Practical, enantiospecific syntheses of 14,15-EET and leukotoxin B (vernolic acid)

Article abstract of DOI:10.1016/S0040-4039(01)00694-3

Cytochrome P450BM3 and its F87V mutant were exploited for a convenient, laboratory scale (1 mmol) preparation of 14(S),15(R)-epoxyeicosatrienoic acid [14(S),15(R)-EET] from arachidonic acid and (+)-leukotoxin B [(+)-12(S),13(R)-vernolic acid] from linoleic acid, respectively. Their enantiomers were accessed via a four-step chemical inversion.

Full text of DOI:10.1016/S0040-4039(01)00694-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 4131–4133
Practical, enantiospecific syntheses of 14,15-EET and
leukotoxin B (vernolic acid)
J. R. Falck,* Y. Krishna Reddy, Donovan C. Haines, Komandla Malla Reddy, U. Murali Krishna,
Sandra Graham, Barbara Murry and Julian A. Peterson
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038, USA
Received 4 April 2001; revised 19 April 2001; accepted 20 April 2001
Abstract—Cytochrome P450BM3 and its F87V mutant were exploited for a convenient, laboratory scale (1 mmol) preparation of
14(S),15(R)-epoxyeicosatrienoic acid [14(S),15(R)-EET] from arachidonic acid and (+)-leukotoxin B [(+)-12(S),13(R)-vernolic
acid] from linoleic acid, respectively. Their enantiomers were accessed via a four-step chemical inversion. © 2001 Elsevier Science
Ltd. All rights reserved.
The cytochrome P450 branch1 of the eicosanoid cas-
cade has emerged as a physiologically significant source
of autacoids. Amongst these, the epoxyeicosatrienoic
acids (EETs) and leukotoxins (LTXs), derived from
arachidonic acid and linoleic acid, respectively, are
amongst the most prominent and widely studied. The
EETs induce mitogenesis in epithelial cells,2 stimulate
the release of numerous hormones, biogenic amines,
and other cellular mediators,3 modulate kinases,4,5 and
regulate gene transcription.6,7 Recent mechanistic stud-
ies have linked their mode of action to Gs-proteins8 and
control of ion channels.9,10 The EETs are also prime
candidates for endothelium-derived hyperpolarizing
factors (EDHFs) in several species.11–13 The LTXs are
bactericidal/antifungal14 and have been implicated in a
variety of pathophysiologic responses in plants15 and
animals16,17 including humans.18,19
polyunsaturated fatty acids by the soluble, catalytically
self-sufficient cytochrome P450BM3 and its genetically
engineered mutants.27,28 This approach is also applica-
ble to isotopically labeled derivatives using commercial
fatty acids.
For preparative incubations, NADP (2 mL of 50 mM
soln in 1% KHCO3), glucose-6-phosphate (10 mL of
200 mM soln in 50 mM MOPS buffer, pH 7.4), and
glucose-6-phosphate dehydrogenase (400 mL of 2860
U/mL soln) were added to an oxygen saturated MOPS
buffer (3.8 L of 50 mM soln, pH 7.4) forming a
NADPH generation and recycling system in a flask
open to the atmosphere.29 After 2 min, arachidonic acid
(500 mg in 250 mL deoxygenated 50 mM K2CO3) was
added followed quickly by 600 units of P450BM3
(F87V).27 A unit is defined as the amount of enzyme
that catalyses the consumption of one micromole of
NADPH per minute in the presence of 250 mM arachi-
donate, 250 mM oxygen, and 250 mM NADPH. After
stirring for 15 minutes at room temperature, the incu-
bation was quenched by the dropwise addition of 1 M
oxalic acid to pH 4, and extracted with Et2O (3×250
mL). The combined ethereal extracts were washed with
distilled water (3×150 mL), dried over Na2SO4, and
evaporated to dryness in vacuo. The residue was
esterified in ether/methanol (100 mL, 4:1) using excess
diazomethane. Silica gel chromatography (10% ethyl
Recent
reports
of
enantiospecific
biological
responses20,21 and stereoselective, high affinity binding
sites22 have evoked considerable demand for the indi-
vidual epoxide antipodes. Since chiral chrom-
atography23,24 and asymmetric total syntheses25,26 are
expensive and/or tedious, we report herein a conve-
nient, cost effective preparation of both enantiomers of
14,15-EET (1) and leukotoxin B (2) (LTX-B; vernolic
acid). Our strategy exploits the inherently high enzy-
matic turnover rate and stereoselective metabolism of
Keywords: eicosanoids; epoxides; inversion reactions; asymmetric syn-
thesis; biosynthesis.
* Corresponding author. Tel.: 214-648-2406; fax: 214-648-6455;
e-mail: j.falck@utsouthwestern.edu
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00694-3

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J. R. Falck et al. / Tetrahedron Letters 42 (2001) 4131–4133
Scheme 1. Reagents and conditions: (a) Al2O3, PhS(CH2)2OH (1.2 equiv.), (n-Bu)2O, 80°C, 16 h; (b) TsCl, DABCO, CH2Cl2, 23°C,
8 h; (c) TPAP (0.1 equiv.), NMO (4 equiv.), CH3CN, 81°C, 24 h; (d) DBU, CH3CN, 81°C, 14 h.
acetate/hexane) of the residue left after evaporation of
all volatiles afforded methyl 14(S),15(R)-EET30 (1) (249
mg, 45%) and unreacted methyl arachidonate (260 mg,
50%).
4. Node, K.; Huo, Y.; Ruan, X.; Yang, B.; Ley, K.; Zeldin,
D. C.; Liao, J. K. Science 1999, 285, 1276–1279.
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7. Toniato, E.; Flati, V.; Cifone, M. G.; Grosso, E. D.;
Roncaioli, P.; Cilenti, L.; Tessitore, A.; Lista, F.; Frati,
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1998, 19, L432–L441.
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A convenient chemical sequence for the inversion of
configuration of an epoxide is outlined in Scheme 1, as
illustrated by the transformation of 1 into its antipode.
Nucleophilic addition of 2-(phenylthio)ethanol to 1
under the influence of alumina31 afforded nearly equal
amounts of alcohol 3 and its regioisomer.32 Without
separation, this mixture was tosylated and then oxi-
dized using catalytic TPAP, recycled by NMO, to give
sulfone 4 and the companion regioisomer. DBU
induced b-elimination of the sulfone with concomitant
displacement of the tosylate generated methyl
14(R),15(S)-EET (5) in good overall yield.
12. Kaley, G. Circ. Res. 1998, 83, 772–773.
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Am. J. Physiol. 1999, 45, H1527–H1535.
14. Ozawa, T.; Nichikimi, M.; Sugiyama, S.; Taki, F.;
Hayakawa, M.; Shionoya, H. Biochem. Int. 1988, 16,
369–373.
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Comparable enzymatic epoxidation of linoleic acid,
preferably with wild type P450BM3,33,34 and dia-
zomethane esterification provided methyl (+)-leuko-
toxin B30 (2) [methyl (+)-12(S),13(R)-vernolate] with a
similar efficiency as above. Likewise, repetition of the
inversion described in Scheme 1 using 2 gave rise to
methyl (−)-leukotoxin B (6). Saponification (NaOH,
THF/H2O, 23°C, 6 h; 1N HCl to pH 4.5) of the above
esters afforded the corresponding free acids in nearly
quantitative yields.
16. Stimers, J. R.; Dobretsov, M.; Hastings, S. L.; Jude, A.
R.; Grant, D. F. Biochem. Biophys. Acta 1999, 1438,
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17. Review: Ishizaki, T.; Ozawa, T.; Voelkel, N. F. Pulm.
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Acknowledgements
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Supported financially by the USPHS NIH (GM31278,
DK38226, GM50858), the Robert A. Welch Founda-
tion, and an unrestricted grant from Taisho
Pharmaceutical.
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(m, 8H), 1.70 (quintet, J=7.3 Hz, 2H), 2.11 (dd, J=7.3,
7.3 Hz, 2H), 2.25–2.38 (m, 4H), 2.50 (d, J=4.6 Hz, D2O
exchangeable, 1H), 2.78–2.83 (m, 4H), 3.08–3.18 (m, 3H),
3.54–3.63 (m, 1H), 3.66 (s, 3H), 3.80–3.90 (m, 1H), 3.90
(dd, J=7.0, 7.0 Hz, 1/2H), 4.23 (dd, J=7.0, 7.0 Hz,
1/2H), 5.31–5.50 (m, 6H), 7.17–7.37 (m, 5H). Tosylate of
3 and its regioisomer: l 0.92 (t, J=7.0 Hz, 3H), 1.26–1.56
(m, 8H), 1.64–1.72 (m, 2H), 2.08–2.33 (m, 6H), 2.42 (s,
3H), 2.76–2.79 (m, 4H), 2.92–2.99 (m, 2H), 3.13 (dd,
J=7.0, 7.0 Hz, 1/2H), 3.42 (dd, J=7.0, 7.0 Hz, 1/2H),
3.54–3.64 (m, 2H), 3.66 (s, 3H), 4.23 (dd, J=7.0, 7.0 Hz,
1/2H), 4.54 (dd, J=7.0, 7.0 Hz, 1/2H), 5.30–5.43 (m, 6H),
7.17–7.37 (m, 7H), 7.74 (d, J=8.2 Hz, 2H). 4 and its
regioisomer: l 0.92 (t, J=7.0 Hz, 3H), 1.26–1.56 (m, 8H),
1.64–1.72 (m, 2H), 2.08–2.33 (m, 6H), 2.42 (s, 3H),
2.76–2.79 (m, 4H), 3.27 (t, J=6.4 Hz, 2H), 3.37 (quintet,
J=4.3 Hz, 1/2H), 3.66 (s, 3H), 3.76–3.84 (m, 3H), 4.47
(quintet, J=4.3 Hz, 1/2H), 5.25–5.43 (m, 6H), 7.34 (d,
J=8.2 Hz, 2H), 7.53–7.58 (m, 2H), 7.63–7.67 (m, 1H),
7.76 (d, J=8.2 Hz, 2H), 7.90 (d, J=8.2 Hz, 2H).
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hexane, 1 mL/min, Rt=17 and 20.8 min for 1 and 5,
respectively.
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