Enlarged analogues of uniconazole, new azole containing inhibitors of ABA 8′-hydroxylase CYP707A

Article abstract of DOI:10.1016/j.bmcl.2009.07.137

We enlarged the uniconazole (UNI) molecule to find a specific inhibitor of abscisic acid (ABA) 8′-hydroxylase, and synthesized various UNI derivatives that were substituted with hydrophilic and hydrophobic groups at the 4-chlorine of the phenyl group of U

Full text of DOI:10.1016/j.bmcl.2009.07.137

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5782–5786
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Bioorganic & Medicinal Chemistry Letters
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Enlarged analogues of uniconazole, new azole containing inhibitors of ABA
80-hydroxylase CYP707A
a
a
a
b
Yasushi Todoroki a, , Hikaru Aoyama , Saori Hiramatsu , Minaho Shirakura , Hataitip Nimitkeatkai ,
*
Satoru Kondo b, Kotomi Ueno c, , Masaharu Mizutani d,à, Nobuhiro Hirai e
a Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan
b Division of Bioresource Science, Graduate School of Horticulture, Chiba University, Matsudo 271-8510, Japan
c The United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan
d Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
e Division of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8501, Japan
a r t i c l e i n f o
a b s t r a c t
We enlarged the uniconazole (UNI) molecule to find a specific inhibitor of abscisic acid (ABA) 80-hydrox-
ylase, and synthesized various UNI derivatives that were substituted with hydrophilic and hydrophobic
groups at the 4-chlorine of the phenyl group of UNI using click chemistry. Considering its potency in ABA
80-hydroxylase inhibition, its small effect on seedling growth, and its ease of application, UT4, the UNI
derivative containing the C4 alkyltriazole, was the best candidate for a highly selective inhibitor of ABA
80-hydroxylase.
Article history:
Received 24 June 2009
Revised 28 July 2009
Accepted 29 July 2009
Available online 3 August 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Plant growth regulator
P450 inhibitor
Abscisic acid
Abscisic acid (ABA) is a plant hormone involved in stress toler-
ance, stomatal closure, seed dormancy, and other physiological
events.1–4 The endogenous levels of ABA in plants are cooperatively
controlled by biosynthesis, transportation, and catabolic inactiva-
tion in response to environmental changes.1–4 A natural or artificial
chemical that perturbs this highly controlled system is promising
not only as a chemical probe for the mechanism of ABA action,5
but also because of its potential use in agriculture and horticulture.
Although ABA is registered as a farm chemical (plant growth regu-
lator), its practical use has been limited mainly due to its weak ef-
fect in field trials,6 which is considered to be due to its rapid
inactivation through biodegradation. Catabolic inactivation of
ABA is mainly controlled by ABA 80-hydroxylase, which is the cyto-
chrome P450 catalyzing the C80-hydroxylation of ABA into 80-hy-
droxy-ABA and its more stable tautomer, phaseic acid, which has
much lower hormonal activity than ABA (Fig. 1).4 ABA 80-hydroxy-
lase was identified as CYP707A1-4 in the model plant Arabidopsis
thaliana in 2004,7,8 and since then many CYP707A isozymes have
been found in various plants.9–12 Gene knockdown and overex-
pression studies suggest that ABA 80-hydroxylase is a key enzyme
for controlling ABA concentration during water deficit stress or
dormancy maintenance and breaking.13,14
To chemically control ABA 80-hydroxylation in plants, we are
developing ABA analogues that function as competitive inhibitors
against ABA 80-hydroxylase.15–17 AHI4, which was developed in a
previous study, requires a very high concentration (>400 lM) to
confer drought tolerance by its exogenous application.17 On the
other hand, uniconazole (UNI), which is a well-known plant retar-
dant developed in the 1980s,18,19 strongly inhibits ABA 80-hydrox-
ylase both in vitro and in vivo.20–22 Saito et al. reported that UNI
induces drought tolerance in Arabidopsis plants at 50 l
M.21 The
main site of action of UNI is suggested to be ent-kaurene oxidase
(CYP701A),23 which catalyzes the three-step oxidation of ent-kau-
rene to ent-kaurenoic acid, a biosynthetic precursor of the plant
hormone gibberellin (Fig. 1). Thus, UNI has been used as a chemical
tool inhibiting gibberellin biosynthesis. UNI also inhibits brassinos-
teroid biosynthesis24,25 and alters the level of other plant hor-
mones, such as auxins, cytokinins, and ethylene.26 Thus, we
cannot use UNI in expectation of only inhibiting ABA catabolism,
in spite of its strong effect on ABA levels.
* Corresponding author. Tel./fax: +81 54 238 4871.
E-mail address: aytodor@agr.shizuoka.ac.jp (Y. Todoroki).
Present address: Graduate School of Agricultural and Life Sciences, The University
of Tokyo, Tokyo 113-8657, Japan.
Present address: Graduate School of Agricultural Science, Kobe University, Kobe
657-8501, Japan.
Azole-type P450 inhibitors including UNI bind to the target
P450 active site by both coordinating to the heme-iron atom and
interacting with surrounding protein residues. Because heme coor-
dination is a common property of azole containing inhibitors, their
affinity and specificity for individual P450 enzymes depend on
à
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.137

Y. Todoroki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5782–5786
5783
Figure 1. S-UNI is a potent inhibitor of ABA 80-hydroxylase and ent-kaurene oxidase.
structural properties other than the azole group. UNI may be small
and flexible enough to embed itself into the various substrate-
binding pockets. This led us to the possibility that enlarging or con-
formationally freezing the UNI molecule would result in a narrow
inhibition spectrum. For this research, we focused on enlarging the
molecule.
of Funaki et al.19,29 and Hallahan et al.30 The azidation reaction30 of
1 gave the azide 2 to be used in a click reaction31 with commer-
cially available acetylene compounds, which have linear alkyl
chains including three alcohols and an acid, to give 11 kinds of
UT (4A, 1H, 2H, 4H, 3, 4, 7, 9, 11, 13, and 15) as racemic compounds
(Scheme 1).32 The click reactions were performed with sodium
ascorbate and CuSO4 in THF. Although most click reactions be-
tween azide and acetylene derivatives quantitatively produce only
one coupling product, the reactions using 2 gave at least three
products, including UT, the main one. Although the structure of
byproducts was not established, NMR analysis indicated that the
conjugated system of 2 was involved in the side reaction. All of
the racemic UT compounds were isolated using preparative-scale
HPLC before conducting enzyme and biological assays.
Although the crystal structures of P450s indicate an overall sim-
ilarity in structural folding in spite of limited sequence identity, re-
gions responsible for substrate binding differ substantially. These
regions correspond to the substrate recognition sites, SRS1-6.27 In
particular, SRS1-3 and 6, which are far from the heme in the active
site, are structurally quite variable and are involved significantly in
substrate specificity. In the case of UNI, the portion of the molecule
far from the triazole interacting with the heme is the 4-chlorine of
the phenyl ring, which might interact with SRS1-3 and 6. Thus, we
selected C4 of the phenyl in UNI as a scaffold for enlarging the UNI
molecule. This paper describes the synthesis, CYP707A-inhibitory
properties and biological activities of the enlarged UNI analogues,
UT.
Inhibitory activity against ABA 80-hydroxylase (CYP707A) was
examined using recombinant Arabidopsis CYP707A3 coexpressed
with Arabidopsis P450 reductase (AR2) in E. coli.33 The activity
was evaluated based on the decrease of the enzyme product, pha-
seic acid, caused by addition of a test compound at a concentration
We used click chemistry,28 by which a variety of compounds are
easily prepared, to enlarge the UNI molecule. A known compound 1
(a racemic mixture) was synthesized by a slightly modified method
two times higher (10 lM) than the substrate S-ABA (5 l
M).34 The
alkyl-type UT, except for the longest-chain UT15, exhibited high
inhibitory activity (greater than 90%), which was equivalent to that
Scheme 1. Synthesis of UT. R: see Table 1. Reagents and conditions: (i) HCl, NaNO2, urea, NaN3, 0 °C, 75%; (ii) alkyne, THF, CuSO4, sodium ascorbate, rt, 30–40%.

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Y. Todoroki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5782–5786
Table 1
Inhibitory activity of UT against recombinant CYP707A3 and rice seedling elongation
Compound
Structural properties
CYP707A3
Rice
c
–R
—
log Pa
Inhibitionb (%)
IC50
0.18
NId
NI
NI
NI
21
42
(l
M)
S-UNI
3.13
100 (KI = 10 nM)
UT4A
UT1H
UT2H
UT4H
UT3
–(CH2)3–COOH
–CH2OH
–CH(OH)–CH3
–CH(OH)–(CH2)2–CH3
–(CH2)2–CH3
–(CH2)3–CH3
2.64
1.55
2.12
3.08
3.51
3.96
63
83
77
92
92
UT4
100 (KI = 195 nM)
UT7
UT9
UT11
UT13
UT15
–(CH2)6–CH3
–(CH2)8–CH3
–(CH2)10–CH3
–(CH2)12–CH3
–(CH2)14–CH3
5.29
6.18
7.07
7.96
8.85
96
100
100
95
NTe
NT
NT
NT
NT
54
a
Calculated partition coefficient (Marvin, ChemAxon, http://www.chemaxon.com/).
Inhibition ratio of compounds (10
The concentration for 50% inhibition (average of at least two sets of experiments).
No significant inhibition even at 100 lM (max concentration tested).
Not tested because the compound was not dissolved in water.
b
c
l lM) (average of two sets of experiments).
M) in the 80-hydroxylation of ABA (5
d
e
involved in seedling growth; the hydrophilic substitution is espe-
cially crucial.
Considering its potency in ABA 80-hydroxylase inhibition, its
small effect on seedling growth, and its ease of application, UT4
was the best candidate for a highly selective inhibitor of ABA 80-
Figure 2. Inhibitory effect of UT and S-UNI on rice seedling growth.
of S-UNI (Table 1). The mode of inhibition of UT4 was determined
to be competitive by plotting the reaction velocities in the presence
and absence of inhibitor on a double-reciprocal plot; the inhibition
constant (KI) was 195 nM. Although this is larger than the value for
S-UNI (10 nM), UT4 has strong affinity to the active site of ABA 80-
hydroxylase, considering that the KM value for (+)-ABA was 3.4 lM
under the same experimental conditions. The alcohol-types UT1H,
UT2H, and UT4H were also effective (75–85% inhibition) but were
less potent than the alkyl-types. The acid-type UT4A was much less
effective than the alcohol-types. These results suggest the follow-
ing: (1) linear elongation at C4 of the phenyl in UNI is generally
not crucial to binding to the active site of ABA 80-hydroxylase;
(2) the protic group in the elongated portion of the molecule
may have a negative effect on binding to the enzyme, and (3) there
is an upper limit to the length and hydrophobicity of the elongated
portion that allows binding to the enzyme.
The alkyl-types of UT are highly hydrophobic; UT7-15 had an
especially high estimated log P value (>5) (Table 1). Compounds
with very high log P value are too poorly water soluble. In fact,
UT7-15 did not dissolve in water, although it dissolved in the buf-
fer used in the enzyme assay. These compounds are not practical to
use as a plant regulator although they may be useful as chemical
probes for in vitro enzymatic studies. Thus, only the UT compounds
with a log P value <4 were assayed for rice seedling arrest.35 The
acid- and alcohol-types of UT were not effective even at 100
whereas the alkyl-types showed a significant effect at a concentra-
tion greater than 30 M (Fig. 2). These results suggest that elonga-
tion at C4 of the phenyl in UNI depresses the affinity to enzymes
lM,
l
Figure 3. Drought tolerance of Akitabeniakari apple seedlings treated with (a) UT4
and (b) S-UNI.

Y. Todoroki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5782–5786
5785
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18. Funaki, Y.; Ishiguri, T.; Kato, T.; Tanaka, S. J. Pesticide Sci. 1984, 9, 229.
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21. Saito, S.; Okamoto, M.; Shinoda, S.; Kushiro, T.; Koshiba, T.; Kamiya, Y.; Hirai,
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Figure 4. Drought tolerance of Fuji apple seedlings treated with distilled water
(left), 10 M of UT (center), and 10 M of S-UNI (right).
l
l
27. Gotoh, O. J. Biol. Chem. 1992, 267, 83.
28. Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128. and references
cited therein.
hydroxylase. To test the effect of UT4 on drought tolerance, 90-
day-old apple seedlings were sprayed with an aqueous solution
29. Funaki, Y.; Ishiguri, Y.; Kato, T.; Tanaka, S. J. Pesticide Sci. 1984, 9, 229.
30. Hallahan, D. L.; Heasman, A. P.; Grossel, M. C.; Quigley, R.; Hedden, P.; Bowyer,
J. R. Plant Physiol. 1988, 88, 1425. 2: 1H NMR (270 MHz, acetone-d6): d 0.69 (9H,
s, t-butyl), 4.77 (1H, d, J = 6.3 Hz, H-3), 5.10 (1H, d, J = 6.3 Hz, HO-3), 7.09 (1H, s,
H-1), 7.18 and 7.52 (each 2H, m, 4-N3-phenyl), 8.02 (1H, s, H-300), 8.82 (1H, s, H-
500); HRMS (FAB): calcd for C15H19ON6 [M+H]+ 299.1620, found 299.1628.
31. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004.
containing UT4 at concentrations of 10, 50, and 100
lM for cultivar
Akitabeniakari ( Fig. 3) and 10
l
M for Fuji ( Fig. 4).36 The UT4-
sprayed seedlings showed drought tolerance at all tested concen-
trations. Although UNI showed a similar effect, browning of Akita-
32. To a stirred solution of 2 (20 mg, 67 lmol) and alkyne (67 lmol) in THF
beniakari leaves was observed at 100
may be caused by inhibition of other enzymes including P450s,
was never observed at 100 M UT4 treatment. This result suggests
lM. This side effect, which
(10 mL) was added aqueous CuSO4 (100 mM, 10 mL) and aqueous sodium
ascorbate (100 mM, 10 mL) at room temperature. The mixture was stirred for
1 h before extraction with EtOAc (30 mL ꢀ 3). The organic layer was washed
with brine, dried over Na2SO4, and concentrated in vacuo. After filtering
l
that UT4 functions as a more selective inhibitor of ABA 80-hydrox-
ylase than UNI. In this research, we used racemic UT compounds. In
the case of UNI, the S-enantiomer are effective in plants, whereas
the R-enantiomer are ineffective in plants but act as fungicides.37,38
Therefore, the activity of racemic UT compounds in present assays
may have been caused by the S-enantiomers. We plan to prepare
optically pure UT compounds to verify whether UT has the same
trend observed for UNI.
through a 0.2-lM acetyl cellulose membrane filter (YMC Duo-Filter QDUO 15),
the filtrate was purified by semipreparative HPLC with a reverse phase C18
column (YMC ODS-AQ, 150 ꢀ 20 mm) to obtain UT (30–40% yield). UT4A: 1H
NMR (270 MHz, acetone-d6): d 0.68 (9H, s, t-butyl), 2.19 (2H, m, –CH2–CH2–
CH2–COOH), 2.34 (2H, m, –CH2–CH2–CH2–COOH), 2.84 (2H, t, J = 7.6 Hz, –CH2–
CH2–CH2–COOH), 4.78 (1H, s, H-3), 7.18 (1H, s, H-1), 7.63 and 7.93 (each 2H, m,
phenyl), 8.10, 8.38, and 8.95 (each 1H, s, 1,2,4-triazole and 1,2,3-triazole);
HRMS (ESI-TOF, positive mode): calcd for C21H26N6O3Na [M+Na]+ 433.1964,
found 433.1962. UT1H: 1H NMR (270 MHz, acetone-d6): d 0.68 (9H, s, t-butyl),
4.71 (1H, s, H-3), 7.19 (1H, s, H-1), 7.65 and 7.93 (each 2H, m, phenyl), 8.11,
8.51, and 8.96 (each 1H, s, 1,2,4-triazole and 1,2,3-triazole); HRMS (ESI-TOF,
positive mode): calcd for C18H22N6O2Na [M+Na]+ 377.1701, found 377.1705.
UT2H: 1H NMR (270 MHz, acetone-d6): d 0.68 (9H, s, t-butyl), 1.60 (3H, d,
J = 6.6 Hz, –CH(OH)–CH3), 4.71 (1H, s, H-3), 5.06 (1H, q, J = 6.6 Hz, –CH(OH)–
CH3), 7.18 (1H, s, H-1), 7.64 and 7.94 (each 2H, m, phenyl), 8.11, 8.46, and 8.95
(each 1H, s, 1,2,4-triazole and 1,2,3-triazole); HRMS (ESI-TOF, positive mode):
calcd for C19H24N6O2Na [M+Na]+ 391.1858, found 391.1865. UT4H: 1H NMR
(270 MHz, acetone-d6): d 0.67 (9H, s, t-butyl), 0.93 (3H, t, J = 7.6 Hz, –CH(OH)–
(CH2)2–CH3), 1.47 and 2.08 (each 2H, m, –CH(OH)–(CH2)2–CH3), 4.78 (1H, s, H-
3), 4.89 (1H, t, J = 6.6 Hz, –CH(OH)–(CH2)2–CH3), 7.16 (1H, s, H-1), 7.67 and 7.97
(each 2H, m, phenyl), 8.02, 8.43, and 8.87 (each 1H, s, 1,2,4-triazole and 1,2,3-
triazole); HRMS (ESI-TOF, positive mode): calcd for C21H28N6O2Na [M+Na]+
419.2171, found 419.2175. UT3: 1H NMR (270 MHz, CD3OD): d 0.68 (9H, s, t-
butyl), 1.02 (3H, t, J = 7.6 Hz, –CH2–CH2–CH3), 1.78 (2H, tq, J = 7.6 and 7.6 Hz, –
CH2–CH2–CH3), 2.77 (2H, t, J = 7.6 Hz, –CH2–CH2–CH3), 4.71 (1H, s, H-3), 7.17
(1H, s, H-1), 7.64 and 7.92 (each 2H, m, phenyl), 8.11, 8.35, and 8.95 (each 1H, s,
1,2,4-triazole and 1,2,3-triazole); HRMS (ESI-TOF, positive mode): calcd for
C20H26N6ONa [M+Na]+ 389.2065, found 389.2065. UT4: 1H NMR (270 MHz,
CD3OD): d 0.68 (9H, s, t-butyl), 0.98 (3H, t, J = 7.6 Hz, –CH2–CH2–CH2–CH3),
1.44 (2H, tq, J = 7.6 and 7.6 Hz, –CH2–CH2–CH2–CH3), 1.74 (2H, tt, J = 7.6 and
7.6 Hz, –CH2–CH2–CH2–CH3), 2.79 (2H, t, J = 7.6 Hz, –CH2–CH2–CH2–CH3), 4.71
(1H, s, H-3), 7.17 (1H, s, H-1), 7.63 and 7.92 (each 2H, m, phenyl), 8.11, 8.34,
Acknowledgments
We thank Toray Industries, Inc. (Tokyo, Japan) for the gift of (+)-
ABA. This research was supported by a Grant-in-Aid for Scientific
Research (No. 18380192) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan.
References and notes
1. Hirai, N. Abscisic acid. In Comprehensive Natural Products Chemistry; Mori, K.,
Ed.; Elsevier: Amsterdam, 1999; Vol. 8, pp 72–91.
2. Davies, W. J.; Jones, H. G. Abscisic Acid; BIOS Scientific Publishers: Oxford, 1991.
3. Zeevaart, J. A. D.; Creelman, R. A. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998,
39, 439.
4. Nambara, E.; Marion-Poll, A. Annu. Rev. Plant Biol. 2005, 56, 165.
5. Huang, D.; Jaradat, MR.; Wu, W.; Ambrose, S. J.; Ross, A. R.; Abrams, S. R.; Cutler,
A. J. Plant J. 2007, 50, 414.
6. Loveys, B. R. In Abscisic Acid; Davies, W. J., Jones, H. G., Eds.; BIOS Scientific
Publishers: Oxford, 1991.

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Y. Todoroki et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5782–5786
and 8.95 (each 1H, s, 1,2,4-triazole and 1,2,3-triazole); HRMS (ESI-TOF, positive
2330g for 20 min at 4 °C. Pelleted cells were suspended in 2.5 mL of a 50 mM
potassium phosphate buffer (pH 7.25) containing 20% glycerol, 1 mM EDTA,
and 0.1 mM dithiothreitol. The suspension was sonicated for 30 s, centrifuged
at 23,470g for 30 min at 4 °C, and the supernatant (enzyme solution) was
collected. The P450 content was determined by spectrophotometric analysis
mode): calcd for C21H28N6ONa [M+Na]+ 403.2281, found 403.2273. UT7: 1H
NMR (270 MHz, CD3OD): d 0.68 (9H, s, t-butyl), 0.88 (3H, t, J = 6.6 Hz, –CH2–
CH2–(CH2)4–CH3), 1.32 (8H, m, –CH2–CH2–(CH2)4–CH3), 1.76 (2H, m, –CH2–
CH2–(CH2)4–CH3), 2.79 (2H, t, J = 7.3 Hz, –CH2–CH2–(CH2)6–CH3), 4.71 (1H, s,
H-3), 7.17 (1H, s, H-1), 7.63 and 7.92 (each 2H, m, phenyl), 8.11, 8.34, and 8.95
(each 1H, s, 1,2,4-triazole and 1,2,3-triazole); HRMS (ESI-TOF, positive mode):
calcd for C24H34N6ONa [M+Na]+ 445.2692, found 445.2690. UT9: 1H NMR
(270 MHz, CD3OD): d 0.68 (9H, s, t-butyl), 0.88 (3H, t, J = 6.6 Hz, –CH2–CH2–
(CH2)6–CH3), 1.30–1.38 (12H, m, –CH2–CH2–(CH2)6–CH3), 1.74 (2H, tt, J = 7.6
and 7.6 Hz, –CH2–CH2–(CH2)6–CH3), 2.78 (2H, t, J = 7.6 Hz, –CH2–CH2–(CH2)6–
CH3), 4.78 (1H, s, H-3), 7.17 (1H, s, H-1), 7.63 and 7.92 (each 2H, m, phenyl),
8.11, 8.34, and 8.95 (each 1H, s, 1,2,4-triazole and 1,2,3-triazole); HRMS (ESI-
TOF, positive mode): calcd for C26H38N6ONa [M+Na]+ 473.3004, found
473.3003. UT10: 1H NMR (270 MHz, CD3OD): d 0.68 (9H, s, t-butyl), 0.89 (3H,
t, J = 6.9 Hz, –CH2–CH2–(CH2)7–CH3), 1.29 (14H, m, –CH2–CH2–(CH2)7–CH3),
1.76 (2H, tt, J = 7.6 and 7.6 Hz, –CH2–CH2–(CH2)7–CH3), 2.79 (2H, t, J = 7.6 Hz, –
CH2–CH2–(CH2)7–CH3), 4.71 (1H, s, H-3), 7.17 (1H, s, H-1), 7.63 and 7.92 (each
2H, m, phenyl), 8.10, 8.34, and 8.95 (each 1H, s, 1,2,4-triazole and 1,2,3-
triazole); HRMS (ESI-TOF, positive mode): calcd for C27H40N6ONa [M+Na]+
487.3161, found 487.3167. UT11: 1H NMR (270 MHz, CD3OD): d 0.68 (9H, s, t-
butyl), 0.89 (3H, t, J = 6.9 Hz, –CH2–CH2–(CH2)8–CH3), 1.29 (16H, m, –CH2–
CH2–(CH2)8–CH3), 1.76 (2H, tt, J = 7.6 and 7.6 Hz, –CH2–CH2–(CH2)8–CH3), 2.79
(2H, t, J = 7.6 Hz, –CH2–CH2–(CH2)8–CH3), 4.71 (1H, s, H-3), 7.18 (1H, s, H-1),
7.63 and 7.92 (each 2H, m, phenyl), 8.11, 8.35, and 8.95 (each 1H, s, 1,2,4-
triazole and 1,2,3-triazole); HRMS (ESI-TOF, positive mode): calcd for
C28H42N6ONa [M+Na]+ 501.3317, found 501.3318. UT12: 1H NMR (270 MHz,
CD3OD): d 0.68 (9H, s, t-butyl), 0.87 (3H, t, J = 6.9 Hz, –CH2–CH2–(CH2)9–CH3),
1.33 (18H, m, –CH2–CH2–(CH2)9–CH3), 1.75 (2H, tt, J = 7.6 and 7.6 Hz, –CH2–
CH2–(CH2)9–CH3), 2.78 (2H, t, J = 7.6 Hz, –CH2–CH2–(CH2)9–CH3), 4.71 (1H, s,
H-3), 7.17 (1H, s, H-1), 7.63 and 7.92 (each 2H, m, phenyl), 8.11, 8.35, and 8.95
(each 1H, s, 1,2,4-triazole and 1,2,3-triazole); HRMS (ESI-TOF, positive mode):
calcd for C29H44N6ONa [M+Na]+ 515.3474, found 515.3480. UT13: 1H NMR
(270 MHz, CD3OD): d 0.68 (9H, s, t-butyl), 0.89 (3H, t, J = 6.9 Hz, –CH2–CH2–
(CH2)10–CH3), 1.33 (20H, m, –CH2–CH2–(CH2)10–CH3), 1.75 (2H, tt, J = 7.6 and
using the extinction coefficient of
a reduced CO difference spectrum
(91.1 mMꢁ1 cmꢁ1).
34. Kinetic analysis was performed according to detailed protocols described
previously.15 A reaction mixture containing 25 g mLꢁ1 CYP707A3 microsomes
coexpressed with AR2 in E. coli, (+)-ABA (final concn: 0.5–128 M), inhibitors
(2–1000 nM in 5 L DMF) and 50 M NADPH in 50 mM potassium phosphate
l
l
l
l
buffer (pH 7.25) were incubated for 10 min at 30 °C; controls contained no
inhibitor. Reactions were initiated by adding NADPH, and stopped by addition
of 50 lL of 1 M NaOH. The reaction mixtures were acidified with 100 lL of 1 M
HCl. To extract the reaction products, the mixtures were loaded onto Oasis HLB
cartridges (1 mL, 30 mg; Waters) and washed with 1 mL of 10% MeOH in a
solution containing 0.5% AcOH. The enzyme products were then eluted with
1 mL of MeOH containing 0.5% AcOH, and the eluate was concentrated in
vacuo. The dried sample was dissolved in 50 lL of MeOH, and 10 lL was
subjected to HPLC. HPLC conditions were: ODS column, Hydrosphere C18
(150 ꢀ 6.0 mm, YMC); solvent, 35% MeOH or 20% MeCN in H2O containing 0.1%
AcOH; flow rate, 1.0 mL minꢁ1
; detection, 254 nm. Enzyme activity was
confirmed by determining the amounts of phaseic acid in control
experiments before each set of measurements. Inhibition constants were
determined using the Enzyme Kinetics module of SigmaPlot 10 software
(2006; Systat Software, Inc.) after determining the mode of inhibition by
plotting the reaction velocities in the presence and absence of inhibitor on a
double-reciprocal plot. For the uninhibited enzymatic reaction, the KM for (+)-
ABA was calculated to be 3.4 0.6 lM based on five separate experiments. All
tests were conducted at least three times.
35. Seeds of rice (Oryza sativa L. cv. Nipponbare) were sterilized with EtOH for
5 min and washed with running tap water. The sterilized seeds were soaked in
water and allowed to germinate for 3 days at 25 °C. The seeds were then placed
in a glass tube (40 mm id) containing 2 mL of a test solution and grown with
the tube sealed with a plastic cap under continuous light (10,000 lux) at 25 °C.
When the seedlings were 7 days old, the length of the second leaf sheath was
measured, and the inhibition ratio was calculated. The inhibition ratio was
defined as [(A ꢁ B)/A] ꢀ 100, where A = the mean length of the second leaf
sheath when water was used, and B = the mean length of the second leaf
sheath when a test compound was used. All tests were conducted at least
twice.
7.6 Hz, –CH2–CH2–(CH2)10–CH3), 2.79 (2H, t, J = 7.6 Hz, –CH2–CH2–(CH2)10
–
CH3), 4.71 (1H, s, H-3), 7.17 (1H, s, H-1), 7.63 and 7.92 (each 2H, m, phenyl),
8.11, 8.34, and 8.95 (each 1H, s, 1,2,4-triazole and 1,2,3-triazole); HRMS (ESI-
TOF, positive mode): calcd for C30H46N6ONa [M+Na]+ 529.3630, found
529.3629. UT15: 1H NMR (270 MHz, CD3OD): d 0.68 (9H, s, t-butyl), 0.88
(3H, t, J = 7.2 Hz, –CH2–CH2–(CH2)12–CH3), 1.33 (24H, m, –CH2–CH2–(CH2)12
–
CH3), 1.73 (2H, tt, J = 7.6 and 7.6 Hz, –CH2–CH2–(CH2)12–CH3), 2.78 (2H, t,
J = 7.6 Hz, –CH2–CH2–(CH2)12–CH3), 4.71 (1H, s, H-3), 7.17 (1H, s, H-1), 7.63 and
7.92 (each 2H, m, phenyl), 8.11, 8.35, and 8.95 (each 1H, s, 1,2,4-triazole and
1,2,3-triazole); HRMS (ESI-TOF, positive mode): calcd for C32H50N6ONa
[M+Na]+ 557.3943, found 557.3953.
36. Cultivar Akitabeniakari apple [Malus sylvestris L. Mill. var. domestica (Borkh.)
Mansf.] seeds were soaked overnight in water, then sown in moist vermiculite
and grown in a greenhouse. Seedlings at 90 days old were divided into two
groups: water-stressed and well-watered. The seedlings of the water-stressed
group were sprayed uniformly with 2 mL of a test solution (10, 50, and
33. A truncated Arabidopsis CYP707A3 (707A3d28), which lacked the putative
membrane-spanning segment of the N-terminus (residues 3–28) was
constructed. Cells of E. coli strain BL21 were transformed with pCW-
CYP707A3d28 and pACYC-AR2 constructs. Cultures (3 mL) were grown
overnight in Luria-Bertani medium supplemented with ampicillin
100
lM) containing UT4, UNI (positive control), or distilled water (untreated
control) before water-stressing them for 150 h, then watering them once, and
then water-stressing them again for an additional 330 h. The well-watered
group was watered once daily so that the vermiculite was saturated. Fuji apple
[Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] seeds were soaked
overnight in water, then sown in moist vermiculite and grown in a greenhouse.
The seedlings were 60 days old at the time of treatment. Each plant was
sprayed uniformly with 2 mL of a test solution. The untreated control group
was sprayed with distilled water, and the treated group was sprayed with a
(50
Broth medium supplemented with ampicillin (50
(100
g mLꢁ1), and aminolevulic acid (0.5 mM) was inoculated with a 0.5-mL
l
g mLꢁ1
)
and chloramphenicol (100
l
g mLꢁ1). Then, 50 mL of Terrific
g mLꢁ1), chloramphenicol
l
l
aliquot of the overnight culture. The culture was incubated at 37 °C with gentle
shaking (225 rpm) until the A600 reached 0.6 (after 2.5–3 h), and then
expression of the P450 enzyme was induced by the addition of isopropyl-b-
solution of UT (10 lM) or S-UNI (10 lM). All test groups were watered before
being treated. The seedlings were water-stressed for 480 h.
37. Tanaka, S. Kagaku to Seibutsu 1986, 24, 180.
D-thiogalactopyranoside (0.1 mM). The culture was shaken continuously
(150 rpm) at 25 °C and cells were harvested 48 h later by centrifugation at
38. Takano, H.; Oguri, Y.; Kato, T. J. Pesticide Sci. 1986, 11, 373.