Synthesis of keto- and hydroxydienoic compounds from linoleic acid

Article abstract of DOI:10.1016/S0009-3084(96)02650-3

A convenient preparative method has been developed for the synthesis of hydroxydienoic (9-HODE and 13-HODE) and ketodienoic compounds (9-KODE and 13-KODE) from natural linoleic acid. Methyl linoleate was treated with 1.25 eq. of m-chloroperbenzoic acid in alcoholic solution, giving a mixture of mono-epoxides (yield 60%), that was treated with a solution of HBr in MeOH to yield a mixture of the bromohydrins (yield 92%). The last was oxidized by Jones reagent to a mixture of bromoketones (yield 64%) and the mixture obtained was dehydrobrominated by DBU to produce a mixture of ketodienoic compounds (yield 94%). Reduction of the ketodienoic compounds by KBH₄ in MeOH led to the corresponding hydroxydienoic (9-HODE and 13-HODE) methyl esters (yield 83%). The synthetic approach described is simple and gives reliable results. The keto- and hydroxy fatty acids obtained were characterized thoroughly by TLC, HPLC, UV, FT-IR, ¹H-, ¹H¹H- and ¹³C-NMR.

Full text of DOI:10.1016/S0009-3084(96)02650-3

Chemistry and Physics of Lipids
85 (1997) 125–134
Synthesis of keto- and hydroxydienoic compounds from
linoleic acid
Dmitry V. Kukleva,*, William W. Christieb, Thierry Durandc, Jean Claude Rossic,
Jean Pierre Vidalc, Sergey P. Kasyanova, Valery N. Akulina, Vladimir V. Bezuglovd
aPacific Research Institute of Fisheries and Oceanography (TINRO), Sche6chenko, 4, Vladi6ostok, 690600, Russia
bDepartment of Chemistry, Scottish Crop Research Institute, In6ergowrie, Dundee DD2 5DA, UK
cLaboratoire de Chimie des Me´diateurs et Physicochimie des Inte´ractions Biologiques associe´ au C.N.R.S., Uni6ersite´ Montpellier I,
Faculte´ de Pharmacie, 15 A6. Ch. Flahault, F-34060 Montpellier, France
dDepartment of PG and LT, Shemyakin-O6chinniko6 Institute of Bioorganic Chemistry, The Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10; 117871 GSP 7 Moscow, Russia
Received 3 July 1996; revised 18 November 1996; accepted 20 November 1996
Abstract
A convenient preparative method has been developed for the synthesis of hydroxydienoic (9-HODE and 13-HODE)
and ketodienoic compounds (9-KODE and 13-KODE) from natural linoleic acid. Methyl linoleate was treated with
1.25 eq. of m-chloroperbenzoic acid in alcoholic solution, giving a mixture of mono-epoxides (yield 60%), that was
treated with a solution of HBr in MeOH to yield a mixture of the bromohydrins (yield 92%). The last was oxidized
by Jones reagent to a mixture of bromoketones (yield 64%) and the mixture obtained was dehydrobrominated by
DBU to produce a mixture of ketodienoic compounds (yield 94%). Reduction of the ketodienoic compounds by
KBH4 in MeOH led to the corresponding hydroxydienoic (9-HODE and 13-HODE) methyl esters (yield 83%). The
synthetic approach described is simple and gives reliable results. The keto- and hydroxy fatty acids obtained were
1
1
characterized thoroughly by TLC, HPLC, UV, FT-IR, H-, H1H- and 13C-NMR. © 1997 Elsevier Science Ireland
Ltd.
Keywords: Linoleic acid; Lipid peroxidation; Ketodienoic compounds; 9-Hydroxy-(10E,12Z)-octadecadien-1-oic acid
(9-HODE); 13-Hydroperoxy-(9Z,11E)-octadecadien-1-oic acid (13-HODE); Synthesis; NMR spectroscopy; Mass
spectrometry; High performance liquid chromatography
* Corresponding author.
0009-3084/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.
PII S0009-3084(96)02650-3

126
D.V. Kukle6 et al. / Chemistry and Physics of Lipids 85 (1997) 125–134
1. Introduction
1988; Honn et al., 1989) have been reported. The
secondary products can be incorporated into
phospholipids (Brezinski and Serhan, 1990), and
by this means they can be involved in regulation
of biological membrane states.
The main goals of the present work were to
develop a preparative synthetic route to both the
secondary products of lipid peroxidation of
Unsaturated fatty acids (UFAs) with a 1,4-
cis,cis-pentadiene structural element (pentadienoic
acids, PD-acids1 are substrates for regio- and
stereospecific addition of molecular oxygen,
catalysed by lipoxygenases (Yamamoto, 1992),
that result in the formation of hydroperoxides.
One of the most abundant unsaturated acid in
cellular systems that forms lipoxygenase products
is (9Z,12Z)-octadecadien-1-oic acid (linoleic acid,
LA) that can be referred to as mono-PD-acid. Via
the established mechanism of enzymatic lipid per-
oxidation by lipoxygenases (Egmond et al., 1973),
linoleic acid can yield products oxygenated in
position 9 or 13 giving 9-hydroperoxy-(10E,12Z)-
octadecadien-1-oic and (9-HPODE) and 13-hy-
linoleic acid, and to perform their characteriza-
2
.
tion
2. Experimental
2.1. Materials and equipment
Acetone, benzene, ether, n-, iso- and c-hexane
were distilled over P2O5, ethanol, iso-propanol
and methanol were distilled from metallic Na, and
methylene chloride was stored over CaCl2 and
DBU over CaH2 in vacuo. All chemicals used
were of analytical reagent grade.
The TLC analysis was performed with the use
of precoated TLC plates (DC-Alufolien Kieselgel
60 F254, Merck, Germany). The plates were devel-
oped by spraying with a 5% solution of phospho-
molybdic acid in EtOH and heating at 110°C for
about 2 min. Visualisation of the spots was done
under a UV lamp at 254 nm. Preparative column
separation and purification were performed with
droperoxy-(9Z,11E)-octadecadien-1-oic
acid
(13-HPODE) (Fig. 1). These primary products of
peroxidation of linoleic acid can be reduced in the
cells (e.g. under catalysis by glutathione-depen-
dent peroxidases (Tappel et al., 1982; Bryant et
al., 1983)) to the more stable hydroxy derivatives
9-hydroxy-(10E,12Z)-octadecadien-1-oic acid (9-
HODE) or 13-hydroxy-(9Z,11E)-octadecadien-1-
oic acid (13-HODE), respectively. Moreover,
under specific conditions 9-HPODE and 13-
HPODE can be transformed by lipoxygenase to
ketodienoic
compounds
namely
9-keto-
(10E,12Z)-octadecadien-1-oic acid (9-KODE) or
13-keto-(9Z,11E)-octadecadien-1-oic acid (13-
KODE) (Vioque and Holman, 1962; Ku¨hn et al.,
1991), respectively. The hydroxydienoic and keto-
dienoic derivatives are the most common sec-
ondary products of lipid peroxidation derived
from linoleic acid.
It should be emphasized that the secondary
products have pronounced biological activity.
Among the different biological responses (Spector
et al., 1988; Needleman et al., 1986), chemotaxis
(Henricks et al., 1991), hyperproliferative effects
(Tsukada et al., 1986; Postoak et al., 1990), regu-
lation of phospholipase activity (Chang et al.,
1985) and regulation of cell adhesion (Honn et al.,
Fig. 1. Peroxidation of linoleic acid.
1 (The term ‘PD-acids’ was introduced to encompass the
polyenoic fatty acids bearing the pentadienoic fragment(s)
(Kuklev et al., 1996).
2 For the preliminary communications see in (Kuklev et al.,
1996).

D.V. Kukle6 et al. / Chemistry and Physics of Lipids 85 (1997) 125–134
127
Kieselgel 63–200 vm (70–230 mesh ASTM)
(Merck, Germany).
of diethyl ether in iso-hexane. This yielded 3.44 g
(65%) of a mixture of isomeric epoxy esters in the
form of a colourless mobile oil. Rf 0.38 (Fig. 3) –
methyl ester of 12,13-epoxy-(9Z)-octadecen-1-oic
acid (2), Rf 0.33 – methyl ester of 9,10-epoxy-
(12Z)-octadecen-1-oic acid (3). From the reaction
mixture 1.2 g of the parent acid was isolated. The
yield of the epoxides was 86% in terms of conver-
sion. Diepoxides were present only in trace
amounts. The mixture of 2 and 3 was used further
without separation. UV spectroscopy of a mixture
of 2 and 3 showed no maximum at u\206 nm.
IR, cm−1: 2928 s, 2856 s, 1741 s, 1460 m., 136 m,
1379 m, 1248 w, 1197 m, 1171 m, 1016 w.
For structure assignment the individual epox-
ides were separated by HPLC (column: Hypersil
H3 (250×4.6 mm), 3 vm; the mobile phase was
iso-hexane/i-propanol (99.6:0.4, v/v), at a flow-
rate of 1 ml/min and with UV-detection at 206
nm). The compound with k%=0.62 was analysed
by GC-MS in the form of the trimethylsilyl
derivatives of the corresponding chlorohydrins
(Section 2.3) and gave fragmentation as follows
(most prominent ions, presumed structures of the
ions): 418 [M], 299 [M – ClCHC5H11], 221 [(TM-
SO)CHCH(Cl)C5H11], 173 [CH(TMSO) C5H11]
and was recognized as (2). The compound with
k%=0.8 (processed as described above) gave frag-
mentation as follows (most prominent ions, pre-
sumed structures of the ions): 418 [M], 307 [M –
C5H11(CHꢀꢀCH)CH2], 259 [M – C5H11(CHꢀꢀ
CH)CH2CH(Cl)], 213 [C5H11(CHꢀꢀCH)CH2CH
(OTMS)] and was recognized as (3).
For high performance liquid chromatography
(HPLC) (analytical and semi-preparative), a Wa-
ters Model 510 pump (Millipore, USA) with a
Waters 490 UV detector were used. The columns
and systems applied for each separation are de-
scribed below.
UV spectra were recorded on a Varian DMS 90
UV/visible spectrophotometer (Varian, Australia)
instrument at a scanning rate of 20 nm/min from
700 down to 200 nm in methanol or ethanol.
IR-spectra of films of the mixtures and individ-
ual compounds were recorded on a Perkin Elmer
FT-IR 1760 instrument (Perkin Elmer, Germany).
1H-(400, 500 MHz) and 13C-NMR (125 MHz)
spectra were recorded on a Bruker WM500 instru-
ment (Bruker, Germany) at 25°C, and the chemi-
cal shifts (l, ppm) are presented relative to an
internal standard (CH3)4Si for solutions in CDCl3.
All signal assignments were carried out with pro-
ton-proton decoupling in support of experiments.
Mass spectra (EI) were recorded on a Jeol
DX300 (Jeol, Japan) instrument at 70 eV. Gas
chromatography-mass spectrometry was carried
out with a Kratos 8/90 double-focusing magnetic
sector instrument, with electron-impact ionization
(EI, at 70 eV), attached to a Varian 2700 gas
chromatograph (Varian Ltd., UK) with a WCOT
fused silica (25 m×0.25 mm) column coated with
OV-1 (Supelco, USA) bonded phase. All mass
spectra were obtained with temperature program-
ming from 100 up to 300°C at a heating rate of
5°C/min.
2.3. Trimethylsilyl (TMS) deri6ati6es of
chlorohydrins of the fatty acids for
mass-spectrometric analysis
2.2. Synthesis of epoxides of linoleic Acid (2,3)
With stirring, a solution of 3.67 g (1.25 eq.) of
m-chloroperbenzoic acid in 50 ml of absolute
ethanol was added dropwise to a solution of 5.0 g
of linoleic acid methyl ester (Fig. 2, 1) in 50 ml
absolute ethanol. The reaction mixture was al-
lowed to stand for 1 h and then 300 mg of
Na2SO3 was added. After vigorous stirring, the
solvent was evaporated under vacuum, and the
residue was taken into a minimal volume of hex-
ane; the resulting solution was chromatographed
on silica gel with elution by a gradient (0“25%)
A sample of the methyl ester of an epoxy acid
(1–3 mg) was dissolved in 200 mkl of dry
pyridine, 50 mkl of trimethylchlorosilane was
added, and the mixture was kept at 50°C for 2 h.
After cooling, it was evaporated in a stream of
nitrogen, and the dry residue was taken into 3 ml
of i-hexane and filtered through cotton wool. The
TMS ethers of the chlorohydrins of the fatty acid
methyl esters so obtained had Rf 0.9 (n-hexane-
ether (80:20, v/v).

128
D.V. Kukle6 et al. / Chemistry and Physics of Lipids 85 (1997) 125–134
Fig. 2. Synthetic scheme.

D.V. Kukle6 et al. / Chemistry and Physics of Lipids 85 (1997) 125–134
129
solved in this solution at −10°C and the mixture
was kept for 1 h at this temperature. The reaction
mixture was diluted with 20 ml of water and
extracted with hexane (3×50 ml). The extract
was washed with a saturated solution of NaCl
(100 ml) and dried over anhydrous Na2SO4. The
dry extract was evaporated under vacuum, the
residue was dissolved in a minimal volume of
hexane and subjected to column chromatography
on 20 g silica gel with elution by a gradient
(10“50%) of diethyl ether in i-hexane. This gave
1.1 g (87%) of a mixture of the isomeric bromohy-
drins in the form of a colourless mobile oil. UV
spectroscopy of the mixture of 4,5,12 and 13
2.4. Synthesis of isomeric bromohydrins
(4,5,12,13)
A solution of 2 ml of AcBr in 20 ml of MeOH
was prepared immediately before the use. One
gram of the mixture of epoxides (2,3) was dis-
showed no maximum at u\206 nm. IR, cm−1
:
3468 s wide, 2928 s, 2856 s, 1740 s, 1460 m, 1437
m, 1377 w, 1248 w, 1199 m, 1173 m, 1077 w. Rf
0.13–0.24 (Fig. 3). The compounds (4,5,12,13)
synthesised were used further without separation.
2.5. Synthesis of isomeric bromoketones
(6,7,14,15)
A solution of the 1.10 g of the bromohydrins
(4,5,12,13) in 50 ml of acetone was cooled to
−40°C and 2.0 ml of standard Jones reagent were
added. Then the reaction temperature was al-
lowed to increase to −15°C, and the reaction
mixture was kept at this temperature for 40 min,
after which 10 ml of iso-propanol were added and
the reaction mixture was warmed up to room
temperature, diluted with 50 ml of water and
extracted with hexane (3×100 ml). The extract
was washed with a saturated solution of NaCl
(100 ml) and dried over anhydrous Na2SO4. The
dried extract was evaporated in vacuo and the
residue was taken into a minimal volume of hex-
ane and subjected to column chromatography on
20 g silica gel with elution by a gradient (0“20%)
of diethyl ether in i-hexane. This gave 700 mg
(64%) of a mixture of the isomeric bromoketones
(6,7,14,15) in the form of a bright yellow mobile
oil with a characteristic odour. UV spectroscopy
of the mixture of 6,7,14 and 15 showed no maxi-
mum at u\206 nm. IR, cm−1: 2928 s, 2856 s,
1739 s, 1599 w, 1460 m, 1435 m, 1363 w, 1248 w,
1197 w, 1170 w, 1019 w. Rf 0.42–0.50 (Fig. 3).
Fig. 3. Thin layer chromatography traces of the reaction
mixtures. Plate A: The developing system used was a mixture
of n-hexane:diethyl ether (80:20, v/v) at +20°C. The plate was
visualized by spraying with phosphomolybdic acid in EtOH
and heating at 110°C for about 2 min. Stripe 1: Methyl
linoleate (1) Rf 0.6. Stripe 2: The reaction mixture of the
epoxidation to the mixture of epoxides (2) and (3). The spot
with Rf 0.6 was produced by the remains of the parent methyl
linoleate; the spot with Rf 0.38 was produced by the 12,13-
epoxy derivative (2); the spot with Rf 0.33 was produced by
the 9,10-epoxy derivative (3). Stripe 3: The reaction mixture of
the bromohydrins synthesis; the diffuse spot with Rf 0.13–0.24
was produced by the four bromohydrins(4,5,12,13). Stripe 4:
The reaction mixture of oxidation of the bromohydrins
(4,5,12,13) to bromoketones (6,7,14,15). Rf 0.42–0.50. Stripe
5: Ketodienoic compounds (8,9); the spot with Rf 0.32 was
produced by the 13-keto-isomer (8) whereas the spot with Rf
0.28 was produced by the 9-keto isomer (9). Plate B: The
developing system used was a mixture of n-hexane:diethyl
ether (50:50, v/v) at +20°C. The plate was visualized by
spraying with phosphomolybdic acid in EtOH and heating at
110°C for about 2 min. Stripe 6: Mixture of methyl esters of
the required HDCs: Rf 0.46: 13-HODE (10). Rf 0.40: 9-HODE
(11).

130
D.V. Kukle6 et al. / Chemistry and Physics of Lipids 85 (1997) 125–134
The compounds synthesised were used further
without separation.
2.6. Synthesis of isomeric conjugated
ketocompounds (8,9,16,17)
Six-hundred-and-fifty milligrams of the bro-
moketones (6,7,14,15) were dissolved into 50 ml
of dry benzene and 1 ml of DBU (4 eq.) was
added. The reaction mixture was kept for 30 min
at room temperature, then it was filtered through
10 g of silica gel, and the cartridge was washed
with 100 ml of dry benzene. The filtrate was
evaporated in vacuo, giving 481 mg (94%) of a
mixture of the isomeric conjugated ketones
(8,9,16,17) in the form of a bright yellow oil. Rf
0.32–0.12 (n-hexane-diethyl ether (80:20, v/v)
(diffuse spot)). The dry oil obtained was taken
into a minimal volume of i-hexane and subjected
to column chromatography on 25 g silica gel with
elution by a gradient (0“20%) of diethyl ether in
i-hexane. This gave 220 mg of a mixture of the
target ketodienoic compounds (8) and (9) in the
form of a viscous colourless oil. Rf 0.28–0.32
(Fig. 3) (diffuse spot). The ketodienoic com-
pounds (8) and (9) were separated by semi-prepar-
Fig. 4. HPLC analysis of standard ketodienoic compounds (I
— 8, II — 9) and hydroxydienoic compounds (III — 10, IV
— 11). Conditions: column Zorbax-SIL (250×4.6 mm), 5
vm, SiO2; the mobile phase was a mixture of n-hexane with
i-propanol (1000:3, v/v), at a flow-rate of 0.9 ml/min, UV-de-
tection at 206 nm).
(CH3O), 127.0 (C-10), 129.4 (C-12), 136.9 (C-9),
142.4 (C-11), 173.2 (C-1), 202.1 (C-13) (Fig. 6).
1H-1H COSY (400 MHz, CDCl3) (Fig. 5): the
1H-1H COSY spectrum of 8 clearly showed the
following connectivities (H-H): 2-3, 3-4, 6-7, 7-8,
8-9, 9-10, 10-11, 11-12, 14-15, 15-16, 17-18. EIMS
of the reduced (NaBH4, H2 over PtO2) methyl
ester and TMS-ether, m/z (I%): 371 [M – Me]+
(7%), 315 [M – C5H11]+ (100%), 173 [TM-
SOCHC5H11]+ (10%).
ative HPLC (column: Hibar®
5 vm, Si60
(10×250 mm) (Merck, Germany), eluent: n-hex-
ane:iso-propanol (1000:2, v/v) at a flow rate of 6
ml/min). Methyl ester of 13-oxo-(9Z,11E)-oc-
tadecadien-1-oic acid (13-KODE methyl ester) (8).
Colorless oil, Rf 0.32 (Fig. 3). HPLC: k%=6.57;
(Fig. 4, peak I). UV (MeOH): umax=278 nm,
‡=20 330. IR (film, cm−1): 2930, 2856, 1740,
1688, 1666, 1630, 1590, 1461, 1436, 1411, 1363,
1260, 1196, 1173, 1085, 996, 961, 870, 799, 725.
1H-NMR (400 MHz, CDCl3, l): 0.89 (t, 3H,
H-18; J18,17 6.9), 1.32 (m, 10H, H-4,5,6,16,17),
1.43 (m, 2H, H-3), 1.59 (m, 4H, H-7,15), 2.28 (t,
2H, H-2; J3,2 7.5), 2.28 (m, 2H, H-8), 2.55 (t, 2H,
H-14, J14,15 7.5), 3.67 (s, 3H, CH3OOC), 5.9 (ddt,
1H, H-9, J9,10 11.1, J9,8 7.8, J9,11 0.9), 6.1 (ddd,
1H, H-10, J10,9 11.1, J10,11 10.6, J10,12 0.9), 6.14
(dd, 1H, H-12, J12,11 15.4, J12,10 0.9), 7.43 (ddd,
1H, H-11, J11,12 15.4, J11,10 11.1, J11,9 0.9) (Fig. 5).
13C-NMR (125 MHz, CDCl3, l): 13.8 (C-18), 22.5
(C-17), 24.2, 24.9, 28.4, 29.2, 29.2, 28.9, 29.2, 32.0
(C-3,4,5,6,7,8,15,16), 34.1 (C-2), 41.1 (C-14), 51.4
Methyl ester of 9-oxo-(10E,12Z)-octadecadien-
1-oic acid (9-KODE methyl ester) (9): Colourless
oil, TLC: Rf 0.28 (Fig. 3). HPLC: k%=7.57, (Fig.
Fig. 5. 1H-NMR and 1H1H-NMR spectra of methyl ester of
13-oxo-(9Z,11E)-octadecadien-1-oic acid (8).

D.V. Kukle6 et al. / Chemistry and Physics of Lipids 85 (1997) 125–134
131
−50°C and 100 mg of KBH4 (2 eq.) were added.
Then the reaction temperature was allowed to
increase up to −10°C during 2 h, after which 10
ml of a saturated solution of NH4Cl were added,
the resulting solution was diluted with water (50
ml), and extracted with n-hexane (3×100 ml). The
extract was washed with a saturated solution of
NaCl (100 ml) and dried over anhydrous Na2SO4.
The dried extract was evaporated in vacuo and the
residue was taken into a minimal volume of hexane
and subjected to column chromatography on 20 g
silica gel with elution by a gradient (10“70%) of
diethyl ether in i-hexane. This gave 236 mg (83%)
of a mixture of the isomeric hydroxy dienoic
compounds in the form of a colourless mobile oil.
Rf 0.4–0.46 (Fig. 3). The hydroxy derivatives
synthesised were analysed by HPLC (Fig. 4) and
separated by semi-preparative HPLC (conditions:
column Hibar® 5 vm, Si60 (10×250 mm) (Merck,
Germany), mobile phase: n-hexane:iso-propanol
(1000:4, v/v); at a flow rate of 6 ml/min). Methyl
ester of 13-hydroxy-(9Z,11E)-octadecadien-1-oic
acid (13-HODE methyl ester). TLC: Rf=0.46
(Fig. 3). UV (EtOH): umax=233 nm, ‡=18 200,
HPLC: k%=23.43 (Fig. 4, peak III). 1H-NMR (500
MHz, CDCl3, l): 0.86 (t, 3H, H-18), 1.2–1.7 (m,
18H, CH2), 2.14 (m, 2H, H-8), 2.27 (t, 2H, H-2, J2,3
7.6), 3.64 (t, 3H, CH3OOC), 4.13 (m, 1H, H-13),
5.40 (ddt, 1H, H-9, J9,10 11.4, J9,8 7.6, J9,12 1), 5.64
(dd, 1H, H-12, J12,11 15.4, J12,13 6.5), 5.94 (dd, 1H,
H-10, J10,11 10.4 J9,10 11.5), 6.45 (ddd, 1H, H-11,
J11,12 15.4, J11,10 10.4, J11,9 1). EIMS of TMS-ether
on hydroxyl group, m/z: 382 [M]+, 311 [M –
Fig. 6. 13C-NMR spectrum of methyl ester of 13-oxo-
(9Z,11E)-octadecadien-1-oic acid (8).
4, peak II). UV (MeOH): umax=278 nm, ‡=
18 700. IR (film) cm−1: 2929, 2856, 1740, 1688,
1665, 1631, 1590, 1462, 1436, 1411, 1364, 1260,
1
1196, 1173, 1085, 1110, 997. H-NMR (400 MHz,
CDCl3, l): 0.89 (t, 3H, H-18; J18,17 6.9), 1.32 (m,
10H, H-4,5,6,16,17), 1.43 (m, 2H, H-3), 1.59 (m,
4H, H-7,15), 2.30 (t, 2H, H-2; J3,2 7.5), 2.33 (m, 2H,
H-14), 2.55 (t, 2H, H-8, J7,8 7.4), 3.67 (s, 3H,
CH3OOC), 5.92 (ddt, 1H, H-13, J13,12 11.1, J13,14
7.9, J13,11 0.9), 6.13 (ddd, 1H, H-12, J12,13 11.1,
J12,11 10.25, J12,10 0.9), 6.14 (dd, 1H, H-10, J10,11
15.1, J10,12 0.9), 7.47 (ddd, 1H, H-11, J11,10 15.1,
J11,12 10.25, J11,13 0.9). 13C-NMR (125 MHz,
CDCl3, l): 13.9 (C-18), 22.5 (C-17), 24.1, 24.9,
28.3, 29.1, 29.1, 29.1, 29.3, 31.5 (C-
3,4,5,6,7,8,15,16), 34.1 (C-2), 41.1 (C-14), 51.5
(CH3O), 127.0 (C-10), 129.4 (C-12), 136.9 (C-13),
142.4 (C-11), 174.2 (C-1), 201.1 (C-9). 1H-1H
COSY (400 MHz, CDCl3) (Fig. 5): the 1H-1H
COSY spectrum of 9 clearly showed the following
connectivities (H-H): 2-3, 3-4, 6-7, 7-8, 10-11,
11-12, 12-13, 13-14, 14-15, 15-16, 17-18. EIMS of
the reduced (NaBH4, H2 over PtO2) methyl ester
and TMS-ether, m/z (I%): 371 [M – Me]+ (10%),
C5H11]+, 225 [M
–
(CH2)7COOMe]+, 173
[CH(TMSO)C5H11]+. Methyl ester of 9-hydroxy-
(10E,12Z)-octadecadien-1-oic acid (9-HODE
methyl ester). TLC: Rf=0.40 (Fig. 3). UV (EtOH):
umax=233 nm, ‡=17 200; HPLC: k%=34.86, (Fig.
1
4, peak 4). H-NMR (500 MHz, CDCl3, l): 0.86
(t, 3H, H-18, J18,17 7.5), 1.2–1.7 (m, 18H, CH2),
2.15 (m, 2H, H-14), 2.27 (t, 2H, H-2, J2,3 7.6), 3.64
(t, 3H, CH3OOC), 4.12 (m, 1H, H-9), 5.42 (ddt,
1H, H-13, J13,12 11.2, J13,14 7.8, J13,11 1), 5.63 (dd,
1H, H-10, J10,11 15.4, J10,9 6.8), 5.94 (dd, 1H, H-12,
J12,13 11.2 J12,11 10.6), 6.46 (ddd, 1H, H-11, J11,10
15.4, J11,12 10.6, J13,11 1). EIMS of TMS-ether on
hydroxyl group, m/z: 382 [M]+, 311 [M –
C5H11]+, 259 [M – C5H11 (CHꢀꢀCH)2]+, 225
[C5H11(CHꢀꢀCH)2CH(TMSO)]+.
259 [M
–
C9H19]+ (100%), 229 [TM-
SOCHC9H19]+ (8%).
2.7. Synthesis of isomeric hydroxydienoic
compounds (10,11)
A solution of 283 mg of the ketodienoic com-
pounds in 50 ml MeOH was cooled down to

132
D.V. Kukle6 et al. / Chemistry and Physics of Lipids 85 (1997) 125–134
3. Results and discussion
byproducts as a result of double elimination of
HBr, first, and ROH as the second step (where R
is acetate, benzoate or TMS) (data not shown). In
the case of bromoketones whose reactivity is very
high toward to dehydrobromination to yield con-
jugated ketones, there is no theoretical possibility
of producing the products of double elimination.
From another standpoint, in our strategy we
had synthesised ketodienoic compounds first and
then we reduced them to obtain allylic alcohols. It
is important to point out the lability of allylic
alcohols toward oxidation. In contrast, h-bro-
moalcohols (4,5,12,13) are sufficiently stable to
oxidation and can be transformed to h-bromoke-
tones (6,7,14,15) in a yield of more than 60% (by
Jones reagent) without major byproducts.
Our goal was to develop a practical approach
for synthesis of ketodienoic and hydroxydienoic
compounds from natural pentadienoic UFAs
(Fig. 1). As the first UFA to be converted to the
oxygenated products we took linoleic acid, which
is a mono-PD-acid and contains the only penta-
dienoic element at the ninth position of its chain.
The first step in our synthetic scheme was syn-
thesis of the mixture of mono-epoxides (2,3 – Fig.
2) by the Prileschajew reaction. We used m-
chloroperbenzoic acid in ethanol for the purpose,
as it gives stable high yields (as can be seen in the
literature (VanRollins et al., 1989; VanRollins,
1990)) and has been used by us for synthesis of
monoepoxides from PUFAs (Kuklev et al., 1993).
Carrying out the reaction in a polar protonic
solvent was essential here because of the lower
activity of m-chloroperbenzoic acid in such sol-
vents (Prileschajewa, 1974). The use of an excess
of the reagent (more than 1.25 eq.) leads to in-
creasing amounts of byproducts, even under the
mild conditions described. The epoxides synthe-
sised (2,3) were separated from the remains of the
parent methyl linoleate (1) and from traces of
diepoxide by column chromatography. In terms
of conversion the yield was 86% at this stage.
The second step was cleavage of the monoepox-
ides (2,3) to bromohydrins (4,5,12,13) with the use
of HBr/MeOH at low temperature (−40 to
−20°C). When we used other reagents, e.g. a
saturated solution of KBr/AcOH/THF (Corey et
al., 1980) or a two-phase system of 32% HBr/n-
heptane (Kuklev et al., 1991) (that we developed
for cleavage 4,5- and 5,6-epoxides of PUFA), the
yields did not exceeded 50–60% due to byproduct
formation (data not shown). Cleavage by HBr/
MeOH at −30°C proceeded smoothly and gave
reliable high yields of more than 90%, almost
without byproducts.
Dehydrobromination of bromoketones (6,7,
14,15) to conjugated ketones (8,9,16,17) by the
action of DBU in dry benzene went smoothly and
with high yields. The ketodienoic compounds
(8,9) and ketomonoenoic compounds (16,17) had
a pronounced difference in chromatographic mo-
bility and could be separated by preparative
column chromatography. We focused our atten-
tion on natural ketodienoic compounds (8,9) and
do not present data on ketomonoenoic derivatives
(16,17). It should be stressed that the double bond
newly created has the trans-configuration, as can
1
be seen from the H- and 13C-NMR data (Figs. 5
and 6). An additional confirmation of the stereo-
chemistry of the products came from the IR-spec-
tra where a band of absorbtion at 965 cm−1 was
characteristic for a trans-double bond. As can be
seen from the 1H-NMR spectrum (Fig. 5), the
proton at C-11 (7.47 ppm) produces a characteris-
tic signal in the form of ddd with the coupling
constants J11,10 15.1 Hz (trans-double bond), J11,12
10.25 Hz (single bond) and J11,13 0.9 Hz (far
constant in conjugated system of double bonds).
It should be emphasized that possibility to see
the far constant with a value of only 0.9 Hz
can be accepted as a confirmation of purity of
the ketodienoic compounds synthesised. At the
present time there is no reliable information about
The key point in our synthetic strategy was
oxidation of the bromohydrins (4,5,12,13) to bro-
moketones (6,7,14,15). This step can be viewed as
a ‘protection’ of the hydroxyl group against elimi-
nation of a proton by DBU. Using different pro-
tective groups (acetate, benzoate, trimethylsilyl)
leads to decreasing of the reaction rate and to
1
the H-NMR characteristics of natural 9-KODE
and 13-KODE. To clarify this point and to prove
our signal assignment we performed a two-
1
dimensional H1H-NMR-experiment (Fig. 5). It is

D.V. Kukle6 et al. / Chemistry and Physics of Lipids 85 (1997) 125–134
133
clearly seen from the spectrum that there are
coupling constants between the proton at 7.47
ppm and protons at 6.13 ppm and 6.14 ppm, and
these are supported by the nature of multiplets of
this signals (Fig. 5); the signal can be assigned to
protons at C-11, C-12 and C-10 respectively. In
the 13C-NMR (Fig. 6) spectrum, there are four
signals of the cis,trans-conjugated system at 127.0
(C-10), 129.4 (C-12), 136.9 (C-13), 142.4 (C-11).
We did not observe an explainable difference in
1H- and 13C-NMR spectra of 9-KODE and 13-
KODE, so we present only spectra of 13-KODE.
No difference in the FT-IR-spectra of 9-KODE
and 13-KODE was found, especially in the finger-
print area. The identity of the ketodienoic com-
pounds (8,9) to the natural ones can be seen from
comparing their UV and IR-data (Vioque and
Holman, 1962; Ku¨hn et al., 1991).
Fig. 8. RP-HPLC analysis of the processed reaction mixture of
the reduction of the ketodienoic compounds (8,9) by KBH4 in
methanol at −30°C (Column: Spherisorb ODS2 5 vm (250×
4.6 mm). Mobile phase: acetonitrile:water (90:10, v/v); flow-
rate of 0.7 ml/min and with UV-detection at 206, 234 nm).
The reduction of the ketodienoic compounds to
the required hydroxy fatty acids (10,11) was per-
formed by the action of potassium borohydride in
methanol at −50°C. Using sodium borohydride
in methanol (or ethanol) as a reagent under the
same conditions led to degradation of the cis-
trans conjugated system of double bonds as
judged by UV-spectra and by HPLC analysis of
the processed reaction mixtures (Figs. 7 and 8).
The expected conjugated hydroxy fatty acids were
only found at trace levels, as shown Fig. 7 (peak
III – unseparated mixture of 9- and 13-HODE,
major peak by detection at 233 nm, but minor
component at 206 nm), judging by the absorbtion
at 206 nm. With the use of potassium borohy-
dride we succeeded in the reduction of the keto-
dienoic compounds (8,9) to the required
conjugated hydroxy fatty acids (10,11) as shown
in Fig. 8 (major peak III – unseparated mixture
of 9- and 13-HODE, absorbing both at 206 nm
and 234 nm as a major component). The hydroxy
derivatives were isolated on a preparative scale by
SP-HPLC and reliably characterized by UV, MS
1
and H-NMR (Section 2.7).
The synthetic approach developed is simple and
gives reliable results. It is of particular interest as
it gives access to both ketodienoic and hydroxy
compounds. We intend to apply it to the synthesis
of a wide range of oxidized derivatives from other
fatty acids. This will give us a ‘library’ of hydroxy
and ketodienoic compounds that will serve us as
standards during biological investigations.
Fig. 7. RP-HPLC analysis of the processed reaction mixture of
the reduction of the ketodienoic compounds (8,9) by NaBH4
in methanol at −30°C (Column: Spherisorb ODS2 5 vm
(250×4.6 mm). Mobile phase: acetonitrile:water (90:10, v/v);
flow-rate of 0.7 ml/min and with UV-detection at 206, 234
nm).
Acknowledgements
We wish to thank The Royal Society (United
Kingdom), The Scottish Office Agriculture, Envi-

134
D.V. Kukle6 et al. / Chemistry and Physics of Lipids 85 (1997) 125–134
Kuklev, D.V., Christie, W.W., Durand, T., Rossi, J.C.,
Girard, J.P., Kasyanov, S.P., Akulin, V.N. and Bezuglov,
V.V. (1996) Synthesis of ketodienoic compounds from
natural unsaturated fatty acids. Russ. J. Bioorg. Chem. 22,
629–634.
ronment and Fisheries Department and French
Ministe`re de l’Education Nationale, de l’Enseigne-
ment Superieur et de la Recherche for financial
support of one of us (Dmitry Kuklev).
Kuklev, D.V., Rybin, V.G., Imbs, A.B. and Bezuglov, V.V.
(1993) Synthesis of monoepoxides of arachdonic, eicos-
apentaenoic and docosahexaenoic acids. Russ. J. Bioorg.
Chem. 19, 675–679.
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