Notoamide R: A Prominent Diketopiperazine Fermentation Metabolite amongst Others of Aspergillus ochraceus in the Absence of Ochratoxins

Abstract

Ochratoxin A is historically the most notable secondary metabolite of Aspergillus ochraceus on account of its toxicity to animals and fish. Currently, over 150 compounds of diverse structure and biosynthesis is a challenge to predict the array for any particular isolate. A brief focus 30 years ago on the failure to produce ochratoxins in foods in Europe and the USA revealed consistent failures to produce ochratoxin A by isolates from some USA beans. Analysis for familiar or novel metabolites particularly focused on a compound for which mass and NMR analyses were inconclusive. Resort to 14C-labelled biosynthetic precursors, particularly phenylalanine, to search for any close alternative to ochratoxins, was combined with conventional shredded-wheat/shaken-flask fermentation. This yielded, for an extract, an autoradiograph of a preparative silica gel chromatogram, which was subsequently analysed for an excised fraction using spectroscopic methodologies. Circumstances then delayed progress for many years until the present collaboration revealed notoamide R. Meanwhile, pharmaceutical discovery around the turn of the millennium revealed stephacidins and notoamides, biosynthetically combining indole, isoprenyl and diketopiperazine components. Later, in Japan, notoamide R was added as a metabolite of an Aspergillus sp. isolated from a marine mussel, and the compound was recovered from 1800 Petri dish fermentations. Renewed attention to our former studies in England has since shown for the first time that notoamide R can be a prominent metabolite of A. ochraceus, sourced from a single shredded wheat flask culture with its structure confirmed by spectroscopic data, and in the absence of ochratoxins. Renewed attention to the archived autoradiographed chromatogram allowed further exploration, but in particular has stimulated a fundamental biosynthetic approach to considering influences redirecting intermediary metabolism to secondary metabolite accumulation.