Cyclopiazonic Acids Research Articles

Origin of the 6/5/6/5 Tetracyclic Cyclopiazonic Acids.

The natural product α-cyclopiazonic acid (α-CPA) is a very potent Ca2+-ATPase inhibitor. The CPA family of compounds comprise over 80 chemical entities with at least five distinct skeletons. While α-CPA features a canonical 6/5/6/5/5 skeleton, the 6/5/6/5 skeleton is the most prevalent among the CPA family. However, the origin of the unique tetracyclic skeleton remains unknown. The 6/5/6/5-type CPAs may derive from a precursor of acetoacetyl-l-tryptophan (AATrp) generated from a hypothetic thioesterase-like pathway. Alternatively, cleavage of the tetramic acid ring would also result in the formation of the 6/5/6/5 scaffold. Aspergillus oryzae HMP-F28 is a marine sponge-associated filamentous fungus known to produce CPAs that act as primary neurotoxins. To elucidate the origin of this subfamily of CPAs, we performed homologous recombination and genetic engineering experiments on strain HMP-F28. Our results are supportive of the ring cleavage pathway through which the tetracyclic 6/5/6/5-type CPAs are generated from 6/5/6/5/5-type pentacyclic CPAs.

Mycotoxins in coffee and chicory: from regulated to emergent

Coffee is a daily basic food product for many people all over the world. In Russia and some European countries, people who try to pursue healthy lifestyle often prefer chicory as a substitute to coffee. Our research goal was to evaluate occurrence of Aspergillus, Penicillium, Fusarium and Alternaria secondary metabolites in coffee and chicory distributed on the RF market. 29 mycotoxins were determined in 48 samples of coffee and chicory using ultra high-performance liquid chromatography coupled with tandem mass-spectrometric detection (UHPLC-MS/MS). The range of analyzed contaminants included regulated mycotoxins (aflatoxins, ochratoxin A, deoxynivalenol, fumonisins, T-2 toxin, and zearalenone), their derivatives and structural analogs (A and B trichothecenes), Alternaria metabolites (alternariol, its methyl ether, altenuene, tentoxin), citrinin and several emergent mycotoxins (citreoviridin, cyclopiazonic and mycophenolic acids, enniatins, beauvericin). To the best of our knowledge, the present study is the first to report results indicating that unregulated emergent mycotoxins occur in the examined products. Chicory samples contained beauvericin (9 of 16 samples, the contents varied from 2.4 to 1173 µg/kg) and enniatin B (6 of 16 samples, 2.8–1109 µg/kg). Green and roasted coffee samples contained mycophenolic acid (11 of 20 samples, 23.5–58.3 µg/kg; 3 of 12 samples, 155.7–712.2 µg/kg accordingly). Several samples were contaminated with aflatoxins, ochratoxin A and fumonisin B2. Their contents in the examined samples did not exceed maximum levels; however, their occurrence indicates a potential health risk for consumers. This requires hygienic assessment and monitoring of these products with the focus on their contamination not only with regulated aflatoxin B1 and ochratoxin A but also with other potentially hazardous mycotoxins

Микотоксины в кофе и цикории: от регламентируемых к эмерджентным

Coffee is a daily basic food product for many people all over the world. In Russia and some European countries, people who try to pursue healthy lifestyle often prefer chicory as a substitute to coffee. Our research goal was to evaluate occurrence of Aspergillus, Penicillium, Fusarium and Alternaria secondary metabolites in coffee and chicory distributed on the RF market. 29 mycotoxins were determined in 48 samples of coffee and chicory using ultra high-performance liquid chromatography coupled with tandem mass-spectrometric detection (UHPLC-MS/MS). The range of analyzed contaminants included regulated mycotoxins (aflatoxins, ochratoxin A, deoxynivalenol, fumonisins, T-2 toxin, and zearalenone), their derivatives and structural analogs (A and B trichothecenes), Alternaria metabolites (alternariol, its methyl ether, altenuene, tentoxin), citrinin and several emergent mycotoxins (citreoviridin, cyclopiazonic and mycophenolic acids, enniatins, beauvericin). To the best of our knowledge, the present study is the first to report results indicating that unregulated emergent mycotoxins occur in the examined products. Chicory samples contained beauvericin (9 of 16 samples, the contents varied from 2.4 to 1173 µg/kg) and enniatin B (6 of 16 samples, 2.8–1109 µg/kg). Green and roasted coffee samples contained mycophenolic acid (11 of 20 samples, 23.5–58.3 µg/kg; 3 of 12 samples, 155.7–712.2 µg/kg accordingly). Several samples were contaminated with aflatoxins, ochratoxin A and fumonisin B2. Their contents in the examined samples did not exceed maximum levels; however, their occurrence indicates a potential health risk for consumers. This requires hygienic assessment and monitoring of these products with the focus on their contamination not only with regulated aflatoxin B1 and ochratoxin A but also with other potentially hazardous mycotoxins.

Mycotoxins contamination of cocoa products and carob marketed in the Russian Federation

Cocoa is a daily basic food for many people all over the world. Also, people engaged in healthy lifestyle often prefer carob. The aim of the present study was to evaluate occurrence of Aspergillus, Penicillium, Fusarium and Alternaria secondary metabolites in cocoa and carob available in the Russian Federation and assess mycotoxin's intake with these products. Material and methods. Concentration of 27 mycotoxins in 63 samples of cocoa and carob products was determined by ultra high-performance liquid chromatography coupled to tandem mass-spectrometric detection (UPLC-MS/MS). The list of mycotoxins included regulated ones (aflatoxins, ochratoxin A, deoxynivalenol, fumonisins, T-2 toxin, zearalenone), their derivatives and structural analogs (A and B trichothecenes), and emergent mycotoxins (alternariol, its monomethyl ether, altenuene, tentoxin, citrinin, sterigmatocistin, cyclopiazonic and mycophenolic acids, enniatins, beauvericin). Results. 29 of 41 cocoa samples were positive for beauvericin, contamination level was from 1.6 to 2184.8 μg/kg. Tentoxin, sterigmatocystin and alternatiol monomethyl ether were detected in 3 samples; their concentration varied in the ranges 0.7-1.2, 1.5-3.3 and 4.0-7.8 μg/kg correspondingly. Carob samples were positive for alternariol (14 of 22 samples, 1.5-43.1 μg/kg); tentoxin (13 samples, 0.5-8.7 μg/kg), mycophenolic acid (6 samples, 6.9-8.2 μg/kg) and for alternatiol monomethyl ether (3 samples, 1.0-1.2 μg/kg). Several samples of cocoa were contaminated with regulated mycotoxins: zearalenone (18 of 41 samples, 2.1-24.6 μg/kg), ochratoxin A (14 samples, 0.75-12.0 μg/kg) and aflatoxin B1 (2 samples, 0.59 and 0.86 μg/ kg). Several carob samples were contaminated with fumonisin B2 (7 of 22 samples, 4.2-5.2 μg/kg), ochratoxin A (5 samples, 0.5-1.4 μg/kg) and aflatoxin B1 (3 samples, 0.15-0.18 μg/kg). The ochratoxin A content in two instant cocoa beverages exceeded its maximum level (5 μg/kg) set for some plant products. Conclusion. To the best of our knowledge, the present study is the first survey devoted to emergent mycotoxin contamination of carob and cocoa alternative products marketed in the Russian Federation. The high occurrence of such mycotoxins as aflatoxins, beauvericin and ochratoxin A in these products indicates a potential health risk and the need for a hygienic assessment of cocoa and carob products' contamination not only with regulated in cocoa products aflatoxin B1, but with other mycotoxin including emergent ones. The risk of dietary intake of aflatoxins, beauvericin and ochratoxin A with cocoa products (especially with instant cocoa beverages) has been revealed for children over 7 years old in organized groups.

Tea: Transfer of Mycotoxins from the Spiked Matrix into an Infusion.

Recent surveys report the occurrence of Aspergillus and Penicillium metabolites (aflatoxins (AFLs), ochratoxin A (OTA), cyclopiazonic and mycophenolic acids (MPA), sterigmatocystin (STC), citrinin), Fusarium (trichothecenes, zearalenone (ZEA), fumonisins (FBs), enniatins (ENNs)) and Alternaria (alternariol (AOH), its methyl ether (AME), tentoxin (TE), and tenuazonic acid (TNZ)) toxins in dry Camellia sinensis and herbal tea samples. Since tea is consumed in the form of infusion, correct risk assessment needs evaluation of mycotoxins’ transfer rates. We have studied the transfer of AFLs, OTA, STC, deoxynivalenol (DON), ZEA, FBs, T-2, and HT-2 toxins, AOH, AME, TE, ENN A and B, beauvericin (BEA), and MPA from the spiked green tea matrix into an infusion under variation of preparation time and water characteristics (total dissolved solids (TDS) and pH). Analytes were detected by HPLC-MS/MS. The main factors affecting transfer rate proved to be mycotoxins’ polarity, pH of the resulting infusion (for OTA, FB2, and MPA) and matrix-infusion contact period. The concentration of mycotoxins increased by 20–50% within the first ten minutes of infusing, after that kinetic curve changed slowly. The concentration of DON and FB2 increased by about 10%, for ZEA, MPA, and STC it stayed constant, while for T-2, TE, AOH, and AFLs G1 and G2 it went down. Maximum transfer correlated well with analytes polarity. Maximum transfer of ENNs, BEA, STC, ZEA, and AOH into infusion was below 25%; AFLs—25–45%; DON, TE, and T-2 toxins 60–90%, FB1—80–100%. The concentration of OTA, MPA, and FB2 in the infusion depended on its pH. At pH about four, 20%, 40%, and 60% of these toxins transferred into an infusion, at pH about seven, their concentrations doubled. Water TDS did not affect transfer significantly.

Dried fruits marketed in Russia: multi-mycotoxin contamination

Mycotoxins (MT) - secondary metabolites of micromycetes - are natural contaminants of plant products. Fruits are particularly susceptible to fungal contamination and MT accumulation due to high sugar content. It can occur at any production stage: during vegetation, drying and storage. The most hazardous MT - aflatoxins (AFLs) and ochratoxin A (OTA) - are regulated in dried fruits in some countries. However, their maximum levels (ML) were not set in Russia yet. The present research was aimed at the evaluation of MT contamination of dried fruits marketed in Russia. Material and methods. 54 samples of dried dates (n=11), apricots (n=9), raisins (n=9), prunes (n=7), figs (n=6), apples (n=3) and mixtures for compote (n=9) were analyzed for 32 MTs by HPLC-MS/MS with positive/negative ESI in the MRM mode. Results. OTA and fumonisins (FBs) were the major regulated contaminants, their occurrence proved to be 10 and 17% correspondingly. Emergent metabolites of Fusarium spp. enniatin A (ENN A, 22%) and beauvericin (BEA, 15%); Penicillium spp. mycophenolic and cyclopiazonic acids (MPA and CPA, about 19%); Alternaria spp. tentoxin (TE, 17%) were detected also. Two-thirds of positive samples were contaminated with two and more MTs. All studied samples could be referred as safe within the EU regulations. Conclusion. Some kinds of dry fruits proved to be susceptible to contamination with particular MTs. Characteristic pattern for raisin was the OTA and MPA combination, for figs - FBs and CPA. According to literature data concerning occurrence and safety of MTs and the results of our survey, the long-term monitoring of AFLs and OTA in dry fruits and AFLs, OTA, FBs and CPA in figs from different regions of Russia is necessary to assess the need for setting of MLs of these MTs.

Genome sequence of Aspergillus flavus A7, a marine-derived fungus with antibacterial activity.

Due to the specific properties of the marine environment, marine microorganisms have exclusive physicochemical characteristics that are different from those of terrestrial microorganisms, which can produce various secondary metabolites (SMs) with considerable structural diversity and biological activity. In this study, three strains of coepiphytic Aspergillus with potential antibacterial activities, A7 (Aspergillus flavus), B27 (Aspergillus flavipes), and R12 (Aspergillus sydowii), were isolated from the South China Sea. Via the Illumina MiSeq sequencing platform, the genomes of the three strains were sequenced, and genome comparison showed the highest diversity of the biosynthetic gene clusters (BGCs) in A7. In addition, a comparison of physiological and genomic characteristics between A7 and other A. flavus strains demonstrated the superior environmental adaptability of A7, which is apparently consistent with the genetic richness of BGCs. By assigning reads to known BGCs, putative BGCs were allocated in A7 that corresponded to various SMs, including naphthopyrone, pyranonigrin E, and cyclopiazonic acids. Based on gene homology analysis, we surmise that a region is involved in the biosynthesis of ustiloxin-like RiPPs, a less thoroughly studied SM in fungi. Our results provide genetic information for the investigation of marine Aspergillus spp., which may help to elucidate their chemical diversity and adaptive strategies.

Stability of Mycotoxins in Individual Stock and Multi-Analyte Standard Solutions.

Standard solutions of mycotoxins prepared in RP HPLC solvents from neat standards are usually used for analytical method development. Multi-mycotoxin HPLC-MS/MS methods necessitate stability estimation for the wide spectrum of fungal metabolites. The stability of individual diluted stock standard solutions of mycotoxins in RP-HPLC solvents and multi-analyte HPLC-MS/MS calibrants was evaluated under standard storage and analysis conditions. Individual stock standard solutions of aflatoxins, sterigmatocystin, A- and B-trichothecenes, zearalenone and its analogues, ochratoxin A, fumonisins, Alternaria toxins, enniatins and beauvericin, moniliformin, citrinin, mycophenolic, cyclopiazonic acids and citreoviridin were prepared in RP-HPLC solvents and stored at −18 °C for 14 months. UV-spectroscopy was utilized to monitor the stability of analytes, excluding fumonisins. The gradual degradation of α-, β-zearalenol and α-, β-zearalanol in acetonitrile was detected. Aflatoxins and sterigmatocystin, zearalenone, Alternaria toxins, enniatins and beauvericin, citrinin, mycophenolic, cyclopiazonic acids and citreoviridin can be referred to as stable. The concentration of the majority of trichothecenes should be monitored. Diluted multi-mycotoxin standard in water/methanol (50/50 v/v) solutions acidified with 0.1% formic acid proved to be stable in silanized glass at 23 °C exposed to light for at least 75 h (CV ≤ 10%). An unexpected manifestation of MS/MS signal suppression/enhancement was discovered in the course of multi-mycotoxin standard solution stability evaluation.

Characterization and abolishment of the cyclopiazonic acids produced by Aspergillus oryzae HMP-F28.

Extracellular alkalinization and H2O2 production are important early events during induced resistance establishment in plants. In a screen for metabolites as plant resistance activators from 98 fungal isolates associated with marine sponge Hymeniacidon perleve, the cyclopiazonic acids (CPAs) produced by Aspergillus oryzae HMP-F28 induced significant extracellular alkalinization coupled with augmented H2O2 production in tobacco cell suspensions. Bioassay-guided fractionation led to the isolation and structural elucidation of a new CPA congener (4, 3-hydroxysperadine A) and three known ones (1-3). To construct a mutasynthetic strain to generate unnatural CPA analogues, a hybrid pks-nrps gene (cpaS) was disrupted to abolish the production of the critical precursor of cyclo-acetoacetyl-L-tryptophan (cAATrp) and all the downstream CPA products. Elimination of cAATrp will allow cAATrp mimics being processed by the CPA biosynthetic machinery to produce CPA derivatives with designed structural features.

Effect of potassium sorbate, sodium benzoate, and sodium nitrite on biosynthesis of cyclopiazonic and mycophenolic acids and citrinin by fungi of the Penicillium genus

The effect of potassium sorbate, sodium benzoate, and sodium nitrite used as preservatives in the food industry in the production of such mytotoxins as citrinin cyclopiazonic and mycophenolic acids by the contaminating fungi Penicillium citrinum, P. commune, and P. brevicompactum, respectively, was investigated. It was shown that the effect of preservatives used at concentrations relevant to the food industry on the synthesis of mycotoxins depended on the species-specific biochemical and physiological features of the cultures. The growth of P. brevicompactum was inhibited to the highest degree by sodium nitrite and potassium sorbate, and the growth of P. commune was so inhibited by sodium benzoate. It was established that the introduction of 0.015% sodium nitrite into the medium resulted in 1.3- and 1.4-fold reductions of the production of citrinin and mycophenolic acid, respectively, while the production of cyclopiazonic acid did not change in comparison with the control. The introduction of 0.015% sodium benzoate caused a more than 1.5-fold increase of the concentration of citrinin, cyclopiazonic, and mycophenolic acids, and the addition of 0.02% potassium sorbate increased the production of cyclopiazonic and mycophenolic acids by 1.7 and 2.6 times, respectively.

О КОНТАМИНАЦИИ МИКОТОКСИНАМИ СЕНАЖА И СИЛОСА В ЖИВОТНОВОДЧЕСКИХ ХОЗЯЙСТВАХ

Improvement of sanitary control of grass fodder, taking into account the whole variety of factors that have a negative impact on the animal, is the most important task of agricultural science. In the present study, we investigated mycotoxin contamination of hayage and silage by the method of indirect competitive enzyme-linked immunosorbent assay. In a survey of 30 commercial feed batches from the livestock farms located in the central regions of the European Russia, namely Bryanskya, Lipetskaya, Moskovskaya, Smolenskaya and Tverskaya provinces, a multiple contamination pattern was shown. In all the samples eight or more mycotoxins were found, and more than half of samples contained 14-15 components. Alternariol was found in all samples of the both types of feeds in amounts from 50 to 1260 mg/kg, while aflatoxin В 1 ochratoxin A, citrinin and ergot alkaloids had extensive distribution with low intensity. In haylage with high incidence of all analyzed fusariotoxins, mass concentration of Т-2 toxin was the lowest (4 to 30 mg/kg), and the levels of diacetoxyscirpenol, deoxynivalenol, zearalenone and fumonisins were in the range of 100-1000 mg/kg. Sterigmatocystin, emodin, cyclopiazonic and mycophenolic acids, PR-toxin occurred everywhere, wherein emodin, cyclopiazonic acid and PR-toxin often present in amounts up to 1000 mg/kg. Silage prepared mainly from corn grass, with frequent detection of T-2 toxin and deoxynivalenol in significant content (to 350 and 2820 mg/kg, respectively) revealed similarities with corn grain from the center of European Russia. Sterigmatocystin and emodin were detected in all silage samples, whereas PR-toxin, cyclopiazonic and mycophenolic acids were slightly inferior to them in frequency. Levels of accumulation of these mycotoxins were lower than those found in silage, and for mycophenolic acid they generally remained the same. A botanical composition of herbage and features of toxin-producing fungi complexes, accompanying the growing season and the subsequent process of fermentation, are discussed among possible reasons for the differences in mycotoxin contamination of haylage and silage.

MYCOTOXIN CONTAMINATIONS IN COMMERCIALLY USED HAYLAGE AND SILAGE

Improvement of sanitary control of grass fodder, taking into account the whole variety of factors that have a negative impact on the animal, is the most important task of agricultural science. In the present study, we investigated mycotoxin contamination of hayage and silage by the method of indirect competitive enzyme-linked immunosorbent assay. In a survey of 30 commercial feed batches from the livestock farms located in the central regions of the European Russia, namely Bryanskya, Lipetskaya, Moskovskaya, Smolenskaya and Tverskaya provinces, a multiple contamination pattern was shown. In all the samples eight or more mycotoxins were found, and more than half of samples contained 14-15 components. Alternariol was found in all samples of the both types of feeds in amounts from 50 to 1260 µg/kg, while aflatoxin Â1, ochratoxin A, citrinin and ergot alkaloids had extensive distribution with low intensity. In haylage with high incidence of all analyzed fusariotoxins, mass concentration of O-2 toxin was the lowest (4 to 30 µg/kg), and the levels of diacetoxyscirpenol, deoxynivalenol, zearalenone and fumonisins were in the range of 100-1000 µg/kg. Sterigmatocystin, emodin, cyclopiazonic and mycophenolic acids, PR-toxin occurred everywhere, wherein emodin, cyclopiazonic acid and PR-toxin often present in amounts up to 1000 µg/kg. Silage prepared mainly from corn grass, with frequent detection of T-2 toxin and deoxynivalenol in significant content (to 350 and 2820 µg/kg, respectively) revealed similarities with corn grain from the center of European Russia. Sterigmatocystin and emodin were detected in all silage samples, whereas PR-toxin, cyclopiazonic and mycophenolic acids were slightly inferior to them in frequency. Levels of accumulation of these mycotoxins were lower than those found in silage, and for mycophenolic acid they generally remained the same. A botanical composition of herbage and features of toxin-producing fungi complexes, accompanying the growing season and the subsequent process of fermentation, are discussed among possible reasons for the differences in mycotoxin contamination of haylage and silage.

A new benzofuran glycoside and indole alkaloids from a sponge-associated rare actinomycete, Amycolatopsis sp.

Three new secondary metabolites, amycofuran (1), amycocyclopiazonic acid (2), and amycolactam (3), were isolated from the sponge-associated rare actinomycete Amycolatopsis sp. Based on combined spectroscopic analyses, the structures of 1–3 were determined to be a new benzofuran glycoside and new indole alkaloids related to cyclopiazonic acids, a class that has previously only been reported in fungi. The absolute configurations of 1 and 3 were deduced by ECD calculations, whereas that of 2 was determined using the modified Mosher method. Amycolactam (3) displayed significant cytotoxicity against the gastric cancer cell line SNU638 and the colon cancer cell line HCT116.

Simultaneous determination of ochratoxin A and cyclopiazonic, mycophenolic, and tenuazonic acids in cornflakes by solid-phase microextraction coupled to high-performance liquid chromatography.

A solid-phase microextraction (SPME) method, coupled to liquid chromatography with diode array UV detection (LC-UV/DAD), for the simultaneous determination of cyclopiazonic acid, mycophenolic acid, tenuazonic acid, and ochratoxin A is described. Chromatographic separation was achieved on a propylamino-bonded silica gel stationary phase using acetonitrile/methanol/ammonium acetate buffer mixture (78:2:20, v/v/v) as mobile phase. SPME adsorption and desorption conditions were optimized using a silica fiber coated with a 60 microm thick polydimethylsiloxane/divinylbenzene film. Estimated limits of detection and limits of quantitation ranged from 3 to 12 ng/mL and from 7 to 29 ng/mL, respectively. The method has been applied to cornflake samples. Samples were subjected to a preliminary short sonication in MeOH/2% KHCO(3) (70:30, v/v); the mixture was evaporated to near dryness and reconstituted in 1.5 mL of 5 mM phosphate buffer (pH 3) for SPME followed by LC-UV/DAD. The overall procedure had recoveries (evaluated on samples spiked at 200 ng/g level) ranging from 74 +/- 4 to 103 +/- 9%. Samples naturally contaminated with cyclopiazonic and tenuazonic acids were found; estimated concentrations were 72 +/- 9 and 25 +/- 6 ng/g, respectively.

Simultaneous determination of tenuazonic and cyclopiazonic acids in tomato products

Abstract A procedure for the simultaneous determination of the mycotoxins tenuazonic acid (TEA) and cyclopiazonic acid (CPA) is presented for the first time. It has been applied to tomato products and involves simple extraction, defatting and partitioning steps followed by metal complexation chromatography on a reverse phase C18 column. The quantification limits in tomato products were 11.0 ng/g for TEA and 8.0 ng/g for CPA and the average recoveries were 88 and 78% for TEA and CPA, respectively.

ChemInform Abstract: Chemical Studies Relevant to the Synthesis of Some Mycotoxins: The Cyclopiazonic Acids and Viridamine

ChemInform Abstract: Chemical Studies Relevant to the Synthesis of Some Mycotoxins: The Cyclopiazonic Acids and Viridamine

Biosynthesis of cyclopiazonic acids in Penicillium cyclopium: The isolation of dimethylallylpyrophosphate: Cyclo-acetoacetyltryptophanyl dimethylallyltransferase

During the production of β-cyclopiazonic acid (βCA) by Penicillium cyclopium, the following points are noted: (a) Dimethylallylpyrophosphate (DMAPP), which is incorporated into α-cyclopiazonic acid (αCA) in vivo, stimulates overall CA synthesis, whereas tryptophan, although incorporated into αCA, inhibits overall CA synthesis. (b) A previously suggested substrate, γ,γ-dimethylallyltryptophan, is not a precursor of αCA. (c) The accumulation of cyclo-acetoacetyl- l-tryptophanyl (cAATrp) is described in both the culture medium and mycelium with increasing growth. (d) A cell-free extract of mycelium will catalyze the conversion of exogenous cAATrp and exogenous DMAPP into βCA in 1:1 stoichiometry, the βCA being bound to a protein.

α-Acetyl-γ-(β-indolyl)methyltetramic acid. A biosynthetic intermediate of cyclopiazonic acid and of bis-secodehydrocyclopiazonic acid

It has been shown that tryptophan is not utilized in a cell-free system of Penicillium cyclopium; α-acetyl-γ-(β-indolyl)methyltetramic acid, a biosynthetic intermediate of the cyclopiazonic acids, has been identified, and the L-configuration at the asymmetric centre of the new indole established.