The selectivity of 6-nor-ABA and 7′-nor-ABA for abscisic acid receptor subtypes

Article abstract of DOI:10.1016/j.bmcl.2015.06.088

Abstract Abscisic acid (ABA), a plant hormone, is involved in many plant development processes and environmental stress responses that are regulated by a Pyrabactin Resistant 1 (PYR)/Pyrabactin Resistant-Like (PYL)/Regulatory Component of ABA Receptor (RC

Full text of DOI:10.1016/j.bmcl.2015.06.088

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3507–3510
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The selectivity of 6-nor-ABA and 70-nor-ABA for abscisic acid receptor
subtypes
Jun Takeuchi a, , Toshiyuki Ohnishi a,b, Masanori Okamoto c, Yasushi Todoroki a,b,d,
⇑
a Graduate School of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
b Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
c Arid Land Research Center, Tottori University, 1390 Hamasaka, Tottori 680-0001, Japan
d Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Abscisic acid (ABA), a plant hormone, is involved in many plant development processes and environmen-
tal stress responses that are regulated by a Pyrabactin Resistant 1 (PYR)/Pyrabactin Resistant-Like
(PYL)/Regulatory Component of ABA Receptor (RCAR) receptor protein-mediated signal transduction
pathway. In Arabidopsis thaliana, PYL proteins constitute a 14-member family comprising two distinct
subclasses: dimeric receptors (PYR1 and PYL1–PYL3) and monomeric receptors (PYL4–PYL13). The indi-
vidual contributions of PYL subclasses/subtypes with specific physiological actions are still poorly under-
stood; consequently, the development of PYL subclass/subtype-selective agonists should be useful to
reveal the different functions of these receptors. In this study, we focused on the ABA analogs 6-nor-
ABA and 70-nor-ABA, which were expected to function as monomeric receptor-selective agonists on
the basis of crystal structures of PYL-ABA complexes and sequence alignments of PYL subtypes. In a pro-
tein phosphatase 2C (PP2C) assay, the agonist activities of both analogs were lower than those of ABA
toward all tested PYL proteins, regardless of subclass/subtype. Nevertheless, we found that 6-nor-ABA
acts as a selective agonist at the physiological level: it induced stomatal closure but did not inhibit seed
germination and root growth. On the basis of observed inhibitory activity against PP2C among different
PYL subtypes, this biological effect of 6-nor-ABA may be attributed to the activity of that agonist on PYL5
and/or PYL6.
Received 2 May 2015
Revised 24 May 2015
Accepted 27 June 2015
Available online 3 July 2015
Keywords:
Abscisic acid
PYR/PYL/RCAR receptor
Agonist
Ó 2015 Elsevier Ltd. All rights reserved.
The plant hormone abscisic acid (ABA, compound 1) plays a key
role in many physiological processes, such as seed dormancy, root
growth, stomatal closure and abiotic stress response.1,2 The physi-
ological actions of ABA are controlled by a signal transduction pro-
studies have indicated that PYL13, which lacks the lysine residue
crucial for ABA binding, does not bind ABA and inhibits specific
PP2Cs independently of ABA.11,12 In contrast, a recent report has
claimed that PYL13 inhibits these PP2Cs in an ABA-dependent
manner.13 Further study is needed to resolve this discrepancy.
Both dimeric and monomeric receptors are involved in ABA-in-
duced physiological responses, with each receptor contributing
additively to regulation of ABA responses.14,15 These receptors dif-
fer substantially in function, as evidenced by the contrasting
expression patterns of the genes encoding various PYL-subtype
members. Although PYL8 is known to play a nonredundant role
in root sensitivity to ABA,15 characterization of the individual PYL
subtypes is generally difficult because of the functional redun-
dancy of the receptors. The details of these functional differences
are thus largely unknown. A chemical compound capable of selec-
tively activating PYL subclasses/subtypes would be a valuable tool
for evaluation of the effect of the specific receptors on the various
roles of ABA. Although some selective agonists of dimeric recep-
tors, such as pyrabactin and quinabactin, have been described,16,17
no reports have appeared of agonists that preferentially activate
cess involving the interaction of two types of proteins:
a
PYR/PYL/RCAR (PYL) receptor and group-A protein phosphatases
2C (PP2Cs)—including HAB1, ABI1 and ABI2—that act as negative
regulators of ABA signaling. By binding to PYL proteins, ABA
induces a conformational change associated with a mobile loop
(gate) closure that enables the receptor to bind and inhibit
PP2Cs.3–6 Arabidopsis PYL proteins, which constitute a 14-member
family,7,8 are divided into two distinct subclasses according to their
oligomeric state: dimeric receptors (PYR1 and PYL1–PYL3) and
monomeric receptors (PYL4–PYL13).9–11 Among these proteins,
the receptor property of PYL13 remains controversial. Several
⇑
Corresponding author. Tel./fax: +81 54 238 4871.
E-mail address: todoroki.yasushi@shizuoka.ac.jp (Y. Todoroki).
Present address: Graduate School of Agricultural and Life Sciences, The University
of Tokyo, Tokyo 113-8657, Japan.
http://dx.doi.org/10.1016/j.bmcl.2015.06.088
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

3508
J. Takeuchi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3507–3510
C6 and/or C70 methyl group of the ABA molecule. A consideration
R1
of steric bulkiness suggests that hydrophobic interactions should
be induced most easily by Leu, followed by Ile and then Val. We
therefore predicted that monomeric receptors can adopt the
gate-closed form; this should be true even in ABA analogs lacking
the C6 or C70 methyl group (6-nor-ABA, compound 2, and 70-nor-
ABA, compound 3, respectively) because of their bulky residues,
which likely compensate for the weakening of the hydrophobic
interaction due to elimination of the C6 or C70 methyl group.
These two compounds have been reported to be weak ABA ago-
nists,21 but their activities toward PYL proteins have not been
investigated. We therefore investigated whether 6-nor-ABA and
70-nor-ABA can function as monomeric receptor-selective agonists.
( )-6-Nor-ABA was prepared according to the method of Ueno
et al., while the synthesis of ( )-70-nor-ABA was carried out follow-
ing a modification of the route of Nanzyo et al., as shown in
Scheme 1.22 In particular, the carbonyl group of compound 4 (pre-
pared as reported previously)23 was protected by treating with
ethylene glycol in the presence of pyridinium p-toluenesulfonate
to afford the ketal 5.24 Allylic oxidation with manganese(III) acet-
ate and tert-butyl hydroperoxide afforded ketone 6.25 The side
chain was introduced by direct addition of (Z)-3-methyl-2-pen-
ten-4-yn-1-ol using n-butyllithium, generating the alcohol 7.
Reduction (generating compound 8), oxidation (generating com-
pound 9) and esterification of 7 resulted in the formation of the
OH
R2
COOH
O
ABA (1): R1 = Me, R2 = Me
6-nor-ABA (2): R1 = H, R2 = Me
7'-nor-ABA (3): R1 = Me, R2 = H
Figure 1. Structures of abscisic acid (ABA) and its analogs.
monomeric receptors. In the present study, we explored the
possibility that a structural analog of ABA could function as a
monomeric receptor-selective agonist, a role suggested by an
examination of the crystal structures of several PYL-ABA com-
plexes18,19 and sequence alignments of PYL subclasses/subtypes.
Although numerous ABA analogs have been designed and synthe-
sized,20 few have been evaluated from this perspective (Fig. 1).
In PYL–ABA complexes, ABA establishes a hydrophobic network
with amino acid residues at the a3 helix and the gate loop to sta-
bilize the gate-closed form of PYL proteins. A comparison of PYL
subtypes reveals only one residue differing between dimeric and
monomeric receptors, namely, a valine (Val) present in dimeric
receptors that is replaced by leucine (Leu) (PYL7-10) or isoleucine
(Ile) (PYL4-6, PYL11 and PYL12) in monomeric receptors
(Supplementary Fig. S1). Because of its location in the a3 helix, this
residue should be involved in the hydrophobic interaction with the
O
Ref. 23
i
ii
O
O
O
O
O
O
O
5,5-dimethylcyclohexane-
1,3-dione
4
5
6
v
iii
iv
OH
OH
O
O
OH
OH
O
O
O
OH
O
O
7
8
9
vi
vii
viii
OH
O
OH
O
O
O
O
OH
O
10
( )-7'-nor-ABA (11)
Scheme 1. Synthesis of ( )-70-nor-ABA. Reagents: (i) ethylene glycol, PPTS; (ii) Mn3O(OAc)9, tBuO2H, O2, EtOAc; (iii) (Z)-3-methylpent-2-en-4-yn-1-ol, n-BuLi, THF; (iv)
SMEAH, THF; (v) MnO2, acetone; (vi) MnO2, NaCN, AcOH, MeOH; (vii) 1 M HCl, acetone; (viii) 1 M NaOH, MeOH.
a
b
DMSO
ABA
6-nor-ABA
7'-nor-ABA
PYL5
120
100
80
60
40
20
0
120
100
80
60
40
20
0
ABA
6-nor-ABA
7'-nor-ABA
0
0.01
No PYR1 PYL1 PYL2 PYL3 PYL4 PYL5 PYL6 PYL8 PYL9 PYL10
PYLs
0.1
1
10
Concentration (µM)
Figure 2. The comparative effects of 6-nor-ABA, 70-nor-ABA and ABA on inhibition of HAB1 by ABA receptors. Chemical inhibition of HAB1 by (a) various ABA receptors in the
presence of 10 M of each test compound or (b) PYL5 in the presence of various concentrations (0.05, 0.1, 0.5, 1, 5 and 10
M) of 6-nor-ABA or 70-nor-ABA.
l
l

J. Takeuchi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3507–3510
3509
Figure 3. Isothermal titration calorimetric analysis of the binding of 6-nor-ABA and 70-nor-ABA to PYL5.
100
ester 10, followed by acidic and basic hydrolysis of the ester to give
( )-70-nor-ABA (11). ( )-6-Nor-ABA and ( )-70-nor-ABA were opti-
cally resolved by HPLC on a chiral column to obtain the corre-
sponding (+)-isomers with the same exciton chirality as that of
S-(+)-ABA.21 Since the unnatural type of ABA analogs showed
weaker activities than the natural type of them (Supplementary
Fig. S2), we used analogs with the same exciton chirality of the nat-
ural type of ABA for all assays.
80
60
40
20
0
To examine the agonist activities of 6-nor-ABA and 70-nor-ABA
toward different subclasses/subtypes of PYL proteins, we assessed
receptor-mediated PP2C inhibition using nine Arabidopsis PYL pro-
teins (dimeric subclass: PYR1 and PYL1–PYL3; monomeric sub-
class: PYL4–PYL6, PYL8 and PYL10) and the PP2C HAB1. 6-Nor-
ABA induced PYL3, PYL5 and PYL6 to inhibit HAB1, while 70-nor-
ABA caused activation of PYL2, PYL3, PYL5, PYL6 and PYL10. The
activities of both analogs were weaker than those of ABA
(Fig. 2a). Dose–response analysis revealed that the IC50 values of
6-nor-ABA and 70-nor-ABA for PYL5, the PYL subtype most sensi-
tive to both compounds, were 510 and 360 nM, respectively
(Fig. 2b). These values were approximately 10-fold higher than that
of ABA (50 nM). Additionally, isothermal titration calorimetry (ITC)
was used to measure the binding affinity of 6-nor-ABA and 70-nor-
ABA for PYL5 (Fig. 3). The Kd values of 6-nor-ABA and 70-nor-ABA
Cont. ABA
1
3
10
30
0.3 µM
6-nor-ABA (µM)
Figure 4. The comparative effects of 6-nor-ABA and ABA on Arabidopsis seed
germination. The seed germination rate in response to ABA or 6-nor-ABA was
measured at 48 h after stratification (n = 3; error bars represent SD).
6-nor-ABA was able to activate PYL5 as strongly as ABA when pre-
sent in a 20-fold excess over ABA. This result suggested that 6-nor-
ABA could be used to examine the effect of PYL5 and PYL6 on the
various physiological actions of ABA. To investigate the relation-
ship between the activation of PYL5/PYL6 and ABA-induced phys-
iological responses, we tested the effect of 6-nor-ABA in three
different physiological assays. In an Arabidopsis seed germination
assay, the inhibitory activity of 6-nor-ABA was at least 100-fold
lower than that of ABA (Fig. 4). A similar trend was observed in root
growth inhibition of 5-day-old seedlings (Fig. 5). In contrast, 6-nor-
ABA showed relatively potent agonistic activity in the induction of
stomatal closure and drought tolerance. Because transpiration low-
ers leaf temperatures through evaporative cooling,27 we tested the
effect of 6-nor-ABA on stomatal apertures using thermal-imaging
methods. We observed that ABA-treated seedlings showed
increased leaf surface temperatures by reducing transpiration.
were 16.7 and 4.7
higher than that of ABA (0.88
l
M, respectively, which were 5- to 20-fold
l
M).26 Thus, 6-nor-ABA and 70-nor-
ABA were less effective than ABA toward all PYL proteins tested,
implying that the C6 and C70 methyl groups of ABA play an impor-
tant role in receptor binding regardless of PYL subclass/subtype.
These results suggest that the PYL-subtype selectivity of ABA ana-
logs is not solely determined by the amino acid residues involved
in the gate closure.
To analyze the plant physiological effects of ABA and its analogs,
we focused on 6-nor-ABA, a more narrow-spectrum agonist for PYL
subtypes (PYL3, PYL5 and PYL6). As PYL3 expression is extremely
low throughout plant development,14 6-nor-ABA may act as a
weak PYL5- and PYL6-selective agonist in plants. With respect to
its potency, the PP2C assay and the ITC experiment revealed that

3510
J. Takeuchi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3507–3510
ABA
6-nor-ABA
selectivity of 6-nor-ABA for PYL subtypes in plants should be inves-
tigated in detail at the genetic levels; however, this selectivity has
potential use, both as a chemical probe and as a practical applica-
tion to induce drought tolerance in plants without inhibiting
growth.
120
100
80
60
40
20
0
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.06.
088.
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surface temperature and (b) drought tolerance.
Treatment with 6-nor-ABA induced stomatal closure (Fig. 6a) and
drought tolerance (Fig. 6b) as effectively as did ABA when the
former was present in 10-fold excess over ABA; this result is con-
sistent with the observations of agonist potency toward PYL5.
Additionally, the profile of this ligand was similar to that of 10-O-
methyl-ABA, which has been found to selectively activate PYL5 in
a PP2C assay as well.28 These results suggest that PYL5 plays an
important role in the induction of stomatal closure, which is in
agreement with the phenotype of the PYL5 over-expression.6 The