Large-scale preparation of (9Z,12E)-[1-13C]-octadeca-9,12-dienoic acid, (9Z,12Z,15E)-[1-13C]-octadeca-9,12,15-trienoic acid and their [1-13C] all-cis isomers

Article abstract of DOI:10.1016/S0009-3084(00)00137-7

Several grams of labelled trans linoleic and linolenic acids with high chemical and isomeric purities (>97%) have been prepared for human metabolism studies. A total of 12.5 g of (9Z,12E)-[1-¹³C]-octadeca-9,12-dienoic acid and 6.3 g of (9Z,12Z,15E)-[1-¹³C]-octadeca-9,12,15-trienoic acid were obtained in, respectively, seven steps (7.8% overall yield) and 11 steps (7% overall yield) from 7-bromo-heptan-1-ol. The trans bromo precursors used for the labelling were synthesised by using copper-catalysed couplings. The trans fatty acids were then obtained via the nitrile derivatives. A total of 23.5 g of (9Z,12Z)-[1-¹³C]-octadeca-9,12-dienoic acid and 10.4 g of (9Z,12Z,15Z)-[1-¹³C]-octadeca-9,12,15-trienoic acid were prepared in five steps in, respectively, 32 and 18% overall yield. Large quantities of bromo and chloro precursors were synthesised from the commercially available acid according to Barton's procedure. In all cases, the main impurities (>0.5%) of each labelled fatty acid have been characterised. Copyright (C) 2000 Elsevier Science Ireland Ltd.

Full text of DOI:10.1016/S0009-3084(00)00137-7

Chemistry and Physics of Lipids
106 (2000) 65–78
www.elsevier.com/locate/chemphyslip
Large-scale preparation of
(9Z,12E)-[1-13C]-octadeca-9,12-dienoic acid,
(9Z,12Z,15E)-[1-13C]-octadeca-9,12,15-trienoic acid and their
[1-13C] all-cis isomers
a,
a
a
b
O. Loreau *, A. Maret , D. Poullain , J.M. Chardigny b, J.L. Se´be´dio ,
B. Beaufre`re c, J.P. Noe¨l a
a CEA/Saclay, Ser6ice des Mole´cules Marque´es, Baˆt. 547, F-91191 Gif sur Y6ette Cedex, France
b INRA, Unite´ de Nutrition Lipidique, 17 rue Sully, F-21034 Dijon Cedex, France
c Laboratoire de Nutrition Humaine, 58 rue Montalembert, BP 321, F-63009 Clermont-Ferrand Cedex 01, France
Received 29 October 1999; received in revised form 9 March 2000; accepted 16 March 2000
Abstract
Several grams of labelled trans linoleic and linolenic acids with high chemical and isomeric purities (\97%) have
been prepared for human metabolism studies. A total of 12.5 g of (9Z,12E)-[1-13C]-octadeca-9,12-dienoic acid and 6.3
g of (9Z,12Z,15E)-[1-13C]-octadeca-9,12,15-trienoic acid were obtained in, respectively, seven steps (7.8% overall yield)
and 11 steps (7% overall yield) from 7-bromo-heptan-1-ol. The trans bromo precursors used for the labelling were
synthesised by using copper-catalysed couplings. The trans fatty acids were then obtained via the nitrile derivatives.
A total of 23.5 g of (9Z,12Z)-[1-13C]-octadeca-9,12-dienoic acid and 10.4 g of (9Z,12Z,15Z)-[1-13C]-octadeca-9,12,15-
trienoic acid were prepared in five steps in, respectively, 32 and 18% overall yield. Large quantities of bromo and
chloro precursors were synthesised from the commercially available acid according to Barton’s procedure. In all cases,
the main impurities (\0.5%) of each labelled fatty acid have been characterised. © 2000 Elsevier Science Ireland Ltd.
All rights reserved.
Keywords: Carbon 13; cis; Labelling; Linoleic; Linolenic; trans
1. Introduction
deodorization (Grandgirard et al., 1984; Se´be´dio
et al., 1988; Ackman and Mag, 1998). Conse-
quently, trans isomers of linoleic and linolenic
acids are found in human foods such as refined
oils, margarines, human milk and infant for-
mulas (Wolff and Se´be´dio, 1991; Wolff,
1993; Chardigny et al., 1996; Ratnayake et al.,
1997).
Geometrical polyunsaturated fatty acid iso-
mers are formed during oil processing such as
* Corresponding author. Tel.: +33-1-69087596; fax: +33-
1-69087991.
E-mail address: olivier.loreau@cea.fr (O. Loreau)
0009-3084/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0009-3084(00)00137-7

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O. Loreau et al. / Chemistry and Physics of Lipids 106 (2000) 65–78
Previous studies (Bre´tillon et al., 1998a,b; Se´be´-
bon 14. The trans bromo precursors were synthe-
sised in small quantities via stereoselective Wittig
reactions (Eynard et al., 1994, 1998; Berdeaux et
al., 1995). Unfortunately, the experimental proce-
dures of these stereoselective Wittig reactions
were not practical in the case of a large-scale
preparation.
Here, we now report convenient large-scale syn-
theses of (9Z,12E)-[1-13C]-octadeca-9,12-dienoic
acid and (9Z,12Z,15E)-[1-13C]-octadeca-9,12,15-
trienoic acid (respectively 7 and 19). These synthe-
ses are suitable for the introduction of a labelled
carbon atom. The trans bromo intermediates 5
and 18 were prepared by synthetic schemes in-
volving copper-catalysed couplings of terminal
acetylenes with allylic bromides and selective par-
tial reductions with Lindlar catalyst. As the all-cis
isomers were needed for the human metabolism
studies, we also describe the large-scale prepara-
tions of (9Z,12Z)-[1-13C]-octadeca-9,12-dienoic
acid and (9Z,12Z,15Z)-[1-13C]-octadeca-9,12,15-
trienoic acid (respectively 22 and 23) by using
Barton’s bromo-decarboxylation.
dio and Chardigny, 1998) reported the use of
trans isomers of linoleic acid labelled with carbon
14 for metabolic studies in animals. In order to
investigate further the metabolism of dietary trans
linoleic acid for comparison with that of the
all-cis compound in healthy volunteers, we syn-
thesised (9Z,12E)-, (9Z,12Z)-[1-13C]-linoleic acid
and (9Z,12Z,15E)-, (9Z,12Z,15Z)-[1-13C]-linolenic
acid on a large scale (several grams) with high
chemical and isomeric purities (\97%). Further-
more, as these labelled fatty acids were used for
human metabolism studies after agreement of an
ethics committee, the main impurities (\0.5%) of
each acid had to be identified.
Fatty acids labelled at the carboxylic position
can be obtained from a halide by well-established
methods (Tulloch, 1979) as shown in Fig. 1.
Several reports have described the syntheses of
all-cis bromo intermediates using a fatty acid as
starting material. The bromo precursors of [1-
13C]- and [1-14C]-oleic acid and (9Z,12Z)-[1-14C]-
linoleic acid could be prepared via a silver salt
degradation (Bergstrom et al., 1952; Howton et
al., 1954). (9Z,12Z)-[1-13C]-Linoleic acid was ob-
tained from an a-bromolinoleate intermediate
(Campbell and Clapp, 1989). Some all-cis fatty
acids labelled with carbon 11 have been synthe-
sised by using Barton’s bromo-decarboxylation
(Barton et al., 1983; Channing and Simpson,
1993). The all-cis bromo precursor can also be
prepared by total organic synthesis. Stoffel syn-
thesised (9Z,12Z)-[1-14C]-linoleic acid by reaction
of an alkali acetylide with a propargylic halide
followed by partial hydrogenation (Stoffel, 1964).
More recently, a few papers have described the
preparation of trans fatty acids labelled with car-
2. Experimental
Both 7-bromo-1-heptanol and (E)-oct-2-en-1-ol
were from Sigma. [1-13C] potassium cyanide (iso-
topic enrichment \99%) was from Isotec. Other
starting materials and chemical reagents were
from Aldrich. 4-(Dimethylamino) pyridine, 2-mer-
captopyridine N-oxide, sodium salt and K13CN
were stored in a desiccator with phosphorus pen-
toxide prior to use. Unless otherwise stated, flash
chromatography was performed using silica gel 60
(0.040–0.063 mm) from Merck. Potassium hy-
droxide solution, hydrochloric acid solution and
sodium chloride were from Labosi. Magnesium
sulphate, ethanol and dimethylsulphoxide were
from Prolabo. Ammonium chloride and other
solvents were from SDS. Acetone and methanol
used for preparative reversed-phase chromatogra-
phy were from Merck. Solvents were purified
before
use:
dimethylsulphoxide,
bro-
motrichloromethane and toluene were distilled;
diethyl ether and tetrahydrofuran were distilled
from sodium benzophenone ketyl. All purified
Fig. 1. Labelling of organic acids from halides. * Denotes
labelling. X=Br, Cl, I.

O. Loreau et al. / Chemistry and Physics of Lipids 106 (2000) 65–78
67
solvents were stored over molecular sieves. Unless
2.2. Non-8-yn-1-ol 2
otherwise stated, reactions were carried out under
argon.
Lithium acetylide, ethylenediamine complex
(90% pure; 61.4 g; 600 mmol; 1.15 eq.) and 350 ml
of dry DMSO were placed in an oven-dried three-
necked flask equipped with a magnetic stirrer and
a thermometer. The mixture was cooled to 10°C
and 7-bromo-heptan-1-ol (101.4 g; 520 mmol) was
added dropwise. After the addition, the dark reac-
tion mixture was stirred vigorously at room tem-
perature for 4 h after which it was slowly added
to a mixture of water and ice. The aqueous solu-
tion was then extracted four times with diethyl
ether. The combined ethereal layers were washed
with saturated sodium chloride solution and wa-
ter, dried over MgSO4 and filtered. After concen-
tration, the residue was chromatographed on
silica gel with pentane/diethyl ether (75/25) giving
35.4 g of a mixture of compound 2 (31.9 g; 228
mmol; 44% yield) and 7-bromo-heptan-1-ol. 2 was
obtained in 48% yield for the synthesis of com-
pound 19 by using the same protocol.
Analytical TLC was performed with silica gel
60F254 (Merck) and visualisation was carried out
with an anisaldehyde solution (anisaldehyde 12.5
ml; acetic acid 5 ml; sulphuric acid 17 ml; 95%
ethanol 450 ml). NMR spectra were recorded on a
Bruker AC 300 spectrometer (7.05 T; 300.13 MHz
(1H); 75.47 MHz (13C)) and the chemical shifts
were reported in ppm. Isotopic enrichment was
determined by mass spectrometry (DCI/NH3) on
a Finnigan instrument (Model 4600). GC/MS
analyses were effected on an HP 6890 Series gas
chromatograph system coupled to an HP 5973
mass selective detector. GC analyses were carried
out on a Varian 3300 gas chromatograph (N2, 10
ml/min) fitted with a flame ionisation detector.
Analytical HPLC analyses were obtained with a
Shimadzu LC-9A pump and a Sedex 65 evapora-
tive light scattering detector. Preparative reversed-
phase chromatography was conducted with a
NovaPrep 5000 apparatus from Merck fitted with
a YRD-883 RI monitor from Uniflows.
1H NMR (CDCl3): 1.25–1.65 (m; 2,3,4,5,6-H2),
1.95 (t; J =2.6 Hz; 9-H1), 2.2 (dt; J =6.9, 2.6 Hz,
7-H2), 3.65 (t; J =6.9 Hz; 1-H2).
2.1. (E)-1-Bromo-oct-2-ene 1
2.3. (E)-Heptadeca-11-en-8-yn-1-ol 3
(E)-Oct-2-en-1-ol (95% pure; 105.2 g; 779
mmol) in 1200 ml of anhydrous diethyl ether and
15 ml of anhydrous pyridine were placed in an
oven-dried three-necked flask equipped with a
magnetic stirrer and a thermometer. The solution
was cooled to −10°C and phosphorus tribromide
(185.9 g; 686 mmol; 0.9 eq.) was added dropwise.
The reaction mixture was stirred at −10°C for 4
h after which it was poured into cold water. The
aqueous layer was extracted four times with di-
ethyl ether. The combined organic layers were
washed twice with brine then with water until the
washes were of neutral pH, dried over MgSO4
and filtered. Partial distillation of diethyl ether
gave a yellow solution (178.9 g) containing com-
pound 1 (124.7 g; 653 mmol; 83% yield). This
ethereal solution of 1 was used without any fur-
ther purification in the next step.
Anhydrous THF (46 ml) and 2 (31.9 g; 228
mmol) were placed in an oven-dried 2-l three-
necked flask equipped with a magnetic stirrer, a
thermometer and a reflux condenser. The mixture
was cooled to 5°C and treated for 1.75 h with 167
ml of 2.94 M CH3MgCl in THF (491 mmol; 2.15
eq.). After the addition, the reaction mixture was
stirred vigorously at 65°C for 1 h and then cooled
to room temperature. CuCl (1.7 g; 17.1 mmol;
0.075 eq.) was added all at once and then 159 g of
the ethereal solution of 1 (110.8 g; 580 mmol; 2.5
eq.) were added over 1 h. The mixture was heated
at 65°C for 2 h giving a homogeneous solution,
cooled to 25°C and quenched with 360 ml of 20%
NH4Cl solution. Sodium cyanide (5.22 g) was
added and the solution became slightly yellow.
The aqueous layer was extracted three times with
diethyl ether and the combined organic layers
were washed with brine, dried over MgSO4 and
concentrated. The crude product was purified on
1H NMR (CDCl3): 0.87 (t; J =6.9 Hz; 8-H3),
1.15–1.4 (m; 5,6,7-H2), 2.03 (m; 4-H2), 3.92 (d;
J =7.3 Hz; 1-H2), 5.55–5.8 (m; 2,3-H1).

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O. Loreau et al. / Chemistry and Physics of Lipids 106 (2000) 65–78
silica gel with pentane/diethyl ether (80/20) giving
compound 3 (45.8 g; 183 mmol; 80% yield) as a
pale yellow oil.
1H NMR (CDCl3): 0.87 (t; J =6.9 Hz; 1-H3),
1.2–1.5 (m; 2,3,4,12,13,14,15-H2), 1.85 (quint.;
J =6.9 Hz; 16-H2), 1.9–2.1 (m; 5,11-H2), 2.71 (bt;
J =5.4 Hz; 8-H2), 3.4 (t; J =6.9 Hz; 17-H2),
5.25–5.5 (m; 6,7,9,10-H1).
1H NMR (CDCl3): 0.87 (t; J =6.9 Hz; 17-H3),
1.2–1.65 (m; 2,3,4,5,6,14,15,16-H2), 2 (m; 13-H2),
2.16 (tt; J = 6.9, 2.3 Hz; 7-H2), 2.86 (m; 10-H2),
3.63 (t; J =6.8 Hz; 1-H2), 5.4 (m; 11-H1), 5.65 (m;
12-H1).
2.6. (9Z,12E)-[1-13C]-Octadeca-9,12-dienenitrile 6
Several attempts were made to optimise the
formation of 3. The different crude materials were
first purified on silica gel with pentane/diethyl
ether (80/20) and then subjected to reversed-phase
chromatography using Partisil ODS 3 with methyl
alcohol/water (85/15) providing pure 3 (8.8 g; 35
mmol)).
The different purified materials were combined
to give 32.3 g of 3 (129 mmol) which were used
for the next step.
A mixture of compound 5 (19.3 g; 61 mmol)
dissolved in 190 ml of dry DMSO and K13CN (5.1
g; 77 mmol; 1.2 eq.) was stirred overnight at 80°C,
and then cooled to room temperature. The reac-
tion mixture was diluted with 300 ml of diethyl
ether and 350 ml of saturated sodium chloride
solution. The aqueous layer was extracted twice
with diethyl ether. The combined organic frac-
tions were washed with water, dried over MgSO4,
filtered and concentrated. The residue was eluted
through silica gel with pentane/diethyl ether (95/5)
to give the nitrile 6 (14 g, 53 mmol; 87% yield).
1H NMR (CDCl3): 0.87 (t; J =6.9 Hz; 18-H3),
1.2–1.5 (m; 4,5,6,7,15,16,17-H2), 1.63 (m; 3-H2),
1.9–2.1 (m; 8,14-H2), 2.32 (dt; J =9.5, 7 Hz;
2-H2), 2.71 (bt; J =5.5 Hz; 11-H2), 5.25–5.5 (m;
9,10,12,13-H1).
2.4. (8Z,11E)-Heptadeca-8,11-dien-1-ol 4
Compound 3 (32.3 g, 129 mmol), Lindlar cata-
lyst (5% wt. Pd; 1.3 g), quinoline (1.3 ml) and 150
ml of ethyl acetate were shaken in hydrogen.
After the absorption of hydrogen had ceased, the
catalyst was removed and the mixture was con-
centrated. Crude material (33.7 g) was combined
with residues obtained from previous partial hy-
drogenation of 3 to give 54.4 g of dienol 4.
Chromatography on silica gel with pentane/di-
ethyl ether (80/20) yielded 4 (51.2 g; 203 mmol;
97% yield).
2.7. (9Z,12E)-[1-13C]-Octadeca-9,12-dienoic acid
7
Compound 6 (14 g; 53 mmol) was placed in 225
ml of ethyl alcohol and 225 ml of 40% KOH. The
mixture was heated at 80°C for 5 h and then
cooled to room temperature. Ground ice was
placed in the flask and the reaction mixture was
acidified to pH 1 with 6 N HCl. The aqueous
layer was extracted three times with 500 ml of
diethyl ether. The combined organic layers were
washed with water until washes were of neutral
pH, dried over MgSO4, filtered and concentrated.
The crude material was chromatographed on sil-
ica gel with pentane/diethyl ether/acetic acid (90/
10/1). Compound 7 (12.5 g; 44 mmol; 83% yield)
was obtained as a yellow oil.
1H NMR (CDCl3): 0.87 (t; J =6.9 Hz; 17-H3),
1.2–1.4 (m; 3,4,5,6,14,15,16-H2), 1.5–1.65 (m; 2-
H2 and OH), 1.9–2.1 (m; 7,13-H2), 2.71 (bt; J =
5.3 Hz; 10-H2), 3.63 (m; 1-H2), 5.25–5.5 (m;
8,9,11,12-H1).
2.5. (6E,9Z)-17-Bromo-heptadeca-6,9-diene 5
The bromide 5 was prepared from compound 4
(50 g; 198 mmol) using phosphorus tribromide (43
g; 159 mmol; 0.8 eq.), anhydrous pyridine (5 ml)
and 300 ml of anhydrous diethyl ether as de-
scribed previously for bromide 1. Purification by
chromatography on silica gel with pentane gave 5
(19.3 g; 61 mmol; 31% yield) as a colourless oil.
TLC: pentane/diethyl ether/acetic acid (90/10/
1). Rf=0.15 (visualisation with an anisaldehyde
solution).
1H NMR (CDCl3): 0.87 (t; J =6.9 Hz; 18-H3),
1.2–1.4 (m; 4,5,6,7,15,16,17-H2), 1.62 (m; 3-H2),

O. Loreau et al. / Chemistry and Physics of Lipids 106 (2000) 65–78
69
1.9–2.1 (m; 8,14-H2), 2.34 (dt; J =7.3, 7.3 Hz,
2.9. (E)-1-Bromo-pent-2-ene 8
2-H2), 2.71 (bt; J =5.4 Hz; 11-H2), 5.25–5.5 (m;
9,10,12,13-H2).
A total of 155 g of a mixture of bromide 8 (84.9
g; 570 mmol; 84% yield) and diethyl ether were
prepared from (E)-pent-2-en-1-ol (95% pure; 58.5
g; 679 mmol) using phosphorus tribromide (142.5
g; 526 mmol; 0.8 eq.), 2.3 ml of dry pyridine and
900 ml of anhydrous diethyl ether as described
previously for bromide 1. This solution of 8 was
used without any further purification in the next
step.
13C NMR (CDCl3): 13.82 (C18), 22.31 (C17),
24.42 (C3), 26.83 (C8), 28.81 and 28.88 (C4, C5
and C6), 28.99 (C15), 29.34 (C7), 30.21 (C11),
31.19 (C16), 32.29 (C14), 33.48 and 34.2 (J =54.3
Hz, C2), 127.62 (C10), 128.01 (C12), 130.05 (C9),
130.61 (C13), 180.13 (C1).
HPLC: column: Hypersil HS C18 (250×4.6
mm). Solvent system: acetone/methanol/water/
acetic acid (50/25/25/0.5). Flow rate: 1.5 ml/min.
Chemical purity: unknown impurity (r.t.=24.5
min; 0.4%), (9Z,12Z)-[1-13C]-linoleic acid (r.t.=26
min; 0.9%), (9Z,12E)-[1-13C]-linoleic acid 7 (r.t.=
27.7 min; 97.7%), (9E,12E)-[1-13C]-linoleic acid
(r.t.=30.6 min; 0.9%), (9Z)-[1-13C]-oleic acid
(r.t.=42 min; 0.1%).
GC/MS: column: HP-23 cis/trans FAME (60
m×0.25 mm×0.25 mm). Column temperature:
200°C. Carrier gas: He (1 ml/min). Injector tem-
perature: 250°C. Identification (methyl ester
derivative): unknown impurity (r.t.=7.1 min; m/
z=295 M+), (9Z)-[1-13C]-oleic acid, methyl ester
(r.t.=8.2 min; m/z=297 M+), (9E,12E)-[1-13C]-
linoleic acid, methyl ester (r.t.=8.5 min; m/z=
295 M+), (9Z,12E)-[1-13C]-linoleic acid, methyl
ester (r.t.=8.8 min; m/z=295 M+), (9Z,12Z)-[1-
13C]-linoleic acid, methyl ester (r.t.=9 min; m/
z=295 M+).
1H NMR (CDCl3): 0.94 (t; J =7.4 Hz; 5-H3), 2
(m; 4-H2); 3.87 (d; J =7.4 Hz; 1-H2), 5.6 (m;
2-H1), 5.75 (m; 3-H1).
2.10. (E)-Oct-5-en-2-yn-1-ol 9
A total of 47 g of a mixture of alcohol 9 (44.6
g; 360 mmol; 94% yield) and diethyl ether were
prepared from prop-2-yn-1-ol (21.3 g; 380 mmol),
260 ml of 3 M methylmagnesium chloride in THF
(780 mmol; 2.05 eq.), Cu(I)Cl (2.7 g; 27.3 mmol;
0.07 eq.) and compound 5 in diethyl ether (570
mmol; 1.5 eq.) as described previously for com-
pound 3.
1H NMR (CDCl3): 0.95 (t, J =7.4 Hz, 8-H3), 2
(m, 7-H2), 2.9 (m, 4-H2), 4.25 (m, 1-H2), 5.35 (m,
5-H1), 5.7 (m, 6-H1).
2.11. (E)-1-Bromo-oct-5-en-2-yne 10
MS (DCI/NH3): m/z (%): 298 (2.7), 299 (100),
300 (16.7), 301 (3.7). Isotopic enrichment: 97.4%.
Compound 10 was obtained from 2.71 g of a
mixture of compound 9 (2.57 g; 20.7 mmol) and
diethyl ether, anhydrous pyridine (0.2 ml), phos-
phorus tribromide (4.3 g; 15.8 mmol; 0.8 eq.) and
anhydrous diethyl ether (20 ml) as described for
bromide 1. After concentration of the ethereal
fractions, the residue was eluted through silica gel
with pentane giving bromide 10 (2.38 g; 12.7
mmol; 61% yield).
2.8. Discoloration of 7
Two typical procedures which can be used for
the discoloration are described:
1. Compound 7 (10.4 g; 37 mmol) was dissolved
in 70 ml of freshly distilled diethyl ether and a
small amount of activated charcoal was added
to the solution. The mixture was stirred at
room temperature for 1.5 h, filtered and con-
centrated to give acid 7 (10.3 g; 99% yield) as
a pale yellow oil.
1H NMR (CDCl3): 0.95 (t, J =7.4 Hz, 8-H3), 2
(m, 7-H2), 2.9 (m, 4-H2), 3.95 (t, J =2.4 Hz,
1-H2), 5.35 (m, 5-H1), 5.7 (m, 6-H1).
2. Compound 7 (1 g) was chromatographed on
Partisil ODS 3 with acetone/methyl alcohol/
water/acetic acid (50/25/25/0.5) giving in a
quantitative yield the acid 7 as a colourless oil.
2.12. (E)-Heptadec-14-ene-8,11-diyn-1-ol 11
Bromide 10 (1.68 g; 9 mmol) and terminal
acetylene 2 (1.26 g; 9 mmol) were placed in 20 ml

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O. Loreau et al. / Chemistry and Physics of Lipids 106 (2000) 65–78
of dry DMF. K2CO3 (2.88 g; 20 mmol), NaI (1.52
g; 10 mmol) and CuI (1.93 g; 10 mmol), each
finely ground and anhydrous, were added. The
suspension was stirred overnight at 40°C, then
quenched with 100 ml of a saturated solution of
NH4Cl. The mixture was extracted three times
with diethyl ether, dried over MgSO4, filtered and
concentrated. The residue was purified by chro-
matography on silica gel with pentane/diethyl
ether (75/25) giving 11 (1.52 g; 61 mmol; 67%).
Then 1 g of purified 11 was eluted through 200 g
of C18 Partisil Prep 20 ODS 3 with methanol/wa-
ter (85/15) and only 0.53 g of 11 were recovered.
1H NMR (CDCl3): 0.95 (t; J =7.4 Hz, 17-H3),
1.2–1.7 (m; 2,3,4,5,6-H2), 2 (m; 16-H2), 2.1 (m;
7-H2), 2.85 (m; 13-H2), 3.1 (quint.; J =2.4 Hz;
10-H2), 3.6 (t; J =6.7 Hz; 1-H2), 5.35 (m; 14-H1),
5.65 (m; 15-H1).
2.15. (2Z,5E)-1-Bromo-octa-2,5-diene 14
A mixture of bromide 14 (32 g; 170 mmol; 94%
yield) and diethyl ether was prepared from 25.8 g
of a mixture of dienol 13 (22.7 g; 180 mmol) and
quinoline, dry pyridine (1.9 ml), anhydrous di-
ethyl ether (230 ml) and phosphorus tribromide
(39.3 g; 145 mmol; 0.8 eq.) as described for bro-
mide 1. This solution was used for the next step
without any further purification.
1H NMR (CDCl3): 0.9 (t; J =7.4 Hz; 8-H3), 1.9
(m; 7-H2), 2.75 (bt; J =6.7 Hz; 4-H2), 3.9 (d;
J =8.2 Hz; 1-H2), 5.2–5.75 (m; 2,3,5,6-H1).
2.16. 2-Non-8-ynyloxy-tetrahydro-pyran 15
A total of 28.5 g of a mixture of 2 (25 g, 178
mmol) and 7-bromo-heptan-1-ol, and 3.8 g of
p-toluene sulphonic acid were placed in 600 ml of
dry dichloromethane. Dihydropyran (27.8 g; 331
mmol) was added dropwise at room temperature.
The reaction mixture was stirred for 5.5 h and
then 500 ml of a saturated NaHCO3 solution were
added. The aqueous layer was extracted with
dichloromethane. The combined organic fractions
were washed with water, dried over MgSO4,
filtered and concentrated. The residue was chro-
matographed on silica gel with pentane/diethyl
ether (95/5) giving 2-non-8-ynyloxy-tetrahydro-
pyran 15 (34.8 g; 155 mmol; 87% yield).
2.13. (8Z,11Z,14E)-Heptadeca-8,11,14-trien-1-ol
12
The triene 12 was prepared from compound 11
(520 mg; 2.1 mmol), purified on silica gel, Lindlar
catalyst (5% wt. Pd; 48 mg), quinoline (40 ml) and
ethyl acetate (15 ml) as described for diene 4.
Chromatography on silica gel with pentane/di-
ethyl ether (75/25) gave 12 (170 mg, 0.68 mmol;
32% yield).
1H NMR (CDCl3): 0.95 (t; J =7.4 Hz; 17-H3),
1.2–1.45 (m; 3,4,5,6-H2), 1.55 (m, 2-H2), 1.9–2.1
(m; 7,16-H2), 2.7–2.85 (m; 10,13-H2), 3.6 (t; J =
6.7 Hz; 1-H2), 5.25–5.55 (m; 8,9,11,12,14,15-H1).
1H NMR (CDCl3): 1.25–1.9 (m; 2,3,4,5,6-H2;
3,4,5-H2 (THP)), 1.95 (t; J =2.6 Hz; 9-H1), 2.15
(dt; J =6.9, 2.6 Hz, 7-H2), 3.36 (dt, J =9.5, 6.6
Hz; 1-Ha), 3.5 (m; 6-Ha (THP)), 3.71 (dt; J =9.5,
6.9 Hz; 1-Hb), 3.85 (m; 6-Hb (THP)), 4.56 (m;
2-H1 (THP)).
2.14. (2Z,5E)-Octa-2,5-dien-1-ol 13
Compound 13 was obtained from 33.2 g of a
mixture of 9 (31.5 g, 254 mmol) and diethyl ether,
Lindlar catalyst (5% wt. Pd; 5.5 g), quinoline (3.4
ml) and ethanol (410 ml) as described for diene 4.
Chromatography on silica gel with pentane/di-
ethyl ether (85/15) gave 32 g of a mixture of the
alcohol 13 (28.2 g; 224 mmol; 88% yield) and
residual quinoline.
2.17. 2-((11Z,14E)-Heptadeca-11,14-dien-
8-ynyloxy)-tetrahydro-pyran 16a and 2-((E)-
10-6inyl-pentadec-12-en-8-ynyloxy)-tetrahydro-
pyran 16b
A mixture of 16a and 16b was obtained from
compound 15 (27.2 g; 121 mmol), 42 ml of 3 M
CH3MgCl in THF (126 mmol; 1.04 eq.), CuCl (1
g; 10.1 mmol; 0.08 eq.) and the ethereal solution
of 14 (74.6 g; 170 mmol; 1.4 eq.) as described for
1H NMR (CDCl3): 0.95 (t; J =7.4 Hz; 8-H3),
1.6 (bt; J =5.1 Hz; OH), 2 (m; 7-H2), 2.75 (bt;
J =6.5 Hz; 4-H2), 4.2 (m; 1-H2), 5.25–5.7 (m;
2,3,5,6-H1).

O. Loreau et al. / Chemistry and Physics of Lipids 106 (2000) 65–78
71
g; 65.2 mmol; 61% yield) was obtained as a
colourless oil.
compound 3. The residue was purified by chro-
matography on silica gel with pentane/diethyl
ether (95/5) giving 31.9 g of a 79:21 mixture of
16a and 16b (96 mmol; 79% yield).
1H NMR (CDCl3): 0.95 (t; J =7.4 Hz; 17-H3),
1.2–1.45 (m; 3,4,5,6-H2), 1.95–2.1 (m; 7,16-H2),
2.65–2.85 (m; 10,13-H2), 3.36 (dt, J =9.5, 6.6 Hz;
1-Ha), 3.5 (m; 6-Ha (THP)), 3.71 (dt; J =9.5, 6.9
Hz; 1-Hb), 3.85 (m; 6-Hb (THP)), 4.56 (m; 2-H1
(THP)), 5.25–5.55 (m; 8,9,11,12,14,15-H1).
1H NMR (CDCl3): 0.95 (t, J =7.4 Hz, 17-H3
(16a)), 0.96 (t, J =7.4 Hz, 17-H3 (16b)), 1.25–1.9
(m; 2,3,4,5,6-H2 (16a and 16b); 3,4,5-H2 (THP)),
1.9–2.05 (m; 16-H2 (16a); 14-H2 (16b)), 2.1–2.25
(m, 7-H2 (16a); 7,11-H2 (16b)), 2.7 (m; 13-H2
(16a)), 2.9 (m; 10-H2 (16a)), 3.05 (m; 10-H1 (16b)),
3.36 (dt, J =9.5,6.6 Hz; 1-Ha (16a and 16b)), 3.5
(m; 6-Ha (THP)), 3.71 (dt; J =9.5, 6.9 Hz; 1-Hb
(16a and 16b)), 3.85 (m; 6-Hb (THP)), 4.56 (m;
2-H1 (THP)), 5.04 (d; J =10 Hz; 2-Hcis (16b:
vinyl)), 5.25 (d, J =17.5 Hz, 2-Htrans (16b: vinyl)),
5.3–5.55 (m; 11,12,14,15-H1 (16a); 12,13-H1
(16b)), 5.75 (ddd, J =17.5, 10, 6 Hz; 1-H1 (16b:
vinyl)).
2.18.1. 2-((E)-10-Vinyl-pentadec-12-en-8-
ynyloxy)-tetrahydro-pyran 16b:
1H NMR (CDCl3): 0.96 (t; J =7.4 Hz; 15-H3),
1.25–1.9 (m; 2,3,4,5,6-H2; 3,4,5-H2 (THP)), 1.95–
2.1 (m; 14-H2), 2.1–2.25 (m; 7,11-H2), 3 (m; 10-
H1), 3.36 (dt; J =9.5, 6.6 Hz; 1-Ha), 3.5 (m; 6-Ha
(THP)), 3.71 (dt; J =9.5, 6.9 Hz; 1-Hb), 3.85 (m;
6-Hb (THP)), 4.56 (m; 2-H1 (THP)), 5.04 (d;
J =10 Hz; 2-Hcis (vinyl)), 5.25 (d; J =17.5 Hz;
2-Htrans (vinyl)), 5.35–5.55 (m; 12,13-H1), 5.75
(ddd; J =17.5, 10, 6 Hz; 1-H1 (vinyl)).
GC/MS: column: HP-5MS (30 m×0.25 mm×
0.25 mm). Column temperature: 42°C (2 min) to
300°C (3 min) at 18°C/min. Carrier gas: He (0.9
ml/min). Injector temperature: 250°C. Compound
16b (r.t.=14.7 min; m/z= 332 M+), compound
17a (r.t.=15 min; m/z=334 M+), compound 17b
(r.t.=14.5 min; m/z= 334 M+).
GC/MS: column: HP-5MS (30 m×0.25 mm×
0.25 mm). Column temperature: 42°C (2 min) to
300°C (3 min) at 18°C/min. Carrier gas: He (0.9
ml/min). Injector temperature: 250°C. Compound
16a (r.t.=15.3 min; m/z=332 M+), compound
16b (r.t.=14.7 min; m/z= 332 M+).
Several purified materials were combined giving
a mixture of 16a and 16b (39.7 g; 119 mmol)
which was used for the next step.
2.18. 2-((8Z,11Z,14E)–Heptadeca-8,11,14-
trienyloxy)-tetrahydro-pyran 17a
2.19. (3E,6Z,9Z)-17-Bromo-heptadeca-3,6,9-
triene 18
A mixture of 16a and 16b (35.7 g; 107 mmol),
Lindlar catalyst (5% wt. Pd; 2.94 g), quinoline
(2.95 ml) and 225 ml of ethyl acetate were shaken
in hydrogen. After the absorption of hydrogen
had ceased, the catalyst was removed and the
mixture was concentrated. Purification on silica
gel with pentane/diethyl ether (95/5) gave a mix-
ture of 17a and 17b and an analytical sample of
pure 16b. Pure compound 17a was obtained by
successive silver ion flash chromatography runs
(silica gel: 300 g; AgNO3: 60 g) with a pentane/di-
ethyl ether gradient ((95/5): 2 l; (85/15): 1 l;
diethyl ether: 1 l). Traces of silver nitrate were
removed by washes with saturated sodium chlo-
ride solution and elution through silica gel with
pentane/diethyl ether (95/5). Compound 17a (21.8
Compound 17a (21.3 g; 63.7 mmol) in 65 ml of
CH2Cl2 was added at 0°C to a slurry of dibromot-
riphenylphosphorane (57.3 g; 136 mmol) in 325
ml of CH2Cl2. After the addition, the mixture was
stirred at room temperature for 1 h, washed with
a saturated solution of sodium bicarbonate, then
with water, dried over Na2SO4, filtered and con-
centrated. The residue was purified by flash chro-
matography on silica gel with pentane. Bromide
18 (18.3 g, 58.5 mmol, 91% yield) was obtained as
a colourless oil.
1H NMR (CDCl3): 0.96 (t; J =7.4 Hz; 1-H3),
1.2–1.5 (m; 12,13,14,15-H2), 1.84 (quint; J =6.9
Hz; 16-H2), 1.95–2.15 (m; 2,11-H2), 2.65–2.85 (m;
5,8-H2), 3.4 (t, J =6.9 Hz, 17-H2), 5.25–5.55 (m;
3,4,6,7,9,10-H1).

72
O. Loreau et al. / Chemistry and Physics of Lipids 106 (2000) 65–78
2.20. (9Z,12Z,15E)-[1-13C]-Octadeca-9,12,15-
trienoic acid 19
MS (DCI/NH3): m/z (%): 296 (3.4), 297 (100),
298 (26.9), 299 (3.8). Isotopic enrichment: 97.4%.
A total of 12.5 g of compound 19 contaminated
with 9% (RP-HPLC, see below for experimental
conditions) of a geometrical isomer with two trans
double bonds were obtained as a pale yellow oil
from the bromide 18 (18.3 g; 58.5 mmol) as
described for acid 7. Successive preparative re-
versed-phase chromatography runs using 1-g sam-
ples with 200 g of Hyperprep 100A HS C18 10m
using acetonitrile/water/acetic acid (75/25/0.5) as
mobile phase gave pure acid 19 (6.3 g; 22.6 mmol,
38% yield from compound 18).
TLC: pentane/diethyl ether/acetic acid (90/10/
1). Rf=0.3 (visualisation with an anisaldehyde
solution).
1H NMR (CDCl3): 0.95 (t; J =7.4 Hz; 18-H3),
1.2–1.4 (m; 4,5,6,7-H2), 1.6 (m; 3-H2), 1.9–2.1 (m;
8,17-H2), 2.34 (dt; J =7.3, 7.3 Hz, 2-H2), 2.65–
2.21. (9Z,12Z)-Octadeca-9,12-dienoyl chloride 20
(9Z,12Z)-Octadeca-9,12-dienoic acid (50 g; 178
mmol) and 400 ml of anhydrous toluene were
placed in an oven-dried 2-l three-necked flask
equipped with a magnetic stirrer, a thermometer
and a gas absorption trap containing 2 N NaOH.
Freshly distilled oxalyl chloride (86 ml; 980 mmol;
5.5 eq.) was added dropwise at 2°C and the yellow
solution was stirred overnight at room tempera-
ture. The solution was then concentrated. Then
3×100 ml of anhydrous toluene were added to
the crude acyl chloride 20 and evaporated under
vacuum to remove traces of oxalyl chloride. The
residue (53 g of 20; 177 mmol; 99% yield) was
used without further purification.
2.85
(m;
14,11-H2),
5.25–5.55
(m;
2.22. (6Z,9Z)-17-Bromo-heptadeca-6,9-diene 21a
and (6Z,9Z)-17-chloro-heptadeca-6,9-diene 21b
9,10,12,13,15,16-H2).
13C NMR (CDCl3): 13.57 (C18), 24.42 (C3),
25.32 (C11 and C17), 26.94 (C8), 28.82 and 28.88
(C4, C5 and C6), 29.32 (C7), 30.17 (C14), 33.49
and 34.22 (J =55.7 Hz, C2), 126.82 (C15), 127.58
(C10), 127.75 (C13), 128.25 (C12), 129.93 (C9),
132.25 (C16), 180.13 (C1).
HPLC: column: Zorbax C18 SB (250×4.6
mm). Solvent system: acetone/methanol/water/
acetic acid (45/27.5/27.5/0.055). Flow rate: 1.5
ml/min. Chemical purity: (9Z,12Z,15Z)-[1-13C]-
4-(Dimethylamino)pyridine (1.74 g; 14.2 mmol;
0.1 eq.), 2-mercaptopyridine N-oxide, sodium salt
(26.5 g; 178 mmol; 1.2 eq.) and 610 ml of bro-
motrichloromethane were introduced into an
oven-dried 2-l three-necked flask equipped with a
magnetic stirrer, a reflux condenser and an oil
bubbler. The mixture was heated to reflux and 20
(43.8 g; 146 mmol) in 220 ml of bro-
motrichloromethane was added dropwise. After
the addition, the mixture was heated to reflux for
1 h and then cooled to room temperature. The
reaction mixture was diluted with 500 ml of di-
ethyl ether and 500 ml of saturated sodium chlo-
ride solution. The aqueous layer was extracted
twice with diethyl ether. The combined ethereal
fractions were washed with water, dried over
MgSO4, filtered and concentrated. The residue
was chromatographed on silica gel with pentane
giving 22.8 g of a mixture of 21a and 21b in 50%
yield ((21a: 20.8 g, 66 mmol); (21b: 2 g, 7 mmol)).
GC: column: Megabore DB5 (30 m×0.53
mm×1.5 mm). Column temperature: 150–250°C
(8°C/min). Injector and detector temperature:
250°C. r.t.(21a)=10.1 min, r.t.(21b)=8.9 min.
linolenic acid (r.t.=
30.2 min; 0.7%),
(9Z,12Z,15E)-[1-13C]-linolenic acid 19 (r.t.=31.4
min; 97.6%), [1-13C]-18:3 fatty acid with two trans
double bonds (r.t.=34.2 min; 1.7%).
GC/MS: column: HP-23 cis/trans FAME (60
m×0.25 mm×0.25 mm). Column temperature:
200°C. Carrier gas: He (1 ml/min). Injector tem-
perature: 250°C. Identification (methyl ester
derivative): [1-13C]-18:3 fatty acid with two trans
double bonds, methyl ester (r.t.=10.5 min; m/
z=293 M+), (9Z,12Z,15E)-[1-13C]-linolenic acid,
methyl ester (r.t.=10.8 min; m/z=293 M+),
(9Z,12Z,15Z)-[1-13C]-linolenic acid, methyl ester
(r.t.=11.2 min; m/z=293 M+).

O. Loreau et al. / Chemistry and Physics of Lipids 106 (2000) 65–78
73
1H NMR (CDCl3): 0.9 (t; J =6.9 Hz; 1-H3,
(21a and 21b)), 1.2–1.5 (m; 2,3,4,12,13,14,15-H2,
(21a and 21b)), 1.75 (quint.; J =6.9 Hz; 16-H2,
(21b)), 1.85 (quint.; J =6.9 Hz; 16-H2, (21a)),
1.9–2.1 (m; 5,11-H2, (21a and 21b)), 2.77 (bt;
J =5.9 Hz; 8-H2, (21a and 21b)), 3.4 (t; J =6.8
Hz; 17-H2 (21a)), 3.52 (t; J =6.8 Hz; 17-H2 (21b)),
5.25–5.45 (m; 6,7,9,10-H1,(21a and 21b)).
Previous attempts were made to optimise the
formation of 21a and the different purified mix-
tures of 21a and 21b were combined to give 39.4
g of halides ((21a: 29.4 g, 93.2 mmol); (21b: 10 g,
37.1 mmol)) which were used for the preparation
of the [1-13C] labelled acid.
TLC: pentane/diethyl ether/acetic acid (85/15/
1). Rf=0.33 (visualisation with an anisaldehyde
solution).
1H NMR (CDCl3): 0.9 (t; J =6.9 Hz; 18-H3),
1.2–1.4 (m; 4,5,6,7,15,16,17-H2), 1.62 (m; 3-H2),
1.95–2.1 (m; 8,14-H2), 2.34 (dt; J =7.3, 7.3 Hz,
2-H2), 2.77 (bt; J =5.9 Hz; 11-H2), 5.25–5.45 (m;
9,10,12,13-H2).
13C NMR (CDCl3): 13.82 (C18), 22.33 (C17),
24.41 (C3), 25.39 (C11), 26.95 (C8 and C14),
28.83 and 28.89 (C4, C5 and C6), 29.12 (C15),
29.34 (C7), 31.29 (C16), 33.49 and 34.22 (J =55.7
Hz, C2), 127.66 (C12), 127.83 (C10), 129.76 (C9),
129.95 (C13), 180.26 (C1).
HPLC: column: Zorbax SB C18 (250×4.6
mm). Solvent system: acetone/methanol/water/
acetic acid (60/20/20/0.4). Flow rate: 1.5 ml/min.
Chemical purity: (9Z,12Z)-[1-13C]-linoleic acid 22
(r.t.=14 min; \99%).
2.23. (9Z,12Z)-[1-13C]-Octadeca-9,12-dienoic acid
22
Compound 22 (31.5 g; 112 mmol; 86% yield
from (21a and 21b)) was obtained as a yellow oil
containing conjugated acids from the mixture of
21a and 21b as described for the acid 7.
MS (DCI/NH3): m/z (%): 299 (100), 300 (18.1),
301 (10.4). Isotopic enrichment: 99%.
TLC: pentane/diethyl ether/acetic acid (85/15/
1). Rf=0.33 (UV-active spot (254 nm)).
2.25. (9Z,12Z,15Z)-[1-13C]-Octadeca-9,12,15-
trienoic acid 23
GC: column: Stalbiwax-DA (30 m×0.53
mm×0.25 mm). Column temperature: 150–250°C
(8°C/min). Injector and detector temperature:
250°C. Chemical purity: 22 (r.t.=12 min; 96.6%),
conjugated acids (r.t.=12.7 min; 3.4%)
Pure acid 23 (10.4 g; 37 mmol) free of conju-
gated acids was prepared in 18% overall yield
from (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid
(57.5 g; 206 mmol) by using the procedure de-
scribed for compound 22.
TLC: pentane/diethyl ether/acetic acid (90/10/
1). Rf=0.3 (visualisation with an anisaldehyde
solution).
2.24. Chemical purification of 22
Compound 22 (31.5 g; 112 mmol) containing
conjugated acids, maleic anhydride (43.9 g; 448
mmol; 4 eq.) and 240 ml of anhydrous o-xylene
were placed in an oven-dried 1-l three-necked
flask equipped with a magnetic stirrer and a reflux
condenser. The mixture was heated to reflux for
4.5 h, then cooled to room temperature and di-
luted with 330 ml of diethyl ether. Hydrolysis with
230 ml of water, addition of LiOH until the pH
was basic and acidification to pH 3 with 4 N HCl
gave a mixture which was extracted three times
with diethyl ether. The combined ethereal frac-
tions were washed with water, dried over MgSO4,
filtered and concentrated. The crude pale yellow
oil was chromatographed on silica gel with pen-
tane/diethyl ether/acetic acid (85/15/1) giving pure
acid 22 (23.5 g; 84 mmol; 75% yield).
1H NMR (CDCl3): 0.96 (t; J =7.4 Hz; 18-H3),
1.2–1.4 (m; 4,5,6,7-H2), 1.6 (m; 3-H2), 2–2.15 (m;
8,17-H2), 2.34 (dt; J =7.3, 7.3 Hz, 2-H2), 2.75–
2.85 (m; 14,11-H2), 5.2–5.45 (m; 9,10,12,13,15,16-
H2).
13C NMR (CDCl3): 14.04 (C18), 20.33 (C17),
24.42 (C3), 25.30 (C14), 25.39 (C11), 26.98 (C8),
28.85 and 28.92 (C4, C5 and C6), 29.35 (C7),
33.52 and 34.25 (J =55.3 Hz, C2), 126.89 (C15),
127.53 (C10), 128 (C12 and C13), 129.95 (C9),
131.66 (C16), 180.39 (C1).
HPLC: column: Zorbax SB C18 (250×4.6
mm). Solvent system: acetone/methanol/water/
acetic acid (50/25/25/0.125). Flow rate: 1.5 ml/
min. Chemical purity: \99%.

74
O. Loreau et al. / Chemistry and Physics of Lipids 106 (2000) 65–78
MS (DCI/NH3): m/z (%): 296 (0.2), 297 (100),
298 (17.5), 299 (4.4). Isotopic enrichment: 99%.
oleic acid (0.1%) were found to be present. One
impurity (0.4%) was identified as a 18:2 fatty acid
but its complete structure remains unknown.
3. Results and discussion
3.2. Synthesis of (9Z,12Z,15E)-[1-13C]-octadeca-
9,12,15-trienoic acid 19 (Figs. 3 and 4)
3.1. Synthesis of [1-13C]-(9Z,12E)-octadeca-9,12-
dienoic acid 7 (Fig. 2)
In a first synthetic scheme, we tried to prepare
(E)-heptadeca-14-ene-8,11-diyn-1-ol 11 on a small
scale by using a copper-catalysed coupling of the
terminal acetylene 2 with the propargylic bromide
The synthesis of the mono trans dienoic acid 7
used (E)-oct-2-en-1-ol and 7-bromoheptan-1-ol as
starting materials.
The trans octenol was converted to its corre-
sponding allylic bromide 1 with phosphorus tri-
bromide in 83% yield (van Liemt et al., 1994) and
the 7-bromoheptan-1-ol was transformed in 45%
yield to non-8-yn-1-ol 2 by reaction with lithium
acetylide, ethylenediamine complex (Porwoll and
Leete, 1985). Compound 2 was treated with
methylmagnesium chloride and coupled with a
molar excess of 1 in the presence of a catalytic
amount of Cu(I)Cl giving the enyne 3 in 80%
yield (Snider and Burbaum, 1983). Partial hydro-
genation of 3 over Lindlar’s catalyst poisoned
with quinoline in ethyl acetate (Rakoff, 1990)
afforded the dienol 4 (97% yield) which was con-
verted into the bromide 5 with PBr3 (31% yield).
As we obtained an unsatisfying yield with phos-
phorus tribromide, dibromotriphenyl phospho-
rane was used with good yield as bromination
reagent at the similar step during the synthesis of
(9Z,12Z,15E)-[1-13C]-octadeca-9,12,15-trienoic
acid. Reaction of 5 with K13CN in DMSO at
80°C provided the nitrile 6 in 87% yield. (9Z,12E)-
[1-13C]-Octadeca-9,12-dienoic acid 7 was obtained
by alkaline hydrolysis of 6 with 40% KOH in
ethanol at 80°C (83% yield). After discoloration
of the acid 7 with activated charcoal, its chemical
purity was determined by RP-HPLC as 97.7%
(isotopic enrichment: 97.4%). NMR data were in
agreement with those reported in the literature for
the methyl ester derivative (Bus et al., 1976;
Berdeaux et al., 1995). The main impurities were
identified by RP-HPLC on an (9Z,12E)-[1-13C]-
linoleic acid sample and GC/MS on its methyl
ester derivative by comparison with authentic
samples. (9Z,12Z)-[1-13C]-Linoleic acid (0.9%),
(9E,12E)-[1-13C]-linoleic acid (0.9%), (9Z)-[1-13C]-
Fig. 2. Synthesis of (9Z,12E)-[1-13C]-octadeca-9,12-dienoic
acid.

O. Loreau et al. / Chemistry and Physics of Lipids 106 (2000) 65–78
75
for 8. In Section 3.1, we described the synthesis of
(9Z,12E)-[1-13C]-linoleic acid via the coupling of 8
with 2.3 equivalents of (E)-1-bromo-oct-2-ene.
Here, compound 2 was coupled with only one
equivalent of propargylic bromide 10 under mild
conditions in the presence of copper(I) iodide,
sodium iodide and potassium carbonate (Lapit-
skaya et al., 1993; Durand et al., 1998). The
alcohol 11 was obtained in 67% yield but was
found to be unstable during purification by flash
chromatography on silica gel or preparative RP-
HPLC on Partisil ODS 3. Moreover, Lindlar re-
duction of purified compound 11 furnished the
trienol 12 in only 32% yield.
10. Compound 11 could give the expected bromo
intermediate 18 after partial hydrogenation and
bromination (Fig. 3).
Prop-2-yn-1-ol was treated with methylmagne-
sium bromide in the presence of a catalytic
amount of copper(I) chloride and coupled with
1.5 equivalents of (E)-1-bromo-pent-2-ene 8 (com-
pound 8 was previously synthesised in 84% yield
from (E)-pent-2-en-1-ol using phosphorus tribro-
mide as bromination reagent). After purification,
the alcohol 9 was obtained in 94% yield and then
transformed into its bromo derivative 10 with
PBr3 (61% yield) by using the procedure described
So we decided to modify our synthetic pathway
(Fig. 4).
Partial hydrogenation of (E)-oct-5-en-2-yn-1-ol
9 over Lindlar’s catalyst poisoned with quinoline
in ethanol (Dogra et al., 1989) afforded the dienol
13 (88% yield) which was converted into its
bromo derivative 14 with PBr3 (94% yield). Sev-
eral attempts were made with terminal acetylenes
2 and 15 in order to reduce the molar excess of 14
used during the copper-catalysed coupling. The
best results were obtained with the protected alco-
hol 15. After coupling of compound 15 with 1.4
equivalents of the allylic bromide 14, the crude
residue was purified by flash chromatography on
silica gel giving a 79:21 mixture of 1,1 and 1,3-
coupling product 16a and 16b (79% yield). Lind-
lar reduction of this mixture furnished a residue
containing the trienes 17a and 17b and a small
amount of 16b. This residue was first purified by
flash chromatography on silica gel giving a mix-
ture of trienes 17a and 17b and an analytical
sample of pure 16b. Compound 17a was obtained
after purification by successive silver-ion flash
chromatography runs (61% yield) and trans-
formed into its corresponding bromide 18 (91%
yield) with dibromo triphenylphosphorane
(Rakoff, 1982; Eynard et al., 1994). Then
(9Z,12Z,15E)-octadeca-9,12,15-trienoic acid 19
(chemical purity: 90% (RP-HPLC); 76% yield
from bromide 18) was prepared by using the
procedure described for the synthesis of the acid
7. GC/MS was then performed on its methyl ester
derivative. The main impurity (9% (RP-HPLC))
was identified as a geometrical isomer with two
Fig. 3. Synthesis of (3E,6Z,9Z)-17-bromo-heptadeca-3,6,9-
triene via (E)-heptadec-14-ene-8,11-diyn-1-ol.

76
O. Loreau et al. / Chemistry and Physics of Lipids 106 (2000) 65–78
Fig. 4. Synthesis of (9Z,12Z,15E)-[1-13C]-octadeca-9,12,15-trienoic acid.
trans double bonds by comparison with a com-
mercial mixture of eight geometrical isomers of
linolenic acid, methyl ester. Purification by succes-
sive preparative reversed-phase chromatography
runs on a gram scale gave (9Z,12Z,15E)-[1-13C]-
linolenic acid 19 (chemical purity: 97.6%; isotopic
enrichment: 97.4%) in 38% yield from bromide 18.
1H and 13C NMR data were in agreement with
those reported for methyl ester (Rakoff and
Emken, 1982; Eynard et al., 1994). (9Z,12Z,15Z)-
[1-13C]-Linolenic acid (0.7%) and the 18:3 fatty
acid with two trans double bonds previously iden-
tified (1.7%) were found to be present as
impurities.
3.3. Synthesis of (9Z,12Z)-[1-13C]-octadeca-9,12-
dienoic acid 22 and (9Z,12Z,15Z)-[1-13C]-
octadeca-9,12,15-trienoic acid 23 (Fig. 5)
Commercially available (9Z,12Z)-octadeca-
9,12-dienoic acid was transformed into its corre-
sponding acyl chloride derivative 20 with freshly
distilled oxalyl chloride in anhydrous toluene. Ac-
cording to the procedure of Barton et al. (1983),
20 was converted to a mixture of bromide 21a and
chloride 21b.
Several attempts were made to optimise the
formation of 21a and we noted that we had a very
low yield (7–13%) and a sharp increase in the

O. Loreau et al. / Chemistry and Physics of Lipids 106 (2000) 65–78
77
(9Z,12Z,15Z)-[1-13C]-Linolenic acid 23 free of
conjugated acids was prepared in 18% overall
yield by using the synthetic pathway described for
(9Z,12Z)-[1-13C]-linoleic acid 22. Chemical purity
of 23 was found to be \99% by RP-HPLC
(isotopic enrichment: 99%). 13C NMR data were
in agreement with those previously obtained for
methyl ester (Rakoff, 1986).
In conclusion, we have developed practical and
efficient large-scale syntheses giving [1-13C] la-
belled (9Z,12E)-, (9Z,12Z)-linoleic acid and
(9Z,12Z,15E)-, (9Z,12Z,15Z)-linolenic acid of
high chemical and isomeric purities (\97%). Our
results show that both the ‘acetylenic route’ using
copper-catalysed coupling and Barton’s bromo-
decarboxylation are useful alternatives for the
preparation of several grams of (9Z,12E)-,
(9Z,12Z)-[1-13C]-linoleic acid and (9Z,12Z,15E)-,
(9Z,12Z,15Z)-[1-13C]-linolenic acid intended for
human studies. Indeed, by-products formed dur-
ing these syntheses could be easily eliminated by
either chromatography or chemical purification
and all the main impurities (\0.5%) present in
the labelled fatty acids could be characterised.
amount of 21b (34–40% of 21a; 60–66% of 21b
(wt.)) when we used concentrated reaction mix-
tures. Using more dilute conditions, we obtained
a mixture of 21a and 21b (50% yield) containing
91.2% (wt.) of 21a. Different purified mixtures of
bromide and chloride compounds were combined
giving a mixture of 21a and 21b (74.6% (wt.) of
21a) which was transformed into [1-13C]-linoleic
acid 22 by using the procedure described for the
synthesis of the mono trans isomers. Unfortu-
nately, compound 22 was contaminated by a
small amount of conjugated acids which were
formed during the alkaline hydrolysis of the ni-
trile intermediate and detected by their UV-active
spot on TLC. These conjugated acids were trans-
formed into adducts by using a Diels Alder reac-
tion with maleic anhydride in refluxing o-xylene
(Viala and Labaudinie`re, 1993). These adducts
were easily eliminated by flash chromatography
on silica gel and pure acid 22 free of conjugated
acids was obtained in 75% yield. The chemical
purity of 22 was found to be \99% by RP-
HPLC (isotopic enrichment: 99%). 13C NMR data
were in agreement with those described for the
methyl ester derivative (Bus et al., 1976).
Acknowledgements
The authors thank M. Alain Valleix (CEA/
Saclay, Service des Mole´cules Marque´es) for
analyses. This work was supported in part by a
grant (No. FAIR 95-0594) from the Commission
of the European Communities.
References
Ackman, R.G., Mag, T.K., 1998. trans Fatty acids and the
potential for less in technical products. In: Se´be´dio, J.L.,
Christie, W.W (Eds.), trans Fatty Acids in Human Nutri-
tion. The Oily Press Ltd., Dundee, Scotland, pp. 35–58.
Barton, D.H.R., Crich, D., Motherwell, W.B., 1983. A practi-
cal alternative to the Hunsdiecker reaction. Tetrahedron
Lett. 24, 4979–4982.
Berdeaux, O., Vate`le, J.P., Eynard, T., Nour, M., Poullain, D.,
Noel, J.P., Se´be´dio, J.L., 1995. Synthesis of (9Z,12E)- and
(9E,12Z)-[1-14C] linoleic acid and (5Z,8Z,11Z,14E)-[1-14C]
arachidonic acid. Chem. Phys. Lipids 78, 71–80.
Bergstrom, S., Paabo, K., Rottenberg, M., 1952. Preparation
of carboxylabelled oleic acid. Acta Chem. Scand. 6, 1127–
1128.
Fig. 5. Synthesis of (9Z,12Z)-[1-13C]-octadeca-9,12-dienoic
acid.

78
O. Loreau et al. / Chemistry and Physics of Lipids 106 (2000) 65–78
Bre´tillon, L., Chardigny, J.M., Noe¨l, J.P., Se´be´dio, J.L.,
1998a. Desaturation and chain elongation of [1-14C] mono-
trans isomers of linoleic and a linolenic acids in perfused
rat liver. J. Lipid Res. 39, 2228–2236.
Porwoll, J.P., Leete, E., 1985. Synthesis of [5,6-13C2,1-14C]
olivetolic acid, methyl [1’-13C] olivetolate and [5,6-13C2,1-
14C] cannabigerolic acid. J. Label. Compd. Radiopharm.
22, 257–271.
Bre´tillon, L., Chardigny, J.M., Se´be´dio, J.L., Poullain, D.,
Noe¨l, J.P., Vate`le, J.M., 1998b. Oxidative metabolism of
[1-14C] mono-trans isomers of linoleic and a linolenic acids
in the rat. Biochim. Biophys. Acta 1390, 207–214.
Bus, J., Sies, I., Lie Ken Jie, M.S.F., 1976. 13C NMR of
methyl, methylene and carbonyl carbon atoms of methyl
alkenoates and alkynoates. Chem. Phys. Lipids 17, 501–
518.
Rakoff, H., 1982. Preparation of fatty acids and esters con-
taining deuterium. Prog. Lipid Res. 21, 225–254.
Rakoff, H., 1986. Syntheses of deuterated methyl 9,15-oc-
tadecadienoate and methyl 9,12,15-octadecatrienoate geo-
metric isomers. J. Label. Compd. Radiopharm. 23,
699–713.
Rakoff, H., 1990. Preparation of deuterated methyl 6,9,12-oc-
tadecatrienoates
and
methyl
6,9,12,15-octadecate-
Campbell, J.R., Clapp, C.H., 1989. A new method for the
preparation of carboxy-labeled unsaturated fatty acids and
its application to linoleic acid. Bioorg. Chem. 17, 281–286.
Channing, M.A., Simpson, N, 1993. Radiosynthesis of 1-[11C]
polyhomoallylic fatty acids. J. Label. Compd. Radio-
pharm. 33, 541–546.
Chardigny, J.M., Wolff, R.L., Mager, E., Bayard, C.C., Se´be´-
dio, J.L., Martine, L., Ratnayake, W.M.N., 1996. Fatty
acid composition of French infant formulas with emphasis
on the content and detailed profile of trans fatty acids. J.
Am. Oil Chem. Soc. 73, 1595–1601.
Dogra, V., Sabharwal, A., Sharma, S., Huq, M.A., Kad, G.L.,
Vig, O.P., 1989. Synthesis of highly unsaturated insect sex
pheromones. Synthesis of (3Z,6Z,9Z)-(3,6,9)-nonadeca-
triene and (3Z,6Z,9Z)-3,6,9-heneicosatriene. J. Indian
Chem. Soc. 66, 169–171.
Durand, S., Parrain, J.L., Santelli, M., 1998. A large scale and
concise synthesis of g-linolenic acid from 4-chlorobut-2-yn-
1-ol. Synthesis, 1015–1018.
Eynard, T., Vate`le, J.M., Poullain, D., Noe¨l, J.P., Chardigny,
J.M., Se´be´dio, J.L., 1994. Synthesis of (9Z,12Z,15E)- and
(9E,12Z,15Z)-octadecatrienoic acids and their [1-14C]-radi-
olabelled analogs. Chem. Phys. Lipids 74, 175–184.
Eynard, T., Poullain, D., Vate`le, J.M., Noe¨l, J.P., Chardigny,
J.M., Se´be´dio, J.L, 1998. Synthesis of methyl
(5Z,8Z,11Z,14Z,17Z)- and (5Z,8Z,11Z,14Z,17E)-[18-14C]
eicosapentaenoate. J. Label. Compd. Radiopharm. 41,
411–421.
traenoates. Lipids 25, 130–134.
Rakoff, H., Emken, E.A., 1982. Synthesis and properties of
methyl 9,12,15-octadecatrienoate geometric isomers.
Chem. Phys. Lipids 31, 215–225.
Ratnayake, W.M.N., Chardigny, J.M., Wolff, R.L., Bayard,
C.C., Se´be´dio, J.L., Martine, L., 1997. Essential fatty acids
and their trans geometrical isomers in powdered and liquid
infant formulas sold in Canada. J. Pediatr. Gastroenterol.
Nutr. 25, 400–407.
Se´be´dio, J.L., Grandgirard, A., Pre´vost, J, 1988. Linoleic acid
isomers in heat treated sunflower oils. J. Am. Oil Chem.
Soc. 65, 362–366.
Se´be´dio, J.L., Chardigny, J.M., 1998. Biochemistry of trans
polyunsaturated fatty acids. In: Se´be´dio, J.L., Christie,
W.W. (Eds.), trans Fatty Acids in Human Nutrition. The
Oily Press Ltd., Dundee, Scotland, pp. 191–216.
Snider, B.B., Burbaum, B.W., 1983. Intramolecular Diels-
Alder reactions of alkenylallenes. A model study for the
bottom half of chlorothricolide. J. Org. Chem. 48, 4370–
4374.
Stoffel, W., 1964. Synthese von [1-14C]-markierten all-cis-
polyenfettsa¨uren. Liebigs Ann. Chem. 673, 26–36.
Tulloch, A.P., 1979. Synthesis of deuterium and carbon-13
labelled lipids. Chem. Phys. Lipids 24, 391–406.
van Liemt, W.B.S., Steggerda, W.F., Esmeijer, R., Lugten-
burg, J., 1994. Synthesis and spectroscopic characterisation
of 13C-labelled ubiquinone-0 and ubiquinone-10. Recl.
Trav. Chim. Pays-Bas 113, 153–161.
Grandgirard, A., Se´be´dio, J.L., Fleury, J., 1984. Geometrical
isomerization of linolenic acid during heat treatment of
vegetable oils. J. Am. Oil Chem. Soc. 61, 1563–1568.
Howton, D.R., Davis, R.H., Nevenzel, J.C., 1954. Unsatu-
rated fatty acids. III. preparation of 1-C14-linoleic acid. J.
Am. Chem. Soc. 76, 4970–4974.
Viala, J., Labaudinie`re, R., 1993. Synthesis of a regioselec-
tively hexadeuterated linoleic acid. J. Org. Chem. 58,
1280–1283.
Wolff, R.L., 1993. Occurrence of artificial trans polyunsatu-
rated fatty acids in refined walnut oils. Sci. Aliment. 13,
155–163.
Lapitskaya, M.A., Vasiljeva, L.L., Pivnitsky, K.K., 1993. A
chemoselective synthesis of functionalized 1,4-alkadiynes
(skipped diacetylenes). Synthesis, 65–66.
Wolff, R.L., Se´be´dio, J.L., 1991. Geometrical isomers of lino-
lenic acid in low-calorie spreads marketed in France. J.
Am. Oil Chem. Soc. 68, 719–725.
.