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.

Keywords: C-H functionalization; biocatalysis; flavin; halogenase; site selective.

Figure 1

Phylogenetic tree of FDHs examined. Branch labels display amino acid substitutions per site.

Figure 2

Substrate activity profiles in heat map form for eight FDHs on substrates in panels 1 (indoles, pyrroles, azoles, anilines, anilides) and 2 (phenols). Maximum conversion is shown for each enzyme-substrate pair (for complete data sets at both time points, see Table S1.).

Figure 3

A) Conversion data from initial activity profile for selected substrates and B) Structures depicted in heat map. Compounds for which >5% conversion was observed are highlighted in green; those halogenated to a trace extent are highlighted in yellow.

Figure 4

Conversion data from initial activity profile and structures for substrates 22–28.

Figure 5

Conversions for substrates with multiple electronically activated sites that provided a single halogenated product. Reactions were conducted on 1–10 mg scale using either RebH (29a–43a) or Rdc2^a (25a, 27a). See Table S2 for full product list. Conversion and selectivity for 27a determined by comparison with authentic material.

Figure 6

Conversion versus halenium affinity (HalA) for each sp carbon on each substrate in Table S2. HalA ranges for panel 1 substrates halogenated by RebH and panel 2 substrates halogenated by Rdc2 are highlighted in blue and green, respectively. The inset shows representative HalA values for common arenes (not substrates).

Figure 7

Poses observed for ROCS pharmacophore overlay. Tryptophan in the RebH-tryptophan complex (PDB 2OA1) is shown in pink. Lys79 is labeled in each model, and the site of halogenation for each substrate is indicated with a red arrow. Structures for each substrate are shown with HalA values for each site and the site halogenated in green.

Scheme 1

(A) General scheme for FDH-catalyzed halogenation. (B) Mechanism for generation of proposed halenium for electrophilic aromatic substitution (EAS) within FDHs.

Scheme 2

General scheme for FDH bioconversions.