Understanding Flavin-Dependent Halogenase Reactivity via Substrate Activity Profiling

Abstract

The activity of four native FDHs and four engineered FDH variants on 93 low molecular weight arenes was used to generate FDH substrate activity profiles. These profiles provided insights into how substrate class, functional group substitution, electronic activation, and binding impact FDH activity and selectivity. The enzymes studied could halogenate a far greater range of substrates than previously recognized, but significant differences in their substrate specificity and selectivity were observed. Trends between the electronic activation of each site on a substrate and halogenation conversion at that site were established, and these data, combined with docking simulations, suggest that substrate binding can override electronic activation even on compounds differing appreciably from native substrates. These findings provide a useful framework for understanding and exploiting FDH reactivity for organic synthesis.

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis

“…This residue could be deprotonating substrate, and thus increasing the electronic activation of substrate. This hypothesis is supported by the observation that halogenation is always observed ortho to the phenol hydroxyl substituent (33, 37, 59), since D325 and K74 are positioned such that deprotonation and chlorination would always occur with this regioselectivity.…”

“…Like RebH (33), several other FDHs, including the Trp-FDHs SttH (56), Thal (33), PrnA (82), PyrH (82), and KrmI (83) as well as the phenol FDHs Rdc2 and GsfI (33, 59, 84), have demonstrated broad substrate scope and interesting site-selectivities (Fig. 4B, 14–20 ).…”

“…Halenium affinities (kJ/mol) for all sp 2 -hybridized C-H bonds are displayed for tryptamine and 1-(4-hydroxyphenyl)pyrrole. Halogenation site selectivity using NCS or FDH is denoted with an arrow (32, 33). B.…”

“…Substrates halogenated by engineered RebH variants that are not halogenated by wtRebH (33, 108). If halogenation site is known, it is denoted with an arrow.…”

Enantioselective Desymmetrization of Methylenedianilines via Enzyme-Catalyzed Remote Halogenation

“…14,15 One of the resulting enantioenriched, halogenated methylenedianiline products was converted to 5-substituted indole with no loss in enantioselectivity at the α-stereogenic center. While only a few substrates were evaluated for this work, we anticipate, given the broad substrate scope of FDHs, 37 that future research efforts will further expand the potential applications of these halogenases in the preparation of a range of useful, bioactive compounds. For instance, similar reactions of 3,3′- or 2,2′-methyelenedianilines would give rise to alternate indole regioisomers, and the amine substituent on the unfunctionalized aniline could be used as a handle for additional substitution.…”