Reprogramming ThDP-Dependent Enzyme for Intermolecular [3 + 2] Annulation via a Homoenolate Intermediate

Abstract Thiamine diphosphate (ThDP)-dependent enzymes are versatile biocatalysts for carbon–carbon bond formation, yet their catalytic repertoire has been largely confined to linear homologation and intramolecular cyclizations that rely on single-site reactive intermediates such as the Breslow or ketyl radical species. Intermolecular annulation─a powerful strategy for constructing cyclic architectures─has remained elusive within this enzyme class. Here, we report the first intermolecular [3 + 2] annulation catalyzed by a ThDP-dependent enzyme, achieved through a homoenolate-mediated nucleophilic addition pathway. A phylogeny-guided enzyme mining identified a previously uncharacterized enzyme from Variovorax boronicumulans(ThDP-5), and subsequent protein engineering yielded the F484S variant, which exhibits markedly enhanced activity and exquisite stereocontrol. This biocatalytic system exhibits a broad substrate scope (30 examples), accommodating diverse substituted cinnamaldehydes and isatins to afford chiral spirooxindole lactones with excellent stereoselectivity (up to > 20:1 dr and 99% ee). Preparative-scale reactions (2 mmol) validate its synthetic utility. This work expands ThDP-dependent biocatalysis to intermolecular annulations, providing a robust and selective platform for the construction of pharmaceutically relevant spirocyclic architectures.