Robert Vleggaar

Structure elucidation of the fumonisins, mycotoxins from Fusarium moniliforme

The structures of the fumonisins, a family of structurally related mycotoxins isolated from cultures of Fusarium moniliforme, were elucidated by mass spectrometry and 1H and 13C n.m.r. spectroscopy as the diester of propane-1,2,3-tricarboxylic acid and either 2-acetylamino- or 2-amino-12,16-dimethyl-3,5,10,14,15-pentahydroxyicosane as well as in each case the C-10 deoxy analogue; in all cases both the C-14 and C-15 hydroxy groups are involved in ester formation with the terminal carboxy group of propane-1,2,3-tricarboxylic acid.

Fumonisins: Isolation, chemical characterization and biological effects

The fumonisin B mycotoxins (FB1 and FB2) have been purified and characterized from corn cultures of Fusarium moniliforme strain MRC 826. Fumonisin B1 (FB1, the major fumonisin produced in culture, has been shown to be responsible for the major toxicological effects of the fungus in rats, horses and pigs. Recent investigations on the purification of compounds with chromatographic characteristics similar to FB1 have led to the identification of two new fumonisins, FB3 and FB4. Fumonisins A1 and A2, the N-acetyl derivatives of FB1 and FB2 respectively, were also purified and shown to be secondary metabolites of the fungus. Short-term carcinogenesis studies in a rat liver bioassay indicated that over a period of 15 to 20 days, at dietary levels of 0.05–0.1%, FB2 and FB3 closely mimic the toxicological and cancer initiating activity of FB1 and thus could contribute to the toxicological effects of the fungus in animals. In contrast, no biological activity could be detected for FA1 under identical experimental conditions. These studies and others have indicated that the fumonisin B mycotoxins, although lacking mutagenicity in the Salmonella test or genotoxicity in the DNA repair assays in primary hepatocytes, appear to induce resistant hepatocytes similar to many known hepatocarcinogens.

Tremorgenic neurotoxins from perennial ryegrass causing ryegrass staggers disorder of livestock: structure elucidation of lolitrem B

The lolitrems, tremorgenic neurotoxins from perennial ryegrass, are implicated in ryegrass staggers disorder in livestock; the assignment of structure (1) to the major neurotoxin, lolitrem B, is based on its spectroscopic properties, particularly a detailed structure of its high-field 1H and 13C n.m.r. spectra, as well as chemical evidence.

Structure-activity relationships of fumonisins in short-term carcinogenesis and cytotoxicity assays☆

A short-term rat liver cancer initiation/promotion model was used to monitor the cancer-initiating activity of the mycotoxins fumonisin B1 (FB1), fumonisin B2 (FB2) and fumonisin B3 (FB3) as well as the N-acetyl derivatives of FB1 and FB2, and their respective hydrolysis products the aminopolyols. The induction of resistant hepatocytes, which develop into hepatocyte nodules on selection by the 2-acetylaminofluorene-partial hepatectomy promoting treatment, was taken as the endpoint for cancer initiation. When fed at a level of 1000 mg/kg diet for 21 days, only the fumonisins B were found to initiate cancer. In addition, these mycotoxins caused a marked reduction in the rat body weight during the initiating treatment. Comparative cytotoxicity studies in primary rat hepatocytes indicated that FB2 exhibited the highest cytotoxic effect followed by FB3 and FB1. In general, the fumonisin B mycotoxins exhibited a low cytotoxic effect in hepatocyte cultures, and the concentrations of FB1 and FB2 that caused a 50% (CD50) release of the total lactate dehydrogenase (LDH) were in the order of 2000 and 1000 microM, respectively. The N-acetyl derivatives also exhibited a cytotoxic effect, but were not as cytotoxic as the parent molecules at high concentrations. The respective aminopolyols exhibited a higher cytotoxicity than did the parent compounds, while tricarballylic acid (TCA) exhibited no dose-response effect despite the fact that it had a higher background cytotoxicity compared with the control. The apparent inability of the aminopolyols to act as cancer initiators could be related to a lack in absorption from the gut.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the mycotoxin fumonisin B1 and identification of its partially hydrolysed metabolites in the faeces of non-human primates

A method has been developed for the determination of fumonisin B1 (FB1) in the faeces of non-human primates (vervet monkeys). The animals were dosed with 14C-labelled FB1, and the radioactive compounds in faeces were recovered by repeated extractions with 0.1 M ethylenediaminetetraacetic acid. The extracts were cleaned-up on a reversed-phase (C18) solid-phase extraction cartridge, and FB1 was determined by o-phthaldialdehyde derivatization and reversed-phase HPLC. The analytical method for the determination of FB1 in the faecal extracts was reproducible [2.6% relative standard deviation (RSD)] and accurate (recovery from spiked blank extracts of 93 +/- 2.9% RSD). Confirmation of the identification of FB1 in faeces was achieved using HPLC and thin-layer chromatography, which showed that the radioactivity extracted corresponded mainly to FB1 and a new metabolite with chromatographic properties similar to those of the mycotoxin. The new metabolite was identified by mass spectrometry and nuclear magnetic resonance spectroscopy to be an equilibrium mixture of the two structural isomers of partially hydrolysed FB1, which are formed by hydrolysis of one of the ester groups of the mycotoxin.

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.

Structure elucidation of fusarin C, a mutagen produced by Fusarium moniliforme

The assignment of structure (1) to fusarin C, a mutagen isolated from cultures of Fusarium moniliforme is based on a detailed study of its high-field 1H and 13C n.m.r. spectra and X-ray crystallography of the 8Z isomer of (1) which defined the substitution pattern and relative configuration of the 2-pyrrolidone moiety; nuclear Overhauser enhancement experiments indicate that the 2E,4E,6E,8E,10E polyene chromophore of (1) exists in solution as an equilibrium between two conformers with s-cis and s-trans topology of the C-5–C-6 single bond.

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.

Flavonoids from Tephrosia—VII : The constitution and absolute configuration of lupinifolin and lupinifolinol, two flavanones from Tephrosia lupinifolia Burch (DC)

Abstract The structure and absolute configuration of lupinifolin, (2S) - 4′,5 - dihydroxy - 8 - (3‴ - methyl - 2‴ - butenyl) - 2″, 2″ - dimethylpyrano[5″.6″ - g]flavanone, and lupinifolinol, (2R,3R) - 8 - (3‴ - methyl - 2‴ - butenyl) - 3,4′,5 - trihydroxy - 2″,2″ - dimethylpyrano[5″.6″ - g]flavanone, have been deduced from spectroscopic and chemical evidence.

Reversed-phase high-performance liquid chromatography of tenuazonic acid and related tetramic acids.

A reversed-phase high-performance liquid chromatographic system for the determination of the fungal toxin, tenuazonic acid, (5S,8S)-3-acetyl-5-sec.-butyltetramic acid, is described. The system utilizes a column packed with deactivated end-capped C18 silica with a high carbon load to overcome the problem of poor chromatographic performance of this beta-diketone on reversed-phase liquid chromatography which previously necessitated the use of anion-exchange, ligand-exchange or ion-pairing methods. The reversed-phase system allows the separation of tenuazonic acid from its (5R,8S)-diastereomer, allo-tenuazonic acid and was applied to the detection of tenuazonic acid in cultures of Alternaria alternata and Phoma sorghina. By means of diode-array ultraviolet detection, (5S)-3-acetyl-5-isopropyltetramic acid was observed in extracts of culture material. This metabolite was purified using the analytical reversed-phase system and was identified by 1H and 13C nuclear magnetic resonance spectroscopy.