Abstact

The lignan (-)-podophyllotoxin possesses significant antiviral and anticancer activities and thus serves as a precursor to several natural products with therapeutic properties. Biosynthesis of podophyllotoxin involves cyclization of (-)-yatein to yield deoxypodophyllotoxin catalyzed by the iron- and alpha-ketoglutarate-dependent (Fe/alpha-KG) oxidase deoxypodophyllotoxin synthase (DPS), which completes the tetracyclic core of podophyllotoxin. Herein, (+)-hydroxy-yatein is also shown to be a substrate for DPS, directly affording (-)-podophyllotoxin as the enzymatic product. Moreover, derivatives of (+)-hydroxy-yatein are also found to be substrates providing synthetic precursors to medicines such as etoposide. Mechanistic analyses utilizing isotopologs and diastereomers of the m-dimethoxy analogue of (+)-hydroxy-yatein indicate antarafacial C-C bond formation during the cyclization reaction and remain consistent with cation-mediated cyclization. A crystallographic study of DPS bound with vanadium(IV) oxide, succinate, and (-)-hydroxy-yatein suggests a subtle interplay of steric interactions between the substrate and the active site that can alter the course of the DPS-catalyzed reaction and thus cyclization versus hydroxylation. Finally, an efficient chemoenzymatic approach to (-)-podophyllotoxin is described that relies only on freeze-dried whole cells after DPS overexpression.