Biomimetic nitration of conjugated linoleic acid: Formation and characterization of naturally occurring conjugated nitrodienes

Article abstract of DOI:10.1021/jo4021562

Nitro-conjugated linoleic acids (NO₂-cLA), endogenous nitrodiene lipids which act as inflammatory signaling mediators, were isolated and single isomers purified from the biomimetic acidic nitration products of conjugated linoleic acid (CLA). Structures were elucidated by means of detailed NMR and HPLC-MS/MS spectroscopic analysis and the relative double bond configurations assigned. Additional synthetic methods produced useful quantities and similar isomeric distributions of these unusual and reactive compounds for biological studies and isotopic standards, and the potential conversion of nitro-linoleic to nitro-conjugated linoleic acids was explored via a facile base-catalyzed isomerization. This represents one of the few descriptions of naturally occurring conjugated nitro dienes (in particular, 1-nitro 1,3-diene), an unusual and highly reactive motif with few biological examples extant.

Full text of DOI:10.1021/jo4021562

Article
pubs.acs.org/joc
Biomimetic Nitration of Conjugated Linoleic Acid: Formation and
Characterization of Naturally Occurring Conjugated Nitrodienes
Steven R. Woodcock,*,† Sonia R. Salvatore,† Gustavo Bonacci,§ Francisco J. Schopfer,†
and Bruce A. Freeman†
†Department of Pharmacology and Chemical Biology, University of Pittsburgh. Pittsburgh, Pennsylvania 15261, United States
§CIBICI-CONICET, Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba,
Cordoba X5000HUA, Argentina
S
* Supporting Information
ABSTRACT: Nitro-conjugated linoleic acids (NO2-cLA), endogenous nitrodiene lipids which act as inflammatory signaling
mediators, were isolated and single isomers purified from the biomimetic acidic nitration products of conjugated linoleic acid
(CLA). Structures were elucidated by means of detailed NMR and HPLC−MS/MS spectroscopic analysis and the relative
double bond configurations assigned. Additional synthetic methods produced useful quantities and similar isomeric distributions
of these unusual and reactive compounds for biological studies and isotopic standards, and the potential conversion of nitro-
linoleic to nitro-conjugated linoleic acids was explored via a facile base-catalyzed isomerization. This represents one of the few
descriptions of naturally occurring conjugated nitro dienes (in particular, 1-nitro 1,3-diene), an unusual and highly reactive motif
with few biological examples extant.
Scheme 1. Nitration and Oxidation of Bis-allylic Linoleic
Acid (3)
INTRODUCTION
■
Nitrated derivatives of unsaturated fatty acids are products of
the reaction of nitrogen oxides with unsaturated lipids.1
Nitrated fatty acids are generated by cardiac tissue following
ischemia/reperfusion2 and ischemic preconditioning,3 oxidative
stress and hypoxia,4 and acid-catalyzed reactions in the gastric
compartment.5 Nitrated lipids6 are a representative subset of
electrophilic lipid mediators,7 a class that includes a number of
oxidized and nitrated lipids produced and detected in vivo that
exert signaling actions8 via Michael addition to thiol-containing
proteins. Through this reversible Michael addition9 they have
been shown to mediate reversible post-translational modifica-
tion of key proteins10 regulating inflammation11 and metabo-
lism10,12 and to modulate the function of inflammatory and
cytoprotective signaling mediators including Keap1/Nrf2,13
HSF-1,14 and NF-κB,11 and PPARγ.15−17
Acidic nitration replicates the environmental conditions that
occur in biological compartments including mitochondrial
membranes, lysosomes, tissues subjected to ischemic con-
ditions, and digestion. Initial work in this field demonstrated
the nitration of bis-allylic linoleic acid (LA (3), Scheme 1) via
biomimetic acidic nitration18,19 (as well as phenylselenation20
for larger scale). Both methods are nonregiospecific, producing
a mixture of positional isomers of NO2-LA with nitration at the
9-, 10-, 12-, and 13-positions (4−7). Studies that isolate and
compare individual isomers,21 synthesize regiospecific iso-
Received: March 1, 2013
Published: December 19, 2013
© 2013 American Chemical Society
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mers22 (5 and analogs23), and characterize the degradation
manifolds of particular isomers24 (4) have been reported.
These initial reports focused on understanding the nitration of
LA because of its natural abundance and the presence of a
reactive bisallylic methylene that favors hydrogen abstraction
reactions.
In a related study, the conjugated diene-containing product
of enzymatic lipid oxidation 13-hydro(per)oxyoctadeca-9,11-
dienoic acid (13-HpODE, 8a, and 13-HODE, 8b)25 was
nitrated in a biphasic reaction to provide the corresponding 9-
nitro-13-hydro(per)oxyoctadeca-9, 11-dienoic acid (9a or 9b);
this product was later shown to be also produced by a base-
catalyzed rearrangement/oxidation of 9-NO2-LA (4). These
were the first reports of conjugated nitrodienes26 derived from
biological lipids.
for production of these lipids to encourage progress in the field.
In the present work, we report the synthesis, isolation, and
spectral characteristics of the biologically relevant nitrodiene-
containing products of the reaction of 10 with nitrite.
RESULTS AND DISCUSSION
■
Acidic nitration of CLA on a preparatory scale (Scheme 2, a)
was investigated using a modification of previous reports of LA
nitration.18,19 As the primary isomer available in vivo, only
9Z,11E-conjugated linoleic acid (10) was used for this study.
Reactions of 10 were typically conducted in a biphasic mixture
with an aqueous acidified nitrite solution in the absence of
oxygen. The reaction was followed by TLC until products were
observed to be UV-active and positive to Griess reagent. Upon
workup, the specific product NO2-cLA (1 + 2) could be
isolated chromatographically in modest yield (8%) from the
resulting mixture of unreacted starting material (45−75%) and
other nitrated and oxidized species and was characterized by
HPLC−MS/MS as a mixture of two regioisomers. Isotopically
labeled sodium nitrite (Na15NO2) was also used for production
of labeled nitro-group products for mass spectrometric
standards.
In a recent work,5 we demonstrated that the direct reaction
of nitrating species with conjugated double-bond-containing
lipids is highly favored when compared to mono- or
polyunsaturated fatty acids (e.g., LA), occurring at a faster
and more extensive rate and yielding higher observed
concentrations in vivo, and is potentially responsible for most
if not all endogenous nitration. Conjugated double-bond-
containing lipids are available from dietary sources27,28 and are
normally present in membranes, plasma, and tissues. 9Z,11E-
Conjugated linoleic acid (10) has been identified29 as the
primary isomer available in vivo (estimated at >80%). CLA was
found5 to be nitrated in vivo, and the products were shown to
display unique potency and signaling activity. In particular, two
predominant positional isomers of naturally occurring nitrated
conjugated linoleic acid (Figure 1), collectively referred to as
After single-isomer isolation (see below) and MS analysis,
structures were assigned on that basis as well as NMR, UV−vis,
and FTIR analysis. On the basis of the formation of specific
fragments upon LC−MS/MS,5,30 it was confirmed that
nitration of 10 occurred only at the 9- and 12-positions. The
products displayed strong UV−vis absorbance as a distinct
chromophore with a significantly higher molar absorptivity in
methanol at longer wavelengths (λmax 312 nm) relative to
isolated nitroalkene-containing NO2-LA (λmax 257 nm), as well
as strong FTIR bands of 1317 and 1510 cm−1, distinct from
nitroalkene (1334, 1522 cm−1 in NO2-LA)31 or nitroalkane
(1379, 1550 cm−1 in nitrostearic acid)32 stretching frequencies.
Aside from features common to all lipids, the 1H NMR
spectrum of each isomer featured in the low-field olefinic region
a doublet at δ 7.52 ppm and two correlated multiplets in the δ
6.0−6.5 ppm region (J = 15 Hz indicating a trans alkene)
(Figure 2). The δ 6.20 signal was coupled to the δ 7.52 doublet
(s-trans) and the δ 6.34 to the methylene quadruplet at δ 2.24;
15N labeling of the nitro group demonstrated coupling to the
low-field doublet and the δ 2.65 methylene triplet by HMQC.
Proton−proton and proton-carbon correlations were confirmed
by 2-D COSY and HMQC. Overall, this resonance spectrum
suggested two nearly identical isomers containing mutually
symmetric, conjugated, electron-deficient diene regions, con-
sistent with an (E,E)-conjugated nitrodiene. On the basis of this
evidence, NO2-cLA was formulated as an approximately
equimolar mixture of 1 and 2.
Figure 1. Nitro-conjugated linoleic acids: (9E,11E)-9-nitrooctadeca-
9,11-dienoic acid (9-NO2-cLA, 1) and (9E,11E)-12-nitrooctadeca-
9,11-dienoic acid (12-NO2-cLA, 2).
NO2-cLA (9- and 12-nitro-conjugated linoleic acid, 1 and 2),
were formed from CLA and act as biologically relevant
electrophilic signaling mediators. Multiple biological conditions
including exposure of CLA to acidified nitrite, myeloperox-
idase/hydrogen peroxide/nitrite, peroxynitrite, and gaseous
nitrogen dioxide (Scheme 2, a−d) were shown5 to form NO2-
cLA at a micromolar scale. However, there has not been
detailed characterization or report of a useful synthetic method
Note that Z-nitroalkene protons33 have a significantly lower
chemical shift, ∼5.6 ppm vs 7.1 ppm for E-nitroalkenes.
Scheme 2. Nitration of Conjugated Linoleic Acid by Multiple Biomimetic and Synthetic Conditions
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which may react with available double bonds by allylic
hydrogen abstraction and direct addition.
Direct addition of nitrogen dioxide to the electron-rich diene
proceeds rapidly, forming a resonance-stabilized allylic radical
intermediate35,36 (I and II). Addition at the terminal positions
is favored by enhanced electron density at the 1,4-positions in
the diene HOMO and maximizes the stability of the resulting
delocalized radical; hence, nitration of 9Z,11E-cLA (10)
produced only species nitrated at the 9- or 12-position. Upon
formation of I and II, free carbon−carbon bond rotation is
allowed and the original bond configuration is lost.
Radical intermediates I and II may proceed to form multiple
products. Nitrodiene formation has been proposed19,37−39 from
either addition/elimination of a second nitrogen dioxide (path
A) or a one-step hydrogen abstraction (path B). Path A is a
two-step route, in which formation of unstable nitrito-nitro
species (III and IV) leads to loss of HNO2 and formation of the
conjugated nitroalkenes 1 and 2. Path B is a one-step radical
hydrogen abstraction, by either nitrogen dioxide or secondary
radicals present. This pathway requires no leaving group but
could potentially produce Z-isomers or nitroalkane−alkenes
(not observed). In plausible pathway C, a second nitration
event may also lead to unproductive dinitro species (V), which
have been previously reported39 in alkene nitration. Low yields
of isolated product (1 + 2) support a number of unproductive
paths being active, perhaps even favored. In the presence of
oxygen pathway D becomes viable, with sequential nitration/
peroxidation leading to formation of multiple oxidation
Figure 2. NMR chemical shifts and correspondences of the nitro diene
moiety of [15N]-NO2-cLA. The double-bond configuration shown
(E,E) based on these coupling values is reproduced in both isomers (1
and 2).
Energetically, nitroalkenes adopt the E-configuration to
minimize allylic repulsion, and E-configuration is also
thermodynamically preferred for a 1,2-disubstituted alkene
such as the γ,δ-double bond. Two isolated E,E-isomers implied
that this is a thermodynamic product and that alternative
(possibly kinetic) products were not observed.
A mechanism for nitration of dienes by acidified nitrite is
proposed in Scheme 3, beginning with formation of nitrogen
−
dioxide. In the presence of strong acid, nitrite (NO2 pKa =
3.4)34 is rapidly protonated to nitrous acid (HNO2), which
rapidly equilibrates with dinitrogen trioxide (N2O3) which may
disproportionate favorably into nitric oxide (·NO) and nitrogen
dioxide radical (·NO2). The latter is a powerful nitrating agent,
Scheme 3. Proposed Mechanism of cLA Nitration via Radical Addition
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Figure 3. (a) Elution profile of individual isomers 12- and 9-NO2-cLA (2 and 1) by preparatory HPLC. Absorbance monitored at 210 nm. (b)
Isomer enrichment of individual fractions collected, as measured by LC/MS (bar thickness proportional to size of fraction). Fractions 1−6 were
combined to afford single-isomer 2 (28.8% recovery of injected material, 8.2% yield based on starting material), and fractions 10−16 likewise pooled
to afford 1 (41.7% recovery, 12.3% from SM).
products (VI, VII) identified previously5 but outside the scope
of this present study.
(Figure 3b), fractions were pooled, subjected to a second
PTLC, and quantified by gravimetric and UV−vis methods.
Recovery of isolated material in this case afforded milligram
quantities of single isomers 2 and 1 each in greater than 90%
purity by LC/MS.
Unfortunately, this method, while a useful model of
biological nitration, was hampered by low (and inconsistent)
yields and difficulty in purification from multiple similar
nitration/oxidation products. This method was intended to
provide a biomimetic model for the production of nitrodienes
in vivo and not otherwise developed in an attempt to increase
yields. Reaction products included unreacted CLA 10 (45−
75%), smaller amounts of nitroalkyl addition products (possibly
III−V which had not reacted further), and NO2-cLA. Overall
yields of 1 and 2 were similar to NO2-LA19 nitration. This work
focused on the electrophilic products of nitration that were
observed to exert biological effects, and products which were
nonactive in biological milieu were not characterized further.
The limitations of the acidic nitration approach encouraged our
search for a scalable NO2-cLA synthetic method that would
closely mimic the nitrated products derived from human
samples (Scheme 2, e,f).
There are limited methods for diene nitration in the
literature.26 Phenylselenation/nitration methods, previously
used for synthesis of nitroalkenes, produced only complex
mixtures consistent with prior reports of nitration−epoxidation
products.40 Instead, the most promising approach developed
from a report41,42 of a two-step trifluoroacetoxy nitration
method. This method was adaptable to a small scale, producing
significantly larger and more consistent quantities (25% yield)
of desired nitro dienes 1 and 2 than acidic nitration. In
addition, the products could be more easily purified, producing
fewer chromatographically similar side products than the
multiplicity of radical nitration products. Subsequent mod-
ification of the method by exchanging ammonium nitrate for
isotopically labeled ammonium nitrate (NH415NO3) gave
similar yields, while more soluble tetrabutylammonium nitrate
improved yield (40%). Analysis by LC/MS and NMR showed
products identical to those obtained by acid nitration and
human derived products, a main goal of the present work. In
particular, LC/MS/MS analysis again showed only 9- and 12-
nitration products 1 and 2 from 10, indicating no isomerization
or double-bond migration.
Both methods of diene nitration allow for facile incorpo-
ration of an 15N-nitro group, as labeled sodium nitrite and
ammonium nitrate are both readily available. Isotopic labeling
of the NO2-cLA allows convenient and efficient standards for
detailed mass spectrometric analyses as well as biological
probes. In addition, it provides the first synthetic route to
obtain isotopically labeled standards for analytical determi-
nation of biological samples containing conjugated nitrated
fatty acids, and given that biological production is expected to
include both regioisomers in the same proportion, the [15N]-
containing standards are appropriate for analysis and
quantitation of all natural product lipids. Labeling by a one-
step procedure43 is preferable economically as well as
practically, and isotopic substitution of the nitro group allows
facile monitoring of product ions via MS/MS where scanning
−
for charged loss of NO2 is used.
Trifluoroacetoxy nitration/deprotonation is reported42 to
occur by an ionic mechanism (Scheme 4) with trifluoroacetyl
nitrate generated in situ from nitrate ion and trifluoroacetic
anhydride. This reactive species rapidly adds a nitronium ion to
the terminus of an electron-rich diene. The resonance-stabilized
intermediate ion pairs (VIII) then combine to form a 1,2- or
1,4-nitrotrifluoroacetoxy ester (intermediate mixtures 11 and
12). For comparison, the ratio of 1,2- to 1,4-addition for 1,3-
butadiene was reported to be approximately 3:2,42 but LC/MS
analysis of the positional preferences of intermediates 11 and
12 proved inconclusive due to facile elimination of labile
trifluoroacetoxy groups upon collision-induced fragmentation
that did not produce informative product ions. However,
hydrolysis of the trifluoroacetoxy nitro intermediates to the
corresponding hydroxy nitro compounds proved successful,
and these were identified as 9- and 12-nitro species based on in-
source fragmentation (see the Supporting Information).
Unfortunately, the position of the hydroxyl was lost on
fragmentation, and thus the ratio of 1,4- to 1,2-addition was not
defined using MS/MS data.
Individual isomers which had been initially identified by LC/
MS were isolated via preparatory HPLC (Figure 3a). Elution of
a product mixture prepurified by PTLC was monitored by
UV−vis and individual fractions collected. After individual LC/
MS analysis to determine relative isomer amounts and purity
Subjecting esters 11 and 12 to mild base eliminated
trifluoroacetate and formed the same regioisomers in the
same double-bond configuration as the acidic nitration-derived
nitrodiene. Base-catalyzed elimination of trifluoroacetate from
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Scheme 4. Proposed Mechanism of cLA Nitration via
Trifluoroacetoxy Nitration
Scheme 5. Base-Promoted Conjugation and Oxidation of
Bis-allylic Nitrolinoleic Acids (4−7)
intermediate XI stabilized by the terminal nitro group. For nitro
groups located at the 10- or 12- (inner) positions (5 and 6),
deprotonation would be slower and would produce inter-
mediate XII; however, inspection of the reaction products by
1H NMR indicated no reaction of 5 and 6.
Acidic quenching of XII would result in a conjugated diene
with the nitro group at the first position, but oxygen insertion
was evidently more rapid than protonation, as only hydro-
peroxy 9a24 and regioisomer 14 were observed along with
unreacted 5 and 6 (Figure 4b). Thus, the observed spectrum
was a result of the mixture of products (5, 6, 9a, and 14).
Despite the suggestively similar maxima we found no evidence
of formation of 1 or 13 under these conditions. Base-catalyzed
conversion of NO2-LA to conjugated species provides some
context for the structural ambiguity of previous biological
descriptions of NO2-LA, but oxidation is evidently highly
preferred.
1,2-addition product 11 is analogous to the well-studied44
formation of nitroalkenes, in which steric considerations allow a
facile E2 mechanism for diastereomers in which the proton and
trifluoroacetate are antiperiplanar (IX) while the nitro group is
opposite the methylene; for the other diastereomers (X) steric
repulsion either results in a kinetic product (Z-nitroalkene) or a
slower E1cb mechanism and internal rotation. The lack of
observed Z-isomers suggests that the slower E1cb is favored in
this case. For either diastereomer of 1,4-addition product 12
elimination is likely to be facile, as deprotonation at an allylic
position (lower pKa) would lead to a stabilized intermediate
(again E1cb), that would in turn allow elimination with minimal
steric repulsion. Based on these observations we can only
speculate whether a similar mechanism confers the (E,E)
configuration in the acidic nitration mechanism (Scheme 3).
To further explore the relationship of the NO2-cLA in this
study with NO2-LA, the UV−vis spectrum of NO2-cLA was
compared to nonconjugated NO2-LA. Prior determinations of
the UV−vis spectra of nonconjugated NO2-LA (4−7) had
employed a base addition step20,45 to simplify and amplify the
poorly defined spectra of the parent NO2-LA to form a
noncharacterized product (Scheme 5). Addition of base to
NO2-LA yielded a new species with λmax 330 nm (Figure 4a)
and significantly higher absorptivity indicative of a significantly
more conjugated species. Subsequent acidic quenching of the
conjugated intermediate resulted in a double-peak with maxima
at 302 and 312 nm (see the Supporting Information). This new
peak was similar but not identical to the spectrum of NO2-cLA
(λmax 312 nm).
Finally, the nitrodiene motif25,42 is highly reactive to both
acids and bases and displays potential for intramolecular
electrocyclic rearrangement46 and intermolecular polymer-
ization.47 Synthesis may be done at scales appropriate to
rapid usage, as long-term storage can be problematic. After
synthesis, neat nitro dienes decompose at ambient temper-
atures (rapidly at elevated temperatures) but can be stable for
up to a week at −20 °C. The NO2-cLA preparations reported
herein were stable for at least 2 months at −80 °C when
solvated in DMSO or methanol (Figure 5a), and these solvents
are preferred for long-term storage. Aqueous buffer (phosphate
or PBS, pH 7.4) solutions below the critical micelle
concentration were stable for several hours (Figure 5b).
Samples were monitored by UV−vis and analyzed by LC/
MS. The appearance of hydroxy or keto species suggested a
parallel to the hydration reaction of nitroalkenes,24 which are in
equilibrium with α-hydroxy (15) nitro species in aqueous
environment (eq 1). Note that the hydrophobic membranes
Rapid deprotonation at the methylene bridge position of
NO2-LA affords a highly conjugated intermediate. For lipids
with nitro groups located at the 9- or 13- (outer) positions (4
and 7), deprotonation is facile and proceeds to conjugated
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Figure 4. (a) UV−vis spectra (methanol) for base-catalyzed conjugation of nonspecific NO2-LA. A 20 μM solution of NO2-LA (4−7, λmax 257 nm)
in MeOH (1 mL) treated with 5.0 μL of 1 M NaOH monitored over 3 min with measurements every 10 s over the region 220−400 nm until
1
equilibrated (λmax 330 nm). (b) H NMR (600 MHz, CDCl3, δ 6.0−8.0 ppm shown) of (a) NO2-LA (4−7); (b) NO2-LA after base-catalyzed
rearrangement; (c) purified 9a + 14; and (d) NO2-cLA 1 + 2 for comparison.
Figure 5. (a) UV−vis absorbance of NO2-cLA compared to initial absorbance. NO2-cLA solution (2 mM) stability over 2 months: methanol at rt
⧫
⧫
□
□
▲
( ) or −80 °C ( ), DMSO at rt ( ) or −80 °C (×). (b) NO2-cLA stability over 24 h in aqueous buffer ( ) or as neat liquid ( ), both at rt.
and similar environments available when applied to cells or
tissue provide both stabilization of the nitroalkene as well as a
variety of alternative potential reaction options.
further develop the field of electrophilic nitro-lipid signaling.
Investigations to dissect the mechanism of diene nitration,
synthesize individual regioisomers, and develop the biological
and pharmacological applications of these lipids are ongoing.
CONCLUSION
■
EXPERIMENTAL SECTION
In summary, both natural isotopic abundance and 15N-labeled
NO2-cLA 1 and 2, representative isomers of nitro-conjugated
linoleic acids found in vivo, were characterized and synthesized.
The structures of the nitrodiene motif, the mechanism of
formation and the relationship with methylene-skipped diene
nitrolinoleic acids were discussed in the context of previous
descriptions of nitrated bis allylic linoleic acid. The structural
identification, facile synthetic methods, and optimized storage
conditions presented herein provide the investigative tools to
■
General Methods. All glassware was oven-dried before use, and
reactions were performed under an atmosphere of dry N2. Specific
9Z,11E-conjugated linoleic acids (approximately 90% purity) and
reagents were purchased and used as received without further
purification. Solvents were purchased dry with molecular sieves or
HPLC-grade and were used as received. Analytical thin-layer
chromatography (TLC) was performed on precoated silica gel F-254
plates and visualized by 254 nm UV light, Greiss reagent (1%
sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine in 5% phosphoric
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acid), or phosphomolybdic acid (10% in ethanol) dip. Flash
chromatography was performed on 40−63 μm silica gel. Preparatory
thin-layer chromatography (PTLC) was performed on silica gel plates
containing gypsum binder and fluorescent indicator. 1H and 13C NMR
spectra were performed at 600 or 400 MHz and 150 or 100 MHz,
respectively, at ambient temperature, and spectra are referenced to
residual nondeuterated solvent (CHCl3: 7.26 ppm, 77.00 ppm). 15N
Note: All nitrodienes were maintained in (organic) solution
whenever possible and fully evaporated only to weigh or switch
solvents. Even slightly elevated temperatures during rotary evaporation
led to rapid formation of impurities. Methanol or DMSO solutions
were preferred for long-term storage of samples for biological
experimentation.
Method B:41,42 Trifluoroacetate Nitration. A 10 mL round-
bottom flask containing a stirbar was charged with dichloromethane (5
mL), 9Z,11E-conjugated linoleic acid (140 mg, 0.5 mmol), trifluoro-
acetic anhydride (0.13 mL, 0.9 mmol), and ammonium nitrate (40 mg
NH4NO3 or NH415NO3; or 152 mg NBu4NO3, 0.5 mmol). The RBF
was stirred for 10 min, and then 10.0 μL hydrofluoroboric acid (48%
aq) was added to the flask. The solution was stirred at room
temperature under nitrogen overnight. The next day, the darkened
solution was quenched with 5 mL of water, stirred 5 min, and then
transferred to a separatory funnel. The organic layer was removed and
the aqueous layer extracted with 3 × 5 mL portions of EtOAc. The
organic layers were then combined, washed once with water and once
with brine, dried over sodium sulfate, and eluted through a small plug
of silica gel. The solvent was removed by rotary evaporation and the
crude oil transferred to a 25 mL RBF and redissolved in 10 mL of
anhydrous diethyl ether. To the stirred flask was added 60 mg of
potassium acetate or proprionate (0.6 mmol, 1.2 equiv) and the
suspension stirred under N2 at rt overnight monitored by TLC. After
24 h (or disappearance of starting material by TLC), the solution was
partitioned with 5 mL 1 M aqueous hydrochloric acid and stirred 20
min. The aqueous layer was extracted twice with 5 mL portions of
Et2O. The organic layers were combined, washed once with water and
once with brine, and dried over sodium sulfate. The solution was
filtered through a silica gel plug, and the solvents were removed by
rotary evaporation. The residual oil was purified as in method A to
yield NO2-cLA as a yellow oil (40 mg, 25%; 66 mg, 40%) or by
preparative HPLC to obtain single isomers as described.
NMR spectra were performed at 50 MHz. H,1H COSY and H,13C
HMBC experiments were performed using standard pulse programs.
FTIR was performed on an FTIR-ATR instrument. Purity of NO2-FA
was assessed by HPLC−UV using a C18 reversed-phase column (2 ×
100 mm, 5 μm) at 220 and 312 nm. LC/MS conditions: C18 reversed-
phase column (2 × 150 mm, 3 μm) using a water/acetonitrile solvent
system containing 0.1% acetic acid as described.5 Structural analysis of
NO2-FA was conducted by HPLC−ESI−MS/MS using a triple
quadrupole mass spectrometer in negative-ion mode in tandem with a
high-resolution hybrid mass spectrometer.
1
1
Separation of individual isomers was performed after synthesis of
NO2-cLA by method B (Figure 3). Crude reaction mixtures were
purified by PTLC and the nitrodiene-containing bands identified by
UV. Subsequent preparative reversed-phase HPLC was used to
separate this mixture of regioisomers into two fractions. For
preparative-scale separation, an LC system with a 5 μm C18 column
(250 mm ×20 mm) and a dual-wavelength UV detector (210 and 312
nm) was used. The mobile phases were water and 0.1% trifluoroacetic
acid (A) and acetonitrile and 0.1% trifluoroacetic acid (B) with a flow
rate of 20 mL/min. A typical reaction mixture (0.5 mmol scale) was
divided into two 48 mg portions each containing approximately 50%
1
product by H NMR. A single portion was redissolved in methanol/
water or acetonitrile/water up to 5 mL and injected. After injection,
separation began at 65% A, 35% B for one minute, then increasing to
35% A, 65% B over 20 min, then increasing to 17% A, 83% B over 80
min. NO2-cLA fractions eluting at 73% B over 45−50 min were
collected and analyzed by LC/MS to identify the regioisomers present
(12-NO2-cLA eluting first, Figure 3b). After fractions were pooled and
extracted into ethyl acetate, a second PTLC purification was
performed to remove hydrolysis products, after which the compounds
were evaporated or solvent-switched to chloroform and then
deuterated chloroform for analysis by NMR. From an estimated 24
mg crude product, 6.9 mg 12-NO2-cLA (28.8% recovery of injected
material, 8.2% yield based on starting material), 10.0 mg 9-NO2-cLA
(41.7% recovery, 12.3% yield), and 1.8 mg mixed-isomer material
(7.5% recovery) were collected.
(9E,11E)-9-Nitrooctadeca-9,11-dienoic Acid (1). 1H NMR (600
MHz, CDCl3) δ (ppm): 10.1 (br s, 1H); 7.54 (−CHCNO2, d, J =
11.4 Hz, 1H); 6.34 (CH2CHCH, dt, J = 14.9, 7.4, 1H); 6.19 (CH
CHCH, dd, J = 14.9, 11.4 Hz, 1H); 2.65 (CNO2CH2, t, J = 7.6 Hz,
2H); 2.34 (CH2CO2, t, J = 7.4 Hz, 2H); 2.24 (CH2CHCH, q, J =
7.3 Hz, 2H); 1.63 (m, 2H); 1.51 (m, 2H); 1.45 (m, 2H); 1.33−1.29
(broad m, 12H); 0.89 (−CH3, t, J = 6.7 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ (ppm): 179.8, 149.25, 149.1, 133.9, 123.5, 33.9, 33.7,
31.6, 28.95, 28.88, 28.81, 28.56, 28.01, 26.5, 24.6, 22.5, 14.0.
1
(9E,11E)-12-Nitrooctadeca-9,11-dienoic acid (2). H NMR (600
Method A:5,25 Acidic Nitration of CLA. A 15 mL culture tube
containing a stirbar was charged with 1.0 mL of 1% sulfuric acid (v/v)
and 2.5 mL of cyclohexane. To the organic layer was added 100 μL of
9Z,11E-conjugated linoleic acid (90 mg, 0.3 mmol) in one portion,
after which the tube was covered with a septum and sparged with
nitrogen with stirring for 15−20 min or until 1 mL of the hexanes had
evaporated. An Eppendorf tube was charged with sodium nitrite
(NaNO2 or Na15NO2, 140 mg, 2 mmol) in 1 mL of deionized water
for a 2 M solution, which was then sparged with N2 for 5−10 min. A
portion (0.75 mL) of the degassed nitrite solution was added to the
biphasic lipid solution upon completion of degassing for a final
concentration of 500 mM nitrite and 100 mM lipid. The solution was
sealed with a septum without inlet or outlet, covered with foil, and
stirred vigorously for 3 h.
Afterward, the biphasic solution was transferred to a separatory
funnel and extracted with three 5 mL portions of EtOAc. The organic
layers were combined and washed once with water and once with
brine, and the solution was dried over sodium sulfate. The solution
was filtered through a Celite/silica gel plug and the solvent removed
by rotary evaporation with minimum heating, and then the residual
crude product was applied to a PTLC plate prewashed with methanol/
dichloromethane (1:1 v/v). The plate was eluted (1% HOAc, 25%
EtOAc/hexanes), and the UV-active band (Rf = 0.70) was removed
and extracted with EtOAc. The extracted product was then applied to
a flash chromatography column for further purification (0.5% HOAc,
0−10% EtOAc/hexanes) to yield NO2-cLA as a yellow oil (12 mg,
8%).
MHz, CDCl3) δ (ppm): 10.1 (br s, 1H); 7.53 (CNO2CH−, d, J =
11.3 Hz, 1H); 6.32 (CHCHCH2, dt, J = 15.0, 7.4, 1H); 6.19 (
CHCHCH, dd, J = 15.0, 11.3 Hz, 1H); 2.65 (CH2CNO2, t, J = 7.5
Hz, 2H); 2.35 (CH2CO2, t, J = 7.4 Hz, 2H); 2.24 (CHCHCH2, q, J
= 7.4 Hz, 2H); 1.63 (m, 2H); 1.51 (m, 2H); 1.45 (m, 2H); 1.33−1.29
(broad m, 12H); 0.88 (−CH3, t, J = 6.7 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ (ppm): 180.0, 149.30, 148.9, 133.8, 123.6, 34.0, 33.6,
31.4, 28.97, 28.91, 28.88, 28.82, 28.51, 28.04, 26.6, 24.6, 22.5, 14.0.
UV−vis: λmax (MeOH) 312 nm, ε = 11,200 M−1 cm−1. FT-IR: 2927,
2856, 1706, 1644, 1559, 1510, 1459, 1434, 1317, 1220, 1169, 971, 862,
727 cm−1. ESI(−)-HRMS: for C18H30O4N calcd 324.2180 [M − 1]−,
found 324.2183. TLC (EtOAc/hexanes, 1:3): Rf = 0.33.
[15N]-(9E,11E)-9-Nitrooctadeca-9,11-dienoic Acid ([15N]-1). 1H
NMR (600 MHz, CDCl3) δ (ppm): 10.1 (br s, 1H); 7.54 (−CH
CNO2, d, J = 11.4 Hz, 1H); 6.34 (CH2CHCH, dt, J = 14.9, 7.4,
1H); 6.19 (CHCHCH, dd, J = 14.9, 11.4 Hz, 1H); 2.65
(CNO2CH2, t, J = 7.6 Hz, 2H); 2.34 (CH2CO2, t, J = 7.4 Hz, 2H);
2.24 (CH2CHCH, q, J = 7.3 Hz, 2H); 1.63 (m, 2H); 1.51 (m, 2H);
1.45 (m, 2H); 1.33−1.29 (broad m, 12H); 0.89 (−CH3, t, J = 6.7 Hz,
3H). 13C NMR (100 MHz, CDCl3) δ (ppm): 179.5, 149.25, 149.06
(JC−N = 11.0 Hz), 134.0, 123.50 (JC−N = 3.4 Hz), 33.9, 33.7, 31.6,
28.96, 28.89, 28.82, 28.57, 28.02, 26.5, 24.6, 22.5, 14.0.
[15N]-(9E,11E)-12-Nitrooctadeca-9,11-dienoic Acid ([15N]-2). 1H
NMR (600 MHz, CDCl3) δ (ppm): 10.1 (br s, 1H); 7.53 (CNO2
CH−, d, J = 11.3 Hz, 1H); 6.32 (CHCHCH2, dt, J = 15.0, 7.4, 1H);
6.19 (CHCHCH, dd, J = 15.0, 11.3 Hz, 1H); 2.65 (CH2CNO2, t,
J = 7.5 Hz, 2H); 2.35 (CH2CO2, t, J = 7.4 Hz, 2H); 2.24 (CH
31
dx.doi.org/10.1021/jo4021562 | J. Org. Chem. 2014, 79, 25−33

The Journal of Organic Chemistry
Article
CHCH2, q, J = 7.4 Hz, 2H); 1.63 (m, 2H); 1.51 (m, 2H); 1.45 (m,
(11) Cui, T.; Schopfer, F. J.; Zhang, J.; Chen, K.; Ichikawa, T.; Baker,
P. R. S.; Batthyany, C. I.; Chacko, B. K.; Feng, X.; Patel, R. P.; Agarwal,
A.; Freeman, B. A.; Chen, Y. E. J. Biol. Chem. 2006, 281, 35686−
35698.
(12) Kelley, E. E.; Batthyany, C. I.; Hundley, N. J.; Woodcock, S. R.;
Bonacci, G.; Del Rio, J. M.; Schopfer, F. J.; Lancaster, J. R., Jr.;
Freeman, B. A.; Tarpey, M. M. J. Biol. Chem. 2008, 283, 36176−36184.
(13) Kansanen, E.; Bonacci, G.; Schopfer, F. J.; Kuosmanen, S. M.;
Tong, K. I.; Leinonen, H.; Woodcock, S. R.; Yamamoto, M.; Carlberg,
C.; Yla-Herttuala, S.; Freeman, B. A.; Levonen, A.-L. J. Biol. Chem.
2011, 286, 14019−14027.
2H); 1.33−1.29 (broad m, 12H); 0.88 (−CH3, t, J = 6.7 Hz, 3H). 13
C
NMR (100 MHz, CDCl3) δ (ppm): 179.2, 149.33 (JC−N = 11.2 Hz),
148.8, 133.77 (JC−N = 1.1 Hz), 123.66 (JC−N = 3.4 Hz), 33.8, 33.6,
31.5, 28.99, 28.93, 28.90, 28.83, 28.54, 28.06, 26.6, 24.6, 22.5, 14.0. 15N
NMR (50.6 MHz, CDCl3) δ (ppm): 377.8, 377.9 (combination of
positional isomers). FT-IR: 2927, 2857, 1708, 1643, 1521, 1481, 1294,
973, 726 cm−1. ESI(−)-HRMS: for C18H30O415N calcd 325.2151 [M
− 1]−, found 325.2153.
ASSOCIATED CONTENT
■
(14) Kansanen, E.; Jyrkkanen, H.-K.; Volger, O. L.; Leinonen, H.;
Kivela, A. M.; Hakkinen, S.-K.; Woodcock, S. R.; Schopfer, F. J.;
Horrevoets, A. J.; Yla-Herttuala, S.; Freeman, B. A.; Levonen, A.-L. J.
Biol. Chem. 2009, 284, 33233−33241.
S
* Supporting Information
1
Copies of H and 13C NMR for compounds 1 and 2 (NO2-
1
cLA). Copies of H, 13C, 1H−1H COSY, 1H−13C HMBC, 15N,
1
and H−15N HMBC NMR for compounds [15N]-NO2-cLA (1
(15) Li, Y.; Zhang, J.; Schopfer, F. J.; Martynowski, D.; Garcia-Barrio,
M. T.; Kovach, A.; Suino-Powell, K.; Baker, P. R. S.; Freeman, B. A.;
Chen, Y. E.; Xu, H. E. Nat. Struct. Mol. Biol. 2008, 15, 865−867.
(16) Schopfer, F. J.; Cole, M. P.; Groeger, A. L.; Chen, C.-S.; Khoo,
N. K. H.; Woodcock, S. R.; Golin-Bisello, F.; Motanya, U. N.; Li, Y.;
Zhang, J.; Garcia-Barrio, M. T.; Rudolph, T. K.; Rudolph, V.; Bonacci,
G.; Baker, P. R. S.; Xu, H. E.; Batthyany, C. I.; Chen, Y. E.; Hallis, T.
M.; Freeman, B. A. J. Biol. Chem. 2010, 285, 12321−12333.
(17) Schopfer, F. J.; Lin, Y.; Baker, P. R. S.; Cui, T.; Garcia-Barrio, M.
T.; Zhang, J.; Chen, K.; Chen, Y. E.; Freeman, B. A. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 2340−2345.
(18) Napolitano, A.; Crescenzi, O.; Camera, E.; Giudicianni, I.;
Picardo, M.; d’Ischia, M. Tetrahedron 2002, 58, 5061−5067.
(19) Napolitano, A.; Camera, E.; Picardo, M.; d’Ischia, M. J. Org.
Chem. 2000, 65, 4853−4860.
(20) Baker, P. R. S.; Schopfer, F. J.; Sweeney, S.; Freeman, B. A. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 11577−82.
and 2). UV−vis spectra and graphs. LC/MS analysis of
products including fragmentation. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 412-648-9319. Fax: 412-648-2229. E-mail: srw22@pitt.
■
edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by NIH Grant Nos. R01-HL058115,
R01-HL64937, and P01-HL103455 (B.A.F.) and R01-
AT006822 (F.J.S.). F.J.S. and B.A.F. acknowledge interest in
Complexa, Inc. We thank Dr. Damodaran Krishnan Achary
(University of Pittsburgh) for assistance with heteronuclear
NMR, Dr. Alessandro Bisello (University of Pittsburgh) for
assistance with purification, and Dr. Andrew Robak (Keuka
College) for helpful discussions.
(21) Alexander, R. L.; Wright, M. W.; Gorczynski, M. J.; Smitherman,
P. K.; Akiyama, T. E.; Wood, H. B.; Berger, J. P.; King, S. B.; Morrow,
C. S. Biochemistry 2008, 48, 492−498.
(22) Dunny, E.; Evans, P. J. Org. Chem. 2010, 75, 5334−5336.
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Napolitano, A.; Barone, V.; d’Ischia, M. J. Org. Chem. 2008, 73, 7517−
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