Noducyclamides A1-A4, B1, and B2 from the Cyanobacterium Nodularia sp. NIES-3585.

A chemical investigation of the hydrophilic fraction of a cultured Nodularia sp. (NIES-3585) afforded six new cyclic lipopeptides, noducyclamides A1-A4 (1-4) containing 10 amino acid residues and dodecapeptides noducyclamides B1 and B2 (5 and 6). The planar structures of these lipopeptides were elucidated based on the combination of HRMS and 1D and 2D NMR spectroscopic data analyses. These peptides are structurally analogous to laxaphycins and contain the nonproteinogenic amino acids 3-hydroxyvaline and 3-hydroxyleucine and a ß-amino decanoic acid residue. The absolute configurations of the noducyclamides (1-6) were determined by acid hydrolysis, followed by advanced Marfey's analysis. Noducyclamide B1 (5) showed cytotoxic activities against MCF7 breast cancer cell lines with an IC50 value of 3.0 µg/mL (2.2 µM).

Cyanoremediation of heavy metals (As(v), Cd(ii), Cr(vi), Pb(ii)) by live cyanobacteria (Anabaena variabilis, and Synechocystis sp.): an eco-sustainable technology.

The cyanoremediation technique for heavy metal (HM) removal from wastewater using live cyanobacteria is promising to reduce the pollution risk both for the environment and human health. In this study, two widely recognized freshwater cyanobacteria, Anabaena variabilis and Synechocystis sp., were used to explore their efficacy in HM (As(v), Cd(ii), Cr(vi), Pb(ii)) removal. The different optimum adsorption conditions were pH 8 and 7.5 for A. variabilis and Synechocystis sp., respectively, but the temperature (25 °C) and contact time (48 hours) were the same for both strains. Under these specified conditions, A. variabilis exhibited the capability to remove 25% of As(v), 78% of Cd(ii), 54% of Cr(vi), and 17% of Pb(ii), whereas Synechocystis sp. removed 77% of As(v), 57% of Cd(ii), 91% of Cr(vi), and 77% of Pb(ii) at different initial concentrations. Metal diversity interfered negatively with cyanobacterial growth, especially Cd(ii) and As(v), as measured by OD730, dry biomass, chlorophyll a, and carotenoid production for both strains. Fourier transform infrared spectrum (FT-IR) analysis revealed the existence of diverse surface binding sites for HM adsorption, stemming from proteins and polysaccharides. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDS) confirmed the presence of HMs on the surface of the cyanobacterial cells. Finally, the zeta potential results indicating alterations in the surface negative charges elucidated the adsorption mechanisms involved in the HM removal by both cyanobacteria. These results provided a comprehensive understanding of the HM adsorption mechanism by cyanobacteria, offering valuable theoretical insights that can be extrapolated to enhance our comprehension of the cyanoremediation mechanisms by various other cyanobacterial strains.

[Up to Date of Cyanobacterial Natural Products].

More than 2000 compounds have been reported from cyanobacteria. The most successful example is dolastatin 10, of which a related compound monomethylauristatin E is used as antibody-drug conjugate (ADC) for Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Recently genome-based analyses by Piel led to the discovery of novel compounds from cyanobacteria. W. H. Gerwick found a potential as anti-SARS-CoV-2 agent in gallinamide A, which was reported as a cathepsin L inhibitor. In our group columbamides were isolated from the marine cyanobacterium Moorena bouillonii. The geometry of the double bond was determined by the coupling constant obtained using non-decoupled heteronuclear single quantum coherence (HSQC). The configuration of chloromethine in a long-chain acyl moiety was determined by the Ohrui method at room temperature using a chiral HPLC column. Columbamide D showed biosurfactant activity. One strain many compounds (OSMAC) is a method to discover new compounds by changing culture conditions. Prior to our experiments, attempts to apply OSMAC in cyanobacteria resulted in the induction or up-regulation of only known compounds. The heat shock culture of the freshwater cyanobacterium Microcystis aeruginosa up-regulated a ribosomal peptide argicyclamide C. At the same time, we discovered bis-prenylated and monoprenylated argicyclamides A and B. More recently iron-limited culture produced hydroxylated argicyclamide A. OSMAC and genome-based screening could lead the discovery of unique biologically active compounds from cyanobacteria.

Syntheses and Biological Activities of Danicalipin A Derivatives.

Synthesis of three derivatives of danicalipin A, tetrachloride, trisulfate and a fluorescent probe was achieved through Wittig reaction strategy. Toxicity of the derivatives against brine shrimp (Artemia salina) as also investigated to provide useful information for the biological activity; i) less chloride derivative showed similar toxicity to danicalipin A, ii) the amphiphilic property, a characteristic feature of danicalipin A, was crucial because trisulfate considerably decreased the toxicity and iii) fluorescent derivative kept brine shrimp toxicity of danicalipin A.

Nostosin G and Spiroidesin B from the Cyanobacterium Dolichospermum sp. NIES-1697.

Chemical investigation of the cyanobacterium Dolichospermum sp. NIES-1697 afforded nostosin G (1), a linear tripeptide, spiroidesin B (2), and two known compounds, anabaenopeptins I (3) and J (4). Planar structures and absolute configurations for 1 and 2 were determined by 2D NMR, HRMS, Marfey's methodology, chiral-phase HPLC, and enzymatic degradation. Nostosin G (1) is a unique example of a linear peptide containing three subunits, 4-hydroxyphenyllactic acid (Hpla), homotyrosine (Hty), and argininal, with potent trypsin inhibitory properties. The biosynthetic gene clusters for nostosin G (1) and spiroidesin B (2) were investigated based on the genome sequence of Dolichospermum sp. NIES-1697.

Argicyclamides A-C Unveil Enzymatic Basis for Guanidine Bis-prenylation.

Guanidine prenylation is an outstanding modification in alkaloid and peptide biosynthesis, but its enzymatic basis has remained elusive. We report the isolation of argicyclamides, a new class of cyanobactins with unique mono- and bis-prenylations on guanidine moieties, from Microcystis aeruginosa NIES-88. The genetic basis of argicyclamide biosynthesis was established by the heterologous expression and in vitro characterization of biosynthetic enzymes including AgcF, a new guanidine prenyltransferase. This study provides important insight into the biosynthesis of prenylated guanidines and offers a new toolkit for peptide modification.

Bioactivities of Lyngbyabellins from Cyanobacteria of Moorea and Okeania Genera.

Cyanobacteria are reported as rich sources of secondary metabolites that provide biological activities such as enzyme inhibition and cytotoxicity. Ten depsipeptide derivatives (lyngbyabellins) were isolated from a Malaysian Moorea bouillonii and a Red Sea Okeania sp.: lyngbyabellins G (1), O (2), P (3), H (4), A (7), 27-deoxylyngbyabellin A (5), and homohydroxydolabellin (6). This study indicated that lyngbyabellins displayed cytotoxicity, antimalarial, and antifouling activities. The isolated compounds were tested for cytotoxic effect against human breast cancer cells (MCF7), for antifouling activity against Amphibalanus amphitrite barnacle larvae, and for antiplasmodial effect towards Plasmodium falciparum. Lyngbyabellins A and G displayed potent antiplasmodial effect against Plasmodium, whereas homohydroxydolabellin showed moderate effect. For antifouling activity, the side chain decreases the activity slightly, but the essential feature is the acyclic structure. As previously reported, the acyclic lyngbyabellins are less cytotoxic than the corresponding cyclic ones, and the side chain increases cytotoxicity. This study revealed that lyngbyabellins, despite being cytotoxic agents as previously reported, also exhibit antimalarial and antifouling activities. The unique chemical structures and functionalities of lyngbyabellin play an essential role in their biological activities.

Biosurfactants from Marine Cyanobacteria Collected in Sabah, Malaysia.

Chemical investigation of the organic extract from Moorea bouillonii, collected in Sabah, Malaysia, led to the isolation of three new chlorinated fatty acid amides, columbamides F (1), G (2), and H (3). The planar structures of 1-3 were established by a combination of mass spectrometric and NMR spectroscopic analyses. The absolute configuration of 1 was determined by Marfey's analysis of its hydrolysate and chiral-phase HPLC analysis after conversion and esterification with Ohrui's acid, (1S,2S)-2-(anthracene-2,3-dicarboximido)cyclohexanecarboxylic acid. Compound 1 showed biosurfactant activity by an oil displacement assay. Related known fatty acid amides columbamide D and serinolamide C exhibited biosurfactant activity with critical micelle concentrations of about 0.34 and 0.78 mM, respectively.

Parthenogenetic female populations in the brown alga Scytosiphon lomentaria (Scytosiphonaceae, Ectocarpales): decay of a sexual trait and acquisition of asexual traits.

In isogamous brown algae, the sexuality of populations needs to be tested by laboratory crossing experiments, as the sexes of gametophytes are morphologically indistinguishable. In some cases, gamete fusion is not observed and the precise reproductive mode of the populations is unknown. In the isogamous brown alga Scytosiphon lomentaria in Japan, both asexual (gamete fusion is unobservable) and sexual populations (gamete fusion is observable) have been reported. In order to elucidate the reproductive mode of asexual populations in this species, we used PCR-based sex markers to investigate the sex ratio of three asexual and two sexual field populations. The markers indicated that the asexual populations consisted only of female individuals, whereas sexual populations are composed of both males and females. In culture, female gametes of most strains from asexual populations were able to fuse with male gametes; however, they had little to no detectable sexual pheromones, significantly larger cell sizes, and more rapid parthenogenetic development compared to female/male gametes from sexual populations. Investigations of sporophytic stages in the field indicated that alternation of gametophytic and parthenosporophytic stages occur in an asexual population. These results indicate that the S. lomentaria asexual populations are female populations that lack sexual reproduction and reproduce parthenogenetically. It is likely that females in the asexual populations have reduced a sexual trait (pheromone production) and have acquired asexual traits (larger gamete sizes and rapid parthenogenetic development).

Synthesis and Structure-Activity Relationship of Omaezallene Derivatives.

Omaezallene derivatives (nor-bromoallene, nor-bromodiene, and bromoenynes) were successfully synthesized. Their antifouling activity and toxicity to the cypris larvae of the barnacle Amphibalanus amphitrite and ecotoxicity to the marine crustacean Tigriopus japonicus were studied. It was revealed that the two side chains of omaezallene were essential to its antifouling activity because the activities of nor-bromoallene and nor-bromodiene were significantly diminished. The bromoenyne was found to exhibit potent antifouling activities comparable to omaezallene with low toxicity and ecotoxicity. Preparation of bromoenyne framework is much easier than that of bromodiene moiety in omaezallene. Based on the antifouling activities of the bromoenynes, the synthesis of fluorescent probes and evaluation of their biological activities were also carried out.

Antioxidants from the Brown Alga Dictyopteris undulata.

An investigation of anti-oxidative compounds from the brown alga Dictyopteris undulata has led to the isolation and identification of isozonarol, isozonarone, chromazonarol, zonaroic acid and isozonaroic acid. Their structures were identified by comparison of MS and NMR spectra. Full NMR assignment and absolute configuration of isozonaroic acid are described. Isozonarol showed the most potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity among the compounds isolated.

Kakeromamide A, a new cyclic pentapeptide inducing astrocyte differentiation isolated from the marine cyanobacterium Moorea bouillonii.

Kakeromamide A (1), a new cyclic pentapeptide encompassing a thiazole ring moiety and a ß-amino acid, was isolated from the marine cyanobacterium Moorea bouillonii. Its structure was elucidated by the spectral analysis and the modified Marfey's method. Compound 1 induced differentiation of neural stem cells into astrocytes at the concentration of 10 µM.

Serinolamides and Lyngbyabellins from an Okeania sp. Cyanobacterium Collected from the Red Sea.

NMR- and MS-guided fractionation of an extract of an Okeania sp. marine cyanobacterium, collected from the Red Sea, led to the isolation of four new metabolites, including serinolamides C (1) and D (2) and lyngbyabellins O (3) and P (4), together with the three known substances lyngbyabellins F (5) and G (6) and dolastatin 16 (7). The planar structures of the new compounds were determined using NMR and MS analyses. The absolute configurations of 1 and 2 were determined by Marfey's analysis of their hydrolysates. The absolute configuration of 3 was ascertained by chiral-phase chromatography of degradation products, while that of 4 was determined by comparison to 3 and 5. The cytotoxic and antifouling activities of these compounds were evaluated using MCF7 breast cancer cells and Amphibalanus amphitrite larvae, respectively. Compounds 3, 4, and 7 exhibited strong antifouling activity, and 3 and 7 were not cytotoxic. A structure-activity relationship was observed for the cytotoxicity of the lyngbyabellins with the presence of a side chain (4 is more active than 3) leading to greater activity. For the antifouling activity, the acyclic form without a side chain (3) was the most active.

Columbamides D and E: Chlorinated Fatty Acid Amides from the Marine Cyanobacterium Moorea bouillonii Collected in Malaysia.

Two new chlorinated fatty acid amides, columbamides D (1) and E (2), along with apratoxins A and C and wewakazole, were isolated from the organic extract of a Moorea bouillonii sample from Sabah, Malaysia. Structure elucidation was accomplished by a combination of MS and NMR analyses. The total synthesis of all four stereoisomers of 1 was completed, and the absolute configuration was determined by chiral-phase HPLC and Marfey's analysis.

New Marine Antifouling Compounds from the Red Alga Laurencia sp.

Six new compounds, omaezol, intricatriol, hachijojimallenes A and B, debromoaplysinal, and 11,12-dihydro-3-hydroxyretinol have been isolated from four collections of Laurencia sp. These structures were determined by MS and NMR analyses. Their antifouling activities were evaluated together with eight previously known compounds isolated from the same samples. In particular, omaezol and hachijojimallene A showed potent activities (EC50 = 0.15-0.23 µg/mL) against larvae of the barnacle Amphibalanus amphitrite.

A quantitative shRNA screen identifies ATP1A1 as a gene that regulates cytotoxicity by aurilide B.

Genome-wide RNA interference (RNAi) with pooled and barcoded short-hairpin RNA (shRNA) libraries provides a powerful tool for identifying cellular components that are relevant to the modes/mechanisms of action (MoA) of bioactive compounds. shRNAs that affect cellular sensitivity to a given compound can be identified by deep sequencing of shRNA-specific barcodes. We used multiplex barcode sequencing technology by adding sample-specific index tags to PCR primers during sequence library preparation, enabling parallel analysis of multiple samples. An shRNA library screen with this system revealed that downregulation of ATP1A1, an α-subunit of Na+/K+ ATPase, conferred significant sensitivity to aurilide B, a natural marine product that induces mitochondria-mediated apoptosis. Combined treatment with ouabain which inhibits Na+/K+ ATPase by targeting α-subunits potentiated sensitivity to aurilide B, suggesting that ATP1A1 regulates mitochondria-mediated apoptosis. Our results indicate that multiplex sequencing facilitates the use of pooled shRNA library screening for the identification of combination drug therapy targets.

Total synthesis and biological activity of dolastatin 16.

The total synthesis of dolastatin 16, a macrocyclic depsipeptide first isolated from the sea hare Dolabella auricularia as a potential antineoplastic metabolite by Pettit et al., was achieved in a convergent manner. Dolastatin 16 was reported by Tan to exhibit strong antifouling activity, and thus shows promise for inhibiting the attachment of marine benthic organisms such as Amphibalanus amphitrite to ships and submerged artificial structures. Therefore, dolastatin 16 is a potential compound for a new, environmentally friendly antifouling material to replace banned tributyltin-based antifouling paints. The synthesis of dolastatin 16 involved the use of prolinol to prevent formation of a diketopiperazine composed of l-proline and N-methyl-d-valine during peptide coupling. This strategy for the elongation of peptide chains allowed the efficient and scalable synthesis of one segment, which was subsequently coupled with a second segment and cyclized to form the macrocyclic framework of dolastatin 16. The synthetic dolastatin 16 exhibited potent antifouling activity similar to that of natural dolastatin 16 toward cypris larvae of Amphibalanus amphitrite.

Wewakazole B, a Cytotoxic Cyanobactin from the Cyanobacterium Moorea producens Collected in the Red Sea.

A mass spectrometry (MS)-guided isolation has led to the purification of a new cyanobactin, wewakazole B (1), along with the known compound curacin D from a Red Sea Moorea producens. The planar structure of 1 was elucidated using a combination of NMR and MS techniques. After ozonolysis and acid hydrolysis, the absolute configurations of the amino acid components of 1 were determined by chiral-phase LC-MS and HPLC analyses. Notably, compound 1 exhibited cytotoxic activity toward human MCF7 breast cancer cells (IC50 = 0.58 µM) and human H460 lung cancer cells (IC50 = 1.0 µM) and was also found to be inactive in a siderophore assay.

cDNA cloning and characterization of vanadium-dependent bromoperoxidases from the red alga Laurencia nipponica.

The marine red alga genus Laurencia is one of the richest producers of unique brominated compounds in the marine environment. The cDNAs for two Laurencia nipponica vanadium-dependent bromoperoxidases (LnVBPO1 and LnVBPO2) were cloned and expressed in Escherichia coli. Enzyme assays of recombinant LnVBPO1 and LnVBPO2 using monochlorodimedone revealed that they were thermolabile but their Km values for Br(-) were significantly lower than other red algal VBPOs. The bromination reaction was also assessed using laurediol, the predicted natural precursor of the brominated ether laurencin. Laurediol, protected by trimethylsilyl at the enyne, was converted to deacetyllaurencin by the LnVBPOs, which was confirmed by tandem mass spectrometry. Native LnVBPO partially purified from algal bodies was active, suggesting that LnVBPO is functional in vivo. These results contributed to our knowledge of the biosynthesis of Laurencia brominated metabolites.

Alnuheptanoid A: a new diarylheptanoid derivative from Alnus japonica.

Extensive chromatographic investigation of the ethanolic extract of Alnus japonica Steud stem bark led to the isolation of a new diarylheptanoid named alnuheptanoid A [(5S)-7-(3,4-dihydroxyphenyl)-1-(4-hydroxyphenyl)-5-methoxyheptan-3-one] (8), together with seven known diarylheptanoid derivatives: platyphyllenone (5), (5S)-1,7-bis(4-hydroxyphenyl)-5-methoxyheptan-3-one (6), 1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-4-hepten-3-one (7), hirsutenone (9), (5R)-O-methylhirsutanonol (10), hirsutanonol (11) and oregonin (13), three triterpenes: α-amyrin (1), betulinaldehyde (3) and betulinic acid (4), and two sterols: ß-sitosterol (2) and daucosterol (12). Compound 6 was isolated for the first time from natural source. The structures of the isolated compounds were determined on the basis of spectroscopic measurements (UV, IR, HR-ESI-MS, 1D and 2D NMR).