Evaluation of the antifouling properties of 3-alyklpyridine compounds

One of the most promising alternative technologies to antifouling (AF) biocides based on toxic heavy metals lies in the development of natural eco-friendly biocides. The present study evaluates the AF potential of structurally different compounds containing a 3-alkylpyridine moiety. The products, namely poly 3-alkylpyridinium salts, saraine, and haminols, were either extracted or derived from natural sources (the sponges Haliclona sp. and Reniera sarai and the mollusc Haminoea fusari), or obtained by chemical synthesis. All the molecules tested showed generally good anti-settlement activity against larvae of the barnacle Amphibalanus (=Balanus) amphitrite (EC50 values between 0.19 and 3.61 μg ml−1) and low toxicity (LC50 values ranging from 2.04 to over 100 μg ml−1) with non-target organisms. For the first time, the AF potential of a synthetic monomeric 3-alkylpyridine was demonstrated, suggesting that chemical synthesis is as a realistic way to produce large amounts of these compounds for future research and development of environmentally-friendly AF biocides.


Introduction
Biofouling affects all man-made surfaces immersed or in contact with fresh or marine water, such as ships' hulls, oil-rigs, mariculture cages, pipelines, heat exchangers, and seawater intakes in general. Many submerged structures are consequently protected by biocidal antifouling (AF) coatings in order to minimize the effects due to the colonization of micro-and macroorganisms (Evans 1999). Recently, environmental issues caused by metal-based AF coatings (in particular organotins) and booster biocides have led to an increased interest in developing non-toxic alternatives. Since marine organisms are able to protect their body surface by means that do not damage the environment (Omae 2003), natural substances with AF properties have received considerable attention in environmentally-friendly AF technologies (Wahl 1989;Abarzua and Jakubowski 1995;Clare 1996;Abarzua et al. 1999;Fusetani 2004;Qian et al. 2010). To date several marine metabolites have shown significant AF activity and potential as natural biocides for AF paints (Hellio et al. 2000;Da Gama et al. 2002;Steinberg and de Nys 2002;Faimali et al. 2003a;Angarano et al. 2007Angarano et al. , 2009Tsokatou et al. 2007;Sjo¨gren et al. 2008;Feng et al. 2009;Zhou et al. 2009a,b;Villa et al. 2010). However, it must be acknowledged that, although substantial progress has been made in identifying novel AF candidates, further endeavors are still needed to explore the potential of molecules for marine applications. Issues related to accessing large amounts of these compounds for evaluating their toxicity and environmental impact have already caused considerable delays.
In a recent review, Qian et al. (2010) discussed the perspectives in the field of natural AF biocides and recommended the selection of non-toxic candidates based on a LC 50 /EC 50 ratio 4 50 and an EC 50 5 5 mg ml 71 against both hard and soft foulers. Compounds which are more toxic should also be considered if they rapidly degrade in the environment. Furthermore, the development of a natural compound into a commercial product also necessitates compliance with environment safety issues (Thomas and Brooks 2010). This requires long and costly studies to fulfill the obligations of national and international regulations, such as the European Biocidal Products Directive (98/8/EC) and CLP Regulation (EC) 1272/2008, or to allow registration for use in North America (by the US EPA and Health Canada), Australia and New Zealand (Thomas and Brooks 2010).
Poly 3-APS have also been reported to exhibit antibacterial activity toward marine bacteria (Chelossi et al. 2006), growth inhibition of marine and freshwater algae (Elersˇek et al. 2008), prevention of biofilm formation (Garaventa et al. 2003), inhibition of acetylcholinesterase (AChE) (Sep cic´et al. 1997b(Sep cic´et al. , 1998 and cytotoxic activity (Sep cic´et al. 1997a;Paleari et al. 2006). Interestingly, other members of the 3-AP family do not seem to possess comparable AF properties although they share common chemical traits that arise from the origin of these molecules from related biosynthetic pathways that, in the case of molluscs of the genus Haminoea, are first dependent on polyketide assembly that uses nicotinic acid as the starter unit (Cutignano et al. 2003(Cutignano et al. , 2004. Here, the AF potential of compounds containing one or more 3-AP moieties (Figure 1) has been assessed by both anti-settlement activity against cyprids and toxicity against nauplii of the barnacle Amphibalanus (¼Balanus) amphitrite and non-target (the copepod T. fulvus) organisms. The compounds tested in this study include a mixture of poly 3-APS (1) newly isolated from an Antarctic sponge of the genus Haliclona sp., together with sarain-1 (2) and haminols (3-6), which have been obtained from natural sources (Haminoea fusari) or by chemical synthesis.

Biological materials
Haliclona sp. was collected by dredging at a depth of 12-20 m off the Italian Station 'Mario Zucchelli' in Terra Nova Bay (Ross Sea, Antarctica) in January 2004. The material was stored in methanol at 48C until extraction. The sponge was identified by Dr Conxita Avila, University of Barcelona, Spain. R. sarai was collected by scuba diving off Sorrento (Naples, Italy) in May 2006. H. fusari (16 specimens) was collected manually from Lake Fusaro (Naples, Italy) at a depth of 1.5-3.0 m in May 2008.

Equipment and reagents
The mass spectrometry (MS) analyses was carried out by a qTOF micro TM spectrometer (Micromass Ltd, UK) equipped with a ESI source operating on positiveion mode (capillary 3500 V, sample cone 30 V, extraction cone 2 V) and by a MALDI-TOF Voyager DE-PRO (PerSeptive BioSystems, Framingham, MA, USA) operating in the positive linear ion mode in the range 1000-10,000 Da. However, the m/z range up to 30,000 Da was explored too. Samples (1 ml) were cocrystallized on the target with 1 ml of 2,5-hydroxybenzoyc acid (DHB) or a-cyano-4-hydroxycinnamic acid (a-CHCA), both dissolved in 50% aqueous acetonitrile containing 0.1% trifluoroacetic acid (10 mg ml 71 ), used as the matrices. External mass calibration was performed with low and intermediate molecular mass peptide standard kits (PerSeptive BioSystems). NMR spectra were acquired by AMX400 and Avance 600 MHz spectrometers operating at 400 and 600 MHz (Bruker). HPLC purifications were carried out by a Gilson apparatus equipped with Spectra SYSTEM UV 2000 detector (Thermo Finnigan). Chemical reagents for organic synthesis were obtained from Aldrich (Milano, Italy). Isolation and structure determination of natural 3-AP compounds Extraction of the Antarctic sponge (25.8 g dry weight) was performed by repeated grinding with methanol followed by sonication (5 min, 50 Watt, 10 KHz). The extract was filtered through paper, which retained residual sponge tissue. After removal of organic solvent at reduced pressure, the concentrated filtrates were recovered in methanol-water 1:1 and partitioned in succession between hexane, diethyl ether and n-butanol. The methanol-soluble part of the butanolic extract was dissolved in water and filtered twice through Centricon-3 (Amicon, Inc.) with 3000 MW cut-off. Retentates contained pure poly 3-APS (1).

Synthesis of haminols
Haminol-A (5, 98 mg) and -B (6, 27 mg) were synthesized according to a slightly modified procedure previously reported by Alvarez and de Lera (1998

Settlement inhibition
Adult specimens of the barnacle A. amphitrite were collected in Genoa harbor (Italy) in May 2008. Larval rearing and bioassays followed the methods described by Faimali et al. (2005). Cyprid anti-settlement bioassays were generally performed with scalar concentrations of compounds from 0.001 to 100 mg ml 71 . Compounds were dissolved in methanol and transferred to 24-well plates followed by evaporation at 258C. Controls contained methanol, followed by evaporation. Cyprids were added to each well (20 + 3) with 2 ml of filtered (0.22 mm) natural seawater (FNSW) at 37% salinity. The plates were incubated in the dark at 288C for 72 h, after which the wells were observed under a stereo-microscope. Cyprids that permanently attached and metamorphosed were scored as settled (Rittschof et al. 2003). Dead larvae were recognizable by the opaque body and the extended thoracic appendages. The assay was performed two or three times with different batches of larvae, and for each batch of larvae, the test was performed in triplicate. Settlement inhibition EC 50 was determined using Probit 1 software.

Naupliar toxicity
Nauplii II of A. amphitrite were used in both mortality and swimming speed alteration (acute and sub-lethal responses) assays, following the methods described in Faimali et al. (2003aFaimali et al. ( , 2005Faimali et al. ( , 2006. The extract was dissolved in methanol and transferred to experimental plates followed by evaporation. Nauplius stage II larvae were added (n ¼ 15 + 5) to each well with 2 ml of FNSW (0.22 mm) at 37% salinity. Controls were performed with methanol only, followed by evaporation. The plates were incubated for 48 h at 20 + 18C in the dark. The bioassays were performed using three to five replicates. The number of dead larvae was counted under a microscope after 24 and 48 h and the LC 50 was determined using Probit software. The therapeutic ratio (T R ), defined as LC 50 /EC 50 (Vitalina et al. 1991;Rittschof et al. 1994;Clare et al. 1999) was calculated using mortality and settlement inhibition values. The aim of T R is to determine whether the mechanism of settlement inhibition was based on a toxic effect.

Swimming speed alteration
Changes of swimming speed of A. amphitrite nauplii II were registered by video-graphic techniques (SBR System) to highlight the sub-lethal levels of toxicity in invertebrate larvae (Garaventa et al. 2010). IC 50 was determined as the concentration that caused a swimming speed inhibition of 50%, using Probit software.
Tigriopus fulvus mortality assay T. fulvus specimens collected in coastal micro-environments of the Ligurian Sea have been maintained in a laboratory acclimatized culture for several generations in FNSW at 37 + 0.5 PSU, 20 + 0.58C and fed with algal cells (Tetraselmis suecica) and yeast (Saccharomyces cerevisiae). Toxicity tests were carried out on nauplii according to Pane et al. (2005Pane et al. ( , 2008.
The assay was performed following the protocol for A. amphitrite nauplii, except for the number of transferred nauplii, which ranged from 8 to 10. Each test was performed using five replicates. The well plates were kept at 208C, and at 24 and 48 h and naulpii were observed under a stereo-microscope to determine the percentage mortality. Living nauplii were easily recognizable as they were swimming, whereas dead nauplii were completely motionless and had an opaque color. LC 50 was determined using Probit software.

Inhibition of acetylcholinesterase (AChE) assay
The inhibition of AChE was found to affect A. amphitrite cyprid settlement, with total inhibition of AChE correlating with total cyprid settlement inhibition (Faimali et al. 2003b). AChE activities were measured using Ellman's colorimetric method (Ellman et al. 1961). AChE from electric eel, alone or in combination with a test compound, was incubated in the presence of the substrate acetylthiocholine iodide (AcTChI) (29 mg ml 71 ) and stained with dithiobisnitrobenzoic acid (DTNB). The colorimetric reaction was recorded for 10 min at 412 nm in a spectrophotometer (6405 Ultra Violet/Visible, Barloworld Scientific Ltd T/A Jenway, Gransmore Green, UK).

Statistical methods
Results are reported as means + standard error. Data obtained from A. amphitrite and T. fulvus assays were analyzed by one-way ANOVA, with the concentration of test compound as the fixed factor, followed by post hoc analysis (Dunett test). The homogeneity of variances was analyzed by the Levene test and normality of data was analyzed by the Kolmogorov-Smirnov test, at a confidence level of 95% prior to the ANOVA test. Statistical calculations were performed with the Statistica 1 software package, Version 8.

Results
Isolation and characterization of natural compounds 1-4 The structure poly 3-APS (1) was determined unambiguously by spectroscopic and spectrometric analysis ( Figure 2) of pure needle-shaped material obtained by size exclusion chromatography of the MeOH soluble part of the BuOH extract of the Antarctic sponge. The 1 H-NMR spectrum of 1 contained four down-shifted signals typical of a 3-substituted pyridine ring between 8.7 and 7.8 ppm (mg ml 71 ), together with the resonances of a saturated alkyl chain (Figure 2A). Within these last signals, those linked to the charged nitrogen atom typically resonated at 4.45 ppm (H 2 -7), whereas the methylene group linked to C-3 of the pyridine ring was observable at 2.75 ppm (H 2 -7 0 ). No methyl groups were present in the spectrum of 1, but integration of the alkyl protons indicated the presence of eight methylenes according to a head-to-tail sequence of unbranched 3-octyl pyridinium units (Davies-Coleman et al. 1993;Sep cic´et al. 1997b;Scott et al. 2000). The NMR data were corroborated by ESI qTOF MS/MS analysis that showed three major fragmentation ions at m/z 190.2, 379.4 and 568.5 assigned to singly charged monomeric (C 13 H 20 N), dimeric (C 26 H 39 N 2 ) and trimeric (C 39 H 58 N 3 ) structures based on the octyl pyridinium unit ( Figure 2C and D). The average size of the molecule was inferred on the basis of MALDI-TOF MS data that showed a main ion cluster around m/z 3674-3675 using both DHB and a-CHCA matrixes ( Figure 2B). In MALDI, macromolecular poly-electrolytes almost exclusively produce singly charged ions due to an as yet unknown mechanism (Chang et al. 2007). Sep cic´et al. (1997b) hypothesized that the absence of multicharged ions of the alkylpyridinium salts is mostly due to partial neutralization of the positive charges by electrons, whereas Scott et al. (2000) maintained that this is the result of the presence of chloride anions that act as counter-ions under conditions of MS analysis. Following these interpretations of the MALDI data, the poly 3-APS (1) isolated from the Antarctic species of Haliclona should consist of an average number of 19 monomeric units according to neutralization by electrons or, alternatively, roughly 16 monomeric units according to the involvement of chloride ions. Spectral data of sarain-1 (2), haminol-2 (3) and haminol-4 (4) were superimposable with those reported in the literature (Cimino et al. 1989;Spinella et al. 1993b;Cutignano et al. 2007).

Synthesis of haminol-A (5) and -B (6)
Haminol-A (5) and -B (6) were obtained by chemical synthesis based on the stereocontrolled palladiumcatalyzed cross-couplings of 1-alkenyl boronic acids (Figure 3). Unlike the original method proposed by Alvarez and de Lera (1998), the reaction yield was significantly improved by use of the alkylborane II (see Supplementary material for synthetic details [Supplementary material is available via a multimedia link on the online article webpage]). The structure and purity of both compounds were determined by spectroscopic methods.

Settlement inhibition
The percentage inhibition of settlement of A. amphitrite cypris larvae at different concentrations of compounds 1-6 are provided in Figure 4 and the EC 50 values in Table 1. Poly 3-APS (1) significantly inhibited  larval settlement at a concentration slightly above 0.1 mg ml 71 (EC 50 0.19 mg ml 71 ). Saraine-1 (2) and haminol-2 (3) were also very active in inhibiting larval settlement at the same order of magnitude (EC 50 s of 0.53 and 0.28 mg ml 71 , respectively), whereas haminol-4 (4) showed less potency (EC 50 of 2.81 mg ml 71 ). Both natural haminols (3 and 4) totally inhibited settlement at 10 mg ml 71 , and were as potent as haminol-A (5) obtained by chemical synthesis. This last compound (EC 50 2.22 mg ml 71 ) was more active than the corresponding acetyl derivative haminol-B (6), which showed a significant settlement inhibition starting at concentrations of 5 mg ml 71 (EC 50 of 3.6 mg ml 71 ) and total inhibition only at 50 mg ml 71 .
Unfortunately, the small amount of haminol-2 (3) did not allow an evaluation of toxicity to non-target species to be performed. The mortality of A. amphitrite cyprids was recorded during the anti-settlement tests, which showed a LC 50 value of 15.5 mg ml 71 , and a calculated T R of 55.5.
Synthetic haminol-B (6) showed low toxicity towards A. amphitrite nauplii II; a concentration of 50 mg ml 71 significantly increased larval mortality, but the calculated LC 50 was 4 50 mg ml 71 , and the T R value was *14.
The effect on the swimming speed of A. amphitrite nauplii was measured for poly 3-APS (1), saraine-1 (2), and synthetic haminol-A (5) and -B (6) at concentrations ranging from 0.1 to 50 mg ml 71 (Supplementary material [Supplementary material is available via a multimedia link on the online article webpage]). At concentrations as high as 50 mg ml 71 , poly 3-APS (1) and synthetic haminol-B (6) produced a significant decrease in swimming speed, but inhibited motility (IC 50 4 50 and 24.2 mg ml 71 , respectively; Table 1; Supplementary material [Supplementary material is available via a multimedia link on the online article webpage]). ANOVA indicated that the other compounds (2 and 5) significantly affected naupliar swimming speed starting at a concentration of 5 mg ml 71 , and showing a sub-lethal effect at low concentrations (IC 50 of 3.4 and 2.66 mg ml 71 respectively; Table 1). At a concentration of 10 mg ml 71 , no movement was registered, in accordance with the results from the barnacle naupliar toxicity assays, in which most of the larvae died.

AChE inhibition
The effect of Haliclona sp. poly 3-APS (1) on AChE from the electric eel was tested at concentrations between 0.01 and 10 mg ml 71 ( Figure 5). Poly 3-APS significantly inhibited AChE activity starting at a concentration of 0.1 mg ml 71 , and total (100%) inhibition was registered at 10 mg ml 71 .

Discussion
Fouling is a complex and dynamic ecological process, involving micro-and macroorganisms from different kingdoms that continuously interact as they colonize surfaces. Barnacles are among the most troublesome macrofouling marine organisms. These arthropods can comprise up to 28% of the macrofouling community (Thomason et al. 1998) and, for this reason, are commonly used as model organisms in anti-macrofouling laboratory bioassays (Briand 2009). In this study, the effect of natural compounds on settlement of larvae of the barnacle A. amphitrite was measured. In order to evaluate the general impact of these compounds on the marine environment, acute toxicity and sub-lethal effects were determined on nauplii of the barnacle as well as on a non-target organism, the copepod T. fulvus, that belongs to the second largest meiofaunal group in marine sediment (Figure 4). Zooplankton plays an important role in the food web by linking the primary producers with higher trophic levels. For this reason, copepods have been suggested as key bio-indicators for qualitative as well as quantitative analysis of marine and freshwater environments (Green et al. 1996;Miliou et al. 2000;Ferdous and Muktadir 2009). In particular, members of the genus Tigriopus are regarded as suitable model species for acute toxicity assessment of marine pollutants (Lee et al. , 2008Raisuddin et al. 2007;Pane et al. 2008;Wang and Wang 2010). The genus offers ideal organisms for evaluating the toxicity of sediments, for example of harbors (Pane et al. 2008), where leaching from AF paints is likely to concentrate biocides in high amounts.
All the compounds tested in this study showed promising AF activity (EC 50 values ranging from 0.19 and 3.61 mg ml 71 ) and generally low toxicity (LC 50 between 2.04 and over 100 mg ml 71 ) ( Table 1). Poly 3-APS (1) isolated from an Antarctic sponge revealed a regular structure based on homo-polymerization of 3-octyl pyridinium units (Figure 2). Such a motif is related structurally to poly 3-APS isolated from the Mediterranean sponge R. sarai (Sep cic´et al. 1997b) and to EGF-active alkaloids reported from the sponge Callyspongia fibrosa (Davies-Coleman et al. 1993). However, the estimated molecular size of this polymer of the Antarctic sponge was unusual, the MW (*3500 amu) being slightly smaller than that of most of the poly 3-APS reported so far (Sep cic´et al. 1997b;Scott et al. 2000). As suggested by the structural similarities, compound 1 exhibited a non-toxic AF activity towards barnacle cyprids (EC 50 0.19 mg ml 71 , T R 4 526), with a potency that was comparable with that reported for other poly-alkylpyridinium compounds , and CuSO 4 (EC 50 of 0.3 mg ml 71 ) (Faimali et al. 2003a). By analogy with other studies, the inhibition of cyprid settlement was reversible (data not shown). The activity of poly-APS on cyprid settlement has been explained in terms of the surfactant properties of these molecules (Malovrh et al. 1999) that can prevent adhesion or induce lysis of the fouling organisms, as well as in terms of interference with the metabolism of acetylcholine. AChE activity has been detected in the cement gland and in the sensory setae of the antennules of cyprids. Total inhibition of AChE activity produces total inhibition of larval settlement, suggesting that the neurotransmitter modulates settlement and adhesion of cypris larvae (Faimali et al. 2003a). Poly 3-APS generally modulate AChE activity (Turk et al. 2007). Recently, synthetic analogues of large 3-AP-based polymers with 12-and 8-membered chains have shown potent reversible noncompetitive inhibition of AChE activity (Houssen et al. 2010). In line with these results, poly 3-APS (1) isolated from the Antarctic sponge totally blocked acetylcholine stimulation ( Figure 5) at a range of concentrations (around 10 mg ml 71 ) that also produced significant inhibition of larval settlement (12% of average settlement; Figure 4). This evident parallelism corroborates the hypothesis of a non-toxic anti-settlement activity based on alteration of the cholinergic signal system.
The degree of polymerization is a key factor for the biological activities of poly 3-APS compounds (Sep cicá nd Turk 2006) and, for this reason, several synthetic strategies have explored laboratory preparation of these polymers, generally with the intention of obtaining macromolecules with MW 4 3000 Da. Nevertheless, the 3-AP family embraces more than 70 structurally different metabolites, including a large number of compounds with low MW (below 1000 Da) (Fontana 2006). Although many of these compounds have promising biological activities that vary with the chemical traits of the molecules, their AF properties have been rarely investigated (for a review see Turk et al. 2008). The characterization of poly APS (1) from the Antarctic sponge prompted the evaluation of the effects on barnacle settlement of two classes of molecules, sarains and haminols, which have been the subject of numerous chemo-ecological studies in the authors' laboratory. Sarains are dimeric 3-AP compounds that have been exclusively isolated from the Mediterranean sponge R. sarai (Cimino et al. 1986(Cimino et al. , 1989. Haminols are based on a single AP unit and serve as chemical mediators in intra-specific interactions within molluscs of the genus Haminoea (Cimino et al. 1991;Spinella et al. 1993a,b;Marin et al. 1999). Interestingly, AF properties have not been reported for either class of compound, although specimens of R. sarai and H. fusari are not fouled in nature.
Saraine-1 (2) showed good results for anti-settlement activity towards A. amphitrite cyprids (EC 50 0.53 mg ml 71 ) and excellent non-toxic properties (T R 10.41), although it was less effective than poly 3-APS (1). Haminol-2 (3) and -4 (4) also showed good AF activity towards A. amphitrite. Haminol-4 (4) possessed high specificity for barnacle larvae, which were killed at a dosage half of that required for the nontarget organism T. fulvus (LC 50 5.5 mg ml 71 vs 12.9 mg ml 71 ). Due to the small amount of haminol-2 (3) in Haminoea, a rigorous evaluation of toxicity could not be performed, even though a non-toxic mechanism (T R 4 55.5) is suggested on the basis of the low mortality of A. amphitrite cyprids during the antisettlement assay.
In comparison with the alkylpyridinium polymers, low MW 3-AP compounds, such as the haminols (3 and 4), possess much simpler structures than those that have been the subject of several laboratory syntheses. In view of the potential application of this class of compounds as natural biocides, the synthetic studies offer the possibility of achieving large quantities of these molecules. Thus, haminol-A (5) and haminol-B (6) were prepared and tested according to the synthetic strategy proposed by Alvarez and de Lera (1998). Both compounds were obtained in high yields (above 30%) through a convergent synthesis based on the independent preparation of two structurally-simple syntons (Figure 3). Compounds 5 and 6, previously isolated from Haminoea orteai (Spinella et al. 1993b), are structural analogs of haminol-4 (4) having the presence of an additional double bond and substitution of the acetyl function with a hydroxy group (Figure 1). These structural changes did not affect the activity against macro-foulers since both synthetic compounds showed inhibition of larval settlement in line with those reported for the natural products (Table 1). The absence of the acetyl group in product 5 led to an apparent increase of toxicity (LC 50 *8 mg ml 71 ) but the results with haminol-B (6) confirmed the non-toxic mechanism of action (T R 4 13.85) observed with natural product 4, which showed a median lethal concentration (LC 50 ) on A. amphitrite larvae above the highest tested concentration (50 mg ml 71 ) and a median sub-lethal concentration (IC 50 ) of 24.2 mg ml 71 . Except for the anti-microfouling activity reported for untenines, nitroalkyl pyridine members isolated from the Okinawan marine sponge Callyspongia sp. (Wang et al. 1996), haminols (3-6) are the first monomeric 3-AP compounds that have shown AF properties.

Conclusions
In addition to the well-documented AF activity of poly 3-APS (1), this study showed that 3-AP compounds of lower MW displayed good anti-settlement activity and low toxicity to non-target organisms. In particular, the results with the natural haminol-4 (4) and synthetic haminol-B (6) indicated that the structural motif of these monomeric AP compounds could be a promising model for the development of a non-toxic class of biocides. Except for generic antimicrobial activities (Caprioli et al. 1992;Pelttari et al. 2002), haminols do not show any toxicity towards eukaryotic cells or fish at micromolar concentrations. There have been no studies on the stability of these molecules in marine ecosystems, although their function as chemical mediators would suggest a transient persistence (Cimino et al. 1991). Large amounts of haminols are easily produced in a pure form by the simple method of synthesis described in this study. The availability of a diverse array of haminol analogs will lead to improvement of therapeutic ratios of compounds that are potential biocides. Structure-activity studies will improve knowledge of some of the poorly understood mechanisms underlying the development of biofouling.
rearing, and the staff of CNR-ICB for technical support. Many thanks also to the three anonymous referees for constructive remarks on the manuscript.