Arabidopsides A and B, two new oxylipins from Arabidopsis thaliana

Article abstract of DOI:10.1016/S0040-4039(03)01148-1

Two new oxylipins, arabidopsides A (1) and B (2), were isolated from the aerial parts of Arabidopsis thaliana, and their structures and absolute stereochemistries were elucidated by spectroscopic data and chemical means. Arabidopsides A (1) and B (2) were rare monogalactosyl diacylglycerides containing 12-oxophytodienoic acid and/or dinor-oxophytodienoic acid.

Full text of DOI:10.1016/S0040-4039(03)01148-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 5553–5556
Arabidopsides A and B, two new oxylipins from
Arabidopsis thaliana
Yosuke Hisamatsu,a Nobuharu Goto,b Koji Hasegawac and Hideyuki Shigemoric,*
aGraduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan
bDepartment of Biology, Miyagi University of Education, Sendai 980-0845, Japan
cInstitute of Applied Biochemistry, University of Tsukuba, Tsukuba 305-8572, Japan
Received 20 March 2003; revised 28 April 2003; accepted 2 May 2003
Abstract—Two new oxylipins, arabidopsides A (1) and B (2), were isolated from the aerial parts of Arabidopsis thaliana, and their
structures and absolute stereochemistries were elucidated by spectroscopic data and chemical means. Arabidopsides A (1) and B
(2) were rare monogalactosyl diacylglycerides containing 12-oxophytodienoic acid and/or dinor-oxophytodienoic acid. © 2003
Elsevier Science Ltd. All rights reserved.
More than 10000 papers have been published on vari-
ous aspects of the biology, chemistry, and genetics of
Arabidopsis thaliana in the last three decades.1 In our
search for bioactive substances from A. thaliana, we
isolated two new oxylipins, named arabidopsides A (1)
and B (2), which were monogalactosyl diacylglycerides
containing 12-oxophytodienoic acid and/or dinor-oxo-
phytodienoic acid, from the aerial parts of this plant. In
this paper we describe the isolation and structure eluci-
dation of 1 and 2.
column (CHCl3/MeOH, 95:5) to afford a glycolipids
fraction, which was purified by a reversed-phase
C18HPLC (CH3CN/H2O, 4:1) to give arabidopsides A
(1, 4.0 mg, 0.004%) and B (2, 1.8 mg, 0.0018%) as
colorless amorphous solids together with known mono-
galactosyl diacylglycerides.
The molecular formula, C43H66O12, of arabidopside A
(1),2 [h]2D4 +56.8° (c 0.95, MeOH), was established by
HRESIMS [m/z 792.4875 (M+NH4)+, D −2.3 mmu].
The IR spectrum implied the presence of hydroxy (3428
cm−1), ester carbonyl (1738 cm−1), and unsaturated
carbonyl (1710 and 1630 cm−1) groups. The gross struc-
The aerial parts (100 g) of A. thaliana ecotype col-0
(Brassicaceae) were extracted with MeOH. The MeOH
extracts were partitioned with EtOAc and H2O. The
EtOAc-soluble portions were subjected to a silica gel
1
ture of 1 was deduced from detailed analysis of the H
and 13C NMR data (Table 1) aided with 2D NMR
Keywords: 12-oxophytodienoic acid; oxylipins; plants; natural products; Arabidopsis thaliana.
* Corresponding author. Fax: +81-29-853-4603; e-mail: hshige@agbi.tsukuba.ac.jp
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)01148-1

5554
Y. Hisamatsu et al. / Tetrahedron Letters 44 (2003) 5553–5556
Table 1. 1H and 13C NMR data of arabidopsides A (1) and B (2) in CD3ODa
1
2
Position
1Hb
J (Hz)
13Cb
64.78
Position
1Hb
J (Hz)
13Cb
64.80
1(a)
1(b)
2
3(a)
3(b)
1%
2%
3%
4%
5%
4.48 dd
4.25 dd
5.30 m
4.03 dd
3.78 dd
4.27 d
3.53 m
3.51 dd
3.87 d
12.0, 3.1
12.0, 6.5
1(a)
1(b)
2
3(a)
3(b)
1%
2%
3%
4%
5%
4.48 dd
4.26 dd
5.30 m
4.03 dd
3.76 dd
4.27 d
3.55 m
3.50 dd
3.87 d
12.2, 3.0
12.2, 6.6
72.67
69.53
72.64
69.54
11.0, 5.5
11.0, 5.7
7.6
11.2, 5.6
11.2, 6.1
7.6
106.16
73.19
75.69
71.03
77.62
63.29
106.16
73.19
75.69
71.04
77.62
63.29
9.7, 3.3
3.3
9.6, 3.2
3.2
3.53 m
3.80 dd
3.76 dd
3.55 m
3.80 dd
3.76 dd
6%(a)
6%(b)
1%%,1%%%
11.4, 7.0
11.4, 5.3
6%(a)
6%(b)
1%%,1%%%
11.4, 7.7
11.4, 5.2
175.81
175.44
35.78
35.69
26.76
26.62
31.08
31.03
29.40
29.17
31.53
30.88
32.61
32.50
46.61
46.55
171.03
170.88
133.73
133.66
214.37
214.28
51.79
51.79
25.76
25.76
129.09
129.03
134.63
134.57
22.57
22.57
15.47
15.23
175.56
2%%,2%%%
3%%,3%%%
4%%,4%%%
5%%,5%%%
2.38 m
1.66 m
1.38 m
1.38 m
2%%,2%%%
3%%,3%%%
4%%,4%%%
5%%,5%%%
2.37 m
1.64 m
1.35 m
1.35 m
35.88
35.70
26.76
31.55
29.43
6%%%
7%%%
1.38 m
1.38 m
1.81 m
1.26 m
3.10 m
6%%,6%%%
7%%,7%%%
8%%(a),8%%%(a)
8%%(b),8%%%(b)
9%%,9%%%
1.35 m
1.35 m
1.81 m
1.21 m
3.09 m
32.63
31.04
33.88
6%%(a),8%%%(a)
6%%(b),8%%%(b)
7%%,9%%%
46.62
171.03
133.67
214.74
51.58
8%%,10%%%
7.960 dd
7.968 dd
6.209 dd
6.210 dd
5.9, 1.9
5.9, 1.9
5.9, 1.9
5.9, 1.9
10%%,10%%%
11%%,11%%%
12%%,12%%%
13%%,13%%%
14%%,14%%%
15%%,15%%%
16%%,16%%%
17%%,17%%%
18%%,18%%%
7.93 dd
6.20 dd
5.7, 2.2
5.7, 2.2
9%%,11%%%
10%%,12%%%
11%%,13%%%
12%%,14%%%
13%%,15%%%
14%%,16%%%
15%%,17%%%
16%%,18%%%
2.56 m
2.52 m
2.47 m
2.21 m
5.45 m
2.46 m
2.21 m
5.44 m
25.76
129.08
134.58
22.57
5.45 m
2.12 m
1.02 t
5.44 m
2.11 m
1.02 t
7.5
7.5
15.22
a Data recorded on a 500 MHz spectrometer with reference to the solvent signals (lH 3.35, lC 49.8).
b In ppm.
experiments (1H–1H COSY, HMQC, and HMBC). The
13C NMR data indicated that the molecule possessed
two unsaturated carbonyl carbons, two ester carbonyl
carbons, four disubstituted olefins, one acetal carbon,
five oxymethines, three oxymethylenes, four methines,
(J1%,2%=7.6 Hz, J2%,3%=9.7 Hz, J3%,4%=3.3 Hz, and J4%,5%=
ꢀ0 Hz). HMBC correlations of H-1% to C-3 (lC 69.53)
and H-3a and H-3b to C-1% (lC 106.16) and the cou-
pling constant (J1%,2%=7.6 Hz) of the anomeric proton
(H-1%) at lH 4.27 revealed that 1 possessed a b-galacto-
1
1
sixteen methylenes, and two methyl groups. The H–1H
sylglycerol moiety. The H–1H COSY connectivities of
COSY connectivities of C-1 to C-3 and C-1% to C-6%
indicated the presence of a glycerol and a sugar compo-
nent. The sugar was assigned to be galactose by
NOESY correlations of H-1% to H-3% and H-5% and H-4%
to H-3% and H-5% and the 1H–1H coupling constants
C-7%% (or C-9%%%) to C-9%%(or C-11%%%) and C-7%% (or C-9%%%)
to C-11%% (or C-13%%%) and HMBC correlations of H-9%%
(or H-11%%%) (lH 6.210 or 6.209) to C-7%% (or C-9%%%) (lC
46.61 or 46.55), C-10%% (or C-12%%%) (lC 214.37 or 214.28),
and C-11%% (or C-13%%%) (lC 51.79) indicated the presence

Y. Hisamatsu et al. / Tetrahedron Letters 44 (2003) 5553–5556
5555
of two cyclopentenone moieties. The 1H–1H COSY
connectivities of C-11%% (or C-13%%%) to C-16%% (or C-18%%%)
and HMBC correlations of Ha-12%% (or Ha-14%%%) (lH
2.47) and Hb-12%% (or Hb-14%%%) (lH 2.21) to C-13%% (or
C-15%%%) (lC 129.09 or 129.03) and C-14%% (or C-16%%%) (lC
134.63 or 134.57) revealed that 2-pentene groups con-
nected to C-11%% and C-13%%%. Z-Geometries of two dis-
ubstituted double bonds at C-13%%ꢀC-14%% and
C-15%%%ꢀC-16%%% were deduced from the carbon chemical
shifts of allylic carbons (C-12%% or C-14%%%, lC 25.76;
C-15%% or C-17%%%, lC 22.57).3 These data and proton and
carbon resonances indicated that 1 possessed two lipids
containing cyclopentenone. The two lipids were pre-
sumed to be cis-12-oxophytodienoic acid and cis-dinor-
oxophytodienoic acid moieties judging from spectral
data of cis-12-oxophytodienoic acid (OPDA)4 and cis-
dinor-oxophytodienoic acid (dinor-OPDA).5 HMBC
correlations of Ha-1 and Hb-1 to ester carbonyl carbon
(lC 175.81 or 175.44) and chemical shifts (lH 5.30; lC
72.67) of C-2 indicated that the OPDA and dinor-
OPDA connected to C-1 and C-2. In order to define the
locations of these lipids in the b-galactosylglycerol moi-
ety of 1, we applied enzymatic hydrolysis. The lipase
type XI (Sigma)-catalyzed hydrolysis of 1 afforded
1-O-deacylarabidopside A (3) [ESIMS m/z 523 (M+
Na)+] (Scheme 1).6 Thus, arabidopside A (1) was
assigned to be sn1-O-(12-oxophytodienoyl)-sn2-O-
presumed to be R, from the basis of a comparison of
the optical rotation of 6 ([h]D −7°) with the reported
values ([h]D −7° for C-2 R and [h]D +2° for C-2 S)
(Scheme 1).9 Therefore, the absolute configuration at
C-2 of 1 has been assigned as S, while the dinor-OPDA
and OPDA of 1 have been enantiomeric mixture (10:3)
of (7%%S,11%%S), (9%%%S,13%%%S)- form and (7%%R,11%%R),
(9%%%R,13%%%R)-form.
Arabidopside B (2),10 [h]2D6 +61.1° (c 0.48, MeOH),
showed the pseudomolecular ion peak at m/z 820 (M+
NH4)+ in the ESIMS. HRESIMS analysis revealed the
molecular formula to be C45H70O12 [m/z 820.5206 (M+
NH4)+, D −0.5 mmu], indicating that 2 assumed to be
an ethylene homolog of 1. The 1H and 13C NMR
spectra of 2 were similar to those of arabidopside A (1),
except for the signals due to the methylene parts.
Treatment of 2 with HCl/MeOH afforded 2 mol of
OPDA. Therefore, arabidopside B (2) was assigned to
be sn1, sn2-di-O-(12-oxophytodienoyl)-monogalactosyl
diglyceride. The chiral GC-analyses of methanolysates
of 2 revealed the mixture (10:3) of enantiomers of
(9%%S,13%%S),
(9%%%S,13%%%S)-form
and
(9%%R,13%%R),
(9%%%R,13%%%R)-form and the absolute configuration of
C-2 was assigned as S judging from optical rotation of
6 ([h]D −10°), which was obtained from methanolysis of
2 with NaOMe.
(dinor-oxophytodienoyl)-monogalactosyl
diglyceride.
The chiral GC-analyses (g-DEX™ 120 Capillary
Column, SUPELCO) of methanolysates of 1 by treat-
ment with HCl/MeOH detected the mixture (10:3) of
(7%%S,11%%S)-4 and (7%%R,11%%R)-4 and the mixture (10:3)
of (9%%%S,13%%%S)-5 and (9%%%R,13%%%R)-5 (Scheme 1).7 The
absolute configuration at C-2 in the b-galactosylglyc-
erol (6), which was derived from 1 with NaOMe,8 was
Arabidopsides A (1) and B (2) are rare oxylipins con-
taining OPDA and dinor-OPDA, although sn1-O-(12-
oxophytodienoyl)-sn2-O-(hexadecatrienoyl)-monogal-
actosyl diglyceride, a chloroplast membrane oxylipin
containing esterified OPDA, has been recently isolated
from A. thaliana.11 The OPDA and dinor-OPDA,
which have been isolated from leaves of A. thaliana on
Scheme 1.

5556
Y. Hisamatsu et al. / Tetrahedron Letters 44 (2003) 5553–5556
wounding, suggested roles in wound signaling and these
compounds are precursors of jasmonic acid. It may
therefore be noted that the OPDA and dinor-OPDA in
A. thaliana are present as arabidopsides A (1) and B
(2), and sn1-O-(12-oxophytodienoyl)-sn2-O-(hexadeca-
trienoyl)-monogalactosyl diglyceride in chloroplast
membranes. The OPDA and dinor-OPDA can be
released from chloroplast membranes enzymatically
and this could account for the rapid transient increase
in free OPDA, dinor-OPDA, and jasmonic acid on the
bioactivity such as wound signaling.12
under reduced pressure and the resulting residue was
purified on a silica gel column (CHCl3/MeOH, 7:1) to
yield 3 (0.4 mg, 62%). Morimoto, T.; Nagatsu, A.;
Murakami, N.; Sakakibara, J.; Tokuda, H.; Nishino, H.;
Iwashima, A. Phytochemistry 1995, 40, 1433–1437.
7. Methanolysis of 1 and the chiral GC analyses of the
methanolysates. After a solution of 1 (0.2 mg) in 0.6
mol/L HCl/MeOH (0.1 mL) was stirred at 60°C for 45
min, the solvent was removed under reduced pressure.
The residue was partitioned with hexane and 90% MeOH
and the hexane-soluble materials were used for chiral GC
analyses. Chiral GC conditions, column: g-DEX™ 120
Capillary Column, SUPELCO; program rate: 40“160°C
(at 10°C/min), 160°C (360 min), 160“200°C (at 10°C/
min), and 200°C (60 min). The absolute configurations of
the OPDA and dinor-OPDA were determined by the
retention time (min). The cis enantiomers, (7%%S,11%%S)-4
(238 min) and (7%%R,11%%R)-4 (238 min) and (9%%%S,13%%%S)-5
(397 min) and (9%%%R,13%%%R)-5 (397 min) were inseparable,
respectively, while the trans enantiomers, (7%%S,11%%R)-4
(157 min) and (7%%R,11%%S)-4 (158 min) (10:3) and
(9%%%S,13%%%R)-5 (359 min) and (9%%%R,13%%%S)-5 (363 min)
(10:3), which were produced from cis forms by enoliza-
tion. Laudert, D.; Hennig, P.; Stelmach, B. A.; Mu¨ller,
A.; Andert, L.; Weiler, E. W. Anal. Biochem. 1997, 246,
211–217.
Acknowledgements
We thank Dr. S. Matsuyama (University of Tsukuba)
for chiral GC analyses and Banyu Pharmaceutical Co.,
Ltd. for measurement of ESIMS. This work was partly
supported by a Grant-in-Aid for Scientific Research
from the Ministry of Education, Science, Sports, and
Culture of Japan.
References
8. Methanolysis of 1 with NaOMe. A solution of 1 (0.9 mg)
and NaOMe (10 equiv.) in anhyd. MeOH (0.1 mL) was
stirred at room temperature for 1 h. The reaction mixture
was partitioned between hexane and water and the water-
soluble materials were purified by C18 column (MeOH/
H2O, 1:1) to give the b-galactosylglycerol (6, 0.27 mg,
92%).
9. Son, B. W.; Cho, Y. J.; Choi, J. S.; Lee, W. K.; Kim,
D.-S.; Choi, H. D.; Choi, J. S.; Jung, J. H.; Im, K. S.;
Choi, W. C. Nat. Prod. Lett. 2001, 15, 299–306.
10. Compound 2: colorless amorphous solid; IR (KBr) wmax
3430, 1738, 1710(sh), 1631, 1166, and 1069 cm−1; UV
(MeOH) umax 225 (m 13200) nm.
1. Veit, M.; Pauli, G. F. J. Nat. Prod. 1999, 62, 1301–1303.
2. Compound 1: IR (KBr) wmax 3428, 1738, 1710, 1630, 1170
and 1073 cm−1; UV (MeOH) umax 222 (m 11000) nm.
3. The allylic carbons of cis- and trans-double bonds in the
aliphatic chains are reported to resonate approximately at
27 and 32 ppm, respectively: Gunstone, F. D.; Pollard,
M. R.; Scrimgeour, C. M.; Vedanayagam, H. S. Chem.
Phys. Lipids 1977, 18, 115–129.
4. Baertschi, S. W.; Ingram, C. D.; Harris, T. M.; Brash, A.
R. Biochemistry 1988, 27, 18–24.
5. Weber, H.; Vick, B. A.; Farmer, E. E. Proc. Natl. Acad.
Sci. USA 1997, 94, 10473–10478.
6. Enzymatic hydrolysis of 1. A solution of 1 (1 mg) and
Lipase type XI (0.72 units, Sigma) in the presence of
Triton X-100 (2.5 mg) in boric acid–borax buffer (0.63
mL, pH 7.7) was stirred at 38°C for 12 h. The reaction
was quenched with AcOH (0.1 mL), and then EtOH was
added to the reaction mixture. The solvent was removed
11. Stelmach, B. A.; Mu¨ller, A.; Hennig, P.; Gebhardt, S.;
Schubert-Zsilavecz, M.; Weiler, E. W. J. Biol. Chem.
2001, 276, 12832–12838.
12. Turner, J. G.; Ellis, C.; Devoto, A. The Plant Cell 2002,
S153–S164.