Project Abstract
Halogenated organic compounds are used extensively as building blocks, synthetic intermediates, and end
use products for pharmaceutical and agrochemical applications. The utility of these compounds, including their
biological activity, arises from the reactivity and physical properties uniquely conferred to them by halogen
substitution. The importance of halogenation and limitations associated with current halogenation methods
prompted us to develop enzymes for biocatalytic halogenation. This proposal outlines the evolution of flavin
dependent halogenases (FDHs) and Fe(II)/a-ketoglutarate dependent halogenases (FeDHs) for a range
selective halogenation and related (i.e. pseudohalogen) atom transfer reactions. Specifically, we will build on
our extensive experience with FDH engineering to expand the range of substrates and sites on those
substrates that can be halogenated. We will focus on enabling halogenation of electron deficient aromatic
substrates with high site- and enantioselectivity. These efforts will benefit from recently characterized single
component flavin reductase/FDH enzymes, and we will optimize conditions for large scale halogenation using
these simplified biocatalysts. Finally, we recently reported that FDHs catalyze enantioselective halocyclization,
and we will expand the substrate scope of these reactions to include systems that cannot be achieved using
small molecule catalysts. In the FeDH space, we will engineer enzymes with expanded substrate scope to
enable site-selective C-H azidation of natural products and pharmaceuticals. This capability will be used for
chemoenzymatic synthesis via fragment coupling reactions and other processes that leverage the unique
reactivity of azides for more extensive remodeling of substrates to generate natural product-like heterocycles.
Finally, we will use both computational design and directed evolution to enable site-selective fluorination using
FeDHs. In addition to addressing a major synthetic challenge, this effort will improve our understanding of non-
native rebound in FeDHs to improve these enzymes for different C-H functionalization processes.