Synthesis of 10(S)-hydroxyeicosatetraenoic acid: A novel cytochrome P-450 metabolite of arachidonic acid

Article abstract of DOI:10.1021/jo951061w

10(S)-(-)-hydroxyeicosa-5(Z),8(Z),11(Z),14(Z)-tetraenoic acid methyl ester (2) was synthesized in eight steps starting from enantiomerically pure (R)-glyceraldehyde acetonide. The 10(R)-(±)enantiomer was prepared using an identical method except that the starting material was (S)-glyceraldehyde acetonide. By use of these authentic standards, it was possible to confirm that arachidonic acid was converted to a mixture of 10(S)-(-)-hydroxyeicosa-5(Z),8(Z),11(Z),14(Z)-tetraenoic acid (1) and the 10(R)-(+)-enantiomer by phenobarbital-induced rat liver microsomes.

Full text of DOI:10.1021/jo951061w

838
J . Org. Chem. 1996, 61, 838-841
Syn th esis of 10(S)-Hyd r oxyeicosa tetr a en oic Acid : A Novel
Cytoch r om e P -450 Meta bolite of Ar a ch id on ic Acid
Suresh N. Yeola, Samir A. Saleh, Alan R. Brash, Chandra Prakash, Douglass F. Taber,1 and
Ian A. Blair*
Departments of Pharmacology and Chemistry, Vanderbilt University, Nashville, Tennessee 37232
Received J une 12, 1995X
10(S)-(-)-hydroxyeicosa-5(Z),8(Z),11(Z),14(Z)-tetraenoic acid methyl ester (2) was synthesized in
eight steps starting from enantiomerically pure (R)-glyceraldehyde acetonide. The 10(R)-(+)-
enantiomer was prepared using an identical method except that the starting material was (S)-
glyceraldehyde acetonide. By use of these authentic standards, it was possible to confirm that
arachidonic acid was converted to a mixture of 10(S)-(-)-hydroxyeicosa-5(Z),8(Z),11(Z),14(Z)-
tetraenoic acid (1) and the 10(R)-(+)-enantiomer by phenobarbital-induced rat liver microsomes.
In tr od u ction
HETE (as its ethyl ester derivative) as a metabolite of
red algae.12 We report the synthesis of 10(S)-(-)-
(5Z,8Z,11Z,14Z)-HETE (1) and its 10(R)-(+)-enantiomer
(Scheme 1). The availability of these compounds has
provided confirmation of the structural assignment of 10-
HETEs from the microsomal incubation and allowed
assignment of their absolute configuration.
Hydroxyeicosatetraenoic acids2 (HETEs) are formed in
numerous cell types by enzymatically-mediated hydroxy-
lation of arachidonic acid. There are three distinct
enzymes involved in HETE biosynthesis:lipoxygenase
(LOX), prostaglandin H (PGH) synthase, and cytochrome
P-450. LOX- and PGH-synthase-mediated arachidonic
acid metabolism is normally highly stereoselective. For
example, platelet 12-LOX produces 12(S)-HETE3,4 and
PGH synthase catalyzes the formation of 11(R)-HETE.5,6
However, cytochrome P-450-mediated metabolism of
arachidonic acid does not generally occur with such high
stereoselectivity or regioselectivity. Thus, incubation of
arachidonic acid with rat liver microsomes results in the
formation of at least eight different HETEs.7,8 Pretreat-
ment of the rats with â-naphthoflavone results in the
conversion of arachidonic acid to an additional three
metabolites: 16-, 17-, and 18-HETEs.9 During a mecha-
nistic study of the P-450-dependent pathway of arachi-
donic acid metabolism using phenobarbital (PB)-induced
rat liver microsomes, we observed three new HETEs as
major products in the microsomal incubation mixture.10
From gas chromatography/mass spectrometry (GC/MS)
analysis, it appeared that the three HETEs had hydroxyl
groups in each of the different bis-allylic positions (C-7,
C-10, and C-13). There have been two previous reports
on the formation of bis-allylic HETEs. Oliw et al.11
identified 13-HETE as an arachidonic acid metabolite in
rat liver microsomes, and Guerriero et al. identified 13-
Resu lts a n d Discu ssion
Chiral HPLC analysis of 10-HETE isolated from an
incubation of arachidonic acid with PB-induced rat liver
microsomes suggested that it was almost a 50:50 mixture
of two enantiomers.10 In order to confirm this observa-
tion, it was decided to first synthesize 10(S)-HETE and
then to use a similar method for preparation of the 10-
(R)-enantiomer. The starting compound for the synthesis
of 10(S)-HETE (1, Scheme 1) was (R)-glyceraldehyde
acetonide (4), prepared by the lead tetraacetate cleavage13
of (D)-mannitol acetonide (3) in 80% yield. A Wittig
condensation of 4 with phosphonium salt 514 using 1
equiv of lithium hexamethyldisilazide and tetrahydro-
furan:toluene (1:1)15 at -78 °C provided the acetonide
diene 6 in 47% yield. Acid hydrolysis of 6 afforded the
diol 7 in 83% yield. The primary hydroxyl group of 7
was selectively protected using tert-butyldimethylsilyl
(TBDMS) chloride in the presence of 4-(dimethylamino)-
pyridine to give 8.16 Protection of the secondary hydroxyl
group was performed using tert-butyldiphenylsilyl (TB-
DPS) chloride to give 9 (in 81% yield). Selective depro-
tection of the bis-silyl ether 9 was performed using
pyridinium p-toluenesulfonate (PPTS). This removed the
TBDMS group and provided the TBDPS-protected sec-
ondary alcohol 10 in 76% yield.16 Oxidation of 10 using
pyridinium chlorochromate gave the aldehyde 11 (73%).
The phosphonium salt17 12 for the Wittig reaction was
* To whom correspondence should be addressed at the Department
of Pharmacology. Tel: (615)-343-8437. Fax: (615)-343-1268
X Abstract published in Advance ACS Abstracts, J anuary 15, 1996.
(1) Department of Chemistry and Biochemistry, University of
Delaware, Newark, DE 19716.
(2) Spector, A. A.; Gordon, J . A.; Moore, S. A. Prog. Lipid. Res. 1988,
27, 271.
(3) Hamberg, M.; Samuelsson, B. Proc. Nat. Acad. Sci. U.S.A. 1974,
71, 3400 and 3824.
(4) Nugteren, D. H. Biochim. Biophys. Acta 1975, 380, 299.
(5) Hamberg, M.; Samuelsson, B. J . Biol. Chem. 1967, 242, 5344.
(6) Nugteren, D. H.; van Dorp, D. A.; Bergstorm, S; Hamberg, M.;
Samuelsson, B. Nature 1966, 212, 38.
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(7) Capdevila, J .; Marnett, L. J .; Chacos, N.; Prough, R. A.; Es-
tabrook R. W. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 767.
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(9) Falck, J . R.; Lumin, S,; Blair, I. A.; Dishman, E.; Martin, M. V.;
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(13) Baer, E. Biochemical Preparations; J ohn Wiley and Sons, Inc.:
New York, 1952; Vol. 2, p 31.
(14) Rokach J .; Girard, Y.; Guindon, Y.; Atkinson, J . G.; Larue, M.;
Young, R. N.; Masson, P.; Holme, G. Tetrahedron Lett. 1980, 1485.
(15) Corey, E. J .; Lansbury, P. T., J r. J . Am. Chem. Soc. 1983, 105,
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(10) Brash, A. R., Boeglin W. E.; Capdevila, J . H.; Yeola, S.; Blair,
I. A. Arch. Biochem. Biophys. 1995, 321,485.
(11) Oliw, E.; Brodowsky, I. D.; Hornsten, L.; Hamberg, M. Arch.
Biochem. Biophys. 1983, 300, 434.
(16) Prakash, C.; Saleh, S.; Blair, I. A. Tetrahedron Lett. 1989, 30,
19.
(17) Pfister, J . R.; Krishna Murthy, D. V. J . Med. Chem. 1983, 26,
1099.
0022-3263/96/1961-0838$12.00/0 © 1996 American Chemical Society

Synthesis of 10(S)-Hydroxyeicosatetraenoic Acid
J . Org. Chem., Vol. 61, No. 3, 1996 839
Sch em e 1
prepared from methyl 8-hydroxy-5-octenoate.18 The Wit-
tig condensation of 11 with 12 gave the TBDPS-protected
10(S)-HETE methyl ester 13 in 49% yield. The TBDPS
group was removed with aqueous tetrabutylammonium
fluoride to give 10(S)-(-)-HETE as its methyl ester
HETE inhibits ion transport in the kidney22 and is a
vasoconstrictor.23 The availability of authentic 10-HETE
enantiomers from the present study will also allow their
biological activity to be assessed. Future studies will be
performed to determine whether 10-HETE is formed in
vivo and whether it is glucuronidated.24 Finally, the
availability of 10-HETE enantiomers will make it pos-
sible to assess their ability to form cyclooxygenase
metabolites by reaction with PGH synthase.25 Such
prostaglandin-like metabolites could conceivably possess
potent biological activities.
derivative 2 ([R]20 -11.6; c 0.1, EtOH). The hydrolysis
D
of methyl ester 2 using lithium hydroxide gave 10(S)-(-
)-HETE (1, 43%) as an unstable oil that had to be used
immediately.
The synthesis of 10(R)-(+)-HETE was completed using
the same reaction sequence (Scheme 1) except that (S)-
glyceraldehyde acetonide was used as the starting mate-
rial. Analyses of 10(S)-HETE and 10(R)-HETE methyl
esters using chiral phase HPLC revealed a single enan-
tiomer (>98% enantiomeric excess) in each case.19 By
comparison of chiral HPLC and GC/MS data, material
isolated from the microsomal incubations was shown to
be a racemic mixture of 10(S)-(-)-HETE and 10(R)-(+)-
HETE.10 It was found that the 10-HETEs were ex-
tremely unstable under acidic conditions, and this may
explain why they have not been observed previously. The
10(S)- and 10(R)-HETEs prepared in the present study
have been used to examine the retention of stereochem-
istry during acid-catalyzed rearrangement and to exam-
ine the enantioselective metabolism of 10-HETE by 15-
LOX.10
Exp er im en ta l Section
Gen er a l Meth od s. Unless otherwise stated, solvents and
reagents were purchased and used without further purifica-
tion. Tetrahydrofuran was distilled from sodium benzophe-
none prior to use. Nuclear magnetic resonance spectra were
recorded using a 200, 300, or 400 MHz spectrometer. Thin
layer chromatography (TLC) was performed using 2.5 × 10
cm, 250 µm analytical plates coated with silica gel G. Column
chromatography was performed under air pressure on TLC
grade 60 Å silica gel. The solvent mixtures indicated for TLC
are volume/volume mixtures.
(Z,Z)-4-Deca-1,4-dien -1-yl-2,2-dim eth yl-1,3-dioxolan e (6).
A portion of 14 mM solution of lithium bis(trimethylsilyl)amide
in THF was added dropwise over a period of 30 min to a
magnetically stirred suspension of phosphonium salt 5 (6.25
g, 13.4 mmol) in THF (10 mL) and toluene (10 mL) at 0 °C.
The reaction mixture was stirred for an additional 30 min at
0 °C. The mixture was cooled to -78 °C, and 4 (2.10 g, 16.2
mmol) in THF (5 mL) was added dropwise. Stirring was
continued at 0 °C for 2 h. The reaction was quenched with a
saturated aqueous solution of NH4Cl (10 mL) and extracted
with ethyl acetate (100 mL). The organic layer was washed
with water (2 × 25 mL), dried (MgSO4), and concentrated to
give a crude oil. This was purified by flash chromatography
using 5% ethyl acetate in hexane to yield 6 as a colorless oil
HETEs have been shown to exhibit a range of potent
biological activities. 12(R)-HETE is a potent vasocon-
strictor in the cornea20 and 12(S)-HETE induces mi-
crovascular endothelial cell retraction,21 whereas 20-
(18) Taber, D. F.; Hoerrner, R. S. J . Org. Chem. 1992, 57, 441.
(19) 10(S)-(-)-hydroxyeicosatetraenoic acid methyl ester was sepa-
rated from 10(R)-(+)-hydroxyeicosatetraenoic acid methyl ester by
HPLC (Chiralcel OD column, 4.6 mm × 250 mm, 5 µm, J . T. Baker)
using hexane/2-propanol (100:2; v/v) as the mobile phase at a flow rate
of 1 mL/min. The retention times for the 10(S)- and 10(R) enantiomers
were 12.61 and 13.65 min, respectively.
(22) Escelante, B.; Erlij, D.; Falck, J . R.; McGiff, J . C. Science 1991,
251, 799.
(20) Murphy, R. C.; Falck, J . R.; Lumin, S.; Yadagiri, P.; Zirolli, J .
A.; Balazy, M.; Masferrer, J . L.; Abraham, N. G.; Schwartzman, M. L.
J . Biol. Chem. 1988, 263, 17197.
(21) Honn, K. V.; Tang, D. G.; Grossi, I.; Duniec, Z. M.; Timar, J .;
Renaud, C.; Leithauser, M.; Blair, I. A.; J ohnson, C. J .; Diglio, C. A.;
Kimler, V. A.; Taylor, J . D., Marnett, L. J . Cancer Res. 1994, 54, 565.
(23) Schwartzman, M. L.; Falck, J . R.; Yadagiri, P.; Escalante, B.
J . Biol. Chem. 1989, 264, 11658.
(24) Prakash, C.; Zhang, J . Y.; Falck. J . R.; Chauhan, K.; Blair, I.
A. Biochem. Biophys. Res. Commun. 1992, 185, 728.
(25) Zhang, J . Y.; Prakash, C.; Yamashita, K.; Blair, I. A. Biochem.
Biophys. Res. Commun. 1992, 183, 138.

840 J . Org. Chem., Vol. 61, No. 3, 1996
Yeola et al.
(1.8 g, 47%): TLC Rf (5% ethyl acetate/hexane) ) 0.69; 1H
NMR (CDCl3) δ 5.25-5.60 (m, 4 H), 4.84 (q, J ) 8.1 Hz, 1 H),
4.04 (dd, J ) 6.0 and 7.9 Hz, 1 H), 3.50 (t, J ) 8.1 Hz, 1 H),
2.83 (t, J ) 7.2 Hz, 2 H), 2.01 (q, J ) 6.9 Hz, 2 H), 1.37 (s, 3
H), 1.33 (s, 3 H) 1.20-1.30 (m, 6 H), 0.86 (t, J ) 6.9 Hz, 3 H);
13C NMR (CDCl3) δ 133.2, 131.1, 127.3, 126.7, 71.9, 69.4, 31.4,
29.2, 27.2, 26.7, 26.1, 25.9, 22.5, 14.0; MS (m/z, electrospray)
239 (19, M + 1), 221 (54), 199 (51), 181 (57), 163 (100), 120
(19), 110 (21).
(2S)-(Z,Z)-3,6-Dod eca d ien e-1,2-d iol (7). Hydrochloric
acid (concd, 1.7 mL) was added dropwise to the solution of
diene 6 (1.3 g, 5.4 mmol) in methanol (30 mL) at 0 °C. Stirring
was continued at 0 °C for 2 h. The reaction mixture was
neutralized to pH ) 7 with concentrated aqueous NH4OH.
Methanol was removed under vacuum. Water (10 mL) was
added to the residue, which was then extracted with ethyl
acetate (2 × 25 mL), dried (MgSO4), and evaporated. The
residue was purified by silica gel flash chromatography using
hexane:acetone (9:1) to give 7 as a colorless oil (0.9 g, 83%):
TLC Rf (30% ethyl acetate/hexane) ) 0.42; 1H NMR (CDCl3) δ
5.20-5.70 (m, 4 H), 4.50-4.65 (m, 1 H), 3.45-3.60 (m, 2 H),
2.70-2.90 (m, 2 H), 1.90-2.10 (m, 2 H), 1.20-1.45 (m, 6 H),
0.85-0.95 (m, 3 H); 13C NMR (CDCl3) δ 132.7, 131.2, 128.1,
126.2, 68.6, 66.3, 31.5, 29.2, 27.2, 26.2, 22.5, 14.0.
2-propanol/acetic acid (99.05:0.9:0.05 v/v/v) as the mobile phase
at a wavelength 235 nm with a flow rate 3 mL/min. The
retention time for the compound 10 was 17.04 min: TLC Rf
1
(10% ethyl acetate/hexane) ) 0.36; H NMR (CDCl3) δ 7.62-
7.78 (m, 4 H), 7.29-7.55 (m, 6 H), 4.91-5.55 (m, 4 H), 4.45-
4.65 (m, 1 H), 3.32-3.62 (m, 2 H), 2.15-2.55 (m, 2 H), 1.71-
2.09 (m, 2 H), 1.15-1.40 (m, 6 H), 1.05 (s, 9 H), 0.86 (m, 3 H);
13C NMR (CDCl3) δ 135.9, 135.8, 133.8, 133.6, 131.1, 130.8,
130.7, 129.8, 129.7, 129.4, 127.7, 127.5, 126.8, 70.7, 67.0, 31.4,
29.2, 27.1, 27.0, 25.9, 22.5, 19.3, 14.0; HRMS calcd for
C24H31O2Si (M - 57) 379.2093, found 379.2089.
(2S)-2-[(ter t-Bu tyld ip h en ylsilyl)oxy]-(Z,Z)-3,6-d od eca -
d ien -1-a l (11). Pyridinium chlorochromate (154 mg, 0.71
mmol), anhydrous sodium acetate (154 mg), and 4 Å molecular
sieves (154 mg) were ground together and then suspended with
stirring in methylene chloride (10 mL) at room temperature.
Alcohol 10 (100 mg) in methylene chloride (1 mL) was added
dropwise to the mixture, which was then stirred for 30 min.
The reaction mixture was diluted with ether (10 mL), and the
mixture was filtered through a Celite pad. The solvent was
evaporated under vacuum, and the residual oil was purified
by flash chromatography using 2% ethyl acetate in hexane to
give 11 as an oil (75 mg, 73%): TLC Rf
(10% ethyl acetate/
hexane) ) 0.47; H NMR (CDCl3) δ 9.50 (s, 1 H), 7.55-7.75
1
(m, 4 H), 7.32-7.50 (m, 6 H), 5.05-5.70 (m, 4 H), 4.75 (d, J )
7.0 Hz, 1 H), 2.40-2.55 (m, 2 H), 1.75-1.95 (m, 2 H), 1.15-
1.40 (m, 6 H), 1.10 (s, 9 H), 0.85 (m, 3 H); 13C NMR (CDCl3) δ
199.2, 135.8, 134.3, 131.2, 130.0, 129.9, 127.8, 127.7, 126.1,
124.9, 76.1, 31.4, 29.2, 27.1, 26.9, 26.5, 22.5, 19.3, 14.1.
(10S)-Meth yl 10-[(ter t-bu tyld ip h en ylsilyl)oxy]eicosa -
5(Z),8(Z),11(Z),14(Z)-t et r a en oa t e (13). Lithium bis(tri-
methylsilyl)amide (580 µL, 0.58 mmol) was added dropwise
to a suspension of phosphonium salt 12 (308 mg, 0.62 mmol)
in dry THF (5 mL) at 0 °C over a period of 20 min. After the
addition was complete, the reaction was stirred for 15 min at
0 °C and for 30 min at room temperature. The reaction
mixture was cooled to -78 °C, aldehyde 11 (180 mg, 0.415
mmol) in THF (3 mL) was added dropwise, and stirring was
continued at 0 °C for 3 h. The reaction was quenched with a
saturated aqueous solution of NH4Cl (10 mL) and extracted
with ethyl acetate (100 mL). The combined organic extract
was washed with water (2 × 25 mL), dried (MgSO4), and
concentrated. The residue was purified by flash chromatog-
raphy using 3% ethyl acetate in hexane to yield 13 as a
colorless oil (116 mg, 49%). Further purification was carried
out by HPLC (silica column, 10 mm × 25 cm, 5 µm) using
hexane/2-propanol/acetic acid (99.05:0.9:0.05 v/v/v) as the
mobile phase at a wavelength of 235 nm with a flow rate 3
mL/min. The retention time for the compound 13 was 15.14
min: TLC Rf
(2S)-1-[(ter t-Bu tyld im eth ylsilyl)oxy]-(Z,Z)-3,6-d od eca -
dien -2-ol (8). 4-(Dimethylamino)pyridine (347 mg, 2.83 mmol)
and tert-butyldimethylsilyl chloride (426 mg, 2.83 mmol) were
added to the solution of diol 7 (560 mg, 2.83 mmol) in
methylene chloride (15 mL). The reaction mixture was stirred
at room temperature for 5 h. Methylene chloride (50 mL) was
added and the mixture washed with saturated aqueous NaCl.
The organic extract was dried over MgSO4 and concentrated.
The residue was purified by flash chromatography with 10%
ethyl acetate in hexane as eluent to give 8 as an oil (660 mg,
75%): TLC Rf (30% ethyl acetate/hexane) ) 0.65; 1H NMR
(CDCl3) δ 5.25-5.60 (m, 4 H), 4.45-4.55 (m, 1 H), 3.50-3.65
(m, 1 H), 3.40-3.50 (m, 1 H), 2.70-2.90 (m, 2 H), 2.20 (bs,
1H), 1.90-2.20 (m, 2 H), 1.22-1.45 (m, 6 H), 0.85-0.95 (m, 12
H), 0.10 (s, 6 H); 13C NMR (CDCl3) δ 132.4, 130.9, 129.1, 127.0,
68.4, 66.6, 32.5, 31.5, 29.3, 27.2, 26.3, 25.9, 22.6, 18.3, 14.0;
HRMS calcd for C14H27O2Si (M - 57) 255.1780, found 255.1773.
(2S)-1-[(ter t-Bu t yld im et h ylsilyl)oxy]-2-[(ter t-b u t yld i-
p h en ylsilyl)oxy]-(Z,Z)-3,6-d od eca d ien e (9). A solution of
diene 8 (295 mg, 0.945 mmol) and 4-(dimethylamino)pyridine
(231 mg, 1.89 mmol) in methylene chloride (5 mL) was stirred
with tert-butyldiphenylsilyl chloride (520 mg, 1.89 mmol) at
room temperature for 15 h. The reaction mixture was parti-
tioned between methylene chloride (50 mL) and saturated
aqueous NaCl. The combined organic exracts were dried
(MgSO4) and concentrated. The residual oil was purified by
flash chromatography using 2% ethyl acetate in hexane to yield
9 as a oil (423 mg, 81%). Further purification was carried out
by HPLC (silica column, 10 mm × 25 cm, 5 µm) using hexane/
2-propanol/acetic acid (99.05:0.9:0.05 v/v/v) as the mobile phase
at a wavelength 235 nm with a flow rate 3 mL/min. The
retention time for the compound 9 was 13.86 min: TLC Rf
(5% ethyl acetate/hexane) ) 0.50; 1H NMR
(CDCl3
) δ 7.64-7.68 (m, 4 H), 7.30-7.42 (m, 6 H), 4.95-5.65
(m, 9 H), 3.62 (s, 3 H), 2.24-2.36 (m, 4 H), 2.20 (t, J ) 7.4 Hz,
2H), 1.81-1.91 (m, 4 H), 1.57-1.65 (m, 2 H), 1.21-1.30 (m, 6
H), 1.01 (s, 9 H), 0.85 (t, J ) 6.9 Hz, 3 H); 13C NMR (CDCl3)
δ 174.3, 135.9, 134.1, 132.1, 131.8, 131.3, 130.6, 129.4, 129.0,
128.9, 128.4, 127.9, 127.4, 127.1, 65.9, 51.3, 33.4, 32.4, 31.4,
29.2, 29.1, 27.1, 26.9, 26.8, 26.4, 25.9, 25.8, 24.7, 22.5, 19.2,
14.0; HRMS calcd for C33H43O3Si (M - 57) 515.2981, found
515.2994.
1
(10% ethyl acetate/hexane) ) 0.74; H NMR (CDCl3) δ 7.62-
7.83 (m, 4 H), 7.25-7.48 (m, 6 H), 4.95-5.48 (m, 4 H), 4.36-
4.64 (m, 1 H), 3.55-3.72 (m, 1 H), 3.38-3.54 (m, 1 H), 2.30-
2.52 (m, 2H), 1.75-2.00 (m, 2 H), 1.15-1.38 (m, 6 H), 1.04 (s,
9 H), 0.80-0.95 (m, 12 H), 0.04 (s, 6H); 13C NMR (CDCl3) δ
136.0, 135.9, 134.3, 130.8, 130.3, 130.1, 129.8, 129.6, 129.4,
127.8, 127.6, 127.4, 127.3, 70.5, 67.7, 31.5, 29.3, 29.2, 29.1, 27.1,
27.0, 26.9, 26.8, 26.1, 25.9, 22.5, 19.3, 18.4, 14.1; HRMS calcd
for C30H45O2Si2 (M - 57) 493.2958, found 493.2944.
10(S)-(-)-H yd r oxyeicosa -5(Z),8(Z),11(Z),14(Z)-t et r a -
en oic Acid Meth yl Ester (2). Tetrabutylammonium fluoride
(300 mL, 0.3 mmol) was added dropwise to a solution of 13
(116 mg, 0.2 mmol) in THF (5 mL) under nitrogen, and stirring
was continued at room temperature for 2 h. The reaction
mixture was partitioned between ethyl acetate and saturated
aqueous NaCl, dried (MgSO4), and concentrated. The residue
was purified by flash chromatography using 3% acetone in
hexane to give 2 as a colorless oil (41 mg, 61%). Further
purification was carried out by HPLC (silica column, 10 mm
× 25 cm, 5 µm) using hexane/2-propanol (100:0.5 v/v) as the
mobile phase at a wavelength 210 nm with a flow rate 3 mL/
(2S)-2-[(ter t-Bu tyld ip h en ylsilyl)oxy]-(Z,Z)-3,6-d od eca -
d ien -1-ol (10). Pyridinium p-toluenesulfonate (97 mg, 0.38
mmol) was added to the solution of 9 (423 mg, 0.77 mmol) in
ethanol (10 mL). The mixture was stirred at 60 °C for 15 h.
The reaction mixture was cooled to room temperature and then
partitioned between methylene chloride (50 mL) and saturated
aqueous NaCl. The combined organic extract was dried
(MgSO4) and concentrated. The residue was purified by flash
chromatography using 5% ethyl acetate in hexane to give 10
as an oil (254 mg, 76%). Further purification was carried out
by HPLC (Silica column, 10 mm × 25 cm, 5 µm) using hexane/
min. The retention time for compound 2 was 38.26 min: [R]20
D
-11.6 (c 0.1, EtOH); TLC Rf (30% ethyl acetate/hexane) ) 0.57;
1H NMR (CD3CN) δ 5.18-5.55 (m, 9 H), 3.62 (s, 3 H), 2.68-
3.05 (m, 4 H), 2.30 (t, J ) 7.4 Hz, 2 H), 1.95-2.25 (m, 4 H),
1.55-1.75 (m, 2 H), 1.15-1.50 (m, 6 H), 0.89 (t, J ) 6.5 Hz, 3

Synthesis of 10(S)-Hydroxyeicosatetraenoic Acid
J . Org. Chem., Vol. 61, No. 3, 1996 841
H); 13C NMR (CD3CN) δ 174.6, 133.1, 130.5, 129.7, 129.5,
129.4, 128.4, 64.0, 51.9, 34.0, 32.3, 30.0, 27.8, 27.3, 26.8, 25.6,
23.3, 14.3; HRMS calcd for C21H33O2 (MH+ - H2O) 317.2480,
found 317.2480.
133.0, 131.6, 130.4, 129.7, 129.5, 129.3, 128.3, 64.0, 33.6, 32.3,
30.0, 27.9, 27.2, 26.8, 25.5, 23.3, 14.3; HRMS calcd for C20H31O2
(MH+ - H2O) 303.2324, found 303.2323. This compound was
unstable on a silica column and so could not be further purified
by HPLC. However, it could be readily prepared in >98%
purity by saponification of methyl ester 2, as described above,
when required.
10(S)-(-)-H yd r oxyeicosa -5(Z),8(Z),11(Z),14(Z)-t et r a -
en oic Acid (1). LiOH (3 N, 600 µL) was added to a solution
of 10(S)-(-)-hydroxyeicosatetraenoic acid methyl ester (2.0 mg,
0.006 mmol) in dimethoxyethane (400 µL), and the mixture
was stirred at 60 °C for 2 h. The reaction mixture was
acidified carefully to pH 6 using dilute HCl (1 N) and extracted
using CH2Cl2 (3 × 5 mL). The organic extracts were combined,
dried over MgSO4, filtered, and concentrated in vacuo to give
1 as a colorless oil (0.82 mg, 43%): TLC Rf (30% ethyl acetate/
hexane) ) 0.46; 1H NMR (CD3CN) δ 5.30-5.47 (m, 8 H), 5.17-
5.25 (m, 1 H), 2.74-2.95 (m, 4 H), 2.27 (t, J ) 7.4 Hz, 2 H),
2.02-2.18 (m, 4 H), 1.58-1.68 (m, 2 H), 1.25-1.40 (m, 6 H),
0.89 (t, J ) 6.7 Hz, 3 H); 13C NMR (CD3CN) δ 175.0 133.1,
Ack n ow led gm en t. Financial support of NIH grant
GM-15431 is gratefully acknowledged.
Su p p or tin g In for m a tion Ava ila ble: NMR spectra for
1a ,b, 6-11, and 13 (18 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 O951061W