Controlled Oxidation of Remote sp3 C–H Bonds in Artemisinin via P450 Catalysts with Fine-Tuned Regio- and Stereoselectivity

• Published in: Journal of the American Chemical Society Vol. 134; no. 45; pp. 18695 - 18704
• Main Authors: Zhang, Kaidong, Shafer, Brian M, Demars, Matthew D, Stern, Harry A, Fasan, Rudi
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 14.11.2012; Amer Chemical Soc
• Subjects: active sites; artemisinin; Artemisinins - chemistry; Artemisinins - metabolism; Biocatalysis; biocatalysts; chemical bonding; Chemistry; Chemistry, Multidisciplinary; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; enantiomers; hydroxylation; Models, Molecular; Molecular Conformation; mutagenesis; Mutagenesis, Site-Directed; oxidation; Oxidation-Reduction; Peptide Mapping; Physical Sciences; prediction; Science & Technology; stereochemistry; Stereoisomerism; FUNCTIONALIZATION; ACTIVATION; ELECTRON-TRANSFER; SELF-SUFFICIENT; MOLECULAR RECOGNITION; CRYSTAL-STRUCTURE; OXYGENATION; HYDROXYLATION; REGIOSELECTIVITY; SELECTIVITY
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/ja3073462

The selective oxyfunctionalization of isolated sp3 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 S and R 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.