5-Oxo-15-HETE: Total synthesis and bioactivity

Article abstract of DOI:10.1016/j.bmcl.2011.01.032

The first total synthesis of 6(E),8(Z),11(Z),13(E) 5-oxo-15-HETE 4 was accomplished. The synthetic material was evaluated with calcium mobilization assay and compared with 5-oxo-ETE the natural ligand for the OXE receptor.

Full text of DOI:10.1016/j.bmcl.2011.01.032

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1857–1860
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Oxo-15-HETE: Total synthesis and bioactivity
Pranav Patel a, Jaganmohan R. Anumolu a, William S. Powell b, Joshua Rokach a,
⇑
a Claude Pepper Institute and Department of Chemistry, Florida Institute of Technology, 150 West University Boulevard, Melbourne, FL 32901, USA
b Meakins-Christie Laboratories, Department of Medicine, McGill University, 3626 St. Urbain Street, Montreal, Quebec, Canada H2X 2P2
a r t i c l e i n f o
a b s t r a c t
Article history:
The first total synthesis of 6(E),8(Z),11(Z),13(E) 5-oxo-15-HETE 4 was accomplished. The synthetic
material was evaluated with calcium mobilization assay and compared with 5-oxo-ETE the natural ligand
for the OXE receptor.
Received 3 December 2010
Revised 6 January 2011
Accepted 10 January 2011
Available online 25 January 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
5-Oxo-15-HETE
5-Oxo-ETE
Calcium mobilization
Lipoxygenase
Chemotaxis
Asthma
Arachidonic acid 1 (AA) is converted to a large number of
biologically active products (eicosanoids) that are important in a
variety of pathological conditions including inflammatory and
allergic diseases. Because of the complex array of products with dif-
ferent chiral centers and double bond configurations, chemical
synthesis of eicosanoids has been critical for their structural
identification and determination of their physiological and phar-
macological properties.
Lipoxygenase (LO) enzymes catalyze the stereospecific oxida-
tion of AA 1 to form hydroperoxyeicosatetraenoic acids (HPETEs);
for example, 5(S)-HPETE is the major initial product formed by
5-LO, 12(S)-HPETE from 12-LO, and 15(S)-HPETE from 15-LO.
However, in the last instance the use of soybean lipoxygenase,
which is widely used as a source of 15-LO, generates a substantial
amount (ꢀ15%) of the R-enantiomer.1 5-HPETE is also converted by
peroxidase to 5-HETE which is further transformed to 5-oxo-ETE 2
by the NADP+-dependent enzyme 5-hydroxy eicosanoid dehydro-
genase (5-HEDH, Scheme 2). This enzyme is highly specific for
OXE receptor.8–10 5-Oxo-ETE is hydroxylated by both lipoxygenase
and cytochrome P450 pathways, being converted to 5-oxo-12-HETE
and 5-oxo-15-HETE 4 by 12-LO11 and 15-LO12, respectively, and to
5-oxo-20-HETE 513 and 5-oxo-19-HETE14 by cytochrome P450
enzymes.
5-Oxo-15-HETE has an OH at C-15 with the S-stereochemistry
and two sets of conjugated dienes (Scheme 1). The diene at C-6
is conjugated with carbonyl compound and show the appropriate
UV at kmax 281, and the diene at C-11 has a kmax 229. Until now,
5-oxo-15-HETE has only been prepared for pharmacological test-
ing from its biological precursors. For example, oxidation of AA
with soybean lipoxygenase gave rise to 5-oxo-15-HETE as one of
the minor products.15 This enzyme also converts 5-oxo-ETE
directly to 5-oxo-15-HETE12 (Scheme 4). We have previously syn-
thesized the latter product for biological testing by incubating
5,15-diHETE with neutrophil microsomes, which contain high
levels of 5-HEDH, in the presence of NADP+.8 In addition, 5-oxo-
15-HETE has also be synthesized by oxidation of 5,15-diHETE with
2,3-dichloro-5,6-dicyanobenzoquinone and separation from the
other dehydrogenase products by HPLC.16
Although 5-oxo-15-HETE appears to activate the OXE receptor,
there are some discrepancies in the literature on its potency
compared to 5-oxo-ETE. Norgauer’s group reported that these
two eicosanoids are equipotent and have equivalent efficacies,17,18
whereas we5,8 and others16 found it to be somewhat less potent
and/or efficacious. It has been suggested that these differences
could have been due to isomerization of 5-oxo-15-HETE to its less
potent 8-trans form.12
eicosanoids containing
a 5(S)-hydroxyl group followed by a
6,7-trans double bond. For example, neither 5(R)-HETE2 nor LTB4
which has a 6,7-cis double bond, is a substrate for this enzyme.2
5-HEDH is present in neutrophils,3 monocytes,4 lymphocytes,4
eosinophils,5 platelets,6 and endothelial and epithelial cells, and
keratinocytes.7
5-Oxo-ETE is the most potent eosinophil chemoattractant
amongst lipid mediators and has similar effects on neutrophils3
and monocytes. Its action is mediated by the highly selective
We have invested substantial efforts in studying structure–
⇑
Corresponding author.
E-mail address: jrokach@fit.edu (J. Rokach).
activity relationships for the 5-oxo-ETE receptor.19 To clarify the
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.032

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P. Patel et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1857–1860
COOH
COOH
COOH
COOH
COOH
OH
OH
O
O
O
OH
OH
5-oxo-15-HETE 4
AA
1
5-oxo-ETE
2
5,15-DiHETE
3
5-oxo-20-HETE 5
Scheme 1. Eicosanoids.
5-HEDH
NADP+
5-LO
COOH
COOH
OH
COOH
AA
1
OOH
O
Peroxidase
keto reductase
2
5-oxo-ETE
7
6
5(S)-HETE
5(S)-HPETE
OXE Receptor
Ca++, Degranulation, F-actin polymerization, L-Selectin,
Adherence, Chemotaxis, migration, tissue infiltration etc.
Scheme 2. Biosynthesis of 5-oxo-ETE.
degree of biological activity of 5-oxo-15-HETE and to avoid any
ambiguity about its structure, we elected to prepare this mediator
by total synthesis.
We approached the synthesis of 5-oxo-15-HETE 4 as described in
detail in Scheme 3. We opted for a convergent synthesis in which
the 5-oxo component and the 15-hydroxy synthon were prepared
and then connected. Synthon 12 was previously synthesized by us
using a known procedure.20 We used a protected 5-oxo group 24
in order to avoid complications later in the synthesis and was pre-
pared in three very efficient steps, as described previously.21 The
synthon 20, which contained the 15-hydroxy component, was pre-
pared from -arabinose as shown in Scheme 3.
D
The synthetic 6(E),8(Z),11(Z),13(E) 5-oxo-15-HETE shows a UV
absorption at kmax 281 for the dienone at C-6 and kmax 229 for
the diene at C-11. The NMR shows the coupling constant for the
proton at C-6 (J = 15.5 Hz, 1H), C-7 (J = 15.1, 11.1 Hz, 1H), C-9
(J = 10.5, 7.8 Hz, 1H), C-11 (J = 10.4, 7.4 Hz, 1H), and C-13
(J = 15.1, 11.1 Hz, 1H).
Human neutrophils were purified from whole blood from
healthy individuals as described previously using dextran 500 to
O
13
Br
O
OH
4 steps
HO
O
O
O
PPh 3
a
H
O
O
O
HO
RO
RO
b
OH
SEt
SEt
D-Arabinose
R = TBDMS
CHO
10
11
12
14
Ph 3P
Br
O
Si(n-Bu3)
O
O
O
Si(n-Bu)3
O
O
CHO
17
Ph 3P
19
RO
c
H
H
e
d
OR
OR
OR
20
18
OHC
1516
COOMe
S
S
O
H
I
I
24
PPh3
COOMe
S
S
h
g
i
f
OR
OR
21
OR
22
23
OR
25
COOH
COOMe
COOMe
l
O
O
j
O
k
OH
OR
OH
26
27
5-oxo-15-HETE
4
Scheme 3. Total synthesis of 5-oxo-15-HETE. Reagents and conditions: (a) LiHMDS, 13, THF/HMPA, rt to À78 °C to rt, 12 h, 97%; (b) Pd/C, H2, ethanol, rt, 5 h, 100%; (c) H5IO6,
THF/Ether, rt, over night, 94%; (d) 17, Benzene, 65 °C, 12 h, 85%; (e) 19, LiHMDS, THF/HMPA, À78 °C to rt, 2 h, 93%; (f) AcOH/THF/H2O (3/2/1), rt, 8 h, 82%; (g) TPP, Im, I2,
CH2Cl2, 0 °C to rt, 3 h, 90%; (h) TPP, CH3CN, 60 °C, 3 days, 100%; (i) 23, n- BuLi, THF/HMPA, 0 °C to À78 °C to rt, 2 h, 85%; (j) PhI(OCOCF3)2, CH3OH/H2O, 0 °C, 2 min, 46%; (k)
PPTS, THF/H2O, rt, 2 days, 78%; (l) LiOH, THF/H2O, rt, 1 h, 72%.

P. Patel et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1857–1860
1859
COOH
OH
5-HEDH
15-LO
COOH
OH
COOH
5,15-DiHETE
5-LO
3
O
AA
1
OH
OH
5-oxo-15-HETE
7
5(S)-HETE
4
15-LO
COOH
5-HEDH
O
5-oxo-ETE
2
Scheme 4. Biosynthesis of 5-oxo-15-HETE.
remove red blood cells, centrifugation over Ficoll–Paque to remove
mononuclear cells and hypotonic lysis to remove any remaining
red blood cells.2 The neutrophils were suspended in phosphate-
buffered saline (PBS) and loaded with indo-1 acetoxymethyl ester
(Invitrogen, as previously described).13 Five minutes prior to data
acquisition, CaCl2 and MgCl2 were added to give final concentra-
tions of 1.8 and 1 mM, respectively. Fluorescence was measured
using a spectrofluorometer with a temperature-controlled cuvette
holder equipped with a magnetic stirrer. After stabilization of the
baseline, various concentrations of 5-oxo-ETE or 5-oxo-15-HETE
were added, followed 1.5 min later by 5-oxo-ETE (10 nM) to eval-
uate receptor desensitization by the initial agonist. After another
0.5 min, digitonin (0.1% final concentration) was added to permit
measurement of the maximal fluorescence.
The response of neutrophils to 10 nM 5-oxo-ETE was unaffected
by the addition of vehicle 90 s earlier (Fig. 1A, top panel). In con-
trast, prior addition of 100 nM 5-oxo-15-HETE, which itself
strongly stimulated Ca2+ mobilization, completely blocked the
response to 5-oxo-ETE (Fig. 1A, lower panel). The ability of 5-
oxo-15-HETE to desensitize neutrophils to 5-oxo-ETE is consistent
with its activity being mediated by the 5-oxo-ETE receptor.
The concentration–response relationships for 5-oxo-ETE and
5-oxo-15-HETE are shown in Figure 1B. 5-Oxo-15-HETE (EC50, of
previous finding that biologically derived 5-oxo-15-HETE was
about one-sixth as potent as 5-oxo-ETE and about 75% as effica-
cious in inducing calcium mobilization in neutrophils.8 This sug-
gests that the lower potency we observed for 5-oxo-15-HETE in
inducing this response was not due to its isomerization to its less
potent 8-trans isomer. Moreover, we found that 5-oxo-15-HETE
could be stored at À80 °C for up to three years without any signif-
icant degradation, as evaluated by reversed-phase-HPLC.
Despite its lower potency in stimulating Ca2+ mobilization in
neutrophils, we previously found that 5-oxo-15-HETE is approxi-
mately equipotent with 5-oxo-ETE in stimulating both neutrophil8
and eosinophil5 chemotaxis, but is only about 40–60% as effica-
cious, consistent with its being a partial agonist. It is possible that
the increased relative potency of 5-oxo-15-HETE in the longer 2 h
chemotaxis assay could be due to resistance to metabolism
(compared to 5-oxo-ETE) by cytochrome P450 and 5-HEDH, which
reduces 5-oxoeicosanoids to 5(S)-hydroxy products.2 Both path-
ways result in dramatic losses in biological activity.22 Thus both
5-oxo-ETE and 5-oxo-15-HETE could play roles as inflammatory
mediators in allergic diseases such as asthma, which are character-
ized by tissue infiltration of eosinophils.
The biosynthesis of 5-oxo-15-HETE is more complicated than
that of 5-oxo-ETE, as it requires an additional step to introduce
the 15-hydroxyl group. It could arise in vivo either by the
15-LO-catalyzed oxidation of 5-oxo-ETE or by the 5-HEDH-cata-
lyzed oxidation of 5,15-diHETE, a product of the combined actions
of 5-LO and 15-LO (Scheme 4). Eosinophils contain all of these
56 10 nM) was about one-eighth as potent as 5-oxo-ETE (EC50
7
,
4 nM) in inducing Ca2+ mobilization and also displayed lower
efficacy, as it induced a maximal response 34 7% lower than that
for 5-oxo-ETE. These results compare very favorably with our
A
B
100
Dig
80
60
5oETE
Veh
5oETE
40
Dig
5o-15h
20
5oETE
5o-15h
0
0
1
2
3
10-10 10-9 10-8 10-7 10-6 10-5
Concentration (M)
Time (min)
Figure 1. Effects of 5-oxo-15-HETE and 5-oxo-ETE on Ca2+ mobilization in human neutrophils. (A) Either vehicle (top panel) or 5-oxo-15-HETE (5o–15h; 100 nM; bottom
panel) were added to a suspension of indo-1-loaded neutrophils whil measuring fluorescence as described above. After 1.5 min, 5-oxo-ETE (5oETE; 10 nM) was added,
followed by digitonin (dig, 0.1%). (B) Concentration–response curves for 5-oxo-ETE (o) and 5-oxo-15-HETE (ꢁ) The results are expressed as percentages of the response to
1 lM 5-oxo-ETE; n = 4).

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P. Patel et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1857–1860
enzymes and produce small amount of both 5-oxo-ETE and 5-oxo-
15-HETE when incubated with calcium ionophore and AA.5
However, cysteinyl leukotrienes are the major 5-LO products
formed by these cells. 5-Oxo-15-HETE could also be synthesized
by transcellular biosynthesis, with 5-HETE or 5-oxo-ETE being
provided by inflammatory cells and then further metabolized by
15-LO in other cells such as epithelial cells, which contain both
15-LO23 and 5-HEDH,24 but little 5-LO activity.
The availability of authentic 5-oxo-15-HETE with the correct
stereochemistry and double bond configuration will be an
important asset in determining the biological role of this proin-
flammatory mediator.
References and notes
1. Falck, J. R.; Manna, S.; Jacobson, H. R.; Estabrook, R. W.; Chacos, N.; Capdevila, J.
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4. Zhang, Y.; Styler, A.; Powell, W. S. J. Leukoc. Biol. 1996, 59, 847.
5. Powell, W. S.; Chung, D.; Gravel, S. J. Immunol. 1995, 154, 4123.
6. Praticò, D.; Ferro, D.; Iuliano, L.; Rokach, J.; Conti, F.; Valesini, G.; FitzGerald, G.
A.; Violi, F. Blood 1999, 93, 3401.
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283, 11234.
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268, 9280.
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156, 336.
Acknowledgments
10. O’Flaherty, J. T.; Taylor, J. S.; Thomas, M. J. J. Biol. Chem. 1998, 273, 32535.
11. Powell, W. S.; Gravel, S.; Khanapure, S. P.; Rokach, J. Blood 1999, 93, 1086.
12. Schwenk, U.; Schroder, J. M. J. Biol. Chem. 1995, 270, 15029.
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Eur. J. Biochem. 1996, 236, 1003.
This work was supported by grants from the National Heart,
Lung, And Blood Institute (J.R. Award Number R01HL081873) and
the Canadian Institutes of Health Research (W.S.P. MOP-6254).
The Meakins-Christie Laboratories—MUHC-RI are supported in part
by a Center grant from Le Fonds de la Recherche en Santé du
Québec as well as by the J. T. Costello Memorial Research Fund.
J.R. also wishes to acknowledge the National Science Foundation
for the AMX-360 (Grant Number CHE-90-13145) and Bruker 400
MHz (Grant Number CHE-03-42251) NMR instruments. The
content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Heart, Lung,
And Blood Institute or the National Institutes of Health.
18. Czech, W.; Barbisch, M.; Tenscher, K.; Schopf, E.; Schroder, J. M.; Norgauer, J. J.
Invest. Dermatol. 1997, 108, 108.
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Rokach, J.; Powell, W. S. J. Pharmacol. Exp. Ther. 2008, 325, 698.
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Supplementary data
24. Erlemann, K. R.; Cossette, C.; Gravel, S.; Lesimple, A.; Lee, G. J.; Saha, G.; Rokach,
J.; Powell, W. S. Free Radic. Biol. Med. 2007, 42, 654.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.032.