Investigation of 44-methylgambierone reactivity with periodate: Structural reassignment, solvent instability and formation of a furanoid analogue.

Gambierones are sulfated polyethers produced by benthic dinoflagellates in the genera Gambierdiscus, Coolia and Fukuyoa. While relative toxicity data for gambierones suggests they are low compared with ciguatoxin analogues, gambierones have been suggested for use as marker compounds for environmental monitoring programs for the presence of Gambierdiscus in marine waters. The published structure of gambierone and analogues of it, including 44-methylgambierone (44-MeGAM), have been reported to possess 1,2- and 4,5-cis diols, while only the 1,2- diol unit has been shown to undergo periodate oxidation.

Structural Characterization of Maitotoxins Produced by Toxic Species.

Identifying compounds responsible for the observed toxicity of the species is a critical step to ascertaining whether they contribute to ciguatera poisoning. Macroalgae samples were collected during research expeditions to Rarotonga (Cook Islands) and North Meyer Island (Kermadec Islands), from which two new species were characterized, CAWD232 and CAWD242. Previous chemical and toxicological investigations of these species demonstrated that they did not produce the routinely monitored Pacific ciguatoxins nor maitotoxin-1 (MTX-1), yet were highly toxic to mice via intraperitoneal (i.

Identification of 24--β-d-Glycosides and 7-Deoxy-Analogues of Okadaic Acid and Dinophysistoxin-1 and -2 in Extracts from Blooms, and Cultures, and Shellfish in Europe, North America and Australasia.

Two high-mass polar compounds were observed in aqueous side-fractions from the purification of okadaic acid () and dinophysistoxin-2 () from blooms in Spain and Norway. These were isolated and shown to be 24--β-d-glucosides of and ( and , respectively) by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and enzymatic hydrolysis. These, together with standards of , , dinophysistoxin-1 (), and a synthetic specimen of 7-deoxy- (), combined with an understanding of their mass spectrometric fragmentation patterns, were then used to identify -, the 24--β-d-glucoside of dinophysistoxin-1 (), , 7-deoxy- (), and 7-deoxy- () in a range of extracts from blooms, cultures, and contaminated shellfish from Spain, Norway, Ireland, Canada, and New Zealand.

Water-soluble polysaccharides from Pleurotus eryngii fruiting bodies, their activity and affinity for Toll-like receptor 2 and dectin-1.

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.

Searching for a UV-filter in the eyes of high-flying birds.

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.

Analysis of total microcystins and nodularins by oxidative cleavage of their ADMAdda, DMAdda, and Adda moieties.

Microcystins (MCs) and nodularins (NODs) exhibit high structural variability, including modifications of the Adda (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) moiety. Variations include 9--desmethylAdda (DMAdda) and 9--acetylDMAdda (ADMAdda) which, unless targeted, may go undetected. Therefore, reference standards were prepared of [ADMAdda]MCs and [DMAdda]MCs, which were analyzed using multiple approaches.

Identification and Structure Elucidation of Epoxyjanthitrems from Infected with the Endophytic Fungus var. and Determination of the Tremorgenic and Anti-Insect Activity of Epoxyjanthitrem I.

Epoxyjanthitrems I-IV (-) and epoxyjanthitriol () were isolated from seed of perennial ryegrass () infected with the endophytic fungus var. Although structures for epoxyjanthitrems I-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 (). Epoxyjanthitrem I induced tremors in mice and gave a dose dependent reduction in weight gain and feeding for porina (), a common pasture pest in New Zealand.

The role of 44-methylgambierone in ciguatera fish poisoning: Acute toxicity, production by marine microalgae and its potential as a biomarker for Gambierdiscus spp.

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.

In vitro hepatic biotransformation of the algal toxin pectenotoxin-2.

We have investigated the 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.

Analysis of free and metabolized microcystins in samples following a bird mortality event.

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.

Identification and Structure Elucidation of Janthitrems A and D from Penicillium janthinellum and Determination of the Tremorgenic and Anti-Insect Activity of Janthitrems A and B.

New compounds, 11,12-epoxyjanthitrem B (1) and 11,12-epoxyjanthitrem C (4), were isolated from Penicillium janthinellum and given the trivial names janthitrem A and janthitrem D, respectively. The known compounds janthitrem B (2) and janthitrem C (3) were also isolated, and NMR assignments were made for all four compounds. This showed that the previously published NMR assignments for 3 needed considerable revision.

The edible mushroom Albatrellus ovinus contains a α-l-fuco-α-d-galactan, α-d-glucan, a branched (1 → 6)-β-d-glucan and a branched (1 → 3)-β-d-glucan.

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.

Identification of 21,22-Dehydroazaspiracids in Mussels ( Mytilus edulis) and in Vitro Toxicity of Azaspiracid-26.

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.

Total Synthesis of (6R,10R,13R,14R,16R,17R,19S,20R,21R,24S, 25S,28S,30S,32R,33R,34R,36S,37S,39R)-Azaspiracid-3 Reveals Non-Identity with the Natural Product.

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.

Stereochemical Definition of the Natural Product (6R,10R,13R, 14R,16R,17R,19S,20S,21R,24S,25S,28S,30S,32R,33R,34R,36S,37S,39R)-Azaspiracid-3 by Total Synthesis and Comparative Analyses.

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.

Characterization of Deoxynivalenol-Glutathione Conjugates Using Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography-High-Resolution Mass Spectrometry.

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.

Preparation and Characterization of Cysteine Adducts of Deoxynivalenol.

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.

Conjugation of Microcystins with Thiols Is Reversible: Base-Catalyzed Deconjugation for Chemical Analysis.

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.

Nucleophilic Addition of Thiols to Deoxynivalenol.

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.

Structure Elucidation, Relative LC-MS Response and In Vitro Toxicity of Azaspiracids 7-10 Isolated from Mussels (Mytilus edulis).

Azaspiracids (AZAs) are marine biotoxins produced by dinoflagellates that can accumulate in shellfish, which if consumed can lead to poisoning events. AZA7-10, 7-10, were isolated from shellfish and their structures, previously proposed on the basis of only LC-MS/MS data, were confirmed by NMR spectroscopy. Purified AZA4-6, 4-6, and 7-10 were accurately quantitated by qNMR and used to assay cytotoxicity with Jurkat T lymphocyte cells for the first time.

Isolation, structure elucidation, relative LC-MS response, and in vitro toxicity of azaspiracids from the dinoflagellate Azadinium spinosum.

We identified three new azaspiracids (AZAs) with molecular weights of 715, 815, and 829 (AZA33 (3), AZA34 (4), and AZA35, respectively) in mussels, seawater, and Azadinium spinosum culture. Approximately 700 μg of 3 and 250 μg of 4 were isolated from a bulk culture of A. spinosum, and their structures determined by MS and NMR spectroscopy.

Epimers of azaspiracids: Isolation, structural elucidation, relative LC-MS response, and in vitro toxicity of 37-epi-azaspiracid-1.

Since azaspiracid-1 (AZA1) was identified in 1998, the number of AZA analogues has increased to over 30. The development of an LC-MS method using a neutral mobile phase led to the discovery of isomers of AZA1, AZA2, and AZA3, present at ~2-16% of the parent analogues in phytoplankton and shellfish samples. Under acidic mobile phase conditions, isomers and their parents are not separated.

Structural characterization of new microcystins containing tryptophan and oxidized tryptophan residues.

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.

Analysis of the flavonoid component of bioactive New Zealand mānuka (Leptospermum scoparium) honey and the isolation, characterisation and synthesis of an unusual pyrrole.

The flavonoid components of New Zealand mānuka (Leptospermum scoparium) honey have been quantified in a series of 31 honeys of varying non-peroxide antibacterial activity to clarify discrepancies between previous studies reported in the literature. Total flavonoid content was 1.16 mg/100 g honey.

Identification of microcystins in a Lake Victoria cyanobacterial bloom using LC-MS with thiol derivatization.

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.