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Summary The cyanobacteria Microcystis proliferate in freshwater ecosystems and produce bioactive compounds including the harmful toxins microcystins (MC). These secondary metabolites play an important role in shaping community composition through biotic interactions although their role and mode of regulation are poorly understood. As natural cyanobacterial populations include producing and non-producing strains, we tested if the production of a range of peptides by coexisting cells could be regulated through intraspecific interactions. With an innovative co-culturing chamber together with advanced mass spectrometry (MS) techniques, we monitored the growth and compared the metabolic profiles of a MC-producing as well as two non-MC-producing Microcystis strains under mono- and co-culture conditions. In monocultures, these strains grew comparably; however, the non-MC-producing mutant produced higher concentrations of cyanopeptolins, aerucyclamides and aeruginosins than the wild type. Physiological responses to co-culturing were reflected in a quantitative change in the production of the major peptides. Using a MS/MS-based molecular networking approach, we identified new analogues of known classes of peptides as well as new compounds. This work provides new insights into the factors that regulate the production of MC and other secondary metabolites in cyanobacteria, and suggests interchangeable or complementary functions allowing bloom-forming cyanobacteria to efficiently colonize and dominate in fluctuating aquatic environments.

The family of polyhalogenated monoterpenes from Plocamium counts over a hundred known members. Using glyceraldehyde acetonide as a chiral pool precursor, an enantioselective and divergent strategy was developed that provides a blueprint for the synthesis of many of the small yet complex acyclic members of this family. The broad applicability of this approach is demonstrated with the short (eight-step) synthesis of four natural products and three analogues. These syntheses are the first of any members of the acyclic polyhalogenated Plocamium monoterpenes, and permitted the evaluation of their selectivity against a range of tumor cell lines.

Five lipopeptides of the lyngbyabellin structure class, four cyclic (1-3, and 5) and one linear (4), were isolated from the extracts of two collections of filamentous marine cyanobacteria obtained from Palmyra Atoll in the Central Pacific Ocean. Their planar structures and absolute configurations were elucidated by combined spectroscopic and chromatographic analyses as well as chemical synthesis of fragments. In addition to structural features typical of the lyngbyabellins, such as two thiazole rings and a chlorinated 2-methyloctanoate residue, these new compounds possess several unique aspects. Of note, metabolites 2 and 3 possessed rare mono-chlorination on the 3-acyloxy-2-methyloctanoate residue while lyngbyabellin N (5) had an unusual N,N-dimethylvaline terminus. Lyngbyabellin N also possessed a leucine statine residue, and showed strong cytotoxic activity against HCT116 colon cancer cell line (IC50 = 40.9 ± 3.3 nM).

Vibrio spp. are the most common and harmful shrimp pathogenic bacteria; however, microalgae and cyanobacteria have the ability to produce antimicrobial substances against these species. In this study, the organic and aqueous extracts of 28 species of marine microalgae and cyanobacteria were screened against Vibrio campbellii M1. Two of these phytoplankton species with antibacterial activity in aqueous extracts (Dunaliella tertiolecta and Skeletonema costatum) and nontoxic to brine shrimp Artemia franciscana nauplii were used to evaluate their anti-Vibrio effect when used as green-water cultures in Vibrio-challenged white shrimp Litopenaeus vannamei cultures. No differences in mortality of juvenile L. vannamei were observed between treatments tested, suggesting that the pathogenicity of V. campbellii could be related to the growth stage of shrimp. The proximal composition of D. tertiolecta and S. costatum was in the recommended range for penaeid shrimp nutrition, allowing shrimp supplemented with these microalgae to have significantly greater total length and weight than control shrimp. Shrimp supplemented with S. costatum presented the highest values of organic mass (11.48 mg/organism) and growth rate (0.31 mg/d) in comparison to D. tertiolecta. These results indicate that microalgae are not only capable of producing antibacterial compounds against Vibrio but can also help shrimp nutrition.

Filamentous marine cyanobacteria are extremely rich sources of bioactive natural products and often employ highly unusual biosynthetic enzymes in their assembly. However, the current lack of techniques for stable DNA transfer into these filamentous organisms, combined with the absence of heterologous expression strategies for nonribosomal cyanobacterial gene clusters, prohibit the creation of mutant strains or the heterologous production of these cyanobacterial compounds in other bacteria. In this study, we evaluated the capability of a derivative of the model actinomycete Streptomyces coelicolor A3(2) to express enzymes involved in the biosynthesis of the protein kinase C activator lyngbyatoxin A from a Hawaiian strain of Moorea producta (previously classified as Lyngbya majuscula). Despite large differences in GC content between these two bacteria and the presence of rare TTA/UUA leucine codons in lyngbyatoxin ORFs we were able to achieve expression of the cytochrome P450 monooxygenase LtxB and reverse prenyltransferase LtxC in S. coelicolor M512 and confirmed the in vitro functionality of S. coelicolor overexpressed LtxC. Attempts to express the entire lyngbyatoxin A gene cluster in S. coelicolor M512 were not successful because of transcript termination observed for the ltxA gene, which encodes a large nonribosomal peptide synthetase. However, these attempts did show a detectable level of cyanobacterial promoter recognition in Streptomyces. Successful expression of lyngbyatoxin A proteins in Streptomyces provides a new platform for biochemical investigation of natural product enzymes from Moorea strains.

DmmA is a haloalkane dehalogenase (HLD) identified and characterized from the metagenomic DNA of a marine microbial consortium. Dehalogenase activity was detected with 1,3-dibromopropane as substrate, with steady-state kinetic parameters typical of HLDs (Km = 0.24 ± 0.05 mM, kcat = 2.4 ± 0.1 s−1). The 2.2-A crystal structure of DmmA revealed a fold and active site similar to other HLDs, but with a substantially larger active site binding pocket, suggestive of an ability to act on bulky substrates. This enhanced cavity was shown to accept a range of linear and cyclic substrates, suggesting that DmmA will contribute to the expanding industrial applications of HLDs.

Marine cyanobacteria are prolific producers of bioactive secondary metabolites responsible for harmful algal blooms as well as rich sources of promising biomedical lead compounds. The current study focused on obtaining a clearer understanding of the remarkable chemical richness of the cyanobacterial genus Lyngbya. Specimens of Lyngbya from various environmental habitats around Curacao were analysed for their capacity to produce secondary metabolites by genetic screening of their biosynthetic pathways. The presence of biosynthetic pathways was compared with the production of corresponding metabolites by LC-ESI-MS² and MALDI-TOF-MS. The comparison of biosynthetic capacity and actual metabolite production revealed no evidence of genetic silencing in response to environmental conditions. On a cellular level, the metabolic origin of the detected metabolites was pinpointed to the cyanobacteria, rather than the sheath-associated heterotrophic bacteria, by MALDI-TOF-MS and multiple displacement amplification of single cells. Finally, the traditional morphology-based taxonomic identifications of these Lyngbya populations were combined with their phylogenetic relationships. As a result, polyphyly of morphologically similar cyanobacteria was identified as the major explanation for the perceived chemical richness of the genus Lyngbya, a result which further underscores the need to revise the taxonomy of this group of biomedically important cyanobacteria.

A collection of Lyngbya bouillonii from Palmyra Atoll in the Central Pacific, a site several thousand kilometers distant from all previous collections of this chemically prolific species of cyanobacterium, was found to contain two new cancer cell cytotoxins of the apratoxin family. The structures of the new compounds, apratoxins F and G, were determined by 1D and 2D NMR techniques in combination with mass spectrometric methods. Stereochemistry was explored by using chromatographic analyses of the hydrolytically released fragments in combination with NMR and optical rotation comparisons with known members of the apratoxin family. Apratoxins F and G add fresh insights into the SAR of this family because they incorporate an N-methyl alanine residue at a position where all prior apratoxins have possessed a proline unit, yet they retain high potency as cytotoxins to H-460 cancer cells with IC(50) values of 2 and 14 nM, respectively. Additional assays using zone inhibition of cancer cells and clonogenic cells give a comparison of the activities of apratoxin F to apratoxin A. Additionally, the clonogenic studies in combination with maximum tolerated dose (MTD) studies provided insights as to dosing schedules that should be used for in vivo studies, and preliminary in vivo evaluation validated the predicted in vivo efficacy for apratoxin A. These new apratoxins are illustrative of a mechanism (the modification of an NRPS adenylation domain specificity pocket) for evolving a biosynthetic pathway so as to diversify the suite of expressed secondary metabolites.

The SSU (16S) rRNA gene was used to investigate the phylogeny of the cyanobacterial genus Lyngbya as well as examined for its capacity to discriminate between different marine species of Lyngbya .W e show that Lyngbya forms a polyphyletic genus composed of a marine lineage and a halophilic ⁄brackish ⁄freshwater lineage. In addition, we found morphological and genetic evidence that Lyngbya spp. often grow in association with other microorganisms, in particular smaller filamentous cyanobacteria such as Oscillatoria, and propose that these associated microorganisms have led to extensive phylogenetic confusion in identification of Lyngbya spp. At the species level, the phylogenetic diversity obtained from the comparison of 16S rRNA genes exceeded morphological diversity in Lyngbya. However, the expectation that this improved phylogeny would be useful to species and subspecies identification was eliminated by the fact that phylogenetic species did not correlate in any respect with the species obtained from current taxonomic systems. In addition, phylogenetic identification was adversely affected by the presence of multiple gene copies within individual Lyngbya colonies. Analysis of clonal Lyngbya cultures and multiple displacement amplified (MDA) single-cell genomes revealed that Lyngbya genomes contain two 16S rRNA gene copies, and that these typically are of variable sequence. Furthermore, intragenomic and interspecies 16S rRNA gene heterogeneity was approximately of the same magnitude. Hence, the intragenomic heterogeneity of the 16S rRNA gene overestimates the microdiversity of different strains and does not accurately reflect speciation within cyanobacteria, including the genus Lyngbya.

The polyhydroxy macrolide (I), possessing a 40-membered ring, is isolated from a new genus of the tropical title marine.

Swinhoeisterols A (1) and B (2), two novel sterols with an unprecedented 6/6/5/7 ring system, were isolated from the sponge Theonella swinhoei. The structures and absolute configurations were elucidated by spectroscopic analysis, X-ray single-crystal diffraction, modified Mosher's method, and TDDFT/ECD calculations. The cytotoxicity of these compounds toward A549 and MG-63 cells encourages studies on their potential target using an inverse virtual screening approach. The predicted inhibitor of h(p300) was corroborated by an in vitro biological test.

The interface between synthetic organic chemistry and natural products was explored in order to unravel the structure of coibacin A, a metabolite isolated from the marine cyanobacterium cf. Oscillatoria sp. that exhibits selective antileishmanial activity and potent anti-inflammatory properties. Our synthetic plan focused on a convergent strategy that allows rapid access to the desired target by coupling of three key fragments involving E-selective Wittig and modified Julia olefinations. CD measurements and comparative HPLC analyses of the natural product and four synthetic stereoisomers led to determination of its absolute configuration, thus correcting the original assignment at C-5 and unambiguously establishing those at C-16 and C-18. Additionally, we synthesized coibacin B on the basis of the assignment of configuration for coibacin A.

A new and improved NMR experiment for F1 scalable, J-Resolved HMBC is presented. This experiment, called J-IMPEACH-MBC, utilizes a minimum number of 180° pulses to preserve maximum signal-to-noise ratio and provides a variable scaling factor (N) that can be used to scale the heteronuclear couplings in F1. It should prove to be useful for the determination of relative stereochemistry and other structural problems. In addition, a description of the advantages of the J-IMPEACH-MBC compared with the J-Resolved HMBC-2 is presented. The method is exemplified with strychnine. Copyright © 2001 John Wiley & Sons, Ltd.

A survey of heteronuclear correlation experiments for the measurement of heteronuclear coupling constants is presented. The purpose of this survey is to evaluate several recent experiments for measuring nJ(C,H) (n = 2, 3) couplings in one comprehensive review. Ten experiments are presented and evaluated relative to one another in terms of experimental usability, ease of data interpretation and number of usable correlations. The experiments compared in this survey are the sensitivity-improved hetero-(ω1)-half-filtered TOCSY (HETLOC), carbon-sorted HETLOC (HSQC-HECADE), coupled/decoupled HSQC-TOCSY, GSQMBC, HSQMBC, G-BIRDR,X-HSQMBC, J-resolved HMBC-2, J-IMPEACH-MBC and methods for extracting heteronuclear coupling constants from gradient-selected variants of the magnitude-mode and phase-sensitive HMBC. The plant alkaloid strychnine was used as a model compound for this study. Copyright © 2001 John Wiley & Sons, Ltd.

A new approach for the acquisition of 1H-detected INADEQUATE-type data is presented. This method uses a decremented variable delay for the evolution of 1J(C,C) couplings. It is demonstrated that this variation of the 1,1-ADEQUATE experiment provides superior performance when compared with the normal static-optimized 1,1-ADEQUATE experiment. This improvement is manifested in both the number and quality of correlations present in the processed data. The technique was validated using the model compound ethyl trans-crotonate and its utility was demonstrated utilizing the structurally unique marine natural product jamaicamide A. Copyright © 2001 John Wiley & Sons, Ltd.

Three new gradient-selected NMR experiments for the analysis of long-range heteronuclear coupling constants are presented. They are demonstrated on a series of compounds including sucrose, strychnine and a complex macrolide, phormidolide, isolated from a marine cyanobacterium. These 1D and 2D heteronuclear single quantum multiple bond correlation (HSQMBC) experiments provide pure absorption, antiphase lineshapes for precise, direct measurement of nJ(C,H) coupling constants. The example compounds were chosen to demonstrate the high sensitivity and ease of spectral interpretation provided by these HSQMBC experiments. In addition, suppression of one-bond CH correlations has been introduced into the HSQMBC pulse sequence. Copyright © 2000 John Wiley & Sons, Ltd.

Curacin A is a thiazoline-containing lipid from the marine cyanobacterium Lyngbya majuscula. Despite being a potent inhibitor of microtubule assembly and of colchicine binding to tubulin, curacin A bears little or no structural resemblance to colchicine or to any other tubulin ligand. We investigated the interaction of curacin A with bovine brain tubulin using three different approaches. We first examined its effect on the intra-chain formation of a cross-link in β-tubulin by N,N′- ethylenebis(iodoacetamide). Formation of this cross-link, between cys239 and cys354, is blocked by colchicine and its A-ring analogues as well as by various other inhibitors of colchicine binding; C-ring analogues do not inhibit its formation. Curacin A strongly inhibited formation of this cross-link. Second, we examined the effect of curacin A on the time-dependent exposure of sulfhydryl groups on tubulin as measured by alkylation with iodo[14C]acetamide. Curacin A inhibited this very strongly, more so than either colchicine or podophyllotoxin. Last, we investigated the effect of curacin A on the time-dependent exposure of hydrophobic areas on the tubulin molecule. We found that curacin A had only a small effect on this process, comparable in magnitude to that of podophyllotoxin. Curacin A thus appears to have an unusual interaction with tubulin. Its binding site on tubulin is likely to overlap with that of the A-ring of colchicine. Drug Dev. Res. 40:223–229, 1997. © 1997 Wiley-Liss, Inc.

Novel polyunsaturated fatty acids with four conjugated double bonds were found in extracts of the green macroalga, Anadyomene stellata. The isolation of five of these with different chain lengths and varying degrees of unsaturation--16:5, 18:4, 20:5, 20:6, and 22:7--was accomplished by organic extraction followed by a combination of vacuum and high-performance liquid chromatography. One of these that was a novel substance (22:7) was characterized as 4Z,7Z,9E,11E,13Z,16Z,19Z-do cosaheptaenoic acid and assigned the trivial name stellaheptaenoic acid. The structure of this new compound, isolated as its methyl ester derivative, was deduced from detailed nuclear magnetic resonance, gas chromatography/mass spectrometry (GC/MS), and other spectroscopic methods. Incubation of a chloroplast preparation, isolated from a crude algal homogenate by differential centrifugation, with six unsaturated fatty acids (palmitoleic, 6Z,9Z,12Z,15Z-octadecatetraenoic acid, arachidonic acid, eicosapentaenoic acid, 7Z,10Z,13Z,16Z-docosatetraenoic acid, and 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoi c acid) resulted in substantially increased synthesis of unique tetraene compounds as detected by ultraviolet spectrophotometry and tentatively identified by GC/MS.

Curacin A is a newly isolated lipid natural product that binds in the colchicine site of tubulin and inhibits mitosis. We have examined its effects on tubulin polymerization, studied by turbidimetry, under three reaction conditions. In 1.0 M glutamate + 1.0 mM MgCl2, with a 37°C reaction temperature, we could find no concentration of curacin A that completely inhibited polymerization. A maximal inhibitory effect on turbidity development (about 50%) was observed with 5 mM drug. Higher drug concentrations induced an abnormal polymerization reaction, which at 40 mM differed little from the control reaction except for slightly delayed depolymerization in response to reducing the temperature to 0°C. In 0.8 M glutamate (no MgCl2), with a 30°C reaction temperature, complete inhibition did occur at 3–5 mM drug, but higher drug concentrations again induced an abnormal polymerization reaction. With 40 mM curacin A this reaction was also similar to the control reaction, except that cold-induced depolymerization was slightly enhanced relative to the control. When polymerization was induced by microtubule-associated proteins, maximal inhibition of turbidity development (about 70%) occurred with 2 mM drug, with higher curacin A concentrations inducing abnormal polymerization reactions that reached about 60% of the control turbidity readings. Under all three reaction conditions the polymer induced by high concentrations of curacin A consisted of large aggregates of tightly coiled spiral fibers that had a 2–3 filament substructure. The implications of these findings with curacin A are discussed in terms of the use of tubulin polymerization assays as a screen for identifying new antimitotic drugs. © 1995 Wiley-Liss, Inc.1

The absolute configuration is determined at C-14, whereas between C-5 and C-7 only a syn relative configuration can be established.

Cyanobacteria are abundant producers of natural products well recognized for their bioactivity and utility in drug discovery and biotechnology applications. In the last decade, characterization of several modular gene clusters that code for the biosynthesis of these compounds has revealed a number of unusual enzymatic reactions. In this article, we review several mechanistic transformations identified in marine cyanobacterial biosynthetic pathways, with an emphasis on modular polyketide synthase(PKS)/non-ribosomal peptide synthetase (NRPS) gene clusters. In selected instances, we also make comparisons between cyanobacterial gene clusters derived from marine and freshwater strains. We then provide an overview of recent developments in cyanobacterial natural products biosynthesis made available through genome sequencing and new advances in bioinformatics and genetics.

The tropical marine cyanobacterium Hormothamnion enteromorphoides produces a suite of cytotoxic and antimicrobial cyclic peptides. The structure of the most lipophilic of these, hormothamnin A, was determined by interpretation of physical data, principally high NMR and FAB MS, in combination with chemical derivitization and degradation schemes. Isolation of a key pentapeptide fragment DPHE-D-LEU-L-ILE-D-allo-ILE-L-LEU, obtained under partial hydrolysis conditions, was instrumental to the final structure determination. β-D-aminooctanoic acid (D-BAOA) was characterized as a per-ester derivative following complete acid hydrolysis and Z-didehydrohomoalanine (DHHA) was spectroscopically in the intact peptide. The remaining residues (HYPRO, 2 × HSER, GLY) were evident from amino acid and spectroscopic analysis. Sequencing of these residues made use of knowledge from fragments, high field NMR (NOESY and ROESY) and FAB MS analysis of the intact peptide. Absolute stereochemistries of the α-amino residues were determined by HPLC analysis of the acid liberated residues derivatized with Marfey's reagent. The absolute stereochemistry of the β-amino residue was shown by circular dichoroism analysis, HPLC analysis of the Marfey derivative, and chiral synthesis of a homolog. By these techniques, hormothamnin A was demonstrated to possess a cyclo-[D-PHE-D-LEU-L-ILE-D-allo-ILE-L-LEU-GLY-D-BAOA-L-HSER-DHHAL-HYPRO-L-HSER] structure.

Curacin A is a structurally novel antimitotic agent isolated from the Caribbean cyanobacterium Lyngbya majuscula. Its planar structure has been previously determined from a spectroscopic investigation. Here, we define the complete relative and absolute configuration of curacin A by comparison of products obtained from chemical degradation of the natural product with the same substances prepared by synthesis. Curacin A is shown to have 2R, 13R, 19R, 21S absolute configuration.

Guided by ichthyotoxic activity against goldfish, a new lipopeptide, malyngamide H [1], and its corresponding free acid, 7-methoxytetradec-4(E)-enoic acid [2], have been isolated from the tropical marine cyanobacterium Lyngbya majuscula. The structure of the new carbon skeleton borne by malyngamide H was elucidated on the basis of spectroscopic analysis, mainly 2D nmr. The absolute stereochemistry of the cyclohexenone moiety of malyngamide H [1] was deduced by a combination of 2D NOESY and exciton chirality circular dichroism spectroscopy.

An ichthyotoxic amide of 7( S )-methoxytetradec-4( E )-enoic acid was isolated from an Okinawan collection of Lyngbya majuscula . Data from the acetate derivative of this amide was used for the probable structure revision of stylocheilamide, a previously reported metabolite from the sea hare Stylocheilus longicauda .

Fractionation of the lipid extract of the marine cyanobacterium Lyngbya majuscula collected from Curacao afforded two quinoline alkaloids in low yield. Their structures were determined as 4,8-dimethyl-6-O-(2'-4'-di-O-methyl-beta-D-xylopyranosyl)-hydroxyquinoli ne and 4,8-dimethyl-6-hydroxyquinoline on the basis of spectroscopic analysis, mainly 2D NMR spectroscopy.

Three new malyngamides, J (1), K (2) and L (3), all amides of 7(S)-methoxytetradec-4(E)-enoic acid, were isolated from Curacao collections of Lyngbya majuscula. The structures of these new malyngamides were elucidated on the basis of 1D and 2D NMR spectroscopic analysis of the natural products and key derivatives. The stereochemistry of malyngamide J (1) was probed by NMR and exciton chirality circular dichroism spectroscopy.

Curacin D is a novel brine shrimp toxic metabolite isolated from a Virgin Islands collection of the marine cyanobacterium Lyngbya majuscula. Structure elucidation of curacin D was accomplished through multidimensional NMR, GC/MS, and comparisons with curacin A. Curacin D provides new insights into structure-activity relationships in this natural product class as well as some aspects of the likely biosynthetic pathway of the curacins.

The first total synthesis of the trichlorinated natural product barbamide is described. The convergent approach involves coupling (S)-3-trichloromethylbutanoyl chloride with Meldrum’s acid (2,2-dimethyl-1,3-dioxane-4,6-dione) to give 15 followed by addition of the novel secondary amine N-methyl-(S)-dolaphenine 2 (prepared in 6 steps and 24% overall yield from N-Cbz-L-phenylalanine) to give the β-keto amide 16 which was converted directly to the required (E)-enol ether.

This Highlight article describes three different imaging mass spectrometry (IMS) approaches, MALDI, DESI and SIMS and their recent applications in the analysis of natural products. IMS has opened up a new avenue for establishing the functional roles of natural products.

Calcium influx through N-methyl D-aspartate receptors (NMDAR) regulates signaling cascades that underlie neuronal development and synaptic plasticity. Earlier we demonstrated that the voltage-gated...

Incubation of [1-14C]linoleic acid with an enzyme preparation obtained from the red algaLithothamnion corallioides Crouan resulted in the formation of 11-hydroxy-9(Z),12(Z)-octadecadienoic acid as well as smaller amounts of 9-hydroxy-10(E),12(Z)-octadecadienoic acid, 13-hydroxy-9(Z),11(E)-octadecadienoic acid and 11-keto-9(Z),12(Z)-octadecadienoic acid. Steric analysis showed that the 11-hydroxyoctadecadienoic acid had the (R) configuration. The 9- and 13-hydroxyoctadecadienoic acids were not optically pure, but were due to mixtures of 75% (R) and 25% (S) enantiomers (9-hydroxyoctadecadienoate), and 24% (R) and 76% (S) enantiomers (13-hydroxy-octadecadienoate). 11-Hydroxyoctadecadienoic acid was unstable at acidic pH. In acidified water, equal parts of 9(R,S)-hydroxy-10(E),12(Z)-octadecadienoate and 13(R,S)-hydroxy-9(Z),11(E)-octadecadienoate, plus smaller amounts of the corresponding (E),(E) isomers were produced. In aprotic solvents, acid treatment resulted in dehydration and in the formation of equal amounts of 8,10,12- and 9,11,13-octadecatrienoates. The enzymatic conversion of linoleic acid into the hydroxyoctadecadienoic acids and the ketooctadecadienoic acid was oxygen-dependent; however, inhibitor experiments indicated that neither lipoxygenase nor cytochrome P-450 were involved in the conversion. This conclusion was supported by experiments with18O2 and H2 18O, which demonstrated that the hydroxyl oxygen of the hydroxy-octadecadienoic acids and the keto oxygen of the 11-ketooctadecadienoic acid were derived from water and not from molecular oxygen.

Marine cyanobacteria are a rich source of biomedically relevant secondary metabolites that are of unique molecular architecture.1 In line with this theme, somocystinamide A (1) was isolated from a mixed assemblage of Schizothrix and Lyngbya and shown to possess remarkable biological properties.2,3 Initially, 1 only showed moderate activity against mouse neuroblastoma cells (Neuro-2a).3 In subsequent studies, however, its IC50 against human umbilical vein endothelial cells (HUVECs) was found to be 500 fM.2 This astonishing in vitro finding was verified in zebra fish wherein antiangiogenetic effects were observed at 80 nM media concentration. Despite this potency, 1 was shown to have no observable adverse effects on zebra fish even at 30 μM. In addition, 1 was shown to trigger apoptosis in tumor cells via caspase-8 activation.2 This activity profile supports the development of 1, or analogs thereof, for potential use in cancer treatment. Biosynthetically, somocystinamide A appears to be assembled through alternating NRPS-PKS elements with a unique termination of a PKS unit via decarboxylation and dehydration to furnish the terminal olefin as seen in curacin A.4 Methylation of the enamide using S-adenosyl methionine, a signature decoration in marine cyano-bacterial natural products,5 produces the tertiary enamide. Secondary enamides have been observed in many natural products and their preparation has been studied extensively in recent years.6 Tertiary enamides, however, are encountered very rarely in natural products and as such, the strategies for their preparation are relatively undeveloped and scarce.6,7 Furthermore, the presence of the disulfide group in 1 requires great care and consideration during the course of synthesis.8 For example, synthetic investigation of epidithiapiperazinedione natural products (such as 2) has met with much difficulty in the installation of the disulfide.8,9 To date, only one complete total synthesis of a compound of this class has been reported.10 Another case in point is psammaplin A (3); in all three of the published syntheses of 3, the sulfur atoms were introduced as a disulfide in the final step so as to avoid side reactions.11 It was envisioned in the synthesis of 1 that the key carbon-carbon connection at the internal olefin would be made by olefin cross metathesis using a ruthenium catalyst.12 Accordingly, terminal olefin 6 was prepared from the known aldehyde 513 via a Wittig reaction.14 Thiazolidine was chosen as the protecting group for the thiol of 4 because of its relative stability and its tandem protection of the carbamate proton. Screening of commercially available ruthenium catalysts revealed that the second generation Hoveyda-Grubbs catalyst (11) was optimal (table 1).12c Furthermore, this reaction was optimized for multi-gram scale by adjusting the concentration and number of equivalents of 7. Good stereoselectivity was observed in all cases (e.g. trans : cis = 18:1, entry 8). Minimizing the amount of 7 facilitated the purification process because the major side product of the reaction, dimer 12, closely eluted with the desired product 8 during chromatography. In line with recent reports that some ruthenium catalysts are more functional group-tolerant than initially suspected,15 alkyl sulfides are apparently very well tolerated by 11, but not by 9, suggesting that there may be competition between tri-cyclohexylphosphine and the sulfide 6 for binding as a ligand on ruthenium.16 Table 1 Olefin cross metathesis with various conditions to produce 8 The methyl ester 8 was hydrolyzed to obtain the carboxylic acid 13 in order to avoid undesired reduction to the primary alcohol. Afterward, the thiol and the carbamate of 13 were reductively deprotected by sodium in liquid ammonia (scheme 2).13a Re-protection of the carboxylic acid as a methyl ester, deprotection of the amine, and acetylation yielded 14 in a good yield. Simultaneous basic hydrolysis of the methyl ester and the thioacetate in the presence of O2 cleanly caused dimerization to the disulfide in one pot to give 15. However, attempts to effect the dimerization by conventional means, such as treatment with I2, did not give good yields.17 Scheme 2 Synthesis of disulfide 15 (somocystinoic acid) With 15 (named “somocystinoic acid”) in hand, various conditions were investigated to couple the in-situ generated imine 16 to the corresponding di-acyl chloride 20, the formation of which was verified by the reaction with methylamine to produce 17. In most cases, the starting material decomposed while in some cases a trace amount of 1 was observed. This result was curious because there are reports of synthesis of simple enamides via acylation of the corresponding acid chloride with imine.7,18 It is possible that the putative acyl iminium ion intermediate 21 is intercepted via an intramolecular reaction due to its dimeric nature (scheme 3).18 In support of this hypothesis, only tautomer 16 and not 19 was observed by 1H and 13C NMR in CD2Cl2.19 Scheme 3 Possible mechanisms for enamide formation We then turned our attention to the recently developed Cu-mediated vinylation reaction.6 It has been reported, although with little experimental evidence, that this approach is inapplicable to acylic secondary amides.6b,d,f Therefore, coupling between a simple amide 23 and a commercially available vinyl bromide 24 was investigated, but found ineffective, giving support to these earlier reports. Observation that the hydrolytic decomposition of 1 to 17 occurs with relatively low activation energy3 inspired us to carry out the opposite reaction,20 specifically condensation of the aldehyde 1821 with 17. A Soxhlet extraction apparatus was found to be a convenient vessel for small scale reactions and allowed the use of solvents heavier than water.22 Observing that the putative intermediate 21 did not yield 1, we hypothesized that the E1 pathway was not viable (scheme 3). In support of this hypothesis, use of a more polar solvent, THF, decreased the yield in comparison to 1,2-dichloroethane, a less polar solvent.23 The best result was obtained when TsOH was used as the catalyst, which gave a 41% yield. Further investigation of this reaction is underway on model systems. The analytical data (1H and 13C NMR, MS, UV, IR and optical rotation) for synthetic 1 were essentially identical to those for natural somocystinamide A,24 thus confirming the originally assigned structure.3 The bioactivity of the synthetic product 1 was evaluated in the murine Neuro-2a cancer cell line, but its activity was highly variable,25 possibly due to the unusual solubility properties of 1, or to factors which we do not currently understand. We also tested synthetic 1 in the brine shrimp toxicity model26 and observed significantly impaired motility in treated (at 1, 10 and 100 μg/mL) versus control shrimps (DMSO). We also noted a much decreased quanitity of intestinal contents in the treated group. These observations underscore the value of a synthetic supply of somocystinamide A for it is clear that it possesses biological properties not yet understood. In conclusion, the first total synthesis of somocystinamide A was achieved despite the presence of two quite challenging functional groups in the molecule. The current synthesis is fairly robust for all steps but the last two such that >1 g of 15 has been prepared smoothly, and scale-up necessary for in vivo testing of this biomedically exciting compound should now be possible. However, our work demonstrates the need for a more robust reaction to prepare tertiary enamides, a development which would open the door for synthesis of other natural products possessing this functional group, such as the laingolides.27

The isolation and structure elucidation of belamide A from the marine cyanobacterium Symploca sp. is described. Belamide A is a highly methylated linear tetrapeptide with structural analogy to the important linear peptides dolastatins 10 and 15. Disruption of the microtubule network in A-10 cells was observed at 20 μM and displayed classic tubulin destabilizing antimitotic characteristics. The moderate cytotoxicity of belamide A (IC 50 0.74 μM vs HCT-116 colon cancer line) provides new insights into structure–activity relationships for this drug class.

Five new lyngbyabellin analogs along with a known compound, dolabellin, have been isolated from the marine cyanobacterium Lyngbya majuscula collected from Papua New Guinea. The structures of lyngbyabellins E–I were elucidated through extensive spectroscopic analysis, including HR-FABMS and 1D and 2D NMR experiments. The absolute configurations of lyngbyabellin E and H were ascertained by chiral HPLC and GC/MS analysis of degradation products, in combination with NMR experiments. All five lyngbyabellins showed cytotoxicity to NCI-H460 human lung tumor and neuro-2a mouse neuroblastoma cell lines with LC 50 values between 0.2 and 4.8 μM.

Four new depsipeptides have been isolated from the marine cyanobacterium Lyngbya semiplena collected from Papua New Guinea. The amino and hydroxy acid partial structures of wewakpeptins A-D (1-4) were elucidated through extensive spectroscopic techniques, including HR-FABMS, 1D (1)H and (13)C NMR, as well as 2D COSY, HSQC, HSQC-TOCSY, and HMBC spectra. The sequence of the residues was determined through a combination of multifaceted approaches including ESI-MS/MS, HMBC, ROESY, and a modified 1D HMBC experiment. The absolute stereochemistry of each residue was determined by chiral HPLC and chiral GC-MS methods. The wewakpeptins represent an unusual arrangement of amino and hydroxy acid subunits relative to known cyanobacterial peptides and possess a bis-ester, a 2,2-dimethyl-3-hydroxy-7-octynoic acid (Dhoya) or 2,2-dimethyl-3-hydroxyoctanoic acid (Dhoaa) residue, and a diprolyl group reminiscent of dolastatin 15. Wewakpeptin A and B were the most cytotoxic among these four depsipeptides with an LC(50) of approximately 0.4 muM to both the NCI-H460 human lung tumor and the neuro-2a mouse neuroblastoma cell lines.