KM-01, a brassinolide inhibitor, its production, isolation and structure from two fungi Drechslera avenae and Pycnoporus coccineus

Article abstract of DOI:10.1016/S0968-0896(98)00088-1

A new brassinolide inhibitor, named KM-01, was isolated from the culture filtrates of two fungal species, Drechslera avenae and Pycnoporus coccineus, and the structure with absolute stereochemistry was elucidated as the fatty acid ester of an eremophilane sesquiterpene, bipolaroxin, based on spectroscopic analysis, chemical degradation, and synthesis of the fatty acid. Copyright (C) 1998 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(98)00088-1

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1975±1982
KM-01, A Brassinolide Inhibitor, Its Production, Isolation and
Structure from Two Fungi Drechslera avenae and
Pycnoporus coccineus
Seong-kie Kim,* Makoto Hatori, Makoto Ojika, Youji Sakagami
and Shingo Marumo
School of Agricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601 Japan
Received 15 April 1998; accepted 28 April 1998
AbstractÐA new brassinolide inhibitor, named KM-01, was isolated from the culture ®ltrates of two fungal species,
Drechslera avenae and Pycnoporus coccineus, and the structure with absolute stereochemistry was elucidated as the
fatty acid ester of an eremophilane sesquiterpene, bipolaroxin, based on spectroscopic analysis, chemical degradation,
and synthesis of the fatty acid. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
previously in the isolation of natural abscisic acid, a
plant hormone, from a fungus, Botrytis cinerea.12
Brassinolide, the ®rst steroidal plant growth regulator,
has been isolated from rape pollen with the bioassay
measuring elongation activity of the second internode of
In the course of our screening program to ®nd new
brassinosteroid-like substances from the agar cultured
materials of fungi, we found that several fungal strains,
including Drechslera avenae and Pycnoporus coccineus,
caused severe inhibition against brassinolide-induced
lamina inclination of rice seedling explants. We were
interested in this unexpected inhibitory activity, because
no brassinolide-inhibitor has hitherto been known.
Hence an active substance, once isolated, may be helpful
to evaluate characteristic activity of brassinosteroids.
We report here the isolation and structure elucidation of
a brassinolide-inhibitor named KM-01 from two fungal
species, Drechslera avenae and Pycnoporus coccineus,
and its structure with absolute stereochemistry was
determined as shown in Figure 3.
1
bean seedlings. After the structure of brassinolide was
2
announced by the USDA group, we have shown that
the rice lamina inclination bioassay, which was origin-
ally devised as an auxin bioassay, was an extremely
sensitive method for bioassay of brassinolide and its 24-
homo analogue.3 By using this bioassay, the second
natural brassinosteroid, castasterone, was then isolated
4
from chestnut insect gall. Since then, an extensive sur-
vey of naturally occurring brassinolide and its related
brassinosteroids using this rice lamina inclination
bioassay has been carried out by many researchers. Up
to now, reports verifying the presence of brassinosteroid
activity in various plant sources have been published.
Among them, structures of thirty kinds of brassinoster-
5
oids have been chemically characterized as well as their
biological activities using various bioassay systems.6±11
In parallel with these investigations from plant resour-
ces, attempts to ®nd brassinosteroid-like substances
from fungal metabolite were also conducted by several
groups. We also commenced similar investigation
among fungal metabolites, since we have succeeded
Results and Discussion
Production of KM-01
We screened fungal products cultured under blue light,
because abscisic acid production by B. cinerea was extre-
mely stimulated by blue-light irradiation during culture.12
The crude extracts of D. avenae and P. coccineus showed
*
Corresponding author.
968-0896/98/$ - see front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00088-1
0

1
976
S. Kim et al./Bioorg. Med. Chem. 6 (1998) 1975±1982
spectrum at 3553, 1694, 1662 and 1636 cm 1. EI mass
�
spectrum gave a molecular ion at m/z 398. The mole-
cular formula C24H O
30 5
was assigned by HREIMS; M+
ion observed at 398.2098 (calcd. 398.2093). The mole-
cular formula indicates the 10 double bond equivalents
in the molecule. The H and 13C NMR spectra showed
all of the protons and carbons in the molecule, and from
these spectral data combined with DEPT analysis the
presence of four methyls, three methylenes, three
methins, two quaternaries, ten ole®ns, one each of
ketone, aldehyde, ester and hydroxyl groups could be
deduced. The de®nitive assignments of all of H and 13C
NMR signals are summarized in Table 1.
1
1
Figure 1. Production of KM-01 under various light irradiation.
The nine partial structures, (1)±(9) (shown in Figure 2)
were deduced as follows. The partial structures (1) and
(2), both of which had connective vicinal protons, were
deduced from 1H± H COSY and coupling constants.
The methyl (3) and methylene (4) should be connected
quaternary carbons. In (5) a conjugated ketonic carbon
at 197.1 ppm was shown to be long range-coupled with
C-9 proton of the trisubstituted double bond as ascer-
tained by HMBC. The conjugated aldehyde carbon
(192.7 ppm) in (6) was long-range coupled with C-12
terminal methylene protons. The ester in (7) should be a
conjugated ester as shown by dc 166.7 ppm and ꢀ
brassinolide-inhibitory activities. Then, we examined
this activity of D. avenae cultured under various wave
length light. As shown in Figure 1, blue light was most
e€ective for the production of the inhibitor. Unexpec-
tedly, dark condition was similarly e€ective as blue light;
however, the amount of inhibitor severely decreased by
BLB irradiation. These light e€ects for production of the
inhibitor were much di€erent from those observed on the
production of abscisic acid by B. cinerea.13 The precise
light e€ects on the inhibitor named KM-01 production
and their mechanism are now under investigation.
1
�
1
1
662 cm . The remaining two carbons were quaternary
shown by DEPT experiments, and one free OH which
disappeared with D O addition exists in the molecule. In
Isolation of KM-01
2
In the previous procedure,14 the yield of KM-01 was
only 0.8 mg/L. Since we noticed that KM-01 was very
unstable under acidic conditions, we changed the par-
tition conditions from the acidic to basic. The acetone
extract of mycelium on agar was partitioned between
water (pH 12) and ether. The ether layer was con-
centrated in vacuo and then puri®ed by silica gel column
chromatography and HPLC monitored with bioassays
as previously described.14,15 The yield of KM-01 was
order to combine the above nine partial structures
together, we employed the HMBC technique, data from
which are summarized in Table 1. Thus, the plane struc-
ture was constructed reasonably as shown in Figure 3.
The bicyclic ring moiety of the postulated structure for
KM-01 was found to be the same as that of bipolaroxin
which had been reported by Sugawara et al.16 as a fungal
metabolite from Bipolaris cynodontis. We tried to obtain
bipolaroxin as a degradation product of KM-01 under
various conditions; however, so far we have not succeeded.
2
0.4 mg/L with this puri®cation procedure.
Structure analysis
KM-01 was obtained as a pale yellow oil, [a] +473
Relative stereochemistry
2
0
d
c 0.027, CHCl ). The UV spectrum (MeOH) showed
(
Relative stereochemistry of KM-01 was clari®ed on
the basis of NOESY and coupling constants data.
3
a maximum absorption at 277 nm (e 55000), and IR
Figure 2. The partial structures of KM-01 (Ð implies quatanary carbons).

S. Kim et al./Bioorg. Med. Chem. 6 (1998) 1975±1982
1977
0
0
0
0
philane sesquiterpenoids.17 The relative stereochemistry
of bipolaroxin was determined by X-ray analysis, but its
absolute stereochemistry remained unclear.
The C-2 ±C-3 and C-4 ±C-5 double bonds of the fatty
acid moiety should be all trans from the values of
J
2
0
0
=15.2 and J
peak between H
0
0
=15.5 Hz. NOESY showed a cross
-14 and H -15, indicating the two
3
4
5
3
3
methyls to be situated in cis-relation. In addition, a
strong NOE was observed on respective protons
between H-3 and H-4, between H-4 and Hax-6, and
between H -6 (dH 2.07) and H -12. These results indi-
Absolute stereochemistry
The absolute stereochemistry of KM-01 was determined
by the CD method. In the CD spectrum (Figure 4), the
large splitting type of Cotton e€ect at 280 and 255 nm
in MeOH was clearly ascribed to the excited interaction
of two chromophores between the dienone ring moiety
and the diene ester side chain. The chiral exciton
ax
2
2
cated that all of H-3, H-4, Hax-6, and H -12 should be
oriented on the same side of the bicyclic ring moiety.
The relative stereochemistry of the ring moiety deduced
was the same as that of bipolaroxin and other eremo-
Table 1. NMR assignments for KM-01 in CDCl
3
1
d
13C HMBC
C no.
d H (multiplicy, J Hz)
1
2
3
4
5
6
CH=
CH=
CH(� O)
CH
6.39 (d, 9.9)
131.1 H-3, H-5, H-9
133.0 H-3
6.27 (dd, 9.9, 4.9)
5.41 (dd, 4.9,4.9)
1.97 (dq, 7.3,4.9)
68.8 H-15, H-14, H-6, H-3, H-2, H-4, H-5
40.9 H- 15, H-14, H-3, H-6, H-2
36.2 H-14, H-15, H-4, H-6, H-3, H-9, H-1
44.3 H-14
C
CH
2
ax 2.07 (d, 14.2)
ex 1.96 (d, 14.2)
7
8
9
C
74.8 H-6, H-9, H-12, H-13
197.1 H-6, H-9
123.9 H-1
C=O
CH=
C
5.98 (s)
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
0
1
2
3
4
161.9 H-14, H-6, H-2, H-1
154.5 H-6, H-12
135.7 H-12
C
CH =
2
CH=O
6.82 (s) 6.29 (s)
9.50 (S)
1.48 (s)
192.7
22.5 H-4
CH
CH
3
5
3
1.02 (d, 7.3)
10.2 H-4, H-6
166.7 H-3, H-2 , H-3
0
0
0
C=O
CH=
CH=
CH=
CH=
CH=
0
0
0
0
0
0
0
0
0
5.79 (d, 15.2)
7.23 (dd, 15.2, 10.6)
6.14 (dd, 15.5, 10.6)
5.99 (dd, 15.5, 7.3)
2.14 (m)
118.7 H-4
145.9 H-4 , H-5
0
0
0
0
0
0
0
126.6 H-6 , H-2 , H-5
0
0
0
0
0
0
0
0
0
0
0
150.8 H-9 , H-7 , H-6 , H4 , H-3
0
0
0
0
38.8 H-8 , H-9 , H-7 , H-6 , H-4 , H-3
0
CH
CH
CH
2
3
3
1.35 (dq, 7.3, 7.3)
0.84 (t, 7.3)
1.01 (d, 6.6)
29.2 H-8 , H-9 , H-6 , H-5
11.6 H-6 , H-7
0
0
19.4 H-7 , H-6 , H-5
OH
2.46 (s)
Figure 3. Carbon-proton connectivity by HMBC analysis (left) and the structures of KM-01 (right).

1
978
S. Kim et al./Bioorg. Med. Chem. 6 (1998) 1975±1982
Figure 4. Splitting CD spectrum of KM-01.
*
coupling theory predicts that the sign of the ®rst CD
band originates from the spatial absolute relationship of
two chromophores.18 The positive sign of the ®rst Cot-
ton e€ect indicates that two chromophores should be
twisted in clockwise sense.19 The conformational energy
of KM-01 was calculated by use of the QUANTA/
CHARMm molecular modeling program,14 and the
most stable conformation obtained showed the diene
ester side chain to be oriented perpendicularly upward
from the ring moiety. The most stable conformation was
also calculated by the AMBER* force ®eld (in water as
a solvent) in the molecular modeling package Macro-
Model 6.020 (Figure 5) and found to be similar to that
obtained by the QUANTA/CHARMm. The S con®g-
uration at C-3 of KM-01 was con®rmed by applying the
modi®ed Mosher method.21 On basic methanolysis KM-
ester, which was calculated by the AMBER force ®eld
(in CHCl as a solvent), showed that the H -14 was
3
3
located in the upper side of the MTPA plane as shown
in Figure 6.
The absolute stereochemistry of the ring system was
thus elucidated as C-3 (S), C-4 (R), C-5 (R) and C-7 (R).
0
Determination of C-6 stereochemistry
0
C-6 stereochemistry of the side chain was determined
®rst by obtaining the fatty acid moiety as its methyl
ester by alkaline hydrolysis followed by methylation
with diazomethane. Next, a possible enantiomeric ester
of the natural acid, methyl (2E,4E,6S)-6-methyl-2,4-
octadienoate, was synthesized (Figure 7). (S)-(� )-2-
Methylbutanol was subjected to Swern oxidation to give
(S)-(+)-2-methylbutanal. The aldehyde was then reac-
ted, without isolation, with methyl (E)-4-triphenylphor-
anylidene-2-butenoate which was prepared from methyl
4-bromo-2-butenoate via (3-carbomethoxy-2-propen-1-
yl)triphenylphosphonium bromide, a€ording the desired
(6S)-dienoate.
0
1 gave unstable enol 1 which, without puri®cation, was
converted to stable quinoxaline 2 by treatment with o-
phenylenediamine (Scheme 1). Basic methanolysis of
quinoxaline provided secondary alcohol 3, which was
1
esteri®ed to give (R)- and (S)-MTPA esters (4a,b). H
NMR signals of the two MTPA esters were assigned
and Ád values were calculated. Satisfactory results were
obtained as shown in Scheme 1 except for the abnormal
3
Ád value at H -14. However, this negative value could
be due to the axial orientation of the H -14. For exam-
3
ple, the most stable conformation of the (S)-MTPA
Figure 6. The lowest energy conformer of an analogue of (S)-
MTPA ester 4b. The dotted line indicates the MTPA plane. The
quinoxaline moity of the (S)-MTPA-ester 4b is replaced by
benzene in the analogue.
Figure 5. The most stable conformer of KM-01 calculated by
the AMBER* force ®eld.

S. Kim et al./Bioorg. Med. Chem. 6 (1998) 1975±1982
1979
Scheme 1.
Figure 7. Synthesis of methyl (2E,4E,6S)-6-methyl-2,4-octadienoate.
Figure 8. HPLC Chromatogram and polarimetric diagram for the resolution of racemate, natural and synthetic ester on a CHIR-
ALCEL OD column.

1
980
S. Kim et al./Bioorg. Med. Chem. 6 (1998) 1975±1982
Comparative identi®cation of the natural ester with the
synthetic product was conducted with HPLC equipped
with a polarimeter. When racemates were subjected to
HPLC on a column of CHIRALCEL OD by using n-
hexane and 2-propanol (90:20) as an eluant, the resolu-
tion was attained as shown in Figure 8. The retention
time of the natural compound, monitored with lmax
FL20S BLB lamp (352‹20 nm); green, Toshiba FL20S
G with Fuji Film BPB 55 ®lter (547‹10 nm); red,
FL20S Re66 with Acrylite No. 102 ®lter (663‹20 nm).
Each cultured material under the various light condi-
tions was extracted with acetone, concentrated and par-
titioned with ether at pH 2. The ether extracts were
bioassayed using modi®ed Raphanus test with brassino-
lide as described in the previous paper.14
2
54 nm, was well coincident with that of the synthetic
compound, and the former's upward signal, monitored
polarimetrically at 365 nm, was the same as that of the
synthetic compound. Thus it is concluded that the C-6
Enol 1. To a stirred solution of KM-01 (2.1 mg) in
ꢀ
0
MeOH (0.9 mL) at 0 C was added a 1 M solution of
of the fatty acid side chain has the S stereochemistry
and the absolute stereochemistry of KM-01 was estab-
lished as shown in Figure 3.
NaOMe in MeOH (0.1 mL). The mixture was stirred at
ambient temperature for 30 min. The mixture was dilu-
ted with ether (20 mL) and water (20 mL). The organic
layer was separated and the aqueous layer extracted
with ether (2Â20 mL). The combined organic extracts
2 4
were washed with saturated NaCl, dried on Na SO ,
Experimental
1
and concentrated to give 1 (2.2 mg) as a crude oil: H
NMR (270 MHz, CDCl ) d 0.87 (t, J=7.4 Hz, 3 H), 1.04
d, J=6.8 Hz, 3 H), 1.18 (d, J=6.8 Hz, 3 H), 1.26 (s, 3
General methods
3
(
1H (270 MHz) and 13C (67.5 MHz) NMR spectra were
recorded on a JEOL JNM GSX-270 (270 MHz), a Bru-
H), 1.30 (dq, J=6.8 and 6.8 Hz, 2 H), 2.08 (m, 1 H),
2.18 (m, 1 H), 5.55 (dd, J=5.4 and 4.4 Hz, 1 H), 5.82 (d,
15.3 Hz, 1 H), 6.05 (dd, J=15.2 and 7.7 Hz, 1 H), 6.17
(dd, J=15.2 and 10.7 Hz, 1 H), 6.19 (dd, J=9.6 and
5.4 Hz, 1 H), 6.20 (s, 1 H), 6.29 (s, 1 H), 6.33 (s, 1 H),
6.45 (d, J=9.6 Hz, 1 H), and 7.24 (dd, J=15.3 and
ker ARX400 (400 MHz) or
600 MHz) NMR spectrometer. Electron impact mass
spectra (EIMS) were obtained on a JEOL JMS-DX
05L mass spectrometer. High-resolution EIMS was
a Bruker AMX600
(
7
+
performed on a JEOL Mstation JMS-700. UV absorp-
tion spectra were measured with Hitachi 330 spectro-
photometer. Infrared absorption spectra were obtained
with a JASCO FT-IR 7000S infrared spectrometer.
Optical rotations were determined with a JASCO DIP-
10.7 Hz, 1 H); EIMS m/z 342 (M ), 188, 160, 137
(base), and 109.
Quinoxaline 2. o-Phenylenediamine (54 mg) was dis-
solved in 2 M AcOH (0.25 mL) by gentle heating, and 4
M NaOAc (0.125 mL) was added. To the mixture at
3
71 digital polarimeter. The CD spectra were obtained
ꢀ
on a JASCO DP-500N spectropolarimeter. The Macro-
model V6.0 calculations were performed on a Silicon
Graphics O2 R5000SG computer.
60 C was added a solution of the above crude enol 1
(2.2 mg) in MeOH (0.75 mL), and the mixture was stir-
ꢀ
red at 60 C for 30 min. The mixture was diluted with
water (2 mL) and extracted with hexane:EtOAc (4:1)
(
3Â3 mL). The combined organic extracts were washed
Fungal culture
with 2 M AcOH (1 mL) and concentrated. The residual
oil was chromatographed on silica gel (hexane:EtOAc
(5:1)) to give 2 (1.7 mg, 77% yield from KM-01) as a
yellow oil: [a] +770 (c 0.14, CHCl ); UV (MeOH) lmax
265 (e 34100), 278 (30000, sh), 348 (18200, sh), 361
(22900), and 377 (19400) nm. IR (CHCl ) 1701, 1640,
Two fungal species, Drechslera avenae and Pycnoporus
coccineus, were kindly gifted by the late Dr. N. Nishi-
hara, National Grassland Research Institute, and their
morphological identi®cation was conducted by Dr. M.
Ito, Shin Nippon Chem Ind., Anjo City, Japan.
2
3
d
3
3
617, 1261, 1141, 1003, and 911 cm 1
�
;
1H NMR
1
D. avenae and P. coccineus were cultured with a potato-
lactose agar medium that was prepared by dissolving
lactose (20 g) and agar (20 g) in 1 L of a potato extract
(400 MHz, CDCl ) d 0.85 (t, J=7.4 Hz, 3 H), 1.01 (d,
3
J=6.8 Hz, 3 H), 1.18 (d, J=6.8 Hz, 3 H), 1.18 (s, 3 H),
1.36 (dq, J=6.8 and 6.8 Hz, 2 H), 2.16 (m, 1 H), 2.22
(dq, J=4.4 and 6.8 Hz, 1 H), 3.00 (d, J=15.8 Hz, 1 H),
3.34 (d, J=15.8 Hz, 1 H), 5.54 (dd, J=5.4 and 4.4 Hz, 1
H), 5.81 (d, 15.3 Hz, 1 H), 6.00 (dd, J=15.2 and 7.7 Hz,
1 H), 6.14 (dd, J=15.2 and 10.7 Hz, 1 H), 6.21 (dd,
J=9.6 and 5.4 Hz, 1 H), 6.53 (d, J=9.6 Hz, 1 H), 6.67
(s, 1 H), 7.24 (dd, J=15.3 and 10.7 Hz, 1 H), 7.67 (m, 2
(
(
200 g) solution. The medium (30 mL) in a Petri dish
9 cm in diameter) was inoculated at four points with the
ꢀ
fungus, and incubated at 26 C for 10 days under blue
light using Toshiba FL20S B19 lamp combined with an
acrylite No. 302 ®lter (lmax 457‹20 nm, 1.5 W/m ). For
the production of the inhibitor under various light irra-
2
+
diation, the following lights were used with the same
2
H), and 7.98 (m, 2 H); EIMS m/z 414 (M , base), 278,
262, 246, 231, 219, 137, and 109; HREIMS calcd. for
energy (1.5 W/m ): near UV, Toshiba FL20S E lamp
320‹20 nm); black light blue (BLB), with Toshiba
(
27 30 2 2
C H N O 414.2307 (M), found 414.2321.

S. Kim et al./Bioorg. Med. Chem. 6 (1998) 1975±1982
1981
Alcohol 3. To a solution of 2 (1.7 mg) in MeOH (1 mL)
was added K CO (13.8 mg), and the mixture was stirred
Methyl (E)-triphenylphophoranylidene-2-butenoate.22 To
a solution of triphenylphosphine (2.6 g, 10 mmol) in dry
benzene (10 mL), methyl 4-bromo-2-butenoate (1.8 g,
10 mmol) was added by slowly dropping with vigorous
stirring at room temperature and the mixture was stirred
overnight. The white solid was collected by ®ltration,
washed with 100 ml of dry benzene, and dried in
2
3
ꢀ
at room temperature for 3.5 h and then at 50 C for 3 h.
The reaction mixture was diluted with water (4 mL) and
extracted with dichloromethane (3Â4 mL). The com-
bined organic layers were washed with water (1 mL) and
concentrated. The residual oil was chromatographed on
silica gel (hexane:EtOAc (4:1, 3:2, and then 1:1)) to give
alcohol (1.0 mg, 88%) as yellow crystals and methyl (2E,
ꢀ
vacuum oven at 60±80 C. The yield of phosphonium
ꢀ
salt (mp 179±180 C) was 4.06 g. The phosphonium salt
4
E, 6S)-6-methyl-2,4-octadienoate (0.5 mg, 73%) as an oil.
(4.06 g) was dissolved in cold water (200 mL), and 2%-
NaOH (20 mL) was dropped cautiously. The mixture
was stirred overnight at room temperature and the pre-
cipitate was ®ltered by suction. The precipitate was
2
2
3: [a] +370 (c 0.057, CHCl ); UV (MeOH) lmax 287
d
3
(
e 15400), 348 (15600, sh), 362 (20000), and 378 (17300)
nm. IR (CHCl ) 3610, 1594, 1494, 1400, 1373, 1332,
243, 1179, 1134, 1103, 1008, 968, and 884 cm 1; 1
NMR (600 MHz, CDCl ) d 1.15 (s, 3 H), 1.29 (d,
J=7.2 Hz, 3 H), 2.01 (dq, J=4.2 and 7.2 Hz, 1 H), 2.97
d, J=15.8 Hz, 1 H), 3.33 (d, J=15.8 Hz, 1 H), 4.30 (br
ꢀ
3
washed with excess ice-cooled water, dried below 20 C
ꢀ
�
1
H
under vacuum. The yield was 2.58 g, mp 175±179 C.
3
Methyl (2E,4E,6S)-6-methyl-2,4-octadienoate. DMSO
was added to the stirred oxalyl chloride (11 mmol)
(
ꢀ
d, 1 H), 6.24 (dd, J=9.6 and 5.4 Hz, 1 H), 6.47 (d,
J=9.6 Hz, 1 H), 6.65 (s, 1 H), 7.66 (m, 2 H), and 7.97
dissolved in 25 mL dichloromethane at � 50 C to
ꢀ
� 60 C. The reaction mixture was stirred for 2 min
+
(
m, 2 H); EIMS m/z 278 (M ), 276, 260, 245 (base),
and (S)-2-methylbuthanol was added within 5 min,
stirring was continued for an additional 15 min.
Triethylamine (7.0 mL, 50 mmol) was added and the
reaction mixture was stirred for 5 min and then allowed
to warm to room temperature. Water (50 mL) was then
added, the organic layer was separated, and the aqueous
layer was reextracted with additional dichrolomethane
(50 mL). The organic layers were combined, washed
with saturated NaCl solution (100 mL), and dried over
anhydrous magnesium sulfate. To the concentrated
organic layer (25 mL), methyl (E)-triphenylphophor-
anylidene-2-butenoate (1.96 g, 5.5 mmol) was added
rapidly and the reaction mixture was allowed to stand
overnight at room temperature. The mixture was
poured into water and the aqueous phase extracted with
ether. The ethereal fraction was washed with saturated
aqueous NaCl and concentrated to yield a viscous oil
which was chromatographed over silica gel eluted with
the solvent system of n-hexane-ethylacetate. The yield
was 182 mg (1.12 mmol).
2
30, and 218; HREIMS calcd for C18
M), found 278.1438. Methyl (2E, 4E, 6S)-6-methyl-2,4-
18 2
H N O 278.1419
(
octadienoate: [a]d +40 (c 0.03, CHCl
3
); 1H NMR
2
2
data were identical to those for the synthetic sample
described below.
MTPA esters 4a, b. A mixture of alcohol (0.4 mg), (R)-
a-methoxy-a-(tri¯uoromethyl)phenylacetic acid ((R)-
MTPA acid) (2.9 mg), 4-dimetylaminopyridine (1.2 mg),
1
0-camphorsulfonic acid (1.1 mg), and 1,3-dicyclohex-
ylcarbodiimide (3.5 mg) in anhydrous dichloromethane
0.5 mL) was stirred at room temperature for 4 h. The
(
reaction mixture was diluted with hexane (0.5 mL) and
the resulting precipitates were removed by ®ltration and
washed with hexane. The combined ®ltrate and wash-
ings were concentrated and chromatographed on silica
gel (hexane:EtOAc (2:1) to give (R)-MTPA ester
(
0.5 mg) as an oil. (S)-MTPA ester was obtained in the
1
same manner. H NMR (600 MHz) spectra of MTPA
esters were measured in CDCl3.
2
0
ꢀ
20
365
+204.3; 1H NMR
[
a] +52.8 (c 0.3, CHCl
3
), [a]
d
Hydrolysis of KM-01 and methylation. KM-01 (4.9 mg,
0
(
reaction was monitored by TLC until the sample dis-
appeared. MeOH was removed. The aqueous solution
was extracted with ethyl acetate (3Â3 mL), acidi®ed
with HCl (0.1N) and extracted with diethyl ether
3
(270 MHz, CDCl ), d 0.86 (3H, t, J=7.4 Hz), 1.02 (3H,
.012 mmol) dissolved in 0.1N-KOH (1 mL) and MeOH
1 mL) was stirred for 30 min at room temperature. The
d, J=6.7 Hz), 1.37 (2H, dq, J=7.1, 7.1 Hz), 2.16 (1H,
m), 3.73 (3H, s), 5.80 (1H, d, J=15.2 Hz), 6.01 (1H, dd,
J=15.2, 7.4 Hz), 6.14 (1H, dd, J=15.2, 10.6 Hz), 7.27
(1H, dd, J=15.2, 10.6 Hz).
References
(
Na SO and evaporated in vacuo. The residue was fur-
3Â3 mL). The organic layer was dried over anhydrous
2
4
1. Mitchell, J. W.; Mandava, N.; Worley, J. F.; Plimmer, J. R.;
Smith, M. V. Nature, 1970, 225, 1065.
ther puri®ed by preparative TLC, showing R
.23 in the petroleum ether-ethyl ether-acetic acid
80:20:1 (v/v)) system. This fatty acid was methylester-
f
value of
0
(
2. Grove, M. D.; Spencer, G. F.; Rohwedder, W. K.; Man-
dava, N. Nature, 1972, 281, 216.
i®ed with diazomethane. After the puri®cation with
HPLC, the yield was 0.52 mg.
3. Marumo, S.; Hattori, H.; Abe, H.; Nonoyama, Y.; Muna-
kata, K. Agric. Biol. Chem., 1968, 32, 528.

1
4
982
S. Kim et al./Bioorg. Med. Chem. 6 (1998) 1975±1982
. Yokota, T.; Arima, M.; Takahashi, N.; Wada, K.; Marumo,
13. Kanoh, K.; Kohno, E.; Natsume, M.; Marumo, S. Chem.
Abstr. 1989, 110, 189131x.
S.; Mori, K.; Takatsuno, S.; Morisaki, M.; Ikekawa, N. Tetra-
hedron Lett., 1982, 23, 1275±1278.
14. Kim, S.-K.; Mizuno, K.; Hatori, M.; Marumo, S. Tetra-
hedron Lett., 1994, 35, 1731.
5. Yokota, T. Plant Hormone Handbook, (Japanese); Takaha-
shi, N.; Masuda, Y. Eds.; Baifuukan: Tokyo, 1994; Vol. 2, pp
15. Kim, S.-K.; Asano, T.; Marumo, S. Biosci. Biotech. Bio-
chem., 1995, 59, 1394.
2
6
03±204.
. Wada, K.; Marumo, S.; Ikekawa, N.; Morishita, T.; Naka-
16. Sugawara, F.; Strobel, G. Proc. Natl. Acad. Sci. USA,
1985, 82, 8291.
mura, K. Plant Cell Physiol., 1981, 22, 323.
7
8
. Cerena, R.; Colombo, R.; Lado, P. Physiol. Veg., 1983, 21,
75.
17. Tanaka, S.; Wada, K.; Marumo, S.; Hattori, K. Tetra-
hedron Lett., 1981, 22, 323.
8
. Wada, K.; Marumo, S.; Abe, T.; Morishita, T.; Nakamura,
18. Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy,
Exiton Coupling in Organic Stereochemistry. University Science
Books; Mill Valley, California, 1983; pp. 85±88.
K. Agric. Biol. Chem., 1984, 48, 719.
. Cerena, R.; Lado, P.; Anastasia, M.; Ciu€reda, P.; Allevi, P.
J. Plant Physiol., 1984, 114, 221.
9
19. Harada, N.; Chen, S.-M. L.; Nakanishi, K. J. Am. Chem.
Soc., 1975, 97, 2480.
1
0. Wada, K.; Kondo, H.; Marumo, S. Agric. Biol. Chem.,
985, 49, 2249.
1
20. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp,
R.; Lipton, M.; Cau®eld, C.; Chang, G.; Hendrickson, T.; Still,
W. C. J. Comput. Chem., 1990, 11, 440.
11. Hewitt, F. R.; Houghm, T.; O'Neil, P.; Sasse, J. M.;
William, E. G.; Roman, K. S. Aust. J. Plant Physiol. 1985, 12,
2
1
01.
21. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am.
Chem. Soc., 1991, 113, 4092.
2. Marumo, S.; Lee, S.-K.; Kamuro, Y. Shokubutsu Saibo
Kogaku, 1990, 2, 503 (in Japanese).
22. Buchta, E.; Andree, F. Chem. Ber., 1960, 93, 1349.