Reaction Mechanism of Intramolecular C-C Coupling Catalyzed by the Non-heme Deoxypodophyllotoxin Synthase

Deoxypodophyllotoxin synthase (DPS) is a non-heme dioxygenase from Sinopodophyllum hexandrum that catalyzes the intramolecular C-C coupling reaction between the aromatic ring and the methylene group in the synthesis of the antitumor natural product podophyllotoxin. Three possible pathways for the C-C coupling reaction have been previously proposed, however, the reaction derails still remain unclear. In this work, we constructed computational models and performed QM/MM calculations to clarify the DPS-catalyzed intramolecular C-C bond formation mechanism. Our calculation results revealed that the C-C coupling reaction follows the radical electrophilic aromatic substitution (rEAS) mechanism rather than the electrophilic aromatic substitution (EAS) mechanism. The highly reactive species Fe(IV)=O first region-selectively abstracts a hydrogen atom from the C7ʹ (methylene) of substrate to trigger the coupling reaction. In addition, the typical OH rebound reaction is effectively blocked by the ligand exchange reaction within the iron center. The proton coupled electron transfer (PCET) between the substrate and the iron center further promotes the re-aromatization reaction of the intermediate. Based on the above results, we proposed that the non-heme-catalyzed coupling reaction between aromatic ring and methylene group should meet two basic conditions. First, the substrate should be well positioned to facilitate the hydrogen atom abstraction. Second, the special coordination center can effectively inhibit the OH rebound reaction. Third, the PCET between from the substrate to the iron center can greatly promote the re-aromatization of C-C coupled intermediate. These results may provide useful information for further understanding biosynthesis of cyclized natural products catalyzed by the non-heme enzymes.