Effect of the minor ABA metabolite 7′-hydroxy-ABA on Arabidopsis ABA 8′-hydroxylase CYP707A3

Article abstract of DOI:10.1016/j.bmcl.2007.06.018

To examine the effect of the minor abscisic acid (ABA) metabolite 7′-hydroxy-ABA on Arabidopsis ABA 8′-hydroxylase (CYP707A3), we developed a novel and facile, four-step synthesis of 7′-hydroxy-ABA from α-ionone. Structural analogues of 7′-hydroxy-ABA, 1′

Full text of DOI:10.1016/j.bmcl.2007.06.018

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4977–4981
Effect of the minor ABA metabolite 70-hydroxy-ABA
on Arabidopsis ABA 80-hydroxylase CYP707A3
Hajime Shimomura,a Hideo Etoh,a Masaharu Mizutani,b
Nobuhiro Hiraic and Yasushi Todorokia,*
aDepartment of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan
bInstitute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
cInternational Innovation Center, Kyoto University, Kyoto 606-8501, Japan
Received 14 April 2007; revised 4 June 2007; accepted 6 June 2007
Available online 10 June 2007
Abstract—To examine the effect of the minor abscisic acid (ABA) metabolite 70-hydroxy-ABA on Arabidopsis ABA 80-hydroxylase
(CYP707A3), we developed a novel and facile, four-step synthesis of 70-hydroxy-ABA from a-ionone. Structural analogues of 70-
hydroxy-ABA, 10-deoxy-70-hydroxy-ABA, and 70-oxo-ABA were also synthesized to evaluate the role of the 70-hydroxyl group on
binding to the enzyme. The result of enzyme inhibition assay suggests that the local polarity at C-70, neither steric bulkiness nor
overall molecular hydrophilicity, would be the major reason why (+)-70-hydroxy-ABA is not a potent inhibitor of CYP707A3.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Abscisic acid (ABA) 80-hydroxylase (e.g., Arabidopsis
CYP707A1-CYP707A4)1,2 is a cytochrome P450 mono-
oxygenase and a key catabolic enzyme controlling
inactivation of ABA, a plant hormone involved in
stress tolerance. The major ABA metabolite in most
plants is 80-hydroxy-ABA (Fig. 1), the product of
ABA 80-hydroxylase, that is easily and nonenzymatical-
ly converted into phaseic acid (PA), a low ABA
activity compound. In some plants the glucose-conju-
gated form of ABA, ABA 10-glucosyl ester, is the major
metabolite.3–6 The minor oxidative pathways, 70- and
90-hydroxylations, are also found in some plants.7
Because Arabidopsis CYP707A3 converted ABA
into neither 70-hydroxy-ABA nor 90-hydroxy-ABA,1,2
these minor oxidative pathways are considered to be
catalyzed by other enzymes than ABA 80-hydroxylase.
probing cellular and molecular events involving ABA.
Recently, we proposed AHI4 as a non-azole inhibitor
of ABA 80-hydroxylase.8 AHI4 was designed to have
an axial hydroxyl group instead of the geminal methyl
groups found at C-60 of AHI1,9 and without the enone
moiety and 3-methyl (C-6) present in the ABA structure
(Fig. 2). AHI4 exhibited more potent inhibitory effect on
this enzyme than AHI1. This suggests that the hydroxyl
group added on the ring reinforced the affinity with the
active site of the enzyme. Considering that the enzyme
product should egress from the active site, 80-hydroxy-
ABA can probably not bind the active site. In this case,
the additional hydroxyl group will function as a substi-
tuent to decrease the affinity. The location of the addi-
tional hydroxyl group will determine whether it
reinforces the affinity of the inhibitor for the active site
or not. Because this knowledge is significant for
designing new inhibitors of ABA 80-hydroxylase, we
are interested in whether the minor oxidative metabo-
lites can inhibit ABA 80-hydroxylase.
Chemical regulation of ABA catabolism by enzyme
inhibitors is a practical method to control ABA concen-
tration. Because ABA is largely catabolized through
80-hydroxylation by ABA 80-hydroxylase, specific inhib-
itors of this enzyme are likely to be very useful tools for
In this paper, we focused on 70-hydroxy-ABA. Our pre-
vious work revealed that an additional methyl group at
C-70 has little effect on the enzyme inhibitory potency,
whereas for C-80 and C-90 it largely decreased the
affinity for the active site.10 This suggests that 70-hydro-
xy-ABA will not be moved out of the active site owing
to its steric bulkiness. If an additional hydroxyl group
Keywords: Abscisic acid; ABA; 70-Hydroxy-ABA; Cytochrome P450;
ABA 80-hydroxylase.
*
Corresponding author. Tel./fax: +81 54 238 4871; e-mail:
aytodor@agr.shizuoka.ac.jp
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.018

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H. Shimomura et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4977–4981
Figure 1. Catabolic pathway of (+)-ABA in plants. The key step is the 80-hydroxylation catalyzed by ABA 80-hydroxylase. 80-Hydroxy-ABA is
spontaneously isomerized to (ꢀ)-phaseic acid (PA), which is (probably enzymatically) reduced to give (ꢀ)-dihydro-PA. The activity of (ꢀ)-phaseic
acid is one-tenth to one-hundredth of that of (+)-ABA. (ꢀ)-Dihydro-PA exhibits no ABA activity. (+)-70-Hydroxy-ABA is more potent than (ꢀ)-
PA.19 The activity of (+)-90-hydroxy-ABA is one-tenth of that of (+)-ABA, whereas (+)-neoPA is inactive.20 The conjugates are inactive.
and their inhibitory activity against recombinant Ara-
OH
bidopsis ABA 80-hydroxylase with respect to the effect
of the 70-hydroxyl group on their binding to the active
site of the enzyme.
OH
OH
CO2H
CO2H
Nelson et al. reported a total synthesis of 70-hydroxy-
ABA in 1991 using a synthetic route composed of 13
steps from 1,4-cyclohexanedione monoacetal.11 We
found a novel and facile synthetic route for 70-hydroxy-
ABA, which is made of only four steps from a-ionone
(Scheme 1),12 although overall yield is low. a-Ionone
was acetoxylated with iodine and silver acetate in ben-
zene to afford 1. Oxidation of 1 with tert-butyl chromate
in tert-butanol afforded 2. Compound 2 was converted to
3 as a 2Z/2E mixture (5:3). Hydrolysis of 3 with porcine
liver esterase afforded ( )-70-hydroxy-ABA. In this reac-
tion, the 2Z-isomer was completely hydrolyzed, whereas
the hydrolysis of 2E-isomer occurred not at C-1, but only
at C-70, to afford the methyl ester of ( )-(2E)-70-hydroxy-
ABA. Oxidation of 1 with pyridinium dichromate and
tert-butyl hydroperoxide gave the deoxy compound 4.
In a similar manner for 2, the deoxy compound 4 affor-
ded ( )-10-deoxy-70-hydroxy-ABA via 5. The racemic
compounds were resolved into (+)- and (ꢀ)-isomers by
HPLC employing a chiral column. Oxidation of optically
pure 70-hydroxy-ABA afforded optically pure 70-oxo-
ABA. The absolute configuration was determined based
AHI1
AHI4
Figure 2. Chemical structures of non-azole ABA 80-hydroxylase
inhibitors.
at C-70 reduces the affinity for the enzyme, it should
depend on the higher polarity or unfavorable electro-
static interactions via the 70-hydroxyl. Thus we prepared
10-deoxy-70-hydroxy-ABA and 70-oxo-ABA in addition
to 70-hydroxy-ABA (Scheme 1). Because the hydroxyl
group at C-10 has no effect on binding to the enzyme,10
it is considered that the absence of 10-hydroxy and the
addition of 70-hydroxyl groups cancels each other out
in respect to molecular polarity in 10-deoxy-70-hydroxy-
ABA. This implies that we can consider the effect of
the 70-hydroxyl group itself without considering the
molecular polarity. 70-Oxo-ABA will be also less polar
than 70-hydroxy-ABA. Using this molecule as well as
70-methyl-ABA, we discuss the effect of the local
polarity at C-70 on the interaction with the enzyme. This
paper describes a novel and facile synthesis of 70-hydro-
xy-ABA, 10-deoxy-70-hydroxy-ABA, and 70-oxo-ABA,

H. Shimomura et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4977–4981
4979
O
α-ionone
i
iii
vi
O
O
CO2Me
O
O
OAc
OAc
OAc
1
4
5
iv, v
ii
O
OH
CO2H
O
O
OAc
OH
2
(+)-1'-Deoxy-7'-hydroxy-ABA
iii
iv, v
vii
OH
OH
OH
O
OH
CO2H
CO2H
CO2Me
O
O
O
OAc
3
(+)-7'-Hydroxy-ABA
(+)-7'-Oxo-ABA
Scheme 1. Synthesis of (+)-70-hydroxy-ABA and its analogues. Reagents: (i) iodine, silver acetate, benzene, 19%; (ii) tert-butyl chromate in tert-butyl
alcohol, acetic anhydride, 10%; (iii) bis(2,2,2-trifluoroethyl)(methoxycarbonylmethyl)phosphonate, KN(TMS)2, THF, 52%; (iv) porcine liver
esterase, MeOH-phosphate buffer, 89%; (v) chiral HPLC; (vi) pyridinium dichromate, t-BuOOH, CH2Cl2, 20%; (vii) MnO2, acetone, 61%.
Compounds 1–5 are a racemic mixture. The detailed procedure: see Supplementary data.
Table 1. The calculated partition coefficient14 (ClogP) of the undis-
sociated form, the pH-dependent partition coefficient15 (logD), and the
retention factor16 (k) in the ODS HPLC analysis
on the Cotton effect in the CD spectrum13; each (+)-iso-
mer had the same configuration as S-(+)-ABA, a natural
type of ABA.
Compound
ClogP
logD
k
Based on the calculated partition coefficient14 (ClogP) of
the undissociated form and the pH-dependent distribu-
tion coefficient15 (logD), (+)-70-hydroxy-ABA is as polar
as (+)-80-hydroxy-ABA (Table 1). (+)-70-Oxo-ABA is
estimated to be less polar than (+)-70-hydroxy-ABA,
although more polar than (+)-ABA. (+)-10-Deoxy-70-hy-
droxy-ABA is calculated to be similar to (+)-ABA. In the
HPLC analysis with an ODS column,16 the retention
factor (k) is larger in the order of 80-hydroxy-ABA < 70-
hydroxy-ABA < 70-oxo-ABA < ABA < 10-deoxy-70-hy-
droxy-ABA (Table 1). This almost agrees with the
polarity based on the predicted partition or distribution
coefficients.
pH 3 pH 5 pH 7
80-Hydroxy-ABA
70-Hydroxy-ABA
70-Oxo-ABA
ABA
0.0495 0.76 0.23
0.0495 0.76 0.23
0.4088 0.88 0.28
0.8810 2.07 1.62
ꢀ1.60
ꢀ1.60
ꢀ1.58
4.86
6.34
9.63
ꢀ0.17 11.4
ꢀ0.37 12.8
10-Deoxy-70-hydroxy-ABA 1.2805 1.87 1.43
An observed NOE between H-5 and H-50-proS in the
NOESY spectra (Fig. 3) of the C70-modified analogues
suggested that their favored conformation is a chair with
the axial side chain. Therefore, we can discuss the effect
of the local structural difference between these analogues
on the affinity for the enzyme active site.
Figure 3. Favored conformations of 70-hydroxy-ABA, 10-deoxy-70-hydroxy-ABA, and 70-oxo-ABA.

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H. Shimomura et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4977–4981
Table 2. Inhibitory activity of optically pure 70-hydroxy-ABA and its
analogues against recombinant CYP707A3
Acknowledgment
Inhibitiona (%)
·10
We thank Toray Industries Inc., Tokyo, Japan, for the
gift of (+)-ABA.
Compound
·1
(+)-70-Hydroxy-ABA
(+)-70-Oxo-ABA
(+)-10-Deoxy-70-hydroxy-ABA
(+)-70-Methyl-ABA
(+)-10-Deoxy-ABA
0
0
15
0
3
63
24
83c
73c
1
4
Supplementary data
b
—
—
1
Synthetic procedure of new compounds and their H
and 13C NMR, high resolution MS, specific rotation,
and circular dichroism data are given. Supplementary
data associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2007.06.018.
a Inhibition ratio in the 80-hydroxylation for ABA (5 lM). The con-
centrations of compounds are 5 lM (·1) and 50 lM (·10). The
inhibition ratios reported in this paper are the averages and standard
deviations of three sets of experiments.
b Not measured.
c Ref. 10.
References and notes
The inhibitory activity of the optically pure compounds
against recombinant CYP707A3 is summarized in Table
2.17 The activity was evaluated based on the decrease of
the enzyme products, 80-hydroxy-ABA and phaseic
acid, caused by addition of a test compound at a concen-
tration equal to or 10 times higher than the substrate
(+)-ABA. (+)-70-Hydroxy-ABA had no inhibitory
effect on the enzyme reaction at the tested concentra-
tions. (+)-10-Deoxy-70-hydroxy-ABA was also a very
weak inhibitor, although it was a little more potent than
(+)-70-hydroxy-ABA. Only (+)-70-oxo-ABA exhibited
a significant activity, which was a little bit weaker than
that of (+)-70-methyl-ABA and (+)-10-deoxy-ABA.
All the (ꢀ)-isomers exhibited no significant inhibitory
activity (data not shown).
1. 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.
2. Saito, S.; Hirai, N.; Matsumoto, C.; Ohigashi, H.; Ohta,
D.; Sakata, K.; Mizutani, M. Plant Physiol. 2004, 134,
1439.
3. Hirai, N. In Comprehensive Natural Products Chemistry;
Mori, K., Ed.; Elsevier: Amsterdam, 1999; Vol. 8, pp
72–91.
4. Abscisic Acid; Davies, W. J., Jones, H. G., Eds.; BIOS
Scientific Publishers: Oxford, 1991.
5. Zeevaart, J. A. D.; Creelman, R. A. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 1998, 39, 439.
6. Abscisic Acid; Addicott, F. T., Ed.; Praeger: New York,
1983.
7. Walker-Simmons, M. K.; Holappa, L. D.; Abrams, G. D.;
Abrams, S. R. Physiol. Plant. 1997, 100, 474, and
references cited therein.
8. Araki, Y.; Miyawaki, A.; Miyashita, T.; Mizutani, M.;
Hirai, N.; Todoroki, Y. Bioorg. Med. Chem. Lett. 2006,
16, 3302.
9. Ueno, K.; Yoneyama, H.; Saito, S.; Mizutani, M.; Sakata,
K.; Hirai, N.; Todoroki, Y. Bioorg. Med. Chem. Lett.
2005, 15, 5226, We named the lead compound in this ref.
as AHI1.
10. Ueno, K.; Araki, Y.; Hirai, N.; Saito, S.; Mizutani, M.;
Sakata, K.; Todoroki, Y. Bioorg. Med. Chem. 2005, 13,
3359.
The catalytic cycle of a cytochrome P450 enzyme is trig-
gered as the substrate enters into the active site and dis-
places the axial water molecule. After the hydroxylation
reaction, the hydroxylated substrate causes the entrance
of bulk water molecules into the substrate-binding pock-
et which results in the release of the product from the
enzyme.18 This suggests that a substrate affinity for the
pocket decreases as its hydrophilicity is increased. In
fact, (+)-70-hydroxy-ABA, which is the most hydrophilic
compound, was the most inactive in (+)-isomers. How-
ever, (+)-10-deoxy-70-hydroxy-ABA was little active in
spite of its more hydrophobic character compared to
(+)-ABA, whereas (+)-70-oxo-ABA was more potent
than (+)-10-deoxy-70-hydroxy-ABA in spite of its less
hydrophobic character. Steric bulkiness of the 70-substit-
uents is not significant in this case, because introduction
of a methyl group at C-70 has little effect on binding to
the active site.10 Likewise, the presence or absence of the
oxygen at C-10 does not affect the inhibitory potency.10
Thus the local polarity at C-70, neither the steric bulki-
ness nor overall molecular hydrophilicity, would be the
major reason why (+)-70-hydroxy-ABA is not a potent
inhibitor. The potency of (+)-70-oxo-ABA suggests that
the hydrogen rather than the oxygen of the 70-hydroxyl
group causes some disadvantageous interaction with the
substrate-binding cavity of the enzyme, or interferes
with the approach of the compound to the cavity. The
present findings will be useful for designing a specific
inhibitor of CYP707A as a structural analogue of
ABA, in addition to discussing the substrate binding
mechanism of CYP707A.
11. Nelson, A. K.; Shaw, A. C.; Abrams, S. R. Tetrahedron
1991, 47, 3259.
12. The synthetic procedure and optical resolution of 70-
hydroxy-ABA, 10-deoxy-70-hydroxy-ABA, and 70-oxo-
ABA can be found in the supplementary data.
10-Deoxy-70-hydroxy-ABA and 70-oxo-ABA are new
ABA analogues. 10-Deoxy-70-hydroxy-ABA: 1H NMR
(500 MHz, CD3OD): d 1.01 (3H, s, H3-90), 1.06 (3H, s,
H3-80), 2.02 (3H, s, H3-6), 2.13 (1H, d, J = 16.8 Hz,
H-50), 2.56 (1H, d, J = 16.8 Hz, H-50), 2.83 (1H, d,
J = 9.6 Hz, H-10), 4.14 (2H, m, H2-70), 5.72 (1H, s, H-2),
6.11 (1H, dd, J = 15.8 and 9.6 Hz, H-5), 6.18 (1H, s,
H-30), 7.66 (1H, d, J = 15.8 Hz, H-4); HREIMS: [M]+ at
m/z 264.1360 (C15H20O4 requires 264.1362). 70-Oxo-
ABA: 1H NMR (270 MHz, CDCl3): d 1.08 (3H, s,
H3-90), 1.14 (3H, s, H3-80), 2.04 (3H, d, J = 1.0 Hz,
H3-6), 2.32 (1H, d, J = 16.8 Hz, H-50), 2.52 (1H, d,
J = 16.8 Hz, H-50), 4.36 (1H, s, HO-10), 5.76 (1H, s, H-
2), 6.25 (1H, d, J = 16.5 Hz, H-5), 6.69 (1H, s, H-30),
7.56 (1H, d, J = 16.5 Hz, H-4), 9.75 (1H, s, H-70);
HREIMS: [M]+ at m/z 278.1161 (C15H18O5 requires
278.1154).

H. Shimomura et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4977–4981
4981
13. Koreeda, M.; Weiss, G.; Nakanishi, K. J. Am. Chem. Soc.
1973, 95, 239. (+)-70-Hydroxy-ABA: CD kext (MeOH) nm
(De): 262 (+32.6), 229 (ꢀ26.3); (+)-10-deoxy-70-hydroxy-
ABA: CD kext (MeOH) nm (De): 268 (+30.9), 225 (ꢀ11.1);
(+)-70-oxo-ABA: CD kext (MeOH) nm (De): 264 (+15.9),
234 (ꢀ13.8).
equation: k = (tR ꢀ tM)/tM, where tM is the dead time that
is taken as the first deviation of the baseline following a
5 ll injection of 1% sodium nitrite solution and tR is the
retention time.
17. Detailed procedures for the enzyme inhibition assay were
reported previously (see Ref. 10).
18. Meunier, B.; de Visser, S. P.; Shaik, S. Chem. Rev. 2004,
104, 3947.
19. Hill, R. D.; Liu, J.-H.; Durnin, D.; Lamb, N.; Shaw, A.;
Abrams, S. R. Plant Physiol. 1995, 108, 573.
20. Zhou, R.; Cutler, A. J.; Ambrose, S. J.; Galka, M. M.;
Nelson, K. M.; Squires, T. M.; Loewen, M. K.; Jadhav, A.
S.; Ross, A. R. S.; Taylor, D. C.; Abrams, S. R. Plant
Physiol. 2004, 134, 361.
14. Chem 3D Ultra 10.0, Cambridge Soft, 2006.
15. Marvin, ChemAxon, http://www.chemaxon.com/; Csizm-
adia, F.; Tsantili-Kakoulidou, A.; Panderi, I.; Darvas, F.
J. Pharm. Sci. 1997, 86, 865.
16. Column, Hydrosphere C18 (S-5 lm, 12 nm, 150 · 6 mm
id); mobile phase, 40% MeOH (0.1% AcOH); flow
rate, 1.0 ml minꢀ1
factor (k) can be calculated according to the following
;
detection, 254 nm. The retention