Deuterated abscisic acid analogs for mass spectrometry and metabolism studies

Article abstract of DOI:10.1002/jlcr.670

Four analogs of abscisic acid (ABA) with deuterium atoms at non-exchangeable positions have been synthesized to be used as standards for quantitation of the plant hormone ABA by mass spectrometry and also to be employed as substrates for metabolism studies. Deuterium atoms were introduced in the side chain of the molecule, at C-4 and/or C-5, by deuteride or hydride reduction of a propargylic alcohol, an intermediate in the synthesis. As well, deuterium labels were introduced at the C-8′ position by conjugate addition of a Grignard reagent containing the label to a cyclohexadienone intermediate, affording specific isotopically labeled ABA molecules with one to five deuterium atoms. Copyright

Full text of DOI:10.1002/jlcr.670

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 273–283.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.670
Research Article
Deuterated abscisic acid analogs for mass
spectrometry and metabolism studies
Suzanne R. Abrams*, Ken Nelson and Stephen J. Ambrose
Plant Biotechnology Institute, National Research Council of Canada,
110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9
Summary
Four analogs of abscisic acid (ABA) with deuterium atoms at non-exchange-
able positions have been synthesized to be used as standards for quantitation of
the plant hormone ABA by mass spectrometry and also to be employed as
substrates for metabolism studies. Deuterium atoms were introduced in the
side chain of the molecule, at C-4 and/or C-5, by deuteride or hydride
reduction of a propargylic alcohol, an intermediate in the synthesis. As well,
deuterium labels were introduced at the C-80 position by conjugate addition of
a Grignard reagent containing the label to a cyclohexadienone intermediate,
affording specific isotopically labeled ABA molecules with one to five
deuterium atoms. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: abscisic acid; 2H labeled standard; hydride reduction of
propargylic alcohol; 1,4-conjugate addition of Grignard
Introduction
Abscisic acid, ABA, 1, a monocyclic sesquiterpenoid natural product, is
a signaling molecule present at low concentrations in all higher plants.
ABA has been found to play important roles in plant growth and
development in diverse processes including regulation of stomatal
*Correspondence to: S. R. Abrams, Plant Biotechnology Institute, National Research Council of
Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, S7N 0W9 Canada.
E-mail: sue.abrams@nrc.ca
Copyright # 2002 John Wiley & Sons, Ltd.
Received 22 April 2002
Revised 29 August 2002
Accepted 16 September 2002

274
S. R. ABRAMS ET AL.
6
9'
8'
4'
5
2H
2H
3
2
6'
3'
5'
4
1'
2'
OH
C2H3
OH
7'
COOH
COOH
1
O
O
2H
1 S (+)-ABA
2
Figure 1. ABA 1 and [2H6 ]ABA 2
aperture, seed maturation and germination, root and shoot growth,
transpiration and stress tolerance.1,2 For example, ABA regulates genes
involved in the synthesis of storage reserves in developing seeds and
prevents immature seeds from germinating prematurely.3 In response to
abiotic stresses such as heat, freezing or drought, ABA levels in plants
elevate, triggering specific biochemical responses.4 The biosynthesis of
ABA and some catabolic pathways have been elucidated, and a number
of genes involved in ABA biosynthesis have been cloned.5, 6 There is
currently much research into the molecular basis of ABA action and a
growing need for improved tools for studying ABA levels and
turnover.7
The most generally employed methods to measure ABA levels in
plant tissues are gas chromatography combined with mass spectrometry
(GC/MS) of ABA methyl ester, and RIA or ELISA immunoassay of the
free acid using a monoclonal antibody raised against a conjugated
derivative of ABA, verified by GC/MS(Figure 1).8
[2H6]ABA 2 has been most commonly employed as the internal
standard in GC/MS quantitation of ABA. This internal standard,
synthesized by base exchange,9 works well for analytical procedures in
which base exchange is minimal. Efforts have been made to develop an
ABA deuterated at C-6 of the side chain, to provide an internal
standard that would be stable to exchange, with limited success.10 The
synthesis of a 13C labeled ABA has been reported.11
Deuterated ABA analogs have also been employed in plant
metabolism studies to assess the relative roles of ABA and its catabolites
in biological processes affected by ABA.12 The principal oxidative
degradation pathway of ABA (Scheme 1) is through hydroxylation at
the 80-carbon atom, thought to be mediated by a P450 monooxygenase.5
The initial oxidation product, 80-hydroxy ABA, 3 , exists in equilibrium
with the bicyclic metabolite phaseic acid 4. In metabolism studies using
corn cells, the rate of oxidation of the deuterated ABA 5 has been found
to be significantly slower than that of the natural hormone 1. This
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DEUTERATED ABSCISIC ACID ANALOGS
275
HOH2C
OH
OH
O
OH
COOH
COOH
COOH
O
O
O
1
3
4
2H3C
C2H3
OH
C2H3
COOH
O
5
Scheme 1. Principal oxidative pathway of metabolism of ABA
reduced rate was attributed to a primary isotope effect, arising from
cleavage of the stronger C–D versus C–H bond at the 80-carbon atom.9
The ABA (+)-5 was employed as a probe to assess the role of the
hormone in cress seed germination. Nonadeutero (+)-5 was signifi-
cantly more effective than the natural hormone (+)-1, consistent with
the hormone itself being the biologically active species, and that the
oxidation process through the 80-carbon atom is the key degradation
pathway.
In this paper, we report the synthesis of a series of deuterated ABA
molecules, designed as internal standards for GC/MS and HPLC
coupled to electrospray mass spectrometry (HPLCMS/MS). The
molecules were also designed to be starting materials for standards of
ABA metabolites through biotransformation of labeled ABA molecules,
and as probes for studying ABA metabolism pathways in planta.
Results and discussion
The general scheme employed for synthesizing ABA with deuterium
atoms in the side chain and the ring was based on a previous syntheses
of ABA and analogs modified at the 80-carbon atom.13,14 The basic
skeleton of the molecule is constructed by alkylation of the mono-
protected cyclohexadienone 6 with the dilithium salt of cis-3-methyl-
pent-2-en-4-yn-1-ol (Scheme 2). Reduction of the triple bond of the
propargylic alcohol of the product 7 affords the dienol 8. Oxidation of
the alcohol to the ester followed by deprotection of the ketone affords
the cyclohexadienone 9. The 80-methyl group of ABA is introduced by
conjugate addition of methyl magnesium bromide to the enone of 9. It
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276
S. R. ABRAMS ET AL.
O
a
CH2OH
OH
+
O
O
CH2OH
O
O
7
6
b
f
CH2OH
COOCH3
c, d, e
Methyl ABA
OH
9
OH
8
O
O
O
Scheme 2. Reagents (a) n-BuLi, THF; (b) Red-Al1; (c) MnO2, acetone; (d)
MnO2, NaCN, AcOH, MeOH; (e) HCl, THF; (f) CH3MgBr
had earlier been shown that the conjugate addition afforded the product
with alkyl group on the same face of the molecule as the hydroxyl
group. The reaction performed with trideuterated methyl magnesium
bromide afforded 80-trideuteromethyl ABA.13
The scheme was modified to incorporate deuterium specifically at
either the C-4, C-5 or both positions by deuteride reduction of the triple
bond of the propargylic alcohol.15 The deuteride addition takes place on
the carbon alpha to the hydroxyl with the postulated formation of a
cyclic intermediate. The proton or deuteron on the beta position arises
from quenching of the reaction with water or deuterium oxide. Thus,
reduction of 7 with lithium aluminum deuteride followed by quenching
with deuterium oxide afforded the dideuterated dienol that was
converted to the methyl ester 10 (Scheme 3). Reduction of the triple
bond of 7 with deuteride reagent, followed by quenching with water, led
to incorporation of deuterium at the C-5 position, which on further
oxidation afforded 11.
The mono and dideuterated cyclohexadienones 10 and 11 were
converted to the labeled ABA by reaction with methyl magnesium
bromide or trideuteromethyl magnesium bromide, followed by hydro-
lysis of the methyl ester, affording 16 – 19 (Scheme 3).
Analysis of the deuterium incorporation achieved in the procedure
was estimated by examination of the molecular ion clusters in LC/MS/
2
MS spectra of the acids 16 – 19. The ion for ABA H0 (m/z 263) in the
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DEUTERATED ABSCISIC ACID ANALOGS
277
CH2OH
OH
O
O
a, b , d, e , f
a, c, d, e, f
7
2H
COOCH3
COOCH3
2H
OH
2H
OH
O
O
11
10
g
g
h
h
2H
2H
2H3C
2H3C
COOR
2H
OH
COOR
2H
OH
COOR
COOR
2H
2H
OH
OH
O
O
O
O
R = CH3 14
R = H 18
R = CH3 15
R = CH3 12
R = H 16
R = CH3 13
R = H 17
19
R = H
2
Scheme 3. Reagents (a) LiAl2H4, 2 CH3O(CH2)2OH; (b) H2O; (c) H2O; (d)
MnO2, acetone; (e) MnO2, NaCN, AcOH, MeOH; (f) HCl, THF; (g) C2H3MgI
(h) CH3MgBr
preparation of the acid 19 was found to be 0.5% of the ion for the
monodeuterated analog, indicating the introduction of the deuteride at
C-5 gave 99.5% incorporation of the label. The sample of acid 18
2
contained 0.8% ABA H3 indicating that the Grignard addition with
labeled reagent proceeded with essentially quantitative isotopic incor-
poration. The samples of acids 16 and 17 contained 2 and 1.7% of the
ABA with 1 Da lower than the expected value, consistent with a slightly
lower incorporation of label in the quenching with deuterium oxide.
Experimental section
The synthesis of the unlabeled ABA using this procedure has been
published.14 The procedures and spectra for synthesis of the isotopically
labeled ABA molecules are presented below. LCMS/MS spectra were
obtained in negative ion electrospray mode using a capillary voltage of–
2.75 KV, counter electrode 35 V, collision energy (ELAB) of 14 V, and
cell pressure of 1.0 ꢀ 10ꢁ3 mBar with argon.
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S. R. ABRAMS ET AL.
Methyl-(2Z,4E)-[4,5-2H2]-5-(1-hydroxy-2,6-dimethyl-4-oxo-2,5-cyclo-
hexadien-1-yl)-3-methylpenta-2,4-dienoate (10).
2
To a solution of LiAl2H4 (96 atom % H) (22.7 ml 1.0 M in THF,
22.7 mmol) in THF (50 ml) at 08C was added dropwise 2-methoxy-
ethanol (3.57 ml, 45.3 mmol). After 30 min., the solution was cannulated
to a stirred solution of ketal alcohol 7 (4.17 g, 15.1 mmol) in THF
(80 ml) at -788C. The solution was warmed to 08C. After 2 h, the
2
reaction was quenched with 2H2O (99.9 atom % H) (10 ml) and
allowed to stir for 1 h. Saturated NaHCO3 (50 ml) was added and the
solution was extracted with ether (3 ꢀ 150 ml). The combined organic
layers were dried (MgSO4), filtered and concentrated under vacuum.
The crude product was dissolved in acetone (100 ml) and stirred with
MnO2 (26.0 g, 300 mmol) at room temperature. After 2 h, the solution
was filtered through a pad of Celite1 and concentrated under vacuum.
The crude ketal aldehyde was dissolved in methanol (150 ml) and stirred
with NaCN (1.78 g, 36.2 mmol), AcOH (0.86 ml, 15.1 mmol) and MnO2
(19.7 g, 226 mmol) at room temperature. After 1.5 h, the solution was
filtered through a pad of Celite1 and concentrated under vacuum. The
residue was brought up in saturated NaHCO3 (50 ml) and extracted
with ether (3 ꢀ 150 ml). The combined organic layers were dried
(MgSO4), filtered through a pad a silica gel and concentrated under
vacuum. To a solution of the ketal ester (3.76 g, 12.2 mmol) in THF
(100 ml) was added HCl (10%, 1 ml) and water (5 ml). After 15 min., the
acid was quenched with saturated. NaHCO3 (20 ml) and diluted with
ether (200 ml). The organic layer was washed with brine (20 ml), dried
(MgSO4) and concentrated under vacuum. Flash chromatography
(EtOAc-hexane, 1:3) gave methyl ester 10 (2.05 g, 51%). Mp:
136.5–137.58. IR lmax: 3366, 2954, 1715, 1669, 1619, 1438, 1380, 1238,
1
1162 cmꢁ1. H NMR: d 6.03 (s, 2 H), 5.73 (s, 1 H), 3.69 (s, 3 H), 1.97
(s, 6 H), 1.94 (s, 3 H). 13C NMR: 185.6, 166.4, 160.3, 149.1, 136.0 (m),
128.5 (m), 126.4, 118.3, 75.0, 51.2, 20.9, 18.1. HRMS: calcd for
C15H162H2O4 [M]+ m/z 264.1329, found 264.1324.
Methyl-(2Z,4E)-[5-2H]-5-(1-hydroxy-2,6-dimethyl-4-oxo-2,5-cyclohexa-
dien-1-yl)-3-methylpenta-2,4-dienoate (11).
Ketal alcohol 7 (13.6 g, 48.0 mmol) was transformed into methyl ester 11
(6.44 g, 51%) following the same procedure for methyl ester 10, with the
following change. After reduction of the alkyne the reaction was
quenched with H2O (10 ml). Mp: 136.6–136.78. IR lmax: 3358, 2953,
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DEUTERATED ABSCISIC ACID ANALOGS
279
1715, 1668, 1622, 1436, 1380, 1238, 1161 cmꢁ1. 1H NMR: d 8.03 (s, 1 H),
6.06 (s, 2 H), 5.74 (s, 1 H), 3.71 (s, 3 H), 1.97 (s, 6 H), 1.95 (s, 3 H). 13C
NMR: 185.7, 166.4, 160.5, 149.2, 136.0 (m), 128.6, 126.4, 118.3, 75.0,
51.2, 21.0, 18.1. HRMS: calcd for C15H172H2O4 [M]+ m/z 263.1267,
found 263.1266.
Methyl-(2Z,4E)-[4,5-2H2]-5-[(1R,6R)/(1S,6S)-1-hydroxy -2,6-dimethyl-
6-(methyl-2H3)-4-oxo-2-cyclohexen-1-yl]-3-methylpenta-2,4-dienoate (12).
To a stirred solution of methyl ester 10 (2.00 g, 7.58 mmol) in THF
2
(50 ml) at -788C was added dropwise C2H3MgI (99+ atom % H)
(37.9 ml, 1.0 M solution in diethyl ether) using a syringe pump (rate =
0.33mlꢁ1min). After 6 h, the temperature was raised to 08C and the
reaction was quenched with saturated NH4Cl (25 ml). The solution was
extracted with ether (3 ꢀ 150 ml) and the combined organic layers were
dried (MgSO4), filtered and concentrated under vacuum. Flash
chromatography (EtOAc–hexane 1:4) gave methyl 2H5-ABA 12
(1.06 g, 49%). IR lmax: 3478, 2955, 1711, 1660, 1435, 1377, 1238,
1160 cmꢁ1. 1H NMR: d 5.92 (q, J = 1 Hz, 1 H), 5.72 (q, J = 1 Hz, 1 H),
3.67 (s, 3 H), 2.44 (d, J= 17 Hz, 1 H), 2.26 (d, J = 17 Hz, 1 H), 1.98 (d, J
= 1 Hz, 3 H), 1.90 (d, J= 1 Hz, 3 H), 0.98 (s, 3 H). 13C NMR: d 197.8,
166.4, 162.4, 149.3, 135.9 (m), 127.9 (m), 127.0, 118.2, 79.6, 51.2, 49.7,
41.3, 24.2, 22.2 (m), 21.1, 18.9. GCMS (EI) m/z: 192 (65), 191 (52), 163
(39), 136 (71), 127 (100), 113 (39), 109 (26), 93 (35).
Methyl-(2Z,4E)-[4,5-2H2]-5-[(1RS)-1-hydroxy-2,6,6-trimethyl-4-oxo-
2-cyclohexen-1-yl]-3-methylpenta-2,4-dienoate (13).
2
Methyl ester 10 (2.01 g, 7.6 mmol) was transformed into methyl H2-
2
ABA 13 (1.07 g, 49%) following the same procedure for methyl H5-
ABA 12, with the following change. The Grignard reagent used was
CH3MgBr (12.7 ml, 3.0 M solution in diethyl ether). IR lmax: 3478,
1
2953, 1714, 1660, 1621, 1435, 1375, 1238, 1161 cmꢁ1. H NMR: d 5.92
(q, J = 1 Hz, 1 H), 5.72 (q, J = 1 Hz, 1 H), 3.67 (s, 3 H), 2.44 (d, J=
17 Hz, 1 H), 2.26 (d, J = 17 Hz, 1 H), 1.98 (d, J = 1 Hz, 3 H), 1.90 (d, J
= 1 Hz, 3 H), 1.08 (s, 3 H), 0.99 (s, 3 H). 13C NMR: d 197.7, 166.4,
162.4, 149.3, 135.9 (m), 127.7 (m), 127.0, 118.2, 79.6, 51.2, 49.7, 41.5,
24.2, 23.0, 21.1, 18.9. GC/MS (EI): m/z 192 (92), 191 (69), 163 (46), 136
(84), 127 (100), 113 (44), 109 (52), 93 (51).
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Methyl-(2Z,4E)-[5-2H]-5-[(1R,6R)/(1S,6S)-1-hydroxy–2,6-dimethyl-
6-(methyl-2H3)-4-oxo-2-cyclohexen-1-yl]-3-methylpenta-2,4-dienoate
(14).
2
Methyl ester 11 (5.17 g, 19.7 mmol) was transformed into methyl H4-
2
ABA 14 (2.85 g, 51%) following the same procedure for methyl H5-
ABA 12. IR lmax: 3471, 2957, 1714, 1660, 1625, 1436, 1377, 1240, 1161.
1H NMR: d 7.84 (s, 1 H), 5.92 (q, J = 1 Hz, 1 H), 5.73 (q, J = 1 Hz,
1 H), 3.68 (s, 3 H), 2.44 (d, J= 17 Hz, 1 H), 2.26 (d, J = 17 Hz, 1 H), 1.98
(d, J = 1 Hz, 3 H), 1.90 (d, J= 1 Hz, 3 H), 0.99 (s, 3 H). 13C NMR: d
197.7, 166.4, 162.4, 149.3, 135.9 (m), 128.0, 127.1, 118.2, 79.6, 51.2, 49.7,
41.3, 24.2, 21.2, 18.9 . GC/MS (EI) m/z: 191 (100), 163 (39), 135 (71),
126 (84), 112 (45), 92 (37).
Methyl-(2Z,4E)-[5-2H]-5-[(1RS)-1-hydroxy-2,6,6-trimethyl-4-oxo-2-
cyclohexen-1-yl]-3-methylpenta-2,4-dienoate (15).
2
Methyl ester 11 (98.0 mg, 0.37 mmol) was transformed into methyl H-
2
ABA 15 (56 mg, 54%) following the same procedure for methyl H5-
ABA 12, with the following change. The Grignard reagent used was
CH3MgBr (0.62 ml, 3.0 M solution in diethyl ether). IR lmax: 3477,
1
2951, 1714, 1655, 1625, 1438, 1376, 1239, 1161 cmꢁ1. H NMR: d 7.80
(s, 1 H), 5.90 (q, J = 1 Hz, 1 H), 5.71 (q, J = 1 Hz, 1 H), 3.65 (s, 3 H),
2.43 (d, J= 17 Hz, 1 H), 2.25 (d, J = 17 Hz, 1 H), 1.97 (d, J = 1 Hz,
3 H), 1.89 (d, J= 1 Hz, 3 H), 1.07 (s, 3 H), 0.98 (s, 3 H). 13C NMR: d
197.8, 166.4, 162.6, 149.4, 136.1 (m), 127.9, 127.0, 118.1, 79.6, 51.2, 49.7,
41.5, 24.3, 23.0, 21.2, 18.9. GCMS (EI) m/z: 191 (100), 163 (41), 135
(75), 126 (84), 112 (36), 92 (54).
(2Z,4E)-[4,5-2H2]-5-[(1R,6R)/(1S,6S)-1-hydroxy-2,6-dimethyl-6-
(methyl-2H3)-4-oxo-2-cyclohexen-1-yl]-3-methylpenta-2,4-dienoic acid (16).
Methyl ester 12 (1.06 g, 3.75 mmol) was dissolved in ethanol (15 ml) and
water (50 ml). A solution of KOH (300 mg) in water (1 ml) was added
and the solution was heated to 508C. Water (100 ml) was slowly added
over the course of the reaction so that the starting material would
remain in solution. After 3 h, the solution was cooled to room
temperature, acidified with 10% HCl, extracted into EtOAc
(3 ꢀ 100 ml), dried (MgSO4) and concentrated under vacuum. The
residue was brought up in EtOAc (150 ml) and washed with saturated
NaHCO3 (3 ꢀ 25 ml). The aqueous phase was acidified with 10% HCl,
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DEUTERATED ABSCISIC ACID ANALOGS
281
extracted into EtOAc (3 ꢀ 100 ml), dried (MgSO4) and concentrated
2
under vacuum to give H5-ABA 16 (0.91 g, 90%). IR lmax: 3600–2400,
3450, 2961, 1651, 1374, 1258, 1160 cmꢁ1. 1H NMR: d 5.95 (q, J = 1 Hz,
1 H), 5.75 (q, J = 1 Hz, 1 H), 2.47 (d, J = 17 Hz, 1 H), 2.27 (d, J =
17 Hz, 1 H), 2.03 (d, J = 1 Hz, 3H0, 1.91 (d, J = 1 Hz, 3H), 1.01 (s, 3H).
13C NMR: d 197.9, 170.3, 162.5, 151.5, 136.4 (m), 127.9 (m), 127.1,
117.8, 79.8, 49.6, 41.4, 24.2, 23.0 (m), 21.4, 19.0. LCMS/MS m/z: 268
(M-H)ꢁ (13), 224 (55), 209 (14), 206 (18), 205 (6), 157 (100), 156 (93),
155 (16), 114 (5).
Methyl esters 13, 14 and 15 were hydrolyzed following the procedure
2
for compound 16 to give H2-ABA 17, H4-ABA 18 and H-ABA 19,
2
2
respectively.
(2Z,4E)-[4,5-2H2]-5-[(1RS)-1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclo-
hexen-1-yl]-3-methylpenta-2,4-dienoic acid (17).
1
IR lmax: 3600–2400, 3450, 2968, 1658, 1375, 1255, 1161. H NMR
(C2H3O2H): d 5.91 (q, J = 1 Hz, 1H), 5.73 (q, J = 1 Hz, 1H), 2.52
(d, J = 17 Hz, 1 H), 2.18 (d, J = 17 Hz, 1H), 2.03 (d, J = 1 Hz, 3H),
1.92 (d, J = 1 Hz, 3H), 1.06 (s, 3H), 1.02 (s, 3H). 13C NMR (C2H3O2H):
d 201.0, 169.4, 166.5, 151.0, 137.5 (m), 129.0 (m), 127.6, 119.5,
80.5, 50.7, 42.8, 24.7, 23.5, 21.2, 19.6. LCMS/MS m/z: 265 (M-H)ꢁ
(10), 221 (56), 206 (23), 203 (11), 202 (6), 154 (100), 153 (91), 152 (16),
111 (6).
(2Z,4E)-[5-2H]-5-[(1R,6R)/(1S,6S)-1-hydroxy-2,6-dimethyl-6-
(methyl-2H3)-4-oxo-2-cyclohexen-1-yl]3-methylpenta-2,4-dienoic acid (18).
IR lmax: 3600–2400, 3444, 2962, 1660, 1377, 1257, 1162 cmꢁ1. 1H NMR:
d 7.79 (s, 1H), 5.95 (q, J = 1 Hz, 1H), 5.75 (q, J = 1 Hz, 1H), 2.47 (d, J
= 17 Hz, 1H), 2.27 (d, J = 1 Hz, 1H), 2.03 (d, J = 1 Hz, 3H), 1.91 (d, J
= 1 Hz, 3H), 1.01 (s, 3H). 13C NMR: d 198.1, 170.9, 162.7, 151.6, 136.5
(m), 128.2, 127.1, 117.9, 79.8, 49.6, 41.4, 24.2, 23.0 (m), 21.4, 19.0.
LCMS/MS m/z: 267 (M-H)ꢁ (6), 223 (30), 208 (8), 205 (12), 204 (2), 156
(100), 155 (10), 114 (3).
(2Z,4E)-[5-2H]-5-[(1RS)-1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclo-
hexen-1-yl]-3-methylpenta-2,4-dienoic acid (19).
1
IR lmax: 3600–2400, 3450, 2967, 1655, 1255, 1160 cmꢁ1. H NMR
(C2H3O2H): d 7.76 (s, 1H), 5.91 (q, J = 1 Hz, 1H), 5.73 (q, J = 1 Hz,
1H), 2.52 (d, J = 17 Hz, 1H), 2.18 (d, J = 1 Hz, 1H), 2.03 (d, J = 1 Hz,
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S. R. ABRAMS ET AL.
3H), 1.92 (d, J = 1 Hz, 3H), 1.05 (s, 3H), 1.02 (s, 3H). 13C NMR
(C2H3O2H): d 201.0, 169.4, 166.5, 151.1, 137.6 (m), 129.3, 127.6, 119.5,
80.5, 50.6, 42.8, 24.7, 23.5, 21.3, 19.6. LCMS/MS m/z: 264 (M-H)ꢁ (5),
220 (34), 205 (15), 202 (9), 153 (100), 152 (10), 111 (3).
Conclusions
The synthetic scheme yielded ABA molecules with deuterium incor-
poration of high regiochemical and isotopic purity. The isotopic labels
at C-4, -5 and -80 are stable under acid or base conditions. The
procedure would also be amenable for synthesis of tritiated ABA
analogs. The labeled analogs are useful standards for mass spectrometry
analysis of ABA using LCMS/MS but not for GC/MS. In GC/MS, the
ion used for quantitation for unlabeled methyl ABA has m/z 190, which
arises from loss of methanol and 2-butene from the molecular ion. The
loss of methanol arises from the methoxyl group of the ester and a
proton from either the hydroxyl group or C-4.16,17 For the deuterated
analogs with one deuterium in the side-chain at C-5 the base peak has
m/z 191 and for those with two deuterium atoms in the sidechain the
base peak has m/z 192 with m/z 191 about 80% of the intensity of the
base peak. The compounds 16 – 19 prepared in this work can be used
for negative ion mode electrospray LC/MS/MS as the base peak
corresponds to the molecular ion. The complete series of isotopically
labeled ABA analogs have been employed in studies on the fragmenta-
tion of ABA by electrospray mass spectrometry to be reported
elsewhere. The (+)-enantiomer of 17 is a particularly useful molecule
for tracking ABA catabolism as it is labeled at carbons not susceptible
to oxidation by plant enzymes. As the molecular weight of the ABA
analog 17 is 2 Da higher than the natural substance it and the analogous
catabolites can clearly be distinguished from endogenous ABA and
metabolites by mass spectrometry. The ABA analogs 16 and 18 have
utility as precursors through biotransformation by plant cells13 to
labeled 70-hydroxyABA, a mass spectrometry standard for the ABA
catabolite.
Acknowledgements
The authors thank Brock Chatson and Andrew Ross for their
contributions to the spectroscopy.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 273–283

DEUTERATED ABSCISIC ACID ANALOGS
283
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