Evaluation of nitroalkenes as nitric oxide donors

Article abstract of DOI:10.1016/j.bmcl.2007.01.016

Chemiluminescence experiments demonstrate that simple nitroalkenes release low levels of nitric oxide. UV and EPR measurements suggest but cannot confirm direct NO release from nitroalkenes. Given the biological activity of nitrated unsaturated fatty acids, these results suggest the possible metabolic conversion of nitroalkenes to NO.

Full text of DOI:10.1016/j.bmcl.2007.01.016

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2013–2017
Evaluation of nitroalkenes as nitric oxide donors
Michael J. Gorczynski, Jinming Huang, Heather Lee and S. Bruce King*
Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, USA
Received 3 October 2006; revised 4 January 2007; accepted 8 January 2007
Available online 17 January 2007
Abstract—Chemiluminescence experiments demonstrate that simple nitroalkenes release low levels of nitric oxide. UV and EPR
measurements suggest but cannot confirm direct NO release from nitroalkenes. Given the biological activity of nitrated unsaturated
fatty acids, these results suggest the possible metabolic conversion of nitroalkenes to NO.
Ó 2007 Elsevier Ltd. All rights reserved.
In 1998, Furchgott, Ignarro, and Murad received the
Nobel Prize for the identification of nitric oxide (NO)
as the endothelium-derived relaxing factor. In vivo gen-
eration of NO, a free radical signaling mediator, occurs
via the nitric oxide synthase (NOS)-catalyzed, five-elec-
1
tron oxidation of L-arginine. The biological activity
of NO results from its ability to activate soluble guany-
late cyclase, which converts guanosine triphosphate into
cyclic guanosine monophosphate, followed by kinase-
Figure 1. Structures of naturally occurring unsaturated nitrated fatty
acids.
2
mediated signal transduction. NO regulates a number
of biological processes including smooth muscle relaxa-
3
4
5
tion, immune stimulation, neurotransmission, and
6
platelet aggregation.
red blood cells, urine, and plasma, making these com-
pounds the single largest pool of bioactive nitrogen oxi-
8
The medicinal use of gaseous or aqueous solutions of
NO remains difficult due to the instability and inconve-
nient handling of the solutions. These drawbacks and an
increased interest in the importance of NO in biology
and medicine have led to the widespread development
des in the vasculature. Additional studies showed that
nitrated unsaturated fatty acids act as ligands for the
9
peroxisome proliferator-activated receptor c (PPARc),
activate endothelial heme oxygenase 1 expression, and
1
0
covalently modify proteins, i.e., glyceraldehyde-3-phos-
11
phate dehydrogenase.
7
of compounds that release NO in situ, i.e., NO donors.
Currently, a number of different classes of NO-related
therapeutic agents (N-nitro, N-nitroso, O-nitro, O-ni-
troso, S-nitroso, metal-nitrosyl, C-nitroso, and C-nitro)
are being evaluated in preclinical and clinical studies.
Nitrated unsaturated fatty acids also relaxed rat aortic
rings with an increase in cyclic guanosine mono-phos-
phate and inhibition of guanylate cyclase blocked this
1
2
relaxation suggesting a potential role for NO. Chemi-
luminescence analysis by three different research groups
of the reaction headspace from the incubation of various
nitrated unsaturated fatty acids in buffer or buffer/alco-
hol mixtures revealed the generation of small amounts
C-nitro compounds, namely nitrated unsaturated fatty
acids, have recently emerged as a unique class of signal-
ing agents (Fig. 1). Initial studies revealed the presence
of nitrated derivatives of linoleic acid (18:2) and oleic
acid (18:1) in high concentrations (P500 nM) in human
1
2b,13
(<1%) of NO.
(
formation, but could not be quantified.
Electron paramagnetic resonance
EPR) trapping experiments provided evidence of NO
1
2b,13a
Treatment
of oxyhemoglobin (HbFe -O ) with nitrolinoleic acid
Keywords: Nitric oxide; Nitric oxide donors; Nitroalkenes; Free
radicals.
II
2
III
*
Corresponding author. Tel.: +1 336 758 5774; fax: +1 336 758
656; e-mail: kingsb@wfu.edu
formed methemoglobin (HbFe ) suggesting NO forma-
1
3a
4
tion.
This rapid conversion does not reconcile with
0
960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.016

2014
M. J. Gorczynski et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2013–2017
the low levels of NO measured by chemiluminescence
and may reflect heme oxidation by the organic nitro
Table 1. Chemiluminescence detection of NO and nitrite from 1–7
ꢀ
a
Compound
½NO þ NO ꢁ (lM)
2
1
4
group. To date, the NO release properties and mecha-
nisms of nitrated unsaturated fatty acids and nitroalk-
enes remain to be completely described (including our
1
2
3
4
5
6
7
245.34 ± 2.28
262.57 ± 7.28
8.15 ± 1.23
13b
previous report).
Given the intense interest in the
79.59 ± 6.12
8.57 ± 1.43
biology of these unique molecules, we wished to further
characterize the NO donor properties of nitroalkenes
with our hypothesis being that simple nitroalkenes,
which contain the same functional group as the nitrated
unsaturated fatty acids, release NO under similar condi-
tions. We describe the synthesis and NO donor proper-
ties of organic nitroalkenes.
15.96 ± 6.12
12.30 ± 0.49
a
Five microliter injection of 50 mM solution (1:1 EtOH:H
2
O) after
24-h incubation into KI/HOAc solution in NOA.
Nitroalkenes are readily available and versatile synthetic
pounds (nitro-methane, -ethane, -cyclohexane, and
-benzene) do not release NO/nitrite under these condi-
tions after 24 h (data not shown). The amount of ob-
served NO/nitrite varies between compounds and no
predictable structural trends emerge. Under these condi-
tions, the nitrated oleic acid derivatives (6 and 7) gener-
ated significantly less NO/nitrite than the simple
nitroalkenes (1 and 2, Table 1). Solvent and hydropho-
bic effects may influence the release of NO/nitrite from
1
5
intermediates in organic chemistry. 1-Nitro-cyclohex-
ene (1) was purchased from Sigma–Aldrich and tested
as received (Fig. 2). Aromatic nitroalkenes 2 and 3 were
synthesized using a two-step method consisting of the
condensation (Henry reaction) of benzaldehyde with
nitromethane or nitroethane, respectively, followed by
dehydration using acetic anhydride and dimethylamino-
1
6
pyridine. Compounds 4 and 5 were synthesized by a
similar condensation/dehydration sequence using tri-
1
2a
these fatty acids as previously noted.
1
6,17
fluoroacetic anhydride and triethylamine.
nitrated fatty acids (6–7) were prepared as previously
described.
The
Based on these results and its commercial availability,
1-nitro-cyclohexene (1) was selected as a model nitroalk-
ene NO donor. Table 2 shows the results of nitrogen
oxide analysis under a variety of detection conditions
from the room temperature incubation of 1 in 1:1
1
3b
Chemiluminescence experiments show the ability of sim-
ple nitroalkenes to release nitric oxide and/or nitrite, the
stable oxidative decomposition product of NO in aque-
EtOH:H O for 24 h. Direct analysis of the headspace
2
1
8
ous solution (Table 1). Solutions of nitroalkenes
50 mM) were incubated in 1:1 EtOH:H O at room tem-
above 1 showed the formation of small amounts of
NO. The contents of the chemiluminescence NO detec-
tor’s reaction chamber dramatically influenced the
amount of NO or its oxidation products observed
(Table 2). Injection of the incubation solution into phos-
phate buffer or KI/phosphate buffer failed to produce
measurable amounts of NO. However, injection of the
incubation solution into KI/HOAc solution or HOAc
alone resulted in NO and/or nitrite formation, similar
to the above results (Table 2). Addition of a reaction ali-
(
2
perature for 24 h and aliquots of these mixtures were
injected into the reaction chamber of a commercial
chemiluminescence nitric oxide analyzer (NOA) con-
taining a KI/HOAc solution, which reduces nitrite to
NO for detection. The high concentration of nitroalk-
ene, extended incubation time, and organic solvent (to
improve substrate solubility) should reveal the presence
of either NO or nitrite and these conditions have been
utilized in determining the NO releasing properties of
quot to a mixture of VCl /HCl, used to reduce nitrate to
3
1
3b
nitrated oleic acids. Under these conditions, nitroalk-
enes release less than 1% of the possible amount of NO/
nitrite (0.016–0.52%, Table 1). Alkyl and aryl nitro com-
NO, further increased the amount of NO formed (still
<1% of the possible amount). In general, increasing
the acid and reducing strength of the reaction chamber
solution increased the amount of nitrogen oxides
formed.
Another group of chemiluminescence experiments gave
the time and concentration dependence of NO and/or
nitrite release from the incubation of 1-nitro-cyclohex-
ene in buffer (Table 3). Similar to the above results, less
than 1% of the overall expected amount of NO/nitrite
formed during these reactions (Table 3). Analysis of
the reaction headspace again clearly showed the forma-
tion of NO within 5 min (15 min for 1 mM, Table 3), but
the amounts formed did not linearly increase with either
time or concentration. The amount of NO present in
solution was smaller for each condition and also did
not follow a linear trend for time or concentration.
Injection of these solutions into KI/HOAc solution for
nitrite analysis resulted in significant (but not linear)
increases in NO/nitrite (Table 3). Similar to a previous
Figure 2. Structures of nitroalkenes used in this study.

M. J. Gorczynski et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2013–2017
Table 2. Chemiluminescence detection of nitrogen oxide release from 1
2015
a
b
ꢀ
c
½NO þ NOꢀ þ NO ꢁ (lM)
ꢀ
c
Time (h)
NOA
[NO] (lM)
½NO þ NO ꢁ (lM)
2
2
3
24
24
24
24
24
24
H
2
O
1.0 ± 0.1
–
279.2 ± 2.2
179.5 ± 16.8
–
–
–
–
–
KI/HOAc
HOAc
PB
–
–
–
–
–
0
0
–
KI/PB
VCl
3
/HCl
405.3 ± 29.9
a
b
c
Contents of NOA reaction chamber; PB, phosphate buffer (pH 7.4, 50 mM).
Fifty microliter injection of the reaction headspace.
2
Five microliter injection of 50 mM solution of 1 in 1:1 EtOH:H O after 24-h incubation.
Table 3. Time- and concentration-dependent NO and nitrite release from 1
a
b
c
ꢀ
d
Time (min)
(mM)
[NO] (lM)
[NO] (lM)
½NO þ NO ꢁ (lM)
2
5
1
1
0
0.26
0
0
1
3
6
5
0
0
5
5
0
0
5
5
0
0
0
3.42 ± 0.36
3.75 ± 0.18
4.57 ± 0.19
12.00 ± 0.18
16.85 ± 0.86
18.41 ± 4.30
18.95 ± 0.70
36.10 ± 2.52
41.42 ± 3.59
57.96 ± 3.48
77.65 ± 1.98
1
0.25 ± 0.06
0.26 ± 0.05
0.53 ± 0.15
0.72 ± 0.02
0.99 ± 0.04
0.99 ± 0.05
0.69 ± 0.10
1.15 ± 0.16
1.69 ± 0.14
2.02 ± 0.05
0
1
0
10
10
10
10
50
50
50
50
0
1
3
6
0.06 ± 0.02
0.07 ± 0.02
0.09 ± 0.01
0.11 ± 0.01
–
1
3
6
0.20 ± 0.03
0.21 ± 0.04
a
b
c
Concentration of 1 in deoxygenated phosphate buffer (PB).
Two-hundred and fifty microliters injection of the reaction headspace into deionized water in the NOA.
Five microliters injection of incubation solution into deionized water in the NOA.
Five microliters injection of incubation solution into KI/HOAc solution in NOA.
d
report, incubation of 1 with the NO-specific trap car-
boxy-PTIO resulted in the loss of the EPR signal pre-
venting accurate NO detection or quantification (data
1
2b
not shown).
Ultraviolet (UV) spectroscopy in the presence of oxy-
and deoxyhemoglobin was further employed to explore
the NO releasing reactions of 1. NO reacts with deoxy-
II
hemoglobin (HbFe ) to cleanly form iron-nitrosyl-he-
moglobin (HbFe -NO) and with oxyhemoglobin
II
II
III
(
absorbance spectrum for the reaction of 1 (50 mM)
HbFe -O ) to form methemoglobin (HbFe ). The
2
II
and HbFe (70 lM) in phosphate buffer (pH 7.4,
50 mM) over 1 h showed a decrease in the absorbance
at 554 nm with an increase in absorbance at 540 and
II
73 nm characteristic of HbFe -NO (Fig. 3). Careful
5
Figure 3. UV spectrum of the reaction of 1 and deoxyhemoglobin in
phosphate buffer.
examination of this spectrum revealed the formation
of methemoglobin (increase of absorbance at 630 nm),
which indicates separate redox chemistry opposed to
simple NO release from 1 (Fig. 3). EPR spectroscopic
analysis of this reaction mixture showed the characteris-
tic absorbance of an iron nitrosyl complex (data not
shown). These results appear reminiscent of the reaction
of nitrite with deoxyhemoglobin where the ferrous iron
using a lower concentration of 1 (500 lM). While these
results may indicate NO release, the rapid oxidation of
oxyhemoglobin to methemoglobin does not match with
the small amounts of NO measured by chemilumines-
cence detection (Tables 1–3). Other direct chemistry
between the nitroalkene and heme iron cannot be
excluded at this time.
1
9
II
reduces nitrite producing NO. Reaction of HbFe -
O (70 lM) with 1 (50 mM) in phosphate buffer (pH
2
7.4, 50 mM) showed a decrease in absorbance at 540
and 576 nm with a concomitant increase in absorbance
Mechanistically, NO release from nitroalkenes remains
speculative. Similar to nitrated alcohols (nitroglycerin)
and nitrite, the release of NO from nitroalkenes chemi-
cally requires reduction. Currently, two mechanisms
III
at 630 characteristic of HbFe formation over 1 h, sim-
ilar to the reported reaction of nitrolinoleic acid with
3a
oxyhemoglobin (Fig. 4). Similar results were obtained
1

2016
M. J. Gorczynski et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2013–2017
Figure 6. EPR spectrum of the reaction of 1 and DBNBS in phosphate
buffer (top line) and the EPR spectrum of DBNBS in phosphate buffer
Figure 4. UV spectrum of the reaction of 1 and oxyhemoglobin in
phosphate buffer.
(
bottom line).
(
Fig. 6). The mixture of DBNBS and NO-saturated
phosphate buffer gave a much weaker intensity three-
line signal that disappeared over 0.5 h confirming the
observed signal was not due to reactions of the trap
and NO. These results support the formation of carbon
radicals during the decomposition of nitroalkenes to NO
but do not distinguish the two proposed pathways.
Other experiments provide evidence that disfavors a
modified Nef reaction. First, neither cyclohexenone
(NMR) nor N O (gas chromatography), the normal
2
Nef reaction products, was identified from the incuba-
tion of 1 in AcOH or KI/AcOH. Moreover, the styrene
derivatives 2 and 3 do not contain acidic allylic hydro-
gens and cannot undergo the Nef reaction. However,
these compounds released NO/nitrite and these results
strongly suggest other pathways besides a Nef reaction
may account for NO release. The final carbon end prod-
ucts of these reactions (or nitrated unsaturated fatty
acids) have not been identified likely due to the low
percent conversion and also the formation of reactive
radical intermediates.
Figure 5. Proposed mechanisms for NO release from nitrated fatty
acids—modified Nef reaction (Path 1) and nitroalkene rearrangement
(Path 2).
have been proposed to account for NO release from
nitrated unsaturated fatty acids (Fig. 5). The first evokes
a modified Nef reaction, a process that normally con-
verts nitroalkanes to the corresponding aldehyde or ke-
tone and nitroxyl (HNO). HNO quickly dimerizes and
In summary, nitroalkenes release NO in very low yields
as determined by chemiluminescence methods. Other
spectroscopic methods do not clearly identify these com-
pounds as spontaneous NO donors, especially compared
2
1
dehydrates to nitrous oxide (N O). In the modified
2
to known NO donors, such as diazonium diolates. At
these levels of NO formation, low-level, analytically dif-
ficult to detect impurities (nitrite, organic nitrites) in the
samples cannot be eliminated as the ultimate NO source.
While these results may indicate that 1 decomposes into
NO, nitrite, and/or nitrate, it appears plausible that the
highly acidic and reducing analytical conditions trigger
NO-releasing chemistry. Such results should be kept in
mind during the chemiluminescence analysis of biologi-
cal samples for nitrated unsaturated fatty acids or any
other species. Given the ability of nitrated unsaturated
fatty acids to relax pre-constricted rat aortas and the
structural similarity of C-nitro and O-nitro compounds
(nitroglycerin), these results may suggest reductive or
hydrolytic (to nitrite) metabolic pathways of NO forma-
tion in vivo. Studies with compound 1 do not necessarily
eliminate the possibility of some unique structural
Nef sequence, abstraction of an acidic allylic proton
yields the nitronate that subsequently forms a hydro-
xy-nitroso species that undergoes homolytic bond cleav-
age to form NO and an allylic radical. The second
mechanism relies upon nitroalkene rearrangement to a
nitrite ester followed by N–O bond homolysis to form
1
2b
NO and a radical. Both mechanisms produce radicals
as the final carbon product and 3,5-dibromo-4-nitroso-
benzene sulfonate (DBNBS), a water soluble spin trap
2
0
for carbon-centered radicals, has been used to detect
a lipid-derived radical during nitrolinoleic acid decom-
1
2b
position. Time-dependent EPR spectroscopy of the
mixture of 1 (10 mM) with DBNBS (10 mM) in phos-
phate buffer (pH 7.4, 50 mM) at room temperature over
0.5 h showed a six-line EPR signal consistent with
the trapping of a secondary carbon-centered radical

M. J. Gorczynski et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2013–2017
2017
features of the nitrated unsaturated fatty acids to pro-
mote NO formation. Clearly, more work, which we
are currently pursuing, must be achieved to further
define the ability and mechanism of nitroalkenes
(including nitrated unsaturated fatty acids) to act as
NO donors.
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Acknowledgments
This work was supported by the National Institutes of
Health (HL62198, SBK.). The NMR spectrometer used
in this work was purchased with partial support from
the NSF (CHE-9708077) and the North Carolina Bio-
technology Center (9703-IDG-1007). The EPR spec-
trometer was purchased with support from the North
Carolina Biotechnology Center (2003-IDG-1013).
1
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Supplementary data
Experimental procedures for the syntheses of 2–5 and
DBNBS as well as details about chemiluminescence,
EPR, and UV experiments are supplied. Supplementary
data associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2007.01.016.
1
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