Design and Evolution of Photoenzymes for Enantioselective Triplet Energy Transfer Catalysis

Abstract

Triplet energy transfer (EnT) photocatalysis is a powerful mode of reactivity in organic chemistry that allows access to a diverse range of synthetically useful chemical transformations. However, performing EnT reactions with high levels of regio- and stereocontrol is a long-standing challenge for small-molecule photocatalysts. Although enzymes offer exquisite control over reaction stereocontrol, EnT photocatalysis is inaccessible to nature’s enzymes due to the limited range of chemical functionalities present in canonical amino acid side-chains. Here, we demonstrate that genetic code expansion can be used to unlock EnT photocatalysis within enzyme active sites and directed evolution enables the development of highly efficient and enantioselective photoenzymes. In an initial study, genetic incorporation of a noncanonical amino acid (ncAA) with a photosensitising side chain (4-benzoylphenylalanine, BpA) into a computationally designed Diels-Alderase protein scaffold afforded a first-generation photoenzyme for [2+2] cycloadditions. Laboratory evolution of this initial design gave rise to an efficient and enantioselective [2+2] cyclase (EnT1.3), which can promote a range of intramolecular [2+2] cycloadditions with high levels of enantiocontrol (up to 99% enantiomeric excess, (ee)). Additionally, EnT1.3 functions effectively under conditions which are typically prohibitive to small-molecule photosensitisers (aerobic and ambient temperature), highlighting its potential for synthetic applications. Building on this initial study, we further expanded the repertoire of photochemical reactions accessible to EnT photoenzymes to include intermolecular [2+2] cycloadditions of 2(1H)-quinolone with a variety of alkene coupling partners. Three rounds of directed evolution afforded a variant (BR3) which can catalyse highly enantioselective (up to 99% ee) intermolecular [2+2] cycloadditions with electron-rich alkenes, which cannot be performed stereoselectively with small-molecule photosenitisers. Furthermore, selected variants across the evolutionary trajectory can promote cycloadditions with unactivated alkenes with high levels of enantiocontrol, which have currently not been reported. In a final study, we apply EnT photoenzymes to 6-π electrocyclizations of acrylanilides, which to our knowledge, demonstrates the first reported enzymatic electrocyclic reaction. Directed evolution facilitated rapid development of a highly efficient and enantioselective (up to 99% ee) photoenzyme for 6-π electrocyclizations with substrates that have proved challenging to achieve enantiocontrol over with small-molecule photosensitisers. The range of highly selective EnT photocatalysis achieved by EnT photoenzymes reported here demonstrates the synthetic versatility of our approach and presents a promising alternative for stereoselective photocatalysis.

Date of Award	14 Dec 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Anthony Green (Main Supervisor) & Daniele Leonori (Co Supervisor)