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Chemical investigation of a marine sponge-associated Bacillus sp. led to the discovery of bacillibactins E and F (1 and 2). Despite containing the well-established cyclic triester core of iron-binding natural products such as enterobactin, bacillibactins E and F (1 and 2) are the first bacterial siderophores that contain nicotinic and benzoic acid moieties. The structures of the new compounds, including their absolute configurations, were determined by extensive spectroscopic analyses and Marfey's method. A plausible biosynthetic pathway to 1 and 2 is proposed; this route bears great similarity to other previously established bacillibactin-like pathways but appears to differentiate itself by a promiscuous DhbE, which likely installs the nicotinic moiety of 1 and the benzoic acid group of 2.

Paclitaxel (Taxol) is a cornerstone of cancer treatment. However, its mechanism of cytotoxicity is incompletely understood and not all patients benefit from treatment. We show that patients with breast cancer did not accumulate sufficient intratumoral paclitaxel to induce mitotic arrest in tumor cells. Instead, clinically relevant concentrations induced multipolar mitotic spindle formation. However, the extent of early multipolarity did not predict patient response. Whereas multipolar divisions frequently led to cell death, multipolar spindles focused into bipolar spindles before division at variable frequency, and maintaining multipolarity throughout mitosis was critical to induce the high rates of chromosomal instability necessary for paclitaxel to elicit cell death. Increasing multipolar divisions in paclitaxel resulted in improved cytotoxicity. Conversely, decreasing paclitaxel-induced multipolar divisions reduced paclitaxel efficacy. Moreover, we found that preexisting chromosomal instability sensitized breast cancer cells to paclitaxel. Both genetic and pharmacological methods of inducing chromosomal instability were sufficient to increase paclitaxel efficacy. In patients, the amount of pretreatment chromosomal instability directly correlated with taxane response in metastatic breast cancer such that patients with a higher rate of preexisting chromosomal instability showed improved response to taxanes. Together, these results support the use of baseline rates of chromosomal instability as a predictive biomarker for paclitaxel response. Furthermore, they suggest that agents that increase chromosomal instability or maintain multipolar spindles throughout mitosis will improve the clinical utility of paclitaxel.

Lactobacillus crispatus is a well-established probiotic with antimicrobial activity against pathogens across several niches of the human body generally attributed to the production of bacteriostatic molecules, including hydrogen peroxide and lactic acid. Here, we show that the cell-free supernatants of clinical isolates of L. crispatus harbor robust bactericidal activity. We further identify phenyl-lactic acid as a bactericidal compound with properties and a susceptibility range nearly identical to that of the cell-free supernatant. As such, we hypothesize that phenyl-lactic acid is a key active ingredient in L. crispatus supernatant. IMPORTANCE Although Lactobacillus crispatus is an established commensal microbe frequently used in probiotics, its protective role in the bladder microbiome has not been clarified. We report here that some urinary isolates of L. crispatus exhibit bactericidal activity, primarily due to its ability to excrete phenyl-lactic acid into its environment. Both cell-free supernatants of L. crispatus isolates and phenyl-lactic acid exhibit bactericidal activity against a wide range of pathogens, including several that are resistant to multiple antibiotics.

SUMMARYThe mammalian microbiome encodes numerous secondary metabolite biosynthetic gene clusters, yet their role in microbe-microbe interactions is unclear. Here, we characterized two polyketide synthase gene clusters (funandpks) in the gut symbiontLimosilactobacillus reuteri.Thepks, but not thefuncluster, encodes antimicrobial activity. Forty-one out of 51L. reuteristrains tested are sensitive to Pks products, which was independent of strains’ host origin. The sensitivity to Pks was also established in intraspecies competition experiments in gnotobiotic mice. Comparative genome analyses between Pks-resistant and sensitive strains identified an acyltransferase gene (act) that is unique to Pks-resistant strains. Subsequent cell wall analysis of the wild-type and theactmutant strains showed that Act acetylates cell wall components. Thepksmutants lost their competitive advantage andactmutants lost their Pks resistancein vivo. Thus, our findings provide insight into how closely related gut symbionts can compete and co- exist in the gastrointestinal tract.

Bacterial symbionts frequently provide chemical defenses for their hosts, and such systems can provide discovery pathways to new antifungals and structurally intriguing metabolites. This report describes a small family of naturally occurring small molecules with chimeric structures and a mixed biosynthesis that features an unexpected but key nonenzymatic step. An insect-associated Pseudomonas protegens strain's activity in an in vivo murine candidiasis assay led to the discovery of a family of highly hydrogen-deficient metabolites. Bioactivity- and mass-guided fractionation led to the pyonitrins, highly complex aromatic metabolites in which 10 of the 20 carbons are quaternary, and 7 of them are contiguous. The P. protegens genome revealed that the production of the pyonitrins is the result of a spontaneous reaction between biosynthetic intermediates of two well-studied Pseudomonas metabolites, pyochelin and pyrrolnitrin. The combined discovery of the pyonitrins and identification of the responsible biosynthetic gene clusters revealed an unexpected biosynthetic route that would have prevented the discovery of these metabolites by bioinformatic analysis alone.

Two new siderophores, madurastatin D1 and D2, together with (-)-madurastatin C1, the enantiomer of a known compound, were isolated from marine-derived Actinomadura sp. The presence of an unusual 4-imidazolidinone ring in madurastatins D1 and D2 inspired us to sequence the Actinomadura sp. genome and to identify the mad biosynthetic gene cluster, knowledge of which enables us to now propose a biosynthetic pathway. Madurastatin D1 and D2 are moderately active in antimicrobial assays with M. luteus.

Antibiotic resistance and emerging viral pandemics have posed an urgent need for new anti-infective drugs. By screening our microbial extract library against the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the notorious ESKAPE pathogens, an active fraction was identified and purified, leading to an initial isolation of adipostatins A (1) and B (2). In order to diversify the chemical structures of adipostatins toward enhanced biological activities, a type III polyketide synthase was identified from the native producer, Streptomyces davawensis DSM101723, and was subsequently expressed in an E. coli host, resulting in the isolation of nine additional adipostatins 3-11, including two new analogs (9 and 11). The structures of 1-11 were established by HRMS, NMR, and chemical derivatization, including using a microgram-scale meta-chloroperoxybenzoic acid epoxidation-MS/MS analysis to unambiguously determine the double bond position in the alkyl chain. The present study discovered SARS-CoV-2 main protease inhibitory activity for the class of adipostatins for the first time. Several of the adipostatins isolated also exhibited antimicrobial activity against selected ESKAPE pathogens.

International audience; Genomics and metabolomics are widely used to explore specialized metabolite diversity. The Paired Omics Data Platform is a community initiative to systematically document links between metabolome and (meta)genome data, aiding identification of natural product biosynthetic origins and metabolite structures.

Advances in genomics and metabolomics have made clear in recent years that microbial biosynthetic capacities on Earth far exceed previous expectations. This is attributable, in part, to the realization that most microbial natural product (NP) producers harbor biosynthetic machineries not readily amenable to classical laboratory fermentation conditions. Such “cryptic” or dormant biosynthetic gene clusters (BGCs) encode for a vast assortment of potentially new antibiotics and, as such, have become extremely attractive targets for activation under controlled laboratory conditions. We report here that co-culturing of a Rhodococcus sp. and a Micromonospora sp. affords keyicin, a new and otherwise unattainable bis-nitroglycosylated anthracycline whose mechanism of action (MOA) appears to deviate from those of other anthracyclines. The structure of keyicin was elucidated using high resolution MS and NMR technologies, as well as detailed molecular modeling studies. Sequencing of the keyicin BGC (within the Micromonospora genome) enabled both structural and genomic comparisons to other anthracycline-producing systems informing efforts to characterize keyicin. The new NP was found to be selectively active against Gram-positive bacteria including both Rhodococcus sp. and Mycobacterium sp. E. coli-based chemical genomics studies revealed that keyicin’s MOA, in contrast to many other anthracyclines, does not invoke nucleic acid damage.

Here we describe the rapid identification and prioritization of novel active marine natural products using an improved dereplication strategy. During the course of our screening of marine natural product libraries, a new cyclic trihydroxamate compound, thalassosamide, was discovered from the α-proteobacterium Thalassospira prof undimaris. Its structure was determined by 2D NMR and MS/MS experiments, and the absolute configuration of the lysine-derived units was established by Marfey’s analysis, whereas that of C-9, 9′, and 9″ was determined via the circular dichroism data of the [Rh2(OCOCF3)4] complex and DFT NMR calculations. Thalassosamide showed moderate in vivo efficacy against Pseudomonas aeruginosa.

Bacterial strains isolated from attine ants showed activity against the insect specialized fungal pathogen Escovopsis and also against the human protozoan parasite Leishmania donovani. The bioassay guided fractionation of extracts from cultures of Streptomyces sp. ICBG292, isolated from the exoskeleton of Cyphomyrmex workers, led to the isolation of Mer-A2026B (1), piericidin-A1 (2) and nigericin (3). Nigericin (3) presented high activity against intracellular amastigotes of L. donovani (IC50 0.129 ± 0.008 μM). Streptomyces puniceus ICBG378, isolated from workers of Acromyrmex rugosus rugosus, produced dinactin (4) with potent anti-L. donovani activity against intracellular amastigotes (IC50 0.018 ± 0.003 μM). Compounds 3 and 4 showed good selectivity indexes, 88.91 and 656.11 respectively, and were more active than positive control, miltefosine. Compounds 1–4 were also active against some Escovopsis strains. Compounds 1 and 2 were also produced by Streptomyces sp. ICBG233, isolated from workers of Atta sexdens, and detected in ants’ extracts by mass spectrometry, suggesting they are produced in the natural environment as defensive compounds involved in the symbiotic interaction., Author summary Visceral leishmaniasis, caused by Leishmania infantum and L. donovani, is characterized by high rate mortality worldwide. Current treatments for this disease suffer from toxicity, variable efficacy, requirements for parenteral administration and length of treatment regimens. New chemical entities and development of new drugs are important to overcome the impact of this protozoan disease. Actinobacterial strains, such as Streptomyces, have been a source of most naturally derived antibiotics, as well as anticancer, anthelmintic, and antifungal drugs. These microorganisms also produce small molecules important in symbiotic interactions with insects, such as fungus-growing ants, fungus-growing termites, beetles and wasps against pathogens. Several novel compounds have been reported from these microorganisms with promising biological activities. In this work we show an interesting ecologic approach for drug discovery that also shows promise for the identification of antileishmanial natural products from fungus-growing ant ecosystem. Two compounds isolated from Streptomyces strains showed potent activity against L. donovani, higher than the positive control (miltefosine) with high selectivity indexes.

Keywords: NDM-1 inhibitors, marine-derived Streptomyces sp., carbapenem-resistant Enterobacteriaceae, metal chelators

Screening of a marine natural products library for inhibitors of TGF-β revealed five pyrimidinedione derivatives, biemamides A–E (1–5). The structures were determined by 2D NMR and HRMS experiments; absolute configurations were established by advanced Marfey’s analysis and ECD calculations. Biemamides A–E specifically inhibited in vitro TGF-β induced epithelial to mesenchymal transition in NMuMG cells. Additionally, using Caenorhabditis elegans, selected biemmamides were found to influence in vivo developmental processes related to body size regulation in a dose-dependent manner.

Rediscovery of known natural products hinders the discovery of new, unique scaffolds. Efforts have mostly focused on streamlining the determination of what compounds are known vs. unknown (dereplication), but an alternative strategy is to focus on what is different. Utilizing statistics and assuming that common actinobacterial metabolites are likely known, focus can be shifted away from dereplication and towards discovery. LC-MS-based principal component analysis (PCA) provides a perfect tool to distinguish unique vs. common metabolites, but the variability inherent within natural products leads to datasets that do not fit ideal standards. To simplify the analysis of PCA models, we developed a script that identifies only those masses or molecules that are unique to each strain within a group, thereby greatly reducing the number of data points to be inspected manually. Since the script is written in R, it facilitates integration with other metabolomics workflows and supports automated mass matching to databases such as Antibase.

Forazoline A, a novel antifungal polyketide with in vivo efficacy against Candida albicans, was discovered using LCMS-based metabolomics to investigate marine-invertebrate-associated bacteria. Forazoline A had a highly unusual and unprecedented skeleton. Acquisition of (13)C-(13)C gCOSY and (13)C-(15)N HMQC NMR data provided the direct carbon-carbon and carbon-nitrogen connectivity, respectively. This approach represents the first example of determining direct (13)C-(15)N connectivity for a natural product. Using yeast chemical genomics, we propose that forazoline A operated through a new mechanism of action with a phenotypic outcome of disrupting membrane integrity.

International audience; LipL and Cpr19 are nonheme, mononuclear Fe(II)-dependent, alpha-ketoglutarate (alpha KG): UMP oxygenases that catalyze the formation of CO2, succinate, phosphate, and uridine-5'-aldehyde, the last of which is a biosynthetic precursor for several nucleoside antibiotics that inhibit bacterial translocase I (MraY). To better understand the chemistry underlying this unusual oxidative dephosphorylation and establish a mechanistic framework for LipL and Cpr19, we report herein the synthesis of two biochemical probes-[1',3',4',5',5'-H-2] UMP and the phosphonate derivative of UMP-and their activity with both enzymes. The results are consistent with a reaction coordinate that proceeds through the loss of one H-2 atom of [1',3',4',5',5'-H-2] UMP and stereospecific hydroxylation geminal to the phosphoester to form a cryptic intermediate, (5'R)-5'-hydroxy-UMP. Thus, these enzyme catalysts can additionally be assigned as UMP hydroxylase-phospholyases.

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich. This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively. Covering: 1988 to 2012 This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

GilOII has been unambiguously identified as the key enzyme performing the crucial C-C bond cleavage reaction responsible for the unique rearrangement of a benz[a]anthracene skeleton to the benzo[d]naphthopyranone backbone typical for the gilvocarcin type natural anticancer antibiotics. Further investigations of this enzyme led to the isolation of a hydroxy-oxepinone intermediate which allowed important conclusions regarding the cleavage mechanism.

The potential of a uniquely permissive engineered glycosyltransferase (OleD ASP) as a catalyst for steroid glycosylation is highlighted. The ability of OleD ASP to glucosylate a range of cardenolides and bufadienolides was assessed using a rapid LC-UV/MS-SPE-NMR analytical platform. While a bias toward OleD-catalyzed C3 monoglucosylation was observed, subtle alterations of the steroidal architecture, in some cases, invoked diglucosylation or, in one case (digoxigenin), C12 glucosylation. This latter case represents the first, and highly efficient, synthesis of digoxigenin 12-O-β-D-glucoside.

Endocrocin is a simple anthraquinone frequently identified in extracts of numerous fungi. Several biosynthetic schemes for endocrocin synthesis have been hypothesized, but to date, no dedicated secondary metabolite gene cluster that produces this polyketide as its major metabolite has been identified. Here we describe our biosynthetic and regulatory characterization of the endocrocin gene cluster in Aspergillus fumigatus . This is the first report of this anthraquinone in this species. The biosynthetic genes required for endocrocin production are regulated by the global regulator of secondary metabolism, LaeA, and encode an iterative nonreducing polyketide synthase ( encA ), a physically discrete metallo-β-lactamase type thioesterase ( encB ), and a monooxygenase ( encC ). Interestingly, the deletion of a gene immediately adjacent to encC , termed encD and encoding a putative 2-oxoglutarate-Fe(II) type oxidoreductase, resulted in higher levels of endocrocin production than in the wild-type strain, whereas overexpression of encD eliminated endocrocin accumulation. We found that overexpression of the encA transcript resulted in higher transcript levels of encA-D and higher production of endocrocin. We discuss a model of the enc cluster as one evolutionary origin of fungal anthraquinones derived from a nonreducing polyketide synthase and a discrete metallo-β-lactamase-type thioesterase.

Utilization of isotopically labeled proteins and peptides is a routinely employed approach in biomolecular NMR investigations. The widespread availability of inexpensive, uniformly (13) C-enriched glucose now makes it possible to produce uniformly (13) C-labeled natural products by microbial fermentation. In this feature article, the authors describe an experimental approach for the rapid structural characterization of uniformly (13) C-labeled natural products based on the Constant-Time HSQC (CT-HSQC) experiment. Rigorous theoretical evaluation of the CT-HSQC experiment allowed the applicability of the experiment to be expanded from the traditional, narrow scope of labeled amino acids to encompass virtually any small molecule or U-(13) C labeled natural product. A suite of experiments including CT-HSQC, (13) C-(13) C COSY, and COSYLR experiments is sufficient for the structure elucidation of uniformly (13) C-labeled small molecules and natural products. Differences in NMR approaches for structure elucidation of natural abundance and uniformly (13) C-labeled molecules are also discussed. The present work provides a researcher working in this area of natural products chemistry with NMR structure elucidation tools for investigating (13) C-labeled small molecules and natural products.

Exocarpic acid (13 E-octadecene-9,11-diynoic acid) from Exocarpos latifolius R.Br. (Santalaceae) was previously shown to have specific antimycobacterial activity. Microarray data suggested inhibition of fatty acid metabolism as a potential mode of action. Experiments designed to elucidate the mechanism of action showed that exocarpic acid was effective at inhibition of mycolic acid biosynthesis and did not act by dissipating the proton gradient in treated M. tuberculosis. Amide derivatives of exocarpic acid displayed similar properties to exocarpic acid, while other polyacetylenic fatty acids varied in their effects on mycolic acid biosynthesis.

Actinomycetes are prolific producers of pharmacologically important compounds accounting for about 70% of the naturally derived antibiotics that are currently in clinical use. In this study, we report on the isolation of Streptomyces sp. strains from Mediterranean sponges, on their secondary metabolite production and on their screening for anti-infective activities. Bioassay-guided isolation and purification yielded three previously known compounds namely, cyclic depsipeptide valinomycin, indolocarbazole alkaloid staurosporine and butenolide. This is the first report of the isolation of valinomycin from a marine source. These compounds exhibited novel anti-parasitic activities specifically against Leishmania major (valinomycin IC(50)0.11 microM; staurosporine IC(50) 5.30 microM) and Trypanosoma brucei brucei (valinomycin IC(50) 0.0032 microM; staurosporine IC(50) 0.022 microM; butenolide IC(50) 31.77 microM). These results underscore the potential of marine actinomycetes to produce bioactive compounds as well as the re-evaluation of previously known compounds for novel anti-infective activities.

The high-throughput screening and drug discovery paradigm has necessitated a change in preparation of natural product samples for screening programs. In an attempt to improve the quality of marine natural products samples for screening, several fractionation strategies were investigated. The final method used HP20SS as a solid support to effectively desalt extracts and fractionate the organic components. Additionally, methods to integrate an automated LCMS fractionation approach to shorten discovery time lines have been implemented.

Three new sterol sulfates, spheciosterol sulfates A-C (1-3), and the known sterol sulfate topsentiasterol sulfate E (4) have been isolated from the sponge Spheciospongia sp., collected in the Philippines. Structures were assigned on the basis of extensive 1D and 2D NMR studies as well as analysis by HRESIMS. Compounds 1-4 inhibited PKCzeta with IC50 values of 1.59, 0.53, 0.11, and 1.21 microM, respectively. In a cell-based assay, 1-4 also inhibited NF-kappaB activation with EC50 values of 12-64 microM.

Rapidly increasing experimental and clinical data provides evidence for the role of hypoxia inducible factor-1 (HIF-1) as a crucial mediator of tumor survival and progression. In our effort to identify inhibitors of the HIF-1 activation pathway, we screened fractions from marine invertebrates. Fractions from an extract of Petrosia (Strongylophora) strongylata potently inhibited the HIF-1 activation pathway. Strongylophorines 2, 3, and 8 isolated from the active fractions were found to be responsible for the HIF-1 inhibition with EC 50 values of 8, 13, and 6 microM, respectively.

Neoamphimedine, a pyridoacridine alkaloid from Xestospongia sp., is a potent antitumor agent both in vitro and in vivo. Neoamphimedine can efficiently induce topoisomerase II mediated catenation of plasmid DNA in vitro and is the only member of more than one hundred pyridoacridines thus far to have this mechanism of action. Herein we report the first total synthesis of neoamphimedine.

Fungal isolate F01V25 was obtained from the alga Dictyosphaeria versluyii collected near Dravuni, Fiji, in 2001 and represented a previously undescribed Penicillium sp. Fermentation of isolate F01V25 resulted in the production of two new polyketides, dictyosphaeric acids A and B, along with the known anthraquinone carviolin. The relative stereochemistry of dictyosphaeric acids A and B was determined using the J-based configuration analysis method in conjunction with ROE and NOE correlations.

Fractionation of the crude methanol extract of the ascidian Cystodytes dellechiajei collected in Brazil yielded two novel alkaloids, sebastianine A (1) and sebastianine B (2). The structures of both 1 and 2 were established by analysis of spectroscopic data, indicating an unprecedented ring system for both compounds, comprising a pyridoacridine system fused with a pyrrole unit in sebastianine A (1) and a pyridoacridine system fused with a pyrrolidine system condensed with alpha-hydroxyisovaleric acid in sebastianine B (2). Both alkaloids displayed a cytotoxic profile against a panel of HCT-116 colon carcinoma cells indicative of a p53 dependent mechanism.

Investigation of the culture extracts of a marine-derived Streptomyces sp. led to the isolation of three new bohemamine-type pyrrolizidine alkaloids, bohemamine B (1), bohemamine C (2), and 5-chlorobohemamine C (3). The structures were elucidated using NMR methods, and the relative stereochemistry was determined using double-pulsed-field-gradient spin echo (DPFGSE) NOE studies.

This study characterized electrophilic and radical products derived from the metabolism of capsaicin by cytochrome P450 and peroxidase enzymes. Multiple glutathione and β-mercaptoethanol conjugates (a.k.a., adducts), derived from the trapping of quinone methide and quinone intermediates of capsaicin, its analogue nonivamide, and O-demethylated and aromatic hydroxylated metabolites thereof, were produced by human liver microsomes and individual recombinant human P450 enzymes. Conjugates derived from concomitant dehydrogenation of the alkyl terminus of capsaicin were also characterized. Modifications to the 4-OH substituent of the vanilloid ring of capsaicinoids largely prevented the formation of electrophilic intermediates, consistent with the proposed structures and mechanisms of formation for the various conjugates. 5,5'-Dicapsaicin, presumably arising from the bimolecular coupling of free radical intermediates was also characterized. Finally, the analysis of hepatic glutathione conjugates and urinary N-acetylcysteine conjugates from mice dosed with capsaicin confirmed the formation of glutathione conjugates of O-demethylated quinone methide and 5-OH-capsaicin in vivo. These data demonstrated that capsaicin and structurally similar analogues are converted to reactive intermediates by certain P450 enzymes, which may partially explain conflicting reports related to the cytotoxic, pro-carcinogenic, and chemoprotective effects of capsaicinoids in different cells and/or organ systems.

Drug resistant infectious diseases are quickly becoming a global health crisis. While Streptomyces spp. have been a major source of antibiotics over the past 50 years, efficient methods are needed to identify new antibiotics and greatly improve the rate of discovery. LCMS-based metabolomics were applied to analyze extracts of 50 Streptomyes spp. Using this methodology, we discovered bottromycin D and used whole genome sequencing to determine its biosynthesis by a ribosomal pathway.

Investigation of a Philippine specimen of the red alga Ceratodictyon spongiosum and its sponge symbiont Haliclona cymaeformis led to the isolation of p-sulfooxyphenylpyruvic acid, whose structure was elucidated using spectroscopic methods, with the Z-enol geometry determined through analysis of (3)J(C,H) coupling constants. The metabolite was tested for tyrosine kinase inhibition using a (3)H-thymidine incorporation assay, but was found inactive.

A marine Verrucosispora sp. isolated from the sponge Chondrilla caribensis f. caribensis was found to produce thiocoraline, a potent cytotoxic compound. Five new analogs of thiocoraline were isolated and represent the first analogs of thiocoraline. 22'-Deoxythiocoraline (2), thiochondrilline C (5), and 12'-sulfoxythiocoraline (6) demonstrated significant cytotoxicity against the A549 human cancer cell line with EC(50) values of 0.13, 2.86, and 1.26 μM, respectively. The analogs provide insight into the SAR and biosynthesis of thiocoraline. The DP4 probability method was used to analyze ab initio NMR calculations to confirm stereochemical assignments.

The biosynthetic gene cluster for tautomycetin (TTN), a highly potent and selective protein phosphatase (PP) inhibitor isolated from Streptomyces griseochromogenes, has recently been cloned and sequenced. To better understand the transformations responsible for converting the post-polyketide synthase product into the exciting anticancer and immunosuppressive chemotherapeutic candidate TTN, we produced and characterized new analogues resulting from inactivation of two genes, ttnD and ttnF, in S. griseochromogenes. Inactivation of ttnD and ttnF, which encode for putative decarboxylase and dehydratase enzymes, respectively, afforded mutant strains SB13013 and SB13014. The DeltattnD mutant SB13013 accumulated four new TTN analogues, TTN D-1, TTN D-2, TTN D-3, and TTN D-4, whereas the DeltattnF mutant accumulated only one new TTN analogue, TTN F-1. The accumulation of these new TTN analogues defines the function of TtnD and TtnF and the timing of their chemistries in relation to installation of the C5 ketone moiety within TTN. Notably, all new analogues possess a structurally distinguishing carboxylic acid moiety, revealing that TtnD apparently cannot catalyze decarboxylation in the absence of TtnF. Additionally, cytotoxicity and PP inhibition assays reveal the importance of the functional groups installed by TtnDF and, consistent with earlier proposals, the C2''-C5 fragment of TTN to be a critical structural determinant behind the important and unique PP-1 selectivity displayed by TTN.

Incubation of farnesyl diphosphate with recombinant yeast squalene synthase in the absence of NADPH gives a mixture of triterpene hydrocarbons and alcohols, including botryococcene-like compounds with 1'-3 linkages between the farnesyl units. One of these molecules has an unusual cyclopentane structure similar to those recently reported in plant extracts and lakebed sediments.

Three new sulfated sterol dimers, fibrosterol sulfates A-C (1-3), have been isolated from the sponge Lissodendoryx (Acanthodoryx) fibrosa, collected in the Philippines. The structures were assigned on the basis of extensive 1D and 2D NMR studies as well as analysis by HRESIMS. Compounds 1 and 2 inhibited PKCzeta with IC(50) values of 16.4 and 5.6 microM, respectively.

There is a need for diverse molecular libraries for phenotype-selective and high-throughput screening. To make marine natural products (MNPs) more amenable to newer screening paradigms and shorten discovery time lines, we have created an MNP library characterized online using MS. To test the potential of the library, we screened a subset of the library in a phenotype-selective screen to identify compounds that inhibited the growth of BRCA2-deficient cells.

Chemical investigation of the cytotoxic and anti-tuberculosis active butanone extract obtained from the growth media of the marine-derived fungus Beauveria felina led to the isolation of two new destruxins, [beta-Me-Pro] destruxin E chlorohydrin (1) and pseudodestruxin C (3), along with five known cyclic depsipeptides. The structures of the new destruxin derivatives were established by analysis of spectroscopic data, while the absolute configuration of the common amino acid residues was established by Marfey's analysis. The absolute configuration of the 2(R),4(S)-5-chloro-2,4-dihydroxypentanoic acid residue in 1 could be established by application of a J-based configuration method followed by derivatization with R-MPA-Cl and NMR analysis.

Poly-acetylated sesquiterpene pyridine alkaloids are ubiquitously found in the Celastraceae family of plants.1–4 Dihydro-β-agarofuran sesquiterpenes frequently incorporate pyridine dicarboxylic acids to bridge their sesquiterpenoid cores at the C-3 and C-13 positions. The dihydro-β-agarofuran skeleton is comprised of a decalin ring, a tetrahydrofuran ring, and methyl substituents located at the C-4, C-10, and two at the C-11 position. Evoninic, wilfordic, edulic, cathaic, and cassininic (often misnamed cassinic) acids are some of the pyridine carboxylic acids present in this class of sesquiterpenes. These compounds are of interest due to their wide range of biological activities including: cytotoxicity,1 immunosuppression,2 insecticidal,3,5,6 insect-antifeedant,5,7 anti-HIV,4 reversal of the multidrug resistance (MDR) phenotype,8–11 and antitumor.12 As part of an ongoing search for bioactive compounds, two new sesquiterpene alkaloids, oppositines A (1) and B (2), were isolated from the Sri Lankan plant, Pleurostylia opposita (Wallich, 1824) (Celastraceae). The air dried stem bark of P. opposita was extracted with 1:1 MeOH/CH2Cl2. The extract was dried and partitioned between petroleum ether and aqueous MeOH, and the aqueous MeOH was subsequently partitioned with chloroform. The chloroform-soluble material was fractionated using size exclusion chromatography, preparative TLC, and reversed-phase HPLC resulting in oppositines A (1) and B (2). Oppositine A (1) was obtained as a light-yellow glass. The molecular formula, C44H51NO18, was determined by HRESIMS. Initial interpretation of the 1H and 13C NMR data indicated that 1 contained five acetyls and one hydroxyl. The data also indicated that oppositine A (1) contained four quaternary carbons, eight methines, five methylenes, and three methyls. The existence of a benzoate was suggested by the signals at δH 7.80 (dd, J = 7.9, 1.3 Hz, H-18, H-18′), 7.52 (tt, J = 7.9, 1.3 Hz, H-20), 7.38 (t, J = 7.9 Hz, H-19, H-19′), as well as five carbons at δ 164.6 (C-16), 133.5 (C-20), 129.5 (C-18, C-18′), 129.1 (C-17), and 128.5 (C-19, C-19′). The signal at δC 164.6 (C-16) was assigned to the carbonyl carbon of the benzoate based on an HMBC correlation with the proton signal at δH 7.80 (H-18, H-18′). HSQC and HMBC data further supported the assignment of the benzoate group (Supplemental Table 1). The existence of a 2,3-disubstituted pyridine unit was suggested by the signals for three aromatic protons at δH 8.82 (dd, J = 4.6, 1.9 Hz, H-6 ), 8.39 (dd, J = 8.0, 1.9 Hz, H-4 ), 7.26 (dd, J = 8.0, 4.6 Hz, H-5 ), as well as five aromatic carbons at δC 168.1 (C-2 ), 153.8 (C-6 ), 138.2 (C-4 ), 124.9 (C-3 ), and 120.8 (C-5 ). HSQC and HMBC data further supported the assignment of the 2,3-disubstituted pyridine unit (Supplemental Table 1). A dicarboxy side chain containing two esters, one methine, two methylenes, one ethyl group, and the 2,3-disubstituted pyridine unit supported the presence of a cassininic acid moiety in oppositine A (1). The NMR data for this portion of the molecule are consistent with data reported for the analogous protons in the compounds cassinine13 and orthophosphonine.14 Based on COSY data, signals at δH 5.88, 5.22, and 5.01 were assigned to H-1, H-2, and H-3, respectively, and signals at δH 7.00, 2.32, 5.51, and 5.38 were assigned to H-6, H-7, H-8, and H-9, respectively. Taken together, these data indicate that 1 is a sesquiterpene pyridine alkaloid with a dihydro-β-agarofuran skeleton. Oppositines A (1) and B (2) have been drawn with the tetrahydrofuran ring on the top face of the molecule so as to conform to the depiction of other cassininic acid containing molecules.13,14 The tetrahydrofuran ring can also be drawn on the bottom side of the molecule, as is noted in a review of Celastraceae sesquiterpenoids.15 The regiosubstitution of the various groups around the terpene skeleton was established based on HMBC data. In the HMBC spectrum of 1, the methyl singlets at δH 2.31 (H-30), 2.19 (H-24), 2.10 (H-26), 1.38 (H-28), and 2.13 (H-22) correlated with the five acetyl carbonyl carbons at δC 170.4 (C-29), 170.0 (C-23), 170.0 (C-25), 168.9 (C-27), and 168.4 (C-21), respectively. An anisotropic effect caused by the benzoate at the C-1 position is thought to be the cause of H-28 resonating at such high field. The effect on H-28 is observed in similar compounds with a benzoate at the C-1 position.1,3,4 The attachment of the acetyl groups at C-2, C-6, C-8, C-9, and C-15 was established based on HMBC correlations from the proton signals at δH 5.22 (H-2), 7.00 (H-6), 5.51(H-8), 5.38 (H-9), and 4.63 (H-15b) to the acetyl carbonyls at δC 168.4 (C-21), 170.0 (C-23), 170.0 (C-25), 168.9 (C-27), and 170.4 (C-29), respectively. The attachment of the benzoate at C-1 was supported by an HMBC correlation from the proton signal at δH 5.88 (H-1) to the carbonyl signal at δC 164.6 (C-16). The attachment of a methyl and a hydroxyl at C-4 was established on the basis of HMBC correlations between δH 1.56 (H-14) and δC 70.0 (C-4), as well as δH 4.96 (OH-4) and δC 70.0 (C-4). Additionally, HMBC correlations between the proton signals at δH 5.01 (H-3) and 5.96, 3.59 (H-13a, H-13b) and the carbon signals at δC 172.2 (C-12 ) and 166.4 (C-13 ), respectively, supported the attachment of the cassininate moiety at C-3 and C-13. Sesquiterpene pyridine alkaloids characteristically include diastereotopic methylenes attached to C-13 that resonate at very different chemical shifts.1–4 The relative stereochemistry of oppositine A (1), as shown in Figure 1, was established on the basis of coupling constants, observed ROE correlations, and molecular modeling. A ROESY experiment showing ROE effects for H-1 to H-2, H-9, and H-12; H-14 to H-3, H-6, and H-15; and H-9 to H-8, and H-12, established the relative position of the substituent groups. All coupling constants associated with the terpene skeleton were less than 6 Hz (Supplemental Table 1), and supported the arrangements determined by the ROESY data. Therefore, the relative configuration in 1 was established as 1R*, 2R*, 3S*, 4S*, 5S*, 6R*, 7R*, 8R*, 9S*, 10R*, 11S*. The stereochemistry at 7′ was not determined. Figure 1 Key ROE correlations supporting the relative stereochemistry of oppositine A (1). Oppositine B (2) was obtained as a light-yellow glass. The molecular formula, C44H51NO17, was determined by HRESIMS, and suggested that oppositine B (2) had one less oxygen than oppositine A (1). Comparison of the NMR data of 2 with 1 confirmed the absence of the hydroxyl at the C-4 position. Specifically, a methine signal at δH 2.72 (H-4), coupled to a methyl signal at δH 1.24 (H-14), showed HMBC correlations to carbons at δC 90.7 (C-5), 74.9 (C-3), 70.4 (C-2), and 50.9 (C-10). The relative stereochemistry of 2 was established on the basis of coupling constants, observed ROE correlations, and comparison with 1. A ROESY experiment showing ROE effects for H-1 to H-2, and H-9; H-14 to H-3, H-6, and H-15; and H-9 to H-8, and H-12, established the relative position of the substituent groups. All coupling constants were less than 6 Hz (Supplemental Table 1), and supported the arrangements determined from the ROESY data. Therefore, the relative configuration in 2 was established as 1R*, 2S*, 3R*, 4R*, 5S*, 6R*, 7R*, 8R*, 9S*, 10R*, 11S*. The stereochemistry at 7′ was not determined. Oppositines A (1) and B (2) showed moderate cytotoxicity against HCT116 cell lines with EC50 values of 27 ± 2 and 26 ± 3 μM, respectively. Oppositines A (1) and B (2) were also tested for anti-HIV activity and HIF-1α inhibition, but were inactive up to a concentration of 10 μg/mL in both assays. Antifungal activity assays for oppositines A (1) and B (2) are currently in progress. To date only two cassininic acid metabolites have been reported, cassinine13 and orthophosphonine.14 Oppositine A (1) differs from cassinine in hydroxylation at the C-4 carbon and the position of the benzoate at the C-1 carbon rather than the C-6 carbon. Oppositine B (2) differs from cassinine in the position of the benzoate. Previous investigations of P. opposita have yielded lupane and spermidine derivatives.16–18 This is the first reported incidence of dihydro-β-agarofuran sesquiterpenes from P. opposita, although a plethora of such compounds has been reported from the Celastraceae family.

[reaction: see text] Synthesis of a novel class of C-10 halogenated and C-12 oxygenated prostaglandin-A(2) derivatives (6a-6c) has been accomplished. (15S)-Prostaglandin-A(2) (1), from the gorgonian Plexaura homomalla, served as the starting material for the synthesis. The absolute configuration was determined using NMR.

Acanthella spp. sponges have been prolific sources of highly functionalized diterpene antibiotics. Two Acanthella cavernosa sponges were investigated based on the activity of their extracts in a screen designed to detect bacterial folate biosynthesis inhibitors. Bacillus subtilis PY79 strain harboring a lacZ reporter gene fusion to a trimethoprim-responsive promoter (PpanB) was used for the screen. The ability of kalihinols to inhibit bacterial folate biosynthesis was investigated resulting in preliminary structure activity relationships. Eight kalihinol type diterpenes were isolated from two Philippine Acanthella cavernosa specimens including two new 10- and 15-formamido-kalihinol F analogs.

Bioassay-guided fractionation of two Philippine sponges of the genus Petrosia has resulted in the isolation of the novel natural product cribrostatin 7 (1) and the known compounds renierone (2) and O-demethylrenierone (3). The structures of these isoquinoline quinones were determined by interpretation of spectroscopic data. Compounds 1, 2 and 3 were cytotoxic against the HCT 116 human colon carcinoma cell line with IC50 values of 45, 24 and 34 microg/mL, respectively.

Fractionation of the crude methanol extract of the ascidian Cystodytes dellechiajei collected in Brazil yielded two novel alkaloids, sebastianine A (1) and sebastianine B (2). The structures of both 1 and 2 were established by analysis of spectroscopic data, indicating an unprecedented ring system for both compounds, comprising a pyridoacridine system fused with a pyrrole unit in sebastianine A (1) and a pyridoacridine system fused with a pyrrolidine system condensed with alpha-hydroxyisovaleric acid in sebastianine B (2). Both alkaloids displayed a cytotoxic profile against a panel of HCT-116 colon carcinoma cells indicative of a p53 dependent mechanism.