Flavonolignans from <i>Aspergillus iizukae</i>, a Fungal Endophyte of Milk Thistle (<i>Silybum marianum</i>)

Silybin A (<b>1</b>), silybin B (<b>2</b>), and isosilybin A (<b>3</b>), three of the seven flavonolignans that constitute silymarin, an extract of the fruits of milk thistle (<i>Silybum marianum</i>), were detected for the first time from a fungal endophyte, <i>Aspergillus iizukae</i>, isolated from the surface-sterilized leaves of <i>S. marianum</i>. The flavonolignans were identified using a UPLC-PDA-HRMS-MS/MS method by matching retention times, HRMS, and MS/MS data with authentic reference compounds. Attenuation of flavonolignan production was observed following successive subculturing of the original flavonolignan-producing culture, as is often the case with endophytes that produce plant-based secondary metabolites. However, production of <b>1</b> and <b>2</b> resumed when attenuated spores were harvested from cultures grown on a medium to which autoclaved leaves of <i>S. marianum</i> were added. The cycle of attenuation followed by resumed biosynthesis of these flavonolignans was replicated in triplicate

Biosynthesis of Fluorinated Peptaibols Using a Site-Directed Building Block Incorporation Approach

Synthetic biological approaches, such as site-directed biosynthesis, have contributed to the expansion of the chemical space of natural products, making possible the biosynthesis of unnatural metabolites that otherwise would be difficult to access. Such methods may allow the incorporation of fluorine, an atom rarely found in nature, into complex secondary metabolites. Organofluorine compounds and secondary metabolites have both played pivotal roles in the development of drugs; however, their discovery and development are often via nonintersecting tracks. In this context, we used the biosynthetic machinery of Trichoderma arundinaceum (strain MSX70741) to incorporate a fluorine atom into peptaibol-type molecules in a site-selective manner. Thus, fermentation of strain MSX70741 in media containing <i>ortho</i>- and <i>meta</i>-F-phenylalanine resulted in the biosynthesis of two new fluorine-containing alamethicin F50 derivatives. The fluorinated products were characterized using spectroscopic (1D and 2D NMR, including ¹⁹F) and spectrometric (HRESIMS/MSⁿ) methods, and their absolute configurations were established by Marfey’s analysis. Fluorine-containing alamethicin F50 derivatives exhibited potency analogous to the nonfluorinated parent when evaluated against a panel of human cancer cell lines. Importantly, the biosynthesis of fluorinated alamethicin F50 derivatives by strain MSX70741 was monitored <i>in situ</i> using a droplet–liquid microjunction–surface sampling probe coupled to a hyphenated system

Biochemometrics for Natural Products Research: Comparison of Data Analysis Approaches and Application to Identification of Bioactive Compounds

A central challenge of natural products research is assigning bioactive compounds from complex mixtures. The gold standard approach to address this challenge, bioassay-guided fractionation, is often biased toward abundant, rather than bioactive, mixture components. This study evaluated the combination of bioassay-guided fractionation with untargeted metabolite profiling to improve active component identification early in the fractionation process. Key to this methodology was statistical modeling of the integrated biological and chemical data sets (biochemometric analysis). Three data analysis approaches for biochemometric analysis were compared, namely, partial least-squares loading vectors, S-plots, and the selectivity ratio. Extracts from the endophytic fungi <i>Alternaria</i> sp. and <i>Pyrenochaeta</i> sp. with antimicrobial activity against <i>Staphylococcus aureus</i> served as test cases. Biochemometric analysis incorporating the selectivity ratio performed best in identifying bioactive ions from these extracts early in the fractionation process, yielding altersetin (<b>3</b>, MIC 0.23 μg/mL) and macrosphelide A (<b>4</b>, MIC 75 μg/mL) as antibacterial constituents from <i>Alternaria</i> sp. and <i>Pyrenochaeta</i> sp., respectively. This study demonstrates the potential of biochemometrics coupled with bioassay-guided fractionation to identify bioactive mixture components. A benefit of this approach is the ability to integrate multiple stages of fractionation and bioassay data into a single analysis

Dereplicating and Spatial Mapping of Secondary Metabolites from Fungal Cultures <i>in Situ</i>

Ambient ionization mass spectrometry techniques have recently become prevalent in natural product research due to their ability to examine secondary metabolites <i>in situ</i>. These techniques retain invaluable spatial and temporal details that are lost through traditional extraction processes. However, most ambient ionization techniques do not collect mutually supportive data, such as chromatographic retention times and/or UV/vis spectra, and this can limit the ability to identify certain metabolites, such as differentiating isomers. To overcome this, the droplet–liquid microjunction–surface sampling probe (droplet–LMJ–SSP) was coupled with UPLC–PDA–HRMS–MS/MS, thus providing separation, retention times, MS data, and UV/vis data used in traditional dereplication protocols. By capturing these mutually supportive data, the identity of secondary metabolites can be confidently and rapidly assigned <i>in situ</i>. Using the droplet–LMJ–SSP, a protocol was constructed to analyze the secondary metabolite profile of fungal cultures without any sample preparation. The results demonstrate that fungal cultures can be dereplicated from the Petri dish, thus identifying secondary metabolites, including isomers, and confirming them against reference standards. Furthermore, heat maps, similar to mass spectrometry imaging, can be used to ascertain the location and relative concentration of secondary metabolites directly on the surface and/or surroundings of a fungal culture

Dereplicating and Spatial Mapping of Secondary Metabolites from Fungal Cultures <i>in Situ</i>

Ambient ionization mass spectrometry techniques have recently become prevalent in natural product research due to their ability to examine secondary metabolites <i>in situ</i>. These techniques retain invaluable spatial and temporal details that are lost through traditional extraction processes. However, most ambient ionization techniques do not collect mutually supportive data, such as chromatographic retention times and/or UV/vis spectra, and this can limit the ability to identify certain metabolites, such as differentiating isomers. To overcome this, the droplet–liquid microjunction–surface sampling probe (droplet–LMJ–SSP) was coupled with UPLC–PDA–HRMS–MS/MS, thus providing separation, retention times, MS data, and UV/vis data used in traditional dereplication protocols. By capturing these mutually supportive data, the identity of secondary metabolites can be confidently and rapidly assigned <i>in situ</i>. Using the droplet–LMJ–SSP, a protocol was constructed to analyze the secondary metabolite profile of fungal cultures without any sample preparation. The results demonstrate that fungal cultures can be dereplicated from the Petri dish, thus identifying secondary metabolites, including isomers, and confirming them against reference standards. Furthermore, heat maps, similar to mass spectrometry imaging, can be used to ascertain the location and relative concentration of secondary metabolites directly on the surface and/or surroundings of a fungal culture

Dioxomorpholines and Derivatives from a Marine-Facultative <i>Aspergillus</i> Species

Two new dioxomorpholines, <b>1</b> and <b>2</b>, three new derivatives, <b>3</b>–<b>5</b>, and the known compound PF1233 B (<b>6</b>) were isolated from a marine-facultative <i>Aspergillus</i> sp. MEXU 27854. Their structures were established by 1D and 2D NMR and HRESIMS data analysis. The absolute configuration of <b>1</b> and <b>2</b> was elucidated by comparison of experimental and DFT-calculated vibrational circular dichroism spectra. Compounds <b>3</b>, <b>5</b>, and <b>6</b> were noncytotoxic to a panel of human cancer cell lines with different functional status for the tumor-suppressor protein p53, but were inhibitors of P-glycoprotein-reversing multidrug resistance in a doxorubicin-resistant cell line

Phylogenetic and chemical diversity of fungal endophytes isolated from <i>Silybum marianum</i> (L) Gaertn. (milk thistle)

<div><p>Use of the herb milk thistle (<i>Silybum marianum</i>) is widespread, and its chemistry has been studied for over 50 years. However, milk thistle endophytes have not been studied previously for their fungal and chemical diversity. We examined the fungal endophytes inhabiting this medicinal herb to determine: (1) species composition and phylogenetic diversity of fungal endophytes; (2) chemical diversity of secondary metabolites produced by these organisms; and (3) cytotoxicity of the pure compounds against the human prostate carcinoma (PC-3) cell line. Forty-one fungal isolates were identified from milk thistle comprising 25 operational taxonomic units based on BLAST search via GenBank using published authentic sequences from nuclear ribosomal internal transcribed spacer sequence data. Maximum likelihood analyses of partial 28S rRNA gene showed that these endophytes had phylogenetic affinities to four major classes of Ascomycota, the Dothideomycetes, Sordariomycetes, Eurotiomycetes, and Leotiomycetes. Chemical studies of solid–substrate fermentation cultures led to the isolation of four new natural products. In addition, 58 known secondary metabolites, representing diverse biosynthetic classes, were isolated and characterized using a suite of nuclear magnetic resonance and mass spectrometry techniques. Selected pure compounds were tested against the PC-3 cell line, where six compounds displayed cytotoxicity.</p></div