Kinetic Solvent Isotope Effect in P450-Mediated Cyclization in Indolactams: Evidence for Branched Reactions and Guide for Their Modulation in Heterocycle Chemoenzymatic Synthesis

Cytochrome P450s demonstrate potential as biocatalysts for selective C–H bond functionalization in heterocycle synthesis. P450 monooxygenase (HinD) can catalyze cyclization for the biosynthesis of indolactam-like natural products. Moreover, the modification of N13 in the substrates leads to the formation of 6/5/6 tricyclic products as the minor product, which demonstrates potential for cancer treatment. However, the biosynthesis of indole-fused 6/5/6 tricyclic products is challenging as relatively few studies focus on the modulation of the minor products from P450 reactions to become dominant products for their bioproduction. Here, the isotopically sensitive branching method was employed to identify the conversion to 6/5/6 tricyclic products as the minor branch in wild-type HinD catalysis, originating from a common intermediate, in addition to the formation of 9-membered indolactam. The observed large kinetic solvent isotope effect (∼5.6) revealed that catalytic flow between two branches is sensitive and that the original minor product can be inverted to the dominant product by directing catalytic flow from the original major branch to the minor branch. Finally, 6/5/6 tricyclic products can be modulated and chemoenzymatically prepared as the major product. Data offer an insight into the rational design for the chemo-enzymatic synthesis of heterocyclic compounds.