Two new cyclic depsipeptide derivatives, kahalalides R (1) and S (2), together with two known congeners, kahalalides F (3) and D (4), were isolated from the Indian sacoglossan mollusk Elysia grandifolia. The structures of the new compounds were unambiguously established on the basis of NMR spectroscopic (1H, 13C, COSY, HMBC) and mass spectrometric (FABMS, ESIMS, MALDI-TOF/PSD) data, which also included Marfey amino acid analyses. The new derivative kahalalide R was found to exert comparable or even higher cytotoxicity than the potential drug candidate kahalalide F toward the MCF7 human mammary carcinoma cell line.

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The Mediterranean sponges Aplysina aerophoba and A. cavernicola accumulate brominated isoxazoline alkaloids including aplysinamisin-1 (1), aerophobin-2 (2), isofistularin-3 (3) or aerothionin (4) at concentrations up to 10% of their respective dry weights. In laboratory feeding experiments employing the polyphagous Mediterranean fish Blennius sphinx crude extracts of both Aplysina sponges were incorporated into artificial fish food at their physiological concentrations (based on volume) and offered to B. sphinx in choice feeding experiments against untreated control food. In addition to the Aplysina sponges, extracts from nine other frequently occurring Mediterranean sponges were likewise included into the experiments. Both Aplysina species elicited strong feeding deterrence compared to the other sponges tested. Bioassay-guided fractionation of A. cavernicola yielded the isoxazoline alkaloids aerothionin (4) and aplysinamisin-1 (1) as well as the 3,4-dihydroxyquinoline-2-carboxylic acid (8) as major deterrent constituents when tested at their physiological concentrations as present in sponges. Aeroplysinin-1 (5) and dienone (6), however, which are formed in A. aerophoba and A. cavernicola from isoxazoline precursors through bioconversion reactions upon tissue injury showed no or only little deterrent activity. Fractionation of a crude extract of A. aerophoba yielded aerophobin-2 (2) and isofistularin-3 (3) as major deterrent constituents against B. sphinx. We propose that the isoxazoline alkaloids 1-4 of Mediterranean Aplysina sponges as well as the 3,4-dihydroxyquinoline-2-carboxylic acid (8) (in the case of A. cavernicola) act as defensive metabolites against B. sphinx and possibly also against other predators while the antibiotically active bioconversion products aeroplysinin-1 (5) and dienone (6) may protect sponges from invasion of bacterial pathogens.

We report the first spectroscopic observation of substrate analogue binding to the reduced state of iron superoxide dismutase from Escherichia coli (Fe 2+ SOD) and demonstrate that the pH dependence reflects inhibition of anion binding by ionized Tyr34, not loss of a positive contribution on the part of Tyr34's labile proton. This can also explain the pH dependence of the K M of Fe 2+ SOD. Thus, it appears that substrate binding to Fe 2+ SOD occurs in the second sphere and is not strongly coupled to hydrogen bond donation. Parallel investigations of substrate analogue binding to the oxidized state (Fe 3+ SOD) confirm formation of a six-coordinate complex and resolve the apparent conflict with earlier nuclear magnetic relaxation dispersion (NMRD) results. Thus, we propose that two F -ions can bind to the oxidized Fe 3+ SOD active site, either displacing the coordinated solvent or lowering its exchange rate with bulk solvent. We show that neutral Tyr34's unfavorable effect on binding of the substrate analogue N 3 -can be ascribed to steric interference, as it does not apply to the smaller substrate analogues F -and OH -. Finally, we report the first demonstration that HS -can act as a substrate analogue with regard both to redox reactivity with FeSOD and to ability to coordinate to the active site Fe 3+ . Indeed, it forms a novel green complex. Thus, we have begun to evaluate the relative importance of different contributions that Tyr34 may make to substrate binding, and we have identified a novel, redox active substrate analogue that offers new possibilities for elucidating the mechanism of FeSOD.