalysis, we demonstrate the ability of a fungal P450 to catalyze unnatural cross-coupling reactions on a panel of phenolic substrates. Moreover, over five rounds of directed evolution, we engineer the fungal P450 to possess the desired reactivity and site-selectivity by improving a low-yielding, unselective reaction 92-fold for the target cross-coupled product. We further expanded the biocatalytic platform by profiling the cross-coupling activity of a library of natural P450s using a bioinformatics-guided approach, leading to the identification of a bacterial P450 capable of cross-coupling substituted naphthols. Despite demonstrating promiscuous activity, the P450 offered little to no stereocontrol in cross-coupling reactions. To overcome this limitation, the P450 was engineered for improved atroposelectivity, ultimately providing a biocatalyst capable of cross-couplings with up to 95:5 er in the formation of privileged chiral biaryl ligands. Altogether, this streamlined method for constructing sterically hindered biaryl bonds provides a programmable platform for assembling biaryl molecules with catalyst-controlled reactivity and selectivity.
DOI: 10.7302/6234
Author: Zetzsche, Lara
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4294884834