The U.S. Government has rights in this invention pursuant to Contract
No. N00014-88-K-0445 between the Office of Naval Research and the University of
The control of biofouling on artificial surfaces is a significant
problem for structures in contact with the marine environment. Subsequent to the
removal of environmentally hazardous organo-tin compounds from antifouling paints,
control of biofouling accumulation has become the single most expensive maintenance
problem incurred by the U.S. Navy for ship operations. In addition, the recent
introduction of zebra mussels threatens to become a major biofouling hazard in
freshwater environments throughout the United States, particularly in the Great
Lakes region. Thus, the quest for an environmentally-safe and effective antifouling
formulation for structures continues to be the subject of much research and development.
We have prepared a compound (zosteric acid) that has significant antifouling properties
yet has proven to be non-toxic when tested against a wide range of living organisms.
Organisms have evolved a variety of mechanisms that prevent fouling
of their surfaces. Some may reduce the accumulation of fouling agents by physical
means, including the sloughing of the outer tissue layer, and/or the production
of an external surface that minimizes bioadhesion. The production of secondary
metabolites that are capable of deterring potential fouling organisms and predators
is relatively common among marine organisms. However, the chemical species employed
to effectuate this type of protective mechanism are as diverse as the organisms
manufacturing them. Known compounds include elemental vanadium and inorganic acids,
as well as a variety of organic compounds including saponins, terpenes and phenolic
acids (Davis et al. 1989). Although the modest antifouling properties of water
soluble phenolic acids were acknowledged in the 19th Century, practical exploitation
of these compounds has been minimal due to their low effectiveness.
Eelgrass is a source for a number of phenolic acids and derivatives
which include antifouling agents. Several sulfonated anti-biological activity.
Crude aqueous extracts containing phenolic acids, in particular, have been found
to have anti-microbial properties. It has been speculated that these compounds
may be significant ecologically in preventing microbial infections, grazing and
biofouling. The seasonal peaks in soluble phenolic acid abundance, however, coincides
with maximum biofouling abundance, which suggests that phenolics are ineffective
in preventing the accumulation of the biofouling load on eelgrass leaves.
It is therefore an object of the invention to provide an improved
It is another object of the invention to provide a novel use of a
naturally occurring chemical compound.
It is a further object of the invention to provide an improved method
of protecting structures in contact with marine environments from fouling.
It is an additional object of the invention to provide a novel structural
article of manufacture resistant to marine fouling.
It is still another object of the invention to provide an improved
antifouling compound which also is non-toxic.
It is yet another object of the invention to provide a novel method
of disrupting bonding of marine organisms to a structure in contact with a marine
It is still an additional object of the invention to provide an improved
method of isolating zosteric acid under acidic solution conditions.
It is also another object of the invention to provide a novel method
of use of sulfate esters of phenolic acid as marine antifouling compositions.
These and other objects will become apparent from the Detailed Description
and from the Brief Description of the Drawings provided hereinafter. According
to one aspect of the invention we provide a method of using an antifouling composition
to protect against biofouling accumulation of an artificial surface, characterised
by the steps of:
- (a) providing an antifouling composition comprising a sulfooxyphenylcarboxylic
- (b) applying the antifouling composition to the artificial surface.
According to a second aspect of the invention we provide a method
of using an antifouling composition to protect against biofouling accumulation
on an artificial surface, characterised by the steps of:
- (a) providing an antifouling composition comprising a sulfooxyphenylcarboxylic
- (b) dissolving the antifouling composition to form a solution; and
- (c) applying the antifouling compound on the artificial surface.
According to a third aspect of the invention we provide an article
of manufacture having an artificial surface resistant to marine biofouling, characterised
in that an antifouling composition is on the surface and the antifouling composition
comprises a sulfooxyphenylcarboxylic acid.
The invention will now be further described, by way of example only,
with reference to the accompanying drawings:
BRIEF DESCRIPTION OF THE DRAWINGS
Detailed Description of the Preferred Embodiments
- FIGURE 1A illustrates the chemical structure of naturally occurring zosteric
acid; FIG 1B shows phenolic acid sulfate esters synthesized in the laboratory;
and FIG 1C illustrates alternate phenolic acid sulfate esters;
- FIGURE 2A shows bacterial density on glass slides as a function of concentration
for different phenolic acid sulfates; FIG. 2B shows antifouling dose response of
natural zosteric acid (full circles); synthetic p. (sulfooxy) cinnamic acid (open
circles); p-(sulfooxy) ferulic acid (open triangles), and (disulfooxy) caffeic
acid (open squares) all relative to control slides against Acinetobacter
sp.; and FIG. 2C illustrates the antifouling dose response curve for ferulic acid
- FIGURE 3 illustrates barnacle density versus concentration of p-sulfooxy ferulic
- FIGURE 4 shows time versus bacterial density on a glass slide treated with
eelgrass extract and another slide separately treated with methanol solvent;
- FIGURE 5 illustrates density of various organisms on ceramic tiles treated
with methanol (open circles) and crude eelgrass extract (filled or dark circles);
- FIGURE 6 shows a number of bacterial density assays as a function of time performed
with methanol extracts prepared from a variety of marine macrophytes. Open circles
are control slide measurements.
Zosteric acid was isolated from eelgrass by a microbial attachment
assay through a series of chromatographic separations (Sephadex and HPLC). The
purified agent was identified through 13C-NMR, 1H-NMR and
high resolution fast atom bombardment mass spectrometry (HRFAMBS) as a sulfate
ester derivative of cinnamic acid, p-sulfooxy cinnamic acid (FIG. 1A),(zosteric
acid). The compound hydrolyzes under acidic conditions.
In order to verify that zosteric acid was indeed responsible for
the antifouling property of the eelgrass extract, p-sulfooxy cinnamic acid was
synthesized in the laboratory from chlorosulfonic acid and p-coumaric acid in pyridine.
The mixture was extracted with ethanol, water and methanol and purified by HPLC.
The product structure was verified by NMR and HRFAMBS examination.
The relatively simple structure of zosteric acid suggested that the
sulfate group was responsible for the antifouling activity. To test the hypothetical
role of the sulfate group in biofouling activity, other analogs (FIG. 1B) were
prepared as above, using different phenolic acid precursors in the reaction sequence.
Yields from this reaction were about 63% for all compounds. Other synthetic analogs
shown in FIG. 1C also are included within the scope of this invention. Variations
on these fundamental compounds involve lengthening the carboxyl tail to control
the rate of dissolution of the compound from a coating on a marine structure.
Dose effectiveness of zosteric acid and the synthesized sulfate esters
were evaluated in the laboratory using a microbial attachment assay. The ability
of each individual compound to inhibit attachment of bacteria to glass slides was
tested over a concentration range spanning four orders of magnitude. Based on these
tests, purified zosteric acid was sufficiently effective against attachment to
conclude that it is the primary agent responsible for the antifouling properties
of the eelgrass extract (see FIG. 2). It also had sufficient activity to be considered
as an agent for incorporation into antifouling coatings. The synthetic sulfate
esters were as effective as natural zosteric acid in preventing bacterial attachment,
making exploitation of natural eelgrass populations unnecessary for the production
of zosteric acid in industrial quantities.
In contrast with natural zosteric acid, the simple phenolic acid
precursors were ineffective over the same concentration range and in fact appear
to have attracted bacteria to the glass slides in higher density than the controls
in some cases. Without limiting other inventions described herein, this is believed
to show that the presence of the sulfate ester is principally responsible for the
antifouling properties of zosteric acid. The addition of a second sulfate ester
(in the case of caffeic acid disulfate), however, did not increase the antifouling
strength of the compound. The precise mode of action in producing the antifouling
response remains undetermined; however, it should be noted that the extracellular
polysaccharides produced by marine organisms are highly sulfated, and these sulfate
esters play an important role in polymerization (i.e. glue/gel formation). Thus,
without limiting the invention, zosteric acid could be operating at the atomic
level by blocking sulfate-binding surface sensors, or by inhibiting the polymerization
of the extracellular glue.
In addition to the bacterial assays described above, the dose-effectiveness
of ferulic acid sulfate (FAS) was tested using a barnacle attachment assay. The
IC-50 dose in the barnacle assay was similar to the results from the bacterial
assay, suggesting a similar mode of action (see FIG. 3). In these tests, barnacles
stopped swimming when exposed to the active agent, but quickly recovered when transferred
to clean seawater, suggesting, as with the bacteria, that attachment was prevented
by a mechanism other than acute toxicity.
Short tests (< 7d) were performed using crude eelgrass extract
painted onto glass slides and ceramic tiles to see if laboratory results could
be duplicated in the field. Treated slides accumulated significantly fewer bacteria
than controls for 48 h (see FIG. 4). For both treatments, the initial colonization
phase occurred within the first 5 h, after which populations remained stable for
the next 45 h.
The crude extract was also capable of inhibiting the attachment of
spirorbid polychaetes and colonial tunicates to ceramic tiles placed into the temperate
environment of Moss Landing Harbor for as long as seven days (see FIG. 5). The
extract was ineffective, however, against solitary tunicates. No barnacles settled
on any of the plates during the course of this experiment.
In another form of the invention, methanolic extracts were prepared
from freshly collected tissue of other macrophytes to determine whether the antifouling
property of the eelgrass extract had its counterpart in other macrophytes. Representative
species of red, brown and green algae, as well as another seagrass (Phyllospadix
sp.) were included in this survey. A fresh extract of Z. marina was
also prepared for comparative purposes. Bacterial attachment was inhibited by
the eelgrass extract as shown previously, but not by any of the other specimens
tested (FIG. 6). Thus Z. marina may be somewhat unique among marine
plants with regard to the chemistry of antifouling defense. Furthermore, sulfate
esters of phenolic acids do not appear to be widely distributed among disparate
marine taxa, even though many of these species contain numerous phenolic compounds.
The following non limiting examples illustrate various aspects of
Emergent shoots of the seagrass Zostera marina L. were
collected by SCUBA divers from a subtidal bed 5-7 meters deep near Del Monte Beach,
in Monterey Bay, California (36° 30' 40" N, 121° 52' 30" W) in March (dry weight
575 g) and October (dry weight 1700 g) 1990.
The combined dry residue from three MeOH extractions (20°) of freshly
dried ground eelgrass leaves was extracted with H2O prior to being partitioned
between hexanes and 10% aq. MeOH; the MeOH phase was then diluted to 40% aq. MeOH
and extracted with CH2Cl2. Bioassays of these fractions indicated
antifouling activity was principally localized in the H2O extract. Lyophilization
of the aqueous extract gave a hygroscopic solid that was separated batchwise into
three colored bands on a Sephadex LH-20 column (42 cm x 3.2 cm OD, MeOH). The bioactivity
was concentrated in the yellow band, which was identified as zosteric acid (1)
after removal of inorganic salts and other impurities by HPLC. Quantitative estimates
were not calculated from the March collection because preliminary experiments
were performed with various fractions that resulted in acid decomposition of the
zosteric acid (1). The October collection, however, yielded 66 mg of zosteric acid
(I) from 1700 g dry biomass.
Final purification of all phenolic acid sulfates (natural and synthetic)
was carried out by HPLC [Regis ODS irregular column, 25 cm x 10 mm ID; RI detector;
90% aq. MeOH solvent (100% H2O for 5-7); 6895 kPa (1000 psi)]. TLC
Rf values [silica gel; BuOH-HOAC-H2O (4:1:1); UV detection]
: subspecimens 1 (0.63) and other analogs yielded values from (0.29) - (0.83).
Frosted ends (4.5 cm2 area) of glass microscope slides
were treated with candidate fractions or purified compounds dissolved in MeOH
and challenged against a clone of Acinetobacter sp. (a fouling marine bacterium
isolated from the surface of eelgrass leaves). Control slides were treated with
MeOH solvent only. Slides were placed into 50 ml screw-cap plastic tubes containing
30 ml of sterile, 0.2 mm-filtered seawater (FSW) and an inoculum of bacteria from
a log-phase liquid culture (final bacterial concentration was ca. 106
ml-1). Tubes were capped and placed horizontally on a rotary shaker
with the treated surface facing down. Slides were removed at 20 minute intervals,
stained with Hoechst (#2287, Sigma Co.) and cell densities in the frosted regions
were enumerated with the aid of epifluorescence microscopy (1000x). Attached bacterial
densities on treated slides were normalized to control slides at each time point.
Mean density (relative to MeOH control) was calculated for each concentration
from all data in the four hour time series.
Barnacle attachment assays were performed in plastic petri dishes
treated with subspecimen 8 and control solvent (MeOH). Dishes were then filled
with FSW and competent cyprid of Balanus amphitrite were added. After 24
hours, the number of attached cyprids in each dish was determined, and normalized
to the number added initially. Ten replicate dishes were analyzed for each concentration.
Synthesis of subspecimen 1 was as follows: ClSO3H (0.2
ml) was added dropwise to 2 (200 mg) in pyridine (0.5 ml) with stirring
at 20°. Ice water was added, and the acidic aqueous mixture was extracted with
Et2O, basified, extracted with Et2O, and H2O removed
under vacuum. Residue was triturated with H2O, neutralized, dried under
vacuum, and triturated with MeOH. MeOH soluble residue purified by HPLC gave 187
mg for subspecimen 1 (63%). Similar yields obtained for other subspecimens
All mass spectra were negative HRFAB. The matrix used was thiglycerol/glycerol.
NMR * assignments interchangeable.
Zosteric acid from Example III: [M-1]- 242.9963, C9H7O6S,
Δ 0.0 mmu. APT and 13C NMR (62.5 MHz, CD3OD-D2O)
: δ 176.5 (s, C-1), 153.5 (s, C-7), 140.8 (d, C-3), 134.3 (s, C-4), 130.2
(2C, d, *C-5), 125.8 (d, C-2), 122.9 (2C, d, *C-6). 1H NMR (300 MHz,
CD3OD-D2O) : δ 7.59 (2H, d, J=8.7 Hz, *H-5),
7.34 (1H, d, J=16.2 Hz, H-3), 7.27 (2H, d, J=8.4 Hz, *H-6), 6.44
(1H, d, J=16.2 Hz, H-2).
Zosteric acid methyl ester: [M-1]- 257.0118, C10H9O6S,
Δ 0.2 mmu of calculated. 13C NMR (75 MHz, CD3OD) :
δ 167.8, 154.5, 144.3, 130.7, 128.9 (2C), 121.2 (2C), 116.5, 50.8.
1H NMR (300 MHz, CD3OD) : δ 7.65 (d, J=15.9
Hz), 7.57 (d, J=8.7 Hz), 7.31 (d, J=8.7 Hz), 6.45 (d, J=15.9
Hz), 3.72 (s).
Synthetic version of subspecimen 1: [M-1]- 242.9958,
C9H7O6S, Δ 0.5 mmu of calculated. NMR spectra
same as zosteric acid.
Mass spectra for subspecimen 5: [M-2H+Na]- 360.9285,
C9H6NaO10S2, Δ 1.5 mmu of calculated.
13C NMR (62.5 MHz, D2O) : δ 176.5, 144.9, 144.3, 140.3,
135.3, 127.4, 124.4, 123.3, 123.1. 1H NMR (250 MHz, D2O)
: δ 7.81 (br s), 7.62 (br s), 7.45 (d, J=16.0 Hz), 6.61 (d,
J=16.0 Hz). The matrix used was glycerol only.
Mass spectra for subspecimens 6 and 7 mixture: [M-1]-
C9H7O7S, Δ 2.4 mmu of calculated.
13C NMR (62.5 MHz, D2O) : δ 177.0, 176.7, 151.0, 149.4,
141.2, 140.9, 140.1, 135.4, 128.9, 128.1, 127.2, 125.7, 124.1, 123.5, 123.0, 121.4,
118.8, 117.0. 1H NMR (250 MHz, D2O) : δ 7.74 (s), 7.59
(d, J=2.0 Hz), 7.56 (s), 7.42-7.35 (m), 7.30 (d, J=3.2 Hz),
7.22 (d, J=1.9 Hz), 7.18 (d, J=2.0 Hz), 7.15 (d, J=2.0 Hz),
7.03 (d, J=8.4 Hz), 6.53 (d, J=18.3 Hz), 6.41 (d, J=14.8 Hz).
The matrix used was only glycerol.
Subspecimen 8: [M-1]- 273.0083, C10H9O7S,
Δ 1.4 mmu of calculated. 13C NMR (62.5 MHz, CD3OD)
: δ 175.6, 153.1, 143.5, 140.2, 134.8, 126.3, 123.5, 121.2, 112.5, 56.6.
1H NMR (250 MHz, CD3OD) : δ 7.40 (d, J=8.3 Hz),
7.30 (d, J=15.9 Hz), 7.14 (d, J=1.9 Hz), 7.02 (dd, J=8.4,
1.9 Hz), 6.40 (d, J=15.9 Hz), 3.83 (s).