Targeted Discovery of Amantamide B, an Ion Channel Modulating Nonapeptide from a South China Sea Oscillatoria Cyanobacterium.

Amantamide B (1) is a new linear nonapeptide analogue of the cyanobacterial natural product amantamide A (2), and both have methyl ester and butanamide termini. These compounds were discovered in this study from the organic extract of a tropical marine filamentous cyanobacterium, Oscillatoria sp., collected around the Paracel Islands in the South China Sea. The use of LC-MS/MS molecular networking for sample prioritization and as an analytical dereplication tool facilitated the targeted isolation of 1 and 2. These molecules were characterized by spectroscopy and spectrometry, and configurational assignments were determined using chemical degradation and chiral-phase HPLC analysis. Compounds 1 and 2 modulated spontaneous calcium oscillations without notable cytotoxicity at 10 µM in short duration in vitro testing on primary cultured neocortical neurons, a model system that evaluates neuronal excitability and/or the potential activity on Ca2+ signaling. Both molecules were also found to be moderately cytotoxic in longer duration bioassays, with in vitro IC50 values of 1-10 µM against CCRF-CEM human T lymphoblastoid cells and U937 human histiocytic lymphoma cells. These formerly undiscovered bioactivities of known compound 2 expand upon its previously reported function as a selective CXCR7 agonist among 168 GPCR targets.

Luquilloamides, Cytotoxic Lipopeptides from a Puerto Rican Collection of the Filamentous Marine Cyanobacterium Oscillatoria sp.

Luquilloamides A-G (1-7) were isolated from a small environmental collection of a marine cyanobacterium found growing on eelgrass (Zostera sp.) near Luquillo, Puerto Rico. Structure elucidation of the luquilloamides was accomplished via detailed NMR and MS analyses, and absolute configurations were determined using a combination of advanced Mosher's method, J-based configuration analysis, semisynthetic fragment analysis derived from ozonolysis, methylation, Baeyer-Villiger oxidation, Mosher's esterification, specific rotations, and ECD data. Except for 2, the luquilloamides share a characteristic tert-butyl-containing polyketide fragment, ß-alanine, and a proposed highly modified polyketide extension. While compound 1 is a linear lipopeptide with two α-methyl branches and a vinyl chloride functionality in the polyketide portion, compounds 4, 6, and 7 possess a cyclohexanone structure with methylation on the α- or ß-positions of the polyketide as well as an acetyl group. Interestingly, the absolute configuration at C-5 and C-6 on the cyclohexanone unit in 7 is opposite to that of 4-6. Compound 3 was revealed to have a tert-butyl-containing polyketide, ß-alanine, and a PKS/NRPS-derived γ-isopropyl pyrrolinone. Compound 2 may be a hydrolysis product of 3. Of the seven new compounds, 1 showed the most potent cytotoxicity to human H-460 lung cancer cells.

Biosynthesis of Cylindrospermopsin in Cyanobacteria: Characterization of CyrJ the Sulfotransferase.

7-Deoxy-desulfo-cylindrospermopsin was purified at small-scale from the supernatant of a culture of the cyanobacterium Oscillatoria sp. PCC 10702. This metabolite was obtained in a pure form using a three-step chromatographic procedure, and its identity was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS quantification showed that this metabolite was excreted in the culture medium of Oscillatoria sp. PCC 10702. Isotopic incorporation studies using [2-13C,15N]glycine, a cylindrospermopsin precursor, and Oscillatoria sp. PCC 10702 cells showed that glycine was incorporated into 7-deoxy-desulfo-cylindrospermopsin, 7-deoxy-cylindrospermopsin, 7-epi-cylindrospermopsin, and cylindrospermopsin. The isotopic incorporation rate was consistent with the following metabolic flux: 7-deoxy-desulfo-cylindrospermopsin → 7-deoxy-cylindrospermopsin → 7-epi-cylindrospermopsin and cylindrospermopsin. We have cloned the cyrJ gene into an expression vector and overproduced the putative sulfotransferase CyrJ in Escherichia coli. The purified protein CyrJ catalyzed, in vitro, the transfer of a sulfonate group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to 7-deoxy-desulfo-cylindrospermopsin to give 7-deoxy-cylindrospermopsin. Kinetic analysis afforded the following apparent constants: KM app. (PAPS) = 0.12 µM, Vmax app. = 20 nM/min, KM app. (7-deoxy-desulfo-cylindrospermopsin) = 0.12 µM, and KI app. (7-deoxy-desulfo-cylindrospermopsin) = 4.1 µM. Preliminary data suggested that CyrJ catalyzed the reaction through a ternary-complex kinetic mechanism. All these data confirmed that CyrJ catalyzed a sulfotransfer during the penultimate step of the biosynthesis of cylindrospermopsin.

Phycocyanin of marine Oscillatoria sp. inhibits lipoxygenase by protein-protein interaction-induced change of active site entry apace: A model for non-specific biofunctions of phycocyanins.

An overview of the biological properties of phycocyanin (PC) amply illustrates that it may not have any specific functional feature towards any system at which it may elicit a specific function, but for the molecular interactions. Nevertheless, based on existing evidences, it is hypothesized that PC has more than one functional target with the interacting systems; therefore, it has diversity of effects. The mechanism of PC action remains elusive of a comprehensive idea. The present investigation focuses on the pro inflammatory enzyme, lipoxygenase (LOX) inhibiting property of PC purified from Oscillatoria sp. Enzyme kinetics studies show that the molecular composite of PC is required for its inhibition shown on LOX. Isothermal titration calorimetric study proves that one molecule of PC interacts with two molecules of LOX. Molecular dynamics simulation study pertaining to PC-LOX interactions shows it to be appropriate as a model to give molecular mechanistic insight into the varied biological properties of PC, demonstrated elsewhere in experimental studies including animal model studies. It explains that the PC-LOX interaction is of a function-freezing, protein-protein interaction in nature. The wide spectrum of properties of PC might be due to its function as a powerful protein hub showing non-specific protein-protein interactions.

Biosynthesis of Anatoxins in Cyanobacteria: Identification of the Carboxy-anatoxins as the Penultimate Biosynthetic Intermediates.

Anatoxin-a, homoanatoxin-a, and dihydroanatoxin-a are potent cyanobacterial neurotoxins. They are biosynthesized in cyanobacteria from proline and acetate by a pathway involving three polyketide synthases. We report the identification of carboxy-anatoxin-a, carboxy-homoanatoxin-a, and carboxy-dihydroanatoxin-a in acidic extracts of Cuspidothrix issatschenkoi CHARLIE-1, Oscillatoria sp. PCC 6506, and Cylindrospermum stagnale PCC 7417, respectively, using liquid chromatography coupled to mass spectrometry. The structure of these carboxy derivatives was confirmed by mass spectrometry and by isotopic incorporation experiments using labeled proline and acetate. Each of these three cyanobacteria only produce one carboxy-anatoxin, suggesting that these metabolites are the product of the hydrolysis by AnaA, the type II thioesterase, of the thioesters bound to AnaG, the last polyketide synthase of the pathway. By measuring the rate of isotopic incorporation of labeled proline into carboxy-homoanatoxin-a and homoanatoxin-a produced by Oscillatoria sp. PCC 6506, we show that carboxy-homoanatoxin-a is the intracellular precursor of homoanatoxin-a, and that homoanatoxin-a is then excreted into the extracellular medium. The transformation of carboxy-homoanatoxin-a into homoanatoxin-a is a very slow two-step process, with accumulation of carboxy-homoanatoxin-a, suggesting that the decarboxylation is spontaneous and not enzymatically catalyzed. However, an unidentified and extracellular catalyst accelerates the decarboxylation when the cell extracts are prepared at neutral pH.

MS/MS-Based Molecular Networking Approach for the Detection of Aplysiatoxin-Related Compounds in Environmental Marine Cyanobacteria.

Certain strains of cyanobacteria produce a wide array of cyanotoxins, such as microcystins, lyngbyatoxins and aplysiatoxins, that are associated with public health issues. In this pilot study, an approach combining LC-MS/MS and molecular networking was employed as a rapid analytical method to detect aplysiatoxins present in four environmental marine cyanobacterial samples collected from intertidal areas in Singapore. Based on 16S-ITS rRNA gene sequences, these filamentous cyanobacterial samples collected from Pulau Hantu were determined as Trichodesmium erythraeum, Oscillatoria sp. PAB-2 and Okeania sp. PNG05-4. Organic extracts were prepared and analyzed on LC-HRMS/MS and Global Natural Product Social Molecular Networking (GNPS) for the presence of aplysiatoxin-related molecules. From the molecular networking, six known compounds, debromoaplysiatoxin (1), anhydrodebromoaplysiatoxin (2), 3-methoxydebromoaplysiatoxin (3), aplysiatoxin (4), oscillatoxin A (5) and 31-noroscillatoxin B (6), as well as potential new analogues, were detected in these samples. In addition, differences and similarities in molecular networking clusters related to the aplysiatoxin molecular family were observed in extracts of Trichodesmium erythraeum collected from two different locations and from different cyanobacterial species found at Pulau Hantu, respectively.

Widespread anatoxin-a detection in benthic cyanobacterial mats throughout a river network.

Benthic algae fuel summer food webs in many sunlit rivers, and are hotspots for primary and secondary production and biogeochemical cycling. Concerningly, riverine benthic algal assemblages can become dominated by toxic cyanobacteria, threatening water quality and public health. In the Eel River in Northern California, over a dozen dog deaths have been attributed to cyanotoxin poisonings since 2000. During the summers of 2013-2015, we documented spatial and temporal patterns of cyanotoxin concentrations in the watershed, showing widespread distribution of anatoxin-a in benthic cyanobacterial mats. Solid phase adsorption toxin tracking (SPATT) samplers were deployed weekly to record dissolved microcystin and anatoxin-a levels at 10 sites throughout the watershed, and 187 Anabaena-dominated or Phormidium-dominated cyanobacterial mat samples were collected from 27 locations to measure intracellular anatoxin-a (ATX) and microcystins (MCY). Anatoxin-a levels were higher than microcystin for both SPATT (mean MCY = 0.8 and ATX = 4.8 ng g resin-1 day-1) and cyanobacterial mat samples (mean MCY = 0.074 and ATX = 1.89 µg g-1 DW). Of the benthic mats sampled, 58.9% had detectable anatoxin-a (max = 70.93 µg g-1 DW), while 37.6% had detectable microcystins (max = 2.29 µg g-1 DW). SPATT cyanotoxin levels peaked in mid-summer in warm mainstem reaches of the watershed. This is one of the first documentations of widespread anatoxin-a occurrence in benthic cyanobacterial mats in a North American watershed.

Characterization of CyrI, the hydroxylase involved in the last step of cylindrospermopsin biosynthesis: Binding studies, site-directed mutagenesis and stereoselectivity.

Cylindrospermopsin, a cytotoxin from cyanobacteria, is biosynthesized by a complex pathway, which involves CyrI, an iron and 2-oxoglutarate dependent hydroxylase that transforms 7-deoxy-cylindrospermopsin into cylindrospermopsin and its epimer, 7-epi-cylindrospermopsin, in the last step. The activity of CyrI from Oscillatoria sp. PCC 7926 depends on Fe(II) (Km = 2.1 µM), and 2-oxoglutarate (Km = 3.2 µM), and is strongly inhibited by 7-deoxy-cylindrospermopsin at concentration higher than 1 µM. Using tryptophan fluorescence, we measured the binding to CyrI of Fe(II) (KD = 0.02 µM) and 2-oxoglutarate (KD = 53 µM and KD = 1.1 µM in the absence or presence of 10 µM Fe(II), respectively). The Oscillatoria sp. PCC 6506 CyrI mutants H157A, D159A, H247A, and R257A were all inactive, and impaired in the binding of Fe(II) or 2-oxoglutarate, confirming the identity of the iron ligands and the role of R257 in the binding of 2-oxoglutarate. We constructed several chimeric enzymes using the Oscillatoria sp. PCC 7926 CyrI protein (stereoselective) and that from Oscillatoria sp. PCC 6506 (not stereoselective) to help understanding the structural factors that influence the stereoselectivity of the hydroxylation. Our data suggest that a predicted α-helix in CyrI (positions 87-108) seems to modulate the stereoselectivity of the reaction.

Microfouling inhibition of human nosocomial pathogen Pseudomonas aeruginosa using marine cyanobacteria.

Pseudomonas aeruginosa is a Gram negative, opportunistic biofilm forming pathogenic bacterium which is developing as a serious problem worldwide. The pathogenicity of P. aeruginosa mainly depends upon biofilm and quorum sensing (QS) mechanism. Targeting biofilm and QS regulated factor will automatically reduce the pathogenicity of P. aeruginosa. Therefore it is compulsory to identify naturally derived biofilm and QS inhibitors against P. aeruginosa. In the present study Oscillatoria subuliformis, a marine cyanobacterium was used against the biofilm and QS of P. aeruginosa. O. subuliformis intracellular methanolic extract (OME) at different concentration (1.5 µg mL-1, 3 µg mL-1 and 5 µg mL-1) was tested against several virulence factors like Extracellular Polymeric Substance (EPS), Cell Surface Hydrophobicity (CSH), elastase, pyocyanin and swarming motility. OME inhibited biofilm (56%), EPS (40%), CSH (56%), pyocyanin (27%), elastase activity and swarming motility in P. aeruginosa without interfering in their survival. Characterization of the OME using FTIR and GCMS confirmed palmitic acid and oleic acid as active compound. CLSM analysis of catheter coated with OME, palmitic and oleic acid proved biofilm inhibition over urinary catheters. The results postulates that oleic and palmitic acid could be an effective attenuator of P. aeruginosa pathogenesis.

Geosmin production and polyphasic characterization of Oscillatoria limosa Agardh ex Gomont isolated from the open canal of a large drinking water system in Tianjin City, China.

Taste and odor (T & O) episodes always cause strong effects on drinking water supply system. Luanhe River diversion into Tianjin City in China is an important drinking water resource. Massive growth of a benthic filamentous cyanobacterium with geosmin production in the open canal caused a strong earthy odor episode in Tianjin. On the basis of the morphological and molecular identification of this cyanobacterium as Oscillatoria limosa Agardh ex Gomont, the genetic basis for geosmin biosynthesis and factors influencing growth and geosmin production of O. limosa CHAB 7000 were studied in this work. A 2268-bp open reading frame, encoding 755 amino acids, was amplified and characterized as the geosmin synthase gene (geo), followed by a cyclic nucleotide-binding protein gene (cnb). Phylogenetic analysis implied that the evolution of the geosmin genes in O. limosa CHAB 7000 might involve a horizontal gene transfer event. Examination on the growth and geosmin production of O. limosa CHAB 7000 at different light intensities showed that the maximum geosmin production was observed at 10µmol photons m-2s-1, while the optimum growth was at 60µmol photons m-2s-1. Under three temperature conditions (15°C, 25°C, and 35°C), the maximum growth and geosmin production were observed at 25°C. Most amounts of geosmin were retained in cells during the growth phase, but high temperature and low light intensity increased the release of geosmin into the medium, implying that O. limosa CHAB 7000 had a high potential harm for the release of geosmin from its cells at these adverse conditions.

Toward Closing the Gap: Quantum Mechanical Calculations and Experimentally Measured Chemical Shifts of a Microcrystalline Lectin.

NMR chemical shifts are exquisitely sensitive probes for conformation and dynamics in molecules and supramolecular assemblies. Although isotropic chemical shifts are easily measured with high accuracy and precision in conventional NMR experiments, they remain challenging to calculate quantum mechanically, particularly in inherently dynamic biological systems. Using a model benchmark protein, the 133-residue agglutinin from Oscillatoria agardhii (OAA), which has been extensively characterized by us previously, we have explored the integration of X-ray crystallography, solution NMR, MAS NMR, and quantum mechanics/molecular mechanics (QM/MM) calculations for analysis of 13Cα and 15NH isotropic chemical shifts. The influence of local interactions, quaternary contacts, and dynamics on the accuracy of calculated chemical shifts is analyzed. Our approach is broadly applicable and expected to be beneficial in chemical shift analysis and chemical-shift-based structure refinement for proteins and protein assemblies.

Dihydroanatoxin-a Is Biosynthesized from Proline in Cylindrospermum stagnale PCC 7417: Isotopic Incorporation Experiments and Mass Spectrometry Analysis.

LC-MS and GC-MS analytical conditions have been developed to detect the cis- and trans-epimers (relative configuration of the carbon bearing the acetyl or propionyl group) of dihydroanatoxin-a and dihydrohomoanatoxin-a, in biological samples. These compounds epimerize under acidic conditions, yielding a major species that was tentatively assigned as the cis-epimer. Cylindrospermum stagnale PCC 7417 was definitively shown to produce dihydroanatoxin-a (1.2 mg/g dried cells). Oscillatoria sp. PCC 9107, Oscillatoria sp. PCC 6506, and C. stagnale PCC 7417, which produce anatoxin-a, homoanatoxin-a, and dihydroanatoxin-a, respectively, were cultivated in the presence of isotopically labeled proline, and the toxins were extracted. Interpretation of the GC-MS electron ionization mass spectra of these labeled anatoxins showed that they are all biosynthesized from proline and that the positions of the labels in these molecules are identical. These data and the fact that the ana cluster of genes is conserved in these cyanobacteria suggest that dihydroanatoxin-a is formed by the reduction of either anatoxin-a or its precursor in a specific step involving AnaK, an F420-dependent oxido-reductase whose gene is found in the ana gene cluster in C. stagnale PCC 7417. This is the first report of a cyanobacterium producing dihydroanatoxin-a, suggesting that other producers are present in the environment.

Synthesis of a molecularly imprinted sorbent for selective solid-phase extraction of ß-N-methylamino-L-alanine.

The aim of the work was to synthesize a molecularly imprinted material for the selective solid-phase extraction (SPE) of ß-N-methylamino-L-alanine (L-2-amino-3-methylpropionic acid; BMAA) from cyanobacterial extracts. BMAA and its structural analogs that can be used as template are small, polar and hydrophilic molecules. These molecules are poorly soluble in organic solvents that are commonly used for the synthesis of acrylic-based polymers. Therefore, a sol gel approach was chosen to carry out the synthesis and the resulting sorbents were evaluated with different extraction procedures in order to determine their ability to selectively retain BMAA. The presence of imprinted cavities in the sorbent was demonstrated by comparing elution profiles obtained by using molecularly imprinted silica (MIS) and non-imprinted silica (NIS) as a control. The molecularly imprinted solid-phase extraction (MISPE) procedure was first developed in a pure medium (acetonitrile) and further optimized for the treatment of cyanobacterial samples. It was characterized by high elution recoveries (89% and 77% respectively in pure and in real media).The repeatability of the extraction procedure in pure medium, in real medium and the reproducibility of MIS synthesis all expressed as RSD values of extraction recovery of BMAA were equal to 3%, 12% and 5%, respectively. A MIS capacity of 0.34 µmol/g was measured. The matrix effects, which affected the quantification of BMAA when employing a mixed mode sorbent, were completely removed by adding a clean-up step of the mixed-mode sorbent extract on the MIS.

Identification of a molecular target of kurahyne, an apoptosis-inducing lipopeptide from marine cyanobacterial assemblages.

In 2014, we isolated kurahyne, an acetylene-containing lipopeptide, from a marine cyanobacterial assemblage of Lyngbya sp. Kurahyne exhibited growth-inhibitory activity against human cancer cells, and induced apoptosis in HeLa cells. However, its mode of action is not yet clear. To elucidate its mode of action, we carried out several cell-based assays, and identified the intracellular target molecule of kurahyne as sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). In addition, we found that kurahyne inhibited the differentiation of macrophages into osteoclasts.

Structural Elucidation of the Bispecificity of A Domains as a Basis for Activating Non-natural Amino Acids.

Many biologically active peptide secondary metabolites of bacteria are produced by modular enzyme complexes, the non-ribosomal peptide synthetases. Substrate selection occurs through an adenylation (A) domain, which activates the cognate amino acid with high fidelity. The recently discovered A domain of an Anabaenopeptin synthetase from Planktothrix agardhii (ApnA A1) is capable of activating two chemically distinct amino acids (Arg and Tyr). Crystal structures of the A domain reveal how both substrates fit into to binding pocket of the enzyme. Analysis of the binding pocket led to the identification of three residues that are critical for substrate recognition. Systematic mutagenesis of these residues created A domains that were monospecific, or changed the substrate specificity to tryptophan. The non-natural amino acid 4-azidophenylalanine is also efficiently activated by a mutant A domain, thus enabling the production of diversified non-ribosomal peptides for bioorthogonal labeling.

(1)H, (13)C and (15)N resonance assignment of the anti-HIV lectin from Oscillatoria agardhii.

Lectins from different sources are known to interfere with HIV infection. The anti-viral activity is mediated by binding to high mannose sugars present on the viral envelope, thereby inhibiting cell entry. The lectin from Oscillatoria agardhii agglutinin (OAA) specifically recognizes a unique substructure of high mannose sugars and exhibits broad anti-HIV activity. Here we report the assignment of backbone and side-chain (1)H, (13)C and (15)N resonances of free OAA.

Total synthesis of largamide B.

Total synthesis of the cyanobacterial metabolite largamide B and the disproval of its originally assigned stereochemistry as well as confirmation of the revised stereochemistry are reported.

Pharmacological studies confirm neurotoxic metabolite(s) produced by the bloom-forming Cylindrospermopsis raciborskii in Hungary.

A rapid cyanobacterial bloom of Cylindrospermopsis raciborskii (3.2 × 10(4) filaments/mL) was detected early November, 2012, in the Fancsika pond (East Hungary). The strong discoloration of water was accompanied by a substantial fish mortality (even dead cats were seen on the site), raising the possibility of some toxic metabolites in the water produced by the bloom-forming cyanobacteria (C. raciborskii). The potential neuronal targets of the toxic substances in the bloom sample were studied on identified neurons (RPas) in the central nervous system of Helix pomatia. The effects of the crude aqueous extracts of the Fancsika bloom sample (FBS) and the laboratory isolate of C. raciborskii from the pond (FLI) were compared with reference samples: C. raciborskii ACT 9505 (isolated in 1995 from Lake Balaton, Hungary), the cylindrospermopsin producer AQS, and the neurotoxin (anatoxin-a, homoanatoxin-a) producer Oscillatoria sp. (PCC 6506) strains. Electrophysiological tests showed that both FBS and FLI samples as well the ACT 9505 extracts modulate the acetylcholine receptors (AChRs) of the neurons, evoking ACh agonist effects, then inhibiting the ACh-evoked neuronal responses. Dose-response data suggested about the same range of toxicity of FBS and FLI samples (EC50 = 0.397 mg/mL and 0.917 mg/mL, respectively) and ACT 9505 extracts (EC50 = 0.734 mg/mL). The extract of the neurotoxin-producing PCC 6506 strain, however, proved to be the strongest inhibitor of the ACh responses on the same neurons (EC50 = 0.073 mg/mL). The presented results demonstrated an anatoxin-a-like cholinergic inhibitory effects of cyanobacterial extracts (both the environmental FBS sample, and the laboratory isolate, FLI) by some (yet unidentified) toxic components in the matrix of secondary metabolites. Previous pharmacological studies of cyanobacterial samples collected in other locations (Balaton, West Hungary) resulted in similar conclusions; therefore, we cannot exclude that this chemotype of C. raciborskii which produce anatoxin-a like neuroactive substances is more widely distributed in this region.

Structural investigation of the antagonist LPS from the cyanobacterium Oscillatoria planktothrix FP1.

Cyanobacteria are aquatic and photosynthetic microorganisms, which contribute up to 30% of the yearly oxygen production on the earth. They have the distinction of being the oldest known fossils, more than 3.5 billion years old, and are one of the largest and most important groups of bacteria on earth. Cyanobacteria are an emerging source of potentially pharmacologically active products and, among these, there are the lipopolysaccharides. Despite their significant and well documented activity, very little is known about the cyanobacteria lipopolysaccharides (LPS) structure. The aim of this work is to investigate the structure of the highly TLR4-antagonist lipopolysaccharide from the cyanobacterium Oscillatoria plankthotrix FP1. The LPS was purified and analysed by means of chemical analysis and 1H and 13C NMR spectroscopy. The LPS was then degraded by Smith degradation, HF and acetic acid hydrolyses. All the obtained products were investigated in detail by chemical analysis, NMR spectroscopy and by mass spectrometry. The LPS consists of a high molecular mass and very complex molecule lacking Kdo and heptose residues, where the polysaccharide chain is mainly constituted by a backbone of 3-substituted α-l-rhamnose units. The core region is rich in galacturonic acid and mannose residues. Moreover a glycolipid portion, similar to Gram-negative lipid A, was identified. This was built up of a non phosphorylated (1'→6) linked glucosamine disaccharide, acylated with 3-hydroxylated fatty acids. In particular 3-hydroxypentadecanoic and 3-hydroxyesadecanoic acids were found, together with esadecanoic and tetradecanoic ones. Finally the presence of a galacturonic acid residue at 6-position of the distal glucosamine in place of the Kdo residue is suggested.

Coibacins A and B: total synthesis and stereochemical revision.

The interface between synthetic organic chemistry and natural products was explored in order to unravel the structure of coibacin A, a metabolite isolated from the marine cyanobacterium cf. Oscillatoria sp. that exhibits selective antileishmanial activity and potent anti-inflammatory properties. Our synthetic plan focused on a convergent strategy that allows rapid access to the desired target by coupling of three key fragments involving E-selective Wittig and modified Julia olefinations. CD measurements and comparative HPLC analyses of the natural product and four synthetic stereoisomers led to determination of its absolute configuration, thus correcting the original assignment at C-5 and unambiguously establishing those at C-16 and C-18. Additionally, we synthesized coibacin B on the basis of the assignment of configuration for coibacin A.