Controlled Oxidation of
Remote sp<sup>3</sup> C–H
Bonds in Artemisinin via P450 Catalysts with Fine-Tuned Regio- and
Stereoselectivity
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Abstract
The selective oxyfunctionalization of isolated sp<sup>3</sup> C–H
bonds in complex molecules represents a formidable challenge in organic
chemistry. Here, we describe a rational, systematic strategy to expedite
the development of P450 oxidation catalysts with refined regio- and
stereoselectivity for the hydroxylation of remote, unactivated C–H
sites in a complex scaffold. Using artemisinin as model substrate,
we demonstrate how a three-tier strategy involving first-sphere active
site mutagenesis, high-throughput P450 fingerprinting, and fingerprint-driven
P450 reactivity predictions enabled the rapid evolution of three efficient
biocatalysts for the selective hydroxylation of a primary and a secondary
C–H site (with both <i>S</i> and <i>R</i> stereoselectivity) in a relevant yet previously inaccessible region
of this complex natural product. The evolved P450 variants could be
applied to provide direct access to the desired hydroxylated derivatives
at preparative scales (0.4 g) and in high isolated yields (>90%),
thereby enabling further elaboration of this molecule. As an example,
enantiopure C7-fluorinated derivatives of the clinical antimalarial
drugs artesunate and artemether, in which a major metabolically sensitive
site is protected by means of a C–H to C–F substitution,
were afforded via P450-mediated chemoenzymatic synthesis