Aggressive dereplication using UHPLC–DAD–QTOF: screening extracts for up to 3000 fungal secondary metabolites

In natural-product drug discovery, finding new compounds is the main task, and thus fast dereplication of known compounds is essential. This is usually performed by manual liquid chromatography-ultraviolet (LC-UV) or visible light-mass spectroscopy (Vis-MS) interpretation of detected peaks, often assisted by automated identification of previously identified compounds. We used a 15 min high-performance liquid chromatography–diode array detection (UHPLC–DAD)–high-resolution MS method (electrospray ionization (ESI)⁺ or ESI^?), followed by 10–60 s of automated data analysis for up to 3000 relevant elemental compositions. By overlaying automatically generated extracted-ion chromatograms from detected compounds on the base peak chromatogram, all major potentially novel peaks could be visualized. Peaks corresponding to compounds available as reference standards, previously identified compounds, and major contaminants from solvents, media, filters etc. were labeled to differentiate these from compounds only identified by elemental composition. This enabled fast manual evaluation of both known peaks and potential novel-compound peaks, by manual verification of: the adduct pattern, UV–Vis, retention time compared with log D, co-identified biosynthetic related compounds, and elution order. System performance, including adduct patterns, in-source fragmentation, and ion-cooler bias, was investigated on reference standards, and the overall method was used on extracts of Aspergillus carbonarius and Penicillium melanoconidium, revealing new nitrogen-containing biomarkers for both species.     

Hesseltin A, a novel antiviral metabolite from Penicillium hesseltinei

[structure: see text] Hesseltin A 1, a novel compound of mixed polyketide-terpenoid origins was isolated from the filamentous fungus Penicillium hesseltinei. The structure and stereochemistry were determined from extensive one- and two-dimensional NMR and mass spectral data.     

Hesseltins B-G, novel meroterpenoids from a new Penicillium species

Six new meroterpenoid compounds, hesseltins B-G, were isolated from Penicillium species along with the previously described hesseltin A. A further 14 compounds, which turned out to be photoisomers, were detected during purification and were subsequently isolated. The structures of these analogues were elucidated by comparison of their NMR spectral data with that of hesseltin A. None of the new hesseltins showed antiviral activity in a Herpes simplex virus type 2 assay.     

Reconstitution of Biosynthetic Machinery for the Synthesis of the Highly Elaborated Indole Diterpene Penitrem

Penitrem A is one of the most elaborated members of the fungal indole diterpenes. Two separate penitrem gene clusters were identified using genomic and RNA sequencing data, and 13 out of 17 transformations in the penitrem biosynthesis were elucidated by heterologous reconstitution of the relevant genes. These reactions involve 1) a prenylation‐initiated cationic cyclization to install the bicyclo[3.2.0]heptane skeleton (PtmE), 2) a two‐step P450‐catalyzed oxidative processes forming the unique tricyclic penitrem skeleton (PtmK and PtmU), and 3) five sequential oxidative transformations (PtmKULNJ). Importantly, without conventional gene disruption, reconstitution of the biosynthetic machinery provided sufficient data to determine the pathway. It was thus demonstrated that the Aspergillus oryzae reconstitution system is a powerful method for studying the biosynthesis of complex natural products.     

epi-Aszonalenins A, B, and C from Aspergillus novofumigatus

Three new benzodiazepines have been isolated from an unusual chemotype of Aspergillus novofumigatus: epi-aszonalenins A, B, and C. The structures were elucidated by use of one- and two-dimensional NMR spectroscopic techniques and HR ESI MS. The relative configuration was established on the basis of a single crystal X-ray diffraction study of epi-aszonalenin A and the absolute configuration was determined by optical rotation comparison with the literature data. The absolute configurations of epi-aszonalenins B and C were determined by circular dichroism comparison to epi-aszonalenin A.     

Review of secondary metabolites and mycotoxins from the <Emphasis Type="Italic">Aspergillus niger</Emphasis> group

Filamentous fungi in the Aspergillus section Nigri (the black aspergilli) represent some of the most widespread food and feed contaminants known but they are also some of the most important workhorses used by the biotechnological industry. The Nigri section consists of six commonly found species (excluding A. aculeatus and its close relatives) from which currently 145 different secondary metabolites have been isolated and/or detected. From a human and animal safety point of view, the mycotoxins ochratoxin A (from A. carbonarius and less frequently A. niger) and fumonisin B₂ (from A. niger) are currently the most problematic compounds. Especially in foods and feeds such as coffee, nuts, dried fruits, and grape-based products where fumonisin-producing fusaria are not a problem, fumonisins pose a risk. Moreover, compounds such as malformins, naptho-γ-pyrones, and bicoumarins (kotanins) call for monitoring in food, feed, and biotechnology products as well as for a better toxicological evaluation, since they are often produced in large amounts by the black aspergilli. For chemical differentiation/identification of the less toxic species the diketopiperazine asperazine can be used as a positive marker since it is consistently produced by A. tubingensis (177 of 177 strains tested) and A. acidus (47 of 47 strains tested) but never by A. niger (140 strains tested). Naptho-γ-pyrones are the compounds produced in the highest quantities and are produced by all six common species in the group (A. niger 134 of 140; A. tubingensis 169 of 177; A. acidus 44 of 47; A. carbonarius 40 of 40, A. brasiliensis 18 of 18; and A. ibericus three of three).      

Determination of mycophenolic acid in meat products using mixed mode reversed phase-anion exchange clean-up and liquid chromatography-high-resolution mass spectrometry

A method for determination of mycophenolic acid (MPA) in dry-cured ham, fermented sausage and liver paté is described. MPA was extracted from meat with bicarbonate-acetonitrile, further cleaned-up by mixed mode reversed phase-anion exchange and detected using a LC-MS system with electrospray ionisation-time-of-flight detection. The limit of detection was 4 microg/kg in sausage and 6 microg/kg in ham and paté. The method was successfully used for quantification of MPA in dry-cured ham and liver paté artificially inoculated with Penicillium brevicompactum. Levels ranged from 190 microg/kg in centre to 11 mg/kg in surface of ham and from 150 microg/kg in bottom to 14 mg/kg in surface of paté.     

Standardized high-performance liquid chromatography of 182 mycotoxins and other fungal metabolites based on alkylphenone retention indices and UV-VIS spectra (diode array detection)

A general standardized method for the analysis of mycotoxins and other fungal secondary metabolites has been developed, based on high-performance liquid chromatography (HPLC) with an alkylphenone retention index and photodiode-array detection combined with thin-layer chromatography (TLC) in two different eluents. Each fungal secondary metabolite is characterized by its bracketed alkylphenone retention time index, its UV-VIS absorption maxima and its retardation factors relative to griseofulvin in two TLC eluents. This system is effective for the comparison of chemotaxonomic data in different laboratories and for a precise identification of fungi based on organic solvent extracts of fungal cultures. All important groups of mycotoxins and other fungal secondary metabolites could be detected in the HPLC system described and data are listed for 182 metabolites. The fungal secondary metabolites separated and characterized include aflatoxin B1, B2, G1 and G2, ochratoxin A, citrinin, penicillin acid, viomellein, penitrem A, patulin, sterigmatocystin, alternariol, tenuazonic acid, trichothecenes, roquefortines, fusarin C, zearalenone, PR-toxin, citreoviridin, viridicatumtoxin, verruculogen, rugulosin, cyclopiazonic acid, penicillin G and many other alkaloids, polyketides and terpenes.     

Phenotypic taxonomy and metabolite profiling in microbial drug discovery

Microorganisms and in particular actinomycetes and microfungi are known to produce a vast number of bioactive secondary metabolites. For industrially important fungal genera such as Penicillium and Aspergillus the production of these compounds has been demonstrated to be very consistent at the species level. This means that direct metabolite profiling techniques such as direct injection mass spectrometry or NMR can easily be used for chemotyping/metabolomics of strains from both culture collections and natural samples using modern informatics tools. In this review we discuss chemotyping/metabolomics as part of intelligent screening and highlight how it can be used for identification and classification of filamentous fungi and for the discovery of novel compounds when used in combination with modern methods for dereplication. In our opinion such approaches will be important for future effective drug discovery strategies, especially for dereplication of culture collections in order to avoid redundancy in the selection of species. This will maximize the chemical diversity of the microbial natural product libraries that can be generated from fungal collections.