Enantioselective remote functionalisation of cyclic amines by engineered P450BM3 variants

Thesis

P450 enzymes activate C–H bonds via the insertion of an oxygen atom from atmospheric dioxygen under ambient conditions in water and accept a broad array of endogenous and exogenous organic substrates. P450_BM3 (CYP102A1) has a unique structure, with the electron-transfer reductase domain fused to the haem monooxygenase domain. This makes P450_BM3 catalytically self-sufficient and promotes high turnover rates. Functionalised cyclic amines are versatile intermediates in drug synthesis and important fragment molecules in drug discovery. Selective oxidation of unactivated ring aliphatic C–H bonds is an attractive route to cyclic amine derivatives; however, classical synthetic methods are limited in scope and lack enantioselectivity. The application of engineered P450 enzymes for selective functionalisation of cyclic amines is surprisingly under-explored.

Chapter 2 describes the substrate and enzyme engineering to access remote hydroxylation products of the medium-sized cyclic amines azepane and azocane. Variant library screening is deployed to identify key residues affecting product selectivity, substrate engineering is utilised to alter the selectivity patterns, and rational design is conducted to improve product selectivity. In Chapter 3, the iterative docking-guided mutagenesis (IDGM) approach is explored. Comparative analysis of the correct binding poses and unwanted poses leads to targeted mutation design to disfavour unwanted poses and increase both the activity and product selectivity of amine oxidation. In Chapter 4, the combined enzyme evolution strategies are used for more complex cyclic amines – the bicyclic, 7-azabicyclo[2.2.1]heptane, and spirocyclic, 8-azaspiro[5.4]decane. These approaches led to scalable enantioselective oxidation of all but one of the unactivated C–H bonds in these important cores of drugs and fragment libraries.

Authors: Zhang, Y

• Alternative title: Regio- and enantio-selective oxidation of unactivated C–H bonds in cyclic amines by iterative docking-guided mutagenesis of P450BM3 (CYP102A1)
• DOI: 10.5287/ora-brvngedmd
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Iterative docking-guided mutagenesis; MD-simulation; Molecular docking; P450BM3; Cyclic amines; C–H functionalisation; Enzyme engineering
• Subjects: Synthetic biology; Biocatalysis; Computational biology; Chemical biology