Synthesis of Tritiated and Deuterated 5-Oxo-ETE
J . Org. Chem., Vol. 63, No. 12, 1998 4101
over a period of 1 h. It was then quenched by the addition of
aqueous saturated ammonium chloride solution (25 mL) and
extracted with diethyl ether (3 × 75 mL). The combined
extracts were washed with cold water (3 × 25 mL), dried over
acetate (2 × 50 mL). The combined ethyl acetate extracts were
washed with cold water (5 × 30 mL) and brine (1 × 30 mL),
2 4
dried over anhydrous Na SO , and filtered, the solvent was
evaporated under reduced pressure to approximately 2 mL
solution and the product was first purified by reversed-phase
HPLC. Approximately 20 injections were performed, and
every time before loading the product on RP column, ethyl
acetate was evaporated under a stream of argon and the
residue dissolved in acetonitrile and then injected onto the
Spherisorb C-18, 10 × 250 mm column. The mobile system
2 4
anhydrous Na SO , filtered, and concentrated under reduced
pressure to afford the crude product, which was purified by
flash column chromatography using 10% ethyl acetate in
hexane to give pure 13 (352 mg, 71%): R
f
) 0.36 (10% ethyl
) δ 0.87 (t, J ) 7.1 Hz, 3
H), 1.32 (m, 4 H), 1.44 (m, 2 H), 1.75 (m, 2 H), 2.15 (m, 4 H),
1
acetate in hexane); H NMR (CDCl
3
2
3
.37 (t, J ) 7.3 Hz, 2 H), 3.1 (m, 4 H), 3.25 (m, 4 H), 3.62 (s,
3 2
CH CN/H O/AcOH (80:20:0.02%) at a flow rate of 3 mL/min
H), 5.45 (dt, J ) 10.5, 7.0 Hz 1 H), 5.88 (d, J ) 14.8 Hz, 1
was used to give a mixture of cis and trans isomers (retention
time 13.5 min). The eluent containing the mixture of 5-oxo-
ETE (2) and 8,9-trans-5-oxo-ETE (18) was neutralized with
H), 6.11 (t, J ) 10.9 Hz, 1 H), 6.61 (dd, J ) 14.8, 11.2 Hz, 1
H); C NMR (CDCl
2
7
1
3
3
) δ 9.66, 13.89, 17.75, 18.58, 21.94, 22.90,
8.95, 30.96, 38.81, 38.89, 41.42, 51.47, 70.40, 73.95, 74.86,
7.85, 80.51, 123.48, 126.26, 128.28, 128.43, 138.71, 173.42.
,5-(Dim et h ylen ed it h io)-6(E),8(Z)-eicosa d ien -11,14-
3
Et N before evaporation of solvents. The separation of indi-
vidual isomers was carried out by NP HPLC [Spherisorb
S10W, 10 × 250 mm column, solvent system: 4.5% 2-propanol
in hexane containing 0.05% AcOH; flow rate 4.5 mL/min].
Analytical separation showed that 8,9-cis and 8,9-trans iso-
5
d iyn oic Acid (Bisa cetylen ic Dith io a cid ) (14). A solution
of the dithio compound 13 (36.9 mg) in THF (9 mL) and 1 M
LiOH (4.5 mL) was stirred at room temperature for 20 h. To
the reaction mixture was added water (8 mL), and THF was
removed under a stream of argon and acidified by the addition
R R
mers were obtained in a ratio of 87:17, t 8,9-cis 16.49 min, t
8,9-trans 19.73 min. To the each of the eluents containing
the pure 5-oxo-ETE (2) and 8,9-trans-5-oxo-ETE (18) was
added an equal amount of ethyl acetate, and they were washed
with water to remove the 2-propanol and acetic acid to give
2 4
of 5% aqueous KH PO (60 mL) and extracted with ethyl
acetate (3 × 30 mL). The combined ethyl acetate extracts were
washed with cold water (4 × 25 mL) and brine (1 × 25 mL),
pure 5-oxo-ETE (2), 8.8 mg, in 52% yield: 1H NMR (CDCl
) δ
3
dried over anhydrous Na
2
SO
4
, and filtered, and the solvent
0.85 (t, J ) 6.0 Hz, 3 H), 1.25-1.32 (m, 6 H), 1.99 (m, 2 H),
2.03 (m, 2 H), 2.40 (t, J ) 6.1 Hz, 2 H), 2.64 (m, 2 H), 2.79 (m,
2 H), 3.16 (m, 2 H), 5.34-5.4 (m, 4 H), 5.86 (m 2 H), 6.13 (dd,
was evaporated under reduced pressure to afford the bisacety-
lenic dithio acid 14 (35 mg) in nearly quantitative yield, which
1
was used in the next step without further purification: R
f
)
J ) 16.0, 10.7 Hz, 1 H), 7.51 (dd, J ) 15.5, 11.5 Hz, 1 H); H
1
0
.43 (35% ethyl acetate in hexane); H NMR (CDCl
3
) δ 0.91
3 3
NMR (CD COCD ) δ 0.87 (t, J ) 7.0 Hz, 3 H), 1.27-1.39 (m,
(
(
(
(
t, J ) 7.1 Hz, 3 H), 1.35 (m, 4 H), 1.50 (m, 2 H), 1.75-1.90
m, 4 H), 2.15 (m, 4 H), 2.40 (t, J ) 7.4 Hz, 2 H), 3.05-3.15
m, 4 H), 3.2-3.4 (m, 4 H), 5.5 (dt, J ) 10.6, 7.2 Hz, 1 H), 5.82
d, J ) 14.6 Hz, 1 H), 6.07 (t, J ) 11.0 Hz, 1 H), 6.66 (dd, J )
6 H), 1.86 (m, 2 H), 2.1 (m, 2 H, peak overlapped with solvent
acetone peak), 2.34 (t, J ) 7.3 Hz, 2 H), 2.72 (t, J ) 7.2 Hz, 2
H), 2.9 (m, 2 H, peak overlapped with water peak), 3.16 (t, J
) 6.6 Hz, 2 H), 5.35-5.5 (m, 4 H), 5.90 (m 2 H), 6.24 (dd, J )
15.9, 10.7 Hz, 1 H), 7.63 (dd, J ) 15.3, 11.6 Hz, 1 H); UV
1
3
1
1
7
3
4.7, 11.2 Hz, 1 H); C NMR (CDCl ) δ 9.67, 13.88, 17.75,
8.58, 22.1, 22.57, 28.32, 30.97, 33.64, 38.93, 41.31, 70.36,
4.88, 76.86, 123.53, 126.33, 127.65, 128.42, 138.65, 178.5.
(EtOH) λmax 278 (ꢀ ) 23 500). 8,9-trans-5-Oxo-ETE (18) was
1
obtained, 2.2 mg, in 13% yield: H NMR (CD
3
COCD
3
) δ 0.86
5
,5-(Dim eth ylen edith io)-6(E),8(Z),11(Z),14(Z)-eicosatet-
(t, J ) 7.0 Hz, 3 H), 1.18 (m, 6 H), 1.84 (m, 2 H), 2.1 (m, 2 H,
peak overlapped with solvent acetone peak), 2.33 (t, J ) 7.3
Hz, 2 H), 2.66 (t, J ) 7.3 Hz, 2 H), 2.9 (m, 2 H, peak overlapped
with water peak), 3.0 (t, J ) 5.8 Hz, 2 H), 5.3-5.51 (m, 4 H),
6.13 (d, J ) 15.6 Hz, 1 H), 6.28 (m, 2 H), 7.23 (dd, J ) 15.7,
9.7 Hz, 1 H).
r a en oic Acid (15). The bisacetylenic dithio acid 14 (24 mg)
was dissolved in 24 mL of 9:1 hexane/ethyl acetate. The
hydrogenation was performed at 0 °C over a period of 2 h using
Lindlar catalyst (5% Pd) (1.2 g). More catalyst (0.6 g) was
added, and the hydrogenation was continued for 30 min more.
Then hydrogen was removed by applying vacuum and purging
with air. To the reaction mixture was added HPLC-grade
methanol (12 mL). The mixture was stirred for 5 min, and
then 20 mL of methanol/methylene chloride (20:80) was added
and further stirred for 5 min. The mixture including the
catalyst was loaded on a pad of Celite (1 g) (before loading the
product on Celite, it was washed with 10 mL of methanol/
methylene chloride (20:80) and the washings were discarded)
and filtered and washed with 200 mL of methanol/methylene
chloride (20:80). Concentration of the filtrate gave the dithio
acid 15 (21.6 mg, 93%). The crude product 15 can be used as
such in the next step. The pure sample was obtained by RP
HPLC [Spherisorb C-18, 10 × 250 mm column, solvent
Deu ter a tion P r oced u r e a n d Deblock in g of d
Acid . 5-Oxo-6(E),8(Z),11(Z),14(Z)-11,12,14,15-d -eicosa tet-
r a en oic Acid (d -5-Oxo-ETE) (21). In a 25 mL three-neck
4
-Dith io
4
4
flask was placed a solution of dithio acid 14 (12 mg) in ethyl
acetate/hexane 1:9 (12 mL), and 100 µg of 4-hydroxy-TEMPO
in ethyl acetate (100 µL) was added. The solution was purged
with argon and the flask connected to the deuteration ap-
paratus. The argon was removed by applying vacuum and the
flask purged with deuterium; the process was repeated three
times. Then Lindlar catalyst (5% Pd) (0.6 g) was added
quickly, and the reaction mixture was stirred well magnetically
at 0-5 °C for 2 h. More catalyst (0.3 g) was added, and the
deuteration was further continued for 30 min more. Then
deuterium was removed by applying vacuum and purging with
air. To the reaction mixture was added HPLC-grade methanol
(5 mL), the mixture was stirred for 5 min, and then 10 mL of
20:80 methanol/methylene chloride was added and further
stirred for 5 min. The mixture including the catalyst was
loaded on a pad of Celite (1 g) (before loading the product on
Celite, it was washed with 10 mL of 20:80 methanol/methylene
chloride and the washings were discarded), filtered, and
washed with 200 mL of methanol/methylene chloride (20:80).
The filtrate on evaporation of solvent at reduced pressure gave
system: 20% water in acetonitrile containing 0.05% AcOH;
1
flow rate 2 mL/min] in 51% yield: H NMR (CDCl
3
) δ 0.84 (t,
J ) 7.0 Hz, 3 H), 1.25-1.40 (m, 6 H), 1.78-1.88 (m, 2 H), 2.06
(
q, J ) 6.9 Hz, 2 H), 2.12-2.18 (m, 2 H), 2.41 (t, J ) 7.3 Hz,
2
5
6
2
7
1
3
H), 2.83 (m, 2 H), 2.90 (m, 2 H), 3.31-3.37 (m, 4 H), 5.4 (m,
H), 5.80 (d, J ) 14.7 Hz, 1 H), 6.04 (t, J ) 10.9 Hz, 1 H),
1
3
3 3
.71 (dd, J ) 14.6, 11.2 Hz, 1 H); C NMR (CD COCD ) δ 14.8,
3.6, 24.1, 26.6, 27.2, 28.0, 30.4, 33.5, 34.3, 40.0 (2 × C), 42.59,
1.6, 125.0, 128.5, 128.7, 128.9, 129.8, 131.1, 131.3, 139.3,
75.0; EIHRMS calcd for C22
94.1994.
34 2 2
H O S 394.2000, observed
11 mg of the product 17, R
hexane), which was used as such in the next step.
To a 0 °C cooled solution of crude d -dithio acid 17 (11 mg,
0.0267 mmol) in methanol/H O (9:1, 5 mL) was added a
solution of 4-hydroxy-TEMPO (40 µg) in CH Cl (40 µL)
followed by solution of [bis(trifluoroacetoxy)iodo]-
f
) 0.55 (35% ethyl acetate in
5
-Oxo-6(E),8(Z),11(Z),14(Z)-eicosa tetr a en oic Acid (5-
Oxo-ETE) (2). To a 0 °C cooled solution of dithio acid 15 (20.8
mg, 0.0527 mmol) in methanol/H O (9:1, 25 mL) was added a
solution of 4-hydroxy-TEMPO (40 µg) in CH Cl (40 µL)
4
2
2
2
2
2
2
followed by a solution of [bis(trifluoroacetoxy)iodo]benzene (48
mg, 0.111 mmol) in methanol (0.4 mL) and the resulting
mixture stirred at 0 °C for 2.5 min. The reaction mixture was
quenched with cold water (20 mL) and extracted with ethyl
a
benzene (44 mg, 0.1 mmol) in methanol (1 mL) and the mixture
stirred at 0 °C for 2.5 min. The reaction mixture was quenched
with cold water (20 mL) and extracted with ethyl acetate (2 ×