A novel biosurfactant, 2-acyloxyethylphosphonate, isolated from waterblooms of Aphanizomenon flos-aquae

Article abstract of DOI:10.3390/11070539

A novel biosurfactant, 2-acyloxyethylphosphonate, was isolated from waterblooms of Aphanizomenon flos-aquae. Its structure was elucidated by chemical degradation and HRFABMS, GC/EI-MS and 1D- and 2D-NMR spectral analyses. The surfactant contained one mole of 2-hydroxyethylphosphonate and one mole of fatty acid, with hexadecanoic acid accounting for 84.1% of the total fatty acid content. The structure was confirmed by synthesis of 2-oleoyloxyethylphosphonate from ethylene oxide, phosphorus acid and oleic acid chloride. Considering the isolated surfactant molecule as hexadecanoyloxyethylphosphonic acid (mw. 364), the critical micelle concentration (CMC) was about 22 mM.

Full text of DOI:10.3390/11070539

Molecules 2006, 11, 539-548
molecules
ISSN 1420-3049
http://www.mdpi.org
Full Paper
A Novel Biosurfactant, 2-Acyloxyethylphosphonate, Isolated
from Waterblooms of Aphanizomenon flos-aquae
1
,
2
2,
2
Kunimitsu Kaya *, Louise F. Morrison , Geoffrey A. Codd *, James S. Metcalf , Tomoharu
3
3
1
Sano , Hiroo Takagi and Takuya Kubo
1
Graduate School of Environmental Studies, Tohoku University, Aoba-ku, Sendai 980-8579, Japan,
E-mail: kubo@mail.kankyo.tohoku.ac.jp
2
3
Division of Environmental and Applied Biology, School of Life Sciences, University of Dundee,
Dundee DD1 4HN, UK. E-mails: L.F.morrison@dundee.ac.uk; j.s.metcalf@dundee.ac.uk
National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba Ibaraki 305-8506, Japan,
E-mails: sanotomo@nies.go.jp; takakiho@nies.go.jp
*
Authors to whom correspondence should be addressed; E-mail: kaya@mail.kankyo.tohoku.ac.jp,
Tel: (+81)-22-795-4506; Fax: (+81)-22-795-4506; E-mail: g.a.codd@dundee.ac.uk
Received: 16 June 2006; in revised form: 18 July 2006 / Accepted: 18 July 2006 / Published: 22 July
2
006
Abstract: A novel biosurfactant, 2-acyloxyethylphosphonate, was isolated from
waterblooms of Aphanizomenon flos-aquae. Its structure was elucidated by chemical
degradation and HRFABMS, GC/EI-MS and 1D- and 2D-NMR spectral analyses. The
surfactant contained one mole of 2-hydroxyethylphosphonate and one mole of fatty acid,
with hexadecanoic acid accounting for 84.1% of the total fatty acid content. The structure
was confirmed by synthesis of 2-oleoyloxyethylphosphonate from ethylene oxide,
phosphorus acid and oleic acid chloride. Considering the isolated surfactant molecule as
hexadecanoyloxyethylphosphonic acid (mw. 364), the critical micelle concentration
(
CMC) was about 22 mM.

Molecules 2006, 11
540
Keywords: biosurfactant, cyanobacterium, Aphanizomenon flos-aquae, fatty acids,
-hydroxyethanephosphonate, 2-acyloxyethanephosphonate, C-P linkage, surface tension,
toxicity, critical micelle concentration (CMC).
2
Introduction
Cyanobactyeria (blue-green algae), which form water blooms in eutrophic lakes and reservoirs, can
produce various kinds of bioactive compounds [1,2]. During investigations into bioactive compounds
from blooms of the cyanobacterium Aphanizomenon flos-aquae collected from Balgavies Loch in
Scotland, we have found a novel 2-hydroxyethylphosphonate-containing biosurfactant and here
present its isolation and structure elucidation.
C-P linkage-containing compounds are widely distributed in the organisms such as animals [3,4],
plants [5], bacteria [6,7], fungi [8] and protozoa [9-11]. Such compounds commonly contain a C-P
linkage in the form of a 2-aminoethylphosphonate group, whereas the biosurfactant isolated from A.
flos-aquae contains a 2-hydroxyethylphosphonate group. This biosurfactant represents the first case of
2
-hydroxyethylphosphonate identification in a biological sample.
Results and Discussion
The biosurfactant was adsorbed onto DEAE-Sephadex™, eluted with 0.2 M ammonium
acetate-containing solvent [12] and further purified by silica-gel column and thin-layer
chromatography. It was obtained at a yield of 64 mg (0.64%) from 10 g of the lyophilized material.
The surfactant, 2-acyloxyethylphosphonate (AOEP) 1, was a colorless amorphous solid which gave an
Rf value of 0.67 on TLC using chloroform/methanol (95/5, v/v).
O
H2
C
O
O
C
H
P OH
OH
2
1
1
The spot on TLC was stained by iodine vapor. The H-NMR data of the intact AOEP species is
summarized in Table 1. These signals, with the exception of one at 2.80 ppm, suggested that the
surfactant contained a mixture of saturated and unsaturated fatty acyl groups. The triplet signal at 2.80
2
ppm suggested that the CH combined with a hetero atom such as nitrogen or phosphorus.

Molecules 2006, 11
541
1
3
Table 1. H-NMR spectroscopic data for 2-acyloxyethanephosphonate in CD OD
1
1
H- H COSY
1
Signal
H
J (Hz)
(
cross peak)
1
2
3
4
5
6
7
8
5.3 (2.6H)
2.80 (2H)
(m)
2, 4
(t, 5.8)
(t, 7.3)
(m)
1
2.16 (2H)
5
2.07 (0.4H)
1.58 (2H)
1, 6, 7
3, 6
4, 5, 8
4
(q, 7.3)
(broad m)
(t, 7.3)
(t, 7.0)
1.32-1.28 (22H)
0.96 (0.3H)
0.89 (2.7H)
6
In order to identify the fatty acyl group present, the surfactant was subjected to methanolysis using
1
.0M HCl-containing methanol at 80 °C for 20 h. Fatty acid methyl esters were extracted with
n-hexane, and analyzed by GC-MS using chemical ionization (CI) and electron impact ionization (EI).
The CI-MS and EI-MS data indicated the presence of tetradecanoic [Rt, 8.21 min; CI: m/z 243
+
+
+
+
(
M+1) ; EI: m/z 242 (M )], hexadecatrienoic [Rt, 9.20 min; CI: m/z 265 (M+1) ; EI: m/z 264 (M )],
+
+
hexadecanoic [Rt, 9.34 min; CI: m/z 271 (M+1) ; EI: m/z 270 (M )] and octadecatrienoic [Rt, 10.22
+
+
min; CI: 293 (M+1) ; EI: m/z 292 (M )] acids. Fragments of the unsaturated fatty acids were observed
+
+
+
by EI-MS: M - m/z 29 (CH
CH=CH), m/z 107 (CH -CH
CH=CH-CH ). These results suggest that the double bond positions of the unsaturated fatty acids are
3
-CH
2
), M - m/z 55 (CH
3
-CH
2
-CH=CH), M - m/z 81 (CH
3 2
-CH -CH=CH-
3
2
- CH=CH-CH=CH -CH=CH) and m/z 121 (CH
3
-CH - CH=CH-CH=CH-
2
2
located at ω-3, 5 and 7. The fatty acid composition is shown in Table 2. The most abundant fatty acid
was hexadecanoic (palmitic) acid, which amounted to 84.1%. The unsaturated fatty acids accounted
for about 10% of the total fatty acid, and were all ω-3 type.
Table 2. Fatty acid composition of 2-acyloxyethanephosphonate
.
Fatty acid
Composition
%)
(
C
C
C
C
14
5.3
84.1
4.8
16
16:3, ∆
7,10,13
18:3, ∆ 9,12,15
5.8
After the extraction of fatty acid methyl esters from the methanolysate, the phosphonic
acid-containing aqueous solution was condensed under reduced pressure, and analyzed by LC-MS.
The phosphonic acid-containing compound was detected at 9.74 min in the positive mode of total ion
+
+
and the selected ion monitors at m/z 126 (M ) and 125 (M-H) . The results suggested that the

Molecules 2006, 11
542
).
phosphonic acid-containing compound was 2-hydroxyethylphosphonic acid (HO-CH
2
-CH
2
-PO
3
H
2
2 4
The C-P linkage was confirmed by hydrolysis with 5 M H SO for the hydrolysis of ester phosphorus
and with 70% perchloric acid (V/V) for the hydrolysis of phosphonate phosphorus [13]. The
phosphorus linkage of the water soluble compound in the methanolysate could be hydrolyzed with
7
2 4
0% perchloric acid, but not 5 M H SO . From this result, the phosphorus linkage of the surfactant was
identified as phosphonate (C-P).
1
1
To elucidate the structure of the intact surfactant, the H- H COSY spectrum was observed. In the
spectrum (Figure 1), the cross peak between the signals at 5.3 ppm (2.6H, multiplet) and 2.80 ppm (2H,
triplet, J= 5.8 Hz) suggested that the protons on the alcoholic carbon esterified with the carboxyl group
2
of the fatty acid that is located at the neighbouring CH -P, while the cross peak between the signal at
5
.3 ppm and 2.07 ppm (0.4H, multiplet) was assigned to protons on the carbon neighbouring olefin
1
(
CH=CH-CH
assigned as follows: 5.3 ppm (multiplet, olefin protons of fatty acid and -C(=O)-O-CH
ppm (O-CH -CH -P); 2.16 ppm (CH -CH -COO of fatty acid); 2.07 ppm (CH=CH-CH - of fatty acid);
.58 ppm (-CH -CH -COO of fatty acid); 1.32-1.28 ppm (-CH -CH -CH=CH- of unsaturated fatty
acid and CH of saturated fatty acid), 0.96 ppm (CH of unsaturated fatty acid), and 0.89 ppm (CH of
saturated fatty acid).
2
-) of the unsaturated fatty acid. The remaining signals of the H-NMR spectrum were
2
-CH -P); 2.80
2
2
2
2
2
2
1
2
2
2
2
2
3
3
1
1
Figure 1. H- H COSY spectrum of 2-acyloxyethylphosphonate isolated from
cyanobacterial waterbloom material. CD
3
OD was used as the solvent.
6
1
2
3
7 8
5
4
From the above assignment and the identification of 2-hydroxyethylphosphonic acid, the structure
of the surfactant was elucidated as 2-acyloxyethylphosphonate, and the most abundant molecule as
2
-hexadecanolyloxyethylphosphonate. In order to confirm the elucidation, a model derivative was
synthesized. The synthesized/authentic surfactant, 2-oleoyloxyethylphosphonate, gave the same Rf

Molecules 2006, 11
543
value (0.67) as that of the surfactant isolated from A. flos-aquae on a HPTLC plate developed with
1
1
chloroform/methanol (95:5, v/v) as the solvent. In the H- H-COSY spectrum of the authentic
surfactant, the correlation peak between the triplet at 2.80 ppm and the multiplet at 5.3 ppm was
observed. The chemical shifts and the correlation were completely agreed with that of the surfactant
isolated from A. flos-aquae. After methanolysis of the synthetic compound, 2-hydroxyethyl-
phosphonate was detected at 9.74 min in the total ion positive mode and the selected ion monitors at
+
+
m/z 126 (M ) and 125 (M-H) by LC-MS. From the results, the structure of the surfactant was
identified as 2-acyloxyethylphosphonate.
The surfactant activity of AOEP isolated from A. flos-aquae can be measured by demonstrating a
reduction in surface tension. In this study, a drop spreading test modified from Hildebrand [14] was
used to measure the effectiveness of AOEP dissolved in deionised water (dH
at reducing the surface tension of a drop of dH O by measuring the area the drop covers when no
longer spherical. The results are shown in Figures 2A and 2B.
AOEP was dissolved in 10 µL dH O or 10 µL MeOH and added to a 10 µL drop of dH
concentrations in dH O were 50 to 0 mg/mL, and 10 to 0 mg/mL in MeOH. In SigmaScan Pro V5.0.0
SPSS Inc), drops were identified and areas measured in pixels with an auto threshold range for objects
2
O) or methanol (MeOH)
2
2
2
O. The
2
(
7
0% darker than background image. The area of each drop containing the surfactant was compared to
O in Figure 2A and MeOH in Figure 2B). On addition of AOEP in water at
the control (0: dH
2
concentrations of 2-10 mg/mL and 50 mg/mL, the spherical shape of water droplet spread on the Petri
dish surface covering an area that was greater than the water control (Figure 2). The drop areas were
increased to 8 mg/mL, and then reduced. From the concentration of AOEP at the inflection point
(
Figure 2E), the critical micelle concentration (CMC) was calculated to be about 22 mM when the
molecular of AOEP isolated from A. flos-aquae was regarded as hexadecanoyloxyethylphosphonic
acid (mw. 364). The drop areas measured when AOEP was dissolved in MeOH were more irregular
than those with water. However, concentrations of the surfactant from 1- 10 mg/mL in MeOH resulted
in the water droplet spreading to a greater area than the control to which 10 µL of MeOH was added.
When dissolved in water, there was little difference between the area of the control drop and 1 mg/mL
drop but this was not the case when AOEP was dissolved in MeOH.
The solubility of different compounds in solvents can vary. In this case, the surfactant appears to be
more soluble in MeOH than water. This may account for the increased drop area measured at 1 mg/mL
AOEP in MeOH compared to water. AOEP is found to be more soluble in solution as a sodium salt.
When dissolved in sodium carbonate buffer as an aqueous carrier, surfactant properties similar to those
measured in MeOH may be observed, but without the irregularity of MeOH use. Surface tension is the
property of water that allows a water droplet to remain spherical and AOEP clearly has the ability to
reduce surface tension, consistent with its surfactant activity. AOEP was found to be non-toxic in brine
shrimp bioassays at concentrations up to 50 µg/mL. The assays were conducted at AOEP
concentrations between 0.05 and 50 µg/mL. C-P linkage-containing compounds are widely distributed
among many species of protozoa, animals, bacteria, and other biota [15]. However, the C-P compounds
have not been previously identified in cyanobacteria. In many cases, 2-aminoethanephosphonate [9] or
its N-methyl analogues [16,17] have been identified as the C-P linkage-containing moiety of lipids,
whereas 2-hydroxyethane phosphonate was identified as the moiety of the biosurfactant isolated from

Molecules 2006, 11
544
the cyanobacterium, A. flos-aquae. Whether the biosurfactant has a natural role in the ecological
functions of the cyanobacterium is unknown and requires investigation.
Figure 2. Drop spreading test of 2-acyloxyethylphosphonate (AOEP) isolated from
waterblooms of Aphanizomenon floa-aquae. The drop spreading test was used
to measure the effectiveness of AOEP dissolved in deionised water (Figure
2
A) or methanol (Figure 2B) at reducing the surface tension of a drop of
deionised water by measuring the area the drop covers when no longer
spherical.
B
EA
F
3
2
1
000
000
000
0
4
3
2
1
000
000
000
000
0
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
-1
-1
AOEP concentration (mg ml
)
AOEP concentration (mg ml )
Conclusions
The biosurfactant 2-acyloxyethanesulfonate was readily soluble in alkaline aqueous solution,
methanol, ethanol and chloroform and was not hydrolyzed with acid and alkaline phosphatases. The
data presented here suggest that the C-P linkage in the biosurfactant is resistant to phosphatases. The
biosurfactant may be used in applications such as food and pharmaceutical industries.
Experimental
General
Ethylene chlorohydrin, phosphorus acid and oleic acid chloride were obtained from Wako
Chemicals, Osaka, Japan. All solvents were of analytical grade. NMR spectra were recorded on a
1
JEOL JNM A-500 spectrometer (500 MHz). The H chemical shift was referenced to CD
3
OD signal
1
1
(
H, δ 3.30 ppm). Homonuclear H connectivities were determined from COSY experiments. Gas
chromatograms were recorded on a HP-5890 GC (Hewlett-Packard, USA) using a methyl silicone
capillary column (25 m x 0.32 mm x 0.25 µm). The instrument was temperature-programmed from 60
to 250 ºC at 20 ºC/min, then 250 to 300 ºC at 4 ºC/min. Electron impact (EI) and chemical ionization
(
CI) (reagent gas iso-butane) MS spectra were obtained with a JEOL JMS 700 (JEOL, Tokyo, Japan).
MS data were obtained under the following conditions: ionization current, 300mA; ionization energy,
0 eV; accelerating voltage, 3 kV; scan range, m/z 50-500. Liquid chromatographic data were acquired
7

Molecules 2006, 11
545
with an LC10AD system (Shimadzu, Japan) combined with a Shimadzu LC-MS 2010 and equipped
with a Zorbax ODS column. The mobile phase was used 0.1% aqueous formic acid (v/v) and methanol.
The methanol concentration was increased from 0 to 80% over 40 min (0 to 55% methanol for 10 min,
then 55 to 80% methanol for 20 min, and held 80% methanol for 10 min) with a flow rate of 0.2
mL/min and a column temperature of 40 ºC. The MS spectra were detected in the conditions of
positive mode ionization, electrospray ionization (ESI) and scan range, m/z 50-400.
Cyanobacterial bloom samples
Waterblooms of Aphanizomenon flos-aquae were collected in August, 2004 from surface water of
Balgavies Loch, Forfa, Scotland. The collected material was frozen at -20 ºC immediately, lyophilized
and the resulting cell powder stored in the University of Dundee cyanobacterial archive at -20 ºC
(
sample ID: 160903 85).
Extraction and isolation
Lyophilized cells of A. flos-aquae (10 g dry wt.) were extracted with MeOH. The extract was
evaporated under reduced pressure. The remaining residue was suspended with
chloroform/methanol/water (3:7:1, v/v), and applied to a DEAE-Sephadex™ column (20 mm I.D. x
3
00 mm). After the column was washed with chloroform/methanol/0.05 M ammonium acetate (200
mL, 3:7:1, v/v), the surfactant was eluted with chloroform/methanol/0.2 M ammonium acetate (200
mL, 3:7:1, v/v). The surfactant fraction was evaporated under reduced pressure and suspended with
chloroform. The suspension was further applied to a silica gel column (20 mm I.D. x 300 mm,
Iatrobeads, Iatron Laboratories, Tokyo, Japan). The column was washed with chloroform and a
mixture of methanol-chloroform (20:80, v/v). The surfactant was eluted with a methanol-chloroform
mixture (200 mL, 40:60, v/v). The eluted surfactant was further purified by HPTLC (E.Merck, Silica
gel 60 containing fluorescent indicator) using chloroform/methanol (95:5, v/v) as the solvent.
1
2
-Acyloxyethylphosphonate: 64 mg (0.64% yield); colorless amorphous solid; for H-NMR spectra, see
Table 1 and Figure 1.
Methanolysis of 2-acyloxyethylphosphonate
2
-Acyloxyethylphosphonate (100 µg) was methanolyzed with 1.0 M HCl-containing methanol at
8
0 °C for 20 h. After the methanolysis, water (2 mL) was added in the solution and fatty acid methyl
esters were extracted with n-hexane and analyzed by GC-MS using CI and EI. The remaining aqueous
solution was condensed under a reduced pressure for LC-MS analysis of the phosphonate compound.
GC-MS data of fatty acid methyl esters: tetradecanoic acid methyl ester: Rt, 8.30 min.; CI, m/z 243
+
+
(
M+1) ; EI, m/z 232 (M ), 199, 143, 87 and 74 (base peak); hexadecanoic acid methyl ester: Rt, 9.34
+
+
min.; CI, m/z 271 (M+1) ; EI, m/z 227 (M ), 171, 87, 74 (base peak); hexadecatrienoic acid methyl
+
+
ester: Rt, 9.20 min.; CI, m/z 265 (M+1) ; EI, m/z 264 (M ), 235, 163, 149, 135, 121, 107, 95, 79 (base
+
+
peak); octadecatrienoic acid methyl ester: Rt, 10.22 min.; CI, m/z 293 (M+1) ; EI, m/z 292 (M ), 263,

Molecules 2006, 11
546
1
63, 149, 135, 121, 95 (base peak), 79. LC-MS data of 2-hydroxyethylphosphonic acid: Rt, 9.74min.;
+
ESI (positive), m/z 126 (M ) and 125 (base peak).
Analysis of the phosphorus linkage
2 4
The C-P linkage was confirmed by the hydrolysis with 5 M H SO for the hydrolysis of ester
phosphorus and with 70% perchloric acid for the phosphonate phosphorus [13]. The phosphorus
linkage of the water soluble compound in the methanolysate could be hydrolyzed with 70% perchloric
2 4
acid, but not 5 M H SO . From this result, the phosphorus linkage of the biosurfactant was identified
as phosphonate (C-P).
Synthesis of 2-oleoyloxyethylphosphonate
Ethylene oxide was derived from ethylene chlorohydrin in 1M sodium hydroxide solution at 4 ºC,
and distilled at 12.5 ºC. Dropwise addition of phosphorus acid (0.1 mol) to 10% aqueous ethylene
oxide (0.1 mol) at 0 ºC gave 2-hydroxyethylphosphonic acid in 30% yield. Oleic acid
(
9-cis-octadecenoic acid) chloride (0.01 mol) was added to 2-hydroxyethylphosphonic acid (0.01
1
mol)-containing dry pyridine at -20 ºC. The yield of 2-oleoyloxyethylphosphonate was 56%. H-NMR:
5
.3 ppm (4H, m, oleic acid olefin protons and C(=O)-O-CH
C(=O)-O-CH -CH -P ), 2.16 ppm (2H, t, J=7.3 Hz, -CH -C(=O)-), 2.07 ppm (4H, m, -CH
.58 ppm (2H, quart., -CH -CH -CH -(=O)-O), 1.32-1.28 ppm (20H, broad m, -CH -CH
and -CH -CH -CH -CH -CH ), and 0.89 ppm (3H, t, J=7.0 Hz, -CH ).
2
-CH
2
-P), 2.80 ppm (2H, t, J=5.8 Hz,
-CH=CH-),
-CH=CH-
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
3
Surfactant activity measurement
Concentrations of AOEP dissolved in dH
of water on a polystyrene Petri dish and stained with methylene blue (0.5 µL, 5 g/L). Additions of
dH O (10 µL) and MeOH (10 µL ) were used as controls. Samples were photographed and the area
2
O (10 µL) or MeOH (10 µL) were added to 10 µL drops
2
covered by drop measured in pixels by SigmaScan Pro 5.0.0 (SPSS Inc).
Toxicity Test
AOEP was dissolved in water at concentrations of 100, 50, 10, 5, 1, 0.5 and 0.1 µg/mL. The brine
shrimp (Artemia salina) assay was carried out according to Campbell et al. [18]. Briefly, A. salina
cysts (Sciento, Manchester) were inoculated into artificial seawater (ASW, Coral Reef Red Sea Salt,
Red Sea Fish Pharm, Israel) and incubated at room temperature under constant illumination. At 24 h,
hatched nauplii were transferred to fresh ASW and incubated for a further 24 h. At 48 h, 15-20 nauplii
were transferred in 100 µL aliquots of ASW to the wells of a 96-well microtitre plate. A sample (100
µL) was added to each nauplii-containing well. The plates were covered and incubated as before. The
number of dead organisms in each well was noted at 24 h.

Molecules 2006, 11
547
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2006 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.
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