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Incorporations of 13C-labelled acetates and methionine into the mycotoxin austin in cultures of Aspergillus ustus give a labelling pattern consistent with a mixed polyketide-terpenoid pathway. Incorporations of 14C and 2H labelled 3,5-dimethylorsellinate confirm the intermediary of a preformed tetraketide-derived phenolic precursor. Further information on the mechanisms involved in the modifications of both the farnesyl- and tetraketide-derived portions of the molecule are provided by incorporation studies with [1-13C,18O2]acetate, [methyl-13C,2H3] methionine, 13C,18O-labelled dimethylorsellinate,18O2 gas and [6-13C,6-2H3]mevalonic acid lactone.

The fungal metabolite dihydrodeoxy-8-epi-austdiol, elaborated by Aspergillus ustus, has been identified as (7R,8R)-7,8-dihydro-7,8-dihydroxy-3,5,7-trimethyl-2-benzopyran-6-one. The absolute configuration of the azaphilones has been deduced.

The assignment of the natural-abundance 13C n.m.r. spectrum of austocystin D, a mycotoxin produced by Aspergillus ustus MRC 1163, allowed a study of its biosynthetic origin. The complete 13C–13C coupling pattern in austocystin D enriched from [1,2-13C2]acetate has been determined and confirms that no randomisation of labelling in ring A occurs during the anthraquinone → xanthone conversion in the biosynthetic pathway.

The fungal metabolite austdiol, elaborated by Aspergillus ustus, has been identified as (7R,8S)-7,8-dihydro-7,8-dihydroxy-3,7-dimethyl-6-oxo-6H-2-benzopyran-5-carbaldehyde. The absolute configuration was deduced by the ‘partial resolution’ method of Horeau.

Incorporationof [1-13C]-, [1,2-13C2]-and [1-13C,2,2,2-2H3]-acetate, and (3RS)-[2-13C]mevalonolactone into austalide D(2), a metabolite of Aspergillus ustus, shows that the austalides are derived from 6[(2E,6E)farnesyl]-5,7-dihydroxy-4-methylphthalide. The proposed mechanism for the subsequent cyclisation and oxidative modifications of the farnesyl moiety, consistent with the relative stereochemistry of the austalides and the structures of the cometabolites, austalides J (4), K (5), and L (6), is supported by the incorporation of austalide K into austalide D. The addition of ethanol to growing cultures of A. ustus severely inhibits normal metabolite production and induces the formation of a new metabolite, ethyl 6-ethyl-2,4-dihydroxy-3-methylbenzoate (11). The biosynthetic origin of this metabolite was studied using [1-13C]- and [1-13C,2,2,2-2H3]acetate and (2S)-[methyl-13C]methionine as precursors.

The 13C n.m.r, spectrum of austin has been fully assigned by inter alia analysis of long range 1H–13C couplings in the fully 1H-coupled 13C n.m.r. spectrum. Further related metabolites, dehydroaustin, austinol, and isoaustin, have been isolated from Aspergillus ustus, Aspergillus variecolor, and Penicillium diversum. Their structures have been assigned by detailed analysis of their 1H and 13C n.m.r. spectra.