Aliphatic sulfates released from Daphnia induce morphological defense of phytoplankton: Isolation and synthesis of kairomones

Article abstract of DOI:10.1016/j.tetlet.2005.05.027

Six aliphatic sulfates, kairomones released from a crustacean Daphnia pulex induce morphological changes of phytoplankton Scenedesmus gutwinskii at ppb (10^-9 g/mL) concentrations.

Full text of DOI:10.1016/j.tetlet.2005.05.027

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 4765–4767
Aliphatic sulfates released from Daphnia induce morphological
defense of phytoplankton: isolation and synthesis of kairomones
Ko Yasumoto,a Akinori Nishigami,a Mina Yasumoto,b Fumie Kasai,c Yasuhiro Okada,a
Takenori Kusumia and Takashi Ooia,*
aGraduate School of Pharmaceutical Sciences, Tokushima University, Tokushima, Tokushima 770-8505, Japan
bMarine Biotechnology Institute Co., Ltd, Heita, Kamaishi, Iwate 026-0001, Japan
cNational Institute for Environmental Studies, Onogawa, Tsukuba 305-8506, Japan
Received 23 March 2005; revised 9 May 2005; accepted 11 May 2005
Available online 31 May 2005
Abstract—Six aliphatic sulfates, kairomones released from a crustacean Daphnia pulex induce morphological changes of phyto-
plankton Scenedesmus gutwinskii at ppb (10À9 g/mL) concentrations.
ꢀ 2005 Elsevier Ltd. All rights reserved.
In a series of food chain in aquatic world, phytoplank-
ton is the bottom creature that supports the lives of ani-
mals. It has been soundly believed that phytoplankton is
docile and never resists against its fate. This belief has
been disputed by Hessen and van Donk, who reported
that a unicellular green alga, Scenedesmus subspicatus,
achieved morphological change into 2-, 4-, and 8-coeno-
bia (colonies) when the water in which a crustacean
Daphnia magna, a grazer of the alga, had been cultured
(Daphnia water) was added to the cultivation medium.1
They also found that the grazing rate of the coenobium
morph was lower than that of the unicellular morph,
owing to the increased size of the former. This metamor-
phosis was supposed to be a self-defense mechanism
acquired by the green alga and triggered by a kairomone
secreted from D. magna. Their report has aroused the
interest of many scientists to attempt to identify the
kairomone,2–10 although the chemical structure has not
been disclosed thus far.
Here we report identification of the Daphnia kairomones
that cause the morphological change in a unicellular
green alga Scenedesmus gutwinskii var. heterospina
(NIES-802) at 10À1–103 ng/mL concentrations (Fig. 1).
Keywords: Kairomone; Phytoplankton; Daphnia; Scenedesmus;
Aliphatic sulfate.
Figure 1. Microscopic (·200) pictures of S. gutwinskii. (a) Control
(10 days cultivation without active sample). (b) Ten days after the
*
Corresponding author. Tel.: +81 88 633 7289; e-mail: tooi@ph.
tokushima-u.ac.jp
addition of active compound 4 (1 ng/mL). The scales are 50 lm.
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.05.027

4766
K. Yasumoto et al. / Tetrahedron Letters 46 (2005) 4765–4767
OSO
3M
OSO
3M
1
4
2
3
6
OSO3M
OSO
OSO
3M
3M
OSO3M
5
Figure 2. Activities (A = Nt/Nc) of sulfates 1–6. DW indicates Daphnia
water (diluted; 10%, v/v). SDS = sodium dodecyl sulfate. Sulfates 1, 2,
and 4 are synthetic materials (Na salts), and 5 and 6 (Na salts) are
commercial products. All results are expressed as means s.d. for
triplicate determinations.
In our experiment, we selected commercially available
frozen Daphnia (Daphnia pulex) rather than Daphnia
water itself as a starting material, because the concentra-
tion of the kairomone in the latter seemed too low to al-
low elucidation of its chemical structure. Frozen
Daphnia (10 kg; Aso Tropical Fish Co. Ltd, Osaka)
was soaked with methanol (20 L · 3), and the methanol
solution was evaporated, the residue being treated with
water (9 L). The mixture was successively extracted with
hexane, dichloromethane, and butanol, and the most ac-
tive butanol extract was separated by HPLC monitoring
the activity using a newly introduced bioassay system
(see Bioassay). The activity (A) was expressed by the
equation A = Nt/Nc, where Nt and Nc correspond with
total number of cells (e.g., 1-cell; 1, 8-cell; 8) and number
of coenobium (e.g., 1-cell; 1, 8-cell; 1), respectively. The
active substances isolated at first were 1,11,17 2,17 and
6.12 While Daphnia water (10% dilution) showed A =
3.5–3.8, the activities of 1, 2, and 6 were A = 4.2, 4.2,
and 3.0 at 10 ng/mL, respectively. To rule out the possi-
bility that the isolated substances are inactive and they
might still be contaminated with a minute amount of
Ôsuper active compoundÕ, compounds 1 and 2 were syn-
thesized. [Compound 1: catalytic hydrogenation of
3-decynol (Lindler catalyst) followed by sulfation (pyri-
dine–SO3). Compound 2: see Scheme 1.]
10 days, dramatic change of the algal form is observed
as shown in Figure 1b, in which most of the alga exist
as 2-, 4-, and 8-cell types.
It is notable that the aliphatic (unsaturated and satu-
rated) sulfates are produced by a crustacean Daphnia
in relatively large amounts, 1; 6.4 mg, 2; 0.4 mg, 3;
0.7 mg, 4; 0.2 mg, 5; 0.2 mg, 6; 3.2 mg per 100 g of the
frozen Daphnia. It is unlikely that 5 and 6 (common sur-
factants) are contaminants from the environment, be-
cause (1) their contents are so high for environmental
pollutants accumulated in the Daphnia body, and (2)
the concomitant 1, 2, 3, and 4 must be the genuine nat-
ural products, obvious from their unusual structures.
Attempts to detect the active substances (transparent
above 210 nm) in Daphnia water were futile because of
their extremely low concentrations and existence of huge
amounts of contaminants. The Ômethylene blue meth-
odÕ,14 a quantitative analysis of environmental anion
surfactants, was therefore applied to the Daphnia water.
The concentration of total anion surfactants was deter-
mined to be 8 ng/mL, which was large enough to cause
the morphological changes of the microalga.
The activities of synthetic 1 and 2 decreased to A = 2.8–
3.1 at 10 ng/mL, indicating that these substances con-
tained more active components. Further separation of
the fraction composed of 1 and 2 afforded triene and alk-
yl sulfates, 3,17 4,17 and 5,12 the activities of which were
A = 2.2 at 100 ng/mL, 4.2 at 1 ng/mL, and 4.3 at 10 ng/
mL, respectively. Synthetic 4 [LAH reduction of methyl
8-methylnonanoate, followed by sulfation (SO3-pyr)]
exhibited the same activity (A = 4.2 at 1 ng/mL,
A = 3.4 at 0.1 ng/mL) (Fig. 2).
The active substances obtained in this study are closely
related to commonly used anion surfactants. Therefore,
activity of sodium dodecyl sulfate (SDS) and sodium
dodecylbenzenesulfonate (LAS), the representative
detergents, was tested. Although LAS was inactive
(1000–0.01 ng/mL), SDS showed moderate activity
(A = 2.2 at 10 ng/mL) (Fig. 2). The fact that SDS is
active at extremely low concentration may stimulate
ecological controversy on the usage of this famous
detergent.
Figure 1 exhibits the effect of compound 4 on the morphol-
ogy of S. gutwinskii. Figure 1a shows the control (culti-
vated for 10 days without active sample), in which every
alga exists as a 1-cell type. When 4 is added to the culti-
vation medium (1 ng/mL) and the alga is cultured for
Lurling and Beekman reported that artificial anion
¨
surfactants extractable from membrane filters caused
metamorphosis of Scenedesmus obliquus.15
The present findings seem to be closely related to the
aggregation phenomenon of the algal mono-cell recently
reported for the Ulvales macro green algae.16
a
OH
b
Br
OH
Bioassay: Each 200 lL of C medium of S. gutwinskii
(5.0 · 102 cells/mL) is delivered into the central 30–50
wells of 96- well polystyrene tissue culture plate (CELL-
STAR, Greiner Bio-one Co., Ltd) containing the test
samples (1000–0.01 ng/mL), and the outer wells are filled
c
OH
d
OH
2
Scheme 1. Reagents: (a) CBr4, Ph3P, quant; (b) K2CO3, NaI, CuI,
DMF,13 20%; (c) H2,quinoline, Pd–BaSO4, MeOH, quant; (d) SO3–
pyr, THF, 47%.

K. Yasumoto et al. / Tetrahedron Letters 46 (2005) 4765–4767
4767
15. Lurling, M.; Beekman, W. Environ. Toxicol. Chem. 2002,
¨
21, 1213–1218.
16. Matsuo, Y.; Imagawa, H.; Nishizawa, M.; Shizuri, Y.
Science 2005, 307, 1598.
with distilled water to avoid dehydration of the system.
The plate is covered with a plastic lid, and incubated at
20 ꢁC (12 light/12 dark) for 10 days. A drop of the med-
ium is placed on a ThomaÕs hemacytometer, and the
numbers of 1-, 2-, 4-, and 8-cell types were counted
under a microscope (·200).
17. Experimental data: (The structures of the compounds were
established by COSY, HSQC, HMBC, and NOESY
spectra. The countercation of natural sulfates was not
identified and expressed as M+.)
1
Compound 1: H NMR (400 MHz, CD3OD) d 5.53 (1H,
dt, J = 11, 7 Hz; H-4), 5.44 (1H, dt, J = 11, 7 Hz; H-3),
4.00 (2H, t, J = 7 Hz; H-1), 2.46 (2H, q, J = 7 Hz; H-2),
2.11 (2H, q, J = 7 Hz; H-5), 1.38 (2H, overlapped, H-6),
1.34 (6H, envelop, H-7, 8, 9), 0.94 (3H, t, J = 7 Hz; H-10).
NOESY cross-peaks were observed between H-2/H-5 and
H-3/H-4. (Underlined coupling constants and NOEs
support the Z-configuration of the olefin.) 13C NMR
(100 MHz, CD3OD): d 133.4 (C-4), 125.6 (C-3), 68.6 (C-
1), 32.9 (C-8), 30.7 (C-6), 30.0 (C-7), 28.5 (C-2), 28.2 (C-5),
23.7 (C-9), 14.4 (C-10). (Underlined chemical shifts of
allylic carbons support the Z-configurations of the ole-
fins.) FABHRMS(À): m/z 235.1017 (calcd for C10H19O4S:
235.1004).
Acknowledgements
This work was in part supported by a research grant
from Faculty of Pharmaceutical Sciences, The Univer-
sity of Tokushima, and by the Ministry of Education,
Science, Sports, and Culture, Grants-in-Aids for Scien-
tific Research on Priority Area (A)(2), 2001–2004,
12045250, and for Scientific Research (A), 2003-,
14207096-00. The authors are grateful to Dr. H. Miyao-
ka, Tokyo University of Pharmacy and Life Science, for
informing the conditions of the yne coupling reaction.
1
Compound 2: H NMR (400 MHz, CD3OD): d 5.47 (1H,
dt, J = 11, 7 Hz; H-4), 5.45 (1H, dt, J = 11, 7 Hz; H-3),
5.42 (1H, dt, J = 11, 7 Hz; H-7), 5.36 (1H, dt, J = 11, 7 Hz;
H-6), 4.02 (2H, t, J = 7 Hz; H-1), 2.86 (2H, t, J = 7 Hz; H-
5), 2.49 (2H, q, J = 7 Hz; H-2), 2.12 (2H, q, J = 7 Hz; H-
8), 1.40 (2H, overlapped; H-9), 1.37 (4H, envelop; H-10,
11), 0.96 (3H, t, J = 7 Hz; H-12). The underlined coupling
constants support the Z-configurations of the two olefins.
NOESY cross-peaks between H-2/H-5 and H-5/H-8
are consistent with the Z-configurations. 13C NMR (100
MHz, CD3OD): d 131.7 (C-4), 131.3 (C-7), 128.7 (C-6),
125.9 (C-3), 68.4 (C-1), 32.6 (C-10), 30.5 (C-9), 28.6 (C-2),
28.1 (C-8), 26.6 (C-5), 23.6 (C-11), 14.4 (C-12). (Under-
lined chemical shifts of allylic carbons support the Z-
configurations of the olefins.) FABHRMS(À): m/z
261.1150 (calcd for C12H21O4S: 261.1161).
References and notes
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1
Compound 3: H NMR (400 MHz, CD3OD): d 5.45–5.54
(2H, m; H-3,4), 5.30–5.45 (4H, m; H-6,7,9,10), 4.03 (2H, t,
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J = 5 Hz: H-8), 2.50 (2H, q, J = 7 Hz; H-2), 1.01 (2H, t,
J = 7 Hz; H-12). NOESY cross-peaks between H-2/H-5
and H-8/H-11 lead to the Z-configurations of 3- and 9-
olefins. 13C NMR (100 MHz, CD3OD): d 132.8 (C-10),
131.4 (C-4), 129.5 (C-7), 128.8 (C-6), 128.2 (C-9), 126.2 (C-
3), 68.4 (C-1), 28.6 (C-2), 26.6 (C-5), 26.4 (C-8), 21.4 (C-
11), 14.6 (C-12). The underlined chemical shifts of the
allylic methylene carbons support the Z-configurations of
the three olefins. HMBC cross-peaks to/from; C-3/H-1,2,5,
C-4/H-2,5, C-6/H-4, C-9/H-8,11, C-10/H-8,11,12. FAB-
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1
Compound 4: H NMR (400 MHz, CD3OD): d 4.03 (2H,
t, J = 7 Hz; H-1), 1.70 (2H, quint, J = 7 Hz; H-2), 1.57
(1H, nonet, J = 7 Hz; H-8), 1.50–1.40 (2H, m; H-3), 1.47–
1.30 (6H, envelop; H-4,5,6), 1.22 (2H, m; H-7), 0.92 (6H,
d, J = 7 Hz; H-9,10). 13C NMR (100 MHz, CD3OD): d
69.1 (C-1), 40.2 (C-7), 31.0 (C-5), 30.5 (C-2), 30.4 (C-4),
29.2 (C-8), 28.5 (C-6), 26.9 (C-3), 23.1 (C-9,10). FAB-
HRMS(À): m/z 237.1157 (calcd for C10H21O4S: 237.1166).