Sequential substitution of 1,2-dichloro-ethene: A convenient stereoselective route to (9Z,11E)-, (10E,12Z)- and (10Z,12Z)-[1-14C] conjugated linoleic acid isomers

Article abstract of DOI:10.1016/S0009-3084(00)00229-2

Conjugated linoleic acid (CLA) isomers are present in human foods derived from milk or ruminant meat. To study their metabolism, (9Z,11E)-, (10E,12Z)- and (10Z,12Z)-[1-¹⁴C]-octadecadienoic acids with high radiochemical and isomeric purities (> 98%) were prepared by stereoselective multi-step syntheses involving sequential substitution of 1,2-dichloro-ethene. In the case of the (9Z,11E) isomer, a first metal-catalyzed cross-coupling reaction between (E)-1,2-dichloro-ethene and 2-non-8-ynyloxy-tetrahydro-pyran, obtained from 7-bromo-heptan-1-ol, gave a conjugated chloroenyne. A second coupling reaction with hexylmagnesium bromide provided a heptadecenynyl derivative. Stereoselective reduction of the triple bond and bromination afforded (7E,9Z)-17-bromo-heptadeca-7,9-diene. Formation of the Grignard reagent and carbonation with ¹⁴CO₂ gave (9Z,11E)-[1-¹⁴C]-octadeca-9,11-dienoic acid (overall yield from 7-bromo-heptan-1-ol, 14.4%). (10E,12Z)- and (10Z,12Z)-[1-¹⁴C]-octadeca-10,12-dienoic acids were synthesized by the same methodology using 1-heptyne, 8-bromo-octan-1-ol and, respectively, (E)-1,2-dichloro-ethene and its (Z) isomer (overall yield from 8-bromo-octan-1-ol, 13.1% (10E,12Z); 17.2% (10Z,12Z)). Impurities (< 2% if present) were identified as being (E,E) CLA isomers and were removed by RP-HPLC. Metabolism studies in animal are in progress.

Full text of DOI:10.1016/S0009-3084(00)00229-2

Chemistry and Physics of Lipids
110 (2001) 57–67
www.elsevier.com/locate/chemphyslip
Sequential substitution of 1,2-dichloro-ethene: a convenient
stereoselective route to (9Z,11E)-, (10E,12Z)- and
(10Z,12Z)-[1-14C] conjugated linoleic acid isomers
O. Loreau a,*, A. Maret a, J.M. Chardigny b, J.L. Se´be´dio b, 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, BP 86510, 17 rue Sully, F-21065 Dijon Cedex, France
Received 30 October 2000; received in revised form 28 November 2000; accepted 7 December 2000
Abstract
Conjugated linoleic acid (CLA) isomers are present in human foods derived from milk or ruminant meat. To study
their metabolism, (9Z,11E)-, (10E,12Z)- and (10Z,12Z)-[1-14C]-octadecadienoic acids with high radiochemical and
isomeric purities (\98%) were prepared by stereoselective multi-step syntheses involving sequential substitution of
1,2-dichloro-ethene. In the case of the (9Z,11E) isomer, a first metal-catalyzed cross-coupling reaction between
(E)-1,2-dichloro-ethene and 2-non-8-ynyloxy-tetrahydro-pyran, obtained from 7-bromo-heptan-1-ol, gave a conju-
gated chloroenyne. A second coupling reaction with hexylmagnesium bromide provided a heptadecenynyl derivative.
Stereoselective reduction of the triple bond and bromination afforded (7E,9Z)-17-bromo-heptadeca-7,9-diene. Forma-
tion of the Grignard reagent and carbonation with 14CO2 gave (9Z,11E)-[1-14C]-octadeca-9,11-dienoic acid (overall
yield from 7-bromo-heptan-1-ol, 14.4%). (10E,12Z)- and (10Z,12Z)-[1-14C]-octadeca-10,12-dienoic acids were synthe-
sized by the same methodology using 1-heptyne, 8-bromo-octan-1-ol and, respectively, (E)-1,2-dichloro-ethene and its
(Z) isomer (overall yield from 8-bromo-octan-1-ol, 13.1% (10E,12Z); 17.2% (10Z,12Z)). Impurities (B2% if present)
were identified as being (E,E) CLA isomers and were removed by RP-HPLC. Metabolism studies in animal are in
progress. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Carbon 14; Conjugated linoleic acid (CLA); Cross-coupling reaction; Dichloro-ethene; Octadecadienoic; Stereoselective
bonds. CLA isomers are present naturally in milk
fat and ruminant meat (Parodi, 1977; Shantha et
al., 1994). Consequently, they are found in human
foods and their physiological properties and
metabolic pathways are thus of great interest.
Recently, potent beneficial effects of CLAs, such
as anticarcinogenic and antiatherogenic activities
(Nicolosi et al., 1993; Lee et al., 1994; Belury,
1. Introduction
Conjugated linoleic acid (CLA) is a collective
term, which designates positional and geometrical
isomers of linoleic acid with conjugated double
* Corresponding author. Tel.: +33-(0)1-69087596; fax: +
33-(0)1-69087991.
E-mail address: olivier.loreau@cea.fr (O. Loreau).
0009-3084/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0009-3084(00)00229-2

58
O. Loreau et al. / Chemistry and Physics of Lipids 110 (2001) 57–67
1995; Ip et al., 1999), and ability to reduce body
fat (West et al., 1998; Park et al., 1999), have been
reported in animals. However, the mechanisms by
with these fatty acids may act are still unclear. To
investigate further the metabolism of (9Z,11E)-
and (10E,12Z)-octadecadienoic acids, which are
the two major CLA isomers, we have prepared
their individual [1-14C] analogs with high isomeric
and radiochemical purities.
Some publications have reported the synthesis
of CLAs as free acids or methyl ester derivatives.
A complex mixture containing CLAs was ob-
tained from linoleic acid by alkaline isomerization
(Nichols et al., 1951). (9Z,11E)- and (9E,11E)-oc-
tadecadienoate methyl esters labeled with deu-
terium were synthesized as a mixture via a Wittig
reaction and the two compounds were separated
by a combination of reversed-phase and silver
resin chromatography (Adlof, 1997). Substantial
amounts of methyl (9Z,11E)-octadeca-9,11-
dienoate in a relatively pure form (83% purity)
were prepared from methyl ricinoleate (Berdeaux
et al., 1997). Chen and Sih have obtained highly
purified (9Z,11E)- and (10E,12Z)-CLAs from a
mixture using a chemoenzymatic synthesis (Chen
and Sih, 1998). Purcell and co-workers have also
described the preparation of (10Z,12Z)-octadeca-
10,12-dienoic acid but no yield was given (Purcell
et al., 1966). Unfortunately, all these syntheses
were unsatisfactory for the synthesis of pure [1-
14C] CLA isomers.
In this report, we describe the stereocontrolled
preparation of (Z,E)- or (Z,Z)-heptadecadienyl-
bromo precursors of CLAs by using the sequen-
tial substitution of (E)- or (Z)-1,2-dichloro-ethene
which was already employed for the synthesis of
conjugated trienes (Ratovelomanana and Lin-
strumelle, 1984; Chemin and Linstrumelle, 1994;
Alami et al., 1995). A first cross-coupling reaction
between a terminal alkyne and 1,2-dichloro-
ethene followed by a second coupling reaction
with an alkylmagnesium bromide gave a conju-
gated enyne. Stereoselective reduction of the triple
bond and bromination afforded the expected
bromo precursor. After formation of the hep-
tadecadienylmagnesium bromide, (9,11)- and
(10,12)-CLA isomers were obtained by carbona-
tion with 14C carbon dioxide.
2. Experimental
14C Barium carbonate (55 mCi/mmol) was from
Mayak Production Association. Other starting
materials and chemical reagents were from
Aldrich or Fluka. Flash chromatography was per-
formed using silica gel 60 (0.040–0.063 mm) from
Merck. Sodium chloride was from Labosi.
Dimethylsulfoxide and sodium hydroxide solution
were from Prolabo. Ammonium chloride, sodium
sulfate, sodium hydrogen carbonate, sulfuric acid
(95%, d=1.83), pentane, diethyl ether, tetrahy-
drofuran and dichloromethane were from sodium
dodecyl sulfate (SDS). Solvents were purified be-
fore use, dimethylsulfoxide was distilled; diethyl
ether and tetrahydrofuran were distilled from
sodium/benzophenone ketyl; dichloromethane
was distilled from calcium hydride. All purified
solvents were stored over molecular sieves. Unless
otherwise stated, reactions were carried out under
argon.
Analytical TLC was performed with silica gel
60F254 (Merck) and visualization was carried out
with an anisaldehyde solution (anisaldehyde 12.5
ml; acetic acid 5 ml; sulfuric acid 17 ml; 95%
ethanol 450 ml) unless otherwise stated. NMR
spectra were recorded on a Bruker AC 300 spec-
trometer (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 in-
strument (Model 4600). Radioactivity measure-
ments were carried out with a Berthold LB 2040
nuclear spectrometer. Analytical RP-HPLC
analyses were obtained with a Shimadzu LC-
10AS pump fitted with a Shimadzu SCL-10A
system controller. The radioactive detector was a
Berthold LB 503 monitor (liquid scintillation
cocktail, MS2 from SDS) and the UV detector
was an LDC 3200 analytical spectro monitor.
Preparative RP-HPLC was conducted with a
Gilson pump (model 303) fitted with a Gilson
manometric module (model 802C) and a Berthold
LB 2040 nuclear spectrometer. GC/MS analyses
were effected on an HP 6890 Series gas chro-
matograph system coupled to an HP 5973 mass
selective detector.

O. Loreau et al. / Chemistry and Physics of Lipids 110 (2001) 57–67
59
2.1. 2-(7-Bromo-heptyloxy)-tetrahydro-pyran 1
2.3. 2-((E)-11-Chloro-undec-10-en-8-ynyloxy)-
tetrahydro-pyran 3
7-Bromo-heptan-1-ol (10 g; 51.2 mmol) and
0.89 g of p-toluenesulfonic acid were placed in
120 ml of dry dichloromethane. 3,4-Dihydro-2H-
pyran (8 ml; 88.2 mmol) was added dropwise with
stirring at room temperature. The reaction mix-
ture was stirred for 5 h and then 50 ml of a
saturated NaHCO3 solution were added. The
aqueous layer was extracted with 100 ml of
dichloromethane. The combined organic fractions
were washed with water, dried over Na2SO4,
filtered and concentrated. The residue was chro-
matographed on silica gel with pentane/diethyl
ether (97/3) giving 2-(7-bromo-heptyloxy)-tetrahy-
dro-pyran 1 (10.4 g; 37.2 mmol; 72% yield).
TLC (visualization with a KMnO4 solution),
Rf=0.26 (pentane/diethyl ether (97/3)).
In a mixture of (E)-1,2-dichloro-ethene (10.8
ml; 140 mmol) and freshly distilled piperidine (5.6
ml; 56.6 mmol) were placed Pd(PPh3)4 (1.66 g;
1.43 mmol) and CuI (555 mg; 2.91 mmol). The
alkyne 2 (6.14 g; 27.4 mmol) dissolved in 15 ml of
anhydrous THF was added dropwise with stirring
at room temperature. The reaction mixture was
stirred for 5 h and then poured into 150 ml of a
saturated NH4Cl solution. The mixture was ex-
tracted with 150 ml of diethyl ether. The organic
layer was washed twice with 100 ml of a saturated
NaCl solution, dried over Na2SO4 and filtered.
The organic phase was partially concentrated un-
der vacuum and pentane was added in order to
precipitate triphenylphosphine. The crude product
was chromatographed on silica gel with pentane/
diethyl ether (96/4) to give 2-((E)-11-chloro-un-
dec-10-en-8-ynyloxy)-tetrahydro-pyran 3 (6.4 g;
22.5 mmol; 82% yield from 2).
1H NMR (CDCl3), 1.25–1.9 (m; 2,3,4,5,6-H2;
3,4,5-H2 (THP)), 3.3–3.42 (m; 1-Ha; 7-H2), 3.5
(m; 6-Ha (THP)), 3.71 (dt; J=9.7, 6.7 Hz; 1-Hb),
3.85 (m; 6-Hb (THP)), 4.55 (m; 2-H1 (THP)).
TLC, Rf=0.3 (pentane/diethyl ether (96/4)).
1H NMR (CDCl3): 1.2–1.9 (m; 2,3,4,5,6-H2;
3,4,5-H2 (THP)), 2.27 (td; J=6.7, 2 Hz; 7-H2),
3.36 (dt; J=9.6, 6.7 Hz; 1-Ha), 3.5 (m; 6-Ha
(THP)), 3.71 (dt; J=9.6, 6.7 Hz; 1-Hb), 3.85 (m;
6-Hb (THP)), 4.55 (m; 2-H1 (THP)), 5.9 (dt; J=
13.4, 2.4 Hz; 10-H1), 6.4 (d; J=13.4 Hz; 11-H1).
2.2. 2-Non-8-ynyloxy-tetrahydro-pyran 2
Lithium acetylide, ethylenediamine complex
(90% pure; 4.6 g; 44.9 mmol) was placed in 25 ml
of dry DMSO. The mixture was cooled to 10°C
and compound 1 (10 g; 35.8 mmol) was added
dropwise with stirring. After the addition, the
dark reaction mixture was stirred vigorously at
room temperature for 4 h after which it was
slowly added to a mixture of water and ice. The
aqueous solution was extracted twice with 100 ml
of diethyl ether. The combined ethereal layers
were washed with water, dried over Na2SO4,
filtered and concentrated. Purification by chro-
matography on silica gel with pentane/diethyl
ether (95/5) gave 2-non-8-ynyloxy-tetrahydro-py-
ran 2 (6.5 g; 29 mmol; 81% yield).
TLC, Rf=0.28 (pentane/diethyl ether (95/5)).
1H NMR (CDCl3), 1.25–1.9 (m; 2,3,4,5,6-H2;
3,4,5-H2 (THP)), 1.92 (t; J=2.6 Hz; 9-H1), 2.16
(td; J=7, 2.6 Hz; 7-H2), 3.36 (dt; J=9.6, 6.7 Hz;
1-Ha), 3.5 (m; 6-Ha (THP)), 3.71 (dt; J=9.6, 6.7
Hz; 1-Hb), 3.85 (m; 6-Hb (THP)), 4.55 (m; 2-H1
(THP)).
2.4. 2-((E)-Heptadec-10-en-8-ynyloxy)-
tetrahydro-pyran 4
Compound 3 (6.3 g; 22.1 mmol), Fe(acac)3 (106
mg; 0.3 mmol) and 1-methyl-2-pyrrolidinone (20
ml; 208 mmol) were placed in 30 ml of anhydrous
THF. This mixture was cooled to 0°C and then a
2 M solution of hexylmagnesium bromide in di-
ethyl ether (14.5 ml; 29 mmol) was added drop-
wise. Stirring was continued for 45 min, then the
reaction mixture was hydrolyzed at 0°C with 120
ml of water and 100 ml of diethyl ether were
added. After decantation, the aqueous layer was
extracted twice with 100 ml of diethyl ether. The
combined organic layers were washed with water,
dried over Na2SO4, filtered and concentrated.
Chromatography on silica gel with pentane/di-

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O. Loreau et al. / Chemistry and Physics of Lipids 110 (2001) 57–67
ethyl ether (96/4) gave 2-((E)-heptadec-10-en-8-
ynyloxy)-tetrahydro-pyran 4 (5.8 g; 17.3 mmol;
78% yield from 3).
13C NMR (CDCl3), 13.85 (C17), 19.44 (THP
C4), 22.39 (C16), 25.30 (THP C5), 25.98 (C2),
27.44 (C7), 28.66, 28.96, 29.15, 29.44, 29.51,
30.54 (THP C3), 31.51 (C15), 32.64 (C12), 62.02
(THP C6), 67.39 (C1), 98.57 (THP C2), 125.36
(C10), 128.43 (C9), 129.69 (C8), 134.42 (C11).
TLC, Rf=0.41 (pentane/diethyl ether (96/4)).
1H NMR (CDCl3), 0.86 (t; J=6.7 Hz; 17-
H3), 1.2–1.9 (m; 2,3,4,5,6,13,14,15,16-H2; 3,4,5-
H2 (THP)), 2.05 (td; J=6.7, 6.7 Hz; 12-H2),
2.26 (td; J=6.7, 1.8 Hz; 7-H2), 3.36 (dt; J=9.6,
6.7 Hz; 1-Ha), 3.5 (m; 6-Ha (THP)), 3.71 (dt;
J=9.6, 6.7 Hz; 1-Hb), 3.85 (m; 6-Hb (THP)),
4.55 (m; 2-H1 (THP)), 5.42 (br d; J=15.9 Hz;
10-H1), 6.02 (dt; J=15.9, 6.7 Hz; 11-H1).
2.6. (7E,9Z)-17-Bromo-heptadeca-7,9-diene 6
Compound 5 (4.3 g; 12.8 mmol) dissolved in
10 ml of CH2Cl2 was added at 0°C to a slurry
of triphenylphosphine dibromide (11.5 g; 27.2
mmol) in 65 ml of CH2Cl2. After the addition,
the reaction mixture was stirred at room tem-
perature for 1.5 h, washed with a saturated so-
lution of sodium bicarbonate, then with water,
dried over Na2SO4 and filtered. Dichlorometh-
ane was partially removed under vacuum and
pentane was added to precipitate triphenylphos-
phine oxide. The residue was purified by chro-
matography on silica gel with pentane giving
(7E,9Z)-17-bromo-heptadeca-7,9-diene 6 (3.5 g;
11.1 mmol; 86% yield) as colorless oil.
2.5. 2-((8Z,10E)-Heptadeca-8,10-dienyloxy)-
tetrahydro-pyran 5
A 2 M solution of 2-methyl-but-2-ene in THF
(24 ml; 48 mmol) was added dropwise, between
−5 and 0°C, to a 1 M BH3.THF solution (24
ml; 24 mmol). The resulting solution was stirred
for 2.25 h at 0°C and then cooled to −20°C.
Compound 4 (5.7 g; 17 mmol) dissolved in 10
ml of anhydrous THF was added dropwise and
the reaction mixture was allowed to warm to
0°C over 4.25 h. Glacial acetic acid (4.5 ml) was
added and the mixture was stirred overnight.
The mixture was treated in an ice bath with a
7.5 N NaOH solution until the pH was alkaline
and then 15 ml of a 30% H2O2 solution were
added. After stirring for 1 h at room tempera-
ture, 50 ml of water and 100 ml of diethyl ether
were added. The aqueous layer was extracted
with 100 ml of diethyl ether. The combined or-
ganic layers were washed with water, dried over
Na2SO4, filtered and concentrated. The residue
was chromatographed on silica gel with pentane/
diethyl ether (97/3) giving 2-((8Z,10E)-hep-
tadeca-8,10-dienyloxy)-tetrahydro-pyran 5 (4.4 g;
13.1 mmol; 77% yield).
TLC, Rf=0.7 (pentane).
1H NMR (CDCl3), 0.87 (t; J=6.7 Hz; 1-H3),
1.2–1.5 (m; 2,3,4,5,12,13,14,15-H2), 1.84 (quint;
J=6.7 Hz; 16-H2), 2–2.2 (m; 6,11-H2), 3.4 (t;
J=7 Hz; 17-H2), 5.27 (dt; J=11, 7.3 Hz; 10-
H1), 5.63 (dt; J=15.2, 6.7 Hz; 7-H1), 5.92 (dd;
J=11, 11 Hz; 9-H1), 6.27 (m; 8-H1).
13C NMR (CDCl3), 13.91 (C1), 22.42 (C2),
27.40 (C11), 27.92, 28.44, 28.73, 28.79, 29.18,
29.37, 31.54 (C3), 32.61 and 32.71 (C6 and
C16), 33.61 (C17), 125.36 (C8), 128.59 (C9),
129.47 (C10), 134.51 (C7).
2.7. (9Z,11E)-[1-14C]-Octadeca-9,11-dienoic acid
TLC, Rf=0.48 (pentane/diethyl ether (97/3)).
1H NMR (CDCl3), 0.86 (t; J=6.7 Hz; 17-
H3), 1.2–1.9 (m; 2,3,4,5,6,13,14,15,16-H2; 3,4,5-
H2 (THP)), 2–2.2 (m; 7,12-H2), 3.36 (dt; J=9.6,
6.7 Hz; 1-Ha), 3.5 (m; 6-Ha (THP)), 3.71 (dt;
J=9.6, 6.7 Hz; 1-Hb), 3.85 (m; 6-Hb (THP)),
4.55 (m; 2-H1 (THP)), 5.27 (dt; J=11, 7.3 Hz;
8-H1), 5.63 (dt; J=15.2, 6.7 Hz; 11-H1), 5.92
(dd; J=11, 11 Hz; 9-H1), 6.27 (m; 10-H1).
7
Bromide 6 (300 mg; 0.95 mmol) dissolved in 3
ml of anhydrous diethyl ether was added to
magnesium turnings (61 mg; 2.51 mmol) and a
crystal of iodine in 1 ml of anhydrous diethyl
ether. The reaction mixture was heated to reflux
for 3 h. The resulting Grignard solution was
carbonated at −20°C with 14CO2 (liberated

O. Loreau et al. / Chemistry and Physics of Lipids 110 (2001) 57–67
61
from Ba14CO3 (210 mg; 1.05 mmol; 57.9 mCi) by
addition of concentrated sulfuric acid). After stir-
ring at −20°C for 2.5 h, 3 ml of a 5% NH4Cl
were added. The mixture was diluted with 60 ml
of diethyl ether and 40 ml of a saturated NH4Cl
solution. A 1 N H2SO4 solution was added drop-
wise until the aqueous layer was acidified to pH 3.
After decantation, the ethereal layer was washed
with water, dried over Na2SO4, filtered and con-
centrated. Chromatography on silica gel with pen-
tane/diethyl ether/acetic acid (80/20/0.1) gave
GC/MS: column, HP-23 cis/trans FAME (60
m×0.25 mm×0.25 mm). Column temperature,
200°C. Carrier gas, He (0.8 ml/min). Injector tem-
perature, 250°C. (9Z,11E)-[1-14C]-Octadeca-9,
11-dienoic acid, methyl ester, (r.t.=15.4 min; m/
z=296 M+). Coelution with an authentic sample.
HPLC, column Chromspher 5 Lipids (250×4.6
mm). Solvent system, hexane/acetonitrile (1000/1).
Flow rate, 1 ml/min. Detection, UV at 230 nm.
(9Z,11E)-[1-14C]-Octadeca-9,11-dienoic
acid,
methyl ester, (r.t.=12.5 min). Coelution with an
authentic sample.
(9Z,11E)-[1-14C]-octadeca-9,11-dienoic acid
7
(31.3 mCi; 0.56 mmol; 59% yield from 6).
RP-HPLC (analytical conditions), column Zor-
bax SB C18 (250×4.6 mm). Solvent system, etha-
nol/water/trifluoroacetic acid (70/30/0.1). Flow
rate, 1 ml/min. Detection, radioactive detection.
Radiochemical purity, (9Z,11E)-[1-14C] CLA 7
(r.t.=30.7 min; 98.7%), (9E,11E)-[1-14C] CLA
(r.t.=36.15 min; 1.3%).
2.9. (E)-1-Chloro-non-1-en-3-yne 8
Compound 8 was prepared as described previ-
ously for 3 using 1-heptyne (1.92 g; 20 mmol)
dissolved in 10 ml of anhydrous THF, (E)-1,2-
chloro-ethene (7.7 ml; 100 mmol), Pd(PPh3)4 (1.15
g; 1 mmol), CuI (381 mg; 2 mmol), piperidine (4
ml; 40 mmol). Chromatography on silica gel with
pentane gave (E)-1-chloro-non-1-en-3-yne 8 (1.84
g; 11.7 mmol; 59% yield from 1-heptyne).
TLC, Rf=0.85 (pentane).
TLC, Rf=0.36 (pentane/diethyl ether/acetic
acid (80/20/0.1)).
1H NMR (CDCl3): 0.87 (t; J=6.7 Hz; 18-H3),
1.2–1.45 (m; 4,5,6,7,14,15,16,17-H2), 1.55–1.7 (m;
3-H2), 2–2.2 (m; 8,13-H2), 2.34 (t; J=7.4 Hz;
2-H2), 5.29 (dt; J=11, 7.3 Hz; 9-H1), 5.55 (m;
(E,E) isomer), 5.64 (dt; J=14.6, 7.3 Hz; 12-H1),
5.93 (dd; J=11, 11 Hz; 10-H1), 6 (m; (E,E)
isomer), 6.28 (m; 11-H1).
1H NMR (CDCl3): 0.89 (t; J=6.9 Hz; 9-H3),
1.2–1.4 (m; 7,8-H2), 1.5 (m; 6-H2), 2.27 (td; J=
7.1, 2.2 Hz; 5-H2), 5.9 (dt; J=13.6, 2.2 Hz; 2-H1),
6.41 (d; J=13.6 Hz; 1-H1).
Purification by preparative RP-HPLC (column,
Zorbax SB C18 (250×21.2 mm); solvent system,
acetone/methanol/water/acetic acid (55/22.5/22.5/
0.45); flow rate, 10 ml/min) gave the acid 7 free of
(9E,11E) isomer with radiochemical purity greater
than 99%.
2.10. 2-(8-Bromo-octyloxy)-tetrahydro-pyran 9
Compound 9 was obtained as described for the
preparation of 1 using 8- bromo-octan-1-ol (10.2
g; 48.8 mmol), 3,4-dihydro-2H-pyran (8 ml; 88.2
mmol), p-toluenesulfonic acid (0.94 g) in 170 ml
of dry CH2Cl2. Purification by chromatography
on silica gel with pentane/diethyl ether (95/5) gave
2-(8-bromo-octyloxy)-tetrahydro-pyran 9 (12.1 g;
41.3 mmol; 85% yield).
2.8. (9Z,11E)-[1-14C]-octadeca-9,11-dienoic acid,
methyl ester
We describe, here, a typical procedure, 1 ml of
BF3-methanol was added to the free fatty acid 7
(2 mg) and the mixture was stirred for 10 min at
room temperature. The methyl ester of 7 was
extracted into hexane (2 ml) and washed twice
with water (5 ml). The organic layer was dried
over sodium sulfate. After removal of solvent
under a stream of nitrogen, the ester was dis-
solved in hexane for GC/MS and HPLC analysis.
TLC (visualization with a KMnO4 solution),
Rf=0.3 (pentane/diethyl ether (95/5)).
1H NMR (CDCl3), 1.25–1.9 (m; 2,3,4,5,6,7-H2;
3,4,5-H2 (THP)), 3.3–3.42 (m; 1-Ha; 8-H2), 3.5
(m; 6-Ha (THP)), 3.71 (dt; J=9.7, 6.7 Hz; 1-Hb),
3.85 (m; 6-Hb (THP)), 4.55 (m; 2-H1 (THP)).

62
O. Loreau et al. / Chemistry and Physics of Lipids 110 (2001) 57–67
2.11. 2-((E)-Heptadec-9-en-11-ynyloxy)-
tetrahydro-pyran 10
2.13. (10E,12Z)-[1-14C]-Octadeca-10,12-dienoic
acid 12
Compound 10 was obtained as described for
the preparation of 4 using (E)-1-chloro-non-1-en-
3-yne 8 (1.8 g; 11.5 mmol), Fe(acac)3 (47 mg; 0.13
mmol), 1-methyl-2-pyrrolidinone (11.5 ml; 120
mmol) and 10 ml of anhydrous THF. The solu-
tion of Grignard reagent was prepared from 2-(8-
bromo-octyloxy)-tetrahydro-pyran 9 (3.8 g; 13
mmol) in 4 ml of anhydrous THF and 340 mg of
magnesium in 5 ml of anhydrous THF (reflux, 3
h). Purification by chromatography on silica gel
with pentane/diethyl ether (97/3) gave impure 2-
((E)-heptadec-9-en-11-ynyloxy)-tetrahydro-pyran
10 as a yellow oil (2.45 g).
(10E,12Z)-[1-14C]-Octadeca-10,12-dienoic acid
12 (29.6 mCi; 0.53 mmol; 56% yield from 11) was
obtained from bromide 11 (300 mg; 0.95 mmol),
39 mg of magnesium turnings and Ba14CO3 (210
mg; 1.05 mmol; 57.9 mCi) according to the proce-
dure described for the preparation of 7.
RP-HPLC (analytical conditions), column Zor-
bax SB C18 (250×4.6 mm). Solvent system, etha-
nol/water/trifluoroacetic acid (70/30/0.1). Flow
rate, 1.5 ml/min. Radiochemical purity,
(10E,12Z)-[1-14C] CLA 12 (r.t.=17.1 min;
98.5%), (10E,12E)-[1-14C] CLA (r.t.=20 min;
1.5%).
TLC, Rf=0.38 (pentane/diethyl ether (97/3)).
1H NMR (CDCl3), 0.87 (t; J=6.6 Hz; 17-H3);
1.2–1.9 (m; 2,3,4,5,6,7,14,15,16-H2; 3,4,5-H2
(THP)), 2.04 (td; J=6.8, 6.8 Hz; 8-H2), 2.25 (br t;
J=6.9 Hz; 13-H2), 3.36 (dt; J=9.6, 6.7 Hz;
1-Ha), 3.5 (m; 6-Ha (THP)), 3.71 (dt; J=9.6, 6.7
Hz; 1-Hb), 3.85 (m; 6-Hb (THP)), 4.55 (m; 2-H1
(THP)), 4.85–5.05 (m; impurity), 5.42 (br d; J=
15.9 Hz; 10-H1), 5.7–5.85 (m; impurity), 6.02 (dt;
J=15.9, 7 Hz; 9-H1).
TLC, Rf=0.36 (pentane/diethyl ether/acetic
acid (80/20/0.1)).
1H NMR (CDCl3), 0.88 (t; J=6.7 Hz; 18-H3),
1.2–1.45 (m; 4,5,6,7,8,15,16,17-H2), 1.5–1.65 (m;
3-H2), 2–2.2 (m; 9,14-H2), 2.3 (t; J=7.3 Hz;
2-H2), 5.29 (dt; J=11, 7.6 Hz; 13-H1), 5.55 (m;
(E,E) isomer), 5.64 (dt; J=14.7, 7.3 Hz; 10-H1),
5.93 (dd; J=10.7, 10.7 Hz; 12-H1), 6 (m; (E,E)
isomer), 6.28 (m; 11-H1).
Purification by preparative RP-HPLC (column,
Zorbax SB C18 (250×21.2 mm); solvent system,
acetone/methanol/water/acetic acid (55/22.5/22.5/
0.45); flow rate, 10 ml/min) gave the acid 12 free
of (E,E) isomer with radiochemical purity greater
than 99%.
2.12. (6Z,8E)-17-Bromo-heptadeca-6,8-diene 11
Bromide 11 (940 mg; 2.98 mmol; 28% yield
from compound 9 (three steps)) was obtained
from 2 g of impure 2-((E)-heptadec-9-en-11-yny-
loxy)-tetrahydro-pyran 10 according to the proce-
dure described for the preparation of 6.
2.14. (10E,12Z)-[1-14C]-Octadeca-10,12-dienoic
acid, methyl ester
The methyl ester derivative was prepared as
described in Section 2.8.
TLC, Rf=0.7 (pentane).
GC/MS: column, HP-23 cis/trans FAME (60
m×0.25 mm×0.25 mm). Column temperature,
200°C. Carrier gas, He (0.8 ml/min). Injector tem-
perature, 250°C. (10E,12Z)-[1-14C]-Octadeca-
10,12-dienoic acid, methyl ester, (r.t.=15.9 min;
m/z=296 M+). Coelution with an authentic
sample.
1H NMR (CDCl3), 0.88 (t; J=6.7 Hz; 1-H3),
1.2–1.5 (m; 2, 3, 4, 11, 12, 13, 14, 15-H2), 1.84
(quint; J=6.9 Hz; 16-H2), 2–2.2 (m; 5,10-H2), 3.4
(t; J=6.8 Hz; 17-H2), 5.29 (dt; J=10.7, 7.6 Hz;
6-H1), 5.64 (dt; J=15.1, 6.9 Hz; 9-H1), 5.93 (dd;
J=10.9, 10.9 Hz; 7-H1), 6.28 (m; 8-H1).
13C NMR (CDCl3), 13.82 (C1), 22.32 (C2),
27.44 (C5), 27.92, 28.47, 28.86, 29.05, 29.18, 31.25
(C3), 32.61 (C10 and C16), 33.71 (C17), 125.46
(C8), 128.34 (C7), 129.92 (C6), 134.26 (C9).
HPLC, column Chromspher 5 Lipids (250×4.6
mm). Solvent system, hexane/acetonitrile (1000/1).
Flow rate, 1 ml/min. Detection, UV at 230 nm.
(10E,12Z)-[1-14C]-Octadeca-10,12-dienoic
acid,

O. Loreau et al. / Chemistry and Physics of Lipids 110 (2001) 57–67
63
2.17. (6Z,8Z)-17-Bromo-heptadeca-6,8-diene 15
methyl ester, (r.t.=11.3 min). Coelution with an
authentic sample.
Bromide 15 (1.25 g; 4 mmol; 36% yield from 9
(three steps)) was obtained from 2.5 g of impure
2-((Z)-heptadec-9-en-11-ynyloxy)-tetrahydro-pyr-
an 14 according to the procedure described for the
preparation of compound 6.
2.15. (Z)-1-Chloro-non-1-en-3-yne 13
Pd(PPh3)4 (1.15 g; 1 mmol), piperidine (4 ml; 40
mmol) and 1-heptyne (1.92 g; 20 mmol) were
added to a solution of (Z)-1,2-dichloro-ethene (4.5
ml; 60 mmol) in anhydrous THF (40 ml). After
stirring for 10 min at room temperature, CuI (380
mg; 2 mmol) was introduced in the reaction flask.
The mixture was stirred for 5 h. Treatment of the
crude mixture and purification by flash chro-
matography, as described for compound 3, gave
(Z)-1-chloro-non-1-en-3-yne 13 (2.53 g; 16.1
mmol; 80% yield from 1-heptyne).
TLC, Rf=0.7 (pentane).
1H NMR (CDCl3), 0.88 (t; J=6.7 Hz; 1-H3),
1.2–1.5 (m; 2,3,4,11,12,13,14,15-H2), 1.84 (quint;
J=7 Hz, 16-H2), 2–2.2 (m; 5,10-H2), 3.4 (t; J=7
Hz; 17-H2), 5.35–5.55 (m; 6,9-H1), 6.15–6.3 (m;
7,8-H1).
13C NMR (CDCl3), 13.85 (C1), 22.36 (C2),
27.21 (C5 and C10), 27.95, 28.50, 28.92, 29.12,
29.37, 31.29 (C3), 32.61 (C16), 33.71 (C17), 123.32
and 123.45 (C7 and C8), 131.63 and 131.89 (C6
and C9).
TLC, Rf=0.85 (pentane).
1H NMR (CDCl3), 0.88 (t; J=7 Hz; 9-H3),
1.25–1.45 (m; 7,8-H2), 1.55 (m; 6-H2), 2.37 (td;
J=7.1, 2.2 Hz; 5-H2), 5.84 (dt; J=7.3, 2.2 Hz;
2-H1), 6.28 (d; J=7.3 Hz; 1-H1).
2.18. (10Z,12Z)-[1-14C]-Octadeca-10,12-dienoic
acid 16
(10Z,12Z)-[1-14C]-Octadeca-10,12-dienoic acid
16 (42.9 mCi; 0.78 mmol; 58% yield from 15) was
obtained from bromide 15 (425 mg, 1.35 mmol),
80 mg of magnesium turnings and Ba14CO3 (280
mg; 1.4 mmol; 77.2 mCi) according to the proce-
dure described for the preparation of compound 7
(the mixture of bromide 15 and magnesium turn-
ings was stirred for only 1.5 h in refluxing diethyl
ether).
2.16. 2-((Z)-Heptadec-9-en-11-ynyl-
oxy)-tetrahydro-pyran 14
Compound 14 was obtained as described for
the preparation of 4 using (Z)-1-chloro-non-1-en-
3-yne 13 (2.4 g; 15.3 mmol), Fe(acac)3 (80 mg;
0.22 mmol), 1-methyl-2-pyrrolidinone (16 ml; 166
mmol) and 10 ml of anhydrous THF. The solu-
tion of Grignard reagent was prepared from 2-(8-
bromo-octyloxy)-tetrahydro-pyran 9 (6.6 g; 22.5
mmol) in 7 ml of anhydrous THF and 640 mg of
magnesium turnings in 5 ml of anhydrous THF
(reflux, 3 h). Purification by chromatography on
silica gel with pentane/diethyl ether (97/3) gave
impure 2-((Z)-heptadec-9-en-11-ynyloxy)-tetrahy-
dro-pyran 14 as a yellow oil (5.15 g).
TLC, Rf=0.38 (pentane/diethyl ether (97/3)).
1H NMR (CDCl3), 0.89 (t; J=7.3 Hz; 17-H3);
1.2–1.9 (m; 2,3,4,5,6,7,14,15,16-H2; 3,4,5-H2
(THP)), 1.95–2.1 (m; impurity); 2.15–2.35 (m;
8,13-H2), 3.36 (dt; J=9.6, 6.7 Hz; 1-Ha), 3.5 (m;
6-Ha (THP)), 3.71 (dt; J=9.6, 6.7 Hz; 1-Hb), 3.85
(m; 6-Hb (THP)), 4.55 (m; 2-H1 (THP)), 4.85–
5.05 (m; impurity); 5.41 (br d; J=11 Hz; 10-H1),
5.79 (dt; J=10.4, 7.3 Hz; 9-H1).
RP-HPLC (analytical conditions), column Zor-
bax SB C18 (250×4.6 mm). Solvent system, etha-
nol/water/trifluoroacetic acid (70/30/0.1). Flow
rate, 1.5 ml/min. Radiochemical purity,
(10Z,12Z)-[1-14C] CLA 16 (r.t.=20 min; \99%).
TLC, Rf=0.36 (pentane/diethyl ether/acetic
acid (80/20/0.1)).
1H NMR (CDCl3), 0.88 (t; J=6.7 Hz; 18-H3),
1.2–1.5 (m; 4,5,6,7,8,15,16,17-H2), 1.5–1.7 (m; 3-
H2), 2.05–2.2 (m; 9,14-H2), 2.33 (t; J=7.6 Hz;
2-H2), 5.35–5.55 (m; 10,13-H1), 6.15–6.3 (m;
11,12-H1).
13C NMR (CDCl3), 13.81 (C18), 22.29 (C17),
24.43 (C3), 27.21 (C9 and C14), 28.79, 28.92,
29.02, 29.09, 29.34, 31.25 (C16), 33.81 (C2),
123.32 and 123.39 (C11 and C12), 131.73 and
131.89 (C10 and C13).

64
O. Loreau et al. / Chemistry and Physics of Lipids 110 (2001) 57–67
3. Results and discussion
obtained in 59% yield after purification by flash
chromatography. Traces of (E,E) isomer were de-
1
3.1. Synthesis of
tected by H NMR (minor signals at 5.55 and 6
(9Z,11E)-[1-14C]-octadeca-9,11-dienoic acid 7
ppm). The radiochemical purity of the free acid 7
was found to be 98.7% by RP-HPLC and only
one impurity (1.3%) coeluting with an authentic
sample of (9E,11E) isomer was found to be
present. This impurity was easily removed by
preparative RP-HPLC to give the acid 7 with high
The synthesis of (9Z,11E)-[1-14C]-octadeca-
9,11-dienoic acid 7 starts with the preparation of
the terminal alkyne 2 (Fig. 1). 7-Bromo-heptan-1-
ol was transformed in 72% yield to its correspond-
ing tetrahydropyranyl derivative 1 which was
converted in 81% yield to 2-non-8-ynyloxy-te-
trahydro-pyran
2
by reaction with lithium
acetylide, ethylenediamine complex (Porwoll and
Leete, 1985). Coupling between compound 2 dis-
solved in THF and 5 equivalents of (E)-1,2-
dichloro-ethene in presence of Pd(PPh3)4, CuI and
piperidine gave the (E)-chloroenyne 3 in 82% yield
(Chemin and Linstrumelle, 1994). Although
Chemin and Linstrumelle reported that THF gave
slightly lower yields of coupling products than
benzene, we used THF as solvent because of the
toxicity of benzene. According to the procedure
developed by Cahiez and Avedissian, compound 3
was then coupled with hexylmagnesium bromide
in a THF/1-methyl-2-pyrrolidinone mixture using
Fe(acac)3 as catalyst (Cahiez and Avedissian,
1998). The enyne 4 was then obtained in 78%
yield. Stereoselective hydroboration of the triple
bond with disiamylborane at low temperature fol-
lowed by protonolysis with acetic acid under mild
conditions afforded (Z,E) conjugated diene 5 in
77% yield (Cabezas and Oehlschlager, 1999). The
reduction of the internal triple bond was con-
ducted with disiamylborane because some authors
reported the formation of complex mixtures when
Pd catalyst and hydrogen were used (Rossi and
Carpita, 1983). Treatment of compound 5 with
triphenylphosphine dibromide gave the bromo
derivative 6 in 86% yield (Rakoff, 1982; Eynard et
al., 1994). At this stage, no geometric isomers of
1
compounds 5 and 6 were detected by H and 13C
NMR. Bromide 6 was then transformed into its
corresponding heptadecadienylmagnesium bro-
mide by reaction with magnesium turnings in
refluxing anhydrous diethyl ether (3 h). The Grig-
nard reagent was carbonated with 14CO2 liberated
from 14C barium carbonate (Howton et al., 1954).
(9Z,11E)-[1-14C]-Octadeca-9,11-dienoic acid 7 was
Fig. 1. Synthesis of (9Z,11E)-[1-14C]-octadeca-9,11-dienoic
acid.

O. Loreau et al. / Chemistry and Physics of Lipids 110 (2001) 57–67
65
radiochemical and isomeric purities (\99%). The
identification of 7 was confirmed on its methyl
ester derivative (Banni et al., 1994) by comparison
with an authentic sample using GC/MS (HP-23
column) and HPLC (ChromSpher
column) (Sehat et al., 1998a,b).
5 Lipids
3.2. Synthesis of (10E,12Z)- and
(10Z,12Z)-[1-14C]-octadeca-10,12-dienoic acid
The synthesis of (10E,12Z)-[1-14C]-octadeca-
10,12-dienoic acid 11 used 1-heptyne and 8-
bromo-octan-1-ol as (Figs. 2 and 3) starting
materials (Fig. 2). 1-Heptyne was converted to the
(E)-chloroenyne 8 in 59% yield and 8-bromo-oc-
tan-1-ol was transformed to its tetrahydropyranyl
derivative 9 (85% yield). Coupling between com-
pound 8 and the Grignard reagent obtained from
9 gave impure (E)-enyne 10 as a yellow oil. After
stereoselective reduction of the triple bond and
reaction with triphenylphosphine dibromide as
described for the preparation of the bromo pre-
cursor 6, the bromide 11 (28% yield from 9 (three
steps)) was obtained in a pure form (no geometric
1
isomer was detected by H or 13C NMR). Forma-
tion of the corresponding heptadecadienylmagne-
sium bromide (3 h in refluxing diethyl ether)
followed by carbonation with 14CO2 produced
(10E,12Z)-[1-14C]-octadeca-10,12-dienoic acid 12
in 56% yield after purification by flash chro-
matography. The radiochemical purity of the acid
12 was found to be 98.5% by RP-HPLC
((10E,12E) isomer, 1.5%). Compound 12 was ob-
tained in a pure form free of (10E,12E) isomer
(radiochemical purity \99%) by preparative RP-
HPLC. As for (9Z,11E)-[1-14C]-octadec-9,11-
dienoic acid 7, the identification of compound 12
was confirmed on its methyl ester derivative by
comparison with an authentic sample (GC/MS
and HPLC).
Fig. 2. Synthesis of (10E,12Z)-[1-14C]-octadeca-10,12-dienoic
acid.
(10Z,12Z)-[1-14C]-Octadeca-10,12-dienoic acid
was synthesized according to the route described
for the preparation of (10E,12Z)-[1-14C]-CLA 12
but using (Z)-1,2-dichloro-ethene instead of the
(E) isomer. In this case, 1-heptyne was coupled
with three equivalents of (Z)-1,2-dichloro-ethene
in the presence of Pd(0), Cu(I) and piperidine to
give (Z)-chloroenyne 13 in 80% yield. Then, 2-
((Z)-heptadec-9-en-11-ynyloxy)-tetrahydro-pyran
14 was prepared by reaction of 13 with the Grig-
nard reagent derived from 9. Compound 14 was
transformed into its corresponding bromo deriva-
tive 15 (36% yield from 9 (three steps)) as re-
ported for the preparation of the (10E,12Z)
isomer. As for (Z,E) bromo precursors 6 and 10,
1
the H and 13C NMR spectra indicated that com-

66
O. Loreau et al. / Chemistry and Physics of Lipids 110 (2001) 57–67
tion of the heptadecadienyl Grignard reagent, es-
pecially the reaction time in refluxing diethyl
ether, could be optimized to avoid the formation
of geometric isomers). Since no authentic sample
of (10Z,12Z)-CLA isomer was commercially avail-
able, the identification of 16 was not checked by
GC/MS or HPLC on the methyl ester derivative
but by 13C NMR. The 13C NMR data of the
ethylenic carbons were in concordance with those
previously reported for a (9Z,11Z) conjugated
octadecadienoic acid (Davis et al., 1999). The
signals were assigned by analogy with data from
(9Z,11Z) CLA isomer and no geometric isomer of
16 was detected.
pound 15 was obtained free of other geometric
isomers. Formation of the heptadecadienylmagne-
sium bromide in refluxing diethyl ether (1.5 h
instead of 3 h as described for (Z,E) compounds)
and carbonation with Ba14CO3 provided the
(10Z,12Z)-[1-14C]-octadeca-10,12-dienoic acid 16
(58% yield) with a radiochemical purity greater
than 99% (RP-HPLC). In contrast to the synthe-
sis of (Z,E)-octadecadienoic acids, no (E,E)-CLA
isomer was detected by 1H NMR or analytical
RP-HPLC after purification by flash chromatog-
raphy on silica gel (this result seems to indicate
that the experimental conditions of the prepara-
In conclusion, we have developed a practical
and efficient stereoselective route to CLAs.
(9Z,11E)-, (10E,12Z)-, (10Z,12Z)-[1-14C]-CLA
with high radiochemical and stereoisomeric puri-
ties (\98%) have been obtained using only purifi-
cation by flash chromatography and metabolism
studies in animals are in progress. More generally,
this methodology involving sequential substitu-
tion of 1,2-dichloro-ethene can be a useful alter-
native for the synthesis of natural products,
especially insect pheromones, with a conjugated
stereo-defined dienic or enynic system.
Acknowledgements
The authors thank M. Alain Valleix (CEA/
Saclay, Service des Mole´cules Marque´es) for
analyses and Natural Lipids Ltd. AS (Norway)
for the gift of authentic samples of (9Z,11E)- and
(10E,12Z)-CLA isomers. This work was sup-
ported by a grant (No. FAIR 98-3671) from the
Commission of the European Communities.
References
Adlof, R., 1997. Preparation of methyl cis-9, trans-11- and
trans-9, trans-11-octadecadienoate-17,17,18,18-d4, two of
the isomers of conjugated linoleic acid. Chem. Phys. Lipids
88, 107–112.
Alami, M., Gueugnot, S., Domingues, E., Linstrumelle, G.,
1995. An efficient synthesis of 1,3(E),5(Z), 1,3(E),5(E) and
1,3(Z),5(Z)-trienes: application to the synthesis of gal-
banolenes and (9Z,11E)-9,11,13-tetradecatrien-1-yl acetate.
Tetrahedron 51, 1209–1220.
Fig. 3. Synthesis of (10Z,12Z)-[1-14C]-octadeca-10,12-dienoic
acid.

O. Loreau et al. / Chemistry and Physics of Lipids 110 (2001) 57–67
67
Nicolosi, R.J., Courtemanche, K.V., Laitinen, L., Scimeca,
J.A., Huth, P.J., 1993. Effect of feeding diets enriched in
conjugated linoleic acid on lipoproteins and aortic athero-
genesis in hamsters. Circulation 88S, 2458.
Park, Y., Albright, K.J., Storkson, J.M., Liu, W., Cook, M.E.,
Pariza, M.W., 1999. Changes in body composition in mice
during feeding and withdrawal of conjugated linoleic acid.
Lipids 34, 243–248.
Banni, S., Day, B.W., Evans, R.W., Corongiu, F.P., Lom-
bardi, B., 1994. Liquid chromatographic-mass spectromet-
ric analysis of conjugated diene fatty acids in a partially
hydrogenated fat. J. Am. Oil Chem. Soc. 71, 1321–1325.
Belury, M.A., 1995. Conjugated dienoic linoleate: a polyunsat-
urated fatty acid with unique chemoprotective properties.
Nutr. Rev. 53, 83–89.
Berdeaux, O., Christie, W.W., Gunstone, F.D., Sebedio, J.-L.,
1997. Large-scale synthesis of methyl cis-9, trans-11-oc-
tadecadienoate from methyl ricinoleate. J. Am. Oil Chem.
Soc. 74, 1011–1015.
Cabezas, J.A., Oehlschlager, A.C., 1999. Stereospecific synthe-
sis of (E,Z)- and (Z,Z)-hexadeca-10,12-dienal. Sex
pheromone components of diaphania hyalinata. Synthesis,
107–111.
Cahiez, G., Avedissian, H., 1998. Highly stereo- and chemose-
lective iron-catalysed alkenylation of organomagnesium
compounds. Synthesis, 1199–1205.
Chemin, D., Linstrumelle, G., 1994. Palladium-catalysed reac-
tion of (E) and (Z)-dichloroethenes with 1-alkynes. An
efficient stereospecific synthesis of (E) and (Z)-enediynes.
Tetrahedron 50, 5335–5344.
Chen, C.A., Sih, C.J., 1998. Chemoenzymatic synthesis of
conjugated linoleic acid. J. Org. Chem. 63, 9620–9621.
Davis, A.L., Mc Neill, G.P., Caswell, D.C., 1999. Analysis of
conjugated linoleic acid isomers by 13C NMR spec-
troscopy. Chem. Phys. Lipids 97, 155–165.
Eynard, T., Vate`le, J.M., Poullain, D., Noe¨l, J.P., Chardigny,
J.M., Se´be´dio, J.L., 1994. Synthesis of methyl
(9Z,12Z,15E)- and (9E,12Z,15Z)-octadecatrienoic acids
and their [1-14C]-radiolabelled analogs. Chem. Phys. Lipids
74, 175–184.
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.
Parodi, P.W., 1977. Conjugated octadecadienoic acids of milk
fat. J. Dairy Sci. 60, 1550–1553.
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.
Purcell, J.M., Morris, S.G., Susi, H., 1966. Proton magnetic
resonance spectra of unsaturated fatty acids. Anal. Chem.
38, 588–592.
Rakoff, H., 1982. Preparation of fatty acids and esters con-
taining deuterium. Prog. Lipid Res. 21, 225–254.
Ratovelomanana, V., Linstrumelle, G., 1984. Stereoselective
preparation of 1-chloro-(E,E)-1,3-dienes. Application to
the synthesis of methyl eleostearate. Tetrahedron Lett. 25,
6001–6004.
Rossi, R., Carpita, A., 1983. Stereospecific synthesis of (Z)-13-
hexadecen-11-yn-1-yl acetate the sex pheromone of the
processionary moth, and of (5Z,7E)-5,7-dodecadien-1-ol, a
sex pheromone component of the forest tent caterpillar.
Tetrahedron 39, 287–290.
Sehat, N., Kramer, J.K.G., Mossoba, M.M., Yurawecz, M.P.,
Roach, J.A.G., Eulitz, K., Morehouse, K.M., Ku, Y.,
1998a. Identification of conjugated linoleic acid isomers in
cheese by gas chromatography, silver ion high performance
liquid chromatography and mass spectral reconstructed ion
profiles. Comparison of chromatographic elution se-
quences. Lipids 33, 963–971.
Sehat, N., Yurawecz, M.P., Roach, J.A.G., Mossoba, M.M.,
Kramer, J.K.G., Ku, Y., 1998b. Silver-ion high-perfor-
mance liquid chromatographic separation and identifica-
tion of conjugated linoleic acid isomers. Lipids 33,
217–221.
Shantha, N.C., Crum, A.D., Decker, E.A., 1994. Evaluation
of conjugated linoleic acid concentrations in cooked beef.
J. Agr. Food Chem. 42, 1757–1760.
West, D.B., Delany, J.P., Camet, P.M., Blohm, F., Truett,
A.A., Scimeca, J., 1998. Effects of conjugated linoleic acid
on body fat and energy metabolism in the mouse. Am. J.
Physiol. 275, R667–R672.
Ip, C., Banni, S., Angioni, E., Carta, G., Mc Ginley, J.,
Thompson, H.J., Barbano, D., Bauman, D., 1999. Conju-
gated linoleic acid-enriched butter fat alters mammary
gland morphogenesis and reduces cancer risk in rats. J.
Nutr. 129, 2135–2142.
Lee, K.N., Kritchevsky, D., Pariza, M.W., 1994. Conjugated
linoleic acid and atherosclerosis in rabbits. Atherosclerosis
108, 19–25.
Nichols, P.L., Herb, S.F., Riemenschneider, R.W., 1951. Iso-
mers of conjugated fatty acids. I. Alkali-isomerized linoleic
acid. J. Am. Chem. Soc. 73, 247–252.
.