The acid-promoted reaction of ethyl linoleate with nitrite. New insights from 15N-labelling and peculiar reactivity of a model skipped diene

Article abstract of DOI:10.1016/S0040-4020(02)00460-X

The acid-promoted reaction of ethyl linoleate with nitrite ions was re-examined by an integrated approach based on the use of ¹⁵NO₂^- combined with extensive GC-MS (EI, NICI, PICI) and 2D ¹H,¹⁵N and ¹H,¹³C NMR analysis. The less polar products proved to be regioisomeric E-nitroalkenes, novel Z-nitroalkenes, and 3-nitro-1,5-hexadienes derivatives. A medium polarity fraction consisted mainly of stereo- and regioisomeric 1,2-nitronitrates along with 1,5-dinitro-1,3-pentadiene compounds. Novel 5-nitro-2,4-pentadienone products could be identified in the most polar fraction, which featured 1,2-nitroalcohols as the most abundant components. Under similar conditions 1,4-hexadiene gave mainly a nitrofuroxan derivative.

Full text of DOI:10.1016/S0040-4020(02)00460-X

TETRAHEDRON
Pergamon
Tetrahedron 58 ꢀ2002) 5061±5067
The acid-promoted reaction of ethyl linoleate with nitrite. New
15
insights from N-labelling and peculiar reactivity of a model
skipped diene
a,p
b
c
Alessandra Napolitano, Orlando Crescenzi, Emanuela Camera, Italo Giudicianni,
d
c
Mauro Picardo and Marco d'Ischia
a
a
Department of Organic Chemistry and Biochemistry, University of Naples `Federico II', Via Cinthia 4, I-80126 Naples, Italy
b
Department of Chemistry, University of Naples `Federico II', Via Cinthia 4, I-80126 Naples, Italy
c
Istituto Dermatologico S. Gallicano, Via S. Gallicano 25a, 00153 Rome, Italy
Centro Interdipartimentale di Metodologie Chimico-Fisiche ꢀCIMCF), University of Naples `Federico II', Via Cinthia 4,
I-80126 Naples, Italy
d
Dedicated in the memory of Professor Guido Sodano
Received 19 February 2002; revised 4 April 2002; accepted 1 May 2002
AbstractÐThe acid-promoted reaction of ethyl linoleate with nitrite ions was re-examined by an integrated approach based on the use of
1
5
2
1
15
1
13
NO
2
combined with extensive GC±MSꢀEI, NICI, PICI) and 2D H, N and H, C NMR analysis. The less polar products proved to be
regioisomeric E-nitroalkenes, novel Z-nitroalkenes, and 3-nitro-1,5-hexadienes derivatives. A medium polarity fraction consisted mainly of
stereo- and regioisomeric 1,2-nitronitrates along with 1,5-dinitro-1,3-pentadiene compounds. Novel 5-nitro-2,4-pentadienone products could
be identi®ed in the most polar fraction, which featured 1,2-nitroalcohols as the most abundant components. Under similar conditions 1,4-
hexadiene gave mainly a nitrofuroxan derivative. q 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
included isomeric E-nitroalkene, 1,2-nitrohydroxyl, 3-nitro-
1,5-hexadiene and 1,5-dinitro-1,3-pentadiene derivatives.
9
The acid-promoted reaction of polyunsaturated fatty acids
with nitrite ions is a potential mechanism of membrane
modi®cation that may affect cellular permeability in the
gastric compartment ꢀpH 2.5±4.5), following cerebral
ischemia and reperfusion, in certain in¯ammatory reactions
of the skin and in all ꢀpatho)physiological conditions asso-
However, a complete picture of the reaction products was
hampered by the marked complexity of the mixtures and the
failure of conventional chromatographic techniques to
1
1
afford suf®ciently puri®ed fractions amenable to 2D H, H
1
13
and H, C NMR correlation analysis; in consequence,
several aspects of the underlying chemistry concerning the
reactivity of the skipped 1,4-diene system with nitrous acid
and species thereof could not be elucidated.
1
ciated with acidosis and nitrite accumulation. Despite the
biomedical relevance and the intrinsic chemical interest of
2
±8
diene nitrosation/nitration,
several aspects of this reac-
tion have remained so far elusive, because of the many
concurrent pathways and the complex mixtures of products.
Literature data on the acid promoted reactivity of simple
diene substrates with nitrite are also surprisingly scanty.
1
5
In this paper we exploit N-labelling to trace the fate of
nitrogen in the acid-promoted reaction of nitrite with ethyl
linoleate, and the potential of a combined GC±MS/NMR
analysis for the in toto characterisation of the reaction
mixture without making recourse to extensive chromato-
graphic fractionations. The overall pattern of reactivity
was then compared with that of 1,4-hexadiene as a model
skipped diene.
Recently, in the course of a systematic investigation of the
acid-induced reaction of unsaturated lipids with nitrite ions,
we undertook a detailed investigation of the reactivity of
ethyl linoleate, the simplest polyunsaturated fatty acid
derivative. Following extensive chromatographic fractiona-
tions, some prominent nitration products could eventually
be isolated and spectroscopically characterised. These
2. Results and discussion
2
.1. Reaction of ethyl linoleate with acidic nitrite
Keywords: nitration; nitrous acid and derivatives; lipids; labelling.
Corresponding author. Tel.: 139-81-67-4133; fax: 139-81-67-4393;
e-mail: alesnapo@unina.it
p
1
The reaction of ethyl linoleate with NO2 was carried out
5
2
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020ꢀ02)00460-X

5062
A. Napolitano et al. / Tetrahedron 58 ꢀ2002) 5061±5067
1 15
Figure 1. H± N HMBC spectrum of the cyclohexane extractable mixture
1
5
2
obtained by acid-promoted reaction of ethyl linoleate with [ N]NaNO .
Letters denote regions of the spectum comprising cross peaks due to groups
of components identi®ed in fractions I±III. All highlighted correlations
were more clearly apparent in the H± N HMBC spectra of the separated
fractions ꢀnot shown).
1
5
Figure 2. Structures and N resonances assignments of the main compo-
nents of fraction I from the reaction mixture of ethyl linoleate with
[
1
15
15
1
15
2
N]NaNO . Arrows indicate multiple bond H± N correlations.
9
in 1% sulfuric acid using 4 M equiv. of nitrite with respect
to the substrate. After 3 h, when linoleate consumption was
1). This was ascribed to a 3-nitro-1,5-hexadiene ꢀ5). Another
noticeable feature was a proton signal at d 4.46 showing one-
bond correlation with a carbon resonance at d 89.1 and with
protons resonating at d 2.70 and 2.45 in the COSY spectrum.
In the H, N HMBC spectrum cross peaks between proton
resonances at d 4.46, 2.70 and 2.45 and a nitrogen signal at d
393.8 were well discernible. Comparison of the proton±
carbon correlations with those of 5 allowed formulation of
the compound as the regioisomer 6 ꢀFig. 2).
1
about 50% ꢀ H NMR evidence) TLC analysis indicated
formation of a collection of products positive to the Griess
reagent for species with nitrosating properties.
1
15
1 15
Fig. 1 shows the H, N HMBC spectrum of the whole
1
5
reaction mixture. Apparently, N signals fell for the most
part in three regions at d 330±340, 365±375 and 390±395.
1
15
To ease identi®cation of speci®c H± N correlations, the
mixture was chromatographed on silica gel plates to afford
three main UV- and Griess-positive fractions that were
Well discernible cross peaks were also observed between a
nitrogen at d 380.5 and protons at d 5.72 and 3.25; and
between a nitrogen at d 380.9 and protons at d 5.65 and
1
13 15
subjected to H, C, N NMR and GC±MSanalysis.
3.30 ꢀregion Ic of Fig. 1). These data argued for regio-
For comparative purposes, the same data were collected
from analogous fractions obtained by reaction with
unlabelled nitrite.
isomeric Z-nitroalkenes 7 and 8, in which the Z geometry
of the double bond hindered the b alkene proton from the
deshielding in¯uence of the nitro group.
1
0
2
.2. Fraction I
GC±MSanalysis ꢀEI, PICI, NICI) of the fraction allowed
straightforward assignment of the fragmentation patterns of
regioisomeric nitroalkenes. These eluted under the peaks at
The less polar fraction ꢀfraction I, R 0.45±0.60, cyclo-
f
hexane/ethyl acetate 95:5) showed as main feature a distinct
R 47.25±47.50 min and gave detectable ꢀpseudo)molecular
t
ion peaks in the PICI ꢀm/z 355 ꢀM1H) , 383 ꢀM1C H ) )
2 5
15
and NICI ꢀm/z 354) spectra of the N-labelled fraction.
Different fragmentation patterns for isomeric 10- and 12-
nitro isomers 1 and 2 ꢀR 47.49 and 47.41 min, respectively)
t
were noted ꢀFig. 3). Such difference would denote a
destabilizing effect of the nitro group toward adjacent allylic
fragmentation of the skipped diene moiety.
1
5
1
1
group of N signals at d 376±381 correlating with proton
resonances spanning from ca. d 7.0 to 2.0 ꢀregion Ia of
Fig. 1). In this fraction, correlation peaks ascribable to the
four regioisomeric E-nitroalkene derivatives 1±4 could
readily be identi®ed ꢀFig. 2).
A characteristic pattern of cross peaks denoting coupling
between a nitrogen signal at d 396.2 and proton resonances
at d 5.83, 5.63 and 4.85 was also apparent ꢀregion Ib of Fig.
Regioisomeric 5/6 eluted as a single GC peak at R
t

A. Napolitano et al. / Tetrahedron 58 ꢀ2002) 5061±5067
5063
group of cross peaks correlating nitrogen signals at d 390±
95 with protons resonating at d around 2.0 and 2.6 ꢀRegion
3
1
1
1
13
IIc, Fig. 1). On the basis of H, H COSY, H, C HMQC and
HMBC experiments, these products could be assigned the
structures of regio- and stereoisomeric 1,2-nitronitrate
5
derivatives of the type 11/12 ꢀFig. 4).
GC±MSanalysis of the fraction showed cluster of peaks
eluting at R 44.7 min which were identi®ed as 9/10 based
t
on the pseudomolecular ion peaks at m/z 401 in the PICI
spectrum and the peculiar fragments in the EI spectrum at
1
m/z 353 ꢀM2 NO ), 305 ꢀM2H NO ± NO ). Another
5
15
15
2
2
2
group of peaks eluting at R 51.0 min and exhibiting pseudo-
t
molecular ion peaks in the NICI spectrum at m/z 418 and a
prominent fragmentaton peak at m/z 370 for the loss of
labelled HNO were identi®ed as isomeric nitronitrates.
Figure 3. Fragmentation patterns of labelled ꢀunlabelled) nitroalkenes 1
and 2.
2
4
6.02 min, and showed pseudomolecular ion peaks in the
2.4. Fraction III
1
PICI spectrum at m/z 355 ꢀM1H) . Intense fragmentation
peaks at m/z 309/306 and 261 were apparent in the EI/MS
spectrum, due to losses of the OEt radical and/or of labelled
1
15
The H, N HMBC spectrum of fraction III displayed corre-
lation peaks for isomeric 1,2-nitroalcohols 13/14 and 15/16
ꢀFig. 5).
1
5
H NO as well as peaks at m/z 243 and 217 which secured
2
identi®cation in comparison with the fragmentation pattern
obtained from unlabelled compounds.
9
GC±MSanalysis of the fraction revealed a cluster of peaks
at R 45.9 min which gave pseudomolecular ion peaks in the
t
2
.3. Fraction II
PICI ꢀm/z 373) and NICI modalities ꢀm/z 372) and EI frag-
mentation peaks at m/z 325 and 307 due to sequential loss of
NO and H O, and as well as at m/z 255 and 227 ꢀm/z 254
1
15
15
The H, N HMBC spectrum of fraction II displayed cross-
peaks pertaining to the dinitrodiene derivatives 9/10, corre-
lating the proton signalꢀs) at d 7.47/7.48 and 2.69/2.71 with
two nitrogen resonances at d 376.8/377.0 ꢀRegion IIa,
Fig. 1). Less intense but discernible cross peaks were also
observed between protons signals in the d 1.75±1.80 region
and the nitrogen resonance at d 396 for the allylic NO2
group. Scalar coupling of the protons at d 7.47/7.48
2
2
9
and 226 in the unlabelled fraction).
Another major component of fraction III displayed a corre-
lation between a nitrogen signal at d 375.0 and two protons
1
at d 7.51 and 2.81 ꢀregion IIIb, Fig. 1). In the H, H COSY
1
1
13
and H± C HMBC spectra, the former proton signal was
recognised as part of a set of three coupled resonances
appearing at d 7.51 ꢀ1H, d, Jˆ12 Hz), 7.25 ꢀ1H, dd,
Jˆ15.2, 12.0 Hz) and 6.62 ꢀ1H, d, Jˆ15.2 Hz) which
showed one-bond correlations with carbons at d 131.6,
133.6 and 138.2, in that order. The 7.51 proton signal was
1
5
ꢀJˆ3.6 Hz) and 6.18 ꢀJˆ1.2 Hz) with the N nitrogens
was well apparent in the H NMR spectrum.
1
Other typical features of the spectrum were: ꢀa) a group of
1
5
15
N resonances at d 335±340 correlating with protons reso-
nating between d 4.4 and 5.6 ꢀRegion IIb, Fig. 1); and ꢀb) a
additionally split by scalar coupling with the N nucleus
ꢀJˆ3.6 Hz). Moreover, the protons at d 7.51 and 2.81
showed long range correlations with carbon signals at d
138.2 ꢀCH) and a 158.3 ꢀquaternary carbon), whereas the
protons at d 7.25 and 6.62 showed cross peaks with carbon
1
5
13
Figure 4. Structures and N ꢀitalicized), C ꢀenbolded) and H resonances
1
1
15
Figure 5. Structures and H± N correlations of 1,2-nitrohydroxyalkene
1 15
products in fraction III. Arrows indicate H± N correlations.
1 15
of the main components of fraction II. Arrows indicate H± N correlations.

5064
A. Napolitano et al. / Tetrahedron 58 ꢀ2002) 5061±5067
down®eld by ca. 0.8 ppm, suggesting a deshielding group
1
13
on the C-2 carbon. Scrutiny of the H, C HMBC spectrum
revealed correlations between the methylene protons and
four carbon resonances at d 109.4, 120.3, 133.2 and
159.0. GC±MSanalysis of the isolated band showed a
main peak giving a molecular ion peak at m/z 185, con-
sistent with a nitrofuroxan derivative, and a diagnostic
fragmentation peak at m/z 139 due to loss of a nitro
group. On this basis the compound was formulated as
3-ꢀ2-butenyl)-4-nitro-2-oxido-1,2,5 oxadiazole ꢀ20).
Figure 6. Structure and assignment of diagnostic proton, carbon ꢀenbolded)
and nitrogen ꢀitalicized) resonances of the yellow component of fraction III.
The remainder of the mixture was accounted for by the
starting material and nitrated products which eluded
detailed characterisation.
1 15
Arrows highlight H± N correlations.
resonances at d 131.6 and 158.3 as well as with a carbonyl
resonance at d 200.2. Cross peaks with the latter resonance
was also exhibited by a distinct triplet at d 2.81 showing one
bond correlation with a carbon at d 28.5. Repeated TLC of
fraction III eventually allowed isolation of a yellow product
2
.6. Mechanistic remarks
The structures of the main products formed by exposure of
ethyl linoleate to nitrite ions in an acidic medium con®rms
the prevalence of nitration reactions. These would derive
ꢀ
lmax 281, 383) with spectral features identical with those of
the above species. GC±MSanalysis indicated an intense
from homolytic attack of NO to the skipped diene system
2
9
of ethyl linoleate generating b-nitroalkyl radicals. Free
peak at m/z 370 and a fragmentation peak at m/z 352
1
5
radical recombination with another molecule of NO2
ꢀ
isomeric structures 17/18 featuring the 5-nitro-2,4-pentadie-
M2 NO ). On this basis the product was assigned regio-
2
would then lead to 1,2-nitronitrite intermediates which
may suffer HNO elimination and/or hydrolysis to give
none moiety ꢀFig. 6).
2
nitroalkenes 1±4 and/or 1,2-nitroalcohols 13±16, respec-
tively. Alternatively, H-atom abstraction would afford
1 15
The H, N HMBC spectrum showed also weak but well
1
discernible cross peaks between a group of N resonances
5
3-nitro-1,5-hexadiene derivatives ꢀ5/6) and 1,5-dinitro-1,3-
pentadiene products ꢀ9/10).
9
at d 353±359 and proton resonances at d 3.1±2.9 and 2.3±
2.6 ꢀregion IIIc, Fig. 1), and between the latter and nitrogen
resonances at d 378±379. These values were suggestive of
The identi®cation of 1,2-nitronitrates ꢀ11/12), which
account for at least 25% of the reacted material based on
H NMR analysis, is paradigmatic of the improved
2
-oxido-1,2,5-oxodiazole ꢀfuroxan) derivatives of the type
1
1
1,12
1
unsubstantiated because of exceedingly overlapped proton
resonances hindering analysis of the H, C NMR correla-
tion spectra.
9 ꢀFig. 7).
Unfortunately this hypothesis remained
analytical potential of the integrated approach reported in
this paper. These products would arise by oxidation of 1,2-
nitronitrite intermediates. Another viable route would
involve trapping of the initially formed b-nitroalkyl radical
by oxygen followed by coupling with NO to give a nitroso-
1
13
2
.5. Reaction of 1,4-hexadiene with acidic nitrite
1
3±15
peroxyl intermediate which then rearranges to a nitrate.
However, the virtually identical product pattern formed in
an oxygen-free atmosphere rules out the prevalence of
oxygen-dependent routes under the speci®c reaction condi-
tions of this study.
To gain a deeper insight into the acid-promoted reaction of
,4-pentadiene systems with nitrite ions, the study was
1
extended to 1,4-hexadiene ꢀ80% E isomer) as a simple
skipped diene. Exposure of the compound to nitrite ꢀ5 M
equiv.) in 1% sulfuric acid resulted in a fast reaction leading
to a relatively simple pattern of products with a main UV
absorbing species positive to the Griess reagent. The
compound was obtained by preparative TLC as a chromato-
Formation of the Z-nitroalkenes 7/8 does not conform with
the expected stereochemical course of alkene nitration,
4
leading mainly to the E isomers. It is plausible that in the
1
case of ethyl linoleate the Z-nitroalkene route is less
unfavourable because of the steric hindrance of the alkyl
chains on the double bonds, weakening the role of the
nitro group as the main determinant of the stereochemical
outcome of the reaction.
graphically homogeneous band. The H NMR spectrum
showed a mixture of stereoisomers ꢀ90% E and 10% Z) in
which the less substituted alkene moiety was fully substi-
tuted. Moreover the bis-allylic methylene group was shifted
The mechanism of formation of the 5-nitrodienones 17/18 is
not straightforward. In principle, a carbonyl-forming route
from hydroperoxide precursors, preceding or following the
nitration step, would be a tenable hypothesis. However, in
separate experiments it was found that neither
ꢀ9Z,11E,13S)-13-hydroperoxyoctadeca-9,11-dienoic acid
nor ꢀ9Z,11E)-13-oxo-9,11-octadecadienoic acid, the
corresponding ketone, when reacted with acidic nitrite
under the same conditions as ethyl linoleate, were converted
to signi®cant amounts of 17/18 as free acids. Moreover,
Figure 7. Proposed structural assignments for the minor, putative furoxan
components of Fraction III. Arrows indicate H± N correlations.
1
15

A. Napolitano et al. / Tetrahedron 58 ꢀ2002) 5061±5067
5065
Figure 8. Proposed mechanism of formation of 5-nitrodienones 17/18.
ꢀ
9E,12E,13S)-13-hydroperoxy-9-nitro-9,11-octadecadienoic
precursors may proceed by a heterolytic pathway, a guess
supported by the polar nature of the nitro-substituted double
bond.
1
6
acid ꢀ21), prepared as recently reported, failed to give
detectable 17/18 free acids by exposure to acidic nitrite or
atmospheric oxygen in the presence of iron ions for
prolonged periods of time ꢀup to 10 h), i.e. under conditions
1
7
favouring conversion of hydroperoxides to ketones.
3. Conclusions
Considering that the reaction course is not substantially
modi®ed in an oxygen-free medium, and that formation of
the yellow nitrodienone is not appreciable during the very
early stages of the reaction, it can be suggested that this
species originates by a complex pathway involving
H-atom abstraction to afford eventually nitronitrite or nitro-
nitroso ꢀnitrooxime) intermediates susceptible to carbonyl-
forming decomposition or hydrolysis ꢀFig. 8).
The integrated NMR/GC±MSmethodology described in
this paper allowed an unprecedented insight into the acid-
promoted reaction of an unsaturated fatty acid with nitrite
1
5
ions. The N tracer approach has the advantage that few or
no processing steps are required, and ensures that all nitro-
genous reaction products are selectively detected even in the
presence of abundant non-nitrogenous species, minimising
the risk that important nitrosation/nitration products were
lost or overlooked. This methodology overcomes the typical
limitations of GC±MSanalysis due to the volatility and the
intrinsic stability of the analytes, which is particularly
unfavourable in the most polar components.
The different reactivity of 1,4-hexadiene, yielding nitro-
furoxan 20 as main product, would re¯ect the presence of
the singly substituted double bond, which is lacked by ethyl
linoleate. Thus, nitration at this double bond and subsequent
conversion of the resulting 1-nitro-1,4-hexadiene to a 1,2-
dinitro-1-nitroso intermediate ꢀor the 1,2-nitrooxime
tautomer) would be a plausible route to compound 20
The inventory of nitration/nitrosation products reported in
this paper and the rationalisation of their formation integrate
and expand current knowledge of the reactivity of 1,4-
dienes with nitrogen oxides derived from acidic nitrite.
Since the skipped diene is the core structural feature of
prominent groups of naturally occurring lipids, the observed
reactivity toward nitrogen oxides may be of relevance to the
bioactivity of these compounds.
1
2,18
ꢀ
Fig. 9).
terminal sites of 1-alkenes is well documented,
is furoxan formation by reaction of polar double bonds bear-
The proclivity of nitrogen oxides to react at
1
9,20
and so
1
2,21
ing electron-withdrawing groups ꢀe.g. cinnamic acids,
12
1
2
methacrylic acid, cinnamaldehyde ) with acidic nitrite.
Formation of the putative 1,2-nitronitroso ꢀnitrooxime)
Finally, the present work may provide the background to set
future studies on nitrite-mediated nitration/nitrosation
processes in complex matrices and biological systems.
4. Experimental
4.1. General
Ethyl linoleate ꢀ98%), 1,4-hexadiene ꢀ99%, E,Z mixture),
[
1
5
N]NaNO ꢀ.99%) were purchased from Aldrich and
2
used as obtained. GC±MSwas carried out on a HP 5889A
or 5970 instrument coupled with a quadrupole mass spectro-
meter using a 30 m Rtx-5MS-crossbond 5% diphenyl±95%
dimethylpolysiloxane column ꢀ0.25 mm i.d., 0.25 m df).
Figure 9. Proposed formation route of nitrofuroxan 20.

5066
A. Napolitano et al. / Tetrahedron 58 ꢀ2002) 5061±5067
Helium was the carrier gas. CI±MSmeasurements were
carried out using methane as the reagent gas. Data were
processed using G1701AA data analysis software. The
temperature program of the column was as follows: at
hexane) 308 nm; FT-IR ꢀCHCl ) n
3
1635, 1571, 1499,
max
2
1
1
1354 cm ; H NMR ꢀ400 MHz, CDCl ) 1.69 ꢀ2.4 H, d,
3
Jˆ7.6, 0.8 Hz), 1.76 ꢀ0.6 H, d, Jˆ7.6, 0.8 Hz), 3.53 ꢀ1.6
H, dd, Jˆ6.4 Hz), 3.62 ꢀ0.4 H, d, Jˆ6.4 Hz) 5.41 ꢀ1H, m),
1
5.75 ꢀ1H, m); C NMR ꢀ100 MHz, CDCl ) 18.6, 27.0,
3
4
08C, hold timeˆ1 min; from 40 to 2808C, rateˆ58C/min;
3
1
109.4, 120.3, 133.2, 159.0; EI MS m/z 185 ꢀM , 40), 139
hold at 2608C for 11 min. The injector and detector were
taken at 220 and 2508C, respectively. The acquisition started
ꢀM2NO , 72), 97 ꢀM2NO ±C H , 100); HR MScalc for
6
2
2
3
5
min after the injection ꢀsolvent delay 5 min), and was set
C H N O 185.1404, found m/z 185.1415.
6 7 3 4
in scan mode in the range 43±550 amu. The EI spectra were
obtained at 70 eV. For CI spectra the source and quadrupole
were taken at 200 and 1008C, respectively, and a mass range
4.4. Isolation of 17/18 and formation from putative
precursors
4
0±600 amu was chosen.
Fraction III ꢀ40 mg) obtained from the reaction mixture of
ethyl linoleate with NaNO was puri®ed by preparative TLC
UV and IR spectra were performed using a diode array and a
1
13
15
2
FT-IR spectrophotometer, respectively. H, C, N NMR
spectra were recorded at 400.1, 100.6 and 40.5 MHz, in that
order using a Bruker DRX-400 MHz instrument ®tted with a
ꢀ
ꢀ
cyclohexane/ethyl acetate 90:10) to yield a yellow band
10 mg) which was subjected to further fractionation ꢀcyclo-
hexane/ethyl acetate 95:5) to afford 17/18 ꢀ5 mg). UV l
1
max
5 mm H/broadband gradient probe with inverse geometry.
Standard Bruker implementations of gradient-selected
1
2
4
81, 383 nm; H NMR ꢀCDCl ) ꢀsee Fig. 6); NICI MS R
3 t
6.50 min, m/z 370.
1
versions of inverse ꢀ H detected) heteronuclear multiple
quantum coherence ꢀHMQC) and heteronuclear multiple
bond correlation ꢀHMBC) experiments were used. The
HMBC experiments used a 100 ms long-range coupling
delay. Samples analysed by NMR spectroscopy were
ꢀ9E,11E)-13-Hydroperoxy-9-nitrooctadecadienoic acid ꢀ21)
was prepared as previously described. ꢀ9Z,11E, 13S)-13-
16
hydroperoxyoctadeca-9,11-dienoic acid was obtained by
lipoxidase oxidation as described and puri®ed by prepara-
23
dissolved in CDCl . Chemical shifts are recorded in d
3
tive TLC ꢀcyclohexane/ethyl acetate 7:3). The isolated
product was exposed to FeꢀII)/H O in 0.1 M phosphate
buffer to give ꢀ9Z,11E)-13-oxo-9,11-octadecadienoic
1
values ꢀppm) down®eld from tetramethylsilane ꢀ H and
1
3
15
C NMR) or with reference to [ N]urea in DMSO at
2
2
7
ꢀ
6.97 ppm, relative to NH ꢀliquid, 298 K) at 0.0 ppm
3
17
acid as the main product which was isolated by preparative
TLC ꢀcyclohexane/ethyl acetate 7/:3). Purity of the
1
5
22
N NMR). Analytical and preparative TLC analyses
were performed on F254 0.25 and 0.5 mm silica gel and
high performance ꢀHPTLC) plates from Merck. Griess
reagent ꢀ1% sulphanylamide and 0.1% naphthylethylene-
diamine in 5% phosphoric acid), potassium dichromate in
20% sulphuric acid and iodine were used for product detec-
tion on TLC plates.
1
compound was checked by NMR analysis: H NMR
ꢀCDCl ) d ꢀppm) ꢀselected resonances): 0.89 ꢀ3H, m)
3
1.29±1.63 ꢀ16H, m), 2.28 ꢀ4H, m), 2.56 ꢀ2H, t,
Jˆ7.2 Hz), 5.88 ꢀ1H, m), 6.09±6.18 ꢀ2H, m), 7.49 ꢀ1H,
dd, Jˆ14.2, 12 Hz).
Compound 21 or ꢀ9Z,11E,13S)-13-hydroperoxyoctadeca-
9,11-dienoic acid or ꢀ9Z,11E)-13-oxo-9,11-octadecadienoic
1
5
4
.2. Reaction of linoleic acid ethyl ester with [ N]NaNO
2
acid were exposed separately to nitrite in 1% sulphuric acid
as detailed above. Periodically, aliquots of the reaction
mixture were extracted with ethyl acetate and analysed by
TLC ꢀcyclohexane/ethyl acetate 7:3) and H NMR. In other
experiments 21 ꢀ0.5 mM) was exposed to ironꢀII) ions
To linoleic acid ethyl ester ꢀ462 mg, 1.5 mmol) in 1% sul-
1
5
furic acid ꢀ20 mL) [ N]labelled or unlabelled sodium nitrite
420 mg, 6.0 mmol) was added while the mixture was taken
ꢀ
1
under vigorous stirring in a stoppered round-bottom ¯ask at
room temperature. After 3 h, the mixture was extracted with
cyclohexane ꢀ3£10 mL) and the combined organic layers
washed with brine and dried over sodium sulfate to afford
a yellow residue. Fractionation of the resulting residue on
PTLC using cyclohexane/ethyl acetate 95:5 as the eluant
ꢀ
1
0.2 M equiv.) in 0.1 M phosphate buffer ꢀpH 7.4). After
0 h, the reaction mixture was extracted with ethyl acetate
and analysed as above.
afforded four main fractions. The R 0.7 fraction ꢀ200 mg)
f
Acknowledgements
consisted of the starting material and was not further
analysed. Fractions at R 0.45±0.60 ꢀ55 mg, fraction I), Rf
f
This work was ®nancially supported by MURST ꢀPRIN
001) and Ministry of Health ꢀRome). We thank the ªCentro
Interdipartimentale di Metodologie Chimico±Fisiche of
Naples Universityº for NMR facilities. GC±MSfacilities
were available at Istituto Dermatologico S. Gallicano
0.35±0.40 ꢀ32 mg, fraction II) and R 0.15±0.30 ꢀ40 mg,
f
fraction III) were subjected to NMR and GC analysis.
2
4
.3. Reaction of 1,4-hexadiene with nitrite
ꢀRome).
To 1,4-hexadiene ꢀ304 mg, 3.7 mmol) in 1% sulfuric acid
20 mL) sodium nitrite ꢀ1.28 g, 18.5 mmol) was added and
ꢀ
the mixture was taken under vigorous stirring in a stoppered
round-bottom ¯ask at room temperature overnight. The
mixture was extracted with ethyl acetate and the residue
obtained ꢀ151 mg) was fractionated by preparative TLC
using cyclohexane/ethyl acetate 7:3 to give a main band
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