Essentials in the acquisition, interpretation, and reporting of plant metabolite profiles.

Plant metabolite profiling reveals the diversity of secondary or specialized metabolites in the plant kingdom with its hundreds of thousands of species. Specialized plant metabolites constitute a vast class of chemicals posing significant challenges in analytical chemistry. In order to be of maximum scientific relevance, reports dealing with these compounds and their source species must be transparent, make use of standards and reference materials, and be based on correctly and traceably identified plant material. Essential aspects in qualitative plant metabolite profiling include: (i) critical review of previous literature and a reasoned sampling strategy; (ii) transparent plant sampling with wild material documented by vouchers in public herbaria and, optimally, seed banks; (iii) if possible, inclusion of generally available reference plant material; (iv) transparent, documented state-of-the art chemical analysis, ideally including chemical reference standards; (v) testing for artefacts during preparative extraction and isolation, using gentle analytical methods; (vi) careful chemical data interpretation, avoiding over- and misinterpretation and taking into account phytochemical complexity when assigning identification confidence levels, and (vii) taking all previous scientific knowledge into account in reporting the scientific data. From the current stage of the phytochemical literature, selected comments and suggestions are given. In the past, proposed revisions of botanical taxonomy were sometimes based on metabolite profiles, but this approach ("chemosystematics" or "chemotaxonomy") is outdated due to the advent of DNA sequence-based phylogenies. In contrast, systematic comparisons of plant metabolite profiles in a known phylogenetic framework remain relevant. This approach, known as chemophenetics, allows characterizing species and clades based on their array of specialized metabolites, aids in deducing the evolution of biosynthetic pathways and coevolution, and can serve in identifying new sources of rare and economically interesting natural products.

When Synthesis Gets It Wrong: Unexpected Epimerization Using PyBOP in the Synthesis of the Cyclic Peptide Thermoactinoamide A.

Chemical synthesis is commonly seen as the final proof of the structure of complex natural products, but even a seemingly easy and well-established synthetic procedure may lead to an unexpected result. This is what happened with the synthesis of thermoactinoamide A (1a), an antimicrobial and antitumor nonribosomal cyclic hexapeptide produced by the thermophilic bacterium Thermoactinomyces vulgaris. The synthetic thermoactinoamide A outsourced to a company and the one described in a synthetic paper showed spectroscopic data identical to each other but different from those of the natural product. After a detailed spectroscopic, degradative, and synthetic study, the synthetic compound was shown to be an epimer (1b) of the intended target compound, originating during the cyclization reaction by extensive epimerization at the activated C-terminal amino acid. This allowed confirmation of the structure of the natural product.

Enhanced Molecular Networking Shows Microbacterium sp. V1 as a Factory of Antioxidant Proline-Rich Peptides.

Two linear proline-rich peptides (1-2), bearing an N-terminal pyroglutamate, were isolated from the marine bacterium Microbacterium sp. V1, associated with the marine sponge Petrosia ficiformis, collected in the volcanic CO2 vents in Ischia Island (South Italy). Peptide production was triggered at low temperature following the one strain many compounds (OSMAC) method. Both peptides were detected together with other peptides (3-8) via an integrated, untargeted MS/MS-based molecular networking and cheminformatic approach. The planar structure of the peptides was determined by extensive 1D and 2D NMR and HR-MS analysis, and the stereochemistry of the aminoacyl residues was inferred by Marfey's analysis. Peptides 1-8 are likely to arise from Microbacterium V1 tailor-made proteolysis of tryptone. Peptides 1 and 2 were shown to display antioxidant properties in the ferric-reducing antioxidant power (FRAP) assay.

Molecular Networking Revealed Unique UV-Absorbing Phospholipids: Favilipids from the Marine Sponge Clathria faviformis.

Analysis of extracts of the marine sponge Clathria faviformis by high-resolution LC-MS2 and molecular networking resulted in the discovery of a new family of potentially UV-protecting phospholipids, the favilipids. One of them, favilipid A (1), was isolated and its structure determined by mass and tandem mass spectrometry, NMR, electronic circular dichroism (ECD), and computational studies. Favilipid A, which has no close analogues among natural products, possesses an unprecedented structure characterized by a 4-aminodihydropiridinium core, resulting in UV-absorbing properties that are very unusual for a phospholipid. Consequently, favilipid A could inspire the development of a new class of molecules to be used as sunscreen ingredients. In addition, favilipid A inhibited by 58-48% three kinases (JAK3, IKKß, and SYK) involved in the regulation of the immune system, suggesting a potential use for treatment of autoimmune diseases, hematologic cancers, and other inflammatory states.

Bioactivity and Metabolome Mining of Deep-Sea Sediment-Derived Microorganisms Reveal New Hybrid PKS-NRPS Macrolactone from Aspergillus versicolor PS108-62.

Despite low temperatures, poor nutrient levels and high pressure, microorganisms thrive in deep-sea environments of polar regions. The adaptability to such extreme environments renders deep-sea microorganisms an encouraging source of novel, bioactive secondary metabolites. In this study, we isolated 77 microorganisms collected by a remotely operated vehicle from the seafloor in the Fram Strait, Arctic Ocean (depth of 2454 m). Thirty-two bacteria and six fungal strains that represented the phylogenetic diversity of the isolates were cultured using an One-Strain-Many-Compounds (OSMAC) approach. The crude EtOAc extracts were tested for antimicrobial and anticancer activities. While antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium was common for many isolates, only two bacteria displayed anticancer activity, and two fungi inhibited the pathogenic yeast Candida albicans. Due to bioactivity against C. albicans and rich chemical diversity based on molecular network-based untargeted metabolomics, Aspergillus versicolor PS108-62 was selected for an in-depth chemical investigation. A chemical work-up of the SPE-fractions of its dichloromethane subextract led to the isolation of a new PKS-NRPS hybrid macrolactone, versicolide A (1), a new quinazoline (-)-isoversicomide A (3), as well as three known compounds, burnettramic acid A (2), cyclopenol (4) and cyclopenin (5). Their structures were elucidated by a combination of HRMS, NMR, [α]D, FT-IR spectroscopy and computational approaches. Due to the low amounts obtained, only compounds 2 and 4 could be tested for bioactivity, with 2 inhibiting the growth of C. albicans (IC50 7.2 µg/mL). These findings highlight, on the one hand, the vast potential of the genus Aspergillus to produce novel chemistry, particularly from underexplored ecological niches such as the Arctic deep sea, and on the other, the importance of untargeted metabolomics for selection of marine extracts for downstream chemical investigations.

A Cytotoxic Heterodimeric Cyclic Diarylheptanoid with a Rearranged Benzene Ring from the Seagrass Zostera marina.

The widespread seagrass Zostera marina contains a new diarylheptanoid heterodimer, zosterabisphenone C (1), featuring an unprecedented rearrangement of one of its benzene rings to a cyclopentenecarbonyl unit. The planar structure and absolute configuration of zosterabisphenone C were elucidated by a combination of spectroscopic (MS, ECD, and low-temperature NMR) and computational (DFT-NMR and DFT-ECD) evidence. Consistent with the previously isolated zosterabisphenones, compound 1 was selectively cytotoxic against HCT 116 adenocarcinoma colon cancer cells, reducing their viability by 73% at 10 µM (IC50 of 7.6 ± 1.1 µM). The biosynthetic origin of zosterabisphenone C (1) from an oxidative rearrangement of zosterabisphenone A (4) is proposed.

Metabolomics and molecular networking analyses in Arabidopsis thaliana show that extracellular self-DNA affects nucleoside/nucleotide cycles with accumulation of cAMP, cGMP and N6-methyl-AMP.

Extracellular DNA (exDNA) widely occurs in the environment due to release by either cell lysis or active secretion. The role of exDNA in plant-soil interactions has been investigated and inhibitory effects on the growth of conspecific individuals by their self-DNA have been reported. Transcriptome analysis in the model plant Arabidopsis thaliana showed a clear recognition by the plant roots of self- and nonself-exDNA, with inhibition occurring only after exposure to the former. In this study, an untargeted metabolomics approach was used to assess at molecular level the plant reactions to exDNA exposure. Thus, the effects on the metabolites profile of A. thaliana after exposure to self- and nonself-exDNA from plants and fish, were studied by NMR, LC-MS, chemometrics and molecular networking analyses. Results show that self-DNA significantly induces the accumulation of RNA constituents (nucleobases, ribonucleosides, dinucleotide and trinucleotide oligomers). Interestingly, AMP and GMP are found along with their cyclic analogues cAMP and cGMP, and in form of cyclic dimers (c-di-AMP and c-di-GMP). Also methylated adenosine monophosphate (m6AMP) and the dimeric dinucleotide N-methyladenylyl-(3'→5') cytidine (m6ApC) increased only in the self-DNA treatment. Such striking evidence of self-DNA effects highlights a major role of exDNA in plant sensing of its environment.

Application of Feature-Based Molecular Networking for Comparative Metabolomics and Targeted Isolation of Stereoisomers from Algicolous Fungi.

Seaweed endophytic (algicolous) fungi are talented producers of bioactive natural products. We have previously isolated two strains of the endophytic fungus, Pyrenochaetopsis sp. FVE-001 and FVE-087, from the thalli of the brown alga Fucus vesiculosus. Initial chemical studies yielded four new decalinoylspirotetramic acid derivatives with antimelanoma activity, namely pyrenosetins A-C (1-3) from Pyrenochaetopsis sp. strain FVE-001, and pyrenosetin D (4) from strain FVE-087. In this study, we applied a comparative metabolomics study employing HRMS/MS based feature-based molecular networking (FB MN) on both Pyrenochaetopsis strains. A higher chemical capacity in production of decalin derivatives was observed in Pyrenochaetopsis sp. FVE-087. Notably, several decalins showed different retention times despite the same MS data and MS/MS fragmentation pattern with the previously isolated pyrenosetins, indicating they may be their stereoisomers. FB MN-based targeted isolation studies coupled with antimelanoma activity testing on the strain FVE-087 afforded two new stereoisomers, pyrenosetins E (5) and F (6). Extensive NMR spectroscopy including DFT computational studies, HR-ESIMS, and Mosher's ester method were used in the structure elucidation of compounds 5 and 6. The 3'R,5'R stereochemistry determined for compound 6 was identical to that previously reported for pyrenosetin C (3), whose stereochemistry was revised as 3'S,5'R in this study. Pyrenosetin E (5) inhibited the growth of human malignant melanoma cells (A-375) with an IC50 value of 40.9 µM, while 6 was inactive. This study points out significant variations in the chemical repertoire of two closely related fungal strains and the versatility of FB MN in identification and targeted isolation of stereoisomers. It also confirms that the little-known fungal genus Pyrenochaetopsis is a prolific source of complex decalinoylspirotetramic acid derivatives.

Seasonal variation of phenolic compounds in Zostera marina (Zosteraceae) from the Baltic Sea.

Seasonal variations of phenolic compounds, in leaves of Zostera marina L. from the Baltic Sea near Kiel/Germany were investigated. Dominant compounds were mono- and disulfated flavonoids and phenylpropanoic acids, in particular luteolin 7,3'-O-disulfate and diosmetin 7-O-sulfate as well as rosmarinic acid, a dimeric phenylpropanoid. All detected sulfated flavones showed similar seasonal trends: there were two significant concentration peaks in June and November. Moreover, two geographically distinct flavonoid chemotypes were identified based on their respective main flavonoid; one chemotype was characterized by the prevalence of luteolin 7,3'-O-disulfate (German Baltic Sea), and the other by the prevalence of diosmetin 7-O-sulfate (Norwegian North Sea). Furthermore, an undescribed tetrameric phenylpropanoid, 7'',8''-didehydrosalvianolic acid B, was isolated and its structure was established by extensive NMR, MS, and CD experiments. This compound inhibited activity of Na+/K+-ATPase in the micro-molar range without any cytotoxic effects against human cancer and normal cells.

Induction of Isochromanones by Co-Cultivation of the Marine Fungus Cosmospora sp. and the Phytopathogen Magnaporthe oryzae.

Microbial co-cultivation is a promising approach for the activation of biosynthetic gene clusters (BGCs) that remain transcriptionally silent under artificial culture conditions. As part of our project aiming at the discovery of marine-derived fungal agrochemicals, we previously used four phytopathogens as model competitors in the co-cultivation of 21 marine fungal strains. Based on comparative untargeted metabolomics analyses and anti-phytopathogenic activities of the co-cultures, we selected the co-culture of marine Cosmospora sp. with the phytopathogen Magnaporthe oryzae for in-depth chemical studies. UPLC-MS/MS-based molecular networking (MN) of the co-culture extract revealed an enhanced diversity of compounds in several molecular families, including isochromanones, specifically induced in the co-culture. Large scale co-cultivation of Cosmospora sp. and M. oryzae resulted in the isolation of five isochromanones from the whole co-culture extract, namely the known soudanones A, E, D (1-3) and their two new derivatives, soudanones H-I (4-5), the known isochromans, pseudoanguillosporins A and B (6, 7), naphtho-γ-pyrones, cephalochromin and ustilaginoidin G (8, 9), and ergosterol (10). Their structures were established by NMR, HR-ESIMS, FT-IR, electronic circular dichroism (ECD) spectroscopy, polarimetry ([α]D), and Mosher's ester reaction. Bioactivity assays revealed antimicrobial activity of compounds 2 and 3 against the phytopathogens M. oryzae and Phytophthora infestans, while pseudoanguillosporin A (6) showed the broadest and strongest anti-phytopathogenic activity against Pseudomonas syringae, Xanthomonas campestris, M. oryzae and P. infestans. This is the first study assessing the anti-phytopathogenic activities of soudanones.

Stable Catechol Keto Tautomers in Cytotoxic Heterodimeric Cyclic Diarylheptanoids from the Seagrass Zostera marina.

Two diarylheptanoid heterodimers, zosterabisphenones A (1) and B (2), were isolated from the seagrass Zostera marina. They feature unprecedented catechol keto tautomers, stable because of steric constraints. Their structure elucidation was based on extensive low-temperature NMR studies and ECD and MS data, with the essential aid of DFT prediction of NMR and ECD spectra. Zosterabisphenone B (2) was selectively cytotoxic against the adenocarcinoma colon cancer cell line HCT116 with IC50 3.6 ± 1.1 µM at 48 h.

Density Functional Theory (DFT)-Aided Structure Elucidation of Linear Diterpenes from the Irish Brown Seaweed Bifurcaria bifurcata.

Brown alga Bifurcaria bifurcata is an extraordinarily rich source of linear (acylic) diterpenes with enormous structural diversity. As part of our interest into secondary metabolites of the Irish seaweeds, here we report four new acyclic diterpenes (1-4) and seven known terpenoids (5-11) from the CHCl3 extract of B. bifurcata. The planar structures of the new metabolites were elucidated by means of 1D and 2D NMR, HRMS, and FT-IR spectroscopy. Since linear diterpenes are highly flexible compounds, the assignment of their stereochemistry by conventional methods, e.g., NOESY NMR, is difficult. Therefore, we employed extensive quantum-mechanical prediction of NMR chemical shifts and optical rotation analyses to identify the relative and absolute configurations of the new compounds 1-4. Several compounds moderately inhibited the human breast cancer cell line (MDA-MB-231) with IC50 values ranging from 10.0 to 33.5 µg/mL. This study not only demonstrates the vast capacity of the Irish B. bifurcata to produce highly oxygenated linear diterpenoids, but also highlights the potential of new methodologies for assignment of their stereogenic centers.

Cholesterol derivatives make large part of the lipids from epidermal molts of the desert-adapted Gila monster lizard (Heloderma suspectum).

In order to understand the cutaneous water loss in the desert-adapted and venomous lizard Heloderma suspectum, the microscopic structure and lipid composition of epidermal molts have been examined using microscopic, spectroscopic and chemical analysis techniques. The molt is formed by a variably thick, superficial beta-layer, an extensive mesos-region and few alpha-cells in its lowermost layers. The beta-layer contains most corneous beta proteins while the mesos-region is much richer in lipids. The proteins in the mesos-region are more unstructured than those located in the beta-layer. Most interestingly, among other lipids, high contents of cholesteryl-ß-glucoside and cholesteryl sulfate were detected, molecules absent or present in traces in other species of squamates. These cholesterol derivatives may be involved in the stabilization and compaction of the mesos-region, but present a limited permeability to water movements. The modest resistance to cutaneous water-loss of this species is compensated by adopting other physiological strategies to limit thermal damage and water transpiration as previous eco-physiological studies have indicated. The increase of steroid derivatives may also be implicated in the heat shock response, influencing the relative behavior in this desert-adapted lizard.

New Tricks with an Old Sponge: Feature-Based Molecular Networking Led to Fast Identification of New Stylissamide L from Stylissa caribica.

Feature-based molecular networking was used to re-examine the secondary metabolites in extracts of a very well studied marine sponge, Stylissa caribica, known to contain a large array of cyclic peptides and brominated alkaloids. The analysis revealed the presence of 13 cyclic peptides in the sponge that had never been detected in previous work and appeared to be new compounds. The most abundant one was isolated and shown to be a new proline-rich cyclic heptapetide that was called stylissamide L (1). Structure of compound 1, including the cis/trans geometry of the three proline residues, was determined by extensive NMR studies; the l configuration of the seven amino acid residues was determined using Marfey's method. Stylissamide L was tested for activity as a cell growth inhibitor and cell migration inhibitor on two cancer cell lines but, unlike other members of the stylissamide family, it showed no significant activity. This approach showed that even a thoroughly studied species such as S. caribica may contain new chemistry that can be revealed if studied with the right tools.

Isolation of Isotrichophycin C and Trichophycins G-I from a Collection of Trichodesmium thiebautii.

The trichophycin family of compounds are chlorinated polyketides first discovered from environmental collections of a bloom-forming Trichodesmium sp. cyanobacterium. In an effort to fully capture the chemical space of this group of metabolites, the utilization of MS/MS-based molecular networking of a Trichodesmium thiebautii extract revealed a metabolome replete with halogenated compounds. Subsequent MS-guided isolation resulted in the characterization of isotrichophycin C and trichophycins G-I (1-4). These new metabolites had intriguing structural variations from those trichophycins previously characterized, which allowed for a comparative study to examine structural features that are associated with toxicity to murine neuroblastoma cells. Additionally, we propose the absolute configuration of the previously characterized trichophycin A (5). Overall, the metabolome of the Trichodesmium bloom is hallmarked by an unprecedented amount of chlorinated molecules, many of which remain to be structurally characterized.

Antibiotic Activity of a Paraphaeosphaeria sporulosa-Produced Diketopiperazine against Salmonella enterica.

A diketopiperazine has been purified from a culture filtrate of the endophytic fungus Paraphaeosphaeria sporulosa, isolated from healthy tissues of strawberry plants in a survey of microbes as sources of anti-bacterial metabolites. Its structure has been determined by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) analyses and was found to be identical to cyclo(L-Pro-L-Phe) purified from species of other fungal genera. This secondary metabolite has been selected following bioguided-assay fractionation against two strains of Salmonella enterica, the causal agent of bovine gastroenteritis. The diketopiperazine cyclo(L-Pro-L-Phe), isolated for the first time from Paraphaeosphaeria species, showed minimum inhibitory concentration (MIC) values of 71.3 and 78.6 µg/mL against the two S. enterica strains. This finding may be significant in limiting the use of synthetic antibiotics in animal husbandry and reducing the emergence of bacterial multidrug resistance. Further in vivo experiments of P. sporulosa diketopiperazines are important for the future application of these metabolites.

Identification of the Biosynthetic Gene Cluster of Thermoactinoamides and Discovery of New Congeners by Integrated Genome Mining and MS-Based Molecular Networking.

The putative non-ribosomal peptide synthetase (NRPS) gene cluster encoding the biosynthesis of the bioactive cyclohexapeptide thermoactinoamide A (1) was identified in Thermoactinomyces vulgaris DSM 43016. Based on an in silico prediction, the biosynthetic operon was shown to contain two trimodular NRPSs, designated as ThdA and ThdB, respectively. Chemical analysis of a bacterial crude extract showed the presence of thermoactinoamide A (1), thereby supporting this biosynthetic hypothesis. Notably, integrating genome mining with a LC-HRMS/MS molecular networking-based investigation of the microbial metabolome, we succeeded in the identification of 10 structural variants (2-11) of thermoactinoamide A (1), five of them being new compounds (thermoactinoamides G-K, 7-11). As only one thermoactinoamide operon was found in T. vulgaris, it can be assumed that all thermoactinoamide congeners are assembled by the same multimodular NRPS system. In light of these findings, we suggest that the thermoactinoamide synthetase is able to create chemical diversity, combining the relaxed substrate selectivity of some adenylation domains with the iterative and/or alternative use of specific modules. In the frame of our screening program to discover antitumor natural products, thermoactinoamide A (1) was shown to exert a moderate growth-inhibitory effect in BxPC-3 cancer cells in the low micromolar range, while being inactive in PANC-1 and 3AB-OS solid tumor models.

Pyrenosetin D, a New Pentacyclic Decalinoyltetramic Acid Derivative from the Algicolous Fungus Pyrenochaetopsis sp. FVE-087.

The fungal genus Pyrenochaetopsis is commonly found in soil, terrestrial, and marine environments, however, has received little attention as a source of bioactive secondary metabolites so far. In a recent work, we reported the isolation and characterization of three new anticancer decalinoyltetramic acid derivatives, pyrenosetins A-C, from the Baltic Fucus vesiculosus-derived endophytic fungus Pyrenochaetopsis sp. FVE-001. Herein we report a new pentacyclic decalinoylspirotetramic acid derivative, pyrenosetin D (1), along with two known decalin derivatives wakodecalines A (2) and B (3) from another endophytic strain Pyrenochaetopsis FVE-087 isolated from the same seaweed and showed anticancer activity in initial screenings. The chemical structures of the purified compounds were elucidated by comprehensive analysis of HR-ESIMS, FT-IR, [a]D, 1D and 2D NMR data coupled with DFT calculations of NMR parameters and optical rotation. Compounds 1-3 were evaluated for their anticancer and toxic potentials against the human malignant melanoma cell line (A-375) and the non-cancerous keratinocyte cell line (HaCaT). Pyrenosetin D (1) showed toxicity towards both A-375 and HaCaT cells with IC50 values of 77.5 and 39.3 µM, respectively, while 2 and 3 were inactive. This is the third chemical study performed on the fungal genus Pyrenochaetopsis and the first report of a pentacyclic decalin ring system from the fungal genus Pyrenochaetopsis.

Structure and Conformation of Zosteraphenols, Tetracyclic Diarylheptanoids from the Seagrass Zostera marina: An NMR and DFT Study.

Zosteraphenols, two new tetracyclic diarylheptanoids were isolated from the seagrass Zostera marina. The rotameric equilibrium of the strained tetracyclic structures, involving a diastereomeric minor rotamer with opposite axial chirality, resulted in coalescent NMR spectra. Although the elusive minor rotamer was only characterized with 1H chemical shifts, the excellent agreement between experimental and DFT-calculated chemical shifts of both rotamers unequivocally supported this analysis. Absolute configuration of zosteraphenols was determined by DFT prediction of their ECD spectra.

A joint molecular networking study of a Smenospongia sponge and a cyanobacterial bloom revealed new antiproliferative chlorinated polyketides.

The bloom-forming cyanobacteria Trichodesmium sp. have been recently shown to produce some of the chlorinated peptides/polyketides previously isolated from the marine sponge Smenospongia aurea. A comparative analysis of extracts from S. aurea and Trichodesmium sp. was performed using tandem mass spectrometry-based molecular networking. The analysis, specifically targeted to chlorinated metabolites, showed that many of them are common to the two organisms, but also that some general differences exist between the two metabolomes. Following this analysis, six new chlorinated metabolites were isolated and their structures elucidated: four polyketides, smenolactones A-D (1-4) from S. aurea, and two new conulothiazole analogues, isoconulothiazole B (5) and conulothiazole C (6) from Trichodesmium sp. The absolute configuration of smenolactone C (3) was determined by taking advantage of the conformational rigidity of open 1,3-disubstituted alkyl chains. The antiproliferative activity of smenolactones was evaluated on three tumor cell lines, and they were active at low-micromolar or sub-micromolar concentrations.