VERIFICATION OF THE MECHANISM OF OXIDATIVE RING EXPANSION IN THE BIOSYNTHESIS OF STIPITATIC ACID BY TALAROMYCES-STIPITATUS

The pattern of incorporation of C-13 from C-13-labeled acetate into st ipitatic acid by Talaromyces stipitatus was studied by C-13 NMR. The r esults obtained were consistent with the biosynthetic routes previousl y elucidated for similar fungal tropolones. These studies, which both required and enabled the first complete C-13 NMR assignments of stipit atic acid, also demonstrated that the decarboxylation step in the bios ynthesis of stipitatic acid occurs after the formation of the tropolon e ring. The biosynthetic pathway to stipitatic acid includes an oxidat ive expansion of the benzene ring of 3-methylorsellinic acid to a seve n-membered tropolone ring. As a prelude to mechanism-based enzyme inac tivation studies, the mechanism of this step was reinvestigated by the biosynthetic incorporation of O-18 into stipitatic acid from O-18(2) The C-13 NMR spectrum of the resulting [O-18]stipitatic acid showed an O-18-induced isotope shift for the tropolone-ring carbonyl resonance with no evidence of O-18 enrichment at the C-6 hydroxyl group. This re sult provides conclusive evidence in support of the earlier report tha t the ring expansion involves oxidation by a monooxygenase to form a h ydroxymethyl intermediate, which undergoes a pinacol rearrangement to form the tropolone ring. (C) 1995 Academic Press, Inc.