Diastereodivergent Synthesis of 8,12-Guaianolides Using Engineered P450 BM3

Abstract 8,12-Guaianolides are a class of sesquiterpene lactones that exhibit diverse biological activities, which are often strongly influenced by structural variations, including the stereochemistry of the C8 oxygen substituent. Here, we report a concise, diastereodivergent approach to both 8α,12- and 8β,12-guaianolide scaffolds from santonin-derived triene 5 by combining engineered cytochrome P450 BM3-catalyzed allylic C–H hydroxylation with a photochemical rearrangement. The P450 BM3 mutant LG-23 selectively hydroxylates the C8α position of 5, whereas a single additional mutation, L437I, inverts the facial selectivity to favor C8β hydroxylation. Structure-guided mutagenesis and docking studies suggest that the β-methyl group of Ile437 remodels the active site and enforces a flipped substrate binding mode that directs the opposite C8 C–H bond toward the heme iron. The utility of this enzymatic stereochemical switch was demonstrated by the synthesis of the 8α,12-guaianolides xerantholide (1) and mikanokryptin (2) and the 8β,12-guaianolide 11,13-dehydroachalensolide (4). This study demonstrates that a single active-site mutation in P450 BM3 can switch the facial selectivity of C–H hydroxylation at one carbon center, providing a strategy for stereodivergent enzymatic oxidation in complex-molecule synthesis.