11ο Πανελλήνιο Συμπόσιο Ωκεανογραφίας & Αλιείας, Μυτιλήνη, Λέσβος, Ελλάδα 2015

Development of new antifouling agents derived from the red alga Sphaerococcus
coronopifolius
1, 2 1 2 1
Proussis, K. , Ioannou, E. , Calogeropoulou, T. , Roussis, V.
1
Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, University of Athens, kyrprous@mail.ntua.gr,
eioannou@pharm.uoa.gr, roussis@pharm.uoa.gr
2
Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, tcalog@eie.gr

Abstract

Biofouling represents one of the most serious problems in modern maritime industries and its control is one of
the biggest challenges of marine biotechnology. In search of ecologically friendly, biodegradable antifouling
agents, a number of marine natural products were evaluated for their inhibitory activity against the settlement
of barnacle cyprids. Among them, the brominated diterpene bromosphaerol, isolated as the main constituent
of the red alga Sphaerococcus coronopifolius, exhibited prominent inhibitory activity coupled with negligible
toxicity. To further improve the therapeutic ratio of the natural product and its effective incorporation in
marine paints, a number of derivatives was designed and prepared.

Keywords: biofouling, bromosphaerol, Balanus amphitrite, settlement inhibition.

1. Introduction

Biofouling is the process by which any surface submerged in the sea is rapidly covered in a biofilm
composed of microorganisms and subsequently colonized by algae and invertebrate animals,
culminating to its corrosion (Armstrong et al., 2000; Callow & Callow, 2002). This complex process
causes substantial problems to underwater structures and leads to considerable economic
consequences (Aldred & Clare, 2008). Biofouling is particularly detrimental for ships’ hulls where high
levels of fouling result in increased erosion, reduction of speed, increased fuel consumption and
increased air pollution. In addition, biofouling is the major cause for maintenance expenses of any
submerged man-made surface, such as buoys, membrane bioreactors and desalination units, power
plants’ cooling water systems, oil pipelines and aquaculture industries. Efficient antifouling paints are
based on copper compounds and booster biocides that, when submerged, release toxic compounds
causing adverse environmental effects. Synthetic chemical compounds, such as tributyltin
compounds (TBT; (C4H9)3Sn), which were incorporated in marine paints to treat biofouling have been
prohibited from use since 2008. Therefore, there is a demand for research on alternative,
environmentally friendly antifouling compounds (Townsin, 2003; Turner, 2010).
In search of ecologically friendly, biodegradable antifouling agents, a number of natural products
isolated from marine algae collected along the coastlines of Greece were evaluated for their
inhibitory activity against the settlement of barnacle cyprids. Barnacles (Balanus amphitrite) have
become a model organism to study the process of biofouling, both in field observations and
laboratory conditions, owing to their ease of growth and persistent biofouling behaviour (Walker &
Yule, 1984; Dreanno et al., 2006). Among the tested metabolites, the brominated diterpene
bromosphaerol, isolated as the main constituent of the red alga Sphaerococcus coronopifolius,
exhibited significant inhibitory activity coupled with negligible toxicity (Piazza et al., 2011).
Specifically, bromosphaerol was found to be the most active compound exhibiting the lowest EC50
value (0.23 mg/L) without toxic effects on the barnacle cyprids (LC50 >100 mg/L). It has been
suggested that only small molecules with a therapeutic ratio (LC50/EC50) >50 and an EC50 <5 mg/L
against both hard and soft fouling organisms should be considered for commercial applications
(Swain et al., 2007; Qian et al., 2010). Driven by the impressive therapeutic ratio of bromosphaerol
(TR = 434.78), a parameter commonly used as a yardstick for selecting non-toxic antifoulants, it was
decided to undertake a series of synthetic modifications for the preparations of a panel of
bromosphaerol derivatives to be evaluated for their antifouling activity.

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2. Materials and methods

2.1 General experiment procedures

Optical rotations were measured on a Perkin-Elmer model 341 polarimeter with a 1 dm cell. UV
spectra were obtained on a Perkin Elmer Lambda 40 spectrophotometer. IR spectra were obtained
on a Bruker Tensor 27 spectrometer. NMR spectra were recorded on Varian 300 and 600
spectrometers. The 2D experiments (HSQC, HMBC, COSY, NOESY) were performed using standard
Varian pulse sequences. Low resolution ESI mass spectra were recorded on a LC-MSn Fleet Thermo
mass spectrometer. High resolution ESI mass spectra were measured on a Thermo Scientific LTQ
Orbitrap Velos mass spectrometer. Column chromatography separations were performed with
Kieselgel 60 (Merck, 200-400 mesh). Thin-layer chromatography was performed with Kieselgel 60 F254
(Merck glass plates 0.2 mm).

2.2 Collection of the alga and isolation of bromosphaerol

Specimens of S. coronopifolius were collected by SCUBA diving in Parga bay in the West coast of
Greece, at a depth of 10-20 m, in September 2013. A specimen is kept at the Herbarium of the
Laboratory of Pharmacognosy, University of Athens. The wet alga was exhaustively extracted with
mixtures of CH2Cl2/MeOH at room temperature and the extract was concentrated under vacuum to
give a dark green oily residue, which was subjected to vacuum column chromatography on silica gel,
using a 10% step gradient of cyclohexane-EtOAc elution sequence. Fractions 2 and 3 (10% EtOAc in
cyclohexane) was subjected to reversed phase HPLC chromatography, using MeOH 100% as the
mobile phase to yield bromosphaerol.

3. Results

The strategy for the derivatization of bromosphaerol (1) is depicted in Fig. 1. So far, 11
bromosphaerol derivatives have been prepared, purified by silica gel chromatography and
characterized by 1D and 2D NMR spectroscopy and high resolution mass spectrometry.
Epoxidation of the double bond of 1 using m-chloroperbenzoic acid afforded a mixture of the two
isomeric epoxides 2 (1R,2S and 1S,2R) in a ratio of 70:30 and in 75% yield.
Hydroboration of the C1-C2 double bond in 1 afforded the corresponding 1-hydroxy- and 2-
hydroxy derivatives, as well as the tetracyclic compound 5 in 45% yield. The hydroxyl derivatives
were in turn oxidized by pyridinium chlorochromate (PCC) to give the 1-keto and 2-keto derivatives 3
and 4, respectively, in 55% overall yield. Compound 5 which was unreactive under PCC treatment
probably arises from nucleophilic attack of the 1-hydroxyl group on the brominated methylene at
position C17.
Reaction of 1 with N-bromoacetamide in the presence of perchloric acid followed by PCC
oxidation afforded the α,β-unsaturated ketone 6 in 40% yield, accompanied by derivative 7 (in 55%
yield) which possess a tetrahydrofuran ring via an oxygen bridge between carbons C-1 and C-11.
Furthermore, the synthesis of oxime derivatives 8, 9 and 10 was effected by reaction of
unsaturated ketone 6 with methoxyamine hydrochloride, carboxymethoxyamine hydrochloride and
hydroxylamine hydrochloride, respectively (in 98%, 96% and 95% yield, respectively).
Finally a Horner-Emmons reaction of unsaturated ketone 6 with triethyl phosphonoacetate
afforded analogue 11 as a mixture of E/Z isomers (90/10 ratio) in 80%yield.

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 11ο Πανελλήνιο Συμπόσιο Ωκεανογραφίας & Αλιείας, Μυτιλήνη, Λέσβος, Ελλάδα 2015

Fig. 1. Synthetic modifications of bromosphaerol (1).

The bromosphaerol derivatives were evaluated for their inhibitory activity against the settlement
of barnacle cyprids. Among them, three analogues displayed significantly higher levels of inhibitory
activity without exerting toxicity against neither cyprids nor nauplii of B. amphitrite. Detailed analysis
on their structure-activity relationship will be presented.

4. Conclusions/Discussion

Natural products, and especially those derived from marine organisms, are usually isolated in
minute amounts and considered as expensive material for all applications but those related to
human health and the pharmaceutical sector. In the particular case, the red alga S. coronopifolius,
from which the antifouling compound bromosphaerol is isolated as the major metabolite, is a
sizeable seaweed found in high density populations in the marine ecosystem. At the same time, in
the framework of the research program MARIPAINTS, its aquaculture in pilot scale is attempted in
land-based systems to expedite its commercial production for potential incorporation in marine
paints. In view of the development of a cost-effective antifouling agent, all synthetic methodology
was selected for its high yield and its easy up-scale adaptation.

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5. Acknowledgements

This work was supported by the project GSRT-EPANII-11ΣΥΝ-5-1274 “MARIPAINTS”, which is

implemented under the “Cooperation 2011” Action of the “Operational programme Competitiveness
and Enterpreneuship” and is co-funded by the European Social Fund (ESF) and National Resources.

6. References

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