1
3710
K. Oh , N. Murofushi / Bioorg. Med. Chem. 10 (2002) 3707–3711
2
(
7
7
H), 7.41 (s, 1H), 7.45 (s, 1H). ESI-MS m/e 412
M+H)+. Anal. calcd for C H Cl N O: C, 62.66; H,
.36; N, 7.30; Cl, 18.50. Found: C, 62.58; H, 7.29; N,
.25; Cl, 18.55.
(m, 1H), 3.29–3.35 (m, 1H), 3.98 (dd, J=7.5, 14.6 Hz,
1H), 4.08–4.19 (m, 3H), 4.82 (dd, J=2.7, 7.2 Hz, 1H),
6.92 (s, 1H), 7.02 (s, 1H), 7.27 (s, 2H), 7.41 (s, 1H), 7.45
(s, 1H). ESI-MS m/e 470 (M+H). Anal. calcd for
C H Cl N O : C, 61.41; H, 7.30; N, 5.97; Cl, 15.10.
2
0
28
2
2
2
4
34
2
2
3
1
-[2-Butoxy-2-(2,4-Dichlorophenyl)-ethyl]-1H-imidazole
1
Found: C, 61.52; H, 7.35; N, 6.02; Cl, 15.14.
(d). Light yellow oil. H NMR (CDCl ) d 0.88 (t, J=7.5
Hz 3H), 1.25–1.37 (m, 2H), 1.48–1.55 (m, 2H), 3.20–
3
Heptyl 8-[1-(2,4-dichlorophenyl)-2-imidazolylethoxy]-oc-
1
3
.26 (m, 1H), 3.31–3.36 (m, 1H), 3.97 (dd, J=7.5, 14.6
tanoate (k). Light yellow oil. H NMR (CDCl ) d 0.88
3
Hz, 1H), 4.17 (dd, J=2.6, 14.3 Hz, 1H), 4.83 (dd,
J=2.7, 7.3 Hz, 1H), 6.92 (t, J=1.3 Hz,1H), 7.01 (d,
J=1.1 Hz, 1H), 7.25 (s, 2H), 7.41 (s, 1H), 7.45 (s, 1H).
ESI-MS m/e 314 (M+H). Anal. calcd for
C H Cl N O: C, 57.52; H, 5.79; N, 8.94; Cl, 22.64.
(t, J=3.3 Hz, 3H), 1.27–1.40 (m, 10H), 1.51–1.71 (m,
10H), 2.29 (t, J=7.2 Hz, 2H), 3.20–3.24 (m, 1H), 3.29–
3.35 (m, 1H), 3.98 (dd, J=7.4, 14.6 Hz, 1H), 4.03–4.19
(m, 3H), 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/e 498 (M+H). Anal. calcd for C H Cl N O : C,
1
5
18
2
2
Found: C, 57.53; H, 5.70; N, 8.96; Cl, 22.69.
2
6
38
2
2
3
62.77; H, 7.70; N, 5.63; Cl, 14.25. Found: C, 62.85; H,
7.85; N, 5.83; Cl, 14.34.
Ethyl 5-[1-(2,4-dichlorophenyl)-2-imidazolylethoxy] pen-
1
tanoate (e). Light yellow oil. H NMR (CDCl ) d 1.26
3
(t, J=7.1 Hz, 3H), 1.55–1.66 (m, 2H), 2.28 (t, J=7.1
Hz, 2H), 3.21–3.26 (m, 1H), 3.32–3.37 (m, 1H), 3.98 (dd,
J=7.3, 14.6 Hz, 1H), 4.11–4.20 (m, 3H), 4.84 (dd, J=2.6,
Preparation of 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-oc-
tadecatrienoic acid. Linolenic acid (925 mg, 3.3 mmol)
was dissolved in a sodium borate buffer solution (470
mL; 0.1 M, pH=9) under nitrogen atmosphere and
7
1
.3 Hz, 1H), 6.92 (s, 1H), 7.02 (s, 1H), 7.25 (s, 2H), 7.41 (s,
H), 7.45 (s, 1H). ESI-MS m/e 385 (M+H). Anal. calcd
ꢀ
cooled to 0 C in a ice bath. Soybean lipoxygenase I (89
for C H Cl N O : C, 56.11; H, 5.76; N, 7.27; Cl, 18.40.
3
Found: C, 56.11; H, 5.70; N, 7.30; Cl, 18.56.
mg) was added, a gentle stream of oxygen was bubbled
through the solution via a Pasteur pipet, and the mix-
ture was stirred for 30 min. Progress of the reaction was
monitored by UV spectroscopy at 234 nm. After the
reaction, the mixture was adjusted to pH 3 with 0.1 M
hydrochloric acid solution and extracted twice with 300
mL of chloroform/methanol (2:1 v/v). The chloroform
layer was collected and concentrated under reduced
pressure. The samples were resuspended in 1 mL n-hex-
ane and loaded onto a silica gel column (1–20 cm) pre-
washed with n-hexane. The loaded column was washed
with 30 mL of n-hexane/diethyl ether/acetic acid
(90:10:1 v/v/v), and 13(S)-HPOT was eluted with 30 mL
of hexane/diethyl ether/acetic acid (75:25:1 v/v/v). After
1
8
22
2
2
Ethyl 6-[1-(2,4-dichlorophenyl)-2-imidazolylethoxy]-hex-
1
anoate (f). Light yellow oil. H NMR (CDCl ) d 1.25
3
(
t, J=7.2 Hz, 3H), 1.55–1.66 (m, 4H), 2.27 (t, J=7.2
Hz, 2H), 3.19–3.24 (m, 1H), 3.31–3.37 (m, 1H), 3.97
dd, J=7.3, 14.6 Hz, 1H), 4.10–4.29 (m, 3H), 4.83 (dd,
J=2.6, 7.3 Hz, 1H), 6.92 (s, 1H), 7.02 (s, 1H), 7.25 (s,
H), 7.41 (s, 1H), 7.44 (s, 1H). ESI-MS m/e 400
M+H). Anal. calcd for C H Cl N O : C, 57.15; H,
(
2
(
1
9
24
2
2
3
6
7
.06; N, 7.02; Cl, 17.76. Found: C, 57.11; H, 6.15; N,
.10; Cl, 17.80.
Propyl 8-[1-(2,4-dichlorophenyl)-2-imidazolylethoxy]-oc-
1
evaporation of the solvent under N , 13 (S)-HPOT was
2
dissolved in methanol and stored at À80 C.
ꢀ
tanoate (h). Light yellow oil. H NMR (CDCl ), d 0.94
3
(
t, J=7.3 Hz, 3H), 1.24–1.29 (m, 2H), 1.50–1.69 (m,
1
3
0H), 2.30 (t, J=7.3 Hz, 2H), 3.19–3.24 (m, 1H), 3.29–
.35 (m, 1H), 3.98 (dd, J=7.5, 14.7 Hz, 1H), 4.03–4.19
Expression and purification of recombinant AOS in E.
coli. The codingre gi on of AOS cDNA that was restric-
ted by the enzymes BamHI and KpnI, was inserted into
an E. coli expression vector pQE30 (Qiagen). E. coli M15,
transformed with this construct, was kindly provided by
(m, 3H), 4.82 (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/e 442 (M+H). Anal. calcd for C H Cl N O : C,
3
2
2
30
2
2
5
6
9.86; H, 6.85; N, 6.35; Cl, 16.04. Found: C, 59.99; H,
.82; N, 6.39; Cl, 16.06.
Prof. E. W. Weiler, Lehrstuhl fu
¨
Fakultat fur Biologie Ruhr-Universita
r Pflanzenphysiologie,
t, Germany. An
¨
¨
¨
overnight culture of M15 E. coli (15 mL) was added to 1 L
of fresh Turia-Bertani medium with amphicillin (100 mg/
mL), placed in 2-L culture flasks and shaken at 100 rpm
Butyl 8-[1-(2,4-dichlorophenyl)-2-imidazolylethoxy]-oc-
1
tanoate (i). Light yellow oil. H NMR (CDCl ) d 0.93
3
ꢀ
(
t, J=7.3 Hz, 3H), 1.24–1.30 (m, 4H), 1.51–1.71 (m,
and at 37 C. After 3 h of incubation, isopropyl-d-thioga-
1
3
0H), 2.29 (t, J=7.2 Hz, 2H), 3.20–3.24 (m, 1H), 3.29–
.35 (m, 1H), 3.98 (dd, J=7.4, 14.6 Hz, 1H), 4.03–4.19
lacto-pyranoside (IPTG) was added to the culture to a
final concentration of 1 mM to induce expression of the
recombinant Arabidopsis AOS protein. The culture was
(m, 3H), 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
ꢀ
shaken continuously at 100 rpm for 8 h at 16 C.
m/e 456 (M+H)+. Anal. calcd for C H Cl N O : C,
2
3
32
2
2
3
6
6
0.66; H, 7.08; N, 6.15; Cl, 15.57. Found: C, 60.59; H,
.90; N, 6.22; Cl, 15.69.
Cells of E. coli from a 1-L culture were pelleted and
resuspended in 50 mL of phosphate buffer (50 mM,
pH=8.0) plus 0.1% Triton X-100. The cell suspension
was sonicated 15 times for 30 s each time while on ice.
The soluble fraction (supernatant) was prepared by
3
-Methylbutyl
8-[1-(2,4-dichlorophenyl)-2-imidazolyl-
1
ethoxy]octanoate (j). Light yellow oil. H NMR
CDCl ) d 0.87 (s, 3H), 0.89 (s, 3H), 1.24–1.27 (m, 8H),
ꢀ
(
centrifugation at 10,000g for 1 h at 4 C. This super-
natant fraction was then applied to a His-Tagaffinity
3
1
.48–1.73 (m, 6H), 2.30 (t, J=7.2 Hz, 2H), 3.20–3.24