Paralytic Shellfish Poisoning

Paralytic shellfish poisoning (PSP)2225 is one of the most severe forms of food poisoning caused by ingestion of seafood. It is acute and often fatal. There is no effective way to destroy the toxins or to treat the patients. Therefore, it poses serious health problems. Deterring shellfish consumption causes economic problems. The history of the poisoning goes back to prehistoric days and the incidents due to the consumption of toxic shellfish are well documented. The problem exists along both the East and West coasts, often leading to a total ban on shell fishing in a wide area with enormous economic loses. It is now known that the toxic principles responsible for toxic effects are produced by a marine plankton Gonyaulax catenella and some other dinoflagellates. At certain unpredictable times these red plankton multiply and cause fed tide'.' Although, many fishes are killed by this fed tide',' mussels and clams survive but concentrate the toxic principles, thus becoming poisonous to humans. The toxin isolated from the Alaskan butter clam, California mussel and the marine microalga Gonyaulax catenella is called saxitoxin (1).2629 It is now established that saxitoxin (1), neosaxitoxin (7) and their congeners (2-18) are involved in paralytic shellfish poisoning (PSP). The dinoflagellate that produce saxitoxin and its congeners are Alexandrium spp. (formerly Gonyaulax or Protogonyaulax), Gymnodinium catenatum, Pyrodinium bahamense var. compressum.30 The origin of saxitoxin

R1HN-

R1 = R2 = R3 = H (Saxitoxin) R1 = R2 = H, R3 = OSO-R1 = R3 = H, R2 = OSO-R1 = OSO-, R2 = R3 = H R1 = R3 = OSO-, R2 = H R1 = R2 = OSO-, R3 = H

R1 = R2 = R3 = H (Saxitoxin) R1 = R2 = H, R3 = OSO-R1 = R3 = H, R2 = OSO-R1 = OSO-, R2 = R3 = H R1 = R3 = OSO-, R2 = H R1 = R2 = OSO-, R3 = H

R3 = H (Neosaxitoxin) H, R3 = OSO-H, R2 = OSO-= OSO-, R2 = R3 = H

R3 = H (Neosaxitoxin) H, R3 = OSO-H, R2 = OSO-= OSO-, R2 = R3 = H

R1HN

(1) in PSP was found to be bacteria.31 Saxitoxin (1) and neosaxitoxin (7) are also reported to be produced by the fresh water blue-green alga, Aphanizomenen flos-aquae.32334 Structurally the toxins of this class could be divided into two major groups, saxitoxin (1) and neosaxitoxin (7). The members of these two groups are further diversified by the presence of 11-O-sulphate or N-sulphate, the absence of carbamoyl group (13, 14, 15) and oxygen at C-13 (15, 17).

2.1 Transfer of Toxins between Organisms

Originally it was thought that the shellfish accumulate toxins by filter feeding the toxic plankton during blooms. The toxins then enter into the hepatopancreas where most toxicity is normally found. It was expected that the toxicity of the shellfish will be lost over several weeks or months after the disappearance of the plankton. But this did not happen. In fact, the first source of saxitoxin, the toxic Alaska butter clam, S. giganteus was found in water where no noticeable bloom of the toxic plankton was seen. Moreover, the toxicity which is mostly localized in the siphons, did not disappear even after a year in uncontaminated sea water. Paralytic shellfish poisons were found in nonfilter feeding snaits, crabs, and toxic macro algae where the secondary transfer of the toxins was not possible. Thus, the mechanism of toxification of these organisms is still not clearly understood.

2.2 Saxitoxin

2.2.1 Isolation

Schantz et al35 first isolated pure saxitoxin (1) from the Alaskan butter clam using weakly basic Amberlite IRC 50 and alumina chromatography. The Alaskan butter clam is still considered the best source of saxitoxin. The isolation procedure is fairly simple. However, this procedure is not applicable for the isolation of other shellfish toxins, since these are not strongly basic.

A general procedure which is now commonly used had been developed.36,37 In the procedure,37 the mixture of the toxin is separated from the other constituents by selective absorption on Bio Gel P-2 or Sephadex G-15. The toxin fraction is eluted with a dilute acetic acid solution. The mixture of toxins is then applied on a column of weakly acidic carboxylic acid resin, Bio-Rex 70 in acid form, the acetic acid gradient elution furnishes pure toxins in the reverse order of the net positive charge of the molecule. The toxins with negative net charge are not separable by this technique. However, they can be separated by either preparative thin layer chromatography (TLC) or careful chromatography on Bio-Gel P-2.

2.2.2 Assay Methods

There are a number of assay methods for detecting saxitoxins.17 In the mouse assay, each mice weighing 20 g are injected (ip) with 1 ml test solution of adjusted pH and toxicity. Time of death is measured, and the toxicity in mouse units (Mu) is found from the standard table and corrected by factor obtained from control mice injected with the standard saxitoxin dihydrochloride solution and expressed in micrograms equivalent of saxitoxin dihydrochloride. One mouse unit (Mu) is the amount of toxin needed to kill a 20 g mouse in 15 min. The assay is very reliable. However, it does not give the amount of individual toxins and latent sulphated toxins. The serious drawback of the method is the requirement of mouse of uniform size, which is sometimes difficult to obtain.

Chemical Assay: Bates et al38 have developed a fluorometric assay of saxitoxin based on the degradation product formed by treatment with NaOH-H2O2. It is quite sensitive method for saxitoxin derivatives, but not for neosaxitoxin derivatives. The limitation of the method is that the other fluorescence products present in the crude extracts interfere in the measurements.

High-Pressure Liquid Chromatography (HPLC) Assay: The method developed by Sullivan et al39,40 is used for routine analysis. All toxins, including latent toxins, can be quantified in a fairly short time. The method is very useful for metabolic studies of toxins.

Immuno assay: Davio et al41 has developed a radioimmuno assay that is very sensitive in the detection of saxitoxin. However, the utility of the method depends upon the selection of an antibody with desirable cross reactivity to toxins with diverse structure variations.

Fly Bioassay: Ross et al42 have developed a method to substitute the mouse assay. In the method flies are temporarily immobilized at low temperature and injected with a minute quantity of test solution using a micro syringe. However, the method has not been officially recognized.

2.2.3 Chemistry

Chemistry of saxitoxin have been extensively studied.43-47 A tentative structure was proposed in 1971.45 The final structure (1) to saxitoxin was assigned by the X-ray crystallography of its p-bromobenzensulfonate salt46 (19) and its hemiketal. Saxitoxin has several interesting structural features. Its perhydro purine skeleton with an additional five member ring fused at the angular position is unprecedented. It has a ketone hydrate at position 12 stabilized by two neighboring electron withdrawing guanidinium groups. The ketone (21) is also readily enolized to effect the rapid exchange of protons at position 11. The molecule has two pKa values, viz. 11.5 and 8.1. The proton and carbon nuclear magnetic resonance (NMR) chemical shift studies under different pH conditions indicated that the later pH value is associated with the imidazoline guanidinium group.48,49 It is suggested that the abnormally low pKa for a

HolSL

HolSL

HoOo

21, Keto form

21, Keto form

guanidinium group is a result of the insufficient participation of N-7 in the guanidinium resonance, probably due to the stereochemical strain of the five member ring. A high resolution NMR study44 suggested that in the pH range of physiological condition, saxitoxin exist in an equilibrium of three molecular species; divalent cation (1) monovalent cation of the hydrated form (20), and monovalent cation in a keto form (21). Saxitoxin is very stable in acidic solution. For example, it can be kept in dilute hydrochloric acid solutions for a long time without loss of its potency. However, the toxin is extremely unstable under alkaline conditions, especially in the presence of oxygen, and undergoes facile oxidative degradation to yield the aromatized aminopurine derivatives (22) and (23) which can be more efficiently obtained by oxidation of the toxin (1) with H2O2.50

The oxidation products are highly fluorescent, and the reaction is used for quantitative estimation. The hydrolysis of the carbamoyl ester function of saxitoxin can only occur in concentrated acid solutions, such as 7.5 N HCl at 100°C. Structure activity relationship studies have also been carried out on saxitoxin. Hydrogenation of saxitoxin with platinum catalyst afforded the hydroxyl isomer of (12^) dihydro saxitoxin (24), whereas reduction with borohydride gave a mixture of (24) and the ^-isomer (25).44 Decarbamoylation of saxitoxin afforded the compound (26) which retains about 70% original toxicity.50

nh

2.2.4 Neosaxitoxin

Neosaxitoxin (7)37,51 was isolated as a minor product from an Alaskan butter clam. Later on it was isolated as a major constituent from most toxic shellfish, dinoflagallates, blue-green algae and the crab samples. Neosaxitoxin was characterized as 1-N-hydroxy-saxitoxin (7) by spectroscopic and chemical evidence by Shimizu et al52 The structure was later confirmed by 15N-NMR.53 Treatment of neosaxitoxin (7) with zinc-acetic acid yielded saxitoxin (1) and dihydro saxitoxin. Neosaxitoxin was not as stable as saxitoxin in acidic conditions, and tends to decompose in hydrochloric acid solutions. The imidazoline guanidine ring of neosaxitoxin has a pKa value similar to saxitoxin (8.65). However titration and NMR studies indicated the presence of another pKa 6.75, which is suggested due to the hydroxy-guanidine group of purine ring in (7).54 Electrophoretic study confirmed that the net positive charge of the molecule around physiological pH is reduced to about half of saxitoxin.54

2.2.5 Gonyautoxin-I

Gonyautoxin-1 (29) was first obtained from soft-shell clams exposed to G. temerensis blooms.42 Subsequently, it was found to be a major component in hon hon

Zn-AcOH

Ac2O/Pyridine h2n pKa = 6.75

Ac2O/Pyridine

Zn-AcOH

nh many PSP samples. Its structure (29) was established on the basis of spectroscopic data and chemical correlation with gonyautoxin-II, neosaxitoxin and saxitoxin.55-58 Reduction of gonyautoxin-I (29) gives a mixture of neosaxitoxin (27) and gonyautoxin-II (30), which can be further reduced to saxitoxin (1). Similar reductive biotransformation was observed in scallop tissues.56 Gonyautoxin-I and its stereoisomer gonyautoxin-IV are probably the most unstable among the PSP toxins.

2.2.6 Gonyautoxin-II

Gonyautoxin-II (30), as a major toxin, was first isolated from soft-shell clams from the New England coast.36, 59 Subsequently, it was found to be a major toxin in a number of samples. The structure (30) of gonyautoxin-II was assigned by spectroscopic data and extensive chemical degradation methods.60 It was first reported to be a free 11-hydroxy derivative of saxitoxin, but later amended to its sulphate ester.61 Finally, structure (30) was confirmed by correlation with saxitoxin.56

2.2.7 Gonyautoxin-III

Gonyautoxin-III (31) is the 11-epimer of gonyautoxin-II (30).60,61 It forms about 7:3 equilibrium mixture of (31) and (30) in a neutral or higher pH solution. It is believed that thermodynamically less stable gonyautoxin-III (31) is the parent form that exists in the living organism.

2.2.8 Gonyautoxin-IV

Gonyautoxin-IV (32) is the 11-epimer of gonyautoxin-I (29).

2.2.9 Gonyautoxin-V

Gonyautoxin-V was first isolated from the Japanese and Alaskan PSP samples. Subsequently, it was found in a number of other organisms.55,62,63 It is the major toxin in the tropical dinoflagellate, P. bahamense var. compress.64 The structure was established by several groups under different names.65,66 Gonyautoxin-V (33) is the carbamoyl-N-sulphate of neosaxitoxin (27), and almost nontoxic. Hydrolysis of (33) with weak acid yielded neosaxitoxin (27) a potent toxin.

2.2.10 Gonyautoxin-VI

Careful chromatography of the mixture of toxins on Bio-Rex 70 yielded gonyautoxin -VI.50, 58 The toxin was the carbamoyl-N-sulphate of saxitoxin (1) and was found to be identical with B2 toxin.65,66 Treatment of the toxin with dilute mineral acid afforded saxitoxin. A partial synthesis of gonyautoxin-VI was achieved by sulfonylcarbamoylation of saxitoxin and neosaxitoxin.65

2.2.11 Gonyautoxin- VII

Gonyautoxin-VII (34) was first found in a toxin mixture from the sea scallop, Placopecten megallanicus.61 The toxin was subsequently found identical with decarbamoyl saxitoxin.66, 68 An identical compound was later found in the little neck clam and considered to be a product of biotransformation in the shellfish.69 A number of decarbamoyl derivatives of PSP toxins have been isolated from the tropical dinoflagellates P. bahamense var. compressa.68

2.2.12 Gonyautoxin-VIII

Gonyautoxin-VIII is the first toxin that was found to have an N-sulfonyl group and a negative net charge on the molecule.70,71 The toxin was characterized as carbamoyl-N-sulfonylgonyautoxin-III (35). It was easily isomerizes to epigonyautoxin-VIII. Treatment of the toxin with dil. mineral acid easily afforded gonyautoxin-III (30).

Epi Gonyautoxin

2.2.13 C3 and C4 Toxins

C3 And C4 toxins are called latent toxins.63 They are not retained on a cation exchange column because of their net negative charge. The C3 and C4 toxins were assigned the structure (36) and (37), respectively.

The structure of C4 toxin was confirmed by X-ray crystallography. Treatment of the C3 and C4 toxins with dilute acids readily yielded gonyautoxin-I and IV, respectively. The presence of sulphate conjugation is characteristic of saxitoxin class of toxins. In the dinoflagellates, most toxins occur as 11-O-sulphate and/or N-sulphocarbamoyl derivatives, and saxitoxin (1) is a minor component. The occurrence of N-sulphated groups is rather rare among natural products. The N-sulphated groups are easily hydrolysed by weak acids and also possibly by the enzymes in the biological system. It is not yet clear whether the formation of the sulphated toxins precedes the unsulphated ones in the dinoflagellates. However, it has been proved that the reductive cleavage of O-sulphate could take place in shellfish to give unsulphated toxins such as saxitoxin. Similarly, the N1-hydroxy group of neosaxitoxin (7) series can be reductively removed. It is not yet known whether saxitoxin types of compounds are the precursors of neosaxitoxin types or vice-versa in the dinoflagellates.

2.2.14 Total Synthesis of (±)-Saxitoxin

Synthesis of (±) saxitoxin has been achieved by two groups. Kishi's group72 at Harvard was the first to report the synthesis of (±) saxitoxin. The strategy of the synthesis (Scheme 1) was to construct A/B ring first, and then attach the ring C (38-45).

MeOX.

(l)PhCH2OCH2CHO

CH2OCH2Ph HN-"\ ^NHCONHo

(l)PhCH2OCH2CHO

Rearrangement

CH2OCH2Ph HN— V -NHCONH

Curtius

Rearrangement

SH(CH2)3SH

CH2OCH2Ph HN— V -NHCONH

Ac0H/CF3C02H

Ac0H/CF3C02H

S-41

S-41

44 45

Scheme 1

The key step in the synthesis was the acid catalyzed ring closure of the bicylic intermediate (41). Using a mixture of acetic acid and trifluoroacetic acid, the compound (42) was obtained in 50% yield. Removal of the protecting groups gave (44) which on treatment with chlorosulfonyl isocyanate followed by hydrolysis finally furnished (±)-saxitoxin (45).

Jacobi's group73 reported a new synthesis of (±)-saxitoxin in 1984. The strategy in the synthesis (Scheme 2) (46-52) was to construct the C/D ring of saxitoxin first and then to add ring A. Thus the spirobicyclic intermediate (51) was cyclized to the tricyclic compound (52), which was then converted into (±)-saxitoxin (45) as in case of Kishi's synthesis.

COOMe

COOMe

Ph^N

NaOMe, NaBH4

Ph N

NaOMe, NaBH4

(1) bf3dms

Scheme 2

2.3 Detection of Paralytic Shellfish Toxins

Several human ailments, such as ciguatera, paralytic shellfish, and diarrhetic shellfish poisoning are caused by the ingestion of toxins produced by marine organisms. Initially, it was thought that PSP was restricted to temperate coastal areas and involved only filter feeding molluscs, recent evidence, however, indicates that the problem is widespread. It is now apparent that the toxins are present not only in molluscs and dinoflagellates but also in zooplankton, crab, red alga and a variety of interstidal organisms. It was imperative to develop qualitative and quantitative methods of detection of these toxins. The methods available now have been reviewed.74-78

2.3.1 Bioassays

The biological assays for marine toxins are most widely utilized method for their detection. A wide range of organisms are sensitive to the toxins and therefore are potential test organisms for a bioassay, but the mouse and housefly are the only species utilized to date. The mouse bioassay has been adopted by the Association of Official Analytical Chemists as an Official Procedure','and is in use today as the primary analytical technique to support the majority of toxin-monitoring programmes in shellfish.

2.3.2 Sodium Channel Binding Assays

The pharmacological activity of the saxitoxins at the molecular level has been exploited in developing assay techniques. The toxins bind to sodium channels in nerve cell membranes, preventing the influx of sodium and subsequent depolarization of the membrane. A number of electrophysiological systems have been utilized for measuring the binding events. These are frog sciatic nerve,79 voltage clamp of single nerve cell,80 and blockage of sodium conductance through single-sodium channel isolated in lipid bilayers.81 These techniques are useful for determining the pharmacological properties of the toxins. They are, however, unlikely to serve as routine assay techniques.

2.3.3 Immunoassays

Johnson et al82,83 were the first to develop immunological techniques for assaying saxitoxins. Saxitoxin was coupled to bovine serum albumin (BSA) via formaldehyde treatment and antibody prepared from rabbit antiserum. Carlson et al84 developed a radio-immunoassay (RIA) capable of detecting low level of saxitoxin. 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 the toxicity of a shellfish extract is due to the collective effect of a number of different toxins present, the application of immunoassays for accurate detection of Total Toxicity'is very difficult. However, immunoassay methods can be of much use as a rapid Field Test"for detecting the presence of the PSP toxins.

2.3.4 Chemical Assays

Schantz et al86 have developed a colorimetric assay based on the reaction of the saxitoxins with picric acid. The method, however, is not sensitive and prone to interferences. Gershey et al81 have described a colorimetric test based on a reaction with 2,3-butanedione, but this was also subject to interferences. Bates and Rapoport38 have reported a chemical assay for STX based on fluorescence of the 8-amino-6-hydroxymethyl-2-iminopurine 3(2H)-propionic acid a hydrogen peroxide oxidation product of STX. The method is extremely sensitive and fairly specific for the PSP toxins.

2.4 Tetrodotoxin

Tetrodotoxin (TTX) is the best known marine toxin because of its frequent involvement in fatal food poisoning, unique chemical structure, and specific action of blocking sodium channels of excitable membranes.88 The toxin derives its name from the pufferfish family (Tetraodontidae) and occurs widely in both the terrestrial and marine animal kingdom.89 The marked fluctuation of toxin concentration in TTX-containing animals from different regions, and seasons led to the belief an exogenous origin of the toxin in these animals. The primary source of the toxin was traced by Yasumoto et al90 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 a Alteromonas sp. and finally Shewanella alga.91 Subsequently, it was found that the toxin is produced by a broad spectrum of bacteria.92-94 However, the identification of the toxin in bacterial cultures had been made on the basis of rather poor evidence.

2.4.1 Chemistry

Tetrodotoxin is a colorless crystalline compound. It is virtually insoluble in all organic solvents but soluble in acidic media. It is weakly basic having the composition C11H11N3O8. The molecule is small (mol. wt. 319), but possesses the remarkable feature that the number of oxygen and nitrogen atoms are equal to the number of carbon atoms. The chemistry and biology of tetrodotoxin has been extensively studied95-101 and reviewed.102-112 Woodward95 demonstrated that the three nitrogen atoms of tetrodotoxin are present in the molecule as a guanidine moiety by isolating guanidine (as the picrate), following vigorous oxidation of the toxin with aqueous sodium permanganate at 75°C. Drastic degradations of the toxin by warm aqueous sodium hydroxide, pyridine-acetic anhydride followed by vacuum pyrolysis, phosphorus hydrogen iodide followed by potassium ferricyanide, and conc. sulfuric acid, gave closely related quinazoline derivatives of structure (53), where the nature of R depended on the exact mode of degradation. The formation of these key compounds indicated strongly that six of the 11 carbon atoms of tetrodotoxin are contained in a carbocyclic ring. It was surprising that in spite of the presence of the guanidine function in the molecule, the toxin was only weakly basic (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-O/,O/-isopropylidene-tetrodotoxin hydrochloride monohydrate which was given structure (54) on the basis of X-ray crystallographic analysis.95 If one element of acetone and methanol is subtracted from the molecular formula of the toxin derivative (C15H23N3O8) and adds two molecules of water one arrives at C11H17N3O8, the exact formula of tetrodotoxin.

Comparison of the NMR spectra of compound (54) and tetrodotoxin further confirmed their close structural relationship. The two compounds, however, differ in one aspect. The compound (54) was a lactone having IR absorption band at 1751 cm1 while the toxin itself lacked a lactonic infrared band. On the other hand, the IR bands assigned to the guanidine moiety (1638, 1605 cm1 ) remained unchanged in the two compounds, thus demonstrating that the hydrochloride cannot be a guanidinium salt. The basicity of tetrodotoxin (pKa 8.5) was far too weak to be originating from the guanidine moiety. This fact, coupled with the observation that the pKa of the hydrochloride increased to 9.2 in aqueous dioxane, strongly suggested that the basicity of tetrodotoxin must be due to its Zwitterionic nature and that one of the hydroxyl groups is being titrated when the pKa is measured. Increased pKa is characteristic of hydroxyl ionization when one proceeds from a medium of high to one of low dielectric constant. That which of the hydroxyl groups in tetrodotoxin is sufficiently acidic to furnish a proton to nitrogen, was revealed by the NMR spectral measurements of heptaacetyl-anhydrotetrodotoxin. If the methylated precursor of (54) was to undergo acetylation, the product would exhibit three characteristic changes, resonances in the NMR arising from protons on carbon which also bear acetoxy groups, viz. C-5, C-7 and C-8. In fact only one such resonance was present in the NMR spectrum of the heptaacetyl compound which forced the conclusion that two of the three groups cannot be present as free hydroxyls in tetrodotoxin, but must be combined in a new entity. If one of the hydroxyl groups combines with the lactones function to form a hemiacetal, only one characteristic proton should remain. Double resonance experiments proved that it is the C-5 hydroxyl in tetrodotoxin which is part of the hemilacetal (or a two-third orthoester) function. This consideration led to the assignment of structure (55) for tetrodotoxin. The presence of hemiacetal function in tetrodotoxin is unique (55a, 55b). This is the first example where a complex function of this nature is present in a natural product.

55a 55b

The monomeric structure of tetrodotoxin was confirmed by single crystal X-ray diffraction studies by Woodward et al.113 Measurement of the unit cell dimensions and the density of the crystals and consideration of symmetry requirements led to the unambiguous conclusion that crystalline tetrodotoxin is monomeric and contains two molecules per unit cell. The monomeric nature of tetrodotoxin in solution had been ascertained through a careful analysis of its titration curve.99 About 1-2 g of crystalline precipitate (tetrodotoxin) was obtained from 100 kg of puffer ovaries by following Hirata's procedure.99 Total synthesis of tetrodoxin was reported in 2004.100

2.4.2 Tetrodotoxin Derivatives

Detection of tetrodotoxin (TTX) derivatives occurring in puffers, newts, and a frog was facilitated by a highly sensitive TTX analyzer, which separates analogues on a reversed phase column and detects fluorescent products formed upon heating with sodium hydroxide solution.114, 115 Yasumoto et al116 have isolated tetrodotoxin (55), 4-epi-TTX (56), 6-epi-TTX (57), 11-deoxy-TTX (58) and 11-deoxy-4-epi-TTX (59) from newts collected in Okinawa, Japan, and assigned their structure mainly through NMR measurements. 11-Nortetrodotoxin-6 (R)-ol, 6-epi TTX (57) and 11-deoxy TTX (58) have been obtained from the puffers Fugu niphobles.111 Chiriquitoxin,118-23 an unusual analogue of tetrodotoxin in which 11-CH2OH of TTX had been replaced by a CH(OH)CH(NH2)-CO2H group, had been isolated from the Costa Rican frog Atelopus chiriquiensis.118 The puffer Arothron nigropunctatus had

Compound

R1

R2

R3

R4

55, TTX

H

OH

OH

OH

56, 4-epi-TTX

OH

H

OH

OH

57, 6-epi-TTX

H

OH

CH2OH

OH

58, 11-deoxy-TTX

H

OH

OH

CH3

59, 11-deoxy-4-epi

OH

H

OH

CH3

60, 11-oxo-TTX

H

OH

OH

ch2oh

furnished 11-oxotetradotoxin hydrate (60) TTX, 4-epi TTX, 6-epi TTX, 11-deoxy TTX, 11-nor TTX-6(R)-ol.115 Two epimers of 11-nor TTX are likely to be decarboxylation products of a hypothetical 11-CO2H derivative.

Those analogues found in puffers and newts were not found in a Costa Rican frog, Atelops chiriquiensis which contained TTX and chiriquitoxin. Interestingly, l-oxo TTX (60) was more active than TTX in blocking sodium channels.124'125 Other analogues were less potent than TTX. A pocket shaped model has been proposed for binding site in the sodium channel protein. The charge groups in a clevis of channel protein supposedly act as anchoring points by interacting with the toxin's functional groups orienting in different directions.125

2.4.3 Mechanism of Tetrodotoxin and Saxitoxin Action Tetrodotoxin (55) and saxitoxin (1) are the most widely studied marine toxins by physiologists and pharmacologists. In spite of their structural dissimilarities, both are known to inactivate the sodium channel in the skeletal muscles and nerve tissues of various animals. The effect of these toxins is specific as both selectively block the transient Na+ current without any effect on the steady state current by K+ ions. Owing to this specific action of these toxins, many investigators are using them as tools in the characterization of ion channels. These toxins have become an extremely useful and popular chemical tool for the study of neurophysiology and neuropharmacology. Tetrodotoxin binds to the entrance part of the Na+ channel and inhibits Na+ channel and Na+ influx, and generates an active potential, thus, causing the blockade of nerve of muscle function. Narahashi126 has reviewed the mechanism of tetrodotoxin and saxitoxin action. Tetrodotoxin is commercially available and in carefully controlled doses is being used as muscle relaxant and pain killer in neurogenic leprosy and terminal cancer.

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