(R)-Eucomic acid, a leaf-opening factor of the model organism, Lotus japonicus

Article abstract of DOI:10.1016/j.tet.2008.11.097

In the post-genomic era, the biological activity of endogenous metabolites can be linked to genomic information via its target protein. Consequently, studies concerned with the identification of endogenous bioactive metabolites from model organisms should

Full text of DOI:10.1016/j.tet.2008.11.097

Tetrahedron 65 (2009) 2136–2141
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
(R)-Eucomic acid, a leaf-opening factor of the model organism, Lotus japonicus
*
Masahiro Okada, Sungwook Park, Takahiro Koshizawa, Minoru Ueda
Department of Chemistry, Tohoku University, 6-3 Aramakiaza-aoba, Aoba-ku, Sendai 980-8578, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In the post-genomic era, the biological activity of endogenous metabolites can be linked to genomic
information via its target protein. Consequently, studies concerned with the identification of endogenous
bioactive metabolites from model organisms should be undertaken. (R)-Eucomic acid (1) was identified
as an endogenous metabolite concerning leaf movement in the model legume, Lotus japonicus. Absolute
stereochemistry was investigated by comparison of physical characteristics of natural and synthetic
enantiomers of 1. Identification of endogenous metabolites in a model legume would be particularly
advantageous for further studies on the chemical biology of this biologically intriguing phenomenon.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 6 September 2008
Received in revised form 27 November 2008
Accepted 28 November 2008
Available online 6 December 2008
Keywords:
Eucomic acid
Leaf-opening factor
Lotus japonicus
Nyctinasty
Model organism
1. Introduction
2. Results and discussion
Biological studies have become increasingly focused on model
organisms, such as Caenorhabditis elegans,1 Drosophila melano-
gaster,2 Arabidopsis thaliana,3 Mus musculus,4 and Homo sapiens.5
Additionally, Lotus japonicus was previously proposed as a promising
model legume for studies on molecular genetics and genomics.6–8
The use of genetic information available for these species has
accelerated the pace of molecular genetic studies generally and
resulted in marked advances in biology. In an attempt to increase the
potential of these studies, investigations on the chemical charac-
teristics of the endogenous metabolites of these model organisms
should be undertaken.
The most important aspect of isolating a biologically active
endogenous chemical factor is the establishment of a bioassay with
high reproducibility. In the evening, the three leaflets composing
each L. japonicus compound leaf move upward from the horizontal
to a vertical position (Fig. 1). Purification of the LOF from
L. japonicus (Fig. 1) was conducted using a novel, highly re-
producible bioassay that employs an L. japonicus (Gifu B-129)
leaflet (Fig. 2). Briefly, the compound leaf of L. japonicus was
removed from the stem by cutting at the petiole and the two side
leaflets were cut at the base. We found that the single remaining
leaflet with the petiole continued the rhythmic movement
Most legumes close their leaves in the evening, as if sleeping,
and open them in the morning in what is referred to as a nycti-
nasty.9 The pioneering work on plant movement was conducted by
Charles Darwin in the 19th century.10 Our previous studies showed
that five pairs of endogenous chemical factors, also referred to as
leaf-closing factor (LCF) and leaf-opening factor (LOF), are involved
in the control of nyctinastic leaf movements.11–13 However, the lack
of genetic information on the plants used in those studies has made
further studies on the chemical biology of this biological phe-
nomenon difficult. The discovery of LOF in L. japonicus would
therefore help us to further elucidate the chemical characteristics of
nyctinasty by utilizing genomic information of the species.
* Corresponding author. Tel./fax: þ81 22 795 6553.
E-mail address: ueda@mail.tains.tohoku.ac.jp (M. Ueda).
Figure 1. Circadian rhythm leaf movement of L. japonicus (left: in the daytime, right: at
night).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.11.097

M. Okada et al. / Tetrahedron 65 (2009) 2136–2141
2137
Figure 3. Structural determination of LOF 1 (arrows: NOE correlations, bold lines:
HMBC correlations).
between two protons (H4a, H4b) and C4, respectively. HMBC spectra
also showed that the LOF had one additional quaternary carbon and
two carboxylic acids by the correlations between H2a–C1, H2a–C3,
H2a–C5, H4b–C3, and H4b–C5. The HMBC correlations between H4b
–
0
0
0
C2 and H2 –C4, together with NOESY correlations between H4a–H2
0
and H4b–H2 gave the planar structure of LOF as 1 (Fig. 3). Com-
pound (R)-(ꢁ)-1 was a known eucomic acid isolated from bulbs of
´
Eucomis punctata L’Herit (Liliaceae) that is a weak radical scaven-
ger.14,15 Cation analysis of natural LOF by capillary electrophoresis
revealed the presence of Naþ (38%), Ca2þ (18%), and Mg2þ (12%)
with a small amount of Kþ (1.4%). However, the Naþ and Ca2þ
cations in the LOF could be attributed to a contamination from
environment, because the original bioactive aqueous fraction con-
tained mainly Kþ (81.8%) and Mg2þ (16.2%), and a small amount of
Ca2þ(2.0%).
Figure 2. Bioassay using a leaflet of L. japonicus (top: a leaflet of L. japonicus, middle:
a leaflet with petiole in sample solution, bottom right: a leaflet in a bioactive fraction,
bottom left: biologically inactive fraction).
Syntheses of eucomic acid were previously reported by two
groups.14,16 We synthesized both enantiomers of 1 in five steps
from (ꢁ)-2, which was easily prepared from commercially available
observed during the day on the whole plant, with the angle be-
O-benzyl-L
-tyrosine,17 and using both enantiomers of 1 enabled
tween the leaflet and the petiole (
q
) remaining >190ꢀ in the day-
confirmation of the absolute stereochemistry of naturally occurring
1 (Scheme 1, Fig. 4).
time, becoming <170ꢀ at night (Fig. 2). We observed a change in the
angle between the petiole and the leaflet after addition of sample.
We added a sample solution to a leaflet with a petiole at 10:00. The
difference in the position of the leaves observed using the bioactive
and inactive fractions gradually appeared after 16:00. The bio-
activity of the sample was assessed by the status of leaves at 21:00.
When less than 40% of the total number of leaves had closed
a
-Hydroxyl carboxylic acid (ꢁ)-2 was protected
by using benzaldehyde to give a diastereomixture of benzylidene
acetals 3 and 4, which can be separated easily. The configuration of
each diastereomer (cis-3 or trans-4) was determined from NOE
correlations as illustrated in Scheme 1. Treatment of 3 with LDA
followed by the addition of benzyl
a-bromoacetate in HMPA
resulted in diastereoselective C–C bond formation to give (ꢁ)-5 in
64% yield after recrystallization. Similarly, alkylation of 4 gave only
(þ)-5 in 65% yield. Synthetic (ꢁ)-5 and (þ)-5 exhibited highly
similar optical rotation values with opposite signs. Hydrogenolysis
of benzylidene acetal, benzyl ether, and benzyl ester gave (R)-(ꢁ)-1
(q
<170ꢀ), the sample was judged to be bioactive.
The bioassay-guided purification of LOF was conducted as fol-
lows: methanolic extract of the terrestrial part of L. japonicus was
concentrated in vacuo and partitioned with organic solvents. The
bioactive aqueous fraction was separated using an Amberlite XAD-7
column chromatography followed by Toyopearl HW-40S gel fil-
tration column chromatography. Following two-step purifications
by MPLC and HPLC using an ODS column gave the bioactive frac-
tion, which made the leaves of L. japonicus open at 1 mg/L. We
established a novel robust bioassay method using sample solutions
as small as 0.1 mL, which enabled bioassay-guided purification
using a small amount of plant material. By employing this method,
from (ꢁ)-5 in good yields as well as (S)-(þ)-1 from (þ)-5. 1H and 13
C
NMR data for synthetic (R)- or (S)-1 were identical to those of
natural 1.
Successive chiral HPLC analyses on synthetic (R)-(ꢁ)-1 and (S)-
(þ)-1 were then carried out. Under optimized separation condi-
tions, the optical purities of (R)-(ꢁ)-1 and (S)-(þ)-1 were up to 99%
ee (Fig. 4), and they can be used as good references for determining
the stereochemistry of naturally occurring 1. Under optimized
conditions, the retention time of naturally occurring 1 was in good
agreement with that of synthetic (R)-(ꢁ)-1 (Fig. 4), indicating that
natural 1 was (R)-(ꢁ)-1.
we succeeded in purifying 136 mg of LOF fraction from 41 g equiv of
L. japonicus.
The active compound in the bioactive fraction was identified by
ESI-MS and NMR. High resolution mass spectroscopy using a neg-
ative-mode FT-ICR ESI-MS produced a molecular peak at m/z
239.0561 ([MꢁH]ꢁ), which corresponded precisely with the
molecular composition of C11H12O6 (m/z 239.0561 calcd for
Next, the leaf-opening activities of synthetic (R)-(ꢁ)-1 and
(S)-(þ)-1 were examined using the leaf bioassay employing
L. japonicus. We prepared a sodium salt of both enantiomers,
(R)-(ꢁ)-1 and (S)-(þ)-1, and examined their biological activities.
Leaf-opening bioactivity was assessed based on the aforemen-
tioned criteria, and the percentage of opening leaves was calcu-
lated on an hourly basis (Fig. 5).
C11H11O6,
D
¼0.0 mmu). Determination of the LOF structure was
performed by 800 MHz NMR equipped with a cold probe. Corre-
lations in HSQC (Heteronuclear Single Quantum Coherence) and
NOESY (Nuclear Overhauser Effect Spectroscopy) gave the planar
structure of LOF (Fig. 3).
Differences in the status of the leaves gradually appeared around
16:00 (5 h before getting dark). More than 60% of the leaves treated
Existence of two isolated methylene groups was confirmed from
HSQC correlations between two protons (H2a, H2b) and C2, and
with 10
m
M of sodium salt of (R)-(ꢁ)-1 remained open at 22:00 (1 h
after getting dark), and 35% of the leaflets continued to open even at

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M. Okada et al. / Tetrahedron 65 (2009) 2136–2141
Scheme 1. Synthetic route of enantiomers of 1.
0:00 (3 h after getting dark). And also,100
m
M solution of sodium salt
Successively, we prepared a potassium salt of both enantio-
mers of 1 and free form of (R)-(ꢁ)-1, and examined their bio-
logical activities (Fig. 6). More than 60% of the leaves treated with
of (R)-(ꢁ)-1 made 60% of the leaflets open even at 0:00 (3 h after
getting dark). Conversely, approximately 20% of the leaves treated
using the sodium salt of (S)-(þ)-1 as well as H2O remained open at
22:00. The findings of this bioassay clearly demonstrated that the
sodium salt of (R)-(ꢁ)-1 induced leaf opening in L. japonicus at
100 mM of potassium salt of (R)-(–)-1 remained open at 22:00 (1 h
after getting dark), but 20% of the leaves treated using the free
(R)-(ꢁ)-1 as well as the potassium salt of (S)-(þ)-1 remained open
at 22:00. Thus, the potassium salt of (R)-(ꢁ)-1 induced leaf
10
100
m
M, while that of (S)-(þ)-1 showed no biological activity even at
m
M, with a noticeable bioactivity observed between (R)-(ꢁ)-1
opening in L. japonicus at 100
m
M, while that of (S)-(þ)-1 and the
and (S)-(þ)-1. These results suggest the existence of a specific protein
capable of recognizing the stereochemistry of 1 in L. japonicus, and
also that our enantiodifferential approach18,19 would be applicable
for studies on the chemical biology of 1.
free form of (R)-(ꢁ)-1 showed no biological activity even at
100 M. These results indicated that the leaf-opening activity of
m
1 was depended not only on the stereochemistry, but also on the
counter cation.
Figure 5. Time course changes in the number of opening and closing events for 20
leaves treated with sodium salt of (R)-(ꢁ)-1, sodium salt of (S)-(þ)-1, and water. Lights
were off after 21:00.
Figure 4. Chiral HPLC analyses of synthetic (S)-(þ)-1, (R)-(ꢁ)-1, and naturally occur-
ring 1.

M. Okada et al. / Tetrahedron 65 (2009) 2136–2141
2139
3. Conclusions
Eucomic acid ((R)-(ꢁ)-1) was isolated from L. japonicus and
considered as a potential LOF in this species. The identification of
endogenous chemical signals within model organisms is important
for combining studies of chemical and biological research. These
molecules could be useful for developing biochemical reagents and
methods for forward chemical genetics,20 such as enantiodiffer-
ences18,19 in cDNA microarray analysis, in which biologically in-
active enantiomer is used as perfect control.
4. Experimental
4.1. General method
L. japonicus Gifu B-129 was grown from seed on power soil in
a Biotron chamber (LPH-1000S, NK System Co., Ltd., Japan) under
16 h light at 26 ꢀC (from 5:00 to 21:00) then 8 h dark conditions at
22 ꢀC for bioassay.
The fresh whole L. japonicus (20.6 kg) plants were collected in
Sendai, Japan for isolation of LOF, and only 1/20th of aqueous layer
(1 kg FW equiv) was used in the next procedure.
The NMR spectra of natural LOF (0.136 mg) in 0.3 ml of D2O
using an NMR tube matched for D2O (PS, Shigemi, Japan) were
measured with an INOVA-800 NMR spectrometer system equip-
ped with a cold probe (800 MHz for 1H, Varian, Inc., USA). 1H
NMR, DQF-COSY, NOESY, and HSQC spectra were measured using
16 scan times, while the HMBC spectra were measured using 128
scan times. NMR spectra of synthetic compounds were recorded
on a Jeol JNM-Alpha500 [1H (500 MHz) and 13C (125 MHz), Jeol
Inc., Japan] using TMS in CDCl3, CD2HOD in CD3OH (1H; 3.33 ppm,
13C; 49.8 ppm), or t-BuOH (1H; 1.23 ppm, 13C; 32.1 ppm) in D2O as
internal standard at various temperatures. The ESI-MS and HR
ESI-MS spectra were recorded on a Bruker Esquire 4000 (Bruker
Daltonics Inc., Germany) and a Bruker APEX-III (Bruker Daltonics
Inc., Germany), respectively. The IR spectra were recorded on
a JASCO FT/IR-410 (JASCO Inc., Japan). The specific rotations were
measured by JASCO DIP-360 polarimeter (JASCO Inc., Japan). Silica
gel column chromatography was performed on Silica Gel 60 N
(Kanto Chemical Co. Ltd., Japan). Reversed-phase open-column
chromatography was performed on Cosmosil 75C18-OPN (Nakalai
Tesque Co. Ltd., Japan). TLC was performed on Silica gel F254
Figure 6. Time course changes in the number of opening and closing events for 20
leaves treated with potassium salt of (R)-(ꢁ)-1, potassium salt of (S)-(þ)-1, and free
form of (R)-(ꢁ)-1. Lights were off after 21:00.
Although sodium salt of (R)-(–)-1 was identified as the main
bioactive metabolite in the extract of L. japonicus, it is suspected
that the genuine bioactive species of (R)-(ꢁ)-1 in vivo would be
a potassium salt because plants typically use potassium instead of
sodium. In fact, extract of L. japonicus mainly contained Kþ as metal
cation, as mentioned before. If our hypothesis is true, L. japonicus
would contain enough amount of (R)-(ꢁ)-1, which operates as the
potassium salt. Then, we measured an endogenous level of (R)-
(ꢁ)-1 in the extract of L. japonicus (Fig. 7). Quantitative analysis
showed that the content of (R)-(ꢁ)-1 in L. japonicus was 244
per kg fresh weight. This content of (R)-(ꢁ)-1 corresponded to
320 M in concentration when the water content of the fresh plant
mmol
m
is estimated to be 80% (data not shown). This result suggested that
the genuine bioactive species of (R)-(ꢁ)-1 was potassium salt.
Figure 7. LC/MS analysis of the (R)-(ꢁ)-1 and the aqueous fraction. LC/ESI-MS (negative) analysis of (R)-(ꢁ)-1 at 75
0.25 g FW equiv/mL by the extracted ion chromatograms m/z 239 (left) and UV at 235 nm (right).
mM and the bioactive aqueous fraction from L. japonicus at

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M. Okada et al. / Tetrahedron 65 (2009) 2136–2141
(0.25 mm or 0.5 mm, MERCK, Germany) or RP-18F254S (0.25 mm,
MERCK). Elemental analyses were carried out on a CHN-corder
MT-6 (Yanako Co., Ltd., Japan).
(d, J¼13.7 Hz, 1H, H4a), 2.85 (d, J¼15.6 Hz, 1H, H2b), 2.83 (d,
J¼13.7 Hz, 1H, H4b), 2.56 (d, J¼15.6 Hz, 1H, H2a); 13C signals were
determined by HSQC and HMBC correlations,
d 181.7, 179.1, 155.0,
131.3, 128.2, 115.0, 78.1, 45.5, 43.3; FT-ICR ESI-HRMS: m/z found
4.2. Bioassay
239.0561 ([MꢁH]–), calcd for C11H11O6 239.0561.
All bioassays were performed in a Biotron chamber (LPH-1000S,
NK System Co., Ltd., Japan) under 16 h light at 26 ꢀC (from 5:00 to
21:00) then 8 h dark at 22 ꢀC. The leaf of a young L. japonicus plant,
which was grown in the same chamber for 1–2 months, was re-
moved from the stem of the plant at the petiole and the two side
leaflets of the compound leaf were cut at their base. The remaining
central leaflet with petiole was then floated on water overnight. In
the morning (at 9:00), the leaves that had reopened and formed
obtuse angles between the leaf and the petiole were selected for
the bioassay.
4.4. Synthesis of LOF (1) and its enantiomer
a
-Hydroxyl carboxylic acid 2 was prepared from commercially
available O-benzyl-L-tyrosine (Watanabe Chemical, Japan) with
NaNO2 in 1,4-dioxane and aqueous sulfuric acid.17
4.4.1. Synthesis of benzylidene acetals 3 and 4
To a solution of (ꢁ)-2 (343 mg, 1.26 mmol) in ethyl acetate
(13 mL) were added n-pentane (13 mL), benzaldehyde (115 mg,
1.08 mmol), TsOH (240 mg, 1.39 mmol), and one drop of H2SO4. The
reaction mixture was stirred and refluxed for 1 day with azeotropic
removal of water. The reaction was stopped by the addition of
aqueous Na2CO3 to the reaction mixture. The organic layer was
washed with saturated aqueous NaCl, dried over absolute Na2SO4,
and evaporated to dryness. The residue was purified by silica gel
column chromatography (n-hexane/ethyl acetate¼30/1 to 20/1),
and recrystallized from n-pentane/Et2O to give cis-3 (104 mg,
Each leaflet with petiole was placed in a 1.5-mL plastic dis-
posable cuvette (Kartell, Milan Co., Italy) taking care to ensure
that the cut surface of the petiole was continuously immersed
into the sample solution (0.1 mL), which was obtained by each
step of separation procedure, with the change in the angle
between petiole and leaflet observed at hourly intervals. Leaf
opening was considered to have occurred when the angle
between the leaflet and petiole was obtuse (
closing was considered to be when the angle was acute (
q
>190ꢀ), while leaf
<170ꢀ).
0.288 mmol, 23%) as a white powder and trans-4 (148 mg,
q
0.411 mmol, 33%) as a white powder, respectively.
During the course of isolation, each resulting fraction was tested
by this method using a set of more than nine leaflets with
petioles. Leaflets with petioles placed in distilled water were used
as negative controls. The bioactivity of a fraction was assessed by
the status of leaves at 21:00. When less than 40% of the total
(2S,4S)-4-(4-Benzyloxybenzyl)-2-phenyl-5-oxodioxolan 3: 1H
NMR (500 MHz, CDCl3) d 7.45–7.30 (m, 8H), 7.26–7.20 (m, 4H), 6.93
(d, J¼7.6 Hz, 2H), 6.31 (d, J¼1.2 Hz, 1H), 5.05 (s, 2H), 4.64 (ddd,
J¼6.6, 4.1, 1.2 Hz, 1H), 3.26 (dd, J¼14.7, 4.1 Hz, 1H), 3.13 (dd, J¼14.7,
6.6 Hz, 1H); 13C NMR (125 MHz, CDCl3)
d 172.2, 158.0, 137.0, 134.3,
number of leaves had closed (
be bioactive.
q
<170ꢀ), the fraction was judged to
130.9, 130.5, 128.6, 128.5, 127.9, 127.8, 127.4, 126.9, 114.9, 103.4, 76.3,
29
70.0, 35.9; [
a
]
ꢁ74.6 (c 0.50, CHCl3). Anal. Found: C, 76.58; H, 5.69.
D
Calcd for C23H20O4: C, 76.65; H, 5.59.
4.3. Isolation of LOF from L. japonicus
(2R,4S)-4-(4-Benzyloxybenzyl)-2-phenyl-5-oxodioxolan 4: 1H
NMR (500 MHz, CDCl3)
d
7.45–7.29 (m, 10H), 7.25 (d, J¼8.7 Hz, 2H),
The fresh whole L. japonicus (20.6 kg) plants were collected in
Sendai, Japan. Terrestrial part was placed in methanol (200 L) and
extracted for 1 week and the extract was concentrated in vacuo to
give an aqueous solution of (5 L, 4 kg FWequiv/L). The aqueous
solution (1.0 L) was partitioned with n-hexane (3ꢂ500 mL), ethyl
acetate (3ꢂ500 mL), and n-butanol (3ꢂ500 mL). 1/20th of the
aqueous layer (250 mL, 1 kg FW eq) was separated by Amberlite
XAD-7 column chromatography (60ꢂ600 mm, Organo Co., Ltd.,
Japan) by stepwise elution with 0, 10, 30, and 100% aqueous
methanol to give four aqueous fractions (13, 5.5, 0.71, and 0.51 g,
respectively; bioactivity, the second fraction 2 g/L, others >2 g/L).
Part of the second aqueous fraction (50 mg) was further purified
by MPLC using Toyopearl HW-40S (25ꢂ400 mm, Tosoh Co., Ltd.,
Japan) with water (flow rate: 1.0 mL/min, detection: 215 nm) to
give a bioactive fraction (23.2 mg; retention time: 140–160 min,
bioactivity, 1 g/L, others >1 g/L). Part of the bioactive fraction
(5.0 mg) was further purified by MPLC equipped with an ODS
column (24ꢂ360 mm, Lop-ODS, Nomura Chemical Co., Ltd., Japan)
with a stepwise elution using 5, 20, 50, and 100% aqueous
methanol (flow rate: 8.0 mL/min, detection: 215 nm) to give
6.95 (d, J¼8.7 Hz, 2H), 6.12 (d, J¼0.9 Hz, 1H), 5.06 (s, 2H), 4.72 (ddd,
J¼5.8, 4.5, 0.9 Hz, 1H), 3.19 (dd, J¼14.6, 4.5 Hz, 1H), 3.13 (dd, J¼14.6,
5.8 Hz, 1H); 13C NMR (125 MHz, CDCl3)
d 172.5, 158.1, 136.9, 135.5,
130.8, 130.2, 128.6, 128.6, 128.0, 127.5, 127.5, 126.1, 115.1, 103.9, 75.3,
29
70.0, 36.1; [
Calcd for C23H20O4: C, 76.65; H, 5.59.
a
]
þ24.1 (c 0.50, CHCl3). Anal. Found: C, 76.48; H, 5.78.
D
4.4.2. Synthesis of benzyl [(2S,4R)-4-(4-benzyloxybenzyl)-2-
phenyl-5-oxodioxolan-4-yl]acetate (ꢁ)-5
To a solution of 3 (125 mg, 0.347 mmol) in THF (3.5 mL) was
added freshly prepared LDA (0.2 M, 2.4 mL, 0.485 mmol) at ꢁ80 ꢀC
under argon atmosphere. After stirring for 1 h, benzyl a-bromoa-
cetate (111 mg, 0.486 mmol) in HMPA (0.77 mL) was slowly added
to the mixture. After additional stirring for 6 h at ꢁ60 ꢀC, the re-
action was quenched with the addition of 1 M aqueous HCl (10 mL).
The reaction mixture was extracted with diethyl ether (3ꢂ10 mL),
washed with saturated aqueous NaCl, dried over Na2SO4, and
evaporated to dryness. The residue was purified by silica gel col-
umn chromatography (n-hexane/ethyl acetate¼25/1 to 10/1), be-
fore being recrystallized from n-pentane/diethyl ether to give (ꢁ)-5
(113 mg, 0.222 mmol, 64%) as a white powder.
a bioactive 20% eluate (259
mg; bioactivity, 0.05 g/L, others >1 g/L).
This process was repeated and collected 20% eluate (600
m
g) was
Benzyl [(2S,4R)-4-(4-benzyloxybenzyl)-2-phenyl-5-oxodiox-
purified by HPLC on an ODS column (Develosil ODS HG-5,
4.6ꢂ250 mm, Nomura Chemical Co., Ltd.) with 2% aqueous CH3CN
(flow rate: 1.0 ml/min, detection: 215 nm) to give a bioactive
olan-4-yl]acetate (ꢁ)-5: 1H NMR (500 MHz, CDCl3)
d 7.45–7.22 (m,
13H), 7.03 (d, J¼8.7 Hz, 2H), 6.98 (d, J¼7.3 Hz, 2H), 6.79 (d, J¼8.7 Hz,
2H), 6.39 (s, 1H), 5.19 (d, J¼12.0 Hz, 1H), 5.16 (d, J¼12.0 Hz, 1H), 5.02
(s, 2H), 3.13 (d, J¼14.1 Hz, 1H), 3.07 (d, J¼17.0 Hz, 1H), 2.96 (d,
J¼14.1 Hz, 1H), 2.92 (d, J¼17.0 Hz, 1H); 13C NMR (125 MHz, CDCl3)
fraction (136 mg; retention time: 10–12 min; bioactivity, 1 mg/L,
others >10 mg/L). The fraction was concentrated in vacuo to
remove CH3CN and freeze-dried several times to give LOF as
a white powder.
d
173.8, 160.5, 158.3, 140.0, 135.4, 135.1, 131.8, 129.8, 128.7, 128.6,
128.6, 128.6, 128.3, 128.0, 127.4, 126.4, 126.1, 114.7, 103.5, 80.8, 70.0,
30
Eucomic acid (R)-(ꢁ)-1: 1H NMR (800 MHz, D2O)
d
7.17 (d,
67.2, 43.1, 41.3; [
a
]
ꢁ49.6 (c 0.50, CHCl3). Anal. Found: C, 75.32; H,
D
0
0
0
0
J¼8.2 Hz, 2H, H2 and H6 ), 6.84 (d, J¼8.2 Hz, 2H, H3 and H5 ), 3.01
5.65. Calcd for C32H28O6: C, 75.57; H, 5.55.

M. Okada et al. / Tetrahedron 65 (2009) 2136–2141
2141
4.4.3. Synthesis of benzyl [(2R,4S)-4-(4-benzyloxybenzyl)-2-
phenyl-5-oxodioxolan-4-yl]acetate (þ)-5
from the regression curve (R2>0.999). Quantitative analysis was
performed by injection of the bioactive aqueous fraction at
0.25 g FWequiv/mL in the same condition. The peak area was
7.15ꢂ106. The content of (R)-(ꢁ)-1 in L. japonicus was calculated to
According to the abovementioned procedure for (ꢁ)-5, 4
(92.1 mg, 0.256 mmol) gave (þ)-5 (84.0 mg, 0.165 mmol, 65%) as
30
a white powder. [
a
]
þ47.9 (c 0.50, CHCl3).
be 244 mmol/kg fresh weight.
D
4.4.4. Synthesis of (R)-(ꢁ)-1 (eucomic acid)
Acknowledgements
To a solution of (ꢁ)-5 (63.1 mg, 0.124 mmol) in MeOH (5 mL)
was added 5% Pd/C (60 mg). After the reaction mixture was stirred
under a H2 atmosphere overnight, the mixture was filtered through
Celite and washed with 50% aqueous MeOH (3ꢂ5 mL). After
evaporation, the residue was purified by HPLC on an ODS column
(4.6ꢂ250 mm, Develosil ODS-HG-5, Nomura Chemical Co., Ltd.) at
a flow rate of 0.5 mL/min with 15% MeOH (retention time: 22.5–
23.0 min). The purified fraction was concentrated and freeze-dried
to give pure (R)-(ꢁ)-1 (eucomic acid) (26.1 mg, 89%) as a white
We thank the National Bioresource Project for providing seeds
of L. japonicus Gifu B-129. We extend our gratitude to the RIKEN
Genomic Sciences Center for the use of 800 MHz NMR spectrom-
eter system equipped with a cold probe and thank Dr. Hiroshi
Hirota and Dr. Fumiaki Hayashi for conducting NMR experiments.
We also thank Dr. Noboru Takada, Dr. Takashi Tokunaga, and Ms.
Shizuka Ishikawa for the collection of plant material and prepara-
tion of plant extracts.
28
24
powder. [
a]
ꢁ12.9 (c 0.50, MeOH); lit14
[a
]
ꢁ13ꢃ2 (c 1.08, H2O).
D
D
References and notes
4.4.5. Synthesis of (S)-(þ)-1
According to the abovementioned procedure for (R)-(ꢁ)-1, (þ)-5
(36.1 mg, 71.0 mol, 81%) as
mol) gave (S)-(þ)-1 (13.8 mg, 57.5
a white powder. [
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m
m
27
a
]
þ12.3 (c 0.50, MeOH).
D
4.5. Chiral HPLC analyses of synthetic (S)-(D)-1, (R)-(L)-1,
and naturally occurring 1
The purified naturally occurring 1, synthetic (S)-(þ)-1, and (R)-
(ꢁ)-1 were analyzed by HPLC on a chiral column (Sumichiral OA-
3100, 4.6ꢂ250 mm, Sumitomo Chemical Co., Ltd., Japan) with 0.1 M
NH4OAc in MeOH and 1,4-dioxane (1:1) (flow rate: 1.5 mL/min,
detection: 280 nm). These chromatograms and retention times are
shown in Figure 4.
4.6. Cation analysis of naturally occurring 1
The cation’s content in purified natural leaf-opening factor (R)-
(ꢁ)-1 was analyzed by capillary electrophoresis using an Agilent CE
system and Cation Solution Kit (Agilent Co., Ltd., USA). Separations
were performed on 64.5 cm long fused-silica capillaries with 50 mm
I.D. Before each analysis, the capillary was rinsed for 4 min with
running buffer containing 20 mM imidazole and 1.0 mM 18-crown-
6, and 0.5 mM lactic acid adjusted to pH 4.5 with acetic acid.
Sample solution was injected with a pressure of 50 mbar for 4.0 s.
The separation run was conducted over 5 min at 20 ꢀC at a constant
voltage of þ20 kV. Detection was performed by indirect UV moni-
toring; the signal wavelength was set at 310 nm and the reference
at 215 nm. The negative UV peaks of analytes were inverted by
reference to a signal wavelength of 310 nm. The content of each
cation was calculated using peak area compared to internal
standards.
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of L. japonicus
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A
triacontyl column (2.0ꢂ150 mm, Develosil RPAQUEOUS,
Nomura Chemical, Tokyo) was attached to an LC system and 5 L of
m
sample solution was eluted at a flow rate of 0.2 mL/min with 35%
aqueous CH3OH with 0.1% formic acid for 16 min. LC/ESI-MS (neg-
ative) was acquired in extracted ion chromatograms scan mode of
m/z 239. Calibration was established by peak areas for injection of
(R)-(ꢁ)-1 solution at 50, 75, and 100
m
M, which were 5.99ꢂ106,
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9.19ꢂ106, and 1.21ꢂ107 area (retention time: 6.3 min), respectively.
The intensity of (R)-(ꢁ)-1 was calculated to be 2.43ꢂ104 area/pmol