Synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-5,8,11,14-tetraenoic acids and their [5,6,8,9,11,12,14,15-3H8]-analogues through a common synthetic route

Article abstract of DOI:10.1016/j.chemphyslip.2004.02.008

Total synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-5,8,11,14- tetraenoic acids and their [5,6,8,9,11,12,14,15-³H ₈]-analogues via the corresponding p-toluenesulphonates is reported. This synthetic approach allows the preparati

Full text of DOI:10.1016/j.chemphyslip.2004.02.008

Chemistry and Physics of Lipids 130 (2004) 117–126
Synthesis of (5Z,8Z,11Z,14Z)-18- and
19-azidoeicosa-5,8,11,14-tetraenoic acids and their
[5,6,8,9,11,12,14,15-3H8]-analogues through a
common synthetic route
Stepan G. Romanova,b, Igor V. Ivanova,b,∗, Valery P. Shevchenkoc, Igor Yu. Nagaevc,
Alexandr A. Pushkova, Nikolai F. Myasoedovc, Galina I. Myagkovaa, Hartmut Kuhnb
a
M.V. Lomonosov State Academy of Fine Chemical Technology, Pr. Vernadskogo 86, 119571 Moscow, Russia
b
Institute of Biochemistry, University Clinics Charité, Humboldt University, Monbijoustr. 2, D-10117 Berlin, Germany
c
Institute of Molecular Genetics, Kurchatov Square 2, 123182 Moscow, Russia
Received 2 September 2003; received in revised form 12 February 2004; accepted 16 February 2004
Abstract
Total synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-5,8,11,14-tetraenoic acids and their [5,6,8,9,11,12,14,15-3H8]-
analogues via the corresponding p-toluenesulphonates is reported. This synthetic approach allows the preparation of radioactively
labelled arachidonic acid derivatives following a common synthetic route. Activity assays indicated that 15-lipoxygenases may
tolerate the azido group in the substrate binding pocket and thus, radioactively labelled azido compounds may be used as
photo-affinity probes to investigate mechanistic features of eicosanoid biosynthesis.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Eicosanoids; Photo-affinity probes; Cross-coupling; Azido compounds; Tritium-labelled fatty acids
1. Introduction
(Funk, 2001). Although the 3D-structures of several
enzymes involved in mammalian eicosanoid biosyn-
thesis have recently been solved (COX-1; COX-2
(Kurumbail et al., 1996); 15-LOX (Gillmor et al.,
1997); LTA4-hydrolase (Anderson et al., 2003)), there
is a number of open questions on the catalysis mech-
anism and on the substrate alignment at the active
site. To investigate the enzyme–substrate interaction
in more detail specific photo-affinity probes are re-
quired that are suitable for covalent labelling of active
site peptides. For the time being, such research tools
have not been commercially available. Arachidonic
Eicosanoids, such as prostaglandins, leukotrienes
and hydroxy fatty acids are lipid mediators for a va-
riety of physiological and patho-physiological events
in mammals. They are formed via the lipoxyge-
nase (LOX) or the cyclooxygenase (COX) pathway
and exhibit a broad spectrum of biological activities
∗
Corresponding author. Tel.: +49-30-450-528037;
fax: +49-30-450-528905.
E-mail address: ivanov.mitht@mail.ru (I.V. Ivanov).
0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2004.02.008

118
S.G. Romanov et al. / Chemistry and Physics of Lipids 130 (2004) 117–126
acid derivatives carrying a hydroxy group close to the
methyl terminus of the fatty acid chain may constitute
useful intermediates in the synthesis of azido fatty
acids (Lie Ken Jie and Lao, 1987), which can be used
as photo-affinity probes for enzymes and receptors
involved in eicosanoid biology.
The aim of this study was to develop an efficient
procedure for the synthesis of regio-isomeric 18- and
19-azido arachidonic acids and their tritiated deriva-
tives.
temperature program: isothermic pre-run at 180 ◦C
for 2 min then from 180 to 290 ◦C at a rate 5 ◦C/min.
Open bed column chromatography was performed
using Silica Gel 60 (Merck, Darmstadt, Germany par-
ticle size ranging from 70 to 230 mesh). For thin-layer
chromatography we employed precasted Silica Gel 60
F254 sheets (Merck, Darmstadt, Germany). THF was
freshly distilled from sodium/benzophenone ketyl. All
solvents and reagents used were of extra pure grade
and purchased from Merck (Darmstadt, Germany)
or Sigma-Aldrich (Deisenhofen, Germany). Prior to
use all glassware and syringes were dried at 140 ◦C
overnight and all reactions were carried out under
atmosphere of dry argon.
2. Experimental
2.1. General
2.2. 7-(Trimethylsilyl)hept-6-yn-2-one (2)
1H and 13C NMR spectra were recorded either
on a Brucker MSL 200 or Brucker MSL 300 spec-
trophotometers in CDCl3 as solvent. Chemical shifts
are referenced to tetramethylsilane as an internal
To a cooled (−78 ◦C) suspension of CuI (2.406 g,
12.63 mmol) in Et2O (60 mL) an ethereal solution
of MeLi (18.06 mL, 25.26 mmol) was added and the
mixture was allowed to warm up to −10 ◦C. After
about 5 min at −10 ◦C the temperature was lowered
again to −78 ◦C. Then, a pre-cooled ethereal so-
lution (15 mL) of acid chloride (−40 ◦C), prepared
from 1 (2.118 g, 11.49 mmol) and oxalyl chloride
(45.96 mmol) was injected. The resulting mixture was
stirred for 15 min at −78 ◦C, quenched with saturated
aqueous NH4Cl (100 mL) and organic products were
extracted with Et2O (3 × 60 mL). The combined or-
ganic extracts were washed with saturated aqueous
NaCl, dried over Na2SO4 and concentrated under
vacuum. Purification by silica gel chromatography
(hexane/Et2O, 1:1) yielded 1.632 g (68%) of pure
1
standard for H NMR or CDCl3 (δ13C = 77.19 ppm)
for 13C NMR. IR spectra were recorded on Shi-
madzu IR-435. HPLC analysis was carried out on
a Shimadzu LC-10Avp liquid chromatograph con-
nected to SPD-10Advp UV detector. RP-HPLC anal-
ysis was performed on a Nucleosil C18-column;
250 mm × 4 mm, 5 ␮m particle size (Machery-Nagel,
Düren, Germany) with different solvent systems and
a flow rate of 1 mL/min was adjusted. Preparative
HPLC was carried out on a Lichrospher 100 RP18
column; 250 mm × 22.5 mm, 10 ␮m particle size
(Knauer, Berlin, Germany) with MeOH/H2O (95:5,
by vol.) and a flow rate of 10 mL/min. SP-HPLC of
lipoxygenation products was carried out on a Nu-
cleosil 100-7 column (Macherey-Nagel; 250 mm ×
4 mm, 5 ␮m particle size) with the hexane/2-propanol
(100:1.5, by vol.) solvent system containing 0.1%
acetic acid and a flow rate of 1 mL/min. Electron spray
ionisation high resolution mass spectra (ESI HRMS)
were recorded either on a MAT711 mass spectrome-
ter (Finigan MAT) or on a VG Autospec instrument.
GC/MS (EIMS) analysis of lipoxygenation products
was carried out on a Shimadzu GC-MS QP-2000
system equipped with a capillary column (30 m ×
0.32 mm, coating thickness 0.25 ␮m). An injector
temperature of 280 ◦C, an ion source temperature of
180 ◦C and electron energy of 70 eV were set up. The
derivatised fatty acids were eluted with a following
2. TLC: Rf = 0.44 (hexane/Et2O, 1:1). IR (neat): ν
1
≡
=
2240 (C C), 1730 (C O), 1245, 840 (Si(CH3)3). H
NMR (300 MHz, CDCl3): δ 2.54 (t, 2H, J = 7.2 Hz,
3-CH2), 2.24 (t, 2H, J = 6.9 Hz, 5-CH2), 2.13 (s, 3H,
CH3), 1.75 (m, 2H, 4-CH2), 0.11 (s, 9H, (CH3)3Si).
13C NMR (75 MHz, CDCl3): δ 208.56, 106.50, 85.59,
42.34, 30.26, 22.59, 19.35, 0.32. Anal. calcd. for
C10H18OSi: C, 65.87; H, 9.95. Found: C, 65.64; H,
10.15.
2.3. rac-(Trimethylsilyl)hept-6-yne-2-ol (3)
To a cooled (−20 ◦C) suspension of LiAlH4
(0.348 g, 9.20 mmol) in THF (30 mL) a solution of 2
(1.600 g, 8.76 mmol) in THF (20 mL) was added. The

S.G. Romanov et al. / Chemistry and Physics of Lipids 130 (2004) 117–126
119
reaction mixture was stirred for 30 min at −20 ◦C
then allowed to warm up to 0 ◦C. Finally the mixture
was quenched with H2O (100 mL) and acidified with
HCl (1M). Organic products were extracted from the
water phase with Et2O (3 × 50 mL). The combined
organic layers were washed with saturated aqueous
NaCl (70 mL), dried over Na2SO4 and concentrated
under vacuum. Flash chromatography on silica gel
(hexane/Et2O, 2:1) yielded the alcohol 3 (1.358 g,
84%). TLC: Rf = 0.23 (hexane/Et2O, 1:1). IR (neat):
2H, J = 6.8 Hz and J = 2.7 Hz, 5-CH2), 1.94 (t, 1H,
J = 2.7 Hz, CH), 1.73–1.89 (m, 2H, 4-CH2), 1.54–1.71
(m, 2H, 3-CH2), 1.34 (d, 3H, J = 6.3 Hz, CH3). 13C
NMR (75 MHz, CDCl3): δ 166.36, 133.42, 132.96,
129.70 (2C), 128.48 (2C), 84.16, 71.29, 68.87, 35.22,
24.57, 20.27, 18.48. Anal. calcd. for C14H16O2: C,
77.75; H, 7.46. Found: C, 77.99; H, 7.34.
2.5. rac-13-(Benzoyloxy)tetradeca-2,5,8-triyne-1-ol
(6)
≡
ν 3600–3200 (OH), 2240 (C C), 1115 (C–O), 1245,
1
840 (Si(CH3)3). H NMR (300 MHz, CDCl3): δ 3.82
To a suspension of previously dried salts CuI
(1.629 g, 8.55 mmol), NaI (1.282 g, 8.55 mmol) and
K2CO3 (0.885 g, 6.41 mmol) in DMF (25 mL) the bro-
mide 5 (0.800 g, 4.27 mmol) and acetylene 4 (1.018 g,
4.70 mmol) were added under argon atmosphere. Af-
ter stirring overnight at rt, the mixture was quenched
with saturated aqueous NH4Cl (100 mL) and the
products were extracted with Et2O (4 × 100 mL). The
combined organic extracts were washed with saturated
aqueous NaCl (2 × 50 mL), dried over Na2SO4 and
the solvent was evaporated under reduced pressure.
The residue was purified by silica gel flash chromatog-
raphy (hexane/Et2O, 1:2) to afford 0.742g (54%) of
alcohol 6. TLC: Rf = 0.30 (hexane/Et2O, 1:2). IR
(m, 1H, 2-CH), 2.23 (m, 2H, 5-CH2), 1.49–1.65 (m,
4H, 3- and 4-CH2), 1.19 (d, 3H, J = 6.3 Hz, CH3),
0.12 (s, 9H, (CH3)3Si). 13C NMR (75 MHz, CDCl3):
δ 107.44, 84.99, 67.86, 30.52, 25.05, 23.75, 20.02,
0.35. Anal. calcd. for C10H20OSi: C, 65.15; H, 10.94.
Found: C, 65.34; H, 11.15.
2.4. rac-2-(Benzoyloxy)hept-6-yn (4)
A solution of BzCl (1.188 mL, 10.23 mmol) in ben-
zene (25 mL) was added to a solution of 3 (0.943 g,
5.11 mmol) in benzene (25 mL) and pyridine (15 mL).
The mixture was stirred overnight at rt and then
acidified with H2SO4 (1 M, 50 mL). Reaction prod-
ucts were extracted with Et2O (2 × 50 mL) and the
combined extracts were concentrated under reduced
pressure. The residue was filtered through a silica
gel column (hexane/Et2O, 4:1). The reaction product,
which showed an Rf = 0.60 in silica gel thin-layer
chromatography (hexane/Et2O, 1:1), was collected,
the solvent was evaporated and the residue was dis-
solved in THF (35 mL). The silyl group was removed
by stirring for 3 h at rt in the presence of n-Bu4NF
(1.937 g, 6.14 mmol). The mixture was quenched
with H2O (100 mL), organic layer was separated and
lipophilic products were extracted from the water
phase with Et2O (2 × 40 mL). The combined organic
layers were washed with saturated aqueous NaCl
(70 mL), dried over Na2SO4 and concentrated under
vacuum. Silica gel chromatography using gradient
elution with hexane/Et2O (from 1 to 10% Et2O)
gave pure 4 as an amorphous mass (1.088 g, 98%).
TLC: Rf = 0.58 (hexane/Et2O, 1:1). IR (neat): ν 3272
≡
≡
(neat): ν 3600–3200 (OH), 2240, 2170 (C C), 1725
1
=
(C O), 1115 (C–O), 709 (Ph). H NMR (300 MHz,
CDCl3): δ 8.02 (m, 2H, –oBz), 7.52 (m, 1H, –pBz),
7.41 (m, 2H, –mBz), 5.19 (m, 1H, 13-CH), 4.22 (m,
2H, CH2OH), 3.17 (m, 2H, 4-CH2), 3.10 (m, 2H,
7-CH2), 2.19 (m, 2H, 10-CH2), 1.75 (m, 2H, 11-CH2),
1.60 (m, 2H, 12-CH2), 1.32 (d, 3H, J = 6.3 Hz,
CH3). 13C NMR (75 MHz, CDCl3): δ 166.37, 133.43,
132.93, 129.68 (2C), 128.46 (2C), 80.46, 80.19,
78.87, 75.55, 74.33, 73.89, 71.38, 53.03, 35.32, 24.73,
20.25, 18.76, 10.02, 9.90. Anal. calcd. for C21H22O3:
C, 78.23; H, 6.88. Found: C, 78.03; H, 6.69.
2.6. rac-2-(Benzoyloxy)-14-bromotetradeca-
6,9,12-triyne (7)
To a solution of alcohol 6 (724 mg, 2.24 mmol)
and CBr4 (894 mg, 2.69 mmol) in CH2Cl2 (30 mL)
a solution of PPh3 (707 mg, 2.69 mmol) in CH2Cl2
(15 mL) was added at 10 ◦C. The reaction mixture was
stirred for 1 h at rt then quenched with MeOH (2 mL)
and volatile components were removed under vacuum.
Column chromatography on silica gel (hexane/Et2O,
(C C), 1725 (C O), 1273 (C–O), 710 (Ph). 1H
=
NMR (300 MHz, CDCl3): δ 8.01 (m, 2H, –oBz), 7.49
(m, 3H, –(m+p)-Bz), 5.19 (m, 1H, 2-CH), 2.22 (td,

120
S.G. Romanov et al. / Chemistry and Physics of Lipids 130 (2004) 117–126
1:1) afforded pure 7 in a yield of 775 mg (90%). TLC:
Rf = 0.69 (hexane/Et2O, 1:2). 1H NMR (300 MHz,
CDCl3): δ 8.02 (m, 2H, –oBz), 7.52 (m, 1H, –pBz),
7.41 (m, 2H, –mBz), 5.18 (m, 1H, 2-CH), 3.87 (m, 2H,
CH2Br), 3.20 (m, 2H, 11-CH2), 3.11 (m, 2H, 8-CH2),
2.19 (m, 2H, 5-CH2), 1.75 (m, 2H, 4-CH2), 1.61 (m,
2H, 3-CH2), 1.32 (d, 3H, J = 6.3 Hz, CH3). 13C NMR
(75 MHz, CDCl3): δ 166.31, 133.42, 132.92, 129.67
(2C), 128.45 (2C), 81.44, 80.50, 79.49, 75.79, 74.24,
73.40, 71.33, 52.98, 35.35, 24.75, 20.27, 18.76, 14.83,
9.90.
Erlenmeyer flask at rt and then cooled to 10 ◦C. A so-
lution of 9 (520 mg, 1.20 mmol) in benzene (35 mL)
and quinoline (1 mL) were added to the catalyst un-
der a stream of argon. After the argon was exchanged
with H2 the mixture was stirred for 2 h at 10 ◦C, then
filtered, washed with HCl (2M, 2 × 30 mL) and the
solvent was evaporated. The crude residue was fil-
tered over silica gel (hexane/Et2O, 2:1). Preparative
RP-HPLC (solvent system MeOH/H2O, 95:5) gave
of 297 mg of pure tetraenoate. Analytical RP-HPLC:
Rt = 6.88 min (MeOH/H2O/AcOH, 95:5:0.1, by vol.;
detection at 210 nm). After HPLC an aqueous solution
(15 mL) of NaOH (264 mg, 6.60 mmol) was added to a
solution of tetraenoate in methanol (60 mL) under ar-
gon atmosphere. The resulting mixture was stirred for
30 h at rt. After the reaction was completed, methanol
was removed by evaporation under reduced pressure.
The residue was carefully acidified to pH 4.0 using
HCl (1 M) and the lipophilic products were extracted
with Et2O (4 × 50 mL). The combined organic ex-
tracts were dried over Na2SO4 and concentrated in
vacuum. The crude residue was purified by silica gel
column chromatography (hexane/Et2O, 1:3) to give
10 in 224 mg yield (58%). Analytical RP-HPLC:
Rt = 6.10 min (MeOH/H2O/AcOH, 85:15:0.1, by vol.;
2.7. Methyl rac-19-(benzoyloxy)eicosa-5,8,11,14-
tetraynoate (9)
In a dry argon-filled round-bottomed flask equipped
with magnetic stirrer anhydrous K2CO3 (397 mg,
2.87 mmol), NaI (575 mg, 3.83 mmol) and CuI
(731 mg, 3.83 mmol) were suspended in DMF
(15 mL). Methyl 5-hexynoate (8) (242 mg, 1.91 mmol)
was added to the suspension at once followed by bro-
mide 7 (739 mg, 1.91 mmol). The reaction mixture
was vigorously stirred overnight at rt, then quenched
with saturated aqueous solution of NH4Cl (200 mL).
The lipophilic products were extracted with Et2O
(4 × 100 mL). The combined organic extracts were
washed with saturated aqueous solution of NaCl (2 ×
150 mL). After drying over Na2SO4 the ethereal solu-
tion was concentrated in vacuum. The crude residue
was purified by silica gel flash chromatography
(hexane/Et2O, 3:1) under argon to give 625 mg (75%)
of 9 as yellow oil. TLC: Rf = 0.42 (hexane/Et2O, 1:1).
1H NMR (300 MHz, CDCl3): δ 8.01 (m, 2H, –oBz),
7.52 (m, 1H, –pBz), 7.42 (m, 2H, –mBz), 5.17 (m, 1H,
19-CH), 3.61 (s, 3H, OCH3), 3.16 (m, 6H, 7-, 10- and
13-CH2), 2.39 (t, 2H, J = 7.2 Hz, 2-CH2), 2.19 (m,
4H, 4- and 16-CH2), 1.35–1.75 (m, 6H, 3-, 17- and
18-CH2), 1.31 (d, 3H, J = 6.3 Hz, CH3). 13C NMR
(75 MHz, CDCl3): δ 173.78, 166.30, 133.42, 132.90,
129.66 (2C), 128.45 (2C), 80.37, 79.65, 78.45, 75.24,
75.11, 74.85, 74.40, 74.30, 71.34, 51.71, 35.34, 32.99,
24.75, 23.97, 20.25, 18.76, 18.31, 9.89 (3C).
1
detection at 210 nm). H NMR (300 MHz, CDCl3): δ
=
5.34–5.48 (m, 8H, CH CH), 3.78 (m, 1H, 19-CH),
2.79 (m, 6H, 7-, 10- and 13-CH2), 2.30 (t, 2H,
J = 7.5 Hz, 2-CH2), 2.07 (m, 4H, 16- and 4-CH2),
1.68 (m, 2H, 3-CH2), 1.43 (m, 4H, 17- and 18-CH2),
1.16 (d, 3H, J = 6.3 Hz, CH3). 13C NMR (75 MHz,
CDCl3): δ 174.27, 130.14, 129.11, 129.03, 128.58,
128.37, 128.32, 128.20, 128.15, 68.19, 39.06, 33.61,
27.31, 26.72, 25.92, 25.81 (2C), 25.78, 24.94, 23.69.
EI HRMS calcd. for C20H32O3 [M+]: 320.2351.
Found: 320.2367.
2.9. (5Z,8Z,11Z,14Z)-19-Azidoeicosa-5,8,11,14-
tetraenoic acid (11)
A mixture of 19-HETE (10) (46 mg, 0.14 mmol),
p-toluenesulphonyl chloride (219 mg, 1.15 mmol) in
CH2Cl2 (3 mL) and anhydrous pyridine (3 mL) was
stirred for 24 h at rt and the reaction was monitored
by HPLC using MeOH/H2O/AcOH (85:15:0.1, by
vol.; detection at 210 nm) as solvent system. After the
reaction was completed the mixture was poured into
diluted HCl (1M, 50 mL) and extracted with Et2O
2.8. rac-(5Z,8Z,11Z,14Z)-19-Hydroxyeicosa-
5,8,11,14-tetraenoic acid (10)
A suspension of Lindlar’s catalyst (995 mg) in dry
benzene (15 mL) was saturated with H2 in a 100-mL

S.G. Romanov et al. / Chemistry and Physics of Lipids 130 (2004) 117–126
121
(3 × 40 mL). The combined organic extracts were
back-washed with HCl (1M, 50 mL), water (50 mL)
and dried over Na2SO4. The crude residue was purified
by silica gel column chromatography (hexane/Et2O,
1:1) to give 37 mg of the product characterised
by a retention time of Rt = 9.31 min in RP-HPLC
(MeOH/H2O/AcOH, 85:15:0.1, by vol.; detection at
210 nm). The compound was then stirred with sodium
azide (25 mg, 0.39 mmol) in DMF (3 mL) for 2.5 h at
75–80 ◦C. The reaction mixture was quenched with
saturated aqueous solution of NH4Cl (20 mL) and the
products were extracted with Et2O (3 × 30 mL). The
combined organic extracts were washed with saturated
solution of NaCl (20 mL), dried over Na2SO4 and the
solvent was evaporated under reduced pressure. The
residue was purified by silica gel flash chromatog-
raphy (hexane/Et2O, 2:1) to afford pure 11 with an
overall yield of 17 mg (35%). Analytical RP-HPLC:
Rt = 13.50 min (MeOH/H2O/AcOH, 85:15:0.1, by
vol.; detection at 210 nm). 1H NMR (200 MHz,
followed by saponification according to the standard
procedure to give 58% of 12 with specific radioactiv-
ity 160 Ci/mmol. Compound 12 was found to be iden-
tical by HPLC with its non-tritiated analogue. Ana-
lytical RP-HPLC: Rt = 6.10 min (MeOH/H2O/AcOH,
85:15:0.1, by vol.; detection at 210 nm). The radio-
chemical purity of [3H-8]-12 was found to be 95%.
2.11. [5,6,8,9,11,12,14,15-3H]-rac-(5Z,8Z,11Z,14Z)-
19-Azidoeicosa-5,8,11,14-tetraenoic acid (13)
Compound 13 was obtained from the tritiated acid
12 (173 mCi, 1.60 Ci/mmol) according to the pro-
cedure described for unlabeled analogue 11. Com-
pound 13 was obtained in 26% yield (44 mCi) with
a radiochemical purity of 97%. The specific activ-
ity of 1.60 Ci/mmol remained unchanged. Analyti-
cal RP-HPLC: Rt = 13.49 min (MeOH/H2O/AcOH,
85:15:0.1, by vol.; detection at 210 nm).
=
CDCl3): δ 5.30–5.50 (m, 8H, CH CH), 3.40 (m, 1H,
2.12. (5Z,8Z,11Z,14Z)-18-Azidoeicosa-5,8,11,14-
19-CH), 2.80 (m, 6H, 7-, 10- and 13-CH2), 2.34 (t,
2H, J = 7.1 Hz, 2-CH2), 2.09 (m, 4H, 4- and 16-CH2),
1.69 (m, 2H, 3-CH2), 1.43 (m, 4H, 17- and 18-CH2),
1.22 (d, 3H, J = 6.8 Hz, CH3). 13C NMR (75 MHz,
CDCl3): δ 179.64, 129.72, 129.16, 128.95, 128.50,
128.47, 128.35, 128.31, 128.21, 58.09, 35.89, 27.03,
26.62, 26.20, 25.80 (4C), 24.67, 19.64. ESI HRMS
calcd. for C20H31NaO2N3 [M + Na]+: 368.2314.
Found: 368.2312.
tetraenoic acid (16)
Azido acid 16 was prepared from 15 (50 mg,
0.156 mmol) by the same procedure as described
for 11. Yield of pure 16: 14 mg (26%). Analyti-
cal RP-HPLC: Rt = 13.50 min (MeOH/H2O/AcOH,
85:15:0.1, by vol.; detection at 210 nm). 1H NMR
=
(300 MHz, CDCl3): δ 5.30–5.50 (m, 8H, CH CH),
3.25 (m, 1H, 18-CH), 2.78 (m, 6H, 7-, 10- and
13-CH2), 2.37 (t, 2H, J = 7.3 Hz, 2-CH2), 2.15 (m, 2H,
4- and 16-CH2), 1.73 (m, 2H, 3-CH2), 1.56 (m, 4H,
17- and 19-CH2), 0.89 (t, 3H, J = 7.3 Hz, CH3). 13C
NMR (75 MHz, CDCl3): δ 178.90, 129.17, 129.06,
128.95 (2C), 128.34 (3C), 128.28, 64.09, 33.93,
33.39, 27.63, 26.63, 25.79 (3C), 24.69, 24.06, 10.68.
ESI HRMS calcd. for C20H31NaO2N3 [M + Na]+:
368.2314. Found: 368.2316.
2.10. [5,6,8,9,11,12,14,15-3H]-rac-(5Z,8Z,11Z,14Z)-
19-Hydroxyeicosa-5,8,11,14-tetraenoic acid (12)
A solution of 9 (15 mg, 0.034 mmol) in benzene
(1 mL), Lindlar’s catalyst (10 mg) and quinoline
(0.15 mL) were placed in a reaction vessel and the
mixture was frozen with liquid nitrogen. After evacu-
ation the ampoule was filled with tritium gas to reach
a pressure 400 hPa. The reaction mixture was stirred
for 1 h at rt followed by freezing in liquid nitrogen
and the remaining tritium gas was removed. After
thawing the mixture was filtered over silica gel and
the filtrate was concentrated under reduced pressure.
To remove labile tritium the residue was dissolved
in methanol (1 mL) and the solvent was evaporated
again. This procedure was repeated twice. The crude
synthesis product was purified by preparative HPLC
2.13. [5,6,8,9,11,12,14,15-3H]-rac-(5Z,8Z,11Z,14Z)-
18-Hydroxyeicosa-5,8,11,14-tetraenoic acid (17)
Free acid 17 was prepared from methyl rac-18-
(benzoyloxy)eicosa-(5Z,8Z,11Z,14Z)-tetraynoate (14)
(5 mg, 0.01 mmol) as described for acid 12. Yield of
17: 56% with a specific radioactivity 160 Ci/mmol.
Compound 17 was found to be identical with
its non-labelled analogue 15 as revealed analyti-

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S.G. Romanov et al. / Chemistry and Physics of Lipids 130 (2004) 117–126
cal RP-HPLC. Analytical RP-HPLC: Rt = 6.08 min
(MeOH/H2O/AcOH, 85:15:0.1, by vol.; detection at
210 nm).
2.16. Catalytic hydrogenation and GC/MS analysis
of hydrogenated lipoxygenase products
To get more informative mass spectra, the methy-
lated lipoxygenase products formed from 11 and 16
were hydrogenated using 10% Pd/CaCO3 as catalyst.
For this purpose the fatty acid derivatives (20 ␮g)
were dissolved in MeOH (1 mL). Then the Pd-catalyst
(3 mg) was added and hydrogen gas was bubbled
through the mixture for 5 min at rt. The solution was
filtered to remove the catalyst, the solvent was evapo-
rated, the residue was methylated with diazomethane,
silylated with BSTFA and analysed by GC/MS as
described in the Section 2.
2.14. [5,6,8,9,11,12,14,15-3H]-rac-(5Z,8Z,11Z,14Z)-
18-Azidoeicosa-5,8,11,14-tetraenoic acid (18)
Azido acid 18 was obtained from the tritiated
compound 17 (117 mCi, 0.79 Ci/mmol) by the
same method as described for 13. Yield: 30.6 mCi
(26%), specific radioactivity 0.79 Ci/mmol. Analyti-
cal RP-HPLC: Rt = 13.50 min (MeOH/H2O/AcOH,
85:15:0.1, by vol.; detection at 210 nm).
2.15. General procedure for oxygenation of azides
11 and 16 by the soybean 15-lipoxygenase-1 and
rabbit reticulocyte 15-LOX
3. Results and discussion
The oxygenation kinetics of the different fatty acid
derivatives were assayed spectrophotometrically mea-
suring the increase in absorbance at 235 nm in the sub-
strate concentration range between 10 and 100 ␮M.
The assay mixture consisted of 0.1 M sodium borate
buffer (pH 9.0 for soybean LOX) or sodium phosphate
buffer (pH 7.4 for rabbit reticulocyte LOX) containing
various concentrations of substrate, and the reaction
was started by addition of enzyme (4 ␮g). Before ad-
dition of the enzyme, the mixture was sonicated for
30 s to achieve homogeneous substrate dispersion. All
measurements were carried out at room temperature.
For structural analysis of the oxygenation products
an ethanol solution of the fatty acid (10 ␮l, 50 mM)
was added to either sodium borate buffer pH 9.0 or
sodium phosphate buffer pH 7.4 (5 mL, 0.1 M). After
the mixture was sonicated for 30 s to achieve homoge-
nous substrate dispersion, soybean 15-lipoxygenase-1
(40 ␮g) or reticulocyte 15-LOX (40 ␮g) was added.
After 25 min incubation at rt the hydroperoxy fatty
acids formed were reduced to the corresponding alco-
hols by the addition of a saturated solution of NaBH4
(50 ␮l) in ethanol. The mixture was acidified to pH
3 with 0.1 M acetic acid and the lipophylic products
were extracted with ethyl acetate (2 × 3 mL). The
extracts were combined, the solvent was evaporated
and the products were analysed by both RP-HPLC
(MeOH/H2O/AcOH, 85:15:0.1, by vol.; detection at
235 nm) and SP-HPLC (hexane/2-propanol/AcOH,
100:1.5:0.1, by vol.; detection at 235 nm).
3.1. Chemical synthesis
In our synthetic strategy we prepared (5Z,8Z,11Z,
14Z)-19- (10) and (5Z,8Z,11Z,14Z)-18-hydroxy-5,8,
11,14-eicosatetraenoic (15) acids (19- and 18-OH-AA)
as key intermediates for formation of azido deriva-
tives. Although alternative synthetic procedures for
the preparation of 18- and 19-OH-AA have al-
ready been reported (Heckmann et al., 1996), that
method does not allow the preparation of radio-
labelled analogues. In contrast, with our synthe-
sis, which follows the acetylenic approach, it is
possible to prepare radioactively labelled skipped
tetraenoic systems by Pd-assisted tritiation of triple
bonds. In fact, as illustrated in Scheme 1, 19-OH-
AA can be synthesised via a tetraacetylenic precur-
sor 9, which can be synthesised from three building
blocks: rac-2-(benzoyloxy)hept-6-yn (4), 7-bromo-
2,5-heptadiyne-1-ol (5) (Ivanov et al., 2002) and
methyl 5-hexynoate (8). Heptyne
4 was syn-
thesised as shown in Scheme 1 starting from
6-(trimethylsilyl)hex-5-ynoic acid (1). Coupling
of acid chloride, prepared from compound 1 by
treatment with an excess of oxalyl chloride, and
lithium dimethylcuprate at −78 ◦C (Posner et al.,
1972) afforded 7-(trimethylsilyl)hept-6-yn-2-one
(2). Consequent reduction of the carbonyl func-
tion using LiAlH4 followed by protection of
OH-group and fluoride-mediated desylilation af-
forded 4 in an overall yield 56% for four steps.

S.G. Romanov et al. / Chemistry and Physics of Lipids 130 (2004) 117–126
123
Scheme 1. Total synthesis of 19-N3-AA (11) and its radio-labelled analogue 13.
Cross-coupling of methyl 5-hexynoate (8) and propar-
gyl bromide 7 obtained from compound 4 and
7-bromo-2,5-heptadiyne-1-ol (5) resulted in methyl
rac-19-(benzoyloxy)eicosa-5,8,11,14-tetraynoate (9)
in 75% yield. Stereospecific hydrogenation of the
skipped triple bonds of 9 using Lindlar’s catalyst and
quinoline (catalyst/substrate ratio, 1.5:1) in benzene
afforded methyl (5Z,8Z,11Z,14Z)-rac-19-(benzoylo-
xy)eicosa-5,8,11,14-tetraenoate. Simultaneous depro-
tection of both carboxy and hydroxy groups, using
NaOH in MeOH–H2O solution proceeded well to
give 10 in 58% yield. Tritiation of 9 was performed
in benzene with a method used before with slight
modifications (Shevchenko et al., 1999). Subsequent
deprotection yielded 12 with high specific radioactiv-
ity (160 Ci/mmol) and a radiochemical purity of more
than 95%. For the safety reason all further modifica-
tions of the radio-labelled compound 12 were carried
out with a diluted sample (1:100) of the unlabelled
analogue 10.
Among a variety of leaving groups, we elected
p-toluenesulphonates for subsequent substitution by
azide ion (Lie Ken Jie and Lao, 1987). Tosylates
can be obtained from corresponding alcohols with

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S.G. Romanov et al. / Chemistry and Physics of Lipids 130 (2004) 117–126
Scheme 2. Synthesis of 18-N3-AA (16) and the radio-labelled analogue 18.
moderate yields without additional protection of the
carbonyl function. On the other hand, tosylates ap-
peared to be more reactive in SN2 substitution by
azide ion. Thus, conversion of hydroxy compounds
10 and 12 to p-toluenesulphonates followed by sub-
stitution to azide in DMF at 75–80 ◦C afforded
(5Z,8Z,11Z,14Z)-19-azidoeicosa-5,8,11,14-tetraenoic
acid (11) and its [5,6,8,9,11,12,14,15-3H8]-analogue
13 (specific activity 1.6 Ci/mmol) with 35 and 26%
yields, respectively. Final radiochemical purity of
13 was 97%. Reverse phase HPLC followed by
UV-detector indicated identical retention times for
compounds 11 and 13.
eicosa-5,8,11,14-tetraenoate (14) as reported pre-
viously (Romanov et al., 2002). Here we extend
application of the tetraacetylenic precursor 14 for
preparation of tritium-labelled analogue 17. Tritia-
tion of 14 and subsequent deprotection yielded 17
with 56% yield (specific radioactivity 160 Ci/mmol)
and a radiochemical purity of more than 98%. For
further manipulations a final specific radioactivity
of 0.79 Ci/mmol was adjusted by diluting the native
sample with the unlabelled analogue 15.
In contrast to azido derivative 11 18-N3-AA (16) ap-
peared to be more sensitive to heat treatment. In fact,
we found nearly complete tosylate substitution after
1.5 h when the reaction mixture was kept at 75–80 ◦C.
Further exposure to the heat induced a high degree
of azide degradation. Azido acid 18 was obtained
from tritiated compound 17 with a yield of 30.6 mCi
(26%) and specific radioactivity 0.79 Ci/mmol. Ana-
As illustrates Scheme 2 18-N3-AA (16) and
[5,6,8,9,11,12,14,15-3H8]-18-N3-AA (18) were ob-
tained in a similar way as described for the C-19-
substituted analogue. 18-OH-AA (15) was prepared
via methyl rac-(5Z,8Z,11Z,14Z)-18-(benzoyloxy)-
Table 1
Oxygenation of arachidonic acid derivatives by soybean LOX-1 and rabbit reticulocyte 15-LOX
Substrate
Soybean LOX-1
Reticulocyte 15-LOX
Km (␮M)
Vmax (s−1
)
Km (␮M)
Vmax (s−1
)
Arachidonic acid
19-N3-AA (11)
18-N3-AA (16)
8.3
4.5
1.0
0.5
3.95
0.42
<0.1
0.39
0.04
8.1
10.9
34.2
1.0
1.2
3.6
9.48
3.39
6.17
0.95
0.34
0.62
Not determined
The kinetic constants were extracted from the Lineweaver–Burk plots (Fig. 1). For each substrate five measurements at different fatty
acid concentrations and three measurements for each concentration were carried out. Because of the low reaction rate a Km could not be
determined for compound 16.

S.G. Romanov et al. / Chemistry and Physics of Lipids 130 (2004) 117–126
125
lytical HPLC showed that this product was identical
with unlabelled compound 16.
3.2. Biochemical assays and analysis
In order to test whether 15-LOXs are capable of
oxygenating polyenoic fatty acids bearing an azido
group close to the methyl end of the fatty acid chain
we used commercially available soybean 15-LOX-1
and rabbit reticulocyte 15-LOX as catalysts. The latter
enzyme was prepared from a stroma-free hemolysis
supernatant of a reticulocyte-rich blood cell suspen-
sion by ammonium sulphate precipitation and two
consequetive steps of chromatography (Rapoport
et al., 1979).
The kinetic parametres (Table 1), extracted from
Lineweaver–Burk plots (Fig. 1) indicate that, com-
pared with arachidonic acid, the 19-azido fatty acids
binds with a similar affinity to active site of rabbit 15-
lipoxygenase. In contrast, the affinity of the 18-azido
derivative was somewhat lower. Interestingly, for this
enzyme the Vmax values were in the same range as that
for arachidonic acid. For the soybean soybean enzyme
the Km of the 19-azido derivative was even lower than
that for arachidonic acid indicating a high affinity
binding at the active site. However, Vmax was drasti-
cally (10-fold) reduced. An even more drastic reduc-
tion in the binding affinity was seen for the 18-azido
derivative. When we analysed the patterns of oxygena-
tion products formed by the rabbit and the soybean
enzymes we found that both enzymes form one major
compound from the 19-azido derivative. For detailed
structural information this compound was analysed
by SP-HPLC and GC/MS. It was eluted with an Rt of
8.51 min (hexane/2-propanol/acetic acid, 100:1.5:0.1,
by vol.) and exhibited a conjugated diene chro-
mophore with an absorbance maximum at 235 nm (not
shown). For further structural information the product
was hydrogenated, methylated with diazomathane and
silylated using bis(trimethylsilyl)trifluoroacetamide
(BSTFA) (Boeynems et al., 1980). The mass spectra
obtained were characterised by a typical ion of major
intensity at m/z 343 (Brash et al, 1981) suggesting a
15-lipoxygenation of the fatty acid chain. Summaris-
ing these data one may conclude that azido derivatives
of arachidonic acid have similar oxygenation kinetics
as arachidonic acid and show a similar alignment at
the active site.
Fig. 1. Estimation of 19-N3-AA (11) oxygenation parametrs (Km
and Vmax) using Lineweaver–Burk plots: (A) soybean LOX-1, and
(B) rabbit reticulocyte 15-LOX.
In conclusion, here we describe a method for the
synthesis of (5Z,8Z,11Z,14Z)-18- and 19-azidoeicosa-
5,8,11,14-tetraenoic acids and their [5,6,8,9,11,12,
14,15-3H8]-analogues via a common synthetic proce-
dure. It should be stressed that these compounds may
not just be of interest for lipoxygenase research. They
may also be used as labelling ligands for cylooxyge-
nase isoforms and for various eicosanoid receptors.
Acknowledgements
This work was supported in part by the Rus-
sian Foundation for Basic Research (02-03-33138

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S.G. Romanov et al. / Chemistry and Physics of Lipids 130 (2004) 117–126
enantiomers and the corresponding 14,15-dehydro analogues:
role of the 16-hydroxy group for the lipoxygenase reaction.
Bioorg. Med. Chem. 10, 2335–2343.
and 03-03-06586) and grants of Russian Ministry
of Education. Authors also wish to thank Deutsche
Akademischer Austauschdienst (A/03/01297) for fi-
nancial support for S.R.
Kurumbail, R.G., Stevens, A.M., Gierse, J.K., McDonald, J.J.,
Stegeman, R.A., Pak, J.Y., Gildehaus, D., Iyashiro, J.M.,
Penning, T.D., Seibert, K., Isakson, P.C., Stallings, W.C., 1996.
Structural basis for selective inhibition of cyclooxygenase-2 by
anti-inflammatory agents. Nature 384, 644–648.
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