Facile preparation of deuterium-labeled standards of indole-3-acetic acid (IAA) and its metabolites to quantitatively analyze the disposition of exogenous IAA in Arabidopsis thaliana

Article abstract of DOI:10.1271/bbb.70151

[2′,2′-²H₂]-indole-3-acetic acid ([2′,2′-²H₂]IAA) was prepared in an easy and efficient manner involving base-catalyzed hydrogen/deuterium exchange. 1-O-([2′,2′-²H₂]-indole-3-acetyl)-β-D- g

Full text of DOI:10.1271/bbb.70151

Biosci. Biotechnol. Biochem., 71 (8), 1946–1954, 2007
Facile Preparation of Deuterium-Labeled Standards of Indole-3-Acetic
Acid (IAA) and Its Metabolites to Quantitatively Analyze the Disposition
of Exogenous IAA in Arabidopsis thaliana
y
1;3;
Kenji KAI,1;3 Shunsuke NAKAMURA,1 Kyo WAKASA,2;3 and Hisashi MIYAGAWA
1Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2Department of Agriculture, Tokyo University of Agriculture, Kanagawa 243-0034, Japan
3CREST, JST, Tokyo 103-0027, Japan
Received March 15, 2007; Accepted May 13, 2007; Online Publication, August 7, 2007
[doi:10.1271/bbb.70151]
[20,20-2H2]-indole-3-acetic acid ([20,20-2H2]IAA) was
prepared in an easy and efficient manner involving base-
catalyzed hydrogen/deuterium exchange. 1-O-([20,20-
2H2]-indole-3-acetyl)-ꢀ-D-glucopyranose, [20,20-2H2]-2-
oxoindole-3-acetic acid, and 1-O-([20,20-2H2]-2-oxoin-
dole-3-acetyl)-ꢀ-D-glucopyranose were also successfully
synthesized from deuterated IAA, and effectively uti-
lized as internal standards in the quantitative analysis of
IAA and its metabolites in Arabidopsis thaliana by using
liquid chromatography-electrospray ionization-tandem
mass spectrometry (LC-ESI-MS/MS). The use of this
technique shows that these metabolites were accumu-
lated in the roots of Arabidopsis seedlings. Dynamic
changes in the metabolites of IAA were observed in
response to exogenous IAA, revealing that each meta-
bolic action was regulated differently to contribute to
the IAA homeostasis in Arabidopsis.
these conjugates are thought to be storage forms that
reversibly release free IAA depending on the need. The
other is oxidation, typically represented by the con-
version of IAA into 2-oxoindole-3-acetic acid (OxIAA,
5).4) It has been proposed that 5 further undergoes
conjugation1) and in this regard we recently identified 1-
O-(2-oxoindole-3-acetyl)-ꢀ-D-glucopyranose (OxIAA-
Glc, 6) as the result of screening oxidized IAA meta-
bolites in Arabidopsis thaliana.5) The homeostatic reg-
ulation of IAA level in a plant is most likely established
by the coordinated control of these multiple conversions,
although the level of each quantitative contribution
remains unclear.
To fully understand the role of metabolism in the
homeostasis of IAA, an accurate quantification of the
levels of IAA and its metabolites in plant tissues is
essential. Several methods have been developed for this
purpose, based on mass spectrometry coupled with GC
or LC,6–11) in which isotope-labeled preparations of IAA
and IAA metabolites are used as the internal standards.
While a standard for IAA is commercially available as
[13C6]IAA ([13C6]-1) or [2,4,5,6,7-2H5]IAA (1-d5),
major IAA metabolites 3–5 have had to be derived
from either of these two labeled preparations by chemi-
cal or enzymatic means.9,10) Thus, a sufficient and
inexpensive supply of isotope-labeled IAA is needed
for synthesizing the isotope-labeled metabolites of IAA.
[20,20-2H2]IAA (1-d2) is a suitable material for this
purpose, since it can be easily prepared by base-
catalyzed hydrogen/deuterium exchange at C-20 of 1
by using an NaOD/D2O solution.12) However the
deuterium content of 96% reported in the literature is
rather unsatisfactory, so we examined the reaction
conditions to synthesize 1-d2 with a higher deuterium
content.
Key words: Arabidopsis thaliana; indole-3-acetic acid;
metabolism; internal standard; quantitative
analysis
Indole-3-acetic acid (IAA, 1) is an auxin, a plant
hormone, which regulates various processes in plant
growth and development. Its endogenous level is
controlled by the relative rates of biosynthesis and
metabolism, as well as by inter-cellular and/or -tissue
transport. It is generally accepted that metabolism plays
a key role in this regulation, because the total amounts
of IAA metabolites found in plants are usually greater
than those of free IAA.1–4)
To date, two major types of catabolic reaction have
been demonstrated for IAA in plants (Fig. 1). One is
conjugation with a variety of plant components. Forms
of IAA bound with glucose (IAA-Glc, 2), amino acids
(IAA-Asp, 3; IAA-Glu, 4), cyclitol, glucan, and peptides
have been described for various plant species.4) Some of
We present in this paper a facile method for syn-
thesizing IAA labeled with deuterium at the C-20
y
To whom correspondence should be addressed. Division of Applied Life Science, Graduate School of Agriculture, Kyoto University,
Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan; Tel/Fax: +81-75-753-6123; E-mail: miyagawa@kais.kyoto-u.ac.jp

Metabolism of IAA in Arabidopsis
1947
O
COOH
OH
HO
UGT84B1
OH
HO
O
O
N
H
N
H
IAA (1)
GH3
GH3
IAA-Glc (2)
H
N
COOH
COOH
COOH
O
O
N
N
H
H
OxIAA (5)
IAA-Asp (3)
H
N
COOH
O
OH
HO
OH
HO
O
O
O
COOH
O
N
H
N
H
IAA-Glu (4)
OxIAA-Glc (6)
Fig. 1. Schematic Representation of the Metabolism of IAA (1) in Arabidopsis Seedlings.
GH3, IAA-amino acid synthase; UGT84B1, IAA-Glc synthase.
position (1-d2) based on a simple base-catalyzed proton
exchange reaction. 1-d2 was converted into 2-d2, 5-d2,
and 6-d2, and using these labeled preparations as internal
standards, an LC-ESI-MS/MS-based method to quantify
trace amounts of the major IAA metabolites in Arabi-
dopsis was established. The method was used to monitor
the influence of exogenous IAA on the endogenous
levels of IAA metabolites in order to better understand
the IAA homeostasis in plants.
the treatment of IAA with 1.0 M NaOD/D2O for 4 days
at 80 ꢀC afforded [20,20-2H2]IAA (1-d2) in a 66% yield
with >99% isotopic purity, this being used as the
substrate in subsequent reactions. The use of a lower
concentration of NaOD/D2O for a longer time (up to 4
days) was apparently effective for improving the
isotopic purity, compared to the method reported in
the literature.12) To date, deuterated IAA has also been
obtained by the hydrolysis of indole-3-acetonitrile by
NaOD/D2O with 95% isotopic purity.6) Compared to
this, the method employed in the present study has an
advantage in terms of the isotopic purity achieved.
[20,20-2H2]IAA-Glc (2-d2) was synthesized from 1-d2
in accordance with the method of Schmidt et al. with
some modifications (Fig. 2).13) By using 2,3,4,6-tetra-O-
benzyl-ꢁ-D-glucopyranosyl trichloroacetimidate as the
glucosyl donor, the ꢀ-anomer of the protected glucose
ester of 1-d2 was obtained in an 87% yield. Subsequent
deprotection of the benzyl groups was conducted by
catalytic hydrogenation with Pd-C. Glucoside 2-d2 was
obtained without any loss of deuterium at C-20, although
the yield was low (22%). To improve this, we attempted
to synthesize 2-d2 through the condensation of 1-d2 with
2,3,4,6-tetra-O-benzyl-D-glucopyranose using N,N0-di-
cyclohexylcarbodiimide (DCC) under basic conditions
followed by deprotection. The reaction proceeded well
to afford the glucosyl ester in a good yield, but was
inadequate because a significant level of deuterium
exchange occurred under the basic conditions employed,
causing a decrease in the isotopical purity (data not
shown).
Results and Discussion
Synthesis of [20,20-2H2]IAA (1-d2), [20,20-2H2]IAA-Glc
(2-d2), [20,20-2H2]OxIAA (5-d2), and [20,20-2H2]OxIAA-
Glc (6-d2)
IAA was stirred in a NaOD/D2O solution, and the
replacement of the ꢁ-hydrogenes with deuterium was
traced by monitoring the change in the signal intensity
assigned to ꢁ-methylene protons in the 1H-NMR
spectrum. High isotopic purity (>99%) was obtained
by 1.0 M NaOD/D2O for 4 days at 80 ꢀC and in 2.0 M
NaOD/D2O for 2 days at 80 ꢀC. Other NMR signals of
1-d2 were identical with those of unlabeled IAA. The
occurrence of degradation products of IAA was ob-
served after 6 days, indicating that the longer reaction
time was unfavorable. The reaction at higher temper-
ature (>80 ꢀC) with 2 M NaOD/D2O, which has been
employed in the literature,12) was also inappropriate,
causing a significant degradation of IAA, along with a
decrease in isotopic purity. We conclude, therefore, that
satisfactory deuteration of IAA was achieved under the
milder conditions of 1.0 M NaOD/D2O for 4 days at
80 ꢀC and 2.0 M NaOD/D2O for 2 days at 80 ꢀC. Finally,
[20,20-2H2]OxIAA (5-d2) was synthesized by oxida-
tion under strong acidic conditions (Fig. 3).14) Treatment

1948
K. KAI et al.
OBn
BnO
OBn
BnO
D
D
D
O
O
D
D
O
D
H2
Pd-C
COOH Cl C
O
OBn
3
OH
HO
OBn
BnO
O
OH
HO
NH
O
O
O
CH2Cl2
40°C
EtOH
rt
BnO
N
N
N
H
H
H
1-d2
2-d2
Fig. 2. Scheme for Synthesizing [20,20-2H2]IAA-Glc (2-d2).
OBn
1)
OBn
BnO
O
BnO
Cl3C
O
D
D
D
D
O
D
D
NH
OH
COOH
COOH
CH2Cl2, 40°C
OH
HO
O
O
DMSO-HCl-
AcOH
rt
2) H2, Pd-C
EtOH, rt
HO
O
O
N
N
H
N
H
H
1-d2
5-d2
6-d2
Fig. 3. Scheme for Synthesizing [20,20-2H2]OxIAA (5-d2) and [20,20-2H2]OxIAA-Glc (6-d2).
of 1-d2 with DMSO/HCl/AcOH afforded 5-d2 in a 79%
yield. No substitution of the deuterium in the product
was apparent by 1H-NMR or by LC-ESI-MS/MS. [20,20-
2H2]OxIAA-Glc (6-d2) was synthesized from 5-d2
according to the same method as that used for 2-d2
(34% yield in 2 steps) (Fig. 3). No loss of deuterium in
6-d2 was apparent.
6 (352!190), and 6-d2 (354!192). By optimizing the
MRM parameters, good sensitivity was achieved, as
well as good linearity between the relative amount and
the response ratio for each pair of unlabeled and labeled
compounds (data not shown). The detection limits for
compounds 2, 5 and 6 were 0.85, 1.6 and 0.4 fmol,
respectively.
The low yield of glucosides in the cases of 2-d2 and 6-
d2 was due to the concomitant reduction of the indole-
and 2-oxoindole-ring, respectively, during deprotection
of the benzyl groups by catalytic hydrogenation. The
yield could be improved by using unprotected glucosyl
donors such as O-glucosyl trichloroacetimidate.15)
Stability of the deuterium labels of [20,20-2H2]IAA-Glc
(2-d2), [20,20-2H2]OxIAA (5-d2) and [20,20-2H2]OxIAA-
Glc (6-d2)
In earlier studies, [20,20-2H2]IAA (1-d2) was used as
an internal standard for GC–MS.16–18) However, the
deuterium atoms were replaced with hydrogen atoms
during the purification and conversion processes, leading
to inaccurate results.8,16,19) In the case of LC-ESI-MS/
MS, highly selective monitoring of a compound is
possible under relatively mild conditions without any
complicated treatment. Therefore, the deuterium label in
the standard compounds used in this study was expected
to be stable during the analyses.
To investigate the stability of the deuterium label in 2-
d2, 5-d2 and 6-d2 during the analytical procedures, each
labeled compound was subjected to a purification
process (see ‘‘Materials and Methods’’) and subsequent
an analysis by LC-ESI-MS/MS. The MRM chromato-
grams obtained are shown in Fig. 5. In all cases, only the
deuterium-labeled compound could be detected by
MRM, showing that no replacement of deuterium by
hydrogen occurred. It was thus concluded that deute-
rium-labeled 2-d2, 5-d2 and 6-d2 were stable enough to
be used as internal standards in the quantification of
these IAA metabolites in plants. The peak detected by
MRM (192!146) at 7.5 min (Fig. 5B) did not agree
with that for 6, being due to the presence of a
contaminant during the purification procedure.
Optimization of the ESI-MS/MS parameters for
quantification
In order to find an optimum multiple reaction mon-
itoring (MRM) transition for quantification, comparative
ESI-MS/MS-based analyses of deuterium-labeled and
unlabeled compounds were performed for 2, 5 and 6
(Fig. 4). In the negative mode, the deprotonated mole-
cule ½M ꢁ Hꢂꢁ of 2-d2 gave major product ions at m=z
176 and 132 by neutral loss of the Glc moiety and
decomposition into quinolinium ions, respectively. Non-
labeled 2 underwent the same ion transformation,
providing the corresponding ions with m=z values
smaller by 2 mass units at the same intensity. In the
case of the pair 5-d2 and 5, both compounds strongly
yielded 2-oxoquinolinium ions at m=z 148 and 146,
respectively, from the protonated molecule ½M þ Hꢂþ in
the positive mode. Deuterium-labeled 6-d2 gave product
ions at m=z 192 and 148 from ½M ꢁ Hꢂꢁ with high
abundance in the negative mode, which was also the
case for 6. In all experiments, the deuterium label in
these compounds was stable enough not to cause any
loss of deuterium during the fragmentation of ½M þ Hꢂþ
or ½M ꢁ Hꢂꢁ into major product ions during ESI-MS/
MS. Based on these results, we selected the following
pairs of ions for MRM-based quantification: 2 (336!
174), 2-d2 (338!176), 5 (192!146), 5-d2 (194!148),
It has been documented that 1-O-IAA-Glc (2) is easily
isomerized into 2-O-, 4-O- and 6-O-IAA-Glc via acyl-
migration.20) As shown in Fig. 5, however, no conver-
sion was observed during the experimental procedures

Metabolism of IAA in Arabidopsis
1949
174
176
100
100
50
132
130
O
D
O
D
OH
HO
OH
HO
OH
HO
OH
HO
O
O
O
O
N
H
174
N
176
H
50
132
130
2
2-d2
218
216
157
113
338
113
156
59
190
278
188
59
248
276
246
336
60 100 140 180 220 260 300 340
60 100 140 180 220 260 300 340
m/z
m/z
146
148
148
100
50
100
50
146
D
COOH
O
D
COOH
128
N
H
O
N
H
5
5-d2
130
132
128
192
194
174
101
176
102
104
104
132
91
77
93
120
77
60 80 100 120 140 160 180 200 220
60 80 100 120 140 160 180 200 220
m/z
m/z
192
148
190
100
50
100
50
146
O
D
O
D
OH
OH
HO
OH
HO
OH
O
HO
O
148
O
O
HO
O
146
N
O
N
H
190
H
192
6
234
6-d2
354
144
128
144
132
113
173
232
352
172
294
113
264
59
292
59
262
214
60 100 140 180 220 260 300 340 380
60 100 140 180 220 260 300 340 380
m/z
m/z
Fig. 4. Product Ion Analyses of Deuterium-Labeled and Non-Labeled IAA-Glc (top), OxIAA (middle), and OxIAA-Glc (bottom).
The characteristic fragmentation of each metabolite is described in the spectra.
used in the present study, while we did apparent
isomerization in an acidic aqueous solution after a long
period of preservation at room temperature. In this
regard, we have previously found that isomers of 6 were
formed during purification of the extract of Arabidopsis
seedlings by preparative HPLC.5) Since such isomer-
ization causes the production of artifact peaks during LC
and consequently disturbs the quantification, a certain
degree of care during the analytical procedures must be
taken.
ence of internal standards. Quantification with LC-ESI-
MS/MS under the optimized conditions already describ-
ed showed that 1, 2 and 5 were accumulated in the roots
as shown in Fig. 6, which is consistent with previous
reports.10,11) Among the compounds quantified, 6 had the
highest levels and was 4-fold more abundant in the roots
than in the aerial parts. Metabolite 6 has been shown to
be derived from 5 in the seedlings of Arabidopsis.5) The
present data suggest that 6 was produced mainly in the
roots.
Quantification of IAA and its metabolites in Arabi-
dopsis seedlings
Effect of exogenous IAA on its endogenous metabo-
lites in Arabidopsis
In order to determine the levels of 1, 2, 5 and 6
in Arabidopsis, 2-week-old seedlings grown in a
Murashige-Skoog (MS) medium were separated into
their aerial parts and roots, and extracted with 80% (v/v)
acetone-2.5 mM diethyldithiocarbamic acid in the pres-
In spite of the many studies on IAA metabolites, little
is known about the relative contribution of each
metabolic pathway to the homeostatic level of free
IAA in plants. Therefore, IAA was fed to seedlings of
Arabidopsis through the roots and harvested at various

1950
K. KAI et al.
8.10
8.15
A
B
MRM
MRM
MRM
MRM
194 148
336 174
192 146
338 176
2
4
6
8
10 12 14
2
4
6
8
10 12 14
Time (min)
Time (min)
6.93
C
MRM
352 190
MRM
354 192
2
4
6
8
10 12 14
Time (min)
Fig. 5. MRM Chromatograms of [20,20-2H2]IAA-Glc (2-d2) (A), [20,20-2H2]OxIAA (5-d2) (B) and [20,20-2H2]OxIAA-Glc (6-d2) (C) in the Eluates of
Solid-Phase Cartridges.
No loss of deuterium in any compound was apparent during the purification or ionization process.
Arabidopsis. On the other hand, when the concentration
of IAA was increased to 10 mM, a significant rise in the
levels of 1 and 2 was observed. However, both
compounds decreased gradually thereafter to normal
5
Aerial parts
4
3
2
1
Roots
levels. This suggests that the level of 2 was determined
by that of endogenous IAA. The increase in the level of
6 was again remarkable, with a moderate but significant
increase of 5 in this case. However, the accumulation of
3 and 4 under these conditions was much more
prominent, which is in marked contrast with the result
obtained after feeding with 100 nM IAA. Thus, the major
metabolic pathway changed to conjugation with amino
acids when an excess of IAA was given.
It has been demonstrated that GH3 genes encoding
IAA-amino acid synthetases were rapidly expressed in
response to exogenous auxin.21–24) UGT84B1 has also
been identified as a conjugate synthetase for 2,20)
although the gene encoding this enzyme was not
auxin-inducible [‘‘AtGenExpress Visualization Tool’’
(http://jsp.weigelworld.org/expviz/expviz.jsp)]. The reg-
ulation of other types of metabolic conversion as well as
of the reverse regeneration of IAA from the metabolites
remains ambiguous. The present study has shown that
the metabolic profile concerning IAA depended on its
concentration, most likely due to the activation of
multiple metabolic reactions in individually different
ways in response to the IAA level. This reflects a
complicated aspect of the regulation of IAA level in
plants, or homeostasis. A variety of T-DNA Arabidopsis
mutants are now available (‘‘SIGnAL’’ supplied by
SALK Institute Genomic Analysis Laboratory) which
have defects in the genes putatively involved in IAA
metabolism.25–27) The profiling of IAA metabolites in
Fig. 6. Quantification of IAA (1), IAA-Glc (2), OxIAA (5) and
OxIAA-Glc (6) in the Aerial Parts and Roots of Arabidopsis.
Two-week-old seedlings were divided into their aerial parts and
roots. The error bars indicate the standard deviation of three
replicates.
times from 2 to 24 h post-treatment to quantify IAA and
its metabolites. The resulting metabolic dynamics are
shown in Fig. 7. After feeding with 100 nM IAA, the
levels of 3–6 increased in a time-dependent manner,
whereas those of 1 and 2 remained almost constant.
Under these conditions, the plants appeared to be able to
maintain homeostasis in terms of the IAA level.
Throughout this experiment, there was much more 6
than any other metabolite, indicating that oxidation and
subsequent glucosylation were the main metabolic
pathway for exogenous IAA in the seedlings of

Metabolism of IAA in Arabidopsis
1951
A
0.08
0.05
0.30
IAA (1)
IAA-Asp (3)
IAA-Glu (4)
0.25
0.04
0.03
0.02
0.01
0.06
0.04
0.02
0.20
0.15
0.10
0.05
12 16
Time (h)
12 16
Time (h)
8
8
16
0
4
20 24
4
20 24
12
0
0
4
8
20 24
Time (h)
4
3
2
1
0.25
0.20
0.15
0.10
0.05
0.12
0.09
0.06
0.03
IAA-Glc (2)
OxIAA (5)
OxIAA-Glc (6)
12 16
Time (h)
12 16
Time (h)
12 16
Time (h)
8
20 24
8
20 24
8
4
4
0
4
20 24
0
0
B
25
60
50
40
30
IAA (1)
IAA-Asp (3)
IAA-Glu (4)
4
3
2
20
15
10
5
20
10
1
12 16
Time (h)
12 16
Time (h)
8
8
16
0
4
20 24
4
20 24
12
0
0
4
8
20 24
Time (h)
10
8
1.2
1.0
0.8
0.6
25
20
15
10
5
IAA-Glc (2)
OxIAA (5)
OxIAA-Glc (6)
6
4
0.4
0.2
2
12 16
Time (h)
12 16
Time (h)
12 16
Time (h)
8
20 24
8
20 24
8
4
4
0
4
20 24
0
0
Fig. 7. Time-Course Study of the Disposition of Exogenous IAA in Arabidopsis.
The seedlings were incubated in a liquid MS medium containing either 100 nM (A) or 10 mM (B) IAA. The error bars indicate the standard
deviation of three replicates.
those mutants is in progress in order to identify the
enzyme(s) that plays a critical role in regulating the level
of each metabolite, and to better understand the complex
mechanism of IAA homeostasis in plants.
API3000 triple-quadrupole-stage mass spectrometer
(Applied Biosystems/MDS Sciex, Ontario, Canada).
The ESI-MS data were measured with the same mass
spectrometer. The HRFAB-MS analysis was conducted
with a JMS 700 spectrometer (JEOL, Tokyo, Japan)
using glycerol as the matrix. Column chromatography
was conducted with Kieselgel 60 (Merck, Darmstadt,
Germany) as the adsorbent, and preparative HPLC was
performed with a LC-10A HPLC system (Shimadzu,
Kyoto, Japan).
Materials and Methods
General. 1H- and 13C-NMR spectra were recorded by
a Bruker Avance 400 spectrometer with TMS as the
1
internal standard. Assignment of the H and 13C signals
was carried out by using 1H–1H COSY, HMQC and
HMBC spectra. The LC-ESI-MS/MS data were collect-
ed with an Agilent 1100 HPLC system coupled to an
Chemicals. NaOD [40% (w/w), in D2O, 99.5 atom %
D] was purchased from Aldrich (Milwaukee, WI, USA)

1952
K. KAI et al.
and D2O (100.0 atom % D) from Acros (New Jersy,
USA). [13C6]IAA (13C6-1), an internal standard for IAA
(1), was purchased from Cambridge Isotope Laborato-
ries (Andover, MA, USA). IAA-Glc (2), OxIAA (5), and
OxIAA-Glc (6) were synthesized in a previous study.5)
The samples of [20,20-2H2]IAA-Asp (3-d2) and [20,20-
2H2]IAA-Glu (4-d2) were kindly presented by Dr. Jun
Hiratake of the Institute for Chemical Research at Kyoto
University. All other chemicals were obtained commer-
cially.
mm (GL Science, Tokyo, Japan); solvent, water:
MeCN =75:25; flow rate, 3.20 ml/min; detection, 280
nm)] gave [20,20-2H2]IAA-Glc (2-d2) as a colorless oil
1
(37 mg, 22%). H-NMR (400 MHz, CD3OD) ꢂ: 3.30–
3.65 (4H, m), 3.66 (1H, m), 3.81 (1H, d, J ¼ 12:0 Hz),
5.52 (1H, d, J ¼ 7:8 Hz, Glc, H-1), 7.01 (1H, m, IAA,
H-5), 7.09 (1H, m, IAA, H-6), 7.19 (1H, s, IAA, H-2),
7.34 (1H, d, J ¼ 8:1 Hz, IAA, H-7), 7.55 (1H, d, J ¼
7:8 Hz, IAA, H-4). 13C-NMR (100 MHz, CD3OD) ꢂ:
62.2 (Glc, C-6), 71.0 (Glc, C-2 or 4 or 5), 74.0 (Glc, C-2
or 4 or 5), 77.9 (Glc, C-3), 78.8 (Glc, C-2 or 4 or 5), 95.5
(Glc, C-1), 107.8 (IAA, C-3), 122.7 (IAA, C-7), 119.5
(IAA, C-4 or 5), 119.9 (IAA, C-4 or C-5), 122.5 (IAA,
C-6), 124.9 (IAA, C-2), 128.6 (IAA, C-3a), 137.9 (IAA,
C-7a), 172.9 (IAA, C-10). ESI-MS: m=z 338 (M ꢁ Hꢁ).
FAB-MS: m=z 362 (M þ Naþ), 339 (Mþ). HRFAB-MS:
m=z 339.1285 (Mþ, calcd. for C16H17D2NO7, 339.1287).
[20,20-2H2]-2-oxoindole-3-acetic acid (5-d2). 1-d2
(400 mg, 2.27 mmol) was dissolved in 6 ml of DMSO/
conc. HCl/AcOH (1:5:10, v/v), and the solution was
stirred for 3 h at room temperature. The mixture was
quenched in 10 ml of ice-water and then extracted with
EtOAc (3 ꢃ 15 ml). The combined EtOAc solution was
successively washed with water and brine. The organic
layer was dried over anhydrous Na2SO4 and concen-
trated in vacuo. The residue was crystallized from
Study for the deuteration of IAA (1). IAA (100 mg,
0.570 mmol) was dissolved in D2O (5 ml) containing the
specified concentration of NaOD and the mixture was
stirred under specified conditions. Every 2 days, ca.
0.5 ml of the reaction mixture was analyzed by 1H-NMR
spectroscopy.
Chemical synthesis.
[20,20-2H2]-indole-3-acetic acid (1-d2). IAA (1 g, 5.70
mmol) was dissolved in 50 ml of 1 M NaOD/D2O, and
the mixture was stirred for 4 days at 80 ꢀC. After cooling
to room temperature, the mixture was acidified with 2 M
HCl to form crystals. The crystals were washed with
water and recrystallized from EtOAc to yield a white
1
yellow substance (669 mg, 66%). H-NMR (400 MHz,
1
acetone-d6) ꢂ: 7.02 (1H, m, H-5), 7.10 (1H, m, H-6), 7.30
(1H, J ¼ 2:4 Hz, H-2), 7.39 (1H, d, J ¼ 8:1 Hz, H-7),
7.61 (1H, d, J ¼ 7:9 Hz, H-4), 10.21 (1H, br, H-1),
10.62 (1H, br, COOH). ESI-MS: m=z 178 (M þ Hþ).
1-O-([20,20-2H2]-indole-3-acetyl)-ꢀ-D-glucopyranose
(2-d2). 1-d2 (100 mg, 0.564 mmol) and 2,3,4,6-tetra-O-
benzyl-ꢁ-D-glucopyranosyl trichloroacetimidate (386
mg, 0.564 mmol) were dissolved in 3 ml of dry CH2Cl2,
and the solution was stirred overnight at 40 ꢀC. The
reaction mixture was diluted with 10 ml of CHCl3,
successively washed with a saturated aqueous NaHCO3
solution and brine, and dried over anhydrous Na2SO4.
The solvent was evaporated in vacuo, and the residue
was purified by column chromatography (hexane-EtOAc
stepwise elution) to give 1-O-([20,20-2H2]-indole-3-ace-
tyl)-2,3,4,6-tetra-O-benzyl-ꢀ-D-glucopyranose as an or-
CHCl3 to give a white pink crystal (346 mg, 79%). H-
NMR (400 MHz, DMSO-d6) ꢂ: 3.61 (1H, s, H-3), 6.81
(1H, d, J ¼ 7:7 Hz, H-8), 6.92 (1H, m, H-5), 7.16 (1H,
m, H-6), 7.22 (1H, d, J ¼ 7:3 Hz, H-7), 10.3 (1H, s, H-
1), 12.3 (1H, s, COOH). 13C-NMR (100 MHz, DMSO-
d6) ꢂ: 41.8 (C-20), 109.3 (C-4), 121.3 (C-5), 123.8 (C-7),
127.9 (C-6), 129.5 (C-3a), 143.1 (C-7a), 172.4 (C-10),
178.3 (C-2). ESI-MS: m=z 194 (M þ Hþ). HRFAB-MS:
m=z 194.0784 (M þ Hþ, calcd. for C10H8D2NO3,
194.0786).
1-O-([20,20-2H2]-2-oxoindole-3-acetyl)-ꢀ-D-glucopyra-
nose (6-d2). 5-d2 (100 mg, 0.518 mmol) and 2,3,4,6-
tetra-O-benzyl-ꢁ-D-glucopyranosyl trichloroacetimidate
(425 mg, 0.621 mmol) were dissolved in 3 ml of dry
CH2Cl2, and the solution was stirred overnight at 40 ꢀC.
The reaction mixture was diluted with 10 ml of CHCl3,
successively washed with a saturated aqueous NaHCO3
solution and brine, and then dried over anhydrous Na2-
SO4. The solvent was evaporated in vacuo, and the res-
idue was purified by column chromatography (CHCl3–
MeOH stepwise elution) to give 1-O-([20,20-2H2]2-
oxoindole-3-acetyl)-2,3,4,6-tetra-O-benzyl-ꢀ-D-glucopy-
ranose as a yellow powder (149 mg, 40%). 1H-NMR
(400 MHz, CDCl3) ꢂ: 3.54–3.81 (7H, m), 4.47–4.89 (9H,
m), 5.62 (0.5H, d, J ¼ 8:1 Hz, Glc, H-1), 5.67 (0.5H, d,
J ¼ 8:1 Hz, Glc, H-1), 6.87–7.33 (24H, m). ESI-MS:
m=z 717 (M þ Hþ).
1
ange paste (342 mg, 87%). H-NMR (400 MHz, CDCl3)
ꢂ: 3.52–3.58 (2H, m), 3.65–3.76 (4H, m), 4.34 (1H, d,
J ¼ 9:2 Hz), 4.44–4.52 (3H, m), 4.60 (1H, d, J ¼ 12:1
Hz), 4.75–4.84 (3H, m), 5.65 (1H, d, J ¼ 8:1 Hz, Glc, H-
1), 6.98 (2H, dd, J ¼ 1:9, 5.9 Hz), 7.02 (1H, d, J ¼ 2:4
Hz), 7.07–7.32 (21H, m), 7.59 (2H, d, J ¼ 7:8 Hz, IAA,
H-4), 7.99 (1H, br, IAA, H-1). ESI-MS: m=z 701 (M þ
Hþ).
1-O-([20,20-2H2]-indole-3-acetyl)-2,3,4,6-tetra-O-benz-
yl-ꢀ-D-glucopyranose (342 mg, 0.488 mmol) and 10%
(w/w) Pd-C (136 mg) were suspended in 30 ml of EtOH,
and the suspension was stirred for 6 h at room temper-
ature in an H2 atmosphere. The precipitate was removed
by filtration, and the filtrate was purified by column
chromatography (CHCl3:MeOH = 90:10). Subsequent
preparative HPLC [column, Inertsil ODS-3 10 ꢃ 250
1-O-([20,20-2H2]2-oxoindole-3-acetyl)-2,3,4,6-tetra-O-
benzyl-ꢀ-D-glucopyranose (149 mg, 0.208 mmol) and
10% (w/w) Pd-C (59.6 mg) were suspended in 10 ml
of EtOH, and the suspension was stirred for 4 h at room
temperature in an H2 atmosphere. The precipitate was

Metabolism of IAA in Arabidopsis
1953
Table 1. Multiple Reaction Monitoring (MRM) Parameters to Quantify Metabolites 2, 5 and 6
Compound
Polarity
DP (V)
FP (V)
EP (V)
CE (V)
CXP (V)
2
5
6
Negative
Positive
Negative
ꢁ51
36
ꢁ260
230
ꢁ10
10
ꢁ14
17
ꢁ11
8
ꢁ46
ꢁ300
ꢁ10
ꢁ24
ꢁ11
DP, declustering potential; FP, focusing potential; EP, entrance potential; CE, collision energy; CXP, collision cell exit potential
removed by filtration, and the filtrate was purified by
column chromatography (CHCl3–MeOH stepwise elu-
tion) and subsequent preparative HPLC (column,
Inertsil ODS-3 10 ꢃ 250 mm; solvent, water:MeCN =
82.5:17.5; flow rate, 3.20 ml/min; detection, 280 nm) to
give [20,20-2H2]OxIAA-Glc (6-d2) as a colorless paste
(63.4 mg, 86%). This preparation was a mixture of keto-
ing 2.5 mM diethyldithiocarbamic acid and the respec-
tive internal standards (0.1–0.5 nmol) for 2 h. After the
extraction procedure had been repeated, the combined
extract was concentrated under reduced pressure. The
aqueous concentrate was subjected to solid-phase
extraction after adjusting the pH value to approximately
2.6. After removing the solvent, the sample was
analyzed by LC-ESI-MS/MS operated in the MRM
mode to quantify IAA and its metabolites. The following
transitions from precursor to product ions were moni-
tored: 1, 176!130; [13C6]-1, 182!136; 3, 291!130;
3-d2, 293!132; 4, 305!130; 4-d2, 307!132; 2,
336!174; 2-d2, 338!176; 5, 192!146; 5-d2, 194!
148; 6, 352!190; 6-d2, 354!192. Quantification was
based on the ratio of the peak area for each metabolite to
that for the internal standard.
1
enol tautomers. H-NMR (400 MHz, CD3OD) ꢂ: 3.31–
3.32 (3H, m, Glc. H-2,4,5), 3.40 (1H, m, Glc, H-3), 3.66
(1H, dd, J ¼ 4:4, 12.0 Hz, Glc, C-6), 3.78–3.83 (2H, m,
Glc-H-6, OxIAA-H-3), 5.47 (0.5H, d, J ¼ 8:0 Hz, Glc,
H-1), 5.51 (0.5H, d, J ¼ 8:0 Hz, Glc, H-1), 6.89 (1H, d,
J ¼ 7:7 Hz, OxIAA, H-4), 6.98 (1H, m, OxIAA, H-5),
7.18 (1H, m, OxIAA, H-6), 7.30 (1H, d, J ¼ 7:3 Hz,
OxIAA, H-7). 13C-NMR (100 MHz, CD3OD) ꢂ: 43.27
(OxIAA, C-3), 43.37 (OxIAA, C-3), 62.27 (Glc, C-6),
70.96 (Glc, C-2 or 4 or 5), 73.88 (Glc, C-2 or 4 or 5),
73.93 (Glc, C-2 or 4 or 5), 77.80 (Glc, C-3), 78.80 (Glc,
C-2 or 4 or 5), 78.85 (Glc, C-2 or 4 or 5), 96.01 (Glc, C-
1), 96.06 (Glc, C-1), 110.85 (OxIAA, C-4), 123.33
(OxIAA, C-5), 123.38 (OxIAA, C-5), 125.27 (OxIAA,
C-7), 129.30 (OxIAA, C-6), 130.23 (OxIAA, C-3a),
130.28 (OxIAA, C-3a), 143.63 (OxIAA, C-7a), 171.57
(OxIAA, C-10), 181.05 (OxIAA, C-2), 181.11 (OxIAA,
C-2). ESI-MS: m=z 354 (M ꢁ Hꢁ). HRFAB-MS: m=z
356.1321 (M þ Hþ, calcd. for C16H18D2NO8,
356.1314).
IAA treatment. Two-week-old seedlings were trans-
ferred into a liquid MS medium containing the appro-
priate concentration of IAA and incubated for 24 h at
22 ꢀC under continuous light. After the seedlings had
been washed with water, a quantitative analysis was
performed as described above.
We thank Dr. Jun Hiratake (Institute for Chemistry,
Kyoto University, Kyoto, Japan) for advice regarding
the synthesis of deuterium-labeled compounds and for
the HRFAB-MS measurements.
ESI-MS/MS analysis. Specified amounts of 2, 2-d2, 5,
5-d2, 6 and 6-d2 were dissolved in 60% (v/v) MeOH.
The solutions were directly introduced into an API3000
mass spectrometer to obtain product-ion spectra. The
parameters for mass spectrometry related to the ioniza-
tion and fragmentation were optimized by using Analyst
1.3 software (Applied Biosystems) as listed in Table 1.
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