Absolute Configuration of Isosilybin A by X‑ray Crystallography of the Heavy Atom Analogue 7‑(4-Bromobenzoyl)isosilybin A

Isosilybin A (<b>1</b>) is one of the major flavonolignans that constitute silymarin, an extract of the fruits (achenes) of milk thistle (<i>Silybum marianum</i>). The chemistry of the <i>Silybum</i> flavonolignans has been studied for over four decades, and the absolute configuration of <b>1</b> has been determined previously by electronic circular dichroism and X-ray crystallography via correlating the relative configuration of the phenylpropanoid moiety to the established absolute configuration of the 3-hydroxyflavanone portion of the molecule. Herein we report the X-ray crystallographic structure of the product of the reaction of <b>1</b> with 4-bromobenzoyl chloride, and, thus, the absolute configuration of <b>1</b> was established as (2<i>R</i>, 3<i>R</i>, 7″<i>R</i>, 8″<i>R</i>) directly via X-ray crystallography of an analogue that incorporated a heavy atom. The results were consistent with previously reported assignments and verified the absolute configuration of the diastereoisomer of <b>1</b>, isosilybin B, and the related diastereoisomeric regioisomers, silybin A and silybin B

Fungal Identification Using Molecular Tools: A Primer for the Natural Products Research Community

Fungi are morphologically, ecologically, metabolically, and phylogenetically diverse. They are known to produce numerous bioactive molecules, which makes them very useful for natural products researchers in their pursuit of discovering new chemical diversity with agricultural, industrial, and pharmaceutical applications. Despite their importance in natural products chemistry, identification of fungi remains a daunting task for chemists, especially those who do not work with a trained mycologist. The purpose of this review is to update natural products researchers about the tools available for molecular identification of fungi. In particular, we discuss (1) problems of using morphology alone in the identification of fungi to the species level; (2) the three nuclear ribosomal genes most commonly used in fungal identification and the potential advantages and limitations of the ITS region, which is the official DNA barcoding marker for species-level identification of fungi; (3) how to use NCBI-BLAST search for DNA barcoding, with a cautionary note regarding its limitations; (4) the numerous curated molecular databases containing fungal sequences; (5) the various protein-coding genes used to augment or supplant ITS in species-level identification of certain fungal groups; and (6) methods used in the construction of phylogenetic trees from DNA sequences to facilitate fungal species identification. We recommend that, whenever possible, both morphology and molecular data be used for fungal identification. Our goal is that this review will provide a set of standardized procedures for the molecular identification of fungi that can be utilized by the natural products research community

Mechanistic Study of the Biomimetic Synthesis of Flavonolignan Diastereoisomers in Milk Thistle

The mechanism for the biomimetic synthesis of flavonolignan diastereoisomers in milk thistle is proposed to proceed by single-electron oxidation of coniferyl alcohol, subsequent reaction with one of the oxygen atoms of taxifolin’s catechol moiety, and finally, further oxidation to form four of the major components of silymarin: silybin A, silybin B, isosilybin A, and isosilybin B. This mechanism is significantly different from a previously proposed process that involves the coupling of two independently formed radicals

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

High-Resolution MS, MS/MS, and UV Database of Fungal Secondary Metabolites as a Dereplication Protocol for Bioactive Natural Products

A major problem in the discovery of new biologically active compounds from natural products is the reisolation of known compounds. Such reisolations waste time and resources, distracting chemists from more promising leads. To address this problem, dereplication strategies are needed that enable crude extracts to be screened for the presence of known compounds before isolation efforts are initiated. In a project to identify anticancer drug leads from filamentous fungi, a significant dereplication challenge arises, as the taxonomy of the source materials is rarely known, and, thus, the literature cannot be probed to identify likely known compounds. An ultraperformance liquid chromatography–photodiode array–high-resolution tandem mass spectrometric (UPLC-PDA-HRMS-MS/MS) method was developed for dereplication of fungal secondary metabolites in crude culture extracts. A database was constructed by recording HRMS and MS/MS spectra of fungal metabolites, utilizing both positive- and negative-ionization modes. Additional details, such as UV-absorption maxima and retention times, were also recorded. Small-scale cultures that showed cytotoxic activities were dereplicated before engaging in the scale-up or purification processes. Using these methods, approximately 50% of the cytotoxic extracts could be eliminated from further study after the confident identification of known compounds. The specific attributes of this dereplication methodology include a focus on bioactive secondary metabolites from fungi, the use of a 10 min chromatographic method, and the inclusion of both HRMS and MS/MS data

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

Flavonolignans decrease VEGFR1, HIF-1α, phosphorylated and total Akt levels in DU145 xenografts.

<p>DU145 xenograft tissues were analyzed for VEGFR1, HIF-1α, phosphorylated Akt^ser473 and total Akt levels by IHC. Quantitative analyses were performed using Zeiss Axioscope 2 microscope (Carl Zeiss, Germany) and photographs were originally captured (at 400x) with a Carl Zeiss AxioCam MrC5 camera with Axiovision Rel 4.5 software. The data shown in the bar diagrams is the mean ± SEM of 4–5 samples. Abbreviations: Sil A: Silybin A; Sil B: Silybin B; Iso A: Isosilybin A, Iso B: Isosilybin B; *, p ≤ 0.001.</p

Effect of flavonolignans on VEGF-induced signaling cascade in HUVEC.

<p>HUVEC were serum starved for 22 h, treated with diastereoisomers for 2 h and stimulated with VEGF (10 ng/ml) for 10 minutes. Total cell lysates were prepared and analyzed for mentioned signaling molecules. The densitometry values presented below the bands are ‘fold change’ compared to control after loading control (β-actin) normalization.</p

Feeding of pure flavonolignans did not affect angiogenesis and normal histology in non-target organs.

<p>(<b>A–B</b>) Lungs, liver and kidneys from each mouse were collected and analyzed for CD31 immunoreactivity as well as for histopathological analyses. Quantitative analyses were performed using Zeiss Axioscope 2 microscope (Carl Zeiss, Germany) and photographs were originally captured (at 400x) with a Carl Zeiss AxioCam MrC5 camera with Axiovision Rel 4.5 software. Abbreviations: Sil A: Silybin A; Sil B: Silybin B; Iso A: Isosilybin A, Iso B: Isosilybin B.</p

Effect of flavonolignans on viability, cell cycle distribution and apoptosis in HUVEC.

<p>(<b>A–B</b>) HUVEC were treated with DMSO or individual flavonolignan and analyzed for total cell number and cell cycle distribution. (<b>C</b>) HUVEC were treated with flavonolignans, and 24 h later, total cell lysates were prepared and analyzed for cell cycle regulators. The densitometry values presented below the bands are ‘fold change’ compared to control after loading control (α-tubulin) normalization. (<b>D</b>) HUVEC were treated with flavonolignans (at 30 µM dose) for 36 h and analyzed for morphology (representative photomicrographs are shown at 100x), levels of cPARP, cleaved caspase 3 and 9, and percentage apoptotic cells. Abbreviations: Sil A: Silybin A; Sil B: Silybin B; Iso A: Isosilybin A, Iso B: Isosilybin B; *, p ≤ 0.001; #, p ≤ 0.01; $, p ≤ 0.05.</p