Directed Evolution of Flavin-Dependent Halogenases for Atroposelective Halogenation of 3-Aryl-4(3H)-quinazolinones via Kinetic or Dynamic Kinetic Resolution

Abstract

In this study, we engineer a variant of the flavin-dependent halogenase RebH that catalyzes site- and atroposelective halogenation of 3-aryl-4(3H)-quinazolinones via kinetic or dynamic kinetic resolution. The required directed evolution uses a combination of random and site-saturation mutagenesis, substrate walking using two probe substrates, and a two-tiered screening approach involving analysis of variant conversion and then enantioselectivity of improved variants. The resulting variant, 3-T, provides >99:1 e.r. for the (M)-atropisomer of the major brominated product, 25-fold improved conversion, and 91-fold improved site-selectivity relative to the parent enzyme on the probe substrate used in the final rounds of evolution. This high activity and selectivity translates well to several additional substrates with varied steric and electronic properties. Computational modeling and docking simulations are used to rationalize the effects of key mutations on substrate scope and site- and atroposelectivity. Given the range of substrates that have been used for atroposelective synthesis via electrophilic halogenation, these results suggest that FDHs could find many additional applications for atroposelective catalysis. More broadly, this study highlights how RebH can be engineered to accept structurally diverse substrates that enable its use for enantioselective catalysis.

Expanding the Reactivity of Flavin-Dependent Halogenases toward Olefins via Enantioselective Intramolecular Haloetherification and Chemoenzymatic Oxidative Rearrangements

“…2−4 Our group and others have established that variants of these enzymes can be engineered to halogenate different non-native substrates or different sites on a given substrate with high (i.e., > 90%) selectivity. 5 We also established that FDHs can catalyze enantioselective desymmetrization of methylenedianilines 6 and atroposelective 7 halogenation of 3-aryl-4(3H)quinazolinones; however, these transformations share a similar mechanism involving electrophilic attack by an active site halogenating agent, likely HOX bound within the FDH active site, and ipso deprotonation (Figure 1A). 8−10 Inspired by the wide range of transformations known to proceed via electrophilic attack of different nucleophiles by electrophilic halogen species, 11 we recently discovered that engineered FDHs can also catalyze enantioselective halolactonization of olefins (Figure 1B).…”

The Single Component Flavin Reductase/Flavin Dependent Halogenase AetF is a Versatile Catalyst for Selective Bromination and Iodination of Arenes and Olefins

“…AetF also provides good levels of conversion and selectivity toward substrates that undergo enantioselective halogenation processes via topologically distinct mechanisms. [21][22][23] Finally, we find that AetF catalyzes iodination of aromatic compounds at sites that are less activated than those that have been characterized using other FDHs and enables enantioselective cycloiodoetherification. These findings suggest that AetF has great potential for expanding the scope of biocatalytic halogenation.…”

“…Given our ongoing interest in asymmetric FDH catalysis, we evaluated the activity of AetF on substrates capable of undergoing desymmetrization, 22 atroposelective dynamic kinetic resolution, 23 and halolactonization 21 via bromination. Again, AetF provides good conversion and modest-to-high enantioselectivity on each of the substrates examined (Figure 2, 12a-14a).…”

Expanding the Reactivity of Flavin Dependent Halogenases Toward Olefins via Enantioselective Intramolecular Haloetherification and Chemoenzymatic Oxidative Rearrangements

“…>90%) selectivity. 5 We also established that FDHs can catalyze enantioselective desymmetrization of methylenedianilines 6 and atroposelective 7 halogenation of 3-aryl-4(3H)quinazolinones; however, these transformations share a similar mechanism involving electrophilic attack by an active site halogenating agent, likely HOX bound within the FDH active site, and ipso deprotonation (Figure 1A). [8][9][10] Inspired by the wide range of transformations known to proceed via electrophilic attack of different nucleophiles by electrophilic halogen species, 11 we recently discovered that engineered FDHs can also catalyze enantioselective halolactonization of olefins (Figure 1B) 12 .…”