Enantioselective NP-HPLC Resolution of Usnic Acid and Isousnic Acid with Revised Absolute Configuration and NMR Assignment of Isousnic Acid

Usnic acid (UA) enantiomers are abundant lichen compounds with diverse bioactivities, including potent antibiotic effects. In contrast, isousnic acid (isoUA) enantiomers are relatively scarce, and their biological functions and biosynthetic mechanisms remain poorly understood. Future studies on UA isomers would greatly benefit from a robust, optimized, and validated enantioselective chromatographic method that resolves all four isomers in a single run. This study aimed to (i) assign the absolute configuration of isoUA through integrated experimental and computational ECD analyses; (ii) perform a comprehensive structural characterization and clarify NMR assignments of isoUA relative to previous reports; (iii) develop and validate a chiral normal-phase HPLC method for simultaneous separation of (+)-isoUA ( 1 ), (−)-isoUA ( 2 ), (+)-UA ( 3 ), and (−)-UA ( 4 ). The absolute configurations of the isoUA enantiomers were assigned as ( S ) for (+)-isoUA ( 1 ) and ( R ) for (−)-isoUA ( 2 ). Examination of NMR data for isoUA revealed misassignments in some prior studies. Importantly, the developed HPLC method achieved baseline resolution of 1–4, enabling quantification in lichen specimens. This study emphasizes the importance of accurate enantiomeric structural elucidation before chiral method development and paves the way for future biosynthetic investigations and the precise detection of these enantiomers in lichen samples.