In this work, we show the potent antifouling effects of two compounds, barettin (cyclo[(6-bromo-8-entryptophan)arginine]) (1), isolated as a Z/E mixture (87/13), and 8,9-dihydrobarettin (cyclo[(6-bromotryptophan)arginine]) (2), isolated from the marine sponge Geodia barretti. The compounds were isolated guided by their ability to inhibit the settlement of cyprid larvae of the barnacle Balanusimprovisus, and their structures were determined by means of mass spectrometry, NMR, and quantitative amino acid analysis. The activities of these brominated diketopiperazine-like cyclic dipeptides are in the range of antifouling agents in use today, as shown by their EC(50) values of 0.9 and 7.9 microM, respectively. However, contrary to today's antifouling agents, the effects of barettin and 8,9-dihydrobarettin are nontoxic and reversible. A small set of synthetic analogues, including l-arginine, l-tryptophan, 5-bromo-d,l-tryptophan, 6-bromo-d,l-tryptophan, and 6-fluoro-d,l-tryptophan, were tested for possible structure-activity relationships. None of these compounds showed any effect at a concentration of 10 microM. We hypothesize that the isolated compounds are part of the sponge's chemical defense to deter fouling organisms. This theory is supported by the fact that barettin is found in water exposed to living specimens of G. barretti in concentrations that completely inhibit barnacles from settling.

Eighteen brominated sponge-derived metabolites and synthetic analogues were analyzed for antilarval settlement of Balanus improvisus. Only compounds exhibiting oxime substituents including bastadin-3 (4), -4 (1), -9 (2), and -16 (3), hemibastadin-1 (6), aplysamine-2 (5), and psammaplin A (10) turned out to inhibit larval settling at 1 to 10 microM. Analogues of hemibastadin-1 (6) were synthesized and tested for structure activity studies. Debromohemibastadin-1 (8) inhibited settling of B. improvisus, albeit at lower concentrations than hemibastadin-1 (6). Both 6 and 8 also induced cyprid mortality. 5,5'-dibromohemibastadin-1 (7) proved to be nontoxic, but settlement inhibition was observed at 10 microM. Tyrosinyltyramine (9), lacking the oxime function, was not antifouling active and was non-toxic at 100 microM. Hemibastadin-1 (6) and the synthetic products showed no general toxicity when tested against brine shrimp larvae. In contrast to the lipophilic psammaplin A (10), the hydrophilic sulfated psammaplin A derivative (11) showed no antifouling activity even though it contains an oxime group. We therefore hypothesize that the compound needs to cross membranes (probably by diffusion) and that the target for psammaplin A lies intracellularly.

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