Isolation and absolute configuration determination of aliphatic sulfates as the Daphnia kairomones inducing morphological defense of a phytoplankton

Article abstract of DOI:10.1248/cpb.54.271

2,6-Dimethylheptyl sulfate (1) and 6-methyloctyl sulfate (3) were isolated from Daphnia pulex as the Daphnia kairomones that induced morphological defense of a freshwater phytoplankton Scenedesmus gutwinskii var. heterospina (NIES-802). The absolute stere

Full text of DOI:10.1248/cpb.54.271

February 2006
Notes
Chem. Pharm. Bull. 54(2) 271—274 (2006)
271
Isolation and Absolute Configuration Determination of Aliphatic Sulfates
as the Daphnia Kairomones Inducing Morphological Defense of a
Phytoplankton
a
a
b
a
,a
*
Ko YASUMOTO, Akinori NISHIGAMI, Fumie KASAI, Takenori KUSUMI, and Takashi OOI
a Graduate School of Pharmaceutical Sciences, Tokushima University; Tokushima, Tokushima 770–8505, Japan: and
b National Institute for Environmental Studies; Onogawa, Tsukuba 305–8506, Japan.
Received October 20, 2005; accepted November 1, 2005; published online November 8, 2005
2,6-Dimethylheptyl sulfate (1) and 6-methyloctyl sulfate (3) were isolated from Daphnia pulex as the Daph-
nia kairomones that induced morphological defense of a freshwater phytoplankton Scenedesmus gutwinskii var.
heterospina (NIES-802). The absolute stereochemistry at C2 of 1 was determined by 1H-NMR analysis of the (R)-
MTPA ester of alcohol 2. The absolute configuration at C6 of 3 was determined by Ohrui’s method applied to al-
cohol 4.
Key words Daphnia kairomone; Scenedesmus; aliphatic sulfate; absolute configuration
(HSOꢀ4 ) in the negative ion FAB-MS of 1 and 3. The H-
NMR spectrum of 1 exhibited three doublet methyls at d
0.98 (3H, d, Jꢂ6.7 Hz, Me-2) and 0.91 (6H, d, Jꢂ6.6 Hz, H-
7, Me-6), two protons of a methylene bearing a sulfate group
at d 3.90 (1H, dd, Jꢂ9.4, 5.8 Hz, H-1) and 3.81 (1H, dd,
Jꢂ9.4, 6.7 Hz, H-1) and eight methine/methylene protons at
1
In aquatic environments, phytoplankton is the bottom crea-
ture playing an important role in the food chain. It has been
soundly believed that phytoplankton is docile and never re-
sists against its fate. Recently, it became clear that many phy-
toplankton resist their predators using various strategies.
Scenedesmus, a unicellular fresh-water phytoplankton, resists
its grazer by changing its morphology. Addition of filtered
medium of Daphnia, a grazer of the plankton, to unicellular
Scenedesmus subspicatus achieves morphological change
into 2, 4, and 8 colonies within a few days. Such a change of
morphology increases resistance of the colonies against
grazer.1) This metamorphosis was supposed to be a self-de-
fense mechanism acquired by the phytoplankton and trig-
gered by a kairomone secreted from Daphnia.1)
d 1.12—1.88. Interpretation of the H–1H COSY spectrum
1
of 1 led to a gross structure as 2,6-dimethylheptyl sulfate.3—5)
The absolute stereochemistry at C-2 of 1 was determined
by the 1H-NMR analysis of (R)-a-methoxy-a-(trifluoro-
methy)phenylacetic acid (MTPA) ester of 2, a hydrolysis
1
product. It has been established that,6) in the H-NMR of an
MTPA ester of a 2(S)-methyl aliphatic alcohol, the chemical
shift difference of the methylene protons at C1 (appearing as
an ABX pattern) is larger in the (S)-MTPA ester than in the
(R)-MTPA ester. The reverse holds true for a 2(R)-methyl al-
cohol.
Recently, we reported identification of the Daphnia
kairomones that cause the morphological change in a unicel-
lular green alga Scenedesmus gutwinskii var. heterospina
(NIES-802) at 10ꢀ1—103 ng/ml concentrations.2)
Sulfate 1 was hydrolyzed by heating with 3 M HCl, and the
resulting alcohol 2 was treated with an excess of (ꢃ)-(S)-a-
methoxy-a-(trifluoromethy)phenylacetyl chloride (MTPA-
Here we report isolation and absolute configuration deter-
mination of new aliphatic sulfates 1 and 3 as the Daphnia
kairomones. The absolute stereochemistrys at C2 of 1 and
1
Cl) in pyridine-d5 to yield the (R)-MTPA ester (5). In its H-
1
NMR spectrum, two sets of the AB signals of ABX patterns
due to the methylene protons at C1 appear at d 4.33 (1H, dd,
Jꢂ10.7, 5.8 Hz) and 4.16 (1H, dd, Jꢂ10.7, 6.6 Hz) [major:
2(R)-5 (ꢂ10)], and at 4.26 (1H, dd, Jꢂ10.7, 6.1 Hz) and 4.23
(1H, dd, Jꢂ10.7, 5.9 Hz) [minor: 2(S)-5 (ꢂ9)] (Fig. 2).
These H-NMR data for the MTPA esters (5) indicated that
natural 1 was a 4 : 1 mixture of 2(R) and 2(S) enantiomers
(Fig. 2).
We were interested which enantiomar had stronger activ-
ity. Therefore, we synthesized 2(R)- and 2(S)-1 by the reac-
tions shown in Chart 1. The olefin of aldehyde 6 was hydro-
genated over Pd-C catalyst, followed by reduction of the sub-
sequent aldehyde to primary alcohol with LiAlH4. The
racemic alcohol (rac-2)7) was esterified with (S)-methoxy-(1-
naphthyl) acetic acid ((S)-1NMA).8,9) The (S)-1NMA esters
were separated to 7 and 8 by recycling HPLC. Hydrolysis of
7 and 8 afforded 2(S)- and 2(R)-2,7) respectively, the absolute
configuration of which was confirmed by the MTPA method:
(R)-MTPA ester (9) of 2(S)-2 shows an overlapped AB pat-
tern of H2-1, while (R)-MTPA ester (10) of 2(R)-2 shows a
well-separated AB pattern (Experimental). Interestingly, the
C6 of 3 were determined by H-NMR analysis of the (R)-
MTPA ester of alcohol 2 and Ohrui’s method applied to alco-
hol 4, respectively.
Frozen Daphnia (10 kg; Aso Tropical Fish Co. Ltd.,
Osaka) was soaked with methanol (20 lꢁ3), 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 active
butanol extract was separated by HPLC monitoring the activ-
ity2) to afford 1 (9 mg) and 3 (5 mg) (Fig. 1).
The molecular formulae of 1 and 3 were established as
C9H19O4S on the basis of HR-FAB-MS. The presence of a
sulfate group was suggested by a fragment ion peak at m/z 97
1
Fig. 1. The Structure of Daphnia Kairomones 1 and 3
The countercations were not identified and expressed as Mꢃ.
∗ To whom correspondence should be addressed. e-mail: tooi@ph.tokushima-u.ac.jp
© 2006 Pharmaceutical Society of Japan

272
Vol. 54, No. 2
Chart 1. Synthesis of (R)-MTPA Esters 9 and 10, and Sulfates 2(S)-1 and 2(R)-1
Fig. 2. Determination of the Absolute Configuration of 1
Fig. 3. Determination of the Absolute Configuration of 3
(S)-1NMA derivatives 7 and 8 show the 1H-NMR patterns of
H2-1 opposite to those of the MTPA esters: the (S)-1NMA
ester of 2(S)-2 exhibits a narrow AB pattern of H2-1 and (S)-
MTPA ester of 2(S)-2 is a well separated pattern (Experimen-
tal). 2(S)- and 2(R)-2 were sulfated, giving 2(S)-1 and 2(R)-2,
respectively. Then, we examined that the morphological
change activity of the compounds. But, there were no differ-
The alcohol derivative 4, obtained by hydrolysis of 3 with
3 M HCl, was condensed with (1R,2R)-2ACyclo-COOH to
yield ester 11. The methyl proton signals of the ester ap-
peared at d 0.70 (3H, t, Jꢂ7.2 Hz, H-8) and 0.62 (3H, d,
Jꢂ6.6 Hz, Me-6). These chemical shifts are the same with
those of the methyl protons of (1R,2R)-2ACyclo-COOH ester
of 6(S)-methyloctan-1-ol,10) leading to the 6(S)-configuration
of 3. This experiment also indicated that natural 6(S)-3 was
free from its antipode.
ence in the activity between 2(R)- and 2(S)-sulfates.
1
The H-NMR spectrum of 3 reveals two methyl groups
at d 0.92 (3H, t, Jꢂ7.8 Hz, H-8), 0.91 (3H, d, Jꢂ7.4 Hz,
Me-6), methylene protons bearing a sulfate group at d 4.03
(2H, t, Jꢂ6.6 Hz, H-1), and 11 methine/methylene protons at
d 1.73—1.10. The 1H–1H COSY, HSQC and HMBC
NMR spectra of 3 were consistent with the structure of 6-
methyloctyl sulfate. To the best of our knowledge, 3 is a new
compound. The absolute stereochemistry at C-6 of 3
Experimental
High-resolution MS were recorded on a JEOL JMS-SX102A spectrome-
ter. 1H- and 13C-NMR spectra were obtained on a Bruker Avance-400 (1H
and 13C at 400 and 100 MHz, respectively). Assignments of the proton and
carbon signals were established by COSY, HSQC and HMBC spectra. Opti-
cal rotations were determined on a JASCO DIP-370 polarimeter.
Bioassay Each 200 ml of C medium of S. gutwinskii (5.0ꢁ102 cells/ml)
is delivered into the central 30—50 wells of 96-well polystyrene tissue cul-
ture plate (CELLSTAR, Greiner Bio-one Co., Ltd) containing the test sam-
ples (1000—0.01 ng/ml), and the outer wells are filled with distilled water to
avoid dehydration of the system. The plate is covered with a plastic lid, and
1
was determined by the H-NMR analysis of a (1R,2R)-2-
(2,3-anthracenedicarboximido)cyclohexanecarboxylic acid
((1R,2R)-2ACyclo-COOH, Ohrui reagent) ester.10)

February 2006
273
incubated at 20 °C (12 light/12 dark) for 10 d. A drop of the medium is
placed on a Thoma’s hemacytometer, and the numbers of 1-, 2-, 4-, and 8-
cell types were counted under a microscope (ꢁ200).
Hydrolysis of (S)- and (R)-2,6-Dimethylheptyl (S)-Methoxy-(1-naph-
thyl)acetate The first-eluting ester (7) (100 mg, 0.29 mmol) was dissolved
in a dioxane–conc. HCl mixture 3 : 1 (1.5 ml) and the mixture was heated at
Extraction and Isolation Frozen Daphnia (10 kg; Aso Tropical Fish 110 °C for 3 h. Water was added to the cooled solution before extraction
Co. Ltd., Osaka) was soaked with methanol (20 lꢁ3), and the methanol so- with hexane. The organic layer was washed with 5% sodium hydrogen car-
lution was evaporated, the residue being treated with water (9 l). The mixture bonate and H2O, dried over Na2SO4, and concentrated in vacuo to yield (S)-
was successively extracted with hexane (9 l), dichloromethane (9 l), and bu- 2,6-dimethylheptan-1-ol (2(S)-2) (40 mg, 0.27 mmol, 95%). Hydrolysis of
tanol (9 l), and the most active butanol extract (18 g) was chromatographed
on a Cosmosil 75C18-OPN (25 g), eluting with MeOH–H2O in a gradient
manner (1 : 1→10 : 0). The active fractions were further purified by HPLC
(CAPCELLPAK C18 column, 5 mm, 10ꢁ250 mm, MeCN–H2O (40 : 60) con-
taining 250 mM NaClO4 as mobile phase with the flow rate 1.0 ml/min (using
an RI detector)) to afford 1 (9 mg) and 3 (5 mg).
the second-eluting ester (8) (90 mg, 0.26 mmol) was performed under the
same condition, affording (R)-2,6-dimethylheptan-1-ol (2(R)-2) (35 mg,
0.24 mmol, 92%).
(S)-2,6-Dimethylheptyl (R)-a-Methoxy-a-(trifluoromethyl)phenylac-
etate (9) To a pyridine-d5 solution (100 ml) of 2(S)-2 (1 mg) was added
(ꢃ)-(S)-MTPACl (15 ml) and the mixture was allowed to stand at room tem-
perature for 30 min, the solution diluted with pyridine-d5 (400 ml), and the
1H-NMR spectrum was recorded. The 1H-NMR spectrum indicated com-
2,6-Dimethylheptyl Sulfate (1) The spectral data (1H-, 13C-NMR and
MS) were in excellent agreement with those previously reported.3,4)
2,6-Dimethylheptyl (R)-a-Methoxy-a-(trifluoromethyl)phenylacetate plete formation of the (R)-MTPA ester (9). 1H-NMR (C5D5N) d: (The
(5) 1 (2.7 mg) was dissolved in a 3 M HCl solution (0.5 ml) and the solution
was heated at 100 °C for 5 h. Dichloromethane (CH2Cl2) and water were
phenyl protons were obscured by the solvent signals.) 4.26 (1H, dd, Jꢂ10.7,
6.1 Hz, H-1), 4.23 (1H, dd, Jꢂ10.7, 5.9 Hz, H-1), 1.77 (1H, octet, Jꢂ6.8 Hz,
added to the solution. The organic phase was dried over Na2SO4 and evapo- H-2), 1.41 (1H, nonet, Jꢂ6.8 Hz, H-6), 1.34—0.97 (6H, m, H-3, 4, 5), 0.87
rated in vacuo to give alcohol (2). This alcohol was dissolved in pyridine-d5 (3H, d, Jꢂ6.8 Hz, Me-2), 0.81 (6H, d, Jꢂ6.6 Hz, H-7, Me-6).
(100 ml) and to the solution was added (ꢃ)-(S)-MTPA-Cl (15 ml). The mix-
ture was allowed to stand at room temperature for 30 min, diluted with pyri-
dine-d5 (400 ml), and the 1H-NMR spectrum was recorded. The spectrum
showed complete formation of the (R)-MTPA ester (5) without the starting
alcohol. 1H-NMR (C5D5N) d: (The phenyl protons were obscured by the sol-
vent signals.) 4.33 (1H, dd, Jꢂ10.7, 5.8 Hz, major H-1), 4.26 (1H, dd,
Jꢂ10.7, 6.1 Hz, minor H-1), 4.23 (1H, dd, Jꢂ10.7, 5.9 Hz, minor H-1), 4.16
(1H, dd, Jꢂ10.7, 6.6 Hz, major H-1), 1.77 (1H, octet, Jꢂ6.8 Hz, H-2), 1.42
(R)-2,6-Dimethylheptyl (R)-a-Methoxy-a-(trifluoromethyl)phenylac-
etate (10) To a pyridine-d5 solution (100 ml) of 2(R)-2 (1 mg) was added
(ꢃ)-(S)-MTPA-Cl (15 ml) at room temperature for 30 min, the solution di-
luted with pyridine-d5 (400 ml), and the 1H-NMR spectrum was recorded for
the (R)-MPTA ester (10). 1H-NMR (C5D5N) d: (the phenyl protons were ob-
scured by the solvent signals) 4.33 (1H, dd, Jꢂ10.7, 5.8 Hz, H-1), 4.16 (1H,
dd, Jꢂ10.7, 6.6 Hz, H-1), 1.77 (1H, octet, Jꢂ6.8 Hz, H-2), 1.41 (1H, nonet,
Jꢂ7.1 Hz, H-6), 1.35—0.98 (6H, m, H-3, 4, 5), 0.86 (3H, d, Jꢂ6.8 Hz, Me-
(1H, nonet, Jꢂ6.6 Hz, H-6), 1.35—0.98 (6H, m, H-3, 4 , 5), 0.86 (3H, d, 2), 0.81 (6H, d, Jꢂ6.6 Hz, H-7, Me-6).
Jꢂ6.6 Hz, Me-2), 0.81 (6H, d, Jꢂ6.6 Hz, H-7, Me-6).
(S)-2,6-Dimethylheptyl Sulfate (2(S)-1) The alcohol 2(S)-2 (36 mg,
2,6-Dimethylheptan-1-ol (rac-2) To 255 mg (1.8 mmol) of 2,6-di-
methyl-5-hepten-1-al (6: commercially available) in MeOH (5 ml) was
added Pd-C catalyst (24 mg, 10% w/w). The reaction mixture was stirred at
room temperature for 2 h under hydrogen atmosphere. The mixture was fil-
0.25 mmol), obtained by hydrolysis of the 1NMA ester (7) was converted to
the sulfate by treatment with pyridine–SO3 complex (159 mg, 1.00 mmol) in
tetrahydrofuran (3 ml) at room temperature for 24 h. The resulting mixture
was neutralized with 1 M sodium hydroxide solution and the aqueous solu-
tered and the aldehyde (265 mg, 1.8 mmol) was obtained after concentration. tion was extracted with hexane to remove the residual alcohol. The aqueous
To a solution of 265 mg (1.8 mmol) of the aldehyde in THF (6 ml) was added
212 mg (5.5 mmol) of LiAlH4. After 1 h, the reaction was quenched by
layer was passed through an ODS column. Mineral salt was removed by
washing the column with distilled water and elution with methanol gave
adding 200 ml of H2O, 15% NaOH, and then 500 ml of H2O. The mixture 2(S)-1 (58 mg, 0.25 mmol). The 1H- and 13C-NMR spectra of this product
was diluted with 10 ml EtOAc and filtered through Celite. The filtrate was
concentrated under reduced pressure to furnish 265 mg (1.8 mmol, 99%
yield) of the racemic alcohol (rac-2). 1H-NMR (CDCl3) d: 3.45 (1H, dd,
Jꢂ10.4, 5.9 Hz, H-1), 3.35 (1H, dd, Jꢂ10.4, 6.7 Hz, H-1), 1.57 (1H, m, H-
were identical with those of natural 1. HR-FAB-MS m/z: 223.1004 (Calcd
for C9H19O4S: 223.1004). [a]D25 ꢃ2.6° (cꢂ0.9, MeOH).
(R)-2,6-Dimethylheptyl Sulfate (2(R)-1) The sulfate (2(R)-1) was ob-
tained in the same manner as described for (2(S)-1). HR-FAB-MS m/z:
2), 1.49 (1H, nonet, Jꢂ6.6 Hz, H-6), 0.97—1.38 (6H, m, H-3, 4, 5), 0.87 223.0978 (Calcd for C9H19O4S: 223.1004). [a]D25 ꢀ0.9° (cꢂ1.0, MeOH).
(3H, d, Jꢂ6.6 Hz, Me-2), 0.83 (6H, d, Jꢂ6.6 Hz, H-7, Me-6). 13C-NMR
(CDCl3) d: 68.2 (C-1), 39.2 (C-5), 35.7 (C-2), 33.4 (C-3), 27.9 (C-6), 24.7
(C-4), 22.7 and 22.6 (C-7, Me-6), 16.6 (Me-2).
(S)-6-Methyloctyl Sulfate (3) Negative HR-FAB-MS: m/z: 223.0975
(Calcd for C9H19O4S: 223.1004). 1H-NMR (CD3OD) d: 4.03 (2H, t,
Jꢂ6.6 Hz, H-1), 1.70 (2H, quint, Jꢂ6.6 Hz, H-2), 1.48—1.29 (7H, m, H-3,
(S)- and (R)-2,6-Dimethylheptyl (S)-Methoxy-(1-naphthyl)acetates (7) 4, 5a, 6, 7a), 1.24—1.13 (2H, m, H-5b, 7b), 0.92 (3H, t, Jꢂ7.8 Hz, H-8),
and (8) To a CH2Cl2 solution (5 ml) of rac-2 (116 mg, 0.81 mmol) were
0.91 (3H, d, Jꢂ7.4 Hz, Me-6). 13C-NMR (CD3OD) d: 61.9 (C-1), 37.7 (C-
added 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride 5), 35.6 (C-6), 30.6 (C-7), 30.5 (C-2), 27.8 (C-4), 27.2 (C-3), 19.6 (C-9),
(EDC) (470 mg, 2.4 mmol), 4-dimethylaminopyridine (DMAP) (300 mg, 11.7 (C-8). [a]D25 ꢀ1.9° (cꢂ0.15, MeOH).
2.4 mmol), NEt3 (510 ml, 3.6 mmol) and (S)-methoxy-(1-naphthyl)acetic acid
(S)-6-Methyloctyl
(1R,2R)-2-(2,3-Anthracenedicarboximido)cyclo-
(1NMA) (261 mg, 1.2 mmol). After the mixture was stirred for 12 h, 10 ml
hexanecarboxylate (11) Sulfate 3 (1.8 mg, 7.5 mmol) was dissolved in 3 M
of CH2Cl2 was added, and the organic layer was washed with 10% aqueous HCl (0.5 ml) and the solution was heated at 100 °C for 5 h. Dichloromethane
citric acid, 5% sodium hydrogen carbonate, H2O and brine, dried over
and water were added to the solution. The organic phase was dried over
Na2SO4, and concentrated in vacuo to yield a crude ester. The crude ester
Na2SO4, and evaporated in vacuo to give the alcohol (4). To a CH2Cl2 solu-
was purified by SiO2 column chromatography with hexane/EtOAc solvent tion of the alcohol (4), were added EDC (1 mg, 5.2 mmol), DMAP (0.6 mg,
system to afford the (S)-1NMA ester (261 mg, 0.76 mmol, 94%). The di-
astereomeric (S)-1NMA ester (261 mg, 0.76 mmol) was separated by 11
times recycling HPLC with 80% aqueous methanol on CAPCELLPAK C18
to yield the first-eluting ester (7) (100 mg, 0.29 mmol) and the second-elut-
4.9 mmol), NEt3 (1 ml, 6.3 mmol) and (1R,2R)-2-(2,3-anthracenedicarbox-
imido)cyclohexanecarboxylic acid (0.6 mg, 1.5 mmol). After the mixture was
stirred for 16 h at room temperature, the crude ester was purified by SiO2
column chromatography with hexane/EtOAc solvent system to afford the
ing ester (8) (90 mg, 0.26 mmol). 7: 1H-NMR (CDCl3) d: 8.29 (1H, d, ester 11.
Jꢄ8.3 Hz), 7.84 (2H, t, Jꢂ8.8 Hz), 7.61 (1H, d, Jꢂ6.8 Hz), 7.49 (3H, m),
5.39 (1H, s), 3.94 (1H, dd, Jꢂ10.5, 6.6 Hz, H-1), 3.89 (1H, dd, Jꢂ10.5,
5.8 Hz, H-1), 3.45 (3H, s), 1.58 (1H, octet, Jꢂ6.6 Hz), 1.36 (1H, nonet,
Acknowledgments We are grateful to Dr. K. Akasaka and Prof. H.
Ohrui of the Graduate School of Life Sciences, Tohoku University for pro-
Jꢂ6.4 Hz), 1.14—0.82 (6H, m), 0.79 (6H, d, Jꢂ6.6 Hz), 0.67 (3H, d, viding us Ohrui reagents.
Jꢂ6.8 Hz). 13C-NMR (CDCl3) d: 170.8, 133.8, 132.3, 131.1, 129.3, 128.6,
126.6, 126.4, 125.7, 125.1, 124.0, 81.1, 69.9, 57.4, 38.9, 33.1, 32.5, 27.8, References
1
24.4, 22.6, 22.6, 16.6. 8: H-NMR (CDCl3) d: 8.29 (1H, d, Jꢂ8.6 Hz), 7.84
(2H, t, Jꢂ8.6 Hz), 7.62 (1H, d, Jꢂ7.1 Hz), 7.49 (3H, m), 5.39 (1H, s), 3.96
(1H, dd, Jꢂ10.5, 6.3 Hz, H-1), 3.87 (1H, dd, Jꢂ10.5, 6.6 Hz, H-1), 3.46
(3H, s), 1.60 (1H, octet, Jꢂ6.4 Hz), 1.39 (1H, nonet, Jꢂ6.6 Hz), 1.13—0.85
(6H, m), 0.80 (6H, d, Jꢂ6.6 Hz), 0.65 (3H, d, Jꢂ6.6 Hz). 13C-NMR (CDCl3)
d: 170.8, 133.8, 132.2, 131.0, 129.3, 128.6, 126.5, 126.3, 125.7, 125.1,
124.0, 81.0, 69.9, 57.4, 39.0, 33.2, 32.4, 27.8, 24.3, 22.6, 22.6, 16.5.
1) Hessen D. O., van Donk E., Hydrobiologia, 127, 129—140 (1993).
2) Yasumoto K., Nishigami A., Yasumoto M., Kasai F., Okada Y.,
Kusumi T., Ooi T., Tetrahedron Lett., 46, 4765—4767 (2005).
3) Tsukamoto S., Kato H., Hirota H., Fusetani N., J. Nat. Prod., 57,
1606—1609 (1994).
4) Crispino A., De Giulio A., De Rosa S., De Stefano S., Milone A., J.
Nat. Prod., 57, 1575—1577 (1994).

274
5) Salvatore D. R., Alfredo M., Antonio C., Andrej J., Alfonso D. G., J.
Vol. 54, No. 2
Hashimoto T., Kan Y., Tetrahedron Lett., 35, 4397—4400 (1994).
Nat. Prod., 60, 462—463 (1997).
6) Tsuda M., Toriyabe Y., Endo T., Kobayashi J., Chem. Pharm. Bull., 51,
448—451 (2003).
7) Huo S., Negishi E., Org. Lett., 3, 3253—3256 (2001).
8) Kusumi T., Takahashi H., Ping X., Fukushima T., Asakawa Y.,
9) Kusumi T., Takahashi H., Hashimoto T., Kan Y., Asakawa Y., Chem.
Lett., 6, 1093—1094 (1994).
10) Imaizumi K., Terasima H., Akasaka K., Ohrui H., Anal. Sci., 19,
1243—1249 (2003).