Further studies on palytoxin. I.

The structures of two degradation products of N-(p-bromobenzoyl) palytoxin were elucidated, and then the sequences of those fragments were determined.     

The conformational features of palytoxin in aqueous solution

Conformational features of palytoxin and acetylated palytoxin were investigated by detailed analyses of NOESY spectra. The conformational differences between palytoxin and acetylated palytoxin may account for the difference in the assembly state of palytoxin, which exists as an associated dimer, and the acetylated derivative, which exists as a monomer in aqueous solution. Two palytoxin units in the dimer may come in contact with each other at the hydrophobic region (C21-40) and the region around two conjugated double bonds (C60-84). The amino group of palytoxin is important for biological activities via Na/K ATPase, but it was not found to be involved in the contact faces of the two palytoxin units. This information should aid in revealing how palytoxin interacts with Na/K ATPase.     

Structures of two oxidation products obtained from palytoxin

The structures of two oxidation products of N-(p-bromobenzoyl)palytoxin were unambiguously determined by means of X-ray crystallographic analysis.     

Molecular shape of palytoxin in aqueous solution

Palytoxin, one of the most toxic non-peptide substances, formed an associated dimer of 5 nm length in aqueous solution.     

Structural studies on palytoxin,a potent coelenterate toxin

The structural elucidation of palytoxin is reported.     

Synthetic studies on palytoxin. Stereocontrolled,practical synthesis of the C.101–C.115 segment

A stereocontrolled and practical synthetic route to the acetal $\text{2a}$, a degradation product of palytoxin, in optically active form is described.     

A truncated palytoxin analogue,palytoxin carboxylic acid,isolated as an insecticidal compound from the red alga, Chondria armata

Palytoxin (PTX) has been widely studied owing to its outstanding toxic properties and complex structural features. It is a great challenge to identify and isolate analogues of PTX from various organisms and reveal their physiological activities. We previously reported the isolation of PTX as an insecticidal compound from the red alga, Chondria armata. In this study, we investigate another active compound from C. armata, which has similar toxicity and chromatographic behavior as PTX. Our detailed structural analysis reveals that this compound is palytoxin carboxylic acid. We characterized its physicochemical properties and conducted extensive NMR studies. This analogue has a structure devoid of the terminus of PTX, such that its presence in the alga implies the role of symbiotic microorganisms in marine ecosystems.     

Antibody characterization and immunoassays for palytoxin using an SPR biosensor

Palytoxin (PLTX), a polyether marine toxin originally isolated from the zoanthid Palythoa toxica, is one of the most toxic non-protein substances known. Fatal poisonings have been linked to ingestion of PLTX-contaminated seafood, and effects in humans have been associated with dermal and inhalational exposure to PLTX containing organisms and waters. Additionally, PLTX co-occurrence with other well-characterized seafood toxins (e.g., ciguatoxins, saxitoxins, tetrodotoxin) has hindered direct associations of PLTX to seafood-borne illnesses. There are currently no validated methods for the quantitative detection of PLTX(s). As such, a well-characterized, robust, specific analytical technique is needed for the detection of PLTX(s) in source organisms, surrounding waters, and clinical samples. Surface plasmon resonance (SPR) biosensors are ideally suited for antibody characterization and quantitative immunoassay detection. Herein, we describe a newly developed SPR assay for PLTX. An anti-mouse substrate was used to characterize the kinetic values for a previously developed monoclonal anti-PLTX. The characterized antibody was then incorporated into a sensitive, rapid, and selective PLTX assay. Buffer type, flow rate, analyte-binding time, and regeneration conditions were optimized for the antibody–PLTX system. Cross-reactivity to potentially co-occurring seafood toxins was also evaluated. We show that this optimized assay is capable of measuring low- to sub-ng/mL PLTX levels in buffer and two seafood matrices (grouper and clam). Preliminary results indicate that this SPR biosensor assay allows for (1) rapid characterization of antibodies and (2) rapid, sensitive PLTX concentration determination in seafood matrices. Method development information contained herein may be broadly applied to future PLTX detection and/or antibody characterization efforts.     

A two-directional synthesis of the C58-C71 fragment of palytoxin

A two directional approach, in which asymmetric dihydroxylation and reduction reactions were used to control absolute configuration, was exploited in the preparation of a C(2)-symmetrical dipyranone. The homotopic dihydropyran (DHP) rings of this precursor were differentiated statistically using by a Prevost reaction and further functionalisation. A second Prevost reaction was used to functionalise the other DHP; global deprotection and peracetylation gave a protected version of the C(58)-C(71) fragment of palytoxin. Methods which might be of value in future synthetic work were developed for the stereoselective functionalisation of THP rings similar to those found in this fragment.     

Model and simulation of Na+/K+ pump phosphorylation in the presence of palytoxin

The ATP hydrolysis reactions responsible for the Na(+)/K(+)-ATPase phosphorylation, according to recent experimental evidences, also occur for the PTX-Na(+)/K(+) pump complex. Moreover, it has been demonstrated that PTX interferes with the enzymes phosphorylation status. However, the reactions involved in the PTX-Na(+)/K(+) pump complex phosphorylation are not very well established yet. This work aims at proposing a reaction model for PTX-Na(+)/K(+) pump complex, with similar structure to the Albers-Post model, to contribute to elucidate the PTX effect over Na(+)/K(+)-ATPase phosphorylation and dephosphorylation. Computational simulations with the proposed model support several hypotheses and also suggest: (i) phosphorylation promotes an increase of the open probability of induced channels; (ii) PTX reduces the Na(+)/K(+) pump phosphorylation rate; (iii) PTX may cause conformational changes to substates where the Na(+)/K(+)-ATPase may not be phosphorylated; (iv) PTX can bind to substates of the two principal states E1 and E2, with highest affinity to phosphorylated enzymes and with ATP bound to its low-affinity sites. The proposed model also allows previewing the behavior of the PTX-pump complex substates for different levels of intracellular ATP concentrations.     

Investigating the potassium interactions with the palytoxin induced channels in Na+/K+ pump

K(+) has been appointed as the main physiological inhibitor of the palytoxin (PTX) effect on the Na(+)/K(+) pump. This toxin acts opening monovalent cationic channels through the Na(+)/K(+) pump. We investigate, by means of computational modeling, the kinetic mechanisms related with K(+) interacting with the complex PTX-Na(+)/K(+) pump. First, a reaction model, with structure similar to Albers-Post model, describing the functional cycle of the pump, was proposed for describing K(+) interference on the complex PTX-Na(+)/K(+) pump in the presence of intracellular ATP. A mathematic model was derived from the reaction model and it was possible to solve numerically the associated differential equations and to simulate experimental maneuvers about the PTX induced currents in the presence of K(+) in the intra- and extracellular space as well as ATP in the intracellular. After the model adjusting to the experimental data, a Monte Carlo method for sensitivity analysis was used to analyze how each reaction parameter acts during each experimental maneuver involving PTX. For ATP and K(+) concentrations conditions, the simulations suggest that the enzyme substate with ATP bound to its high-affinity sites is the main substate for the PTX binding. The activation rate of the induced current is limited by the K(+) deocclusion from the PTX-Na(+)/K(+) pump complex. The K(+) occlusion in the PTX induced channels in the enzymes with ATP bound to its low-affinity sites is the main mechanism responsible for the reduction of the enzyme affinity to PTX.     

Putative palytoxin and its new analogue,ovatoxin-a,in <Emphasis Type="Italic">Ostreopsis ovata</Emphasis> collected along the ligurian coasts during the 2006 toxic outbreak

In this article we report on the liquid chromatography tandem mass spectrometry (LC-MS) investigation of plankton samples collected in the summer of 2006 along the Ligurian coasts, coinciding with a massive bloom of the tropical microalga Ostreopsis ovata. LC-MS analyses indicated the occurrence of putative palytoxin along with a much more abundant palytoxin-like compound never reported so far, which we named ovatoxin-a. On the basis of molecular formula, fragmentation pattern, and chromatographic behavior, the structure of ovatoxin-a appeared to be strictly related to that of palytoxin. We report also on the analysis of cultured O. ovata, which was necessary to unequivocally demonstrate that putative palytoxin and ovatoxin-a contained in field samples were actually produced by O. ovata itself.     

Herrn Professor Dr. Dr. h. c. mult. J. F. K. Huber

Ohne Zusammenfassung     

Gaspolarographie. II. Mitt.

Zusammenfassung Stickstoffdioxid, Stickstoffmonoxid, Distickstofftrioxid und Distickstoffmonoxid sind in wasserfreiem Dimethylsulfoxid polarographisch erfaßbar.

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Summary Nitrogen dioxide, nitrogen, monoxide, nitrogen trioxide, and nitrous oxide can be detected polarographically in anhydrous dimethylsulfoxide.

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Résumé On peut déceler le peroxyde d'azote, l'oxyde azotique, l'anhydride azoteux et l'oxyde azoteux par polarographie dans le diméthylsulfoxyde anhydre.

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Herrn Prof. Dr.A. A. Benedetti-Pichler zum 70. Geburtstag gewidmet.

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Für die Unterstiitzung der Untersuehungen wird der Regierung der USA gedankt.     

Ein Teleskop-Brenner. D.R.G.M.

Ohne Zusammenfassung