Isolation, Structure Elucidation, and Biological Activity of the Selective TACR2 Antagonist Tumonolide and its Aldehyde from a Marine Cyanobacterium.

The macrocyclic tumonolide (1) with enamide functionality and the linear tumonolide aldehyde (2) are new interconverting natural products from a marine cyanobacterium with a peptide-polyketide skeleton, representing a hybrid of apratoxins and palmyrolides or laingolides. The planar structures were established by NMR and mass spectrometry. The relative configuration of the stereogenically-rich apratoxin-like polyketide portion was determined using J-based configuration analysis. The absolute configuration of tumonolide (1) was determined by chiral analysis of the amino acid units and computational methods, followed by NMR chemical shift and ECD spectrum prediction, indicating all-R configuration for the polyketide portion, as in palmyrolide A and contrary to the all-S configuration in apratoxins. Functional screening against a panel of 168 GPCR targets revealed tumonolide (1) as a selective antagonist of TACR2 with an IC50 of 7.0 µM, closely correlating with binding affinity. Molecular docking studies established the binding mode and rationalized the selectivity for TACR2 over TACR1 and TACR3. RNA sequencing upon treatment of HCT116 colorectal cancer cells demonstrated activation of the pulmonary fibrosis idiopathic signaling pathway and the insulin secretion signaling pathway at 20 µM, indicating its potential to modulate these pathways.

Spatial and Temporal Resolution of Cyanobacterial Bloom Chemistry Reveals an Open-Ocean Trichodesmium thiebautii as a Talented Producer of Specialized Metabolites.

While the ecological role that Trichodesmium sp. play in nitrogen fixation has been widely studied, little information is available on potential specialized metabolites that are associated with blooms and standing stock Trichodesmium colonies. While a collection of biological material from a T. thiebautii bloom event from North Padre Island, Texas, in 2014 indicated that this species was a prolific producer of chlorinated specialized metabolites, additional spatial and temporal resolution was needed. We have completed these metabolite comparison studies, detailed in the current report, utilizing LC-MS/MS-based molecular networking to visualize and annotate the specialized metabolite composition of these Trichodesmium blooms and colonies in the Gulf of Mexico (GoM) and other waters. Our results showed that T. thiebautii blooms and colonies found in the GoM have a remarkably consistent specialized metabolome. Additionally, we isolated and characterized one new macrocyclic compound from T. thiebautii, trichothilone A (1), which was also detected in three independent cultures of T. erythraeum. Genome mining identified genes predicted to synthesize certain functional groups in the T. thiebautii metabolites. These results provoke intriguing questions of how these specialized metabolites affect Trichodesmium ecophysiology, symbioses with marine invertebrates, and niche development in the global oligotrophic ocean.

Zosterabisphenone B, a new diarylheptanoid heterodimer from the seagrass Zostera marina, induces apoptosis cell death in colon cancer cells and reduces tumour growth in mice.

Colorectal cancer (CRC) is one of the most common malignant tumours worldwide. Diarylheptanoids, secondary metabolites isolated from Zostera marina, are of interest in natural products research due to their biological activities. Zosterabisphenone B (ZBP B) has recently been shown to inhibit the viability of CRC cells. The aim of this study was to investigate the therapeutic potential of ZBP B for targeting human CRC cells. Cell viability was determined using the MTT assay. Flow cytometry and Western blot analyses were used to assess apoptosis and autophagy. A CRC xenograft model was used to evaluate the in vivo effect of ZBP B. No cytotoxic effect on HCEC cells was observed in the in vitro experiments. ZBP B caused morphological changes in HCT116 colon cancer cells due to an increase in early and late apoptotic cell populations. Mechanistically, ZBP B led to an increase in cleaved caspase-3, caspase-8, caspase-9, PARP and BID proteins and a decrease in Bcl-2 and c-Myc proteins. In the xenograft model of CRC, ZBP B led to a reduction in tumour growth. These results indicate that ZBP B exerts a selective cytotoxic effect on CRC cells by affecting apoptotic signalling pathways and reducing tumour growth in mice. Taken together, our results suggest that ZBP B could be a lead compound for the synthesis and development of CRC drugs.

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.

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.

Metabolomics and chemometrics of seven aromatic plants: Carob, eucalyptus, laurel, mint, myrtle, rosemary and strawberry tree.

INTRODUCTION: Arbutus unedo L. (strawberry tree), Ceratonia siliqua L. (carob), Eucalyptus camaldulensis Dehnh. (eucalyptus), Laurus nobilis L. (laurel), Mentha aquatica L. (water mint), Myrtus communis L. (common myrtle), and Rosmarinus officinalis L. (rosemary) are aromatic plants from the Mediterranean region whose parts and preparations are used for their nutritional properties and health benefits. OBJECTIVES: To evaluate and compare the metabolites profile, total phenol content (TPC), and antioxidant activity of plant leaves for their future use. Gas chromatography-mass spectrometry (GC-MS) was used for metabolomics. Data comparison was performed by chemometrics. METHODOLOGY: Polar and apolar extracts were analysed using untargeted GC-MS metabolomics followed by chemometrics (principal component analysis, heatmap correlation and dendrogram) to identify, quantify and compare the major organic compounds in the plants. Additionally, nuclear magnetic resonance (NMR) spectroscopy was used for the laurel polar extract to identify d-gluco-l-glycero-3-octulose whose presence was unclear from the GC-MS data. TPC and antioxidant assays were performed using classical methods (Folin-Ciocalteu, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH)) and correlated to the phytochemical profiles. RESULTS: Forty-three metabolites were identified including amino acids, organic acids, carbohydrates, phenols, polyols, fatty acids, and alkanes. Eight metabolites (d-fructose, d-glucose, d-mannose, gallic acid, quinic acid, myo-inositol, palmitic and stearic acids) were in common between all species. d-Gluco-l-glycero-3-octulose (37.29 ± 1.19%), d-pinitol (31.33 ± 5.12%), and arbutin (1.30 ± 0.44%,) were characteristic compounds of laurel, carob, and strawberry tree, respectively. Carob showed the highest values of TPC and antioxidant activity. CONCLUSION: GC-MS metabolomics and chemometrics analyses are fast and useful methods to determine and compare the metabolomics profiling of aromatic plants of food and industrial interest.

Chemical Analysis and Antimicrobial Activity of Moringa oleifera Lam. Leaves and Seeds.

Moringa oleifera is a traditional food crop widespread in Asiatic, African, and South American continents. The plant, able to grow in harsh conditions, shows a high nutritional value and medicinal potential evidencing cardioprotective, anti-inflammatory, antioxidant, and antimicrobial properties. The purpose of this study was the phytochemical analysis of M. oleifera and the identification of the antimicrobial compounds by combining a chemical approach with in vitro tests. The metabolite profile of M. oleifera polar and apolar extracts of leaves and seeds were investigated by using Nuclear Magnetic Resonance spectroscopy and Gas Chromatography-Mass Spectrometry. The antimicrobial activity of all of the obtained extract was evaluated against four bacterial pathogens (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Salmonella enterica). The chemical analysis provided a wide set of metabolites that were identified and quantified. Moreover, apolar extracts from seeds showed a significant concentration-dependent antimicrobial activity against S. aureus and S. epidermidis, (4 mg/mL reduced the viability up to 50%) that was associated to the content of specific fatty acids. Our results remarked the advantages of an integrated approach for the identification of plant metabolites and its use in association with biological tests to recognize the compounds responsible for bioactivity without compounds purification.

Diterpenes from Euphorbia myrsinites and Their Anti-inflammatory Property.

Euphorbia myrsinites is one of the oldest spurges described and used in folk medicine. It is characterized by blue-grey stems similar to myrtle, and it is spread in the Mediterranean region, Asia, and the USA. Chemical analysis of E. myrsinites collected in Turkey afforded the isolation of 4 diterpenes based on the so-called myrsinane skeleton being tetraesters of the tetracyclic diterpene alcohol myrsinol. In this study, the phytochemical analysis of this species collected in Italy has been undertaken to afford the isolation of a new atisane diterpene, named myrsatisane, 3 ingenol derivatives, along with the 4 tetraester derivatives previously found. A triterpene compound based on the euphane skeleton has also been isolated. Structural elucidation of the new myrsatisane was based on spectroscopic techniques, including HR-MS and 1- and 2-dimensional NMR experiments. Its relative configuration was determined by NOE correlations, while absolute stereochemistry was obtained by quantum-mechanical DFT studies. While diterpenes with the atisane skeleton are relatively common in Euphorbia species, this is the first report of an atisane diterpene from E. myrsinites. All the isolated terpenes were tested for anti-inflammatory activity on J774A.1 macrophages stimulated with lipopolysaccharide by evaluation of nitrite and pro-inflammatory cytokine Il-1ß levels. Among tested compounds, the 3 ingenol diterpenes exhibited a dose-dependent (0.001 - 3 µM) significant activity, thus showing their potential as anti-inflammatory drug candidates.

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.

Metabolomic analysis and antioxidant activity of wild type and mutant chia (Salvia hispanica L.) stem and flower grown under different irrigation regimes.

BACKGROUND: Chia (Salvia hispanica L.) is a functional food from Central America. Interest in it is growing rapidly due to the many health benefits from the seed. However, when chia is grown at high latitudes, seed yield may be low whereas a high stem biomass and immature inflorescences are produced. Little is known about the chemical composition and the properties of stems and flowers. In this work, the metabolite profile, the antioxidant activity, and the total polyphenol content of stems and inflorescences were evaluated in a factorial experiment with different chia populations (commercial black chia and long-day flowering mutants G3, G8, and G17) and irrigation (100% and 50% of evapotranspiration). RESULTS: The results show the influence of irrigation and seed source on the antioxidant activity and total polyphenol content of chia flower and stem. Inflorescences exhibit higher antioxidant activity, suggesting their potential use as natural antioxidant. The mutants G3 and G8, at 50% irrigation, contained the highest amounts of compounds with nutraceutical value, especially within the flower. The mutant G17 showed lower antioxidant activity and polyphenol content compared to other seed sources but exhibited high omega 3 content in flowers but low in stems. This indicates that chia varieties should be chosen according to the objective of cultivation. CONCLUSION: These findings, indicating a close relation of metabolite content with irrigation and seed source, may provide the basis for the use of chia flower and stem for their nutraceutical value in the food, feed, and supplement industries. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Computational prediction of chiroptical properties in structure elucidation of natural products.

Covering: up to 2019This review covers the current status of the quantum mechanical prediction of chiroptical properties, such as electronic CD and optical rotation, as needed for stereochemical assignments in new natural products. The reliability of the prediction of chiroptical properties is steadily increasing, with a parallel decrease in the required computational resources. Now, quantum mechanical calculations for a medium-sized natural product can be reliably performed by natural product chemists on a mainstream PC. This review is aimed to guide natural product chemists through the numerous steps involved in such calculations. Through a concise, but comprehensive, discussion of the current computational practice, enriched by a few illustrative examples, this review provides readers with the theoretical background and practical knowledge needed to select the most appropriate parameters for performing the calculations, to anticipate possible problems, and to critically evaluate the reliability of their computational results. Common reasons for mistakes are also discussed; in particular, the importance of the correct evaluation of conformational ensembles of flexible molecules (an aspect often overlooked in current research) is stressed.

Molecular Networking-Based Analysis of Cytotoxic Saponins from Sea Cucumber Holothuria atra.

The saponin composition of a specimen of black sea cucumber, Holothuria atra collected in the Persian Gulf was studied by a combined approach including LC-MS/MS, Molecular Networking, pure compound isolation, and NMR spectroscopy. The saponin composition of Holothuria atra turned out to be more complex than previously reported. The most abundant saponins in the extract (1⁻4) were isolated and characterized by 1D- and 2D-NMR experiments. Compound 1 was identified as a new triterpene glycoside saponin, holothurin A5. The side chain of the new saponin 1, unprecedented among triterpene glycosides, is characterized by an electrophilic enone function, which can undergo slow water or methanol addition under neutral conditions. The cytotoxic activity of compounds 1⁻4, evaluated on the human cervix carcinoma HeLa cell line, was remarkable, with IC50 values ranging from 1.2 to 2.5 µg/mL.

Metabolomics of the alimurgic plants Taraxacum officinale, Papaver rhoeas and Urtica dioica by combined NMR and GC-MS analysis.

INTRODUCTION: The phytoalimurgic plants, common dandelion (Taraxacum officinale), corn poppy (Papaver rhoeas) and stinging nettle (Urtica dioica) are a source of nutraceuticals. OBJECTIVES: To apply a combined metabolomic fingerprinting approach by nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS) to common dandelion, corn poppy and stinging nettles to obtain simultaneous identification and quantitation of the major classes of organic compounds. METHODOLOGY: The whole plants collected in the Cilento National Park were dried and then extracted to obtain non-polar and polar organic extracts. GC-MS was used for non-polar extracts while 1 H-NMR spectroscopy was used for polar extracts. In both cases, simultaneous identification and quantification of the bioactive metabolites was obtained. RESULTS: Non-polar organic extracts of all plants were mainly composed of palmitic, stearic and oleic acids. The two pentacyclic triterpenols α- and ß-amyrin were detected in nettle extract. The analysis of polar organic extracts allowed to detect and quantify organic acids and sugars as main metabolites along with amino acids, caffeoyl derivatives, flavonoids, and nucleotides. In particular, corn poppy leaves contained a huge amount of glyceric acid (55.7% of the total extract). Stinging nettles, instead, exhibited a large amount of choline (19.5%). CONCLUSION: Metabolomic approach coupling GC-MS with NMR spectroscopy allowed to provide a detailed metabolite profile of three alimurgic plants, common dandelion, corn poppy and stinging nettle, from both a qualitative and quantitative point of view.

Fishing for Targets of Alien Metabolites: A Novel Peroxisome Proliferator-Activated Receptor (PPAR) Agonist from a Marine Pest.

Although the chemical warfare between invasive and native species has become a central problem in invasion biology, the molecular mechanisms by which bioactive metabolites from invasive pests influence local communities remain poorly characterized. This study demonstrates that the alkaloid caulerpin (CAU)-a bioactive component of the green alga Caulerpa cylindracea that has invaded the entire Mediterranean basin-is an agonist of peroxisome proliferator-activated receptors (PPARs). Our interdisciplinary study started with the in silico prediction of the ligand-protein interaction, which was then validated by in vivo, ex vivo and in vitro assays. On the basis of these results, we candidate CAU as a causal factor of the metabolic and behavioural disorders observed in Diplodus sargus, a native edible fish of high ecological and commercial relevance, feeding on C. cylindracea. Moreover, given the considerable interest in PPAR activators for the treatment of relevant human diseases, our findings are also discussed in terms of a possible nutraceutical/pharmacological valorisation of the invasive algal biomasses, supporting an innovative strategy for conserving biodiversity as an alternative to unrealistic campaigns for the eradication of invasive pests.

Cyanobacterial ent-Sterol-Like Natural Products from a Deviated Ubiquinone Pathway.

Natural products from marine animals show high potential for the development of new medicines, but drug development based on these compounds is commonly hampered by their low natural abundance. Since many of these metabolites are suspected or known to be produced by uncultivated bacterial symbionts, the rapidly growing diversity of sequenced prokaryotic genomes offers the opportunity to identify alternative, culturable sources of natural products computationally. In this work, we investigated the potential of using this sequenced resource to facilitate the production of meroterpenoid-like compounds related to those from marine sources. This genome-mining strategy revealed a biosynthetic gene cluster for highly modified cytotoxic meroterpenoids related to pelorol and other compounds isolated from sponges. Functional characterization of the terpene cyclase MstE showed that it generates an ent-sterol-like skeleton fused to an aryl moiety from an open-chain precursor and is therefore a promising tool for the chemoenzymatic preparation of synthetically challenging chemical scaffolds.

Erylusamides: Novel Atypical Glycolipids from Erylus cf. deficiens.

Among marine organisms, sponges are the richest sources of pharmacologically-active compounds. Stemming from a previous lead discovery program that gathered a comprehensive library of organic extracts of marine sponges from the off-shore region of Portugal, crude extracts of Erylus cf. deficiens collected in the Gorringe Bank (Atlantic Ocean) were tested in the innovative high throughput screening (HTS) assay for inhibitors of indoleamine 2,3-dioxygenase (IDO) and showed activity. Bioassay guided fractionation of the dichloromethane extract led to the isolation of four new glycolipids, named erylusamide A-D. The structures of the isolated compounds were established by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and chemical derivatization. The metabolites shared a pentasaccharide moiety constituted by unusual highly acetylated ᴅ-glucose moieties as well as ᴅ-xylose and ᴅ-galactose. The aglycones were unprecedented long chain dihydroxyketo amides. Erylusamides A, B and D differ in the length of the hydrocarbon chain, while erylusamide C is a structural isomer of erylusamide B.