Abscinazole-E2B, a practical and selective inhibitor of ABA 8′-hydroxylase CYP707A

Article abstract of DOI:10.1016/j.bmc.2012.03.068

We developed abscinazole-E2B (Abz-E2B), a practical and specific inhibitor of abscisic acid (ABA) 8′-hydroxylase (CYP707A), by structural modification of abscinazole-E1 (Abz-E1), another compound we developed. A butoxy group was introduced to Abz-E2B instead of the tosylate group of Abz-E1, in expectation of better water solubility, because the calculated log P value of Abz-E2B is 3.47, which is smaller than that of Abz-E1 (4.02). The water solubility of Abz-E2B was greater than 90% at a concentration of 100 μM, at which the solubility of Abz-E1 was 20%. The enzyme specificity was improved significantly. In in vitro assays constructed using recombinant enzymes, (±)-Abz-E2B was a considerably weaker inhibitor than (±)-Abz-E1 for CYP701A, a GA biosynthetic enzyme, which is a target of S-uniconazole (S-UNI), a lead compound of Abz-E1. (±)-Abz-E2B application to plants resulted in improved desiccation tolerance and an increase in endogenous ABA, with little retardation of growth. We also prepared optically pure Abz-E2B and determined its absolute configuration. The R-enantiomer of Abz-E2B was the more potent inhibitor of CYP707A, unlike UNI, whereas both enantiomers were markedly less effective than S-UNI in inhibiting CYP701A. Because S-Abz-E2B arrested the growth of rice seedlings at 100 μM, probably because of off-target effects, R-Abz-E2B should be used as a chemical tool for research focusing on CYP707A when 100 μM or higher concentration is required, although (±)-Abz-E2B may be useful as an alternative option at a lower concentration.

Full text of DOI:10.1016/j.bmc.2012.03.068

Bioorganic & Medicinal Chemistry 20 (2012) 3162–3172
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Abscinazole-E2B, a practical and selective inhibitor
of ABA 80-hydroxylase CYP707A
Mariko Okazaki a, , Monrudee Kittikorn b, , Kotomi Ueno c, Masaharu Mizutani c, Nobuhiro Hirai d,
Satoru Kondo b, Toshiyuki Ohnishi e, Yasushi Todoroki a,
⇑
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 Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan
d Division of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8501, Japan
e Division of Global Research Leaders, Shizuoka University, Shizuoka 422-8529, Japan
a r t i c l e i n f o
a b s t r a c t
We developed abscinazole-E2B (Abz-E2B), a practical and specific inhibitor of abscisic acid (ABA) 80-
hydroxylase (CYP707A), by structural modification of abscinazole-E1 (Abz-E1), another compound we
developed. A butoxy group was introduced to Abz-E2B instead of the tosylate group of Abz-E1, in expec-
tation of better water solubility, because the calculated log P value of Abz-E2B is 3.47, which is smaller
than that of Abz-E1 (4.02). The water solubility of Abz-E2B was greater than 90% at a concentration of
Article history:
Received 23 February 2012
Revised 30 March 2012
Accepted 30 March 2012
Available online 5 April 2012
100 lM, at which the solubility of Abz-E1 was 20%. The enzyme specificity was improved significantly.
Keywords:
In in vitro assays constructed using recombinant enzymes, ( )-Abz-E2B was a considerably weaker inhib-
itor than ( )-Abz-E1 for CYP701A, a GA biosynthetic enzyme, which is a target of S-uniconazole (S-UNI), a
lead compound of Abz-E1. ( )-Abz-E2B application to plants resulted in improved desiccation tolerance
and an increase in endogenous ABA, with little retardation of growth. We also prepared optically pure
Abz-E2B and determined its absolute configuration. The R-enantiomer of Abz-E2B was the more potent
inhibitor of CYP707A, unlike UNI, whereas both enantiomers were markedly less effective than S-UNI in
Plant growth regulator
P450 inhibitor
ABA
inhibiting CYP701A. Because S-Abz-E2B arrested the growth of rice seedlings at 100
because of off-target effects, R-Abz-E2B should be used as a chemical tool for research focusing on
CYP707A when 100 M or higher concentration is required, although ( )-Abz-E2B may be useful as an
alternative option at a lower concentration.
lM, probably
l
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
droxy-ABA (Fig. 1) and its more stable tautomer, phaseic acid,
which has considerably lower hormonal activity than ABA.4,7–14
Thus, we have focused on developing a specific inhibitor of ABA
80-hydroxylase.
The plant hormone abscisic acid (ABA) is involved in seed dor-
mancy, stress responses, and other physiological events.1–4 Its bio-
synthesis, transport, and catabolic inactivation cooperatively
regulate the endogenous level of ABA in response to environmental
changes.1–4 A chemical compound that perturbs this highly con-
trolled system is promising as a chemical probe for the mechanism
of ABA action.5 In addition, this chemical is potentially useful in
agriculture and horticulture. Although ABA is registered as an agri-
cultural farm chemical (plant growth regulator), its practical use
has been limited, mainly due to its weak effect in field trials,6 con-
sidered to be due to its rapid inactivation through biodegradation.
Catabolic inactivation of ABA is mainly controlled by ABA 80-
hydroxylase, a member of the CYP707A subfamily, which is the
cytochrome P450 catalyzing the C80-hydroxylation of ABA to 80-hy-
The plant growth retardant S-uniconazole (S-UNI)15,16 functions
as an inhibitor of CYP707A17 in addition to ent-kaurene oxidase
(CYP701A), which catalyzes the three-step oxidation of ent-kau-
rene to ent-kaurenoic acid (KA),18 a biosynthetic precursor of the
plant hormone gibberellin (GA). S-Diniconazole, the R enantiomer
of which is a fungicide with a structure very similar to S-UNI, also
inhibits CYP707A,19 although it arrests plant growth.20 The broad
inhibition spectrum of some azole compounds against P450 en-
zymes has apparently resulted from the heme coordination of an
azole nitrogen, which is a common mechanism of azole P450
inhibitors. In other words, their specificity for individual P450 en-
zymes depends on structural properties other than the azole group.
In early work,21 we modified the structure of S-UNI to develop a
more specific inhibitor against CYP707A by various approaches.22–24
The molecular enlargement approach, which was the most effective,
led us to develop a specific inhibitor of CYP707A, ( )-UTn.22
⇑
Corresponding author. Tel./fax: +81 54 238 4871.
E-mail address: aytodor@ipc.shizuoka.ac.jp (Y. Todoroki).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.068

M. Okazaki et al. / Bioorg. Med. Chem. 20 (2012) 3162–3172
3163
8'
HO
O
OH
OH
CO2H
CO2H
O
ABA catabolism
8'-hydroxy-ABA
S-(+)-ABA
ABA 8'-hydroxylase
(CYP707A)
N
N
N
N
N
N
O
O
n-1
Cl
4'
N
N
N
O
O
3
HO
N
HO
N
HO
HO
TsO
O
R
1"
N
2
N
N
N
N
N
S-(+)-UNI
UTn
Abz-E1
Abz-E2P (R=propyl)
Abz-E2B (R=butyl)
N
N
N
N
4"
ent-kaurene oxidase
(CYP701A)
GA biosynthesis
CO2H
ent-kaurene
KA
Figure 1. Specific inhibitors of ABA 80-hydroxylase (CYP707A). These were developed from S-UNI, which is a potent inhibitor of both CYP707A and ent-kaurene oxidase
(CYP701A). A disadvantage of UTn is its poor water solubility.22 Abz-E1 has improved water solubility but it is potentially unstable owing to the tosylate group.25 This work
describes the development of Abz-E2P and Abz-E2B as novel CYP707A inhibitors that are chemically stable and more water soluble.
However, Because ( )-UTn was difficult to dissolve in water because
of its high hydrophobicity, it was difficult to handle as a chemical
tool. Aiming to overcome this defect, we developed ( )-abscinaz-
ole-E1 (Abz-E1, Fig. 1), which has a tosylate-protected diethylene
glycol chain instead of an alkyl chain.25 This compound has better
water solubility than ( )-UTn. However, its water solubility was still
lower than that of S-UNI. Moreover, because the tosylate group is
chemically reactive and rich in intermolecular interactions, ( )-
Abz-E1 is not practical enough for both research and agricultural
uses, although it may be useful as a precursor for structural evolu-
tion. In the present study, therefore, we improved the chemical
and biological properties of ( )-Abz-E1 to develop a more practical
and specific inhibitor of CYP707A. We describe the design and prep-
aration of a novel CYP707A inhibitor, abscinazole-E2B (Abz-E2B,
Fig. 1), its water solubility, and its effect on CYP707A and CYP701A
both in vitro and in vivo. We also describe the preparation, absolute
configuration, and enzymatic and biological effects of optically pure
Abz-E2B.
the longer carbon chain is unable to dissolve in water in the con-
centration range required for bioassays (1–100 M), although it
l
can be partially dissolved in buffer solution at concentrations re-
quired for enzyme assays (1–100 nM). On the other hand, the
introduction of protic functional groups in the alkyl chain dimin-
ishes the inhibitory activity. Thus, we developed ( )-Abz-E1, which
has a tosylate-protected diethylene glycol chain.25 The inhibition
constant (KI) of ( )-Abz-E1 for recombinant Arabidopsis CYP707A3
co-expressed with Arabidopsis P450 reductase (ATR2) in Escherichia
coli was 27 nM,25 which was considerably smaller than that of ( )-
UT4 (195 nM). This improved activity was considered to result
from either the long chain, ether linkage, or tosylate moiety. Espe-
cially because a tosylate moiety is large and rich in intermolecular
interactions, we first tried to detosylate ( )-Abz-E1 to evaluate the
effect of the tosylate moiety on inhibition of CYP707A.
Treatment of ( )-Abz-E1 with lithium aluminum hydride in THF
at room temperature gave two products (Scheme 1). One is the
detosylated compound 1, and the other is the more reduced com-
pound 2. The KI values of 1 and 2 for recombinant Arabidopsis
CYP707A3 were 850 and 270 nM, respectively, meaning that these
compounds have lower affinity for the CYP707A3 active site than
Abz-E1. This suggests that the tosylate moiety of Abz-E1 plays a
significant role in binding to the CYP707A3 active site. However,
other possibilities are not excluded. Compound 1 has a terminal
hydroxy group, and the chains of both compounds 1 and 2 are
shorter than that of Abz-E1. Because the introduction of protic
functional groups in the alkyl chain of ( )-UTn diminished the
2. Results and discussion
( )-UTn (Fig. 1), an enlarged analogue of S-UNI, has a 4-alkyl-
1,2,3-triazole instead of the chlorine of S-UNI. Our previous study
showed that a longer alkyl chain results in higher inhibitory activ-
ity for CYP707A and lower for CYP701A.22 However, the longer al-
kyl chain encourages hydrophobicity. In fact, ( )-UTn (n > 4) with

3164
M. Okazaki et al. / Bioorg. Med. Chem. 20 (2012) 3162–3172
N
N
N
N
O
N
O
N
O
N
N
N
O
O
O
HO
N
HO
N
HO
N
+
TsO
HO
N
N
N
N
N
N
( )-Abz-E1
1
2
Scheme 1. Synthesis of 1 and 2. Reagents and conditions: LiAlH4, dry THF, 0 °C?RT, 24% (1) and 28% (2).
inhibitory activity and CYP707A3 preferred binding to UTn with
the longer alkyl chain, these structural properties of compounds
1 and 2 may reduce the affinity for the CYP707A3 active site. To
evaluate the effect of the tosylate group of Abz-E1, we had to create
new compounds that have a longer chain with no hydroxy group.
Accordingly, we designed abscinazole-E2P and abscinazole-E2B
(Fig. 1), which respectively have a propoxy and butoxy group in-
stead of a tosylate, considering that both the length of the chain
and the calculated log P value, which should be less than 3.5 for
better water solubility than Abz-E1 (calculated log P = 4.02).
( )-Abz-E2P and ( )-Abz-E2B were prepared from ( )-Abz-E1 by
treatment with sodium hydride in propanol and butanol, respec-
tively (Scheme 2). The KI values for recombinant Arabidopsis
CYP707A3 were 130 nM for ( )-Abz-E2P and 36 nM for ( )-
Abz-E2B. This means that the affinity of Abz-E2B for the CYP707A3
active site is nearly equivalent to that of Abz-E1 (KI = 27 nM).
Therefore, the tosylate moiety of Abz-E1 seems not to be essential
for the high affinity to the active site. Considering that ( )-Abz-E2B
was more potent than ( )-Abz-E2P, the factors responsible for the
high activity of ( )-Abz-E1 are not specific p–p, CH–p, or electro-
static interactions by the tosylate moiety but nonspecific hydro-
phobic interaction of the terminal lipophilic moiety with the
lipophilic region at the active site.
The water solubility of ( )-Abz-E2P and ( )-Abz-E2B was more
than 90% at a concentration of 100 lM, whereas it was 20% for
( )-Abz-E1 and 40% for S-UNI under the same conditions (Table
1), meaning that these new compounds, especially Abz-E2B, are
ideal CYP707A inhibitors with both potent activity and good water
solubility.
We examined the selectivity between CYP707A and CYP701A
by evaluating both inhibitory activity against recombinant rice
CYP701A6 expressed in insect cells using a baculovirus vector
and growth of rice seedlings. Because CYP701A enzymes catalyze
all three steps to KA via the corresponding alcohol and aldehyde,18
we evaluated the IC50 value for formation of the end product, KA,
instead of the KI value, which requires very complicated kinetic
analysis. We were unable to determine the IC50 values of ( )-
Abz-E2P and ( )-Abz-E2B because 50% inhibition was not achieved
N
N
N
O
N
O
N
N
O
O
HO
N
HO
N
TsO
O
R
N
N
even at the highest tested concentration, 1
substrate ent-kaurene was used, whereas the IC50 values of Abz-E1
and UNI were 1 M and 57 nM under the same conditions (Table
lM, when 1 lM of the
N
N
( )- Abz-E1
l
( )- Abz-E2P (R=propyl)
( )- Abz-E2B (R=butyl)
1). This result, together with the strong effect on CYP707A, sug-
gests that the CYP707A/CYP701A selectivity of Abz-E2P and Abz-
E2B is considerably higher than that of Abz-E1. On the other hand,
slight inhibition of rice seedling growth by ( )-Abz-E2P and ( )-
Scheme 2. Synthesis of ( )-Abz-E2P and ( )-Abz-E2B. Reagents and conditions:
NaH, propanol or butanol, RT, 77% (Abz-E2P) and 85% (Abz-E2B).
Table 1
Enzymatic, biological, and physicochemical properties of azole inhibitors of CYP707A
Compound
CYP707A inhibitiona
CYP701A inhibitionb
Growth inhibitionc
clogPd
Solubility (%) in water (2 mL)e
KI (nM)
Ratio (%)
100
—
—
51
0
3.4
20
13
—
IC50
(
lM)
IC50
(lM)
20 nmol
200 nmol
S-UNI
R-UNI
( )-UT4
( )-Abz-E1
1
10
0.057
0.300
—
1.0
—
—
—
—
5.5
1.1
0.18
7.8
42
>100
>100
>100
>100
>100
>100
>100
3.13
3.13
3.96
4.02
1.46
2.50
2.98
3.42
3.42
3.42
79.4
—
40.7
68.9
—
—
—
—
—
39.5
—
—
21.3
95.7
99.5
94.8
95.3
—
f
1450
195
27
850
270
130
36
2
( )-Abz-E2P
( )-Abz-E2B
S-Abz-E2B
R-Abz-E2B
28
360
—
—
—
a
Arabidopsis recombinant CYP707A3 expressed in E. coli.
b
Rice recombinant CYP701A6 expressed in insect cells. Inhibition ratios were determined using 0.1
M ent-kaurene.
Length of the second leaf sheath of rice seedlings.
lM test compounds and 10 lM ent-kaurene. The inhibition assay for
determining the IC50 values was performed using 1
l
c
d
e
f
Calculated partition coefficient (Marvin, ChemAxon, http://www.chemaxon.com/marvin/sketch/index.php).
Calculated based on the in water to in MeOH ratio of HPLC peak area. The amount of solute: 20 and 200 nmol.
Not tested.

M. Okazaki et al. / Bioorg. Med. Chem. 20 (2012) 3162–3172
3165
Abz-E2B was observed only at a high concentration, 100
l
M
CYP707A inhibition, it was enhanced less than expected by co-
treatment with ABA (Fig. 3c and d).
(Fig. 2). This was the same trend observed for ( )-Abz-E1, although,
based on the very weak in vitro inhibition of CYP701A, the growth
retardant effect of ( )-Abz-E2P and ( )-Abz-E2B is expected to be
considerably smaller than that of ( )-Abz-E1. The inhibitory pro-
files in the in vitro enzyme assay using the recombinant enzyme
may not fully correspond to those in the in vivo enzyme reaction.
Otherwise, the observed inhibition of growth may depend on inhi-
bition of CYP707A to increase endogenous ABA, or on inhibition of
off-target enzymes other than CYP701A and CYP707A. This ques-
tion is discussed again below.
Among the tested plants, the effect of ( )-Abz-E2B in the ab-
sence of exogenous ABA was the most significant for Arabidopsis
seeds. Although lettuce seeds and rice seedlings were insensitive
to ( )-Abz-E2B in the absence of exogenous ABA, the lettuce seeds
were more responsive in the presence of ABA. Rice seedlings were
so insensitive to ( )-Abz-E2B that their early growth was not af-
fected by co-treatment with ABA (data not shown). This difference
may depend on a difference in ABA sensitivity, activity and ligand
recognition of CYP707A homologues, or sensitivity to residual
CYP701A inhibitory activity of ( )-Abz-E2B.
The effect of ( )-Abz-E2P and ( )-Abz-E2B on seed germination
and early growth of lettuce was examined in both the presence and
absence of ABA (Fig. 3a and b). In the absence of ABA, neither com-
We also tested the enhancement of ( )-Abz-E2B on desiccation
tolerance under more practical conditions. Nineteen-day-old bent
grass (Agrostis capillaris) was sprayed with an aqueous solution
pound had an effect at a concentration of 100
hand, in the presence of ABA, they enhanced the effect of ABA.
The effect of ( )-Abz-E2P was slight in the presence of 3 M ABA
and significant in the presence of 10 M ABA, whereas that of
( )-Abz-E2B was slight in the presence of 1 M ABA and significant
in the presence of 3 and 10 M ABA. The greater effect of ( )-Abz-
lM. On the other
containing ( )-Abz-E2B at 50 lM. Water was not supplied for
l
10 days, and the plants were then rehydrated. Application of ( )-
Abz-E2B before dehydration induced significant drought tolerance
during dehydration (Fig. 4a). The endogenous amount of ABA dur-
ing dehydration increased significantly at day 9 after treatment
with ( )-Abz-E2B (Fig. 4b). The increase in ABA content on days
7–9 suggests that plants activated ABA biosynthesis by sensing
water deficit stress after 7 days. Inhibition of ABA catabolism by
Abz-E2B must have increased the ABA content.
l
l
l
E2B than ( )-Abz-E2P was consistent with results of in vitro
CYP707A3 assays. This suggests that the enhancement of ABA
activity depends on inhibition of ABA inactivation. We also tested
the effect of ( )-Abz-E2B on germination and leaf emergence of
Arabidopsis thaliana in both the presence and absence of ABA. In
contrast to the lettuce assay, ( )-Abz-E2B exhibited an inhibitory
In the above-mentioned enzymatic and biological assays, Abz-
E2B was applied in racemic form. The S-isomer of the parent lead
compound UNI is more potent than the R-isomer in inhibiting
CYP707A. To verify that the selective inhibitory activity of ( )-
Abz-E2B for CYP707A is also caused by the S-isomer, optically pure
Abz-E2B was prepared from optically pure Abz-E1, which was pre-
pared by optical resolution of the racemate using semipreparative
HPLC with a chiral column. The absolute stereochemistry of both
enantiomers of Abz-E2B was determined by an advanced Mosher’s
effect at 100 and 300 lM in the absence of ABA. Although this
activity may arise from an increase in endogenous ABA due to
( )-Abz-E1
method26 using 1H NMR spectra of their R-
a-methoxy-a-(trifluoro-
methyl)phenyl acetates (MTPA). The R-MTPA esters were prepared
with S-MTPA-Cl in pyridine. For convenience, we prepared MTPA
esters of optically pure Abz-E2B by diastereomeric resolution of
R-MTPA-( )-Abz-E2B using preparative HPLC with a silica gel col-
umn. The rotational sign of Abz-E2B in the diastereomers was
identified on the basis of chiral HPLC analysis of Abz-E2B released
by hydrolysis of the diastereomers after the 1H NMR analysis. To
exclude the possibility that the MTPA plane is not formed owing
to the bulky t-butyl group, we constructed a molecular model of
the MTPA esters. The geometries, optimized with B3LYP/6-31G(d)
from the initial geometries with the ideal MTPA plane, retained
the plane sufficiently to apply the advanced Mosher’s method,
even though the plane was slightly distorted (Fig. 5). In addition,
we experimentally confirmed that the advanced Mosher’s method
gave the correct absolute configuration of UNI (see Supplementary
data). The phenyl moiety in MTPA is located so as to have a shield-
ing effect on the vinylazole moiety for S-Abz-E2B and the t-butyl
group for R-Abz-E2B. 1H NMR analysis showed that the vinyltriaz-
ole protons of the (+)-isomer and the t-butyl protons of the (ꢀ)-iso-
mer were shielded. Thus, the absolute configuration of the (+)- and
(ꢀ)-isomers was determined to be S and R, respectively.
( )- Abz-E2P
( )- Abz-E2B
control
S-UNI
Both enantiomeric isomers of Abz-E2B were assayed enzymati-
cally and biologically. R-Abz-E2B was a more effective inhibitor of
recombinant Arabidopsis CYP707A3 than the S-isomer by a factor of
10 on the basis of the KI values (Table 1). This is inconsistent with
the case of UNI, which had an S-isomer that was more potent than
the R-isomer by a factor of more than 100.23 The enhancement of
exogenous ABA activity on seed germination and early growth of
lettuce and Arabidopsis showed the same trend. R-Abz-E2B en-
hanced the inhibition of lettuce seed germination and early growth
of lettuce and Arabidopsis more than S-Abz-E2B (Fig. 6a–d),
although neither enantiomer had a significant enhancement of
0
0.3
1
3
10
30
100 μM
Figure 2. Comparison of effect of ( )-Abz-E2P, ( )-Abz-E2B and S-UNI on rice
seedling growth on day 7 after treatment. S-UNI was not tested at a concentration
greater than 30
lM in this experiment. Images of seedlings treated with ( )-Abz-E1,
which were obtained in earlier work,25 are shown for comparison.

3166
M. Okazaki et al. / Bioorg. Med. Chem. 20 (2012) 3162–3172
a
b
100
50
no inhibitor
( )-Abz-E2P 100 μM
( )- Abz-E2B 100 μM
( )- Abz-E2B
100 μM
( )- Abz-E2P
100 μM
no
inhibitor
0
0
1
3
10
30
0
1
3
ABA (μM)
ABA (μM)
c
d
100
no inhibitor
300
( )- Abz-E2B 10 μM
( )- Abz-E2B 30 μM
( )- Abz-E2B 100 μM
( )- Abz-E2B 300 μM
100
30
0
50
0
0
0.1
0.3
0
0.1
0.3
ABA (μM)
ABA (μM)
Figure 3. Enhancement of ( )-Abz-E2P and ( )-Abz-E2B on ABA activity: (a) inhibition of lettuce seed germination at 24 h (n = 2), (b) inhibition of lettuce seedling growth at
168 h, (c) inhibition of Arabidopsis seed germination at 24 h (n = 2), (d) leaf emergence in Arabidopsis at 96 h after imbibition.
exogenous ABA activity in Arabidopsis seed germination, consistent
with the case of racemic Abz-E2B (Fig. 3c).
ties to UNI, the remaining portion of the structure must be respon-
sible for the inverted enantiomeric property. The long side chain of
R-Abz-E2B may interact more effectively with the CYP707A active
site than that of S-Abz-E2B. Because the length of the side chain of
Abz-E2B is comparable to that of the UNI molecule, the energetic
contribution to binding the CYP707A active site may be large en-
ough to override the disadvantage of the UNI portion of the R-iso-
mer in fitting into the active site. Such an inversion accompanying
structural modification of chiral compounds has also been ob-
served for an ABA analogue, AHI1, which functions as an inhibitor
of CYP707A.28
Comparing S- and R-UNI in the same most stable conformation
(A), the largest structural difference between them is the relative
space occupied by the triazole, especially N400, which would coor-
dinate the heme iron in the CYP707A active site, and the t-butyl
(Fig. 7). If R-UNI adopts a different conformation from S-UNI, this
steric difference may be considerably canceled. The UNI molecule
is relatively flexible, which allows it to adopt at least four specific
conformers by rotation of two single bonds, C2-N100 and C2-C3.27
The conformers B and C, rather than A, of R-UNI resemble con-
former A of S-UNI in their spatial relationship between N400 and
the t-butyl (Fig. 7). The theoretical energy difference between A/
B or A/C is respectively calculated to be 2.5 or 4.0 kcal molꢀ1 in
the gas phase, meaning that B and C are theoretically less advanta-
geous in binding to the CYP707A active site than A by a factor of
100–1000, which fluctuates according to interactions with the ac-
tive site residues. From this point of view, the activity of R-UNI,
which is lower than S-UNI by a factor of 140, may be reasonable,
although the A conformer of R-UNI may merely have lower affinity
to the active site than S-UNI by a factor of 140. The structure of
Abz-E2B is the same as that of UNI except for the 1,2,3-triazole,
which possesses a long ether chain. Because the UNI portion in
the Abz-E2B molecule must have similar conformational proper-
Conversely, in the inhibition of rice seedling growth, S-Abz-E2B
showed slightly stronger average activity than the R-isomer at a
concentration of 100
E2B at 100 M fluctuated, unlike that of the R-isomer. Some rice
seedlings treated with 100 M S-Abz-E2B were markedly arrested
in growth and were comparable to dwarf seedlings resulting from
30–100 M S-UNI treatment (Fig. 8b). The rescue by co-treatment
lM (Fig. 8a). The retardant effect of S-Abz-
l
l
l
with GA3 and S-Abz-E2B was less effective than co-treatment with
GA3 and S-UNI (Fig. 9). Considered together with the very low ef-
fect of S-Abz-E2B on CYP701A, this unusual dwarfing may depend
on inhibition of not only CYP701A but also other enzymes. Thus, R-
Abz-E2B should be used as a chemical tool for research focusing on
CYP707A when 100 lM or higher concentration is required. At a

M. Okazaki et al. / Bioorg. Med. Chem. 20 (2012) 3162–3172
3167
a
day 7
t-butyl
t-butyl
1
1
S
R
5″
3″
5″
3″
day 12
R
R
no inhibitor
dehydration
( )- Abz-E2B 50 μM
rehydration
b
N
N
1.0
0.8
0.6
0.4
0.2
0.0
N
N
no inhibitor
( )- Abz-E2B
N
N
t-butyl
t-butyl
O
O
H
H
F3C
F3C
R
R
1
O
O
S
R
2
O
O
N
N
N
N
5″
N
N
3″
R-MTPA-S-Abz-E2B
R-MTPA-R-Abz-E2B
0
2
4
6
8
10
12
days
δ (ppm)
Figure 4. Effect of Abz-E1 (50 lM) on drought tolerance of bent grass (a) and ABA
content of the leaves (b).
H
R-MTPA ester of
(+)-Abz-E2B (−)-Abz-E2B
lower concentration than 100
alternative option.
lM, ( )-Abz-E2B may be useful as an
3
1
6.15
6.21
3. Conclusions
7.26
7.82
7.94
0.74
7.35
7.99
8.08
0.67
We developed Abz-E2B, a novel inhibitor of ABA 80-hydroxylase
(CYP707A). Abz-E2B, a structural analogue of UNI that substitutes a
4-(2-(2-butoxyethoxy)ethoxy)methyl-1H-1,2,3-triazole for a chlo-
rine, is more specific, chemically stable, and water soluble than
Abz-E1, another inhibitor we reported on,25 which has a tosylate in-
stead of the butoxy in Abz-E2B. In an in vitro recombinant P450
inhibition assay, ( )-Abz-E2B was more potent against CYP707A3
and less potent against CYP701A6 than ( )-Abz-E1. This superior
inhibitory specificity of CYP707A was confirmed in bioassays. Bent
grass sprayed with ( )-Abz-E2B solution exhibited stronger drought
tolerance than control grass sprayed with distilled water. The opti-
cally active Abz-E2B exhibited different enantiomeric properties
from UNI in CYP707A inhibition and related biological assays. The
R-enantiomer of Abz-E2B was the more potent inhibitor of
CYP707A, unlike UNI, whereas the S-enantiomer arrested the
growth of rice seedlings at high concentration, probably because
of off-target effects. Therefore, R-Abz-E2B should be used as a chem-
ical tool for research focusing on CYP707A, although ( )-Abz-E2B
may be useful as an alternative option at low concentration.
3″, 5″
t-butyl
Figure 5. Determination of the absolute configuration of optically pure Abz-E2B by
an advanced Mosher’s method. Molecular models of the MTPA esters were
optimized with B3LYP/6-31G(d) from the initial geometries with the ideal MTPA
plane. Proton signals of H300 and H500 of 1,2,4-triazole rings were not identified,
which is because no HMBC cross-peak was observed between these protons and C2.
Stick models: carbons, gray; hydrogens, white; oxygens, red; nitrogens, blue; and
fluorines, light green. The structural formulas correspond to views from the right
side of the models. The MTPA plane, defined by the trifluoromethyl group, the
carbonyl group, and the carbinol proton, are colored in purple in the structural
formula.
Toyomasu (Yamagata University) and Dr. Shinjiro Yamaguchi (RI-
KEN Plant Science Center), respectively. ent-Kaurenoic acid was
also purchased from OlChemIm Ltd, Czech Republic. 1H NMR spec-
tra were recorded with tetramethylsilane as the internal standard
using JEOL JNM-EX270 (270 MHz) and JNM-LA500 (500 MHz) NMR
spectrometers. 13C NMR and 2D-correlation NMR experiments
were recorded using a JNM-LA500 (500 MHz) NMR spectrometer.
High resolution mass spectra were obtained with a JEOL JMS-
T100LC AccuTOF mass spectrometer. Column chromatography
was performed on silica gel (Wakogel C-200).
4. Experimental
4.1. General
(+)-ABA was a gift from Toray Industries Inc., Tokyo, Japan. ent-
Kaurene and ent-kaurenoic acid were gifts from Prof. Tomonobu

3168
M. Okazaki et al. / Bioorg. Med. Chem. 20 (2012) 3162–3172
a
b
no inhibitor
R-Abz-E2B 100 μM
S-Abz-E2B 100 μM
100
50
S-Abz-E2B
100 μM
R-Abz-E2B
100 μM
no
inhibitor
0
0
1
3
10
30
0
1
3
ABA (μM)
ABA (μM)
c
d
no inhibitor
100
S-Abz-E2B
100 μM
R-Abz-E2B 30 μM
R-Abz-E2B 100 μM
S-Abz-E2B 100 μM
R-Abz-E2B
100 μM
50
0
R-Abz-E2B
30 μM
no
inhibitor
0
0.1
0.3
0
0.1
0.3
ABA (μM)
ABA (μM)
Figure 6. Enhancement of R- and S-Abz-E2B on ABA activity: (a) inhibition of lettuce seed germination at 24 h (n = 3), (b) inhibition of lettuce seedling growth at 216 h, (c)
inhibition of Arabidopsis seed germination at 24 h (n = 3), (d) leaf emergence in Arabidopsis at 192 h after imbibition.
4.2. Synthesis of chemicals
(270 MHz, acetone-d6): d 0.71 (9H, s, t-butyl), 1.14 (3H, t, J =
6.9 Hz, –OCH2CH3), 3.49 (2H, q, J = 6.9 Hz, –OCH2CH3), 3.58 and
3.70 (each 2H, m, –OCH2CH2O–), 4.70 (2H, m, 1,2,3-triazole-
CH2O–), 4.82 (1H, d, J = 5.9 Hz, HO-3), 5.22 (1H, d, J = 5.9 Hz, H-3),
7.20 (1H, s, H-1), 7.70 and 8.01 (each 2H, m, 1-phenyl), 8.04, 8.56,
and 8.88 (each 1H, s, 1,2,4-triazole and 1,2,3-triazole); UV kmax
4.2.1. (E)-1-(4-(4-((2-(2-hydroxyethoxy)ethoxy)methyl)-1H-1,2,
3-triazol-1-yl)phenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)
pent-1-en-3-ol (1) and (E)-1-(4-(4-((2-ethoxyethoxy)methyl)-
1H-1,2,3-triazol-1-yl)phenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-
1-yl)pent-1-en-3-ol (2)
(MeOH) nm (e): 269.8 (21,000); HRMS (ESI-TOF, positive mode):
To a stirred solution of Abz-E1 (100 mg, 168
lmol) and molecu-
calcd for C22H30N6O3Na [M+Na]+ 449.2277, found 449.2276.
lar sieves 4A in dry THF (25 mL) was added LiAlH4 (536 mg,
14.1 mmol) at 0 °C under Ar. The mixture was stirred for 40 min
at the same temperature, and for 3 h at room temperature. After
quenched with 1 M HCl at 0 °C, the resulting mixture was extracted
with EtOAc (90 mL ꢁ 3). The organic layer was washed with brine,
dried over Na2SO4, and concentrated in vacuo. The residual oil was
purified by silica gel column chromatography with 0–20% acetone
in EtOAc to obtain 1 (17.5 mg, 24%) and 2 (20.0 mg, 28%) as colorless
oils. A portion of these samples was further purified for bio-
assays by HPLC (YMC Hydrosphere C18, 150 ꢁ 20 mm, 55% MeOH,
4.2.2. (E)-1-(4-(4-((2-(2-propoxyethoxy)ethoxy)methyl)-1H-1,2,
3-triazol-1-yl)phenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)
pent-1-en-3-ol (Abz-E2P)
To a stirred solution of 1-propanol (2 mL) was added NaH (60%
in oil, 77 mg, 1.9 mmol) at room temperature under Ar. After stir-
red for 15 min, Abz-E1 (100 mg, 168 lmol) dissolved in 1-propanol
(3 mL) was added. The mixture was stirred for 18 h at room tem-
perature. After quenched with sat. NH4Cl, the resulting mixture
was extracted with EtOAc (15 mL ꢁ 3). The organic layer was
washed with brine, dried over Na2SO4, and concentrated in vacuo.
The residual oil was purified by silica gel column chromatography
with 3% MeOH in CH2Cl2 to obtain Abz-E2P (71.2 mg, 77%) as a
pale yellow oil. 1H NMR (270 MHz, CDCl3): d 0.68 (9H, s, t-butyl),
0.90 (3H, t, J = 7.3 Hz, –OCH2CH2CH3), 1.60 (2H, qt, J = 7.3 and
6.9 Hz, –OCH2CH2CH3), 3.42 (2H, t, J = 6.9 Hz, –OCH2CH2CH3),
3.59–3.79 (8H, m, –OCH2CH2OCH2CH2O–), 4.37 (1H, d, J = 8.6 Hz,
HO-3), 4.60 (1H, d, J = 8.6 Hz, H-3), 4.81 (2H, s, 1,2,3-triazole-
8.0 ml minꢀ1, 254 nm) to obtain 7.7 mg of 1 and 8.8 mg of 2. 1: 1
H
NMR (270 MHz, acetone-d6): d 0.71 (9H, s, t-butyl), 3.54-3.71 (9H,
m, ꢀOCH2CH2OCH2CH2OH), 4.72 (2H, s, 1,2,3-triazole-CH2O–),
4.82 (1H, d, J = 5.9 Hz, HO-3), 5.23 (1H, d, J = 5.9 Hz, H-3), 7.20
(1H, s, H-1), 7.70 and 8.01 (each 2H, m, 1-phenyl), 8.04, 8.59, and
8.88 (each 1H, s, 1,2,4-triazole and 1,2,3-triazole); UV kmax (MeOH)
nm (
C
e): 269.6 (19,000); HRMS (ESI-TOF, positive mode): calcd for
22H30N6O4Na [M+Na]+ 465.2223, found 465.2227. 2: 1H NMR

M. Okazaki et al. / Bioorg. Med. Chem. 20 (2012) 3162–3172
3169
A
A
t-butyl
GA3 1 μM
N4"
S-UNI
R-UNI
B
C
GA3 0.1 μM
no GA3
Figure 7. Comparison of the most stable conformation (A) of S- and R-UNI and the
rotamers of R-UNI in the C2-N100 bond (B) and C2–C3 bond (C). Stick models:
carbons, gray; hydrogens, white; oxygens, red; nitrogens, blue; and chlorines,
green.
no
inhibitor
S-UNI
0.1 μM
S-Abz-E2B
100 μM
a
Figure 9. Rescue of rice seedlings on exogenous GA3 in the presence of S-UNI and S-
Abz-E2B.
control R- Abz-E2B
(ESI-TOF, positive mode): calcd for
507.2696, found 507.2698.
C
25H36N6O4Na [M+Na]+
4.2.3. (E)-1-(4-(4-((2-(2-butoxyethoxy)ethoxy)methyl)-1H-1,2,3-
triazol-1-yl)phenyl)-4,4-dimethyl-2-(1H-1,2,4-triazol-1-yl)pent-
1-en-3-ol (Abz-E2B)
S- Abz-E2B
To a stirred solution of 1-butanol (2 mL) was added NaH (60% in
oil, 86 mg, 2.1 mmol) at room temperature under Ar. After stirred
for 15 min, Abz-E1 (100 mg, 168 lmol) dissolved in 1-propanol
(3 mL) was added. The mixture was stirred for 19 h at room temper-
ature. After quenched with sat. NH4Cl, the resulting mixture was
extracted with EtOAc (15 mL ꢁ 3). The organic layer was washed
with brine, dried over Na2SO4, and concentrated in vacuo. The
residual oil was purified by silica gel column chromatography with
3% MeOH in CH2Cl2 to obtain ( )-Abz-E2B (71.4 mg, 85%), which
was further purified for bioassays by HPLC (YMC Hydrosphere
C18, 150 ꢁ 20 mm, 55% MeOH, 9.0 ml minꢀ1, 254 nm) to obtain a
colorless oil (70.5 mg). 1H NMR (270 MHz, CDCl3): d 0.68 (9H, s, t-
butyl), 0.90 (3H, t, J = 7.3 Hz, –O CH2CH2CH2CH3), 1.35 (2H, m, –
OCH2CH2CH2CH3), 1.56 (2H, m, –OCH2CH2CH2CH3), 3.46 (2H, t,
J = 6.6 Hz, –OCH2CH2CH2CH3), 3.60–3.78 (8H, m, –OCH2CH2OCH2-
CH2O–), 4.35 (1H, d, J = 8.9 Hz, HO-3), 4.60 (1H, d, J = 8.9 Hz, H-3),
4.80 (2H, s, 1,2,3-triazole-CH2O–), 7.00 (1H, s, H-1), 7.56 (2H, m,
1-phenyl), 7.82 (2H, m, 1-phenyl), 8.08 (2H, m, 1,2,4-triazole and
1,2,3-triazole), 8.54 (1H, s, 1,2,4-triazole); UV kmax (MeOH) nm
S-UNI
0
3
10
30
100 μM
b
(
C
e
): 269.2 (21,000); HRMS (ESI-TOF, positive mode): calcd for
26H38N6O4Na [M+Na]+ 521.2852, found 521.2855.
Figure 8. Comparison of effect of R-Abz-E2B, S-Abz-E2B, and S-UNI on rice seedling
growth on day 7 after treatment (a), and the unstable retardant effect of S-Abz-E2B
at 100 lM.
4.3. Determination of the absolute configuration of optically
pure Abz-E2B by advanced Mosher’s method
CH2O–), 7.00 (1H, s, H-1), 7.56 (2H, m, 1-phenyl), 7.82 (2H, m, 1-
4.3.1. Optical resolution of Abz-E1
phenyl), 8.07 (2H, m, 1,2,4-triazole and 1,2,3-triazole), 8.54 (1H,
s, 1,2,4-triazole); UV kmax (MeOH) nm (e): 269.8 (22,000); HRMS
( )-Abz-E1 (27 mg) was subjected to chiral HPLC under the fol-
lowing conditions: column, Chiralpak AD-H (250 ꢁ 10 mm, Dai-

3170
M. Okazaki et al. / Bioorg. Med. Chem. 20 (2012) 3162–3172
cel); solvent, 35% EtOH in hexane; flow rate, 5 mL minꢀ1; detec-
tion, 254 nm. The materials at tR 29.5-32.5 and 36.7–40.5 min were
collected to give (+)-Abz-E1 (12.9 mg) and (ꢀ)-Abz-E1 (13.6 mg),
respectively, with an optical purity of each 99.9%. (+)-Abz-E1:
(+)-Abz-E2B, 5.6 mg). Although the sign of the rotation of Abz-
E2B in each diastereomer was specified in parentheses to avoid
confusion, it was yet unknown at this time and determined by
identifying the rotational sign of Abz-E2B released from the MTPA
esters (see Section 4.3.4). 1H NMR (500 MHz, CDCl3): diastereomer
A (R-MTPA-(+)-Abz-E2B): d 0.74 (9H, s, t-butyl), 0.90 (3H, t,
J = 7.3 Hz, –O CH2CH2CH2CH3), 1.35 (2H, m, –OCH2CH2CH2CH3),
½
a 2D3
ꢂ
+18.6 (MeOH, c 0.860); (ꢀ)-Abz-E1: ½a D23
ꢀ23.7 (MeOH, c
ꢂ
0.907). 1H NMR and HRMS data of optically pure Abz-E1 agreed
with those of racemic Abz-E1.
1.56 (2H, m, –OCH2CH2CH2CH3), 3.47 (2H, t, J = 6.6 Hz,
–
4.3.2. Preparation of optically pure Abz-E2B
OCH2CH2CH2CH3), 3.60-3.77 (11H, m, 4 ꢁ –CH2O– and –OMe),
4.81 (2H, s, 1,2,3-triazole-CH2O–), 6.15 (1H, s, H-3), 7.26 (1H, s,
H-1), 7.44 (3H, m, phenyl in MTPA), 7.60 (2H, m, phenyl in MTPA),
7.67 and 7.87 (each 2H, m, 1-phenyl), 7.82 and 7.94 (each 1H, s,
1,2,4-triazole), 8.09 (1H, s, 1,2,3-triazole); diastereomer B (R-
MTPA-(ꢀ)-Abz-E2B): d 0.67 (9H, s, t-butyl), 0.90 (3H, t, J = 7.3 Hz,
–O CH2CH2CH2CH3), 1.36 (2H, m, –OCH2CH2CH2CH3), 1.56 (2H,
m, –OCH2CH2CH2CH3), 3.47 (2H, t, J = 6.6 Hz, –OCH2CH2CH2CH3),
3.51 (3H, s, –OMe), 3.60-3.77 (11H, m, 4 ꢁ –CH2O–), 4.81 (2H, s,
1,2,3-triazole-CH2O–), 6.21 (1H, s, H-3), 7.35 (1H, s, H-1), 7.48
(3H, m, phenyl in MTPA), 7.60 (2H, m, phenyl in MTPA), 7.68 and
7.86 (each 2H, m, 1-phenyl), 7.80 and 8.08 (each 1H, s, 1,2,4-tria-
zole), 8.09 (1H, s, 1,2,3-triazole). Protons of 1,2,4-triazole and
1,2,3-triazole were assigned on the basis of an HMBC correlation:
-N-CH@C(N@N–)-CH2O–.
(+)-Abz-E2B (3.6 mg) and (ꢀ)-Abz-E2B (5.1 mg) were prepared
from (+)-Abz-E1 (10 mg) respectively in the same manner as ( )-
Abz-E2B. (+)-Abz-E2B: 1H NMR (270 MHz, CDCl3): d 0.68 (9H, s,
t-butyl), 0.90 (3H, t, J = 7.5 Hz, –OCH2CH2CH2CH3), 1.36 (2H, m, –
OCH2CH2CH2CH3), 1.56 (2H, m, –OCH2CH2CH2CH3), 3.46 (2H, t,
J = 6.6 Hz, –OCH2CH2CH2CH3), 3.61–3.77 (8H, m, –OCH2CH2OCH2-
CH2O–), 4.35 (1H, d, J = 8.9 Hz, HO-3), 4.60 (1H, d, J = 8.9 Hz, H-3),
4.80 (2H, s, 1,2,3-triazole-CH2O–), 7.00 (1H, s, H-1), 7.56 (2H, m,
1-phenyl), 7.82 (2H, m, 1-phenyl), 8.07 (1H, s, 1,2,4-triazole),
8.08 (1H, s, 1,2,3-triazole), 8.54 (1H, s, 1,2,4-triazole); 13C NMR
(67.8 MHz, CDCl3): d 13.9 (–OCH2CH2CH2CH3), 19.3 (–OCH2CH2
CH2CH3), 26.1 (methyls in t-butyl), 31.7 (–OCH2CH2CH2CH3), 36.3
(tertiary carbon in t-butyl), 64.7, 70.0, 70.1, 70.6, and 70.7 (diethyl-
ene glycol linker), 71.2 (–OCH2CH2CH2CH3), 75.8 (C3), 120.6 (1,2,3-
triazole), 120.7 (phenyl), 127.5 (C1), 130.3 (phenyl), 134.3 (phe-
nyl), 136.7 (phenyl), 137.8 (C2), 143.2 (1,2,4-triazole), 146.4
(1,2,3-triazole), 151.7 (1,2,4-triazole); HRMS (ESI-TOF, positive
4.3.4. Hydrolysis of the diastereomers A and B
To a stirred solution of the diastereomer A (5.6 mg, 7.8 lmol) in
mode): calcd for
C
26H38N6O4Na [M+Na]+ 521.2852, found
MeOH (1.2 mL) was added 1 M NaOH (1 mL). After stirred for 1.5 h,
1 M HCl (1.5 mL) was added to quench the reaction. The resulting
mixture was extracted with EtOAc (8 mL ꢁ 3). The organic layer
was washed with brine, dried over Na2SO4, and concentrated in va-
cuo. The residual oil was purified by silica gel column chromatog-
raphy with 5% MeOH in CH2Cl2 to obtain a crude oil containing
Abz-E2B. The crude oil was further purified by HPLC (YMC Hydro-
sphere C18, 150 ꢁ 20 mm, 70% MeOH, 8.0 ml minꢀ1, 254 nm) to
521.2843; [
a
]26D +24.7 (MeOH, c 0.146). (ꢀ)-Abz-E2B: 1H NMR
(270 MHz, CDCl3): d 0.68 (9H, s, t-butyl), 0.90 (3H, t, J = 7.3 Hz, –
OCH2CH2CH2CH3), 1.34 (2H, m, –OCH2CH2CH2CH3), 1.56 (2H, m,
–OCH2CH2CH2CH3), 3.46 (2H, t, J = 6.6 Hz, –OCH2CH2CH2CH3),
3.60–3.76 (8H, m, –OCH2CH2OCH2CH2O–), 4.35 (1H, d, J = 8.9 Hz,
HO-3), 4.60 (1H, d, J = 8.9 Hz, H-3), 4.80 (2H, s, 1,2,3-triazole-
CH2O–), 7.00 (1H, s, H-1), 7.56 (2H, m, 1-phenyl), 7.82 (2H, m, 1-
phenyl), 8.07 (2H, s, 1,2,4-triazole and 1,2,3-triazole, overlapped),
8.54 (1H, s, 1,2,4-triazole); 13C NMR (67.8 MHz, CDCl3): d 13.9
(–OCH2CH2CH2CH3), 19.3 (–OCH2CH2CH2CH3), 26.1 (methyls in
t-butyl), 31.7 (–OCH2CH2CH2CH3), 36.3 (tertiary carbon in t-butyl),
64.7, 70.0, 70.1, 70.6, and 70.7 (diethylene glycol linker), 71.2
(–OCH2CH2CH2CH3), 75.8 (C3), 120.6 (1,2,3-triazole), 120.7 (phe-
nyl), 127.5 (C1), 130.3 (phenyl), 134.3 (phenyl), 136.7 (phenyl),
137.7 (C2), 143.2 (1,2,4-triazole), 146.4 (1,2,3-triazole), 151.7
(1,2,4-triazole); HRMS (ESI-TOF, positive mode): calcd for
obtain Abz-E2B (2.8 mg, 5.6
same manner as the diastereomer A, the diastereomer B (15.6
mg, 21.8 mol) was hydrolyzed to release Abz-E2B (4.5 mg, 9.0
mol, 41%) as a colorless oil. Abz-E2B obtained from the diastereo-
lmol, 72%) as a colorless oil. In the
l
l
mers A and B were determined to be the (+)- and (ꢀ)-isomers,
respectively, on the basis of chiral HPLC analysis (see Section 4.3.2).
4.4. Water-solubility test
C
26H38N6O4Na [M+Na]+ 521.2852, found 521.2857; (+)-Abz-E2B:
MeOH solutions of test samples were put in a glass vial and con-
centrated in vacuo. Distilled water (2 mL) or MeOH (2 mL) was
added to the vial. After shaking several times and leaving to stand
½
a 2D7
ꢂ
ꢀ24.1 (MeOH, c 0.157). Protons of 1,2,4-triazole and 1,2,3-
triazole were assigned on the basis of an HMBC correlation: –N–
CH@C(N@N–)–CH2O–.
for 1 h, 5 lL of the solution was subjected to HPLC. HPLC conditions
were: ODS column, Hydrosphere C18 (150 ꢁ 6.0 mm, YMC); sol-
vent, 85% MeOH in H2O; flow rate, 1.0 mL minꢀ1; detection,
254 nm. Solubility (%) in water was calculated based on the in
water/in MeOH ratio of the HPLC peak area.
4.3.3. Preparation of R-MTPA-esters of the optically pure Abz-
E2B
To a stirred solution of ( )-Abz-E2B (50 mg, 100
pyridine-CH2Cl2 (1:1, 450 l) was added dimethylaminopyridine
(78.9 mg, 703 mol) and S- -methoxy- -(trifluoromethyl)phenyl-
acetyl chloride (MTPA-Cl) (55 l, 293 mol). After stirred for 6 h,
lmol) in dry
l
l
a
a
4.5. Preparation of recombinant enzymes
l
l
water was added to quench the reaction. The resulting mixture
was extracted with EtOAc (7 mL ꢁ 3). The organic layer was
washed with brine, dried over Na2SO4, and concentrated in vacuo.
The residual oil was purified by silica gel column chromatography
with 70% EtOAc in hexane to obtain R-MTPA-( )-Abz-E2B (55 mg,
4.5.1. Coexpression of recombinant Arabidopsis CYP707A3 and
Arabidopsis P450 reductase (ATR2) in E. coli
A truncated Arabidopsis CYP707A3 (707A3d28), which lacked
the putative membrane-spanning segment of the N-terminus, res-
idues 3-28, was constructed. Cells of E. coli strain BL21 were trans-
formed with the constructs pCW-CYP707A3d28 and pACYC-AR2.
Cultures (3 mL) were grown overnight in Luria-Bertani medium
63 lmol, 63%) as a pale yellow oil. R-MTPA-( )-Abz-E2B (55 mg)
was diastereomerically resolved by silicagel HPLC (YMC-Pack SIL-
06, 150 ꢁ 20 mm, 30% 2-propanol in hexane, 4.0 ml minꢀ1
,
supplemented with ampicillin (50
l
g mLꢀ1) and chloramphenicol
254 nm) and subsequently purified by chiral HPLC (Chiralpak AD-
H, 250 ꢁ 10 mm, Daicel; solvent, 20% EtOH in hexane; flow rate,
4 mL minꢀ1; detection, 254 nm) to obtain the diastereomer A (R-
MTPA-(ꢀ)-Abz-E2B, 15.6 mg) and the diastereomer B (R-MTPA-
(100
l
g mLꢀ1). Then, 50 mL of Terrific Broth medium supple-
mented with ampicillin (50 l lg
g mLꢀ1), chloramphenicol (100
mLꢀ1), and aminolevulic acid (0.5 mM) was inoculated with an ali-
quot of the overnight culture (0.5 mL). The culture was incubated

M. Okazaki et al. / Bioorg. Med. Chem. 20 (2012) 3162–3172
3171
with gentle shaking (225 rpm) at 37 °C until A600 reached 0.6 (at
2.5–3 h), and expression of the P450 enzyme was induced by the
addition of isopropyl b- -thiogalactopyranoside (0.1 mM). The cul-
NADPH, and stopped by addition of 50
tion mixture was acidified with 100
reaction products, the mixture was loaded onto an Oasis HLB car-
tridge (1 mL, 30 mg; Waters) and washed with 1 mL of 10% MeOH
in H2O containing 1% AcOH. The enzyme products were then eluted
with 1 mL of MeOH containing 1% AcOH, and the eluate was con-
l
L of 1 M NaOH. Each reac-
l
L of 1 M HCl. To extract the
D
ture was shaken continuously (150 rpm) at 25 °C and cells were
harvested 48 h later by centrifugation at 2,330ꢁg for 20 min at
4 °C. Pelleted cells were suspended in 2.5 mL of buffer A (50 mM
potassium phosphate buffer, pH 7.25, 20% glycerol, 1 mM EDTA,
0.1 mM dithiothreitol). The suspension was sonicated for 30 s,
and the enzyme solution in the supernatant was collected by cen-
trifugation at 23,470ꢁg for 30 min at 4 °C. The P450 content was
determined by spectrophotometric analysis, using the extinction
coefficient of a reduced CO difference spectrum (91.1 mMꢀ1 cmꢀ1).
centrated in vacuo. The dried sample was dissolved in 50
MeOH, and 20 L was subjected to HPLC. HPLC conditions were:
ODS column, Hydrosphere C18 (150 ꢁ 6.0 mm, YMC); solvent,
20% MeCN in H2O containing 0.1% AcOH; flow rate, 1.0 mL minꢀ1
lL of
l
;
detection, 254 nm. Enzyme activity was confirmed by deter-
mining the amount of PA in control experiments before each set
of measurements. Inhibition constants were determined using
the Enzyme Kinetics module of SigmaPlot 10 software30 after
determining the mode of inhibition by plotting the reaction veloc-
ity in the presence and absence of inhibitor on a double-reciprocal
plot. All assays were conducted at least twice.
4.5.2. Cloning and heterologous expression of OsCYP701A6 with
a baculovirus-insect cell system
cDNA containing the entire open-reading frame of OsCYP701A6
was amplified by RT-PCR. The full-length OsCYP701A6 (AK066285)
was obtained from the Rice Genome Resource Center (the National
Institute of Agrobiological Sciences, Japan), and the OsCYP701A6
ORF was amplified by PCR with the full-length cDNA as template.
Nucleotide sequences of gene-specific primers were as follows:
TO-35: 50-AAAACTAGTATGGAGGCGTTCGTGC-30 (SpeI site in italics
and start codon underlined) and TO-36: 50-AAAATTCGAATCACAT-
CCTTCCTCTGCG-30 (HindIII site in italics and stop codon under-
lined). A 1-ng aliquot of the full-length cDNA was used as a
4.6.2. CYP701A6
The microsomal fractions of OsCYP701A6 were combined with
purified NADPH-P450 reductase.31 A reaction mixture containing
OsCYP701A6 microsomal fraction (25 pmol), ATR2 (0.125 U), ent-
kaurene (10
and inhibitor (0.03–10
50 mM potassium phosphate buffer (pH 7.25, 380
bated at 30 °C for 5 min. Reactions were initiated by addition of
20 mM NADPH (20 L), and were carried out at 30 °C for 30 min.
After termination by adding 1 M HCl (100 L) and EtOAc
(200 L), 5 L of 1 mM abietic acid was added as an internal stan-
l
M, final conc. after addition of NADPH) as substrate,
M, final conc. after addition of NADPH) in
L) was preincu-
l
l
template for the PCR reaction in a 20-
l
L reaction mixture contain-
L), PrimeSTAR HS DNA
M of the
ing 5ꢁ PrimeSTAR Buffer (Mg2+ plus) (4
l
l
Polymerase (0.5 U), 200
lM dNTP mixture, and 0.2
l
l
gene-specific primers described above. The PCR product was gel-
purified and cloned into pJET1.2/blunt using a CloneJET PCR Clon-
ing Kit (Fermentas, Canada). The cloned inserts were sequenced
with pJET1.2 forward and reverse sequencing primers to confirm
the absence of PCR errors in the inserts. Full-length cDNA of OsCY-
P701A6 in pJET1.2/blunt plasmid vector was excised with the
restriction enzymes SpeI and HindIII (TaKaRa Bio) and purified by
1% (w/v) agarose gel electrophoresis. This cDNA was ligated into
pFastBac1 vector (Invitrogen, USA) digested with the same set of
restriction enzymes. The pFastBac1-OsCYP701A6 constructs were
used for the preparation of recombinant bacmid DNA by transfor-
mation of E. coli strain DH10Bac (Invitrogen). Preparation of re-
combinant baculovirus DNA containing OsCYP701A6 cDNA and
transfection of Sf9 (Spodoptera frugiperda 9) cells were performed
according to the manufacturer’s protocol (Invitrogen). For expres-
sion of OsCYP701A6, Sf9 cells infected by baculovirus containing
OsCYP701A6 cDNA were incubated in Grace’s insect cell medium,
0.1% (w/v) Pluronic F-68 (Invitrogen), 10% (v/v) fetal bovine serum,
l
l
dard. The reaction products were extracted three times with an
equal volume of ethyl acetate and the organic phase was collected.
Anhydrous Na2SO4 was added to remove residual water. To deriv-
atize the reaction products, methanol (100
(100 L) (2.0 M in Et2O) were added and the reaction mixture
was incubated at room temperature for 15 min. Organic solvent
was removed under Ar and samples were adjusted to 200 L with
lL) and TMS–CHN2
l
l
Et2O before GC-MS analysis (QP2010-plus, Shimadzu Corp., Japan).
GC conditions were: column, DB-5 ms (0.25 mm id ꢁ 15 m;
0.25 lm film thickness; J&W Scientific); carrier gas, He; flow rate,
1.84 ml minꢀ1; injection port temperature, 280 °C; splitless injec-
tion; column oven temperature, 80 °C (1 min), 80-200 °C
(18 °C min-1), 200–210 °C (2 °C minꢀ1), 210–280 °C (30 °C minꢀ1),
280 °C (3 min). The content of KA was calculated from the area ra-
tio of molecular and fragment ions of methyl KA (m/z 316, 273, and
257) to those of methyl abietate (m/z 316 and 256).
100
l
M 5-aminolevulinic acid and 100
l
M ferrous citrate on a ro-
4.7. Bioassays
tary shaker at 27 °C and 150 rpm for 96 h. Insect cells were then
harvested by centrifugation at 3,000ꢁg for 10 min and washed
with PBS buffer three times. After centrifuging again, insect cells
were sonicated in buffer A containing 50 mM potassium phosphate
(pH 7.3), 20% (v/v) glycerol, 1 mM EDTA, 0.1 mM dithiothreitol and
centrifuged at 8,000ꢁg for 10 min. The supernatant was further
centrifuged at 100,000ꢁg for 1 h and the resulting pellet (micro-
somal fraction) was homogenized with buffer A. The concentration
of active P450 was estimated from the carbon monoxide difference
spectrum.29
4.7.1. Arabidopsis seed germination assay
Twenty-five seeds (Col-0) were sterilized successively with 70%
(v/v) EtOH for 30 min and reagent grade EtOH for 1 min. The ster-
ilized seeds were soaked in 250 lL of a test solution and incubated
in the dark for 3 days at 5 °C. The vernalized seeds in the test solu-
tion were transferred to 24-well plates placed on two sheets of fil-
ter paper, and allowed to germinate under continuous light for
24 h at 22 °C. All assays were conducted at least twice.
4.7.2. Lettuce seed germination and growth assays
4.6. Enzyme assay
Five seeds (Lactuca sativa L. cv. Grand Rapids) were placed in
24-well plates on two sheets of filter paper soaked in 0.2 mL of a
test solution and allowed to germinate and grow under continuous
light for 23 days at 22 °C. All assays were conducted at least twice.
4.6.1. CYP707A3
A reaction mixture containing 25
somes coexpressed AR2 in E. coli, (+)-ABA (final conc. 1–64
inhibitors (0 for control, 5–10,000 nM in 5 L DMF) and 130
l
g mLꢀ1 of CYP707A3 micro-
M),
l
l
lM
4.7.3. Rice seedling elongation assay
NADPH in 100 mM potassium phosphate buffer (pH 7.25) were
incubated for 10 min at 30 °C. Reactions were initiated by adding
Seeds of rice (O. sativa L. cv. Nipponbare) were sterilized with
EtOH for 5 min and washed with running tap water. The sterilized

3172
M. Okazaki et al. / Bioorg. Med. Chem. 20 (2012) 3162–3172
seeds were soaked in water for 3 days at 25 °C to germinate. The
seeds were then placed in a glass tube containing 2 mL of a test
solution and grown with the tube sealed with a plastic cap under
continuous light at 25 °C. When the seedlings were 7 days old,
the length of the second leaf sheath was measured. All assays were
conducted at least twice.
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, M. R.; Wu, W.; Ambrose, S. J.; Ross, A. R.; Abrams, S. R.;
Cutler, A. J. Plant J. 2007, 50, 414.
4.7.4. Bent grasses drought tolerance assay
6. Loveys, B. R. In Abscisic Acid; Davies, W. J., Jones, H. G., Eds.; BIOS Scientific
Publishers: Oxford, 1991.
7. Saito, S.; Hirai, N.; Matsumoto, C.; Ohigashi, H.; Ohta, D.; Sakata, K.; Mizutani,
M. Plant Physiol. 2004, 134, 1439.
8. Kushiro, T.; Okamoto, M.; Nakabayashi, K.; Yamagishi, K.; Kitamura, S.; Asami,
T.; Hirai, N.; Koshiba, T.; Kamiya, Y.; Nambara, E. EMBO J. 2004, 23, 1647.
9. Yang, S. H.; Choi, D. Biochem. Biophys. Res. Commun. 2006, 350, 685.
10. Yang, S. H.; Zeevaart, J. A. D. Plant J. 2006, 47, 675.
11. Millar, A. A.; Jacobsen, J. V.; Ross, J. J.; Helliwell, C. A.; Poole, A. T.; Scofield, G.;
Reid, J. B.; Gubler, F. Plant J. 2006, 45, 942.
Bent grass (Agrostis capillaris), which was planted in a plastic
box with moist vermiculite and grown in a greenhouse covered
with polyvinyl film, was used in theses experiments. A 0.2% (v/v)
Hyponex nutrient solution (Hyponex Japan Co., Ltd, Osaka, Japan)
was supplied at 2 L mꢀ2 to vermiculite at 14 day intervals. The
temperature ranged from 25 to 33 °C during the day (6 a.m. to 6
p.m.) and 23 to 25 °C at night (6 p.m. to 6 a.m.). The relative humid-
ity (RH) was 45–65% during the day and 65–75% at night. Ninety-
day-old bent grass was planted uniformly in a plastic box 40 cm
wide, 40 cm long and 5 cm deep, watered, then sprayed with a test
12. Destefano-Beltran, L.; Knauber, D.; Huckle, L.; Suttle, J. C. Plant Mol. Biol. 2006,
61, 687.
13. Umezawa, T.; Okamoto, M.; Kushiro, T.; Nambara, E.; Oono, Y.; Seki, M.;
Kobayashi, M.; Koshiba, T.; Kamiya, Y.; Shinozaki, K. Plant J. 2006, 46, 171.
14. Okamoto, M.; Kuwahara, A.; Seo, M.; Kushiro, T.; Asami, T.; Hirai, N.; Kamiya,
Y.; Koshiba, T.; Nambara, E. Plant Physiol. 2006, 141, 97.
15. Funaki, Y.; Ishiguri, T.; Kato, T.; Tanaka, S. J. Pesticide Sci. 1984, 9, 229.
16. Funaki, Y.; Oshita, H.; Yamamoto, S.; Tanaka, S.; Kato, T. (Sumitomo Chemical
Co.). Ger. Offen. DE3010560, 1980.
17. Saito, S.; Okamoto, M.; Shinoda, S.; Kushiro, T.; Koshiba, T.; Kamiya, Y.; Hirai,
N.; Todoroki, Y.; Sakata, K.; Nambara, E.; Mizutani, M. Biosci. Biotechnol.
Biochem. 2006, 70, 1731.
18. Izumi, K.; Kamiya, Y.; Sakurai, A.; Oshio, H.; Takahashi, N. Plant Cell Physiol.
1985, 26, 821.
19. Kitahata, N.; Saito, S.; Miyazawa, Y.; Umezawa, T.; Shimada, Y.; Min, Y. K.;
Mizutani, M.; Hirai, N.; Shinozaki, K.; Yoshida, S.; Asami, T. Bioorg. Med. Chem.
2005, 13, 4491.
solution (50
put in a controlled room at 350
l
M Abz-E2B) or distilled water. The boxes were then
l
mol mꢀ2 s-1 photosynthetic pho-
ton flux at 25 °C and 60% RH. The bent grass was grown for 10 days
under dehydration conditions (water not supplied to the vermicu-
lite) until rehydration (a sufficient amount of water was supplied
to the vermiculite). Under dehydration and rehydration conditions,
leaves of bent grass were sampled for analysis of endogenous ABA
content. The extraction and quantification of ABA in apple seed-
lings were performed according to a method similar to that re-
ported previously in apple seedlings32 using HPLC and gas
chromatography–mass spectrometry-selective ion monitoring
(GC-MS-SIM) (Shimadzu QP5000). ABA was calculated by the ratio
of peak areas for m/z 190(d0)/194(d6). 30,50,50,70,70,70-Hexadeuterat-
ed ABA (ABA-d6) was purchased from Shoko Co. (Tokyo, Japan).
20. Fletcher, R. A.; Hofstra, G.; Gao, J.-G. Plant Cell Physiol. 1986, 27, 367.
21. Todoroki, Y.; Ueno, K. Cur. Med. Chem. 2010, 17, 3230.
22. Todoroki, Y.; Aoyama, H.; Hiramatsu, S.; Shirakura, M.; Nimitkeatkai, H.;
Kondo, S.; Ueno, K.; Mizutani, M.; Hirai, N. Bioorg. Med. Chem. Lett. 2009, 19,
5782.
23. Todoroki, Y.; Kobayashi, K.; Shirakura, M.; Aoyama, H.; Takatori, K.;
Nimitkeatkai, H.; Jin, M.-H.; Hiramatsu, S.; Ueno, K.; Kondo, S.; Mizutani, M.;
Hirai, N. Bioorg. Med. Chem. 2009, 17, 6620.
Acknowledgments
24. Todoroki, Y.; Naiki, K.; Aoyama, H.; Shirakura, M.; Ueno, K.; Mizutani, M.; Hirai,
N. Bioorg. Med. Chem. Lett. 2010, 20, 5506.
25. Okazaki, M.; Nimitkeatkai, H.; Muramatsu, T.; Aoyama, H.; Ueno, K.; Mizutani,
M.; Hirai, N.; Kondo, S.; Ohnishi, T.; Todoroki, Y. Bioorg. Med. Chem. 2011, 19,
406.
26. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113,
4092.
27. Todoroki, Y.; Kobayashi, K.; Yoneyama, H.; Hiramatsu, S.; Jin, M.-H.; Watanabe,
B.; Mizutani, M.; Hirai, N. Bioorg. Med. Chem. 2008, 16, 3141.
28. Ueno, K.; Yoneyama, H.; Mizutani, M.; Hirai, N.; Todoroki, Y. Bioorg. Med. Chem.
2007, 15, 6311.
We thank Prof. Akira Kitajima (Kyoto University) for kindly pro-
viding rice (Nipponbare) seeds. We thank Toray Industries Inc., To-
kyo, Japan, for a gift of (+)-ABA. Part of this research was carried
out using an instrument at the Center for Instrumental Analysis
of Shizuoka University. This research was supported by a Grant-
in-Aid for Scientific Research (No. 22580118) from the Ministry
of Education, Culture, Sports, Science and Technology of Japan.
29. Omura, T.; Sato, R. J. Biol. Chem. 1964, 239, 2370.
Supplementary data
30. SigmaPlot 10; Systat Software, Inc.: Richmond CA, 2006.
31. Mizutani, M.; Ohta, D. Plant Physiol. 1998, 116, 357.
32. Kondo, S.; Ponrod, W.; Kanlayanarat, S.; Hirai, N. J. Am. Soc. Hort. Sci. 2002, 127,
737.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.03.068.