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.It is, suggested that the residual labelling of the carbamoylgroup presumably arises from metabolism of the precursor, which is a commonproblem during biosynthesis of toxins in these marine organisms.Feedingwith [2-13C] glycine demonstrated20 that the carbon atoms C-11 and C-12 of13gonyautoxin were specifically labelled with C, whereas the labels werefound scrambled when [13C2]-acetate was fed to G.tamarensis.It was proposedthat arginine combines with acetate such as at C(5)-C(6) and to a lesser extentof C10-C11, while [2-13C] acetate labelled C-6 and C-11 but not C-5.Theresults are consistent in which a Claisen condensation occurs at the ±-carbonof arginine and decarboxylation takes place in the presence of the adjacentamino function.The incorporation of doubly labelled [2-13C, 2-15N] ornithineinto neosaxitoxin revealed that the C4-N bond remains intact in the biosynthesisof the toxin.21 The hydroxymethyl function at C-6 has been assumed to bederived from acetate.Feedings of [13C] carbon dioxide, of [13C] formate, andof 3-hydroxy[1-13C] propionate were unsuccessful, suggesting that this carbonatom is not derived via C1 or C3 unit.Feeding of [1,2-13C2] glycine, [3-13C]serine, or [methyl-13C] methionine led to labelled neosaxitoxin (2) enrichedat C-13 with 13C label, consistent with the operation of the C1-tetrahydrofolatepathway.19 Experiments have shown that (2) is converted into (1) in shellfish,demonstrating thereby that N-hydroxylated toxins are converted into N-nonhydroxylated ones.22The origin of the saxitonins is precisely not known.Some believe thatthese toxins result from the bacterial contaminants within the dinoflagellates.Shimizu et al21 have shown that the toxicity is an inherent character of eachof these algae species and is not the result of symbiotic microorganisms.The Biosynthesis of Bioactive Metabolites of Marine Organisms 129cyanobacterium A.flosaquae, used in the experiments, did not containsymbionts.The chemistry, biochemistry and biogenesis of saxitoxin (1), itsrelatives and tetrodotoxin (23) have been reviewed.184.2 BrevetoxinsThe dinoflagellate Gymnodinium breve Davis (syn.Ptychodiscus brevis Davis)causes heavy fish mortality in the Gulf of Mexico.A number of toxins,known as the brevetoxins (GB-toxins), have been isolated from thisdinoflagellate.23-28 The brevetoxin-B (13), brevetoxin-C (14) and brevetoxin-3 (15) all contain 11 transfused ether rings and differ only in the substituentsat C-39.Other toxins, GB-5 and GB-6, have acetoxy or epoxy functions atthe periphery of the molecules.Brevetoxin-A (16) differs in the nature of theA,B ring systems.29 The brevetoxins may be derived in nature from atranspolyene precursor by an epoxide-mediated cyclisation, giving the desiredabsolute configuration at every olefinic center.3013, X = O, Y = CH2, R = H14, X = HCl, Y = O, R = H15, X = HOH, Y = CH2, R = H16Feeding experiments have been carried out on neurotoxin brevetoxin-B(13) in the ten day old cultures of G.breve.30,31 The organism was treated withantibiotics to remove bacterial contaminants and then supplied with the levelledprecursors.During feeding with the precursors, it was observed that the 130 Bioactive Marine Natural Productsgrowth of cultures was often highly erratic.Acetates stimulated the growthin some experiments, while in some other there was a rapid deterioration too.The labeling pattern of brevetoxin-B (13) as obtained by feeding experimentswith [1-13C], [2-13C] and [13C2] acetates and [methyl-13C] methionine is asfollows : 16 carbon atoms of (13) were found to be derived from [1-13C]acetate; 30 carbons from [2-13C] acetate and four carbons (the methyl groupsat C-8, C-22, C-25 and C-36) from [methyl-13C] methionine.The experimentsfurther revealed that the carbon backbone is not a simple polyketide andcontains six sets of adjacent carbon atoms that are both labelled by [2-13C]acetate plus two sets of three adjacent carbons which are labelled.Similarresults were obtained on the biosynthesis of (13) by Schimizu and Chou32 whoalso found that prolonged incubation produced random, but differential labelling.Short-term incubation (for two days) with [2-13C] acetate furnished brevetoxin-B in which the same 30 carbon atoms were labelled, but 18 carbon atomsshowed splitting patterns due to the presence of an adjacent 13C atom.Studieswith [13C2] acetate revealed the presence of 5 intact acetate units.These datawere rationalized by the involvement of succinate, 2-oxoglutarate and propionate.The remaining six-, five- and four-carbon fragments were rationalized as aunit that had been derived from 3-hydroxy-3-methylmalonate.Schimizu andKobayashi33 have suggested that the biosynthesis of brevetoxin-B is mixedtype.The fragments may be formed by the Claisen-type condensation ofdicarboxylic acids followed by decarboxylation.4.3 TetrodotoxinTetrodotoxin (17) a highly toxic compound, has been isolated from a varietyof marine organisms, including pufferfish, newts and blue-ringed octopus.Itis believed to be a product of bacterial metabolism.21,34 Feeding experiments14with C-labelled citrulline, arginine, glucose and acetate to newts by injectionor oral administration did not furnish labelled tetrodotoxin, although theseprecursors were metabolised by the organisms to produce sterols and aminoacids.It is postuated that they are produced only in response to aggression ordevelopmental needs.However, the biosynthesis of (17) remains an unsolvedproblem.109+8A7 182114536 4A17 Biosynthesis of Bioactive Metabolites of Marine Organisms 131CarotenesThe red or pink colours of halophilic bacteria are due to a wide variety ofisoprenoid compounds (phytoene, phytofluene, lycopene and ²-carotene).An unusual character of halophilic bacteria is that they need at least 15-25%salt for their normal growth.The biosynthesis of carotenoids of Halobacteriumcutirubrum, carried out with cell-free preparation using labelled precursors,proceeds as follows: Isopentyl phyrophosphate-trans-phytoene-trans-phytofluene-carotene ²-carotene.35 It has also been demonstrated that theC50 red pigments bacterioruberins are formed from C40 carotene, lycopene.364.4 SterolsAlgal sterols play a key role in the marine environment because algae are atthe bottom of the food chain.The status of marine sterols has been reviewedby Kerr and Baker
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