Portobelamides A and B and Caciqueamide, Cytotoxic Peptidic Natural Products from a Caldora sp. Marine Cyanobacterium.

Three new compounds, portobelamides A and B (1 and 2), 3-amino-2-methyl-7-octynoic acid (AMOYA) and hydroxyisovaleric acid (Hiva) containing cyclic depsipeptides, and one long chain lipopeptide caciqueamide (3), were isolated from a field-collection of a Caldora sp. marine cyanobacterium obtained from Panama as part of the Panama International Cooperative Biodiversity Group Program. Their planar structures were elucidated through analysis of 2D NMR and MS data, especially high resolution (HR) MS2/MS3 fragmentation methods. The absolute configurations of compounds 1 and 2 were deduced by traditional hydrolysis, derivative formation, and chromatographic analyses compared with standards. Portobelamide A (1) showed good cytotoxicity against H-460 human lung cancer cells (33% survival at 0.9 µM).

Dragocins A-D, Structurally Intriguing Cytotoxic Metabolites from a Panamanian Marine Cyanobacterium.

Dragocins A-D (1-4) were isolated from a dark-red wooly textured marine cyanobacterium collected in Boca del Drago, Panama. Dragocins A-C (1-3) possessed 2,3-dihydroxypyrrolidine, 1-hydroxy-5- O-Me-benzoyl, and 4'-substituted-ß-ribofuranose moieties that connected to form a nine-membered macrocyclic ring. Dragocins A-C are members of a unique hybrid structural class with substitution at the C-4' position of a ribofuranose unit. Of the four new compounds, dragocin A was the most potent cytotoxin to human H-460 lung cancer cells.

Enzyme inhibition by hydroamination: design and mechanism of a hybrid carmaphycin-syringolin enone proteasome inhibitor.

Hydroamination reactions involving the addition of an amine to an inactivated alkene are entropically prohibited and require strong chemical catalysts. While this synthetic process is efficient at generating substituted amines, there is no equivalent in small molecule-mediated enzyme inhibition. We report an unusual mechanism of proteasome inhibition that involves a hydroamination reaction of alkene derivatives of the epoxyketone natural product carmaphycin. We show that the carmaphycin enone first forms a hemiketal intermediate with the catalytic Thr1 residue of the proteasome before cyclization by an unanticipated intramolecular alkene hydroamination reaction, resulting in a stable six-membered morpholine ring. The carmaphycin enone electrophile, which does not undergo a 1,4-Michael addition as previously observed with vinyl sulfone and α,ß-unsaturated amide-based inhibitors, is partially reversible and gives insight into the design of proteasome inhibitors for cancer chemotherapy.

Lipopeptides from the tropical marine cyanobacterium Symploca sp.

A collection of the tropical marine cyanobacterium Symploca sp., collected near Kimbe Bay, Papua New Guinea, previously yielded several new metabolites including kimbeamides A-C, kimbelactone A, and tasihalide C. Investigations into a more polar cytotoxic fraction yielded three new lipopeptides, tasiamides C-E (1-3). The planar structures were deduced by 2D NMR spectroscopy and tandem mass spectrometry, and their absolute configurations were determined by a combination of Marfey's and chiral-phase GC-MS analysis. These new metabolites are similar to several previously isolated compounds, including tasiamide (4), grassystatins (5, 6), and symplocin A, all of which were isolated from similar filamentous marine cyanobacteria.

Parguerene and precarriebowmide, two classes of lipopeptides from the marine cyanobacterium Moorea producens.

Two new marine cyanobacterial natural products, parguerene (1) and precarriebowmide (2), were isolated from a collection of Moorea producens obtained from La Parguera, Puerto Rico. The planar structures of both were deduced by 2D NMR spectroscopy and mass spectrometry. Parguerene is a modified acyl amide with some structural similarity to the bacterial metabolite stipiamide (3), whereas precarriebowmide is a lipopeptide and represents a minor modification compared to two other known metabolites, carriebowmide (4) and carriebowmide sulfone (5). The identification of 2 led to an investigation into whether carriebowmide and carriebowmide sulfone were true secondary metabolites or isolation artifacts.

Five chemically rich species of tropical marine cyanobacteria of the genus Okeania gen. nov. (Oscillatoriales, Cyanoprokaryota).

An adverse consequence of applying morphology-based taxonomic systems to catalog cyanobacteria, which generally are limited in the number of available morphological characters, is a fundamental underestimation of natural biodiversity. In this study, we further dissect the polyphyletic cyanobacterial genus Lyngbya and delineate the new genus Okeania gen. nov. Okeania is a tropical and subtropical, globally distributed marine group abundant in the shallow-water benthos. Members of Okeania are of considerable ecological and biomedical importance because specimens within this group biosynthesize biologically active secondary metabolites and are known to form blooms in coastal benthic environments. Herein, we describe five species of the genus Okeania: O. hirsuta (type species of the genus), O. plumata, O. lorea, O. erythroflocculosa, and O. comitata, under the provisions of the International Code of Nomenclature for Algae, Fungi, and Plants. All five Okeania species were morphologically, phylogenetically, and chemically distinct. This investigation provides a classification system that is able to identify Okeania spp. and predict their production of bioactive secondary metabolites.

Viequeamide A, a cytotoxic member of the kulolide superfamily of cyclic depsipeptides from a marine button cyanobacterium.

The viequeamides, a family of 2,2-dimethyl-3-hydroxy-7-octynoic acid (Dhoya)-containing cyclic depsipeptides, were isolated from a shallow subtidal collection of a "button" cyanobacterium (Rivularia sp.) from near the island of Vieques, Puerto Rico. Planar structures of the two major compounds, viequeamide A (1) and viequeamide B (2), were elucidated by 2D-NMR spectroscopy and mass spectrometry, whereas absolute configurations were determined by traditional hydrolysis, derivative formation, and chromatography in comparison with standards. In addition, a series of related minor metabolites, viequeamides C-F (3-6), were characterized by HRMS fragmentation methods. Viequeamide A was found to be highly toxic to H460 human lung cancer cells (IC(50) = 60 ± 10 nM), whereas the mixture of B-F was inactive. From a broader perspective, the viequeamides help to define a "superfamily" of related cyanobacterial natural products, the first of which to be discovered was kulolide. Within the kulolide superfamily, a wide variation in biological properties is observed, and the reported producing strains are also highly divergent, giving rise to several intriguing questions about structure-activity relationships and the evolutionary origins of this metabolite class.

Honaucins A-C, potent inhibitors of inflammation and bacterial quorum sensing: synthetic derivatives and structure-activity relationships.

Honaucins A-C were isolated from the cyanobacterium Leptolyngbya crossbyana which was found overgrowing corals on the Hawaiian coast. Honaucin A consists of (S)-3-hydroxy-γ-butyrolactone and 4-chlorocrotonic acid, which are connected via an ester linkage. Honaucin A and its two natural analogs exhibit potent inhibition of both bioluminescence, a quorum-sensing-dependent phenotype, in Vibrio harveyi BB120 and lipopolysaccharide-stimulated nitric oxide production in the murine macrophage cell line RAW264.7. The decrease in nitric oxide production was accompanied by a decrease in the transcripts of several proinflammatory cytokines, most dramatically interleukin-1ß. Synthesis of honaucin A, as well as a number of analogs, and subsequent evaluation in anti-inflammation and quorum-sensing inhibition bioassays revealed the essential structural features for activity in this chemical class and provided analogs with greater potency in both assays.

Biosynthetically intriguing chlorinated lipophilic metabolites from geographically distant tropical marine cyanobacteria.

Five new vinylchlorine-containing metabolites, the lipoamides janthielamide A and kimbeamides A-C and the ketide-extended pyranone kimbelactone A, have been isolated from collections of marine cyanobacteria made in Curaçao and Papua New Guinea. Both janthielamide A and kimbeamide A exhibited moderate sodium channel blocking activity in murine Neuro-2a cells. Consistent with this activity, janthielamide A was also found to antagonize veratridine-induced sodium influx in murine cerebrocortical neurons. These lipoamides represent the newest additions to a relatively rare family of marine cyanobacterial-derived lipoamides and a new structural class of compounds exhibiting neuromodulatory activities from marine cyanobacteria.

The carmaphycins: new proteasome inhibitors exhibiting an α,ß-epoxyketone warhead from a marine cyanobacterium.

Two new peptidic proteasome inhibitors were isolated as trace components from a Curaçao collection of the marine cyanobacterium Symploca sp. Carmaphycin A (1) and carmaphycin B (2) feature a leucine-derived α,ß-epoxyketone warhead directly connected to either methionine sulfoxide or methionine sulfone. Their structures were elucidated on the basis of extensive NMR and MS analyses and confirmed by total synthesis, which in turn provided more material for further biological evaluations. Pure carmaphycins A and B were found to inhibit the ß5 subunit (chymotrypsin-like activity) of the S. cerevisiae 20S proteasome in the low nanomolar range. Additionally, they exhibited strong cytotoxicity to lung and colon cancer cell lines, as well as exquisite antiproliferative effects in the NCI60 cell-line panel. These assay results as well as initial structural biology studies suggest a distinctive binding mode for these new inhibitors.

Moorea producens gen. nov., sp. nov. and Moorea bouillonii comb. nov., tropical marine cyanobacteria rich in bioactive secondary metabolites.

The filamentous cyanobacterial genus Moorea gen. nov., described here under the provisions of the International Code of Botanical Nomenclature, is a cosmopolitan pan-tropical group abundant in the marine benthos. Members of the genus Moorea are photosynthetic (containing phycocyanin, phycoerythrin, allophycocyanin and chlorophyll a), but non-diazotrophic (lack heterocysts and nitrogenase reductase genes). The cells (discoid and 25-80 µm wide) are arranged in long filaments (<10 cm in length) and often form extensive mats or blooms in shallow water. The cells are surrounded by thick polysaccharide sheaths covered by a rich diversity of heterotrophic micro-organisms. A distinctive character of this genus is its extraordinarily rich production of bioactive secondary metabolites. This is matched by genomes rich in polyketide synthase and non-ribosomal peptide synthetase biosynthetic genes which are dedicated to secondary metabolism. The encoded natural products are sometimes responsible for harmful algae blooms and, due to morphological resemblance to the genus Lyngbya, this group has often been incorrectly cited in the literature. We here describe two species of the genus Moorea: Moorea producens sp. nov. (type species of the genus) with 3L(T) as the nomenclature type, and Moorea bouillonii comb. nov. with PNG5-198(R) as the nomenclature type.

Lyngbyabellins K-N from Two Palmyra Atoll Collections of the Marine Cyanobacterium Moorea bouillonii.

Five lipopeptides of the lyngbyabellin structure class, four cyclic (1-3, and 5) and one linear (4), were isolated from the extracts of two collections of filamentous marine cyanobacteria obtained from Palmyra Atoll in the Central Pacific Ocean. Their planar structures and absolute configurations were elucidated by combined spectroscopic and chromatographic analyses as well as chemical synthesis of fragments. In addition to structural features typical of the lyngbyabellins, such as two thiazole rings and a chlorinated 2-methyloctanoate residue, these new compounds possess several unique aspects. Of note, metabolites 2 and 3 possessed rare mono-chlorination on the 3-acyloxy-2-methyloctanoate residue while lyngbyabellin N (5) had an unusual N,N-dimethylvaline terminus. Lyngbyabellin N also possessed a leucine statine residue, and showed strong cytotoxic activity against HCT116 colon cancer cell line (IC50 = 40.9 ± 3.3 nM).

Cymatherelactone and cymatherols A-C, polycyclic oxylipins from the marine brown alga Cymathere triplicata.

An investigation of the oxylipin chemistry of the temperate brown alga Cymathere triplicata led to the isolation of several secondary metabolites, cymatherelactone (1) and cymatherols A-C (2-4), the latter as their methyl ester derivatives (5-7), which contained cyclopentyl, cyclopropyl, epoxide and lactone rings. Their structures were elucidated using a combination of spectroscopic techniques and synthetic chemistry. Cymatherelactone (1), as well as R- and S-Mosher's esters of its seco acid, exhibited moderate sodium channel blocking activity.

Phylogeny-guided isolation of ethyl tumonoate A from the marine cyanobacterium cf. Oscillatoria margaritifera.

The evolutionary relationships of cyanobacteria, as inferred by their SSU (16S) rRNA genes, were used as predictors of their potential to produce varied secondary metabolites. The evolutionary relatedness in geographically distant cyanobacterial specimens was then used as a guide for the detection and isolation of new variations of predicted molecules. This phylogeny-guided isolation approach for new secondary metabolites was tested in its capacity to direct the search for specific classes of new natural products from Curaçao marine cyanobacteria. As a result, we discovered ethyl tumonoate A (1), a new tumonoic acid derivative with anti-inflammatory activity and inhibitory activity of calcium oscillations in neocortical neurons.

Cytotoxic veraguamides, alkynyl bromide-containing cyclic depsipeptides from the marine cyanobacterium cf. Oscillatoria margaritifera.

A family of cancer cell cytotoxic cyclodepsipeptides, veraguamides A-C (1-3) and H-L (4-8), were isolated from a collection of cf. Oscillatoria margaritifera obtained from the Coiba National Park, Panama, as part of the Panama International Cooperative Biodiversity Group program. The planar structure of veraguamide A (1) was deduced by 2D NMR spectroscopy and mass spectrometry, whereas the structures of 2-8 were mainly determined by a combination of 1H NMR and MS2/MS3 techniques. These new compounds are analogous to the mollusk-derived kulomo'opunalide natural products, with two of the veraguamides (C and H) containing the same terminal alkyne moiety. However, four veraguamides, A, B, K, and L, also feature an alkynyl bromide, a functionality that has been previously observed in only one other marine natural product, jamaicamide A. Veraguamide A showed potent cytotoxicity to the H-460 human lung cancer cell line (LD50=141 nM).

Temporal dynamics of natural product biosynthesis in marine cyanobacteria.

Sessile marine organisms are prolific sources of biologically active natural products. However, these compounds are often found in highly variable amounts, with the abiotic and biotic factors governing their production remaining poorly understood. We present an approach that permits monitoring of in vivo natural product production and turnover using mass spectrometry and stable isotope ((15)N) feeding with small cultures of various marine strains of the natural product-rich cyanobacterial genus Lyngbya. This temporal comparison of the amount of in vivo (15)N labeling of nitrogen-containing metabolites represents a direct way to discover and evaluate factors influencing natural product biosynthesis, as well as the timing of specific steps in metabolite assembly, and is a strong complement to more traditional in vitro studies. Relative quantification of (15)N labeling allowed the concurrent measurement of turnover rates of multiple natural products from small amounts of biomass. This technique also afforded the production of the neurotoxic jamaicamides to be more carefully studied, including an assessment of how jamaicamide turnover compares with filament growth rate and primary metabolism and provided new insights into the biosynthetic timing of jamaicamide A bromination. This approach should be valuable in determining how environmental factors affect secondary metabolite production, ultimately yielding insight into the energetic balance among growth, primary production, and secondary metabolism, and thus aid in the development of methods to improve compound yields for biomedical or biotechnological applications.

The hoiamides, structurally intriguing neurotoxic lipopeptides from Papua New Guinea marine cyanobacteria.

Two related peptide metabolites, one a cyclic depsipeptide, hoiamide B (2), and the other a linear lipopeptide, hoiamide C (3), were isolated from two different collections of marine cyanobacteria obtained in Papua New Guinea. Their structures were elucidated by combining various techniques in spectroscopy, chromatography, and synthetic chemistry. Both metabolites belong to the unique hoiamide structural class, characterized by possessing an acetate extended and S-adenosyl methionine modified isoleucine unit, a central triheterocyclic system comprised of two alpha-methylated thiazolines and one thiazole, and a highly oxygenated and methylated C-15 polyketide unit. In neocortical neurons, the cyclic depsipeptide 2 stimulated sodium influx and suppressed spontaneous Ca(2+) oscillations with EC(50) values of 3.9 microM and 79.8 nM, respectively, while 3 had no significant effects in these assays.

Evolved diversification of a modular natural product pathway: apratoxins F and G, two cytotoxic cyclic depsipeptides from a Palmyra collection of Lyngbya bouillonii.

A collection of Lyngbya bouillonii from Palmyra Atoll in the Central Pacific, a site several thousand kilometers distant from all previous collections of this chemically prolific species of cyanobacterium, was found to contain two new cancer cell cytotoxins of the apratoxin family. The structures of the new compounds, apratoxins F and G, were determined by 1D and 2D NMR techniques in combination with mass spectrometric methods. Stereochemistry was explored by using chromatographic analyses of the hydrolytically released fragments in combination with NMR and optical rotation comparisons with known members of the apratoxin family. Apratoxins F and G add fresh insights into the SAR of this family because they incorporate an N-methyl alanine residue at a position where all prior apratoxins have possessed a proline unit, yet they retain high potency as cytotoxins to H-460 cancer cells with IC(50) values of 2 and 14 nM, respectively. Additional assays using zone inhibition of cancer cells and clonogenic cells give a comparison of the activities of apratoxin F to apratoxin A. Additionally, the clonogenic studies in combination with maximum tolerated dose (MTD) studies provided insights as to dosing schedules that should be used for in vivo studies, and preliminary in vivo evaluation validated the predicted in vivo efficacy for apratoxin A. These new apratoxins are illustrative of a mechanism (the modification of an NRPS adenylation domain specificity pocket) for evolving a biosynthetic pathway so as to diversify the suite of expressed secondary metabolites.

Structure revision and absolute configuration of malhamensilipin A from the freshwater chrysophyte Poterioochromonas malhamensis.

Malhamensilipin A (2), a bioactive chlorosulfolipid initially reported in 1994 from the freshwater alga Poterioochromonas malhamensis, was reinvestigated for its structural and stereochemical features. HRESIMS data revealed that 2 possesses two sulfate groups rather than the one originally reported. A combination of J-based configurational and Mosher's analyses led us to assign its absolute configuration as 11R, 12S, 13S, 14R, 15S, and 16S. Finally, comparison of (1)H and (13)C NMR chemical shifts with synthetic standards confirmed that malhamensilipin A (2) possesses a terminal double bond of E configuration.

Palmyramide A, a cyclic depsipeptide from a Palmyra Atoll collection of the marine cyanobacterium Lyngbya majuscula.

Bioassay-guided fractionation of the extract of a consortium of a marine cyanobacterium and a red alga (Rhodophyta) led to the discovery of a novel compound, palmyramide A, along with the known compounds curacin D and malyngamide C. The planar structure of palmyramide A was determined by one- and two-dimensional NMR studies and mass spectrometry. Palmyramide A is a cyclic depsipeptide that features an unusual arrangement of three amino acids and three hydroxy acids; one of the hydroxy acids is the rare 2,2-dimethyl-3-hydroxyhexanoic acid unit (Dmhha). The absolute configurations of the six residues were determined by Marfey's analysis, chiral HPLC analysis, and GC/MS analysis of the hydrolysate. Morphological and phylogenetic studies revealed the sample to be composed of a Lyngbya majuscula-Centroceras sp. association. MALDI-imaging analysis of the cultured L. majuscula indicated that it was the true producer of this new depsipeptide. Pure palmyramide A showed sodium channel blocking activity in neuro-2a cells and cytotoxic activity in H-460 human lung carcinoma cells.