Besarhanamides A and B from the marine cyanobacterium Lyngbya majuscula

Besarhanamides A (1) and B (2) are fatty acid amides purified from the marine cyanobacterium, Lyngbya majuscula, collected from Pulau Hantu, Singapore. The structure determination of these secondary metabolites was carried out using extensive 2D NMR spectral data as well as chemical manipulations including the Marfey's method. In addition, besarhanamide A exhibited moderate toxicity with LD(50) at 13 microM in the brine shrimp toxicity bioassay.     

Two malyngamides from the caribbean cyanobacterium Lyngbya majuscula

Chemical examination of two collections of the Caribbean cyanobacterium, Lyngbya majuscula, led us to isolate several antimicrobial agents. The most active was elemental sulphur, followed by the previously reported fatty acid (−)-7(S)-methoxytetradec-4(E)-enoate, and then by two new amides of this fatty acid, malyngamide D and malyngamide D acetate. The new compounds possess vinyl chloride and α,β-unsaturated enone functional groups. The structures of the new compounds are based on spectroscopic evidence including several 2D NMR techniques at high field as well as chemical interconversion.     

Hantupeptins B and C,cytotoxic cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula

Hantupeptins B (2) and C (3) were isolated, along with the previously reported hantupeptin A (1), from the marine cyanobacterium, Lyngbya majuscula, collected from Pulau Hantu Besar, Singapore. Their structures were elucidated by interpretation of extensive 1D and 2D NMR spectroscopic data. Compounds 2 and 3 are cyclic depsipeptides consisting of five α-amino/hydroxy acid residues, including phenyllactic acid, proline, N-methyl-valine, valine, N-methyl-isoleucine, and a β-hydroxy acid unit with different degrees of unsaturation at the terminal end of each molecule. The absolute configurations of the common amino acids and phenyllactic acid were determined by the advanced Marfey’s and chiral HPLC analyses, respectively. The complete stereochemistry of 3-hydroxy-2-methyl-7-octynoic acid moiety in hantupeptin A was elucidated by a combination of homonuclear J-resolved 2D NMR experiments and by Mosher’s method. Hantupeptins B and C showed moderate in vitro cytotoxicity when tested against MOLT-4 (leukemic) and MCF-7 (breast cancer) cell lines.     

Pharmacology and toxicology of pahayokolide A, a bioactive metabolite from a freshwater species of Lyngbya isolated from the Florida Everglades

The genus of filamentous cyanobacteria, Lyngbya, has been found to be a rich source of bioactive metabolites. However, identification of such compounds from Lyngbya has largely focused on a few marine representatives. Here, we report on the pharmacology and toxicology of pahayokolide A from a freshwater isolate, Lyngbya sp. strain 15-2, from the Florida Everglades. Specifically, we investigated inhibition of microbial representatives and mammalian cell lines, as well as toxicity of the compound to both invertebrate and vertebrate models. Pahayokolide A inhibited representatives of Bacillus, as well as the yeast, Saccharomyces cerevisiae. Interestingly, the compound also inhibited several representatives of green algae that were also isolated from the Everglades. Pahayokolide A was shown to inhibit a number of cancer cell lines over a range of concentrations (IC50 varied from 2.13 to 44.57 microM) depending on the cell-type. When tested against brine shrimp, pahayokolide was only marginally toxic at the highest concentrations tested (1 mg/mL). The compound was, however, acutely toxic to zebrafish embryos (LC50=2.15 microM). Possible biomedical and environmental health aspects of the pahayokolides remain to be investigated; however, the identification of bioactive metabolites such as these demonstrates the potential of the Florida Everglades as source of new toxins and drugs.     

7-methoxy-9-methylhexadeca-4(E),8(E)-dienoic acid from Lyngbya majuscula

A new fatty acid, 7-methoxy-9-methylhexadeca- 4(E),8(E)-dienoic acid, has been isolated from a toxic, deep-water variety of the marine blue-green alga Lyngbya majuscula.     

Porpoisamides A and B, two novel epimeric cyclic depsipeptides from a Florida Keys collection of Lyngbya sp

NMR-guided fractionation of a non-polar extract of a Florida Keys collection of Lyngbya sp. resulted in the isolation of two novel epimeric cyclic depsipeptides, porpoisamides A (1) and B (2). The planar structures of these compounds were determined using NMR spectroscopic techniques. The absolute configurations of amino and hydroxy acid subunits were assigned by enantioselective HPLC analysis. These compounds showed weak cytotoxicity towards HCT-116 colorectal carcinoma and U2OS osteosarcoma cells. The porpoisamides are a unique pair of cyclic depsipeptides that are epimeric at C-2 of the β-amino acid, 3-amino-2-methyloctanoic acid.     

Pitipeptolides C-F, antimycobacterial cyclodepsipeptides from the marine cyanobacterium Lyngbya majuscula from Guam

Pitipeptolides A (1) and B (2) are cyclic depsipeptides isolated from the marine cyanobacterium Lyngbya majuscula from Piti Bomb Holes, Guam. Additional analogues have now been isolated by revisiting larger collections of the same cyanobacterium. The four identified analogues, pitipeptolides C–F (3–6), are the tetrahydro analogue (3), an analogue with a lower degree of methylation (4) as well as two homologues (5 and 6) of pitipeptolide A. Their structures were elucidated using 2D NMR experiments, chiral HPLC analysis and comparison with pitipeptolide A. The identified analogues showed weaker cytotoxic activities compared to the two major parent compounds, pitipeptolides A (1) and B (2), against HT-29 colon adenocarcinoma and MCF7 breast cancer cells. On the other hand, pitipeptolide F (6) was the most potent pitipeptolide in a disc diffusion assay against Mycobacterium tuberculosis. The latter finding suggests that the structure of pitipeptolides could be optimized for selective antibacterial activity.     

Morphological, chemical, and genetic diversity of tropical marine cyanobacteria Lyngbya spp. and Symploca spp. (Oscillatoriales)

Although diverse natural products have been isolated from the benthic, filamentous cyanobacterium Lyngbya majuscula, it is unclear whether this chemical variation can be used to establish taxonomic relationships among disparate collections. We compared morphological characteristics, secondary-metabolite compositions, and partial 16S ribosomal DNA (rDNA) sequences among several collections of L. majuscula Gomont, Lyngbya spp., and Symploca spp. from Guam and the Republic of Palau. The morphological characteristics examined were cell length, cell width, and the presence or absence of a calyptra. Secondary metabolites were analyzed by two-dimensional thin-layer chromatography. Each collection possessed a distinct cellular morphology that readily distinguished Lyngbya spp. from Symploca spp. Each collection yielded a unique chemotype, but common chemical characteristics were shared among four collections of L. majuscula. A phylogeny based on secondary-metabolite composition supported the reciprocal monophyly of Lyngbya and Symploca but yielded a basal polytomy for Lyngbya. Pairwise sequence divergence among species ranged from 10 to 14% across 605 bp of 16S rDNA, while collections of L. majuscula showed 0 to 1.3% divergence. Although the phylogeny of 16S rDNA sequences strongly supported the reciprocal monophyly of Lyngbya and Symploca as well as the monophyly of Lyngbya bouillonii and L. majuscula, genetic divergence was not correlated with chemical and morphological differences. These data suggest that 16S rDNA sequence analyses do not predict chemical variability among Lyngbya species. Other mechanisms, including higher rates of evolution for biosynthetic genes, horizontal gene transfer, and interactions between different genotypes and environmental conditions, may play important roles in generating qualitative and quantitative chemical variation within and among Lyngbya species.     

Steroidal saponins from the aerial parts of Tribulus pentandrus Forssk

Seven new steroidal glycosides named pentandrosides A(1)-G(7) were isolated from the EtOH extract of the aerial parts of Tribulus pentandrus. Pentandrosides A(1)-E(5) possess cholestane aglycones, pentandroside F(6) a furostan-type aglycone and pentandroside G(7) an unusual acyloxypregnane aglycone probably derived from the degradation of a furostan skeleton. Structure elucidation of 1-7 was accomplished through the extensive use of 1D- and 2D NMR experiments including 1H-1H (DQF-COSY, 1D-TOCSY) and 1H-13C (HSQC, HMBC) spectroscopy along with ESIMS and HRESIMS.     

Malyngamide 4, a new lipopeptide from the Red Sea marine cyanobacterium Moorea producens (formerly Lyngbya majuscula)

In our continuing effort to discover new drug leads from Red Sea marine organisms, a sample of the marine cyanobacterium Moorea producens (previously Lyngbya majuscula) was investigated. Bioassay-directed purification of a tumor cell-growth inhibitory fraction of the organic extract of the Red Sea cyanobacterium afforded a new compound, malyngamide 4 (1), together with five previously reported compounds, malyngamide A (2) and B (3), (S)-7-methoxytetradec-4(E)-enoic acid (lyngbic acid, 4), aplysiatoxin (5) and debromoaplysiatoxin (6). Assignment of the planar structures of these compounds was based on extensive analysis of one- and two-dimensional NMR spectra and high-resolution mass spectrometric data. The isolated compounds were evaluated for their inhibitory activity against three cancer cell lines. In addition, the antibacterial activity of the compounds against Mycobacterium tuberculosis H₃₇Rv ATCC 27294 (H₃₇Rv) was evaluated. Lyngbic acid (4) was the most active against M. tuberculosis, while malyngamides 4 (1) and B (3) moderately inhibited the cancer cell lines. The other compounds were deemed inactive at the test concentrations.     

Constituents of Acacia cedilloi and Acacia gaumeri. Revised structure and complete NMR assignments of resinone

The rare lupene derivative named resinone has only been isolated before from Fluorensia resinosa. We now report the isolation of this compound from the bark of the new recently described Acacia cedilloi (Fabaceae), and the revision of its structure to 16beta-hydroxylup-20(29)-en-3-one, based on NMR and MS spectral data. The detailed 1H and 13C NMR assignments of resinone and its acetate achieved by 1D and 2D NMR experiments (including DEPT, COSY, HMQC and HMBC) are reported. In addition, the study of A. cedilloi and A. gaumeri afforded the known related lupenes lupeol and lupenone, the acyclic squalene, the sterols beta-sitosterol, stigmasta-7,22-dien-3beta-ol (spinasterol) and stigmasta-5,22,25-trien-3beta-ol (22-dehydroclerosterol) as well as alpha-tocopherol and beta-carotene.     

Antioxidant potential of C-phycocyanin isolated from cyanobacterial species Lyngbya, Phormidium and Spirulina spp

The antioxidant activity of C-Phycocyanin (C-PC) isolated from three cyanobacterial species Lyngbya (marine), Phormidium (marine) and Spirulina (fresh water) was studied in vitro. The results demonstrate that C-PCs from Lyngbya, Phormidium and Spirulina spp. are able to scavenge peroxyl radicals (determined by crocin bleaching assay) with relative rate constant ratio of 3.13, 1.89 and 1.8, respectively. C-PCs also scavenge hydroxyl radicals (determined by deoxyribose degradation assay) with second order rate constant values of 7.87 x 10(10), 9.58 x 10(10) and 6.42 x 10(10), respectively. Interestingly, Lyngbya C-PC is found to be an effective inhibitor of peroxyl radicals (IC50 6.63 microM), as compared to Spirulina (IC50 12.15 microM) and Phormidium C-PC (IC50 12.74 microM) and is close to uric acid (IC50 2.15 microM). Further, the studies suggest that the covalently-linked tetrapyrrole chromophore phycocyanobilin is involved in the radical scavenging activity of C-PC. The electron spin resonance (ESR) spectra of C-PCs indicate the presence of free radical active sites, which may play an important role in its radical scavenging property. This is the first report on the ESR activity of native C-PCs without perturbations that can cause radical formation.     

Dihydroagarofuran alkaloid and triterpenes from Maytenus heterophylla and Maytenus arbutifolia

The antimicrobially active EtOH extracts of Maytenus heterophylla yielded a new dihydroagarofuran alkaloid,1beta-acetoxy-9alpha-benzoyloxy-2beta,6alpha-dinicotinoyloxy-beta-dihydroagarofuran, together with the known compounds beta-amyrin, maytenfolic acid, 3alpha-hydroxy-2-oxofriedelane-20alpha-carboxylic acid, lup-20(29)-ene-1beta,3beta-diol, (-)-4'-methylepigallocatechin, and (-)-epicatechin. In addition, beta-amyrin, (-)-epicatechin and (-)-4'-methylepigallocatechin were isolated from Maytenus arbutifolia. The structure elucidation of the isolated compounds was based primarily on 1D and 2D NMR analyses, including HMQC, HMBC, and NOESY correlations. Maytenfolic acid showed moderate antimicrobial activity by inhibiting the growth of Candida albicans, Cryptococcus neoformans, Staphylococcus aureus and Pseudomonas aeruginosa.     

Persipeptides A and B, two cyclic peptides from Streptomyces sp. UTMC 1154

Two new N-methylated cyclopeptides, persipeptide A (1) and B (2), have been isolated from Streptomyces sp. UTMC1154. Their structures were established using 1D and 2D NMR experiments. 2D TOCSY experiments were applied to identify the amino acid residues, while HMBC correlations were used to determine their sequence. According to Marfey's method, all amino acids had the l-configuration. The two cyclic peptides had the same ring size and amino acid composition, but differed in their sequence; they did not show activity against the tested bacteria, fungi and algae. Molecular identification experiments placed the strain in the genus Streptomyces closely related to Streptomyces coerulescens DSM40146(T) (99.45%) and Streptomyces varsoviensis DSM40346(T) (99.25%).     

A sesquiterpene acid and flavonoids from Polygonum viscosum

4-Isobutyl-6-methyl-5-oxo-3a,4,5,7a-tetrahydro-1H-inden-13-oic acid (named viscosumic acid) and quercetin 3-O-(6"-feruloyl)-beta-D-galactopyranoside, and the known 3',5-dihydroxy-3,4',5',7-tetramethoxyflavone have been isolated from Polygonum viscosum. The structures of these isolates were determined primarily on the basis of extensive 1D and 2D NMR spectral analyses, notably, 13C PENDANT, COSY45, TOCSY, GOESY, NOESY, HMQC and HMBC.     

The structure of syringomycins A1, E and G

By a combination of 1D and 2D 1H- and 13C-NMR, FAB-MS, and chemical and enzymatic reactions carried out at the milligram level, it has been demonstrated that syringomycin E, the major phytotoxic antibiotic produced by Pseudomonas syringae pv. syringae, is a new lipodepsipeptide. Its amino acid sequence is Ser-Ser-Dab-Dab-Arg-Phe-Dhb-4(Cl)Thr-3(OH)Asp with the beta-carboxy group of the C-terminal residue closing a macrocyclic ring on the OH group of the N-terminal Ser, which in turn is N-acylated by 3-hydroxydodecanoic acid. Syringomycins A1 and G, two other metabolites of the same bacterium, differ from syringomycin E only in their fatty acid moieties corresponding, respectively, to 3-hydroxydecanoic and 3-hydroxytetradecanoic acid.     

Morphological and genetic evidence that the cyanobacterium Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck encompasses at least two species

Dense blooms of the cyanobacterium Lyngbya wollei are increasingly responsible for declining water quality and habitat degradation in numerous springs, rivers, and reservoirs. This research represents the first molecular phylogenetic analysis of L. wollei in comparison with the traditional morphological characterization of this species. Specimens were collected from several springs in Florida and a reservoir in North Carolina. Segments of the small-subunit (SSU) rRNA and nifH genes were PCR amplified, cloned, and sequenced. The phylogenetic analysis of the SSU rRNA gene revealed sequences that fell into three distinct subclusters, each with >97% sequence similarity. These were designated operational taxonomic unit 1 (OTU1), OTU2, and OTU3. Similarly, the nifH sequences fell into three distinct subclusters named S1, S2, and S3. When either bulk samples or individual filaments were analyzed, we recovered OTU1 with S1, OTU2 with S2, and OTU3 with S3. The coherence between the three SSU rRNA gene and nifH subclusters was consistent with genetically distinct strains or species. Cells associated with subclusters OTU3 and S3 were significantly wider and longer than those associated with other subclusters. The combined molecular and morphological data indicate that the species commonly identified as L. wollei in the literature represents two or possibly more species. Springs containing OTU3 and S3 demonstrated lower ion concentrations than other collection sites. Geographical locations of Lyngbya subclusters did not correlate with residual dissolved inorganic nitrogen or phosphorus concentrations. This study emphasizes the need to complement traditional identification with molecular characterization to more definitively detect and characterize harmful cyanobacterial species or strains.     

Emerging Lyngbya wollei toxins: A new high resolution mass spectrometry method to elucidate a potential environmental threat

Mass spectrometric methods for the quantitative and qualitative analyses of algal biotoxins are often complicated by co-eluting compounds that present analytically as interferences. This issue is particularly critical for organic polyamines, where co-eluting materials can suppress the formation of cations during electrospray ionization. Here we present an extraction procedure designed specifically to overcome matrix-derived ion suppression of algal toxins in samples of Lyngbya wollei, a filamentous benthic algae known to produce several saxitoxin analogues. Lyngbya wollei samples were collected from a large, persistent harmful algal bloom in Lake Wateree, SC. Six known Lyngbya wollei-specific toxins (LWT1–6) were successfully resolved and quantified against saxitoxin using hydrophilic interaction liquid chromatography coupled with triple quadrupole and quadrupole time-of-flight mass spectrometry. The parent ions [M²⁺ – H⁺]⁺ were observed for LWTs 1–6 and the [M]²⁺ ion was observed for LWT5. High resolution mass spectra and unique fragmentation ions were obtained for LWTs 1–6. A dilution factor of 50 resulted in a linear calibration of saxitoxin in the algae matrix. Ion suppression was resolved by sample dilution, which led to linear, positive correlations between peak area and mass of the extracted sample (R² > 0.96). Optimized sample extraction method and instrument parameters are presented.     

Chemical constituents of leaves and stem bark of Plumeria obtusa

The continued studies on the constituents of the fresh leaves and stem bark of Plumeria obtusa Linn. have led to the isolation and characterization of four new triterpenoids, dammara-12,20(22)Z-dien-3-one (1), dammara-12,20(22)Z-dien-3beta-ol (2), olean-12-en-3beta,27-diol (3), and 27-hydroxyolean-12-en-3-one (4) and 12 known compounds, which included eight triterpenoids; dammara-3beta,20(S),25-triol (5), urs-12-en-3beta-hydroxy-27-Z-feruloyloxy-28-oic acid (6), 3beta-hydroxyolean-12-en-28-oic acid (7), 3beta,27-dihydroxylupan-29-ene (8), 3beta-hydroxylupan-29-en-28-oic acid (9), 3beta-hydroxyursan-12-en-28-oic acid (11), 3beta-hydroxy-27-p-coumaroyloxy-olea-12-en-28-oic acid (12) and urs-12-en-3-one (15); an iridoid 1alpha-plumieride (10); a cardenolide 3alpha,14beta-dihydroxy-17beta-card-20(22)-enolide (13); a fatty acid ester methyl n-octadecanoate (14) and a steroid 3beta-hydroxy-delta5-stigmastane (16). The new constituents were characterized through spectroscopic studies including 1D (1H and 31C NMR) and 2D (COSY-45, NOESY, J-resolved, HMQC and HMBC) NMR and chemical transformations. This is the first report on the isolation of dammarane triterpenoids from P. obtusa. Compounds 5 and 6 are hitherto unreported from P. obtusa. The known compounds were identified by comparison of their spectral data with those reported in the literature.     

Contemporaneous N(2) Fixation and Oxygenic Photosynthesis in the Nonheterocystous Mat-Forming Cyanobacterium Lyngbya aestuarii

The nonheterocystous filamentous cyanobacterial genus Lyngbya is a widespread and frequently dominant component of marine microbial mats. It is suspected of contributing to relatively high rates of N(2) fixation associated with mats. The ability to contemporaneously conduct O(2)-sensitive N(2) fixation and oxygenic photosynthesis was investigated in Lyngbya aestuarii isolates from a North Carolina intertidal mat. Short-term (<4-h) additions of the photosystem II (O(2) evolution) inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea stimulated light-mediated N(2) fixation (nitrogenase activity), indicating potential inhibition of N(2) fixation by O(2) production. However, some degree of light-mediated N(2) fixation in the absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea was observed. Electron microscopic immunocytochemical localization of nitrogenase, coupled to microautoradiographic studies of CO(2) fixation and cellular deposition of the tetrazolium salt 2,4,5-triphenyltetrazolium chloride, revealed that (i) nitrogenase was widely distributed throughout individual filaments during illuminated and dark periods, (ii) CO(2) fixation was most active in intercalary regions, and (iii) daylight 2,4,5-triphenyltetrazolium chloride reduction (formazan deposition) was most intense in terminal regions. Results suggest lateral partitioning of photosynthesis and N(2) fixation during illumination, with N(2) fixation being confined to terminal regions. During darkness, a larger share of the filament appears capable of N(2) fixation.