Synthesis and biotransformation of plausible biosynthetic intermediates of salicylaldehyde-type phytotoxins of rice blast fungus, Magnaporthe grisea

Article abstract of DOI:10.1002/ejoc.201100771

Rice blast fungus, Magnaporthe grisea, produces a series of salicylaldehyde-type phytotoxins such as pyriculol, pyricuol, and pyriculariol. Plausible biosynthetic intermediates of these phytotoxins were synthesized in deuterium labeled forms using the Sti

Full text of DOI:10.1002/ejoc.201100771

FULL PAPER
DOI: 10.1002/ejoc.201100771
Synthesis and Biotransformation of Plausible Biosynthetic Intermediates of
Salicylaldehyde-Type Phytotoxins of Rice Blast Fungus, Magnaporthe grisea
Koji Tanaka,*[a] Ayaka Sasaki,*[a] Hai-Qun Cao,[a,b] Teiko Yamada,[a] Masahiro Igarashi,[a]
Isao Komine,[a] Hirokazu Nakahigashi,[a] Naoko Minami,[a] Shigefumi Kuwahara,[a]
Manabu Nukina,[c] and Hiromasa Kiyota*[a]
Keywords: Phytochemistry / Biosynthesis / Isotopic labeling / Rice blast disease
Rice blast fungus, Magnaporthe grisea, produces a series of
salicylaldehyde-type phytotoxins such as pyriculol, pyricuol,
and pyriculariol. Plausible biosynthetic intermediates of
these phytotoxins were synthesized in deuterium labeled
forms using the Stille coupling reaction as the key step, and
subjected to biotransformation by the fungus. Heptatrienyl-
salicylaldehyde was converted into dihydropyriculol by the
fungus by a shaking culture. A new biosynthetic pathway
comprising both oxidation and reduction is proposed.
tatrienylsalicyl alcohol (7) is an important biosynthetic in-
termediate of salicylaldehyde-type polyketide synthesis,[8,12]
because the fungus could oxidize (Z)- and (E)-1-phenylpro-
penes to racemic erythro-diol and optically active threo-diol,
respectively.[13] We also assumed that heptatrienylsalicylal-
dehyde (8) could be an intermediate, because Iwasaki et al.
reported that aldehyde 2 was converted to alcohol 1 by the
fungus.[14] We began the biosynthetic studies using possible
labeled intermediates. In this paper, synthesis and biotrans-
formation of deuterium labeled 7 and 8 are described.
Introduction
Rice blast fungus Magnaporthe grisea (Hebert) Barr. (the
perfect stage of Pyricularia oryzae Cavara), which causes
rice blast disease, is the most serious pest of rice.[1] The
fungus produces several main phytotoxic compounds ac-
cording to the fermentation method, i.e. (12R,13S)-di-
hydropyriculol (1),[2] (12R,13S)-pyriculol (2),[3] and pyr-
iculone (3)[4] by a shaking culture, racemic dihydropyricular-
iol (4)[6] and pyriculariol (5)[7] by an aeration and agitation
culture, and tenuazonic acid by a stationary culture in a soy
sauce–sucrose medium (Scheme 1). (R)-Pyricuol (6)[5] to-
gether with 2 have been produced by a shaking culture with
a potato dextrose medium. These salicylaldehyde-type phy-
totoxins are common metabolites of blast fungi of various
host plants.[8] To develop new methods to control the blast
disease a better knowledge of its biosynthesis is necessary.
Although a number of gene clusters of the fungus poten-
tially involved in pathogenicity have been clarified, that en-
coding the biosynthesis of these toxins has not yet been
specified.[9] Little is known about the biosynthesis of these
phytotoxic compounds (Scheme 1), except that they are de-
rived from seven units of acetyl CoA like other salicylalde-
hyde-type congeners.[10,11] Nukina et al. suggested that hep-
[a] Laboratory of Applied Bioorganic Chemistry, Graduate School
of Agricultural Science, Tohoku University,
1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan
Fax: +81-22-717-8785
E-mail: kiyota@biochem.tohoku.ac.jp
[b] College of Plant Protection, Anhui Agricultural University,
130 Changjiang West Rd., Hefei 230036, P. R. China
Fax: +86-551-5786790
E-mail: caohaiqun@ahau.edu.cn
[c] Faculty of Agriculture, Yamagata University,
1-23 Wakaba, Tsuruoka 997-8555, Japan
Fax: +81-235-28-2867
Scheme 1. Salicylaldehyde-type phytotoxins from rice blast fungus,
Magnaporthe grisea.
E-Mail: mnukina@tds1.tr.yamagata-u.ac.jp
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Biosynthesis of Rice Blast Fungus Phytotoxin Intermediates
sucrose medium) of M. grisea and the culture was incu-
bated using a reciprocal shaker for 1 d at 24 °C. Each broth
was filtered, the filtrate extracted into EtOAc, and the ex-
tract analyzed by liquid chromatography– mass spectrome-
try (LC–MS). A substantial amount of 1 was detected in
each extract (A: +EtOAc, B: +7-d2 in EtOAc, C: +8-d in
EtOAc, and D: control). The LC–MS [ESI(+)] showed that
extract B contained only unlabeled 1, which is quite similar
to that of extracts A and D, but almost equal amounts of
unlabeled and labeled 1 and 1-d were detected in extract C
(Figure 1). The presence of EtOAc and 7-d2 did not affect
the pattern of fermentation products.
Results and Discussion
Synthesis of Substrates
Synthesis of the side chain fragment started from the
known dibromide 9[15] (Scheme 2), which was prepared
from sorbic acid in three steps. Treatment of 9 with
NaHMDS afforded the bromo-alkyne 10, which was sub-
jected to palladium-catalyzed hydrostannylation[16] to give
(E)-stannane 12 accompanied by a small amount of regio-
and stereoisomers. Hydrostannylation of the corresponding
terminal alkyne 11 was not reproducible. The Stille cou-
pling reaction with the known triflate 13[17,18] successfully
furnished unstable lactone 14. The carbonyl moiety of 14
[19]
was reduced with LiAlH4
at –78 °C to give 7 in 43%
yield. In addition, half-reduced 8 was also obtained in 34%
yield. For biotransformation the corresponding α-deuterio
derivatives 7-d2 and 8-d were prepared in a similar manner
using LiAlD4.
Figure 1. Regions of LC–MS [ESI(+)] of extracts A (+EtOAc), B
(+7-d2 in EtOAc), and C (+8-d in EtOAc).
1
The production of 1 and 1-d was also confirmed by H
NMR analysis of extract C. A pair of broad singlets (δH
=
5.01 ppm, 1 H for 1; and δH = 4.99 ppm, 0.5 H for 1-d) were
observed. These results indicate that 8 is the most possible
biosynthetic intermediate for the phytotoxins. Thus, the di-
hydro derivatives 1 and 4 are produced at the later stages
by the reduction of 2 and 5, respectively. Although there
still remains the possibility that 7-d2 was too polar to per-
meate the cell membrane, it did not seem to inhibit the bio-
synthetic pathway. New biosynthetic pathways are proposed
in Schemes 3 and 4. When the fungus is fermented by a
shaking culture, epoxidation of the 10,12-double bond of 8
occurs to give (10R,11S)-15 and/or (10S,11R)-15
(Scheme 3). For (10R,11S)-15, after the formation of 2 by
hydration of the epoxy ring at C-11 (route a), reduction and
oxidation afford 1 and 3, respectively. (10S,11R)-15 can also
furnish 2 by route aЈ by hydration at C-10. Thus, the bio-
synthetic conversion could be performed by way of stereo-
Scheme 2. Synthetic scheme of plausible biosynthetic intermediates
(dba = dibenzylideneacetone). a) NaHMDS, THF –78 °C (80% for
10). b) BuLi, THF, –78 °C. c) Bu3SnH, PPh3, Pd2dba3, THF. d)
P(2-furyl)3, Pd2dba3, LiCl, N,N-dimethylformamide (DMF, 35%
from 10). e) LiAlH4, THF, –78 °C (43% for 7 and 34% for 8). f)
LiAlD4, THF, –78 °C (36% for 7-d2 and 20% for 8-d).
Biotransformation of 7-d2 and 8-d by Magnaporthe grisea
Compounds 7-d2 and 8-d were each subjected to bio- selective epoxidation followed by regioselective hydration,
transformation. A solution of each substrate in ethyl acetate and/or nonstereoselective epoxidation followed by S-carbon
(EtOAc) was introduced into the culture broth (soy sauce– specific hydration. Our stereoselective synthesis of 6[12]
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K. Tanaka, A. Sasaki, H. Kiyota et al.
FULL PAPER
showed that it is presumably formed not from 2 (through and hydration occur in a nonselective manner. Reduction
pinacol rearrangement) but from (10R,11S)- and/or of the formyl group leads to 4. Both oxidation and re-
(10S,11R)-15 by epoxide rearrangement followed by re- duction would occur during the later stage of the polyketide
duction when the fungus is fermented in a potato dextrose synthesis.
medium (route b and/or bЈ).
It is often the case with fungi that they produce different
secondary metabolites under different fermentation condi-
tions. The rice blast fungus also has the ability to produce
various metabolites according to natural nutritional status.
It would be interesting to know the relationships between
the order of phytotoxicity (3 Ͼ 6 Ͼ 2 Ͼ 5) and the environ-
mental conditions that produce them. It is somewhat curi-
ous that both oxidation and reduction occur during the
later stage of the biosynthesis. However, oxidation from sal-
icylalcohols to salicylaldehydes is restricted. Further syn-
thetic and fermentative studies along with genetic analysis
will clarify the whole biosynthetic pathway of these salicyl-
aldehyde-type phytotoxins.
Conclusions
Plausible biosynthetic intermediates for the salicylalde-
hyde-type phytotoxins of the rice blast fungus were synthe-
sized using the Stille coupling reaction as the key step. Deu-
terium-labeled heptatrienylsalicylaldehyde (8-d) was bio-
transformed into deuteriodihydropyriculol by the fungus,
and a new biosynthetic pathway containing both oxidation
and reduction is proposed.
Experimental Section
General: FTIR spectra were recorded as films with a Jasco 4100
spectrometer (ATR, Zn-Se). 1H and 13C NMR spectra were re-
1
corded with Varian Inova 600 (600 MHz for H and 150 MHz for
13C) and Varian Inova 500 (500 MHz for 1H and 125 MHz for 13C)
spectrometers with tetramethylsilane (δ = 0 ppm for 1H) and CDCl3
(δ = 77.0 ppm for 13C) as internal standards. Mass spectra were
recorded with a Jeol JMS–700 spectrometer. LC–MS data were re-
corded with a Thermo-LCQ Deca system. Merck silica gel 60 (70–
230 mesh) was used for column chromatography.
Scheme 3. A newly proposed biosynthetic pathway for the salicylal-
dehyde-type phytotoxins (shaking culture).
(3E,5E)-1-Bromohepta-3,5-dien-1-yne (10): To a solution of 9
(4.12 g, 16.4 mmol) in dry THF (80 mL) was added NaHMDS
(1.07 m in hexane, 25.0 mL, 26.8 mmol) at –78 °C, and the mixture
was stirred at this temperature for 1 h. The reaction was quenched
with sat. aq. NH4Cl solution and extracted into pentane. The or-
ganic layer was washed with H2O and brine, dried with MgSO4,
and concentrated in vacuo. The residue was chromatographed on
silica gel. Elution with pentane/Et2O (100:1) gave 10 (2.25 g,
13.2 mmol, 80%) as a yellow oil. Rf = 0.80 (SiO2, hexane/EtOAc
= 10:1). FTIR (ATR): ν = 3024, 2962, 2934, 2911, 2870, 2849, 2189
˜
(CϵC), 2157 (CϵC) 1641, 1445, 1291, 980 (C–Br), 928 cm–1. 1H
NMR (500 MHz, CDCl3): δ = 1.79 (d, J = 7.3 Hz, 3 H, 7-H), 5.45
(d, J = 15.7 Hz, 1 H, 3-H), 5.85 (dq, J = 15.1, 7.3 Hz, 1 H, 6-H),
6.08 (dd, J = 15.1, 10.7 Hz, 1 H, 5-H), 6.60 (dd, J = 15.7, 10.7 Hz,
1 H, 4-H) ppm. 13C NMR (125 MHz, CDCl3): δ = 18.3 (C-7), 50.3,
Scheme 4. A newly proposed biosynthetic pathway for the salicyl-
aldehyde-type phytotoxins (aeration and agitation culture).
On the other hand, racemic pyriculariol (5)[7c] is formed 79.6, 107.8, 130.6, 133.5, 143.6 ppm. EI-MS: m/z
by an aeration and agitation culture (Scheme 4). If trans-
epoxide 16 is produced in enantiomerically pure form, com-
pletely non-regioselective hydration is necessary to give ra-
= 172
[M(81Br)]+·, 170 [M(79Br)]+·, 91 [M – Br]+, 89, 65, 39. HRMS (EI):
calcd. for C7H779Br [M(79Br)]+· 169.9726; found 169.9732.
(1E,3E,5E)-Tributylhepta-1,3,5-trienylstannane (12): To a solution
cemic 5. Thus, it is rather plausible that both epoxidation of 10 (80 mg, 0.47 mmol), PPh3 (10 mg, 0.04 mmol), and Pd2dba3
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Biosynthesis of Rice Blast Fungus Phytotoxin Intermediates
(3.7 mg, 0.0040 mmol) in dry THF (5 mL) was added Bu3SnH
(314 mg, 1.08 mmol) dropwise and the mixture was stirred at 20 °C
for 0.5 h. The mixture was filtered through a Celite pad, and the
filtrate was concentrated in vacuo to give 12 as a dark brown oil.
1H NMR (500 MHz, CDCl3): δ = 0.78–1.03 (m, 15 H, 1Ј, 4Ј-H),
1.25–1.40 (m, 6 H, 3Ј-H), 1.45–1.55 (m, 6 H, 2Ј-H), 1.78 (d, J =
6.7 Hz, 3 H, 7-H), 5.73 (dq, J = 14.9, 6.7 Hz, 1 H, 6-H), 6.03–6.13
(m, 3 H), 6.20 (d, J = 18.6 Hz, 1 H, 1-H), 6.56 (dd, J = 18.6, 9.3 Hz,
1 H, 2-H) ppm. Crude 12 was used in the next step without further
purification.
[M – H2O – CH3]+, 149, 148, 91, 84, 77, 39, 18. HRMS (EI): calcd.
for C14H13DO2 [M]+· 215.1052; found 215.1052.
(1ЈE,3ЈE,5ЈE)-6-(Hepta-1Ј,3Ј,5Ј-trienyl)-2-hydroxybenzenemethanol
(7) and (1ЈE,3ЈE,5ЈE)-6-(Hepta-1Ј,3Ј,5Ј-trienyl)-2-hydroxybenzalde-
hyde (8): In the same manner as described for the deuterio deriva-
tives, reduction of 12 with LiAlH4 furnished alcohol 7 (43%) and
aldehyde 8 (34%). 7: FTIR (ATR): ν = 3382 (br., O–H), 3012,
˜
2958, 2923, 2852, 1712, 1593, 1577, 1464, 1362, 1258, 1224, 1193,
1000, 981, 961, 912, 801, 769, 735 cm–1. 1H NMR (500 MHz,
CDCl3): δ = 1.81 (d, J = 6.8 Hz, 3 H, 7Ј-H), 2.30 (br. s, 1 H,
CH2OH), 4.99 (s, 2 H, α-H), 5.79 (dq, J = 15.1, 6.8 Hz, 1 H, 6Ј-
H), 6.16 (pseudo dd, J = 15.1, 10.3 Hz, 1 H, 5Ј-H), 6.25 (dd, J =
15.1, 6.8 Hz, 1 H, 3Ј-H), 6.32 (dd, J = 14.8, 9.8 Hz, 1 H, 4Ј-H),
6.60–6.67 (m, 2 H, 2Ј,1Ј-H), 6.76 (d, J = 7.8 Hz, 1 H, 3-H), 7.02
(d, J = 7.8 Hz, 1 H, 5-H), 7.14 (t, J = 7.8 Hz, 1 H, 4-H), 7.55 (br.
(1ЈE,3ЈE,5ЈE)-6-(Hepta-1Ј,3Ј,5Ј-trienyl)-2,2-dimethyl-4H-1,3-benzo-
dioxin-4-one (14): To a solution of Pd2dba3 (15 mg, 0.017 mmol),
LiCl (101 mg, 2.38 mmol), P(2-furyl)3 (9.9 mg, 0.043 mmol), and
13 (383 mg, 1.17 mmol) in dry DMF (5 mL) was added a solution
of crude 12 (63 mg) in dry DMF (7.5 mL), and the mixture was
stirred at 20 °C for 36 h. After addition of KF (1 g), the mixture
was extracted with EtOAc. The organic layer was washed with H2O
and brine, dried with MgSO4, and concentrated in vacuo. The resi-
due was chromatographed on silica gel. Elution with hexane/EtOAc
(100:1 to 25:1) gave 14 (44.8 mg, 0.166 mmol, 35% from 10) as a
1
s, 1 H, ArOH) ppm. H NMR (600 MHz, CD2Cl2): δ = 1.80 (d, J
= 6.7 Hz, 3 H, 7Ј-H), 2.30 (br. s, 1 H, CH2OH), 5.00 (s, 2 H, α-H),
5.81 (dq, J = 14.7, 6.7 Hz, 1 H, 6Ј-H), 6.16 (ddd, J = 14.7, 10.2,
1.2 Hz, 1 H, 5Ј-H), 6.29 (dd, J = 15.0, 7.3 Hz, 1 H, 3Ј-H), 6.33 (dd,
J = 15.0, 10.2 Hz, 1 H, 4Ј-H), 6.64–6.70 (m, 2 H, 1Ј,2Ј-H), 6.74 (d,
J = 8.4 Hz, 1 H, 3-H), 7.04 (d, J = 7.8 Hz, 1 H, 5-H), 7.14 (t, J =
7.8 Hz, 1 H, 4-H), 7.60 (br. s, 1 H, ArOH) ppm. 13C NMR
(150 MHz, CD2Cl2): δ = 18.5 (C-7Ј), 60.4 (C-α), 115.8 (C-3), 118.1
(C-5), 122.1 (C-1), 127.9 (C-1Ј), 129.2 (C-4), 130.6 (C-3Ј), 131.4 (C-
6Ј), 131.9 (C-5Ј), 132.8 (C-2Ј), 134.7 (C-4Ј), 137.1 (C-6), 157.1 (C-
2) ppm. MS (EI): m/z = 216 [M]+·, 198 [M – H2O]+, 183 [M –
H2O – CH3]+, 165, 149, 144, 128, 115, 91, 81, 76, 54, 40. HRMS
(EI): calcd. for C14H16O2 [M]+· 216.1145; found 216.1149. 8: color-
colorless oil. FTIR (ATR): ν = 3071, 2997, 2938, 2853, 1727
˜
(C=O), 1572, 1474, 1379, 1318, 1269, 1251, 1207, 1080, 1044, 998,
967, 925, 690 cm–1. 1H NMR (500 MHz, CDCl3): δ = 1.71 (s, 6 H,
gem-Me2), 1.81 (d, J = 6.9 Hz, 3 H, 7Ј-H), 5.81 (dq, J = 15.1,
6.8 Hz, 1 H), 6.17 (dd, J = 15.1, 7.8 Hz, 1 H), 6.33–6.42 (m, 2 H),
6.79–6.87 (m, 2 H), 7.33 (d, J = 7.8 Hz, 1 H), 7.42 (dd, J = 8.3,
7.8 Hz, 1 H), 7.69 (d, J = 15.6 Hz, 1 H, 1Ј-H) ppm. Lactone 14 was
unstable and used in the next steps without further purification.
less crystals. FTIR (ATR): ν = 3015, 2955, 2926, 2854 (H–CO),
˜
1733, 1645 (C=O), 1601, 1456, 1331, 1311, 1236, 997, 772 cm–1. 1H
NMR (500 MHz, CDCl3): δ = 1.82 (d, J = 6.8 Hz, 3 H, 7Ј-H), 5.86
(dq, J = 14.6, 6.8 Hz, 1 H, 6Ј-H), 6.14–6.20 (m, 1 H), 6.30 (dd, J
= 15.1, 10.7 Hz, 1 H), 6.40 (dd, J = 15.1, 10.7 Hz, 1 H), 6.70 (dd,
J = 15.1, 10.7 Hz, 1 H), 6.84 (d, J = 8.3 Hz, 1 H), 6.995 (d, J =
8.8 Hz, 1 H), 7.00 (d, J = 15.1 Hz, 1 H, 1Ј-H), 7.44 (dd, J = 8.3,
7.8 Hz, 1 H, 4-H), 10.34 (s, 1 H, CHO), 11.93 (s, 1 H, OH) ppm.
(1ЈE,3ЈE,5ЈE)-6-(Hepta-1Ј,3Ј,5Ј-trienyl)-2-hydroxybenzenemethanol-
α,α-d2 (7-d2) and (1ЈE,3ЈE,5ЈE)-6-(Hepta-1Ј,3Ј,5Ј-trienyl)-2-hy-
droxybenzaldehyde-α-d (8-d): To a suspension of LiAlD4 (38 mg,
0.91 mmol) in dry THF (6 mL) was added a solution of 14 (61 mg,
0.23 mmol) in dry THF (6 mL) at –78 °C and the mixture was
stirred at this temperature for 3 h. A mixture of 0.1 m aq. HCl/
MeOH was added, and the resulting mixture was diluted with Et2O.
The organic layer was washed with H2O and brine, dried with
MgSO4, and concentrated in vacuo. The residue was chromato-
graphed on silica gel. Elution with hexane/EtOAc (4:1) gave 7-d2
(18 mg, 0.082 mmol, 36%) and 8-d (9.7 mg, 0.045 mmol, 20%). 7-
Biotransformation: The fungus M. grisea, stored at 10 °C on a slant
agar culture, was cultured in darkness at 24 °C on a reciprocal
shaker, using 500 mL Sakaguchi flasks each containing 100 mL of
soy sauce–sucrose medium. The medium contained 50 mL of com-
mercial soy sauce and 50 g of sucrose in 1 liter of tap water and
0.01% silicone antifoaming agent. On the sixth day of the culture,
when the growth of the fungus had almost reached its maximal
stage, a solution of (A) EtOAc (100 μL), (B) 7-d2 (7.0 mg) in EtOAc
(100 μL), (C) 8-d (9.7 mg) in EtOAc (100 μL), or (D) nothing was
introduced into each flask with a sterile microsyringe. Cultivation
was continued for a further day under the same conditions. Each
culture broth was filtered, and the filtrates were independently and
repeatedly extracted into EtOAc. Each extract was washed with
H2O and brine, dried with Na2SO4, and concentrated in vacuo to
give crude extracts (A) 13.6 mg, (B) 21.7 mg, (C) 16.9 mg, and (D)
19.8 mg. Each residue was diluted with MeOH and analyzed with
LC–MS (L-column2 ODS, Φ = 4.6ϫ150 mm, eluting solvent: a
linear gradient of MeOH/H2O from 95:5 to 100:0 in 15 min at a
flow rate of 1.0 mL/min). Compound 1 eluted at 10.6 min, ESI-MS
d : colorless crystals. FTIR (ATR): ν = 3200 (br., O–H), 3011,
˜
2
2959, 2927, 2852, 1721, 1591, 1577, 1464, 1284, 1079, 1035, 997,
1
945, 791, 743 cm–1. H NMR (500 MHz, CDCl3): δ = 1.81 (d, J =
6.8 Hz, 3 H, 7Ј-H), 2.21 (br. s, 1 H, CD2OH), 5.79 (dq, J = 14.7,
6.8 Hz, 1 H, 6Ј-H), 6.17 (dd, J = 14.7, 11.3 Hz, 1 H), 6.25 (dd, J
= 15.1, 7.3 Hz, 1 H), 6.32 (dd, J = 15.1, 9.7 Hz, 1 H), 6.60–6.67
(m, 2 H), 6.77 (d, J = 8.3 Hz, 1 H), 7.03 (d, J = 7.8 Hz, 1 H), 7.14
(dd, J = 8.3, 7.8 Hz, 1 H, 4-H), 7.5 (s, 1 H, ArOH) ppm. 13C NMR
(125 MHz, CDCl3): δ = 18.4 (C-7Ј), 115.6, 118.1, 127.5, 129.0,
130.2, 131.0, 131.6, 132.7, 134.4, 156.9 (C-2) ppm. MS (EI): m/z =
218 [M]+·, 200 [M – H2O]+, 185 [M – H2O – CH3]+, 167, 149,
146, 117, 68, 54, 31, 27. HRMS (EI): calcd. for C14H16D2O2 [M]+·
218.1270; found 218.1270. 8-d: colorless crystals. FTIR (ATR): ν
˜
= 3019, 2905 (D–CO), 1637 (C=O), 1598, 1456, 1339, 1311, 1242,
1210, 1177, 991, 784, 739, 714 cm–1. 1H NMR (500 MHz, CDCl3):
δ = 1.83 (d, J = 6.8 Hz, 3 H, 7Ј-H), 5.86 (dq, J = 15.1, 6.8 Hz, 1 (+): m/z = 273.09 [M + Na]+, and ESI-MS (–): m/z = 249.10 [M –
H, 6Ј-H), 6.17 (dd, J = 14.6, 7.8 Hz, 1 H), 6.31 (dd, J = 15.1, H]– in each extract. Compound 1-d was eluted at 10.4 min, ESI-
10.7 Hz, 1 H), 6.70 (dd, J = 15.1, 10.7 Hz, 1 H), 6.84 (d, J = 8.3 Hz,
1 H), 6.93 (d, J = 13.2 Hz, 1 H, 1Ј-H), 6.99 (d, J = 7.8 Hz, 1 H),
7.44 (dd, J = 8.3, 7.8 Hz, 1 H), 11.95 (s, 1 H, OH) ppm. 13C NMR
(125 MHz, CDCl3): δ = 18.5 (C-7Ј), 116.5, 118.1, 124.7, 129.5,
MS (+): m/z = 274.08 [M + Na]+, and ESI-MS (–): m/z = 250.09
1
[M – H]– in extract B. H NMR (600 MHz, CDCl3) of a mixture
of 1 and 1-d (1:1): δ = 1.75 (d, J = 6.0 Hz, 3 H, 14-H), 4.17–4.21
(m, 1 H, 11-H), 4.30–4.34 (m, 1 H, 10-H), 4.99 (br. s, 0.5 H, 1-H
131.4, 132.5, 136.2, 136.3, 137.0, 142.8, 163.0 (C-2), 200.5 (C-α) of 1-d), 5.01 (s, 1 H, 1-H for 1), 5.56 (dd, J = 15.0, 6.7 Hz, 1 H,
ppm. MS (EI): m/z = 215 [M]+·, 200, 197 [M – H2O]+, 186, 182
12-H), 5.83 (dq, J = 15.5, 7.0 Hz, 1 H, 13-H), 6.04 (dd, J = 15.3,
Eur. J. Org. Chem. 2011, 6276–6280
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1
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Financial support from the Japan Society for the Promotion of
Science (No. 17580092 and 19580120), the Agricultural Chemical
Research Foundation, the Intelligent Cosmos Foundation, and the
Naito Foundation is gratefully acknowledged.
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Received: May 31, 2011
Published Online: September 27, 2011;
6280
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 6276–6280