The total synthesis of 5-oxo-12(s)-hydroxy-6(e),8(z),10(e),14(z)- eicosatetraenoic acid and its 8,9-trans-isomer and their identification in human platelets

Article abstract of DOI:10.1021/jo9813282

The first total synthesis of 5-oxo-12(S)-hydroxy-6(E),8(Z),10(E), 14(Z)- eicosatetraenoic acid (5-oxo-12-HETE) 6 and its 8-trans-isomer 7 is reported. The synthetic 5-oxo-12-HETE 6 and its 8,9-trans-isomer 7 were used to identify their formation in mixtures of platelets and neutrophils by transcellular metabolism.

Full text of DOI:10.1021/jo9813282

8976
J . Org. Chem. 1998, 63, 8976-8982
Th e Tota l Syn th esis of
5-Oxo-12(S)-h yd r oxy-6(E),8(Z),10(E),14(Z)-eicosa tetr a en oic Acid
a n d Its 8,9-tr a n s-Isom er a n d Th eir Id en tifica tion in Hu m a n
P la telets
Subhash P. Khanapure, William S. Powell,† and J oshua Rokach*
Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology,
150 W. University Boulevard., Melbourne, Florida 32901
Received J uly 8, 1998
The first total synthesis of 5-oxo-12(S)-hydroxy-6(E),8(Z),10(E), 14(Z)-eicosatetraenoic acid (5-oxo-
12-HETE) 6 and its 8-trans-isomer 7 is reported. The synthetic 5-oxo-12-HETE 6 and its 8,9-
trans-isomer 7 were used to identify their formation in mixtures of platelets and neutrophils by
transcellular metabolism.
In tr od u ction
biochemical pathways by which 6 is produced by trans-
cellular metabolism. Scheme 2 shows another possible
mechanism for the formation of the 8-trans-isomer 7.
We have recently been interested in oxoeicosanoids
because these have been shown to possess significant
biological activity, for example, 5-oxoeicosatetraenoic acid
(5-oxo-ETE),1 12-oxo-ETE.2 In addition, many of the
oxoeicosanoids, for example, 12-oxo-ETE and 12-oxoleu-
kotriene B4 (12-oxo-LTB4), have been shown to be inter-
mediates in the formation of other bioactive molecules
such as 12(R)-HETE,2 12-HETrE,2-4 and c-LTB3.5 The
strategy we have followed in every case involved the total
synthesis of the target oxoeicosanoid1,2,5-7 and its use as
a standard for the positive identification of the biological
products. The larger amount available through total
synthesis has allowed the evaluation of the biological
properties of these molecules.
The purpose of this report is to describe the total
synthesis of 5-oxo-12-hydroxyeicosatetraenoic acid (5-oxo-
12-HETE) (the abbreviation 5-oxo-12-HETE refers to the
5-oxo derivative of 5,12-diHETE) 6 and its trans-isomer
7. These synthetic materials have been used to identify
their formation by mixtures of platelets and neutrophils
as a result of a transcellular metabolism between neu-
trophils and platelets. Neutrophils that contain 5-LO
make 5-HPETE, 5-oxo-ETE, LTA4, and LTB4. Platelets
do not contain 5-lipoxygenase (5-LO), but do contain 12-
LO, the major product of which is 12-hydroperoxyeico-
satetraenoic acid (12-HPETE). Coincubation of platelets
and neutrophils results in the formation of two new oxo
metabolites, 6 and 7, in addition to the expected and
known metabolites. Scheme 1 shows the two possible
Resu lts a n d Discu ssion
The oxoeicosanoids of interest have in common a
conjugated dienone or trienone functional group, which
renders them unstable and difficult to handle. Any cis-
double bonds present in the conjugated system are highly
prone to isomerization. We have found that they can be
stored for prolonged periods in solution at -20 °C. The
purer the oxo-eicosanoid, the better and longer its chance
for survival. We have been able to keep several of these
oxoeicosanoids in acetonitrile or ethyl acetate for more
than a year without noticeable deterioration. Except for
a cis-trans isomerization, we are not certain of the
decomposition products. We only observed a decline in
concentration as judged by UV absorbance in 12-oxo-
LTB4. This indicates that the decomposition products
have lost their conjugated polyenone chromophore. High-
performance liquid chromatography (HPLC) shows no
other significant product than the oxo-eicosanoid itself.
This is reminiscent of the peptido leukotrienes LTC4,
LTD4, and LTE4, which can be kept intact when stored
under carefully defined conditions, but will deteriorate
rapidly, as judged by UV absorbance, with a drop in
concentration, when in use, for example, in a biological
experiment lasting a day or more.
For this reason we have, as illustrated in the total
synthesis of 5-oxo-12-HETE 6 described here, designed
a synthesis in which the carbonyl function is protected
through the whole synthesis and deblocking is the last
step in the synthesis. There is a price to pay for this
strategy. In the deprotection of the carbonyl group at
the end of the synthesis, it is impossible to prevent a
partial cis-trans isomerization; however, we have been
able to keep it to a minimum. As it turned out, however,
this disadvantage has been turned to a benefit. In all
the cases we studied, for example, 5-oxo-ETE,1 12-oxo-
† Meakins-Christie Laboratories, McGill University, 3626 St-Urbain
St., Montreal, Quebec H2X 2P2, Canada.
(1) Khanapure, S. P.; Shi, X. X.; Powell, W. S.; Rokach, J . J . Org.
Chem. 1998, 63, 337-342.
(2) Wang, S. S.; Rokach, J .; Powell, W. S.; Dekle, C.; Feinmark, S.
J . Tetrahedron Lett. 1994, 35, 4051-4054.
(3) Schwartzman, M. L.; Balazy, M.; Masferrer, J .; Abraham, N. G.;
McGiff, J . C.; Murphy, R. C. Proc Natl. Acad. Sci. USA 1987, 84, 8125-
8129.
(4) Schwartzman, M. L.; Lavrovsky, Y.; Stoltz, R. A.; Conners, M.
S.; Falck, J . R.; Chauhan, K.; Abraham, N. G. J . Biol. Chem. 1994,
269, 24321-24327.
(5) Khanapure, S. P.; Manna, S.; Rokach, J .; Murphy, R. C.;
Wheelan, P.; Powell, W. S. J . Org. Chem. 1995, 60, 1806-1813.
(6) Wang, S. S.; Shi, X.-X.; Powell, W. S.; Tieman, T.; Feinmark, S.
J .; Rokach, J . Tetrahedron Lett. 1994, 36, 513-516.
(7) Khanapure, S. P.; Wang, S. S.; Powell, W. S.; Rokach, J . J . Org.
Chem. 1997, 62, 325-330.
ETE,2 12-oxo-LTB4 , and the present case, the isolation
5
of the cis-oxo-eicosanoid from biological sources invari-
ably is accompanied by substantial amounts of the trans-
isomers, and we needed the synthetic authentic material
to identify them and study their biological properties.
10.1021/jo9813282 CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/10/1998

Synthesis of 5-Oxo-12-HETE and Its 8,9-trans-Isomer
J . Org. Chem., Vol. 63, No. 24, 1998 8977
Sch em e 1
Sch em e 2
thereof in vivo, in 5-oxo-12-HETE 6 and 8-trans-5-oxo-
12-HETE 7, remains to be done. Cis-trans isomerization
of oxoeicosanoids is likely to affect the biological activity
of these compounds. For example, 8-trans-5-oxo-ETE is
less potent than 5-oxo-ETE in activating neutrophils.8
The strategy we used to construct the 5-oxo-12-HETE
is shown in Scheme 3. We elected to use two synthons
12 and 13 and construct the 8,9-cis double bond last.
Synthon 12 was prepared as shown in Scheme 4 from
the dithioacetonide derivative 15, itself prepared from
D-arabinose in four steps. Derivative 15, which we used
extensively in the past,9,10 is the equivalent of a 1,4-
dialdehyde 14 in which the two aldehyde poles can be
used separately and independently. As shown in Scheme
4, we reworked the chemistry completely and to our
advantage compared with that used in the synthesis of
12-epi-LTB4.9 The use of periodic acid, as we described
recently,11 saves steps and, more importantly, simplifies
Another related thought worth discussing here is the
following. Do these isomerizations and, in the present
case, the formation of the all-trans compound 7, occur in
vivo? We do not know whether any enzyme is performing
such isomerization in vivo, and in view of the very facile
acid-catalyzed isomerization, we cannot be sure whether
the isomerization, if it is happening in vivo, is enzymatic
or chemical. In addition, it is possible that some of the
all-trans-compound formed is due to an artifact of the
isolation procedure. In one instance, however, in 12-oxo-
ETE and its 8,9-trans-isomer isolated from aplysia
nervous tissue, we have shown by incubating the two
synthetic isomers with aplysia neural homogenates that
the formation of the trans-compound is not an artifact of
the experiment.2 These experiments not only suggest
that the trans-isomer in this case is formed in vivo, but
may be suggestive of the involvement of an enzyme.
The ease with which the chemical cis-trans isomer-
ization occurs varies somewhat with the different struc-
tures, and the biochemical precedent described above for
8,9-trans-12-oxo-ETE may not be a general phenomenon.
Independent verification of this isomerization, or the lack
(8) Powell, W. S.; MacLeod, R. J .; Gravel, S.; Gravelle, F.; Bhakar,
A. J . Immunol. 1996, 156, 336-342.
(9) Zamboni, R.; Rokach, J . Tetrahedron Lett. 1982, 23, 2631-2634.
(10) Leblanc, Y.; Fitzsimmons, B. J .; Adams, J .; Perez, F.; Rokach,
J . J . Org. Chem. 1986, 51, 789-793.
(11) Shi, X.-X.; Khanapure, S. P.; Rokach, J . Tetrahedron Lett. 1996,
37, 4331-4334.

8978 J . Org. Chem., Vol. 63, No. 24, 1998
Khanapure et al.
Sch em e 3
Sch em e 4
the workup of the reaction. Typically the substrate is
dissolved in tetrahydrofuran (THF)/ether; the periodic
acid, dissolved in THF, is added to the reaction, stirred
for a short time, and filtered through Celite. The solvents
are evaporated, and in many instances, the products are
pure and can be used as such for the next step. As can
be seen, we used the periodic acid twice in the prepara-
tion of synthon 12 to generate aldehydes 17 and 20. We
previously have performed transformation of 19 to 20 in
two steps.9 The first is the aqueous acid hydrolysis of
the acetonide, and the second, the lead tetraacetate
cleavage of the diol followed by chromatographic purifica-
tion. Although the yields of the two steps were good, the
one-step procedure described here is clearly superior
because it saves time and avoids unnecessary chromato-
graphic purifications.
Synthon 131 was prepared from aldehyde 24 as shown.
We previously used the stabilized Wittig reagent 25.9,12-15
For example, in our synthesis of LTA4 we used either 1
or 2 equiv of 25 in benzene reflux to prepare a monoene
aldehyde or diene aldehyde. Although the use of 2 equiv
was convenient to prepare diene aldehyde, contamination
with some triene aldehyde and monoene aldehyde oc-
curred and the product was difficult to purify. One
equivalent, however, can be used conveniently and ef-
ficiently for the formation of the monoadduct. In some
cases yields were very good,12,15 in others the yields were
moderate,9,14 but the starting material could be recovered
and recycled. Because aldehyde 24 is more reactive than
the R,â-unsaturated aldehyde 13, we anticipated that the
Wittig reaction of 24 and 25 could be performed selec-
tively to give 13 with minimum diene aldehyde contami-
nation.
27, do not separate at this stage, but are easily separated
by column chromatography after fluoride deprotection of
the silyl group to give pure 28 and 29. After the ester
hydrolysis of 28, the thiolane deprotection of the cis-
product 30 is effected as shown in Scheme 5, and the pure
cis- and trans-products 6 and 7 are obtained in 41% and
5% yield, respectively. Separately, the trans-dithiolate
derivative 31 was converted to 7 in 71% yield. The
structural proof of the two end products 6 and 7 is based
on proton NMR and mass spectral data. Electrospray
MS analysis revealed an M-1 ion at m/z 333 for both
5-oxo-12-HETE 6 and 8-trans-5-oxo-12-HETE 7.
The objective of the synthesis described herein was to
determine whether platelets can synthesize and/or me-
tabolize 5-oxoeicosanoids. Using synthetic 6 and 7 we
have shown that these compounds are metabolites of
endogenous arachidonic acid in mixtures of platelets and
neutrophils.16 5-Oxo-12-HETE 6 is formed by the com-
bined actions of 5-lipoxygenase (neutrophils), 12-lipoxy-
genase (platelets), and 5-hydroxyeicosanoid dehydro-
genase (platelets and neutrophils) as shown in Scheme
1. 8-trans-5-Oxo-12-HETE 7 could be formed either by
isomerization of the 8-cis double bond of 6 as described
earlier or by a nonenzymatic degradation of LTA4.
Examination of Scheme 2 reveals that there is a
different biochemical pathway potentially leading to
8-trans-5-oxo-12-HETE (5-oxo-12-epi-6-trans-LTB4) 7. 5,12-
The condensation of synthons 12 and 13 afforded the
two Wittig products 26 and 27 as shown in Scheme 5. It
is evident that there is an unusually high amount of the
trans-product 27. This is not unlike an observation we
9,14
made in the synthesis of LTB4
and is the subject of a
separate investigation. The two triene products, 26 and
(12) Rokach, J .; Young, R. N.; Kakushima, M.; Lau, C.-K.; Seguin,
R.; Frenette, R.; Guindon, Y. Tetrahedron Lett. 1981, 22, 979-982.
(13) Rokach, J .; Zamboni, R.; Lau, C.-K.; Guindon, Y. Tetrahedron
Lett. 1981, 22, 2759-2762.
(14) Guindon, Y.; Zamboni, R.; Lau, C.-K.; Rokach, J . Tetrahedron
Lett. 1982, 23, 739-742.
(15) Zamboni, R.; Rokach, J . Tetrahedron Lett. 1983, 24, 999-1002.
(16) Powell, W. S.; Gravel, S.; Khanapure, S. P.; Rokach, J . Blood
1998, submitted for publication.

Synthesis of 5-Oxo-12-HETE and Its 8,9-trans-Isomer
J . Org. Chem., Vol. 63, No. 24, 1998 8979
Sch em e 5
Sch em e 6
Somewhat to our surprise, 5-oxo-12-HETE and its
8-trans-isomer have virtually no biological activity on
neutrophils. In contrast, another 5-oxo-HETE, namely
5-oxo-15-HETE, displays substantial activity on both
neutrophils17 and eosinophils.18 5-Oxo-12-HETE was
more than 10,000 times less active than 5-oxo-ETE in
mobilizing calcium in neutrophils,16 and preliminary
experiments indicate that, in contrast to 5-oxo-ETE and
5-oxo-15-HETE, it does not induce neutrophil migration
at concentrations as high as 10 µM.19 Comparison of the
structures of 5-oxo-ETE (5), 5-oxo-15-HETE (32), and
5-oxo-12-HETE (6) (Scheme 6) suggests that a saturated
carbon at position 10 is essential for biological activity
mediated by the activation of the 5-oxo-ETE receptor. The
region from carbon 11 and beyond of 5-oxo-15-HETE is
considerably different from 5-oxo-ETE, yet a substantial
degree of biological activity is retained, presumably
because of the presence of the methylene group at carbon
10. In contrast, the conjugated triene in 6, which would
be flat in conformation, confers rigidity at carbons 10 and
11, resulting in substantial distortion from the flexibility
at carbon 10 of 5 and 32, which is presumably required
for interaction with the putative 5-oxo-ETE receptor.
Exp er im en ta l Section
Rea gen ts a n d Meth od s. Unless stated otherwise, all
reagents and chemicals were obtained from commercial sources
and used without further purification. 1H NMR spectra were
recorded on a 360-MHz spectrometer with tetramethylsilane
as an internal standard J values are given in Hertz. All
reactions were carried out under an inert (nitrogen or argon)
atmosphere with dry, freshly distilled solvents under anhy-
drous conditions unless otherwise noted. Yields refer to
chromatographically and spectroscopically (1H NMR) homo-
geneous materials.
Me t h yl-5,5-(d im e t h yle n e d it h io)-6(E )-8-oxo-7-oct e n -
oa te (13). A solution of aldehyde 24 (2.8 g, 11.95 mmol) and
triphenylphosphoraniledene acetaldehyde 25 (3.7 g) in benzene
(30 mL) was refluxed for 18 h. (The amount of the triphenyl-
phosphoraniledene acetaldehyde was added in 3 stages: 1.3
g at the start of the reaction; 1.2 g after 2 h, and a further 1.2
g after another 2 h.) The reaction mixture was cooled to room
temperature, filtered through Celite, and washed with ethyl
acetate; the combined filtrate was concentrated under reduced
pressure to afford the crude product, which was purified by
flash column chromatography using 20% ethyl acetate in
hexane. The less polar fractions gave 706 mg of starting
material 24, and from the more polar fractions pure product
13 (1.75 g, 56%) was obtained (yield based on consumed
starting material 75%). 1H NMR (CDCl3) δ 1.80 (m, 2 H), 2.14
Dihydroxy products 9 and 10 are found in cells as a result
of LTA4 hydrolysis and often have been used qualitatively
and quantitatively as an index of LTA4 production in
cells, because LTA4 is very unstable and cannot be
measured intact in vivo. On oxidation by a dehydrogen-
ase, these dihydroxy derivatives 9 and 10 will yield 5-oxo
compounds 7 and 11. Derivative 7 is identical with the
trans-product isolated in vivo and shown in Scheme 1.
In addition, 11 is enantiomeric with 7 and can only be
assessed and identified by special studies. Hence it is
not impossible that 5-oxo products 7 and 11, derived from
LTA4 hydrolysis as shown in Scheme 2, contribute to the
amount of 7 derived by the biosynthetic sequence shown
in Scheme 1. We have not yet performed the special
studies necessary to completely clarify the biochemical
origin of the trans-oxo derivative 7.
(17) Powell, W. S.; Gravel, S.; MacLeod, R. J .; Mills, E.; Hashefi,
M. J . Biol. Chem. 1993, 268, 9280-9286.
(18) Schwenk, U.; Morita, E.; Engel, R.; Schroder, J . M. J . Biol.
Chem. 1992, 267, 12482-12488.
(19) Powell, W. S.; Ahmed, S.; Gravel, S.; Khanapure, S. P.; Rokach,
J . unpublished results.

8980 J . Org. Chem., Vol. 63, No. 24, 1998
Khanapure et al.
(m, 2 H), 2.32 (t, J ) 7.2 Hz, 2 H), 3.21 (m, 2 H), 3.32 (m, 2 H),
3.61 (s, 3 H), 6.25 (dd, J ) 15.2 and 7.7 Hz, 1 H), 6.78 (d, J )
15.2, 1 H), 9.58 (d, J ) 7.8, 1 H): 13C NMR (CDCl3) δ 22.3,
33.2, 39.0 (2 × C), 39.5, 51.2, 68.5, 128.5, 158.9, 172.8, 193.1;
HREIMS calcd (C11H16O3S2, M+) 260.0541, obsd 260.0545.
2-Deoxy-3-O-(ter t-b u t yld ip h en ylsilyl)-1,2-b is-O-(iso-
p r op ylid en e)-D-er yth r o-p en tose (17). To a 0 °C cooled
solution of dithioacetal 15 (10.4 g, 20 mmol) in anhydrous ethyl
ether (200 mL) and THF (80 mL) was added a solution of
periodic acid 16 (5.472 g, 24 mmol) in dry THF (20 mL) for 3
min under argon. The ice bath was removed, and the reaction
mixture was stirred at room temperature for 5 min. A white
solid was precipitated. Potassium carbonate (4 g) was added
and stirred for 1 min. The reaction mixture was then diluted
with ether (100 mL), filtered through Celite/florisil into a flask
containing saturated aqueous Na2SO3 solution (60 mL), and
washed with ether (50 mL). The combined filtrate was washed
with aqueous Na2SO3 (2 × 60 mL), water (4 × 200 mL), and
brine (100 mL); dried over anhydrous Na2SO4; and filtered and
concentrated under reduced pressure to afford the crude
product (8.4 g) which was purified by flash column chroma-
tography with hexane/ethyl acetate (8:2) to afford the pure
aldehyde 17, 7.0 g in 84% yield. 1H NMR (CDCl3) δ 1.05 (s, 9
H), 1.28 (s, 3 H), 1.29 (s, 3 H), 2.58 (m, 2 H), 3.63 (m, 1 H),
3.95 (m, 1 H), 4.13 (m, 2 H), 7.44 (m, 6 H), 7.70 (m, 4 H), 9.6
(t, J ) 2.3 Hz, 1 H). 13C NMR (CDCl3) δ 19.3, 25.1, 26.2, 26.9,
48.0, 67.2, 70.1, 76.7, 78.6, 109.5, 127.7 (2 × C), 127.8 (2 × C),
130.0, 130.1, 133.0, 135.5, 135.8 (2 × C), 135.9, (2 × C).
3(S)-[(ter t-Bu t yld ip h en ylsilyl)oxy]-1,2-b is-O-(isop r o-
p ylid en e)-5(Z)-u n d ecen e-1,2(R)-d iol (19). To a stirred
suspension of the hexyltriphenylphosphonium bromide 18
(23.485 g, 55 mmol) in THF (550 mL) was added dropwise
sodium hexamethyldisilazide (1 M, 51 mL, 51 mmol) under
argon. The reaction mixture turned orange, after being stirred
for 30 min at room temperature. It was then cooled to -78
°C, and anhydrous hydroxymethylphosphoramide (HMPA)
(115 mL) was added. The reaction mixture was stirred for 2
min, and then aldehyde 17 (7 g, 16.99 mmol) in THF (215 mL)
was added dropwise at -78 °C to the resulting red solution.
The reaction mixture was stirred for 2 h at -78 °C, and then
allowed to warm slowly to 0 °C for 2 h. It was then quenched
by the addition of aqueous saturated ammonium chloride
solution (150 mL). The THF layer was separated, the solvent
evaporated at reduced pressure, and the residue extracted with
diethyl ether (3 × 150 mL). The combined extracts were
washed with cold water (3 × 25 mL), dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure to
afford the crude product, which first was purified quickly by
flash column chromatography using 5% ethyl acetate in hexane
and then purified further by column chromatography using
(d, J ) 1.6 Hz, 1 H). 13C NMR (CDCl3) δ 14.0, 14.5, 19.3, 22.5,
26.9, 27.3, 29.1, 31.1, 31.5,77.8, 122.6, 127.7 (2 × C), 127.8 (2
× C), 129.9 (2 × C), 130.0 (2 × C), 133.0, 133.1, 133.4, 135.8
(2 × C), 203.3.
E t h yl-4(S )-[(t er t -b u t yld ip h e n ylsilyl)oxy]-2(E ),6(Z)-
d od eca d ien oa te (22). Sodium hydride (60% dispersion) (223
mg, 5.58 mmol) and dry hexane (3 mL) were placed in a 50-
mL round-bottom flask under argon. After the mixture was
stirred for 3 min, hexane was removed, and dry benzene (20
mL) was added. To this stirred suspension was added ethyl
phosphonoacetate 21 (1.3 mL, 6.53 mmol), stirring continued
at room temperature for 1 h, and then aldehyde 20 (810 mg,
1.98 mmol) in benzene (10 mL) was added at room tempera-
ture. The reaction mixture was stirred at room temperature
for 3 h and then quenched with saturated aqueous ammonium
chloride solution (20 mL), the benzene layer was separated,
and the aqueous layer was extracted with ether (2 × 50 mL).
The combined benzene and ether extracts were dried over
anhydrous Na2SO4 and filtered, and the solvent was evapo-
rated at reduced pressure to give the crude product which was
purified by column chromatography over silica gel using
hexane/ethyl acetate (19:1) to yield the pure product 22, 908
mg as a colorless oil in 96% yield. 1H NMR (CDCl3) δ 0.87 (t,
J ) 7.2 Hz, 3 H), 1.08 (s, 9 H), 1.2-1.3 (m, 6 H), 1.28 (t, J )
7.1 Hz, 3 H), 1.77 (m, 2 H), 2.1-2.3 (m, 2 H), 4.17 (q, J ) 7.1
Hz, 2 H), 4.34 (m, 1 H), 5.21 (m, 1 H), 5.39 (m, 1 H), 5.94 (d,
J ) 15.6 Hz, 1 H), 7.72 (dd, J ) 15.5, 4.9 Hz, 1 H), 7.3-7.4
(m, 6 H), 7.6-7.7 (m, 4 H). 13C NMR (CDCl3) δ 14.3, 14.5,
19.5, 22.7, 27.2, 27.4, 29.3, 31.6, 35.4, 60.4, 72.6, 120.5, 123.5
127.8 (4 × C), 129.9 (2 × C), 133.2, 133.6, 134.1, 136.0 (4 ×
C), 150.0, 166.7.
4(S)-[(ter t-Bu tyld ip h en ylsilyl)oxy]-2(E),6(Z)-d od eca d i-
en yl-1-ol (23). To a -60 °C cooled solution of silyl ester 22
(520 mg, 1.05 mmol) in anhydrous dichloromethane (12 mL)
was slowly added dropwise a 1 M solution (in dichloromethane)
of DIBAL-H (2.5 mL, 2.5 mmol) under argon. The reaction
mixture was stirred for 15 min at -60 °C and then allowed to
warm to -20 °C for 1 h. The reaction mixture was then
quenched with water, neutralized with 10% HCl, and extracted
with dichloromethane (2 × 25 mL). The combined extracts
were washed with water (3 × 25 mL) and brine (1 × 25 mL),
dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure to afford the crude product which was
purified by flash column chromatography with hexane/ethyl
acetate (19:1) to give the pure alcohol 23 (438 mg, 96%). 1H
NMR (CDCl3) δ 0.85 (t, J ) 7.3 Hz, 3 H), 1.04 (s, 9 H), 1.2-
1.3 (m, 6 H), 1.85 (m, 2 H), 2.18 (m, 1 H), 2.27 (m, 1 H), 3.90
(d, J ) 5.1 Hz, 2 H), 4.19 (s, 1 H), 5.29 (m, 1 H), 5.38 (m, 1 H),
5.45 (m, 1 H), 5.55 (dd, J ) 14.5, 7.0 Hz, 1 H), 7.37 (m, 6 H),
7.65 (m, 4 H). 13C NMR (CDCl3) δ 14.3, 19.5, 22.8, 27.2, 27.5,
29.4, 31.7, 36.1, 63.2, 73.8, 124.8, 127.6 (2 × C), 127.7 (2 × C),
129.5, 129.7 (2 × C), 129.8 (2 × C), 132.3, 134.3, 134.4, 134.7,
136.2 (2 × C), 136.3 (2 × C).
1
1% ether in hexane to give pure product 19 (6.75 g, 83%). H
NMR (CDCl3) δ 0.90 (t, J ) 7.1 Hz, 3 H), 1.11 (s, 9 H), 1.2-
1.3 (m, 6 H), 1.35 (m, 3 H), 1.38 (m, 3 H), 1.85 (m, 2 H), 2.05-
2.15 (m, 1 H), 2.23-2.35 (m, 1 H), 3.85 (t, J ) 7.4 Hz, 1 H),
3.96 (m, 1 H), 4.12 (q, J ) 5.5 Hz, 1 H), 5.39 (m, 2 H), 7.4 (m,
6 H), 7.75 (m, 4 H). 13C NMR (CDCl3) δ 14.0, 19.4, 22.5, 25.3,
26.5, 27.0, 27.2, 29.1, 31.5, 32.1, 66.0, 73.2, 77.83, 108.7, 124.1,
127.4 (2 × C), 127.5 (2 × C), 129.6, 129.7, 132.4, 133.7, 134.1,
136.0 (2 × C), 136.1 (2 × C).
4(S)-[(ter t-Bu tyldiph en ylsilyl)oxy]-2(E),6(Z)-dodecadien -
1-yl-tr is(3-m eth oxyp h en yl)p h osp h on iu m br om id e (12).
To a cooled (0-5 °C), stirred solution of alcohol 23 (210 mg,
0.492 mmol) and 1,2-bis(diphenylphosphino)ethane (DIPHOS)
(215 mg, 0.541 mmol) in dry CH2Cl2 (8 mL) carbon tetrabro-
mide (244 mg, 0.738 mmol) in dry CH2Cl2 (8 mL) was slowly
added under argon. The reaction mixture was stirred for 30
min at 0 °C and then diluted with CH2Cl2 (8 mL), and the
resulting solution of the bromide was filtered through a small
pad of silica/Celite and washed with CH2Cl2 (20 mL). The
combined filtrate was dried over anhydrous Na2SO4 and
filtered, and the solution was used as such in the next step.
To the above-mentioned solution of labile bromide was added
tris(3-methoxy)phenylphosphine (909 mg, 2.5 mmol), and the
mixture was stirred at room temperature for 18 h under argon
atmosphere. The solvent was evaporated in vacuo. The
phosphonium salt was purified by flash column chromatogra-
phy with methylene chloride/methanol (19:1) to afford the
phosphonium salt 12 (248 mg, 65%) as a colorless fluffy solid.
1H NMR (CDCl3) δ 0.81 (t, J ) 7.2 Hz, 3 H), 0.83 (s, 9 H),
1.1-1.2 (m, 6 H), 1.8-2.0 (m, 4 H), 3.86 (m, 9 H), 4.19 (s, 1
2(S)-[(ter t-Bu tyld ip h en ylsilyl)oxy]-4(Z)-d ecen a l (20).
To a solution of acetonide 19 (960 mg, 1.9 mmol) in anhydrous
ethyl ether (20 mL), a solution of periodic acid (910 mg, 4
mmol) in dry THF (5 mL) was slowly added dropwise under
argon. The reaction mixture was stirred overnight at room
temperature. A white solid was precipitated. The reaction
mixture was diluted with ether (50 mL), filtered through
Celite, and washed with ether (50 mL). The combined filtrate
was washed with aqueous Na2SO3 (40 mL), water (2 × 40 mL),
dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure to afford the crude product which was
purified by flash column chromatography with hexane/ethyl
acetate (19:1) to afford the pure aldehyde 20 (810 mg, 98%).
1H NMR (CDCl3) δ 0.85 (t, J ) 7.1 Hz, 3 H), 1.09 (s, 9 H), 1.26
(m, 6 H), 1.87 (m, 2 H), 2.3 (m, 1 H), 2.4 (m, 1 H), 4.03 (s, 1 H),
5.35 (m, 1 H), 5.5 (m, 1 H), 7.38 (m, 6 H), 7.65 (m, 4 H), 9.53

Synthesis of 5-Oxo-12-HETE and Its 8,9-trans-Isomer
J . Org. Chem., Vol. 63, No. 24, 1998 8981
H), 4.71 (m, 1 H), 4.94 (m, 2 H), 5.15 (m, 1 H), 5.51 (m, 1 H),
6.24 (m, 1 H), 7.3-7.7 (m, 22 H).
14.7, 10.2 Hz, 1 H). 13C NMR (CD3COCD3) δ 14.8, 23.7, 24.4,
28.5, 30.5, 32.7, 34.5, 36.9, 40.3, (2 × C), 42.9, 71.8, 72.7, 125.4,
125.9, 126.9, 129.2, 130.9, 132.9, 139.9, 140.1, 174.9.
Wittig Con d en sa tion of 12 a n d 13: Meth yl-5,5-(d i-
m eth ylen edith io)-12(S)-[(ter t-bu tyldiph en ylsilyl)oxy]-6(E),
8(Z),10(E),14(Z)-icosa t et r a en oa t e (26) a n d Met h yl-5,5-
(d im eth ylen ed ith io)-12(S)-[(ter t-bu tyld ip h en ylsilyl)oxy]-
6(E),8(E),10(E),14(Z)-icosa t et r a en oa t e (27). To a cooled
(-98 °C), stirred solution of the phosphonium salt 12 (248 mg,
0.291 mmol) in THF (4 mL) sodium hexamethyldisilazide (1
M, 233 µL, 0.233 mmol) was added dropwise under argon.
After stirring for 15 min at -98 °C, HMPA (1.2 mL) was added,
the reaction mixture was stirred for 2 min, and then aldehyde
13 (70 mg, 0.267 mmol) in THF (2 mL) was added dropwise
at -98 °C to the resulting red solution. The reaction mixture
was stirred for 15 min at -98 °C, and then allowed to warm
slowly to 0 °C for 1 h. It was then quenched by the addition
of aqueous saturated ammonium chloride solution (1 mL) and
extracted with diethyl ether (3 × 50 mL). The combined
extracts were washed with cold water (3 × 25 mL), dried over
anhydrous Na2SO4, filtered, and concentrated under reduced
pressure to afford the crude product which was purified by
flash column chromatography using 5% ethyl acetate in hexane
to give a mixture of cis- and trans-isomers 26 and 27 (152 mg,
71%). This mixture was difficult to separate as such, so it was
desilylated and the individual isomers were separated.
Met h yl-5,5-(d im et h ylen ed it h io)-12(S)-h yd r oxy-6(E),
8(Z),10(E),14(Z)-eicosa -tetr a en oa te (28) a n d Meth yl-
5,5-(d im et h ylen ed it h io)-12(S)-h yd r oxy-6(E),8(E),10(E),
14(Z)-eicosa tetr a en oa te (29). A solution of the preceding
mixture of 26 and 27 (37 mg, 0.05 mmol) in THF (4.5 mL)
was treated with tetra-n-butylammonium fluoride (180 µL of
1 M solution in THF, 0.18 mmol), and the mixture was stirred
at room temperature overnight. Then the reaction mixture
was diluted with saturated aqueous ammonium chloride
solution (2 mL) and extracted with ether (3 × 20 mL). The
combined organic layers were washed with water (3 × 10 mL)
and brine (10 mL), dried (Na2SO4), and concentrated in vacuo.
The residue was purified by flash chromatography (elution
with ethyl acetate/hexane 1:9) to give the less polar product
28 (9 mg, 43%) as an oil. 1H NMR (C6D6) δ 0.93 (t, J ) 7.0
Hz, 3 H), 1.31 (m, 6 H), 1.9 (m, 2 H), 2.04 (m, 4 H), 2.13 (m, 2
H), 2.35 (m, 2 H), 2.78 (s, 4 H), 3.36 (s, 3 H), 4.14 (m, 1 H,
C12-H), 5.55 (m, 2 H, C14 and C15-H), 5.76 (dd, J ) 15.1, 5.8
Hz, 1 H, C11-H), 5.98 (d, J ) 14.7 Hz, 1 H, C6-H), 6.09 (m, 2
H, C8 and C9-H), 7.10 (dd, J ) 14.6, 10.1 Hz, 1 H, C10-H),
7.28 (dd, J ) 14.8, 10.2 Hz, 1 H, C7-H). 13C NMR (C6D6) δ
14.7, 23.3, 23.6, 28.1, 30.1, 32.2, 34.1, 36.3, 39.6, (2 × C), 42.3,
51.5, 71.4, 72.3, 125.0, 125.7, 125.8, 129.2, 130.2, 133.4, 138.4,
139.7, 173.5. Of the more polar trans-isomer 29, 5.2 mg was
obtained in 24% yield. 1H NMR (C6D6) δ 0.94 (t, J ) 7.0 Hz,
3 H), 1.44 (m, 8 H), 1.94 (m, 2 H), 2.10 (m, 6 H), 2.35 (m, 2 H),
2.80 (s, 4 H), 3.35 (s, 3 H), 5.57 (m, 2 H), 5.68 (dd, J ) 14.5,
6.0 Hz, 2 H), 5.91 (d, J ) 14.7 Hz, 1 H), 6.24 (m, 2 H), 6.29 (m,
1 H), 6.68 (m, 1 H). 13C NMR (C6D6) δ 14.6, 23.3, 23.8, 28.1,
30.1, 30.5, 32.2, 34.2, 36.3, 39.5 (2 × C), 42.4, 51.3, 71.5, 72.4,
125.5, 129.9, 130.6, 132.4, 133.3, 133.5, 137.2, 139.0, 173.2.
5,5-(D im e t h y le n e d it h io )-12(S )-h y d r o x y -6(E ),8(Z),
10(E),14(Z)-eicosa tetr a en oic Acid (30). To a solution of the
dithio compound 28 (29 mg, 0.0683 mmol) in THF (6 mL) and
1 M LiOH (1.6 mL) was added a solution of 4-hydroxy-2,2,6,6-
tetramethylpiperidinyloxy, free radical (100 µg) in methanol
(50 µL), and the solution was stirred at room temperature for
18 h. The solvent THF was removed by a stream of argon,
then water (4 mL) and 5% KH2PO4 (80 mL) were added and
extracted with ethyl acetate (2 × 40 mL). The combined ethyl
acetate extracts were washed with cold water (4 × 20 mL) and
brine (1 × 10 mL), dried over anhydrous Na2SO4 and filtered;
the solvent evaporated under reduced pressure to afford 28
mg of the dithio acid 30 in nearly quantitative yield, which
was pure enough to use as such in the next step. 1H NMR
(CD3COCD3) δ 0.88 (t, J ) 6.8 Hz, 3 H), 1.34.40 (m, 8 H), 1.77
(m, 2 H), 2.16 (m, 2 H), 2.29 (m, 2 H), 2.36 (t, J ) 7.3 Hz, 2 H),
3.32 (m, 4 H), 4.21 (dd, J ) 12.2, 6.0 Hz 1 H), 5.46 (m, 2 H),
5.81 (dd, J ) 15.1, 6.0 Hz, 1 H), 5.90 (d, J ) 14.7 Hz, 1 H),
6.04 (m, 2 H), 6.78 (dd, J ) 14.8, 10.2 Hz, 1 H), 6.9 (dd, J )
5,5-(D im e t h y le n e d it h io )-12(S )-h y d r o x y -6(E ),8(E ),
10(E),14(Z)-eicosa tetr a en oic Acid (31). To a solution of the
dithio compound 29 (12.5 mg, 0.0295 mmol) in THF (3 mL)
and 1 M LiOH (0.8 mL) was added a solution of 4-hydroxy-
TEMPO (50 µg) in methanol (50 µL), and the reaction mixture
was stirred at room temperature for 18 h. The solvent THF
was removed by a stream of argon, then water (2 mL) and 5%
KH2PO4 (40 mL) were added and extracted with ethyl acetate
(3 × 25 mL). The combined ethyl acetate extracts were
washed with cold water (4 × 20 mL) and brine (1 × 10 mL),
dried over anhydrous Na2SO4, and filtered; the solvent was
evaporated under reduced pressure to afford 12 mg of the
dithio acid 31 in nearly quantitative yield, which was pure
enough to use as such in the next step. 1H NMR (CD3COCD3)
δ 0.88 (t, J ) 6.9 Hz, 3 H), 1.29 (m, 8 H), 1.75 (m, 2 H), 2.14
(m, 2 H), 2.28 (m, 2 H), 2.35 (t, J ) 7.4 Hz, 2 H), 3.30 (m, 4 H),
3.91 (br s, 1 H, OH), 4.15 (m, 1 H), 5.43 (m, 2 H), 5.77 (m, 1
H), 5.88 (d, J ) 14.6 Hz, 1 H), 6.28 (m, 3 H), 6.44 (m, 1 H). 13
C
NMR (CD3COCD3) δ 14.9, 23.8, 24.4, 28.6, 30.6, 32.8, 34.6,
37.0, 40.2 (2 × C), 42.9, 71.8, 72.8, 127.0, 130.4, 130.7, 132.6,
132.9, 134.3, 139.1, 139.3, 174.8.
5-Oxo-12(S)-h yd r oxy-6(E),8(Z),10(E),14(Z)-eicosa t et -
r a en oic Acid (5-Oxo-12-HETE) 6. To a solution of dithio
acid 30 (14 mg, 0.0341 mmol) in methanol/H2O (9:1, 5 mL)
was added a solution of 4-hydroxy-TEMPO (100 µg) in metha-
nol (50 µL) followed by [bis(trifluoroacetoxy)iodo]benzene (22
mg, 0.051 mmol) under argon, and the reaction mixture was
stirred at room temperature for 2 min. The reaction mixture
was quenched with cold water (45 mL) and extracted with
ethyl acetate (2 × 25 mL). The combined ethyl acetate extracts
were washed with cold water (8 × 25 mL) and brine (1 × 25
mL), dried over anhydrous Na2SO4, and filtered. 4-Hydroxy-
TEMPO (50 µg) in ethyl acetate (50 µL) was added, and the
product in ethyl acetate was kept at -20 °C. Analytical
separation on Waters Nova-pak C-18, 3.9 × 150 mm column,
using MeOH/H2O/AcOH (70:30:0.1%) as the mobile phase and
a flow rate of 0.5 mL/min, showed that 8,9-cis- and 8,9-trans-
isomers 6 and 7 were obtained in the ratio of 87:13, tr 8,9-cis
24.43 min, tr 8,9-trans 18.62 min. The individual isomers were
separated by reversed-phase HPLC (Sperisorb S10W, C-18, 10
× 250 mm column) using MeOH/H2O/AcOH (70:30:0.1%) as
the mobile phase and a flow rate of 6 mL/min to give pure cis-
and trans-isomers. To the eluant (180 mL) containing the pure
cis-isomer 6 was added triethylamine (600 µL) to neutralize
the acetic acid. The mixture was stirred at room temperature
for 1 min and then methanol was evaporated under high
vacuum to concentrate the solution to approximately 60 mL.
A 5% aqueous solution of KH2PO4 (80 mL) was added,
extracted with ethyl acetate (2 × 40 mL), and washed with
water (4 × 25 mL). The pure product (4.657 mg) was obtained
in 40.5% yield. 1H NMR (CD3COCD3) δ 0.866 (t, J ) 6.8 Hz,
3 H), 1.29 (m, 8 H), 1.88 (t, J ) 7.4 Hz, 2 H), 2.34 (m, 4 H),
2.72 (t, J ) 7.2 Hz, 2 H), 4.27 (m, 1 H), 5.46 (m, 2 H), 6.03 (dd,
J ) 15.0, 6.6 Hz, 1 H), 6.16 (t, J ) 11.3 Hz, 1 H), 6.22 (d, J )
15.4 Hz, 1 H), 6.44 (t, J ) 11.2 Hz, 1 H), 6.97 (dd, J ) 14.7,
11.9 Hz, 1 H), 7.36 (dd, J ) 15.1, 11.9 Hz, 1 H). Electrospray
MS calcd for C20H29O4 (M-1) 333, obsd 333. Similarly, to the
eluant (120 mL) containing the pure trans-isomer 7 was added
triethylamine (400 µL) to neutralize the acetic acid. The
mixture was stirred at room temperature for 1 min, and then
methanol was evaporated under high vacuum to concentrate
the solution to approximately 40 mL. A 5% aqueous solution
of KH2PO4 (40 mL) was added, extracted with ethyl acetate
(2 × 25 mL), and washed with water (4 × 10 mL). The pure
product 7 (560 µg) was obtained in 5% yield. 1H NMR (CD3-
COCD3) δ 0.93 (t, J ) 7.4 Hz, 3 H), 1.34 (m, 8 H), 1.85 (m, 2
H), 2.29-2.36 (m, 4 H), 2.67 (t, J ) 7.3 Hz, 2 H), 4.05 (br s, 1
H, OH), 4.21 (m, 1 H), 5.45 (m, 2 H), 6.03 (dd, J ) 15.4, 5.7
Hz, 1 H), 6.20 (d, J ) 15.5 Hz, 1 H), 6.42 (m, 2 H), 6.75 (dd, J
) 14.7, 11.0 Hz, 1 H), 7.28 (dd, J ) 14.4, 11.1 Hz, 1 H).
Electrospray MS calcd for C20H29O4 (M-1) 333, obsd 333.

8982 J . Org. Chem., Vol. 63, No. 24, 1998
Khanapure et al.
5-Oxo-12(S)-h yd r oxy-6(E),8(E),10(E),14(Z)-eicosa t et -
r a en oic a cid (7): (8,9-tr a n s-5-oxo-12-HETE) (7). To a
solution of dithio acid 31 (8 mg, 0.0195 mmol) in methanol/
H2O (9:1, 3 mL) was added a solution of 4-hydroxy-TEMPO
(50 µg) in methanol (50 µL) followed by [bis(trifluoroacetoxy)-
iodo]benzene (12 mg, 0.028 mmol) under argon and the
reaction mixture was stirred at room temperature for 3 min.
The reaction mixture was quenched with cold water (25 mL)
and extracted with ethyl acetate (2 × 30 mL). The combined
ethyl acetate extracts were washed with cold water (8 × 10
mL) and brine (1 × 10 mL), dried over anhydrous Na2SO4,
and filtered. 4-Hydroxy-TEMPO (50 µg) in ethyl acetate (50
µL) was added and the product in ethyl acetate was kept at
-20 °C. The product was purified by reversed-phase HPLC
(Sperisorb S10W, C-18, 10 × 250 mm column) using MeOH/
H2O/AcOH (70:30:0.1%) as the mobile phase and a flow rate
of 6 mL/min to give a pure trans-isomer 7. Triethylamine (500
µL) was added to the eluant (75 mL) containing the pure trans-
isomer 7 to neutralize the acetic acid. The mixture was stirred
at room temperature for 1 min and then methanol was
evaporated under high vacuum to concentrate the solution to
approximately 25 mL. A 5% aqueous solution of KH2PO4 (40
mL) was added, extracted with ethyl acetate (2 × 30 mL), and
washed with water (4 × 15 mL). The pure product 4.639 mg
was obtained in 71% yield. 1H NMR (CD3COCD3) δ 0.93 (t, J
) 7.4 Hz, 3 H), 1.34 (m, 8 H), 1.85 (m, 2 H), 2.29-2.36 (m, 4
H), 2.67 (t, J ) 7.3 Hz, 2 H), 4.05 (br s, 1 H, OH), 4.21 (m, 1
H), 5.45 (m, 2 H), 6.03 (dd, J ) 15.4, 5.7 Hz, 1 H), 6.20 (d, J
) 15.5 Hz, 1 H), 6.42 (m, 2 H), 6.75 (dd, J ) 14.7, 11.0 Hz, 1
H), 7.28 (dd, J ) 14.4, 11.1 Hz, 1 H).
Ack n ow led gm en t. We acknowledge the National
Institutes of Health for support under Grant DK-44730
(J . R.), the National Science Foundation for an AMX-
360 NMR instrument (Grant CHE-9013145), the Medi-
cal Research Council of Canada for support under Grant
MT-6254 (W. S. P.), and for support from the Quebec
Heart and Stroke Foundation (W. S. P.).
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H NMR
and 13C NMR spectral data for new compounds described
herein (23 pages). This material is contained in libraries on
microfiche, immediately follows this article in the microfilm
version of the journal, and can be ordered from the ACS; see
any current masthead page for ordering information.
J O9813282