Directed Evolution of an FeII-Dependent Halogenase for Asymmetric C(sp3)–H Chlorination

By using structure-guided directed evolution, the substrate scope of the FeII and α-ketoglutarate dependent halogenase Wi-WelO15 from Westiella intricata HT-29-1 was engineered to enable chemo-, regio-, and diastereoselective chlorination of unactivated C­(sp3)–H bonds using NaCl as chlorine source. While FeII dependent enzymes are often oxygen sensitive, variants of this halogenase could be screened in lysates under aerobic conditions. The developed biocatalysts offer a route to mild, late-stage chlorination on milligram scale of non-natural hapalindoles containing a ketone instead of an isonitrile functionality, thereby unlocking them for preparative biocatalysis.