A Maldiisotopic Approach to Discover Natural Products: Cryptomaldamide, a Hybrid Tripeptide from the Marine Cyanobacterium Moorea producens.

Genome sequencing of microorganisms has revealed a greatly increased capacity for natural products biosynthesis than was previously recognized from compound isolation efforts alone. Hence, new methods are needed for the discovery and description of this hidden secondary metabolite potential. Here we show that provision of heavy nitrogen 15N-nitrate to marine cyanobacterial cultures followed by single-filament MALDI analysis over a period of days was highly effective in identifying a new natural product with an exceptionally high nitrogen content. The compound, named cryptomaldamide, was subsequently isolated using MS to guide the purification process, and its structure determined by 2D NMR and other spectroscopic and chromatographic methods. Bioinformatic analysis of the draft genome sequence identified a 28.7 kB gene cluster that putatively encodes for cryptomaldamide biosynthesis. Notably, an amidinotransferase is proposed to initiate the biosynthetic process by transferring an amidino group from arginine to serine to produce the first residue to be incorporated by the hybrid NRPS-PKS pathway. The maldiisotopic approach presented here is thus demonstrated to provide an orthogonal method by which to discover novel chemical diversity from Nature.

Biosynthesis-assisted structural elucidation of the bartolosides, chlorinated aromatic glycolipids from cyanobacteria.

The isolation of the bartolosides, unprecedented cyanobacterial glycolipids featuring aliphatic chains with chlorine substituents and C-glycosyl moieties, is reported. Their chlorinated dialkylresorcinol (DAR) core presented a major structural-elucidation challenge. To overcome this, we discovered the bartoloside (brt) biosynthetic gene cluster and linked it to the natural products through in vitro characterization of the DAR-forming ketosynthase and aromatase. Bioinformatic analysis also revealed a novel potential halogenase. Knowledge of the bartoloside biosynthesis constrained the DAR core structure by defining key pathway intermediates, ultimately allowing us to determine the full structures of the bartolosides. This work illustrates the power of genomics to enable the use of biosynthetic information for structure elucidation.

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.

Antitumor activity of hierridin B, a cyanobacterial secondary metabolite found in both filamentous and unicellular marine strains.

Cyanobacteria are widely recognized as a valuable source of bioactive metabolites. The majority of such compounds have been isolated from so-called complex cyanobacteria, such as filamentous or colonial forms, which usually display a larger number of biosynthetic gene clusters in their genomes, when compared to free-living unicellular forms. Nevertheless, picocyanobacteria are also known to have potential to produce bioactive natural products. Here, we report the isolation of hierridin B from the marine picocyanobacterium Cyanobium sp. LEGE 06113. This compound had previously been isolated from the filamentous epiphytic cyanobacterium Phormidium ectocarpi SAG 60.90, and had been shown to possess antiplasmodial activity. A phylogenetic analysis of the 16S rRNA gene from both strains confirmed that these cyanobacteria derive from different evolutionary lineages. We further investigated the biological activity of hierridin B, and tested its cytotoxicity towards a panel of human cancer cell lines; it showed selective cytotoxicity towards HT-29 colon adenocarcinoma cells.

Heterologous production of 4-O-demethylbarbamide, a marine cyanobacterial natural product.

Heterologous expression of the barbamide biosynthetic gene cluster, obtained from the marine cyanobacterium Moorea producens, in the terrestrial actinobacterium Streptomyces venezuelae, resulted in the production of a new barbamide congener 4-O-demethylbarbamide, demonstrating the potential of this approach for investigating the assembly and tailoring of complex marine natural products.

Exploiting adaptive laboratory evolution of Streptomyces clavuligerus for antibiotic discovery and overproduction.

Adaptation is normally viewed as the enemy of the antibiotic discovery and development process because adaptation among pathogens to antibiotic exposure leads to resistance. We present a method here that, in contrast, exploits the power of adaptation among antibiotic producers to accelerate the discovery of antibiotics. A competition-based adaptive laboratory evolution scheme is presented whereby an antibiotic-producing microorganism is competed against a target pathogen and serially passed over time until the producer evolves the ability to synthesize a chemical entity that inhibits growth of the pathogen. When multiple Streptomyces clavuligerus replicates were adaptively evolved against methicillin-resistant Staphylococcus aureus N315 in this manner, a strain emerged that acquired the ability to constitutively produce holomycin. In contrast, no holomycin could be detected from the unevolved wild-type strain. Moreover, genome re-sequencing revealed that the evolved strain had lost pSCL4, a large 1.8 Mbp plasmid, and acquired several single nucleotide polymorphisms in genes that have been shown to affect secondary metabolite biosynthesis. These results demonstrate that competition-based adaptive laboratory evolution can constitute a platform to create mutants that overproduce known antibiotics and possibly to discover new compounds as well.

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.

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.

Cannabinomimetic lipid from a marine cyanobacterium.

NMR-guided fractionation of two independent collections of the marine cyanobacteria Lyngbya majuscula obtained from Papua New Guinea and Oscillatoria sp. collected in Panama led to the isolation of the new lipids serinolamide A (3) and propenediester (4). Their structures were determined by NMR and MS data analysis. Serinolamide A (3) exhibited a moderate agonist effect and selectivity for the CB1 cannabinoid receptor (Ki=1.3 µM, >5-fold) and represents the newest addition to the known cannabinomimetic natural products of marine origin.

Molluscicidal metabolites from an assemblage of Palmyra Atoll cyanobacteria.

Molluscicides can play an important role in the control of schistosomiasis because snails of the genus Biomphalaria act as intermediate hosts for the parasite. Schistosomiasis is one of 13 neglected tropical diseases with high morbidity and mortality that collectively affect one billion of the world's poorest population, mainly in developing countries. Thiopalmyrone (1) and palmyrrolinone (2), metabolites isolated from extracts of a Palmyra Atoll environmental assemblage of two cyanobacteria, cf. Oscillatoria and Hormoscilla spp., represent new and potent molluscicidal chemotypes against Biomphalaria glabrata (LC50=8.3 and 6.0 µM, respectively). A slight enhancement in molluscicidal effect (LC50=5.0 µM) was observed when these two natural products were utilized as an equimolar binary mixture.

Synthesis of phosphatidylinositol 3-kinase (PI3K) inhibitory analogues of the sponge meroterpenoid liphagal.

Analogues of the sponge meroterpenoid liphagal have been synthesized and evaluated for inhibition of PI3Kα and PI3Kγ as part of a program aimed at developing new isoform-selective PI3K inhibitors. One of the analogues, compound 24, with IC50 values of 66 nM against PI3Kα and 1840 nM against PI3Kγ, representing a 27-fold preference for PI3Kα, exhibited enhanced chemical stability and modestly enhanced potency and selectivity compared with the natural product liphagal.

Palmyrolide A, an unusually stabilized neuroactive macrolide from Palmyra Atoll cyanobacteria.

Palmyrolide A (1) is a new neuroactive macrolide isolated from a marine cyanobacterial assemblage composed of Leptolyngbya cf. and Oscillatoria spp. collected from Palmyra Atoll. It features a rare N-methyl enamide and an intriguing t-butyl branch; the latter renders the adjacent lactone ester bond resistant to hydrolysis. Consistent with its significant suppression of calcium influx in cerebrocortical neurons (IC(50) = 3.70 µM), palmyrolide A (1) showed a relatively potent sodium channel blocking activity in neuro-2a cells (IC(50) = 5.2 µM), without appreciable cytotoxicity.

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.

Synergistic allelochemicals from a freshwater cyanobacterium.

The ability of cyanobacteria to produce complex secondary metabolites with potent biological activities has gathered considerable attention due to their potential therapeutic and agrochemical applications. However, the precise physiological or ecological roles played by a majority of these metabolites have remained elusive. Several studies have shown that cyanobacteria are able to interfere with other organisms in their communities through the release of compounds into the surrounding medium, a phenomenon usually referred to as allelopathy. Exudates from the freshwater cyanobacterium Oscillatoria sp. had previously been shown to inhibit the green microalga Chlorella vulgaris. In this study, we observed that maximal allelopathic activity is highest in early growth stages of the cyanobacterium, and this provided sufficient material for isolation and chemical characterization of active compounds that inhibited the growth of C. vulgaris. Using a bioassay-guided approach, we isolated and structurally characterized these metabolites as cyclic peptides containing several unusually modified amino acids that are found both in the cells and in the spent media of Oscillatoria sp. cultures. Strikingly, only the mixture of the two most abundant metabolites in the cells was active toward C. vulgaris. Synergism was also observed in a lung cancer cell cytotoxicity assay. The binary mixture inhibited other phytoplanktonic organisms, supporting a natural function of this synergistic mixture of metabolites as allelochemicals.

A concise enantioselective synthesis of the chlorosulfolipid malhamensilipin A.

The first enantioselective synthesis of a member of the chlorosulfolipid family of natural products is reported. All of the polar substituents of malhamensilipin A are introduced with high stereoselectivity, and the unique (E)-chlorovinyl sulfate is created by a chemo-, regio-, and stereoselective E2 elimination of HCl in a reaction that likely has a counterpart in the biosynthesis of this fascinating natural product.

Cyanolide A, a glycosidic macrolide with potent Molluscicidal activity from the Papua New Guinea cyanobacterium Lyngbya bouillonii.

Over the last 50 years, molluscicides have played a critical role in the control of schistosomiasis transmission. Cyanolide A (2), isolated from extracts of a Papua New Guinea collection of Lyngbya bouillonii, is a new and highly potent molluscicidal agent against the snail vector Biomphalaria glabrata (LC(50) = 1.2 microM). The structure of cyanolide A (2) was elucidated through extensive NMR spectroscopic analyses, yielding a symmetrical dimer that represents the newest addition to the family of glycosidic macrolides from cyanobacteria.

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.

Alotamide A, a novel neuropharmacological agent from the marine cyanobacterium Lyngbya bouillonii.

Alotamide A (1), a structurally intriguing cyclic depsipeptide, was isolated from the marine mat-forming cyanobacterium Lyngbya bouillonii collected in Papua New Guinea. It features three contiguous peptidic residues and an unsaturated heptaketide with oxidations and methylations unlike those found in any other marine cyanobacterial metabolite. Pure alotamide A (1) displays an unusual calcium influx activation profile in murine cerebrocortical neurons with an EC50 of 4.18 microM.