Identification of a degradation intermediate of the momilactone A rice phytoalexin by the rice blast fungus

Article abstract of DOI:10.1271/bbb.110756

We have already shown that major rice diterpene phytoalexin, momilactone A, was detoxified by Magnaporthe oryzae. We report here the identification by NMR, MS, and chemical synthesis of 3,6-dioxo-19-nor-9α-pimara-7,15-diene (1) as the degradation intermediate. Compound 1 exhibited similar antifungal activity to that of momilactone A, indicating 1 to be a precursor of possible detoxified compounds.

Full text of DOI:10.1271/bbb.110756

Biosci. Biotechnol. Biochem., 76 (2), 414–416, 2012
Note
Identification of a Degradation Intermediate
of the Momilactone A Rice Phytoalexin by the Rice Blast Fungus
Takuya IMAI,1 Yuko OHASHI,2 Ichiro MITSUHARA,2 Shigemi SEO,2
y
1;
Hiroaki TOSHIMA,1 and Morifumi HASEGAWA
1College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki 300-0393, Japan
2Plant-Microbe Interactions Research Unit, National Institute of Agrobiological Sciences,
Tsukuba, Ibaraki 305-8602, Japan
Received October 11, 2011; Accepted November 6, 2011; Online Publication, February 7, 2012
[doi:10.1271/bbb.110756]
We have already shown that major rice diterpene
phytoalexin, momilactone A, was detoxified by Magna-
porthe oryzae. We report here the identification by
NMR, MS, and chemical synthesis of 3,6-dioxo-19-nor-
9ꢀ-pimara-7,15-diene (1) as the degradation intermedi-
ate. Compound 1 exhibited similar antifungal activity to
that of momilactone A, indicating 1 to be a precursor of
possible detoxified compounds.
except for ꢀ-lactone, had been preserved in the structure
of M286 (Table 1, Fig. 1). A methyl doublet signal at ꢁH
1.21 ppm (H-18), a methine signal at ꢁC 40.55 ppm, and
the absence of a carbonyl carbon signal at around ꢁC
170 ppm suggested a modification to the ꢀ-lactone ring
of momilactone A, the carbonyl carbon of which
appeared at ꢁC 174.25 ppm.6) Two carbonyl signals (ꢁC
198.81 and 212.26 ppm) were observed in the 13C-NMR
data of M286. The carbonyl signal at ꢁC 212.26 ppm was
assigned to a C-3 ketone by HMBC correlations from H-
2/H-18 to C-3. The carbonyl signal at ꢁC 198.81 ppm
was reasonable for an ꢂ,ꢃ-unsaturated ketone, and an
HMBC correlation with H-5 supported the assignment
of this signal as a C-6 ketone. COSY correlation
between H-5 and a methine signal at ꢁH 2.69 ppm
suggested that the methine proton was H-4. The large
J4;5 coupling (10.7 Hz) should represent an anti relation-
ship between H-4 and H-5. NOESY cross-peaks be-
tween H-4 and H-20, and between H-5 and H-18
supported this configuration. M286 was thus identified
as 3,6-dioxo-19-nor-9ꢃ-pimara-7,15-diene (Fig. 1, 1).
Compound 1 was chemically synthesized from mo-
milactone A to confirm the spectroscopic elucidation of
the structure. 6ꢃ-Hydroxy-3-oxo-19-nor-9ꢃ-pimara-
7,15-diene (2) has already been reported as a decarbo-
xylation product of momilactone A which was desig-
nated as 19-nor-MA in the original report.7) Compound
2 was converted to 1 by Dess-Martin oxidation. The
identity of compound 1 obtained by the chemical
synthesis and by fungal biotransformation was con-
firmed by a comparison of the NMR, MS, tR in GC/MS,
and specific rotation data.
Key words: phytoalexin; rice; blast fungus; detoxifica-
tion; momilactone
Phytoalexins are low-molecular-weight antimicrobial
compounds which accumulate in plants invaded by
pathogenic microorganisms.1,2) The accumulation of
phytoalexins is recognized as one of the mechanisms
for the induced resistance of higher plants against
pathogens. However, pathogenic microorganisms have
the ability to avoid the resistance mechanisms of plants.
The detoxification of phytoalexins is an effective solu-
tion to evade the growth inhibition of pathogenic
microorganisms caused by phytoalexins.3,4)
We have recently reported that momilactone A
(Fig. 1), a major diterpene phytoalexin, was detoxified
by the rice blast fungus, Magnaporthe oryzae, and that a
possible metabolite of momilactone A was maximized
after 4 h and then lost after 8 h in a fungal suspension
culture containing momilactone A.5) We therefore tried
to purify the metabolite and elucidate its structure. A
GC/MS analysis showed the metabolite to exhibit a
plausible molecular ion at m=z 286, so we have
designated it as M286. Fractionation guided by a GC/
MS analysis, using solvent extraction and ODS-HPLC
separation, gave 1.32 mg of M286 from two batches of a
blast fungal suspension culture (80 mL) 4 h after adding
5 mg of momilactone A.
Compound 1 and momilactone A were extracted after
different incubation times from a rice blast fungus
suspension culture containing 300 mM (ca. 100 ppm)
momilactone A, and were quantified by LC/MS/MS.
The results are summarized in Fig. 2. The level of
momilactone A rapidly decreased up to 8 h after the
addition of momilactone A, and then gradually decreased
up to 24 h. The level of 1 reached a maximum 4 h after
the addition of momilactone A. The concentration of 1
at the maximum level was approximately 70 mM, this
being approximately 1/4 of the initial concentration of
momilactone A. However, the level of momilactone A
The high-resolution mass spectrum of M286 gave
a molecular weight of 286.1909, agreeing with the
molecular formula C19H26O2 (calcd. as 286.1933). This
formula indicated the loss of one carbon and one oxygen
1
from momilactone A (C20H26O3). H- and 13C-NMR,
1
COSY, HSQC, HMBC and a comparison of H- and
13C-NMR data with those of momilactone A6) suggested
that the original carbon skeleton of momilactone A,
y
To whom correspondence should be addressed. Fax: +81-29-888-8525; E-mail: morifumi@mx.ibaraki.ac.jp
Abbreviations: SRM, selected reaction monitoring; PDB, potato dextrose broth

Degradation Intermediate of Momilactone A by M. oryzae
415
Fig. 1. Structures of 3,6-Dioxo-19-nor-9ꢃ-pimara-7,15-diene (1) and Momilactone A, and COSY, Selected HMBC, and Selected NOESY
Correlations for 1.
COSY correlations are represented by bold lines, HMBC correlations are represented by single-headed arrows from H to C, and NOESY
correlations are represented by double-headed arrows. COSY correlations between H-9 and H-11 were not clearly observed because of the
overlapping signals.
Table 1. 13C–1H-NMR Data for 3,6-Dioxo-19-nor-9ꢃ-pimara-7,15-
diene (1)
maximal level for 1 h, and then decreased to almost the
zero level 8 h after the addition of momilactone A. This
result indicates that 1 must have been rapidly converted
to other compounds by the rice blast fungus. The
identification of the converted products is one objective
of our future research.
The inhibitory activity against spore germination of
the rice blast fungus was almost the same as that of
momilactone A. IC50values for 1 and momilactone A
were 0.50 mM and 0.54 mM, respectively. We had
concluded in our previous report5) that the rice blast
fungus could detoxify the phytoalexin, momilactone A,
because the antifungal activity of a rice blast fungus
suspension culture became lower after 8 h of incubation
with momilactone A. This result suggested that 1 could
be an intermediate in the detoxification pathway for
momilactone A, and that further conversion of 1 would
be necessary for detoxification.
Position
1
ꢁC
ꢁH (multiplicity, J in Hz)
35.69
1.63 (1H, m, overlapped)
2.01 (1H, m, overlapped)
2.40 (1H, ddd, 14.8, 4.3, 2.8)
2.55 (1H, dddd, 14.8, 14.8, 5.4, 0.9)
—
2
36.95
3
4
212.26
40.55
55.44
198.81
124.25
160.42
51.61
38.19
23.76
2.69 (1H, dqd, 10.7, 6.4, 0.9)
2.45 (1H, d, 10.7)
—
5
6
7
5.82 (1H, br s)
—
8
9
1.97 (1H, m, overlapped)
—
10
11
1.61 (1H, m, overlapped)
1.88 (1H, m)
12
13
14
36.83
40.94
47.58
1.63 (2H, m, overlapped)
—
2.13 (1H, dd, 12.0, 2.2)
2.28 (1H, br d, 12.0)
5.86 (1H, dd, 17.5, 10.7)
4.97 (1H, dd, 10.7, 1.0)
5.01 (1H, dd, 17.5, 1.0)
0.95 (3H, s)
15
16
148.49
110.51
Experimental
General analytical methods. 1H-NMR (400.13 MHz), 13C-NMR
(100.61 MHz) and 2D-NMR spectra were measured in CDCl3 with
TMS as an internal standard by an AVANCE III FT-NMR spectrom-
eter (Bruker BioSpin) equipped with a 5-mm BBFO probe. The IR
spectrum was measured as a KBr pellet by an FT/IR-4100 spectrom-
eter (Jasco) and the UV spectrum was measured in MeOH by a V-550
spectrometer (Jasco). The specific rotation was measured in CHCl3 by
a P-2100 polarimeter equipped with a halogen lamp and a 589 nm filter
(Jasco). The mass spectra were measured by a JMS-BU25 (GCmate II)
mass spectrometer (Jeol) in the direct inlet EI mode (70 eV).
Chemical. Momilactone A was purified from rice husks according
to the method reported by Kato et al.8) with some modifications.
Fungal material. The rice blast fungus (Magnaporthe oryzae strain
P-2) had been maintained on potato dextrose agar (Nissui Pharma-
ceutical) as a stock culture in our laboratory. A small piece of this
stock culture was inoculated and grown on potato dextrose agar or
oatmeal agar in a Petri dish (9 mm in diameter) at 26 ꢀC in the dark
before inoculating into the suspension culture.
17
18
20
22.05
14.32
20.52
1.21 (3H, d, 6.4)
1.15 (3H, s)
ꢁH values for the overlapped signals were estimated by an HSQC
experiment.
decreased to approximately 2/3 of its initial level after
4 h of incubation. Compound 1 could not be detected
in a suspension culture without momilactone A, in a
suspension culture heated in boiling water, or in a
culture without the fungus (data not shown). These
results suggest that 1 would have been one of the major
intermediates during the fungal degradation of momi-
lactone A. The accumulation of 1 remained at the

416
T. IMAI et al.
82%). NMR and MS data were identical to those of 1 which had been
purified from an M. oryzae culture containing momilactone A. 1. UV
ꢄmax (MeOH) nm ("): 243 (14,300); IR ꢅ~max (KBr) cmꢂ1: 1712, 1673;
26
½ꢂꢃD ꢂ254ꢀ (c 0.41, CHCl3).
Momilactone A
Quantification of 3,6-dioxo-19-nor-9ꢃ-pimara-7,15-diene (1) in a
rice blast fungus culture containing momilactone A. The fungal layers
on an oatmeal medium were cut into squares of approximately 1 mm2.
The small pieces were suspended in a PDB liquid medium (150 mL)
and then incubated for 6 d in the dark at 27 ꢀC with rotary shaking at
150 rpm. A portion of the culture (30 mL) was transferred to a fresh
PDB medium (120 mL) and further incubated for 1 d under the same
conditions. Approximately ten fungal clusters were collected from the
culture and transferred to a fresh PDB medium (1 mL), to which 20 mM
momilactone A in MeOH (15 mL) was added. The medium was
incubated for 1–24 h at 27 ꢀC with reciprocal shaking at 200 strokes per
min. MeOH (2.3 mL) was added to the culture after this incubation. A
portion of the supernatant (30 mL) was diluted with MeOH (0.87 mL),
and 5 mL of the solution was subjected to an LC/MS/MS analysis to
quantify 1 and momilactone A. The LC/MS/MS analysis was
performed with an AB SCIEX 3200 QTRAP LC/MS/MS system
coupled with a Shimadzu Prominence UFLC system. Liquid chromato-
graphic separation of the analytes was achieved in a Shim-pack XR-
ODS column (2.0 mm i.d. ꢁ 30 mm, 2.2 mm particle size; Shimadzu)
with a linear binary gradient of 0.1% (v/v) aqueous acetic acid
containing 10 mM ammonium acetate (solvent A) and MeOH (solvent
B) at a flow rate of 0.2 mL minꢂ1 at 40 ꢀC. The graded solvent was
programmed as follows: initial, 20% B; 0–5 min, a linear gradient from
20% B to 100% B; 5–7 min, isocratic elution by 100% B. The
equilibration time between two runs was 3 min. The parameters of the
mass spectrometer were optimized for detecting 1 and momilactone A
by using Analyst 1.5.1 software (AB SCIEX) for selective reaction
monitoring (SRM). The ion source (Turbo V) was operated in the
positive ESI mode. The following SRM transitions were monitored: 1,
287!175; momilactone A, 315!271. Calibration curves were
obtained from the SRM peak areas of standards for 1 and momilactone
A in respective concentration ranges of 10–10000 ng mLꢂ1 and 1–
Compound 1
Fig. 2. Time-Dependent Accumulation of 3,6-Dioxo-19-nor-9ꢃ-
pimara-7,15-diene (1) and Degradation of Momilactone A in a Rice
Blast Fungus Suspension Culture Containing Momilactone A.
Compound 1 and momilactone A were extracted from the rice
blast fungus suspension culture at different times after adding
momilactone A. Their concentrations were quantified by using LC/
MS/MS. Values are presented as the mean ꢄ SD (n ¼ 3). The
concentrations of 1 and momilactone A are respectively represented
by circles and triangles.
Purification of 3,6-dioxo-19-nor-9ꢃ-pimara-7,15-diene (1) from the
rice blast fungus suspension culture containing momilactone A. The
mycelia of rice blast fungus grown on potato dextrose agar were
inoculated into 80 mL of a potato dextrose broth medium (PDB;
Sigma-Aldrich). After the suspension culture had been incubated at
27 ꢀC for 2 months in the dark while shaking at 150 rpm, 5 mg of
momilactone A (1 mL of an MeOH solution) was added to the culture.
After a further 4 h of incubation, 373 mL of MeOH was added to the
culture. The aqueous MeOH suspension was heated in a boiling water
bath for 5 min. The suspension was filtered through cotton to remove
the mycelia, and the resulting filtrate was evaporated in vacuo. After
adding 100 mL of distilled water to the residue, the aqueous solution
was extracted with EtOAc (100 mL ꢁ 3). The EtOAc extract was
evaporated in vacuo to dryness to give a crude extract (ca. 25 mg). A
170 mL amount of a 10,000 ppm MeOH solution of the crude extract
was repeatedly separated by ODS-HPLC under the following con-
ditions: column, Cosmosil 5C18AR (1 cm i.d. ꢁ 25 cm, 5 mm particle
size; Nacalai Tesque); solvent, 50% (v/v) aq. MeCN; flow rate,
2.0 mL minꢂ1; column oven temperature, 40 ꢀC; detection, UV 210 nm.
Compound 1 was eluted immediately after momilactone A (tR
44.0 min) as an isolated peak at tR 46.9 min. The elution of compound
1 was confirmed by a GC/MS analysis under the previously described
conditions.5) The foregoing procedure was conducted twice to purify
compound 1 (1.32 mg). 1. HRMS m=z (Mþ): calcd. for C19H26O2,
286.1933; found, 286.1909; NMR: see Table 1; EIMS m=z (rel. int.):
81 (72), 91 (59), 105 (55), 119 (53), 147 (71), 203 (74), 215 (39),
1000 ng mLꢂ1
.
Assay of the inhibitory activity against spore germination of the rice
blast fungus. A spore suspension of the rice blast fungus (strain P-2)
was prepared by the method described previously.9) The antifungal
activity was evaluated by using the spore suspension according to the
previously reported method.10)
Acknowledgment
This work was supported by JSPS KAKENHI
(22580113).
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