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.However, neosaxitoxin exhibited no cross reactivity.Chu et al85 have developed an enzyme linked immunosorbent assay[ELISA] to the PSP toxins that is sensitive to about 2-10 pg STX.Since thetoxicity of a shellfish extract is due to the collective effect of a number ofdifferent toxins present, the application of immunoassays for accurate detectionof otal Toxicity is very difficult.However, immunoassay methods can beTof much use as a rapid Field Test for detecting the presence of the PSPtoxins. 164 Bioactive Marine Natural Products2.3.4 Chemical AssaysSchantz et al86 have developed a colorimetric assay based on the reaction ofthe saxitoxins with picric acid.The method, however, is not sensitive andprone to interferences.Gershey et al87 have described a colorimetric testbased on a reaction with 2,3-butanedione, but this was also subject tointerferences.Bates and Rapoport38 have reported a chemical assay for STXbased on fluorescence of the 8-amino-6-hydroxymethyl-2-iminopurine 3(2H)-propionic acid a hydrogen peroxide oxidation product of STX.The methodis extremely sensitive and fairly specific for the PSP toxins.2.4 TetrodotoxinTetrodotoxin (TTX) is the best known marine toxin because of its frequentinvolvement in fatal food poisoning, unique chemical structure, and specificaction of blocking sodium channels of excitable membranes.88 The toxinderives its name from the pufferfish family (Tetraodontidae) and occurswidely in both the terrestrial and marine animal kingdom.89 The markedfluctuation of toxin concentration in TTX-containing animals from differentregions, and seasons led to the belief an exogenous origin of the toxin inthese animals.The primary source of the toxin was traced by Yasumoto etal90 from fish to a dietary alga and finally to an epiphytic, or symbiotic,bacterium.The bacterium was first thought to be a Pseudomonas sp.then aAlteromonas sp.and finally Shewanella alga.91 Subsequently, it was foundthat the toxin is produced by a broad spectrum of bacteria.92-94 However, theidentification of the toxin in bacterial cultures had been made on the basis ofrather poor evidence.2.4.1 ChemistryTetrodotoxin is a colorless crystalline compound.It is virtually insoluble inall organic solvents but soluble in acidic media.It is weakly basic having thecomposition C11H17N3O8.The molecule is small (mol.wt.319), but possessesthe remarkable feature that the number of oxygen and nitrogen atoms areequal to the number of carbon atoms.The chemistry and biology of tetrodotoxinhas been extensively studied95-101 and reviewed.102-112 Woodward95demonstrated that the three nitrogen atoms of tetrodotoxin are present in themolecule as a guanidine moiety by isolating guanidine (as the picrate), followingvigorous oxidation of the toxin with aqueous sodium permanganate at 75C.Drastic degradations of the toxin by warm aqueous sodium hydroxide, pyridine-acetic anhydride followed by vacuum pyrolysis, phosphorus hydrogen iodidefollowed by potassium ferricyanide, and conc.sulfuric acid, gave closelyrelated quinazoline derivatives of structure (53), where the nature of Rdepended on the exact mode of degradation.The formation of these keycompounds indicated strongly that six of the 11 carbon atoms of tetrodotoxinare contained in a carbocyclic ring.It was surprising that in spite of the Bioactive Marine Toxins 165presence of the guanidine function in the molecule, the toxin was only weaklybasic (pKa 8.5) and attempts to prepare crystalline salts did not succeed.However, treatment of the toxin with 0.2 N hydrogen chloride in methanol-acetone did furnish a crystalline O-methyl-O2 ,O2 -isopropylidene-tetrodotoxinhydrochloride monohydrate which was given structure (54) on the basis ofX-ray crystallographic analysis.95 If one element of acetone and methanol issubtracted from the molecular formula of the toxin derivative (C15H23N3O8)and adds two molecules of water one arrives at C11H17N3O8, the exact formulaof tetrodotoxin.5354Comparison of the NMR spectra of compound (54) and tetrodotoxin furtherconfirmed their close structural relationship.The two compounds, however,differ in one aspect.The compound (54) was a lactone having IR absorption1band at 1751 cm while the toxin itself lacked a lactonic infrared band.On the1other hand, the IR bands assigned to the guanidine moiety (1638, 1605 cm )remained unchanged in the two compounds, thus demonstrating that thehydrochloride cannot be a guanidinium salt.The basicity of tetrodotoxin(pKa 8.5) was far too weak to be originating from the guanidine moiety.Thisfact, coupled with the observation that the pKa of the hydrochloride increasedto 9.2 in aqueous dioxane, strongly suggested that the basicity of tetrodotoxinmust be due to its Zwitterionic nature and that one of the hydroxyl groups isbeing titrated when the pKa is measured.Increased pKa is characteristic ofhydroxyl ionization when one proceeds from a medium of high to one of lowdielectric constant.That which of the hydroxyl groups in tetrodotoxin issufficiently acidic to furnish a proton to nitrogen, was revealed by the NMRspectral measurements of heptaacetyl-anhydrotetrodotoxin.If the methylatedprecursor of (54) was to undergo acetylation, the product would exhibit threecharacteristic changes, resonances in the NMR arising from protons on carbonwhich also bear acetoxy groups, viz.C-5, C-7 and C-8.In fact only one such 166 Bioactive Marine Natural Productsresonance was present in the NMR spectrum of the heptaacetyl compoundwhich forced the conclusion that two of the three groups cannot be presentas free hydroxyls in tetrodotoxin, but must be combined in a new entity.Ifone of the hydroxyl groups combines with the lactones function to form ahemiacetal, only one characteristic proton should remain.Double resonanceexperiments proved that it is the C-5 hydroxyl in tetrodotoxin which is partof the hemilacetal (or a two-third orthoester) function.This considerationled to the assignment of structure (55) for tetrodotoxin.The presence ofhemiacetal function in tetrodotoxin is unique (55a, 55b).This is the firstexample where a complex function of this nature is present in a naturalproduct.55a 55bThe monomeric structure of tetrodotoxin was confirmed by single crystalX-ray diffraction studies by Woodward et al
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