Acyloxy nitroso compounds as nitroxyl (HNO) donors: Kinetics, reactions with thiols, and vasodilation properties

Article abstract of DOI:10.1021/jm101432z

Acyloxy nitroso compounds hydrolyze to nitroxyl (HNO), a nitrogen monoxide with distinct chemistry and biology. Ultraviolet-visible spectroscopy and mass spectrometry show hydrolysis rate depends on pH and ester group structure with the observed rate being trifluoroacetate (3) > acetate (1) > pivalate (2). Under all conditions, 3 rapidly hydrolyzes to HNO. A combination of spectroscopic, kinetic, and product studies show that addition of thiols increases the decomposition rate of 1 and 2, leading to hydrolysis and HNO. Under conditions that favor thiolates, the thiolate directly reacts with the nitroso group, yielding oximes without HNO formation. Biologically, 3 behaves like Angeli's salt, demonstrating thiol-sensitive nitric oxide-mediated soluble guanylate cyclase-dependent vasorelaxation, suggesting HNO-mediated vasorelaxation. The slow HNO-donor 1 demonstrates weak thiol-insensitive vasorelaxation, indicating HNO release kinetics determine HNO bioavailability and activity. These results show that acyloxy nitroso compounds represent new HNO donors capable of vasorelaxation depending on HNO release kinetics.

Full text of DOI:10.1021/jm101432z

J. Med. Chem. 2011, 54, 1059–1070 1059
DOI: 10.1021/jm101432z
Acyloxy Nitroso Compounds as Nitroxyl (HNO) Donors: Kinetics, Reactions with Thiols,
and Vasodilation Properties
Mai E. Shoman,† Jenna F. DuMond,† T. S. Isbell,‡ J. H. Crawford,‡ Angela Brandon,‡ Jaideep Honovar,‡ Dario A. Vitturi,‡
C. R. White,§ R. P. Patel,‡ and S. Bruce King*,†
†Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109, United States, ‡Department of Pathology and Center
for Free Radical Biology, University of Alabama;Birmingham, Birmingham, Alabama, United States, and §Department of Medicine and Center
for Free Radical Biology, University of Alabama;Birmingham, Birmingham, Alabama, United States
Received November 8, 2010
Acyloxy nitroso compounds hydrolyze to nitroxyl (HNO), a nitrogen monoxide with distinct chemistry
and biology. Ultraviolet-visible spectroscopy and mass spectrometry show hydrolysis rate depends on
pH and ester group structure with the observed rate being trifluoroacetate (3) > acetate (1) > pivalate (2).
Under all conditions, 3 rapidly hydrolyzes to HNO. A combination of spectroscopic, kinetic, and
product studies show that addition of thiols increases the decomposition rate of 1 and 2, leading to
hydrolysis and HNO. Under conditions that favor thiolates, the thiolate directly reacts with the nitroso
group, yielding oximes without HNO formation. Biologically, 3 behaves like Angeli’s salt, demonstrat-
ing thiol-sensitive nitric oxide-mediated soluble guanylate cyclase-dependent vasorelaxation, suggesting
HNO-mediated vasorelaxation. The slow HNO-donor 1 demonstrates weak thiol-insensitive vasor-
elaxation, indicating HNO release kinetics determine HNO bioavailability and activity. These results
show that acyloxy nitroso compounds represent new HNO donors capable of vasorelaxation depending
on HNO release kinetics.
Introduction
proposed to mediate this biochemistry including ferric heme
proteins, flavins, and quinones.21,22
Nitroxyl (HNOa) is a nitrogen-containing compound chem-
ically related to the well-known signaling agent nitric oxide
(NO) through one-electron reduction and protonation.1,2
Nitroxyl demonstrates separate chemistry and biology com-
pared to NO, which has driven studies to better understand its
chemical reactions, potential endogenous formation, and the
development of HNO-based therapeutics.1-10 Endogenous
mechanisms of HNO formation have been proposed includ-
ing direct enzymatic production from nitric oxide synthases,
heme protein-mediated oxidation of various substrates, and
nonenzymatic reactions between thiols and S-nitrosothiols.11-15
Possible specific functions for endogenously produced
HNO range from antioxidant16 to vasorelaxant17-25 to cyto-
toxic mediator.26 Some of these HNO associated activities
may occur via oxidation to NO, and subsequent stimulation
of NO-signaling and various 1-electron acceptors have been
Irrespective of endogenous HNO formation, a parallel line
of work entails the development of HNO donors as ther-
apeutics.1-10 This area stems from previous studies demon-
strating that HNO mediates the action of cyanamide,27,28
a
drug used to treat alcoholism. Exciting newer studies show the
HNO donor (sodium trioxodinitrate, Angeli’s salt, AS) exerts
positive inotropic effects in both normal and failing hearts,
and decreases venous pressures, therapeutic properties optimal
for the treatment of heart failure.5,29 An increasing under-
standing of HNO reactivity reveals NO-independent mechan-
isms of biological action and identifies thiols as critical reaction
targets. Recent studies suggest thiols represent the primary
reaction target for HNO and likely mediate much of their
biological activity.5 For example, nitroxyl inhibits the thiol
containing enzymes aldehyde dehydrogenase (ALDH) and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH), show-
ing both reversible and irreversible components of inhibition.27
The majority of the actions of HNO on cardiac tissue (in-
creasing inotropy and calcium sensitivity) have also been
attributed to HNO interaction with key thiols.29-31 Consis-
tent with this concept, we showed HNO selectively modifies
mitochondrial thiols.32
*To whom correspondence should be addressed. Phone: 336 758
5774. Fax: 336 758 4656. E-mail: kingsb@wfu.edu.
a Abbreviations: ALDH, aldehyde dehydrogenase; AS, Angeli’s salt;
C-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-
oxidecarboxy-PTIO; DMSO, dimethyl sulfoxide; DTT, dithiothreitol;
GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GSH, reduced
glutathione; GSSG, oxidized glutathione; HNO, nitroxyl; KH,
Krebs-Henseleit buffer; L-NAME, L-NG-nitroarginine methyl ester;
MNO, MahmaNonoate; MnIIITSPP, manganese(III) meso-(tetrakis-
(4-sulfonato-phenyl)) porphyrinate; PE, phenylephrine; NAC, N-acetyl-
L-cysteine; NO, nitric oxide; NAP, N-acetyl-DL-penicillamine; ODQ,
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; sGC, soluable guanylate
cyclase; SNP, sodium nitroprusside; SOD, superoxide dismutase; TP,
thiophenol.
The chemical behavior of HNO (rapid dimerization and
dehydration to nitrous oxide, N2O) requires the use of HNO
donors in these studies.6 A current major limitation in HNO-
based therapeutic development and our understanding of HNO
biology is the reliance on a select few HNO donors.2,5,10,33
The most widely utilized is Angeli’s salt, which also pro-
duces nitrite that possesses its own biological activity.34,35
r
2011 American Chemical Society
Published on Web 01/19/2011
pubs.acs.org/jmc

1060 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Shoman et al.
Scheme 1
added dropwise with stirring to a solution of [bis(trifluoroa-
cetoxy)iodo]benzene (2.15 g, 5 mmol) in CH2Cl2 (10 mL) at 0 °C.
A blue color appeared with the addition of the oxime. After 1 h
at 0 °C, the reaction mixture was allowed to warm to room
temperature and stirred for another 1 h. After disappearance of
the oxime as judged by TLC, the solvent was evaporated and the
residue was purified through a short pad of silica gel (hexanes) to
give 3 as blue oil (0.74 g, 65% yield) and characterized as pre-
viously described.36
Gas Chromatographic Detection of Nitrous Oxide.36 Sub-
strate (0.1 mmol) was placed in a 25 mL round-bottom flask,
sealed with rubber septa, and flushed with argon. Solvent (2 mL,
either MeOH, 1:1 MeOH:Tris buffer (50 mM, pH = 7.6), 1:1
MeOH:0.1 M NaOH, 1:1 MeOH:0.1 M KOH, or 1:1 MeOH:1
M HCl) was added via syringe. In some samples, GSH or NAP
(0.2 mmol) was added to the solvent before injection. After
certain times, headspace (250 μL) was injected into a 7890A
Development of structurally distinct HNO donors with diverse
release rates would complement the majority of studies using
AS and form the basis of more efficient formulations.
Previously, we reported acyloxy nitroso compounds as new
hydrolytic HNO donors that relax preconstricted rat aorta
(Scheme 1).36 Here, we further characterize the kinetics, prod-
ucts, mechanism of HNO release, and the vasoactive proper-
ties of three (1-3, Scheme 1) acyloxy nitroso compounds.
These results provide detailed decomposition rate informa-
tion and reveal the ability of acyloxy nitroso compounds to
directly react with thiolate ions to form oximes and disulfides.
The chemical reactivity of this new class of HNO donors pro-
vides a basis to explain the observed ability of these com-
pounds to relax preconstricted rat aorta.
Agilent Technologies gas chromatograph equipped with a ther-
1
mal conductivity detector and a 6 ft ꢀ
/ in. Porapack Q
8
column. The oven operated at 40 °C for 5 min and then
150 °C for 4.5 min. The purged packed inlet with a total flow (He
carrier gas) at 18 mL/min and a septum purge flow of 3 mL/min
was kept constant at 140 °C, and the back detector with a
reference flow of 9 mL/min and a makeup flow of 6 mL/min was
kept constant at 150 °C. The retention time of N2O was 2.5 min,
and the yields were calculated from a standard curve of N2O
purchased from Matheson Tri-Gas. This experiment was re-
peated measuring headspace injections every 30 min for 9 h to
evaluate kinetic measurements.
Materials and Methods
Chemistry. General. Cyclohexanone oxime, 2,2-dimethylpro-
panoic acid, lead(IV) tetraacetate, [bis(trifluoroacetoxy)iodo]-
benzene, reduced glutathione (GSH), oxidized glutathione (GS-
SG), N-acetyl-DL-penicillamine (NAP), N-acetyl-L-cysteine (NAC),
dithiothreitol (DTT), and thiophenol (TP) were purchased from
Sigma-Aldrich Chemical Co. and used as received. Analytical
thin layer chromatography (TLC) was performed on silica gel
plates with C-4 Spectroline 254 indicator. Visualization was
accomplished with UV light and 20% phosphomolybdic acid
solution in EtOH. Solvents for extraction and purification were
technical grade and used as received. 1H NMR and 13C NMR
spectra were recorded using a Bruker Advance 300 MHz NMR
spectrometer. Chemical shifts are given in ppm (δ); multiplicities
are indicated by s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), and br (broadened). HT Laboratories, San Diego,
CA, performed ESI analyses. Combustion elemental analysis
(performed by Atlantic Microlab, Inc., Norcross, GA) provided
>95% purity of any newly described compounds. UV-vis spec-
trometry was performed on a Cary 100 Bio UV-vis spectro-
photometer (Varian, Walnut Creek, CA).
UV-Vis Assay for the Decomposition of 1 and 2.36 A solution
of Tris buffer (40 mM, pH = 7.0, 1.0 mL) was added to a solution
of 1 (70 μmol, 12 mg) in MeOH (1 mL) at room temperature in a
sealed cuvette. UV-vis measurements were taken every 5 min
for 60 min at 665 nm. In other experiments, a solution of
NaOH (70 mM, 1 mL) was used and similar measurements
made. Similarly, a solution of Tris buffer (40 mM, pH = 7.0,
0.5 mL) was added to a solution of 2 (51 μmol, 10.9 mg) in
MeOH (1 mL) at room temperature in a sealed cuvette. UV-vis
measurements were taken every 30 min for 4 h at 665 nm. In
other experiments, a solution of NaOH (0.1, 0.2, 0.5 M, 1 mL)
was used and similar measurements made.
UV-Vis Assay for the Reaction of 1 and 2 with Thiols. A
solution of GSH (35.1 μmol, 10.8 mg) in Tris buffer (40 mM,
pH = 7.0, 0.5 mL) was added to a solution of 1 (35.1 μmol, 6 mg)
in MeOH (0.5 mL) at room temperature in a sealed cuvette.
UV-vis measurements were taken every 5 min for 4 h at 665 nm.
Similar experiments were performed using varying concentra-
tions of GSH (70.2 μmol, 21.6 mg, or 175 μmol, 53.7 mg), GSH
(175 μmol, 53.7 mg) in NaOH (35.1 mM, 0.5 mL), or GSSG
(35.1 μmol, 21.5 mg). Other experiments were done using NAC
(35.1, 70.2, 175 μmol). Similarly, a solution of GSH (51 μmol,
15.6 mg) in Tris buffer (40 mM, pH = 7.0, 0.5 mL) was added to
a solution of 2 (51 μmol, 10.9 mg) in MeOH (1 mL) at room
temperature in a sealed cuvette. UV-vis measurements were
taken every 30 min for 4 h at 665 nm. Similar experiments were
performed using varying concentrations of GSH (102 μmol,
31.2 mg or 260 μmol, 79.8 mg). In other experiments, a solution
of NaOH (0.5 M, 1 mL) was used instead of buffer and GSSG
(51 μmol, 21.5 mg) instead of GSH.
1-Nitrosocyclohexyl Acetate (NCA, 1).36 Synthesized as pre-
viously described to give 1 as a blue oil (4.67 g, 57%).
1-Nitrosocyclohexyl Pivalate (NCP, 2). A solution of cyclo-
hexanone oxime (4.88 g, 43.16 mmol) in CH2Cl2 (50 mL) was
added dropwise with stirring to a solution of lead(IV) tetra-
acetate (19.14 g, 43.16 mmol) and 2,2-dimethylpropanoic acid
(44.08 g, 431.6 mmol) in CH2Cl2 (200 mL) at 0 °C. A blue color
appeared with the addition of the oxime. The mixture was
stirred at 0 °C for 1 h and at room temperature for 3 h. Water
(50 mL) was added, the layers separated, and the CH2Cl2 layer
was washed with saturated sodium bicarbonate (10 ꢀ 50 mL),
dried over MgSO4, filtered, and concentrated. The crude residue
was purified by passing through a short pad of silica gel (20:1
hexanes:EtOAc, Rf = 0.77) to give 2 as a blue oil (4.32 g, 47%).
UV/vis (MeOH): λmax = 665 nm, ε = 20.7 M-1 cm-1; 1H NMR
(benzene-d6) 1.2 (s, 9H) 1.2-1.8 (m, 10H). 13C NMR (benzene-d6)
21.9, 24.9, 27.2, 29.3, 39.2, 122.8, 175.5. ESI m/z 212 [100%,
M - 1]. Anal. Calcd. For C11H19NO3: C, 61.95; H, 8.98, N, 6.57.
Found: C, 61.71, H, 9.01, N, 6.45.
Product Isolation. Following UV-vis analysis, the MeOH
was removed to leave an aqueous fraction. This fraction was
extracted with CH2Cl2 (3 ꢀ 10 mL), the organic layers were
combined, dried, filtered, and concentrated to give a residue that
was analyzed using TLC, and 1H, and 13C NMR spectroscopy.
In other experiments, DTT (340 μmol, 52.4 mg) in MeOH
(12 mL) was added and stirred at room temperature for 24 h
until the disappearance of the blue color. The MeOH was
removed and the residue dissolved in EtOAc and extracted with
1 M NaOH. After the organic layer was dried with MgSO4, the
products of the reaction were identified by TLC and chemical
1-Nitrosocyclohexyl Trifluoroacetate (NCTFA, 3). A solution
of cyclohexanone oxime (0.57 g, 5 mmol) in CH2Cl2 (10 mL) was

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1061
shift analysis of the 1H and 13C NMR as compared to the
standards: cyclohexanone, cyclohexanone oxime, GSH, GSSG,
reduced and oxidized DTT.
facilities, exposed to 12 h light/dark cycles and provided food
and water ad libitum. All experiments were conducted according
to Institutional Animal Care and Use Committee approved
protocols.13
GC-MS Analysis of the Reaction of 1-3 with Thiols. A
solution of acyloxy nitroso compound 1, 2, or 3 (0.3 mmol) in
solvent (10 mL, MeOH, 1:1 MeOH:Tris buffer (50 mM, pH =
7.6), 1:1 MeOH:1 M NaOH) was stirred overnight. In some
experiments GSH, NAC, TP (0.75 mmol), and/or cyclopenta-
none (1.5, 3, or 6 mmol) was added. The MeOH was evaporated,
CH2Cl2 (2 mL) was added, the organic layer was separated, and
an aliquot (1 μL) was injected onto a 7890A Agilent Technol-
ogies gas chromatograph coupled to a 5957C Agilent Technol-
ogies inert XL EI/CI MSD with triple axis detector run in
electron impact mode, and a 5-MS capillary column (30 m,
0.25 μm film, 0.25 mm OD) were used. The oven operated at
50 °C for 1 min and ramped to 250 °C for 24 min for a total run
time of 25 min. The purged packed inlet was kept constant at
250 °C and had a total flow (He carrier gas) of 61 mL/min with a
pressure of 7.65 psi and a septum purge flow of 1 mL/min °C.
Products were identified by retention time analysis compared to
standard cyclohexanone, cyclohexanone oxime, and cyclopen-
tanone oxime (0.1 mg/mL) each. The amount of the cyclohex-
anone and cyclohexanone oxime formed were calculated using
area under the peak. For the kinetic analysis of these reactions,
MeOH, Tris buffer (50 mM, pH = 7.6), or 1 M NaOH (5 mL)
was added to a solution of 1, 2, or 3 (0.3 mmol) and cyclopenta-
none oxime (internal standard, 500 μL, 0.5 mg/mL) in MeOH
(4.5 mL). In other experiments, NAC or TP (0.75 mmol) was
added. Aliquots (1 μL) of these solutions were injected into the
GC-MS spectrometer using the previous method every 30 min
for 24 h. The amount of cyclohexanone, cyclohexanone oxime,
and acyloxy nitroso compound was calculated by comparing
their area under the peak to the peak of the cyclopentanone oxime
internal standard.
Vessel Bioassay Studies. For all vessel experiments, thoracic
aortas from male Sprague-Dawley rats were used as previously
described after cardiac puncture.38 Briefly, aortas were isolated and
submerged in KH buffer maintained at room temperature. Vessels
were cleansed of fat and connective tissue and segmented into 2-4
mm rings. Vessel rings were placed in vessels bioassay chambers
(Radnoti Glass Technologies, Monrovia, CA, USA) containing
15 mL of KH buffer equilibrated at 37 °C and perfused with a gas
mixture comprising of 21% O2/5% CO2/74% N2, pH 7.45. A basal
tension of 2 g was applied, and vessel rings were allowed to
equilibrate for 1 h. Following equilibration, a hyperpolarizing
concentration of KCl (70 mM) was added to the baths to check
for viability and assess maximum constriction.
To assess the effects of O2 on HNO-donor mediated vasodila-
tion, vessels were equilibrated to 21% or 1% O2 gas mixtures
(containing 5% CO2 and balanced with N2). Gas was precisely
delivered by mass flow controllers (Sierra Instruments, California,
USA) set to 0.15 L/min as previously described.38 In all
experiments, vessels were pretreated with nitric oxide synthase
inhibitor L-NG-nitroarginine methyl ester (L-NAME, 100 μM)
and indomethacin (5 μM) to inhibit cyclooxygenase. Vessels
were preconstricted to approximately 50-75% of maximal KCl
constriction with phenylephrine (PE, 100 nM). AS, 1, or 3
dependent vasodilation was assessed by cumulative administra-
tion of increasing concentrations. Experiments were performed
in the absence or presence of C-PTIO (200 μM), superoxide
dismutase (SOD, 100 U/mL), GSH (0-1 mM), NAC (1 mM),
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 μM), or
MnIIITSPP (20 μM), which were added after PE and 5 min
before initiating MNO, AS, 1, or 3 concentration response.
Vasodilatory effects of cumulative MNO, AS, 1, or 3 additions
were determined by measuring the δ tension and expressing this
as a percent relaxation with respect to the maximal PE constric-
tion. Only one donor was used per aorta preparation with
between 1 and 2 concentration response curves evaluated for
each vessel segment; no differences in responses observed in
consecutive runs (not shown), consistent with recent data show-
ing no tolerance using AS.17 EC50s were obtained by fitting
concentration-dependent dilation data nonlinear curve fitting
using sigmoidal curve with variable slope algorithm using Graph-
Pad Prism software.
13C NMR Measurements of the Reaction of 1-3 with Thiols.
A solution of GSH (2.5 mmol, 767.5 mg) in Tris buffer (50 mM,
pH = 7.6) or 1 M NaOH (5 mL) and compound (1-3, 1 mmol)
and cyclopentanone (10 mmol, 884 μL) in MeOH (5 mL) was
stirred overnight at room temperature. The MeOH was evapo-
rated, and the aqueous layer extracted with CH2Cl2 (2 mL). The
organic layer was separated, dried with MgSO4, and evaporated.
The products were dissolved in CDCl3 for 13C NMR measure-
ments. The products were identified by chemical shift analysis
compared to standards of cyclopentanone, cyclopentanone oxime,
cyclohexanone, and cyclohexanone oxime.
Pharmacology. Materials. Angeli’s salt (AS) and Mahma-
Nonoate (MNO) were purchased from Cayman Chemicals.
Manganese(III) meso-(tetrakis(4-sulfonato-phenyl))porphyrinate
(MnIIITSPP) was purchased from Frontier Scientific. All other
reagents were purchased from Sigma Aldrich Chemical Co..
Indomethacin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimi-
dazoline-1-oxyl-3-oxidecarboxy-PTIO (C-PTIO) were dissolved in
100% EtOH or MeOH, respectively. Acyloxy nitroso compounds
(1 and 3) were solubilized in 100% dimethyl sulfoxide (DMSO).
The concentration of 1 was determined spectrophotometrically
(ε667 = 20.7 M-1 cm-1), while the concentration of 3 was
calculated by formula weight.36 All HNO-donor solutions were
diluted in vessel relaxation experiments to a final solvent con-
centration of 0.1% (v/v). No effects of these solvents were
observed on vasoactive responses at these concentrations (not
shown). AS and MNO were dissolved in 0.1N NaOH and
In a subset of experiments, after exposure to different NO/
HNO-donors, the effects of sodium nitroprusside (SNP), an
endothelium independent vasodilator were evaluated. In all
cases SNP was able to fully dilate vessels indicating that different
NO or HNO-donors did not compromise the ability of vessels to
respond to vasoactive stimuli (not shown).
Statistics. Concentration-dependent vessel relaxation curves
were fitted to sigmoidal dose-response algorithms by nonlinear
regression curve fitting and analyzed by 2-way RM-ANOVA
with Bonferonni post-test to evaluate significance. When di-
rectly comparing measured EC50, unpaired t test was used.
Significance was set at P < 0.05. All data analysis was per-
formed on GraphPad Prism 5.
Results
its concentration determined spectrophotometrically (ε237
=
Synthesis. Oxidation of cyclohexanone oxime (5) with lead
tetraacetate generates 1-nitrosocyclohexyl acetate (1) and a
similar oxidation in the presence of excess 2,2-dimethyl-
propanoic acid or trifluoroacetic acid (5-10 equiv) yields 1-
nitrosocyclohexyl pivalate or trifluoroacetate (2-3, Scheme 2).
Oxidation of cyclohexanone oxime (5) with [bis(trifluoro-
acetoxy)iodo]benzene provides 1-nitrosocyclohexyl trifluor-
oacetate (3, Scheme 2).39 1H and 13C NMR spectroscopy,
6100 M-1 cm-1 and ε250 = 8 mM-1 cm-1 respectively).37
Krebs-Henseleit buffer (KH) was used for all studies and
consisted of the following composition (final concentrations:
118.0 mM NaCl, 27.2 mM NaHCO3, 4.8 mM KCl, 1 mM
KH2PO4, 1.2 mM MgSO4, 11.1 mM D-glucose, 0.03 mM
EDTA) with a pH of 7.4 at 37 °C and 5% CO2 perfusion. Male
Sprague-Dawley rats (200-250 g) were purchased from Harlan
(Indianapolis, IN, USA). Animals were housed in approved

1062 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Shoman et al.
Table 1. Product Formation from the Decomposition of 1-3
Scheme 2
N2O (%)
organic
4:5
entry compd
conditions
2 h 24 h
1
1
1
1
2
2
2
3
3
3
3
MeOH/Tris (50 mM, pH 7.6) 12
39
36
29
4
84:16
97:3
91:9
2
MeOH/0.1 NaOH
MeOH/HCl
50
14
2
3
4
MeOH/Tris (50 mM, pH 7.6)
MeOH/0.1 NaOH
MeOH/HCl
86:14
85:15
94:6
5
2
10
8
6
2
Scheme 3
7
MeOH/Tris (50 mM, pH 7.6) 57
54
17
10
97:3
8
Tris (50 mM, pH 7.6)
MeOH
MeOH/0.1 NaOH
8
1
9
10
100:0
100:0
HNO formation (entry 1 vs entry 3). Addition of GSH (1 or
2 equiv) to these incubations quenches N2O production in
each case (<2%), providing further evidence for the inter-
mediacy of HNO in these reactions. 1-Nitrosocyclohexyl piva-
late (2) does not produce N2O (HNO) under neutral buffered,
basic, or acidic conditions (entries 4-6) (Supporting Infor-
mation).
mass spectrometry, and elemental analysis confirm the
structure of these acyloxy nitroso compounds, which exist
as bright-blue oils.
Acyloxy Nitroso Compound Decomposition Kinetics and
Products. Previous work shows that 1-nitrosocyclohexyl
acetate (1) demonstrates reasonable stability at neutral pH.36
Monitoring the disappearance of the absorption at 667 nm
using ultraviolet-visible (UV-vis) spectroscopy or the peak
representing the major mass fragment (Mþ - NO) of the
acyloxy nitroso compound by gas chromatography-mass
spectrometry (GC-MS) provides kinetic information regard-
ing the decomposition of 1. Incubation of 1 in a 1:1 mixture
of MeOH:Tris buffer (50 mM, pH = 7.6) at room tempera-
ture results in the exponential decrease of 1 over time with
a rate constant of k = 8.6 ꢀ 10-4 min-1 and a t1/2 = 800 min
(UV-vis spectroscopy) and k = 7.8 ꢀ 10-4 min-1 and a t1/2
= 890 min (GC-MS). Compound 1 decomposes much slower
in MeOH with a rate constant of k = 2.1 ꢀ 10-4 min-1 and a
t1/2 = 3261 min. These kinetic results stand in contrast to the
previously reported rapid decomposition of 1 in MeOH:0.1
N NaOH (t1/2 = 0.8 min). 1-Nitrosocyclohexyl pivalate (2)
decomposes very slowly in a 1:1 mixture of MeOH:Tris
buffer (50 mM, pH = 7.6) at room temperature as judged
by UV-vis or GC-MS experiments with a rate constant k =
2.5 ꢀ 10-4 min-1 and a t1/2 = 2268 min. UV-vis experi-
ments also show that 2 does not appreciably decompose over
4 h in a mixture of MeOH:1 equiv NaOH (Supporting In-
formation). 1-Nitrosocyclohexyl trifluoroacetate (3) decom-
poses immediately upon addition of water or buffer to a
MeOH solution of 3 as determined by the visual disappear-
ance of the deep-blue color.
Gas chromatographic headspace analysis of N2O, the
dimerization and dehydration product of HNO, provides
evidence for HNO intermediacy during the decomposition of
1-3 (Scheme 3, Table 1). The results with 1 and 3 appear
similar to those previously reported under neutral and basic
conditions (entries 1, 2, and 7-10).36 Under buffered condi-
tions (entry 1), the rate of N2O formation matches the rate of
decomposition of 1 for approximately the first 3 h but de-
creases as decomposition of the acyloxy nitroso compound
continues (Supporting Information). Under basic condi-
tions, N2O formation occurs within the first hour, consistent
with the observed decomposition kinetics (entry 2, Support-
ing Information). Acid-catalyzed hydrolysis of 1 appears
slightly less efficient compared to basic hydrolysis in terms of
A combination of TLC, NMR spectroscopy, and GC-MS
experiments reveals the formation of cyclohexanone (4) and
cyclohexanone oxime (5) from the decomposition of 1-3 over
time (much longer for 2) in MeOH/buffer and MeOH/basic
conditions (Scheme 3, Table 1). In general, cyclohexanone (4)
represents the major product from the decomposition of 1-3
with smaller amounts of cyclohexanone oxime (5) beingformed
(Table 1). No other cyclohexanone-derived products were iden-
tified, and starting material was completely consumed during
these incubations as judged by both UV-vis spectroscopy and
GC-MS.
Acyloxy Nitroso Compound Decomposition Kinetics and
Products in the Presence of Thiols. Kinetic UV-vis spectro-
scopic experiments monitoring the disappearance of the ab-
sorption at 667 nm show that the addition of GSH to a
solution of 1 in MeOH:TRIS buffer (40 mM, pH = 7.0)
increases the rate of acyloxy nitroso compound decomposi-
tion (Supporting Information). The addition of GSH (5 equiv)
completely consumes 2, which does not appreciably decom-
pose under neutral or basic conditions over 4 h (Supporting
Information). Table 2 summarizes more detailed kinetic
information regarding the decomposition of 1 in the presence
of thiols from GC-MS experiments monitoring the disap-
pearance of the major mass fragment (Mþ - NO) of the
acyloxy nitroso compound. Reaction of 1 occurs much more
rapidly in a MeOH:buffer mixture than in MeOH alone
(Table 2, entries 1 and 5). Addition of NAC approximately
doubles the rate of decomposition in MeOH:buffer and has
little effect on the rate in MeOH (Table 2, entries 2, 3, 6 and 7).
Addition of thiophenol (TP) to the reaction mixtures increases
the rate of decomposition nearly 200-fold in MeOH:buffer
and 30-fold in MeOH (Table 2, entries 4 and 8). Similar to 1,
treatment of 2 with TP results in its exponential decrease over
time with a rate constant of k = 1.5 ꢀ 10-1 min-1 and a
t1/2 = 4.6 min.
Addition of thiols to the decomposition of 1-3 under
buffered conditions dramatically alters the observed reaction
products by ((1) abolishing N2O formation, (2) generating a
disulfide product, and (3) changing the ratio of cyclohexanone
(4):cyclohexanone oxime (5) produced (Scheme 3). Table 3
shows the ratio of 4:5 for the decomposition of 1-3 in the
presence of different thiols in a mixture of MeOH:Tris buffer

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1063
Table 2. Kinetics of the Decomposition of 1 in the Presence of Thiols
Table 4. Product Formation from the Decomposition of 1-3 in the
Presence of Thiols under Basic Conditions
thiol
(equiv)
k
t1/2
(min)
(min-1
)
thiol
entry compd (2.5 equiv)
4:5
(with cyclopentanone 10 equiv)
entry
conditions
4:5
1
2
3
4
5
6
7
8
MeOH/Tris (50 mM, pH 7.6)
7.8 ꢀ 10-4 890
MeOH/Tris (50 mM, pH 7.6) NAC (2.5) 1.5 ꢀ 10-3 453
1
2
3
4
5
6
7
1
1
1
2
2
2
3
GSH
TP
2:98
21:79
10:90
9:91
12:88
21:79
10:90
9:91
MeOH/Tris (50 mM, pH 7.6) NAC (5)
MeOH/Tris (50 mM, pH 7.6) TP (2.5)
MeOH
1.6 ꢀ 10-3 434
1.5 ꢀ 10-1
4.6
NAC
GSH
TP
2.1 ꢀ 10-4 3261
NAC (2.5) 2.1 ꢀ 10-4 3261
14:86
2:98
97:3
11:89
6:94
MeOH
MeOH
MeOH
5.2 ꢀ 10-4 1324
NAC
GSH
NAC (5)
TP (2.5)
6.0 ꢀ 10-3 113
pearance of the absorption at 667 nm of 2 upon the addition of
AS within 20 min at room temperature in MeOH:buffer (Sup-
porting Information). Control experiments show that nitrite,
another product of AS decomposition, does not affect the
UV-vis absorbance of 2. Addition of copper(II) sulfate to
this reaction mixture generates a species with a UV-vis
absorbance at 653 nm, similar to the spectrum of Cu(II) sul-
fate and Cupferron (Supporting Information).
Vasodilation Assays. Figure 1 shows that Angeli’s salt, 1, 3,
and MNO stimulated vasodilation in a concentration de-
pendent manner with the potency being MNO > AS > 3 >
1 consistent with our previous studies.36 Figure 1 shows that
in each case, vasodilation occurs by NO-dependent activation
of soluble guanylate cyclase (sGC) as indicated by inhibition of
relaxation in the presence of either the sGC inhibitor ODQ or
the NO-scavenger C-PTIO.
Effect of Oxygen on HNO-Donor Dependent Dilation.
Recent studies underscore the importance of varying oxygen
tensions and specifically lower oxygen tensions (that are more
consistent with in vivo situations) in modulating vasodilatory
mechanisms of nitrovasodilators.38 HNO represents an in-
triguing nitrosovasodilator due to the potential competing
reaction between HNO and oxygen, leading to the formation
of reactive nitrogen species that could affect HNO signal-
ing.40,41 Figure 2 shows the HNO-donor dependent dilation
assessed at 21% O2 and 1% O2 and reveals that lowering
oxygen tensions inhibited AS dependent dilation but had no
effect on 1 or 3 dependent-vasodilation.
Effects of SOD on HNO-Dependent Relaxation at High
and Low Oxygen Tension. Previous studies suggest that the
cupric ion (Cu2þ) in SOD oxidizes HNO to NO, possibly
mediating NO-signaling from HNO.25 A second distinct
mechanism by which SOD may potentiate HNO-dependent
vasodilation is superoxide scavenging, thereby preventing
rapid NO-scavenging (and peroxynitrite formation) and im-
proving NO-dependent vasodilation.42 Figure 3 shows the
effects of SOD on AS, 1, and 3 dependent-vasodilation. SOD
significantly potentiated vasodilation by AS (indicated by
leftward shift in concentration-dependent relaxation) but did
not significantly change the vasodilation efficiency of 1 or 3.
Selective Effects of Thiols toward HNO-Donor Dependent
Vasodilation. Rapid and selective reactivity with reduced thiols
likely underlies many of the biological effects of HNO, and
thiols have been used experimentally to inhibit and implicate a
role for HNO. Parts A and B of Figure 4 show the effects of
NAC on MNO, AS, 1, and 3 dose-dependent vasodilation.
Figure 4C shows the respective EC50 for vasodilation in the
presence and absence of NAC. NAC had no significant effect
on MNO nor 1 mediated vasodilation and elicited small but
significant right shifts in AS and 3 mediated vasodilation, as
indicated by significant increases in EC50. To further probe
thiol dependence of HNO-donor dependent stimulation of
Table 3. Product Formation from the Decomposition of 1-3 in the
Presence of Thiols
thiol
entry compd (2.5 equiv)
4:5
(with cyclopentanone 10 equiv)
4:5
1
2
3
4
5
6
7
1
1
1
2
2
2
3
GSH
TP
26:74
7:92
5:95
87:13
25:75
95:5
NAC
GSH
TP
5:95
88:12
33:67
90:10
98:2
18:82
4:96
73:28
NAC
GSH
(50 mM, pH 7.6). In general and in contrast to reactions in the
absence of thiol (Table 1), cyclohexanone oxime (5) represents
the major product from these decompositions of 1 and 2 with
smaller amounts of 4 being formed (Table 3). The decomposi-
tion of 3 in the presence of GSH produces cyclohexanone (4) as
the major product, although the percentage of 5 formed
increases compared to the nonthiol reaction (Table 3, entry 7
and Table 1, entry 7). No other cyclohexanone-derived pro-
ducts were identified, and starting material was completely
consumed during these incubations as judged by both UV-vis
spectroscopy and GC-MS. Kinetic GC-MS experiments reveal
the formation of 4 and 5 occurs at the same rate as the
disappearance of starting material (Supporting Information).
Addition of cyclopentanone to these reactions further influ-
ences the ratio of cyclohexanone (4):cyclohexanone oxime (5)
produced (Table 3). Incubation of 1 or 2 with GSH or NAC
under in the presence of cyclopentanone produces 4 as the major
product with only small amounts of 5(Table3,entries1,3,4,and
6) and increases the amount of 4 formed during the decomposi-
tion of 3 in the presence of GSH (Table 3, entry 7). Incubation of
1 or 2 with TP in the presence of cyclopentanone increases the
amount of 4 formed, but 5 remains the major component of the
reaction mixture (Table 3, entries 2 and 5). GC-MS and 13C
NMR experiments confirm the formation of cyclopentanone
oxime in these reactions (Supporting Information).
Similar experiments conducted in a 1:1 mixture of MeOH:1.0
N NaOH reveal further changes in the ratio of cyclohexanone
(4):cyclohexanone oxime (5) produced in the presence and
absence of cyclopentanone (Table 4). Generally, incubation
of 1 or 2 with GSH, TP, or NAC under basic conditions
produces cyclohexanone oxime (5) as the major product
with only small amounts of cyclohexanone (4) being formed
(Table 4, entries 1-6). Exposure of 3 to these conditions
(with GSH) yields mainly cyclohexanone (4) (Table 4, entry 7).
Unlike the results from reactions in buffered solution, the
addition of cyclopentanone to these reactions did not sig-
nificantly increase the amount of 4 produced and had little
change on the overall ratio of 4:5 formed (Table 4).
Reaction of HNO with Acyloxy Nitroso Compounds.
Kinetic UV-vis spectroscopic experiments show the disap-

1064 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Shoman et al.
Figure 1. Role of NO in HNO-donor mediated vasodilation. MNO (A), AS (B), 1 (C), or 3 (D) dependent vasodilation was assessed using rat
thoracic aorta in the absence (0) or presence of either ODQ (1 μM, b) or CPTIO (200 μM, 2). Data show mean ( SEM (n = 3-4) by *P < 0.05,
**P < 0.01, # P < 0.001 by 2-way RM-ANOVA with Bonferroni post-test for ODQ effect relative to control condition. NAC effect was not
significantly different relative to control. In some cases, error bars are not observed and were smaller than symbol size.
Figure 2. Effect of oxygen tension on HNO-donor mediated vasodilation. AS (A), 1 (B), or 3 (C) mediated vasodilation was assessed at either
21% O2 (filled symbols) or 1% O2 (open symbols). Data show mean ( SEM (n = 3). *P < 0.001, by 2-way RM-ANOVA with Bonferroni post-
test relative to 21% O2 condition.
Figure 3. Effect of SOD on HNO-donor mediated vasodilation. AS (A), 1 (B), or 3 (C) mediated vasodilation was assessed at 21% O2 in the
presence (filled symbols) or absence of (open symbols) SOD (100U/ml). Data show mean ( SEM (n = 3-4). *P < 0.001 by 2-way RM-
ANOVA with Bonferroni post-test relative to with SOD condition.
NO-signaling, we assessed the effects of differing doses of
GSH, which were added to vessel bioassay chambers prior to
HNO donors. Figure 4D shows that in a concentration
dependent manner, GSH inhibited (indicated by increased
EC50) vasodilation stimulated by AS and 3 but had no effect
on 1 mediated dilation.
Effects of MnIIITPPS on MNO and HNO Donor Mediated
Vasodilation. Figure 5 shows that the proposed HNO sca-
venger MnIIITPPS in fact potentiates vasodilation elicited by
AS and 3 but had no effect on 1 nor MNO dependent effects
response, paralleling the distinct vasodilation mechanisms of
AS and 3 versus 1 observed in Figure 4.43

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1065
Figure 4. Effects of NAC or GSH on MNO and HNO-donor dependent dilation. Vasodilation induced by the MNO, Angelis salt (AS), 1, or 3
was determined in the absence or presence of either N-acetylcysteine (NAC, 1 mM) (A-C) or varying concentrations of GSH (0-10 mM) (D).
(C) EC50 in the presence and absence of NAC. P-values show significance by unpaired t test. (D) fold-changes in EC50 relative to the control
(i.e., absence of GSH) with an increase in the fold indicating inhibition of vasodilation. Data show mean ( SEM (n = 3-4). P-values show
significance by 1-way ANOVA. Clear bars = AS, black bars = 1, gray bars = 3.
Figure 5. Effects of MnIIITPPS on MNO and HNO-donor dependent vasodilation. Vasodilation stimulated by either MNO (A), AS (B), 1 (C),
or 3 (D) was assessed in the absence (O) or presence (b) of MnIIITPPS (20 μM). Data show mean ( SEM (n = 2-3). *P < 0.001 by 2-way RM-
ANOVA with Bonferronni post-test relative to corresponding with MnIIITPPS condition.
Discussion
HNO donor compounds that are structurally distinct from
Angeli’s salt and release HNO at different rates upon hydro-
lysis at neutral pH.36 Here, we detail the chemical decomposi-
tion of acyloxy nitroso compounds to HNO, describe their
reactions with thiols and thiolate ions, and compare the
A more comprehensive array of HNO donor compounds is
needed to provide better experimental tools to study HNO
biology and aid in the development of HNO therapeutics. We
and others have recently synthesized and characterized novel

1066 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Shoman et al.
Scheme 4
reactivity of 1 and 3 with AS and an NO-donor (MNO) using
vasodilation as a biological end point.17-19,21,22,24
Decomposition and HNO Release from Acyloxy Nitroso
Compounds. Acyloxy nitroso compounds (1-3) hydrolyze to
yield an unstable R-hydroxy nitroso intermediate that col-
lapses to cyclohexanone and HNO (Scheme 1, Scheme 5,
path 1). This mechanism predicts that the rate and amount of
HNO formation should directly relate to the ease of ester
hydrolysis for 1-3 (with the prediction of 3 > 1 > 2).
Kinetic decomposition studies show the water-sensitive tri-
fluoroacetate group facilitates hydrolysis and 3 rapidly
decomposes upon mixing with any aqueous solvent.44 The
t-butyl ester resists hydrolysis, and 2 decomposes very slowly
in either neutral (t1/2 = 2268 min) or basic conditions.
1-Nitrosocyclohexyl acetate (1) rapidly hydrolyzes under
basic aqueous conditions favoring hydrolysis (t1/2 =0.8 min)
but predictably hydrolyzes slowly in neutral buffered
conditions (t1/2 = 800-890 min).36 Regardless of the rate,
cyclohexanone represents the major organic product for each
of these reactions, indicating a hydrolytic pathway (Table 1).
These results generally segregate 1-3 into three groups
regarding kinetic decomposition (and presumably HNO
release) at neutral pH, with 1 being a slow HNO donor, 2
not being an HNO donor (or extremely slow HNO donor),
and 3 being a rapid HNO donor similar to Angeli’s salt
(t1/2 = 2.3 min).45
Scheme 5
Gas chromatographic headspace identification of N2O
from the aqueous-based decomposition of 1-3 parallels the
kinetic studies. 1-Nitrosocyclohexyl trifluoroacetate (3), which
decomposes fastest, yields the largest amount of N2O (57%)
within 2 h in a mixture of MeOH:buffer compared to 1 (12%)
and 2 (2%, Table 1). Incubation of 1 under basic conditions
facilitates hydrolysis of the acetate group and subsequent
N2O formation but has little effect on N2O release from 2
that contains the hydrolysis resistant t-butyl ester (Table 1).
Identification of N2O, the dimerization/dehydration prod-
uct of HNO,6 provides evidence for HNO intermediacy,
and N2O quenching by glutathione, which rapidly reacts
with HNO,46 supports this assertion. Earlier work also shows
potassium ferricyanide reduces N2O formation from the
decomposition of 1 and 3, presumably through the iron(III)-
mediated oxidation of HNO to NO.36 The decomposition of
3 in the presence of methemoglobin yields iron(II) nitrosyl
hemoglobin, a characteristic reaction of HNO, which supports
HNO release from 3.36
While these gas chromatographic results strongly imply
HNO release from 1 and 3, the less than expected quantita-
tive amounts of N2O clearly reveals limitations to this indirect
method of HNO detection. This assay relies on HNO dimer-
ization, making the rate and amount of N2O formation
dependent on the square of HNO concentration. As the
amount of donor and HNO decrease with time, the rate of
dimerization decreases, which may explain the decrease in
rate of N2O formation compared to rate of donor decom-
position observed with 1 (Supporting Information). Also, a
very slow HNO donor, such as 2, would only produce a low
steady-state HNO concentration (and low N2O formation).
The reaction of HNO with other species, including residual
oxygen, solvent components, or other organic byproduct,
would also reduce N2O formation. Indeed, Angeli’s salt-
generated HNO directly reacts with the nitroso group of the
acyloxy nitroso compound (2), similar to C-nitroso com-
pounds, to give a proposed “cupferron-like” derivative (6,
Scheme 4).47 Addition of copper(II) to this complex generates
a species with absorption properties similar to a Cupferron-
copper complex. The observation of small amounts of
cyclohexanone oxime (5) in the neutral decomposition of
1-3 clearly indicates the occurrence of non-HNO producing
reactions (Table 1). Such reactions may contribute to decreased
N2O yields and clearly show the ability of HNO to react with
other organic molecules including the parent donor. The gas
chromatographic identification of N2O provides a reasonable
marker for HNO formation, but further evidence should be
gathered for new HNO donors and improvements in HNO
detection remains a priority for advancing HNO chemistry and
biology.
Reactions of Acyloxy Nitroso Compounds with Thiols. The
addition of GSH to the decomposition of 1-3 under neutral
or basic conditions quenches N2O formation, presumably
through thiol trapping of the nascent HNO. While the decom-
position of 1-3 in the absence of thiol gives cyclohexanone
(Table 1), the decomposition of 1 and 2 under neutral and basic
conditions in the presence of GSH, NAC, or TP forms disulfide
and cyclohexanone oxime (5) as the major organic products
(Tables 3 and 4). Decomposition of 3 in the presence of GSH
predominately gives cyclohexanone under both neutral and
basic conditions (Table 3, entry 7 and Table 4, entry 7).
Scheme 5 (path 1) shows a reasonable pathway for the forma-
tion of these products via HNO. Hydrolysis of the acyloxy
nitroso compound produces HNO, the corresponding car-
boxylic acid, and cyclohexanone (4). In the presence of GSH
or NAC, the thiol reacts with HNO to generate the proposed
N-hydroxysulfenamide intermediate and further thiol reduc-
tion gives disulfide and hydroxylamine (Scheme 5). Hydroxyl-
amine condenses with cyclohexanone to form cyclohexanone
oxime (Scheme 5, path 1).
However, the addition of NAC or GSH (thiol pKas = 9.5
and 8.7, respectively)48 under buffered conditions to 1 or 2
increases the rate of acyloxy nitroso compound decomposition

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1067
as judged by UV-vis spectroscopy (Table 2 and Supporting
Information). Addition of thiophenol (pKa = 6.6)49 to 1 or 2
under buffered conditions further increases their rate of
decomposition (Table 2). The rapid reaction of 2 under these
conditions, which does not produce N2O over the same time
period in the absence of thiol, suggests a nonhydrolytic (and
non-HNO) mediated decomposition pathway. On the basis
of the pKas of the thiols examined and the pH of the
incubation buffer, these results strongly suggest the direct
reaction of thiolates with the acyloxy nitroso compounds
(Scheme 5, path 2). Thiolate addition to the nitroso group
would disrupt the n to π* transition, resulting in decreased
absorbance and generate an organic N-hydroxysulfenamide
intermediate (7, Scheme 5, path 2). Attack of a second thiol
(or thiolate) on 7 would generate disulfide and cyclohexa-
none oxime (5) without the intermediacy of HNO (Scheme 5,
path 2). The increased rate of reaction with TP compared to
GSH or NAC provides evidence for thiolate ion involve-
ment. Overall, these results show that in the presence of
thiols, acyloxy nitroso compounds can decompose through
two different paths, only one of which involves HNO, to
form identical products (disulfides and oximes).
Experiments using cyclopentanone as a trap for hydro-
xylamine provide a method for separating the two decom-
position paths of acyloxy nitroso compounds in the presence
of thiols. Only path 1 (Scheme 5) includes HNO and hydro-
xylamine, and the addition of a second ketone (cyclopenta-
none) could trap any hydroxylamine to generate cyclopen-
tanone oxime (8) and preserve any originally generated
cyclohexanone (4, Scheme 5). The addition of excess cyclo-
pentanone to the decomposition of 1 under neutral buffered
conditions in the presence of GSH reverses the ratio of 4:5
from 26:74 to 87:13, a similar ratio to decomposition in the
absence of thiol (Tables 1 and 3). Similar experiments using
NAC also reverse the ratio of 4:5 formed from 1 and the
addition of cyclopentanone to the decomposition of 2 in the
presence of these thiols yields a similar reversal in the ratio of
4:5 (Table 3). GC-MS experiments show the formation of 8
during these reactions and provide strong evidence that 1 and
2 primarily decompose through a path that involves free
hydroxylamine (Scheme 5, path 1). However, the decom-
position of 1 and 2 in the presence of excess cyclopentanone
and TP predominantly forms cyclohexanone oxime (5,
Table 3), which suggests these reactions do not generate
hydroxylamine and proceed through the thiolate of thiophe-
nol via path 2. Decomposition of 1 or 2 in the presence of
GSH, NAC, or TP under basic conditions (that ensure thiolate
formation) yields cyclohexanone oxime (5) as the major
product regardless of the presence of cyclopentanone, pro-
viding strong evidence that acyloxy nitroso compounds
directly react with thiolate ions through path 2 (Table 4).
Regardless of conditions (pH, presence/absence of thiol, or
cyclopentanone), 3 primarily forms cyclohexanone (4), sug-
gesting a primarily hydrolytic decomposition (path 1, Scheme 5).
On the basis of these results, the reactivity and HNO-
donating ability appears to depend on the structure of the
acyloxy nitroso compound, the presence of thiols, the pKa of
these thiols, and the pH of the reaction medium. Compound
3, which contains the hydrolytically sensitive trifluoroacetate
group, rapidly hydrolyzes (generating HNO) regardless of
the reaction conditions or presence of thiols (path 1, Scheme 5).
Compounds 1 and 2 hydrolyze (at different rates) to HNO in
the absence of thiols or under conditions that favor the thiol
(rather than thiolate) ionization state (path 1, Scheme 5).
However, these compounds appear to directly react through
thiolate addition to the nitroso group (path 2, Scheme 5)
under conditions that favor the thiolate ionization state. These
findings indicate acyloxy nitroso compounds may demon-
strate different reactivity (including their ability to release
HNO) depending on their structure and the presence of
thiols/thiolates. This differing reactivity with thiols com-
pared to thiolates could have important implications in
targeting specific highly reactive low pKa thiol residues in
various proteins.
Vasorelaxation Properties and Structure-Activity Rela-
tionships. Angeli’s salt (AS) elicits vasodilation of rabbit tho-
racic aorta and bovine intrapulmonary arteries through soluble
guanylate cyclase (sGC) activation.50 The sGC inhibitor
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and
NO-scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimi-
dazoline-1-oxyl-3-oxide (C-PTIO) block this relaxation,
providing evidence for NO-mediated sGC activation, sug-
gesting some oxidative metabolism of HNO to NO.18,51
While early work indicates that NO is the only nitrogen
monoxide capable of sGC activation,52-54 recent reports
provide evidence for HNO-mediated sGC activation,55
a
finding that has also been challenged.56 Our previous studies
show that acyloxy nitroso compounds 1 and 3 relax precon-
stricted rat aorta similar to other HNO donors including
AS.36 Compared to AS (EC50 = 0.082 μM), 1 is much less
potent (EC50 = 10 μM) and 3 slightly (∼10-fold) less potent
(EC50 = 0.88 μM), indicating some differences in the biolo-
gical actions of these HNO donors.50
The current vasorelaxation studies show differences be-
tween the acyloxy nitroso compounds 1 and 3 compared to
AS and each other. The very poor water solubility and ex-
tremely slow decomposition rate of 2 prevented the reliable
assay of its activity. Both ODQ and C-PTIO inhibit vasodi-
lation induced by 1 and 3, revealing that this activity likely
occurs through NO-mediated activation of sGC, similar to
AS (Figure 1). Experiments with bovine lung sGC indicate
that 3 acts as a competent sGC activator, presumably through
HNO formation,55 but the C-PTIO inhibition observed in
these studies suggests NO-mediated activity. Overall, the
acyloxy nitroso compounds 1 and 3 appear to elicit vasodila-
tion through NO mediated activation of sGC in this system.
The most surprising and interesting results were the sim-
ilarity in vasodilatory mechanisms between AS and 3 com-
pared to 1 (Figures 4 and 5). Reduced thiols (NAC and GSH
(Figure 4)) and the putative HNO trapping porphyrin
(MnIIITPPS) differentially altered the observed biological
response of AS and 3 compared to 1 (Figures 4 and 5).
Multiple studies show that HNO rapidly reacts with thiols
with a measured rate constant for reaction with GSH of
∼106 M-1 s-1.8 Addition of thiols also experimentally discerns
HNO and NO-dependent pathways, as thiols selectively
scavenge HNO with little effect on NO (confirmed by the
lack of any effect of NAC on MNO-dependent vasodilation).
Consistent with thisconcept and supporting previous work,17,24
NAC and GSH inhibited AS-mediated vasodilation (Figure 4).
Surprisingly, these thiols failed to modulate dilation by 1,
suggesting a distinct vasodilatory mechanism for this HNO
donor compared to AS. Moreover, GSH did inhibit dilation
mediated by 3, a compound structurally similar to 1 but
which releases HNO at a faster rate. Further evidence illus-
trating a distinct mechanism of action of 1 was provided by
the lack of effect of the addition of MnIIITPPS. This water-
solubleporphyrinreactswithHNO viaa reductive nitrosylation

1068 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Shoman et al.
donors. Compounds that release HNO quickly (AS and 3)
will generate a relatively greater concentration of HNO at
certain times compared to a slow HNO donor at the same
concentration. The dimerization pathway will also be more
prevalent for a rapid HNO donor. Given the high reactivity
of HNO, larger concentrations of HNO from rapid HNO
donors may alter biological targets (protein thiols, heme, or
metal groups) in a different fashion than a slow HNO donor.
For example, a rapid HNO donor generating higher con-
centrations of HNO will modify more thiols/targets in an
unselective manner in a given protein as compared to a slow
HNO donor that would likely modify the most reactive thiol/
target and perhaps lead to a specific effect. In this work, the
observed activity of AS and 3 appear to be the result of rapid
HNO release and that of 1 from a slow HNO donor that may
be eliciting the observed effects through different mechan-
isms. Also, the level of oxygen or the presence of SOD had
little effect on the activity of 1 and 3, but SOD enhanced the
activity of Angeli’s salt and decreased oxygen tension de-
creased AS ability to elicit vasorelaxation (Figures 2 and 3).
Given the differences in the rate of HNO release, the dif-
ferences between AS and 1 may be expected but the differ-
ences between AS and 3 with regard to oxygen tension and
SOD remain unclear if both are acting as rapid HNO donors.
Possible explanations include that oxygen and/or SOD react
differently with the donor molecule (AS or 3) or that the
different mechanisms (protonation vs hydrolysis) results in
the difference. These results clearly show that the overall
chemistry of the donor molecule (including HNO, NO, or
other donors) must be accounted for when evaluating the
observed biological actions for a given compound.
Scheme 6
reaction, yielding the corresponding MnII-NO complex.43
Figure 5 shows that MnIIITPPS potentiates vasodilatation in
the presence of AS and 3, suggesting reductive nitrosylation
yields bioactive NO. However, MnIIITPPS had no effect
on 1, similar to the reactivity profile observed with GSH.
MnIIITPPS did not affect MNO-dependent responses, sug-
gesting direct reactions with NO do not play a role. Together,
these data highlight the similarities in the biological activity
of AS and the trifluoroacetate (3) and illustrate the differ-
ences compared to the structurally similar but slower HNO
releasing acetate (1).
The chemical findings appear to provide some insight to
the biological action of the acyloxy nitroso compounds
(1 and 3) relative to AS. Variation of the ester group structure
changes the HNO release profile and separates 1-3 into
three groups of HNO donors (Scheme 6). Compound 3 gen-
erates HNO rapidly with a release profile most similar to
Angeli’s salt, the most commonly used HNO donor. Even in
the presence of thiols, 3 rapidly hydrolyzes and the lack of
cyclohexanone oxime formation suggests some interaction
of the thiol with the trifluoroacetate group (perhaps during
hydrolysis). Compound 1 slowly hydrolyzes under neutral
conditions, making it a slow HNO donor, while under these
conditions 2 does not hydrolyze to a significant extent in the
same time frame. Both 1 and 2 preferentially react with
thiolates at the nitroso group under appropriate conditions.
These chemical properties identify 3 as an HNO donor
similar to Angeli’s salt, a compound that releases HNO rapidly
upon introduction to water. Compound 1 releases HNO
much more slowly (and 2 even slower) and both possess
potential reactivity with thiolates providing other mecha-
nisms for biological activity. Given this chemistry, AS and 3
would be expected to demonstrate similar biological proper-
ties attributed to the rapid release of HNO (they exhibit
similar potencies). Compound 1 releases HNO much more
slowly, reacts with thiolates, and induces vasorelaxation.
Confirmed HNO traps (thiols and metal porphyrins) affect
AS and 3 in a similar fashion, with thiols quenching activity
and the manganese porphyrin enhancing activity presum-
ably through thiol reduction of HNO and metal heme-
mediated oxidation of HNO to NO, respectively (Scheme 6).
Neither of these agents alters the activity of 1, which strongly
suggests that under these conditions 1 does not generate
HNO or very little HNO and acts through a separate
mechanism.
Conclusions
Nitroxyl (HNO) has emerged as a unique nitrogen mon-
oxide demonstrating different chemistry and biology from the
better-known redox related compound, nitric oxide. Acyloxy
nitroso compounds release HNO through hydrolysis and the
rate of hydrolysis depends on pH and the structure of the ester
portion of the molecule with the observed rate being trifluor-
oacetate (3) > acetate (1) > pivalate (2). Under all condi-
tions, 3 rapidly hydrolyzes to HNO, making this compound
most similar to Angeli’s salt. Thiol addition to the decom-
position of the acetate (2) and pivalate (3) increases the rate of
reaction and alters product distribution. Conditions that
favor thiols lead to hydrolysis and HNO, while conditions
that favor thiolates generate oximes without HNO formation.
Biologically, the trifluoroacetate (3) behaves similarly to
Angeli’s salt, demonstrating thiol-sensitive nitric oxide-mediated
soluble guanylate cyclase-dependent vasorelaxation, which
strongly suggests HNO-mediated vasorelaxation. In contrast,
the slow HNO-donor acetate (1) demonstrates weak thiol-
insensitive vasorelaxation, indicating that the rate of HNO
release from these structurally similar compounds determines
HNO bioavailability and activity. In general, these results
show that (1) acyloxy nitroso compounds act as HNO donors
upon hydrolysis, (2) the rate of hydrolysis clearly dictates the
observed biological response, and (3) the overall chemistry of
the HNO donor (regardless of structural similarity) must be
considered in evaluation of the biological properties.
The rate of HNO release appears as the major chemical
difference between AS and the acyloxy nitroso compound (3)
compared to the structurally similar 1. Similar to NO donors,
differences in the rate of HNO release could greatly alter
both the observed chemical and biological behavior of HNO
Acknowledgment. R.P.P. acknowledges funds from the
National Institutes of Health (HL092624) and a cardiovas-
cular pathophysiology training fellowship to T.S.I. S.B.K.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1069
acknowledges support from the National Institutes of Health
(HL62198) and the Egyptian Ministry of Higher Education
through a Joint Supervision Channel Program (M.E.S.).
(22) Buyukafsar, K.; Nelli, S.; Martin, W. Formation of nitric oxide
from nitroxyl anion: role of quinones and ferricytochrome c. Br.
J. Pharmacol. 2001, 132 (1), 165–172.
(23) Ellis, A.; Li, C. G.; Rand, M. J. Differential actions of L-cysteine on
responses to nitric oxide, nitroxyl anions and EDRF in the rat
aorta. Br. J. Pharmacol. 2000, 129 (2), 315–322.
Supporting Information Available: 1H and 13C NMR data for
compound 2, along with UV-vis and GC-MS kinetic decom-
position data for compounds 1-3. This material is available free
of charge via the Internet at http://pubs.acs.org.
(24) Pino, R. Z.; Feelisch, M. Bioassay Discrimination between Nitric-
Oxide (NO and Nitroxyl (NO-) Using L-Cysteine. Biochem.
3
Biophys. Res. Commun. 1994, 201 (1), 54–62.
(25) Fukuto, J. M.; Hobbs, A. J.; Ignarro, L. J. Conversion of
Nitroxyl (HNO) to Nitric-Oxide (NO) in Biological-Systems;
the Role of Physiological Oxidants and Relevance to the Biolo-
gical-Activity of HNO. Biochem. Biophys. Res. Commun. 1993,
196 (2), 707–713.
(26) Wink, D. A.; Feelisch, M.; Fukuto, J.; Chistodoulou, D.; Jourd’heuil,
D.; Grisham, M. B.; Vodovotz, Y.; Cook, J. A.; Krishna, M.;
DeGraff, W. G.; Kim, S.; Gamson, J.; Mitchell, J. B. The cyto-
toxicity of nitroxyl: possible implications for the pathophysiologi-
cal role of NO. Arch. Biochem. Biophys. 1998, 351 (1), 66–74.
(27) DeMaster, E. G.; Redfern, B.; Nagasawa, H. T. Mechanisms
of inhibition of aldehyde dehydrogenase by nitroxyl, the active
metabolite of the alcohol deterrent agent cyanamide. Biochem.
Pharmacol. 1998, 55 (12), 2007–2015.
References
(1) DuMond, J. F., King, S. B. The Chemistry of Nitroxyl (HNO)
Releasing Compounds. Antioxid. Redox Signaling 2010.
(2) Miranda, K. M. The chemistry of nitroxyl (HNO) and implications
in biology. Coord. Chem. Rev. 2005, 249 (3-4), 433–455.
(3) Fukuto, J. M.; Bianco, C. L.; Chavez, T. A. Nitroxyl (HNO)
signaling. Free Radical Biol. Med. 2009, 47 (9), 1318–1324.
(4) Irvine, J. C.; Ritchie, R. H.; Favaloro, J. L.; Andrews, K. L.;
Widdop, R. E.; Kemp-Harper, B. K. Nitroxyl (HNO): the Cinderella
of the nitric oxide story. Trends Pharmacol. Sci. 2008, 29 (12),
601–608.
(5) Paolocci, N.; Jackson, M. I.; Lopez, B. E.; Miranda, K.; Tocchetti,
C. G.; Wink, D. A.; Hobbs, A. J.; Fukuto, J. M. The pharmacology
of nitroxyl (HNO) and its therapeutic potential: not just the Janus
face of NO. Pharmacol. Ther. 2007, 113 (2), 442–458.
(6) Bonner, F. T.; Hughes, M. N. The Aqueous Solution Chemistry of
Nitrogen in Low Positive Oxidation States. Comments Inorg.
Chem. 1988, 7, 215–234.
(7) Bartberger, M. D.; Fukuto, J. M.; Houk, K. N. On the acidity and
reactivity of HNO in aqueous solution and biological systems.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (5), 2194–2198.
(28) Nagasawa, H. T.; Demaster, E. G.; Redfern, B.; Shirota, F. N.;
Goon, J. W. Evidence for Nitroxyl in the Catalase-Mediated
Bioactivation of the Alcohol Deterrent Agent Cyanamide. J. Med.
Chem. 1990, 33 (12), 3120–3122.
(29) Tocchetti, C. G.; Wang, W.; Froehlich, J. P.; Huke, S.; Aon, M. A.;
Wilson, G. M.; Di Benedetto, G.; O’Rourke, B.; Gao, W. D.; Wink,
D. A.; Toscano, J. P.; Zaccolo, M.; Bers, D. M.; Valdivia, H. H.;
Cheng, H. P.; Kass, D. A.; Paolocci, N. Nitroxyl improves cellular
heart function by directly enhancing cardiac sarcoplasmic reticu-
lum Ca2þ cycling. Circ. Res. 2007, 100 (1), 96–104.
(8) Miranda, K. M.; Paolocci, N.; Katori, T.; Thomas, D. D.; Ford, E.;
Bartberger, M. D.; Espey, M. G.; Kass, D. A.; Feelisch, M.;
Fukuto, J. M.; Wink, D. A. A biochemical rationale for the discrete
behavior of nitroxyl and nitric oxide in the cardiovascular system.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (16), 9196–9201.
(9) King, S. B.; Nagasawa, H. T. Chemical approaches toward gen-
eration of nitroxyl. In Methods in Enzymology, Part C: Biological
and Antioxidant Activities; Packer, L., Ed.; Academic Press: New
York, 1999; Nitric Oxide , pp 211-220
(30) Dai, T. Y.; Tian, Y.; Tocchetti, C. G.; Katori, T.; Murphy, A. M.;
Kass, D. A.; Paolocci, N.; Gao, W. D. Nitroxyl increases force
development in rat cardiac muscle. J. Physiol. (Oxford, U.K.) 2007,
580 (3), 951–960.
(31) Paolocci, N.; Saavedra, W. F.; Miranda, K. M.; Martignani, C.;
Isoda, T.; Hare, J. M.; Espey, M. G.; Fukuto, J. M.; Feelisch, M.;
Wink, D. A.; Kass, D. A. Nitroxyl anion exerts redox-sensitive
positive cardiac inotropy in vivo by calcitonin gene-related peptide
signaling. Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (18), 10463–10468.
(32) Shiva, S.; Crawford, J. H.; Ramachandran, A.; Ceaser, E. K.;
Hillson, T.; Brookes, P. S.; Patel, R. P.; Darley-Usmar, V. M.
Mechanisms of the interaction of nitroxyl with mitochondria.
Biochem. J. 2004, 379 (Pt 2), 359–366.
(33) Miranda, K. M.; Katori, T.; de Holding, C. L. T.; Thomas, L.;
Ridnour, L. A.; MeLendon, W. J.; Cologna, S. M.; Dutton, A. S.;
Champion, H. C.; Mancardi, D.; Tocchetti, C. G.; Saavedra, J. E.;
Keefer, L. K.; Houk, K. N.; Fukuto, J. M.; Kass, D. A.; Paolocci,
N.; Wink, D. A. Comparison of the NO and HNO donating
properties of diazeniumdiolates: primary amine adducts release
HNO in vivo. J. Med. Chem. 2005, 48 (26), 8220–8228.
(34) Gladwin, M. T.; Raat, N. J. H.; Shiva, S.; Dezfulian, C.; Hogg, N.;
Kim-Shapiro, D. B.; Patel, R. P. Nitrite as a vascular endocrine
nitric oxide reservoir that contributes to hypoxic signaling, cyto-
protection, and vasodilation. Am. J. Physiol.: Heart Circ. Physiol.
2006, 291 (5), H2026–H2035.
(10) Miranda, K. M.; Nagasawa, H. T.; Toscano, J. P. Donors of HNO.
Curr. Top. Med. Chem. 2005, 5 (7), 647–664.
(11) Wong, P. S. Y.; Hyun, J.; Fukuto, J. M.; Shirota, F. N.; DeMaster,
E. G.; Shoeman, D. W.; Nagasawa, H. T. Reaction between
S-nitrosothiols and thiols: generation of nitroxyl (HNO) and
subsequent chemistry. Biochemistry 1998, 37 (16), 5362–5371.
(12) Spencer, N. Y.; Patel, N. K.; Keszler, A.; Hogg, N. Oxidation
and nitrosylation of oxyhemoglobin by S-nitrosoglutathione via
nitroxyl anion. Free Radical Biol. Med. 2003, 35 (11), 1515–1526.
(13) Schmidt, H. H. H. W.; Hofmann, H.; Schindler, U.; Shutenko,
Z. S.; Cunningham, D. D.; Feelisch, M. No NO from NO
3
synthase. Proc. Natl. Acad. Sci. U.S.A. 1996, 93 (25), 14492–
14497.
(14) Arnelle, D. R.; Stamler, J. S. NOþ, NO , and NO- Donation by
3
S-Nitrosothiols;Implications for Regulation of Physiological
Functions by S-Nitrosylation and Acceleration of Disulfide
Formation. Arch. Biochem. Biophys. 1995, 318 (2), 279–285.
(15) Wei, C. C.; Wang, Z. Q.; Hemann, C.; Hille, R.; Stuehr, D. J. A
tetrahydrobiopterin radical forms and then becomes reduced dur-
ing N-omega-hydroxyarginine oxidation by nitric-oxide synthase.
J. Biol. Chem. 2003, 278 (47), 46668–46673.
(35) Gladwin, M. T.; Schechter, A. N.; Kim-Shapiro, D. B.; Patel, R. P.;
Hogg, N.; Shiva, S.; Cannon, R. O.; Kelm, M.; Wink, D. A.; Espey,
M. G.; Oldfield, E. H.; Pluta, R. M.; Freeman, B. A.; Lancaster,
J. R.; Feelisch, M.; Lundberg, J. O. The emerging biology of the
nitrite anion. Nature Chem. Biol. 2005, 1 (6), 308–314.
(16) Lopez, B. E.; Shinyashiki, M.; Han, T. H.; Fukuto, J. M. Anti-
oxidant actions of nitroxyl (HNO). Free Radical Biol. Med. 2007,
42 (4), 482–491.
(36) Sha, X.; Isbell, T. S.; Patel, R. P.; Day, C. S.; King, S. B. Hydrolysis
of acyloxy nitroso compounds yields nitroxyl (HNO). J. Am.
Chem. Soc. 2006, 128 (30), 9687–9692.
(17) Irvine, J. C.; Favaloro, J. L.; Widdop, R. E.; Kemp-Harper, B. K.
Nitroxyl anion donor, Angeli’s salt, does not develop tolerance in
rat isolated aortae. Hypertension 2007, 49 (4), 885–892.
(18) Favaloro, J. L.; Kemp-Harper, B. K. The nitroxyl anion (HNO) is
a potent dilator of rat coronary vasculature. Cardiovasc. Res. 2007,
73 (3), 587–596.
(37) He, X. J.; Azarov, I.; Jeffers, A.; Presley, T.; Richardson, J.; King,
S. B.; Gladwin, M. T.; Kim-Shapiro, D. B. The potential of
Angeli’s salt to decrease nitric oxide scavenging by plasma hemo-
globin. Free Radical Biol. Med. 2008, 44 (7), 1420–1432.
(38) Isbell, D. C.; Voros, S.; Yang, Z.; DiMaria, J. M.; Berr, S. S.;
French, B. A.; Epstein, F. H.; Bishop, S. P.; Wang, H.; Roy, R. J.;
Kemp, B. A.; Matsubara, H.; Carey, R. M.; Kramer, C. M.
Interaction between bradykinin subtype 2 and angiotensin II type
2 receptors during post-MI left ventricular remodeling. Am.
J. Physiol.: Heart Circ. Physiol. 2007, 293 (6), H3372–3378.
(39) Calvet, G.; Dussaussois, M.; Blanchard, N.; Kouklovsky, C. Lewis
acid-promoted hetero Diels-Alder cycloaddition of R-acetoxyni-
troso dienophiles. Org. Lett. 2004, 6 (14), 2449–2451.
(19) Irvine, J. C.; Favaloro, J. L.; Kemp-Harper, B. K. NO- activates
soluble guanylate cyclase and K-v channels to vasodilate resistance
arteries. Hypertension 2003, 41 (6), 1301–1307.
(20) Crawford, J. H.; White, C. R.; Patel, R. P. Vasoactivity of
S-nitrosohemoglobin: role of oxygen, heme, and NO oxidation
states. Blood 2003, 101 (11), 4408–4415.
(21) Nelli, S.; McIntosh, L.; Martin, W. Role of copper ions and
cytochrome P450 in the vasodilator actions of the nitroxyl anion
generator, Angeli’s salt, on rat aorta. Eur. J. Pharmacol. 2001, 412
(3), 281–289.
(40) Espey, M. G.; Miranda, K. M.; Thomas, D. D.; Wink, D. A.
Ingress and reactive chemistry of nitroxyl-derived species within
human cells. Free Radical Biol. Med. 2002, 33 (6), 827–834.

1070 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Shoman et al.
(41) Kirsch, M.; de Groot, H. Formation of peroxynitrite from reaction
of nitroxyl anion with molecular oxygen. J. Biol. Chem. 2002, 277
(16), 13379–13388.
(42) White, C. R.; Brock, T. A.; Chang, L. Y.; Crapo, J.; Briscoe, P.; Ku,
D.; Bradley, W. A.; Gianturco, S. H.; Gore, J.; Freeman, B. A.
Superoxide and peroxynitrite in atherosclerosis. Proc. Natl. Acad.
Sci. U.S.A 1994, 91 (3), 1044–1048.
(49) Pankratov, N.; Shalabai, A. V. Quantum-Chemical Evaluation of
the Protolytic Properties of Thiophenols. J. Struct. Chem. 2004, 45 (5),
756–761.
(50) Fukuto, J. M.; Chiang, K.; Hszieh, R.; Wong, P.; Chaudhuri, G.
The Pharmacological Activity of Nitroxyl;A Potent Vasodilator
with Activity Similar to Nitric-Oxide and or Endothelium-Derived
Relaxing Factor. J. Pharmacol. Exp. Ther. 1992, 263 (2), 546–551.
(43) Marti, M. A.; Bari, S. E.; Estrin, D. A.; Doctorovich, F. Discrimi-
nation of nitroxyl and nitric oxide by water-soluble Mn(III) por-
phyrins. J. Am. Chem. Soc. 2005, 127 (13), 4680–4684.
(44) Bender, M. L. Mechanisms of Catalysis of Nucleophilic Reac-
tions of Carboxylic Acid Derivatives. Chem. Rev. 1960, 60 (1),
53–113.
(45) Maragos, C. M.; Morley, D.; Wink, D. A.; Dunams, T. M.;
Saavedra, J. E.; Hoffman, A.; Bove, A. A.; Isaac, L.; Hrabie,
J. A.; Keefer, L. K. Complexes of NO with Nucleophiles as Agents
for the Controlled Biological Release of Nitric-Oxide;Vasorelaxant
Effects. J. Med. Chem. 1991, 34 (11), 3242–3247.
(46) Doyle, M. P.; Mahapatro, S. N.; Broene, R. D.; Guy, J. K.
Oxidation and Reduction of Hemoproteins by Trioxodinitrate-
(II);the Role of Nitrosyl Hydride and Nitrite. J. Am. Chem. Soc.
1988, 110 (2), 593–599.
(47) Shoeman, D. W.; Nagasawa, H. T. The reaction of nitroxyl (HNO)
with nitrosobenzene gives Cupferron (N-nitrosophenylhydroxylamine).
Nitric Oxide: Biol. Chem. 1998, 2 (1), 66–72.
(51) Favaloro, J. L.; Kemp-Harper, B. K. Redox variants of NO (NO
3
and HNO) elicit vasorelaxation of resistance arteries via distinct
mechanisms. Am. J. Physiol.: Heart Circ. Physiol. 2009, 296 (5),
H1274–H1280.
(52) Hobbs, A. J. Soluble guanylate cyclase: the forgotten sibling.
Trends Pharmacol. Sci. 1997, 18 (12), 484–491.
(53) Griffiths, C.; Wykes, V.; Bellamy, T. C.; Garthwaite, J. A new and
simple method for delivering clamped nitric oxide concentrations in the
physiological range: Application to activation of guanylyl cyclase-coupled
nitric oxide receptors. Mol. Pharmacol. 2003, 64 (6), 1349–1356.
(54) Poulos, T. L. Soluble guanylate cyclase. Curr. Opin. Struct. Biol.
2006, 16 (6), 736–743.
(55) Miller, T. W.; Cherney, M. M.; Lee, A. J.; Francoleon, N. E.;
Farmer, P. J.; King, S. B.; Hobbs, A. J.; Miranda, K. M.; Burstyn,
J. N.; Fukuto, J. M. The Effects of Nitroxyl (HNO) on Soluble
Guanylate Cyclase Activity Interactions at Ferrous Hemes and
Cysteine Thiols. J. Biol. Chem. 2009, 284 (33), 21788–21796.
(56) Zeller, A.; Wenzl, M. V.; Beretta, M.; Stessel, H.; Russwurm, M.;
Koesling, D.; Schmidt, K.; Mayer, B. Mechanisms Underlying
Activation of Soluble Guanylate Cyclase by the Nitroxyl Donor
Angeli’s Salt. Mol. Pharmacol. 2009, 76 (5), 1115–1122.
(48) Podhradsky, D.; Drobnica, L.; Kristian, P. Reactions of Cysteine,
Its Derivatives, Glutathione, Coenzyme-a, and Dihydrolipoic Acid
with Isothiocyanates. Experientia 1979, 35 (2), 154–155.