Free radical oxidation of 15-(S)-hydroxyeicosatetraenoic acid with the Fenton reagent: characterization of an epoxy-alcohol and cytotoxic 4-hydroxy-2E-nonenal from the heptatrienyl radical pathway

Article abstract of DOI:10.1016/j.chemphyslip.2006.02.015

The oxidation of (5Z,8Z,11Z,13E,15S)-15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-(S)-HETE, 1a) with the Fenton reagent (Fe²⁺/EDTA/H₂O₂) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with 75% substrate consumption after 1 h to give a mixture of products, one of which was identified as (2E,4S)-4-hydroxy-2-nonenal (3a, 18% yield). Methylation of the mixture with diazomethane allowed isolation of another main product which could be identified as methyl (5Z,8Z,13E)-11,12-trans-epoxy-15-hydroxy-5,8,13-eicosatrienoate (2a methyl ester, 8% yield). A similar oxidation carried out on (15-²H)-15-HETE (1b) indicated complete retention of the label in 2b methyl ester and 3b, consistent with an oxidation pathway involving as the primary event H-atom abstraction at C-10. Overall, these results support the recently proposed role of 1a as a potential precursor of the cytotoxic γ-hydroxyalkenal 3a and disclose a hitherto unrecognized interconnection between 1a and the epoxy-alcohol 2a, previously implicated only in the metabolic transformations of the 15-hydroperoxy derivative of arachidonic acid.

Full text of DOI:10.1016/j.chemphyslip.2006.02.015

Chemistry and Physics of Lipids 142 (2006) 14–22
Free radical oxidation of 15-(S)-hydroxyeicosatetraenoic
acid with the Fenton reagent: characterization of an
epoxy-alcohol and cytotoxic 4-hydroxy-2E-nonenal
from the heptatrienyl radical pathway
P. Maninia, S. Brigantib, C. Fabbrib, M. Picardob, A. Napolitanoa,∗, M. d’Ischiaa
a
Department of Organic Chemistry and Biochemistry, University of Naples “Federico II”, Via Cinthia 4, I-80126 Naples, Italy
b
Institute of Dermatology S. Gallicano, Via S. Gallicano 25a, 00153 Rome, Italy
Received 25 October 2005; received in revised form 17 February 2006; accepted 21 February 2006
Available online 20 March 2006
Abstract
The oxidation of (5Z,8Z,11Z,13E,15S)-15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-(S)-HETE, 1a) with the Fenton reagent
(Fe2+/EDTA/H2O2) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with 75% substrate consumption after
1 h to give a mixture of products, one of which was identified as (2E,4S)-4-hydroxy-2-nonenal (3a, 18% yield). Methylation of
the mixture with diazomethane allowed isolation of another main product which could be identified as methyl (5Z,8Z,13E)-11,12-
trans-epoxy-15-hydroxy-5,8,13-eicosatrienoate (2a methyl ester, 8% yield). A similar oxidation carried out on (15-2H)-15-HETE
(1b) indicated complete retention of the label in 2b methyl ester and 3b, consistent with an oxidation pathway involving as the
primary event H-atom abstraction at C-10. Overall, these results support the recently proposed role of 1a as a potential precursor
of the cytotoxic ␥-hydroxyalkenal 3a and disclose a hitherto unrecognized interconnection between 1a and the epoxy-alcohol 2a,
previously implicated only in the metabolic transformations of the 15-hydroperoxy derivative of arachidonic acid.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Arachidonic acid; Fenton reagent; 4-hydroxynonenal; Lipid peroxidation; Deuterium labeling
1. Introduction
(5Z,8Z,11Z,13E,15S)-15-Hydroxy-5,8,11,13-eicosa-
tetraenoic acid (15-(S)-HETE, 1a) is a major product of
lipid metabolism which is generated via the regiospe-
Abbreviations: 15-(S)-HETE, (5Z,8Z,11Z,13E,15S)-15-hydroxy-
cific and stereospecific oxidation of arachidonic acid
5,8,11,13-eicosatetraenoic acid; PPAR, peroxysome proliferator-
catalyzed by 15-lipoxygenase, followed by a reduction
activated receptor; 13-(S)-HODE, (9Z,11E,13S)-13-hydroxy-9,11-
¨
step (Kuhn, 1996). Additional routes to 1a involve
octadecadienoic acid; EDTA, ethylendiaminetetraacetic acid; BSTFA,
bis(trimethylsilyl)trifluoroacetamide; IBX, o-iodoxybenzoic acid;
TMS, tetramethylsilane;COSY, correlationspectroscopy;HMQC, het-
eronuclear multiple quantum coherence; HMBC, heteronuclear multi-
ple bond correlation; ESI(+)/MS, electrospray positive ion mode mass
spectrometry; HR, high resolution; EI, electron impact
non-enzymatic peroxidation processes (Nemann and
Khenkin, 1997) in which, however, the R enantiomer is
also formed to a comparable extent, along with other
positional isomers.
In mammalian cells 1a acts as a physiological medi-
ator, being involved in signal transduction and gene
∗
Corresponding author. Tel.: +39 081674133; fax: +39 081674393.
E-mail address: alesnapo@unina.it (A. Napolitano).
0009-3084/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2006.02.015

P. Manini et al. / Chemistry and Physics of Lipids 142 (2006) 14–22
15
expression. It has been shown to be chemotactic and
chemokinetic for polymorphonuclear leukocytes and
vascularsmoothmusclecells(Fischeretal., 1992). Addi-
tional biological activities of 1a include stimulation of
insulin secretion by pancreatic tissue (Laychock, 1985),
induction of phosphorylation of cytoskeletal proteins,
enhancement of surface expression of integrin receptors
and platelet aggregation in tumor cells (Buchanan et al.,
1998), and a role as second messenger in angiotensin-II
induced aldosterone production (Natarajan et al., 1988).
Moreover, 1a is an important ligand to peroxysome
proliferator-activated receptor (PPAR)-␥, (Nagy et al.,
1998; Marx et al., 1999) a nuclear receptor that has
been implicated in the modulation of critical aspects
of development and homeostasis, including adipocyte
differentiation, glucose metabolism, and macrophage
development and function. Binding of 1a to PPAR-␥
nuclear receptors has been shown to induce apoptosis
(Chen et al., 2003).
In addition to being biologically active per se, 1a
is the key precursor to a range of potent lipid medi-
ators, including the lipoxins, which arise by enzy-
matic oxidation mediated by 5-lipoxygenase (Chavis
et al., 1992). This latter process has been extensively
investigated following recognition of the key role of
lipoxins in cell–cell interactions and the regulation of
inflammatory signals. Very little is known, by contrast,
about non-enzymatic oxidative transformations of 1a
induced by reactive oxygen species. Because such path-
ways may contribute to affect the biological activity of
this important lipid mediator in settings of oxidative
stress, an understanding of the nature of the products
formed by oxidation of 1a under biomimetic conditions
is of considerable interest. A recent study (Schneider
et al., 2004) has shown that 1a, like (9Z,11E,13S)-
13-hydroxy-9,11-octadecadienoic acid (13-(S)-HODE)
(Sun and Salomon, 2004), can undergo autoxidative
transformation at 37 ◦C as dry film to give cytotoxic
4-hydroxynonenal as a prominent polar product. The dis-
covery of the unexpected tendency of ω6-hydroxy fatty
acidsto undergooxidativefragmentation disclosed novel
possible pathways of formation of bioactive aldehydes
in vivo.
ical conditions different from those reported in previ-
ous studies (Schneider et al., 2004; Sun and Salomon,
2004).
2. Materials and methods
2.1. Materials
Arachidonic acid (99%) and soybean lipoxidase
(linoleate oxygen reductase, E.C. 1.13.11.12) type IB
were from Sigma. Hydrogen peroxide (water solu-
tion, 33%), sodium borohydride, sodium borodeuteride
and bis(trimethylsilyl)trifluoroacetamide (BSTFA) were
from Aldrich Chemie; Fe(NH4)2(SO4)2·6H2O was from
Carlo Erba; ethylenediaminetetraacetic acid (EDTA)
was from Fluka. Organic solvents were HPLC qual-
ity; phosphate buffer (0.1 M, pH 7.4) and borate
buffer (0.1 M, pH 9.0) were treated with Chelex-100
resin before use to remove transition metal contami-
nants.
(5Z,8Z,11Z,13E,15S)-15-Hydroxy-5,8,11,13-eicosa-
tetraenoic acid (1a, 15-(S)-HETE) was synthesized
and purified as described (Napolitano et al., 2002). 1a
methyl ester was obtained by treatment of the free acid
with diazomethane. o-Iodoxybenzoic acid (IBX) was
freshly prepared from 2-iodobenzoic acid according to
a reported procedure (Frigerio et al., 1999).
Diazomethane was prepared by reaction of N-methyl-
N-nitroso-p-toluenesulfonamide in ethanolic KOH and
collected in peroxide-free ether in a dry ice/acetone bath.
Caution! Diazomethane is explosive and must be kept at
−20 ◦C.
IR spectra were obtained using a Nicolet 5700 FT-
IR spectrophotometer. Optical rotations were measured
using a JASCO P-1010 polarimeter. NMR spectra were
recorded in CDCl3 with a Bruker DRX-400 MHz instru-
ment. 1H and 13C NMR spectra were recorded at
400.1 and 100.6 MHz, respectively, using tetramethyl-
In the present paper, we have investigated the reac-
tion of 1a with Fe2+/ethylendiaminetetraacetic acid
(EDTA)/H2O2 (the Fenton reagent), which is widely
used to model non-enzymatic oxidative processes. Aim
of the work was to provide a complete spectral char-
acterization of the main oxidation products of this
important lipid mediator, and to assess its tendency
to undergo oxidative fragmentation to cytotoxic ␥-
hydroxyalkenals under physiologically relevant free rad-
1
silane (TMS) as the internal standard. H–1H COSY,
1H–13C HMQC and 1H–13C HMBC NMR experi-
ments were run at 400.1 MHz using standard pulse
programs.
Analytical and preparative TLC analyses were per-
formedonF254 silicagelplates(0.25and0.5 mm, respec-
tively) using cyclohexane/ethyl acetate 70:30 (v/v)

16
P. Manini et al. / Chemistry and Physics of Lipids 142 (2006) 14–22
2.3. Synthesis of (15-2H,5Z,8Z,11Z,13E)-
15-hydroxy-5,8,11,13-eicosatetraenoic acid (1b)
(eluant A) and chloroform/methanol 95:5 (v/v) (eluant
B). Ce(SO4)2 (0.05 M in 10% H2SO4) and iodine were
used for product detection on TLC plates.
Electrospray positive ion mode mass spectrometry
(ESI(+)/MS) were run on a Waters ZQ quadrupole mass
spectrometer. High resolution (HR) ESI mass spec-
tra were obtained with a Finnegan MAT 90 instru-
ment. GC/MS was carried out on a Trace GC Ultra-
ThermoFinnigan instrument coupled with a Trace DSQ-
ThermoFinnigan mass spectrometer. Helium was the
carrier gas with a 1 mL/min flow rate. Data were
processed using QualBrowser software. The following
analytical conditions were used: 30 m cross-bond 5%
diphenylpolysiloxane column (0.25 mm i.d., 0.25 ␮m
df). Temperature program: 110 ◦C, hold time 0.8 min;
up to 280 ◦C, rate 5 ◦C/min, hold time 40 min. The inlet,
transfer line, and ion source were taken at 280, 250, and
220 ◦C, respectively. The acquisition started 15 min after
the injection (solvent delay 15 min), with scan time set
at 0.62 s, sampling at 1.6 scans/s.
A solution of 4 (100 mg, 0.31 mmol) in CHCl3 (1 mL)
was added to 0.1 M phosphate buffer, pH 7.4 (282 mL)
and treated under vigorous stirring at room temperature
with sodium borodeuteride (214 mg, 5.1 mmol). After
5 min the mixture was acidified to pH 3 with HCl and
extracted with CHCl3 (3 × 150 mL). The organic lay-
ers were combined, treated with anhydrous sodium sul-
fate, and dried under reduced pressure to afford pure 1b
(80 mg, 80% yield).
1b: 1H NMR (400 MHz, CDCl3), δ (ppm): 0.89 (3H, t
J = 7.2 Hz, H-20), 1.2–1.5 (4H, m, H-18, H-19), 1.5–1.6
(4H, m, H-3, H-17), 1.71 (2H, t J = 7.2 Hz, H-16), 2.11
(2H, t J = 7.2 Hz, H-4), 2.32 (2H, t J = 7.2 Hz, H-2), 2.81
(2H, t J = 5.6 Hz, H-7), 2.96 (2H, t J = 6.4 Hz, H-10),
5.3–5.6 (5H, m, H-5, H-6, H-8, H-9, H-11), 5.70 (1H, d
J = 15.2 Hz, H-14), 6.00 (1H, t J = 10.8 Hz, H-12), 6.58
(1H, dd J = 15.2, 10.8 Hz, H-13).
Prior to GC/MS analysis, all the samples were
treated with 200 ␮L of ethereal diazomethane solution
to give the corresponding methyl ester derivative. When
required, the samples were treated with 50 mL of a 50%
solution of BSTFA in pyridine and kept at 50 ◦C for
30 min to give the corresponding trimethylsilyl deriva-
tive.
2.4. Isolation of compounds 2a methyl ester and 3a
Compound 1a (100 mg, 0.31 mmol) was dissolved
in CHCl3 (1 mL) and emulsified in 0.1 M phosphate
buffer, pH 7.4 (60 mL). The mixture was treated
under vigorous stirring at room temperature with
Fe(NH4)2(SO4)2·6H2O (62 mg, 0.16 mmol) and EDTA
(59 mg, 0.16 mmol), and the oxidation was started by
additionofhydrogenperoxide(61 ␮L, 0.62 mmol). After
1 h the reaction mixture was extracted with ethyl acetate
(2 × 30 mL), and the organic layers were combined,
dried over sodium sulfate and treated with an ethereal
solution of diazomethane (2 mL). After 15 min the mix-
ture was dried under reduced pressure and the residue
was fractionated by preparative thin layer chromatog-
raphy (eluant A) to give, besides unreacted 1a methyl
ester (Rf = 0.56, eluant A, 26 mg, 25% yield), 2a methyl
ester (Rf = 0.40, eluant A, 8 mg, 8% yield) and 3a
(Rf = 0.32, eluant A, 8 mg, 18% yield) (Gioacchini et
al., 1999).
2a methyl ester: FT-IR (CHCl3) νmax 3500–3200,
1732, 968, 870 cm−1; [␣]d25 = +4.2◦ (c 1.34, CHCl3);
1H NMR (400 MHz, CDCl3), δ (ppm): see Table 1;
13C NMR (100 MHz, CDCl3) δ (ppm): see Table 1;
GC/MS (tetramethylsilane (TMS) derivative) tR
32.4 min; EI m/z (relative intensity) 422 (M+, 15),
407 (M+-CH3, 11), 349 (M+-Si(CH3)3, 78), 281
(M+-CH2CH:CH(CH2)3CO2CH3, 22), 249 (M+-C6
H12OSi(CH3)3, 100), 173 (•C6H12OSi(CH3)3, 44).
ESI(+)/HRMS for C21H35O4 calcd. 351.2535 [M + H]+,
found 351.2529
2.2. Synthesis of (5Z,8Z,11Z,13E)-15-oxo-
5,8,11,13-eicosatetraenoic acid (4)
Compound 1a (140 mg, 0.44 mmol) was dissolved
in ethyl acetate (82 mL), treated with o-iodoxybenzoic
acid (1.13 g, 4.1 mmol) and kept under reflux at 85 ◦C.
After 1 h the mixture was filtered to remove unreacted
o-iodoxybenzoic acid as a white solid and afforded pure
4 (133 mg, 95% yield).
4: FT-IR (CHCl3) νmax 1710 cm−1
;
1H NMR
(O’Flaherty et al., 1994); 13C NMR (100 MHz, CDCl3)
δ (ppm): 14.9 (CH3), 23.4 (CH2), 25.7 (CH2), 27.4
(CH2), 30.5 (CH2), 32.3 (CH2), 32.6 (CH2), 34.2
(CH2), 34.9 (CH2), 41.9 (CH2), 127.2 (CH), 129.2
(2 × CH), 130.1 (CH), 130.4 (2 × CH), 137.3 (CH),
140.4 (CH), 179.6 (C), 202.1 (C); GC/MS (methyl ester
derivative) tR 28.2 min; Electron impact (EI) m/z (rel-
ative intensity) 332 (M+, 15), 317 (M+-CH3, 13), 261
(M+-CH3(CH2)4, 7), 205 (M+-CH:CH(CH2)3CO2CH3,
22), 191 (M+-CH2CH:CH(CH2)3CO2CH3, 31), 181
(M+-CH3(CH2)4COCH:CHCH:CH, 37), 165 (M+-CH:
CHCH2CH:CH(CH2)3CO2CH3, 53), 151 (CH3(CH2)4
COCH:CHCH:CH, 100).

P. Manini et al. / Chemistry and Physics of Lipids 142 (2006) 14–22
17
Table 1
Spectral data of compound 2a methyl ester (CDCl3)
Carbon
δC
δH(mult. J (Hz))
1H–1H COSY
1H–13C HMBC
1
2
3
4
5
6
7
8
175.2
34.2
25.5
–
–
1.70
2.11, 2.32
1.70, 5.38
2.11
2.78
5.38
2.78
2.40
2.88, 5.38
2.40, 3.15
2.88, 5.43
3.15, 5.94
4.12, 5.43
1.30, 1.53, 5.94
–
2.32 (2H, t 7.2)
1.70 (2H, t 7.2)
2.11 (2H, dt 7.2, 5.6)
5.38 (1H, m)
5.38 (1H, m)
2.78 (2H, t 5.6)
5.38 (1H, m)
27.4, 25.5
27.4, 34.2, 130.0
25.5, 34.2, 129.3, 130.0
25.5
27.4
129.3, 130.0
30.5
27.4
129.3
130.0
32.6
130.0
129.3
30.5
60.4
58.2
123.1
136.4
72.7
9
5.38 (1H, m)
2.40 (2H, m)
32.6
10
11
12
13
14
15
58.2, 60.4, 123.1, 129.3, 130.0
30.4, 129.3
123.1, 129.3, 136.4
58.2, 60.4, 72.7
58.2, 72.7
2.88 (1H, dt 5.6, 2.4)
3.15 (1H, dd 7.6, 2.4)
5.43 (1H, dd, 15.6, 7.6)
5.94 (1H, dd 15.6, 6.4)
4.12 (1H, m)
–
16
37.9
1.30 (1H, m)
1.53 (1H, m)
1.53, 4.12
1.30, 4.12
25.8
25.8, 136.4
17
18
19
20
26.6
25.8
23.4
14.8
52.3
1.53 (2H, m)
1.30 (2H, m)
1.30 (2H, m)
0.89 (3H, t 7.2)
3.67 (3H, s)
1.30
1.53
0.89
1.30
–
25.8
14.8, 23.4, 37.9
14.8, 25.8
23.4, 25.8
–
–OCH3
ESI(+)/MS m/z (relative intensity) 173 ([M + CH3]+, 24),
158 ([M + H]+, 100), 140 ([M + H − H2O]+, 6).
2.5. Isolation of compound 2b methyl ester and 3b
Compound 1b was subjected to oxidation with the
Fenton reagent under the same conditions adopted for
1a. After work up and treatment with an ethereal solution
of diazomethane, preparative thin layer chromatography
(eluant A) afforded, besides unreacted 1b methyl ester
(Rf = 0.56, eluant A, 26 mg, 25% yield), 2b methyl ester
(Rf = 0.40, eluant A, 8 mg, 8% yield) and 3b (Rf = 0.32,
eluant A, 8 mg, 18% yield).
3. Results
The reaction of 1a with the Fenton reagent
(0.5 mequiv of the Fe2+/EDTA complex and 2 mequiv of
H2O2) was typically carried out with the lipid emulsified
in 0.1 M phosphate buffer, pH 7.4 (Spiteller and Spiteller,
1997). After 1 h, TLC analysis of the reaction mixture
extracted with ethyl acetate after methylation with dia-
zomethane revealed the presence of a complex mixture
of species, two of which, eluted at Rf = 0.40 (A, posi-
tive to Ce(SO4)2) and Rf = 0.32 (B, λ = 254 nm, positive
to Ce(SO4)2), appeared to be relatively more abundant.
Preparative TLC of a mixture obtained by preparative
scale oxidation of 1a eventually afforded the products in
sufficient amounts and pure form for spectral character-
ization.
2b methyl ester: 1H NMR (400 MHz, CDCl3), δ
(ppm): 0.89 (3H, t J = 7.2 Hz, H-20), 1.30 (5H, m, H-
16, H-18, H-19), 1.53 (3H, m, H-16, H-17), 1.70 (2H,
t J = 7.2 Hz, H-3), 2.11 (2H, dt J = 7.2, 5.6 Hz, H-4),
2.32 (2H, t J = 7.2 Hz, H-2), 2.40 (2H, m, H-10), 2.78
(2H, t J = 5.6 Hz, H-7), 2.88 (1H, dt J = 5.6, 2.4 Hz, H-
11), 3.15 (1H, dd J = 7.6, 2.4 Hz, H-12), 3.67 (3H, s,
–OCH3), 5.38 (4H, m, H-5, H-6, H-8, H-9), 5.43 (1H,
dd J = 15.6, 7.6 Hz, H-13), 5.94 (1H, d J = 15.6 Hz, H-
14); GC/MS (TMS derivative) tR 32.4 min; EI m/z 423
(M+). ESI(+)/HRMS for C21H342HO4 calcd. 352.2598
[M + H]+, found 352.2602.
3b: 1H NMR (400 MHz, CDCl3), δ (ppm): 0.89 (3H, t
J = 7.2 Hz, H-9), 1.2–1.4 (6H, m, H-6, H-7, H-8), 1.5–1.7
(2H, m, H-5), 6.34 (1H, dd J = 15.6, 7.6 Hz, H-2), 6.82
(1H, d J = 15.6 Hz, H-3), 9.60 (1H, d J = 7.6 Hz, H-1);

18
P. Manini et al. / Chemistry and Physics of Lipids 142 (2006) 14–22
1
The H NMR spectrum of product A displayed as
most significant features a group of signals centered at δ
5.38, due to the protons on the skipped cis double bonds,
and two signals at δ 5.43 and 5.94, the latter showing
coupling with a signal at δ 4.12 (1H, m), suggesting an
allylic alcohol moiety with an E configuration at the dou-
ble bond (J = 15.6 Hz).
Scheme 1. Preparation of 1b from 1a.
The 1H–1H COSY spectrum revealed correlation of
the olefin proton at δ 5.43 with a resonance at δ 3.15
which in turn gave a cross peak with a signal at δ 2.88.
The latter two signals were strongly suggestive of pro-
tons on a trans epoxide functionality (J = 2.4 Hz). More-
over, the proton signal at δ 2.88 correlated with a –CH2
resonance at δ 2.40 which was coupled to one of the
cis olefin protons at δ 5.38. In the high field region of
the spectrum two signals at δ 2.78 and 2.11 were clearly
ascribable to bisallylic- and allylic-CH2 protons, respec-
tively.
25
The compound exhibited optical activity ([␣]D
=
+4.2◦(c 1.34, CHCl3)). Close inspection of the 1H NMR
spectrum of 2a methyl ester indicated a single species,
ruling out the presence of a mixture of diastereoiso-
meric trans epoxide derivatives. Unfortunately, attempts
to define the stereochemical features of 2a met with fail-
ure, not was it possible to isolate other diastereoisomers.
However, evidence for the formation of trace amounts of
other diastereoisomeric 11,12-epoxide derivatives was
obtained by 1H NMR analysis of the crude reaction
mixture which revealed the presence of a species fea-
turing two signals at δ 2.93 and 3.15 (J = 2.4 Hz), typical
of a trans epoxide functionality. These signals gave in
the 1H–1H COSY spectrum distinct correlations with an
adjacent allylic alcohol moiety with an E configuration
at the double bond closely similar to those observed for
2a methyl ester.
Product B was identified as (2E)-4-hydroxy-2-
nonenal (3a) by comparison of the spectral properties
with literature data (Gioacchini et al., 1999). The identity
of compound B with 3a was also secured by comparing
its chromatographic and spectral properties with those of
an authentic sample prepared by a synthetic procedure
(Gioacchini et al., 1999).
The 1H–13C HMBC spectrum displayed correlations
between the trans double bond proton resonances at δ
5.43 and 5.94 and the carbon resonances at δ 58.2, due
to the epoxide ring, and δ 72.7, relative to the alco-
holic functionality; the epoxide proton at δ 3.15 and
the carbon resonances at δ 123.1 and 136.4 belong-
ing to the trans double bond; between the methylene
protons at δ 2.11, 2.40, and 2.78 and the carbon sig-
nals at δ 129.3 and 130.0 of the cis double bonds. On
the basis of these data, the product was assigned the
structure of methyl (5Z,8Z,13E)-11,12-trans-epoxy-15-
hydroxy-5,8,13-eicosatrienoate (2a methyl ester). Struc-
tural assignments for 2a methyl ester based on extensive
2D NMR spectral analysis are reported in Table 1.
In accord with this conclusion, the FT-IR spectrum
displayed intense bands in the range 3500–3200 cm−1
consistent with an hydroxyl group; at 968 cm−1(C–H
bending of trans olefins) and at 870 cm−1 typical of the
C–H stretching of trans epoxides (Gardner and Kleiman,
1981). The EI/MS spectrum of the O-TMS derivative
gave a molecular ion peak at m/z 422. Diagnostic frag-
mentation peaks in the EI/MS spectrum corroborated
the structural conclusions based on NMR analysis (see
Fig. 1).
Overall yields of products 2a methyl ester and 3a
accounted for about 35% of reacted 1a; 1H NMR anal-
ysis of other fractions of the mixture indicated that the
remainder of the mixture consisted mainly of hydroxy-,
oxo- and epoxy-derivatives.
To inquire into the mechanism of the Fenton-induced
oxidation and to determine whether the asymmetric cen-
ter at C-15 was affected in the conversion of 1a to
2a and 3a, the reaction was carried out on the 15-
2H derivative of 15-HETE (1b). The latter compound
was synthesized by reaction of 1a with o-iodoxybenzoic
acid (IBX) to give (5Z,8Z,11Z,13E)-15-oxo-5,8,11,13-
eicosatetraenoic acid (4) followed by reduction with
NaBD4, according to a previously reported procedure
(Manini et al., 2005) (Scheme 1).
1H NMR analysis of the main reaction products indi-
cated complete retention of the label, consistent with
structures 2b methyl ester and 3b (Figs. 2 and 3). This
was apparent from (a) the lack of detectable –CHOH sig-
Fig. 1. Origin of salient fragmentation peaks in the EI/MS spectrum
of the O-TMS derivative of compound 2a methyl ester.

P. Manini et al. / Chemistry and Physics of Lipids 142 (2006) 14–22
19
4. Discussion
Formerly regarded as relatively stable products of
lipid peroxidation (Spiteller and Spiteller, 1997), the
ω6-hydroxy derivatives of linoleic acid (13-(S)-HODE)
and arachidonic acid (15-(S)-HETE, 1a) have recently
been proposed to play a role as potential precur-
sors of biologically active lipid compounds (Schneider
et al., 2004; Sun and Salomon, 2004). In a previ-
ous study we showed that 13-(S)-HODE can undergo
free radical oxidation induced by the Fenton reagent
to give 13-oxo-9,11-octadecadienoic acid, 9,10-epoxy-
13-hydroxy-11-octadecenoic acid and 9-hydroxy-13-
oxo-octadecenoic acid (Manini et al., 2005). We have
now extended the study to 1a and have shown
that this important inflammatory mediator can like-
wise undergo free radical oxidation when exposed to
the Fenton reagent to give a collection of products,
the major of which could be isolated and spectrally
characterized.
Fig. 2. Selected region of the 1H NMR spectrum of 2b methyl ester.
Inset shows the same spectral region for the unlabelled compound.
A major outcome of the present study is the iden-
tification of the cytotoxic ␥-hydroxyalkenal 3a, as the
main fragmentation product of 1a under the conditions
of the Fenton-mediated oxidation. Compound 3a is one
of the most prominent cytotoxic products generated dur-
ing lipid peroxidation, which may contribute to various
disease processes secondary to oxidative stress through
covalent adduct formation with proteins and nucleic
acids(Benedettietal., 1980;Cohnetal., 1996;Comporti,
1998; Ji et al., 2001; Nair et al., 1999). The generation of
3a from 1a may therefore represent a pathologically sig-
nificant process that contributes to the accumulation of
cytotoxic aldehydes subsequent to non-enzymatic oxida-
tive lipid transformation in vivo.
1
nals in the region of the H NMR spectrum comprised
between δ 4.0–4.5; (b) the multiplicity of the signals at δ
5.94 (d, J = 15.6 Hz) due to the H-14 proton of 2b methyl
esterandatδ6.82(d, J = 15.6 Hz)duetotheH-3of3b;(c)
the mass spectrometric analysis of the products, giving
pseudomolecular ion peaks consistent with the presence
of one deuterium atom.
Several epoxy alcohols have been described from the
oxidation of arachidonic acid and/or its hydroperoxides
(Chang et al., 1996; Pfister et al., 1998; Pfister et al.,
2003), but only two have so far been structurally charac-
terized by NMR techniques, namely (13R,14R,15S)-13,
14-epoxy-15-hydroxy-5Z,8Z,11Z-eicosatrienoic acid
and
(11S,12R,15S)-11,12-epoxy-15-hydroxy-5Z,8Z,
13E-eicosatrienoic acid (Hamberg et al., 1986); this
latter, which was isolated by oxidative conversion of
arachidonic acid by the fungus Saprolegnia parasitica,
differs from 2a only in the cis geometry of the epoxy
functionality as apparent from the H11-H12 coupling
constant (J = 4.2 Hz versus 2.4 Hz in the case of 2a).
For these epoxy alcohols a biosynthetic origin via
lipoxygenase-mediated conversion of arachidonic
acid to the 15-hydroperoxy derivative was suggested,
followed by the action of a hydroperoxide isomerase
(Pfister et al., 1998). Formation pathways of epoxy
Fig. 3. Selected region of the 1H NMR spectrum of 3b. Inset shows
the same spectral region for the unlabelled compound.

20
P. Manini et al. / Chemistry and Physics of Lipids 142 (2006) 14–22
Scheme 2. Proposed mechanism of formation of compounds 2a and 3a.
alcohol derivatives by oxidation of 1a have not so far
been reported.
Plausible mechanisms for the free radical oxidation
of 1a to 2a and 3a are outlined in Scheme 2.
these positions such as the peroxyl radical II would be
mainly the kinetic products, formed under the relatively
high H atom donor conditions of reaction ensured by
the presence of unreacted 1a during oxidation. H-atom
abstraction from a donor converts peroxyl radical II
to the corresponding hydroperoxide III that could be
engaged with two main reaction routes. A Hock-type
rearrangement of hydroperoxide III seems likely and
is invoked here to account for the generation of 3a
(Schneider et al., 2004; Frimer, 1979). The other route
involvestheFe2+-mediated reduction of IIIto thealkoxyl
radical IV by a Fenton-type mechanism, followed by
intramolecular cyclization and hydrogen abstraction to
give the epoxy-derivative 2a. This latter is akin to that
proposed for the formation of 14,15-leukotriene A4 by
cyclization of the 15-alkoxy derivative generated in the
hematin-catalyzed decomposition of 15-hydroperoxy
One route involves HO•-induced H-atom abstraction
at C-10 carbon, leading to formation of the highly
delocalized heptatrienyl radical I. Subsequent coupling
with oxygen would give rise to peroxyl radicals. The
factors that govern the partitioning of oxygen to the
several possible reactive sites of the heptatrienyl radical
I and the relevance of such factors to product distribu-
tion have been addressed recently (Pratt et al., 2003).
Based on theoretical analysis and model studies, it was
shown that a heptatrienyl radical related to I would
display the greatest spin densities on the inner positions,
corresponding to the C-10 and C-12 positions in I. This
suggests that products resulting from oxygen addition at

P. Manini et al. / Chemistry and Physics of Lipids 142 (2006) 14–22
21
derivative of arachidonic acid (Schreiber et al., 1986).
5. Conclusions
Other evolution pathways of the radical IV should also
be considered involving coupling with oxygen to give
eventually ␣-or ␥-hydroxyl/oxo epoxides by mecha-
nisms similar to those described for the iron- (Gardner
and Kleiman, 1981) and hematin- (Dix and Marnett,
1985) induced decomposition of 13-hydroperoxy-
9,11-octadecadienoic acid (HPODE). Evidence for the
formation of epoxy and hydroxy derivatives of 1a as
minor constituents of the Fenton oxidation mixture was
obtained by NMR analysis.
Analternativerouteto2ainvolveshomolyticattackof
peroxyl radical intermediates to 1a followed by decom-
position of the resulting radical. Such processes are
rather common and have been invoked in the epoxi-
dation of the unsaturated components of plasmalogens
(Murphy, 2001; Stadelmann-Ingrand et al., 2001) and of
cholesterol linoleate (Spiteller, 2002). The preferential
formation of the 11,12 epoxide 2a observed under the
reaction conditions examined may be accounted for in
terms of the higher stability of the allylic radical result-
ing from addition at the 11- position. Despite the rather
poor mass balance, 2a and 3a represent the most sig-
nificant components of the oxidation mixture of 1a and
are likely to reflect the main reaction routes. All other
components of the reaction mixture were present in very
low amounts and could not be identified despite several
efforts.
This study represents the first investigation of the
reactivity of 1a with the Fenton reagent. We have shown
that 1a may be an important source of the cytotoxic
aldehyde 3a by free radical oxidation processes under
conditions of physiological relevance, thus confirming
previous observations relating to the transformation of
1aunderautoxidativeconditions(Schneideretal., 2004).
In addition, we have been able to demonstrate that 1a
can be converted to a signficant extent to the epoxy
alcohol 2a. Although there is a vast literature on the
formation of epoxy alcohols in mammalian systems by
both enzymatic and non-enzymatic routes from fatty
acid hydroperoxides (Chang et al., 1996; Pfister et al.,
1998; Pfister et al., 2003), these compounds have never
been related to the oxidative metabolism of 1a. The
physiological significance of 2a and its isomers is also
an attractive issue for future studies. It may be rel-
evant to mention here the identification of 11,12,15-
trihydroxyeicosatrienoic acid, produced by hydrolysis
of the corresponding epoxy alcohols, as endothelium
derived relaxing factor of rabbit aorta (Pfister et al., 1998;
Pfister et al., 2003), whereby a possible involvement of
the transformation products of 2a in the mechanisms of
regulation of the vascular tone may be suggested.
Acknowledgments
The reaction paths proposed in Scheme 2 are consis-
tent with the observed retention of deuterium in products
2b and 3b derived from oxidation of 1b. This finding
clearly indicates a significant change of mechanism with
respect to the oxidation of 13-(S)-HODE: whereas in
the latter case H-atom abstraction at the allylic -CHOH
center was the prevailing reaction channel, as evidenced
fromappreciableproton–deuteriumexchangeandlossof
optical activity of the epoxy alcohol product (Manini et
al., 2005), in the case of 1a this route would hardly com-
pete with oxidation at the bis-allylic C-10 center. This
could be explained considering that the C–H bond dis-
sociation enthalpy (BDE) associated with the formation
of a heptatrienyl radical such as I is ca. 9 kcal/mol lower
than that associated with the formation of a pentadienyl
radical such as that centered on the C-8 of 13-(S)-HODE
(Pratt et al., 2003).
On this basis, it is concluded that all products isolated
by oxidation of 1a retain the original S configuration at
C-15 of the parent lipid. This is in accord with the results
oftheprevious study onthegeneration of 3a by oxidation
of 1a, in which carbon chain cleavage was shown to leave
the stereochemistry at C-15 unaffected (Schneider et al.,
2004).
This study was carried out in the frame of MIUR
(“Natural compounds and synthetic analogs with anti-
tumor activity”, PRIN 2003) and Ministry of Health
(IFO no.125) projects. We thank the “Centro Interdi-
partimentalediMetodologieChimico-Fisiche”(CIMCF,
University of Naples Federico II) for NMR and mass
facilities. We thank Mrs. Silvana Corsani for technical
assistance.
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