Site-specific 2H-labeled oleic acid and derived esters for use as tracers of ethyl oleate metabolism in honey bees

Article abstract of DOI:10.1002/jlcr.1953

The inventory of labeled fatty acids and protocols for their syntheses are constantly increasing, but site-specific labeled precursors in biosynthetic studies are still needed. Ethyl oleate (EO) is an important primer pheromone in honeybees, which is responsible for the regulation of behavioral maturation. During our biosynthetic studies on EO, a site-specific labeled oleic acid precursor was required. In this report, a synthetic route adaptable to the preparation of [9,10,11,11-D₄] oleic acid and its derived esters for use as tracers of EO metabolism in honey bees is presented. Copyright

Full text of DOI:10.1002/jlcr.1953

Research Article
Received 24 June 2011,
Revised 13 September 2011,
Accepted 2 November 2011
Published online 8 December 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1953
2
Site-specific H-labeled oleic acid and derived
esters for use as tracers of ethyl oleate
metabolism in honey bees
Hao Chen and Erika Plettner*
The inventory of labeled fatty acids and protocols for their syntheses are constantly increasing, but site-specific labeled pre-
cursors in biosynthetic studies are still needed. Ethyl oleate (EO) is an important primer pheromone in honeybees, which is
responsible for the regulation of behavioral maturation. During our biosynthetic studies on EO, a site-specific labeled oleic
acid precursor was required. In this report, a synthetic route adaptable to the preparation of [9,10,11,11-D
derived esters for use as tracers of EO metabolism in honey bees is presented.
4
] oleic acid and its
Keywords: (Z)-9-octadecenoic acid; ethyl ester; N-acetyl cysteamide thioester; triacylglycerol
Introduction
exist in the molecule or not. Third, we also wished to potentially
monitor beta-oxidation and omega oxidation of the oleate and
derived fatty acids in the first a few oxidation cycles and, to
detect products, we needed to place the deuterium labels after
position 8 in oleate and before the end of the chain.
The cohesion and distribution of tasks in a honey bee (Api1s
mellifera L.) hive is controlled mainly by pheromone blends.
For example, the transition of worker bees from nursing to
foraging tasks is influenced by the presence of old workers: a large
number of foragers delay the transition of the younger workers,
whereas a scarce population of foragers causes the opposite effect.
An inhibitory primer pheromone that controls this process was
By the combination of the above three requirements, a
4
[9,10,11,11-D ] oleic acid precursor was highly desirable. In this
report, we present a synthetic route that was developed to pro-
vide us with the specifically labeled molecular probe and its
derived esters.
2
identified as ethyl oleate (EO). In that report, it has been
demonstrated that EO is biosynthesized de novo by foragers from
glucose. In order to identify and characterize the possible enzymes Experimental
involved the metabolism of EO, stable isotope labeled oleic
acid was required, as a substrate and also as a synthetic precursor
General
to the N-acetylcysteamide thioester, monoglycerides, and
triglycerides and of EO standards.
Commercial-grade solvents were distilled under nitrogen prior to
use, and reagents were used without further purification with
There are three reasons for the choice of site-specific deutera-
tion of oleic acid. First, a GC-MS method for the quantitative ana-
lysis of deuterium-labeled oleate in biological samples was
developed in our studies (unpublished results). In order to opti-
mally discriminate the tracer from naturally occurring fatty acids
present in bee samples, a minimum incorporation of four deuter-
ium atoms was required because of good isotope fractionation
from non-labeled endogenous material. Second, on the basis of
our genetic and bioconversion studies, a desaturase gene is
highly expressed in the esophagus of worker bees and is upregu-
lated upon exposure of honey bees to high doses of EO (unpub-
lished results), suggesting a possible metabolic pathway shown
in Scheme 1. In our hypothesis, the EO in honey bees could be
further desaturated to the ethyl ester of an unsaturated fatty acid
the following exceptions: triethylamine was distilled and stored
over NaOH. CH Cl was distilled over CaCl and stored over
2 2 2
molecular sieves 3 Å. Dried THF (MBraun Inc. Stratham, New
Hampshire, USA) was obtained from a MBRAUN LTS 350 solvent
1
13
purification system. The H and C NMR spectra were recorded
in CDCl on Bruker 400 and 500 MHz spectrometers. The refer-
3
ence for NMR chemical shifts was the residue peaks of solvents.
Data are given as chemical shift (d), multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet), and coupling con-
stants (s) (Hz). Mass spectra were acquired on a Varian Saturn
2000 ion trap mass spectrometer, interfaced with a Varian GC,
in EI mode [2 mscans (0.55 s/scan), emission current (30 mA),
with one (Z) double bond at the position 9 and the second and Department of Chemistry, Simon Fraser University, 8888 University Drive,
Burnaby, BC V5A 1S6, Canada
third double bonds at positions 6, 12, or 15. With the
[
9,10,11,11-D
4
] labeling pattern, a labeled tri-unsaturated fatty
*
Correspondence to: Erika Plettner, Department of Chemistry, Simon Fraser
3,4
acid ethyl ester would give diagnostic fragment ions, which
University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada.
could tell us if the three methylene-interrupted double bonds E-mail:plettner@sfu.ca
J. Label Compd. Radiopharm 2012, 55 66–70
Copyright © 2011 John Wiley & Sons, Ltd.

H. Chen and E. Plettner
by flash chromatography on silica gel eluting with 5% EtOAc
in hexanes to afford the title compound as light red oil (4.3g,
8
3%). Deuterium incorporation rate: 98% (calculated by the
1
residual signal of -CH I at 3.20 ppm) H NMR (400MHz, CDCl ) d
1
2
3
1
3
.81 (2H, t, J = 6.8 Hz), 1.40–1.28 (10H, m), (0.88, t, J = 6.8 Hz);
C
NMR (100MHz, CDCl ) d 33.33, 31.74, 30.44, 29.07, 28.51, 22.61,
3
14.06, 7.10 (quintet).
Synthesis of dec-9-yn-1-ol (6)
Compound 6 was synthesized from 2-decyn-1-ol (5) (1 g,
9
6
.5 mmol) with the previously described procedure. The title
compound was obtained as a yellow oil (0.9 g, 90%), which was
1
used directly for the next step without further purification. H
NMR (500 MHz, CDCl
J = 5.0 Hz, OH), 2.32 (1H, t, J = 2.7 Hz), 2.19 (2H, td, J = 7.0,
.5 Hz), 1.55–1.49 (4H, m), 1.34–1.28 (8H, m).
3
) d 3.54 (2H, q, J = 6.1 Hz), 3.40 (1H, t,
2
Scheme 1. Formation of EO from oleic acid in honey bee gut fluid and desa-
turation of excess EO in honey bee tissue to an ethyl ester of an unsaturated
fatty acid with a Δ9 double bond and two more double bonds at unknown
positions.
Synthesis tert-butyl (dec-9-ynyloxy)dimethylsilane (7)
Compound 7 was synthesized from 6 (1 g, 6.5 mmol) with the
9
previously described procedure. The title compound was
obtained as a light yellow oil (1.2 g, 75%). H NMR (500 MHz,
scanning SIS (49–375 m/z)]. The GC-MS instrument was also
1
ꢀ
equipped with a SPB-5 column and was programmed 100 C
CDCl ) d 3.65 (2H, t, J = 6.4 Hz), 2.32 (1H, t, J = 2.7 Hz), 2.19 (2H,
ꢀ
ꢀ
ꢀ
ꢀ
3
(
(
5 min), 10 C /min, 200 C (4.0 min), 15 C/min, and 250 C
14.0 min). Flash column chromatography was performed with
td, J = 6.9, 2.6 Hz), 1.55–1.49 (4H, m), 1.45–1.31 (8H, m), 0.92
(9H, d, J = 2.7 Hz), 0.07 (6H, s).
Kieselgel 60 (230–400 mesh ASTM).
2
Synthesis of [11,11-D ] octadec-9-yn-1-yloxy-tert-butyl
dimethylsilane (8)
Synthesis of 1-[1,1-D ] octanol (2)
2
Compound 2 was synthesized from octanoic acid (1) (4 g,
9
The general procedure of Cran and co-workers was utilized. The
2
7.7 mmol) according to the procedure reported by Rolla and
5
title compound was obtained from 4 (1.2 g, 5.1 mmol) and 7
1.2 g, 4.3 mmol) as a light yellow oil (1.0 g, 61%). Deuterium
incorporation analyzed by GC-MS: D2 97.8%; D1 1.5%; D0
3
) d 3.62 (2H, t, J = 6.8 Hz); 1.98
(2H, t, J = 6.8 Hz); 1.68–1.30 (24H, m); 0.92 (9H, s), 0.90
co-workers. The title compound was obtained as a colorless oil
3.6 g, 97%). Total deuterium incorporation rate: 98% (calculated
(
(
1
by the residual signal of -CH
CDCl ) d 1.54 (2H, t, J = 6.8 Hz), 1.35–1.27 (10H, m), (0.87, t,
J = 6.8 Hz); C NMR (100 MHz, CDCl
2
OH at 3.61 ppm). H NMR (400 MHz,
1
0
.6%. H NMR (400 MHz, CDCl
3
1
3
3
) d 62.24 (quintet), 32.55,
1.78, 29.37, 29.24, 25, 67, 22.61, 14.04.
13
(
3H, t, J = 6.8 Hz), 0.07 (6H, s); C NMR (100 MHz, CDCl
3
) d
8.04, 63.33, 63.31, 32.88, 32.86, 31.86, 29.36, 29.29, 29.23,
9.17, 29.15, 28.83, 28.70, 28.48, 25.99, 25.76, 22.67, 18.76, 18.40
3
6
2
2
Synthesis of [1,1-D ] octane-1-yl-methanesulfonyl ester (3)
(quintet), 14.11, ꢁ5.25.
Compound 2 was synthesized from 3 (3 g, 22.7 mmol) accord-
6
ing to the procedure that Mosher and Williams was utilized
Synthesis of [9,10,11,11-D
4
] (9Z)-octadec-9-en-1-yloxyl-tert-
for the unlabeled 3. The title compound was obtained as light
yellow oil (4.5 g, 96%). Total deuterium incorporation rate: 98%
butyl dimethylsilane (9)
9
The general procedure of Cran and co-workers was utilized. The
title compound was obtained from 8 (150 mg, 0.39 mmol) as a
light yellow oil (140 mg, 92%). Total deuterium incorporation
rate: 97% (calculated by the residual signal of vinylic at
(
calculated by the residual signal of -CH OMs at 4.23 ppm). The
2
1
6 1
H NMR of 3 was compared to literature. H NMR (400 MHz,
CDCl ) d 3.00 (3H, s), 1.73 (2H, t, J = 6.8 Hz), 1.41–1.27 (10H,
m), 0.88 (3H, t, J = 6.8 Hz); C NMR (100 MHz, CDCl ) d 69.41
3
13
3
5
9
.42 ppm); Deuterium incorporation analyzed by GC-MS: D4
(quint), 37.38, 31.69, 29.06, 28.97, 28.90, 25.34, 22.59, 14.04.
1
8.4%; D 0.2%; D 0.9%; D 0.4%; D 0.1%. H NMR (400 MHz,
3
2
1
0
CDCl ) d 3.60 (2H, t, J = 6.8 Hz); 2.04 (2H, t, J = 6.8 Hz); 1.68–1.30
Synthesis of 1-iodo-[1,1-D ] octane (4)
3
2
13
(24H, m); 0.92 (9H, s), 0.90 (3H, t, J = 6.8 Hz), 0.07 (6H, s);
C
A similar procedure to that of Organ and co-workers was uti-
NMR (100 MHz, CDCl ) d 63.34, 32.90, 31.91, 29.76, 29.66, 29.59,
3
7
lized. A solution of 3 (4.5 g, 21.6 mmol) in dry acetone was
2
2
9.58, 29.54, 29.46, 29.43, 29.33, 29.28, 29.27, 25.99, 25.81,
2.69, 18.39, 14.11, ꢁ5.21.
added sodium iodide (7.9, 52.8 mmol). The reaction mixture
was heated at gentle reflux for 4 h. The mixture was allowed
to cool to room temperature. The acetone was removed by
Synthesis of [9,10,11,11-D ] (9Z)-octadec-9-en-1-ol (10)
4
ꢀ
rotary evaporation in a 20–25 C water bath. The residue was
9
partitioned between 50 mL of hexanes and 50 mL of 10% The general procedure of Cran and co-workers was utilized.
sodium thiosulfate solution by swirling the flask until all the The title compound was obtained from 9 (150 mg, 0.39 mmol)
precipitate dissolved. The aqueous phase was discarded, and as a light yellow oil (75 mg, 70%). Deuterium incorporation
the organic layer was washed with 50 mL of brine, dried over rate: 97% (calculated by the residual signal of vinylic H at
1
Na
2
SO
4
and concentrated in vacuo. The residue was purified 5.42 ppm). H NMR (400 MHz, CDCl
3
) d 3.66 (2H, t, J = 6.8 Hz);
J. Label Compd. Radiopharm 2012, 55 66–70
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

H. Chen and E. Plettner
1
2
0
.03 (2H, t, J = 6.8 Hz); 1.62–1.55 (2H, m), 1.39–1.29 (22H, m); 5.38 ppm); H NMR (500 MHz, CDCl
3
) d 5.26 (1H, quintet, J = 5.0,
13
3
.90 (3H, t, J = 6.8 Hz); C NMR (100 MHz, CDCl ) d 129.39 10.0 Hz), 4.29 (2H, dd, J = 5.0, 15.0 Hz); 4.14 (2H, dd, J = 5.0,
(
2
m), 63.33, 32.82, 31.91, 29.75, 29.58, 29.54, 29.51, 29.41, 15.0 Hz); 2.31 (6H, m, J = 5.0 Hz); 2.00 (6H, t, J = 5.0 Hz); 1.61–1.57
1
3
9.33, 29.28, 29.24, 27.07, 25.74, 22.69, 14.11.
(6H, m), 1.30–1.26 (60H, m); 0.88 (9H, t, J = 5.0 Hz); C NMR
125 MHz, CDCl ) d 173.29 (overlapped), 172.87, 129.40 (m),
(
3
Synthesis of [9,10,11,11-D
4
] oleic acid (11)
68.80, 62.11 (overlapped), 34.21, 34.04, 31.92, , 29.58, 29.55,
9.34, 29.29, 29.22, 29.20, 29.14, 29.10, 29.07; 27.05, 26.34
m, weak signal), 24.90, 24.86, , 22.70, 14.13.
2
(
9
The procedure described by Cran and co-workers was utilized.
The title compound was obtained from 10 (75 mg, 0.26 mmol)
as a light yellow oil (60 mg, 82%). Deuterium incorporation rates:
9
5
7% (calculated by the residual signal of vinylic protons at
.42 ppm); 98% (calculated by the residual signal of -CD¼H;
Synthesis of isoproylideneglycerol (16)
CDCH protons at 2.12 ppm); total D4 incorporation at 97.5%.
2
Deuterium incorporation analyzed by GC-MS: D4 82.7%; D3 Compound 16 was synthesized from acetone (47.4 g, 0.82 mol),
1
2 1 0 3
1
1
1
5.4%; D 0.9%; D 0.2%; D 0.9%. H NMR (400 MHz, CDCl ) d glycerol (20 g, 0.22 mol), p-TsOH (0.6 g, 3.1 mmol), and 100 mL
ꢀ
1.19 (1H, bs), 2.35 (2H, t, J = 6.8 Hz); 2.01 (2H, t, J = 6.8 Hz); of petroleum ether (30–60 C) with the previously described pro-
1
3
11
.67–1.60 (2H, m), 1.31–1.27 (20H, m); 0.88 (3H, t, J = 6.8 Hz);
C
cedure. The tile compound was obtained as a colorless oil
1
NMR (100 MHz, CDCl ) d 180.32, 129.45 (m), 34.09, 31.92, 29.68, (23 g, 80%). H NMR (400 MHz, CDCl
3
) d 4.23 (1H, m), 4.03 (1H,
3
2
9.58, 29.54, 29.33, 29.28, 29.15, 29.07, 29.04; 27.03, 26.41 (weak dd, J = 8.0 Hz), 3.78 (1H, dd, J = 8.0 Hz), 3.72 (1H, m), 3.58 (1H,
13
signal), 24.67, 22.69, 14.11. GC-MS (derivatized with BSTFA): m), 2.04 (OH, t, J = 8.0 Hz), 1.42 (3H, s), 1.36 (3H, s); C NMR
+
m/z 359.4 (M + 1 , 57.5%), 343.5 (70%), 129.1 (95%), 117.1 (100 MHz, CDCl
100%), 75.1 (82.5%).
3
) d 109.40, 76.16, 65.70, 63.00, 26.69, 25.25.
(
Synthesis of 13
Synthesis of 17
A solution of d
4
-oleic acid (50 mg, 0.18 mmol) in 5 mL of A solution of d
Cl
lowed by DMAP (2 mg, 0.018 mmol) and EDC (69 mg, (2 mg, 0.014 mmol) and EDC (27 mg, 0.14 mmol). The mixture
.36 mmol). The mixture was allowed to stir under N
at room was allowed to stir under N at room temperature overnight.
temperature overnight. The reaction solution was diluted with The reaction solution was diluted with 20 mL of CH Cl , which
0 mL of CH Cl , which was washed with saturated NH
Cl. The was washed with saturated NH Cl. The organic phase was
organic phase was separated. The remaining aqueous phase separated. The remaining aqueous phase was extracted with
was extracted with two portions of CH Cl
. The combined two portions of CH Cl . The combined organic phase was
organic phase was washed with brine, dried over Na SO
washed with brine, dried over Na SO , and then concentrated
4
-oleic acid (11) (20 mg, 0.07 mmol) in 5 mL of
CH
2
Cl
2
was added N-acetyl cysteamine (31 mg, 0.26 mmol), fol- CH
2
2
was added 16 (14 mg, 0.10 mmol), followed by DMAP
0
2
2
2
2
2
2
2
4
4
2
2
2
2
2
4
,
2
4
and then concentrated in vacuo. The residue was purified by in vacuo. The residue was purified by flash chromatography
flash chromatography on silica gel (hexanes/EtOAc 4:1) to on silica gel (hexanes/EtOAc 9:1) to afford the tile compound
afford the tile compound as a light yellow solid (57 mg, as a colorless oil (24 mg, 85%). Deuterium incorporation
8
2%). Deuterium incorporation rates: 95% (calculated by the percentages: 95% (calculated by the residual signal of vinylic
1
1
residual signal of vinylic protons at 5.36 ppm). H NMR protons at 5.40 ppm);
H NMR (400 MHz, CDCl ) d 4.31
3
(
(
(
500 MHz, CDCl ) d 5.91 (1H, bs, -NH), 3.42 (2H, q, J = 5.0 Hz), 3.01 (1H, quintet, J = 4.0, 12.0Hz), 4.16 (1H, dd, J = 4.0, 12.0 Hz), 4.08
3
2H, t, J = 5.0Hz), 2.56 (2H, t, J = 5.0Hz), 1.99–1.93 (2H, m), 1.95 (2H, m, J = 4.0, 12.0 Hz), 3.73 (1H, dd, J = 4.0, 12.0 Hz), 2.34 (2H, t,
1
3
3H, s), 1.64 (2H, m), 1.30–1.25 (20H, m), 0.87 (3H, t, J = 5.0 Hz);
C
J = 8.0 Hz), 2.00 (2H, t, J = 8.0Hz), 1.64 (2H, t, J = 8.0Hz), 1.43 (3H,
13
NMR (125 MHz, CDCl ) 200.25, 170.22, 129.87 (m, the residue of s), 1.37 (3H, s); 1.26–1.30 (20H, m), 0.88 (3H, t, J = 8.0 Hz); C NMR
3
3
vinylic protons), 44.09, 39.74, 32.34, 31.87, 29.46, 29.42, 29.28, (100 MHz, CDCl ) d 173.60, 109.81, 73.66, 66.36, 64.52, 34.10,
2
9.10, 29.05, 28.86, 28.40, 25.62, 23.18, 22.65, 14.09, (note: the 31.89, 29.67, 29.56, 29.52, 29.31, 29.26, 29.14, 29.09, 27.02, 26.69,
signal of -CD was not observed).
25.39, 24.88, 22.67, 14.10, (note: signals of carbon labeled by
deuterium were not observed).
2
Synthesis of 15
A solution of d -oleic acid (11) (38 mg, 0.132 mmol) in 5 mL of
CH Cl was added glycerol (3 mg, 0.033 mmol), followed by
2 2
4
Synthesis of 18
DMAP (4 mg, 0.033 mmol) and EDC (25 mg, 0.132 mmol). The Compound 18 was prepared from 17 by following a reported
12
mixture was allowed to stir under N
night. The reaction solution was diluted with 20 mL of CH
which was washed with saturated NH Cl. The organic phase (400 MHz, CDCl
was separated. The remaining aqueous phase was extracted with (1H, t, J = 8.0 Hz), 4.18 (2H, m, J = 4.0, 12.0 Hz), 3.96 (1H, m,
two portions of CH Cl . The combined organic phase was J = 4.0, 12.0 Hz), 2.37 (2H, t, J = 8.0 Hz), 2.02 (2H, t, J = 8.0 Hz),
washed with brine, dried over Na SO , and then concentrated 1.65 (2H, t, J = 8.0 Hz); 1.20–1.31 (20H, m), 0.90 (3H, t, J = 8.0 Hz);
in vacuo. The residue was purified by flash chromatography on
2
at room temperature over- procedure. Deuterium incorporation rates: 96% (calculated
1
2
Cl
2
,
by the residual signal of vinylic protons at 5.36 ppm). H NMR
4.61 (1H, m, J = 4.0, 12.0 Hz), 4.28
4
3
) d
2
2
2
4
13
3
C NMR (100 MHz, CDCl ) d 173.59, 73.47, 66.77, 65.42, 61.23,
silica gel (hexanes/EtOAc 9:1) to afford the title compound as a 34.11, 31.91, 29.69, 29.57, 29.54, 29.32, 29.28, 29.16, 29.10,
colorless oil (17 mg, 60%). Deuterium incorporation percentages: 27.04, 24.88, 22.69, 14.10 (note: signals of carbon labeled by
9
5% (calculated by the residual signal of vinylic protons at deuterium were not observed).
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 66–70

H. Chen and E. Plettner
Synthesis of 19
based bioanalytical assay. To meet the requirement for our
research plan, we chose to assemble the labeled acid from
two components, in which the first two site-specific deuterium
atoms at position 11 were introduced by lithium aluminum
A solution of oleic acid (63 mg, 0.22 mmol) in 5 mL of CH Cl was
2
2
added 18 (20 mg, 0.055 mmol), followed by DMAP (3 mg,
.022 mmol) and EDC (42 mg, 0.22 mmol). The mixture was
allowed to stir under N at room temperature overnight. The
reaction solution was diluted with 20 mL of CH Cl , which was
washed with saturated NH Cl. The organic phase was separated.
The remaining aqueous phase was extracted with two portions
of CH Cl . The combined organic phase was washed with brine,
dried over Na SO , and then concentrated in vacuo. The residue
0
deuteride (LiAlD ) reduction, followed by the catalytic semi-
4
2
deuteration using D2 and Lindlar catalyst to introduce the
second two deuterium atoms (Figure 1).
2
2
4
The synthesis (Scheme 2) starts with a reduction of octanoic
5
acid (1) with LiAlD to give d -octanol (2). After mesylation of
4
2
2
2
d -octanol, d -octane iodide (4) was prepared by the treatment
2
2
2
4
6,7
of mesylate 3 with NaI in acetone. With the first deuterium-
labeled fragment 4 in hand, we turned our attention to the
second fragment. Treatment of commercially available dec-
was purified by flash chromatography on silica gel (hexanes/
EtOAc 9:1) to afford the title compound as a colorless oil
1
(
30 mg, 63%). H NMR (400 MHz, CDCl
3
) d 5.37 (4H, m,), 5.26
2-yn-ol (5) with NaH in 1,3-diaminopropane by an alkyne zipper
(
1H, m) 4.61 (1H, m, J = 4.0, 12.0 Hz), 4.32 (1H, dd, J = 8.0 Hz),
.17 (2H, dd, J = 4.0, 12.0 Hz), 2.33 (6H, m, J = 4.0 Hz), 2.04 (10H,
m), 1.61–1.58 (6H, m), 1.32–1.28 (60H, m), 0.90 (9H, t, J = 8.0 Hz);
NMR (100 MHz, CDCl ) d 173.27, 172.85, 130.04, 130.02, 129.72,
29.69, 68.88, 68.21, 34.21, 34.04, 31.91, 30.93, 29.78, 29.71,
9.54, 29.19, 29.13, 29.10, 27.28, 27.13, 24.85, 22.69, 14.12 (note:
reaction that gave rise to isomerization of the internal alkyne to
4
8,9
the terminal position afforded dec-9-yn-1-ol (6). The alcohol
9
functionality on 6 was then protected as the TBDMS silyl ether 7.
3
Treatment of the protected alcohol 7 with n-BuLi generated the
1
2
corresponding lithium acetylide, which was coupled to iodide
9,10
4
to afford 8 in 80% yield.
The obtained key intermediate 8
signals of carbon labeled by deuterium were not observed).
9
was then partially reduced with D
After removing the silyl group, the resulting alcohol 10 was
treated with Jones reagent to give the desired d
2
to 9 over Lindlar catalyst.
9
4
-oleic acid (11).
Results and discussion
With the site-specific d -oleic acid in hand, three derived
4
As discussed in the Introduction section, our plan was to esters were also prepared. Thioester 13 and ester 15 were
develop a d -labeled oleic acid precursor with two of deuterium synthesized directly by coupling 11 with the N-acetyl cysteamine
4
located at position11 specifically for use as a tracer in a GC-MS (12) and glycerol (14), respectively (Scheme 3).
Figure 1. Strategy for the preparation the site-specific deuterium labeled oleic acid.
ꢀ
Scheme 2. (a) LiAlD
THF-HMPA, -78 C-0 C; (g) Lindlar catalyst, D
4
, THF, reflux; (b) TEA, MsCl, CH
2
Cl
2
, rt; (c) NaI, acetone, reflux; (d) 1,3-diaminopropane, NaH, 60 C; (e) TBDMSCl, TEA, DMAP, CH
2
Cl
2
, rt; (f) n-BuLi,
ꢀ
ꢀ
ꢀ
2
, hexanes, rt; (h) TBAF, THF, rt; (i) Jones reagent, acetone, 0 C.
J. Label Compd. Radiopharm 2012, 55 66–70
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

H. Chen and E. Plettner
ꢀ
Scheme 3. (a) EDC, DMAP, CH
2
Cl
2
, rt; (b); EDC, DMAP, CH
2
Cl
2
, rt; (c) acetone, p-TsOH, petroleum ether (30–60 C), reflux; (d) d
4 2 2
-oleic acid, EDC, DMAP, CH Cl , rt; (e)
triethylborate, trifluoroethanol, and TFA in 8:1:1 ratio, rt; (f) oleic acid, EDC, DMAP, CH
2
Cl , rt.
2
To synthesize compound 19, glycerol (14) was first treated with References
acetone in the presence of catalytic p-TsOH to afford 2,3-isopropy-
1
1
[1] Y. Le Conte, A. Hefetz, Annu. Rev. Entomol. 2008, 53, 523–542.
lideneglycerol (16). Compound 16 was coupled with d -oleic
4
[
2] I. Leoncini, Y. Le Conte, G. Costaglialo, E. Plettner, A. L. Toth, M. Wang,
Z. Huang, J-M. Becard, D. Crauser, K. N. Slessor, G. E. Robinson, Proc.
Natl. Acad. Sci. USA 2004, 101, 17559–17564.
acid (11) in the presence of EDC and DMAP to give 17. The product
7 was treated with TFA in trifluoroethanol/triethyl borate (1:8v/v)
1
12
[
3] A. J. Fellenberg, D. W. Johnson, A. Poulos, P. Sharp, Biomed. Environ.
Mass Spectrom. 1987, 14, 127–130.
solution at room temperature to afford 18, which was subse-
quently coupled with oleic acid to yield the final product 19.
In conclusion, a synthetic route for the efficient preparation of
a site-specific deuterium labeled oleic acid from readily available
starting materials and reagents has been developed. Several
derived esters were also prepared. The labeled precursors have
been used as powerful probes in the study of biosynthesis of
EO in honeybees. The results of these investigations will be
reported in due course.
[
4] A. Brauner, H. Budzikiewicz, W. Boland, Org. Mass Spectrom. 1982, 17,
1
61–164.
[5] D. Landini, A. Maia, F. Montanari, F. Rolla, J. Org. Chem. 1983, 48,
3774–3777.
[6] H. R. Williams, H. S. Mosher, J. Am. Chem. Soc. 1954, 76, 2984.
[7] M. G. Organ, J. Wang, J. Org. Chem. 2003, 68, 5568.
[8] S. R. Macaulay, J. Org. Chem. 1980, 45, 734–735.
[9] S. N. Crane, K. Bateman, S. Gagne, J. F. Levesque, J. Label Compd
Ridiopharm. 2006, 49, 1273–1285.
[
10] (a) E. F. Magoon, L. H. Slaugh, Terahedron 1967, 23, 4509–4515; (b)
G. Ohloff, C. Vial, F. Naf, M. Pawlak, Helv. Chim. Acta 1977, 60,
1161–1174.
Conflict of Interest
[
11] M. Renoll, M. S. Newman, Organic Syntheses, Coll. 1955, 3, 502.
The authors did not report any conflict of interest.
[12] P. R. J. Gaffney, C. B. Reese, J. Chem. Soc., Perkin Trans. 1 2001, 2, 192.
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J. Label Compd. Radiopharm 2012, 55 66–70