Synthesis of 14,15-EET from Arachidonic Acid Using Urea-Hydrogen Peroxide as the Oxidant

Article abstract of DOI:10.1080/00397911.2014.956369

14,15- Epoxyeicosatrienoic acid (14,15-EET) is an endogenous bioactive lipid with pharmacological benefits in multiple cardiovascular diseases. We describe here a practical synthesis of 14,15-EET from arachidonic acid using urea-hydrogen peroxide (UHP) as the oxidant.

Full text of DOI:10.1080/00397911.2014.956369

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Synthesis of 14,15-EET From Arachidonic
Acid Using Urea–Hydrogen Peroxide as
the Oxidant
Fuchun Xie a b , Bingbing X. Li a b , Nabil J. Alkayed c d & Xiangshu
Xiao a b
a Program in Chemical Biology, Department of Physiology and
Pharmacology , Oregon Health and Science University , Portland ,
Oregon , USA
b Knight Cancer Institute , Oregon Health and Science University ,
Portland , Oregon , USA
c Department of Anesthesiology and Perioperative Medicine , Oregon
Health and Science University , Portland , Oregon , USA
d Knight Cardiovascular Institute , Oregon Health and Science
University , Portland , Oregon , USA
Accepted author version posted online: 10 Sep 2014.Published
online: 20 Oct 2014.
To cite this article: Fuchun Xie , Bingbing X. Li , Nabil J. Alkayed & Xiangshu Xiao (2014): Synthesis
of 14,15-EET From Arachidonic Acid Using Urea–Hydrogen Peroxide as the Oxidant, Synthetic
Communications: An International Journal for Rapid Communication of Synthetic Organic Chemistry,
DOI: 10.1080/00397911.2014.956369
To link to this article: http://dx.doi.org/10.1080/00397911.2014.956369
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Synthetic Communications1, 0: 1–6, 2014
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2014.956369
SYNTHESIS OF 14,15-EET FROM ARACHIDONIC
ACID USING UREA–HYDROGEN PEROXIDE
AS THE OXIDANT
Fuchun Xie,1,2 Bingbing X. Li,1,2 Nabil J. Alkayed,3,4 and
Xiangshu Xiao1,2
1Program in Chemical Biology, Department of Physiology and
Pharmacology, Oregon Health and Science University, Portland,
Oregon, USA
2Knight Cancer Institute, Oregon Health and Science University, Portland,
Oregon, USA
3Department of Anesthesiology and Perioperative Medicine, Oregon Health
and Science University, Portland, Oregon, USA
4Knight Cardiovascular Institute, Oregon Health and Science University,
Portland, Oregon, USA
GRAPHICAL ABSTRACT
Abstract 14,15- Epoxyeicosatrienoic acid (14,15-EET) is an endogenous bioactive lipid
with pharmacological benefits in multiple cardiovascular diseases. We describe here a
practical synthesis of 14,15-EET from arachidonic acid using urea–hydrogen peroxide
(UHP) as the oxidant.
Keywords Arachidonic acid; 14,15-EET; epoxidation; urea–hydrogen peroxide
INTRODUCTION
Epoxyeicosatrienoic acids (EETs) are a group of endogenous bioactive
lipids synthesized from the precursor arachidonic acid (1, Scheme 1) by cytochrome
P450 (CYP) epoxygenases.[1] Depending on the regiochemistry of epoxidation on the
Received July 9, 2014.
Address correspondence to Xiangshu Xiao, Department of Physiology and Pharmacology, Oregon
Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA. E-mail:
xiaoxi@ohsu.edu
Color versions of one or more of the figures in the article can be found online at www.tandfonline.
com/lsyc.
1

2
F. XIE ET AL.
Scheme 1. Synthesis of 14,15-EET (4) from arachidonic acid (1).
double bonds, four different regioisomeric EETs, 5,6-EET, 8,9-EET, 11,12-EET, and
14,15-EET, can be generated from 1 by different CYP isoforms.[1,2] We and others
have shown that EETs display a wide range of biological activities including anti-
inflammatory, proangiogenic, and vasodilatory effects.[3–6] Among the four
regioisomers, 14,15-EET (4, Scheme 1) has been shown to be one of the more potent
mediators of their cardiovascular-protective effects.[7,8] To reap the pharmacological
benefits of 4 towards cardiovascular disease, intensive efforts have been undertaken
to investigate its structure–activity relationship (SAR) and improve its chemical and
metabolic stability.[9]
One of the limitations on the investigation of the SAR of 4 is that its chemical
synthesis is not trivial. Two primary methods have been developed to prepare 4 from
1, which contains all the carbon atoms present in 4. The first one involves nonselec-
tive epoxidation of the olefins present in 1 by 3-chloroperoxybenzoic acid
(MCPBA).[10] The tedious purification of 4 from other isomers, along with 1, and
the necessary inclusion of radioactive arachidonic acid make this method imprac-
tical. Alternatively, Corey et al. designed an elegant and highly regioselective syn-
thesis of 4[11] by taking advantage of the unique conformation of 1.[12] In this
latter method, an anhydrous ethereal solution of 90% H2O2 was required.[11] Because
90% H2O2 is explosive and no longer commercially available, several attempts have
been made to employ commercially available aqueous H2O2 solutions. For example,
Perrier et al. described a modified procedure using 30% aqueous H2O2 to prepare 4
from 1.[13] However, Ryan et al. later reported that this method did not consistently
generate desired product 4 with a reasonable yield.[14] Therefore, they developed
another modified procedure using a solution of LiOH.H2O in 50% H2O2 but with
lower overall yield (30–35%).[14]
RESULTS AND DISCUSSION
We hypothesized that urea–hydrogen peroxide (UHP) adduct would be a more
suitable surrogate for the anhydrous and hazardous 90% H2O2 because it can release
oxidative species in organic media.[15] Compared to the highly concentrated H2O2
solution, the UHP solid is easy to handle, safe, and stable at room temperature.

CONVENIENT SYNTHESIS OF 14,15-EET
3
Herein we report our practical and convenient synthesis of 4 from 1 using UHP as
the oxidant.
Our modified procedure is overall similar to Corey’s original method as shown
in Scheme 1. The key was to generate peroxyarachidonic acid (3) that would selec-
tively epoxidize D14,15-double bond presumably under the control of the substrate’s
unique conformation, where the acid group is in close proximity to D14,15-double
bond as revealed by molecular dynamics simulations.[11,12] Thus, acid 1 was first con-
verted into arachidonylimidazole (2) by carbonyldiimidazole (CDI, 1.05 equiv) in
dichloromethane at room temperature. Then the solution containing 2 was slowly
added to a suspension of UHP (20 equiv) in ethyl ether containing 0.01 equiv of lith-
ium imidazolide, which was freshly prepared from imidazole and n-BuLi at 0 ꢀC and
served as a basic catalyst. After a brief period of stirring (3 min), finely powdered
anhydrous KHSO4 (15 equiv) was added and the resulting mixture was stirred for
another 3 min to generate peroxy acid 3. The solid was removed by filtration and
the organic filtrate containing 3 was dried over Na2SO4. During standing at room
temperature, the peroxy acid 3 was spontaneously converted into the epoxy acid
4. This procedure gave a mixture of 1 and 4 in a combined total yield of 90% after
1
silica-gel column chromatography. The specific epoxide proton signals in the H
NMR spectrum at 2.95 ppm (for 4) and allylic protons at 2.05 ppm (for 1) were used
to quantify the ratio of 4:1, which was determined to be 4.82:2 or 12:5 (Figure 1).
Although 1 and 4 could be partially separated by careful silica-gel column chroma-
tography, we found it was much more efficient to purify the methyl ester 5
(Scheme 1). Therefore, the mixture containing 1 and 4 was treated with CDI and
methanol to give their corresponding methyl esters 5 and 6.[16] The pure ester 5
was obtained after silica-gel column chromatography in 52% yield, whose 1H
Figure 1. 1H NMR (400 MHz) spectra of a mixture of 1 and 4 (a), pure 4 (b), and pure 1 (c). The 1H NMR
spectra were collected in CDCl3. The distinct proton signals in 1 and 4 are labeled with # and ꢁ, respect-
ively. The integration values for these protons and –CH3 signals are shown.

4
F. XIE ET AL.
Figure 2. Observed 1H-1H COSY (in bold lines) in 4.
NMR data were identical to previously reported values.[16] Finally, pure 4 was regen-
erated in quantitative yield after saponification of the methyl ester 5 with an aqueous
solution of NaOH.[10] The deficiency of NMR information on 4 in the literature led
1
1
us to further assign its H NMR signals with the aid of H-1H correlation spec-
troscopy (COSY) (Figs. 2 and S1 in the Supplementary Information). The presence
of 4 bis-allylic protons (H7 and H10) at ꢂ 2.80 ppm and a coupling between H3 and
allylic protons at 2.12 ppm unambiguously established the regiochemistry of 4 as the
14,15-epoxide. The complete assignment of the protons is shown in Fig. S1.
In conclusion, we have developed a convenient and practical procedure to
synthesize 4 from 1 using UHP as the oxidant with an overall yield of 46%. UHP
is commercially available, inexpensive, safe, and easy to handle. This synthetic
procedure should further facilitate SAR and biological studies of 4.
EXPERIMENTAL
The solvents used for reactions were purified from the Glass Contout solvent
1
purification system. All H and 13C NMR spectra were recorded on Bruker Avance
400-MHz spectrometer using CDCl3 as solvent and the chemical shifts of the
residual CHCl3 (d 7.24) was taken as reference. The following abbreviations were
used to describe the splitting pattern of individual peaks if applicable: s ¼ singlet,
d ¼ doublet, t ¼ triplet, q ¼ quartet, and m ¼ multiplet. The coupling constants (J)
are reported in hertz (Hz). Silica-gel flash chromatography was performed using
230- to 400-mesh silica gel (EMD).
(ꢃ)-Methyl 14,15-Epoxyeicosa-cis-5,8,11-trienoate (5)
CDI (77 mg, 0.476 mmol) was added to a stirred solution of 1 (138 mg,
0.45 mmol) in CH2Cl2 (3 mL) under argon at room temperature for 20 min. The
resulting solution was then added dropwise over 3 min to a stirred suspension of
UHP (846 mg, 9 mmol) in Et2O (15 mL) containing 0.01 equiv of lithium imidazolide
at 0 ꢀC. The reaction mixture was stirred for 3 min, when CH2Cl2 (7 mL) and finely
powdered anhydrous KHSO4 (0.92 g, 6.74 mmol) were added. The resulting mixture
was vigorously stirred for another 3 min. The solid was removed by filtration and the
filtrate was dried over Na2SO4 overnight. The solvents were removed and the residue
was purified by flash column chromatography, eluting with CH2Cl2-MeOH (20:1) to
give a ratio of ꢂ 12:5 mixture of product 4 and 1 (127 mg, 90%) as a colorless oil.
CDI (133 mg, 0.82 mmol) was added to a stirred solution of this mixture (127 mg,

CONVENIENT SYNTHESIS OF 14,15-EET
5
0.40 mmol) in CH2Cl2 (2 mL) under argon at room temperature. The mixture was
stirred for 30 min, when MeOH (170 mL, 4.1 mmol) was added and the reaction mix-
ture was stirred for another 4 h at room temperature. The solvents were removed and
the residue was purified by flash column chromatography, eluting with hexanes–
1
EtOAc (10:1) to give 5 (70 mg, 0.21 mmol, 52%) as a colorless oil. The H NMR
characterization data are identical to the literature values.[16] 1H NMR (400 MHz,
CDCl3) d 5.55–5.41 (m, 2 H), 5.40–5.31 (m, 4 H), 3.67 (s, 3 H), 2.97–2.80 (m,
2 H), 2.86–2.76 (m, 4 H), 2.45–2.36 (m, 1 H), 2.32 (t, J ¼ 7.5 Hz, 2 H), 2.27–2.17
(m, 1 H), 2.10 (q, J ¼ 7.0 Hz, 2 H), 1.70 (quintet, J ¼ 7.5 Hz, 2 H), 1.57–1.41 (m, 4
H), 1.37–1.27 (m, 4 H), 0.90 (t, J ¼ 7.0 Hz, 3 H).
ACKNOWLEDGMENTS
We are grateful for Dr. Sanjiv Kaul for discussions and the Knight
Cardiovascular Institute.
FUNDING
We are thankful for the philanthropic support from Philip and Penny Knight.
SUPPLEMENTAL MATERIAL
Supplemental data for this article can be accessed on the publisher’s website.
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