Syntheses of deuterated jasmonates for mass spectrometry and metabolism studies

Article abstract of DOI:10.1002/jlcr.994

Jasmonic acid and its metabolites play an essential role in the regulation of plant development and systemic defense responses. Isotopically labeled standards are required to quantify plant hormones for metabolism studies using mass spectrometry. A conven

Full text of DOI:10.1002/jlcr.994

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 797–809.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.994
Research Article
Syntheses of deuterated jasmonates for mass
spectrometry and metabolism studies
Patrycja W. Galka, Stephen J. Ambrose, Andrew R. S. Ross and
Suzanne R. Abrams*
Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium
Place, Saskatoon, Sask., Canada S7N 0W9
Summary
Jasmonic acid and its metabolites play an essential role in the regulation of plant
development and systemic defense responses. Isotopically labeled standards are
required to quantify plant hormones for metabolism studies using mass spectrometry.
A convenient method for the preparation of deuterated analogs of jasmonates is
demonstrated. Modification of commercially available methyl jasmonate by base-
catalyzed proton/deuterium exchange or Wittig reaction introduces either two or three
heavy atoms into a molecule. Copyright # 2005 John Wiley & Sons, Ltd.
2
Key Words: jasmonates; 12-OH-jasmonates; H-labeled standards; a-proton
exchange; Wittig reaction
Introduction
(ꢀ)ꢀMethyl jasmonate [(ꢀ)ꢀMJA] 1 and (ꢀ)ꢀjasmonic acid [(ꢀ)ꢀJA] 2 are
phytohormones widespread in the plant kingdom. Biosynthesis of jasmonates
has been well studied.1 The biosynthetic pathway starts from a-linolenic acid
and yields (+)ꢀ7-iso-JA (3) as the final product. Because of greater steric
hindrance of the cis-isomer, (+)ꢀ7-iso-JA epimerizes to a more stable trans
configuration via the keto-enol tautomerization (Figure 1) during the isolation
process or even within the plant. Consequently, JA is analyzed as the more
stable epimer.
These natural compounds are known to be involved in many physiological
and biochemical processes in plants, in particular in the response to
biotic stress, promotion of senescence, inhibition of growth and chemical
defense.2–4 The volatility of MJA led to the suggestion that it is also
*Correspondence to: Suzanne R. Abrams, Plant Biotechnology Institute, National Research Council of
Canada, 110 Gymnasium Place, Saskatoon, Sask., Canada S7N 0W9. E-mail: sue.abrams@nrc.ca
Copyright # 2005 John Wiley & Sons, Ltd.
Accepted 4 June 2005

798
P. W. GALKA ET AL.
O
6
O
11
12
7
2
5
8
10
9
4
3
COOR
COOR
3 R =H
1
1 R = CH3
2 R =H
Figure 1. JA tautomerization
involved in plant to plant communication.5 Whereas jasmonate biosynthetic
pathway has been extensively studied, metabolism downstream of JA is
less understood. A number of JA metabolites have been isolated such
as cyclopentenone derivatives and amino acid conjugates.4 Recent reports
show that 11-hydroxy-JA and 12-hydroxy-JA are the predominant hydro-
xylated compounds observed in studies in which JA was fed to plant cell
culture.6
Quantification of jasmonates in plant materials is an essential step
for elucidation of their transport, distribution and metabolism. Several
methods have been used to determine jasmonate levels in plant extracts.
Earlier, bioassays7 based on JA’s biological properties and radioimmuno-
assays8 were used. An HPLC method was developed based on the
derivatization of JA with fluorescent hydrazide giving a stable fluorescent
product.9 The application of these methods, however, is limited because they
lack specificity, sensitivity and ability to estimate recovery. More recent
improved methods for jasmonates quantification involve the use of stable,
isotope-labeled analogs as internal standards and analysis by GC-MS10,11 or
HPLC-MS.12
For jasmonates several methods of labeling have been developed so far.13–18
For example Seto et al. incorporated deuterium atoms by catalytic semi-
deuteriogenation of a 9,10-acetylene analog.14 Ozonolysis of jasmonate and
reconstruction of the pentenyl side chain by a Wittig reaction with
[2H5]propylidenetriphenylphosphorane has been used to prepare [10-2H,
11-2H2, 12-2H3]jasmonate.13 Others15–17 have prepared 13C- and 14C-labeled
analogs.
In order to expand the plant hormone profiling method established earlier in
our laboratory19 to JA and its metabolites, we needed to develop simple and
versatile synthesis of jasmonates, to be employed as internal and external
standards. In this paper we present the synthesis of new deuterated racemic
compounds (Figure 2) labeled at the C-2 and C-12 positions: MJA (4, 6), JA
(5, 7) and 12-OH-JA derivatives (8, 9) designed as internal standards for GC-
MS and HPLC-coupled to electrospray ionization tandem mass spectrometry
(ESI-MS/MS).
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SYNTHESES OF DEUTERATED JASMONATES
799
2H
OH
O
O
O
2H
2H
COOR
2H
COOR
COOR
2H
2H
2H
6 R = CH3
7 R = H
4 R = CH3
5 R = H
8 R = CH3
9 R = H
Figure 2. Deuterated jusmonates
Results and discussion
Two different approaches were applied to synthesize deuterium-labeled
jasmonates with high isotopic incorporation: (a) base-catalyzed hydrogen/
deuterium exchange in position a to the ester and (b) jasmonate ozonolysis and
reconstruction by a Wittig reaction. Commercially available racemic MJA was
used as a convenient starting material.
Synthesis of [2,2-2H2]-MJA and [2,2-2H2]-JA
When a NaOMe-CH3O2H solution of ketal 10 was refluxed for 5 days the
a-hydrogen atoms were replaced by deuterium. Higher isotopic purity was
obtained simply by repeating the reaction with a fresh portion of CH3O2H.
Subsequent ketal deprotection led to [2,2-2H2]-MJA (4). Deuterium incorpora-
tion was examined by LC-MS analysis using positive ion ESI, which showed
no response for the protonated molecular ion [M+H]+ of unlabeled MJA
2
ðm=z ¼ 225Þ. However, the intensity of the corresponding ion for MJA H1
ðm=z ¼ 226Þ was found to be 4% of that for the 2H2-analog ðm=z ¼ 227Þ. Thus
the calculated value for 2H2-incorporation in 4 was found to be 96%. The 1H
NMR spectrum of 4 was identical to that of unlabeled compound 1 except for
the absence of the signal at 2.67 ppm, which was assigned to the C-2 proton. In
order to obtain [2,2-2H2]-JA (5), the methyl ester (4) was hydrolyzed using
porcine liver esterase20 under slightly basic conditions (Scheme 1). The level of
O
O
O
O
O
O
a
b
c, d
COOCH3
COOR
COOCH3
COOCH3
2H
2H
2H
11
2H
4 R = CH3
5 R = H
( ) 1
10
Scheme 1. Reagents and conditions: (a) 2,2-dimethyl-1,3-propanediol, pyridi-
nium p-tosylate, C6H6; (b) NaOCH3, CH3O2H; (c) 10% aq. HCl; (d) porcine
liver esterase, phosphate buffer, pH 7.5, THF
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P. W. GALKA ET AL.
2H2 incorporation at C-2 was determined to be 94%. Isotopically labeled and
non-labeled JA analogs were examined for the corresponding fragmentation
pathways using negative ion ESI-MS/MS. In multiple reaction monitoring
(MRM) mode, each ionized compound gives a distinct precursor-to-product
ion transition that is diagnostic for the presence of that particular compound.
For [2,2-2H2]-JA (5) the most intense transition observed is from m=z ¼ 211 to
61 ð211 > 61Þ, whereas for JA (2) it is 209 > 59, due to the loss of a 2-
pentenylcyclopentenone fragment through a McLafferty rearrangement. This
shows that monitoring for specific precursor-to-product ion transitions,
utilizing isotopically labeled internal standards, can provide selective
quantification of jasmonates. This is especially advantageous when quantify-
ing low levels of target compounds in the presence of a high level of
background from sample matrix or co-extractives.
Synthesis of [12,12,12-2H3]-MJA and [12,12,12-2H3]-JA
The tri-deuterated jasmonate analogs (6, 7) were prepared by a method similar
to the procedure of Miersh (Scheme 2).13
Ozonolysis of a protected MJA 10 gave a convenient precursor 13 for the
Wittig reaction. The Wittig reagent (ylide) required for the reaction was
prepared by deprotonation of the triphenyl-[1,1,1-2H3]-propyl phosphonium
bromide generated by the quaternization of the phosphine with organic halide.
Thus, Z-selective Wittig reaction of 12 followed by the deprotection step led to
MJA in its isotopically labeled form (6). GC-MS analysis of 6 indicated the
presence of 7% of the E-isomer. [12,12,12-2H3]-MJA saponification with 1 M
KOH gave [12,12,12-2H3]-JA (7). Both compounds 6 and 7 were examined for
2H3-incorporation by LC-MS and contained, respectively, 98% of
12,12,12-2H3-MJA and 97% of [12,12,12-2H3]-JA. Positive ion ESI-MS of 6
generated a strong molecular ion [M+H]+ ðm=z ¼ 228Þ. Collision-induced
2H
2H
2H
2H
O
O
2H
2H
d,e
O
O
O
O
a,b
O
c
H
COOCH3
COOCH3
COOR
H3COOC
14
6 R = CH3
7 R = H
12
( ) 1
Scheme 2. Reagents and conditions: (a) 2,2-dimethyl-1,3-propanediol, pyridi-
nium p-tosylate, C6H6; (b) O3, CH3OH, ꢀ788C; (c) Brꢀ +Ph3PCH2CH2C2H3
(13), KN[Si(CH3)3]2, THF; (d) 10% aq. HCl, acetone; (e) 1 M aq. KOH,
CH3OH, 458C
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SYNTHESES OF DEUTERATED JASMONATES
801
dissociation (CID) of this ion generated a fragment of m=z ¼ 154 due to the
loss of methyl acetate, whereas 225 > 151 was the predominant transition for
the unlabeled MJA. These transitions could be used for MRM analysis.
Similarly, MS analysis of [12,12,12-2H3]-JA and JA in the positive ion mode
showed major transitions for each compound (214 > 154 and 211 > 151,
respectively).
Synthesis of 12-OH-[2,2-2H2]-MJA and 12-OH-[2,2-2H2]-JA
Following our protocols, and applying a combination of both synthetic
pathways, deuterated hydroxy-metabolites of JA 8, 9 were synthesized for the
first time (Scheme 3). As expected, compound 12 readily underwent Wittig
reaction with the (3-tert-butyldimethylsilyloxy-propyl)(triphenyl)phosphonium
bromide in the presence of a base, led to the pentenyl side chain with a TBDMS
(tert-butyldimethylsilyl) protected 12-OH-group. Subsequent base-catalyzed
a-proton exchange introduced two deuterium atoms into the molecule.
The attempts to obtain detailed analytical data for intermediate 17 failed
because this compound is not stable in solution. Under these conditions, the
ketone group is partially deprotected. Nevertheless, it can be stored neat at
–208C without decomposition. Mild acid hydrolysis of 17 readily removed
ketone- and hydroxy-protecting groups, giving the desired 12-OH-[2,2-2H2]-
MJA (8). GC-MS analysis of 8 showed that the ions at m=z ¼ 260, 243 and
225 contained two deuterium atoms, which were, respectively, assigned to
[M+NH4]+, [M+H]+, and [MꢀH2O]+. These results were consistent with
the 1H NMR data. Hydrolysis of hydroxylated MJA derivative 8 with porcine
liver esterase at pH 7.5 produced 12-OH-[2,2-2H2]-JA (9). The isotopic purity
was established by LC-MS. The incorporation of 2H2 was found to be 95% for
12-OH-[2,2-2H2]-MJA (8) and 96% for 12-OH-[2,2-2H2]-JA (9). Further
OH
OTBDMS
d
OTBDMS
e, f
O
O
O
O
O
O
a,b,c
COOCH3
COOR
COOCH3
COOCH3
2H
2H
2H
2H
17
( ) 1
8 R = CH3
9 R = H
16
Scheme 3. Reagents and conditions: (a) 2,2-dimethyl-1,3-propanediol,
pyridinium p-tosylate, C6H6; (b) O3, CH3OH, ꢀ788C; (c) Brꢀ
+Ph3PCH2CH2CH2OTBDMS (15), KN[Si(CH3)3]2, THF; (d) NaOCH3,
CH3O2H; (e) 10% aq. HCl; (f) porcine liver esterase, phosphate buffer, pH
7.5, THF
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P. W. GALKA ET AL.
analysis of these compounds by tandem mass spectrometry provided the
information about their fragmentation patterns. A molecular ion of 12-OH-
[2,2-2H2]-MJA (8) ðm=z ¼ 243Þ was examined using positive ion electrospray
ionization, and peaks at m=z ¼ 225, 193 and 165 were assigned to the fragments
generated after loss of water, methanol and carbon monoxide. A similar pattern
was observed for 12-OH-MJA (m=z ¼ 223, 191, 163). Thus the transitions
243 > 225, 243 > 193 or 243 > 165 could be used for MRM analysis of 8.
Negative ion ESI-MS of 12-OH-[2,2-2H2]-JA showed a strong molecular ion at
m=z ¼ 227 for which minimal fragmentation was observed under the
experimental conditions. The product ion MS/MS spectrum showed fragments
at m=z ¼ 147 and 61. The fragment ion at m=z ¼ 61 generated by CID of 9
corresponds to loss of a pentenylcyclopentenone fragment from the deproto-
nated molecular ion ðm=z ¼ 227Þ. Because the deuterium labeling is retained in
the observed fragment, this transition could also be used for MRM studies.
Experimental section
All chemicals and reagents were used as received from the suppliers unless
otherwise noted. Tetrahydrofuran was distilled from sodium metal/benzophe-
none. All other solvents were purchased from Aldrich in Sure-SealTM
containers. Melting points (m.p.) are uncorrected and were recorded on
Electrothermal Digital Melting Point Apparatus IA 9000 Series. IR spectra
were recorded using KBr pellets on Perkin-Elmer Paragon 1000. NMR spectra
were recorded on a Bruker AMX 500 MHz spectrometer. Chemical shifts d are
given in ppm relative to the solvent peak (chloroform) of 7.24 for 1H and 77.0
for 13C and H3PO4 for 31P. J values are given in Hertz (Hz). Merck silica gel 60
F254 plates (0.2 mm) with aluminum sheet backing were used in analytical
TLC. The spots were visualized by dipping in a solution of phosphomolybdic
acid and heating on a hot plate. Preparative TLC was performed on
UniplateTM Silica Gel (1000 mm) with UV254 indicator. Flash column
chromatography was performed using Merck silica gel 60 (230-400 mesh).
Low-resolution GC-MS spectra were obtained using a DB-5 MS column
(30 m) and a 6890N Network GC system (Agilent, Palo Alto, CA) equipped
with a 5973N mass selective detector, using either electron impact (EI) or
chemical ionization (CI) mode. High-resolution mass spectra were acquired on
a QSTAR hybrid quadrupole-time of flight mass spectrometer (ABI/Sciex,
Concord, Ont.). GC was performed on a HP 6890 Series system with FID
detector. LC-MS was performed on an Alliance 2695 HPLC (Waters, Milford,
MA) equipped with a Genesis C18 column (2:1 ꢁ 100 mm, 4 mm; Jones
Chromatography, Hengoed, UK) at various isocratic compositions with a flow
rate of 0.2 ml/min, coupled to a Quattro Ultima quadrupole tandem mass
spectrometer (Micromass, Manchester, UK) fitted with a Z-spray ESI source
(Micromass).
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SYNTHESES OF DEUTERATED JASMONATES
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Methyl {1-[(2Z)-pent-2-en-1-yl]-6,10-dioxa-8,8-dimethylspiro[4.5]dec-2-yl}
acetate (10)
A round-bottomed flask, equipped with a Dean-Stark trap was charged under
argon with MJA 1 (10.00 g, 44.6 mmol), neopentyl glycol (9.29 g, 89.0 mmol,
2 eq.), pyridinium p-tosylate (0.17 g, 0.7 mmol, 1.5% mol) and 300 ml of dry
benzene. The reaction was completed after 18 h of reflux. After the solution
was cooled to room temperature the organic layer was washed with 100 ml of
water, dried over anhydrous MgSO4 and filtered. The evaporation of the
solvent under reduced pressure gave the crude product which was further
purified by flash column chromatography (20% EtOAc in hexanes). Yield
13.26 g (96%) of colorless oil. IR (KBr) nmax 2955, 2905, 2851, 1740, 1450,
1
1435, 1394, 1135, 1016, 908, 871 cm –1; H NMR (500 MHz, d, CDCl3): 5.43
(1H, m, CH), 5.37 (1H, m, CH), 3.64 (3H, s, OCH3), 3.52 (2H, m, OCH2), 3.39
(2H, m, OCH2), 2.61 (1H, dd J1 ¼ 4:0, J2 ¼ 14:8 Hz, CH2), 2.36 (1H, m, CH2),
2.18–1.84 (8H, m), 1.61 (1H, m, CH), 1.29 (1H, m, CH2), 1.14 (3H, s, CH3),
0.96 (3H, t J ¼ 7:5 Hz, CH3), 0.76 (3H, s, CH3); 13C NMR (d, CDCl3): 172.5
(C=O), 132.0 (CH), 128.3 (CH), 108.2 (C), 72.2, (OCH2), 71.1 (OCH2), 53.1
(CH), 51.3 (OCH3), 38.8 (CH2), 38.7 (CH), 34.5 (C), 29.2 (CH2), 28.0 (CH2),
25.4 (CH2), 22.8 (CH3), 22.2 (CH3), 20.6 (CH2), 14.2 (CH3); GCMS (EI) m/z
(relative intensity) 310 ([M]+, 5), 237 (10), 195 (26), 141 (100), 128 (31), 109
(15), 69 (47), 55 (34), 41 (32).
Methyl {1-[(2Z)-pent-2-en-1-yl]-6,10-dioxa-8,8-dimethylspiro[4.5]dec-2-yl}
acetic acid-2H2 (11)
A solution of 10 (1.00 g, 3.2 mmol) in CH3O2H (10 ml) was added dropwise to
a solution of CH3ONa (0.43 g, 8.0 mmol) in 2 ml of CH3O2H. The mixture was
refluxed for 5 days at 658C. NH4Cl was added, the pH was adjusted to 7 with
10% aqueous HCl and the organic solvent was evaporated. The residue was
taken up in water (5 ml) and after the product was extracted with Et2O the
organic layer was dried over anhydrous MgSO4 and concentrated in vacuo
after filtration. The product was purified by flash column chromatography
(15% EtOAc in hexanes). Yield 0.82 g (82%). IR (KBr) nmax 2940, 2870, 1736,
1
1462, 1434, 1257, 1136, 1064, 1002, 908; H NMR (d, CDCl3): 5.43 (1H, m,
CH), 5.37 (1H, m, CH), 3.64 (3H, s, OCH3), 3.54 (2H, m, OCH2), 3.39 (2H, m,
OCH2), 2.37 (1H, m, CH2), 2.16–1.84 (7H, m), 1.61 (1H, m, CH2), 1.30 (1H,
m, CH2), 1.14 (3H, s, CH3), 0.95 (3H, t J ¼ 7:5 Hz, CH3), 0.76 (3H, s, CH3);
13C NMR (d, CDCl3): 173.4 (C=O), 132.1 (CH), 128.3 (CH), 108.2 (C), 72.3
(OCH2), 71.1 (OCH2), 53.1 (CH), 51.4 (OCH3), 38.6 (CH), 30.1 (C), 29.3
(CH2), 28.0 (CH2), 25.8 (CH2), 22.8 (CH3), 20.8 (CH3), 20.6 (CH2), 14.1
(CH3); GCMS (EI) m/z (relative intensity) 312 ([M]+, 21), 283 (10), 237 (36),
195 (66), 151 (20), 141 (100), 128 (36), 109 (17), 69 (53), 55 (25).
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P. W. GALKA ET AL.
Methyl {3-oxo-2-[(2Z)-pent-2-en-1-yl]cyclopentyl}acetate-2H2 (4)
The crude compound 11 (0.40 g, 1.3 mmol) was dissolved in acetone followed
by the addition of aqueous 10% HCl (1 ml). The reaction mixture was stirred
at room temperature for 0.5 h and then neutralized with 10% aqueous NaOH.
The organic product was extracted with Et2O and the etheral layer was dried
over anhydrous MgSO4, filtered and concentrated under reduced pressure.
The product was purified by flash column chromatography (20% Et2O in
hexanes). Yield 0.19 g (65%). IR (KBr) nmax 3024, 2937, 2913, 1735, 1450,
1259, 1156, 1067, 991, 820; 1H NMR (d, CDCl3): 5.45 (1H, m, CH), 5.25 (1H,
m, CH), 3.69 (3H, s, OCH3), 2.39–2.02 (8H, m), 1.89 (1H, m, CH2), 1.50 (1H,
m, CH2), 0.96 (3H, t J ¼ 7:5 Hz, CH3); 13C NMR (d, CDCl3): 219.0 (C=O),
172.9 (C=O), 134.1 (CH), 124.9 (CH), 53.9 (CH), 51.6 (OCH3), 37.9 (CH),
37.7 (CH2), 27.1 (CH2), 25.5 (CH2), 20.6 (CH2), 14.1 (CH3); GCMS (EI) m/z
(relative intensity) 226 ([M]+, 77), 195 (29), 151 (81), 133 (33), 95 (47), 83
(100), 67 (24), 55 (25).
{3-Oxo-2-[(2Z)-pent-2-en-1-yl]cyclopentyl}acetic acid-2H2 (5)
[2,2-2H2]-MJA 4 (90 mg, 0.39 mmol) was dissolved in THF (1 ml) and
KH2PO4/K2HPO4 buffer (pH 7.5) was added (1 ml) followed by the addition
of a solution of porcine liver esterase in 3 ml of the phosphate buffer. The
reaction mixture was stirred for 2 h at room temperature, acidified to pH 5
with 10% aqueous HCl solution and extracted with EtOAc. After the organic
layer was dried over anhydrous MgSO4, filtered and evaporated the product
was purified by preparative TLC plate (30% EtOAc in hexanes). Yield 67 mg
(80%) of colorless oil. IR (KBr) nmax 3250 (br), 3028, 2908, 1734, 1541, 1406,
1295, 1156, 1070, 1028, 827; 1H NMR (d, CDCl3): 5.46 (1H, m, CH), 5.25 (1H,
m, CH), 2.45–2.25 (5H, m), 2.16–2.03 (3H, m), 1.92 (1H, m, CH2), 1.53 (1H,
m, CH2), 0.96 (3H, t J ¼ 7:5 Hz, CH3); 13C NMR (d, CDCl3): 218.8 (C=O),
177.7 (C=O), 134.2 (CH), 124.8 (CH), 53.8 (CH), 37.7 (CH), 37.6 (CH2), 27.1
(CH2), 25.5 (CH2), 20.6 (CH2), 14.1 (CH3); GCMS (EI) m/z (relative intensity)
212 ([M]+, 51), 151 (56), 144 (44), 133 (20), 109 (31), 95 (37), 83 (100), 67 (19),
55 (21).
Methyl [1-(2-oxoethyl)-6,10-dioxa-8,8-dimethylspiro[4.5]dec-2-yl]acetate (12)
A solution of 10 (7.00 g, 22.5 mmol) in methanol (250 ml) was cooled to –788C.
Ozonized oxygen was passed through for 2 h. Dimethyl sulfide (2.25 ml,
30.6 mmol, 1.36 eq.) was then added dropwise to quench the resulting ozonide.
The reaction mixture was brought to room temperature and stirred for
another 1.5 h. After the solvent was removed the product was purified by flash
column chromatography (20% EtOAc in hexanes). Yield 4.51 g (71%) of
colorless oil that solidifies when stored in the fridge. IR (KBr) nmax 2938, 2847,
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SYNTHESES OF DEUTERATED JASMONATES
1
805
2723, 2019, 1734, 1436, 1137, 1018 cmꢀ1; H NMR (d, CDCl3): 9.81 (1H, s,
CHO), 3.65 (3H, s, OCH3), 3.52 (2H, m, OCH2), 3.37 (2H, m, OCH2), 2.71
(1H, m, CH2), 2.51 (1H, m, CH2), 2.37 (1H, m, CH2), 2.26–1.92 (6H, m), 1.39
(1H, CH2), 1.11 (3H, s, CH3), 0.72 (3H, s, CH3); 13C NMR (d, CDCl3): 218.9
(C=O), 172.8 (C=O), 109.2 (C), 72.4 (OCH2), 70.8 (OCH2), 51.6 (CH), 50.0
(OCH3), 38.7 (CH), 38.4 (CH2), 37.3 (CH2), 34.2 (C), 33.1 (CH2), 27.4 (CH2),
23.1 (CH3), 21.9 (CH3); GCMS (EI) m/z (relative intensity) 284 ([M]+, 4), 283
(20), 211 (28), 181 (30), 167 (54), 129 (98), 128 (100), 115 (98), 97 (39), 83 (30),
69 (99), 55 (47).
Triphenyl(3,3,3-2H3-propyl)phosphonium bromide (13)
A pressure tube with threaded Teflon cap (Ace Glass) was charged with
[3,3,3-2H3]-bromopropane (1.00 g, 7.9 mmol) and triphenylphosphine (1.04 g,
4.0 mmol).
The threaded cap on a pressure tube was finger tight and the tube was
placed in an oil bath (1108C). After the reaction mixture was stirred for 48 h
the white precipitate appeared. The tube was cooled to ambient temperature
and the phosphonium salt was washed with Et2O and dried under high
vacuum. Yield 1.32 g (85%) of white solid. IR (KBr) nmax 3043, 2850, 2670,
2467, 2212, 1914, 1829, 1586, 1490, 1439, 1343, 1112, 1022, 943, 822; 1H NMR
(d, CDCl3): 7.72 (9H, m, CH), 7.63 (6H, m, CH), 3.61 (2H, m, CH2), 1.61 (2H,
m, CH2); 13C NMR (d, CDCl3): 134.9 (C), 133.41 (CH), 130.7 (CH), 117.9
(d, CH2), 24.2 (d, CH2), 16.0 (C2H3); 31P{1H} NMR (d, CDCl3): 24.6 (s);
HRMS calculated for C21H219H3P [M]+ m=z 308.1647 found 308.1647; m.p.
238.5–238.88C.
Methyl {1-[(2Z)-pent-2-en-5,5,5-2H3-1-yl]-6,10-dioxa-8,8-dimethylspiro[4.5]-
dec-2-yl}acetate (14)
A phosphonium salt 13 (0.90 g, 2.3 mmol) was suspended in freshly distilled
THF (150 ml). 0.5 M solution of potassium bis(trimethylsilyl)-amide in toluene
(4.6 ml, 2.3 mmol) was added dropwise at room temperature. The reaction
mixture was stirred for 1 h and then was cooled to –788C. After a solution of
aldehyde 12 (0.44 g, 1.5 mmol) in THF was added, the mixture was warmed up
to –408C and the stirring was continued for another 1.5 h. Next the reaction
was brought to room temperature, water was added and the organic material
was extracted with Et2O. Combined organic extracts were washed with brine,
dried over anhydrous MgSO4 and concentrated in vacuo after filtration.
Purification of the crude material by flash column chromatography (10%
1
EtOAc in hexanes) gave 0.28 g (61%) of colorless oil. H NMR (d, CDCl3):
5.43 (1H, m, CH), 5.36 (1H, m, CH), 3.64 (3H, s, OCH3), 3.53 (2H, m, OCH2),
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P. W. GALKA ET AL.
3.39 (2H, m, CH2), 2.61 (1H, m, CH2), 2.37 (1H, m, CH2), 2.18–1.84 (8H, m),
1.61 (1H, m, CH2), 1.29 (1H, m, CH2), 1.14 (3H, s, CH3), 0.76 (3H, s, CH3).
Methyl {3-oxo-2-[(2Z)-pent-2-en-5,5,5-2H3-1-yl]cyclopentyl}acetate (6)
To a solution of 14 (0.28 g, 0.9 mmol) in acetone (25 ml), aqueous 10% HCl
(1 ml) was added at room temperature. After the reaction mixture was stirred
for 0.5 h the solution was neutralized with aqueous 10% NaOH. The product
was extracted with Et2O and the organic layer was dried over anhydrous
MgSO4, filtered and concentrated under reduced pressure. Yield 120 mg
(60%). IR (KBr) nmax 3028, 2936, 2909, 2217, 2076, 1738, 1437, 1335, 1163,
1
986 cmꢀ1; H NMR (d, CDCl3): 5.44 (1H, m, CH), 5.25 (1H, m, CH), 3.69
(3H, s, OCH3), 2.70 (1H, m, CH2), 2.44–1.83 (10H, m), 1.48 (1H, m, CH2); 13
C
NMR (d, CDCl3): 219.0 (C=O), 172.5 (C=O), 134.1 (CH), 125.0 (CH), 54.0
(CH), 51.6 (OCH3), 38.8 (CH2), 38.0 (CH), 37.7 (CH2), 27.2 (CH2), 25.4
(CH2), 20.33 (CH2); GCMS (EI) m/z (relative intensity) 227 ([M]+, 74), 196
(27), 154 (81), 135 (39), 109 (25), 95 (44), 83 (100), 67 (22), 55 (19).
{3-Oxo-2-[(2Z)-pent-2-en-5,5,5-2H3-1-yl]cyclopentyl}acetic acid (7)
[12,12,12-2H3]-MJA (6) (40 mg, 0.18 mmol) was dissolved in CH3OH (5 ml)
and the solution was adjusted to pH 9 with aqueous 1 M KOH. The reaction
mixture was warmed up to 458C and was stirred for 2 h. Then it was acidified
with 10% aqueous HCl to pH 3 at 08C and the organic product was extracted
with EtOAc. Combined organic extracts were dried over anhydrous MgSO4,
filtered and concentrated under vacuum. [12,12,12-2H3]-JA was purified by
preparative TLC plate (AcOH:Et2O:hexanes 1:39:60). Yield 31 mg (84%). IR
(KBr) nmax 3250 (br), 3028, 2935, 2218, 2077, 1735, 1406, 1166, 1057; 1H NMR
(d, CDCl3): 5.46 (1H, m, CH), 5.25 (1H, m, CH), 2.77 (1H, m, CH2), 2.43–1.87
(10H, m), 1.52 (1H, m, CH2); 13C NMR (d, CDCl3): 218.8 (C=O), 177.2
(C=O), 134.2 (CH), 124.8 (CH), 53.8 (CH), 38.5 (CH2), 37.7 (CH), 37.3
(CH2), 27.2 (CH2), 25.4 (CH2), 20.3 (CH2); GCMS (EI) m/z (relative intensity)
213 ([M]+, 15), 154 (30), 142 (28), 95 (27), 83 (100), 67 (14), 55 (17).
[(3-Tert-butyldimethyl)silyloxy-propyl](triphenyl)phosphonium bromide (15)
To an ice-water bath cooled solution of 3-bromopropanol (2.00 g, 14.4 mmol),
tert-butyl-dimethylsilylchloride (2.38 g, 15.8 mmol) and imidazole (1.18 g,
17.2 mmol) were added. After the ice bath was removed, the mixture was
stirred at room temperature overnight. Next water was added and the product
was extracted with three portions of Et2O. The combined organic extracts
were dried over anhydrous MgSO4, filtered and concentrated in vacuo. Yield
3.60 g (99%). Without further purification the product was used in the next
step. 1H NMR (d, CDCl3): 3.71 (2H, t J ¼ 5:7 Hz, CH2), 3.49 (2H,
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SYNTHESES OF DEUTERATED JASMONATES
807
t J ¼ 6:5 Hz, CH2), 2.01 (2H, m, CH2), 0.87 (9H, s, CH3), 0.04 (6H, s, CH2). A
pressure tube with threaded Teflon cap (Ace Glass) was charged with
1-bromo-3-tert-butyl-dimethylsilyloxypropane (3.60 g, 14.2 mmol) and triphe-
nylphosphine (3.73 g, 14.2 mmol). The threaded cap on a pressure tube was
finger tight and the tube was placed in an oil bath (1208C). After the reaction
mixture was stirred for 48 h a white solid appeared. The mixture was then
cooled to ambient temperature and a glass-like solid was formed. The product
was purified by crystallization (CH2Cl2/Et2O). Yield 4.24 g (58%) of a white
solid. IR (KBr) nmax 3318, 2940, 2867, 2428, 2218, 1992, 1918, 1838, 1618,
1586, 1485, 1253, 1116, 940, 836; 1H NMR (d, CDCl3): 7.73 (9H, m, CH), 7.63
(6H, m, CH), 3.76 (2H, m, CH2), 3.69 (2H, m, CH2), 1.82 (2H, m, CH2), 0.77
(9H, s, CH3), ꢀ0.05 (6H, s, CH3); 13C NMR (d, CDCl3): 134.9 (C), 133.4
(CH), 130.3 (CH), 118.0 (d, CH2), 61.45 (OCH2), 25.71 (CH3), 18.8 (CH2),
17.9 (C), ꢀ5.5 (SiCH3); 31P{1H} NMR (d, CDCl3): 25.0 (s); HRMS calculated
for C27H36OPSi [M]+ m=z 435.2273 found 435.2267; m.p. 127.3–128.28C.
Methyl {2-[(2Z)-5-(tert-butyldimethyl)silyloxy-pent-2-en-1-yl]-6,10-dioxa-
8,8-dimethylspiro[4.5]dec-2-yl}acetate (16)
A phosphonium salt 15 (1.54 g, 3.0 mmol) was suspended in freshly distilled
THF (90 ml). 0.5 M solution of potassium bis(trimethylsilyl)-amide in toluene
(6.0 ml, 3.0 mmol) was added dropwise at room temperature. The reaction
mixture was stirred for 1 h and then was cooled to –788C. After a solution of
aldehyde 13 (0.56 g, 2.0 mmol) in THF was added, the mixture was warmed up
to –408C and the stirring was continued for another 1.5 h. Next the reaction
was brought to room temperature, water was added and the organic material
was extracted with Et2O. Combined organic extracts were washed with brine,
dried over anhydrous MgSO4 and concentrated in vacuo after filtration.
Purification of the crude material by flash column chromatography (10%
EtOAc in hexanes) gave 0.60 g (68%) of colorless oil. IR (KBr) nmax 3021,
2938, 2853, 1740, 1451, 1361, 1252, 1094, 1006, 927, 835; 1H NMR (d, CDCl3):
5.56 (1H, m, CH), 5.38 (1H, m, CH), 3.63 (3H, s, OCH3), 3.60 (2H, t
J ¼ 7:1 Hz, OCH2), 3.52 (2H, m, OCH2), 3.38 (2H, m, OCH2), 2.59 (1H, m,
CH2), 2.38 (1H, m, CH2), 2.30 (2H, m, CH2), 2.17–1.83 (6H, m), 1.61 (1H, m,
CH2), 1.29 (1H, m, CH2), 1.13 (3H, s, CH3), 0.88 (9H, s, CH3), 0.75 (3H, s,
CH3), 0.04 (6H, s, CH3); 13C NMR (d, CDCl3): 173.3 (C=O), 130.9 (CH),
126.1 (CH), 108.1 (C), 72.3 (OCH2), 71.1 (OCH2), 62.9 (OCH2), 53.1 (CH),
51.3 (OCH3), 39.9 (CH2), 38.7 (CH), 31.3 (CH2), 30.1 (C), 29.2 (CH2), 28.1
(CH2), 26.0 (CH2), 25.9 (CH3), 22.8 (CH3), 22.2 (CH3), 18.3 (C), ꢀ5.30
(SiCH3); GCMS (EI) m/z (relative intensity) 440 ([M]+, 21), 383 (97), 367 (30),
325 (59), 297 (63), 265 (25), 223 (86), 149 (30), 141 (100), 128 (44), 119 (22), 105
(20), 89 (59), 73 (66), 55 (24).
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808
P. W. GALKA ET AL.
Methyl {2-[(2Z)-5-hydroxypent-2-en-1-yl]-3-oxocyclopentyl}acetate-2H2 (8)
A solution of 17 (0.45 g, 1.0 mmol) in CH3O2H (5 ml) was added dropwise to a
solution of CH3ONa (0.13 g, 2.5 mmol) in 2 ml of CH3O2H. The mixture was
refluxed for 5 days at 658C. NH4Cl was added, the pH was adjusted to 7 with
1% aqueous HCl and the organic solvent was evaporated. The residue was
taken up in water (5 ml) and after the product was extracted with Et2O the
organic layer was dried over anhydrous MgSO4 and concentrated in vacuo
after filtration. Without delay, the crude product was dissolved in acetone
(25 ml) and 1 ml of aqueous 10% HCl was added at room temperature. After
the reaction mixture was stirred for 0.5 h the solution was brought to pH 5–6
with aqueous 10% NaOH. The products were extracted with Et2O then the
organic layer was dried over anhydrous MgSO4, filtered and concentrated
under reduced pressure. Purification by preparative TLC plate (EtOAc:hex-
anes 1:1) gave pure 7. Yield 10 mg. IR (KBr) nmax 3458, 3025, 2930, 2883, 1734,
1
1435, 1261, 1048, 798; H NMR (d, CDCl3): 5.47 (2H, m, CH), 3.70 (3H, s,
OCH3), 3.67 (2H, t J ¼ 6:2 Hz, OCH2), 2.46–2.07 (8H, m), 1.92 (1H, m, CH2),
1.50 (1H, m, CH2); 13C NMR (d, CDCl3): 219.1 (C=O), 174.5 (C=O), 128.6
(CH), 128.3 (CH), 62.1 (OCH2), 54.0 (CH), 51.7 (OCH3), 37.8 (CH2), 37.7
(CH), 30.9 (CH2), 27.2 (CH2), 25.4 (CH2); GCMS (CI) m/z (relative intensity)
260 ([M+NH4]+, 100), 258 (21), 243 (88), 242 (31), 228 (24), 225 (68), 223
(24), 176 (47).
{2-[(2Z)-5-hydroxypent-2-en-1-yl]-3-oxocyclopentyl}acetic acid-2H2 (9)
12-OH-2,2-2H2-MJA 8 (12 mg, 0.39 mmol) was dissolved in THF (0.2 ml) and
KH2PO4/K2HPO4 buffer (pH 7.5) was added (1.0 ml) followed by the addition
of a solution of esterase in 3.0 ml of the phosphate buffer. The reaction mixture
was stirred for 12 h at room temperature, acidified to pH 5 with 1% aqueous
HCl solution and extracted with EtOAc. After the organic layer was dried over
anhydrous MgSO4, filtered and evaporated the product was purified by
preparative TLC (EtOAc:hexanes 6: 4). Yield 4.7 mg (40%). IR (KBr) nmax
1
3300 (br), 3021, 1730, 1543, 1403, 1260, 1049, 867; H NMR (d, CDCl3): 5.76
(1H, brs, OH), 5.44 (2H, m, CH), 3.66 (2H, m, OCH2), 2.55–2.24 (7H, m), 2.12
(1H, m, CH2), 1.94 (1H, m, CH2), 1.52 (1H, m, CH2); 13C NMR (d, CDCl3):
219.1 (C=O), 176.6 (C=O), 128.7 (CH), 128.3 (CH), 62.1 (OCH2), 54.1 (CH),
37.8 (CH2), 37.6 (CH), 30.7 (CH2), 27.3 (CH), 25.4 (CH2); LCMS (negative
ion ESI) m/z 228 ([M]ꢀ, 19), 227 ([M–H]ꢀ, 100), 221 (48), 217 (12), 125 (19),
120 (16), 116 (11), 107 (10).
Conclusions
Deuterated jasmonates were efficiently prepared from commercially available
( ꢂ )-MJA in high isotopic purity. The analogs are two and three mass units
Copyright # 2005 John Wiley & Sons, Ltd.
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SYNTHESES OF DEUTERATED JASMONATES
809
higher than the naturally occurring compounds and are used as standards for
mass spectrometry analysis. Volatility of the methyl ester analogs (4, 6, 8)
makes them useful standards for GC-MS as well. Because the molecular
weight of the deuterated derivatives are different from the natural compounds
they can be clearly distinguished from the endogenous jasmonates by mass
spectrometry and used for quantification of jasmonates in plant material.
Acknowledgements
We would like to thank Brock Chatson for his contributions to the
spectroscopy.
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