Asymmetric synthesis and stereochemical structure-activity relationship of (R)- and (S)-8-[1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethoxy] octanoic acid heptyl ester, a potent inhibitor of allene oxide synthase

Article abstract of DOI:10.1016/j.bmc.2007.10.095

The preparation of both enantiomers of 8-[1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethoxy] octanoic acid heptyl ester (JM-8686), a potent inhibitor of allene oxide synthase, has been achieved using 2,4-dichlorophenacyl bromide as a starting material. The key step was the asymmetric reduction of 1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethanone with chiral BINAL-H. The products were purified by chiral high-performance liquid chromatography (HPLC) to afford pure (R)-JM-8686 and (S)-JM-8686. The inhibitory activities and binding affinities of these enantiomers toward allene oxide synthase were determined. We found that the inhibition potency of (R)-JM-8686 is approximately 200 times greater than that of (S)-JM-8686, with IC₅₀ values of approximately 5 ± 0.2 nM and 950 ± 18 nM, respectively. The dissociation constants of (R)-JM-8686 and (S)-JM-8686 with respect to the recombinant allene oxide synthase were approximately 1.4 ± 0.3 μM and 4.8 ± 0.6 μM, respectively.

Full text of DOI:10.1016/j.bmc.2007.10.095

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1090–1095
Asymmetric synthesis and stereochemical structure–activity
relationship of (R)- and (S)-8-[1-(2,4-dichlorophenyl)-
2-imidazol-1-yl-ethoxy] octanoic acid heptyl ester,
a potent inhibitor of allene oxide synthase
Keimei Oh,a,* Yoichiro Shimura,a Kyoko Ishikawa,a Yudai Ito,a Tadao Asami,b
Noboru Murofushia and Yuko Yoshizawaa
aDepartment of Biotechnology, Faculty of Bio-resource Sciences, Akita Prefectural University, Akita 010-0195, Japan
bDepartment of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Received 19 September 2007; revised 28 October 2007; accepted 29 October 2007
Available online 4 November 2007
Abstract—The preparation of both enantiomers of 8-[1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethoxy] octanoic acid heptyl ester (JM-
8686), a potent inhibitor of allene oxide synthase, has been achieved using 2,4-dichlorophenacyl bromide as a starting material. The
key step was the asymmetric reduction of 1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethanone with chiral BINAL-H. The products were
purified by chiral high-performance liquid chromatography (HPLC) to afford pure (R)-JM-8686 and (S)-JM-8686. The inhibitory
activities and binding affinities of these enantiomers toward allene oxide synthase were determined. We found that the inhibition
potency of (R)-JM-8686 is approximately 200 times greater than that of (S)-JM-8686, with IC50 values of approximately
5
0.2 nM and 950 18 nM, respectively. The dissociation constants of (R)-JM-8686 and (S)-JM-8686 with respect to the recom-
binant allene oxide synthase were approximately 1.4 0.3 lM and 4.8 0.6 lM, respectively.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
(DES).1 AOS catalyzes the first reaction in the specific
biosynthetic pathway of jasmonic acid related com-
pounds, collectively called Jasmonates (JAs).3 JAs are
essential signal mediators in the defense system of plants
against pest attack, in mechanical injury, and also in
several developmental processes.4–7 Correlations be-
tween endogenous JA levels in specific tissues and the ef-
fects of the applied JAs have provided evidence that JAs
have a role in fruit ripening, embryo development,5
senescence,8 and the accumulation of storage proteins.9
Analysis of JA-deficient mutants of Arabidopsis has pro-
vided evidence that JAs play a key role in flower devel-
opment. The critical requirement of JAs in male fertility
was established by the characterization of an Arabidop-
sis mutant that fails to produce linolenic acid, the fatty
acid precursor of JAs.10 Analysis of JA-deficient mu-
tants of tomato has indicated that JAs are key regulators
involved in the defense response to herbivore attack and
mechanical wounding.11,12
The oxylipin biosynthetic pathway in plants leads to the
production of a group of bioactive compounds.1 The
initial step of most phyto-oxylipin biosyntheses is cata-
lyzed by lipoxygenase (LOX), which adds molecular
oxygen to either the C-9 or C-13 position of linolenic
or linoleic acid.2 The resulting hydroperoxides are fur-
ther metabolized by several enzymes including three clo-
sely related members of the CYP74 family of
cytochrome P-450: allene oxide synthase (AOS), hydro-
peroxide lyase (HPL), and divinyl ether synthase
Abbreviations: AOS, allene oxide synthase; BINAL-H, 2,20-dihydroxy-
1,10-binaphthyl-lithium aluminum hydride; DES, divinyl ether
synthase; HPL, hydroperoxide lyase; 13(S) HPOT, 13(S)-hydroper-
oxy-9(Z),11(E),15(Z)-octadecatrienoic acid; JAs, jasmonates; JM-86-
86, 8-[1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethoxy] octanoic acid
heptyl ester; LOX, lipoxygenase.
Keywords: Allene oxide synthase inhibitor; Imidazole; Jasmonic acid
biosynthesis.
The importance of JAs in the plant life cycle has pro-
moted increasing interest in understanding the mecha-
nisms by which JAs biosynthesis is regulated.7
*
Corresponding author. Tel.: +81 18 872 1590; fax: +81 18 872
1670; e-mail: jmwang@akita-pu.ac.jp
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.095

K. Oh et al. / Bioorg. Med. Chem. 16 (2008) 1090–1095
1091
JM-8686 {(S)-8-[1-(2,4-dichlorophenyl)-2-imidazol-1-yl-
ethoxy] octanoic acid heptyl ester (4b)}, the starting
material 2,4-dichlorophenacyl bromide (1) was used.
Compound 1 was coupled with imidazole according to
the previously described method17 to give 2. (R)-1-(2,4-
Dichlorophenyl)-2-imidazol-1-yl-ethanol (3a) and (S)-
1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethanol
(3b)
Figure 1. Chemical structure of JM-8686.
were prepared by asymmetric reduction of 2 using chiral
BINAL-H.18 The target compounds 4a and 4b were pre-
pared from their corresponding (R) and (S) precursors
(3a and 3b) by the method we previously described.15
The optical purity of the synthesized compounds was
examined by chiral HPLC (described in Section 4). Data
Available evidence indicates that AOS is an important
site of regulation. Herbivore attack and mechanical
wounding positively regulate AOS expression.13 Muta-
tions that disrupt AOS function in Arabidopsis caused
male sterility and attenuated responses of leaves to
mechanical wounding.14 Thus, specific inhibitors inter-
fering with AOS functions may be useful for dissecting
the functions associated with JA biosynthesis.
1
from the chiral HPLC and H NMR analyses indicate
that the yield of the asymmetric reduction of 2 by (R)-
BINAL-H is approximately 56%, and the enantiomeric
excess (ee) of 3a is approximately 48.6%. The yield
and optical purity of the product for the reaction to pre-
pare 3b are approximately 62% and 56.3%, respectively
(Table 1). The relationships of the absolute configura-
tions and specific rotations of the individual stereoiso-
mers (3a and 3b) were confirmed by comparison with
the data reported previously.19
We have previously reported the first JA biosynthesis
inhibitor, JM-8686 (the chemical structure is shown in
Fig. 1), that exhibits potent inhibitory activity against
AOS.15 Kinetic analysis of AOS inhibition by JM-8686
indicated that it binds to the active site of AOS. In addi-
tion, JM-8686 exhibits high selectivity between AOS and
HPL, a closely related member of the CYP74 family of
cytochrome P-450.16 Thus, using the stereoisomers of
JM-8686 to investigate the properties of the inhibitor–
enzyme interaction may provide stereochemical infor-
mation about the binding site of AOS, which is useful
for designing new AOS inhibitors as well as for under-
standing the structural functions of AOS. Here, we re-
port the asymmetric synthesis of (R)-(ꢀ)-JM-8686 (4a)
and (S)-(+)-JM-8686 (4b), and their biological activities
against AOS.
The target compounds, 4a and 4b, were prepared from
3a and 3b and purified by chiral HPLC. The optical
purities are greater than 99% (ee) as determined from
the chiral HPLC peak areas (Table 1). The absolute ste-
reo configurations of 4a and 4b were determined by
comparison of their CD spectra with those of the corre-
Table 1. Specific rotations and retention times on chiral HPLC of 3a,
3b, 4a, and 4b
Compound Specific rotation
Retention
timea (min)
24
½aꢁD +992 24 (C = 1, MeOH)
2. Results and discussion
2.1. Chemistry
3a
3b
4a
4b
8.81 0.05
10.95 0.07
8.29 0.08
24
½aꢁD ꢀ998 42 (C = 1, MeOH)
24
½aꢁD +416 25 (C = 0.01, MeOH)
24
½aꢁD ꢀ435 29 (C = 0.01, MeOH) 17.42 0.3
As shown in Scheme 1, for the asymmetric syntheses of
(R)-JM-8686 {(R)-8-[1-(2,4-dichlorophenyl)-2-imidazol-
1-yl-ethoxy] octanoic acid heptyl ester (4a)} and (S)-
a To separate 3a and 3b, a flow rate of 4 mL/min was used on a chiral
HPLC column.
Scheme 1. Reagents and conditions: (a) imidazole (5 equiv), DMF; (b) (R)-BINAL-H, THF, ꢀ100 °C; (c) (S)-BINAL-H, THF, ꢀ100 °C; (d) NaH,
DMF; (e) Br(CH2)7COO(CH2)6CH3.

1092
K. Oh et al. / Bioorg. Med. Chem. 16 (2008) 1090–1095
sponding precursors, 3a and 3b. As shown in Figure 2,
the CD spectral pattern of 3a is the same as 4a, and that
of 3b is the same as 4b, indicating that 4a is (R)-(ꢀ)-JM-
8686 and 4b is (S)-(+)-JM-8686.
2.2. Inhibitory activity of the enantiomers against allene
oxide synthase
The inhibitory activity of the two isomers against puri-
fied recombinant AOS was investigated by continuously
observing the dehydration process of the substrate. As
shown in Figure 3, without inhibitor (squares), the
absorbance of the substrate was decreased by approxi-
mately 0.1 within 120 s, which is associated with the
AOS activity. At concentrations of 1 (lM) for the two
isomers, (R)-(ꢀ)-JM-8686 (triangles) exhibits approxi-
mately 93% inhibition while (S)-(+)-JM-8686 (circles)
shows approximately 50% inhibition, indicating that
(R)-(ꢀ)-JM-8686 is more potent than (S)-(+)-JM-8686.
The IC50 values of (R)-(ꢀ)-JM-8686 and (S)-(+)-JM-
8686 calculated from the dose-dependent inhibition
curves of AOS were approximately 5 0.2 nM and
950 18 nM, respectively (Table 2).
Figure 3. Inhibitory activity of (R)- and (S)-JM-8686 against AOS.
The experiments were carried out by incubation of 13(S) HPOT
(20 lM) with 1 lM (R)-(ꢀ)-JM-8686 (triangles), 1 lM (S)-(+)-JM-
8686 (circles), or without adding inhibitor (diamonds). The reaction
was initiated by adding the enzyme. The progress curve was recorded
for 120 s. Data are shown as plots of the average of three independent
experiments.
2.3. Binding affinity of the stereoisomers to allene oxide
synthase
Binding of JM-8686 to AOS was determined by measur-
ing the optical difference spectra upon addition of JM-
8686 to recombinant AOS. AOS exhibited a soret
absorption peak at 419 nm, which is characteristic of
low-spin P450s (Fig. 4A, solid line). The addition of
(R)-(ꢀ)-JM-8686 to AOS induced an absorbance shift
of the heme soret band from 419 to 429 nm (Fig. 4A,
broken line, data for (S)-(+)-JM-8686 are not shown).
The difference spectrum patterns induced by (R)-(ꢀ)-
JM-8686 and (S)-(+)-JM-8686 are shown in Figures
Table 2. I50 values of the stereoisomers of JM-8686 upon AOS
inhibition and binding affinities
a
I50 (nM)
b
Compound
Kd (lM)
(S)-(+)-JM-8686
(R)-(ꢀ)-JM-8686
950 18
5
4.8 0.6
1.4 0.3
0.2
a The concentration that produces 50% inhibition of AOS activity
when compared to the control (untreated) condition.
b The dissociation constant Kd was determined by titrating the
observed spectral absorbance difference DA (429–391 nm) versus the
inhibitor concentration (Fig. 4).
4B and C. Both stereoisomers induced typical type II
binding spectra, which are characteristic of a change
from a low to a high spin state of the ferric iron. This
behavior is usually associated with the direct coordina-
tion of the imidazole group of JM-8686 to the heme iron
of AOS.20 The dissociation constant (Kd) was deter-
mined by titrating the observed spectral absorbance dif-
ference DA (429–391 nm) versus the concentration of
inhibitor (Fig. 4D). The Kd values for (R)-(ꢀ)-JM-
8686 and (S)-(+)-JM-8686 were approximately
1.4 0.3 lM and 4.8 0.6 lM, respectively (Table 2).
These results clearly indicate a positive correlation be-
tween inhibitory activity and binding affinity of the
two stereoisomers.
AOS (CYP74A) belongs to a unique class of P450s. The
catalytic action of AOS in the biosynthesis of epoxyalco-
hols has been well characterized.21 The hydroperoxide
substrate is thought to interact with the ferric ion of
AOS. One-electron transfer from AOS Fe (III) to the
O–O bond of 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octa-
decatrienoic acid [13(S) HPOT] results in the formation
of AOS Fe (IV)–OH and a substrate alkoxy radical. The
Figure 2. Induced circular dichroism spectra of 3a (triangles), 3b
(circles), 4a (diamonds), and 4b (squares) in a methanol solution
(10ꢀ4 M).

K. Oh et al. / Bioorg. Med. Chem. 16 (2008) 1090–1095
1093
Figure 4. Binding of JM-8686 to AOS. (A) Absorption spectra of oxidized AOS (solid line) and its (R)-(ꢀ)-JM-8686 complex (broken line).
Recombinant AOS (2.5 lM) was dissolved in 50 mM NaH2PO4 (pH = 7.0) with 0.1% Tween 20 containing 20% glycerol, and (R)-(ꢀ)-JM-8686 was
added to AOS at a final concentration of 12 lM. (B) Spectrophotometric titration of AOS with (R)-(ꢀ)-JM-8686. (R)-(ꢀ)-JM-8686 was added to
AOS (2.5 lM) at various final concentrations (a, 2; b, 4; c, 6; d, 8; e, 10; f, 12 lM). (C) Spectrophotometric titration of AOS with (S)-(+)-JM-8686
induced spectral changes in AOS. The amount of (S)-(+)-JM-8686 added was: a, 2; b, 4; c, 6; d, 8; e, 10; f, 12; g, 18 lM. (D) A plot of absorbance
differences DA (429–391 nm) against (R)-(ꢀ)-JM-8686 (circles) and (S)-(+)-JM-8686 (open circles) concentrations. Data are shown as plots of the
average of two independent experiments.
subsequent free radical oxidation and loss of a b-proton
result in the formation of an unstable allene oxide,
which is the product of the AOS reaction. These obser-
vations suggest the asymmetric carbon of the substrate
plays a key role in the catalytic action of AOS. Data ta-
ken from binding assays of the two stereoisomers of JM-
8686 to AOS clearly indicate that (R)-(ꢀ)-JM-8686
exhibits a high binding affinity toward AOS, which sug-
gests there is a stereochemical recognition mechanism
for inhibitor–enzyme interaction. In addition, AOS has
no absolute requirement for molecular oxygen and
NADPH in catalyzing the biochemical conversion of
13(S)-hydroperoxylinolenic acid. Instead, it uses hydro-
peroxylinolenic acid both as an oxygen donor (using the
peroxy moiety) and as a substrate.21,22 Included in this
class of P450 enzymes are thromboxane synthase and
PGI2 synthase.23 Since JM-8686 exhibits potent inhibi-
tion of AOS activity, we expect that experimental use
of this synthetic series will provide important informa-
tion about the catalytic mechanism of this class of cyto-
chrome P450s. The structure of crystallized plant AOS
has not been investigated. Oldham et al. recently
reported the crystal structure of coral allene oxide syn-
thase,24 the enzyme that uses 8-(R)-hydroperoxyeicosa-
tetraenoic acid as a substrate. With the aid of a
structure-based sequence alignment, we found the se-
quence identity between Arabidopsis AOS and coral
AOS is only about 10% (data not shown), so that the
3D structure of Arabidopsis AOS may be different from
that of coral AOS. Data obtained from this study

1094
K. Oh et al. / Bioorg. Med. Chem. 16 (2008) 1090–1095
indicate that the AOS–JM-8686 interaction is highly re-
stricted by the stereochemical configuration of the inhib-
itor. This observation implies that JM-8686 is a useful
tool for determining the structure of AOS. Further stud-
ies of the in vivo actions of this synthetic inhibitor are
now in progress.
1H), 6.87 (s, 1H), 6.91 (s, 1H), 7.31 (dd, J = 8.4,
2.2 Hz, 1H), 7.41 (d, J = 4.8 Hz, 1H), 7.42 (s, 1H),
7.59 (d, J = 8.4 Hz, 1H). ESI-MS m/z 257.2 (M+H),
Calcd 257.1 (M+H).
4.2.4. Preparation of (S)-1-(2,4-dichlorophenyl)-2-imida-
zol-1-yl-ethanol (3b). The 3b isomer was prepared in a
similar way using (S)-BINAL-H. White rod crystal:
1
3. Conclusion
mp 111.7–112.7. H NMR (CDCl3) d 3.86 (dd, J = 8.3,
14.2 Hz, 1H), 4.22 (dd, J = 2.2, 13.9 Hz, 1H), 5.23 (dd,
J = 1.8, 8.4 Hz, 1H), 6.87 (s, 1H), 6.92 (s, 1H), 7.31
(dd, J = 8.4, 2.2 Hz, 2H), 7.41 (d, J = 2.2 Hz, 2H), 7.59
(d, J = 8.4 Hz, 1H). ESI-MS m/z 257.2 (M+H), Calcd
257.2 (M+H).
We synthesized two enantiomers of JM-8686 by asym-
metric reduction of 1-(2,4-dichlorophenyl)-2-imidazol-
1-yl-ethanone using chiral BINAL-H derivatives
coupled with chiral HPLC. Analyses of the binding
affinity and inhibitory activity of both enantiomers indi-
cate that the potency of the (R)-isomer is greater than
that of the (S)-isomer.
4.2.5. Preparation of (R)-8-[1-(2,4-dichlorophenyl)-2-imi-
dazol-1-yl-ethoxy] octanoic acid heptyl ester (4a).
Compound 4a was prepared using (R)-1-(2,4-dichloro-
phenyl)-2-imidazol-1-yl-ethanol (3a) by the method de-
1
4. Experimental
4.1. General
scribed previously.15 Colorless oil. H NMR (CDCl3) d
0.88 (t, J = 6.6 Hz, 3H), 1.24–1.32 (m, 16H), 1.45–1.65
(m, 4H), 2.29 (t, J = 7.7 Hz, 2H), 3.18–3.23 (m, 1H),
3.29–3.35 (m, 1H), 3.98 (dd, J = 7.4, 14.4 Hz, 1H),
4.05 (t, J = 6.6 Hz, 2H), 4.17 (dd, J = 2.5, 14.4 Hz,
1H), 4.83 (dd, J = 2.5, 7.4 Hz, 1H), 6.92 (s, 1H), 7.01
(s, 1H), 7.26 (s, 2H), 7.41 (s, 1H), 7.45 (s, 1H). ESI-
MS m/z 498.3 (M+H), Calcd 498.2 (M+H).
Melting points (mp) were determined with a Yanako
melting point apparatus. 1H NMR spectra were re-
corded with a JOEL ECP-400 spectrometer, chemical
shifts being expressed in ppm downfield from TMS as
an internal standard. Electrospray ionization mass spec-
tra (ESI-MS) were recorded on a PE Sciex API-2000
LC/MS System. Optical rotations were measured on a
JASCO P-1020 polarimeter using methanol solutions
at concentrations given in g/100 cm3. Solvents were
either dehydrated reagent or of HPLC grade. Ampicillin
and isopropyl-D-thiogalactopyranoside were purchased
from Wako Chemical Inc. (Tokyo, Japan). Linolenic
acid and soybean lipoxygenase were obtained from Sig-
ma (Tokyo, Japan). Other reagents of the highest qual-
ity were purchased from Tokyo Kasei Co. (Tokyo,
Japan).
4.2.6. Preparation of (S)-8-[1-(2,4-dichlorophenyl)-2-imi-
dazol-1-yl-ethoxy] octanoic acid heptyl ester (4b). Com-
pound 4b was prepared in a similar way by using 3b.
1
Colorless oil. H NMR (CDCl3) d 0.88 (t, J = 6.6 Hz,
3H), 1.24–1.32 (m, 16H), 1.49–1.69 (m, 4H), 2.29 (t,
J = 7.7 Hz, 2H), 3.18–3.24 (m, 1H), 3.29–3.35 (m, 1H),
3.98 (dd, J = 7.3, 14.3 Hz, 1H), 4.05 (t, J = 6.6 Hz,
2H), 4.17 (dd, J = 2.6, 14.3 Hz, 1H), 4.81 (dd, J = 2.6,
7.3 Hz, 1H), 6.92 (s, 1H), 7.02 (s, 1H), 7.26 (s, 2H),
7.41 (s, 1H), 7.45 (s, 1H). ESI-MS m/z 498.2 (M+H),
Calcd 498.2 (M+H).
4.2. Chemistry
4.2.7. Circular dichroism (CD). CD measurements were
performed by using a J-720 spectropolarimeter (Jasco)
as described previously.25 Methanol was used as a
blank. Samples were dissolved in methanol at a concen-
tration of 0.1 mg/mL and placed in a cuvette with a 1 cm
path length. Spectra were scanned at 25 °C in the region
220–300 nm. The observed specific ellipticity (difference
between sample and blank) was converted to the mean
residue ellipticity h (deg/cm2/dmol).
4.2.1.
Preparation
of
13(S)-hydroperoxy-
9(Z),11(E),15(Z)-octadecatrienoic acid. 13(S) HPOT
was prepared according to the method described previ-
ously.15 The stock solution of 13(S) HPOT was pre-
pared by dissolving 13(S) HPOT in methanol (10 mM)
and was stored at ꢀ20 °C.
4.2.2. Preparation of 1-(2,4-dichlorophenyl)-2-imidazol-1-
yl-ethanone (2). Compound 2 was prepared by using 2,4-
dichlorophenacyl bromide as described previously.17
White needle crystal: mp 78.3–79.3 °C. 1H NMR
(CDCl3) d 5.33 (s, 2H), 6.92 (s, 1H), 7.02 (s, 1H), 7.38
(d, J = 9.1 Hz, 1H), 7.51 (s, 2H), 7.57 (d, J = 9.1 Hz,
1H). ESI-MS m/z 255.1 (M+H), Calcd 255.0 (M+H).
4.2.8. HPLC separation of stereoisomers. The optical
purity of the synthesized compounds was examined
by HPLC with a chiral stationary phase column (Dai-
cel Chem. Ltd, CHIRALPAK OJ, 4.6 mm 250 mm),
using n-hexane/2-propanol (15:1) as the eluent, at a
flow rate of 2 mL/min with a detection wavelength of
254 nm.
4.2.3. Preparation of (R)-1-(2,4-dichlorophenyl)-2-imida-
zol-1-yl-ethanol (3a). Preparation of 3a was carried out
by the method using (R)-BINAL-H as described previ-
ously.18 White rod crystal: mp 115.6–117.1 °C. 1H
NMR (CDCl3) d 3.87 (dd, J = 8.4, 4.3 Hz, 1H), 4.22
(dd, J = 2.2, 14.3 Hz, 1H), 5.24 (dd, J = 2.0, 8.4 Hz,
4.3. Biology
4.3.1. Expression and purification of recombinant AOS.
The coding region of AOS cDNA that was restricted by
the enzymes BamHI and KpnI was inserted into an

K. Oh et al. / Bioorg. Med. Chem. 16 (2008) 1090–1095
1095
2. Feussner, I.; Wasternack, C. Annu. Rev. Plant Physiol.
Plant Mol. Biol. 2002, 53, 275.
Escherichia coli expression vector pQE30 (Qiagen).
E. coli M15, transformed with this construct, was kindly
3. Laudert, D.; Pfannschmidt, U.; Lottspeich, F.; Hollander-
Czytko, H.; Weiler, E. W. Plant Mol. Biol. 1996, 31, 323.
4. Kessler, A.; Baldwin, I. T. Annu. Rev. Plant Biol. 2002, 53,
299.
5. Browse, J. Vitam. Horm. 2005, 72, 431.
6. Weber, H. Trends Plant Sci. 2002, 7, 217.
provided by Prof. E.W. Weiler, Lehrstuhl fur Pflanzen-
¨
physiologie, Fakulta¨t fur Biologie Ruhr-Universita¨t,
¨
Germany. Expression and purification of recombinant
AOS were performed as described previously.16
4.3.2. In vitro assays for AOS. The enzyme reaction mix-
ture contained 50 mM sodium phosphate buffer (pH 7.0,
0.1% Tween 20), enzyme (AOS, 5 nM), and designated
concentrations of substrate (13(S) HPOT) at 25 °C.
Activity was measured by following the decrease in
absorption at 235 nm using a Shimadzu UV3100 spec-
trophotometer (Shimadzu, Kyoto, Japan). An absorp-
tion coefficient of 22.8 mMꢀ1/cmꢀ1 was used.
7. Creelman, R. A.; Mullet, J. E. Annu. Rev. Plant Physiol.
Plant Mol. Biol. 1997, 48, 355.
8. He, Y.; Fukushige, H.; Hildebrand, D. F.; Gan, S. Plant
Physiol. 2002, 128, 876.
9. Staswick, P. E. Plant Cell 1990, 2, 1.
10. McConn, M.; Browse, J. Plant Cell 1996, 8, 403.
11. Li, C.; Liu, G.; Xu, C.; Lee, G. I.; Bauer, P.; Ling, H. Q.;
Ganal, M. W.; Howe, G. A. Plant Cell 2003, 15, 1646.
12. Li, C.; Schilmiller, A. L.; Liu, G.; Lee, G. I.; Jayanty, S.;
Sageman, C.; Vrebalov, J.; Giovannoni, J. J.; Yagi, K.;
Kobayashi, Y.; Howe, G. A. Plant Cell 2005, 17, 971.
13. Laudert, D.; Weiler, E. W. Plant J. 1998, 15, 675.
14. Park, J. H.; Halitschke, R.; Kim, H. B.; Baldwin, I. T.;
Feldmann, K. A.; Feyereisen, R. Plant J. 2002, 31, 1.
15. Oh, K.; Murofushi, N. Bioorg. Med. Chem. 2002, 10, 3707.
16. Oh, K.; Asami, T.; Matsui, K.; Howe, G. A.; Murofushi,
N. FEBS Lett. 2006, 580, 5791.
4.3.3. Binding assay of JM-8686 to recombinant AOS.
Binding of JM-8686 to AOS was measured by optical
difference spectroscopy of the purified recombinant
AOS using a Shimadzu UV3100 spectrophotometer as
described previously.16
17. Aren, E. J.; Wuis, E. W.; Veringa, E. J.; Eveline, W. J.
Biolochem. Pharmacol. 1988, 50, 3486.
Acknowledgments
18. Noyori, R. J. Am. Chem. Soc. 1979, 101, 3129.
19. Liao, Y. W.; Li, H. X. Yaoxue Xuebao 1993, 28, 22.
20. Jefcoate, C. R. Methods Enzymol. 1978, 52, 258.
21. Song, W. C.; Baertschi, S. W.; Boeglin, W. E.; Harris, T.
M.; Brash, A. R. J. Biol. Chem. 1993, 268, 6293.
22. Oldham, M. L.; Brash, A. R.; Newcomer, M. E. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 297.
We thank Prof. Dr. Elmar W. Weiler, Lehrstuhl fur
¨
Pflanzenphysiologie, Fakulta¨t fur Biologie Ruhr-Uni-
¨
versita¨t, Germany, for his generous supply of the
pQE30-AOS clone.
23. Porter, T. D.; Coon, M. J. J. Biol. Chem. 1991, 266,
13469.
References and notes
24. Ullrich, V.; Fraf, H. Trends Pharmacol. Sci. 1984, 8, 352.
25. Itoh, H.; Tashima, Y. Biochem. J. 1997, 326, 567.
1. Howe, G. A.; Schilmiller, A. L. Curr. Opin. Plant Biol.
2002, 5, 230.