Isolation and structural elucidation of a new cyclohexenone compound from lasiodiplodia theobromae

Article abstract of DOI:10.1271/bbb.90218

A new cyclohexenone compound was isolated as a mixture of enantiomers from a culture filtrate of Lasiodiplodia theobromae. The relative structure was determined to be 4,5-dihydroxy-3-methyl-cyclohex-2-enone on the basis of MS, ¹-NMR, and ¹

Full text of DOI:10.1271/bbb.90218

Biosci. Biotechnol. Biochem., 73 (8), 1890–1892, 2009
Note
Isolation and Structural Elucidation of a New Cyclohexenone Compound
from Lasiodiplodia theobromae
y
Naoki KITAOKA, Kensuke NABETA, and Hideyuki MATSUURA
Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
Received March 25, 2009; Accepted April 24, 2009; Online Publication, August 7, 2009
[doi:10.1271/bbb.90218]
A new cyclohexenone compound was isolated as a
mixture of enantiomers from a culture filtrate of
Lasiodiplodia theobromae. The relative structure was
determined to be 4,5-dihydroxy-3-methyl-cyclohex-2-
enone on the basis of MS, 1H-NMR, and 13C-NMR
spectroscopic analyses, including 2D-NMR experiments.
Resolution of the enantiomers was conducted by a
coupling reaction with (S)-MTPA-Cl followed by HPLC
separation.
methyl group, an sp3 methylene, two sp3 oxymethines,
an sp2 methine, and two sp2 quaternary carbons,
including a ketone moiety. The HMQC spectrum
established the connections between carbons and direct-
ly attached protons. Based on the molecular formula and
the carbon chemical shift values for C-4 (ꢀ 74.8 ppm)
and C-5 (ꢀ 72.7 ppm), these carbons were concluded to
be attached to hydroxyl groups. The correlations
between CH3/C-2 and CH3/C-3 in the HMBC spectra
suggested a double bond connected to a methyl group at
1
Key words: theobroxide; Lasiodiplodia theobromae;
Nicotiana tabacum
C-3. The H-1H spin coupling system of H-4, H-5, and
H-6 was deduced from COSY correlations between H-4
and H-5, and H-5 and H2-6 (Fig. 1). The linkage of the
two partial structures just described was confirmed by
HMBC correlations of H-6/C-1 and CH3/C-4 which
suggested that the planar structure of 1 was 4,5-
dihydroxy-3-methyl-cyclohex-2-enone. The coupling
constants for H-5/H-6a and H-5/H-6b were 4.5 and
9.7 Hz, respectively. In addition to these coupling
constants, NOE correlations between H-4 and H-6b
suggested that H-4 and H-6b resided on the same face of
the cyclohexanone ring of 1, while H-5 and H-6a were
on the other side of the ring. To determine the absolute
stereochemistry at C-4 and C-5, compound 1a was
synthesized from theobroxide (2) by a reaction with
LiAlH4 to open the epoxide ring, yielding a mixture of
11a and 11b. After separating 11a and 11b by using
HPLC, 11a was oxidized to afford expected cyclo-
hexenone 1a together with 12; these were separated
according to the procedure described in the Experimen-
Lasiodiplodia theobromae is a pathogenic fungus that
colonizes a large variety of host plants in the tropics and
subtropics, and causes diseases in fruits and root crops
during storage. However, L. theobromae is also a source
of many organic compounds with interesting biological
activities, such as jasmonic acid, mellein, lasiodiplodin,
and theobroxide ((1S,2R,5S,6R)-3-methyl-7-oxa-bicyclo-
[4.1.0]hept-3-en-2,5-diol, 2),1,2) the latter having been
reported to stimulate flower bud formation in morning
glory (Pharbitis nil) and tuber formation in potato
(Solanum tuberosum) under non-inductive conditions.3)
Compound 2 has also been shown to inhibit stem
elongation in morning glory and spinach through the
suppression of gibberellin biosynthesis and stimulation
of jasmonic acid biosynthesis.4–7) We have subsequently
encountered its co-metabolites, (1S,4R,5S,6R)-7,9-
dioxa-3-methyl-8-oxobicyclo[4.3.0]-2-nonene-4,5-diol (3),
(4S,5S)-4,5-dihydroxy-2-methyl-cyclohex-2-enone (4),
and (3aS,4R,5S,7aR)-7,9-dioxa-3-methyl-8-oxobicyclo-
[4.3.0]-2-nonene-4,5-diol (5).8,9) In this paper, we report
the isolation, structural elucidation, and biological
activities of the novel theobroxide-related compound,
4,5-dihydroxy-3-methyl-cyclohex-2-enone (1).
A culture filtrate (36 liters) of Lasiodiplodia theo-
bromae OCS71 was evaporated under reduced pressure,
and the aqueous residue was extracted with EtOAc. This
extract was then subjected to flash column chromatog-
raphy on silica gel and HPLC to yield compounds 1
(39 mg), 2 (1.0 g), 3 (83 mg), and 4 (21 mg).
Compound 1 was obtained as brown oil, and a
molecular formula of C7H10O3 was obtained by high-
resolution FD-mass spectrometry. Three degrees of
unsaturation were inferred from the molecular formula
of 1. The value for specific rotation was þ11:3ꢀ (c 0.01,
MeOH). The 1H- and 13C-NMR spectra of 1 closely
resembled those of 4, and showed the presence of a
1
tal section. The EI-MS, H-NMR, and 13C-NMR data
for 1a (Fig. 2) prepared from theobroxide were consis-
tent with those of 1 isolated from L. theobromae.
However, the specific optical rotation values for 1 and
1a synthesized from theobroxide were þ11:3ꢀ and
ꢁ79:0ꢀ, respectively. We presumed that 1 isolated from
L. theobromae was a mixture of enantiomers. In order to
prove this hypothesis, we carried out a coupling reaction
of 1 with (S)-MTPA-Cl. A mixture of 1 and (S)-MTPA-
Cl in anhydrous pyridine was stirred for 48 h to afford
13a and 13b, and the disappearance of 1 was confirmed
by a TLC analysis. Compounds 13a and 13b were
separable by HPLC, the ratio of peak areas being
13a:13b = 5:7. However, a coupling reaction of 1a
prepared from 2 and (S)-MTPA-Cl in anhydrous
pyridine gave only 13a. These results indicated that
natural product 1 was a mixture of enantiomers
(1a:1b = 5:7). We could not explain why 1 was isolated
as a mixture of 1a and 1b, while 4 was isolated as a
y
To whom correspondence should be addressed. Tel: +81-11-706-2495; Fax: +81-11-706-2505; E-mail: matsuura@chem.agr.hokudai.ac.jp

New Cyclohexenone Compound from Lasiodiplodia theobromae
1891
OR
OR
HO
H
H
R
O
3
H3C
H
OR
R
4
1
S
5
6
R
S
OH
Ha
2
Hb
O
O
O
1
1a : R = H
13a : R = (R)-MTPA
1b : R = H
13b : R = (R)-MTPA
(mixture of enantiomers)
COSY :
HMBC (H C) :
OH
O
O
O
1
3
OH
5
OH
4
2
O
6
6
2
1
O
5
O
7
4
3
8
OH
9 O
OH
OH
OH
O
OH
2
3
4
5
OH
OH
OH
OH
H3CO
H3CO
O
OH
R
O
O
HOH2C
O
O
O
O
6
R = H : 7
9
10
R = OH : 8
Fig. 1. Important 2D-NMR Correlations for 1 and Chemical Structures of 1–10, 1a, 1b, 13a and 13b.
800
OH
OH
OH
700
600
500
400
300
200
100
0
OH
a
O
*
OH
*
**
OH
OH
OH
2
11a (21%)
11b (16%)
OH
O
Control
1
2
3
4
OH
OH
b
Fig. 2. Growth Inhibitory Activities of 1–4 against Seedlings of
Nicotiana tabacum.
11a
Data are mean ꢂ SD (n ¼ 5). Asterisks denote a significant
difference between each compound and the control (t-test
ꢃ p < 0:05; ꢃꢃ p < 0:01; ꢃꢃꢃ p < 0:001).
OH
O
1a (24%)
12 (21%)
4 against seedlings of N. tabacum. Three other related
oxygenated cyclohexenone derivatives, 6, 7 and 8, have
been isolated from unpolished rice fermented with a
xylariaceous endophytic fungus (strain YUA-026) by
Shiono et al.10) Some of these compounds exhibited
phytotoxic activity against lettuce (Lactuca sativa L.) at
a concentration of 50 mg/ml. Nagata et al. have also
isolated cyclohexenone compounds 9 and 10 from tea
gray blight fungi, Pestalotiopsis longiesta and P. theae,
respectively, and compounds 9 and 10 induced leaf
necrosis of cv. Yabukita at concentrations of 60 mg/ml
and 4 mg/ml, respectively.11) It is interesting that these
cyclohexenone compounds had such activities, and it is
quite possible that fungi may use these oxygenated
cyclohexenone compounds as a tool to invade other
organisms.
Scheme 1. Preparation of 1a from Theobroxide (2).
a) LiAlH4, THF, ꢁ78 ꢀC, 3 h; b) Dess-Martin periodinane, THF,
room temperature, 3 h.
single component, as confirmed by CD measurements8)
and by chemical synthesis from theobroxide (unpub-
lished data).
The biological activities of 1–4 were evaluated from
their growth inhibitory effects on seedlings of Nicotiana
tabacum. All of the compounds showed inhibitory
effects at a concentration of 0.5 mM, and the mixture
of 1a and 1b showing the strongest activity. Although
there have been some previous reports on the biological
activity of 2,3–7) this is the first report of the growth
inhibitory activity of 2 and related compounds 1, 3, and

1892
N. KITAOKA et al.
CD3OD): 201.6 (C-4), 145.2 (C-2), 135.4 (C-3), 70.0 (C-1), 64.3 (C-5),
Experimental
40.5 (C-6), 15.6 (C-7). IR ꢂmax (KBr) cmꢁ1: 3330 (OH), 1681 (C=O),
1437, 1351, 1037.
General. Data were obtained using the following instruments:
NMR, Bruker AMX-500 FT-NMR spectrometer and Jeol JNM-EX 270
FT-NMR spectrometer; FD-, EI-, and HRFD-MS, Jeol JMS SX-102A
mass spectrometer; IR, Digilab FTS-50A instrument.
Coupling reaction of 1 with (S)-MTPA-Cl. Compound 1, which had
been isolated from L. theobromae (5.6 mg, 0.039 mmol), and (S)-(þ)-ꢁ-
methoxy-ꢁ-trifluoromethylphenylacetyl chloride (32 mg, 0.126 mmol)
were dissolved in pyridine (2 ml) and stirred at room temperature for
24 h. The reaction mixture was diluted with EtOAc and successively
washed with 1 N HCl, saturated aq. NaHCO3, and saturated aq. NaCl.
After drying over Na2SO4, the organic layer was concentrated in vacuo,
and the resulting residue was purified by preparative thin-layer
chromatography (Merck silica gel 60 F254; hexane:EtOAc = 1:1,
v/v) and HPLC (Capcellpack C18, 4:6 ꢄ 250 mm; MeOH:H2O = 3:2,
v/v, 0.4 ml/min, A250) to give MTPA esters 13a (4.1 mg, 0.007 mmol,
tR ¼ 26 min) and 13b (5.7 mg, 0.010 mmol, tR ¼ 24 min).
Isolation. Lasiodiplodia theobromae OCS71 was grown statically in
twelve 5-liter flasks each containing 3 liters of a potato-sucrose
medium at 25 ꢀC in the dark for 14 d. The culture filtrate was
concentrated in vacuo, and the concentrated culture filtrate was
extracted with EtOAc (3 liters ꢄ 3). The extract was dried over
Na2SO4 and evaporated to give a crude extract which was subjected to
silica gel flash column chromatography (Kanto Chemical, silica gel
60N, 270 g; EtOAc) to give fractions A–D. Fraction B was subjected
to silica gel flash column chromatography (Kanto Chemical silica
gel 60N, 270 g; MeOH:CHCl3 = 5:95, v/v) to give 2 (1.07 g) and
3 (83 mg). Fraction D was subjected to silica gel flash column
chromatography (Kanto Chemical silica gel 60N, 270 g; MeOH:
CHCl3 = 5:95, 10:90, v/v), and then purified by HPLC (YMC Pack
ODS-AM, 10 ꢄ 300 mm; MeOH:H2O = 5:95, v/v, 2 ml/min, A250) to
give 1 (39 mg) and 4 (21 mg).
Compound 13a
FD-MS m=z (rel. int., %): 574 [M]þ (69), 340 (100); HRFD-MS
m=z (Mþ): calcd. for C27H24F6O6, 574.1426; found, 547.1435; ꢀH
(270 MHz, CDCl3): 7.51–7.58 (2H, m), 7.37–7.47 (8H, m), 5.97 (1H,
m), 5.87 (1H, d, J ¼ 6:6 Hz), 5.55 (1H, ddd, J ¼ 8:2, 6.6, 4.6 Hz), 3.54
(3H, s), 3.43 (3H, s), 2.85 (1H, dd, J ¼ 16:5, 4.6 Hz), 2.53 (1H, dd,
J ¼ 16:5, 8.2 Hz), 1.67 (3H, m).
Compound 1
25
½ꢁꢅD þ11:3ꢀ (c 0.01, MeOH); EI-MS m=z (rel. int., %): 142 [M]þ
Compound 13b
(4), 124 (8), 98 (100), 70 (39), 69 (46), 45 (47), 43 (23), 42 (42), 41
(34); FD-MS m=z (rel. int., %): 142 [M]þ (100); HRFD-MS m=z (Mþ):
calcd. for C7H10O3, 142.0629; found, 142.0634; ꢀH (500 MHz,
CD3OD): 5.85 (1H, m, H-2), 4.10 (1H, d, J ¼ 7:1 Hz, H-4), 3.91
(1H, ddd, J ¼ 9:7, 7.1, 4.5 Hz, H-5), 2.72 (1H, ddd, J ¼ 16:3, 4.5,
0.7 Hz, H-6a), 2.40 (1H, dd, J ¼ 16:3, 9.7 Hz, H-6b), 2.06 (3H, d,
J ¼ 6:6 Hz, H-7); ꢀC (125 MHz, CD3OD): 199.7 (C-1), 164.7 (C-3),
127.3 (C-2), 74.8 (C-4), 72.7 (C-5), 44.6 (C-6), 20.8 (C-7); IR ꢂmax
(KBr) cmꢁ1: 3355 (OH), 1655 (C=O), 1508, 1377, 1026.
FD-MS m=z (rel. int., %): 574 [M]þ (100); HRFD-MS m=z (Mþ):
calcd. for C27H24F6O6, 574.1426; found, 547.142; ꢀH (270 MHz,
CDCl3): 7.52–7.48 (2H, m), 7.46–7.36 (8H, m), 6.00 (1H, m), 5.60
(1H, d, J ¼ 4:6 Hz), 5.49 (1H, ddd, J ¼ 5:6, 4.6, 4.0 Hz), 3.46 (3H, d,
J ¼ 1:0 Hz), 3.44 (3H, d, J ¼ 1:0 Hz), 2.70 (1H, dd, J ¼ 17:1, 4.0 Hz),
2.58 (1H, dd, J ¼ 17:1, 5.6 Hz), 1.85 (3H, d, J ¼ 0:7 Hz).
Bioassay. Compounds 1–4 were each dissolved in water to a
concentration of 0.5 mM. Tobacco (Nicotiana tabacum cv. Xanthi-nc)
seeds were planted on Jiffy-7 (’ 42 mm) which had been treated with a
test solution (50 ml) of each compound. Control plants were prepared
by using a pot treated with 50 ml of water. The temperature and
moisture of the chambers were set at 25 ꢀC and 60%, respectively, and
the conditions of day length were 10 h of dark and 14 h of light. Water
was given once a day. In order to make sure one plant grew in each pot,
any excess plants were removed during the period of the test. After 21 d,
the plants were harvested, and the fresh weight of each was measured.
Chemical conversion of 2 to 11a and 11b. To solution of 2 (367 mg,
2.58 mmol) in dry THF (15 ml) was added LiAlH4 (141 mg,
3.71 mmol) in dry THF (15 ml) dropwise under nitrogen at ꢁ78 ꢀC,
and the mixture was stirred at ꢁ78 ꢀC for 3 h. The reaction mixture was
quenched with EtOAc/H2O and then concentrated in vacuo, and the
residue was subjected to silica gel flash column chromatography
(Kanto Chemical silica gel 60N, 40 g; MeOH:CHCl3 = 1:9, v/v),
before being purified by HPLC (Intertsil ODS, 20 ꢄ 250 mm;
MeOH:H2O = 5:95, v/v, 7 ml/min, A210) to afford 11a (76.8 mg,
colorless oil, 21%) and 11b (58.9 mg, colorless oil, 16%).
Acknowledgments
Compound 11a
25
½ꢁꢅD þ29:3ꢀ (c 0.41, MeOH); FD-MS m=z (rel. int., %): 144 [M]þ
We thank Dr. E. Fukushi and Mr. K. Watanabe of GC-
MS and NMR Laboratory, Faculty of Agriculture,
Hokkaido University for measuring the mass spectral data.
(18), 126 (100); ꢀH (270 MHz, CD3OD): 5.49 (1H, m, H-2), 4.18 (1H,
m), 3.82 (1H, ddd, J ¼ 8:5, 6.3, 4.3 Hz), 3.67 (1H, dd, J ¼ 6:3,
0.7 Hz), 1.80 (1H, m, H-6a), 1.75 (1H, m, H-6b), 1.74 (3H, m, H-7); ꢀC
(68 MHz, CD3OD): 138.8, 127.2, 75.2, 70.8, 65.3, 37.5, 19.9. IR ꢂmax
(KBr) cmꢁ1: 3299 (OH), 1424, 1257, 1010.
References
Compound 11b
25
½ꢁꢅD ꢁ17:1ꢀ (c 0.23, MeOH); FD-MS m=z (rel. int., %): 144 [M]þ
1) Aldridge DC, Galt S, Giles D, and Turner WB, J. Chem. Soc. C,
1623–1627 (1971).
(3), 100 (100); ꢀH (270 MHz, CD3OD): 5.36 (1H, m, H-2), 4.02 (1H,
m, H-4), 3.81 (1H, m, H-1), 3.74 (1H, ddd, J ¼ 10:8, 7.3, 3.5 Hz, H-6),
1.95 (1H, ddd, J ¼ 13:2, 3.5, 3.5 Hz, H-5a), 1.77 (3H, m, H-7), 1.74
(1H, ddd, J ¼ 13:2, 10.8, 4.6 Hz, H-5b); ꢀC (68 MHz, CD3OD): 138.3,
127.7, 74.0, 70.1, 69.1, 39.0, 20.3. IR ꢂmax (KBr) cmꢁ1: 3249 (OH),
1364, 1255, 1016.
2) Nakamori K, Matsuura H, Yoshihara T, Ichihara A, and Koda
Y, Phytochemistry, 45, 835–839 (1994).
3) Yoshihara T, Ohmori F, Nakamori K, Amanuma M, Tsutsumi
T, Ichihara A, and Matsuura H, J. Plant Growth Regul., 19,
457–461 (2000).
Chemical conversion of 11a into 1a and 12. To a stirred solution of
11a (14.4 mg, 0.10 mmol) in THF (5 ml) was added Dess-Martin
periodinane (42.4 mg, 0.10 mmol), and the mixture was stirred at room
temperature for 1 h. The reaction mixture was quenched with saturated
aq. Na2SO3 and concentrated in vacuo, and the residue was poured into
water and extracted with n-BuOH. The combined n-BuOH layers were
concentrated in vacuo, and the residue was subjected to silica gel flash
column chromatography (Kanto Chemical silica gel 60N, 40 g;
MeOH:CHCl3 = 5:95, v/v), and then purified by HPLC (Intertsil
ODS, 20 ꢄ 250 mm; MeOH:H2O = 1:9, v/v, 7 ml/min, A250) to afford
1a (3.4 mg, colorless oil, 24%) and 12 (3.0 mg, colorless oil, 21%).
Compound 12
FD-MS m=z (rel. int., %): 142 [M]þ (100); HRFD-MS m=z (Mþ):
calcd. for C7H10O3, 142.0629; found, 142.0643; ꢀH (500 MHz,
CD3OD): 6.67 (1H, m, H-2), 4.46 (2H, m, H-1, H-5), 2.25 (1H, dddd,
J ¼ 13:4, 4.9, 3.6, 1.3 Hz, H-6a), 2.14 (1H, ddd, J ¼ 13:4, 10.9,
4.2 Hz, H-6b), 1.79 (3H, dd, J ¼ 1:3, 1.3 Hz, H-7); ꢀC (68 MHz,
4) Kong F, Gao X, Nam K, Takahashi K, Matsuura H, and
Yoshihara T, Plant Sci., 169, 721–725 (2005).
5) Kong F, Abe J, Takahashi K, Matsuura H, Yoshihara T, and
Nabeta K, Biochem. Biophys. Res. Commun., 336, 1150–1156
(2005).
6) Kong F, Gao X, Nam K, Takahashi K, Matsuura H, and
Yoshihara T, J. Plant Physiol., 163, 557–561 (2005).
7) Gao X, Kong F, Wang F, Matsuura H, and Yoshihara T, J. Plant
Physiol., 163, 398–404 (2006).
8) Takei R, Takahashi K, Matsuura H, and Nabeta K, Biosci.
Biotechnol. Biochem., 72, 2069–2073 (2008).
9) Matsuura H, Obara N, Chisaka N, Ichihara A, and Yoshihara T,
Biosci. Biotechnol. Biochem., 62, 2460–2462 (1998).
10) Shiono Y, Murayama T, Takahashi K, Okada K, Katohda S, and
Ikeda M, Biosci. Biotechnol. Biochem., 69, 287–292 (2005).
11) Nagata T, Ando Y, and Hirota A, Biosci. Biotechnol. Biochem.,
56, 810–811 (1992).