Anti-Barnacle Activities of Isothiocyanates Derived from ß-Citronellol and Their Structure-Activity Relationships.

Antifouling agents with low toxicity are in high demand for sustaining marine industries and the environment. This study aimed to synthesize 15 isothiocyanates derived from ß-citronellol and evaluate their antifouling activities and toxicities against cypris larvae of the barnacle Amphibalanus amphitrite. The synthesized isothiocyanates exhibited effective antifouling activities (EC50 =0.10-3.33 µg mL-1 ) with high therapeutic ratios (LC50 /EC50 >30). Four isothiocyanates with an amide or isocyano group showed great potential as effective antifouling agents (EC50 =0.10-0.32 µg mL-1 , LC50 /EC50 =104-833). The enantiomers of the isothiocyanates only slightly differed in their antifouling activities. These results may serve as a basis for further research and development of ß-citronellol-derived isothiocyanates as effective low-toxic antifouling agents. To the best of our knowledge, this study is the first to report the antifouling activities of isothiocyanates derived from accessible natural products.

Effective Synthesis and Antifouling Activity of Dolastatin 16 Derivatives.

Some derivatives of dolastatin 16, a depsipeptide natural product first obtained from the sea hare Dolabella auricularia, were synthesized through second-generation synthesis of two unusual amino acids, dolaphenvaline and dolamethylleuine. The second-generation synthesis enabled derivatizations such as functionalization of the aromatic ring in dolaphenvaline. The derivatives of fragments and whole structures were evaluated for antifouling activity against the cypris larvae of Amphibalanus amphitrite. Small fragments inhibited the settlement of the cypris larvae at potent to moderate concentrations (EC50 = 0.60-4.62 µg/mL), although dolastatin 16 with a substituent on the aromatic ring (24) was much less potent than dolastatin 16.

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.

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.

Isocyanides Derived from α,α-Disubstituted Amino Acids: Synthesis and Antifouling Activity Assessment.

Herein, we contribute to the development of environmentally friendly antifoulants by synthesizing eighteen isocyanides derived from α,α-disubstituted amino acids and evaluating their antifouling activity/toxicity against the cypris larvae of the Balanus amphitrite barnacle. Almost all isocyanides showed good antifouling activity without significant toxicity and exhibited EC50 values of 0.07 - 7.30 µg/mL after 120-h exposure. The lowest EC50 values were observed for valine-, methionine-, and phenylalanine-derived isocyanides, which achieved > 95% cypris larvae settlement inhibition at concentrations of less than 30 µg/mL without exhibiting significant toxicity. Thus, the prepared isocyanides should be useful for further research focused on the development of environmentally friendly antifouling agents.

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.

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.

Design, Synthesis, and Antifouling Activity of Glucosamine-Based Isocyanides.

Biofouling, an undesirable accumulation of organisms on sea-immersed structures such as ship hulls and fishing nets, is a serious economic issue whose effects include oil wastage and clogged nets. Organotin compounds were utilized since the 1960s as an antifouling material; however, the use of such compounds was later banned by the International Maritime Organization (IMO) due to their high toxicity toward marine organisms, resulting in masculinization and imposex. Since the ban, there have been extensive efforts to develop environmentally benign antifoulants. Natural antifouling products obtained from marine creatures have been the subject of considerable attention due to their potent antifouling activity and low toxicity. These antifouling compounds often contain isocyano groups, which are well known to have natural antifouling properties. On the basis of our previous total synthesis of natural isocyanoterpenoids, we envisaged the installation of an isocyano functional group onto glucosamine to produce an environmentally friendly antifouling material. This paper describes an effective synthetic method for various glucosamine-based isocyanides and evaluation of their antifouling activity and toxicity against cypris larvae of the barnacle Amphibalanus amphitrite. Glucosamine isocyanides with an ether functionality at the anomeric position exhibited potent antifouling activity, with EC50 values below 1 µg/mL, without detectable toxicity even at a high concentration of 10 µg/mL. Two isocyanides had EC50 values of 0.23 and 0.25 µg/mL, comparable to that of CuSO4, which is used as a fouling inhibitor (EC50 = 0.27 µg/mL).

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.

Anti-barnacle Activity of Isocyanides Derived from Amino Acids.

Creation of new potent antifouling active compounds is important for the development of environmentally friendly antifouling agents. Fifteen isocyanide congeners derived from proteinogenic amino acids were synthesized, and the antifouling activity and toxicity of these compounds against cypris larvae of the barnacle Balanus amphitrite were investigated. All synthesized amino acid-isocyanides exhibited potent anti-barnacle activity with EC50 values of 0.07 - 10.34 µg/ml after 120 h exposure without significant toxicity. In addition, seven compounds showed more than 95% settlement inhibition of the cypris larvae at 10 µg/ml after 120 h exposure without any mortality observed. Considering their structure, these amino acid-isocyanides would eventually be biodegraded to their original nontoxic amino acids. These should be useful for further research focused on the development of environmentally friendly antifoulants.

Omaezallene from red alga Laurencia sp.: structure elucidation, total synthesis, and antifouling activity.

Natural antifouling products have been the subject of considerable attention. We screened marine algae for antifouling activity and discovered omaezallenes, the new bromoallene-containing natural products isolated from the red alga Laurencia sp. Described is the isolation, structure elucidation, and total syntheses of omaezallenes. The relative and absolute configurations of natural omaezallenes were unambiguously established through total synthesis. The antifouling activities and ecotoxicity of omaezallenes were also evaluated.

Total synthesis of 10-isocyano-4-cadinene and its stereoisomers and evaluations of antifouling activities.

The first enantioselective total synthesis of 10-isocyano-4-cadinene, a marine sesquiterpene isolated from nudibranchs of the family Phyllidiidae, and determination of its absolute stereochemistry were achieved. 10-Isocyano-4-cadinene is expected to be a novel nontoxic antifouling agent. In the synthesis, intermolecular Diels-Alder reaction and samarium diiodide induced Barbier-type cyclization were employed as key steps. The absolute configuration of 10-isocyano-4-cadinene was determined as (1S,6S,7R,10S) by comparison of the optical rotations between natural and synthetic samples. In addition, the authors successfully synthesized 10-epi- and di-1,6-epi-10-isocyano-4-cadinene through the same synthetic pathway. Antifouling activities against Balanus amphitrite with the cadinenes were also evaluated.

Anti-barnacle activity of novel simple alkyl isocyanides derived from citronellol.

Twenty novel simple alkyl isocyanides derived from citronellol were synthesized and evaluated for their antifouling activity and toxicity against cypris larvae of the barnacle, Balanus amphitrite. The anti-barnacle activity of the synthesized isocyanides was in the EC(50) range of 0.08-1.49 µg ml(-1). Simple isocyanides containing a benzoate and chloro group showed the most potent anti-barnacle activity. In addition, none of the synthesized compounds showed significant toxicity and LC(50) values were <10 µg ml(-1). The LC(50)/EC(50) ratios of almost all of the synthesized compounds were >10(2). The results indicate that these simple isocyanides are promising low-toxicity antifouling agents.

Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real-time PCR.

Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.

Total synthesis of 10-isocyano-4-cadinene and determination of its absolute configuration.

The first enantioselective total synthesis of 10-isocyano-4-cadinene, a marine sesquiterpene isolated from nudibranchs of the family Phyllidiidae, was achieved. The cadinene is expected to be a novel nontoxic antifouling agent. In the synthesis, an intermolecular Diels-Alder reaction and a SmI(2)-induced Barbier-type reaction were employed as key steps. The absolute configuration of 10-isocyano-4-cadinene was determined to be (1S, 6S, 7R, 10S) on the basis of the total synthesis. Antifouling activities against Balanus amphitrite with both enantiomers of 10-isocyano-4-cadinene were also evaluated.

Purification and partial amino acid sequence analysis of the larval settlement-inducing pheromone from adult extracts of the barnacle, Balanus amphitrite (=Amphibalanus amphitrite).

A previously undescribed larval settlement-inducing protein was purified from adult extracts of the barnacle, Balanus amphitrite (=Amphibalanus amphitrite). Results of SDS-PAGE indicated that the relative molecular mass of the protein in reduced and denatured form is 31,600 +/- 500 kDa, and that it is distinct from the Settlement Inducing Protein Complex (SIPC) which has previously been determined as a larval settlement-inducing pheromone. The N-terminal 33-residue sequence of the intact protein showed no similarity with previously reported proteins in the EMBL/Genbank/DDBJ databases. The purified protein at a concentration of 10 microg ml(-1) induced approximately four times more larval settlement than the control (filtered natural seawater). In addition, results of the assay using both 24-well polystyrene plates and agarose gels indicated that this protein is probably released into seawater and attracts cypris larvae. These results suggest that the purified protein is a waterborne type pheromone which induces settlement of larvae of B. amphitrite.

Synthesis and anti-barnacle activities of novel isocyanocyclohexane compounds containing an ester or an ether functional group.

Ten novel isocyanocyclohexane compounds that possess an oxygenic functional group at the 4-position were synthesized and evaluated for their antifouling activity against the larvae of the barnacle Balanus amphitrite with the aim of exploring the structure-activity relationships further. The anti-barnacle effect of the synthesized compounds was in the EC50 range of 0.0096-17.0 microg ml(-1). Some ester derivatives exhibited extremely high antifouling activities, and none of the synthesized isocyanocyclohexane compounds showed significant toxicity. The results suggest that the ester function is one of the important groups in the expression of potent antifouling activity in isocyano compounds.

Antifouling activity of simple synthetic isocyanides against larvae of the barnacle Balanus amphitrite.

Twelve simple linear isocyanides were synthesized and examined for antifouling activity and toxicity against cyprid larvae of the barnacle, Balanus amphitrite. Larval settlement was inhibited, with EC50 values of 0.046-1.90 microg ml(-1), and they were much less toxic (LD50 values ranging over 21.28 microg ml(-1)) than CuSO4 (EC50 0.30 microg ml(-1) and LD50 2.95 microg ml(-1)). The data indicate that simple linear isocyanides are promising non-toxic antifouling agents.

Synthesis and anti-barnacle activities of novel 3-isocyanotheonellin analogues.

An antifouling active natural compound, 3-isocyanotheonellin, and its analogues were synthesized and evaluated for antifouling activity against the larvae of the barnacle Balanus amphirite in order to explore structure-activity relationships. The anti-barnacle effect of the 3-isocyanotheonellin analogues was in the EC50 range 0.18-7.20 micrograms ml-1. Some synthesized analogues exhibited potent antifouling activity as high as 3-isocyanothoenllin, and none of the synthesized isocyano compounds showed significant toxicity. The results of the present study suggest that an isocyano group and a hydrophobic site at a suitable position are important for potent antifouling activity without toxicity.

Antifouling substances against larvae of the barnacle Balanus amphitrite from the marine sponge, Acanthella cavernosa.

A total of 118 Japanese marine invertebrates were extracted with methanol, and their chloroform-soluble materials tested for antifouling activity as well as toxicity against barnacle (Balanus amphitrite) larvae. Among 86 species that showed more than 80% larval settlement inhibition, 13 species, including 8 sponges were weakly toxic to the barnacle larvae. The sponge Acanthella cavernosa, which showed most promising activity, was extracted with ethanol and fractionated by solvent partitioning, silica gel column chromatography, gel filtration, and ODS HPLC to give two active compounds which on the basis of spectral data and chemical transformation were identified as a new compound, 10-formamido-4-cadinene and a known compound, T-cadinol, They inhibited larval settlement at concentrations of 0.5 microgram ml-1.