Biosynthesis of marine natural products: microorganisms and macroalgae

Bradley S. Moore Department of Chemistry, Box 351700, University of Washington, Seattle, WA, 98195-1700, USA
Received (in Cambridge, UK) 16th June 1999 Covering: 1989 through 1998 Previous review: 1989, 6, 143 1 2 2.1 2.2 2.3 2.4 2.5 2.6 3 3.1 3.2 3.3 4 5 Introduction Marine microorganisms Marine bacteria Cyanobacteria Dinoflagellates Diatoms Symbiotic microorganisms Miscellaneous microorganisms Macroalgae Green algae Brown algae Red algae Acknowledgements References

Fig. 1

2 2.1

Marine microorganisms Marine bacteria

Introduction

This review covers the literature published on the biosynthesis of marine microbial and macroalgal natural products over a 10-year period from 1989 through 1998. A companion review covering the same time period on the biosynthesis of natural products from marine macroorganisms will appear at a later date in this journal. An earlier report by Garson published in this journal surveyed the whole field of marine natural product biosynthesis through mid 1988.1 The field through mid 1992 was reviewed elsewhere in an updated report,2 and several reviews covering specific aspects of marine microbial and macroalgal natural product biosynthesis have been published during this period. Reviewed biosynthetic topics include dinoflagellate and algal sterol side chains,3,4 algal oxylipins,58 and microalgal metabolites.9,10 Some of the microorganisms discussed in this report are not strictly marine. Bacteria isolated from coastal waters may have originated from terrestrial habitats and washed into the ocean. Many of these bacteria found at the interface of terrestrial and marine environments, especially the actinobacteria, tolerate wide ranges of salinities. The biosynthesis of natural products from freshwater cyanobacteria and microalgae are described for compounds that are structurally related to marine products. The biosynthetic origins of many marine invertebrate-derived natural products are not clear and have been proposed to involve associated microorganisms. A section on symbiotic microorganisms highlights our current knowledge on the involvement of invertebrate-hosted microorganisms in natural product biosynthesis through cellular localization studies. The review is organized on the basis of a similar taxonomic system used in previous marine natural product biosynthesis1, 2 and structure11 reviews. General labeling patterns consistently used throughout this report are outlined in Fig. 1.
Present address: College of Pharmacy, Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ 85721-0207, USA. E-mail: moore@pharmacy.arizona.edu; Tel: +1 520 626 6931; Fax: +1 520 626 2466.

Marine bacteria have recently emerged as an entirely new source of structurally novel natural products12,13 for the development of new drug candidates.14,15 The rich variety of chemically novel and biologically active metabolites serves to indicate that marine bacteria are a genetically rich resource for recombinant technologies. Biosynthetic studies with cultured marine bacteria have increased over the past few years and in a few cases have expanded beyond simple feeding experiments with labeled precursors to studies at the biochemical and genetic levels. The first marine bacterial natural product to be reported was the highly brominated pyrrole antibiotic pentabromopseudiline 1 by Burkholder and coworkers in 1966 from a culture of Pseudomonas bromoutilis.16,17 Pentabromopseudiline has since been identified together with the blue pigment violacein 2 from several other seawater-derived bacteria, including Chromobacteria sp.18 and Alteromonas luteoviolaceus.19 Three different syntheses1921 and a structureactivity relationship study22 have been reported. The biosynthesis of this highly unusual metabolite, which is composed of more than 70% bromine by weight, was not apparent from its structure, leading to a study by Laatsch and coworkers with the A. luteoviolaceus strain.23 An acetate origin of 1 was excluded from several 13C-acetate feeding experiments. Rather, the benzene ring of 1 was shown to be carbohydrate-derived, whereas the origin of the pyrrole ring was not deduced in this report involving numerous feeding experiments with labeled acetate, glucose, and amino acids. Feeding experiments with differently labeled 13C-labeled glucoses are summarized in Scheme 1 and surreptitiously implied that shikimic acid 3 is converted into a symmetrical

Scheme 1

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

derived from C2 of acetate. The feature of an acetate origin for methyl branches is uncommon and has been observed in some bacterial,3034 cyanobacterial35 and dinoflagellate3639 (see Section 2.3) metabolites. The process has been proposed to involve the addition of malonate to a polyketide keto group to give a b-hydroxyacyl acid intermediate, which is decarboxylated and dehydrated in a similar manner to the loss of carbon dioxide and water from mevalonic acid in terpenoid biosynthesis (Scheme 4). Reduction of the resulting exomethylene

Scheme 2

were further complicated because this strain lacks the glycolytic enzyme triosephosphate isomerase, thus preventing the conversion of dihydroxyacetone phosphate into glyceraldehyde3-phosphate and hence not into phosphoenolpyruvate. Although [2-13C]shikimic acid and its methyl ester were not incorporated into 1, neither labeled the co-produced alkaloid 2, which was shown in this organism23 and in bacteria of the genus Chromobacteria2426 to be derived from the dimerization of two units of tryptophan (Scheme 3). As shikimic acid and its methyl

Scheme 4

group leads to the methyl group. This pathway was corroborated by a feeding experiment with [2-13C,2H3]acetate, which showed that just two of the three hydrogens at each methyl group were enriched with deuterium. Although the labeling pattern of the five-carbon fragment C11C14 and C16 is consistent with an alternative pathway involving a mevalonate-derived starter unit, Andersen suggests that mevalonate is not a likely intermediate in 4 biosynthesis due to the combined observation that [2-13C]mevalonolactone is not incorporated into 4 and that 4 is uniformly labeled by acetate. The biosynthesis of the antibiotic andrimid 5 has additionally been reported by the Andersen group.40 Andrimid and the related metabolites moiramides AC were produced by fermentation of the bacterium Pseudomonas fluorescens obtained from

Scheme 3

ester were not transported into the cells of A. luteoviolaceus, an observation documented in other microbial systems as well,27 the deduced intermediacy of shikimic acid in 1 biosynthesis could not be directly verified. Feeding experiments with phydroxy-[2,3,5,6-2H4]- and p-hydroxy-[3,5-13C2]benzoic acids, however, demonstrated that p-hydroxybenzoic acid is converted very efficiently (approximately 85% specific incorporation) into the benzene moiety of 1 (Scheme 1). It is probably transformed through decarboxylation into the benzene ring of 1, and the hydroxy group provides activation for further substitutions (Scheme 2). The Andersen group in British Columbia has been very active in elucidating the biosynthesis of several marine microbial metabolites. Their first venture into the study of microbial biosynthesis involved the novel lactone oncorhyncolide 4,28 a biologically inactive metabolite produced by a seawaterderived Gram-negative bacterium.29 Several plausible biosynthetic pathways were postulated, and these were tested through feeding experiments with 13C-labeled acetate. All of the carbons in 4 are acetate-derived as summarized in the structure of 4. The incorporation data are consistent with a polyketide origin of 4, whose pendant methyl groups (C15 and C16) are 654 Nat. Prod. Rep., 1999, 16, 653674

the tissues of an unidentified Alaskan tunicate. Andrimid was first reported from cultures of an Enterobacter sp. intracellular symbiont of the brown planthopper Nilaparvata lugens41 and since from a Vibrio obtained from an unidentified Hyatella sponge.42 Stable isotope feeding experiments demonstrated that the unusual acylsuccinimide unit, which was shown to be required for its antimicrobial activity,43 is derived from an interesting combination of acetate and amino acid building blocks. The proposed pathway involves the homologation of valine with malonyl-CoA to the corresponding g-amino-b-keto acid, followed by the addition of glycine which in turn is chain extended with a second malonyl-CoA to give a putative dipeptide intermediate derived from two g-amino-b-keto acids (Scheme 5). Condensation followed by decarboxylation, dehydration and reduction of the resulting exomethylene group, analogous to that in Scheme 4, gives the g-lactam. Andrimid is probably synthesized by a novel mixed polyketide/peptide

Scheme 5

synthetase that is capable of utilizing both malonyl-CoA and amino acids as substrates. Two additional pseudomonads recently isolated from a marine alga and a marine tube worm by the Andersen group led to the discovery of the antimycobacterial cyclic depsipeptides massetolides AH44 and the previously known compound viscosin 6.45, 46 Branched amino acids comprise five positions of the massetolide/viscosin family of nonapeptolides, two of which at positions AA4 and AA9 are naturally varied in the series. Precursor-directed biosynthesis47 with nonproteinogenic amino acids, including l- and d-butyrine, l- and d-norvaline, land d-tert-leucine, and l-cyclopropylalanine, was used to generate unnatural massetolides in order to extend the structural diversity of this series. Massetolide analogs, including massetolides IK 79, were only generated from the l-butyrine, lnorvaline, and l-cyclopropylalanine substitution experiments at positions AA1, AA4, and AA9. Unfortunately, the yields of unnatural peptides were too low to provide sufficient sample for antimycobacterial testing. Although the biosynthesis of edaphic streptomycete products is well documented and has contributed to our basic knowledge of secondary metabolism, only two studies have been reported with streptomycetes isolated from the marine environment. The

first study by Floss and coworkers involved the boroncontaining ionophore aplasmomycin from the sediment-derived Streptomyces griseus SS-204850 and is discussed in conjunction with the structurally related marine cyanobacterial metabolite borophycin51 in Section 2.2. More recently, the biosynthesis of the bicyclic depsipeptide salinamide A 10, a potent antiinflammatory agent52 and bacterial RNA polymerase inhibitor,53 has been examined.54 The 10-producing strain Streptomyces sp. CNB-091 was isolated from the surface of the jellyfish Cassiopeia xamachana.52 The proposed pathway involves the non-ribosomal peptide synthetase product Thr-dIle-Hpg-MePhe-d-aThr-Ser (Hpg = p-hydroxyphenylglycine) and proceeds through the desmethyl analogs of the naturally occurring salinamides E 11 and C 12 (Scheme 6). Extensive feeding experiments with 13C-labeled intermediates indicated that both of the seven-carbon, non-amino acid residues of 10 are formed by a single chain extension of a carboxylic acid derived from a branched amino acid. The (2S,3S)-3-hydroxy-2,4-dimethylpentanoate unit is derived from isobutyrate via valine with methylmalonyl-CoA extension, whereas the fragment bridging the p-hydroxyphenylglycine and glycine residues is derived from the condensation of the isoleucine product tiglic acid with malonyl-CoA. The mode of cyclization of 12 to 10 has been proposed to involve either an epoxide intermediate which is opened by the Hpg phenol followed by dehydration and a second epoxidation (Scheme 7, path a) or an Fe(ii)-dependent oxygenase mediated [2 + 2] cycloaddition followed by dehydrogenation (Scheme 7, path b). Such Fe(ii)-dependent oxygenase reactions have precedence in b-lactam chamistry and have also been proposed to account for polyether formation in polyketides such as monensin and brevetoxin.55 Recent advances in genetic engineering has transformed the field of natural products, making it now possible to engineer novel small molecules56 and to functionally express large biosynthetic gene clusters in a heterologous host.57 Yazawa and coworkers report the first cloning and expression of a marine

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Scheme 6

bacterial product in a marine cyanobacterium.58 Shewanella putrefaciens strain SCRC-2738, isolated from Pacific mackerel intestines, produces high concentrations of the polyunsaturated w-3 fatty acid eicosa-5,8,11,14,17-pentaenoic acid (EPA).59,60 A 38 kb clone containing the EPA biosynthetic gene cluster was functionally expressed in Escherichia coli61 and in the marine cyanobacterium Synechococcus sp.58 The cosmid carrying the EPA gene cluster contains eight open reading frames (ORFs), three of which are homologous to genes encoding enzymes involved in fatty acid de novo synthesis or in carbon chain elongation.61 Deletion of any ORF other than ORF1 in the cluster containing the EPA biosynthesis genes resulted in the loss of EPA production and did not accumulate intermediate products 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 and in the Synechococcus transconjugant58 was higher at a lower temperature than the optimal growth temperature. However, EPA was produced in lower titers in the cyanobacterial transconjugant than in the E. coli recombinant, even though the EPA biosynthesis gene cluster contains its natural promoters.58 The proposed EPA biosynthetic pathway in S. putrefaciens involves the chain elongation and aerobic desaturation of palmitoyl-CoA, and is distinct from the anaerobic pathway involved in the synthesis of n-7 type monounsaturated fatty acids.62 The ketocarotenoid pigment astaxanthin 13 is produced by a multitude of microorganisms, including the marine bacteria Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1,63 the yeast Phaffia rhodozyma,64 and the freshwater alga Haematococcus pluvialis.65 Several marine animals, including crustaceans and salmon, attain their red coloration from carotenoid pigments derived from their diet. Consequently, the metabolic engineering of 13 for the production of carotenoidenriched feed supplements for cultured fish and shellfish has gained considerable industrial attention.66,67 In addition, 13 exhibits diverse biological activities, including anti-cancer properties,68 enhancement of immune responses,69 and quenching of free-radicals,70 thus making 13 a very attractive candidate for metabolic engineering. The biosynthesis of carotenoids, including 13 and its intermediates, is well characterized in several microbial systems at the biochemical and genetic levels and has recently been extensively reviewed by Armstrong.71 The A. aurantiacum carotenoid biosynthesis gene cluster was identified by Misawa and coworkers and consists of five ORFs designated crtW (b-carotene ketolase), crtZ (b-carotene hydroxylase), crtY (lycopene cyclase), crtI (phytoene desaturase) and crtB (phytoene synthase) and organized in a crtWZYIB operon.72 Homologous genes have been identified in Alcaligenes sp. strain PC-1 by colony hybridization.73 A crtW homolog has been isolated from the alga H. pluvialis and termed crtO74 and bkt.75 A non-homologous ketolase gene from the cyanobacterium Synechocystis sp. PCC 6803, also designated crtO, codes for an asymmetrically acting b-carotene ketolase which introduces just one keto group on only one of the two ionone rings of b-carotene 14 to produce echinenone 15.76 The crtW homolog in the cyanobacterium, crtR, rather codes for a symmetrically acting hydroxylase that catalyzes the hydroxylation of 14 to zeaxanthin 16.77 Interestingly, all bacterial crt gene clusters analyzed to date, with the exception of A. aurantiacum, contain a crtE gene which encodes a geranylgeranyl pyrophosphate (GGPP) synthase which converts farnesyl pyrophosphate (FPP) to GGPP.71 The analysis of carotenoid biosynthesis genes from the nonphotosynthetic bacteria Erwinia uredova78 and Erwinia herbicola,7981 which encode the synthesis of 16 and its b-d-diglucoside, has led to a basic

Scheme 7

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understanding of carotenoid biosynthesis. The functions of the crt genes were partially deduced through the structural analysis of accumulated carotenoids in a genetically amenable noncarotenogenic heterologous host, E. coli, carrying various combinations of the Erwinia genes.78 Similarly, the functions of the A. aurantiacum crt genes were analyzed in E. coli transformants.72 The crtB, crtI and crtY genes, which code for the synthesis of the carotenoid branchpoint intermediate 14 from two moles of GGPP, are functionally equivalent in A. aurantiacum and Erwinia (Scheme 8).72 The further conversion of 14 to 13 formally involves the addition of a hydroxy group to C3 and a keto group to C4 on each ionone ring. The marine bacterial crtZ and crtW gene products CrtZ and CrtW are bifunctional in their activity and possess significantly broad substrate specificities allowing for multiple pathways leading to 13 (Scheme 8).82,83 The Erwinia crtZ gene product is functionally similar to the corresponding marine bacterial enzyme, but their substrate affinities, b-carotene versus canthaxanthin 17, are different.82 Astaxanthin and other new and known carotenoids have been metabolically engineered in several hosts that naturally do not synthesize carotenoids, such as E. coli,72,78,84,85 Zymomonas mobilis,86 Agrobacterium tumefaciens,86 and the food yeasts Saccharomyces cerevisiae87 and Candida utilis.8890 The level of carotenoid biosynthesis in E. coli transformants was increased 1.54.5 fold when isopentenyl diphosphate (IPP) isomerase genes from H. pluvialis, P. rhodozyma, or S. cerevisiae were co-expressed.91 This gene enhances the carbon flux of the isoprenoid pathway leading to the formation of FPP in E. coli. Similarly, metabolic engineering of the isoprenoid pathway in C. utilis, in which the 3-hydroxy methylglutaryl coenzyme A reductase was overexpressed, lead to an increase of carotenoid biosynthesis in transformants carrying exogenous crt genes.90 The cyanobacterium Synechococcus PCC7942, which normally accumulates 14 and 16, is capable of synthesizing 13 and other ketocarotenoids when expressing the H. pluvialis crtO oxygenase gene.92 2.2 Cyanobacteria

Cyanobacteria (blue-green algae) are prokaryotic, photosynthetic microorganisms that are very rich in biologically active secondary metabolites. Although research on the natural products chemistry of cyanobacteria is very active and has been recently reviewed,9395 biosynthetic studies have been few, especially with the marine strains. The biosyntheses of marine and freshwater cyanobacterial metabolites are discussed. Shimizus work on saxitoxin and neosaxitoxin 18 biosynthesis in the freshwater cyanobacterium Aphanizomenon flos-aquae was largely reviewed in the previous review in this series.1 Feeding experiments with 13C- and 2H-labeled precursors have shown that neosaxitoxin is biosynthesized from arginine and acetate and involves a Claisen-type condensation between C2 of arginine and C1 of acetate (Scheme 9).9698 The cyclic heptapeptide microcystin-LR 19 is the major hepatotoxin associated with toxic waterblooms of Microcystis aeruginosa found in the Northern Hemisphere.99 MicrocystinLR, a potent inhibitor of both type 1 and type 2A protein phosphatases,100 has been implicated in net-pen liver disease, a common toxicopathic disease of Atlantic salmon reared in seawater in British Columbia and Washington State.101 Stable isotope feeding experiments by the Moore group in Hawaii established the origins of the unusual (2S,3S,8S,9S)-3-amino9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (Adda) and (2R,3S)-3-methylaspartic acid (Masp) residues in 19.102 The Adda unit is synthesized by the polyketide pathway involving a putative phenylacetyl-CoA starter unit and four malonyl-CoA extensions. Sodium [1,2-13C2]acetate was incorporated at C1 through C8 and the remaining Adda backbone carbons were derived from l-[U-13C]phenylalanine. l-[methyl13C]Methionine labeled the 2-, 6-, and 8-methyl and 9-methoxy

carbons. A second pathway to the C1C2 unit exists which was proposed to involve propionate. Conversely, all of the Adda side chain methyl groups in the related cyclic pentapeptide nodularin 20 from the brackish water cyanobacterium Nodularia spumigena103 were clearly shown to be derived from methionine.104 The Masp unit in 19 as well as 20 was shown to be derived from acetate and pyruvate and probably involves the formation and rearrangement of citramalic acid 21 (Scheme 10). The proposed formation of Masp is similar to the biosynthesis of leucine and glutamic acid. The structurally related motuporin ([l-Val2]nodularin, 22), recently isolated from the Papua New Guinea sponge Theonella swinhoei, has been proposed to be a product of an associated blue-green alga.105 The occurrence of many isoforms of the microcystins and the content of unusual and modified amino acids suggest that 19 is synthesized nonribosomally by peptide synthetases.106 In the presence of the protein synthesis inhibitor chloramphenicol, microcystin synthesis in M. aeruginosa is not inhibited, thus supporting a nonribosomal thio-template mechanism.107 Brner and coworkers isolated and sequenced a 2982 bp fragment, mapep1, of M. aeruginosa DNA which encodes a complete non-epimerizing peptide synthetase module that hybridized exclusively to DNA from hepatotoxic strains.108 DNA flanking this fragment was homologous to additional modules constituting a peptide synthetase gene cluster.109 Insertional replacement of mapep1 with the chloramphenicol resistance gene cassette by homologous recombination resulted in a mutant that was lacking specific peptide synthetase activities and was unable to produce microcystins, including 19.110 The biosynthesis of the cyanopeptolins, 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 is part of a biosynthesis gene cluster that encodes a peptide synthetase complex involved in microcystin biosynthesis. This is the first report of genetic transformation and mutation by homologous recombination in a bloom-forming blue-green alga. Anatoxin-a(s) 23, a unique phosphate ester of a cyclic Nhydroxyguanidine moiety, is a potent neurotoxin produced by the freshwater cyanophyte Anabaena flos-aquae.111 Feeding experiments with stable and radiolabeled precursors established that all of the carbons of the triaminopropane backbone and the guanidino unit in 23 are derived from l-arginine and that the three methyl carbons arise from l-methionine or other donors to the tetrahydrofolate C1 pool.112 During the conversion of larginine to 23, as shown in a feeding experiment with l-[U13C]arginine, the elements of glycine are lost. The intermediacy of (2S,4S)-4-hydroxyarginine 24, a minor constituent in A. flosaquae,112 was established in a feeding experiment with [3,3,4,5,6-2H5]24.113 Deuterium atoms were retained at C4 and C5 in the resulting 23, but lost at C3, implying that an intermediate having a keto functionality is formed at C3 in the course of replacing the C-glycyl unit with a dimethylamino residue. Retention of deuterium at C5 further suggests that the ring closure proceeds via an SN2-type process. Moores proposed biosynthesis of 23 is summarized in Scheme 11.114 Anabaena flos-aquae also produces the structurally unrelated toxic alkaloid anatoxin-a 25.115 The biosynthesis of 25 in A. flos-aquae and its homolog homoanatoxin-a116 26 in Oscillatoria formosa was investigated by Hemscheidt and coworkers.117 Based on feeding experiments with 13C-labeled acetate and (S)-glutamate, the pyrrolidine ring of 25 and 26 is derived in a different manner than that found in structurally related tropane alkaloids of higher plants. Glutamic semialdehyde is postulated as the primer unit for the triketide fragment (Scheme 12). The biosynthesis of borophycin 27, a cytotoxic boroncontaining polyketide from Nostoc linckia, was also examined by Moore and coworkers.51 Borophycin is structurally related to two streptomycete antibiotics, boromycin118 28 from a terresNat. Prod. Rep., 1999, 16, 653674 657

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Scheme 8

Scheme 9

trial strain of Streptomyces antibioticus and aplasmomycin48250 29 from a marine strain of S. griseus, and to tartrolon B119 30 from the myxobacterium Soangium cellulosum. All four ionophores are acetate-derived polyketides whose methyl groups are derived from methionine. While polyketide methyl branches are often introduced by C-methylation in cyanobacteria, the biosynthesis of the streptomycete products 28 and 29 is unusual as methyl groups are commonly derived from the utilization of methylmalonyl-CoA in macrolide assembly. Each metabolite additionally has an unusual C3 starter unit. In the case of 27, methionine methylated acetate, and not propionate, serves as the primer unit,51 whereas 28, 29 and 30 use a glycerol-derived starter unit such as phosphoglycerate or phosphoenolpyruvate.4850,118,119 Scytophycins120 contain structural features that are related to several marine natural products from derived from sponges, nudibranchs and sea hares such as the swinholides, bistheonellides/misakinolides, ulapualides, kabiramides, halichondrimides and mycalolides.11 The biosynthesis of tolytoxin 31, a scytophycin-like polyketide from a terrestrial strain of Scytonema mirabile utilizes a glycine starter unit and is extended by 15 acetate units.121 The one-carbon branches are derived from the tetrahydrofolate C1 pool. Lyngbya majuscula is a common source of structurally diverse secondary metabolites possessing broad ranges of biological activities. Gerwick and coworkers have identified several novel metabolites from the Curaaoan (Caribbean) strain122127 and have recently probed the origin of the trichloromethyl group in the molluscicidal metabolite barbamide 32.128 Feeding experiments with differently 13C-labeled lleucines are summarized in Scheme 13. Leucine is probably catabolized to 3-methylbutyryl-CoA that is chain extended by malonyl-CoA to give the 7-carbon enolether fragment in a similar fashion to that in 10 biosynthesis (Section 2.1). Chlorination exclusively occurs at the pro-S methyl group of leucine, which is not activated by a double bond, as incorporation experiments with l-[U-2H]leucine showed that leucine hydrogens were retained at C1, C2, and C3 in 32. Gerwick speculates that related sponge-derived metabolites containing chlorinated leucine residues may consequently be of cyanobacterial origin from l-leucine. Unson and Faulkner have shown that the chlorinated leucine metabolite 13-demethylisoNat. Prod. Rep., 1999, 16, 653674 659

Scheme 10

Scheme 11

Scheme 12

dysidenin 33 is localized in the cells of the cyanobacterium Oscillatoria spongeliae,129 which is a symbiont of the marine sponge Dysidea herbacea (Section 2.5). 2.3 Dinoflagellates

Toxic blooms of dinoflagellates known as red tides are responsible for massive fish kills, shellfish contamination, and human poisoning. The toxic metabolites are often characterized as large polycyclic ethers and represent amazing structural, synthetic, and biosynthetic targets. The chemistry, function, and biosynthesis of a broad range of microalgal metabolites, 660 Nat. Prod. Rep., 1999, 16, 653674

Scheme 13

including the polyethers, have recently been reviewed.9,10,130,131 The biosyntheses of the neurotoxins brevetoxin A132,133 34 and B134 35 from the red tide organism Gymnodinium breve were examined by the Nakanishi36,135 and Shimizu136 groups and have previously been reviewed.1,2 Their biosyntheses serve to illustrate the unique mode of polyketide assembly that appears to be the norm in dinoflagellates. The all-trans ring systems of 34 and 35 are proposed to be formed by a cascade of epoxide openings of the respective all-trans epoxides which are derived from the all-trans linear polyenes. The discovery of hemibrevetoxin B137 36, which structurally resembles the eastern half of the brevetoxins, suggests that the cyclization event is initiated by the opening of an epoxide on the eastern end of the molecule. The carbon backbone of the brevetoxins (and all other dinoflagellate polyketides examined to date) is uniquely synthesized by novel polyketide synthases. Feeding experiments with 13C-labeled acetate and methionine resulted in uncharacteristic incorporation patterns, prompting speculation that acetate-derived dicarboxylic acids from the citric acid pathway were biosynthetic intermediates.36,135,136 Unusual 1,4-polyketides, amphidinoketides I 37 and II 38, recently isolated from the dinoflagellate Amphidinium sp., were similarly suggested on the basis of the substitution pattern of the keto groups to be derived from the condensation of dicarboxylic acids.138 Unfortunately, this proposed mechanism has not been verified in the brevetoxin system as feeding experiments with

and 14C-labeled succinate, propionate and mevalonate were unsuccessful.36 An alternate mechanism for polyketide assembly in dinoflagellates has recently been forwarded by Wright and coworkers38 for the dinophysistoxin (DTX) family of polyether metabolites. This group of diarrhetic shellfish poisoning toxins, which includes okadaic acid139, 140 39, okadaic acid diol ester 40,141 DTX-1142 41, DTX-2,143 DTX-4144 42, and DTX-5a/ 5b,145 are produced by dinoflagellates belonging to the genera Dinophysis and Prorocentrum. Norte and coworkers demonstrated that all of the carbons in 39 and 41 from Prorocentrum lima, except for the polyketide starter unit-derived C37 and C38, originate from acetate.146,147 The starter unit of the okadaic acid and diol units is rather derived intact from glycolate.148,149 The origins of the oxygen atoms were deduced indirectly149 by NMR on the basis of 18O-induced shifts in 13NMR of 40 and 42 labeled with [1-13C,18O ]acetate and 2 [2-13C,18O]glycolate and directly150 by MS analysis of 39 enriched with 18O2 and [18O2]acetate. Although the structures of these toxins are related to the terrestrial polyether antibiotics such as monensin A, the acetate labeling pattern is more related to that of the brevetoxins in which the nascent polyketide chain is occasionally interrupted with carbons derived from the methyl group of acetate. This labeling pattern initially prompted similar speculation for the involvement of citric acid pathway biosynthetic intermediates, such as succinate and glutarate.146,147 The Wright group in Halifax demonstrated that the absolute 13C incorporation in 42 labeled with [2-13C]acetate was uniform in the okadaic acid, diol, and sulfated chain moieties, suggestive that all of the acetate-derived carbons originate from the same biosynthetic pool.38 If citric acid pathway intermediates were involved in okadaic acid/DTX biosynthesis, Wright reasons that non-uniform isotope enrichment would likely occur. This result with uniform enrichment was suggested38 to be consistent with typical polyketide assembly followed by rearrangements to remove specific carboxy-derived acetate carbons from the nascent chain and the

13C-

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Scheme 15

introduction of pendant methyl and exomethylene groups by the aldol-type condensation pathway depicted in Scheme 4. Additionally, the uniform enrichment throughout 42 suggested that 39 is quickly converted to 42 as the ultimate biosynthetic product.149 The high average retention of deuterium from [2-2H3, 2-13C]acetate at the pendant methyl and exomethylene groups was further indicative of a single pool of acetate supplying the acetate or malonate biosynthetic precursors. The terminal carbons in the okadaic acid (C1), the diol (C51), and the sulfated chain (C66) all arise through cleavage of an acetate unit.38 Retention of deuterium at C51 was lower than that at C66, suggesting different cleavage mechanisms. Baeyer Villiger oxidation was proposed to account for the introduction of the oxygen atom across the intact acetate-derived carbons at C51 and C53; 18O from [1-13C,18O2]acetate was retained only at the ester carbonyl C53 and not at the diol oxygen, and C51 and C53 originated from the same acetate unit based on long range 13C13C coupling of [1,2-13C ]acetate-labeled 42.38 Similar 2 BaeyerVilliger oxidations have been described in the biosyntheses of a few other polyketides.151154 Cleavage at C1 and C66 may similarly proceed via a BaeyerVilliger oxidation or occur through b-oxidation; the C1 carbonyl of 39 is derived from molecular oxygen.150 A third type of oxygenation reaction has been proposed by Wright to account for the interrupted pattern of acetate units in the chain.38 Oxidation of a methylderived carbon to the a-diketide followed by a Favorskii-type rearrangement,155158 peroxide attack by a flavin monooxygenase, and collapse of the cyclopropanone in which the carboxy-derived carbon is eliminated as carbon dioxide, would give a shortened polyketide chain containing an oxidized methyl-derived carbon (Scheme 14). Dinoflagellates may have

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

Scheme 14

evolved this unique oxidation and deletion mechanism in order to arrange the necessary oxidation pattern for the subsequent cyclization and methylation reactions in polyether synthesis. Wright suggests38 that this oxidation and deletion process, which accounts for uniform acetate labeling and the interrupted labeling pattern, may be operative in other dinoflagellate polyketides as well. The 18O incorporation patterns in 39 further suggest that the cyclization of the tricyclic ether rings occurs via a b-epoxide intermediate (Scheme 15).150 The origins of the carbons in the polyether macrolide goniodomin A 43, a potent antifungal agent from Alexandrium 662 Nat. Prod. Rep., 1999, 16, 653674

methyl (methionine-derived) and the C35/C36 starter unit (glycolate-derived?) originate from acetate. The acetate-labeling pattern was characteristic of that observed in the brevetoxins and the DTXs in which the nascent polyketide chain is occasionally interrupted with carbons derived from the methyl group of acetate. Murakami thus proposes either involvement of citric acid pathway intermediates or a single carbon deletion process in 43 biosynthesis. Amphidinolides are cytotoxic macrolides from an unidentified Amphidinium that was isolated from the Okinawan flatworm Amphiscolops sp.160 The characteristic feature of an odd-numbered macrocyclic lactone present in this series of metabolites cannot be accounted for by typical polyketide assembly. Hence, Kobayashi and coworkers established the biosynthetic origin of the carbons in the 15-membered macrolide amphidinolide J 44, the most abundant macrolide in this dinoflagellate,161 with 13C-labeled acetates.37 As with the polyethers from dinoflagellates, the acetate labeling pattern of 44 was interrupted, resulting in the odd-numbered lactone. Kobayashi proposes that 44 is synthesized through nonsuccessive mixed polyketides involving normal polyketide units and dicarboxylic acid precursors from the citric acid cycle (Scheme 16). The four carbon fragment labeled with one intact acetate unit and two methyl-derived carbons was proposed to originate from a-ketoglutarate and the three carbon fragment composed of one intact acetate unit and a lone methyl-derived carbon from succinate. However, no incorporation of 13Clabeled succinate was observed in a separate feeding experiment to substantiate this proposal.162 Wright rather suggests that the carbon deletion process that was proposed in DTX biosynthesis may be involved in the formation of 44 as well.38 Sulfonium compounds occur in a wide range of unicellular algae as well as marine and terrestrial plants and are proposed to be a major source of sulfur flux in the environment.163 Gonyaulax polyedra produces the cyclopropane sulfonium gonyauline 45 which causes the period-shortening of bioluminescent circadian rhythmicity in the photosynthetic dinoflagellate.164,165 Nakamura and coworkers in Hokkaido have investigated the biosynthesis of 45 and other sulfoniums in G. polyedra and other dinoflagellates with 13C-labeled methio-

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 microalgae in the oceans, very few secondary metabolites, let alone their biosyntheses, have been described. Domoic acid 48, a potent neuroexcitatory toxin produced by Nitzschia pungens forma multiseries, has been implicated in shellfish and bird poisonings.176 Labeling experiments with 13C-labeled acetates by the Wright group indicated that the prenylated amino acid 48 is derived from the condensation of a geranyl unit with a C5 citric acid cycle derivative (Scheme 18).177 Incorporation levels

Scheme 16

nine.166,167 Although 45 and methionine are structurally similar, suggesting that 45 is directly derived from methionine by methylation and deaminationcyclopropanation reactions, methionine is not incorporated intact into 45. The carboxy carbons of methionine or S-methylmethionine are lost upon incorporation,166 whereas the remaining carbons of methionine, as demonstrated by feeding experiments with [methyl-13C]- and [2,3-13C2]methionine, are retained (Scheme 17).167 These

Scheme 18

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

Scheme 17

experiments implied the intermediacy of 3-dimethylsulfoniopropionate (dimethyl-b-propiothetin) 46, the most widely distributed sulfonium metabolite in dinoflagellates,168 in the formation of 45 and this was corroborated in a feeding experiment with uniformly 13C-labeled 46.167 The origin of 46 from methionine was previously established in the green alga Ulva lactuca,169,170 the red alga Chondria coerulescens,171 the heterotrophic dinoflagellate Crypthecodinium cohnii,172 and in lower and higher plants.173 The biosynthetic route to 46 has recently been established in the green macroalga Enteromorpha intestinalis by Hanson and coworkers174,175 and is discussed in Section 3.1. The carboxylate carbon of 45 was specifically enriched with NaH13CO3 fed in the presence of 46, suggesting an origin from CO2.167 The sulfonium gonyol166 47, which is accumulated in G. polyedra when methionine is added to the

probably derived in an analogous manner by the condensation of isopentenyl pyrophosphate with the common C5 unit. The involvement of a related C5 precursor has similarly been deduced in the biosynthesis of the alkaloid lycopodine179 and the streptomycete antibiotics tautomycin and tautomycetin.180 The biosynthesis of algal pheromones in the freshwater diatoms Gomphonema parvulum and Asterionella formosa is discussed in Section 3.2. 2.5 Symbiotic microorganisms Symbiotic microorganisms are often proposed as the true producers of natural products isolated from marine invertebrates.181 Numerous examples exist in which structurally related or identical compounds have been reported from taxonomically distinct invertebrates or from invertebrates and cultured microorganisms alike. Several recent examples (structures 5068) of metabolites isolated from sponges and ascidians that are structurally related to microbial products are listed in Table 1. These findings support the hypothesis that many invertebrate-derived products are of microbial origin. Nat. Prod. Rep., 1999, 16, 653674 663

Table 1

Examples of structurally related metabolites from marine invertebrates and cultured microorganisms and their sources Source Sponge (Theonella swinhoei) Sponge (Jaspis sp.) Sponge (Theonella sp.) Ascidian (Ecteinascidia turbinata) Sponge (Reniera sp.) Ascidian (Eudisoma sp.) Sponge (Discodermia dissoluta) Ascidian (Lissoclinum patella) Ascidian (Didemnum sp.) Ascidian (Polysyncraton lithostrotum) Sponge (Neosiphonia superstes) Sponge (Theonella swinhoei) Related compound Nodularin103 20 Chondramide D184 51 Ferintoic acid A186 53 Saframycin B190 55 Saframycin B190 55 Staurosporine193 58 Alteramide A195 60 Tetrenolin197 62 Enterocin199, 200 (vulgamycin155) 63 Calicheamicin gI1202 65 Scytophycin C120 67 Scytophycin C120 67 Microbial source of related compound Cyanobacterium (Nodularia spumigena) Myxobacterium (Chondromyces crocatus) Cyanobacterium (Microcystis aeruginosa) Bacterium (Streptomyces lavendulae) Bacterium (Streptomyces lavendulae) Bacterium (Streptomyces staurosporeus) Marine bacterium (Alteromonas sp.) Bacteria (Actinomycetales) Bacteria (Streptomyces spp.) Bacterium (Micromonospora echinospora) Cyanobacterium (Scytonema pseudohofmanni) Cyanobacterium (Scytonema pseudohotmanni)

Marine natural product Motuporin105 22 Jaspamide182 (jasplakinolide183) 50 Keramamide A185 52 Ecteinascidin 743187189 54 Renieramycin E191 56 11-Hydroxystaurosporine192 57 Discodermide194 59 Lissoclinolide196 61 Enterocin198 63 Namenamicin201 64 Sphinxolide B203 66 Swinholide A204 68

664

Nat. Prod. Rep., 1999, 16, 653674

Table 1

continued

Faulkner and coworkers at Scripps propose that secondary metabolites are biosynthesized within the cells in which they are localized and have demonstrated in several cases that secondary metabolites are cellularly located within sponge-associated microorganisms.205207 These elegant experiments serve as a model of invertebrate-microbe symbiosis for the production of natural products. In the first experiment to demonstrate that sponge secondary metabolites are localized in prokaryotic symbiont cells, Unson and Faulkner examined the tropical sponge Dysidea herbacea, which supports the filamentous cyanobacterium Oscillatoria spongeliae as its major prokaryotic symbiont.129,208a Two chemotypes of Dysidea exist, one containing both polychlorinated amino acid-derived metabolites and sesquiterpenes, while the second contains only brominated diphenyl ethers. In both cases, cyanobacteria were separated from the sponge cells by flow cytometry, and the isolated cells were chemically analyzed. A unique group of polychlorinated compounds, including the major metabolite 13-demethylisodysidenin 33, was limited to the cyanobacterial symbiont from an Australian specimen of D. herbacea.129 Related metabolites from cultured cyanobacteria include barbamide 32 from Lyngbya majuscula, which also contains an uncommon chlorinated leucine-derived residue (Section 2.2). Accompanying sesquiterpenes herbadysidolide 69 and spirodysin 70, which are commonly found in other species of Dysidea, were isolated only

of the sponge.129 Similarly, Garson and coworkers recently reported on the chemistry of sorted D. herbacea/O. spongeliae by using Percoll gradients.208b They also found that 70 was associated with the sponge cells, either the choanocytes or the archaeocytes, whereas chlorinated diketopiperazines composed of chlorinated leucine residues were constricted to the cyanobacterium. Dysidea herbacea from Palau, on the other hand, contains 2-(2A,4A-dibromophenyl)-4,6-dibromophenol 71 as its major metabolite. Similar cell sorting experiments by Faulkner and coworkers revealed that 71 was also localized in the symbiont O. spongeliae and not in the sponge cells or associated heterotrophic bacteria.208a The finding that 71 is localized in the

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

cyanobacterium and not the associated heterotrophic bacteria contradicts an earlier report by Elyakov and coworkers.209 The Russian group reported that a related brominated diphenyl ether 72, which had previously been isolated from a species of Dysidea, was produced by a cultured bacterium of the genus Vibrio that had been isolated from a Dysidea. The production level of 72 by the cultured bacterium, however, was very small in comparison to the large amounts of brominated diphenyl ethers that are commonly isolated from D. herbacea and further adds to the suspicion that the Dysidea-associated Vibrio may not be the genuine producer of brominated diphenyl ethers.206 The tropical marine sponge Haliclona sp. from Heron Island in Australia characteristically contains a large population of dinoflagellates that occupy roughly 10% of the cellular volume.208c The dinoflagellate is morphologically related to the coral symbiont Symbiodinium microadriaticum. Cell sorting by Nat. Prod. Rep., 1999, 16, 653674 665

Percoll density gradient fractionation by Garson and coworkers demonstrated that the cytotoxic alkaloids haliclonacyclamines A and B are localized within the sponge cells rather than the dinoflagellate.208c Garson proposes that these cytotoxins may assist the sponge in preserving its habitat on exposed coral reef locations. The lithistid sponge Theonella swinhoei contains a diverse series of biologically active metabolites, many of which are structurally related to microbial products.207 Bewley and Faulkner demonstrated that the Palauan T. swinhoei contains three distinct microbial cell populations, viz. unicellular heterotrophic bacteria, unicellular cyanobacteria, and filamentous heterotrophic bacteria.210 Sorting of the four cell types on the basis of cell density allowed for the chemical analysis of each cell population. The macrolide swinholide A204 68, which Kitagawa suggested to be a cyanobacterial product due to its striking structural similarity to scytophycin C120 67, was localized in the unicellular bacteria. The anti-fungal bicyclic glycopeptide theopalauamide211 73, again a suggested cyanobacterial product due to its unique blue-green algal-like aromatic b-amino acid212 (compare with the Adda unit of 19, 20, and 22, Section 2.2), was rather located in the filamentous bacterial fraction. Bewley and Faulkner further note a strong correlation between other lithistid sponge cyclic peptides containing aromatic b-amino acids and the presence of filamentous bacteria and cautiously suggest a potential relationship.207 Contrary to earlier speculation, 68 and 73 were not located in the cyanobacterial or sponge cells. These experiments illustrate the complexity of marine invertebrates and their microflora and caution against predicting the correct source of marine natural products based simply on structural comparison with microbial products.

Scheme 19

merase catalyzes the formation of 2-C-methyl-d-erythritol 4-phosphate 75 in a single step by intramolecular rearrangement and reduction.224,225 The remaining enzymatic steps to IPP are still uncharacterized. The mevalonate-independent pathway has been found by the Rohmer and Lichtenthaler groups exclusively in all three freshwater unicellular green algae (Scenedesmus obliquus, Chlorella fusca, and Chlamydomonas reinhardtii) investigated to date.226228 All isoprenoids examined, including sterols, carotenoids, and the prenyl-side chains of chlorophylls and plastoquinone, were only synthesized by the mevalonateindependent pathway. On the other hand, in the red alga Cyanidium caldarium and in the chrysophyte Ochromonas danica, both terpenoid biosynthetic pathways were detected.227,228 As in higher plants,217 sterols were synthesized via the mevalonate pathway, whereas the chloroplast isoprenoids phytol and b-carotene were formed by the mevalonateindependent route. The biosynthesis of the carotenoid astaxanthin 13 has been characterized in the freshwater unicellular green alga H. pluvialis65,229231 and is discussed in relation to 13-biosynthesis in marine bacteria (Section 2.1). Ethylene biosynthesis in H. pluvialis is related to that in higher plants.232 3 Macroalgae

2.6

Miscellaneous microorganisms

The classical mevalonic acid pathway was, until recently, widely assumed to be universally responsible for terpenoid biosynthesis. Recent 13C-labeled feeding studies by Rohmer and coworkers, however, demonstrated initially that eubacteria synthesize terpenoids via an additional independent pathway involving the intermediacy of d-1-deoxyxylulose-5-phosphate 74.213216 Subsequently, this newly discovered mevalonateindependent pathway has since been firmly established in higher plants, microalgae, cyanobacteria, and a variety of bacteria. 217220 The intermediates and enzymatic steps have been largely characterized in E. coli for the first two reactions. The initial reaction involves the condensation of glyceraldehyde-3-phosphate with a C2-unit derived from pyruvate to yield 74 by the thiamin-dependent d-1-deoxyxylulose-5-phosphate synthase (Scheme 19).221223 A subsequent reductioiso666 Nat. Prod. Rep., 1999, 16, 653674

Marine algae are an extremely rich source of novel oxidized fatty acid-derived compounds called oxylipins. Several biosynthetic pathways based on structural motifs common within oxylipins have been postulated for algal and other marine organism-derived oxylipins. Gerwick has published several extensive reviews58 on the structure and biosynthesis of marine algal oxylipins and recently proposed8 a hydroperoxide generated epoxy allylic carbocation as the central intermediate in oxylipin biosynthesis (Scheme 20). In this section, recent experiments involving algal oxylipin biosynthesis, as well as other algal products, are reviewed. Hypothetical pathways purely based on structural analysis are largely omitted; the reader is rather referred to Gerwicks series of comprehensive reviews. 3.1 Green algae As discussed in Section 2.3, sulfonium compounds are synthesized and accumulated by a variety of marine micro and macroalgae. Proceedings of the First International Symposium on DMSP (45) and Related Sulfonium Compounds have been published in a 36-chapter book and cover a broad range of topics related to 45.233 The sulfonium 45, which is biodegraded to the atmospheric gas dimethyl sulfide,163 acts as an osmoprotectant234 and a cryoprotectant.235 The steps involved in 45 biosynthesis in the marine macroalga Enteromorpha intestinalis have recently been characterized at the chemical174 and biochemical175 levels by Hanson and coworkers. From methio-

Scheme 22

Scheme 20

Scheme 23

nine, the steps include transamination to 4-methylthio-2-oxobutyrate 76, ketoreduction to 4-methylthio-2-hydroxybutyrate 77, S-methylation to the novel sulfonium 4-dimethylsulfonio2-hydroxybutyrate 78, and oxidative decarboxylation to 45 (Scheme 21).174 Substrate-specific enzymes catalyzing the

double bond into a conjugated E,E-diene within the conjugated tetraene system is an oxidative enzymatic process and occurs at a position in relationship to the carboxy terminus.238 The mechanistic features of conjugated tetraene biosynthesis has previously been examined by Hamberg in a cell free system of the red marine alga Lithothamnion corallioides.239 3.2 Brown algae Female gametes of marine brown algae release nonfunctionalized acyclic and/or alicyclic C11 olefins as chemical signals to attract flagellated, motile males.240 Boland and coworkers have shown that these pheromones originate from cleaved polyunsaturated C20 fatty acids.241 Transformation of 2H-labeled natural eicosanoids and shortened unnatural fatty acids indicated that the C11H18 hydrocarbon dictyotene 82 is derived from

Scheme 21

conversion of l-methionine to 78 were partially characterized in cell-free extracts and involve a 2-oxoglutarate-dependent aminotransferase, an NADPH-linked reductase, and an Sadenosylmethionine-dependent methyltransferase.175 In vivo isotope tracer experiments indicate that the first two steps are reversible.174 d-Enantiomers of 77 and 78 were preferred in vitro and in vivo.175 The key intermediate 78 was identified in several diverse phytoplankton species (the prymnesiophyte Emiliania huxleyi, the diatom Melosira nummuloides, and the prasinophyte Tetraselmis sp.), indicating that the same pathway is operative in other important algal sources of 45.174 The higher plant (Wollastonia biflora) pathway to 45 is completely different than in E. intestinalis and in other 45-rich chlorophyte algae and involves the intermediates S-methylmethionine and dimethylsulfoniopropionaldehyde.236,237 Novel polyunsaturated fatty acids with four conjugated double bonds, including (4Z,7Z,9E,11E,13Z,16Z,19Z)-docosaheptaenoic acid (stellaheptaenoic acid) 79, were isolated by Gerwick and coworkers from Anadyomene stellata (Scheme 22).238 A chloroplast preparation produced increased levels of these tetraene fatty acids when unsaturated fatty acids were added. 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

(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (arachidonic acid), while the series of C11H16 hydrocarbons, including ectocarpene 83, multifidene 84, hormosirene 85, finavarrene 86, and giffordene 87, are produced from (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid 88.241,242 Some of the resulting hydrocarbons are thermally labile and undergo spontaneous electrocyclic and sigmatropic reactions to thermostable products. The biosynthesis of 83 in Ectocarpus siliculosus involves a spontaneous Cope rearrangement of the divinylcyclopropane 89.243, 244 Presumed functionalization of 88 by a 9-lipoxygenase yields the hydroperoxide 90 which Pohnert and Boland propose may rearrange to the Hock-oxycarbenium ion 91 before Nat. Prod. Rep., 1999, 16, 653674 667

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

Scheme 25

(5Z,8Z,11Z,14Z)-eicosatetraen-17-ynoic acid 96 and (5Z,8Z,11Z,14Z,19)-eicosapentaen-17-ynoic acid 97, structural analogs 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 (Scheme 26).250 These experiments established 88 as the natural

Scheme 26 Scheme 24

similar rearrangement was detected in the freshwater diatom Gomphonema parvulum which alternatively produces a significant amount of the thermostable C11 cyclopropane isomer 85 and the same C9 fragment 92 (Scheme 24).245 The aldehyde oxygen atom of 92 was demonstrated in a crude, cell-free extract of G. parvulum to be derived from molecular oxygen, thus supporting the proposed functionalization of 88 at C9. The activation energies of the Cope rearrangements of thermolabile bis-alkenylcyclopropane precursors (such as 89) to 6-substituted cyclohepta-1,4-dienes (such as 83) were examined at temperatures typical for the Mediterranean and the Arctic in the spring.243,244 Half-lives of ca. 2030 minutes were found at 18 C (Mediterranean) and over 1 hour at 8 C (Arctic), suggesting that the immediate precursor 89, and not the cyclized product 83, is the actual pheromone. Comparative bioassays of 83 and 89 with male gametes of E. siliculosus verified that the thermolabile cyclopropane 89 is the true male-attracting signal, thus establishing that the spontaneous Cope rearrangement serves as an environmentally controlled mechanism for the deactivation of the pheromone.244 Once formed, these unstable cyclohepta-1,4-dienes are oxidized to a complex mixture of oxygenated derivatives, which themselves act as anti-feedants against algae grazers.240,246,247 In contrast to lower plants, the terrestrial plant Senecio isatideus utilizes (3Z,6Z,9Z)-dodecatrienoic acid 93 for the production of related C11 hydrocarbons. The C12 acid 93 is derived from linolenic acid via three successive b-oxidations and undergoes an oxidative decarboxylation/cyclization to 83 (Scheme 24).248,249 In a similar fashion, the C8 hydrocarbon (3E,5Z)-octa1,3,5-triene (fucoserratene) 94 is derived from 88 by oxidative cleavage of the corresponding 12-hydroperoxy intermediate 95 (Scheme 25)250 The biosynthesis of 94, which was first identified as the sexual pheromone of the brown alga Fucus serratus,251, 252 was examined in the freshwater diatom Asterionella formosa.253, 254 The highly unsaturated eicosanoids 668 Nat. Prod. Rep., 1999, 16, 653674

precursor to 94 and that the pathway proceeds via 95, which is cleaved by a hydroperoxide lyase to yield 94 and the polar fragment 12-oxododeca-5,8,10-trienoic acid 100 (Scheme 25). Hombeck and Boland speculate that 100, which is structurally related to the defensive agent 92, serves a similar role in the diatom and alga.250 Support for the involvement of a 12-lipoxygenase was further provided from inhibition experiments in which the formation of 94 was suppressed under anaerobic conditions but could be reversed by addition of dioxygen.254 The acyclic C11 tetraene 87, which is a major product of Giffordia mitchellae, results from a spontaneous antarafacial [1,7]-sigmatrophic hydrogen shift of the thermolabile (1,3Z,5Z,8Z)-undecatetraene 101 intermediate derived from cleavage of 88 (Scheme 27).242 A low temperature synthesis of

Scheme 27

the postulated intermediate 101 was developed, and the kinetic data furnished a half-life of approximately 2.5 hours at environmental temperature (18 C) and an activation energy among the lowest values currently known for natural pericyclic reactions.255 This rearrangement in nature has thus been proposed to be spontaneous and not catalyzed by an enzyme. Another pericyclic reaction proposed by Pohnert and Boland accounts for the C9 hydrocarbon 7-methylcycloocta-1,3,5-triene 102,255 a minor component of the hydrocarbons from the Mediterranean phaeophyte Cutleria multifida. The authors

propose that (3Z,5Z,7Z)-nona-1,3,5,7-tetraene 103 is produced by cleavage of a suitable fatty acid hydroperoxide and undergoes an 8pe electrocyclic ring closure (Scheme 27). The half-life of synthetic 103 was limited to just a few minutes at ambient temperature and the activation energy of the 8pe electrocyclization was significantly low. NMR studies indicated that the monocyclic 102 was the only isomer present and provided no evidence for a bicyclic cyclohexadiene equilibrium intermediate. Macrophytic brown algae within the genus Laminaria produce a host of divinyl ethers and hydroxy fatty acids putatively derived from w-6 lipoxygenase (LOX) metabolism.256 The Laminaria saccharina gametophyte cell suspension culture produces three monohydroxy fatty acids derived from w-6 oxidation (5Z,8Z,11Z,13E)-[(15S)-hydroxyeicosa5,8,11,13-tetraenoic acid 104, (6Z,9Z,11E,15Z)-(13S)-hydroxyoctadeca-6,9,11,15-tetraenoic acid 105, and (9Z,11E)-(13S)hydroxyoctadeca-9,11-dienoic acid 106], whereas the field-collected sporophyte also contains (9Z,11E,15Z)-(13S)hydroxyoctadeca-9,11,15-trienoic acid 107.257 Yields of 104106 were increased 24 fold when linoleic 108 and glinolenic 109 acids were exogenously added to the cell suspension (Scheme 28). a-Linolenic acid 110, on the other hand, was reportedly toxic to the culture. The lipid metabolism of Dictyopteris membranacea was investigated with 14Clabeled acetate and fatty acids.258

Scheme 29

catalyzed by a 8489 kDa sodium-dependent 12-lipoxygenase into (5Z,8Z,10E,14Z)-(12S)-hydroperoxyeicosa-5,8,10,14-tetraenoic acid followed by hydroperoxide isomerase-catalyzed conversion to 113. The stereochemistry of the conversion was characterized with the alternate substrate (6Z,9Z,12Z)-octadecatrienoic acid 115, stereospecifically deuterated in the 8R, 8S, 11R, and 11S positions. The lipoxygenase-catalyzed reaction occurred with abstraction of the pro-8R hydrogen and addition of oxygen at C-10 in an antarafacial relationship to provide

Scheme 30

Scheme 28

3.3

Red algae

Oxylipin distribution and biosynthesis in marine red algae has been extensively reviewed by Gerwick.58 Further studies on the biosynthesis of vicinal dihydroxy fatty acids from the temperate red alga Gracilariopsis lemaneiformis were reported by Hamberg and Gerwick.259 Experiments with fractionated tissue homogenates of the alga extended prior results with an acetone powder preparation260 on the mode of conversion of arachidonic acid 111 and eicosapentaenoic acid 112 to the diols (5Z,8Z,10E,14Z)-(12R,13S)-dihydroxyeicosa-5,8,10,14-tetraenoic acid 113 and (5Z,8Z,10E,14Z,17Z)-(12R,13S)-dihydroxyeicosa-5,8,10,14,17-pentaenoic acid 114, respectively (Scheme 29). The pathway consists of an initial oxygenation of 111

(10S)-hydroperoxide 116 (Scheme 30). Arachidonic acid 12-lipoxygenase from human platelets, which does not require sodium for its catalytic activity, catalyzes the same reaction.261 The hydroperoxide isomerase (vicinal diol synthase262) reaction proceeds with the intramolecular260 hydroxylation of C-11 and concomitant loss of the pro-11S hydrogen to the diol 117 with overall retention of absolute stereochemistry (Scheme 30).259 The cell-free conversion of arachidonic acid 111 to (13R)hydroxyarachidonic acid 118 by Lithothamnion corallioides proceeds by an unusual mechanism, as its hydroxy group is derived from water as shown by H218O labeling.263 While not incorporated, molecular oxygen is required for the oxidation, prompting Gerwick and coworkers to speculate on the formation of 83 by direct displacement of an enzyme-111 complex 119 by water (Scheme 31). Under mild acid conditions, 118 readily undergoes allylic rearrangement to racemic mixtures of the co-produced oxylipins (15S)- 104 and (11R)hydroxyeicosatetraenoic 120 acids. 18O-Incorporation studies with molecular oxygen implicated that 104 and 120 are largely derived from lipoxygenase metabolism. The conjugated tetraene bosseopentaenoic acid264 121, also generated from incubation experiments with 111 in L. corallioides263 and Bossiella orbigniana,264 is formed by an independent pathway not involving the intermediacy of the hydroxy acids 104 or 120. Hamberg previously reported239 that a unique L. corallioides Nat. Prod. Rep., 1999, 16, 653674 669

Scheme 32

Scheme 31

oxidase catalyzes the formation of the conjugated tetraene system in a related C18 polyunsaturated fatty acid without the involvement of oxygenated intermediates. An arachidonate 9(S)-lipoxygenase has been proposed to account for the diverse oxylipins of Polyneura latissima.265 Conjugated triene-containing fatty acids in Ptilota filicina are biosynthesized in a completely different manner. Gerwick and coworkers demonstrated that the novel enzyme polyenoic fatty acid isomerase catalyzes the formation of conjugated trienes from a variety of fatty acid precursors, including arachidonic acid 111 and (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid 112 to the corresponding 5Z,7E,9E-conjugated triene regioisomers 122 and 123.266 Using stereospecifically deuterium labeled glinolenates, the regio- and stereochemistry of the proton transfers involved in the two sequential isomerizations were elucidated. The authors propose a 1,3-allylic shift of the pro10S hydrogen (Ha) to C-12 of the enzyme-bound diene intermediate 124 followed by a second isomerization involving removal of the pro-7R hydrogen (Hc) and reprotonization at C11 with a solvent-derived proton (Scheme 32). A broad range of alternative substrates were tested, and on the basis of the deduced products, Gerwick and coworkers hypothesize that polyenoic acid isomerase preferentially orients the protonated form of the substrate in the catalytic site with respect to the methyl terminus.267 Many species of Laurencia contain characteristic cyclic bromo ethers, which are derived from bromonium ion-induced cyclization of acylic polyene precursors. Murai and coworkers previously reported the biomimetic conversion of (3E)- and (3Z)-laurediol 125 to (E)- and (Z)-prelaureatin 126 with commercial lactoperoxidase268, 269 (LPO) and, more recently, with a partially purified bromoperoxidase (BPO) from Laurencia nipponica (Scheme 33).270 Further treatment of (Z)-126 with LPO or BPO afforded laureatin 127 and isolaureatin 128, whereas the (E)-isomer furnished the allene laurallene 129 upon treatment with LPO.271 The biosynthesis of halomethanes in the marine red alga Endocladia muricata and in higher plants in the family Brassicaceae via a novel methyltransferase reaction was reported.272 670 Nat. Prod. Rep., 1999, 16, 653674

Scheme 33

Acknowledgements

Research on exploring and engineering natural products diversity from marine microorganisms in the authors laboratory has been generously supported by the National and Washington Sea Grant Programs through the National Oceanic and Atmospheric Administration (R/B-20 and R/B-28). 5 References
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