Intracellular abundance, localization, and enzymatic activity of a saxitoxin biosynthesis enzyme, SxtG, in two sister subclones of the dinoflagellate Alexandrium catenella with extremely different levels of paralytic shellfish toxins.

Paralytic shellfish poisoning is caused by saxitoxin (STX), and its analogues (paralytic shellfish toxins (PSTs)) produced by marine dinoflagellates. SxtA and SxtG are the most essential enzymes in STX biosynthesis. Previous studies investigated the abundance and subcellular localization (i.

Synthesis of Saxitoxin Biosynthetic Intermediates: Reveal the Mechanism for Formation of its Tricyclic Skeleton in Biosynthesis.

The synthesis and biosynthesis of the complex saxitoxin (STX) structure have garnered significant interest. Previously, we hypothesized that the tricyclic skeleton of STX originates from the monocyclic precursor 11-hydroxy-IntC'2 during biosynthesis, although direct evidence has been lacking. In this study, we identified conditions to synthesize a proposed tricyclic biosynthetic intermediate, 12,12-dideoxy-decarbamoyloxySTX (dd-doSTX), along with its 6-epimer (6-epi-dd-doSTX) and a bicyclic compound, in a single step from di-Boc protected 11-hydroxy-IntC'2.

Assay for okadaic acid O-acyl transferase using HPLC-FLD.

Dinophysistoxin 1 (DTX1, 1) and okadaic acid (OA, 2), produced by the dinoflagellates Dinophysis spp. and Prorocentrum spp., are primary diarrhetic shellfish toxins (DSTs), which may cause gastric illness in people consuming such as bivalves.

Synthesis and Identification of decarbamoyloxySaxitoxins in Toxic Microalgae and their Reactions with the Oxygenase, SxtT, Reveal Saxitoxin Biosynthesis.

Saxitoxin (STX, 1) is a representative compound of paralytic shellfish toxins (PSTs) that are produced by marine dinoflagellates and freshwater cyanobacteria. Although several pathways have been proposed for the biosynthesis of STX, the order of ring and side chain hydroxylation, and formation of the tricyclic skeleton have not been well established. In this study, 12,12-dideoxy-decarbamoyloxySTX (dd-doSTX, 2), the most reduced STX analogue having the tricyclic skeleton, and its analogues, 12β-deoxy-doSTX (12β-d-doSTX, 3), 12α-deoxy-doSTX (12α-d-doSTX, 4), and doSTX (5), were synthesized, and these compounds were screened in the toxic microalgae using high-resolution LCMSMS.

The quite low cross-reactivity of Kawatsu's anti-tetrodotoxin monoclonal antibody to 5,6,11-trideoxytetrodotoxin, 11-nortetrodotoxin-6(S)-ol, and 11-oxotetrodotoxin, the major tetrodotoxin analogues in pufferfish.

The monoclonal antibody against tetrodotoxin (TTX), prepared by Kawatsu et al. (1997), has been used in several TTX-related studies. Herein, we confirmed the quite low cross-reactivity of this antibody to three major TTX analogues in pufferfish using competitive ELISA: 5,6,11-trideoxyTTX (<2.

Metabolic inhibitor induces dynamic changes in saxitoxin biosynthesis and metabolism in the dinoflagellate Alexandrium pacificum (Group IV) under in vivo labeling condition.

In paralytic shellfish toxin-producing dinoflagellates, intracellular levels of saxitoxin and its analogues (STXs) are controlled by a balance between degradation and biosynthesis in response to marine environmental fluctuations and stresses. The purpose of this study was to demonstrate the utility of statistical analysis of in vivo labeling data for the dynamic analysis of variations in toxin production under stress. A toxic strain of the dinoflagellate Alexandrium pacificum (Group IV) was cultured in colchicine-containing N-labeled sodium nitrate-medium and metabolite levels were analyzed over time by liquid chromatography-mass spectrometry.

State-Dependent Inhibition of Voltage-Gated Sodium Channels in Neuroblastoma Neuro-2A Cells by Arachidonic Acid from .

Voltage-gated sodium channels (Na) are closely associated with epilepsy, cardiac and skeletal muscle diseases, and neuropathic pain. Several toxic compounds have been isolated from the marine sponge ; however, toxic substances that modulate Na are yet to be identified. This study aimed to identify Na inhibitors from two snake venoms and using mouse neuroblastoma Neuro-2A cells (N2A), which primarily express the specific Na subtype Na1.

Isolation and Biological Activity of 9-Tetrodotoxin and Isolation of Tb-242B, Possible Biosynthetic Shunt Products of Tetrodotoxin from Pufferfish.

Tetrodotoxin (TTX, ) is a potent voltage-gated sodium channel blocker detected in certain marine and terrestrial organisms. We report here a new TTX analogue, 9-TTX (), and a TTX-related compound, Tb-242B (), isolated from the pufferfish and , respectively. NMR analysis suggested that exists as a mixture of hemilactal and 10,8-lactone forms, whereas other reported TTX analogues are commonly present as an equilibrium mixture of hemilactal and 10,7-lactone forms.

Mass spectrometry-guided discovery of new analogs of bicyclic phosphotriester salinipostin and evaluation of their monoacylglycerol lipase inhibitory activity.

Natural products containing the highly unusual phosphotriester ring are known to be potent serine hydrolase inhibitors. The long-chain bicyclic enol-phosphotriester salinipostins (SPTs) from the marine actinomycete Salinispora have been identified as selective antimalarial agents. A potential regulatory function has been suggested for phosphotriesters based on their structural relationship with actinomycete signaling molecules and the prevalence of spt-like biosynthetic gene clusters across actinomycetes.

First Identification of 12β-Deoxygonyautoxin 5 (12α-Gonyautoxinol 5) in the Cyanobacterium (TA04) and 12β-Deoxysaxitoxin (12α-Saxitoxinol) in (TA04) and the Dinoflagellate (Group IV) (120518KureAC).

Saxitoxin and its analogues, paralytic shellfish toxins (PSTs), are potent and specific voltage-gated sodium channel blockers. These toxins are produced by some species of freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates of PSTs, as well as new analogues, from such organisms and proposed the biosynthetic and metabolic pathways of PSTs.

Preparation of domoic acid analogues using a bioconversion system, and their toxicity in mice.

Domoic acid (1, DA), a member of the natural kainoid family, is a potent agonist of ionotropic glutamate receptors in the central nervous system. The chemical synthesis of DA and its derivatives requires considerable effort to establish a pyrrolidine ring containing three contiguous stereocenters. Recently, a biosynthetic cyclase for DA, DabC, was identified.

Two new skeletal analogues of saxitoxin found in the scallop, Patinopecten yessoensis, as possible metabolites of paralytic shellfish toxins.

The scallop, Patinopecten yessoensis, was screened for new saxitoxin analogues to study the metabolism of paralytic shellfish toxins (PSTs), and this resulted in the discovery of two new analogues: M5-hemiaminal (HA) and M6-HA. M5-HA was isolated and its structure was determined by using NMR spectroscopy. It contains hydrogen at C-4 with opposite stereochemistry to that in saxitoxin, and a hemiaminal was formed between 9-NH and the hydrated ketone at C-12 in α-orientation.

SxtA localizes to chloroplasts and changes to its 3'UTR may reduce toxin biosynthesis in non-toxic Alexandrium catenella (Group I).

SxtA is the enzyme that catalyses the first step of saxitoxin biosynthesis. We developed an immunofluorescent method to detect SxtA using antibodies against SxtA peptides. Confocal microscopy revealed the presence of abundant, sub-cellularly localized signal in cells of toxic species and its absence in non-toxic species.

Synthesis of C12-Keto Saxitoxin Derivatives with Unusual Inhibitory Activity Against Voltage-Gated Sodium Channels.

A novel series of C12-keto-type saxitoxin (STX) derivatives bearing an unusual nonhydrated form of the ketone at C12 has been synthesized, and their Na -inhibitory activity has been evaluated in a cell-based assay as well as whole-cell patch-clamp recording. Among these compounds, 11-benzylidene STX (3 a) showed potent inhibitory activity against neuroblastoma Neuro 2A in both cell-based and electrophysiological analyses, with EC and IC values of 8.5 and 30.

Temporal Variation of the Profile and Concentrations of Paralytic Shellfish Toxins and Tetrodotoxin in the Scallop, Cultured in a Bay of East Japan.

Paralytic shellfish toxins (PSTs) are the major neurotoxic contaminants of edible bivalves in Japan. Tetrodotoxin (TTX) was recently detected in bivalve shellfish around the world, drawing widespread attention. In Japan, high levels of TTX were reported in the digestive gland of the scallop, , in 1993; however, no new data have emerged since then.

Identification of a Novel Saxitoxin Analogue, 12β-Deoxygonyautoxin 3, in the Cyanobacterium, (TA04).

Saxitoxin (STX) and its analogues, the potent voltage-gated sodium channel blockers, are biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates in the extract of the cyanobacterium, (TA04), that are primarily produced during the early and middle stages in the biosynthetic pathway to produce STX. These findings allowed us to propose a putative biosynthetic pathway responsible for STX production based on the structures of these intermediates.

Molecular Determinants of Brevetoxin Binding to Voltage-Gated Sodium Channels.

Brevetoxins are produced by dinoflagellates such as in warm-water red tides and cause neurotoxic shellfish poisoning. They bind to voltage-gated sodium channels at neurotoxin receptor 5, making the channels more active by shifting the voltage-dependence of activation to more negative potentials and by slowing the inactivation process. Previous work using photoaffinity labeling identified binding to the IS6 and IVS5 transmembrane segments of the channel α subunit.

Possible Biosynthetic Products and Metabolites of Kainic Acid from the Red Alga Digenea simplex and Their Biological Activity.

Four kainic acid (KA, 1)-related compounds, 4-hydroxykainic acid (2), allo-4-hydroxykainic acid (3), N-dimethylallyl-l-glutamic acid (4), and N-dimethylallyl- threo-3-hydroxyglutamic acid (5), were isolated from the red alga Digenea simplex. The structures of these compounds were elucidated using spectroscopic methods. Compounds 2 and 3 are possible oxidative metabolites of KA and allo-KA (6), respectively.

Metabolomic study of saxitoxin analogues and biosynthetic intermediates in dinoflagellates using N-labelled sodium nitrate as a nitrogen source.

A stable-isotope-labelling method using N-labelled sodium nitrate as a nitrogen source was developed for the toxic dinoflagellate Alexandrium catenella. The labelled saxitoxin analogues (STXs), their precursor, and the biosynthetic intermediates were analyzed by column-switching high-resolution hydrophilic interaction liquid chromatography with mass spectrometry. The low contents on Day 0, high N incorporation % of Int-C'2 and Int-E' suggested that their turn-over rates are high and that the sizes of the pool of these compounds are smaller than those of the other intermediates.

Pufferfish Saxitoxin and Tetrodotoxin Binding Protein (PSTBP) Analogues in the Blood Plasma of the Pufferfish , , , and .

Pufferfish saxitoxin and tetrodotoxin (TTX) binding protein (PSTBP) is a glycoprotein that we previously isolated from the blood plasma of the pufferfish ; this protein was also detected in seven species of the genus . We proposed that PSTBP is a carrier protein for TTX in pufferfish; however, PSTBP had not yet been found in genera other than . In this study, we investigated the presence of PSTBP-like proteins in the toxic pufferfish , , , and .

Spiro Bicyclic Guanidino Compounds from Pufferfish: Possible Biosynthetic Intermediates of Tetrodotoxin in Marine Environments.

Tetrodotoxin (TTX, 1) is a potent neurotoxin that is widely found in both terrestrial and marine animals; however, the biosynthetic pathway and genes for TTX have not yet been elucidated. Previously, we proposed that TTX originated from a monoterpene; this hypothesis was based on the structures of cyclic guanidino compounds that are commonly found in toxic newts. However, these compounds have not been detected in marine organisms.

Six domoic acid related compounds from the red alga, Chondria armata, and domoic acid biosynthesis by the diatom, Pseudo-nitzschia multiseries.

Domoic acid (DA, 1), a potent neurotoxin that causes amnesic shellfish poisoning, has been found in diatoms and red algae. While biosynthetic pathway towards DA from geranyl diphosphate and L-glutamate has been previously proposed, its late stage is still unclear. Here, six novel DA related compounds, 7'-methyl-isodomoic acid A (2) and B (3), N-geranyl-L-glutamic acid (4), 7'-hydroxymethyl-isodomoic acid A (5) and B (6), and N-geranyl-3(R)-hydroxy-L-glutamic acid (7), were isolated from the red alga, Chondria armata, and their structures were determined.

Syntheses and Biological Activities of the LMNO, ent-LMNO, and NOPQR(S) Ring Systems of Maitotoxin.

Structure-activity relationship studies of maitotoxin (MTX), a marine natural product produced by an epiphytic dinoflagellate, were conducted using chemically synthesized model compounds corresponding to the partial structures of MTX. Both enantiomers of the LMNO ring system were synthesized via aldol reaction of the LM ring aldehyde and the NO ring ketone. These fragments were derived from a common cis-fused pyranopyran intermediate prepared through a sequence involving Nozaki-Hiyama-Kishi reaction, intramolecular oxa-Michael addition, and Pummerer rearrangement.

Differential binding of tetrodotoxin and its derivatives to voltage-sensitive sodium channel subtypes (Na 1.1 to Na 1.7).

Background And Purpose: The development of subtype-selective ligands to inhibit voltage-sensitive sodium channels (VSSCs) has been attempted with the aim of developing therapeutic compounds. Tetrodotoxin (TTX) is a toxin from pufferfish that strongly inhibits VSSCs. Many TTX analogues have been identified from marine and terrestrial sources, although their specificity for particular VSSC subtypes has not been investigated.