Amelia E. de Jesus

Tremorgenic mycotoxins from Penicillium crustosum: isolation of penitrems A–F and the structure elucidation and absolute configuration of penitrem A

The isolation and characteristics of six tremorgenic mycotoxins, penitrems A–F from cultures of Penicillium crustosum are reported. The assignment of structure (2) to penitrem A is based on a detailed study of its high-field 1H and 13C n.m.r. spectra. The conformation and relative configuration of penitrem A was deduced from the observed proton–proton nuclear Overhauser effects (n.O.e.) and the magnitude of the proton–proton coupling constants. The chirality of C-25 was determined as S by the ‘partial resolution’ method of Horeau and penitrem A must therefore have the (12R, 14S, 15R, 18S, 19R, 22S, 23S, 24R, 25S, 26R, 28S, 31R, 32S) absolute configuration.

Tremorgenic mycotoxins from Penicillium crustosum. Biosynthesis of penitrem A

The biosynthesis of penitrem A has been studied with both 13C- and 2H-labelled precursors, viz. [1-13C]-, [2-13C]-, [1,2-13C2]-, and [1-13C,2-2H3]-acetate, [2-13C]-, [2,3-13C2]-, [2-2H2]- and [5-2H2]-mevalonate. The results show that penitrem A is derived from tryptophan, which contributes the indole moiety of the metabolite, geranylgeranylpyrophosphate, and two isopentenylpyrophosphate units. A 1,2-bond migration, involving the 2,3-bond of a mevalonate unit, occurs in the course of the biosynthesis and results in the observation of a one-bond (C,C) coupling between two [1-13C]acetate-derived carbon atoms and between two [2-13C]acetate-derived carbon atoms. Analysis of the one-bond (C,C)coupling constants in [2-13C]acetate-derived penitrem A showed that [1,2-13C2]acetate was formed during the fermentation. Although loss of water from an hydroxyisopropyl group to form the isopropenyl function present in penitrem A should proceed with retention of the stereochemical integrity of the two methyl groups, isomerization of the double bond causes equal distribution of 13C label between C-36 and C-38 and precludes any stereochemical deductions.

Structure and biosynthesis of the penitrems A–F, six novel tremorgenic mycotoxins from Penicillium crustosum

The structures of six new mycotoxins, penitrems A–F, isolated from cultures of Penicillium crustosum are deduced from their 13C and 1H n.m.r. spectra and from biosynthetic results.

Tremorgenic mycotoxins from Penicillium crustosum. Structure elucidation and absolute configuration of penitrems B–F

The assignment of structures (2)–(6) to penitrems B–F, tremorgenic mycotoxins isolated from cultures of Penicillium crustosum, is based on a detailed study of their high field 1H and 13C n.m.r. spectra. The relative configuration of penitrems B–F was deduced from the magnitude of the proton–proton coupling constants. The absolute configuration of these metabolites is based on comparisons with that of penitrem A and on the absolute configuration of penitrem D determined by the ‘partial resolution’ method of Horeau.

Structure elucidation of the janthitrems, novel tremorgenic mycotoxins from Penicillium janthinellum

The assignment of the structures (1)–(3) to janthitrems E-G, tremorgenic mycotoxins isolated from cultures of Penicillium janthinellum is based on a detailed study of their highfield 1H and 13C n.m.r. spectra. In the structure elucidation extensive use was made of heteronuclear 13C-{1H} selective population inversion (SPI) and homonuclear 1H-{1H} n.O.e. experiments.

Biosynthesis of viridicatumtoxin, a mycotoxin from Penicilium expansum

Incorporation of sodium [1-13C]-, [2-13C]-, [1,2-13C2]-, and [1-13C, 18O]-acetate, sodium [1,2,3-13C3]- malonate, and (2S)-[Me-13C]methionine as well as sodium hydrogen[14C]carbonate and sodium[1-14C]pyruvate into viridicatumtoxin, a metabolite from Penicillium expansum, indicates its formation by a mixed polyketide–terpenoid biosynthetic pathway.

Biosynthesis of α-cyclopiazonic acid. Steric course of proton removal during the cyclisation of β-cyclopiazonic acid in Penicillium griseofulvum

Feeding experiments with (3R)- and (3S)-[3-3H] tryptophan have shown that, in Penicillium griseofulvum Dierckx, incorporation of tryptophan into α-cycopiazonic acid (1) proceeds with loss of the 3-pro-S hydrogen atom. [3-2H2]Tryptophan was converted by P. griseofulvum into α-cyclopiazonic acid which was shown by 2H n.m.r. spectroscopy to contain deuterium only at the expected C-4 position. (2RS)-[2-3H,3-14C]Tryptophan was incorporated into α-cyclopiazonic acid (1) with loss of ca. 50% of the tritium. These findings are consistent with cyclisation of β-cyclopiazonic acid (4) to α-cyclopiazonic acid (1)via a 1,4-didehydro-derivative, with C–C bond formation at C-4 occurring from the side of the molecule opposite to that from which proton removal takes place.

Biosynthesis of aflatoxins. Incorporation of [4′-2H2]averufin into aflatoxin B1 by Aspergillus flavus

The observation of an NIH shift on incorporation of [2H3]acetate into aflatoxin B1 in cultures of Aspergillus flavus provides evidence for the timing of deoxygenation and hydroxylation steps which are an essential part of the biosynthetic pathway.

Biosynthesis of PR toxin by Penicillium roqueforti

The biosynthesis of PR toxin, a metabolite produced by Penicillium roqueforti, has been studied utilizing both 14C- and 13C-labelled precursors. The results indicate that PR toxin is formed via an isoprene biosynthetic pathway.

Large-scale purification of the mycotoxins aflatoxin B1, B2 and G1.

The isolation and purification of gram quantities of the important mycotoxins aflatoxin B1, B2 and G1 are described. The method involves final purification on a Waters Prep LC-500 instrument, loaded with silica cartridges, and elution with chloroform.