Site-Selective C–H Halogenation Using Flavin-Dependent Halogenases Identified via Family-Wide Activity Profiling

Enzymes
are powerful catalysts for site-selective C–H bond
functionalization. Identifying suitable enzymes for this task and
for biocatalysis in general remains challenging, however, due to the
fundamental difficulty of predicting catalytic activity from sequence
information. In this study, family-wide activity profiling was used
to obtain sequence–function information on flavin-dependent
halogenases (FDHs). This broad survey provided a number of insights
into FDH activity, including halide specificity and substrate preference,
that were not apparent from the more focused studies reported to date.
Regions of FDH sequence space that are most likely to contain enzymes
suitable for halogenating small-molecule substrates were also identified.
FDHs with novel substrate scope and complementary regioselectivity
on large, three-dimensionally complex compounds were characterized
and used for preparative-scale late-stage C–H functionalization.
In many cases, these enzymes provide activities that required several
rounds of directed evolution to accomplish in previous efforts, highlighting
that this approach can achieve significant time savings for biocatalyst
identification and provide advanced starting points for further evolution.