Structural Diversification of 14-Membered Macrolides by Chemoenzymatic Synthesis

The pikromycin polyketide synthase (PKS) catalyzes formation of 12-membered macrolactone 10-deoxymethynolide, and 14-membered macrolactone narbonolide. Herein, we show the efficient diversification of novel 14-membered macrolactones and identification of 6-membered δ-lactones from a series of unnatural pentaketides using the PikAIII/PikAIV PKS <i>in vitro</i> system. New macrocycles were further elaborated by the addition of D-desosamine and late-stage C-H hydroxylation. Molecular dynamics (MD) simulations and density functional theory (DFT) calculations were conducted to probe the reactivity and selectivity of this terminal catalytic step on the assembled unnatural macrolides. This approach highlights the flexibility of the PikAIII/PikAIV bimodule system in processing non-native substrates and demonstrates the utility of sequential biocatalytic steps for the chemoenzymatic synthesis of complex antibiotic scaffolds.