Synthesis of (6Z,9Z,11E)-octadecatrienoic and (8Z,11Z,13E)-eicosatrienoic acids and their [1-14C]-radiolabeled analogs

Article abstract of DOI:10.1016/S0009-3084(01)00170-0

In order to study the metabolic pathway and the physiological effects of 9c,11t-18:2 (major isomer of conjugated linoleic acid) and its C_18:3 and C_20:3 metabolites, 6c,9c,11t-18:3 and 8c,11c,13t-20:3 and their [1-¹⁴C]-radiolabeled analogs were prepared stereoselectively by total synthesis. The 8c,11c,13t-20:3 was obtained in 11 steps. The synthesis involves a highly stereoselective Wittig reaction between 3-(t-butyldiphenylsilyloxy)propanal and the ylide of 7-(2-tetrahydropyranyloxy)heptanylphosphonium salt which gave (3Z)-1-(t-butyldiphenylsilyloxy)-10-(2-tetrahydropyranyloxy)dec-3-ene in a first step. Then the t-butyldiphenylsilyl derivative was deprotected selectively and the resulting alcohol function was converted via a bromide into a phosphonium salt. The second stereoselective Wittig condensation between the phosphonium salt and commercial (2E)-non-2-enal under cis-olefinic conditions using Lithium hexamethyldisilazide as base afforded the (7Z,10Z,12E)-1-(2-tetrahydropyranyloxy)nonadeca-7,10,12-triene in a very good isomeric purity. The intermediate product was brominated and transformed by reaction with magnesium into Grignard reagent, which was one-carbon elongated by unlabeled or labeled carbon dioxide to obtain the 8c,11c,13t-20:3 in good isomeric purity (95%) and high radiochemical purity for its [1-¹⁴C]-radiolabeled analog (99%). 6c,9c,11t-18:3 was synthesized in a similar way by using 5-(2-tetrahydropyranyloxy)pentanylphosphonium salt in place of 7-(2-tetrahydropyranyloxy)heptanylphosphonium salt in a first step. Other reactions were unchanged and products were obtained in similar yields. Similar to 8c,11c,13t-20:3, the 6c,9c,11t-18:3 was obtained in a very good isomeric purity (95%) and its [1-¹⁴C]-radiolabeled analog in a high radiochemical purity (95%).

Full text of DOI:10.1016/S0009-3084(01)00170-0

Chemistry and Physics of Lipids
112 (2001) 121–135
www.elsevier.com/locate/chemphyslip
Synthesis of (6Z,9Z,11E)-octadecatrienoic and
(8Z,11Z,13E)-eicosatrienoic acids and their
[1-14C]-radiolabeled analogs
Silke Gna¨dig a, Olivier Berdeaux a,*, Olivier Loreau b, Jean-Pierre Noe¨l b,
Jean-Louis Se´be´dio a
a Unite´ de Nutrition Lipidique, Institut National de la Recherche Agronomique (INRA), 17 rue Sully, BP 86510,
F-21065 Dijon Cedex, France
b CEA Saclay, Ser6ice des Mole´cules Marque´es, Baˆt 547, F-91191 Gif-sur-Y6ette Cedex, France
Received 23 March 2001; received in revised form 8 June 2001; accepted 15 June 2001
Abstract
In order to study the metabolic pathway and the physiological effects of 9c,11t-18:2 (major isomer of conjugated
linoleic acid) and its C18:3 and C20:3 metabolites, 6c,9c,11t-18:3 and 8c,11c,13t-20:3 and their [1-14C]-radiolabeled
analogs were prepared stereoselectively by total synthesis. The 8c,11c,13t-20:3 was obtained in 11 steps. The synthesis
involves a highly stereoselective Wittig reaction between 3-(t-butyldiphenylsilyloxy)propanal and the ylide of
7-(2-tetrahydropyranyloxy)heptanylphosphonium salt which gave (3Z)-1-(t-butyldiphenylsilyloxy)-10-(2-tetrahy-
dropyranyloxy)dec-3-ene in a first step. Then the t-butyldiphenylsilyl derivative was deprotected selectively and the
resulting alcohol function was converted via a bromide into a phosphonium salt. The second stereoselective Wittig
condensation between the phosphonium salt and commercial (2E)-non-2-enal under cis-olefinic conditions using
Lithium hexamethyldisilazide as base afforded the (7Z,10Z,12E)-1-(2-tetrahydropyranyloxy)nonadeca-7,10,12-triene
in a very good isomeric purity. The intermediate product was brominated and transformed by reaction with
magnesium into Grignard reagent, which was one-carbon elongated by unlabeled or labeled carbon dioxide to obtain
the 8c,11c,13t-20:3 in good isomeric purity (95%) and high radiochemical purity for its [1-14C]-radiolabeled analog
(99%). 6c,9c,11t-18:3 was synthesized in a similar way by using 5-(2-tetrahydropyranyloxy)pentanylphosphonium salt
in place of 7-(2-tetrahydropyranyloxy)heptanylphosphonium salt in a first step. Other reactions were unchanged and
products were obtained in similar yields. Similar to 8c,11c,13t-20:3, the 6c,9c,11t-18:3 was obtained in a very good
isomeric purity (95%) and its [1-14C]-radiolabeled analog in a high radiochemical purity (95%). © 2001 Elsevier
Science Ireland Ltd. All rights reserved.
Keywords: Conjugated linoleic acid; Fatty acid; Wittig reaction; [1-14C]-labeled fatty acids; Stereoselective syntheses
* Corresponding author. Tel.: +33-380-693-110; fax: +33-380-693-223.
E-mail address: berdeaux@dijon.inra.fr (O. Berdeaux).
0009-3084/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0009-3084(01)00170-0

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S. Gna¨dig et al. / Chemistry and Physics of Lipids 112 (2001) 121–135
1. Introduction
arachidonic acid pathway and prostaglandin
metabolism as one possible way of action.
In order to clarify the metabolic pathway and
to study the physiological effects of 9c,11t-18:2
and its desaturation and elongation metabolites,
synthesis of 9c,11t-18:2; 6c,9c,11t-18:3 and
8c,11c,19t-20:3 and their [1-14C]-radiolabeled
analogs is necessary. Synthesis of also small quan-
tities of pure metabolites allow the use in biologi-
cal in vitro experiments. Moreover, the utilization
of radiolabeled analogs permits the detection of
small changes in metabolism products.
The aim of this work was to develop two
efficient stereoselective syntheses for 8c,11c,13t-
20:3 and 6c,9c,11t-18:3 which allowed their prepa-
ration up to a gram scale, in high chemical and
isomeric purities.
Conjugated linoleic acid (CLA) is a collective
term that refers to a mixture of positional and
geometrical isomers of linoleic acid (9c,12c-18:2),
which have two conjugated double bonds in cis or
trans configuration. The most important isomer of
CLA is the 9c,11t-18:2 as it is found naturally in
ruminant animal tissues, ruminant animal fat and
milk fat, as major sources of CLA in human
nutrition (Chin et al., 1992). It is considered that
CLA is formed as a consequence of microbial
hydrogenation in the rumen and by D9 desatura-
tion of trans vaccenic acid in the mammary gland
(Kepler et al., 1966; Griinari et al., 2000). For
example, about 85–90% of total CLA found in
milk fat is 9c,11t-18:2 (Jiang et al., 1996; Lin et
al., 1995).
CLA has reached scientific interest because of
possible beneficial effects for human health. It has
been reported to have anticarcinogenic and an-
tiatherogenic effects in various animal experi-
ments (Banni and Martin, 1998; Se´be´dio et al.,
1999a,b; Pariza et al., 2000). Furthermore, it has
been shown in several feeding studies that CLA
influenced body composition in different animals,
as it reduced body fat and increases lean body
mass (Park et al., 1999; West et al., 1998).
Until now, the way of action of CLA is still
unclear. To ensure the effects of CLA, including
possible interactions of one CLA-isomer to an-
other and to clarify their reaction pathways, the
syntheses of pure CLA-isomers were necessary.
Recently, methods to synthesize large quantities
of purified isomers, in particular, 9c,11t-18:2 and
its stable isotopes were developed in order to
carry out animal and human experiments on iso-
lated isomers (Berdeaux et al., 1997; Lie Ken Jie
et al., 1997; Adlof, 1997).
Moreover, these syntheses should give the pos-
sibility to prepare the corresponding [1-14C]
analogs.
2. Experimental procedures
2.1. Laboratory materials
Starting materials and chemical reagents were
purchased from Acros Organic (Noisy-Le-Grand,
France) or Sigma–Aldrich (Saint Quentin
Fallavier, France) and [14C]-barium carbonate (55
mCi mmol−1) from Mayak Production Associa-
tion. Silica gel (35–70 mesh) was purchased from
SDS (Peypin, France).
All solvents were purified before use:
dichloromethane,
hexamethylphosporamide
(HMPA) and acetonitrile were distilled from cal-
cium hydride; tetrahydrofuran (THF) and diethyl
ether from sodium benzophenone ketyl.
It was recently shown by the working groups of
Se´be´dio and Banni that CLA could be metabo-
lized in vivo into conjugated C18:3 and C20:3
fatty acids by desaturation and followed elonga-
tion (Banni et al., 1999; Se´be´dio et al., 1997,
1999a,b). Also a diminution of the PGE2 level
after CLA-treatment was found by several work-
ing groups (Liu and Belury, 1998). These results
hypothesized an interaction of CLA with the
2.2. Nuclear magnetic resonance
Nuclear magnetic resonance (NMR) spectra
were obtained on a Bruker FT-NMR spectrome-
1
ter operating at 500 MHz for H and 125.8 MHz
for 13C. Unless otherwise stated spectra were
recorded in CDCl3, and chemical shifts were re-
ported (in ppm) downfield from tetramethylsilane
(l).

S. Gna¨dig et al. / Chemistry and Physics of Lipids 112 (2001) 121–135
123
2.3. Gas chromatography
i.d., film thickness 0.25 mm, SGE, Melbourne,
Australia). The instrument was fitted with a split-
less injector maintained at 250 °C and coupled
with a Fourier-transform infrared spectrometer
(FTS 60A, Bio-Rad, Cambridge, MA). The two
instruments were connected by a Digibal tracer®
direct-deposition interface. The oven temperature
was programmed from 60 to 200 °C (20 °C
min−1).
2.3.1. Analytical gas chromatography analysis
Two gas chromatographs were used for gas
chromatography (GC) analyses. The first one was
a Carlo Erba HRGC 5300 Mega Series equipped
with a ‘on column’ injector and a flame ionization
detector. Helium was the carrier gas (1 ml min−1).
The column used was a DB5 fused silica capillary
column (J & W Scientific, Folsom, CA; 0.25 mm of
film thickness, 8-m length, 0.25-mm i.d.). It was
programmed from 60 to 250 °C at 20 °C min−1
and held at this temperature until completion of
the analysis. The second one was a Packard model
438A equipped with a ‘Ross’ injector and a flame
ionization detector. Helium was the carrier gas (1
ml min−1). The column used was a HP1 fused
silica capillary column (Hewlett-Packard, Les Ulis,
France; 0.25-mm film thickness, 30-m length, 0.25-
mm i.d.). The oven temperature was programmed
2.6. Thin layer chromatography
Thin layer chromatography (TLC) was per-
formed on a 0.25 mm pre-coated silica gel plate
containing a fluorescent indicator. Spots were vi-
sualized using one or more of the following tech-
niques: (a) UV illumination; (b) spraying with a
solution containing anisaldehyde (Ka¨gi-Miescher
reagent: anisaldehyde 12.5 ml; acetic acid 5 ml;
sulfuric acid 17 ml; 95% ethanol 450 ml); and (c)
iodine vapor.
from 60 to 200 °C at 10 °C min−1
.
2.4. Gas chromatography–mass spectrometry
2.7. Re6ersed phase-high performance liquid
chromatography (Banni et al., 1994)
A Hewlett-Packard 5890 gas chromatograph
coupled to an Hewlett-Packard model 5970 mass
spectrometer instrument was used for the gas
chromatography–mass spectrometry (GC–MS)
analyses. The latter was operated in the electron
impact mode at 70 eV with a source temperature
of 250 °C. The GC separation was performed on
a DB5 capillary column as described above, and
helium was used as carrier gas. The oven tempera-
ture was programmed from 60 to 190 °C at 20 °C
min−1. Splitless injection was used with the injec-
tion port maintained at 250 °C. GC–MS analysis
of 4,4-dimethyloxazoline (DMOX) were carried
out using a BPX column (Melbourne, Australia;
30-m length, 0.25-mm i.d. and 0.25-mm film thick-
ness), which was programmed from 60 to 170 °C
Separation of the 6c,9c,11t-18:3 and 6c,9t,11t-
18:3 were performed with a Varian 9010 liquid
chromatograph, equipped with a JASCO MD-
1510 photodiode array detector. A C-18 Nucleosil
(SFCC, Eragny, France; 250-mm length, 10-mm
i.d. and 5-mm particle size) was used for separa-
tion. The mobile phase was CH3CN/H2O/
CH3CO2H (70:30:0.12, v/v/v), flowing at a rate of
4 ml min−1. The photodiode array detector was
set at a primary monitoring wavelength of 234 nm
to detect fatty acids containing conjugated diene
and a secondary monitoring wavelength of 200 nm
(to detect non-conjugated dienic fatty acids).
at 20 °C min−1
.
2.8. Re6ersed phase-high performance liquid
2.5. Gas–liquid
chromatography for labeled compounds
chromatography–Fourier-transform infrared
spectroscopy
Analytical reversed phase-high performance liq-
uid chromatography (RP-HPLC) analyses were
obtained with a Shimadzu LC-10AS pump fitted
with a Shimadzu SCL-10A system controller. The
Samples were analyzed by GC (HP 5890 gas
chromatograph; column BPX 70, 30 m×0.25 mm

124
S. Gna¨dig et al. / Chemistry and Physics of Lipids 112 (2001) 121–135
radioactive detector was a Berthold LB 503 moni-
tor (liquid scintillation cocktail, MS2 from SDS).
A Zorbax SB C18 column (250×4.6 mm2) was
used and the mobile phase was ethanol/water/trifl-
uoroacetic acid (70:30:0.1, v/v/v) flowing at a rate
of 1.5 ml min−1).
min, diluted with a mixture of petroleum ether/di-
ethyl ether (3:1, v/v) and filtered on a pad of silica
gel. After evaporation of solvents, chromatogra-
phy of the residue on silica gel using petroleum
ether/diethyl ether (90:10, v/v) gave the aldehyde
4 as a colorless oil (11.28 g, 36 mmol, 75.5%
yield). TLC: Rf=0.84 (petroleum ether/diethyl
ether, 60:40, v/v). GC/MS: m/z=255 [M-57]+.
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. A Zorbax SB C18
column (250×21.2 mm2) was used for all labeled
separations. The mobile phase was acetonitrile/
water/acetic acid (70:30:0.1, v/v/v); flowing at rate
3.1.3. 1-Iodo-7-(2-tetrahydropyranyloxy)heptane
To a solution containing triphenylphosphine
(20.1 g, 76.3 mmol), imidazole (10.34 g, 152
mmol) and 7-(2-tetrahydropyranyloxy)heptan-1-ol
1 (10.67, 50.8 mmol) in 140 ml of THF, iodine
(19.36 g, 76.3 mmol) was added at −10 °C. The
solution was allowed to warm up to room temper-
ature and then stirred for 30 min. The reaction
mixture was diluted with diethyl ether, washed
successively with a saturated solution of sodium
thiosulfate and a saturated solution of sodium
hydrogen carbonate. The organic layer was dried
over Na2SO4 and evaporated to dryness. The
residue was stirred with petroleum ether to precip-
itate triphenyl phosphine oxide. After filtration
and concentration, the residue was chro-
matographed on silica gel (petroleum ether/di-
ethyl ether, 97:3, v/v) to give 2 (13.1 g, 40.2 mmol,
79.1% of yield) as a clear oil. TLC: Rf=0.43
(petroleum ether/diethyl ether, 95:5, v/v). GC/MS:
m/z=101 [OTHP]+, 325 [MꢀH]+, 326 [M]+. GC/
FTIR: 2932.7, 2855.5, 1136.1, 1078.3, 1026.6
of 10 ml min−1
.
3. Synthetic procedure
Unless otherwise stated, all reactions were car-
ried out under nitrogen as inert atmosphere.
3.1. Synthesis of (8Z,11Z,13E)-eicosa-8,11,13-tri-
enoic acid
3.1.1. 3-(t-Butyldiphenylsilyloxy)propanol
To a mixture of propane-1,3-diol (22.13 g, 0.29
mol), dimethylaminopyridine (1.28 g, 0.01 mmol)
and triethylamine (9.6 g, 0.095 mol) in 250 ml of
CH2Cl2 was added dropwise at room temperature
t-butyldiphenylchlorosilane (20 g, 0.072 mol) in
60 ml of CH2Cl2. After stirring at room tempera-
ture for 18 h, the mixture was diluted with diethyl
ether, washed with brine, several times with water
and then dried over Na2SO4. After concentration
in vacuo, flash chromatography of the residue on
silica gel (petroleum ether/diethyl ether, 60:40,
v/v) gave the monoprotected diol (19.02 g, 60.5
mmol, 84% yield) as a colorless oil. TLC: Rf=
0.68 (petroleum ether/diethyl ether, 50:50, v/v).
GC/MS: m/z=257 [M-57]+.
cm−1 1H NMR (CDCl3): 1.25–1.35 (m, 6H,
,
3,4,5-H2), 1.4–1.9 (m, 10H, 2, 6-H2, 3%,4%,5%-H2
(THP)), 3.11 (t, 2H, J=7 Hz, 1-H2) 3.25–3.85
(m, 4H, 7-H2, 6%-H2 (THP)), 4.5 (t, 1H, J=3.4
Hz, 2%H (THP)). 13C NMR (CDCl3): 98.6 (THP
C2%), 67.3 (C7), 62.1 (THP C6%), 33.3 (C2), 30.6
(THP C3%), 30.3 (C6), 29.5 (C3), 28.2 (C4), 25.9
(THP C5%), 25.3 (C5), 19.5 (THP C4%), 7.0 (C1).
3.1.2. 3-(t-Butyldiphenylsilyloxy)propanal
To a mixture of pyridinium dichromate (PDC;
26.95 g, 70 mmol) and powdered molecular sieves
3.1.4. [7-(2-Tetrahydropyranyloxy)heptyl]tri-
phenylphosphonium iodide
A solution containing triphenylphosphine (19.3
g, 73.6 mmol), 1-iodo-7-(2-tetrahydropyrany-
loxy)heptane 2 (12 g, 36.7 mmol) and calcium
carbonate (2 g) in 100 ml of acetonitrile was
heated at 80–85 °C for 22 h. After filtration of
,
4 A (90 g) in 190 ml of CH2Cl2, was added
dropwise 3-(t-butyldiphenylsilyloxy)propan-1-ol
(15 g, 47 mmol) in 130 ml of CH2Cl2 at 0 °C. The
mixture was stirred at room temperature for 45

S. Gna¨dig et al. / Chemistry and Physics of Lipids 112 (2001) 121–135
125
calcium carbonate and concentration, the oily
residue was dissolved in a minimum of CH2Cl2
and precipitated by addition of diethyl ether.
The phosphonium salt 3, obtained as a gum
(19.9 g, 34 mmol, 92.6% yield), was dried under
vacuum (0.05 mmHg) at room temperature.
TLC: Rf=0.69 (CH2Cl2/MeOH, 95:5, v/v).
3%,4%,5%-H2 (THP)), 1.85–2.40 (m, 5H, OH, 2,5-
H2), 3.25–3.9 (m, 6H, 1,10-H2, 6%-H2 (THP)) 4.5
(t, 1H, J=3.5 Hz, 2%-H (THP)), 5.3–5.55 (m,
2H, HCꢁCH). 13C NMR (CDCl3): 133.1 (C4),
125.1 (C3), 98.8 (THP C2%), 67.5 (C10), 62.3
(THP C6%), 62.2 (C1), 30.7 (THP C3%, C9), 29.6
(C9), 29.5 (C6), 29.0 (C2), 27.2 (THP C5%), 26.0
(C5), 25.4 (C8), 19.6 (THP C4%).
3.1.5. (3Z)-1-(t-butyldiphenylsilyloxy)-10-(2-
tetrahydropyranyloxy)dec-3-ene
3.1.7. (3Z)-1-iodo-10-(2-tetrahydropyranyl-
To a solution of phosphonium salt 3 (15.48,
26.3 mmol) in a mixture of THF/HMPA (150
ml, 5:1, v/v), n-BuLi (2.5 M in hexane, 10.52 ml,
26.3 mmol) was added dropwise at −78 °C.
oxy)dec-3-ene
To a solution containing triphenylphosphine
(6.14 g, 23.4 mmol), imidazole (3.18 g, 46.8
mmol) and (3Z)-10-(2-tetrahydropyranyloxy)dec-
3-en-1-ol 6 (5.94 g, 23.4 mmol) in 45 ml of
THF, cooled to −30 °C, iodine (5.94 g, 23.4
mmol) was added. The solution was allowed to
warm up to room temperature and stirred at
this temperature for 30 min. The reaction mix-
ture was diluted with diethyl ether (250 ml),
washed successively with a saturated solution of
sodium thiosulfate (250 ml) and water (2×250
ml). The organic layer was dried (Na2SO4) and
evaporated. The residue was stirred with
petroleum ether to precipitate triphenylphos-
phine oxide and filtered. After concentration in
vacuum, flash chromatography of the residue on
silica gel (petroleum ether/diethyl ether, 95:5, v/
v) gave the iodide 7 (4.83 g, 13.1 mmol, 84.2%
of yield) as an oil. TLC: Rf=0.55 (petroleum
ether/diethyl ether, 95:5, v/v). GC/MS: m/z=
101 [OTHP]+, 239 [M-I]+, 366 [M]+. GC/FTIR:
After stirring for
1
h
at −78 °C, 3-(t-
butyldiphenylsilyloxy)propanal 4 (8.61 g, 27.5
mmol) in 25 ml of THF was added slowly, and
the temperature was allowed to warm up to
0 °C within 90 min. After stirring at room tem-
perature for 1 h, the reaction mixture was di-
luted with diethyl ether/petroleum ether (1:3,
v/v) and filtered on a pad of silica gel. After
evaporation of the solvents, the residue was
chromatographed on silica gel (petroleum ether/
diethyl ether, 95:5, v/v) and afforded 8.31 g of 5
as an oil (16.8 mmol, 63% of yield). TLC: Rf=
0.42 (petroleum ether/diethyl ether, 95:5, v/v).
GC/MS: m/z=101 [OTHP]+, 437 [M-57]+.
3.1.6. (3Z)-10-(2-tetrahydropyranyloxy)dec-
3-en-1-ol (Freeman and Kim, 1992)
To a solution of 5 (8.10 g, 16.4 mmol) in 60
ml of THF, was added at room temperature to
3007.1, 2931.9, 2854.1, 1137.1, 1032.2 cm−1, H
1
a
1
M
solution of tetra-n-butylammonium
NMR (CDCl3): 1.2–1.9 (m, 14H, 6,7,8,9-H2,
3%,4%,5%-H2 (THP)), 2.0 (m, 2H, 5-H2), 2.59 (q,
2H, J=7.3 Hz, 2-H2) 3.1 (t, 2H, J=7.3 Hz,
1-H2), 3.3–3.9 (m, 4H, 10-H2, 6%-H2 (THP), 4.55
(t, 1H, J=2.5 Hz, 2%-H (THP)), 5.2–5.6 (m, 2H,
HCꢁCH).
fluoride in THF (9.75 and 32.8 ml). The result-
ing solution was stirred for 1 h, diluted with
diethyl ether and washed with water. The
aqueous layer was extracted with diethyl ether.
The organic layers were combined and dried
(Na2SO4). The solvent was evaporated in vacuo
and the residue was chromatographed on silica
gel (petroleum ether/diethyl ether, 60:40, v/v) to
give 6 (4.18 g, 16 mmol, 99% of yield) as a clear
oil. TLC: Rf=0.65 (petroleum ether/diethyl
ether, 50:50, v/v). GC/MS: m/z=101 [OTHP]+,
255 [M-1]+, 256 [M]+. GC/FTIR: 3386.0,
3.1.8. (3Z)-[10-(2-tetrahydropyranyloxy)dec-
3-enyl]triphenylphosphonium iodide
A solution containing triphenylphosphine (6.87
g, 26.2 mmol) (3Z)-1-iodo-10-(2-tetrahydropy-
ranyloxy)dec-3-ene 7 (4.82 g, 13.1 mmol) and
calcium carbonate (0.7 g) in 36 ml of acetonitrile
was heated at 75 °C for 20 h. After filtration of
calcium carbonate and evaporation of acetoni-
3005.3, 2934.9, 2856.4, 1138.9, 1025.4 cm−1, H
NMR (CDCl3): 1.2–1.85 (m, 14H, 6,7,8,9-H2,
1

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S. Gna¨dig et al. / Chemistry and Physics of Lipids 112 (2001) 121–135
trile, the oily residue was dissolved in a minimum
of CH2Cl2 and precipitated by addition of diethyl
ether. The phosphonium salt 8 obtained as a gum
(7.47 g, 11.9 mmol, 91% yield) was dried under
vacuum (0.05 mmHg) for 12 h at room tempera-
ture. TLC: Rf=0.80 (CH2Cl2/MeOH, v/v).
3.1.10. (7Z,10Z,12E)-1-bromononadeca-7,10,12-
triene
9 (2.33 g, 6.43 mmol) diluted in 20 ml of
CH2Cl2, was added at 0 °C to a slurry of
triphenylphosphine dibromide (5.41 g, 12.82
mmol) in 20 ml of CH2Cl2. The mixture was
stirred at room temperature for 15 min, diluted
with diethyl ether, washed with a saturated solu-
tion of sodium bicarbonate, then with water. The
organic layer was dried over Na2SO4 and concen-
trated. Flash chromatography of the residue on
silica gel (hexane) gave the bromide 10 (1.7 g, 4.98
mmol, 77.4% yield) as a colorless oil. TLC: Rf=
0.94 (petroleum ether/diethyl ether, 95:5, v/v).
GC/MS: m/z=262/264 [MꢀBr]+, 340/342 [M]+.
1H NMR (CDCl3): 0.85 (t, 3H, J=6.9 Hz, 19-
H3), 1.1–1.5 (m, 14H, 3,4,5,15,16,17,18-H2), 1.8–
1.9 (m, 2H, 2-H2), 2.0–2.15 (m, 4H, 6,14-H2), 2.90
(t, 2H, J=6.5, 9-H2), 3.38 (t, 2H, J=6.5, 1-H2),
5.23 (dt, 1H, J7–6=7.6 Hz, J7–8=10.6 Hz, 7-H),
5.36 (m, 2H, 8,10-H), 5.67 (dt, 1H, J13–12=15 Hz,
3.1.9. (7Z,10Z,12E)-1-(2-tetrahydropy-
ranyloxy)nonadeca-7,10,12-triene
At −78 °C, 3.66 ml (9.15 mmol) of a 2.5 M
solution of n-BuLi in hexane was added to a
solution of 1.67 g (10.3 mmol) of hexamethyldisi-
lazane (HMDS) in 32 ml of THF and 8 ml of
HMPA. The reaction temperature was raised to
0 °C for 3 min, and cooled back to −78 °C,
then a solution of 6.4 g (10.2 mmol) of the
phosphonium salt 8 in 42 ml of THF was added.
The reaction mixture turned to orange. After
stirring at −78 °C for 30 min, (2E)-non-2-enal
(1.86 g, 1.33 mmol) in 5 ml of THF was added
(the orange reaction mixture became yellow). Af-
ter 45 min at −78 °C, the cooling bath was
removed and the reaction was stirred at room
temperature for 90 min. The mixture was diluted
with diethyl ether/petroleum ether (1:3, v/v) and
filtered on a pad of silica gel. After evaporation of
the solvents, the residue was chromatographed on
silica gel (petroleum ether/diethyl ether, 98:2, v/v)
and afforded 1.9 g of 9 as an oil (5.25 mmol, 51%
of yield). TLC: Rf=0.60 (petroleum ether/diethyl
ether, 95:5, v/v). GC/MS: m/z=101 [OTHP]+,
J13–14=7 Hz, 13-H), 5.94 (dd, 1H, J11–10=10.9
Hz, J11–12=10.9 Hz, 11-H), 6.30 (dd, 1H, J12–
11=10.9 Hz, J12–13=15.0 Hz, 12-H). 13C NMR
(CDCl3): 135.3 (C13), 130.1 (C7), 128.8 (C11),
127.8 (C12), 127.6 (C10), 125.3 (C8), 33.9 (C2),
32.9 (C14), 32.8 (C17), 31.7 (C1), 29.4 (C15),
29.33 (C5), 28.9 (C16), 28.4 (C4), 28.0 (C3), 27.1
(C6), 26.0 (C9), 22.6 (C18), 14.1 (C19).
3.1.11. (8Z,11Z,13E)-eicosa-8,11,13-trienoic acid
Bromide 10 (670 mg, 1.96 mmol) dissolved in
1.5 ml of anhydrous diethyl ether was added to
magnesium turnings (120 mg, 4.93 mmol) and a
crystal of iodine in 3.5 ml of anhydrous diethyl
ether. The reaction mixture was refluxed for 90
min at 35 °C. The resulting Grignard compound
was carbonated at −20 °C with CO2 (liberated
from 500 mg (2.5 mmol) of barium carbonate by
addition of concentrated sulfuric acid). After stir-
ring at −20 °C for 2.5 h, 10 ml of 5% NH4Cl
were added. The mixture was diluted with 50 ml
of saturated NH4Cl solution and 50 ml of ether.
A 1 N H2SO4 solution was added dropwise until
the aqueous layer was acidified to pH 3. After
decantation, the etheral layer was washed with
water, dried (Na2SO4), filtered and concentrated.
Flash chromatography of the residue on silica gel
1
278 [M]+. H NMR (CDCl3): 0.88 (t, 3H, J=7
Hz, 19-H3), 1.18–1.90 (m, 22H, 2,3,4,5,15,16,17,
18-H2, 3%,4%,5%-H2 (THP)), 2.0–2.15 (m, 4H, 6,14-
H2), 2.90 (t, 2H, J=7, 9-H2), 3.33–3.91 (m, 4H,
1-H2, 6%-H2 (THP)), 4.57 (t, 1H, J=2.8 Hz, 2%-H
(THP)), 5.25 (dt, 1H, J7–6=7.6 Hz, J7–8=10.6
Hz, 7-H), 5.37 (m, 2H, 8,10-H), 5.68 (dt, 1H,
J
J
13–12=15 Hz, J13–14=7 Hz, 13-H), 5.95 (dd, 1H,
11–10=10.9 Hz, J11–12=10.9 Hz, 11-H), 6.32
(dd, 1H, J12–11=10.9 Hz, J12–13=15.0 Hz, 12-H).
13C NMR (CDCl3): 135.3 (C13), 130.4 (C7), 128.8
(C11), 127.7 (C12), 127.6 (C10), 125.3 (C8), 98.8
(THP C2%), 67.6 (C1), 62.3 (THP C6%), 32.9 (C14),
31.7 (THP C3%), 30.8 (C2), 29.7 (C17), 29.6 (C15),
29.36 (C4), 19.2 (C5), 28.9 (C16), 27.2 (THP C5%),
26.2 (C6), 26.0 (C3), 25.5 (C9), 22.6 (C18), 19.7
(THP C4%), 14.1 (C19).

S. Gna¨dig et al. / Chemistry and Physics of Lipids 112 (2001) 121–135
127
(hexane/diethyl ether/acetic acid, 90:10:0.1, v/v/v)
gave the free fatty acid 11 (328.5 mg, 1.07 mmol,
55% yield) as an oil. TLC: Rf=0.25 (hexane/di-
5-H2, 6%-H2 (THP)), 4.57 (t, 1H, J=3.3 Hz, 2%-H
(THP)). 13C NMR (CDCl3): 98.8 (THP C2%), 67.6
(C5), 62.3 (THP C6%), 33.1 (C2), 31.0 (THP C3%),
28.5 (C4), 27.2 (THP C5%), 25.5 (C3), 19.7 (THP
C4%), 7.1 (C1).
1
ethyl ether/acetic acid, 90:10:0.1, v/v/v). H NMR
(CDCl3): 0.85 (t, 3H, J=7 Hz, 20-H3), 1.2–1.45
(m, 14H, 4,5,6,16,17,18,19-H2), 1.62 (quint, 2H,
J=7.5 Hz, 3H2), 2.0–2.15 (m, 4H, 7,15-H2), 2.34
(t, 2H, J=7.5, 2-H2), 2.88 (t, 2H, J=6.8, 10-H2),
5.23 (dt, 1H, J8–7=7.6 Hz, J8–9=10.6 Hz, 8-H),
5.28 (s, 1H, COOH), 5.36 (m, 2H, 9,11-H), 5.66
(dt, 1H, J15–14=15 Hz, J15–16=7 Hz, 15-H), 5.94
(dd, 1H, J12–11=10.9 Hz, J12–13=10.9 Hz, 12-H),
6.30 (dd, 1H, J13–12=10.9 Hz, J13–14=15.0 Hz,
13-H). 13C NMR (CDCl3): 180.3 (C1), 135.3
(C14), 130.2 (C8), 128.7 (C12), 127.7 (C13), 127.6
(C11), 125.3 (C9), 34.0 (C2), 32.9 (C15), 31.7
(C18), 29.4 (C16), 29.3 (C6), 28.9 (C5), 28.9
(C17), 28.8 (C4), 27.1 (C7), 26.0 (C3), 24.6 (C10),
22.6 (C19).
3.2.2. [5-(2-Tetrahydropyranyloxy)pentyl]tri-
phenylphosphonium iodide
14.03 g (25.03 mmol) [5-(2-tetrahydropyrany-
loxy)pentyl]triphenylphosphonium iodide 14 was
obtained in 95% yield using the procedure previ-
ously described for the preparation of the pho-
phonium salt 3. The reaction time was 24 h and
temperature was 80–85 °C. TLC: Rf=0.69
(CH2Cl2/MeOH, v/v). 1H NMR (CDCl3): 1.4–
1.85 (m, 12H, 2,3,4-H2, 3%,4%,5%-H2 (THP)), 3.30–
3.85 (m, 4H, 5-H2, 6%-H2 (THP)), 4.5 (m, 1H, 2%-H
(THP)), 5.3 (m, 2H, 1-H), 5.37 (m, 2H, 8,10-H),
7.68–7.88 (m, 15H, ꢀC6H5).
3.1.12. (8Z,11Z,13E)-[1-14C]-eicosa-8,11,13-tri-
enoic acid
3.3. (3Z)-1-(t-butyldiphenylsilyloxy)-8-(2-tetra-
hydropyranyloxy)oct-3-ene
(8Z,11Z,13E)-[1-14C]-eicosa-8,11,13-trienoic
acid 11% (30 mCi; specific activity: 50 mCi
mmol−1, 0.6 mmol, 60% yield from 10) was ob-
tained from bromide 10 (340 mg; 1 mmol), mag-
nesium turnings (65 mg) and Ba14CO3 (216 mg,
1.1 mmol, 60.5 mCi) according to the procedure
described for the preparation of the unlabeled
acid 11. The radiochemical purity of the [1-14C]
acid was found to be 93.9% by analytical RP-
HPLC.
A Wittig reaction between the ylide of 14 and
the aldehyde 4 was effected as described for the
preparation of compound 5. The (3Z)-1-(t-
butyldiphenylsilyloxy)-8-(2-tetrahydropyranyloxy)
oct-3-ene 15 was obtained in 54.7% yield (4.35 g,
9.33 mmol). TLC: Rf=0.44 (petroleum ether/di-
ethyl ether, 90:10, v/v). 1H NMR (CDCl3): 1.05 (s,
9H, ꢀCH3), 1.15–1.9 (m, 10H, 3,4-H2, 3%,4%,5%-H2
(THP)), 2.0 (q, 2H, J=7 Hz, 5-H2), 2.3 (q, 2H,
J=6.8, 2-H2), 3.65 (t, 2H, J=7, 1-H2), 3.32–3.90
(m, 4H, 8-H2, 6%-H2 (THP)), 4.57 (t, 1H, J=3.3
Hz, 2%-H (THP)), 5.34–5.50 (m, 2H, ꢀHCꢁCHꢀ),
7.3–7.75 (m, 10H, ꢀC6H5).
3.2. Synthesis of (6Z,9Z,11E)-octadeca-6,9,11-
trienoic acid
3.2.1. 1-Iodo-5-(2-tetrahydropyranyloxy)pentane
7.85 g (26.34 mmol) of 1-iodo-5-(2-tetrahy-
dropyranyloxy)pentane 13 was obtained from 7-
(2-tetrahydropyranyloxy) pentan-1-ol in 72.6%
yield using the procedure previously described for
the preparation of 2. TLC: Rf=0.43 (petroleum
ether/diethyl ether, 95:5, v/v). GC/MS: m/z=101
[OTHP]+, 298 [M]+. GC/FTIR: 2938, 2863.9,
1135.6, 1030.1 cm−1. 1H NMR (CDCl3): 1.45–2.0
(m, 10H, 3,4-H2, 3%,4%,5%-H2 (THP)), 2.4 (m, 2H,
2-H2), 3.20 (t, 2H, J=7, 1-H2), 3.70–3.90 (m, 4H,
3.3.1. (3Z)-8-(2-tetrahydropyranyloxy)oct-3-
en-1-ol
To a solution of the THP ether 15 (4.32 g, 9.27
mmol) in 60 ml of THF, was added at room
temperature to a 1 M solution of tetra-n-butylam-
monium fluoride in THF (9.75 ml, 32.8 mmol).
The solution was stirred for 90 min, diluted with
500 ml of diethyl ether and washed with H2O
(3×500 ml). The water layer was extracted with
diethyl ether, and the organic layers were com-

128
S. Gna¨dig et al. / Chemistry and Physics of Lipids 112 (2001) 121–135
bined and dried (Na2SO4). After concentration,
the residue was chromatographed over silica gel
(petroleum ether/diethyl ether, 60:40, v/v) to give
16 (2.07 g, 9.1 mmol, 98.1% yield) as a clear oil.
TLC: Rf=0.16 (petroleum ether/diethyl ether,
90:10, v/v). GC/MS: m/z=101 [OTHP]+, 228
[M]+. GC/FTIR: 3366.5, 3006.1, 1657.27,
preparation of compound 9. The (5Z,8Z,12E)-1-
(2-tetrahydropyranyloxy)heptadeca-5,8,10-triene
19 was obtained in 67.8% (1.74 g, 5.21 mmol).
TLC: Rf=0.49 (petroleum ether/diethyl ether,
98:2, v/v). GC/MS: m/z=101 [OTHP]+, 334
[M]+. 1H NMR (CDCl3): 0.89 (t, 3H, J=6.7
Hz, 17-H3), 1.20–1.90 (m, 18H, 2,3,13,14,15,16-
H2, 3%,4%,5%-H2 (THP)), 2.07–2.18 (m, 4H, 4,12-
H2), 2.91 (t, 2H, J=7, 7-H2), 3.35–3.90 (m, 4H,
1-H2, 6%-H2 (THP)), 4.6 (m, 1H, 2%-H (THP)),
5.25 (dt, 1H, J5–4=7.6 Hz, J5–6=10.6 Hz, 5-
1
1139.4, 1032.9 cm−1. H NMR (CDCl3): 1.35–
1.9 (m, 11H, 6,7-H2, 3%,4%,5%-H2 (THP), ꢀOH),
2.1 (q, 2H, J=7.3 Hz, 5-H2), 2.32 (q, 2H, J=
6.7, 2-H2), 3.3–3.95 (m, 6H, 1,8-H2, 6%-H2
(THP)), 4.55 (t, 1H, J=3.6 Hz, 2%-H (THP)),
5.3–5.6 (m, 2H, ꢀHCꢁCHꢀ).
H), 5.37 (m, 2H, 6,8-H), 5.68 (dt, 1H, J11–10
15 Hz, J11–12=7 Hz, 11-H), 5.95 (dd, 1H,
9–8=10.9 Hz, J9–10=10.9 Hz, 9-H), 6.35 (dd,
=
J
1H, J10–9=10.9 Hz, J10–11=15.0 Hz, 10-H). 13C
NMR (CDCl3): 135.3 (C11), 130.4 (C5), 128.8
(C9), 127.7 (C10), 127.6 (C8), 125.3 (C6), 98.8
(THP C2%), 67.6 (C1), 62.3 (THP C6%), 32.9
(C12), 31.7 (THP C3%), 30.8 (C2), 29.6 (C13),
29.36 (C14), (THP C5%), 26.2 (C4), 26.0 (C3),
22.6 (C16), 19.7 (THP C4%), 14.1 (C17).
3.3.2. (3Z)-1-iodo-8-(2-tetrahydropyranyloxy)oct-
3-ene
2.58 g (7.64 mmol) of (3Z)-1-iodo-8-(2-te-
trahydropyranyloxy)oct-3-ene 17 was obtained
from alcohol 16 in 84% yield using the proce-
dure previously described for the preparation of
7. TLC: Rf=0.58 (petroleum ether/diethyl ether,
50:50, v/v). GC/MS: m/z=101 [OTHP]+, 211
[MꢀI]+, 338 [M]+. GC/FTIR: 2938.7, 2862.9,
3.3.5. (5Z,8Z,10E)-1-bromoheptadeca-5,8,10-tri-
ene
1719.6, 1652.1, 1137.1, 1032.5 cm−1 1H NMR
.
(CDCl3): 1.17–1.90 (m, 10H, 3,4-H2, 3%,4%,5%-H2
(THP)), 2.06 (q, 2H, J=7.2 Hz, 5-H2), 2.63 (q,
2H, J=7.3 Hz, 2-H2), 3.13 (t, 2H, J=7.3, 1-
H2), 3.35–3.92 (m, 4H, 8-H2, 6%-H2 (THP)), 4.57
(t, 1H, J=3.5 Hz, 2%-H (THP)), 5.1–5.6 (m,
2H). 13C NMR (CDCl3): 131.9 (C4), 127.7 (C3),
98.5 (THP C2%), 67.0 (C8), 62.0 (THP C6%), 31.1
(THP C3%), 30.4 (C7), 29.0 (C2), 26.9 (THP C5%),
25.8 (C5), 25.1 (C6), 19.3 (THP C4%), 5.1 (C1).
The tetrahydropyranyl ether 19 was converted
to the bromide 20 in 53.3% yield (\97% purity
by GC) by the procedure described for the
preparation of 10. TLC: Rf=0.84 (petroleum
ether/diethyl ether, 98:8, v/v). 1H NMR
(CDCl3): (t, 3H, J=6.7 Hz, 17-H3), 1.2–1.60
(m, 10H, 3,13,14,15,16-H2), 1.8–2.2 (m, 6H,
2,4,12-H2), 2.90 (t, 2H, J=7, 7-H2), 3.4 (t, 2H,
1-H2), 5.25 (dt, 1H, J5–4=7.6 Hz, J5–6=10.6
Hz, 5-H), 5.37 (m, 2H, 6,8-H), 5.68 (dt, 1H,
J
11–10=15 Hz, J11–12=7 Hz, 11-H), 5.95 (dd,
3.3.3. (3Z)-[8-(2-tetrahydropyranyloxy)oct-3-enyl]
triphenylphosphonium iodide
1H, J9–8=10.9 Hz, J9–10=10.9 Hz, 9-H), 6.35
(dd, 1H, J10–9=10.9 Hz, J10–11=15.0 Hz, 10-
H). 13C NMR (CDCl3): 135.2 (C11), 129.0 (C5),
128.8 (C9), 128.4 (C10), 127.3 (C8), 125.0 (C6),
33.8 (C2), 33.0 (C12), 32.0 (C1), 31.6 (C15), 29.0
(C13), 28.9 (C14), 28.0 (C3), 26.1 (C4), 26.0
(C7), 22.5 (C16), 14.0 (C17).
5.61 g (9.35 mmol) (3Z)-[8-(2-tetrahydropy-
ranyloxy)oct-3-enyl]triphenylphosphonium iodide
18 was obtained in 90% yield using the proce-
dure previously described for the preparation of
the phophonium salt 8. Reaction time was 20 h.
TLC: Rf=0.53 (CH2Cl2/MeOH, 95:5).
3.3.4. (5Z,8Z,120E)-1-(2-tetrahydropyranyloxy)
heptadeca-5,8,10-triene
A Wittig reaction between the ylide of 18 and
(2E)-non-2-enal was effected as described for the
3.3.6. (6Z,9Z,11E)-octadeca-6,9,11-trienoic acid
Bromide 20 (420 mg, 1.3 mmol) dissolved in 2
ml of anhydrous diethyl ether was added to mag-
nesium turning (86 mg, 3.5 mmol) and a crystal of

S. Gna¨dig et al. / Chemistry and Physics of Lipids 112 (2001) 121–135
129
iodine in 1 ml of anhydrous diethyl ether. The
reaction was refluxed for 90 min at 35 °C. The
resulting Grignard compounds was carbonated at
−20 °C with CO2 (liberated from 2.4 g (12.1
mmol) of barium carbonate by addition of con-
centrated sulfuric acid). After stirring at −20 °C
for 2 h, 7 ml of 5% NH4Cl were added. The
mixture was diluted with saturated NH4Cl solu-
tion. A 1 N H2SO4 solution was added dropwise
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. Flash chromatography of the residue
on silica gel (hexane/diethyl ether/acetic acid,
80:20:0.1, v/v/v) gave the acid 21 (106 mg, 0.38
mmol, 30% yield) as an oil.
BF3/MeOH were added. The solution was stirred
for 10 min at room temperature. The solution was
diluted with 2 ml of pentane and washed with a
saturated solution of sodium bicarbonate. The
organic layer was removed and was used for GC
analysis.
3.3.9. Preparation of 4,4-dimethyloxazoline
deri6ati6es
FAME (500 mg) were converted to their
DMOX derivatives by treatment with 2-amino-2-
methylpropanol (0.25 ml) in a sealed ampoule
under N2 at 170 °C for 8 h (Fay and Richli, 1991;
Luthria and Sprecher, 1993; Zhang et al., 1988).
The reaction mixture was cooled, dissolved in 3
ml of dichloromethane and washed twice with 1
ml of water. After drying the organic phase, the
solvent was removed under a stream of nitrogen
and the sample was dissolved in hexane. The
sample was applied to a short column of Florisil
which was subsequently washed with hexane prior
to elute the DMOX derivatives with a mixture of
hexane/acetone (96:4, v/v; Dobson and Christie,
1996).
TLC: Rf=0.26 (hexane/diethyl ether/acetic
1
acid, 90:10:0.1, v/v/v). H NMR (CDCl3): 0.9 (t,
3H, J=7 Hz, 18-H3), 1.2–1.5 (m, 10H,
4,14,15,16,17-H2), 1.68 (m, 2H, 3H2), 2.05–2.15
(m, 4H, 7,13-H2), 2.37 (t, 2H, J=7.5, 2-H2), 2.9
(t, 2H, J=6.8, 8-H2), 5.25 (dt, 1H, J6–5=7.6 Hz,
J
6–7=10.6 Hz, 6-H), 5.4 (m, 2H, 7,9-H), 5.69 (dt,
1H, J13–12=15 Hz, J13–14=7 Hz, 13-H), 5.94 (dd,
1H, J12–11=10.9 Hz, J12–13=10.9 Hz, 12-H), 6.30
(dd, 1H, J11–10=10.9 Hz, J11–12=15.0 Hz, 11-H),
7.3 (s, 1H, COOH).
4. Results and discussion
3.3.7. (6Z,9Z,11E)-[1-14C]-octadeca-6,9,11-tri-
Our synthetic strategy was to form the conju-
gated double bond system at the end of the syn-
thesis and also just before labeling in order to
avoid degradation and isomerization.
enoic acid
A
mixture of (6Z,9Z,11E)-[1-14C]-octadeca-
6,9,11-trienoic acid and its geometrical isomers
(8.5 mCi, 53 mCi mmol−1, 0.16 mmol, 19.3%
yield from 20) was obtained from bromide 20 (260
mg; 0.83 mmol), magnesium turnings (58 mg) and
Ba14CO3 (370 mg, 1.88 mmol, 103.4 mCi) accord-
ing to the procedure described for the preparation
of the unlabeled acid 21. The radiochemical purity
4.1. Synthesis of (8Z,11Z,13E)-eicosa-8,11,13-tri-
enoic acid
As shown in Fig. 1, the synthesis of the eicosa-
trienoic acid 11 used heptane-1,7-diol as a starting
material, which is readily available in large quan-
tities. Heptane-1,7-diol was tetrahydropyranylated
in the presence of pyridinium p-toluenesulfonate
to give the monoprotected heptane-1,7-diol 1 ac-
cording to Danieli et al. (1984). The phosphonium
salt 3 was prepared from the monoprotected hep-
tanediol 1 by reaction with iodine in modified
Garegg–Samuelsson conditions (Garegg and Sa-
muelsson, 1980; Berlage et al., 1987) to give 2
(79.1% yield) followed by reaction with
of
acid was found to be 46% by RP-HPLC. 0.2 mCi
of
(8Z,11Z,13E)-[1-14C]-eicosa-8,11,13-trienoic
(6Z,9Z,11E)-[1-14C]-octadeca-6,9,11-trienoic
acid 21% (radiochemical purity: 95%) were ob-
tained by preparative RP-HPLC.
3.3.8. Preparation of fatty acid methyl esters
Free fatty acids were converted to fatty acid
methyl esters using boron trifluoride in methanol
(14%). To free fatty acids in pentane 500 ml

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S. Gna¨dig et al. / Chemistry and Physics of Lipids 112 (2001) 121–135
triphenylphosphine in the presence of calcium car-
bonate. 1,3-Propanediol was monoprotected with
tert-butyldiphenylsilyl chloride to give 3-(t-
butyldiphenylsilyloxy)propanol (84% yield) which
ment of the iodine atom of 7 by triphenylphos-
phine in refluxing acetonitrile gave the ten-carbon
chain unsaturated phosphonium salt 8. The ylide
from 8 was reacted with commercially available
(2E)-non-2-enal to give (5Z,8Z,10E)-1-(2-tetrahy-
dropyranyloxy)heptadeca-5,8,10-triene 9 in 51%
yield. Therefore, the synthesis of the conjugated
double bond system with specific cis stereochem-
istry at the newly formed double bond was carried
out using a Wittig reaction. Lithium hexamethyld-
isilazide was chosen as a weak base in the pres-
ence of HMPA in THF at −78 °C according to
Labelle et al. (1990), to avoid isomerization of the
existing trans-double bond of the (2E)-non-2-
enal. Replacement of the tetrahydropyranyloxy
function of 9 by a bromine atom was accom-
plished by triphenylphosphine dibromide (Sonnet,
1976) in 77.4% yield. At this stage, no geometrical
was
oxidized
to
3-(t-butyldiphenylsily-
loxy)propanal 4 with PDC in 79.1% yield (Abdel-
Baky and Giese, 1986; Moody et al., 1992;
Herscovici et al., 1982). Highly stereoselective
Wittig condensation between the ylide of the
phosphonium salt 3 and aldehyde 4 in presence of
HMPA (Maryanoff and Reitz, 1989) furnished
the (3Z)-1-(t-butyldiphenylsilyloxy)-10-(2-tetrahy-
dropyranyloxy)dec-3-ene 5 in 63% yield. Selective
deprotection (desilylation) of compound 5 af-
forded alcohol 6 (Freeman and Kim, 1992) which
was transformed to iodide 7 using modified
Garegg–Samuelsson conditions (Garegg and
Samuelsson, 1980; Berlage et al., 1987). Displace-
Fig. 1. Synthesis of (8Z,11Z,13E)-eicosa-8,11,13-trienoic acid and of its corresponding [1-14C]-labeled acid.

S. Gna¨dig et al. / Chemistry and Physics of Lipids 112 (2001) 121–135
131
isomers of compounds 9 and 10 were detected by
Ken Jie et al., 1997; Berdeaux et al., 1998). For
the 8c,11c,13t-20:3 the same shift was found for
the 11-H, but it was overlaid by the shift of the
10-H.
Analysis of the impurity showed that it was a
geometrical isomer of (8Z,11Z,13E)-eicosa-
8,11,13-trienoic acid. In fact, the DMOX deriva-
1
GC, H and 13C NMR.
In previous syntheses of labeled fatty acids
(Vate`le et al., 1994; Eynard et al., 1994; Berdeaux
et al., 1995), [1-14C] fatty acids were prepared by
reaction at 80 °C of bromo precursors with
K14CN and alkaline hydrolysis at 80 °C of the
resulting nitriles. Unfortunately, in the case of
conjugated fatty acids, isomerization of the conju-
gated system occurred with the heat treatment.
Therefore, to avoid degradation and isomeriza-
tion of the cis–trans conjugated double bond
system, the carbonation of the corresponding
Grignard reagent with 14C-carbon dioxide was
chosen for the labeling (Channing and Simpson,
1993).
Firstly, the unlabeled fatty acid was prepared.
The bromide-compound 10 was transformed into
its corresponding nonadecatrienylmagnesium bro-
mide by reaction with magnesium turnings in
refluxing anhydrous diethyl ether (90 min). The
Grignard reagent was carbonated with CO2 liber-
ated from barium carbonate (Howton et al.,
1954). (5Z,8Z,10E)-eicosa-8,11,13-trienoic acid 11
was obtained in 51% yield after purification by
flash chromatography. GC analysis showed a 95%
purity and small quantities of an impurity. The
structure of (8Z,11Z,13E)-eicosa-8,11,13-trienoic
acid was confirmed by GC–MS and NMR. An
aliquot of the product was converted into its
DMOX derivative for GC–MS analysis which
gave a spectrum with a molecular ion of m/z=
359. A mass interval of 12 units (instead of 14)
occurred between m/z 182 (C7) and 194 (C8),
between m/z 222 (C10) and 234 (C11) and between
248 (C12) and 260 (C13), indicating the presence of
a double bond in the position 8 and two conju-
gated double bonds in positions 11 and 13. The
1H NMR spectrum of free 11 (Fig. 2) showed four
distinct signals for olefinic protons, one multiplet
and one double-triplet (lH=5.36 and 5.66 ppm),
which correspond to the shifts of the protons of
the outer trans–cis conjugated diene system (14-H
and 11-H), and two doublet–doublet (lH=5.94
and 6.30 ppm), which correspond to the shifts of
the protons of the inner trans–cis diene system
(13-H and 12-H). The 9c,11t-18:2 showed a dou-
ble-triplet for the proton 11-H at lH=5.36 (Lie
tive
of
the
impurity
and
of
(8Z,11Z,13E)-eicosa-8,11,13-trienoic acid gave
the same spectrum. Moreover, traces of
(8Z,11E,13E)-eicosa-8,11,13-trienoic acid were
detected by 1H NMR (two minor multiplets at
lH=5.55 for the outer positioned protons (11-H,
14-H) and lH=6 ppm for the inner positioned
1
protons (12-H, 13-H). H NMR showed a minor
triplet at lH=2.77 for the protons 10-H of the
methylene carbon which is between the E,E-diene
system and the Z-double bond of (8Z,11E,13E)-
eicosa-8,11,13-trienoic acid, while the triplet at
lH=2.88 corresponded at the protons 10-H of
the methylene carbon which is between the E,Z-
diene system and the Z-double bond of
(8Z,11Z,13E)-octadeca-8,11,13-trienoic acid (Fig.
2). These two triplets allowed a quantitation of
the (8Z,11E,13E)-eicosa-8,11,13-trienoic acid
which represented 5% of the mixture, according to
the GC and HPLC results.
The synthesis of the [1-14C]-radiolabeled analog
11% from the bromo precursor 10 followed the
same procedure of that described for the fatty
acid, using 14CO2 liberated from 14C barium car-
bonate. Its radiochemical purity was determined
by TLC and RP-HPLC as being 94% (specific
activity: 31.3 mCi mmol−1). However, 0.4 mCi of
(8Z,11Z,13E)-[1-14C]-eicosa-8,11,13-trienoic acid
with a radiochemical purity greater than 99%
were obtained by preparative RP-HPLC.
4.2. Synthesis of (6Z,9Z,11E)-octadeca-6,9,11-
trienoic acid
The octadecatrienoic acid was synthesized in a
similar way by using pentane-1,5-diol instead of
heptane-1,7-diol as starting material (Fig. 3). A
highly stereoselective Wittig condensation be-
tween the 5-(2-tetrahydropyranyloxy) pentylphos-
phonium salt 14 and the aldehyde 4 furnished the

132
S. Gna¨dig et al. / Chemistry and Physics of Lipids 112 (2001) 121–135

S. Gna¨dig et al. / Chemistry and Physics of Lipids 112 (2001) 121–135
133
Fig. 3. Synthesis of (6Z,9Z,11E)-octadeca-6,9,11-trienoic acid and of its corresponding [1-14C]-labeled acid.
(3Z)-1-(t-butyldiphenylsilyloxy)-8-(2-tetrahydro-
pyranyloxy)oct-3-ene 15 in 55% yield. After
preparation of the phosphonium salt 18 from 15
in 74% yield (three steps) as described for com-
pound 8, Wittig condensation of 18 with (2E)-
non-2-enal in similar conditions as for the
analysis showed that the 6c,9c,11t-18:3 had a
purity of \88% and it was accompanied by the
6c,9t,11t-18:3 (11%) and small amounts of
6c,9t,11c-18:3 and 6c,9c,11c-18:3. Direct analysis
on the free fatty acids by RP-HPLC confirmed
this analysis. Structures of the 6c,9c,11t-18:3 and
its isomers were determined by GC–MS. There-
fore, an aliquot of the mixture was converted to
its DMOX derivatives. GC–MS analysis of both
isomers gave a spectrum with a molecular ion m/z
331, an intense ion at m/z 152 as well as ions of
about equal intensity at m/z 166 and 167. A
similar type of fragmentation has been reported
for unsaturated fatty acids having the first double
bond in D6 position (Dobson and Christie, 1996).
The other two double bonds of the conjugated
system in the positions 9 and 11 were located by
fragments m/z 194 (C8) and 206 (C9), and frag-
ments at m/z 220 (C10) and 232 (C11). The forma-
preparation of compounds
9
gave the
(5Z,8Z,120E)-1-(2-tetrahydropyranyloxy)hepta-
deca-5,8,10-triene 19 in 68% yield (no geometric
1
isomers was detected by GC, H or 13C NMR).
The bromo precursor 20 was obtained in 53%
yield by reaction with triphenylphosphine dibro-
mide as described for the preparation of the bro-
mide 10. Formation of the corresponding
heptadecatrienylmagnesium bromide (90 min in
refluxing diethyl ether) followed by carbonation
with CO2 produced (6Z,9Z,11E)-octadeca-6,9,11-
trienoic acid 21. After purification by flash chro-
matography and derivatization to FAME, GC

134
S. Gna¨dig et al. / Chemistry and Physics of Lipids 112 (2001) 121–135
References
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Acknowledgements
The authors are grateful to Christian Ginies
and Etienne Semon for NMR, GC–FTIR and
GC–MS analyses. Silke Gna¨dig was funded by
a
Marie-Curie-Fellowship (FAIR-CT98-5071)
from the European Union.

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