Chemistry of nitrated lipids: Remarkable instability of 9-nitrolinoleic acid in neutral aqueous medium and a novel nitronitrate ester product by concurrent autoxidation/nitric oxide-release pathways

Article abstract of DOI:10.1021/jo801364v

(Chemical Equation Presented) Despite the mounting interest in nitrolinoleic acids and related nitrated polyunsaturated fatty acids as a novel class of bioactive signaling lipids, their chemistry and metabolic fate have remained poorly elucidated. Herein, we report an expedient nitroselenenylation/ oxidation route to 9-nitrolinoleic acid (1) and 10-nitrolinoleic acid (2), which enabled comparative product studies under physiologically relevant conditions. Under biomimetic conditions, 1 decayed at an unusually fast rate to give the hydroxy-, keto-, and nitronitrate ester derivatives 3, 4, and 5 as main products, identified by ESI-MS and 2D NMR spectroscopy, including ¹H,¹⁵N HMBC experiments on the ¹⁵N-labeled derivatives. The 13-nitrato functionality in 5 suggested partitioning of 1 between concurrent peroxidation and nitric oxide (NO)-release pathways. Lipid 2 decayed at a much slower rate giving only the hydroxynitro derivative 6 as an isolable product. Diphenylpicrylhydrazide (DPPH) radical quenching experiments and DFT computations concurred to support a higher H-atom donating ability of 1 versus 2, due to more effective stabilization of the resulting pentadienyl radical by the terminal nitro group. The markedly different stability of isomeric nitrolinoleic acids disclosed in the present study may provide an explanation for the previous identification of 2, but not 1, in body fluids and offers a key for future insights into the biological activities of nitrated lipids.

Full text of DOI:10.1021/jo801364v

Chemistry of Nitrated Lipids: Remarkable Instability of
9-Nitrolinoleic Acid in Neutral Aqueous Medium and a Novel
Nitronitrate Ester Product by Concurrent Autoxidation/Nitric
Oxide-Release Pathways
Paola Manini,† Luigia Capelli,† Samantha Reale,‡ Marianna Arzillo,†,§ Orlando Crescenzi,§
Alessandra Napolitano,† Vincenzo Barone,§,| and Marco d’Ischia*,†
Department of Organic Chemistry and Biochemistry, UniVersity of Naples Federico II, Via Cinthia 4,
I-80126 Naples, Italy, Dipartimento di Chimica, Ingegneria Chimica e Materiali, UniVersita` degli Studi di
L’Aquila, Via Vetoio Coppito II, I-67100 Coppito, L’Aquila, “Paolo Corradini” Department of Chemistry
and INSTM, UniVersity of Naples Federico II, Via Cinthia 4, I-80126 Naples, Italy, and Institute for
Physico-Chemical Processes (IPCF)-CNR, Via G. Moruzzi 1, I-56124 Pisa, Italy
dischia@unina.it
ReceiVed June 23, 2008
Despite the mounting interest in nitrolinoleic acids and related nitrated polyunsaturated fatty acids as a
novel class of bioactive signaling lipids, their chemistry and metabolic fate have remained poorly elucidated.
Herein, we report an expedient nitroselenenylation/oxidation route to 9-nitrolinoleic acid (1) and 10-
nitrolinoleic acid (2), which enabled comparative product studies under physiologically relevant conditions.
Under biomimetic conditions, 1 decayed at an unusually fast rate to give the hydroxy-, keto-, and
nitronitrate ester derivatives 3, 4, and 5 as main products, identified by ESI-MS and 2D NMR spectroscopy,
1
including H,15N HMBC experiments on the 15N-labeled derivatives. The 13-nitrato functionality in 5
suggested partitioning of 1 between concurrent peroxidation and nitric oxide (NO)-release pathways.
Lipid 2 decayed at a much slower rate giving only the hydroxynitro derivative 6 as an isolable product.
Diphenylpicrylhydrazide (DPPH) radical quenching experiments and DFT computations concurred to
support a higher H-atom donating ability of 1 versus 2, due to more effective stabilization of the resulting
pentadienyl radical by the terminal nitro group. The markedly different stability of isomeric nitrolinoleic
acids disclosed in the present study may provide an explanation for the previous identification of 2, but
not 1, in body fluids and offers a key for future insights into the biological activities of nitrated lipids.
Introduction
acids generated in vivo remain to be defined, a significant
proportion of them appears to be present in the form of
nitroalkene derivatives, such as the nitrooleic acids and the
nitrolinoleic acids.3,4 These latter comprise four isomeric
derivatives of (Z,Z)-9,12-octadecadienoic acid exhibiting a
Nitrated derivatives of unsaturated fatty acids have emerged
during the past decade as important products of lipid modifica-
tion under (patho)physiological conditions associated with
oxidative stress and elevated production of nitric oxide (NO).1,2
Although the detailed structural features of the nitrated fatty
(1) Freeman, B. A.; Baker, P. R. S.; Schopfer, F. J.; Woodcock, S. R.;
Napolitano, A.; d’Ischia, M. J. Biol. Chem. 2008, 283, 15515–15519.
(2) (a) Trostchansky, A.; Rubbo, H. Free Radic. Biol. Med. 2008, 44, 1887–
1896. (b) Trostchansky, A.; Souza, J. M.; Ferreira, A.; Ferrari, M.; Blanco, F.;
Trujillo, M.; Castro, D.; Cerecetto, H.; Baker, P. R. S.; O’Donnell, V. B.; Rubbo,
H. Biochemistry 2007, 46, 4645–4653.
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Batthyany, C.; Chacko, B. K.; Feng, X.; Patel, R. P.; Agarwal, A.; Freeman,
B. A.; Chen, Y. E. J. Biol. Chem. 2006, 281, 35686–35698.
† Department of Organic Chemistry and Biochemistry, University of Naples
Federico II.
‡ Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita` degli
Studi di L’Aquila.
§ “Paolo Corradini” Department of Chemistry and INSTM, University of
Naples Federico II.
| Institute for Physico-Chemical Processes (IPCF)-CNR.
10.1021/jo801364v CCC: $40.75
Published on Web 09/04/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 7517–7525 7517

Manini et al.
conjugated nitro group on the terminal (9- and 13-) or inner
(10- and 12-) positions of the (Z,Z)-1,4-pentadienyl system. So
far, 10-nitrolinoleic acid ((9E,12Z)-10-nitrooctadecadienoic acid,
2) and the 12-nitro isomer have been detected in human plasma
and urine by LC-MS techniques,5-7 whereas 9-nitrolinoleic
acid ((9E,12Z)-9-nitrooctadecadienoic acid, 1) and its 13-nitro
isomer have been described only as products of chemical
nitration of linoleic acid in an organic solvent.8,9
stabilized and become highly resistant to NO-release. This
finding suggests that nitrolinoleic acids provide a hydrophobi-
cally stabilized NO reserve and, in view of their accumulation
to detectable levels in lipophilic biological compartments,
represent the single largest pool of bioactive oxides of nitrogen
in the vasculature.10 Apart from the above studies, current
knowledge of the chemical behavior and fate of these nitroalkene
fatty acid derivatives under physiologically relevant conditions
is scanty and limited to the decomposition routes supposedly
associated to their NO donor abilities. Surprising is also the
lack of general information about the free radical oxidation
pathways of the nitro-1,4-pentadienyl system, the reactive core
of nitrated polyunsaturated fatty acids. A detailed elucidation
of the chemical properties of nitrolinoleic acids under physi-
ologically relevant conditions is therefore of paramount impor-
tance for further progress toward an understanding of the
signaling capabilities of these bioactive lipids. To this aim,
access to the individual isomers is essential to advancing
structure-activity knowledge and to rationally design new more
active structural variants for therapeutic purposes. In this paper
we report the first convenient access route to pure 1 and 2 as
free acids and a comparative investigation of their chemical
behavior in aqueous phosphate buffer at pH 7.4. By integrating
product analysis with mechanistic experiments, we provide a
comparative description of the basic chemistry of nitrolinoleic
acids under physiologically relevant conditions. The remarkable
position-dependent influence of the nitro group on the oxidation
behavior of the 1,4-pentadienyl moiety is also addressed with
the aid of a density functional theory (DFT) investigation.
A substantial body of evidence indicates that nitrolinoleic
acids behave as pluripotent signaling molecules that transduce
the actions of NO via multiple mechanisms,7,10,11 which in part
are receptor-mediated and in part reflect the electrophilic and
NO-releasing behavior of the nitropentadiene reactive moiety
in a physiological milieu. The electrophilic nature of nitroli-
noleates is deduced by their ability to cause post-translational
modifications of protein residues9,12,13 and by the occurrence
in vivo of a significant proportion of vicinal hydroxynitro
derivatives,7,10 suggesting addition of water onto the double
bond. The ability of nitrolinoleic acids to release NO is the focus
of much interest. Experimental evidence derived from different
experiments (EPR, chemiluminescence, deoxy- and oxymyo-
globin assays)10,14-17 suggests the release of NO as well as
nitrite,15,16 but the detailed pathways remain to be definitively
assessed. In the presence of other lipids or amphiphiles at levels
above the critical micellar concentration, nitrolinoleic acids are
Results and Discussion
Preparation of Nitrolinoleic Acids. Unlike nitrooleic acids,
for which simple access routes based on standard Henry
condensation chemistry are available,16,18 nitrolinoleic acids are
difficult to synthesize. Their preparation currently relies on direct
nitration of the parent fatty acid4,6,8,9,19,20 because of the lack
of synthetic routes assembling the C-18 nitrated fatty acid chain.
Early methods based on acidic nitrite or NO2+-forming reagents
led invariably to mixtures of regio- and stereoisomers in low
yields, along with abundant side products, and only small
amounts of 2 and the 12-nitro isomer as carboxylate esters could
be obtained. More recently, a nitrophenylselenenylation/oxida-
tion protocol has been reported to produce a statistical distribu-
tion of the four regioisomeric nitrolinoleic acids in good overall
yields,9 but their separation as free acids remained a difficult
task. To provide access to individual nitrolinoleic acid isomers
needed for probing the structural basis of their biological
activities and fate, to determine if certain isomers are more
reactive than others in competing autoxidation/NO-release and
water addition pathways, and to understand why unsaturated
lipids with the nitro group on terminal positions of the polyene
systems (e.g., 1) have so far eluded identification in vivo, it
was necessary to devise expedient methods for preparation of
representative isomeric nitrolinoleates as pure free acids. Ni-
trolinoleates 1 and 2 were thus selected for the purposes of the
(4) Lim, D. G.; Sweeney, S.; Bloodsworth, A.; White, C. R.; Chumley, P. H.;
Krishna, N. R.; Schopfer, F.; O’Donnell, V. B.; Eiserich, J. P.; Freeman, B. A.
Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15941–15946.
(5) Lima, E. S.; Di Mascio, P.; Rubbo, H.; Abballa, D. S. P. Biochemistry
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(6) Baker, P. R. S.; Schopfer, F. J.; Sweeney, S.; Freeman, B. A. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 11577–11582.
(7) Baker, P. R. S.; Lin, Y.; J.; Schopfer, F.J.; R.; Woodcock, S. R.; Groeger,
A. L.; Batthyany, C.; Sweeney, S.; Long, M. H.; Iles, K. E.; Baker, L. M. S.;
Branchaud, B. P.; Chen, Y. E.; Freeman, B. A. J. Biol. Chem. 2005, 280, 42464–
42475.
(8) (a) Napolitano, A.; Camera, E.; Picardo, M.; d’Ischia, M. J. Org. Chem.
2000, 65, 4853–4860. (b) d’Ischia, M. Tetrahedron Lett. 1996, 37, 5773–5774.
(c) d’Ischia, M.; Rega, N.; Barone, V. Tetrahedron 1999, 55, 9297–9308.
(9) Alexander, R. L.; Bates, D. J. P.; Wright, M. W.; King, S. B.; Morrow,
C. S. Biochemistry 2006, 45, 7889–7896.
(10) Schopfer, F. J.; Baker, P. R. S.; Giles, G.; Chumley, P.; Batthyany, C.;
Crawford, J.; Patel, R. P.; Hogg, N.; Branchaud, B. P.; Freeman, B. A. J. Biol.
Chem. 2005, 280, 19289–19297.
(11) Wright, M. M.; Schopfer, F. J.; Baker, P. R. S.; Vidyasagar, V.; Powell,
P.; Chumley PIles, K. E.; Freeman, B. A.; Agarwal, A. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 4299–4304.
(12) Batthyany, C.; Schopfer, F. J.; Baker, P. R. S.; Duran, R.; Baker,
L. M. S.; Huang, Y.; Cervenansky, C.; Branchaud, B. P.; Freeman, B. A. J. Biol.
Chem. 2006, 281, 20450–20463.
(13) Baker, L. M. S.; Baker, P. R. S.; Golin-Bisello, F.; Schopfer, F. J.; Fink,
M.; Woodcock, S. R.; Branchaud, B. P.; Radi, R.; Freeman, B. A. J. Biol. Chem.
2007, 282, 31085–31093.
(14) Gorczynski, M. J.; Huang, J.; Lee, H.; King, S. B. Bioorg. Med. Chem.
Lett. 2007, 17, 2013–2017.
(15) Chakrapani, H.; Gorczynski, M. J.; King, S. B. J. Am. Chem. Soc. 2006,
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(16) Gorczynski, M. J.; Huang, J.; King, S. B. Org. Lett. 2006, 8, 2305–
2308.
(17) Lima, E. S.; Bonini, M. G.; Augusto, O.; Barbeiro, H. V.; Souza, H. P.;
Abballa, D. S. P. Free Radic. Biol. Med. 2005, 39, 532–539.
(18) Woodcock, S. R.; Marwitz, A. J. V.; Bruno, P.; Branchaud, B. P. Org.
Lett. 2006, 8, 3931–3934.
(19) O’Donnell, V. B.; Eiserich, J. P.; Chumley, F. H.; Jablonsky, M. J.;
Krishna, N. R.; Kirk, M.; Barnes, S.; Darley-Usmar, V. M.; Freeman, B. A.
Chem. Res. Toxicol. 1999, 12, 83–92.
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1999, 301, 454–470.
7518 J. Org. Chem. Vol. 73, No. 19, 2008

Chemistry of Nitrated Lipids
SCHEME 1. Preparation of 1
FIGURE 1. Consumption of linoleic acid ([), 1 (2) or 2 (9) with
reaction time during incubation in 0.1 M phosphate buffer at pH 7.4.
All experiments have been carried out in triplicate. Data are expressed
as average ( SD.
present study because of their appropriate positional isomerism.
After an extensive search of reaction conditions with a variety
of nitrating systems, it was found that the target nitroalkenes 1
and 2 could be conveniently obtained by a modification of the
reported nitrophenylselenenylation/oxidation procedure9 through
a judicious combination of experimental conditions and protec-
tion strategies.
SCHEME 2. Preparation of 2
Compound 1 was prepared by a modified protocol in which
linoleic acid was reacted with PhSeBr in THF at -78 °C and
then with HgCl2 and AgNO2 in dry acetonitrile to produce
regioisomeric nitrophenylselenenyl adducts (Scheme 1).21 Pre-
parative HPLC then afforded a main fraction containing a
mixture of the appropriate stereoisomeric nitrophenylselenenyl
adducts, which were treated with H2O2 to remove phenylsele-
ninic acid and give the desired 1 in 12% overall yield. Complete
spectral data of 1 are given in the Supporting Information. The
proposed structural assignment was secured by TOCSY experi-
ments showing cross peaks between the terminal methyl proton
signal at δ 0.89 and resonances at δ 2.05, 5.35, and 5.52, due
to H-14 allylic protons and H-12 and H-13 double bond protons,
in that order. The same procedure was successfully extended
to obtain 15N-labeled 1 in 9% isolated yield using Na15NO2 in
the place of AgNO2. Preparation of 2 as free acid could not be
achieved by the same protocol because of difficulties in the
separation of the appropriate nitrophenylselenenyl intermediates
or the final nitrolinoleates. Accordingly, an alternate strategy
was devised which involved the use of the allylic ester of linoleic
acid as the substrate. This ester group was preferred oversimple
alkyl groups because of the facile removal by hydrogenolysis18
and the presence of an additional double bond expected to
improve chromatographic separation over silver nitrate-impreg-
nated silica gel.8 Indeed, nitrophenylselenenylation/oxidation of
linoleic acid allylester as above followed by a chromatographic
step gave pure 2 allyl ester, from which 2 was eventually
obtained in ca. 10% overall yield after a deprotection step
(Scheme 2). Spectral data of 2 are reported in the Supporting
Information.
particular, access to 1 was made possible by preparative HPLC
separation of the nitrophenylselelenyl adducts prior to H2O2
treatment, whereas preparation of 2 capitalized on the use of
the linoleic acid allyl ester as the substrate of the nitrophe-
nylselenenylation protocol, allowing better separation of the final
nitrolinoleates and facile deprotection of the carboxyl group.
Stability of 1 and 2 in Aqueous Phosphate Buffer at
pH 7.4. Prior to product investigation, the relative stability of
regioisomeric nitrolinoleic acids under physiologically relevant
conditions was investigated. Accordingly, freshly prepared pure
1 and 2 at 20 µM concentration were dissolved in phosphate
buffer at pH 7.4 containing 20% ethanol, under the conditions
used in previous and related studies,10,22 and their rates of
decomposition at 20 °C were monitored by HPLC. Data in
Figure 1 revealed a markedly faster decay of 1 relative to 2:
after 15 min nearly 80% 1 had decayed versus little or no 2.
Under the same conditions, linoleic acid remained virtually
unchanged over more than 5 h.
To summarize, the synthetic protocols described in Schemes
1 and 2 provide the first expedient access to single nitrolinoleic
acids as free acids and in sufficient amounts for biological
studies. They differ from previously published reports of
nitrophenylselenenylation of fatty acids9 in the modified ex-
perimental protocol, which relies on a low-temperature proce-
dure favoring selective nitration of the ∆9 double bond, and in
the carboxyl protection and product separation strategies. In
The faster decay of 1 relative to 2 was also apparent from
NMR analyses of the crude ethyl acetate extracts of the reaction
mixtures from the nitrolinoleic acids suspended in 0.1 M
phosphate buffer, pH 7.4. After 2 h incubation, complete
conversion of 1 to a complex mixture of products was noted,
whereas in the case of 2 a substantial proportion of unchanged
(21) Hayama, Y.; Tomoda, S.; Takeuchi, Y.; Nomura, Y. Tetrahedron Lett.
1982, 23, 4733–4734.
(22) Lin, D.; Zhang, J.; Sayre, L. M. J. Org. Chem. 2007, 72, 9471–9480.
J. Org. Chem. Vol. 73, No. 19, 2008 7519

Manini et al.
starting material was present. Similar data were obtained when
the NMR analysis was run on an equimolar mixture of 1 and 2.
Notably, even when stored dry in the cold, 1 was unstable
compared to 2: whereas pure samples of the former underwent
extensive degradation after a month or so, the latter remained
virtually unaffected during the same period. In separate experi-
ments, the NO-releasing abilities of 1 and 2 in phosphate buffer
were investigated using the oxymyoglobin assay.10,14,15 The
results (see the Supporting Information) indicated a faster
decrease in the 580 and 543 nm maxima (characteristic of the
visible R- and ꢀ-band absorbance of oxymyoglobin) and a more
rapid increase in the 630 and 503 nm maxima (characteristic
of metmyoglobin) in the case of 1, suggesting faster NO-release
from this fatty acid. A detailed analysis of NO-release from
nitrolinoleic acids was out of the scope of this paper and was
not pursued further. Overall, these preliminary data highlighted
a marked instability of 1 in phosphate buffer when compared
to 2 and a consistently higher tendency to release NO. With
this information available, the main reaction products generated
under the same conditions were then investigated.
due to [(M - NO2) - H]- and [NO2]-. The pattern of three
1
olefin proton resonances in the H NMR spectrum resembling
that of 3 (δ 7.51, 7.28 and 6.64), with in addition a deshielded
methylene proton signal at δ 2.79. A salient feature of the 13C
NMR spectrum was a signal at δ 199.2 suggestive of a
conjugated keto group. The 1H,15N HMBC spectrum of the 15N-
labeled compound showed distinct cross peaks between the
proton signals at δ 7.51 and 2.79 and a nitrogen resonance at δ
374.9, while the MS spectrum indicated a pseudomolecular ion
peak [M + H]+ at m/z 341. Based on these data, the compound
was identified as (9E,11E)-9-nitro-13-oxo-9,11-octadecadienoic
acid (4) (9% isolated yield). The least polar compound C
displayed in the 1H NMR spectrum, besides three olefinic proton
signals, a double triplet at δ 5.41 giving a cross peak in the
1H,13C HMQC spectrum with a carbon signal at δ 83.0. Quite
unexpectedly, the 1H, 15N HMBC spectrum of the labeled
derivative revealed the presence of two nitrogen groups, one
resonating at δ 375.3 and correlating with proton signals at δ
7.48 and 2.70, and the other resonating at δ 338.4, suggestive
of a nitrate ester,24,25 correlating with the proton signal at δ
5.41. Accordingly, the compound was identified as (9E,11E)-
9-nitro-13-nitrate-9,11-octadecadienoic acid (5) (6% isolated
yield). The ESI(+) mass spectrum of 5 showed peaks for the
[M + Na]+ and [M + K]+ ions at m/z 409 and 425, as well as
peaks at m/z 324, 346, and 362, suggesting loss of HNO3 from
the [M + H]+, [M + Na]+, and [M + K]+ pseudomolecular
ion peaks. ESI(-)/MS/MS analysis showed the pseudomolecular
ion peak [M - H]- at m/z 385 and its daughter ion at m/z 62
due to [NO3]-. The 15N-labeled derivative gave expected
pseudomolecular ion peaks at m/z 411 and 427 ([M + Na]+
and [M + K]+) and peaks at m/z 325, 347, and 363 consistent
with loss of H15NO3 from pseudomolecular ion peaks. ESI(-)/
MS/MS experiments gave the pseudomolecular ion peak [M -
H]- at m/z 387 and its daughter ion at m/z 63 assigned to
[15NO3]-. Although products 3-5 account for a modest mass
balance, data refer to isolated yields and therefore underestimate
actual product yields. Scrutiny of the 1H and 13C NMR spectra
of the crude reaction mixtures after 1 had completely disap-
peared revealed the expected patterns of resonances for 3-5
and traces of other unknown species. The same products 3-5
were also formed by decomposition of 20 µM 1 (HPLC),
suggesting that their formation is independent of substrate
concentration. In a subsequent set of control experiments, it was
found that: (a) in unbuffered water (pH around 5) formation of
peroxidation products of 1 was markedly slower with no
detectable 5, (b) under an argon atmosphere formation of
products 3-5 was inhibited, and (c) when 1 was left to
decompose at room temperature as a dry film formation of 3
and 4 occurred but no detectable 5.
Product Studies. Incubation of 1 in 0.1 M phosphate buffer,
pH 7.4, resulted in the generation of a very complex mixture
of products. After 2 h, the mixture was worked up and
chromatographed on silica gel plates to give three main UV-vis
bands, A (Rf ) 0.20), B (Rf ) 0.30), and C (Rf ) 0.36), positive
to the Griess reagent for nitrite-releasing substances. The same
products were also obtained by reacting 15N-labeled 1 prepared
as above. Compound A exhibited pseudomolecular ion peaks
in the ESI(+)-MS spectrum at m/z 364 and 380 ([M + Na]+
and [M + K]+),5 whereas ESI(-)/MS/MS analysis (see the
Supporting Information) showed the pseudomolecular ion peak
[M - H]- at m/z 340 and its daughter ions at m/z 322, 294,
293, due to loss of H2O, NO2, or HNO2, and m/z 46 indicating
[NO2]-. The compound was thus formulated as (9E,11E)-13-
hydroxy-9-nitro-9,11-octadecadienoic acid (3) (15% isolated
yield) on the basis of extensive 2D NMR experiments. The 15N-
labeled 3 consistently exhibited pseudomolecular ion peaks ([M
+ Na]+ and [M + K]+) at m/z 365 and 381 and distinct cross
peaks between proton signals at δ 7.55 and 2.69 and the nitrogen
1
resonance at δ 376.7 in the H,15N HMBC spectrum. Spectro-
scopic data were in good agreement with those reported in the
literature for the corresponding methyl ester isolated from the
reaction of 13-hydroxyoctadecadienoic acid with acidic nitrite.23
To gain a deeper insight into the products resulting from
aqueous decomposition of 1, the crude reaction mixtures
obtained from both the unlabeled and 15N-labeled substrates
were investigated by LC-MS. Analysis of the LC traces at 2 h
reaction time indicated the presence, besides 3, 4, and 5, of
several additional species, one of which exhibited distinct [M
+ Na]+ and [M + K]+ pseudomolecular ion peaks at m/z 380
and 396, suggesting the 13-hydroperoxy derivative of 1. Another
species displayed a [M + H]+ pseudomolecular ion peak at m/z
295, compatible with a dienone in which the nitro group was
Compound B showed in the ESI(+) mass spectrum a
pseudomolecular ion peak [M + H]+ at m/z 340, whereas the
ESI(-)/MS/MS analysis revealed the pseudomolecular ion peak
[M - H]- at m/z 338 and its daughter ions at m/z 292 and 46
(24) Napolitano, A.; Crescenzi, O.; Camera, E.; Giudicianni, I.; Picardo, M.;
d’Ischia, M. Tetrahedron 2002, 58, 5061–5067.
(23) Napolitano, A.; Camera, E.; Picardo, M.; d’Ischia, M. J. Org. Chem.
2002, 67, 1125–1132.
(25) Napolitano, A.; Panzella, L.; Savarese, M.; Sacchi, R.; Giudicianni, I.;
Paolillo, L.; d’Ischia, M. Chem. Res. Toxicol. 2004, 17, 1329–1337.
7520 J. Org. Chem. Vol. 73, No. 19, 2008

Chemistry of Nitrated Lipids
SCHEME 3. Mechanistic Pathway Proposed for the Formation of Compounds 3-5 from 1
evidently lost. The lack of nitrogen was corroborated by the
analysis of the mixture from 15N-labeled 1, revealing the same
pseudomolecular ion for the same peak. Similar species gener-
ated by decomposition of nitrolinoleic acids had been reported
previously.10 In contrast to 1, decay of 2 in aqueous phosphate
buffer at pH 7.4 gave after 2 h a single isolable species which
was obtained by preparative TLC and was identified as (12Z)-
9-hydroxy-10-nitrooctadec-12-enoic acid (6, 9% isolated yield)
by extensive spectral characterization. Diagnostic features in
the 1H NMR spectrum were two sets of multiplets at δ
4.44-4.46 and 4.06/3.90, showing one-bond correlations with
carbon resonances at δ 91.7/92.5 and at δ 72.4/71.5, respec-
peroxidation steps. This path is supported by the mass spectrometric
identification of the hydroperoxy derivative and the substantial
inhibition of product formation in the absence of oxygen. NO-
release is apparently a pH-dependent route triggered by a
deprotonation equilibrium and is involved in the formation of
5 at pH 7.4. A substantial deprotonation of nitrolinoleic acids
can be predicted at pH 7.4 based on a suggested pKa value of
5 for the allylic methylene group10 and would be favored in
the case of 1, from which a highly conjugated, resonance-
stabilized nitronate structure can be generated. One viable
mechanism for NO-release is based on the recently proposed
modified Nef sequence10 and is sketched in path B. In this route,
the first formed nitronate anion would be converted to a
hydroxynitroso intermediate which can undergo homolytic
cleavage to form NO and an allylic radical. The latter might
then react with oxygen to give eventually a dienone product,
as suggested by the presence of a species in the LC-MS
chromatogram with a pseudomolecular ion peak at m/z 295.
Alternative mechanisms, mutually nonexclusive, can account
for NO-release, for example, nitroalkene rearrangement to a
nitrite ester followed by N-O bond homolysis to form NO and
a carbon-centered radical,14 but they cannot be distinguished
1
1
1
tively. These and H,1H COSY, H,13C HMQC and H,13C
HMBC data indicated that the product was in fact a 1:1 mixture
of diastereoisomers (pairs of enantiomers). This conclusion was
confirmed by LC-MS analysis of the isolated band showing
two species eluted under very close peaks (16.8 and 17.2 min)
with pseudomolecular ion peaks [M + H]+ at m/z 344.
based on the present results. Fast and effective coupling of NO
26
with peroxyl radicals (k > 1010 M-1 s-1
)
produced by
Unreacted 2 accounted for some 80% of the mixture after
2 h reaction time. Although no other single product could be
isolated from the mixture, NMR and LC-MS investigations
indicated the formation, besides 6, of several species all in
minute amounts.
Mechanistic and Computational Studies. The product
studies reported above revealed three main degradation channels
of the nitropentadienyl system in neutral aqueous buffer, namely:
(a) H-atom abstraction from the bis-allylic methylene group
triggering classic peroxidation chain reactions, (b) NO-release,
and (c) nucleophilic addition of water onto the nitroalkene
moiety. Whereas the latter path accounts for the observed
formation of 6 by decay of 2, the first two routes may lead to
3-5, as outlined in Scheme 3.
peroxidation of 1 followed by rearrangement of the resulting
peroxynitrite ester intermediate is likely to be involved in the
formation of 5. It may be relevant to notice, in this connection,
that effective trapping of NO by the peroxyl radicals present in
the mixture may bias estimation of NO release by the chemi-
luminescence and other assays. Product analysis suggested a
higher oxidizability of 1 relative to 2. Since H-atom abstraction
from the 1,4-pentadiene system (C-H bond dissociation en-
thalpy (BDE) ) 76.4 kcal mol-1 27-29 dictates the oxidizability
)
of polyunsaturated fatty acids, at least a rough estimate of the
(26) Padmaja, S.; Huie, R. E. Biochem. Biophys. Res. Commun. 1993, 195,
539–544.
(27) Trenwith, A. B. J. Chem. Soc., Faraday Trans. 1 1982, 78, 3131–3136.
(28) Pratt, D. A.; Mills, J. H.; Porter, N. A. J. Am. Chem. Soc. 2003, 125,
5801–5810.
(29) Tallman, K. A.; Pratt, D. A.; Porter, N. A. J. Am. Chem. Soc. 2001,
123, 11827–11828.
H-atom abstraction from the bis-allylic methylene group (path
A) would result in a stabilized nitropentadienyl radical which would
undergo coupling with oxygen to give 3 and 4 via the classic lipid
J. Org. Chem. Vol. 73, No. 19, 2008 7521

Manini et al.
FIGURE 3. Isodensity surfaces of the singly occupied molecular orbital
and total spin density of the main conformer of 7R, computed at the
UPBE0/6-31+G(d,p) level.
7 and 8, as well as the corresponding bis-allylic radicals (dubbed
7R and 8R), were sampled by relaxed grid searches of the
C3-C4 and C4-C5 dihedrals (methyl rotations were not
systematically explored). For both 7 and 8, four minima were
identified, arranged in enantiomeric pairs which, within each
compound, are separated by 0.2-0.4 kcal mol-1: the energy
difference between the absolute minimum of 8 with respect to
7 is also rather small (0.5 kcal mol-1). The bis-allylic radical
7R gives rise to six minima (two enantiomeric pairs, and two
structures of Cs symmetry), one of the planar conformers being
definitely more stable than the others (by at least 3.9 kcal mol-1).
By contrast, planar conformers were not found among the stable
conformers of 8R, which features instead four pairs of enan-
tiomeric minima: even in those conformers in which the carbon
skeleton is approximately planar, the nitro group is significantly
distorted out of the molecular plane. A first indication of the
relative stabilities of the two radicals can be gained by
comparison of the energies of their most stable conformers: then,
7R is favored by 9.0 kcal mol-1 with respect to 8R. Such a
direct comparison is significant, since, as stated before, 7 and 8
are almost isoenergetic at this level. Moreover, the overall
picture is confirmed when more refined treatments are adopted,
including single point energy evaluations with the recently
developed M05-2X density functional,34 which has shown
remarkable performance for energetics involving radicals,35 use
of a large basis set, and introduction of rotational/harmonic
vibrational contributions (see the Supporting Information): in
terms of computed enthalpies at 298.15 K, 8 is more stable than
7 by 0.4 kcal mol-1, while 8R is less stable than 7R by 8.4
kcal mol-1. In view of the relatively apolar nature of the models,
it is not surprising that introduction of the aqueous environment
by means of an implicit model (namely the polarizable
continuum model)36-39 involves only minor changes in com-
puted geometries and relative stabilities (see the Supporting
Information). Figure 3 depicts the SOMO and the total spin
density for the main conformer of 7R: the 2, 4, and 6 positions
appear almost equivalent, with significant involvement of the
nitro group.
FIGURE 2. Absorbance decay at 515 nm of a 100 µM solution of
DPPH in ethanol after addition of: 1 (9), 2 ([), and linoleic acid (2).
Final fatty acid concentration: 7.7 mM. All experiments have been
carried out in triplicate. Data are expressed as average ( SD.
relative abilities of nitrolinoleic acids to act as H-atom donors
was desirable. Accordingly, the activity of 1 and 2 in the
diphenylpicryl hydrazide (DPPH) radical quenching spectro-
photometric test was determined against linoleic acid as a
reference fatty acid.30 This test, which is run in ethanol and is
therefore independent of pH-related effects, enables measure-
ment of the radical-scavenging properties of a given substance
and is often used in the food industry to establish the rank order
of antioxidants. The results in Figure 2 indicated more effective
H-atom transfer to the DPPH radical from 1 than from 2 or
linoleic acid. This trend can be taken to suggest a larger radical-
stabilizing effect of the nitro group on the terminal pentadienyl
positions, making 1 more prone to H-atom loss and free radical
formation.
To support this conclusion and to investigate in some detail
the position-dependent influence of the nitro group on radical
stability, a computational (DFT) investigation of the isomeric
nitro-1,4-pentadienyl systems was undertaken. A theoretical
description of the 1,4-pentadiene system and the corresponding
radical, and a discussion of how theoretical methodologies
perform in modeling these systems, including determination of
bond dissociation enthalpies (BDEs), has appeared recently.28
For the purposes of the present study two simplified truncated
structures featuring the isomeric nitropentadiene moieties typical
of 1 and 2 were investigated, namely 7 and 8, respectively.
A more quantitative comparison (see the Supporting Informa-
tion) based on SOMO localization and on use of a larger basis
set shows a slightly lower relative weight at position C-6 with
respect to C-2 and C-4. A note of caution here is in order
Geometry optimizations were based on the “hybrid” PBE0
functional,31 which has provided quite satisfactory energies and
geometries for a wide range of organic and biological systems
(including notably radical species) in combination with the
6-31+G(d,p) basis set.32,33 The unrestricted formulation was
used to describe radical species. The conformational spaces of
(33) For a general introduction to basis sets, see: Foresman, J. B.; Frisch, A.
Exploring Chemistry with Electronic Structure Methods, 2nd ed.; Gaussian, Inc.;
Pittsburgh, PA, 1996.
(34) Zhao, Y.; Schultz, N. E.; Truhlar, D. G. J. Chem. Theory Comput. 2006,
2, 364–382.
(35) Zhao, Y.; Truhlar, D. G. J. Phys. Chem. A 2008, 112, 1095–1099.
(36) Miertus, S.; Scrocco, E.; Tomasi, J. J. Chem. Phys. 1981, 55, 117–129.
(37) Cossi, M.; Scalmani, G.; Rega, N.; Barone, V. J. Chem. Phys. 2002,
117, 43–54.
(38) Scalmani, G.; Barone, V.; Kudin, K. N.; Pomelli, C. S.; Scuseria, G. E.;
Frisch, M. J. Theor. Chem. Acc. 2004, 111, 90–100.
(39) Tomasi, J.; Mennucci, B.; Cammi, R. Chem. ReV. 2005, 105, 2999–
3093.
(30) Yu, L.; Adams, D.; Gabel, M. J. Agric. Food Chem. 2002, 50, 4135–
4140.
(31) Adamo, C.; Barone, V. J. Chem. Phys. 1999, 110, 6158–6170.
(32) Francl, M. M.; Petro, W. J.; Hehre, W. J. S.; Binkley, J.; Gordon, M. S.;
DeFrees, D. J.; Pople, J. A. J. Chem. Phys. 1982, 77, 3654–3665.
7522 J. Org. Chem. Vol. 73, No. 19, 2008

Chemistry of Nitrated Lipids
the literature with modifications.4,6,9 In brief, a solution of 1 (1 g,
3.6 mmol) in dry THF (6 mL) was added to a solution of PhSeBr
(843 mg, 3.6 mmol) in dry THF (6 mL) at -78 °C under an argon
atmosphere. After 10 min, HgCl2 (1.26 g, 4.6 mmol) was added to
the mixture followed by a solution of AgNO2 (550 mg, 3.6 mmol)
in dry acetonitrile (12 mL, aliquots of 1 mL over 30 min). After
2 h, the reaction mixture was taken at room temperature for 1.5 h,
then filtered on Celite and washed extensively with diethyl ether.
The surnatant was evaporated to dryness, taken up in CHCl3,
extracted with brine, and evaporated under reduced pressure after
drying over anhydrous sodium sulfate. The residue was subjected
concerning the validity of the truncated structure to model lipid
peroxidation. In particular, steric interactions between the long
tails of the real lipids are evidently ill reproduced by the small
methyl groups of the models. However, for both 7 and 8, the
most stable conformer is characterized by a skew arrangement
of the terminal C-CH3 units, which should be compatible even
with the complete chains. The chain termini are almost
co-oriented in the secondary conformers identified, but again
this holds for both 7 and 8. The situation is slightly more
involved for the radical species: in the most stable conformer
of 7R, the C-CH3 units are coplanar and point in the same
direction; in the conformer which comes next, the backbone is
still planar, but the methyl groups are now on opposite sides
(however, as hinted above, this conformer is significantly less
stable). The most stable conformer identified for 8R corresponds,
with minor distortions, to this latter chain fold; conversely, the
second conformer of 8R resembles the geometry of the absolute
minimum of 7R (but is much closer in energy to its own
minimum). While the relative orientation of the terminal C-CH3
groups can admittedly provide only a rough indication as to
the stability of the untruncated compounds, overall it would
appear that longer terminal tails could be better accommodated
in the floppier 8R than in 7R; in turn, this would imply a smaller
energy separation between the two radicals, and would to some
extent qualify the rather drastic reactivity differences predicted
by use of the heptadiene models.
to preparative HPLC (eluant I) to afford two fractions, I (tR
)
18-20 min) and II (tR ) 21-23 min). Fractions I and II were
evaporated under reduced pressure and worked up as described
above. The residues were taken up in CHCl3 and treated separately
with H2O2 (4 molar equiv) under vigorous stirring for 30 min at 4
°C and for additional 30 min at room temperature. After workup
as above, residues were subjected to preparative TLC (eluant a) to
afford from fraction I a 1:1 mixture of 1 and 2 (264 mg) and from
fraction II pure 1 (135 mg, 12% yield, Rf ) 0.50, eluant a).
1
1: H NMR (400 MHz, CDCl3) δ (ppm) 2.05 (2H, m, H-14),
2.59 (2H, t, J ) 7.6 Hz, H-8), 2.95 (2H, t, J ) 7.6 Hz, H-11), 5.34
(1H, m, H-12), 5.52 (1H, m, H-13), 7.02 (1H, t, J ) 7.6 Hz, H-10);
13C NMR (100 MHz CDCl3) δ (ppm) 26.9 (CH2), 27.1 (CH2), 28.2
(CH2), 123.3 (CH), 133.3 (CH), 134.4 (CH), 151.8 (C) 180.0 (C);
LC-MS tR ) 43 min; ESI(+)-MS m/z 326 ([M + H]+), 348 ([M
+ Na]+), 364 ([M + K]+); ESI(+)-HRMS for C18H31NO4 calcd
325.2253 [M + H]+, found 325.2249.
Synthesis of [15N]-(9E,12Z)-9-Nitroottadecadienoic Acid ([15N]1).
The title compound was prepared as described for 1, but using
Na15NO2 (250 mg, 3.6 mmol) instead of AgNO2. Two fractions
were isolated, one consisting of pure [15N]1 (198 mg, 17% yields)
and the other of a 1:1 mixture of [15N]1 and [15N]2 (105 mg).
[15N]1: 1H NMR (300 MHz, CDCl3) δ (ppm) 2.05 (2H, m, H-14),
2.59 (2H, dt, J ) 7.6, 3.6 Hz, H-8), 2.95 (2H, t, J ) 7.6 Hz, H-11),
5.34 (1H, dt, J ) 7.2, 10.0 Hz, H-12), 5.52 (1H, dt, J ) 7.2, 10.5
Hz, H-13), 7.02 (1H, dt, J ) 7.6, 3.6 Hz, H-10); LC-MS tR ) 43
min; ESI(+)-MS m/z 327 ([M + H]+), 349 ([M + Na]+), 365 ([M
+ K]+); ESI(+)-HRMS for C18H3115NO4 calcd 326.2223 [M + H]+,
found 326.2220.
Conclusions
The preparation of two isomeric nitrolinoleic acids in pure
form is reported for the first time (only the ester derivative of
2 was obtained previously),8 and their markedly different
chemical behavior is disclosed. Compound 1, bearing a nitro
group on a terminal position of the pentadiene moiety, is
markedly unstable when exposed to phosphate buffer at pH 7.4,
and partitions mainly between two concurrent degradation
pathways, one resulting in NO-release presumably via a base-
dependent Nef-type reaction, and the other involving a typical
peroxidation process. A remarkable outcome is formation of
the nitronitrate ester 5 involving probably trapping of NO by
transient peroxyl radicals. Compound 2 decays at slower rate
and gives mainly the stereoisomeric hydroxynitro derivatives 6
by nucleophilic addition of water. The differential behavior of
1 and 2 can be rationalized on the basis of DPPH radical
quenching experiments and DFT calculations, indicating a higher
H-atom donating ability and a larger radical stabilization when
the nitro group is located on the terminal rather than inner
positions of the 1,4-pentadienyl core. These results provide an
underpinning for ongoing investigations of the physiological
properties of nitrolinoleic acids and may orient the design of
new pharmacologically active nitrated lipids. They also offer a
plausible explanation as to why 1 and the isomeric 13-
nitrolinoleic acid have so far eluded detection in biological
systems.
Synthesis of allyl (9E,12Z)-10-Nitrooctadecadienoate (2 Allyl
Ester). The title compound was prepared following the nitrophe-
nylselenenylation protocol described for 1 with allyl (9Z,12Z)-
octadecadienoate as the substrate. After workup, the residue was
fractionated on silica gel using cyclohexane/ethyl acetate (gradient
mixture from pure cyclohexane to cyclohexane/ethyl acetate 98:2)
to afford the mixture of nitrophenylselenenyl-adducts of allyl
(9Z,12Z)-octadecadienoate (1.0 g, 60% yield). This latter was taken
up in CHCl3 and treated with H2O2 (4 molar equiv) under vigorous
stirring at 4 °C. After 30 min, the mixture was taken at room
temperature for additional 30 min. The reaction mixture was then
extracted with brine, and the organic layers were dried over
anhydrous sodium sulfate, filtered, and evaporated. The residue was
fractionated on silver-impregnated silica gel (eluant cyclohexane)
to afford two fractions, one consisting of a mixture of 1 allyl ester
and 2 allyl ester (98 mg) and the other of pure 2 allyl ester (119
mg, 17% yield, Rf ) 0.62, eluant b).
2 allyl ester: 1H NMR (400 MHz, CDCl3) δ (ppm) 2.12 (dt, J )
7.2 Hz, H-14), 2.23 (2H, dt, J ) 7.6 Hz, H-8), 3.33 (2H, d, J )
7.2 Hz, H-11), 4.56 (2H, d, J ) 6.0 Hz, -OCH-), 5.2-5.3 (1H,
m, H-12), 5.23 (1H, dd, J ) 10.5, 0.9 Hz, -OCH2CHdCH2), 5.31
(1H, dd, J ) 17.1, 0.9 Hz, -OCH2CHdCH2), 5.48 (1H, m, H-13),
5.90 (1H, ddt, J ) 17.1, 10.5, 6.0 Hz, -OCH2CHdCH2), 7.09 (1H,
dt, J ) 7.6, 3.9 Hz, H-9); ESI(+)-HRMS for C21H35NO4 calcd.
365.2566 [M + H]+, found 325.2563.
Deprotection of 2 Allyl Ester. A solution of 2 allyl ester (119
mg, 0.3 mmol) in dry THF (5.84 mL) was treated with HCOOH
(123 µL, 3.3 mmol) and Pd(PPh3)4 (18.8 mg, 16.3 µmol) under an
argon atmosphere at 80 °C. After 24 h, the reaction mixture was
Experimental Section
For all isolated compounds, 1H and 13C NMR resonances due to
C-2 (CH2), C-3/C-4/C-5/C-6/C-7/C-15/C-16/C-17 (CH2) and C-18
(CH3) groups appear in the 1H/13C NMR spectra at δ (CDCl3) 2.34
(2H, t, J ) 7.4 Hz)/33.6, 1.2-1.6 (16H, m)/22-32, 0.89 (3H, t, J
) 7.2 Hz)/14.0, in that order.
Synthesis of (9E,12Z)-9-Nitroottadecadienoic Acid (1). The
title compound was prepared according to a procedure reported in
J. Org. Chem. Vol. 73, No. 19, 2008 7523

Manini et al.
filtered on Celite, evaporated to dryness, and fractionated by
preparative TLC (eluant a) to afford 2 (87 mg, 82% yield, Rf )
0.48, eluant a).
MS m/z 341 ([M + H]+), 363 ([M + Na]+), 379 ([M + K]+);
ESI(+)-HRMS for C18H2915NO5 calcd 340.2016 [M + H]+, found
340.2020.
[15N]5: 1H NMR (400 MHz, CDCl3) δ (ppm) 1.80 (2H, m, H-14),
2.70 (2H, dt, J ) 7.6, 3.6 Hz, H-8), 5.41 (1H, ddt, J ) 7.2, 6.8, 3.6
Hz, H-13), 6.17 (1H, dd, J ) 15.2, 7.2 Hz, H-12), 6.49 (1H, dd, J
) 15.2, 11.6 Hz, H-11), 7.48 (1H, dd, J ) 11.6, 3.6 Hz, H-10);
LC-MS tR ) 39 min; ESI(+)-MS m/z 325 ([M - H15NO3 + H]+),
411 ([M + Na]+), 347 ([M - H15NO3 + Na]+), 427 ([M + K]+),
363 ([M - H15NO3 + K]+); ESI(-)/MS/MS m/z 387 ([M - H]-),
63 (15[NO3]-); ESI(+)-HRMS for C18H3015N2O7 calcd 388.1994
[M + H]+, found 388.1996.
Isolation of (12Z)-9-hydroxy-10-nitro-12-ottadecenoic Acid (6)
(Mixture of Stereoisomers). The reaction for compound 2 was
carried out as described for 1. After workup, preparative TLC
afforded 6 (mixture of stereoisomers) (7 mg, 9% yield, Rf ) 0.35,
eluant a).
6: 1H NMR (400 MHz, CDCl3) δ (ppm) (the superscripts a and
b are referred to the two diastereoisomers) 1.2-1.6 (2H, m, H-8),
2.02 (2H, m, H-14), 2.51b (1H, m, H-11), 2.60a (1H, m, H-11),
2.78b (1H, m, H-11), 2.91a (1H, m, H-11),4.44-4.46a,b (1H, m,
H-10), 3.90b (1H, m, H-9), 4.06a (1H, m, H-9), 5.29 (1H, m, H-12),
5.57 (1H, m, H-13); 13C NMR (100 MHz CDCl3) δ (ppm) 25.0
(CH2), 26.4a (CH2), 28.2 (CH2), 28.8b (CH2), 71.5b (CH), 72.4a
(CH), 91.7a (CH), 92.5b (CH), 121.9 (CH), 136.0 (CH), 179.1 (C);
LC-MS tR ) 16.8 e 17.2 min; ESI(+)-MS m/z 344 ([M + H]+),
366 ([M + Na]+), 382 ([M + K]+); ESI(+)-HRMS for C18H33NO5
calcd 343.2359 [M + H]+, found 343.2355.
Computational Methods. All calculations were performed with
the Gaussian package of programs.40,41 Geometries were optimized
at the DFT level of theory using the PBE0 functional with the
6-31+G(d,p) basis set.32,33 The PBE0 (also referred to as PBE1PBE)
is a hybrid functional obtained by combining a predetermined
amount of exact exchange with the Perdew-Burke-Ernzerhof
exchange and correlation functionals.31 In control calculations, the
polarizable continuum model (PCM)36-39 was used to simulate the
aqueous environment. In view of the faster convergence, a scaled
Van der Waals cavity based on universal force field (UFF) radii42
was used, and surface elements were modeled by spherical Gaussian
1
2: H NMR (300 MHz, CDCl3) δ (ppm) 2.12 (2H, dt, J ) 7.2
Hz, H-14), 2.21 (2H, dt, J ) 7.6 Hz, H-8), 3.33 (2H, d, J ) 6.9
Hz, H-11), 5.26 (1H, m, H-12), 5.48 (1H, m, H-13), 7.08 (1H, dt,
J ) 7.6, 3.9 Hz, H-9); 13C NMR (50 MHz CDCl3) δ (ppm) 25.1
(CH2), 27.9 (CH2), 28.0 (CH2), 123.8 (CH), 132.7 (CH), 136.2 (CH),
150.8 (C) 179.3 (C); LC-MS tR ) 42 min; ESI(+) m/z 326 ([M
+ H]+), 348 ([M + Na]+), 364 ([M + K]+).
Isolation of (9E,11E)-13-Hydroxy-9-nitro-9,11-octadecadienoic
Acid (3), (9E,11E)-9-Nitro-13-oxo-9,11-octadecadienoic Acid (4),
and (9E,11E)-9-Nitro-13-nitrate-9,11-octadecadienoic Acid (5). A
solution of 1 (75 mg) in ethanol (44 mL) was added to 0.1 M
phosphate buffer, pH 7.4 (178 mL) and kept at 37 °C and under
vigorous stirring. After 2 h, the reaction mixture was acidified to
pH 3 with HCl 3 M and extracted with chloroform. After drying
over anhydrous sodium sulfate and evaporation of the solvent, the
residue was fractionated by preparative TLC (eluant a) to afford 3
(11 mg, 15% yield, Rf ) 0.20, eluant a), 4 (7 mg, 9% yield, Rf )
0.30, eluant a), and 5 (5 mg, 6% yield, Rf ) 0.36, eluant a).
3: 1H NMR (400 MHz,CDCl3) δ (ppm) 1.2-1.6 (2H, m, H-14),
2.69 (2H, t, J ) 7.6 Hz, H-8), 4.33 (1H, m, H-13), 6.32 (1H, dd,
J ) 14.8, 5.2 Hz, H-12), 6.45 (1H, dd, J ) 14.8, 11.6 Hz, H-11),
7.55 (1H, d, J ) 11.6 Hz, H-10); 13C NMR (100 MHz CDCl3) δ
(ppm) 27.2 (CH2), 37.8 (CH2), 72.7 (CH), 122.8 (CH), 133.4 (CH),
150.0 (CH), 151.6 (C), 178.6 (C); LC-MS tR ) 20 min; ESI(+)-
MS5 m/z 324 ([M - H2O + H]+), 364 ([M + Na]+), 380 ([M +
K]+); ESI(-)/MS/MS m/z 340 ([M - H]-), 322 ([(M - H2O) -
H]-), 294 ([(M - NO2) - H]-), 293 ([(M - HNO2) - H]-), 46
([NO2]-).
4: 1H NMR (400 MHz,CDCl3) δ (ppm) 2.62 (2H, t, J ) 7.6 Hz,
H-14), 2.79 (2H, t, J ) 7.6 Hz, H-8), 6.64 (1H, d, J ) 15.2 Hz,
H-12), 7.28 (1H, dd, J ) 15.2, 12.0 Hz, H-11), 7.51 (1H, d, J )
12.0 Hz, H-10); 13C NMR (125 MHz CDCl3) δ (ppm) 27.2 (CH2),
42.3 (CH2), 129.6 (CH), 137.3 (CH), 156.1 (CH), 158.2 (C), 177.9
(C), 199.2 (C); LC-MS tR ) 26 min; ESI(+)-MS m/z 340 ([M +
H]+), 362 ([M + Na]+), 378 ([M + K]+); ESI(-)/MS/MS: m/z
338 ([M - H]-), 292 ([(M - NO2) - H]-), 46 ([NO2]-); ESI(+)-
HRMS for C18H29NO5 calcd. 339.2046 [M + H]+, found 339.2049.
(40) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
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Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci,
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M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.;
Honda, Y. ; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian,
H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.;
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M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Scalmani, G.;
Kudin, K. N.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.;
Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.;
Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai,
H.; Li, X.; Hratchian, H. P., Peralta, J. E.; Izmaylov, A. F.; Heyd, J. J.; Brothers,
E.; Staroverov, V.; Zheng, G.; Kobayashi, R.; Normand, J.; Burant, J. C.; Millam,
J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo,
J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli,
C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.;
Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.;
Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.;
Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.;
Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.;
Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.;
Chen, W.; Wong, M. W.; Pople, J. A. Gaussian DeVelopment Version, ReVision
F.01; Gaussian, Inc.: Wallingford, CT, 2006.
1
5: H NMR (400 MHz, CDCl3) δ (ppm) 1.80 (2H, m, H-14),
2.70 (2H, t, J ) 7.6 Hz, H-8), 5.41 (1H, dt, J ) 7.2, 6.8 Hz, H-13),
6.17 (1H, dd, J ) 15.2, 7.2 Hz, H-12), 6.49 (1H, dd, J ) 15.2,
11.6 Hz, H-11), 7.48 (1H, d, J ) 11.6 Hz, H-10); 13C NMR (100
MHz CDCl3) δ (ppm) 27.2 (CH2), 26.4 (CH2), 83.0 (CH), 127.6
(CH), 129.3 (CH), 132.6 (CH), 153.8 (C), 179.0 (C); LC-MS tR )
39 min; ESI(+)-MS m/z 324 ([M - HNO3 + H]+), 409 ([M +
Na]+), 346 ([M - HNO3 + Na]+), 425 ([M + K]+), 362 ([M -
HNO3 + K]+); ESI(-)/MS/MS: m/z 385 ([M - H]-), 62 ([NO3]-);
ESI(+)-HRMS for C18H30N2O7 calcd 386.2053 [M + H]+, found
386.2055.
Isolation of [15N]-(9E,11E)-13-Hydroxy-9-nitro-9,11-octadeca-
dienoic ([15N]3), [15N]-(9E,11E)-9-Nitro-13-oxo-9,11-octadecadi-
enoic ([15N]4) and [15N]-(9E,11E)-9-Nitro-13-nitrate-9,11-octadec-
adienoic Acids ([15N]5). Compounds [15N]3, [15N]4, and [15N]5 were
prepared from [15N]1 following the same procedure described above
for 1.
1
[15N]3: H NMR (400 MHz,CDCl3) δ (ppm) 1.2-1.6 (2H, m,
H-14), 2.69 (2H, dt, J ) 7.6, 3.6 Hz, H-8), 4.34 (1H, m, H-13),
6.32 (1H, dd, J ) 14.2, 5.2 Hz, H-12), 6.45 (1H, dd, J ) 14.8,
11.6 Hz, H-11), 7.55 (1H, dd, J ) 11.6, 3.6 Hz, H-10); LC-MS tR
) 20 min; ESI(+)-MS5 m/z 325 ([M - H2O + H]+), 365 ([M +
Na]+), 381 ([M + K]+).
1
[15N]4: H NMR (400 MHz,CDCl3) δ (ppm) 2.61 (2H, t, J )
7.6 Hz, H-14), 2.79 (2H, dt, J ) 7.6, 3.6 Hz, H-8), 6.64 (1H, d, J
) 15.2 Hz, H-12), 7.28 (1H, dd, J ) 15.2, 12.0 Hz, H-11), 7.51
(1H, dd, J ) 12.0, 3.6 Hz, H-10); LC-MS tR ) 26 min; ESI(+)-
(42) Rappe´, A. K.; Casewit, C. J.; Colwell, K. S.; Goddard, W. A., III.; Skiff,
W. M. J. Am. Chem. Soc. 1992, 114, 10024–10035.
7524 J. Org. Chem. Vol. 73, No. 19, 2008

Chemistry of Nitrated Lipids
functions.43,44 All minima were checked by computing the harmonic
vibrational frequencies. Single-point energy evaluations were also
performed with the recently developed M05-2X density functional34
and the large 6-311+G(2df,p) basis set.
Supporting Information Available: General methods and
experimental procedures. NMR spectra of compounds 1, [15N]1,
2, 3, [15N]3, 4, [15N]4, 5, [15N]5, 6, allyl (9Z,12Z)-octadecadi-
enoate, and 2 allylester. ESI(+) mass spectra of compounds 1,
2, 3, [15N]3, 4, [15N]4, 5, [15N]5, 6, (9 E,11 E)-13-hydroperoxy-
9-nitrooctadecadienoic acid, and (10 E,12 Z)-9-oxooctadecadi-
enoic acid. ESI(-)/MS/MS analysis of compounds 3, 4, 5, and
[15N]5. LC-MS profile of the autoxidation mixture of 1.
UV-vis spectra for oxymyoglobin assay of compounds 1 and
2. Computational data for compounds 7 and 8 and the corre-
sponding bis-allylic radicals 7R and 8R. This material is
available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. This work was supported in part by grants
from Italian Ministry of Health. Access to the computer facilities
of the VILLAGE network (http://village.unina.it) is kindly
acknowledged. We thank Miss Silvana Corsani for technical
assistance.
(43) York, D. A.; Karplus, M. J. Phys. Chem. A 1999, 103, 11060–11079.
(44) Caricato, M.; Scalmani, G.; Frisch, M. J. In Continuum SolVation Models
in Chemical Physics; Mennucci, B., Cammi, R., Eds.; John Wiley & Sons:
Chichester, 2007; pp 64-81.
JO801364V
J. Org. Chem. Vol. 73, No. 19, 2008 7525