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Ciguatera poisoning (CP), caused by ciguatoxins (CTXs), is one of the most common food-borne diseases, affecting more than 50,000 people each year. In most cases, CP are managed with symptomatic and supportive remedies, and no specific treatment has been devised. In this study, toward the development of therapeutic antibodies for CP, we examined to humanize mouse anti-CTX3C antibody 10C9 (m10C9), which exhibited neutralizing activity against ciguatoxin in vitro and in vivo. The complementarity determining regions were grafted onto a human germline sequence with high sequence identity to m10C9, and the backmutations were examined to maintain the binding affinity. The optimized humanized antibody, Opt.h10C9Fab, showed a strong binding affinity to CTX3C with a high affinity (K D  = 19.0 nM), and only two backmutations of Arg L46 and Cys H94 in the framework regions were involved in determining the antigen binding affinity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ciguatoxins (CTXs) are neurotoxins responsible for ciguatera poisoning (CP), which affects more than 50,000 people worldwide annually. The development of analytical methods to prevent CP is a pressing global issue, and the N2a assay is one of the most promising methods for detecting CTXs. CTXs are highly toxic, and an action level of 0.01 μg CTX1B equivalent (eq)/kg in fish has been proposed. It is desirable to further increase the detection sensitivity of CTXs in the N2a assay to detect such low concentrations reliably. The opening of voltage-gated sodium channels (Na V channels) and blocking of voltage-gated potassium channels (K V channels) are thought to be involved in the toxicity of CTXs. Therefore, in this study, we developed an assay that could detect CTXs with higher sensitivity than conventional N2a assays, using K V channel inhibitors as sensitizing reagents for N2a cells. The addition of the K V channel inhibitors 4-aminopyridine and tetraethylammonium chloride to N2a cells, in addition to the traditional sensitizing reagents ouabain and veratridine, increased the sensitivity of N2a cells to CTXs by up to approximately 4-fold. This is also the first study to demonstrate the influence of K V channels on the toxicity of CTXs in a cell-based assay.

Rikō Majima published seven papers in this journal, and seeing these papers and their surrounding contexts allows us to glance at the birth of a galaxy of scientists.

Ciguatera fish poisoning (CFP) is a foodborne illness affecting > 50,000 people worldwide annually. It is caused by eating marine invertebrates and fish that have accumulated ciguatoxins (CTXs). Recently, the risk of CFP to human health, the local economy, and fishery resources have increased; therefore, detection methods are urgently needed. Functional assays for detecting ciguatoxins in fish include receptor binding (RBA) and neuroblastoma cell-based assay (N2a assay), which can detect all CTX congeners. In this study, we made these assays easier to use. For RBA, an assay was developed using a novel near-infrared fluorescent ligand, PREX710-BTX, to save valuable CTXs. In the N2a assay, a 1-day assay was developed with the same detection performance as the conventional 2-day assay. Additionally, in these assays, we used calibrated CTX standards from the Pacific determined by quantitative NMR for the first time to compare the relative potency of congeners, which differed significantly among previous studies. In the RBA, there was almost no difference in the binding affinity among congeners, showing that the differences in side chains, stereochemistry, and backbone structure of CTXs did not affect the binding affinity. However, this result did not correlate with the toxic equivalency factors (TEFs) based on acute toxicity in mice. In contrast, the N2a assay showed a good correlation with TEFs based on acute toxicity in mice, except for CTX3C. These findings, obtained with calibrated toxin standards, provide important insights into evaluating the total toxicity of CTXs using functional assays., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Dinoflagellates of the genera Gambierdiscus and Fukuyoa are able to produce potent neurotoxins like ciguatoxins (CTXs), which, after biooxidation in fish, are responsible for ciguatera intoxication. An isolate of G. australes from the Canary Islands, that revealed the presence of CTX-like compounds by immunosensing tools, was studied by immunocytochemistry to localize intracellular CTX-like compounds, using 8H4 monoclonal antibody that specifically recognizes the right wing of CTX1B and CTX3C analogues. Confocal microscopy observations of immunostained whole cells revealed a strong positive reaction on cell surface and all along the cell outline, while no reaction was detected inside the cells, probably because the antibody was not able to pass through thecal plates. Cell sections showed a positive antibody staining not only on thecal plates, but also inside cytoplasm, with numerous small dots and larger tubule-like reticulate structures. Small fluorescent dots were detected also on the nuclear surface. These observations indicate that CTX-like compounds are present in G. australes cytoplasm, and then are, at least in part, released to cover the cell surface., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ciguatoxins (CTXs) are marine neurotoxins produced by microalgae of the genera Gambierdiscus and Fukuyoa. CTXs may reach humans through food webs and cause ciguatera fish poisoning (CFP). An immunosensor for the detection of Pacific CTXs in fish was developed using multiwalled carbon nanotube (MWCNT)-modified carbon electrodes and a smartphone-controlled potentiostat. The biosensor attained a limit of detection (LOD) and a limit of quantification (LOQ) of 6 and 27 pg/mL of CTX1B, respectively, which were 0.001 and 0.005 μg/kg in fish flesh. In the analysis of fish samples from Japan and Fiji, excellent correlations were found with sandwich enzyme-linked immunosorbent assays (ELISAs), a cell-based assay (CBA) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Stability of at least 3 months at -20 °C was predicted. In just over 2 h, the biosensor provides reliable, accurate and precise Pacific CTX contents in fish extracts, being suitable for monitoring and research programs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The importance of ciguatoxins (CTXs) in seafood safety and their emerging occurrence in locations far away from tropical areas highlight the need for simple and low-cost methods for the sensitive and rapid detection of these potent marine toxins to protect seafood consumers. Herein, an electrochemical immunosensor for the detection of CTXs is presented. A sandwich configuration is proposed, using magnetic beads (MBs) as immobilization supports for two capture antibodies, with their combination facilitating the detection of CTX1B, CTX3C, 54-deoxyCTX1B, and 51-hydroxyCTX3C. PolyHRP-streptavidin is used for the detection of the biotinylated detector antibody. Experimental conditions are first optimized using colorimetry, and these conditions are subsequently used for electrochemical detection on electrode arrays. Limits of detection at the pg/mL level are achieved for CTX1B and 51-hydroxyCTX3C. The applicability of the immunosensor to the analysis of fish samples is demonstrated, attaining detection of CTX1B at contents as low as 0.01 μg/kg and providing results in correlation with those obtained using mouse bioassay (MBA) and cell-based assay (CBA), and confirmed by liquid chromatography coupled to high-resolution mass spectrometry (LC-ESI-HRMS). This user-friendly bioanalytical tool for the rapid detection of CTXs can be used to mitigate ciguatera risk and contribute to the protection of consumer health.

"Ciguatera" fish poisoning (CFP) is one of the well-known food poisoning caused by the ingestion of fish that have accumulated trace amounts of ciguatoxins (CTXs). CFP affects more than 50,000 individuals annually. The difficulty in preventing CFP comes from the lack of reliable methods for analysis of CTXs in contaminated fish, together with the normal appearance, taste, and smell of CTX-contaminated fish. Thus, a sensitive, accurate, routine, and portable analytical method to detect CTXs is urgently required. Monoclonal antibodies (mAbs) specific against either wing of major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) were generated by immunizing mice with rationally designed synthetic haptens-KLH conjugates instead of the CTXs. Haptenic groups with a surface area greater than 400 Å 2 are required to produce mAbs that can strongly bind to CTXs. Furthermore, a highly sensitive fluorescence-based sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This protocol can detect and quantify four major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) with a limit of detection (LOD) of less than 1 pg/mL. The LOD determined for this sandwich ELISA is sufficient to detect CTX1B-contaminated fish at the FDA guidance level of 0.01 ppb.

The kedarcidin chromophore is a formidible target for total synthesis. Herein, we describe a viable synthesis of this highly unstable natural product. This entailed the early introduction and gram-scale synthesis of 2-deoxysugar conjugates of both L-mycarose and L-kedarosamine. Key advances include: (1) stereoselective allenylzinc keto-addition to form an epoxyalkyne; (2) α-selective glycosylations with 2-deoxy thioglycosides (AgPF 6 /DTBMP) and Schmidt donors (TiCl 4 ); (3) Mitsunobu aryl etherification to install a hindered 1,2-cis-configuration; (4) atropselective and convergent Sonogashira-Shiina cyclization sequence; (5) Ohfune-based amidation protocol for naphthoic acid; (6) Ce(III)-mediated nine-membered enediyne cyclization and ester/mesylate derivatisation; (7) SmI 2 -based reductive olefination and global HF-deprotection end-game. The longest linear sequence from gram-scale intermediates is 17-steps, and HRMS data of the synthetic natural product was obtained for the first time.

Ciguatera fish poisoning (CFP) caused by the consumption of fish that have accumulated ciguatoxins (CTXs) affects more than 50000 people annually. The spread of CFP causes enormous damage to public health, fishery resources, and the economies of tropical and subtropical endemic regions. The difficulty in avoiding CFP arises from the lack of sensitive and reliable analytical methods for the detection and quantification of CTXs in contaminated fish, along with the normal appearance, smell, and taste of fish contaminated with the causative toxins. Thus, an accurate, sensitive, routine, and portable detection method for CTXs is urgently required. We have successfully developed a highly sensitive fluorescent sandwich ELISA, which can detect, differentiate, and quantify four major CTX congeners (CTX1B, CTX3C, 51-hydroxyCTX3C, and 54-deoxyCTX1B) with a detection limit of less than 1 pg/mL. The ELISA protocol, using one microtiter plate coated with two mAbs (10C9 and 3G8), and ALP-linked 8H4, can detect any of the four CTX congeners in a single operation. CTX1B spiked into fish at the FDA guidance level of 0.01 ppb CTX1B equivalent toxicity in fish from Pacific regions was also proven to be reliably detected by this ELISA. Furthermore, the efficiency of extraction/purification procedures and the matrix effect of contaminants in fish were evaluated in detail, since pretreatment and matrix effects are critical for ELISA analysis.

Ciguatoxins are polyether marine toxins that act as sodium channel activators. These toxins cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents several symptoms in humans including long-term neurological alterations. Earlier work has shown that both acute and chronic exposure of primary cortical neurons to synthetic ciguatoxin CTX3C have profound impacts on neuronal function. Thus, the present work aimed to identify relevant neuronal genes and metabolic pathways that could be altered by ciguatoxin exposure. To study the effect of ciguatoxins in primary neurons in culture, we performed a transcriptomic analysis using whole mouse genome microarrays, for primary cortical neurons exposed during 6, 24, or 72 h in culture to CTX3C. Here, we have shown that the effects of the toxin on gene expression differ with the exposure time. The results presented here have identified several relevant genes and pathways related to the effect of ciguatoxins on neurons and may assist in future research or even treatment of ciguatera. Moreover, we demonstrated that the effects of the toxin on gene expression were exclusively consequential of its action as a voltage-gated sodium channel activator, since all the effects of CTX3C were avoided by preincubation of the neurons with the sodium channel blocker tetrodotoxin.

A synthesis of the core framework of presporolide, which possesses both a strained bicyclo[7.3.0]dodecadiyne moiety and a distinctive macrolactone structure, is reported. This synthesis features: (i) a Cu-mediated O-arylation of a hindered tertiary alcohol using triarylbismuth reagent; (ii) stereoselective construction of the strained nine-membered diyne ring; and (iii) atroposelective formation of the macrolactone.

Valuable synthetic routes to the Lycopodium alkaloid lycodine (1) and its unsymmetric dimers, complanadines A (4) and B (5), have been developed. Regioselective construction of the bicyclo[3.3.1]nonane core structure of lycodine was achieved by a remote functionality-controlled Diels-Alder reaction and subsequent intramolecular Mizoroki-Heck reaction. A key coupling reaction of the lycodine units, pyridine N-oxide (66) and aryl bromide (65), through C-H arylation at the C1 position of 66 provided the unsymmetric dimer structure at a late stage of the synthesis. This strategy greatly simplified the construction of the dimeric architecture and functionalization. Complanadines A (4) and B (5) were synthesized by adjusting the oxidation level of the bipyridine mono-N-oxide (67). The diverse utility of this common intermediate (67) suggests a possible biosynthetic pathway of complanadines in Nature. Both enantiomers of lycodine (1) and complanadines A (4) and B (5) were prepared in sufficient quantities for biological evaluation. The effect on neuron differentiation of PC-12 cells upon treatment with culture medium, in which human astrocytoma cells had been cultured in the presence of 1, 4, or 5 was evaluated., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Avermectins were isolated as compounds possessing anthelmintic activity from the culture broth of Streptomycesavermitilis by Ōmura and co-workers. Owing to their potent anthelmintic and insecticidal activities, as well as their unique pentacyclic architecture, the avermectin family attracted keen interest from synthetic organic chemists. We have recently completed a more efficient and straightforward total synthesis of avermectin B1a, as compared with previous syntheses.

Our chemical syntheses and related scientific investigations of natural products with complex architectures and powerful biological activities are described, focusing on the very large 3 nm-long polycyclic ethers called the ciguatoxins, highly strained and labile chromoprotein antitumor antibiotics featuring nine-membered enediyne cores, and extremely potent anthelmintic macrolides called the avermectins.

Limonoids are highly oxygenated C13α-triterpenes and common secondary metabolites. Several hundred congeners have been isolated to date. The first total synthesis of (±)-limonin, the flagship congener of the limonoids, is now reported and features 1) a tandem radical cyclization generating the BCD ring system with the C13α configuration that is essential to the limonoids and a Robinson annulation to construct the limonoid androstane framework, 2) a singlet-oxygen cycloaddition and a Baeyer-Villiger oxidation to synthesize the highly oxidized D ring, and 3) a Suárez reaction to construct the unique AA' ring system., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ciguatoxins are sodium channels activators that cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents with long-term neurological alterations. In central neurons, chronic perturbations in activity induce homeostatic synaptic mechanisms that adjust the strength of excitatory synapses and modulate glutamate receptor expression in order to stabilize the overall activity. Immediate early genes, such as Arc and Egr1, are induced in response to activity changes and underlie the trafficking of glutamate receptors during neuronal homeostasis. To better understand the long lasting neurological consequences of ciguatera, it is important to establish the role that chronic changes in activity produced by ciguatoxins represent to central neurons. Here, the effect of a 30 min exposure of 10-13 days in vitro (DIV) cortical neurons to the synthetic ciguatoxin CTX 3C on Arc and Egr1 expression was evaluated using real-time polymerase chain reaction approaches. Since the toxin increased the mRNA levels of both Arc and Egr1, the effect of CTX 3C in NaV channels, membrane potential, firing activity, miniature excitatory postsynaptic currents (mEPSCs), and glutamate receptors expression in cortical neurons after a 24 h exposure was evaluated using electrophysiological and western blot approaches. The data presented here show that CTX 3C induced an upregulation of Arc and Egr1 that was prevented by previous coincubation of the neurons with the NaV channel blocker tetrodotoxin. In addition, chronic CTX 3C caused a concentration-dependent shift in the activation voltage of NaV channels to more negative potentials and produced membrane potential depolarization. Moreover, 24 h treatment of cortical neurons with 5 nM CTX 3C decreased neuronal firing and induced synaptic scaling mechanisms, as evidenced by a decrease in the amplitude of mEPSCs and downregulation in the protein level of glutamate receptors that was also prevented by tetrodotoxin. These findings identify an unanticipated role for ciguatoxin in the regulation of homeostatic plasticity in central neurons involving NaV channels and raise the possibility that some of the neurological symptoms of ciguatera might be explained by these compensatory mechanisms.

Ciguatera is a human global disease caused by the consumption of contaminated fish that have accumulated ciguatoxins (CTXs), sodium channel activator toxins. Symptoms of ciguatera include neurological alterations such as paraesthesiae, dysaesthesiae, depression, and heightened nociperception, among others. An important issue to understand these long-term neurological alterations is to establish the role that changes in activity produced by CTX 3C represent to neurons. Here, the effects of synthetic ciguatoxin CTX 3C on membrane potential, spontaneous spiking, and properties of synaptic transmission in cultured cortical neurons of 11-18 days in vitro (DIV) were evaluated using electrophysiological approaches. CTX 3C induced a large depolarization that decreased neuronal firing and caused a rapid inward tonic current that was primarily GABAergic. Moreover, the toxin enhanced the amplitude of miniature postsynaptic inhibitory currents (mIPSCs), whereas it decreased the amplitude of miniature postsynaptic excitatory currents (mEPSCs). The frequency of mIPSCs increased, whereas the frequency of mEPSCs remained unaltered. We describe, for the first time, that a rapid membrane depolarization caused by CTX 3C in cortical neurons activates mechanisms that tend to suppress electrical activity by shifting the balance between excitatory and inhibitory synaptic transmission toward inhibition. Indeed, these results suggest that the acute effects of CTX on synaptic transmission could underlie some of the neurological symptoms caused by ciguatera in humans.

Ciguatoxins, the principal causative agents of ciguatera seafood poisoning, are extremely large polycyclic ethers. We report herein a reliable route for constructing the left wing of CTX1B, which possesses the acid/base/oxidant-sensitive bisallylic ether moiety, by a 6-exo radical cyclization/ring-closing metathesis strategy. This new route enabled us to achieve the second-generation total synthesis of CTX1B and the first synthesis of 54-deoxyCTX1B., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The cyanosporasides A-F are a collection of monochlorinated benzenoid derivatives isolated from the marine actinomycetes Salinispora and Streptomyces sp. All derivatives feature one of two types of cyanocyclopenta[a]indene frameworks, which are regioisomeric in the position of a single chlorine atom. It is proposed that these chloro-substituted benzenoids are formed biosynthetically through the cycloaromatization of a bicyclic nine-membered enediyne precursor. Herein, we report the synthesis of such a bicyclic precursor, its spontaneous transannulation into a p-benzyne, and its differential 1,4 hydrochlorination reactivity under either organochlorine or chloride-salt conditions. Our bioinspired approach culminated in the first regiodivergent total synthesis of the aglycons A/F and B/C, as well as cyanosporasides D and E. In addition, empirical insights into the site selectivity of a natural-like p-benzyne, calculated to be a ground-state triplet diradical, to hydrogen, chlorine, and chloride sources are revealed., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ciguatoxins (CTXs) and maitotoxins (MTXs) are polyether ladder shaped toxins derived from the dinoflagellate Gambierdiscus toxicus. Despite the fact that MTXs are 3 times larger than CTXs, part of the structure of MTXs resembles that of CTXs. To date, the synthetic ciguatoxin, CTX 3C has been reported to activate voltage-gated sodium channels, whereas the main effect of MTX is inducing calcium influx into the cell leading to cell death. However, there is a lack of information regarding the effects of these toxins in a common cellular model. Here, in order to have an overview of the main effects of these toxins in mice cortical neurons, we examined the effects of MTX and the synthetic ciguatoxin CTX 3C on the main voltage dependent ion channels in neurons, sodium, potassium, and calcium channels as well as on membrane potential, cytosolic calcium concentration ([Ca(2+)]c), intracellular pH (pHi), and neuronal viability. Regarding voltage-gated ion channels, neither CTX 3C nor MTX affected voltage-gated calcium or potassium channels, but while CTX 3C had a large effect on voltage-gated sodium channels (VGSC) by shifting the activation and inactivation curves to more hyperpolarized potentials and decreasing peak sodium channel amplitude, MTX, at 5 nM, had no effect on VGSC activation and inactivation but decreased peak sodium current amplitude. Other major differences between both toxins were the massive calcium influx and intracellular acidification produced by MTX but not by CTX 3C. Indeed, the novel finding that MTX produces acidosis supports a pathway recently described in which MTX produces calcium influx via the sodium-hydrogen exchanger (NHX). For the first time, we found that VGSC blockers partially blocked the MTX-induced calcium influx, intracellular acidification, and protected against the short-term MTX-induced cytotoxicity. The results presented here provide the first report that shows the comparative effects of two prototypical ciguatera toxins, CTX 3C and MTX, in a neuronal model. We hypothesize that the analogies and differences in the bioactivity of these two toxins, produced by the same microorganism, may be strongly linked to their chemical structure.

Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the consumption of fish that have accumulated a type of sodium channel activator toxin called ciguatoxins (CTXs), which are produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50000 people each year. The extremely low level of CTXs in tainted fish has hampered the development of antibodies for the detection of these toxins. Monoclonal antibodies (mAbs) specific against major congeners of CTX3C, 51-hydroxyCTX3C, CTX1B, and 54-deoxyCTX1B were prepared by immunization of mice with protein conjugates of rationally designed synthetic haptens in place of the natural toxins. We found that haptenic groups possessing a surface area larger than 400 angstroms2 were required to produce mAbs that can bind strongly to CTXs. Direct sandwich ELISA utilizing two different monoclonal antibodies that bind specifically to one of the two wings of a CTX were established to detect CTXs. No cross-reactivity was observed against the other marine toxins tested, including brevetoxin A, brevetoxin B, okadaic acid, and maitotoxin.

Ciguatera fish poisoning (CFP) is a form of food poisoning caused by the ingestion of a variety of reef fish that have accumulated trace amounts of ciguatoxins produced by dinoflagellates of the genus Gambierdiscus through the food chain. CFP affects more than 50,000 people each year. The extremely low level of the causative neurotoxins, ciguatoxins, in fish has hampered the preparation of antibodies for detecting the toxins. In this paper, we describe a thiol strategy for synthesizing a keyhole limpet hemocyanin (KLH)-conjugate (20) of the ABCDE-ring fragment of the Pacific ciguatoxins, CTX1B (1) and 54-deoxyCTX1B (4). We succeeded in producing a monoclonal antibody (3G8) against the left wings of these ciguatoxins by immunizing mice with the hapten-KLH conjugate (20) as the synthetic antigen. The most promising mAb, 3G8, does not cross-react with other related marine toxins. Sandwich enzyme-linked immunosorbent assay (ELISA) utilizing 3G8 and the previously prepared monoclonal antibody (8H4) enabled us to detect 1 specifically at less than 0.28 ng/mL., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A highly efficient total synthesis of the 11-membered cyclic aspercyclides A (1) and B (2) has been achieved by chemo- and regioselective intramolecular oxidative C-O bond formation from differently substituted diphenols.

Toxin profiles of representative ciguatera species caught at different locations of Japan were investigated in fish flesh by high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Identification and quantification of 16 toxins were facilitated by the use of 14 reference toxins prepared by either synthesis or isolation from natural sources and the previous LC-MS data thereof. Sodium adduct ions [M + Na](+) were used as parent and product ions. Distinct regional differences were unveiled: ciguatoxin-1B type toxins were found in snappers and groupers from Okinawa, ciguatoxin-3C type toxins were found in a spotted knifejaw, Oplegnathus punctatus, from Miyazaki located 730 km north of Okinawa, and both types of toxins were found in a red snapper, Lutjanus bohar, from Minamitorishima (Marcus) Island. Twelve toxins were identified in a dinoflagellate, Gambierdiscus toxicus, collected as the primary toxin source in French Polynesia. Occurrence of M-seco-toxins in fish and oxidized toxins in the dinoflagellate was confirmed for the first time. The present LC-MS/MS method is rapid, specific, and accurate. It not only outperforms the currently employed mouse bioassays but also enables the study of the toxin dynamics during the food chain transmission.

Ciguatera is a global disease caused by the consumption of certain warm-water fish (ciguateric fish) that have accumulated orally effective levels of sodium channel activator toxins (ciguatoxins) through the marine food chain. The effect of ciguatoxin standards and contaminated ciguatoxin samples was evaluated by electrophysiological recordings in cultured cerebellar neurons. The toxins affected both voltage-gated sodium (Nav) and potassium channels (Kv) although with different potencies. CTX 3C was the most active toxin blocking the peak inward sodium currents, followed by P-CTX 1B and 51-OH CTX 3C. In contrast, P-CTX 1B was more effective in blocking potassium currents. The analysis of six different samples of contaminated fish, in which a ciguatoxin analogue of mass 1040.6, not identical with the standard 51-OH CTX 3C, was the most prevalent compound, indicated an additive effect of the different ciguatoxins present in the samples. The results presented here constitute the first comparison of the potencies of three different purified ciguatoxins on sodium and potassium channels in the same neuronal preparation and indicate that electrophysiological recordings from cultured cerebellar neurons may provide a valuable tool to detect and quantify ciguatoxins in the very low nanomolar range.

This paper describes the details of our synthetic studies on the marine steroidal alkaloids cortistatins A and J. The key features of our strategy include (i) an efficient Knoevenagel/electrocyclic strategy to couple the diketone and the CD-ring fragment, (ii) a chemoselective radical cyclization to construct the oxabicyclo[3.2.1]octene B-ring system, (iii) a highly stereocontrolled installation of the isoquinoline unit, and (iv) a late-stage functionalization of the A-ring.

Ciguatoxins, the principal causative toxins of ciguatera seafood poisoning, are large ladder-like polycyclic ethers. We report a highly stereoselective 6-exo radical cyclization/ring-closing olefin metathesis sequence to construct the syn/trans-fused polyether system. The new method was applied to the practical synthesis of ciguatoxin CTX3C.

The mechanism by which anti-ciguatoxin antibody 10C9Fab recognizes a fragment of ciguatoxin CTX3C (CTX3C-ABCDE) was investigated by mutational analysis based on structural data. 10C9Fab has an extraordinarily large and deep antigen-binding pocket at the center of its variable region. We mutated several residues located at the antigen-binding pocket to Ala, and kinetic analysis of the interactions between the mutant proteins and the antigen fragment was performed. The results indicate that some residues associated with the rigid antigen-binding pocket are structural hot-spots and that L-N94 is an energetic hot-spot for association of the antibody with the antigen fragment CTX3C-ABCDE, suggesting the importance of structural complementarity and energetic hot-spot interactions for specific recognition of polycyclic ethers.

Okinawa being located in the subtropical region has the highest incidence of ciguatera in Japan. Officially, 33 outbreaks involving 103 patients have been reported between 1997 and 2006. The implicated species were Variola louti, Lutjanus bohar, Lutjanus monostigma, Epinephelus fuscoguttatus, unidentified Lutjanus sp., Plectropomus areolatus, Oplegnathus punctatus, Epinephelus polyphekadion, Caranx ignobilis and moray eel. Toxicities of the leftover meals, as determined by mouse bioassays, ranged from 0.025 to 0.8 MU/g or above (equivalent to 0.175-5.6 ngCTX1B/g). We collected 612 specimens of fish belonging to L. monostigma, L. bohar, Lutjanus argentimaculatus, Lutjanus russellii, V. louti, Variola albimarginata, and E. fuscoguttatus from the coasts around Okinawa and examined the toxicity of the flesh by the mouse bioassay. The rate of toxic fish was as follows: L. monostigma: 32.3%, L. bohar: 11.9%, V. louti: 14.3%, E. fuscoguttatus: 20.8%. Only one out of 36 samples of V. albimarginata and two of 74 samples of L. russellii were found toxic. None of the 35 samples of L. argentimaculatus was toxic. Nor the L. bohar samples weighing less than 4 kg were toxic. In all toxic samples, CTX1B was detected by LC/MS analysis but CTX3C and 51-hydroxyCTX3C were not., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Ciguatoxins are the major causative toxins of ciguatera seafood poisoning. Limited availability of ciguatoxins has hampered the development of a reliable and specific immunoassay for detecting these toxins in contaminated fish. Monoclonal antibodies (mAbs) specific against both ends of Pacific ciguatoxins CTX3C and 51-hydroxyCTX3C were prepared by immunization of mice with the protein conjugates of rationally designed synthetic haptens in place of the natural toxin. Haptenic groups that possess a surface area larger than 400 A(2) were required to produce mAbs that can bind strongly to CTX3C or 51-hydroxyCTX3C. A direct sandwich enzyme-linked immunosorbent assay (ELISA) using these mAbs was established to detect CTX3C and 51-hydroxyCTX3C at the ppb level with no cross-reactivity against the other marine toxins, including brevetoxin A, brevetoxin B, okadaic acid, or maitotoxin., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Ciguatera fish poisoning (CFP) is a human foodborne intoxication caused by ingestion of tropical fishes contaminated with the potent polyether toxins known as ciguatoxins (CTXs). These toxins are issued from Gambierdiscus species of dinoflagellates. Herbivorous fish accumulate these toxins in their musculature and viscera after ingesting dinoflagellates. Epidemiological studies showed that CFP has been present in areas between 35 degrees North and 35 degrees South latitude, mainly, Indo-pacific and Caribbean areas, but not in waters closed to European and African continent. In the present paper, a specimen of Seriola dumerili weighing 70 kg and a smaller Seriola fasciata specimen, captured in waters belonging to Selvagens Islands (Madeira Arquipelago), were analyzed. Fishes from this genus were implicated in previous suspected ciguatera poisoning outbreaks in the Portuguese Madeira Arquipelago in the North Atlantic Ocean. Analysis was performed by two approaches, a functional method using cerebellar granule cells and by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) method. The study was carried out in one portion of the tail muscle of Seriola fasciata and five parts of the body of Seriola dumerili (tail muscle, head, ventral muscle, mid muscle, and liver). The functional method consisted in the modification of the inward sodium current in cerebellar granule cells and the chemical method was a high resolution chromatography, which allowed elucidating the toxin profile in the samples. In addition, UPLC-MS technique was optimized and used for detecting and quantifying CTXs for the first time. After fish extraction and clean up, the chromatograms revealed the presence of CTX-1B at 1111.6 m/z, CTX-3C at 1023.5 m/z, a CTX analogue at 1040.6 m/z, and a CTX from the Caribbean or Indic waters at 1141.6 m/z. Therefore, the results obtained in the present paper for both methods confirm, for the first time, the presence of CTX in fish from Madeira Arquipelago.

Ciguatoxins, the principal causative toxins of ciguatera seafood poisoning, are potent neurotoxic polycyclic ethers. We report herein the total synthesis of a 10-membered F-ring analogue of 51-hydroxyCTX3C, which constitutes the first example of an F-ring modified ciguatoxin that exhibits potent cytotoxicity as well as mouse acute toxicity.

The proposed structure of the maduropeptin chromophore, the biologically active component of the highly potent chromoprotein antitumor antibiotics, was stereoselectively synthesized but did not satisfy the spectra of the natural product. We demonstrated that the correct structure is diastereomeric, which possesses an antipodal sugar moiety.

Ciguatera is a global food poisoning caused by the consumption of fish that have accumulated sodium channel activator toxins, ciguatoxins. At present, most diagnosed cases of ciguatera are treated with symptomatic and supportive remedies, and no specific therapy has been devised. Here we report that ciguatoxin CTX3C can be effectively neutralized in vitro and in vivo by simultaneous use of two anti-ciguatoxin monoclonal antibodies, providing the first rational approach toward directly preventing and treating ciguatera.

The synthetic ciguatoxin CTX3C has been shown to activate tetrodotoxin (TTX)-sensitive sodium channels (Na(v)1.2, Na(v)1.4, and Na(v)1.5) by accelerating activation kinetics and shifting the activation curve toward hyperpolarization (Yamaoka, K., Inoue, M., Miyahara, H., Miyazaki, K., and Hirama, M. (2004) Br. J. Pharmacol. 142, 879-889). In this study, we further explored the effects of CTX3C on the TTX-resistant sodium channel Na(v)1.8. TTX-resistant channels have been shown to be involved in transducing pain and related sensations (Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257-262). Thus, we hypothesized that ciguatoxin-induced activation of the Na(v)1.8 current would account for the neurological symptoms of ciguatera poisoning. We found that 0.1 mum CTX3C preferentially affected the activation process of the Na(v)1.8 channel compared with those of the Na(v)1.2 and Na(v)1.4 channels. Importantly, without stimulation, 0.1 mum CTX3C induced a large leakage current (I (L)). The conductance of the I (L) calculated relative to the maximum conductance (G (max)) was 10 times larger than that of Na(v)1.2 or Na(v)1.4. To determine the molecular domain of Na(v)1.8 responsible for conferring higher sensitivity to CTX3C, we made two chimeric constructs from Na(v)1.4 and Na(v)1.8. Chimeras containing the N-terminal half of Na(v)1.8 exhibited a large response similar to wild-type Na(v)1.8, indicating that the region conferring high sensitivity to ciguatoxin action is located in the D1 or D2 domains.

Formation of an epoxide before 9-membered ring cyclization and SmI2 mediated reductive olefination in the presence of the epoxide successfully produced the epoxybicyclo[7.3.0]dodecadienediyne core of the kedarcidin chromophore.

In the Masamune-Bergman cyclization of a nine-membered non-conjugated enediyne with an internal, maduropeptin-like nucleophile, the exocyclic alkene migrated to form the nine-membered conjugated enediyne, triggered by the intramolecular addition of the amide group; final aromatized products showed up to 85% yield.

Zoanthenol, isolated from Zoanthus sp., possesses an extremely complex architecture including contiguous quaternary carbons. An enantioselective synthesis of the fully functionalized ABC-ring of zoanthenol has been achieved and is described herein. The key features of the synthesis are the enzymatic kinetic optical resolution and the Mizoroki-Heck/Simmons-Smith reaction strategy used to construct the congested asymmetric quaternary carbons.

An efficient synthesis of the pentacyclic framework of cortistatins has been developed. The key strategy comprises assembly of the A- and the CD-ring fragments by Knoevenagel reaction, facile formation of the pyran ring via electrocyclization, and construction of the seven-membered B-ring by radical addition to an alpha,beta-unsaturated ketone.

Ciguatoxins are a family of marine toxins composed of transfused polycyclic ethers. It has not yet been clarified at the atomic level on the pathogenic mechanism of these toxins or the interaction between a polycyclic ether compounds and a protein. Using the crystal structures of anti-ciguatoxin antibody 10C9 Fab in ligand-free form and in complexes with ABCD-ring (CTX3C-ABCD) and ABCDE-ring (CTX3C-ABCDE) fragments of the antigen CTX3C at resolutions of 2.6, 2.4, and 2.3 angstroms, respectively, we elucidated the mechanism of the interaction between the polycyclic ethers and the antibody. 10C9 Fab has an extraordinarily large and deep binding pocket at the center of the variable region, where CTX3C-ABCD or CTX3C-ABCDE binds longitudinally in the pocket via hydrogen bonds and van der Waals interactions. Upon antigen-antibody complexation, 10C9 Fab adjusts to the antigen fragments by means of rotational motion in the variable region. In addition, the antigen fragment lacking the E-ring induces a large motion in the constant region. Consequently, the thermostability of 10C9 Fab is enhanced by 10 degrees C upon complexation with CTX3C-ABCDE but not with CTX3C-ABCD. The crystal structures presented in this study also show that 10C9 Fab recoginition of CTX3C antigens requires molecular rearrangements over the entire antibody structure. These results further expand the fundamental understanding of the mechanism by which ladder-like polycyclic ethers are recognized and may be useful for the design of novel therapeutic agents by antibodies, marine toxins, or new diagnostic reagents for the detection and targeting of members of the polycyclic ether family.

To address how proteins recognize polyether toxin compounds, we focused on the interaction between the ABC ring compound of ciguatoxin 3C and its specific antibody, 1C49. Surface plasmon resonance analyses indicated that Escherichia coli-expressed variable domain fragments (Fv) of 1C49 had the high affinity constants and slow dissociation constants typical of antigen-antibody interactions. Linear van't Hoff analyses suggested that the interaction is enthalpy-driven. We resolved the crystal structure of 1C49 Fv bound to ABC ring compound of ciguatoxin 3C at a resolution of 1.7A. The binding pocket of the antibody had many aromatic rings and bound the antigen by shape complementarity typical of hapten-antibody interactions. Three hydrogen bonds and many van der Waals interactions were present. We mutated several residues of the antibody to Ala, and we used surface plasmon resonance to analyze the interactions between the mutated antibodies and the antigen. This analysis identified Tyr-91 and Trp-96 in the light chain as hot spots for the interaction, and other residues made incremental contributions by conferring enthalpic advantages and reducing the dissociation rate constant. Systematic mutation of Tyr-91 indicated that CH-pi and pi-pi interactions between the aromatic ring at this site and the antigen made substantial contributions to the association, and van der Waals interactions inhibited dissociation, suggesting that aromaticity and bulkiness are critical for the specific recognition of polyether compounds by proteins.

An efficient route to the trans-fused tetrahydrooxepin corresponding to the E ring of ciguatoxin was developed. Wide screening of allylation reactions of sulfur or fluoro-substituted tetrahydrooxepin revealed that the optimum method for obtaining the beta-allylation product selectively was the use of a combination of allyltrimethylsilane and TiCl(4) with 6-fluoro-7-hydroxytetrahydrooxepin.

Maduropeptin, an extremely potent antitumor agent, consists of a 1:1 complex of a carrier protein and a chromophore. We report herein a general and efficient route for the synthesis of the highly strained bicyclo[7.3.0]dodecadiyne core of the chromophore. The key feature of the synthetic strategy is the use of two Sonogashira coupling reactions in a stepwise manner to construct the conjugated dienyne substructure of the fused-ring system, including the Z alkene at C4,C13.

In advanced studies directed toward the total synthesis of the kedarcidin chromophore, we have successfully achieved the late-stage installation of the nine-membered diyne ring in the presence of the highly functionalised ansamacrocyclic bridge.