Abstract
Alternapyrone, a bioactive polyketide produced by the fungal host Aspergillus oryzae, is biosynthesized by a polyketide synthase encoded by the alt1-5 gene cluster. Despite its known bioactivity, the stereochemical configuration of the three stereogenic centers in its polyketide backbone has remained unresolved. In this study, we determined the complete stereostructure of alternapyrone using an integrative approach that combines predictive, rule-based stereochemical analysis with experimental validation through total synthesis. The efficient total synthesis enabled the precise assignment of the hypothesized stereochemistry by matching the synthetic product to the natural compound. This comprehensive study conclusively established the absolute configuration of alternapyrone.