Biocatalytic P450 Oxidative Phenolic Coupling Reactions for Biaryl-bond Formation
Yazarians, Jessica
2022
Abstract
Atropisomers, derived from the Greek word “atropos,” meaning “without turn”, are chiral molecules that possess a bond that is sterically hindered, impeding free rotation around that bond. This structural feature impacts the three-dimensional shape of atropisomers, which can influence the physical and biological properties of the compounds that contain this form of stereoisomerism. Atropisomers are structurally diverse, found in natural products, drugs, materials, and privileged scaffolds in asymmetric catalysis. The selective formation of chiral biaryl bonds in the form of sterically hindered atropisomers persists as an open challenge in modern synthesis. Synthetic strategies to forge hindered biaryl bonds often require either prefunctionalization at the desired site of bond formation, or the direct transformation of two C–H bonds into a C–C bond through oxidative coupling. However, control over the chemoselectivity, site-selectivity and atroposelectivity in direct oxidative coupling reactions remains challenging. Nature employs site- and stereoselective oxidative coupling in the synthesis of atropoisomeric natural products with enzymes such as cytochromes P450 (P450s) implicated in the intermolecular dimerization reactions of phenols.1 Despite the growing number of enzymes capable of catalyzing oxidative coupling reactions site- and stereoselectively, most are not studied beyond their native reactivity, with few examples of enzymatic cross-coupling reactions reported. The catalyst-controlled site-selectivity displayed by the P450 enzymes KtnC and DesC in the oxidative dimerization of coumarins provided inspiration for developing an enzymatic platform for oxidative phenolic coupling reactions.2 We hypothesized that this class of enzymes could be developed into tunable biocatalysts for intermolecular fragment coupling, with the potential to overcome the limitations inherent to established synthetic methods for the direct oxidative coupling of phenolic compounds. Furthermore, applying P450s in biocatalysis for atroposelective cross-coupling reactions has the potential to compliment current methods but remains underexplored. In Chapter 2, a biocatalytic platform for oxidative phenolic coupling catalyzed by the fungal P450s KtnC and DesC is disclosed. The engineering and heterologous expression of the genes encoding P450s in Saccharomyces cerevisiae and Pichia pastoris are described. Analytical methods for reaction profiling by liquid chromatography mass spectrometry were developed allowing for rapid method optimization. In chapter 3, the substrate scope of KtnC was further explored. The ability to catalyze atroposelective oxidative dimerization and cross-coupling reactions on a panel of coumarin substrates is demonstrated, with wild-type KtnC displaying promiscuity with non-native coumarins and phenolic substrates. In Chapter 4, a directed evolution strategy was developed to engineer P450 variants with improved activity and selectivity, overcoming the challenges accessing cross-coupled coumarin products bearing the 6,6'-connectivity. Enzyme libraries were generated through semi-rational mutagenesis,3 wherein beneficial mutations were identified through site-scanning saturation mutagenesis of active site residues.4 Over two rounds of evolution, the chemoselectivity and site-selectivity of the KtnC-mediated reaction were enhanced in comparison to wild-type. A variant possessing two substitutions, P333G and L422V, provided a 3-fold increase in the percent yield of the desired product. This biocatalytic method for constructing sterically hindered biaryl bonds provides an engineerable platform for assembling molecules with catalyst-controlled reactivity and selectivity. 1. Huttel, W.; Muller, M., Nat. Prod. Rep. 2021, 38, 1408-1408. 2. Mazzaferro, L. S.; Huttel, W.; Fries, A.; Muller, M., J. Am. Chem. Soc. 2015, 137, 12289-12295. 3. Lutz, S., Curr. Opin. Biotech. 2010, 21, 734-743. 4. Chen, M. M. Y.; Snow, C. D.; Vizcarra, C. L.; Mayo, S. L.; Arnold, F. H., Protein Eng. Des. Sel. 2012, 25, 171-178.Deep Blue DOI
Subjects
biocatalysis oxidative coupling of phenols atroposelective biaryl-bond formation atropisomer P450 enzyme catalyzed C-C bond formation
Types
Thesis
Metadata
Show full item recordCollections
Remediation of Harmful Language
The University of Michigan Library aims to describe library materials in a way that respects the people and communities who create, use, and are represented in our collections. Report harmful or offensive language in catalog records, finding aids, or elsewhere in our collections anonymously through our metadata feedback form. More information at Remediation of Harmful Language.
Accessibility
If you are unable to use this file in its current format, please select the Contact Us link and we can modify it to make it more accessible to you.