Biosynthesis of marine natural products: microorganisms andmacroalgae

Bradley S. Moore†

Department of Chemistry, Box 351700, University of Washington, Seattle, WA, 98195-1700, USA

Received (in Cambridge, UK) 16th June 1999Covering: 1989 through 1998Previous review: 1989, 6, 143

1 Introduction2 Marine microorganisms2.1 Marine bacteria2.2 Cyanobacteria2.3 Dinoflagellates2.4 Diatoms2.5 Symbiotic microorganisms2.6 Miscellaneous microorganisms3 Macroalgae3.1 Green algae3.2 Brown algae3.3 Red algae4 Acknowledgements5 References

1 Introduction

This review covers the literature published on the biosynthesisof marine microbial and macroalgal natural products over a10-year period from 1989 through 1998. A companion reviewcovering the same time period on the biosynthesis of naturalproducts from marine macroorganisms will appear at a laterdate in this journal. An earlier report by Garson published in thisjournal surveyed the whole field of marine natural productbiosynthesis through mid 1988.1 The field through mid 1992was reviewed elsewhere in an updated report,2 and severalreviews covering specific aspects of marine microbial andmacroalgal natural product biosynthesis have been publishedduring this period. Reviewed biosynthetic topics includedinoflagellate and algal sterol side chains,3,4 algal oxylipins,5–8

and microalgal metabolites.9,10

Some of the microorganisms discussed in this report are notstrictly marine. Bacteria isolated from coastal waters may haveoriginated from terrestrial habitats and washed into the ocean.Many of these bacteria found at the interface of terrestrial andmarine environments, especially the actinobacteria, toleratewide ranges of salinities. The biosynthesis of natural productsfrom freshwater cyanobacteria and microalgae are described forcompounds that are structurally related to marine products. Thebiosynthetic origins of many marine invertebrate-derivednatural products are not clear and have been proposed to involveassociated microorganisms. A section on symbiotic micro-organisms highlights our current knowledge on the involvementof invertebrate-hosted microorganisms in natural productbiosynthesis through cellular localization studies.

The review is organized on the basis of a similar taxonomicsystem used in previous marine natural product biosynthesis1, 2

and structure11 reviews. General labeling patterns consistentlyused throughout this report are outlined in Fig. 1.

2 Marine microorganisms

2.1 Marine bacteria

Marine bacteria have recently emerged as an entirely newsource of structurally novel natural products12,13 for thedevelopment of new drug candidates.14,15 The rich variety ofchemically novel and biologically active metabolites serves toindicate that marine bacteria are a genetically rich resource forrecombinant technologies. Biosynthetic studies with culturedmarine bacteria have increased over the past few years and in afew cases have expanded beyond simple feeding experimentswith labeled precursors to studies at the biochemical and geneticlevels.

The first marine bacterial natural product to be reported wasthe highly brominated pyrrole antibiotic pentabromopseudiline1 by Burkholder and coworkers in 1966 from a culture ofPseudomonas bromoutilis.16,17 Pentabromopseudiline has sincebeen identified together with the blue pigment violacein 2 fromseveral other seawater-derived bacteria, including Chromo-bacteria sp.18 and Alteromonas luteoviolaceus.19 Three differ-ent syntheses19–21 and a structure–activity relationship study22

have been reported. The biosynthesis of this highly unusualmetabolite, which is composed of more than 70% bromine byweight, was not apparent from its structure, leading to a study byLaatsch and coworkers with the A. luteoviolaceus strain.23 Anacetate origin of 1 was excluded from several 13C-acetatefeeding experiments. Rather, the benzene ring of 1 was shownto be carbohydrate-derived, whereas the origin of the pyrrolering was not deduced in this report involving numerous feedingexperiments with labeled acetate, glucose, and amino acids.Feeding experiments with differently labeled 13C-labeledglucoses are summarized in Scheme 1 and surreptitiouslyimplied that shikimic acid 3 is converted into a symmetrical

† Present address: College of Pharmacy, Department of Pharmacology andToxicology, University of Arizona, Tucson, AZ 85721-0207, USA. E-mail:moore@pharmacy.arizona.edu; Tel: +1 520 626 6931; Fax: +1 520 6262466.

Fig. 1

Scheme 1

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intermediate before assimilation into the benzene ring of 1(Scheme 2). The results of the glucose feeding experiments

were further complicated because this strain lacks the glycolyticenzyme triosephosphate isomerase, thus preventing the conver-sion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate and hence not into phosphoenolpyruvate. Although[2-13C]shikimic acid and its methyl ester were not incorporatedinto 1, neither labeled the co-produced alkaloid 2, which wasshown in this organism23 and in bacteria of the genusChromobacteria24–26 to be derived from the dimerization of twounits of tryptophan (Scheme 3). As shikimic acid and its methyl

ester were not transported into the cells of A. luteoviolaceus, anobservation documented in other microbial systems as well,27

the deduced intermediacy of shikimic acid in 1 biosynthesiscould not be directly verified. Feeding experiments with p-hydroxy-[2,3,5,6-2H4]- and p-hydroxy-[3,5-13C2]benzoic acids,however, demonstrated that p-hydroxybenzoic acid is convertedvery efficiently (approximately 85% specific incorporation)into the benzene moiety of 1 (Scheme 1). It is probablytransformed through decarboxylation into the benzene ring of 1,and the hydroxy group provides activation for further substitu-tions (Scheme 2).

The Andersen group in British Columbia has been very activein elucidating the biosynthesis of several marine microbialmetabolites. Their first venture into the study of microbialbiosynthesis involved the novel lactone oncorhyncolide 4,28 abiologically inactive metabolite produced by a seawater-derived Gram-negative bacterium.29 Several plausible bio-synthetic pathways were postulated, and these were testedthrough feeding experiments with 13C-labeled acetate. All of thecarbons in 4 are acetate-derived as summarized in the structureof 4. The incorporation data are consistent with a polyketideorigin of 4, whose pendant methyl groups (C15 and C16) are

derived from C2 of acetate. The feature of an acetate origin formethyl branches is uncommon and has been observed in somebacterial,30–34 cyanobacterial35 and dinoflagellate36–39 (seeSection 2.3) metabolites. The process has been proposed toinvolve the addition of malonate to a polyketide keto group togive a b-hydroxyacyl acid intermediate, which is decarboxy-lated and dehydrated in a similar manner to the loss of carbondioxide and water from mevalonic acid in terpenoid bio-synthesis (Scheme 4). Reduction of the resulting exomethylene

group leads to the methyl group. This pathway was corroboratedby a feeding experiment with [2-13C,2H3]acetate, which showedthat just two of the three hydrogens at each methyl group wereenriched with deuterium. Although the labeling pattern of thefive-carbon fragment C11–C14 and C16 is consistent with analternative pathway involving a mevalonate-derived starter unit,Andersen suggests that mevalonate is not a likely intermediatein 4 biosynthesis due to the combined observation that[2-13C]mevalonolactone is not incorporated into 4 and that 4 isuniformly labeled by acetate.

The biosynthesis of the antibiotic andrimid 5 has additionallybeen reported by the Andersen group.40 Andrimid and therelated metabolites moiramides A–C were produced by fermen-tation of the bacterium Pseudomonas fluorescens obtained from

the tissues of an unidentified Alaskan tunicate. Andrimid wasfirst reported from cultures of an Enterobacter sp. intracellularsymbiont of the brown planthopper Nilaparvata lugens41 andsince from a Vibrio obtained from an unidentified Hyatellasponge.42 Stable isotope feeding experiments demonstrated thatthe unusual acylsuccinimide unit, which was shown to berequired for its antimicrobial activity,43 is derived from aninteresting combination of acetate and amino acid buildingblocks. The proposed pathway involves the homologation ofvaline with malonyl-CoA to the corresponding g-amino-b-ketoacid, followed by the addition of glycine which in turn is chainextended with a second malonyl-CoA to give a putativedipeptide intermediate derived from two g-amino-b-keto acids(Scheme 5). Condensation followed by decarboxylation, dehy-dration and reduction of the resulting exomethylene group,analogous to that in Scheme 4, gives the g-lactam. Andrimid isprobably synthesized by a novel mixed polyketide/peptide

Scheme 2

Scheme 3

Scheme 4

654 Nat. Prod. Rep., 1999, 16, 653–674

synthetase that is capable of utilizing both malonyl-CoA andamino acids as substrates.

Two additional pseudomonads recently isolated from amarine alga and a marine tube worm by the Andersen group ledto the discovery of the antimycobacterial cyclic depsipeptidesmassetolides A–H44 and the previously known compoundviscosin 6.45, 46 Branched amino acids comprise five positionsof the massetolide/viscosin family of nonapeptolides, two ofwhich at positions AA4 and AA9 are naturally varied in theseries. Precursor-directed biosynthesis47 with nonproteinogenicamino acids, including l- and d-butyrine, l- and d-norvaline, l-and d-tert-leucine, and l-cyclopropylalanine, was used togenerate unnatural massetolides in order to extend the structuraldiversity of this series. Massetolide analogs, including masseto-lides I–K 7–9, were only generated from the l-butyrine, l-norvaline, and l-cyclopropylalanine substitution experiments atpositions AA1, AA4, and AA9. Unfortunately, the yields ofunnatural peptides were too low to provide sufficient sample forantimycobacterial testing.

Although the biosynthesis of edaphic streptomycete productsis well documented and has contributed to our basic knowledgeof secondary metabolism, only two studies have been reportedwith streptomycetes isolated from the marine environment. The

first study by Floss and coworkers involved the boron-containing ionophore aplasmomycin from the sediment-derivedStreptomyces griseus SS-2048–50 and is discussed in conjunc-tion with the structurally related marine cyanobacterial metabo-lite borophycin51 in Section 2.2. More recently, the biosynthesisof the bicyclic depsipeptide salinamide A 10, a potent anti-inflammatory agent52 and bacterial RNA polymerase in-hibitor,53 has been examined.54 The 10-producing strainStreptomyces sp. CNB-091 was isolated from the surface of thejellyfish Cassiopeia xamachana.52 The proposed pathwayinvolves the non-ribosomal peptide synthetase product Thr-d-Ile-Hpg-MePhe-d-aThr-Ser (Hpg = p-hydroxyphenylglycine)and proceeds through the desmethyl analogs of the naturallyoccurring salinamides E 11 and C 12 (Scheme 6). Extensivefeeding experiments with 13C-labeled intermediates indicatedthat both of the seven-carbon, non-amino acid residues of 10 areformed by a single chain extension of a carboxylic acid derivedfrom a branched amino acid. The (2S,3S)-3-hydroxy-2,4-dime-thylpentanoate unit is derived from isobutyrate via valine withmethylmalonyl-CoA extension, whereas the fragment bridgingthe p-hydroxyphenylglycine and glycine residues is derivedfrom the condensation of the isoleucine product tiglic acid withmalonyl-CoA. The mode of cyclization of 12 to 10 has beenproposed to involve either an epoxide intermediate which isopened by the Hpg phenol followed by dehydration and asecond epoxidation (Scheme 7, path a) or an Fe(ii)-dependentoxygenase mediated [2 + 2] cycloaddition followed bydehydrogenation (Scheme 7, path b). Such Fe(ii)-dependentoxygenase reactions have precedence in b-lactam chamistry andhave also been proposed to account for polyether formation inpolyketides such as monensin and brevetoxin.55

Recent advances in genetic engineering has transformed thefield of natural products, making it now possible to engineernovel small molecules56 and to functionally express largebiosynthetic gene clusters in a heterologous host.57 Yazawa andcoworkers report the first cloning and expression of a marine

Scheme 5

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bacterial product in a marine cyanobacterium.58 Shewanellaputrefaciens strain SCRC-2738, isolated from Pacific mackerelintestines, produces high concentrations of the polyunsaturatedw-3 fatty acid eicosa-5,8,11,14,17-pentaenoic acid (EPA).59,60

A 38 kb clone containing the EPA biosynthetic gene cluster wasfunctionally expressed in Escherichia coli61 and in the marinecyanobacterium Synechococcus sp.58 The cosmid carrying theEPA gene cluster contains eight open reading frames (ORFs),three of which are homologous to genes encoding enzymesinvolved in fatty acid de novo synthesis or in carbon chainelongation.61 Deletion of any ORF other than ORF1 in thecluster containing the EPA biosynthesis genes resulted in theloss of EPA production and did not accumulate intermediateproducts when cloned into E. coli, further verifying that this

gene cluster codes for EPA synthesis.61 As in the wild type S.putrefaciens, EPA production in the E. coli recombinant61 andin the Synechococcus transconjugant58 was higher at a lowertemperature than the optimal growth temperature. However,EPA was produced in lower titers in the cyanobacterialtransconjugant than in the E. coli recombinant, even though theEPA biosynthesis gene cluster contains its natural promoters.58

The proposed EPA biosynthetic pathway in S. putrefaciensinvolves the chain elongation and aerobic desaturation ofpalmitoyl-CoA, and is distinct from the anaerobic pathwayinvolved in the synthesis of n-7 type monounsaturated fattyacids.62

The ketocarotenoid pigment astaxanthin 13 is produced by amultitude of microorganisms, including the marine bacteriaAgrobacterium aurantiacum and Alcaligenes sp. strain PC-1,63

the yeast Phaffia rhodozyma,64 and the freshwater algaHaematococcus pluvialis.65 Several marine animals, includingcrustaceans and salmon, attain their red coloration fromcarotenoid pigments derived from their diet. Consequently, themetabolic engineering of 13 for the production of carotenoid-enriched feed supplements for cultured fish and shellfish hasgained considerable industrial attention.66,67 In addition, 13exhibits diverse biological activities, including anti-cancerproperties,68 enhancement of immune responses,69 and quench-ing of free-radicals,70 thus making 13 a very attractive candidatefor metabolic engineering. The biosynthesis of carotenoids,including 13 and its intermediates, is well characterized inseveral microbial systems at the biochemical and genetic levelsand has recently been extensively reviewed by Armstrong.71

The A. aurantiacum carotenoid biosynthesis gene cluster wasidentified by Misawa and coworkers and consists of five ORFsdesignated crtW (b-carotene ketolase), crtZ (b-carotene hydrox-ylase), crtY (lycopene cyclase), crtI (phytoene desaturase) andcrtB (phytoene synthase) and organized in a crtWZYIBoperon.72 Homologous genes have been identified in Alcali-genes sp. strain PC-1 by colony hybridization.73 A crtWhomolog has been isolated from the alga H. pluvialis and termedcrtO74 and bkt.75 A non-homologous ketolase gene from thecyanobacterium Synechocystis sp. PCC 6803, also designatedcrtO, codes for an asymmetrically acting b-carotene ketolasewhich introduces just one keto group on only one of the twoionone rings of b-carotene 14 to produce echinenone 15.76 ThecrtW homolog in the cyanobacterium, crtR, rather codes for asymmetrically acting hydroxylase that catalyzes the hydroxyla-tion of 14 to zeaxanthin 16.77 Interestingly, all bacterial crt geneclusters analyzed to date, with the exception of A. aurantiacum,contain a crtE gene which encodes a geranylgeranyl pyro-phosphate (GGPP) synthase which converts farnesyl pyro-phosphate (FPP) to GGPP.71 The analysis of carotenoidbiosynthesis genes from the nonphotosynthetic bacteria Erwi-nia uredova78 and Erwinia herbicola,79–81 which encode thesynthesis of 16 and its b-d-diglucoside, has led to a basic

Scheme 6

Scheme 7

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understanding of carotenoid biosynthesis. The functions of thecrt genes were partially deduced through the structural analysisof accumulated carotenoids in a genetically amenable non-carotenogenic heterologous host, E. coli, carrying variouscombinations of the Erwinia genes.78 Similarly, the functions ofthe A. aurantiacum crt genes were analyzed in E. colitransformants.72 The crtB, crtI and crtY genes, which code forthe synthesis of the carotenoid branchpoint intermediate 14from two moles of GGPP, are functionally equivalent in A.aurantiacum and Erwinia (Scheme 8).72 The further conversionof 14 to 13 formally involves the addition of a hydroxy group toC3 and a keto group to C4 on each ionone ring. The marinebacterial crtZ and crtW gene products CrtZ and CrtW arebifunctional in their activity and possess significantly broadsubstrate specificities allowing for multiple pathways leading to13 (Scheme 8).82,83 The Erwinia crtZ gene product is function-ally similar to the corresponding marine bacterial enzyme, buttheir substrate affinities, b-carotene versus canthaxanthin 17,are different.82 Astaxanthin and other new and known carote-noids have been metabolically engineered in several hosts thatnaturally do not synthesize carotenoids, such as E. coli,72,78,84,85

Zymomonas mobilis,86 Agrobacterium tumefaciens,86 and thefood yeasts Saccharomyces cerevisiae87 and Candida uti-lis.88–90 The level of carotenoid biosynthesis in E. colitransformants was increased 1.5–4.5 fold when isopentenyldiphosphate (IPP) isomerase genes from H. pluvialis, P.rhodozyma, or S. cerevisiae were co-expressed.91 This geneenhances the carbon flux of the isoprenoid pathway leading tothe formation of FPP in E. coli. Similarly, metabolic engineer-ing of the isoprenoid pathway in C. utilis, in which the3-hydroxy methylglutaryl coenzyme A reductase was overex-pressed, lead to an increase of carotenoid biosynthesis intransformants carrying exogenous crt genes.90 The cyanobacte-rium Synechococcus PCC7942, which normally accumulates 14and 16, is capable of synthesizing 13 and other ketocarotenoidswhen expressing the H. pluvialis crtO oxygenase gene.92

2.2 Cyanobacteria

Cyanobacteria (blue-green algae) are prokaryotic, photosyn-thetic microorganisms that are very rich in biologically activesecondary metabolites. Although research on the naturalproducts chemistry of cyanobacteria is very active and has beenrecently reviewed,93–95 biosynthetic studies have been few,especially with the marine strains. The biosyntheses of marineand freshwater cyanobacterial metabolites are discussed.

Shimizu’s work on saxitoxin and neosaxitoxin 18 bio-synthesis in the freshwater cyanobacterium Aphanizomenonflos-aquae was largely reviewed in the previous review in thisseries.1 Feeding experiments with 13C- and 2H-labeled pre-cursors have shown that neosaxitoxin is biosynthesized fromarginine and acetate and involves a Claisen-type condensationbetween C2 of arginine and C1 of acetate (Scheme 9).96–98

The cyclic heptapeptide microcystin-LR 19 is the majorhepatotoxin associated with toxic waterblooms of Microcystisaeruginosa found in the Northern Hemisphere.99 Microcystin-LR, a potent inhibitor of both type 1 and type 2A proteinphosphatases,100 has been implicated in net-pen liver disease, acommon toxicopathic disease of Atlantic salmon reared inseawater in British Columbia and Washington State.101 Stableisotope feeding experiments by the Moore group in Hawaiiestablished the origins of the unusual (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid(Adda) and (2R,3S)-3-methylaspartic acid (Masp) residues in19.102 The Adda unit is synthesized by the polyketide pathwayinvolving a putative phenylacetyl-CoA starter unit and fourmalonyl-CoA extensions. Sodium [1,2-13C2]acetate was incor-porated at C1 through C8 and the remaining Adda backbonecarbons were derived from l-[U-13C]phenylalanine. l-[methyl-13C]Methionine labeled the 2-, 6-, and 8-methyl and 9-methoxy

carbons. A second pathway to the C1–C2 unit exists which wasproposed to involve propionate. Conversely, all of the Addaside chain methyl groups in the related cyclic pentapeptidenodularin 20 from the brackish water cyanobacterium Nodu-laria spumigena103 were clearly shown to be derived frommethionine.104 The Masp unit in 19 as well as 20 was shown tobe derived from acetate and pyruvate and probably involves theformation and rearrangement of citramalic acid 21 (Scheme 10).The proposed formation of Masp is similar to the biosynthesisof leucine and glutamic acid. The structurally related motuporin([l-Val2]nodularin, 22), recently isolated from the Papua NewGuinea sponge Theonella swinhoei, has been proposed to be aproduct of an associated blue-green alga.105 The occurrence ofmany isoforms of the microcystins and the content of unusualand modified amino acids suggest that 19 is synthesized non-ribosomally by peptide synthetases.106 In the presence of theprotein synthesis inhibitor chloramphenicol, microcystin syn-thesis in M. aeruginosa is not inhibited, thus supporting anonribosomal thio-template mechanism.107 Börner and cowork-ers isolated and sequenced a 2982 bp fragment, mapep1, of M.aeruginosa DNA which encodes a complete non-epimerizingpeptide synthetase module that hybridized exclusively to DNAfrom hepatotoxic strains.108 DNA flanking this fragment washomologous to additional modules constituting a peptidesynthetase gene cluster.109 Insertional replacement of mapep1with the chloramphenicol resistance gene cassette by homolo-gous recombination resulted in a mutant that was lackingspecific peptide synthetase activities and was unable to producemicrocystins, including 19.110 The biosynthesis of the cyano-peptolins, cyclic peptides which are also produced by M.aeruginosa and share various constituent amino acids with 19,was not altered in this mutant. Thus, the mapep1 fragment ispart of a biosynthesis gene cluster that encodes a peptidesynthetase complex involved in microcystin biosynthesis. Thisis the first report of genetic transformation and mutation byhomologous recombination in a bloom-forming blue-greenalga.

Anatoxin-a(s) 23, a unique phosphate ester of a cyclic N-hydroxyguanidine moiety, is a potent neurotoxin produced bythe freshwater cyanophyte Anabaena flos-aquae.111 Feedingexperiments with stable and radiolabeled precursors establishedthat all of the carbons of the triaminopropane backbone and theguanidino unit in 23 are derived from l-arginine and that thethree methyl carbons arise from l-methionine or other donors tothe tetrahydrofolate C1 pool.112 During the conversion of l-arginine to 23, as shown in a feeding experiment with l-[U-13C]arginine, the elements of glycine are lost. The intermediacyof (2S,4S)-4-hydroxyarginine 24, a minor constituent in A. flos-aquae,112 was established in a feeding experiment with[3,3,4,5,6-2H5]24.113 Deuterium atoms were retained at C4 andC5 in the resulting 23, but lost at C3, implying that anintermediate having a keto functionality is formed at C3 in thecourse of replacing the C-glycyl unit with a dimethylaminoresidue. Retention of deuterium at C5 further suggests that thering closure proceeds via an SN2-type process. Moore’sproposed biosynthesis of 23 is summarized in Scheme 11.114

Anabaena flos-aquae also produces the structurally unrelatedtoxic alkaloid anatoxin-a 25.115 The biosynthesis of 25 in A.flos-aquae and its homolog homoanatoxin-a116 26 in Oscil-latoria formosa was investigated by Hemscheidt and cowork-ers.117 Based on feeding experiments with 13C-labeled acetateand (S)-glutamate, the pyrrolidine ring of 25 and 26 is derivedin a different manner than that found in structurally relatedtropane alkaloids of higher plants. Glutamic semialdehyde ispostulated as the primer unit for the triketide fragment (Scheme12).

The biosynthesis of borophycin 27, a cytotoxic boron-containing polyketide from Nostoc linckia, was also examinedby Moore and coworkers.51 Borophycin is structurally related totwo streptomycete antibiotics, boromycin118 28 from a terres-

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Sche

me

8

658 Nat. Prod. Rep., 1999, 16, 653–674

trial strain of Streptomyces antibioticus and aplasmomycin48250

29 from a marine strain of S. griseus, and to tartrolon B119 30from the myxobacterium Soangium cellulosum. All fourionophores are acetate-derived polyketides whose methylgroups are derived from methionine. While polyketide methylbranches are often introduced by C-methylation in cyano-bacteria, the biosynthesis of the streptomycete products 28 and29 is unusual as methyl groups are commonly derived from theutilization of methylmalonyl-CoA in macrolide assembly. Eachmetabolite additionally has an unusual C3 starter unit. In thecase of 27, methionine methylated acetate, and not propionate,serves as the primer unit,51 whereas 28, 29 and 30 use aglycerol-derived starter unit such as phosphoglycerate orphosphoenolpyruvate.48–50,118,119

Scytophycins120 contain structural features that are related toseveral marine natural products from derived from sponges,nudibranchs and sea hares such as the swinholides, bistheo-nellides/misakinolides, ulapualides, kabiramides, halichondri-mides and mycalolides.11 The biosynthesis of tolytoxin 31, ascytophycin-like polyketide from a terrestrial strain of Scyto-nema mirabile utilizes a glycine starter unit and is extended by15 acetate units.121 The one-carbon branches are derived fromthe tetrahydrofolate C1 pool.

Lyngbya majuscula is a common source of structurallydiverse secondary metabolites possessing broad ranges ofbiological activities. Gerwick and coworkers have identifiedseveral novel metabolites from the Curaçaoan (Caribbean)strain122–127 and have recently probed the origin of thetrichloromethyl group in the molluscicidal metabolite barba-mide 32.128 Feeding experiments with differently 13C-labeled l-leucines are summarized in Scheme 13. Leucine is probablycatabolized to 3-methylbutyryl-CoA that is chain extended bymalonyl-CoA to give the 7-carbon enolether fragment in asimilar fashion to that in 10 biosynthesis (Section 2.1).Chlorination exclusively occurs at the pro-S methyl group ofleucine, which is not activated by a double bond, as incorpora-tion experiments with l-[U-2H]leucine showed that leucinehydrogens were retained at C1, C2, and C3 in 32. Gerwickspeculates that related sponge-derived metabolites containingchlorinated leucine residues may consequently be of cyano-bacterial origin from l-leucine. Unson and Faulkner haveshown that the chlorinated leucine metabolite 13-demethyliso-

Scheme 9

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dysidenin 33 is localized in the cells of the cyanobacteriumOscillatoria spongeliae,129 which is a symbiont of the marinesponge Dysidea herbacea (Section 2.5).

2.3 Dinoflagellates

Toxic blooms of dinoflagellates known as “red tides” areresponsible for massive fish kills, shellfish contamination, andhuman poisoning. The toxic metabolites are often characterizedas large polycyclic ethers and represent amazing structural,synthetic, and biosynthetic targets. The chemistry, function, andbiosynthesis of a broad range of microalgal metabolites,

Scheme 10

Scheme 11

Scheme 12

660 Nat. Prod. Rep., 1999, 16, 653–674

including the polyethers, have recently been re-viewed.9,10,130,131

The biosyntheses of the neurotoxins brevetoxin A132,133 34and B134 35 from the red tide organism Gymnodinium brevewere examined by the Nakanishi36,135 and Shimizu136 groupsand have previously been reviewed.1,2 Their biosyntheses serveto illustrate the unique mode of polyketide assembly thatappears to be the norm in dinoflagellates. The all-trans ringsystems of 34 and 35 are proposed to be formed by a cascade ofepoxide openings of the respective all-trans epoxides which arederived from the all-trans linear polyenes. The discovery ofhemibrevetoxin B137 36, which structurally resembles theeastern half of the brevetoxins, suggests that the cyclizationevent is initiated by the opening of an epoxide on the eastern endof the molecule. The carbon backbone of the brevetoxins (andall other dinoflagellate polyketides examined to date) isuniquely synthesized by novel polyketide synthases. Feedingexperiments with 13C-labeled acetate and methionine resulted inuncharacteristic incorporation patterns, prompting speculationthat acetate-derived dicarboxylic acids from the citric acidpathway were biosynthetic intermediates.36,135,136 Unusual1,4-polyketides, amphidinoketides I 37 and II 38, recentlyisolated from the dinoflagellate Amphidinium sp., were sim-ilarly suggested on the basis of the substitution pattern of theketo groups to be derived from the condensation of dicarboxylicacids.138 Unfortunately, this proposed mechanism has not beenverified in the brevetoxin system as feeding experiments with

13C- and 14C-labeled succinate, propionate and mevalonatewere unsuccessful.36

An alternate mechanism for polyketide assembly in dino-flagellates has recently been forwarded by Wright and cowork-ers38 for the dinophysistoxin (DTX) family of polyethermetabolites. This group of diarrhetic shellfish poisoning toxins,which includes okadaic acid139, 140 39, okadaic acid diol ester40,141 DTX-1142 41, DTX-2,143 DTX-4144 42, and DTX-5a/5b,145 are produced by dinoflagellates belonging to the generaDinophysis and Prorocentrum. Norte and coworkers demon-strated that all of the carbons in 39 and 41 from Prorocentrumlima, except for the polyketide starter unit-derived C37 andC38, originate from acetate.146,147 The starter unit of theokadaic acid and diol units is rather derived intact fromglycolate.148,149 The origins of the oxygen atoms were deducedindirectly149 by NMR on the basis of 18O-induced shifts in13NMR of 40 and 42 labeled with [1-13C,18O2]acetate and[2-13C,18O]glycolate and directly150 by MS analysis of 39enriched with 18O2 and [18O2]acetate. Although the structures ofthese toxins are related to the terrestrial polyether antibioticssuch as monensin A, the acetate labeling pattern is more relatedto that of the brevetoxins in which the nascent polyketide chainis occasionally interrupted with carbons derived from themethyl group of acetate. This labeling pattern initially promptedsimilar speculation for the involvement of citric acid pathwaybiosynthetic intermediates, such as succinate and glutar-ate.146,147 The Wright group in Halifax demonstrated that theabsolute 13C incorporation in 42 labeled with [2-13C]acetatewas uniform in the okadaic acid, diol, and sulfated chainmoieties, suggestive that all of the acetate-derived carbonsoriginate from the same biosynthetic pool.38 If citric acidpathway intermediates were involved in okadaic acid/DTXbiosynthesis, Wright reasons that non-uniform isotope enrich-ment would likely occur. This result with uniform enrichmentwas suggested38 to be consistent with typical polyketideassembly followed by rearrangements to remove specificcarboxy-derived acetate carbons from the nascent chain and the

Scheme 13

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introduction of pendant methyl and exomethylene groups by thealdol-type condensation pathway depicted in Scheme 4. Addi-tionally, the uniform enrichment throughout 42 suggested that39 is quickly converted to 42 as the ultimate biosyntheticproduct.149 The high average retention of deuterium from[2-2H3, 2-13C]acetate at the pendant methyl and exomethylenegroups was further indicative of a single pool of acetatesupplying the acetate or malonate biosynthetic precursors. Theterminal carbons in the okadaic acid (C1), the diol (C51), andthe sulfated chain (C66) all arise through cleavage of an acetateunit.38 Retention of deuterium at C51 was lower than that atC66, suggesting different cleavage mechanisms. Baeyer–Villiger oxidation was proposed to account for the introductionof the oxygen atom across the intact acetate-derived carbons atC51 and C53; 18O from [1-13C,18O2]acetate was retained only atthe ester carbonyl C53 and not at the diol oxygen, and C51 andC53 originated from the same acetate unit based on long range13C–13C coupling of [1,2-13C2]acetate-labeled 42.38 SimilarBaeyer–Villiger oxidations have been described in the bio-syntheses of a few other polyketides.151–154 Cleavage at C1 andC66 may similarly proceed via a Baeyer–Villiger oxidation oroccur through b-oxidation; the C1 carbonyl of 39 is derivedfrom molecular oxygen.150 A third type of oxygenation reactionhas been proposed by Wright to account for the interruptedpattern of acetate units in the chain.38 Oxidation of a methyl-derived carbon to the a-diketide followed by a Favorskii-typerearrangement,155–158 peroxide attack by a flavin mono-oxygenase, and collapse of the cyclopropanone in which thecarboxy-derived carbon is eliminated as carbon dioxide, wouldgive a shortened polyketide chain containing an oxidizedmethyl-derived carbon (Scheme 14). Dinoflagellates may have

evolved this unique oxidation and deletion mechanism in orderto arrange the necessary oxidation pattern for the subsequentcyclization and methylation reactions in polyether synthesis.Wright suggests38 that this oxidation and deletion process,which accounts for uniform acetate labeling and the interruptedlabeling pattern, may be operative in other dinoflagellatepolyketides as well. The 18O incorporation patterns in 39 furthersuggest that the cyclization of the tricyclic ether rings occurs viaa b-epoxide intermediate (Scheme 15).150

The origins of the carbons in the polyether macrolidegoniodomin A 43, a potent antifungal agent from Alexandrium

hiranoi (formerly Goniodoma pseudogoniaulax),159 were sim-ilarly investigated with 13C-labeled acetate and methionine byMurakami and coworkers.39 All of the cabons in 43 but the C34

methyl (methionine-derived) and the C35/C36 starter unit(glycolate-derived?) originate from acetate. The acetate-label-ing pattern was characteristic of that observed in the brevetoxinsand the DTXs in which the nascent polyketide chain isoccasionally interrupted with carbons derived from the methylgroup of acetate. Murakami thus proposes either involvement ofcitric acid pathway intermediates or a single carbon deletionprocess in 43 biosynthesis.

Amphidinolides are cytotoxic macrolides from an uni-dentified Amphidinium that was isolated from the Okinawanflatworm Amphiscolops sp.160 The characteristic feature of anodd-numbered macrocyclic lactone present in this series ofmetabolites cannot be accounted for by typical polyketideassembly. Hence, Kobayashi and coworkers established thebiosynthetic origin of the carbons in the 15-membered macro-lide amphidinolide J 44, the most abundant macrolide in thisdinoflagellate,161 with 13C-labeled acetates.37 As with thepolyethers from dinoflagellates, the acetate labeling pattern of44 was interrupted, resulting in the odd-numbered lactone.Kobayashi proposes that 44 is synthesized through non-successive mixed polyketides involving normal polyketideunits and dicarboxylic acid precursors from the citric acid cycle(Scheme 16). The four carbon fragment labeled with one intactacetate unit and two methyl-derived carbons was proposed tooriginate from a-ketoglutarate and the three carbon fragmentcomposed of one intact acetate unit and a lone methyl-derivedcarbon from succinate. However, no incorporation of 13C-labeled succinate was observed in a separate feeding experimentto substantiate this proposal.162 Wright rather suggests that thecarbon deletion process that was proposed in DTX biosynthesismay be involved in the formation of 44 as well.38

Sulfonium compounds occur in a wide range of unicellularalgae as well as marine and terrestrial plants and are proposed tobe a major source of sulfur flux in the environment.163

Gonyaulax polyedra produces the cyclopropane sulfoniumgonyauline 45 which causes the period-shortening of bio-luminescent circadian rhythmicity in the photosynthetic dino-flagellate.164,165 Nakamura and coworkers in Hokkaido haveinvestigated the biosynthesis of 45 and other sulfoniums in G.polyedra and other dinoflagellates with 13C-labeled methio-

Scheme 14

Scheme 15

662 Nat. Prod. Rep., 1999, 16, 653–674

nine.166,167 Although 45 and methionine are structurally similar,suggesting that 45 is directly derived from methionine bymethylation and deamination–cyclopropanation reactions, me-thionine is not incorporated intact into 45. The carboxy carbonsof methionine or S-methylmethionine are lost upon incorpora-tion,166 whereas the remaining carbons of methionine, asdemonstrated by feeding experiments with [methyl-13C]- and[2,3-13C2]methionine, are retained (Scheme 17).167 These

experiments implied the intermediacy of 3-dimethylsulfonio-propionate (dimethyl-b-propiothetin) 46, the most widelydistributed sulfonium metabolite in dinoflagellates,168 in theformation of 45 and this was corroborated in a feedingexperiment with uniformly 13C-labeled 46.167 The origin of 46from methionine was previously established in the green algaUlva lactuca,169,170 the red alga Chondria coerulescens,171 theheterotrophic dinoflagellate Crypthecodinium cohnii,172 and inlower and higher plants.173 The biosynthetic route to 46 hasrecently been established in the green macroalga Enteromorphaintestinalis by Hanson and coworkers174,175 and is discussed inSection 3.1. The carboxylate carbon of 45 was specificallyenriched with NaH13CO3 fed in the presence of 46, suggestingan origin from CO2.167 The sulfonium gonyol166 47, which isaccumulated in G. polyedra when methionine is added to the

culture medium and is a major metabolite in Amphidinium sp.Y-5, is derived from 46 and acetate (Scheme 17).167

2.4 Diatoms

Although diatoms comprise the largest population of micro-algae in the oceans, very few secondary metabolites, let alonetheir biosyntheses, have been described. Domoic acid 48, apotent neuroexcitatory toxin produced by Nitzschia pungensforma multiseries, has been implicated in shellfish and birdpoisonings.176 Labeling experiments with 13C-labeled acetatesby the Wright group indicated that the prenylated amino acid 48is derived from the condensation of a geranyl unit with a C5

citric acid cycle derivative (Scheme 18).177 Incorporation levels

were substantially higher in the putative 3-hydroxyglutamicacid unit than in the isoprenoid precursor, indicative of themixed biosynthesis of 48. Domoic acid is structurally related tothe marine macroalgal metabolite, kainic acid 49,178 which is

probably derived in an analogous manner by the condensationof isopentenyl pyrophosphate with the common C5 unit. Theinvolvement of a related C5 precursor has similarly beendeduced in the biosynthesis of the alkaloid lycopodine179 andthe streptomycete antibiotics tautomycin and tautomycetin.180

The biosynthesis of algal pheromones in the freshwaterdiatoms Gomphonema parvulum and Asterionella formosa isdiscussed in Section 3.2.

2.5 Symbiotic microorganisms

Symbiotic microorganisms are often proposed as the trueproducers of natural products isolated from marine inverte-brates.181 Numerous examples exist in which structurallyrelated or identical compounds have been reported fromtaxonomically distinct invertebrates or from invertebrates andcultured microorganisms alike. Several recent examples (struc-tures 50–68) of metabolites isolated from sponges and ascidiansthat are structurally related to microbial products are listed inTable 1. These findings support the hypothesis that manyinvertebrate-derived products are of microbial origin.

Scheme 16

Scheme 17

Scheme 18

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Table 1 Examples of structurally related metabolites from marine invertebrates and cultured microorganisms and their sources

Marine natural product Source Related compound Microbial source of related compound

Motuporin105 22 Sponge (Theonella swinhoei) Nodularin103 20 Cyanobacterium (Nodularia spumigena)Jaspamide182 (jasplakinolide183) 50 Sponge (Jaspis sp.) Chondramide D184 51 Myxobacterium (Chondromyces crocatus)Keramamide A185 52 Sponge (Theonella sp.) Ferintoic acid A186 53 Cyanobacterium (Microcystis aeruginosa)Ecteinascidin 743187–189 54 Ascidian (Ecteinascidia turbinata) Saframycin B190 55 Bacterium (Streptomyces lavendulae)Renieramycin E191 56 Sponge (Reniera sp.) Saframycin B190 55 Bacterium (Streptomyces lavendulae)11-Hydroxystaurosporine192 57 Ascidian (Eudisoma sp.) Staurosporine193 58 Bacterium (Streptomyces staurosporeus)Discodermide194 59 Sponge (Discodermia dissoluta) Alteramide A195 60 Marine bacterium (Alteromonas sp.)Lissoclinolide196 61 Ascidian (Lissoclinum patella) Tetrenolin197 62 Bacteria (Actinomycetales)Enterocin198 63 Ascidian (Didemnum sp.) Enterocin199, 200

(vulgamycin155) 63Bacteria (Streptomyces spp.)

Namenamicin201 64 Ascidian (Polysyncraton lithostrotum) Calicheamicin gI1202 65 Bacterium (Micromonospora echinospora)

Sphinxolide B203 66 Sponge (Neosiphonia superstes) Scytophycin C120 67 Cyanobacterium (Scytonema pseudohofmanni)Swinholide A204 68 Sponge (Theonella swinhoei) Scytophycin C120 67 Cyanobacterium (Scytonema pseudohotmanni)

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Faulkner and coworkers at Scripps propose that secondarymetabolites are biosynthesized within the cells in which they arelocalized and have demonstrated in several cases that secondarymetabolites are cellularly located within sponge-associatedmicroorganisms.205–207 These elegant experiments serve as amodel of invertebrate-microbe symbiosis for the production ofnatural products.

In the first experiment to demonstrate that “sponge” secon-dary metabolites are localized in prokaryotic symbiont cells,Unson and Faulkner examined the tropical sponge Dysideaherbacea, which supports the filamentous cyanobacteriumOscillatoria spongeliae as its major prokaryotic sym-biont.129,208a Two chemotypes of Dysidea exist, one containingboth polychlorinated amino acid-derived metabolites andsesquiterpenes, while the second contains only brominateddiphenyl ethers. In both cases, cyanobacteria were separatedfrom the sponge cells by flow cytometry, and the isolated cellswere chemically analyzed. A unique group of polychlorinatedcompounds, including the major metabolite 13-demethylisody-sidenin 33, was limited to the cyanobacterial symbiont from anAustralian specimen of D. herbacea.129 Related metabolitesfrom cultured cyanobacteria include barbamide 32 fromLyngbya majuscula, which also contains an uncommon chlorin-ated leucine-derived residue (Section 2.2). Accompanyingsesquiterpenes herbadysidolide 69 and spirodysin 70, which arecommonly found in other species of Dysidea, were isolated only

from the sponge cells. Unson and Faulkner hence propose thatthe polychlorinated compounds are biosynthesized by thecyanobacterial symbiont while the sesquiterpenes are products

of the sponge.129 Similarly, Garson and coworkers recentlyreported on the chemistry of sorted D. herbacea/O. spongeliaeby using Percoll gradients.208b They also found that 70 wasassociated with the sponge cells, either the choanocytes or thearchaeocytes, whereas chlorinated diketopiperazines composedof chlorinated leucine residues were constricted to the cyano-bacterium.

Dysidea herbacea from Palau, on the other hand, contains2-(2A,4A-dibromophenyl)-4,6-dibromophenol 71 as its majormetabolite. Similar cell sorting experiments by Faulkner andcoworkers revealed that 71 was also localized in the symbiontO. spongeliae and not in the sponge cells or associatedheterotrophic bacteria.208a The finding that 71 is localized in the

cyanobacterium and not the associated heterotrophic bacteriacontradicts an earlier report by Elyakov and coworkers.209 TheRussian group reported that a related brominated diphenyl ether72, which had previously been isolated from a species ofDysidea, was produced by a cultured bacterium of the genusVibrio that had been isolated from a Dysidea. The productionlevel of 72 by the cultured bacterium, however, was very smallin comparison to the large amounts of brominated diphenylethers that are commonly isolated from D. herbacea and furtheradds to the suspicion that the Dysidea-associated Vibrio maynot be the genuine producer of brominated diphenyl ethers.206

The tropical marine sponge Haliclona sp. from Heron Islandin Australia characteristically contains a large population ofdinoflagellates that occupy roughly 10% of the cellularvolume.208c The dinoflagellate is morphologically related to thecoral symbiont Symbiodinium microadriaticum. Cell sorting by

Table 1 continued—

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Percoll density gradient fractionation by Garson and coworkersdemonstrated that the cytotoxic alkaloids haliclonacyclaminesA and B are localized within the sponge cells rather than thedinoflagellate.208c Garson proposes that these cytotoxins mayassist the sponge in preserving its habitat on exposed coral reeflocations.

The lithistid sponge Theonella swinhoei contains a diverseseries of biologically active metabolites, many of which arestructurally related to microbial products.207 Bewley andFaulkner demonstrated that the Palauan T. swinhoei containsthree distinct microbial cell populations, viz. unicellular hetero-trophic bacteria, unicellular cyanobacteria, and filamentousheterotrophic bacteria.210 Sorting of the four cell types on thebasis of cell density allowed for the chemical analysis of eachcell population. The macrolide swinholide A204 68, whichKitagawa suggested to be a cyanobacterial product due to itsstriking structural similarity to scytophycin C120 67, waslocalized in the unicellular bacteria. The anti-fungal bicyclicglycopeptide theopalauamide211 73, again a suggested cyano-bacterial product due to its unique blue-green algal-likearomatic b-amino acid212 (compare with the Adda unit of 19,20, and 22, Section 2.2), was rather located in the filamentousbacterial fraction. Bewley and Faulkner further note a strongcorrelation between other lithistid sponge cyclic peptidescontaining aromatic b-amino acids and the presence offilamentous bacteria and cautiously suggest a potential relation-ship.207 Contrary to earlier speculation, 68 and 73 were notlocated in the cyanobacterial or sponge cells. These experimentsillustrate the complexity of marine invertebrates and theirmicroflora and caution against predicting the correct source ofmarine natural products based simply on structural comparisonwith microbial products.

2.6 Miscellaneous microorganisms

The classical mevalonic acid pathway was, until recently,widely assumed to be universally responsible for terpenoidbiosynthesis. Recent 13C-labeled feeding studies by Rohmerand coworkers, however, demonstrated initially that eubacteriasynthesize terpenoids via an additional independent pathwayinvolving the intermediacy of d-1-deoxyxylulose-5-phosphate74.213–216 Subsequently, this newly discovered mevalonate-independent pathway has since been firmly established inhigher plants, microalgae, cyanobacteria, and a variety ofbacteria. 217–220 The intermediates and enzymatic steps havebeen largely characterized in E. coli for the first two reactions.The initial reaction involves the condensation of glycer-aldehyde-3-phosphate with a C2-unit derived from pyruvate toyield 74 by the thiamin-dependent d-1-deoxyxylulose-5-phos-phate synthase (Scheme 19).221–223 A subsequent reductioiso-

merase catalyzes the formation of 2-C-methyl-d-erythritol4-phosphate 75 in a single step by intramolecular rearrangementand reduction.224,225 The remaining enzymatic steps to IPP arestill uncharacterized. The mevalonate-independent pathway hasbeen found by the Rohmer and Lichtenthaler groups exclusivelyin all three freshwater unicellular green algae (Scenedesmusobliquus, Chlorella fusca, and Chlamydomonas reinhardtii)investigated to date.226–228 All isoprenoids examined, includingsterols, carotenoids, and the prenyl-side chains of chlorophyllsand plastoquinone, were only synthesized by the mevalonate-independent pathway. On the other hand, in the red algaCyanidium caldarium and in the chrysophyte Ochromonasdanica, both terpenoid biosynthetic pathways were de-tected.227,228 As in higher plants,217 sterols were synthesized viathe mevalonate pathway, whereas the chloroplast isoprenoidsphytol and b-carotene were formed by the mevalonate-independent route.

The biosynthesis of the carotenoid astaxanthin 13 has beencharacterized in the freshwater unicellular green alga H.pluvialis65,229–231 and is discussed in relation to 13-biosynthesisin marine bacteria (Section 2.1). Ethylene biosynthesis in H.pluvialis is related to that in higher plants.232

3 Macroalgae

Marine algae are an extremely rich source of novel oxidizedfatty acid-derived compounds called oxylipins. Several bio-synthetic pathways based on structural motifs common withinoxylipins have been postulated for algal and other marineorganism-derived oxylipins. Gerwick has published severalextensive reviews5–8 on the structure and biosynthesis of marinealgal oxylipins and recently proposed8 a hydroperoxide gen-erated epoxy allylic carbocation as the central intermediate inoxylipin biosynthesis (Scheme 20). In this section, recentexperiments involving algal oxylipin biosynthesis, as well asother algal products, are reviewed. Hypothetical pathwayspurely based on structural analysis are largely omitted; thereader is rather referred to Gerwick’s series of comprehensivereviews.

3.1 Green algae

As discussed in Section 2.3, sulfonium compounds aresynthesized and accumulated by a variety of marine micro andmacroalgae. Proceedings of the “First International Symposiumon DMSP (45) and Related Sulfonium Compounds” have beenpublished in a 36-chapter book and cover a broad range of topicsrelated to 45.233 The sulfonium 45, which is biodegraded to theatmospheric gas dimethyl sulfide,163 acts as an osmopro-tectant234 and a cryoprotectant.235 The steps involved in 45biosynthesis in the marine macroalga Enteromorpha intestinalishave recently been characterized at the chemical174 andbiochemical175 levels by Hanson and coworkers. From methio-

Scheme 19

666 Nat. Prod. Rep., 1999, 16, 653–674

nine, the steps include transamination to 4-methylthio-2-oxo-butyrate 76, ketoreduction to 4-methylthio-2-hydroxybutyrate77, S-methylation to the novel sulfonium 4-dimethylsulfonio-2-hydroxybutyrate 78, and oxidative decarboxylation to 45(Scheme 21).174 Substrate-specific enzymes catalyzing the

conversion of l-methionine to 78 were partially characterized incell-free extracts and involve a 2-oxoglutarate-dependentaminotransferase, an NADPH-linked reductase, and an S-adenosylmethionine-dependent methyltransferase.175 In vivoisotope tracer experiments indicate that the first two steps arereversible.174 d-Enantiomers of 77 and 78 were preferred invitro and in vivo.175 The key intermediate 78 was identified inseveral diverse phytoplankton species (the prymnesiophyteEmiliania huxleyi, the diatom Melosira nummuloides, and theprasinophyte Tetraselmis sp.), indicating that the same pathwayis operative in other important algal sources of 45.174 The higherplant (Wollastonia biflora) pathway to 45 is completelydifferent than in E. intestinalis and in other 45-rich chlorophytealgae and involves the intermediates S-methylmethionine anddimethylsulfoniopropionaldehyde.236,237

Novel polyunsaturated fatty acids with four conjugateddouble bonds, including (4Z,7Z,9E,11E,13Z,16Z,19Z)-docosa-heptaenoic acid (stellaheptaenoic acid) 79, were isolated byGerwick and coworkers from Anadyomene stellata (Scheme22).238 A chloroplast preparation produced increased levels ofthese tetraene fatty acids when unsaturated fatty acids wereadded. Increased levels of 79 were observed when(4Z,7Z,10Z,13Z,16Z,19Z)-docosahexaenoic acid 80 or(7Z,10Z,13Z,16Z)-docosatetraenoic acid 81 were added(Scheme 23). The authors suggest that the conversion of a Z

double bond into a conjugated E,E-diene within the conjugatedtetraene system is an oxidative enzymatic process and occurs ata position in relationship to the carboxy terminus.238 Themechanistic features of conjugated tetraene biosynthesis haspreviously been examined by Hamberg in a cell free system ofthe red marine alga Lithothamnion corallioides.239

3.2 Brown algae

Female gametes of marine brown algae release nonfunction-alized acyclic and/or alicyclic C11 olefins as chemical signals toattract flagellated, motile males.240 Boland and coworkers haveshown that these pheromones originate from cleaved polyunsat-urated C20 fatty acids.241 Transformation of 2H-labeled naturaleicosanoids and shortened unnatural fatty acids indicated thatthe C11H18 hydrocarbon dictyotene 82 is derived from

(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (arachidonic acid),while the series of C11H16 hydrocarbons, including ectocarpene83, multifidene 84, hormosirene 85, finavarrene 86, andgiffordene 87, are produced from (5Z,8Z,11Z,14Z,17Z)-eicosa-pentaenoic acid 88.241,242 Some of the resulting hydrocarbonsare thermally labile and undergo spontaneous electrocyclic andsigmatropic reactions to thermostable products. The bio-synthesis of 83 in Ectocarpus siliculosus involves a sponta-neous Cope rearrangement of the divinylcyclopropane89.243, 244 Presumed functionalization of 88 by a 9-lipoxygenaseyields the hydroperoxide 90 which Pohnert and Boland proposemay rearrange to the Hock-oxycarbenium ion 91 before

Scheme 20

Scheme 21

Scheme 22

Scheme 23

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cleavage into the labile C11 hydrocarbon 89, the cis-dis-ubstituted cyclopropane of the algal pheromone 85, and thehighly water soluble C9 by-product 92 (Scheme 24).244, 245 A

similar rearrangement was detected in the freshwater diatomGomphonema parvulum which alternatively produces a sig-nificant amount of the thermostable C11 cyclopropane isomer85 and the same C9 fragment 92 (Scheme 24).245 The aldehydeoxygen atom of 92 was demonstrated in a crude, cell-freeextract of G. parvulum to be derived from molecular oxygen,thus supporting the proposed functionalization of 88 at C9. Theactivation energies of the Cope rearrangements of thermolabilebis-alkenylcyclopropane precursors (such as 89) to 6-substi-tuted cyclohepta-1,4-dienes (such as 83) were examined attemperatures typical for the Mediterranean and the Arctic in thespring.243,244 Half-lives of ca. 20–30 minutes were found at18 °C (Mediterranean) and over 1 hour at 8 °C (Arctic),suggesting that the immediate precursor 89, and not the cyclizedproduct 83, is the actual pheromone. Comparative bioassays of83 and 89 with male gametes of E. siliculosus verified that thethermolabile cyclopropane 89 is the true male-attracting signal,thus establishing that the spontaneous Cope rearrangementserves as an environmentally controlled mechanism for thedeactivation of the pheromone.244 Once formed, these unstablecyclohepta-1,4-dienes are oxidized to a complex mixture ofoxygenated derivatives, which themselves act as anti-feedantsagainst algae grazers.240,246,247 In contrast to lower plants, theterrestrial plant Senecio isatideus utilizes (3Z,6Z,9Z)-dodeca-trienoic acid 93 for the production of related C11 hydrocarbons.The C12 acid 93 is derived from linolenic acid via threesuccessive b-oxidations and undergoes an oxidative decarbox-ylation/cyclization to 83 (Scheme 24).248,249

In a similar fashion, the C8 hydrocarbon (3E,5Z)-octa-1,3,5-triene (fucoserratene) 94 is derived from 88 by oxidativecleavage of the corresponding 12-hydroperoxy intermediate 95(Scheme 25)250 The biosynthesis of 94, which was firstidentified as the sexual pheromone of the brown alga Fucusserratus,251, 252 was examined in the freshwater diatom Aster-ionella formosa.253, 254 The highly unsaturated eicosanoids

(5Z,8Z,11Z,14Z)-eicosatetraen-17-ynoic acid 96 and(5Z,8Z,11Z,14Z,19)-eicosapentaen-17-ynoic acid 97, structuralanalogs of 88, were converted to the expected 94-analogs (3E)-octa-1,3-dien-5-yne 98 and (5E)-octa-1,5,7-trien-3-yne 99,respectively, by crude homogenates of A. formosa (Scheme26).250 These experiments established 88 as the natural

precursor to 94 and that the pathway proceeds via 95, which iscleaved by a hydroperoxide lyase to yield 94 and the polarfragment 12-oxododeca-5,8,10-trienoic acid 100 (Scheme 25).Hombeck and Boland speculate that 100, which is structurallyrelated to the defensive agent 92, serves a similar role in thediatom and alga.250 Support for the involvement of a 12-lipox-ygenase was further provided from inhibition experiments inwhich the formation of 94 was suppressed under anaerobicconditions but could be reversed by addition of dioxygen.254

The acyclic C11 tetraene 87, which is a major product ofGiffordia mitchellae, results from a spontaneous antarafacial[1,7]-sigmatrophic hydrogen shift of the thermolabile(1,3Z,5Z,8Z)-undecatetraene 101 intermediate derived fromcleavage of 88 (Scheme 27).242 A low temperature synthesis of

the postulated intermediate 101 was developed, and the kineticdata furnished a half-life of approximately 2.5 hours atenvironmental temperature (18 °C) and an activation energyamong the lowest values currently known for natural pericyclicreactions.255 This rearrangement in nature has thus beenproposed to be spontaneous and not catalyzed by an enzyme.Another pericyclic reaction proposed by Pohnert and Bolandaccounts for the C9 hydrocarbon 7-methylcycloocta-1,3,5-tri-ene 102,255 a minor component of the hydrocarbons from theMediterranean phaeophyte Cutleria multifida. The authors

Scheme 24

Scheme 25

Scheme 26

Scheme 27

668 Nat. Prod. Rep., 1999, 16, 653–674

propose that (3Z,5Z,7Z)-nona-1,3,5,7-tetraene 103 is producedby cleavage of a suitable fatty acid hydroperoxide andundergoes an 8pe electrocyclic ring closure (Scheme 27). Thehalf-life of synthetic 103 was limited to just a few minutes atambient temperature and the activation energy of the 8peelectrocyclization was significantly low. NMR studies indicatedthat the monocyclic 102 was the only isomer present andprovided no evidence for a bicyclic cyclohexadiene equilibriumintermediate.

Macrophytic brown algae within the genus Laminariaproduce a host of divinyl ethers and hydroxy fatty acidsputatively derived from w-6 lipoxygenase (LOX) metabo-lism.256 The Laminaria saccharina gametophyte cell suspen-sion culture produces three monohydroxy fatty acids derivedfrom w-6 oxidation (5Z,8Z,11Z,13E)-[(15S)-hydroxyeicosa-5,8,11,13-tetraenoic acid 104, (6Z,9Z,11E,15Z)-(13S)-hydroxy-octadeca-6,9,11,15-tetraenoic acid 105, and (9Z,11E)-(13S)-hydroxyoctadeca-9,11-dienoic acid 106], whereas thefield-collected sporophyte also contains (9Z,11E,15Z)-(13S)-hydroxyoctadeca-9,11,15-trienoic acid 107.257 Yields of104–106 were increased 2–4 fold when linoleic 108 and g-linolenic 109 acids were exogenously added to the cellsuspension (Scheme 28). a-Linolenic acid 110, on the otherhand, was reportedly toxic to the culture. The lipid metabolismof Dictyopteris membranacea was investigated with 14C-labeled acetate and fatty acids.258

3.3 Red algae

Oxylipin distribution and biosynthesis in marine red algae hasbeen extensively reviewed by Gerwick.5–8 Further studies onthe biosynthesis of vicinal dihydroxy fatty acids from thetemperate red alga Gracilariopsis lemaneiformis were reportedby Hamberg and Gerwick.259 Experiments with fractionatedtissue homogenates of the alga extended prior results with anacetone powder preparation260 on the mode of conversion ofarachidonic acid 111 and eicosapentaenoic acid 112 to the diols(5Z,8Z,10E,14Z)-(12R,13S)-dihydroxyeicosa-5,8,10,14-tetra-enoic acid 113 and (5Z,8Z,10E,14Z,17Z)-(12R,13S)-dihydroxy-eicosa-5,8,10,14,17-pentaenoic acid 114, respectively (Scheme29). The pathway consists of an initial oxygenation of 111

catalyzed by a 84–89 kDa sodium-dependent 12-lipoxygenaseinto (5Z,8Z,10E,14Z)-(12S)-hydroperoxyeicosa-5,8,10,14-tet-raenoic acid followed by hydroperoxide isomerase-catalyzedconversion to 113. The stereochemistry of the conversion wascharacterized with the alternate substrate (6Z,9Z,12Z)-octadeca-trienoic acid 115, stereospecifically deuterated in the 8R, 8S,11R, and 11S positions. The lipoxygenase-catalyzed reactionoccurred with abstraction of the pro-8R hydrogen and additionof oxygen at C-10 in an antarafacial relationship to provide

(10S)-hydroperoxide 116 (Scheme 30). Arachidonic acid 12-li-poxygenase from human platelets, which does not requiresodium for its catalytic activity, catalyzes the same reaction.261

The hydroperoxide isomerase (vicinal diol synthase262) reactionproceeds with the intramolecular260 hydroxylation of C-11 andconcomitant loss of the pro-11S hydrogen to the diol 117 withoverall retention of absolute stereochemistry (Scheme 30).259

The cell-free conversion of arachidonic acid 111 to (13R)-hydroxyarachidonic acid 118 by Lithothamnion corallioidesproceeds by an unusual mechanism, as its hydroxy group isderived from water as shown by H2

18O labeling.263 While notincorporated, molecular oxygen is required for the oxidation,prompting Gerwick and coworkers to speculate on the forma-tion of 83 by direct displacement of an enzyme-111 complex119 by water (Scheme 31). Under mild acid conditions, 118readily undergoes allylic rearrangement to racemic mixtures ofthe co-produced oxylipins (15S)- 104 and (11R)-hydroxyeicosatetraenoic 120 acids. 18O-Incorporation studieswith molecular oxygen implicated that 104 and 120 are largelyderived from lipoxygenase metabolism. The conjugated tetra-ene bosseopentaenoic acid264 121, also generated from incuba-tion experiments with 111 in L. corallioides263 and Bossiellaorbigniana,264 is formed by an independent pathway notinvolving the intermediacy of the hydroxy acids 104 or 120.Hamberg previously reported239 that a unique L. corallioides

Scheme 28

Scheme 29

Scheme 30

Nat. Prod. Rep., 1999, 16, 653–674 669

oxidase catalyzes the formation of the conjugated tetraenesystem in a related C18 polyunsaturated fatty acid without theinvolvement of oxygenated intermediates. An arachidonate9(S)-lipoxygenase has been proposed to account for the diverseoxylipins of Polyneura latissima.265

Conjugated triene-containing fatty acids in Ptilota filicina arebiosynthesized in a completely different manner. Gerwick andcoworkers demonstrated that the novel enzyme polyenoic fattyacid isomerase catalyzes the formation of conjugated trienesfrom a variety of fatty acid precursors, including arachidonicacid 111 and (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid 112to the corresponding 5Z,7E,9E-conjugated triene regioisomers122 and 123.266 Using stereospecifically deuterium labeled g-linolenates, the regio- and stereochemistry of the protontransfers involved in the two sequential isomerizations wereelucidated. The authors propose a 1,3-allylic shift of the pro-10S hydrogen (Ha) to C-12 of the enzyme-bound dieneintermediate 124 followed by a second isomerization involvingremoval of the pro-7R hydrogen (Hc) and reprotonization at C-11 with a solvent-derived proton (Scheme 32). A broad range ofalternative substrates were tested, and on the basis of thededuced products, Gerwick and coworkers hypothesize thatpolyenoic acid isomerase preferentially orients the protonatedform of the substrate in the catalytic site with respect to themethyl terminus.267

Many species of Laurencia contain characteristic cyclicbromo ethers, which are derived from bromonium ion-inducedcyclization of acylic polyene precursors. Murai and coworkerspreviously reported the biomimetic conversion of (3E)- and(3Z)-laurediol 125 to (E)- and (Z)-prelaureatin 126 withcommercial lactoperoxidase268, 269 (LPO) and, more recently,with a partially purified bromoperoxidase (BPO) from Laur-encia nipponica (Scheme 33).270 Further treatment of (Z)-126with LPO or BPO afforded laureatin 127 and isolaureatin 128,whereas the (E)-isomer furnished the allene laurallene 129 upontreatment with LPO.271 The biosynthesis of halomethanes in themarine red alga Endocladia muricata and in higher plants in thefamily Brassicaceae via a novel methyltransferase reaction wasreported.272

4 Acknowledgements

Research on exploring and engineering natural productsdiversity from marine microorganisms in the author’s labo-ratory has been generously supported by the National andWashington Sea Grant Programs through the National Oceanicand Atmospheric Administration (R/B-20 and R/B-28).

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