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A new analogue of gambierone, 44-methylgambierone, was isolated from the benthic dinoflagellate Gambierdiscus australes collected from Raoul Island (Rangitahua/Kermadec Islands). This molecule has been previously reported as maitotoxin-3. The structure of 44-methylgambierone was elucidated using 1D- and 2D-nuclear magnetic resonance spectroscopy and mass spectrometry techniques. The nine-ring polyether backbone (A–I) and functional groups (carbonyl, terminal diol, 1,3-diene and monosulphate) are the same for both compounds with the addition of an olefinic methyl group being the only modification in 44-methylgambierone.

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid (1) and dinophysistoxin-2 (2) from Dinophysis blooms in Spain and Norway. These were isolated and shown to be 24-O-β-d-glucosides of 1 and 2 (4 and 5, respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of 1, 2, dinophysistoxin-1 (3), and a synthetic specimen of 7-deoxy-1 (7), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify 1–5, the 24-O-β-d-glucoside of dinophysistoxin-1 (6), 7, 7-deoxy-2 (8), and 7-deoxy-3 (9) in a range of extracts from Dinophysis blooms, Dinophysis cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand. A range of Prorocentrum lima cultures was also examined by liquid chromatography–high resolution tandem mass spectrometry (LC–HRMS/MS) and was found to contain 1, 3, 7, and 9. However, although 4–6 were not detected in these cultures, low levels of putative glycosides with the same exact masses as 4 and 6 were present. The potential implications of these findings for the toxicology, metabolism, and biosynthesis of the okadaic acid group of marine biotoxins are briefly discussed.

The mushroom cell wall contains polysaccharides that can activate cells of the innate immune system through receptors such as Toll-like receptors (TLR) and dectin-1. In the present study, Pleurotus eryngii polysaccharide fractions containing a 3-O methylated mannogalactan and (1→3)/(1→6)-β-d-glucans were isolated and extensively characterized by 2D NMR and methylation analysis. Traces of a (1→3)-α-d-glucan and a (1→2)-α-d-mannan were also observed. Affinity for TLR2, TLR2-TLR6 and dectin-1 using HEK-cells expressing the relevant receptor genes was tested. PeWN, containing the 3-O methylated mannogalactan, was inactive towards TLR2, whereas fraction PeWB, containing more β-glucan, activated the TLR2-TLR6 heterodimer. Activation of the human β-glucan receptor dectin-1 correlated with the amount of β-glucan in each fraction. Nitric oxide and cytokine supernatant levels of D2SC/1 dendritic cells stimulated with the P. eryngii fractions and interferon-γ were low to moderate. The results indicate that the immunomodulatory activity of water-soluble P. eryngii polysaccharide fractions is modest.

The eye lens is a unique organ as no cells can be replaced throughout life. This makes it decisive that the lens is protected against damaging UV-radiation. An ultraviolet (UV)-absorbing compound of unknown identity is present in the aqueous humor of geese (wild and domestic) and other birds flying at high altitudes. A goose aqueous humor extract, that was believed to contain the UV protective compound which was designated as “compound X”, was fractionated and examined using a variety of spectroscopic techniques including LC–MS and high field one- and two dimensional-NMR methods. A series of compounds were identified but none of them appeared to be the UV protective “compound X”. It may be that the level of the UV protective compound in goose aqueous humor is much less than the compounds identified in our investigation, or it may have been degraded by the isolation and chromatographic purification protocols used in our investigations.

Microcystins (MCs) and nodularins (NODs) exhibit high structural variability, including modifications of the Adda (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid) moiety. Variations include 9-O-desmethylAdda (DMAdda) and 9-O-acetylDMAdda (ADMAdda) which, unless targeted, may go undetected. Therefore, reference standards were prepared of [ADMAdda5]MCs and [DMAdda5]MCs, which were analyzed using multiple approaches. The cross-reactivities of the [DMAdda5]- and [ADMAdda5]MC standards were similar to that of MC-LR when analyzed with a protein phosphatase 2A (PP2A) inhibition assay, but were, Graphical abstract Image 1, Highlights • Toxic ADMAdda/DMAdda microcystins (MCs) exhibit low cross-reactivity with Adda ELISA. • A method for oxidative cleavage was expanded to test for ADMAdda/DMAdda MCs. • Two sites of oxidative cleavage of the Adda/DMAdda/ADMAdda were observed. • Oxidation products were identified and analytically tested to quantitate ‘total MCs’.

Epoxyjanthitrems I&ndash, IV (1&ndash, 4) and epoxyjanthitriol (5) were isolated from seed of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Epichlo&euml, festucae var. lolii. Although structures for epoxyjanthitrems I&ndash, IV have previously been proposed in the literature, this is the first report of a full structural elucidation yielding NMR (Nuclear magnetic resonance) assignments for all five epoxyjanthitrem compounds, and additionally, it is the first isolation of epoxyjanthitriol (5). Epoxyjanthitrem I induced tremors in mice and gave a dose dependent reduction in weight gain and feeding for porina (Wiseana cervinata), a common pasture pest in New Zealand. These data suggest that epoxyjanthitrems are involved in the observed effects of the AR37 endophyte on livestock and insect pests.

Ciguatera fish poisoning (CFP) is prevalent around the tropical and sub-tropical latitudes of the world and impacts many Pacific island communities intrinsically linked to the reef system for sustenance and trade. While the genus Gambierdiscus has been linked with CFP, it is commonly found on tropical reef systems in microalgal assemblages with other genera of toxin-producing, epiphytic and/or benthic dinoflagellates – Amphidinium, Coolia, Fukuyoa, Ostreopsis and Prorocentrum. Identifying a biomarker compound that can be used for the early detection of Gambierdiscus blooms, specifically in a mixed microalgal community, is paramount in enabling the development of management and mitigation strategies. Following on from the recent structural elucidation of 44-methylgambierone, its potential to contribute to CFP intoxication events and applicability as a biomarker compound for Gambierdiscus spp. was investigated. The acute toxicity of this secondary metabolite was determined by intraperitoneal injection using mice, which showed it to be of low toxicity, with an LD50 between 20 and 38 mg kg−1. The production of 44-methylgambierone by 252 marine microalgal isolates consisting of 90 species from 32 genera across seven classes, was assessed by liquid chromatography-tandem mass spectrometry. It was discovered that the production of this secondary metabolite was ubiquitous to the eight Gambierdiscus species tested, however not all isolates of G. carpenteri, and some species/isolates of Coolia and Fukuyoa.

We have investigated the in vitro metabolism of pectenotoxin-2 (PTX-2) using primary hepatocytes from Wistar rats in suspension. Purified PTX-2 was rapidly metabolized. Two major and several minor oxidized PTX-2 metabolites were formed, none of which had retention times corresponding to PTX-1, -11, or −13. Hydrolysis products, such as PTX-2 seco acid, were not observed. Preliminary multi-stage LC-MS analyses indicated that the major hepatic PTX-2 metabolites resulted from the insertion of an oxygen atom at the positions C-19 to C-24, or at C-44. The rapid oxidative metabolism may explain the low oral toxicity of PTXs observed in vivo studies., Highlights • PTX-2 is rapidly metabolized in rat hepatocytes. • Two major and several minor oxidized PTX-2 metabolites were formed. • The results may explain the low oral toxicity of PTXs observed in vivo studies.

In the summer of 2012, over 750 dead and dying birds were observed at the Paul S. Sarbanes Ecosystem Restoration Project at Poplar Island, Maryland, USA (Chesapeake Bay). Clinical signs suggested avian botulism, but an ongoing dense Microcystis bloom was present in an impoundment on the island. Enzyme-linked immunosorbent assay (ELISA) analysis of a water sample indicated 6000 ng mL −1 of microcystins (MCs). LC-UV/MS analysis confirmed the presence of MC-LR and a high concentration of an unknown MC congener ( m/z 1037.5). The unknown MC was purified and confirmed to be [D-Leu 1 ]MC-LR using NMR spectroscopy, LC-HRMS and LC–MS 2 , which slowly converted to [D-Leu 1 ,Glu(OMe) 6 ]MC-LR during storage in MeOH. Lyophilized algal material from the bloom was further characterized using LC-HRMS and LC–MS 2 in combination with chemical derivatizations, and an additional 24 variants were detected, including MCs conjugated to Cys, GSH and γ-GluCys and their corresponding sulfoxides. Mallard ( Anas platyrhynchos ) livers were tested to confirm MC exposure. Two broad-specificity MC ELISAs and LC–MS 2 were used to measure free MCs, while ‘total’ MCs were estimated by both MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) and thiol de-conjugation techniques. Free microcystins in the livers (63–112 ng g −1 ) accounted for 33–41% of total microcystins detected by de-conjugation and MMPB techniques. Free [D-Leu 1 ]MC-LR was quantitated in tissues at 25–67 ng g −1 (LC–MS 2 ). The levels of microcystin varied based on analytical method used, highlighting the need to develop a comprehensive analysis strategy to elucidate the etiology of bird mortality events when microcystin-producing HABs are present.

Tumor heterogeneity is one of the hallmarks of glioblastoma multiforme (GBM). Morphology within a given GBM tumor can be extremely variable where some regions of the tumor have a soft, gel-like structure while other areas are dense and fibrous. Abnormal mechanical stress and tissue stiffening caused by cancer proliferation are believed to affect vascularity by compressing structurally weak blood vessels and restricting the supply of nutrients and oxygen to the tissue. These effects contribute to a hypoxic microenvironment that promotes disease progression and chemoresistance. The genetic and molecular mechanisms that govern tissue stiffness within GBM tumors, however, are largely unknown. Magnetic Resonance Elastography (MRE) is an emerging technique for quantifying tissue stiffness non-invasively. We have evaluated 10 GBM patients by MRE imaging obtained prior to surgical resection. During surgery, 2–7 stereotactically navigated biopsies were collected from locations within the tumor with varying degrees of measured stiffness. Biopsies were processed to extract RNA, proteins, polar metabolites and lipids. Biomolecules were analyzed on relevant -omics platforms (RNA sequencing, MS-proteomics and lipidomics, NMR of polar metabolites). Differential expression and gene set enrichment analysis of patient paired biopsies indicate an overall increase in macrophage infiltration and extracellular matrix re-organization associated with increased tumor stiffness. Among the most highly upregulated genes in stiff tumor tissue were lymphatic endothelial hyaluronic acid receptor 1 (LYVE-1) and macrophage receptor with collagenous structure (MARCO), both of which have been associated with immune cell infiltration and tissue stiffness. Our preliminary findings offer novel insights into tumor morphology in GBM that can be inferred from imaging prior to surgery. This can be used to identify tumor regions with high risk of progression and infiltration, thereby informing and guiding surgical strategy and may ultimately lead to novel treatment strategies.

The previously accepted structure of the marine toxin azaspiracid-3 is revised based upon an original convergent and stereoselective total synthesis of the natural product. The development of a structural revision hypothesis, its testing, and corroboration are reported. Synthetic (6R,10R,13R,14R,16R,17R,19S,20S,21R,24S,25S,28S,30S,32R, 33R,34R,36S,37S,39R)-azaspiracid-3 chromatographically and spectroscopically matched naturally occurring azaspiracid-3, whereas the previously assigned 20R epimer did not.

A convergent and stereoselective total synthesis of the previously assigned structure of azaspiracid-3 has been achieved by a late-stage Nozaki-Hiyama-Kishi coupling to form the C21-C22 bond with the C20 configuration unambiguously established from l-(+)-tartaric acid. Postcoupling steps involved oxidation to an ynone, modified Stryker reduction of the alkyne, global deprotection, and oxidation of the resulting C1 primary alcohol to the carboxylic acid. The synthetic product matched naturally occurring azaspiracid-3 by mass spectrometry, but differed both chromatographically and spectroscopically.

Glutathione (GSH) conjugates of the mycotoxin 4-deoxynivalenol (DON), 1, have been detected in plants by LC-MS, but their identities were not confirmed due to a lack of standards. We have synthesized DON-GSH conjugates in alkaline solution. The major products 2 and 5 were isolated and their structures determined by mass spectrometry and NMR spectroscopy as GSH adducts at C-13 and C-10 (via epoxide and Michael addition, respectively) of 1. Other Michael addition products were also tentatively identified by LC-MS. Concentrations of 2 and 5 were determined by quantitative NMR and are suitable for use as quantitative standards for LC-MS studies of plant and animal metabolism of 1. LC-MS showed that in the presence of human glutathione S-transferases of the alpha and mu classes, the reaction of DON and GSH proceeded with a half-life of 17 h, identical with the rate of the uncatalyzed reaction rate, indicating an absence of catalysis.

Microcystins are potent cyclic heptapeptide toxins found in many freshwater cyanobacteria. Most microcystins contain an α,β-unsaturated amide that can react with thiol-containing amino acids, peptides, and proteins in vivo and in vitro. While soluble conjugates formed from small peptides can be extracted and analyzed directly by LC-MS, microcystins conjugated to proteins are analyzed after oxidative cleavage of their Adda side chains, but information on which microcystin analogues were present is lost. Observations during the development of thiol-derivatization-based LC-MS methods for microcystin analysis indicated that the reaction of thiols with microcystins was reversible. The kinetics of deconjugation was investigated with mercaptoethanol as a model thiol to identify suitable reaction conditions. A range of microcystins conjugated to mercaptoethanol, methanethiol, cysteine, and glutathione were then successfully deconjugated, demonstrating the feasibility of releasing conjugated forms of microcystins for chemical analysis. Reagents for removing the released thiols or for trapping the released microcystins increased the reaction rate. Optimization of methodologies based on this reaction should increase the method's utility for measuring free and conjugated microcystins. The results also indicate that thiol-conjugated microcystins slowly release free microcystins, even at neutral pH, with consequences for assessment of toxin exposure, metabolism, and trophic transfer. A range of other common natural and environmental toxins, such as deoxynivalenol and acrylamide, also contain α,β-unsaturated carbonyl groups and can be expected to behave in a similar manner.

Albatrellus ovinus, the sheep polypore, is a large, dense mushroom being rich in cell wall material. Polysaccharides were isolated by sequential extraction, enzymatic treatment and analyzed with respect to monosaccharide composition, glycosidic linkages by methylation and GC-MS as well as NMR spectroscopy. A fucogalactan composed of an (1 → 6)-α-d-galactan backbone with single α-l-Fucp residues attached at O-2 was identified in the hot water extract obtained after treatment with a protease and size exclusion chromatography. Both the hot water extract and the hot alkali extract contained an (1 → 4)-α-d-glucan whereas β-d-glucans were mainly present in the latter. Structural analysis suggested the presence of two different β-d-glucan backbone structures; a (1 → 6)-linked β-d-glucan with single β-d-Glcp residues at O-3 and also a (1 → 3)-linked β-d-glucan with branches in O-6. In addition there were identified short (1 → 2)-linked β-d-xylan and (1 → 3)-α-d-mannan chains.

Azaspiracids (AZAs) are marine biotoxins produced by the genera Azadinium and Amphidoma, pelagic marine dinoflagellates that may accumulate in shellfish resulting in human illness following consumption. The complexity of these toxins has been well documented, with more than 40 structural variants reported that are produced by dinoflagellates, result from metabolism in shellfish, or are extraction artifacts. Approximately 34 μg of a new AZA with MW 823 Da (AZA26 (3)) was isolated from blue mussels ( Mytilus edulis), and its structure determined by MS and NMR spectroscopy. AZA26, possibly a bioconversion product of AZA5, lacked the C-20-C-21 diol present in all AZAs reported thus far and had a 21,22-olefin and a keto group at C-23. Toxicological assessment of 3 using an in vitro model system based on Jurkat T lymphocyte cells showed the potency to be ∼30-fold lower than that of AZA1. The corresponding 21,22-dehydro-23-oxo-analogue of AZA10 (AZA28) and 21,22-dehydro analogues of AZA3, -4, -5, -6, -9, and -10 (AZA25, -48 (4), -60, -27, -49, and -61, respectively) were also identified by HRMS/MS, periodate cleavage reactivity, conversion from known analogues, and NMR (for 4 that was present in a partially purified sample of AZA7).

The previously accepted structure of the marine toxin azaspiracid-3 is revised based upon an original convergent and stereoselective total synthesis of the natural product. The development of a structural revision hypothesis, its testing, and corroboration are reported. Synthetic (6R,10R,13R,14R,16R,17R,19S,20S,21R,24S,25S,28S,30S,32R, 33R,34R,36S,37S,39R)-azaspiracid-3 chromatographically and spectroscopically matched naturally occurring azaspiracid-3, whereas the previously assigned 20R epimer did not.

A convergent and stereoselective total synthesis of the previously assigned structure of azaspiracid-3 has been achieved by a late-stage Nozaki-Hiyama-Kishi coupling to form the C21-C22 bond with the C20 configuration unambiguously established from l-(+)-tartaric acid. Postcoupling steps involved oxidation to an ynone, modified Stryker reduction of the alkyne, global deprotection, and oxidation of the resulting C1 primary alcohol to the carboxylic acid. The synthetic product matched naturally occurring azaspiracid-3 by mass spectrometry, but differed both chromatographically and spectroscopically.

Two new hydrophobic microcystins, microcystin-FA ( 1 ) and microcystin-WA ( 2 ), were isolated from the cyanobacterium Microcystis sp. (CAWBG11). The structures were deduced using one- and two-dimensional nuclear magnetic resonance spectroscopy and tandem mass spectrometry. The absolute stereochemistry of the amino acid residues in 1 and 2 was determined using the Advanced Marfey's method.

Microcystins are cyclic heptapeptides from cyanobacteria which are responsible for poisonings of livestock and humans. Cyanobacteria also produce a range of peptides and other compounds that can result in complex chromatograms when samples are analysed by LC-MS. Thiol derivatization of the α,β-unsaturated amide present in most microcystins was recently shown to simplify analysis of LC-MS chromatograms of a Microcystis culture, making it easier to identify peaks corresponding to microcystins in complex mixtures. This method was applied to analysis of extracts taken from a natural cyanobacteria bloom in Mwanza Gulf, Lake Victoria, Tanzania, in 2010, revealing the presence of numerous putative microcystin analogues in the sample. Results were verified using LC-MS², LC-MS/MS with precursor-ion scanning, and LC-HRMS, leading to identification of 8 major and 17 minor microcystins in the sample, including analogues of microcystin-RY, -RL and -RA. Microcystin-YR (2), -RR (3), and -RY (9) were isolated from bloom material from Lake Victoria, and the structure of 9 was confirmed by NMR spectroscopic analysis and NMR spectral comparison with 2 and 3. Confirmation of the structure of MC-RY (9) facilitated detailed analysis of its MS² spectrum, thereby supporting the structures of related analogues tentatively established on the basis of MS analyses.

Microcystins are cyclic peptides produced by cyanobacteria, which can be harmful to humans and animals when ingested. Eight of the (more than) 90 microcystin variants presently characterized, contain the amino acid tryptophan. The well-researched oxidation products of tryptophan; kynurenine, oxindolylalanine, and N-formylkynurenine, have been previously identified in intact polypeptides but microcystin congeners containing oxidized tryptophan moieties have not been reported. Liquid chromatography-tandem mass spectrometric analysis of an extract of Microcystis CAWBG11 led to the tentative identification of two new tryptophan-containing microcystins (MC‑WAba and MC-WL), as well as eight other microcystin analogs containing kynurenine, oxindolylalanine and N‑formylkynurenine (Nfk). Investigation of one of these congeners (MC‑NfkA) by nuclear magnetic resonance spectroscopy was used to verify the presence of Nfk in the microcystin. Liquid chromatography-mass spectrometry analysis of a tryptophan oxidation experiment demonstrated that tryptophan-containing microcystins could be converted into oxidized tryptophan analogs and that low levels of oxidized tryptophan congeners were present intracellularly in CAWBG11. MC-NfkR and MC-LNfk were detected in standards of MC-WR and MC-LW, indicating that care during storage of tryptophan-containing microcystins is required.

Conjugation with the biologically relevant thiol glutathione is one of the metabolic pathways for the mycotoxin deoxynivalenol (DON) in wheat. The occurrence of putative DON–cysteine conjugates has also been shown in wheat, likely in part as a result of degradation of the glutathione conjugates. It was reported that thiols react in vitro with DON at two positions: reversibly at C-10 of the α,β-unsaturated ketone and irreversibly at C-13 of the epoxy group. We synthesized pure DON–cysteine adducts and made analytical standards using quantitative NMR experiments. Compounds were characterized using NMR and LC–HRMS/MS and tested in vitro for toxicity. Cysteine conjugates were much less toxic than DON at the same concentration, and LC–HRMS analysis demonstrated that there was no detectable metabolism of the conjugates in human monocytes or human macrophages.

A simple, yet robust analytical method was developed to detect and quantify three sulfonamides (SA), namely sulfamethoxazole (SMO), sulfachloropyridazine (SCP), and sulfamethazine (SM), and a macrolide tylosin (TT) in aqueous (calcium chloride and leachate solutions) and solid (agricultural soils) matrices using high performance liquid chromatography and ultra violet detection at 290 nm (TT) and 275 nm (SA) respectively. Chromatography was performed using a Phenomenex Onyx Monolithic C(18) column for TT and a C(18) Luna column for sulfonamides as single analytes eluted isocratically with a mobile phase consisting of acetonitrile: trifluoroacetic acid: tetrahydrofuran in the ratio 22.5:68:9.5 for TT, 40:55:5 for SMO, 32:63:5 for SCP and 31:64:5 for SM (v/v) at 1.0 mL min(-1) and an injection volume of 20 μL. A gradient method to detect all three sulfonamides in a single run was also developed. The soil residue analysis consisted of extraction with dichloromethane and pre-concentration steps as the aqueous phase was measured directly. The limits of detection at an S/N (signal: noise) ratio of 3 were 20.0 μg L(-1) and 50 μg L(-1) for all sulfonamides and tylosin respectively. The average recoveries for all sulfonamides and tylosin in aqueous matrices ranged from 95 to 105% across the six concentrations investigated. Recoveries from the soils were slightly lower for sulfonamides and tylosin. The isocratic method was used to determine the sorption and degradation of sulfonamides in soils, while the gradient method was used to determine degradation kinetics and leachate concentrations in soils and aqueous systems.

Microcystins are a group of cyclic heptapeptides originating from cyanobacteria. Cyanobacteria also produce a range of peptides and other compounds that can result in complex chromatograms when samples are analyzed by LC-MS. Derivatization with appropriate thiols (e.g., mercaptoethanol) of the olefin in the α,β-unsaturated amide present in most microcystins was shown to simplify analysis of LC-MS chromatograms of sample extracts, making it much easier to identify peaks corresponding to candidate microcystins. Furthermore, interpretation of MS(2) spectra was facilitated by addition of the mass associated with the thiol to the α,β-unsaturated amide of microcystins. Cyanotoxins containing Mdha or Dha reacted readily with thiols, whereas Mser, Ser, Mdhb, and thiol-derivatives of Mdha or Dha did not react under the conditions used. This approach therefore provides a convenient LC-MS method to obtain evidence for the presence of Mdha or Dha and can likely be used to differentiate between the isobaric amino acids Mdha and Dhb in candidate cyanotoxin peaks. When O-(2-mercaptoethyl)-O'-methyl-hexa(ethylene glycol) (MEMHEG) (M(w)t. 356) was used as the thiol, the resulting derivatives eluted in an LC-MS mass window that was largely free of interferences. This approach simplifies detection of candidate microcystin analogues even in the presence of complex mixtures of coeluting components. The method was used for qualitative analysis of a Microcystis aeruginosa culture from Lake Naivasha, Kenya, and the results were verified using precursor-ion scanning and high-resolution mass spectrometry.

Cyanolichens are usually stated to be bipartite (mycobiont plus cyanobacterial photobiont). Analyses revealed green algal carbohydrates in supposedly cyanobacterial lichens (in the genera Pseudocyphellaria, Sticta and Peltigera). Investigations were carried out to determine if both cyanobacteria and green algae were present in these lichens and, if so, what were their roles.The types of photobiont present were determined by light and fluorescence microscopy. Small carbohydrates were analysed to detect the presence of green algal metabolites. Thalli were treated with selected strengths of Zn(2+) solutions that stop cyanobacterial but not green algal photosynthesis. CO(2) exchange was measured before and after treatment to determine the contribution of each photobiont to total thallus photosynthesis. Heterocyst frequencies were determined to clarify whether the cyanobacteria were modified for increased nitrogen fixation (high heterocyst frequencies) or were normal, vegetative cells.Several cyanobacterial lichens had green algae present in the photosynthetic layer of the thallus. The presence of the green algal transfer carbohydrate (ribitol) and the incomplete inhibition of thallus photosynthesis upon treatment with Zn(2+) solutions showed that both photobionts contributed to the photosynthesis of the lichen thallus. Low heterocyst frequencies showed that, despite the presence of adjacent green algae, the cyanobacteria were not altered to increase nitrogen fixation.These cyanobacterial lichens are a tripartite lichen symbiont combination in which the mycobiont has two primarily photosynthetic photobionts, 'co-primary photobionts', a cyanobacterium (dominant) and a green alga. This demonstrates high flexibility in photobiont choice by the mycobiont in the Peltigerales. Overall thallus appearance does not change whether one or two photobionts are present in the cyanobacterial thallus. This suggests that, if there is a photobiont effect on thallus structure, it is not specific to one or the other photobiont.

Orchard soils can contain elevated concentrations of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p′-DDE), 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (p,p′-DDT), and heavy metals as a result of historical agrichemical applications. The bioavailability of p,p′-DDE, p,p′-DDT, As, Cd, Cu, and Pb from five field-aged New Zealand orchards and three grazing soils was assessed by using a 28-d bioassay with Aporrectodea caliginosa and chemical assays. Significant relationships were found between total soil and earthworm tissue concentrations for p,p′-DDE (p

The reactions of cis -[PtCl 2 L 2 ] [L = PPh 3 , PMe 2 Ph or L 2 = Ph 2 P(CH 2 ) 2 PPh 2 (dppe)] with endo -8-camphanylphosphonic acid (CamPO 3 H 2 ) and Ag 2 O in refluxing dichloromethane gave platinum(II) phosphonate complexes [Pt(O 3 PCam)L 2 ]. The X-ray crystal structure of [Pt(O 3 PCam)(PPh 3 ) 2 ]·2CHCl 3 shows that the bulky camphanyl group, rather than being directed away from the platinum, is instead directed into a pocket formed by the Pt and the two PPh 3 ligands. This allows the O 3 P–CH 2 group to have a preferred staggered conformation. The complexes were studied in detail by NMR spectroscopy, which demonstrates non-fluxional behaviour for the sterically bulky PPh 3 and dppe derivatives, which contain inequivalent phosphine ligands in their 31 P NMR spectra. These findings are backed up by theoretical calculations on the PPh 3 and PPhMe 2 derivatives, which show, respectively, high and low energy barriers to rotation of the camphanyl group in the PPh 3 and PPhMe 2 complexes. The X-ray crystal structure of CamPO 3 H 2 is also reported, and consists of hydrogen-bonded hexameric aggregates, which assemble to form a columnar structure containing hydrophilic phosphonic acid channels surrounded by a sheath of bulky, hydrophobic camphanyl groups.

Alkylation of [Pt2(µ-S)2(PPh3)4] with 2,4-dinitrophenylhydrazone-functionalized alkylating agents XC6H4C{=NNHC6H3(NO2)2}CH2Br (X = H, Ph) gives monoalkylated cations [Pt2(µ-S){µ-SCH2C{=NNHC6H3(NO2)2}C6H4X}(PPh3)4]+. An X-ray diffraction study on [Pt2(µ-S){µ-SCH2C{=NNHC6H3(NO2)2}Ph}(PPh3)4]BPh4 shows the crystal to be the Z isomer, with the phenyl ring and NHC6H3(NO2)2 groups mutually trans. 1H- and 31P{1H} NMR spectroscopic methods indicate a mixture of Z (major) and E (minor) isomers in solution, which slowly convert mainly to the E isomer. Reaction of [Pt2(µ-S)2(PPh3)4] with the dinitrophenylhydrazone of chloroacetone [ClCH2C{=NNH(C6H3(NO2)2}Me] and NaBPh4 gives [Pt2(µ-S){µ-SCH2C{=NNHC6H3(NO2)2}Me}(PPh3)4]BPh4, which exists as a single (E) isomer.

Pinnatoxin H was isolated from a culture of the dinoflagellate Vulcanodinium rugosum isolated from the South China Sea. The structure of pinnatoxin H was elucidated by LC–MS/MS and NMR spectroscopy. It was found to have the same macrocyclic structure and substituents as pinnatoxins D, E and F, but the side chain on the cyclohexenyl ring was an ethenyl group, as found in pinnatoxin G and portimine. The observation that this strain of V. rugosum produced only pinnatoxin H and portimine is consistent with previous findings that the profile of pinnatoxins can vary significantly among strains. The acute toxicity of pinnatoxin H to mice was 67 μg/kg (intraperitoneal) and 163 μg/kg (gavage).

Pinnatoxins and pteriatoxins are a group of cyclic imine toxins that have hitherto only been isolated from Japanese shellfish. As with other cyclic imine shellfish toxins, pinnatoxins cause rapid death in the mouse bioassay for lipophilic shellfish toxins, but there is no evidence directly linking these compounds to human illness. We have identified the known pinnatoxins A (1) and D (6), and the novel pinnatoxins E (7), F (8) and G (5), in a range of shellfish and environmental samples from Australia and New Zealand using LC-MS. After isolation from the digestive glands of Pacific oysters, the structures of the novel pinnatoxins were determined by mass spectrometry and NMR spectroscopy, and their LD(50) values were evaluated by ip administration to mice. Examination of the toxin structures, together with analysis of environmental samples, suggests that pinnatoxins F and G are produced separately in different dinoflagellates. Furthermore, it appears probable that pinnatoxin F (8) is the progenitor of pinnatoxins D (6) and E (7), and that pinnatoxin G (6) is the progenitor of both pinnatoxins A-C (1 and 2) and pteriatoxins A-C (3 and 4), via metabolic and hydrolytic transformations in shellfish.

Brevetoxins (BTXs) are a class of cyclic polyether toxins produced by the dinoflagellate Karenia brevis. These substances are subject to extensive conjugative metabolism in shellfish. BTX-B forms a conjugate with cysteine and is oxidized and reduced to yield BTX-B2, which is further modified by fatty acid addition via cysteine amide linkage to give biologically active brevetoxin metabolites. In this study, we evaluated the commonly used in vitro (ELISA, radioimmunoassay, receptor binding assay and N2A cytotoxicity assay) and in vivo mouse brevetoxin bioassays for the detection of the brevetoxin fatty acid conjugate N-palmitoylBTX-B2, and compared the results to those for dihydroBTX-B and BTX-B2. The receptor binding assay for N-palmitoylBTX-B2 showed comparable sensitivity to that for dihydroBTX-B, and an 11-fold higher sensitivity than for BTX-B2. Although the ELISA showed similarly high sensitivity to dihydroBTX-B and BTX-B2, with EC50 values of ca. 0.26 ng/ml, it was 23 times less sensitive to N-palmitoylBTX-B2. On the other hand, the N2A cytotoxicity assay was highly sensitive to N-palmitoylBTX-B2, with an EC50 of 0.15 ng/ml, but was 12- and 40-fold less sensitive to dihydroBTX-B and BTX-B2, respectively. The relative sensitivity of the N2A cytotoxicity assay for each of these metabolites paralleled that of the mouse bioassay (relative LD50 values 1:20:30 for N-palmitoylBTX-B2:dihydroBTX-B:BTX-B2). We conclude that the most sensitive bioassay for dihydroBTX-B and BTX-B2 is the ELISA, whereas the N2A cytotoxicity assay is most sensitive for N-palmitoylBTX-B2.

Penitrem A is a potent neurotoxin produced by several species in the genus Penicillium, which primarily affects the central nervous system. The toxin has several effects on neurotransmitter release, both at the central and peripheral level, as well as on ion channels. We have evaluated the hepatic metabolism of penitrem A by rat hepatocytes and rat-liver microsomes in vitro. In addition, we have conducted an in vivo study in mice and determined metabolites in several organs. According to our results, penitrem A is extensively metabolized in the liver to at least five metabolites more hydrophilic than the parent compound.

Brevetoxins are neurotoxins associated with blooms of marine algae such as Karenia brevis and can accumulate in the marine food chain, causing intoxication of marine animals and people consuming seafood. Brevetoxin-B2 ( 5) is a toxic metabolite produced in shellfish exposed to algae that contain brevetoxin-B ( 1). S-Desoxybrevetoxin-B2 ( 4) has been proposed as a cometabolite produced during this transformation, and while LC-MS analyses suggest its presence in shellfish, it has not yet been isolated and characterized. Studies on these materials are severely constrained by the difficulty of obtaining and purifying them from natural sources. We have developed a convenient one-pot conversion of commercially available brevetoxin-B ( 1) into S-desoxybrevetoxin-B2 ( 4), and a simple method for converting 4 into brevetoxin-B2 ( 5). Full NMR and mass-spectral characterization of 4 and 5 confirmed their structures and showed that the ratio of diastereoisomers in the synthetic 4 and 5 was similar to that observed in naturally contaminated shellfish. The LD 50 values for 4, 5, and dihydrobrevetoxin-B ( 6) by ip injection in mice were 211, 400, and 250 microg/kg, respectively. The methodology for synthesis of brevetoxin metabolites should greatly facilitate toxicological, biochemical and immunochemical studies of these substances, as well as the production of analytical standards.

Marine algal toxins of the okadaic acid group can occur as fatty acid esters in blue mussels, and are commonly determined indirectly by transformation to their parent toxins by alkaline hydrolysis. Some data are available regarding the identity of the fatty acid esters, mainly of palmitic acid (16:0) derivatives of okadaic acid (OA), dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2). Other fatty acid derivatives have been described, but with limited mass spectral data. In this paper, the mass spectral characterization of the [MH]− and [M+Na]+ ions of 16 fatty acid derivatives of each of OA, DTX1 and DTX2 is presented. The characteristic fragmentation of [M+Na]+ ions of OA analogues provided a useful tool for identifying these, and has not been described previously. In addition, a set of negative ion multiple reaction monitoring (MRM) methods was developed for direct determination of 16 fatty acid esters of OA, 16 fatty acid esters of DTX1 and 16 fatty acid esters of DTX2 in shellfish extracts. The MRM methods were employed to study the profiles of fatty acid esters of OA analogues in blue mussels and to compare these with fatty acid ester profiles reported for other groups of marine algal toxins. Copyright © 2008 John Wiley & Sons, Ltd.

Yucca schidigera juice in doses of 1.5 g (63 mg sapogenin) and 3.0 g (126 mg sapogenin) per kg live weight was administered intraruminally to 30 lambs for 21 days to investigate whether the saponins in Y. schidigera were toxic to lambs and whether they could cause hepatogenous photosensitisation. Twelve lambs died or had to be euthanised. The main pathological findings in the diseased lambs were acute tubular necrosis in the kidneys, dehydration and watery content in the gastrointestinal tract. Fifteen lambs were euthanised at the end of the study, and the main pathological findings in dosed animals were accumulation of homogeneous pale PAS-positive material in the hepatocytes. There was a rise in serum creatinine and urea concentrations in the lambs with renal lesions the day before they died. Major Y. schidigera-related saponins were found in the liver and kidney samples from all lambs that were dosed with Y. schidigera juice. The results of the present study demonstrate that un-hydrolysed saponins can be absorbed from the gastrointestinal tract. The possible role of saponins in causing nephrotoxicity is discussed.

Yessotoxins from a large-scale culture (226 L) of Protoceratium reticulatum strain CAWD129 were harvested by filtration followed by solid-phase extraction. The extract was purified by column chromatography over basic alumina and reverse-phase flash chromatography to afford pure yessotoxin (193 mg). Isolation of yessotoxin was greatly facilitated by selection of a strain which did not produce analogues that interfered with yessotoxin isolation. In addition to yessotoxin, numerous minor yessotoxins were detected by LC-MS in other fractions. From one of these, an early eluting minor analogue with the same molecular weight as yessotoxin and a similar mass spectrometric fragmentation pattern was isolated. This analogue was identified by NMR and mass spectrometry as a novel yessotoxin analogue containing a furan ring in the side chain. This finding reveals biosynthetic flexibility of the yessotoxin pathway in P. reticulatum and confirms earlier findings of production of many minor yessotoxin analogues by this alga. Production of these analogues appeared to be a constitutive trait of P. reticulatum CAWD129.

Conjugation of deoxynivalenol (DON) with sulfur compounds is recognized as a significant reaction pathway, and putative DON–glutathione (DON–GSH) conjugates have been reported in planta. To understand and control the reaction of trichothecenes with biologically important thiols, we studied the reaction of DON, T-2 tetraol, and de-epoxy-DON with a range of model thiols. Reaction conditions were optimized for DON with 2-mercaptoethanol. Major reaction products were identified using HRMS and NMR spectroscopy. The results indicate that thiols react reversibly with the double bond (Michael addition) and irreversibly with the epoxide group in trichothecenes. These reactions occurred at different rates, and multiple isomers were produced including diconjugated forms. LC-MS analyses indicated that glutathione and cysteine reacted with DON in a similar manner to the model thiols. In contrast to DON, none of the tested mercaptoethanol adducts displayed toxicity in human monocytes or induced pro-inflammatory cytokines in human macrophages.

Pectenotoxins from marine dinoflagellates of the genus Dinophysis are rapidly hydrolyzed by many shellfish to give pectenotoxin-2 seco acid, which isomerizes to 7-epi-pectenotoxin-2 seco acid. Three series of fatty acid esters of pectenotoxin-2 seco acid (PTX-2 seco acid) and 7-epi-PTX-2 seco acid were detected by LC-MS analysis of extracts from blue mussels (Mytilus edulis) from Ireland. The locations of the fatty acid ester linkages were identified by a combination of LC-MSn in positive- and negative-ion modes, LC-MS analysis of the products from reaction of the esters with sodium periodate, and NMR analysis of purified samples of the two most abundant ester derivatives. The 37-O-acyl esters of PTX-2 seco acid were the most abundant, followed by the corresponding 11-O-acyl esters, accompanied by low levels of the 33-O-acyl esters. The most abundant fatty acid esters in the fractionated sample were, in order, the 16:0, 22:6, 14:0, 16:1, 18:4, and 20:5 fatty acids, although a wide array of other PTX-2 seco acid fatty acid esters were also present at low levels.

The complexes [CpCo{P(OR′)2O}3]SnR3 − nCln [R′ = Me, Et; R = Ph, Me] are readily prepared from the corresponding organotin chloride and the sodium salt of the Klaui ligands. The X-ray crystal structures of the full series are reported for R = Ph, n = 0–3, and these show that they are all six-coordinate, including the Ph3Sn derivative which is the first example of a SnC3O3 coordination sphere. 1H, 13C, 31P and 119Sn NMR spectra are reported, and interpreted in terms of significant second-order effects and fluxional processes.

Yessotoxin 32-O-[beta-L-arabinofuranosyl-(5'--1'')-beta-L-arabinofuranoside] (3) was isolated from extracts of Protoceratium reticulatum during a large scale isolation of yessotoxin (1). The structure was characterized by mass spectrometry and NMR spectroscopy. Di-glycoside-3, along with the corresponding mono-glycoside (2) were detected in cultures of P. reticulatum originating from Europe and New Zealand, suggesting that production of arabinosides of 1 is a normal feature of this alga. Formation of multiply charged anions and fragmentation of 3 occurred much more readily than for 1 and 2 under the LC-MS conditions used in this study.

Pectenotoxins (PTXs) accumulate in shellfish feeding on dinoflagellates of the genus Dinophysis, so that humans can be exposed to these toxins through shellfish consumption. Some PTXs are toxic to experimental animals, whereas others are of much lower toxicity. Pectenotoxin-2, the most abundant PTX from most Dinophysis spp., is rapidly metabolized by most shellfish to a mixture of pectenotoxin-2 seco acid (2) and 7-epi-pectenotoxin-2 seco acid (1). A mixture of 1 and 2 was produced during purification of an extract from in vitro enzymatic hydrolysis of pectenotoxin-2. These were separated by preparative HPLC, and the structure of 1 was confirmed by one- and two-dimensional 1H and 13C NMR spectroscopy and LC-MS3 analyses. No toxic changes were recorded in mice injected intraperitoneally with 1 or 2 at a dose of 5000 μg/kg. PTX seco acids are therefore unlikely to be of consequence to human consumers at the concentrations found in contaminated shellfish. Keywords: pectenotoxin; seco acid; PTX-2 seco acid; D...

A solid-phase extract from Protoceratium reticulatum was partitioned between water and butanol and the two fractions purified on an alumina column. Fractionation was monitored by ELISA and LC–MS. Results indicate that while almost all yessotoxin (1) was extracted into butanol, large amounts of yessotoxin analogs remained in the aqueous extract along with lesser amounts in the butanolic extract. NMR analysis of selected fractions from reverse-phase chromatography of the extracts confirmed the presence of yessotoxin analogs, although structure determinations were not possible due to the complexity of the mixtures. Analysis of fractions with LC–MS3 and neutral-loss LC–MS/MS indicated the presence of more than 90 yessotoxin analogs, although structures for most of these have not yet been determined. These analogs provide a mechanism to rationalise the discrepancy between ELISA and LC–MS analyses of algae and shellfish.

Cyclometallated gold(III) complexes derived from 2-benzylpyridine, 2-anilinopyridine [AuCl2(C5H4N-X-C6H4)] [X = CH2 (2-bp) or NH (2-anp)] or 2-p-tolylpyridine (tolpy) [AuCl2(C5H4NC6H3CH3)] react with the sulfones RCH2SO2CH2R (R = CN or COPh) in hot methanol with added trimethylamine base to give new aurathietanedioxide complexes containing the Au(CHRSO2CHR) four-membered ring system. The previously reported cyclo-aurated dimethylbenzylamine (MeO-damp) complex [Au{CH(COPh)SO2CH(COPh)}(Me2NCH2C6H3OMe)] was also synthesised by this method, and found to exist as a mixture of major (trans) and minor (cis) isomers with respect to the orientation of the COPh substituents across the four-membered ring. The reaction of one complex with tBuNC in refluxing dichloromethane results in insertion of the isonitrile into the Au–C bond trans to the phenyl ring, followed by proton transfer. An X-ray crystal structure determination on this product was carried out.

44,55-Dihydroxyyessotoxin (1) was isolated from extracts of Protoceratium reticulatum and identified by analysis of its one- and two-dimensional NMR and mass spectra. In addition, LC–MS methods revealed the presence of compounds tentatively identified as (44-R,S)-44,55-dihydroxy-41a-homoyessotoxin (2) and (44-R,S)-44,55-dihydroxy-9-methyl-41a-homoyessotoxin (3). LC–MS analyses indicate that 1 is a constituent of P. reticulatum in New Zealand and Norway, and it was present in three species of mussels from New Zealand, Norway, and Canada.