New potato micro-tuber-inducing cyclohexene compounds related to theobroxide from lasiodiplodia theobromae

Article abstract of DOI:10.1271/bbb.80171

Two new cyclohexene compounds related to theobroxide (3) were isolated from the mycelia of Lasiodiplodia theobromae OCS71. The structures of these compounds were determined to be (4S,5S)-4,5-dihydroxy-2-methyl-cyclohex-2-enone (1) and (3aS,4R,5S,7aR)-4,5-dihydroxy-7-methyl-3a,4,5,7a-tetrahydrobenzo[1,3] dioxol-2-one (2) by means of spectroscopic analyses and chemical correlation to 3. Compound 2 was shown to take up the carbonate ion to form a carbonic acid ester non-enzymatically. The compounds also showed potato micro-tuber-inducing activities at a concentration of 10^-3 M, using a culture of single-node segments of potato stems in vitro.

Full text of DOI:10.1271/bbb.80171

Biosci. Biotechnol. Biochem., 72 (8), 2069–2073, 2008
New Potato Micro-Tuber-Inducing Cyclohexene Compounds Related
to Theobroxide from Lasiodiplodia theobromae
y
Ryo TAKEI, Kosaku TAKAHASHI, Hideyuki MATSUURA, and Kensuke NABETA
Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan
Received March 17, 2008; Accepted May 1, 2008; Online Publication, August 7, 2008
[doi:10.1271/bbb.80171]
Two new cyclohexene compounds related to theobr-
oxide (3) were isolated from the mycelia of Lasiodiplodia
O
OH
O
OH
O
OH
theobromae OCS71. The structures of these compounds
were determined to be (4S,5S)-4,5-dihydroxy-2-methyl-
cyclohex-2-enone (1) and (3aS,4R,5S,7aR)-4,5-dihy-
droxy-7-methyl-3a,4,5,7a-tetrahydrobenzo[1,3]dioxol-2-
one (2) by means of spectroscopic analyses and chemical
correlation to 3. Compound 2 was shown to take up the
carbonate ion to form a carbonic acid ester non-
enzymatically. The compounds also showed potato
micro-tuber-inducing activities at a concentration of
10À3 M, using a culture of single-node segments of potato
stems in vitro.
O
O
O
OH
OH
O
OH
1
OH
2
OH
3
O
4
Fig. 1. New Compounds 1 and 2 and Their Related Compounds 3
and 4.
Results and Discussion
Key words: theobroxide related compounds; potato mi-
cro-tuber-inducing activity; Lasiodiplodia
theobromae
The L. theobromae OCS71 fungus was statically
grown in 500-ml flasks containing 200 ml of a 2%
potato-dextrose medium at 25 ꢁC in the dark for 21 d.
Compounds 1 (colorless oil, 1.5 mg) and 2 (colorless oil,
6.1 mg) were isolated from an acetone extract of mycelia
(DW = 139 g) of L. theobromae OCS71 by silica gel
column chromatography and HPLC.
Lasiodiplodia theobromae (synonym Botryodiplodia
theobromae) is a common pathogenic fungus found in
the tropics and subtropics on a great variety of host
plants.1) This fungus causes various diseases in fruit and
root crops during storage. The fungus is also known to
produce jasmonic acid related compounds and some
other metabolites that exhibit interesting biological
activities. Theobroxide (3) and its related compound
(4) produced by L. theobromae have induced potato
micro-tuber formation under non-inductive condi-
tions.2–4) A biosynthetic study of theobroxide has
recently shown that four acetate units were incorporated
into it by a polyketide biosynthetic pathway.5) L. theo-
bromae is believed to produce other theobroxide-related
compounds that possess biological activity. In our
screening of L. theobromae OCS71, new cyclohexene
compounds 1 and 2, which are structurally related to
theobroxide, were found in the mycelia of the fungus.
We report here the isolation, structural elucidation, and
biological activities of 1 and 2. Non-enzymatic forma-
tion of compounds 2 and 4 from 3 in the presence of
The molecular formula of 1 was established as
C7H10O3 on the basis of HREI-MS data. The value for
the specific rotation was +128ꢁ (c 0.2, MeOH). The IR
absorption peaks at 3452 and 1674 cmꢀ1 respectively
indicated the presence of hydroxyl groups and a car-
bonyl group. The UV absorption maximum at 231 nm
suggested the presence of a conjugated enone group in
the structure. The NMR spectral data for 1 resembled
those of theobroxide. The 1H- and 13C-NMR spectra
showed the presence of a methyl, sp3 methylene, two sp3
oxymethines, sp2 methine, and two sp2 quaternary
carbons, including a ketone. Three degrees of unsatura-
tion were inferred from the molecular formula of 1.
HMBC correlations between CH3/C-1 and C-2, and
H-3/C-2 suggested a double bond connecting a methyl
1
group at C-2. The H-1H spin coupling system of H-4,
H-5, and H2-6 was deduced by COSY correlations
between H-4/H-5 and H-5/H2-6. Considering the
molecular formula, IR spectral data, and the carbon
2ꢀ
CO3 is also reported.
y
To whom correspondence should be addressed. Tel/Fax: +81-11-706-2505; E-mail: knabeta@chem.agr.hokudai.ac.jp

2070
R. TAKEI et al.
O
O
O
O
O
O
H
2
1
3O
H
Ha
Hb
O
O
O
O
t-Bu
H3C
H
H3C
H
3a
7a
1
4
2
3
6
5
7
6
4
H
OH
H
OH
5
b
COSY:
NOESY:
HMBC:
H
OH
H
OH
OH
OH
1
2
OH
3a (18 %)
2 (28 %)
Fig. 2. Important 2D-NMR Correlations of 1 and 2.
a
3
+
OH
OH
OH
C4
b
O
O
C7
O
C
O
O
large 3JCH
small 3JCH
O
O
t-Bu
O
H3a
H7a
O
O
3JHH = 8.0 Hz
4 (25 %)
3b (38 %)
Fig. 3. Newman Projection of the Carbonic Acid Ester Moiety of 2.
Fig. 4. Preparation of 2 and 4 from 3.
a, (Boc)2O, DMAP, CH3CN, room temperature, 24 h. b, BF3-
OEt2, CH2Cl2, ꢀ40 ꢁC, 30 min.
chemical shift values at C-4 (ꢀ 73.4) and C-5 (ꢀ 73.6),
each carbon was attached to a hydroxyl group. The
linkage of the two partial structures just described was
confirmed by HMBC correlations of H2-6/C-1 and H-4/
C-3, suggesting that the planar structure of 1 was 4,5-
dihydroxy-2-methyl-cyclohex-2-enone. The coupling
constant values between H-4/H-5 and H-5/H-6b were
7.7 and 11.4 Hz, respectively. NOESY correlations
observed between H-4/H-6b suggested that H-4 and
H-6b resided on the same face of 1, while H-5 and H-6a
resided on the other side of the molecule. To determine
the absolute stereochemistry at C-4 and C-5 of 1,
compound 1 was converted to its dibenzoyl derivative
(1a). The CD spectra of 1a showed positive and negative
Cotton effects at 239.0 and 221.5 nm, respectively.
The absolute structure of 1 was therefore deduced
to be (4S,5S)-4,5-dihydroxy-2-methyl-cyclohex-2-enone
(Fig. 2).
COSY correlations between H-3a/H-4 and H-7a, and
H-4/H-5. HMBC correlations of H-5/C-6 suggested the
presence of a cyclohexene ring in the structure of 2.
The proton chemical shift values for the oxymethine
protons, H-7a (ꢀ 5.06) and H-3a (ꢀ 4.64), suggested that
these protons were deshielded by an ester group.
Considering the HMBC relationship of H-3a/C-2 and
the unsaturation degree, a carbonic acid ester had been
formed at C-3a and C-7a as a five-membered ring,
enabling the planar structure of 2 to be determined as
4,5-dihydroxy-7-methyl-3a,4,5,7a-tetrahydrobenzo[1,3]-
dioxol-2-one. To determine the absolute configurations
at C-4 and C-5, compound 2 was converted to its
dimethylaminobenzoate derivative (2a). The CD spectra
for 2a showed positive and negative Cotton effects at
324.5 nm and 301.0 nm, respectively, allowing the
absolute configurations at C-4 and C-5 to be respec-
tively deduced as R and S. To narrow down the possible
structure of the carbonic acid ester moiety, a J-based
configuration analysis6) was conducted by the NMR
The molecular formula of 2 [ꢀ12:1ꢁ (c 0.5, MeOH)]
was determined to be C8H10O5 by HREI-MS data,
which is the same as that of theobroxide-related
compound 4. The IR absorption peaks at 3406 and
1784 cmꢀ1 respectively indicated the presence of
hydroxyl groups and an ester group. Four degrees of
unsaturation were inferred from the molecular formula
of 2. The NMR spectral data for 2 were similar to those
3
spectral data. HMBC data revealed that J (H-3a/C-7)
3
and J (H-7a/C-4) were small and large, respectively
3
(Fig. 3). The J (H-3a/H-7a) value of 8.0 Hz indicated
that the relative configuration of H-3a and H-7a was cis.
The NOESY correlation of H-5/H-3a demonstrated
that H-5 and H-3a resided on the same face of the mole-
cule. Accordingly, the absolute structure of 2 was deter-
mined to be (3aS,4R,5S,7aR)-4,5-dihydroxy-7-methyl-
3a,4,5,7a-tetrahydrobenzo[1,3]dioxol-2-one (Fig. 2). To
confirm this structure of 2, chemical conversion of
3 into 2 was conducted (Fig. 4). Compound 3 was
reacted with di-t-butyl dicarbonate to introduce a
carbonic acid ester into the structure,7,8) yielding 3a
and 3b as a mixture. After HPLC separation, com-
pounds 3a and 3b were cyclized to afford expected
1
of 4.4) The H- and 13C-NMR spectra showed the pres-
ence of a methyl, four sp3 oxymethines, sp2 methine,
and two sp2 quaternary carbons, including a carbonyl
1
carbon. The most significant difference in the H-NMR
spectra between these compounds was observed at
around 5.0 ppm, i.e., a broad doublet signal at ꢀH 5.06
in 2 changed to a multiplet signal at ꢀH 5.13 in 4.
HMBC correlations between H-6 and CH3/C-7, and
H-7a/C-7 and CH3 suggested a double bond connecting
a methyl group at C-7. The 1H-1H spin coupling
sequence of H-3a, H-4, H-5, and H-7a was deduced by

New Cyclohexene Compounds from Lasiodiplodia theobromae
2071
O
O
O
HO
H
O
OH
O
O
-
O
OH
O
OH+
-H2O
trans-diaxial opening
OH
OH
OH
OH
OH
3
2
Fig. 5. Proposed Biosynthetic Reaction from 3 to 2.
bicyclic carbonates 2 and 4, respectively. The EI-MS
and 1H-NMR data for chemically prepared 2 were
consistent with those of 2 isolated from L. theobromae,
supporting the absolute structure of 2 as shown in
Fig. 2.
dark for 21 d. The mycelia were washed with distilled
water, filtered, and dried at room temperature in the dark
(DW = 139 g). The dried mycelia were extracted with
acetone (2 l ꢂ 2), and concentrated in vacuo to give
an aqueous residue. This was extracted with EtOAc
(350 ml ꢂ 5). The extract was dried over Na2SO4 and
evaporated to give a crude extract (1.86 g). This was
subjected to silica gel column chromatography (Kanto
Chemical, silica gel 60N, 200 g; MeOH:CHCl3 =
0:100, 3:97, 10:90, 20:80, v/v). The fraction from
MeOH:CHCl3 = 10:90 (v/v) was applied to silica gel
column chromatography (Kanto Chemical, silica sel
60N, 30 g; MeOH:CHCl3 = 10:90), and then purified
by HPLC (Inertsil ODS, 20 ꢂ 250 mm; MeOH:H2O =
10:90, v/v, 5 ml/min, A210 nm) to afford 1 (1.5 mg) and
2 (6.1 mg).
Our previous study showed that a theobroxide (3) was
formed by a polyketide biosynthetic pathway,5) and that
the carbon of the carbonic acid ester in the carbon-
yldioxy derivative (4) originated from the carbonyl
carbon of an acetate. We examined whether the
conversion of 3 into 2 and 4 occurred enzymatically in
the reaction mixture containing Na2CO3. It was found
that 2 and 4 were produced with or without a cell-free
extract prepared from L. theobromae. Accordingly, 2
and 4 were postulated to be non-enzymatically gener-
ated by CO32ꢀ, which was enzymatically formed from
carboxylic acids such as an acetate within the culture of
L. theobromae. Taking into consideration the stereo-
chemistry of 2, 3, and 4, one of the epoxycarbons was
22
Compound 1: ½ꢁꢃD +128ꢁ (c 0.21, MeOH); EI-MS
m=z (rel. int., %): 142[M]þ (8), 124 (5), 98 (100), 70
(38), 69 (35), 43 (17), 41 (15); HREI-MS m=z: 142.0616
[M]þ (calcd. for C7H10O3: 142.0629); 1H-NMR
(500 MHz, CD3OD) ꢀ (ppm): 6.64 (1H, m, H-3), 4.21
(1H, ddq, 7.7, 2.1, 2.1 Hz, H-4), 3.81 (1H, ddd,
J ¼ 11:4, 7.7, 4.6 Hz, H-5), 2.71 (1H, dd, J ¼ 15:9,
4.6 Hz, H-6a), 2.40 (1H, dd, J ¼ 15:9, 11.4 Hz, H-6b),
1.72 (3H, m, 2-CH3); 13C-NMR (125 MHz, CD3OD)
ꢀ (ppm): 199.7 (C-1), 148.0 (C-3), 136.5 (C-2), 73.6
(C-5), 73.4 (C-4), 45.5 (C-6), 15.3 (2-CH3); IR ꢂmax
cmꢀ1: 3452, 2924, 1674, 1438, 1368, 1078, 982, 898;
UV (MeOH) ꢃmax nm ("): 231 (5980).
ꢀ
probably attacked by the HCO3 anion via trans-diaxial
opening, and the resulting monocarbonate intermediate
was further converted to the carbonyl dioxy compound
with the elimination of H2O (Fig. 5).9,10)
The potato micro-tuber-inducing activity of 1 and 2
was evaluated by the method of Koda et al.11) While the
activity of the theobroxide (3) was observed at a
concentration of 10ꢀ5 M, the activity of 1 and 2 appeared
at a concentration of 10ꢀ3 M, showing that both
compounds had weaker activity than 3. No obvious
difference between potato micro-tubers treated with 1
and 2 was apparent in the experiment.
22
Compound 2: ½ꢁꢃD ꢀ12:1ꢁ (c 0.49, MeOH); EI-MS
m=z (rel. int., %): 186[M]þ (8), 124 (11), 113 (9), 100
(100), 95 (33), 71 (54), 55 (33), 41 (32); HREI-MS m=z:
186.0525 [M]þ (calcd. for C8H10O5: 186.0528); 1H-
NMR (500 MHz, CD3OD) ꢀ (ppm): 5.69 (1H, m, H-6),
5.06 (1H, br d, J ¼ 8:0 Hz, H-7a), 4.64 (1H, dd, J ¼ 8:0,
9.0 Hz, H-3a), 3.95 (1H, m, H-5), 3.44 (1H, dd, J ¼ 9:0,
9.0 Hz, H-4), 1.83 (3H, m, 7-CH3); 13C-NMR (125 MHz,
CD3OD) ꢀ (ppm): 155.9 (C-2), 132.7 (C-6), 129.7 (C-7),
80.3 (C-3a), 78.5 (C-7a), 74.8 (C-4), 69.7 (C-5), 19.5
(7-CH3); IR ꢂmax cmꢀ1: 3406, 2924, 1784, 1440, 1365,
1174, 1062.
Materials and Methods
General. Data were obtained with the following
instruments: IR, Hitachi 270-30 Infrared spectropho-
tometer; optical rotation, Jasco DPI-370 polarimeter;
NMR, Bruker AMX-500 FT-NMR and Jeol JNM-EX
270 FT-NMR spectrometers; FD-, EI- and HREI-MS,
Jeol JMS SX-102A mass spectrometer; UV, Hitachi
U-2800 spectrophotometer; CD, Jasco J-720W spectro-
polarimeter.
Preparation of the dibenzoyl derivative (1a). Com-
pound 1 (1.5 mg, 0.01 mmol) and benzoyl chloride
(25 ml, 0.2 mmol) were dissolved in pyridine (1 ml).
The reaction mixture was stirred at room temperature for
Isolation. Lasiodiplodia theobromae OCS71 was
grown statically in fifty 500-ml flasks each containing
200 ml of a 2% potato-dextrose medium at 25 ꢁC in the

2072
R. TAKEI et al.
24 h and then treated with MeOH (1 ml). After removing
the solvent in vacuo, the residue was purified by HPLC
(Inertsil ODS, 20 ꢂ 250 mm; MeOH:H2O = 80:20, v/v,
5 ml/min, A280 nm) to give the dibenzoate of 1 (1a;
3.1 mg, 89%). 1a: EI-MS m=z (rel. int., %): 350[M]þ (2),
229 (6), 228 (20), 105 (100), 77 (22); UV (MeOH) ꢃmax
nm ("): 232 (21700); 1H-NMR (270 MHz, CDCl3) ꢀ
(ppm): 7.98 (4H, m), 7.52 (2H, m), 7.40 (4H, m), 6.70
(1H, m), 6.04 (1H, m), 5.71 (1H, ddd, J ¼ 10:4, 7.8,
4.8 Hz), 3.17 (1H, dd, J ¼ 16:5, 4.8 Hz), 2.75 (1H, dd,
J ¼ 16:5, 10.4 Hz), 1.88 (3H, m).
m, H-2), 5.15 (1H, m, H-3), 4.16 (1H, br d, J ¼ 8:9 Hz,
H-6), 3.38 (1H, m, H-5), 3.30 (1H, m, H-4), 1.81 (3H, m,
H-7), 1.45 [9H, s, C(CH3)3]. Compound 3b: FD-MS
1
m=z, 242 [M]þ; H-NMR (500 MHz, CDCl3) ꢀ (ppm):
5.64 (1H, m, H-2), 5.05 (1H, m, H-6), 4.35 (1H, m, H-3),
3.33 (2H, m, H-4, 5), 1.72 (3H, m, H-7), 1.46 [9H, s,
C(CH3)3].
In vitro conversion of 3 into 2 and 4. Frozen mycelia
of L. theobrome (1.83 g) were ground in liquid nitrogen
and suspended in 20 ml of a 100 mM phosphate buffer
(pH 6.4, 1 mM DTT). After centrifuging the suspension
(13;000 ꢂ g), the resulting supernatant was used for a
cell-free experiment. In vitro conversion was carried
out in a final volume of 6 ml containing the 100 mM
phosphate buffer (pH 6.4), 1 mM theobroxide, and 1 mM
Na2CO3 with or without 1 ml of the cell-free extract.
The reaction mixture was incubated at 25 ꢁC. Seven days
after starting the reaction, the reaction mixture (6 ml)
was extracted with the same volume of EtOAc and then
evaporated. The residue was dissolved in a small amount
of MeOH and subjected to HPLC (Inertsil ODS, 20 ꢂ
250 mm, MeOH:H2O = 10:90, 5 ml/min, A210 nm). The
retention times of the products in this experiment and
of authentic samples were same (2, retention time,
7.5 min: 4, retention time, 7.0 min). The molecular
ion peaks of both products (m=z 186) were observed
by EI-MS.
Preparation of the dimethylaminobenzoyl derivative
(2a). Compound 2 (1 mg, 0.005 mmol) and 4-(dimethyl-
amino)benzoyl chloride (19.9 mg, 0.11 mmol) were
dissolved in pyridine (1 ml). The reaction mixture was
stirred at room temperature for 24 h and then treated
with MeOH (1 ml). After removing the solvent in vacuo,
the residue was purified by HPLC (Inertsil ODS,
20 ꢂ 250 mm; CH3CN:H2O = 70:30, v/v, 5 ml/min,
A280 nm) to give the dibenzoate of 2 (2a; 0.6 mg, 25%).
2a: EI-MS m=z (rel. int., %): 480[M]þ (8), 316 (29), 164
(51), 148 (100), 120 (11); UV (MeOH) ꢃmax nm ("): 316
(37200); 1H-NMR (270 MHz, CDCl3) ꢀ (ppm): 7.83
(4H, m), 6.61 (4H, m), 5.91 (1H, m), 5.68 (1H, dd,
J ¼ 7:3, 7.3 Hz), 5.62 (1H, m), 5.07 (1H, m), 4.98
(1H, dd, J ¼ 7:3, 7.3 Hz), 3.00 (6H, m), 2.99 (6H, m),
1.97 (3H, m).
Chemical conversion of theobroxide (3) into 2 and 4.
Theobroxide
Bioassay. The potato micro-tuber-inducing activity
was assayed by cultures of single–node segments of the
potato stem in vitro as previously described.9)
3 (100 mg, 0.7 mmol) and (Boc)2O
(152.6 mg, 0.7 mmol) were dissolved in CH3CN
(5 ml).7) DMAP (7 mg) was added, and the reaction
mixture was stirred at room temperature for 24 h. After
removing the solvent in vacuo, the residue was purified
by silica gel column chromatography (Kanto Chemical,
silica gel 60N, 20 g; MeOH:CHCl3 = 3:97, v/v) and
HPLC (Inertsil ODS, 20 ꢂ 250 mm; CH3CN:H2O =
30:70, v/v, 8 ml/min, A210 nm) to give 3a (30.0 mg,
0.12 mmol, 18%; retention time, 63.4 min) and 3b
(64.6 mg, 0.27 mmol, 38%; retention time, 65.7 min).
Compound 3a (25.2 mg, 0.10 mmol) and BF3-OEt2/
CH2Cl2 (0.2 mM, 0.5 ml, 0.10 mmol) were dissolved in
CH2Cl2 (3 ml).8) The reaction mixture was stirred at
ꢀ40 ꢁC for 30 min. The reaction solution was poured
into saturated NaHCO3 aq. (6 ml) and successfully
extracted with CH2Cl2 and EtOAc (6 ml ꢂ 3). Each
extract was washed with saturated brine (5 ml ꢂ 1) and
dried over Na2SO4. The EtOAc extract was evaporated
and the resulting residue (5.7 mg) was purified by HPLC
(Inertsil ODS, 20 ꢂ 250 mm; MeOH:H2O = 10:90, v/v,
5 ml/min, A210 nm) to give compound 2 (5.3 mg, 0.03
mmol, 28%). Compound 4 was converted from 3 in the
same manner above (yield, 25%). The specific rotation
Acknowledgments
We thank to Dr. E. Fukushi and Mr. K. Watanabe for
measuring the mass spectral data.
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