Asymmetrical ligand binding by abscisic acid 8′-hydroxylase

Bioorganic & Medicinal Chemistry 15 (2007) 6311–6322

Asymmetrical ligand binding by abscisic acid 8⁰-hydroxylase

Kotomi Ueno,^aHidetaka Yoneyama,^bMasaharu Mizutani,^c

Nobuhiro Hirai^dand Yasushi Todoroki^b,*

^aThe United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, Japan

^bDepartment of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan

^cInstitute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

^dInternational Innovation Center, Kyoto University, Kyoto 606-8501, Japan

Received 15 May 2007; revised 4 June 2007; accepted 5 June 2007

Available online 8 June 2007

Abstract—Abscisic acid (ABA), a plant stress hormone, has a chiral center (C1⁰) in its molecule, yielding the enantiomers (1⁰S)-(+)-

ABA and (1⁰R)-(ꢀ)-ABA during chemical synthesis. ABA 8⁰-hydroxylase (CYP707A), which is the major and key P450 enzyme in

ABA catabolism in plants, catalyzes naturally occurring (1⁰S)-(+)-enantiomer, whereas it does not recognize naturally not occurring

(1⁰R)-(ꢀ)-enantiomer as either a substrate or an inhibitor. Here we report a structural ABA analogue (AHI1), whose both enanti-

omers bind to recombinant Arabidopsis CYP707A3, in spite of stereo-structural similarity to ABA. The diﬀerence of AHI1 from

ABA is the absence of the side-chain methyl group (C6) and lack of the a,b-unsaturated carbonyl (C2⁰@C3⁰–C4⁰@O) in the six-

membered ring. To explore which moiety is responsible for asymmetrical binding by CYP707A3, we synthesized and tested ABA

analogues that lacked each moiety. Competitive inhibition was observed for the (1⁰R) enantiomers of these analogues in the potency

order of (1⁰R,2⁰R)-(ꢀ)-2⁰,3⁰-dihydro-4⁰-deoxo-ABA (K_I= 0.45 lM) > (1⁰R)-(ꢀ)-4⁰-oxo-ABA (K_I= 27 lM) > (1⁰R)-(ꢀ)-6-nor-ABA

and (1⁰R,2⁰R)-(ꢀ)-2⁰,3⁰-dihydro-ABA (no inhibition). In contrast to the (1⁰R)-enantiomers, the inhibition potency of the (1⁰S)-ana-

logues declined with the saturation of the C2⁰,C3⁰-double bond or with the elimination of the C4⁰-oxo moiety. These ﬁndings suggest

that the C4⁰-oxo moiety coupled with the C2⁰,C3⁰-double bond is the signiﬁcant key functional group by which ABA 8⁰-hydroxylase

distinguishes (1⁰S)-(+)-ABA from (1⁰R)-(ꢀ)-ABA.

1. Introduction

to its weak eﬀect in ﬁeld trial.⁶The weak eﬀect is consid-

ered to be due to the rapid inactivation through biodeg-

radation. 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-hydroxy-ABA and its more stable tautomer

phaseic acid which has much lower hormonal activity

than ABA.⁷ABA 8⁰-hydroxylase was identiﬁed as

CYP707A1-4 in the model plant, Arabidopsis thaliana,

in 2004,^8,9and since then many CYP707A isozymes

have been found in various plants.^10–13Gene knock-

down and overexpression studies suggest that ABA 8⁰-

hydroxylase is a key enzyme for controlling ABA

concentration during water deﬁcit stress or dormancy

maintenance and breaking.^14,15To chemically control

ABA 8⁰-hydroxylation in plants, we are developing sta-

ble ABA analogues^16–18and speciﬁc inhibitors^19–21

against ABA 8⁰-hydroxylase. For eﬀectively designing

these chemicals, we need to know the substrate binding

and enzyme reaction mechanisms in detail. Because the

3D structure of ABA 8⁰-hydroxylase has still not been

Abscisic acid (ABA) is a plant hormone involved in

stress tolerance, stomatal closure, ﬂowering, seed dor-

mancy, and other physiological events.^1–3In addition,

it was reported recently that ABA should function as

an endogenous proinﬂammatory cytokine in human.⁴

Endogenous levels of ABA in plants are properly and

cooperatively controlled by the biosynthesis, transporta-

tion, and catabolic inactivation, in response to environ-

mental changes.^1–3A natural or artiﬁcial chemical which

perturbs this highly controlled system is promising not

only as chemical probes for the mechanism of ABA ac-

tion,⁵but also for practical purposes because of its po-

tential use in agriculture, horticulture, or clinic.

Although ABA is registered as a pesticide (plant growth

regulator), its practical use has been limited mainly due

Keywords: ABA; Abscisic acid; P450.

*

Corresponding author. Tel./fax: +81 54 238 4871; e-mail: aytodor@

agr.shizuoka.ac.jp

doi:10.1016/j.bmc.2007.06.010

6312

K. Ueno et al. / Bioorg. Med. Chem. 15 (2007) 6311–6322

investigated, we have attempted to explore the active site

from the ligand side using many ABA analogues that we

have developed.^19–21In the process of this research, we

found AHI1, a compound which can be a key molecule

to uncover the mechanism of asymmetrical ligand recog-

nition by ABA 8⁰-hydroxylase.

be in cis between the side chain and the C2⁰-methyl

(C7⁰); in this case, C7⁰of AHI1 mimics well the steric

orientation of that of ABA (Fig. 1). Therefore, an enan-

tiomer of AHI1 has the (1⁰S,2⁰S) or (1⁰R,2⁰R) conﬁgura-

tion; the former corresponds to (1⁰S)-(+)-ABA. As

described in detail later, we found that both the enanti-

omers of AHI1 bind to the active site of ABA 8⁰-hydrox-

ylase, contrary to the case of ABA, in spite that the 2D

and 3D structures of AHI1 are very similar to those of

ABA (Fig. 1). The structural diﬀerences between AHI1

and ABA are the absence of the methyl group (C6) in

the side chain and the a,b-unsaturated carbonyl group

(C2⁰@C3⁰–C4⁰@O) in the six-membered ring. Therefore,

either of these moieties should play an important role in

chiral recognition of ABA by ABA 8⁰-hydroxylase. For

clarifying which moiety is mainly responsible, we pre-

pared optically pure 6-nor-ABA, 2⁰,3⁰-dihydro-ABA,

4⁰-deoxo-ABA, 2⁰,3⁰-dihydro-4⁰-deoxo-ABA, and epi-

AHI1 in addition to AHI1, and examined their binding

potency to the ABA 8⁰-hydroxylase active site. Because

cytochrome P450 enzymes perform numerous functions

including metabolism of drugs in human, sterol synthe-

sis in microorganisms, as well as biosynthesis of natural

products and hormone metabolisms in plants, they are

targets for the development of drugs for human thera-

pies or pesticides for agricultural purposes.²⁶Many

drugs and pesticides targeting P450 enzymes are asym-

metrical, and generally each enantiomer has a diﬀerent

eﬀect on the target enzyme reaction. Although the pres-

ent study is an example for probing a mechanism of

asymmetrical recognition of a speciﬁc ligand by a spe-

ciﬁc enzyme, it may give a general idea for asymmetric

interaction of a small molecule with an endogenous

macromolecule.

ABA has a chiral center (C1⁰) in its molecule, yielding

the enantiomers (1⁰S)-(+)-ABA and (1⁰R)-(ꢀ)-ABA

during chemical synthesis (Fig. 1). All the naturally

occurring ABA has the (1⁰S)-conﬁguration, and its

counterpart has never been detected in plants.^1–3Nev-

ertheless, many reports demonstrated that both enanti-

omers showed similar hormonal activities in many

assay systems,²²suggesting that plants have a mecha-

nism which permits the isomer that is not occurring

naturally to mimic the endogenous hormone. However,

the mechanism by which this occurs is not yet known.

All the ABA-binding proteins ever identiﬁed, including

three ABA receptors,^23–25cannot bind (1⁰R)-(ꢀ)-ABA.

ABA 8⁰-hydroxylase also stereospeciﬁcally catalyzes

naturally occurring (1⁰S)-(+)-ABA.⁹(1⁰R)-(ꢀ)-ABA is

not recognized by ABA 8⁰-hydroxylase as either a sub-

strate or an inhibitor. If we understand what structural

properties of ABA cause the asymmetrical recognition

by ABA 8⁰-hydroxylase, we can speculate on a mecha-

nism by which an ABA-binding protein does or does

not discriminate between each enantiomer of ABA.

This knowledge will be very useful to design an asym-

metrical probe (agonist or antagonist) for an ABA-

binding protein.

AHI1 is a lead compound for the development of an

ABA 8⁰-hydroxylase inhibitor.²⁰AHI1 was designed to

Figure 1. Structural properties of optically pure ABA and AHI1. (a) 2D-structures of both enantiomers. The arrow indicates the site of oxidation by

CYP707A3. (b) 3D-structures of both enantiomers and their overlay structures. In the stick models, carbons, hydrogens, and oxygens are colored

gray, white, and red, respectively. In the overlay structures, the (+)-enantiomer is depicted in blue, whereas the (ꢀ)-enantiomer is depicted in red.

K. Ueno et al. / Bioorg. Med. Chem. 15 (2007) 6311–6322

6313

2. Results and discussion

mixture including little diastereomers. Oxidative cleav-

age of the ester with NaIO₄and OsO₄after alkaline

hydrolysis yielded the single compound, which agreed

with 2 derived from (+)-AHI1, by spectrometric and chi-

ral HPLC analyses. Therefore, the absolute conﬁgura-

tion of C1⁰in 2 is S. Finally we determined the

absolute conﬁguration of (+)-AHI1 as (1⁰S,2⁰S); that is,

(+)-AHI1 corresponds to (1⁰S)-(+)-ABA, the naturally

occurring ABA, whereas (ꢀ)-AHI1 corresponds to

(1⁰R)-(ꢀ)-ABA, the naturally not occurring form.

2.1. Preparation of optically pure AHI1 and determina-

tion of the absolute conﬁgurations

Racemic AHI1, which was synthesized as reported pre-

viously,²⁰was optically resolved into (+)- and (ꢀ)-enan-

tiomers using HPLC with a chiral column. As described

above, the absolute conﬁgurations of optically pure

AHI1 are (1⁰S,2⁰S) or (1⁰R,2⁰R); the (1⁰S,2⁰S)-AHI1 cor-

responds to (1⁰S)-(+)-ABA, whereas the (1⁰R,2⁰R)-AHI1

corresponds to (1⁰R)-(ꢀ)-ABA (Fig. 1). We determined

the absolute conﬁguration of AHI1 by converting it to

the same compound 2 as that derived from ABA. The

overall scheme is shown in Figure 2. Because the reac-

tions in this scheme do not include any substitution

reactions at C1⁰to induce chiral inversion or racemiza-

tion, the C1⁰conﬁgurations should be maintained. Oxi-

dative cleavage of (+)-AHI1 gave the aldehyde 1 which

was converted to the ketone 2 with methylmagnesium

bromide and subsequent pyridinium dichromate (PDC)

oxidation. These reactions have no eﬀect on the absolute

conﬁguration at C2⁰; therefore, the relative conﬁguration

of compound 2 derived from (+)-AHI1 is (1⁰S^*,2⁰S^*). On

the other hand, sodium borohydride reduction of the

methyl ester of (+)-ABA (Me ABA) in tetrahydrofuran

(THF) proceeded to give 3 as a diastereomeric mixture

at C4⁰and the methyl esters of 1⁰,4⁰-trans-diol-ABA (4)

and 1⁰,4⁰-cis-diol-ABA (5). Deoxygenation of 3 with tri-

butyltin hydride via thiono esteriﬁcation yielded the

methyl ester of 2⁰,3⁰-dihydro-4⁰-deoxo-ABA as a 2E/2Z

The fact that compound 2 was obtained from (+)-Me

ABA with little diastereomeric impurity means that

the reduction at C2⁰of (+)-Me ABA proceeded stereo-

speciﬁcally. The hydride addition must have occurred

from the same side as the 1⁰-hydroxyl group, which is

less bulkier than the side chain.

The favored conformation of AHI1 is similar to that of

ABA; it is a chair form with the axial side chain and

equatorial C7⁰, as previously reported (Fig. 1b).²⁰There-

fore, we can discuss the eﬀect of the local structural dif-

ference between AHI1 and ABA on the aﬃnity for the

enzyme active site.

2.2. Both enantiomers of AHI1 act as competitive

inhibitors

We estimated binding potency of optically pure AHI1 to

the active site of ABA 8⁰-hydroxylase, by examining its

competitive inhibition potency against the recombinant

ii,iii

i

O

OH

CO₂H

(+)-AHI1

1

2

i

v,vi,vii

OH

CO₂Me

CO₂H

HO

3

(1'S,2'S)-2',3'-dihydro-

4'-deoxo-ABA

iii,vii

+

iv

OH

CO₂Me

CO₂H

O

HO

O

4

(1'S,2'S)-2',3'-dihydro-ABA

(1'S)-Me ABA

Me 1',4'-trans-diol-ABA ( )

+

viii,ix,vii

OH

CO₂Me

CO₂H

HO

Me 1',4'-cis-diol-ABA (5)

(1'S)-4'-deoxo-ABA

Figure ₀2. Dete₀rmination of the ₀absolute conﬁguration of (+)-AHI1 (red and green arrows) and synthesis of enone-modiﬁed ABA analogues:

(1⁰S^*,2 S^*)-2⁰,3 -dihydro-ABA, 4 -deoxo-ABA, and (1⁰S^*,2⁰S^*)-2⁰,3⁰-dihydro-4⁰-deoxo-ABA (blue and green arrows). The absolute conﬁguration of

(+)-AHI1 was determined by the chemical correlation method. To clarify the conﬁguration at C2⁰, the synthetic scheme of enone-modiﬁed ABA

analogues was depicted using (1⁰S)-enantiomer. Compound 3 was a diastereomeric mixture derived from the stereochemical impurity at C4⁰. The

optically pure enone-modiﬁed compounds were gained by optical resolution of the racemates or synthesis from (+)-ABA. Reagents: (i) NaIO₄, OsO₄;

(ii) MeMgBr; (iii) PDC, Celite; (iv) NaBH₄; (v) ClC(S)OPh, 4-DMAP; (vi) n-Bu₃SnH, AIBN; (vii) NaOH; (viii) ClCO₂Me, 4-DMAP; and (ix)

[Pd₂dba₃]ÆCHCl₃, n-Bu₃P, NaBH₄. DMAP, dimethylaminopyridine; AIBN, 2,2⁰-azobisisobutyronitrile; dba, dibenzylideneacetone.

6314

K. Ueno et al. / Bioorg. Med. Chem. 15 (2007) 6311–6322

Table 1. Inhibition constants for both enantiomers of AHI1, 6-nor-

ABA, enone-modiﬁed ABA analogues and epi-AHI1 of recombinant

CYP707A3

Compounds 3, 4, and 5 were prepared from Me ABA

with NaBH₄in THF described above. Oxidation of

(1⁰S,2⁰S)-3 with PDC and subsequent alkaline

hydrolysis produced (1⁰S,2⁰S)-(+)-2⁰,3⁰-dihydro-ABA.

(1⁰R,2⁰R)-(ꢀ)-2⁰,3⁰-dihydro-ABA was prepared from

(ꢀ)-ABA by the same method as the (+)-enantiomer.

(1⁰S,2⁰S)-(+)-2⁰,3⁰-dihydro-4⁰-deoxo-ABA was synthe-

sized described above. (1⁰S)-(+)-4⁰-Deoxo-ABA was

synthesized by elimination of the 4⁰-hydroxyl group of

5 with palladium-catalyzed borohydride reduction via

the allylic carbonate before alkaline hydrolysis. The

(ꢀ)-enantiomers of 2⁰,3⁰-dihydro-4⁰-deoxo-ABA and

4⁰-deoxo-ABA were prepared by optical resolution of

their racemates that were synthesized from ( )-ABA in

the same manner as the (+)-enantiomer. The NMR

and MS spectral data of these analogues are consistent

with those reported previously.^27–29Compared to the

absolute conﬁguration of the starting compound ABA,

all the (+)-probes have the 1⁰S conﬁguration, whereas

all the (ꢀ)-probes have the 1⁰R conﬁguration.

Compound

K_I[lM]

(+)-Enantiomer (ꢀ)-Enantiomer

AHI1

1.28 0.32

0.16 0.01^a

5.80 1.33

2.32 0.49

0.30 0.04

6-Nor-ABA

2⁰,3⁰-Dihydro-ABA

4⁰-Deoxo-ABA

NI^b

NI

27.2 8.8

0.45 0.11

1.63 0.82

2⁰,3⁰-Dihydro-4⁰-deoxo-ABA 5.27 0.84

epi-AHI1

2.50 0.54

^aPublished value.¹⁹

^bNo measurable inhibition.

Arabidopsis CYP707A3 enzyme. (+)-AHI1 acted as a

competitive inhibitor of CYP707A3 with a K_Ivalue of

1.28 lM (Table 1), which is slightly higher than the

K_Mvalue (0.71 lM) of (+)-ABA. This suggests that

the aﬃnity of (+)-AHI1 for the enzyme active site is

somewhat weaker than that of (+)-ABA. AHI1 does

not have the methyl group (C6) in the side chain and

the enone moiety (C2⁰@C3⁰–C4⁰@O) in the ring of

ABA (Fig. 1). Because the elimination of C6 in (+)-

ABA does not aﬀect binding to the active site,¹⁹the

low aﬃnity of (+)-AHI1 is likely a consequence of the

absence of the enone moiety in the ring. (+)-AHI1 was

converted into unidentiﬁed UV-absorbing compounds

during the enzymatic reaction. Because this conversion

was reduced by the addition of (+)-ABA, the UV-

absorbing compounds are presumed to be the enzymatic

reaction products. This suggests that (+)-AHI1 acts as a

substrate of CYP707A3.

The favored conformation of the ring-modiﬁed probes

was estimated to be a chair (half-chair in the case of

4⁰-deoxo-ABA) with the axial side chain on the basis

of the NMR (NOE diﬀerence spectra and NOESY

experiments) and theoretical analysis (Fig. 3 and Table

2). Therefore, we can discuss the eﬀect of the local struc-

tural diﬀerence between these probes and ABA on the

aﬃnity for the enzyme active site.

2.4. The binding potency of (+)- and (ꢀ)-probes to the

CYP707A3 active site

Both enantiomers of the probes were tested for their

ability to inhibit the 8⁰-hydroxylation reaction by re-

combinant CYP707A3, except for (+)-6-nor-ABA

whose activity was reported previously.¹⁹The results

are summarized in Table 1. The mode of inhibition of

all analogues with inhibition potency was competitive

(Fig. 4a and b). (ꢀ)-6-Nor-ABA did not inhibit the en-

zyme, unlike (+)-6-nor-ABA. This suggests that the

methyl group of the side chain has nothing to do with

chiral recognition by CYP707A3. Therefore, the key

moieties aﬀecting the aﬃnity for CYP707A3 should be

the C4⁰-oxo, the C2⁰,C3⁰-double bond, or both.

(ꢀ)-AHI1 also acted as a competitive inhibitor, in spite

of its similarity to (ꢀ)-ABA. Its K_Ivalue was 0.30 lM

(Table 1), which was lower than that of (+)-AHI1 and

the K_Mvalue of (+)-ABA. This means that (ꢀ)-AHI1

binds to the active site more strongly than (+)-AHI1

and (+)-ABA. (ꢀ)-AHI1 yielded no enzyme reaction

product, meaning that it was an inhibitor but not a sub-

strate of CYP707A3. The absence of the ring enone may

allow (1⁰R)-enantiomers to bind to the active site, in a

manner not equal to that of (+)-ABA, as described later

in detail.

Each enone-modiﬁed (ꢀ)-probe exhibited a diﬀerent

aﬃnity. The most active one was (1⁰R,2⁰R)-(ꢀ)-2⁰,3⁰-

dihydro-4⁰-deoxo-ABA with a K_Ivalue of 0.45 lM (Ta-

ble 1), indicating that this probe binds to the active site

as eﬀectively as (ꢀ)-AHI1. The structural diﬀerence of

(ꢀ)-2⁰,3⁰-dihydro-4⁰-deoxo-ABA from (ꢀ)-AHI1 is the

existence of the side-chain methyl (C6), suggesting that

C6 in the (ꢀ)-enantiomer has little eﬀect on binding to

the active site, similarly to the (+)-enantiomer. (ꢀ)-

2⁰,3⁰-Dihydro-ABA exhibited no inhibitory activity,

suggesting that the C4⁰-oxo moiety rather than the

C2⁰,C3⁰-double bond in (ꢀ)-ABA disturbs binding to

the active site. (ꢀ)-4⁰-Deoxo-ABA functioned weakly

as an inhibitor, although its activity was one-sixtieth

that of (ꢀ)-2⁰,3⁰-dihydro-4⁰-deoxo-ABA. This suggests

that the presence of the C2⁰,C3⁰-double bond also results

in loss of aﬃnity. Thus (ꢀ)-ABA cannot bind to the

2.3. Preparation of probes for exploring which functional

group of (ꢀ)-ABA disturbs binding to the active site

We prepared four ABA analogues to identify the key

functional group of (ꢀ)-ABA that is responsible for

disturbing binding to the active site: 6-nor-ABA¹⁹;

2⁰,3⁰-dihydro-ABA²⁷; 4⁰-deoxo-ABA;²⁸and 2⁰,3⁰-dihy-

dro-4⁰-deoxo-ABA.²⁹Optically pure 6-nor-ABA was

synthesized as reported previously.¹⁹4⁰-Deoxo-ABA

and 2⁰,3⁰-dihydro-4⁰-deoxo-ABA have never been asym-

metrically prepared, and the known synthesis of

optically pure 2⁰,3⁰-dihydro-ABA was complex and time

consuming. We synthesized these three analogues using

the new synthetic routes (Fig. 2), in which the starting

compound is ABA; therefore, the absolute conﬁguration

of the analogues at C1⁰depends on that of ABA.

K. Ueno et al. / Bioorg. Med. Chem. 15 (2007) 6311–6322

6315

active site of CYP707A3 owing to the 4⁰-oxo moiety

coupled with the C2⁰,C3⁰-double bond.

All the enone-modiﬁed (+)-probes acted as weak inhib-

itors of CYP707A3. The absence of the ring enone in

(+)-ABA results in a decrease in aﬃnity, although not

necessarily signiﬁcant.

2.5. A putative mechanism of asymmetrical ligand binding

by CYP707A3

Biological chiral recognition toward a substrate with

one stereocenter involves at least three locations inter-

acting with one and more receptor sites.³⁰ABA has

three oxygenated functional groups: C1-carboxyl, C1⁰-

hydroxyl, and C4⁰-oxo moiety. The C1-carboxyl in

(+)-ABA is an absolutely essential moiety, including

the (2Z,4E) geometry of the side chain, for binding to

the CYP707A3 active site.¹⁹As described above, the

C4⁰-oxo in (+)-ABA is also related to binding to the

active site. On the other hand, the C1⁰-hydroxyl has

nothing to do with binding to the active site.¹⁹Thus

two of the three locations in (+)-ABA are the C1-car-

boxyl and C4⁰-oxo moieties, and the third location is

not an oxygenated functional group. As shown in Figure

1b, the major diﬀerence between (+)- and (ꢀ)-ABA is

the location of the axial C6⁰-methyl (C8⁰) in the ring.

Because C8⁰is the site hydroxylated by CYP707A3, it

is reasonable to assume that C8⁰functions as a third

location for the chiral recognition by CYP707A3. If

the C1- and C4⁰-moieties of (ꢀ)-ABA interact with the

active site similarly to (+)-ABA, the C8⁰of (ꢀ)-ABA

cannot occupy the same location as that of (+)-ABA.

However, the binding interaction is not required to

occur in all three locations if the asymmetrical discrim-

ination is not necessary. Simply, this may be the reason

why (ꢀ)-AHI1 and (ꢀ)-2⁰,3⁰-dihydro-4⁰-deoxo-ABA,

which lacks the C4⁰-oxo function, can bind to the active

site. These two probes may be mainly anchored to the

active site through the C1-carboxyl, and reinforce the

aﬃnity by relatively non-speciﬁc, hydrophobic interac-

tions by the lipophilic ring, although this binding man-

ner should be diﬀerent from that of (+)-ABA. In that

case, (ꢀ)-ABA cannot bind to the active site in the sim-

ilar manner to these probes, probably because the C4⁰-

oxo moiety decreases the lipophilicity of the ring, or it

has an unfavorable electrostatic interaction with the ac-

tive site. This hypothesis led us to speculate that an ABA

analogue with the same side chain as that of ABA, if its

six-membered ring has no oxygenated functional group

except for the C1⁰-hydroxyl group, can bind to the

CYP707A3 active site, independent of its C1⁰-conﬁgura-

tion. Therefore, we synthesized and tested the epimer of

AHI1 (epi-AHI1). epi-AHI1 should adopt a chair con-

formation with the equatorial side chain because of

the 1,3-diaxial repulsion between C7⁰and C8⁰(Fig. 5a

and Table 2). Our previous study¹⁹suggests that ABA

analogues with the C4⁰-oxo moiety are required to have

the axial side chain in the favored conformation for a

good aﬃnity to the active site. Therefore, epi-AHI1

can be a good probe for testing our hypothesis about

the binding mechanism of ABA analogues with no

C4⁰-oxo moiety.

Figure 3. Favored conformations of 2⁰,3⁰-dihydro-ABA, 4⁰-deoxo-

ABA, and 2⁰,3⁰-dihydro-4⁰-deoxo-ABA. Blue arrows represent the

observed NOEs.

Table 2. Conformational energy proﬁles of 2⁰,3⁰-dihydro-ABA, 4⁰-

deoxo-ABA, 2⁰,3⁰-dihydro-4⁰-deoxo-ABA, and epi-AHI1

Compound

Relative total energy^a[kcal mol^ꢀ1

]

2⁰,3⁰-Dihydro-ABA

4⁰-Deoxo-ABA

ꢀ2.91

ꢀ1.02

2⁰,3⁰-Dihydro-4⁰-deoxo-ABA ꢀ3.71

epi-AHI1

+4.78

^aValues are relative total energies of a chair form with the axial side

chain when energies of a chair form with the equatorial side chain are

set to zero. Total energies are based on single point energies plus

zero-point energies. Geometry optimizations and frequencies were

calculated at the B3LYP/6-31G(d) level of theory, and single point

energies were calculated at the B3LYP/6-311++G(2df,2p) level of

theory.

6316

K. Ueno et al. / Bioorg. Med. Chem. 15 (2007) 6311–6322

Figure 4. Competitive inhibition of CYP707A3 by (ꢀ)-2⁰,3⁰-dihydro-4⁰-deoxo-ABA (a), (ꢀ)-4⁰-deoxo-ABA (b), and (+)-epi-AHI1 (c). Assays

contained (+)-ABA (open square), (+)-ABA and indicative concentration of ABA analogues (0.5, 20, and 4 lM in (a, b, and c), respectively; closed

triangle). The inset is a double reciprocal plot of the same data.

Figure 5. Structure and synthesis of racemic epi-AHI1. (a) 2D- and 3D-structure of (1⁰S,2⁰R)-epi-AHI1. In the stick model, carbons, hydrogens, and

oxygens are colored grey, white, and red, respectively. (b) The route of epi-AHI1 synthesis. Although all compounds are racemates, one enantiomer is

shown to indicate the relative conﬁgurations. Acetyli₀de anion attack to trimethylcyclohexanone generated two diastereomers at a ratio of ₀10:1; the

major diastereomer whose relative conﬁguration is (1 S^*,2⁰S^*) was converted to AHI1 described previously (Ref. 20), whereas the minor, (1 S^*2⁰R^*)-

diastereomer 6 was converted to epi-AHI1 via compound 7. Reagents: (i) n-BuLi, 2-propynyl-TBS ether; (ii) SMEAH; (iii) AcOH:H₂O (3:1, v/v); (iv)

PDC, Celite; (v) ethyl di-o-tolylphosphonoacetate, NaH; (vi) NaOH.

2.6. Synthesis of epi-AHI1

using chiral HPLC, although the absolute conﬁgura-

tions of optically pure epi-AHI1 have never been

determined.

Racemic epi-AHI1 was prepared using the same syn-

thetic route of AHI1 that was reported previously

(Fig. 5b).²⁰Brieﬂy, the racemic compound 6 generated

by acetylide anion attack to trimethylcyclohexanone

was reduced with sodium bis(methoxyethoxy)alumi-

num dihydride (SMEAH), de-protected with AcOH

and H₂O, and oxidized with PDC to give the racemic

aldehyde 7. The Horner–Emmons reaction of 7 and

subsequent alkaline hydrolysis gave epi-AHI1 with a

total yield of 1.5%. The relative conﬁgurations of

epi-AHI1 were determined on the basis of those of

the compound 6. The introduction of the side chain

to trimethylcyclohexanone yielded the compound 6

as the minor diastereomer. Because in this reaction

the major diastereomer was the synthetic precursor

of AHI1, the end product derived from the compound

6 must be the epimer of AHI1. Racemic epi-AHI1 was

optically resolved to obtain (+)- and (ꢀ)-enantiomers

2.7. Both enantiomers of epi-AHI1 act as a substrate of

CYP707A3

In the inhibition assay using recombinant CYP707A3,

both enantiomers of epi-AHI1 competitively inhibited

the enzyme reaction. The K_Ivalues for (+)- and (ꢀ)-

epi-AHI1 were 2.50 and 1.63 lM, respectively (Table

1 and Fig. 4c). This suggests that the aﬃnity of both

epi-AHI1 enantiomers for the enzyme active site is al-

most equivalent to that of AHI1 and 2⁰,3⁰-dihydro-4⁰-

deoxo-ABA. Each enantiomer of epi-AHI1 was

converted into distinct UV-absorbing product during

the enzyme reaction. The product from (+)-epi-AHI1

showed a quasimolecular ion [M+Na]⁺at m/z 275 in

the ESI-TOF-MS (positive mode) analysis; it has 14

additional mass unit compared to (+)-epi-AHI1. This

K. Ueno et al. / Bioorg. Med. Chem. 15 (2007) 6311–6322

6317

suggests that a methylene group of epi-AHI1 was oxi-

dized to a carbonyl group. Cytochrome P450s catalyze

not only hydroxylation but also carbonylation and car-

boxylation.²⁶In fact, P450-catalyzed oxidation of ABA

includes the further oxidation of the ﬁrst product 8⁰-

hydroxy-ABA to 8⁰-oxo-ABA in maize cells.³¹There-

fore, it is reasonable to assume that CYP707A3

catalyzes the two-step oxidation of epi-AHI1. On the

other hand, the enzyme product of (ꢀ)-epi-AHI1

showed a quasimolecular ion at m/z 269 (positive

mode), which cannot lead us to the estimated molecular

structure.

one-modiﬁed ABA analogues were synthesized from

( )-ABA, and (+)-ABA was also converted to enone-

modiﬁed ABA analogues by the same manners to deter-

mine the absolute conﬁguration of these analogues.

(1⁰R,2⁰R)-2⁰,3⁰-dihydro-ABA was prepared from (ꢀ)-

ABA by the same method as the counterpart, and

(1⁰R)-enantiomers of 4⁰-deoxo-ABA and 2⁰,3⁰-dihydro-

4⁰-deoxo-ABA were obtained by optical resolution of

the racemates. ¹H NMR spectra were recorded with tet-

ramethylsilane as the internal standard using a JEOL

JNM-EX 270 NMR spectrometer (270 MHz). For clar-

ity, the atoms of all the compounds with the carbon

skeleton of ABA were numbered as in ABA for peak

assignments. High resolution mass spectra (HRMS)

were obtained with a JEOL JMS-DX 303 HF mass spec-

trometer and with a JEOL JMS-T100LC ‘AccuTOF’.

Optical rotation was recorded with a Jasco DIP-1000

digital polarimeter. Column chromatography was per-

formed on silica gel (Wakogel C-200). Purity in two sol-

vent systems (H₂O–MeOH and H₂O–MeCN) was

determined using a Shimadzu LC-10ADVP instrument,

and all ﬁnal compounds were >97% pure.

In spite of its diﬀerent conformation from that of AHI1

and other C4⁰-deoxo-probes prepared in this study, both

enantiomers of epi-AHI1 occupied the active site of

CYP707A3 to be converted to the enzyme reaction

products. These results conﬁrm our speculation that

an ABA analogue with the same side chain as that of

ABA, if its six-membered ring has no oxygenated func-

tional group except for the C1⁰-hydroxyl group, can

bind to the CYP707A3 active site, independent of its

C1⁰-conﬁguration. In the previous study, we developed

AHI4 as a strong non-azole inhibitor of ABA 8⁰-hydrox-

ylase.²¹The epimer of AHI4 at C2⁰, epi-AHI4, adopts

the similar ring conformation to epi-AHI1. Neverthe-

less, epi-AHI4 did not exhibit the enzyme inhibition

activity.²¹epi-AHI4 has a hydroxyl group instead of

the C8⁰of epi-AHI1; therefore, the ring of AHI4 should

be more hydrophilic than that of AHI1. This may be the

reason why epi-AHI4 did not act as an inhibitor of the

enzyme.

4.2. (+)- and (ꢀ)-AHI1

( )-AHI1²⁰(45 mg, 0.19 mmol) were subjected to chi-

ral HPLC under the following conditions: column,

Chiralpak AD-H (250 · 4.6 mm, Daicel); solvent, 8%

2-propanol in n-hexane containing 0.1% triﬂuoroace-

tic acid (TFA); ﬂow rate, 1.0 mL min^ꢀ1; detection,

254 nm. The materials at t_R8.2 and 11.6 min were col-

lected to give (+)-AHI1 (22 mg, 92 lmol) and its (ꢀ)-

enantiomer (22 mg, 92 lmol) with an optical purity of

29

99.9% and 95.9%, respectively. (+)-AHI1: ½aꢁ +71.4

D

3. Conclusions

29

D

(c 1.083, MeOH); (ꢀ)-AHI1; ½aꢁ ꢀ69.2 (c 1.117,

MeOH).

Plant P450 CYP707A3, ABA 8⁰-hydroxylase, binds

enantioselectively (+)-ABA but not (ꢀ)-ABA, whereas

the enzyme binds both enantiomers of AHI1. We focused

on the structural diﬀerences between the two compounds,

and designed and synthesized four asymmetrical molecu-

lar probes for exploring a key functional group of (ꢀ)-

ABA responsible for disturbing binding to the active site:

6-nor-ABA; 2⁰,3⁰-dihydro-ABA; 4⁰-deoxo-ABA; and

2⁰,3⁰-dihydro-4⁰-deoxo-ABA. The structure–activity stud-

ies using these probes suggested that the C4⁰-carbonyl

moiety coupled with the C2⁰,C3⁰-double bond is a signif-

icant key function for asymmetrical binding of ABA by

ABA 8⁰-hydroxylase.

4.3. Synthesis of 2⁰,3⁰-dihydro-4⁰-deoxo-ABA

4.3.1. (E)-3-((1⁰S,2⁰S)-1⁰-Hydroxy-2⁰,6⁰,6⁰-trimethylcyclo-

hexyl)acrylaldehyde (1). NaIO₄(20 mg, 93 lmol) was

added to a solution of (+)-AHI1 (2.2 mg, 9.2 lmol) in

1,4-dioxane (0.1 mL) and H₂O (0.1 mL). A solution of

OsO₄in H₂O (7.1 lM solution, 5 lL, 36 lmol) was added,

the mixture was stirred for 14 h at room temperature.

NaIO₄(25 mg, 0.12 mmol) and a solution of OsO₄in

H₂O (0.11 mL, 0.78 mmol) were then added to a mixture,

and stirred for 12 h at room temperature. After being

quenched with 2-propanol (0.5 mL), the resulting mixture

was extracted with EtOAc (10· 1.5 mL). The organic layer

was washed with brine and H₂O, dried over Na₂SO₄, and

concentrated. The residual oil was puriﬁed by column

chromatography (Sep-Pak Plus Silica cartridge) with 5%

EtOAc in hexane to obtain 1 (1.0 mg, 5.1 lmol, 55%) as

4. Experimental

4.1. Chemicals

1

white amorphous solid. H NMR (270 MHz, CDCl₃): d

(+)-ABA was a gift from Toray Industries Inc., Tokyo,

Japan. Phaseic acid was prepared as reported previ-

ously.³²Synthesis of ( )-AHI1 was reported previ-

ously.²⁰Compounds 1–7, enone-modiﬁed ABA

analogues, and ( )-epi-AHI1 were prepared by the syn-

thetic route shown as Figures 2 and 5, and as described

below. Optical resolutions of racemic AHI1 and epi-

AHI1 were performed using chiral HPLC. Racemic en-

0.79 (3H, d, J = 6.9 Hz, H₃-7⁰), 0.82 (3H, s, H₃-9⁰), 1.09

(3H, s, H₃-8⁰), 1.20–1.32 and 1.45–1.72 (6H, m, H₂-3⁰,

H₂-4⁰and H₂-5⁰), 1.96 (1H, ddq, J = 13.2, 6.9, and

3.6 Hz, H-2⁰), 6.43 (1H, dd, J = 15.5 and 7.9 Hz, H-2)

7.12 (1H, d, J = 15.5 Hz, H-3), and 9.63 (1H, d,

J = 7.9 Hz, H-1); HRMS (EI) Calcd for C₁₂H₂₀O₂[M]⁺

196.1463. Found: 196.1461.

6318

K. Ueno et al. / Bioorg. Med. Chem. 15 (2007) 6311–6322

4.3.2. (E)-4-((1⁰S,2⁰S)-1⁰-Hydroxy-2⁰,6⁰,6⁰-trimethylcyclo-

hexyl)but-3-en-2-one (2) from 1. A solution of methylmag-

nesium bromide (35% in Et₂O, ca. 3 M, 80 lL, 0.24 mmol)

was added to a solution of the aldehyde 1 (1.0 mg,

5.1 lmol) in dry THF (0.1 mL) at ꢀ10 ꢁC under Ar. The

mixture was stirred for 20 min, and H₂O (10 mL) was then

added to the mixture. The solution was extracted with

EtOAc (5· 2 mL), the organic layer was washed with brine

andH₂O, driedoverNa₂SO₄, andconcentrated.Theresid-

ualoilwaschromatographed on silica gelwith 40%EtOAc

in hexane to give the alcohol (0.8 mg, 3.8 lmol) as color-

less oil. PDC (3 mg, 8 lmol) and Celite were added to a

solution of the alcohol (0.8 mg, 3.8 lmol) in dry CH₂Cl₂

(0.2 mL), andthemixturewasstirredfor16 hatroomtem-

perature. The mixture was ﬁltered with Celite and the ﬁl-

trate was concentrated. The residual oil was puriﬁed by

column chromatography on silica gel with 15% EtOAc

in hexane and with a Sep-Pak Plus C18 cartridge using

70% MeOH in H₂O to obtain 2 (0.4 mg, 2.1 lmol, 40%)

as colorless oil. ¹H NMR (270 MHz, CDCl₃): d 0.77

(3H, d, J = 6.6 Hz, H₃-7⁰), 0.80 (3H, s, H₃-9⁰), 1.07 (3H,

s, H₃-8⁰), 1.16–1.67 (6H, m, H₂-3⁰, H₂-4⁰, and H₂-5⁰),

1.93 (1H, ddq, J = 13.5, 6.6, and 3.2 Hz, H-2⁰), 2.28 (3H,

s, H₃-1), 6.40 and 7.10 (each 1H, d, J = 15.8 Hz, H-3,

and H-4); HRMS (EI) Calcd for C₁₃H₂₂O₂[M]⁺

210.1620. Found: 210.1618.

4.3.5. (1⁰S,2⁰S)-(+)-3. (+)-ABA (11 mg, 41 lmol) was

converted to (1⁰S,2⁰S)-(+)-3 (5.0 mg, 18 lmol, 44%) by

the same method described above.

4.3.6. (1⁰S^*,2⁰S^*)-( )-2⁰,3⁰-Dihydro-4⁰-deoxo-ABA. (1⁰S^*,

2⁰S^*)-( )-3 (0.16 g, 0.57 mmol) was dissolved in dry

CH₂Cl₂(3 mL), and dry pyridine (0.7 mL) and 4-dim-

ethylaminopyridine (4-DMAP, 76 mg, 0.62 mmol) were

added to the solution. Phenyl chlorothioxoformate

(1 mL, 7.3 mmol) was added to the solution at 0 ꢁC,

and the resulting mixture was stirred for 105 min at

room temperature in the dark. After quenching the reac-

tion with brine, the CH₂Cl₂layer was separated, and the

aqueous layer was extracted with EtOAc (5· 10 mL).

The CH₂Cl₂and EtOAc layers were combined, washed

with saturated NaHCO₃, 0.1 M HCl, brine, and H₂O,

dried over Na₂SO₄, and concentrated. The residual oil

was puriﬁed by column chromatography on silica gel

with 5% EtOAc in hexane to give the thiono ester

(0.21 g) as yellow oil. The thiono ester (0.11 g) was dis-

solved in dry toluene (3 mL) and warmed to 70 ꢁC. A

solution of 2,2⁰-azobisisobutyronitrile (7.3 mg, 45 lmol)

and tri-n-butyltin hydride (0.2 mL, 0.76 mmol) in tolu-

ene (1.5 mL) was added dropwise to the solution over

3 min, and the resulting mixture was stirred for 2 h at

70 ꢁC under Ar. The toluene solution was applied onto

silica gel column and products were eluted with 8%

EtOAc in hexane. The eluate was concentrated, the res-

idue (34 mg) was redissolved in MeOH (1.5 mL), and

1 M NaOH (1.5 mL, 1.5 mmol) was added to the solu-

tion. After stirring for 16.5 h, the solution was ﬁlled

up to 50 mL with brine. The resulting solution was ex-

tracted with hexane (3· 5 mL), and the aqueous layer

was extracted with EtOAc (5· 8 mL) after acidifying

to pH 1 with 1 M HCl. The EtOAc layer was washed

with H₂O, dried over Na₂SO₄, and concentrated. The

residual oil was puriﬁed using silica gel column

chromatography with 15% EtOAc in hexane containing

0.1% AcOH and Sep-Pak Plus C18 cartridges with 65–

80% MeOH in H₂O containing 0.05% AcOH to obtain

(1⁰S^*,2⁰S^*)-( )-2⁰,3⁰-dihydro-4⁰-deoxo-ABA (9.6 mg,

4.3.3. (1⁰S^*,2⁰S^*)-( )-2. Compound ( )-1²⁰(4.3 mg, 22 lmol)

gave ( )-2 (3.3 mg, 16 lmol, 73%) in the similar manner

to 1.

4.3.4. Methyl (1⁰S^*,2⁰S^*)-( )-2⁰,3⁰-dihydro-4⁰-hydroxy-ab-

scisate (3) and methyl (1⁰S^*)-( )-1⁰,4⁰-cis/trans-diol-absci-

sate (4 and 5). Racemic ABA (0.36 g, 1.4 mmol) was

dissolved in MeOH (10 mL) and

a solution of

(trimethylsilyl)diazomethane (2.0 M in hexanes, 5 mL,

10 mmol) was added to the solution with cooling. After

removal of MeOH and hexane in vacuo, the residue

was redissolved in dry THF (15 mL), and NaBH₄

(0.22 g, 5.9 mmol) was added to the solution. The mixture

was stirred for 30 h at room temperature in the dark, and

brine was added to the resulting mixture with cooling to

quench the reaction. After separating the THF layer, the

aqueous layer was extracted with EtOAc (5· 20 mL). The

THF and EtOAc layers were combined, washed with sat-

urated NH₄Cl and H₂O, dried over Na₂SO₄, and concen-

trated. The residual oil was puriﬁed by column

chromatography on silica gel with 20–40% EtOAc in hex-

ane to give methyl ( )-1⁰,4⁰-trans-diol-abscisate (4, 0.13 g,

0.48 mmol, 35%) as colorless oil, (1⁰S^*,2⁰S^*)-( )-3 (0.18 g,

0.65 mmol, 47%) as white amorphous solid, and methyl

( )-1⁰,4⁰-cis-diol-abscisate (5, 0.054 g, 0.19 mmol, 14%)

as colorless oil. ¹H NMR and MS spectra of 4 and 5 were

considered with a previous report.³³Data of 3 (1:1 diaste-

reomeric mixture at C4⁰). ¹H NMR (270 MHz, CDCl₃): d

0.81 (3H, d, J = 6.6 Hz, H₃-7⁰), 0.82 (3H, s, H₃-9⁰), 1.10

and 1.27 (3H, s, H₃-8⁰), 1.22–1.31, 1.45–1.56, 1.69–1.77,

and 1.87–1.92 (4H, m, H₂-3⁰, and H₂-5⁰), 2.01 and 2.03

(3H, d, J = 1.0 Hz, H₃-6), 2.05 and 2.37 (1H, m, H-2⁰),

4.03 and 4.13 (1H, m, H-4⁰), 5.70 (1H, broad s, H-2),

6.28 and 6.42 (1H, d, J = 16.2 Hz, H-5), 7.76 and 7.80

(1H, d, J = 16.2 Hz, H-4); HRMS (EI) Calcd for

C₁₆H₂₆O₄[M]⁺282.1831. Found: 282.1829.

1

38 lmol, 15%) as colorless oil. The H NMR data were

consistent with those reported previously.²⁹UV k_max

(MeOH) nm (e): 262.2 (17,400).

4.3.7. (1⁰S,2⁰S)-(+)-2⁰,3⁰-Dihydro-4⁰-deoxo-ABA. (1⁰S,

2⁰S)-(+)-3 (3.8 mg, 14 lmol) was converted to (2E/2Z)-

(2.8 mg,

(1⁰S,2⁰S)-(+)-2⁰,3⁰-dihydro-4⁰-deoxo-ABA

11 lmol, 79%) by the same method. The 2E/2Z isomer

(350 lg, 1.3 lmol) was puriﬁed by column to obtain

(1⁰S,2⁰S)-(+)-2⁰,3⁰-dihydro-4⁰-deoxo-ABA

(267 lg,

1.1 lmol, 85%) as a colorless oil. HRMS (EI) Calcd

for C₁₅H₂₄O₃[M]⁺252.1725. Found: 252.1726.

4.3.8. (1⁰R,2⁰R)-(ꢀ)-2⁰,3⁰-Dihydro-4⁰-deoxo-ABA. (1⁰S^*,

2⁰S^*)-( )-2⁰,3⁰-Dihydro-4⁰-deoxo-ABA (2.7 mg, 11 lmol)

was subjected to chiral HPLC using the following condi-

tions: column, Chiralpak AD-H (250 · 4.6 mm, Daicel);

solvent, 11% 2-propanol in n-hexane containing 0.1%

TFA; ﬂow rate, 0.6 mL min^ꢀ1; detection, 254 nm. The

materials at t_R9.4 and 12.1 min were collected to give

(1⁰S,2⁰S)-(+)-2⁰,3⁰-dihydro-4⁰-deoxo-ABA (0.68 mg,

2.7 lmol) and its (1⁰R,2⁰R)-(ꢀ)-enantiomer (0.65 mg,

K. Ueno et al. / Bioorg. Med. Chem. 15 (2007) 6311–6322

6319

2.6 lmol) with an optical purity of 99.9% and 99.6%,

4.5.3. (1⁰R,2⁰R)-(ꢀ)-2⁰,3⁰-Dihydro-ABA. (ꢀ)-ABA (3.1 mg,

respectively. (1⁰S,2⁰S)-(+)-2⁰,3⁰-dihydro-4⁰-deoxo-ABA:

12 lmol) was converted to (1⁰R,2⁰R)-(ꢀ)-2⁰,3⁰-dihydro-

28

D

½aꢁ +79.7 (c 0.104, MeOH); (1⁰R,2⁰R)-(ꢀ)-2⁰,3⁰-dihy-

ABA (0.84 mg, 3.2 lmol, 26%) via (ꢀ)-3 in the similar man-

28

dro-4⁰-deoxo-ABA; ½aꢁ ꢀ82.0 (c 0.0651, MeOH).

26

D

ner to (+)-enantiomer. ½aꢁ ꢀ59.8 (c 0.0461, MeOH) (lit.²⁷

D

HRMS (EI) Calcd for C₁₅H₂₄O₃[M]⁺252.1725. Found:

252.1726.

[a]_Dꢀ65.2); HRMS (EI) Calcd for C₁₅H₂₂O₄[M]⁺

266.1518. Found: 266.1521.

4.6. Synthesis of 4⁰-deoxo-ABA

4.4. (E)-4-((1⁰S,2⁰S)-1⁰-hydroxy-2⁰, 6⁰,6⁰-trimethylcyclo-

hexyl)but-3-en-2-one (2) from (2E/2Z)-(1⁰S,2⁰S)-(+)-2⁰,3⁰-

dihydro-4⁰-deoxo-ABA

4.6.1. ( )-4⁰-Deoxo-ABA. Anhydrous pyridine (0.5 mL)

and 4-DMAP(23 mg, 0.19 mmol)were addedtoa solution

of 5 (54 mg, 0.19 mmol) in dry CH₂Cl₂(1 mL), which was

cooled to 0 ꢁC. Chloroformic acid methyl ester (0.4 mL,

5.2 mmol) was added to the solution, and the mixture

was stirred for 30 min at room temperature. After quench-

ing with H₂O at 0 ꢁC and adding brine, the resulting mix-

ture was extracted with EtOAc (6· 6 mL). The organic

layer was suﬃciently washed with 0.1 M HCl, saturated

NaHCO₃and H₂O, dried over Na₂SO₄, and concentrated.

The residual oil containing the methyl carbonate (58 mg)

was dissolved in EtOAc, added hexane, and ﬁltered

through a short column of silica gel to remove pyridine.

The eluate was concentrated, and the methyl carbonate

(46 mg) was dissolved in 1,4-dioxane (1 mL).

[Pd₂(dba)₃]ÆCHCl₃2.7 mg (2.6 lmol), n-Bu₃P (10% v/v in

dioxane, 7 ll, 2.8 lmol), and a solution of NaBH₄

(15 mg, 0.39 mmol) in H₂O (0.15 mL) were added to the

solution, and the mixture was stirred for 40 min at room

temperature in the dark. Brine was added to the reaction

mixture, and the solution was extracted with EtOAc (8·

4 mL). The organic layer was washed with saturated

NH₄Cl and H₂O, dried over Na₂SO₄, and concentrated.

The residual oil was puriﬁed by column chromatography

on silica gel with 10% EtOAc in hexane to give the ester

(26 mg), which dissolved in MeOH (1 mL). An aqueous

solution of NaOH (1 M, 1 mL, 1 mmol) was added to the

solution and stirred for 13.5 h at room temperature in the

dark. The resulting mixture was ﬁlled up to 50 mL with

brine, the solution was extracted withhexane. The aqueous

layer was acidiﬁed to pH 1 with 1 M HCl before extracting

with EtOAc (4· 5 mL). The EtOAc layer was washed with

H₂O, dried over Na₂SO₄, and concentrated. The residual

oil was puriﬁed by column chromatography on silica gel

with 20% EtOAc in hexane to obtain 4⁰-deoxo-ABA

(18 mg, 70 lmol, 46%) as colorless oil. The ¹H NMR and

mass spectral data were consistent with those reported pre-

viously.²⁸UV k_max(MeOH) nm (e): 262.2 (19,500).

(2E/2Z)-(1⁰S,2⁰S)-(+)-2⁰,3⁰-Dihydro-4⁰-deoxo-ABA (2.0 mg,

7.9 lmol) was converted to 2 (0.3 mg, 1.4 lmol, 18%) by

oxidative cleavage with NaIO₄and OsO₄(described

above). Compound 2 was analyzed by HPLC with the fol-

lowing conditions. ODS HPLC: column, Hydrosphere

C18 (150 · 6.0 mm, YMC); solvent, 75% MeOH in

H₂O; ﬂow rate, 1.0 mL min^ꢀ1; detection, 254 nm. The

peak of compound 2 prepared from (+)-AHI1 agreed with

that from (1⁰S)-(+)-ABA upon coinjection analysis (t_R

6.8 min). Chiral HPLC conditions: column, Chiralpak

AD-H (250 · 4.6 mm, Daicel); solvent, 8% 2-propanol

in n-hexane containing 0.1% TFA; ﬂow rate,

1.0 mL min^ꢀ1; detection, 254 nm. The peaks at t_R6.7

and 7.5 min were detected when the ( )-2 was injected,

whereas the peak from injected 2 was detected at t_R

6.5 min.

4.5. Synthesis of 2⁰,3⁰-dihydro-ABA

4.5.1. (1⁰S^*,2⁰S^*)-( )-2⁰,3⁰-Dihydro-ABA. A mixture of

PDC (51 mg, 0.14 mmol) and Celite wa₀s we₀tted with

CH₂Cl₂and added a solution of (1 S^*,2 S^*)-( )-3

(31 mg, 0.11 mmol) in dry CH₂Cl₂(0.5 mL). The mix-

ture was stirred for 15.5 h at room temperature, ﬁltered

with Celite, and eluted with CH₂Cl₂and EtOAc. The

eluate was concentrated and ﬁltered through a short col-

umn of silica gel to remove Celite before the residual

crude oil was dissolved in MeOH (1 mL). A solution

of NaOH in H₂O (1 M, 1.5 mL, 1.5 mmol) was added

to the solution and the mixture was stirred for 5 h at

room temperature in the dark. The resulting mixture

was ﬁlled up to 40 mL with brine and extracted with

hexane (3· 5 mL). The aqueous layer was acidiﬁed with

1 M HCl to pH 1 and extracted with EtOAc (7· 7 mL).

The EtOAc layer was washed with H₂O, dried over

Na₂SO₄, and concentrated. The residual oil was puriﬁed

by silica gel column chromatography with 50–70%

EtOAc₀in hexane containing 0.1% AcOH to give

(1⁰S^*,2 S^*)-( )-2⁰,3⁰-dihydro-ABA (18 mg) as white

amorphous solid, which was recrystallized from ace-

tone–toluene to give colorless crystal (16 mg, 60 lmol,

4.6.2. (1⁰S)-(+)- and (1⁰R)-(ꢀ)-4⁰-Deoxo-ABA. ( )-4⁰-Deo-

xo-ABA (2.1 mg, 8.4 lmol) was optically resolved by chiral

HPLC using the following conditions: column, Chiralpak

AD-H (250 · 4.6 mm, Daicel); solvent, 12% 2-propanol in

n-hexane containing 0.1% TFA; ﬂow rate, 0.6 mL min^ꢀ1

;

1

55%). The H NMR data were consistent with those re-

ported previously.²⁷UV k_max(MeOH) nm (e): 259.8

(21,600).

detection, 254 nm. The materials at t_R9.4 and 10.6 min

were collected to give (+)-4⁰-deoxo-ABA (0.84 mg,

3.4 lmol) and its (ꢀ)-enantiomer (0.94 mg, 3.7 lmol) with

an optical purity of 99.8% and 99.9%, respectively. (+)-4⁰-

4.5.2. (1⁰S,2⁰S)-(+)-2⁰,3⁰-Dihydro-ABA. Compound (+)-

3 (4.1 mg, 15 lmol) was converted to (1⁰S,2⁰S)-(+)-

29

deoxo-ABA: ½aꢁ +210.6 (c 0.0843, MeOH); HRMS (EI)

29

D

Calcd for C₁₅H₂₂O₃[M]⁺250.1569. Found: 250.1570. (ꢀ)-

2⁰,3⁰-dihydro-ABA (2.2 mg, 8.2 lmol, 57%) by the same

26

manner. ½aꢁ +66.7 (c 0.145, MeOH) (lit.²⁷[a]_D+63.5);

4⁰-deoxo-ABA; ½aꢁ ꢀ215.5 (c 0.0937, MeOH); HRMS

D

HRMS (EI) Calcd for C₁₅H₂₂O₄[M]⁺266.1518. Found:

266.1519.

(EI) Calcd for C₁₅H₂₂O₃[M]⁺250.1569. Found: 250.1564.

By use of the same chiral HPLC conditions, the peak of

6320

K. Ueno et al. / Bioorg. Med. Chem. 15 (2007) 6311–6322

(1⁰S)-4⁰-deoxo-ABA prepared from (+)-ABA was consis-

tent with that of (+)-4⁰-deoxo-ABA, elucidating the (1⁰S)

conﬁguration of (+)-4⁰-deoxo-ABA.

1.49–1.72 (6H, m, H₂-3⁰, H₂-4⁰, and H₂-5⁰), 2.01 (1H,

ddd, J = 12.6, 6.6, and 4.0 Hz, H-2⁰), 6.39 (1H, dd,

J = 15.5 and 7.9 Hz, H-2), 7.82 (1H, d, J = 15.5 Hz, H-

3), 9.61 (1H, d, J = 7.9 Hz, H-1); HRMS (EI) Calcd for

C₁₂H₂₀O₂[M]⁺196.1463. Found: 196.1466.

4.7. Synthesis of epi-AHI1

4.7.1. (1⁰S^*,2⁰R^*)-( )-3⁰-(1⁰-Hydroxy-2⁰,6⁰,6⁰-trimethylcycloh-

exan-1⁰-yl)-propynol TBS ether (6). A solution of n-buty-

llithium in n-hexane (1.6 mol l^ꢀ1, 9.0 mL, 14 mmol) was

added dropwise to a solution of 2-propynyl-tert-butyl-

dimethylsilyl (TBS) ether (3.6 g, 21 mmol) in dry THF

(50 mL) at ꢀ78 ꢁC under Ar. The mixture was stirred

for 40 min, and a solution of 2,2,6-trimethylcyclohexa-

none (1.0 g, 7.1 mmol) in dry THF (10 mL) was then

added dropwise to the mixture at ꢀ78 ꢁC. The reaction

mixture was allowed to warm to ꢀ28 ꢁC over 60 min be-

fore being quenched with water and extracted with

EtOAc (4· 30 mL). The organic layer was washed with

brine and H₂O, dried over Na₂SO₄, and concentrated.

The residual oil was puriﬁed by column chromatogra-

phy on silica gel with 2% EtOAc in hexane to obtain

(1⁰₀S^*,2⁰₀R^*)-6 (0.13 g, 0.41 mmol, 5.7%) and its

(1 S^*,2 S^*)-diastereomer (1.4 g, 4.6 mmol, 65%) as color-

less oils. ¹H NMR (270 MHz, CDCl₃): d 0.12 (6H, s, Si–

Me₂), 0.91 (9H, s, t-Bu–Si), 1.05 (3H, d, J = 6.9 Hz, H₃-

7⁰), 1.07 and 1.08 (each 3H, s, H₃-8⁰, and H₃-9⁰), 1.15–

1.60 (6H, m, H₂-3⁰, H₂-4⁰, and H₂-5⁰), 1.89 (1H, m, H-

2⁰), 4.16 (2H, s, H₂-1); HRMS (EI) Calcd for

C₁₈H₃₄O₂Si [M]⁺310.2328. Found: 310.2326.

4.7.3. (1⁰S^*,2⁰R^*)-( )-epi-AHI1. A solution of ethyl di-o-

tolylphosphonoacetate (0.39 g, 1.1 mmol) in dry THF

(1 mL) was added to a solution of NaH (61 mg,

1.5 mmol) in dry THF (1 mL) at 0 ꢁC under Ar. After

being stirred for 25 min at 0 ꢁC, the mixture was cooled

to ꢀ78 ꢁC and a solution of 7 (70 mg, 0.36 mmol) in dry

THF (1 mL) was added. The reaction mixture was al-

lowed to warm to ꢀ14 ꢁC over 2 h before it was poured

into saturated NH₄Cl and extracted with EtOAc (5·

8 mL). The organic layer was washed with H₂O, dried

over Na₂SO₄, and concentrated. The residual oil was

chromatographed using silica gel with 4–6% EtOAc in

hexane to obtain a colorless oil (0.12 g) as crude prod-

ucts. The oil (0.12 g) was dissolved in MeOH (1.5 mL)

and 1 M NaOH (1.5 mL, 1.5 mmol) was added to a

solution. The mixture was stirred for 4.5 h at room tem-

perature in the dark before it was ﬁlled up with H₂O to

30 mL and extracted with CH₂Cl₂(3· 4 mL). The solu-

tion was extracted with EtOAc (5· 7 mL) after being

acidiﬁed with 1 M HCl to pH 1. The EtOAc layer was

washed with H₂O, dried over Na₂SO₄, and concen-

trated. The residual oil was prepuriﬁed by silica gel col-

umn chromatography with 20% EtOAc in hexane

containing 0.1% AcOH and Sep-Pak Plus C18 cartridges

with 80% MeOH in H₂O containing 0.05% AcOH to

give a mixture of 2E/2Z isomers of epi-AHI1 (69 mg,

0.29 mmol) as colorless oil. The isomers were separated

using HPLC under the following conditions: column,

YMC-Pack AQ311 (ODS, 100 · 6.0 mm I.D., YMC);

solvent, 65% MeOH in H₂O containing 0.1% AcOH;

ﬂow rate, 1.0 mL min^ꢀ1; detection, 254 nm. The materi-

als at t_R15.8 and 22.4 min were collected to give 2E-( )-

epi-AHI1 (18 mg, 75 lmol, 21%) and 2Z-( )-epi-AHI1

(50 mg, 0.21 mmol, 59%), respectively, as colorless oils.

4.7.2. (1⁰S^*,2⁰R^*)-( )-3⁰-(1⁰-Hydroxy-2⁰,6⁰,6⁰-trimethylcyclo-

hexan-1⁰-yl)-propenal (7). A solution of sodium bis(2-

methoxyethoxy)aluminum dihydride (SMEAH) in tolu-

ene (ca. 70%, 0.85 mL, 3.1 mmol) was added dropwise

to a solution of 6 (0.27 g, 0.88 mmol) in dry THF

(25 mL) at 0 ꢁC under Ar. The mixture was stirred for

2 h at room temperature before it was poured into

H₂O. The solution was extracted with EtOAc

(20 mL · 8) after saturated NaHCO₃was added to the

solution. The organic layer was washed with saturated

NH₄Cl and H₂O, dried over Na₂SO₄, and concentrated.

After the residual oil (0.24 g) was dissolved in THF

(1 mL), 75% AcOH in H₂O (4 mL) was added to the solu-

tion, and the mixture was then stirred for 19 h at room

temperature. The resulting mixture was ﬁlled up with

brine to 40 mL before being extracted with EtOAc (8·

7 mL). The organic layer was washed substantially with

saturated NaHCO₃and H₂O, dried over Na₂SO₄, and

concentrated. The residual oil was puriﬁed by column

chromatography on silica gel with 30% EtOAc in hexane

to give the alcohol (0.11 g, 0.54 mmol, 62%) as white

amorphous solid. After PDC (0.25 g, 0.66 mmol) and

Celite (0.5 g) were dissolved in dry CH₂Cl₂(2 mL), a

solution of the alcohol (0.11 g, 0.54 mmol) in dry CH₂Cl₂

(2 mL) was added and stirred for 4 h at room tempera-

ture. The mixture was ﬁltered with Celite and the residue

was eluted with CH₂Cl₂(20 mL) and EtOAc (40 mL).

The combined organic solution was concentrated and

the residual oil was puriﬁed by column chromatography

on silica gel with 10–15% EtOAc in hexane to give 7

(76 mg, 0.39 mmol, 73%) as colorless oil. ¹H NMR

(270 MHz, CDCl₃): d 0.77 (3H, d, J = 6.6 Hz, H₃-7⁰),

0.86 (3H, s, H₃-8⁰), 1.07 (3H, s, H₃-9⁰), 1.20–1.38 and

1

Data of 2Z-epi-AHI1. H NMR (500 MHz, CDCl₃): d

0.76 (3H, d, J = 6.7 Hz, H₃-7⁰), 0.87 (3H, s, H₃-8⁰),

1.00 (3H, s, H₃-9⁰), 1.18 (1H, broad d, J = 12.8 Hz, H-

5⁰proS), 1.35 (1H, m, H-3⁰proR), 1.49 (1H, m, H-3⁰

proS), 1.53 (2H, m, H₂-4⁰), 1.68 (1H, m, H-5⁰proR),

1.91 (1H, ddd, J = 12.8, 6.7, and 3.6 Hz, H-2⁰), 5.65

(1H, d, J = 11.3 Hz, H-2), 6.09 (1H, d, J = 15.6 Hz, H-

5), 6.70 (1H, dd, J = 11.3 and 11.3 Hz, H-3), 7.51 (1H,

dd, J = 15.6 and 11.3 Hz, H-4); ¹³C NMR (125 MHz,

CDCl₃): d 16.4 (C-7⁰), 21.4 (C-4⁰), 23.7 (C-9⁰), 25.5 (C-

8⁰), 29.7 (C-3⁰), 34.6 (C-2⁰), 36.0 (C-5⁰), 38.2 (C-6⁰),

79.0 (C-1⁰), 115.8 (C-2), 125.9 (C-4), 146.2 (C-3), 149.9

(C-5), 170.8 (C-1); UV k_max(MeOH) nm (e): 260.6

(21,900); HRMS (EI) calcd for C₁₄H₂₂O₃[M]⁺

238.1569. Found: 238.1568.

4.7.4. (+)- and (ꢀ)-epi-AHI1. A Chiralpak AD-H HPLC

column (250 · 4.6 mm ID, Daicel; solvent, 5% 2-propa-

nol in hexane containing 0.1% TFA; ﬂow rate,

1.0 mL min^ꢀ1; detection, 254 nm) was injected with ( )-

epi-AHI1 (40 mg, 0.17 mmol). The materials at t_R11.2

and 12.2 min were collected to give (ꢀ)-epi-AHI1

(19 mg, 80 lmol) and its (+)-enantiomer (19 mg,

K. Ueno et al. / Bioorg. Med. Chem. 15 (2007) 6311–6322

6321

80 lmol) with an optical purity of 99.2% and 99.9%,

containing 0.1% AcOH; ﬂow rate, 1.0 mL min^ꢀ1; detec-

tion, 254 nm. The materials at t_R20.7 and 22.7 min were

collected to give the enzyme products from (ꢀ)-epi-

AHI1 and its (+)-enantiomer, respectively.

28

D

respectively. (ꢀ)-epi-AHI1: ½aꢁ ꢀ57.7 (c 1.250, MeOH);

28

(+)-epi-AHI1: ½aꢁ +56.6 (c 1.278, MeOH).

D

4.8. Microsomal assay

4.10. Computational method

Kinetic analysis was performed using the detailed proto-

cols described previously.^8,19,20Before assay, the inhibi-

tors that were obtained less than 1 mg were re-quantiﬁed

using the molar absorption coeﬃcient of each com-

pound, which has the same conjugated system. A reac-

tion mixture containing 25 lg mL^ꢀ1CYP707A3

microsomes, (+)-ABA (ﬁnal concentration: 0.5, 1, 2, 4,

8, and 24 lM), inhibitors (0 for control, 0.5–20 lM),

and 50 lM NADPH in 50 mM potassium phosphate

buﬀer (pH 7.25) was incubated for 10 min at 30 ꢁC.

Reactions were initiated by adding NADPH, and

stopped by addition of 50 lL of 1 M HCl. To extract

the reaction products, reaction mixtures were loaded

onto Oasis HLB cartridges (1 mL, 30 mg; Waters) and

washed with 1 mL of 10% MeOH in H₂O 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 dis-

solved in 50 lL of MeOH, and 10 lL was subjected to

HPLC. HPLC conditions were: ODS column, YMC

Hydrosphere C18 (150 · 6.0 mm, ID); solvent, 40%

MeOH in H₂O containing 0.1% AcOH; ﬂow rate,

1.0 mL min^ꢀ1; detection, 254 nm. Enzyme activity was

conﬁrmed by determining the amounts of 8⁰-hydroxy-

ABA (8⁰-HOABA) and phaseic acid (PA) in control

experiments before each set of measurements. The

amount of 8⁰-HOABA was estimated on the basis of a

calibration curve for ABA (19, 38, 77, and 191 pmol) be-

cause oxygenation at C-8⁰has little eﬀect on the molar

absorbance coeﬃcient of ABA.¹⁶The amount of PA

was determined on the basis of the relative ratio (1.18)

of the molar absorption coeﬃcient for 8⁰-HOABA/

PA.³⁴Inhibition constants were determined using the

Enzyme Kinetics module of SigmaPlot 10 software³⁵

after determining the mode of inhibition by plotting

the reaction velocities in the presence and absence of

inhibitor on a double-reciprocal plot. Values are re-

ported as mean values with standard errors of the entire

datasets. For the non-inhibited enzymatic reaction, the

K_Mfor (+)-ABA was calculated to be 0.71 0.08 lM,

based on 16 separate experiments. All tests were con-

ducted at least three times.

All of the minimum-energy conformers of ABA ana-

logues were generated and minimized using MM3 com-

bined with the molecular dynamics simulation built into

CAChe 3.11.³⁶These MM3-minimized structures were

fully optimized with density functional theory, using

the Becke three parameter hybrid functional (B3LYP)

method and the 6-31G(d) basis set in Gaussian 03,³⁷fol-

lowed by a calculation of the harmonic vibrational fre-

quencies at 298 K at the same level. The single point

energies

were

calculated

with

B3LYP/6-

311++G(2df,2p) level of theory. The zero-point energies

were scaled by 0.9804.³⁸

Acknowledgments

We thank Toray Industries Inc., Tokyo, Japan, for the

gift of (+)-ABA. This research was supported by a

Grants-in-Aid for Scientiﬁc Research (No. 17.661) from

the Ministry of Education, Culture, Sports, Science and

Technology of Japan.

References and notes

1. Hirai, N. Abscisic acid. In Comprehensive Natural Prod-

ucts 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, 439–473.

4. Bruzzone, S.; Moreschi, I.; Usai, C.; Guida, L.; Damonte,

G.; Salis, A.; Scarﬁ, S.; Millo, E.; De Flora, A.; Zocchi, E.

Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 5759–5764.

5. Huang, D.; Jaradat, MR.; Wu, W.; Ambrose, S. J.; Ross,

A. R.; Abrams, S. R.; Cutler, A. J. Plant J. 2007, 50, 414–

428.

6. Loveys, B. R. In Abscisic Acid; Davies, W. J., Jones, H.

G., Eds.; BIOS Scientiﬁc Publishers: Oxford, 1991.

7. Nambara, E.; Marion-Poll, A. Annu. Rev. Plant. Biol.

2005, 56, 165–185.

8. Saito, S.; Hirai, N.; Matsumoto, C.; Ohigashi, H.; Ohta,

D.; Sakata, K.; Mizutani, M. Plant Physiol. 2004, 134,

1439–1449.

4.9. Isolation of enzyme products of epi-AHI1

9. Kushiro, T.; Okamoto, M.; Nakabayashi, K.; Yamagishi,

K.; Kitamura, S.; Asami, T.; Hirai, N.; Koshiba, T.;

Kamiya, Y.; Nambara, E. EMBO J. 2004, 23, 1647–1656.

10. Yang, S. H.; Choi, D. Biochem. Biophys. Res. Commun.

2006, 350, 685–690.

11. Yang, S. H.; Zeevaart, J. A. D. Plant J. 2006, 47, 675–686.

12. 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, 942–954.

13. Destefano-Beltran, L.; Knauber, D.; Huckle, L.; Suttle, J.

C. Plant Mol. Biol. 2006, 61, 687–697.

14. Umezawa, T.; Okamoto, M.; Kushiro, T.; Nambara, E.;

Oono, Y.; Seki, M.; Kobayashi, M.; Koshiba, T.; Kamiya,

Y.; Shinozaki, K. Plant J. 2006, 46, 171–182.

A reaction mixture containing 25 lg mL^ꢀ1CYP707A3

microsomes, 10 lM (+)- or (ꢀ)-epi-AHI1, and 50 lM

NADPH in 50 mM potassium phosphate buﬀer (pH

7.25) was incubated for 4 d at 30 ꢁC. CYP707A3 micro-

somes and NADPH were added to the mixture every 2

and 6 h, respectively. The reaction products were puri-

ﬁed described above after being added 50 ll of 1 M

HCl to the mixture. The enzyme products were sub-

jected to HPLC under the following conditions: column,

YMC Hydrosphere C18 (150 · 6.0 mm, ID); solvent,

45% MeOH (0–10 min), 45–100% MeOH (linear gradi-

ent, 10–37.5 min), 100% MeOH (37.5–43 min) in H₂O

6322

K. Ueno et al. / Bioorg. Med. Chem. 15 (2007) 6311–6322

15. Okamoto, M.; Kuwahara, A.; Seo, M.; Kushiro, T.;

Asami, T.; Hirai, N.; Kamiya, Y.; Koshiba, T.; Nambara,

E. Plant Physiol. 2006, 141, 97–107.

16. Todoroki, Y.; Hirai, N.; Koshimizu, K. Biosci. Biotechnol.

Biochem. 1994, 58, 707–715.

17. Todoroki, Y.; Hirai, N.; Koshimizu, K. Phytochemistry

1995, 38, 561–568.

18. Todoroki, Y.; Sawada, M.; Matsumoto, M.; Tsukada, S.;

Ueno, K.; Isaka, M.; Owaki, M.; Hirai, N. Bioorg. Med.

Chem. 2004, 12, 363–370.

25. Liu, X.; Yue, Y.; Li, B.; Nie, Y.; Li, W.; Wu, W. H.; Ma,

L. Science 2007, 315, 1712–1716.

26. Cytochrome P450: Structure, Mechanism, and Biochemis-

try; Ortiz de Montellano, P. R., Ed., 3rd ed.; Kluwer

Academic/Plenum Publishers: New York, 2005.

27. Lamb, N.; Abrams, S. R. Can. J. Chem. 1990, 68, 1151–1162.

28. Oritani, T.; Yamashita, K. Agric. Biol. Chem. 1974, 38,

801–808.

29. Oritani, T.; Yamashita, K. Agric. Biol. Chem. 1970, 34,

830–837.

19. Ueno, K.; Araki, Y.; Hirai, N.; Saito, S.; Mizutani, M.;

Sakata, K.; Todoroki, Y. Bioorg. Med. Chem. 2005, 13,

3359–3370.

20. Ueno, K.; Yoneyama, H.; Saito, S.; Mizutani, M.; Sakata,

K.; Hirai, N.; Todoroki, Y. Bioorg. Med. Chem. Lett.

2005, 15, 5226–5229.

21. Araki, Y.; Miyawaki, A.; Miyashita, T.; Mizutani, M.;

Hirai, N.; Todoroki, Y. Bioorg. Med. Chem. Lett. 2006,

16, 3302–3305.

30. Sundaresan, V.; Abrol, R. Chirality 2005, 17, S30–S39.

31. Zaharia, L. I.; Gai, Y.; Nelson, K. M.; Ambrose, S. J.;

Abrams, S. R. Phytochemistry 2004, 65, 3199–3209.

32. Kondo, S.; Ponrod, W.; Kanlayanarat, S.; Hirai, N. Plant

Growth Regul. 2003, 39, 119–124.

33. Hirai, N.; Okamoto, M.; Fujimura, M.; Ichiyama, T.;

Todoroki, Y.; Ohigashi, H.; Takeda, N.; Yoshizumi, H.;

Tatematsu, A. J. Mass Spectrom. Soc. Jpn. 2000, 48,

8–22.

22. Lin, B. L.; Wang, H. J.; Wang, J. S.; Zaharia, L. I.;

Abrams, S. R. J. Exp. Bot. 2005, 56, 2935–2948.

23. Razem, F. A.; El-Kereamy, A.; Abrams, S. R.; Hill, R. D.

Nature 2006, 439, 290–294.

24. Shen, Y. Y.; Wang, X. F.; Wu, F. Q.; Du, S. Y.; Cao, Z.;

Shang, Y.; Wang, X. L.; Peng, C. C.; Yu, X. C.; Zhu, S.

Y.; Fan, R. C.; Xu, Y. H.; Zhang, D. P. Nature 2006, 443,

823–826.

34. Todoroki, Y.; Hirai, N.; Ohigashi, H. Tetrahedron 2000,

56, 1649–1653.

35. SigmaPlot 10; Systat Software, Inc.: Richmond CA; 2006.

36. CAChe, 3.11; Oxford Molecular Ltd: London; 1998.

37. Gaussian 03 Revision D.01; Gaussian, Inc.: Wallingford

CT; 2004.

38. Bauschlicher, C. W., Jr.; Partridge, H. J. Chem. Phys.

1995, 103, 1788–1791.

Article Doi

Asymmetrical ligand binding by abscisic acid 8′-hydroxylase

DOI: 10.1016/j.bmc.2007.06.010

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2007.06.010

Full text of DOI:10.1016/j.bmc.2007.06.010

Products guided by the article

R&D Labs maybe for 951319-45-2

Relevant to this article

Hot Product