Verification of the Mechanism of Oxidative Ring Expansion in the Biosynthesis of Stipitatic Acid by Talaromyces stipitatus

The pattern of incorporation of 13C from 13C-labeled acetate into stipitatic acid by Talaromyces stipitatus was studied by 13C NMR. The results obtained were consistent with the biosynthetic routes previously elucidated for similar fungal tropolones. These studies, which both required and enabled the first complete 13C NMR assignments of stipitatic acid, also demonstrated that the decarboxylation step in the biosynthesis of stipitatic acid occurs after the formation of the tropolone ring. The biosynthetic pathway to stipitatic acid includes an oxidative expansion of the benzene ring of 3-methylorsellinic acid to a seven-membered tropolone ring. As a prelude to mechanism-based enzyme inactivation studies, the mechanism of this step was reinvestigated by the biosynthetic incorporation of 18O into stipitatic acid from 18O2. The 13C NMR spectrum of the resulting [18O]stipitatic acid showed an 18O-induced isotope shift for the tropolone-ring carbonyl resonance with no evidence of 18O enrichment at the C-6 hydroxyl group. This result provides conclusive evidence in support of the earlier report that the ring expansion involves oxidation by a monooxygenase to form a hydroxymethyl intermediate, which undergoes a pinacol rearrangement to form the tropolone ring.