Hydrolysis of acyloxy nitroso compounds yields nitroxyl (HNO)

Article abstract of DOI:10.1021/ja062365a

Nitroxyl (HNO/NO^-), the reduced form of nitric oxide, has gained attention based on its separate chemistry and biology from nitric oxide. The inherent reactivity of HNO requires new and mechanistically unique donors for the detailed study of HN

Full text of DOI:10.1021/ja062365a

Published on Web 07/12/2006
Hydrolysis of Acyloxy Nitroso Compounds Yields Nitroxyl
(HNO)
Xin Sha,† T. Scott Isbell,‡ Rakesh P. Patel,‡ Cynthia S. Day,† and S. Bruce King*,†
Contribution from the Department of Chemistry, Wake Forest UniVersity,
Winston-Salem, North Carolina 27109 and the Department of Pathology, Center for Free
Radical Biology, UniVersity of AlabamasBirmingham, Birmingham, Alabama 35294
Received April 6, 2006; E-mail: kingsb@wfu.edu
Abstract: Nitroxyl (HNO/NO-), the reduced form of nitric oxide, has gained attention based on its separate
chemistry and biology from nitric oxide. The inherent reactivity of HNO requires new and mechanistically
unique donors for the detailed study of HNO chemistry and biology. Oxidation of cyclohexanone oxime
with lead tetraacetate yields 1-nitrosocyclohexyl acetate, whereas oxidation of oximes in the presence of
excess carboxylic acid gives various acyloxy nitroso compounds. These bright blue compounds exist as
monomers as indicated by their infrared, proton, and carbon NMR spectra, and X-ray crystallographic
analysis reveals the nitroso groups possess a “nitroxyl-like” bent configuration. Hydrolysis of these
compounds produces nitrous oxide, the dimerization and dehydration product of HNO, and provides evidence
for the intermediacy of HNO. Both thiols and oxidative metal complexes inhibit nitrous oxide formation.
Hydrolysis of these compounds in the presence of ferric heme complexes forms ferrous nitrosyl complexes
providing further evidence for the intermediacy of HNO. Kinetic analysis shows that the rate of hydrolysis
depends on pH and the structure of the acyl group of the acyloxy nitroso compound. These compounds
relax pre-constricted rat aortic rings similar to known HNO donors. Together, these results identify acyloxy
nitroso compounds as a new class of HNO donors.
Introduction
Recent studies indicate the potential of nitroxyl donors for
the treatment of heart failure, a condition characterized by the
Nitroxyl (HNO/NO-), the reduced form of nitric oxide (NO),
has emerged as an important constituent in nitric oxide chemistry
and biochemistry in recent years.1,2 HNO and NO- form an
acid/base pair with NO- being isoelectronic with dioxygen.
HNO possesses a singlet ground state (1HNO) and a triplet
excited state (3HNO) with an energy gap of about 18 kcal/mol,
whereas NO- exists in a triplet ground state (3NO-) and a singlet
excited state (1NO-) with a singlet-triplet energy gap around
17-21 kcal/mol.3,4 The pKa value of 1HNO/3NO- has recently
been corrected to 11.4 making 1HNO the exclusive species under
physiological conditions. In addition, the high negative reduction
heart’s inability to maintain adequate blood circulation to the
peripheral tissues and the lungs.16,17 Heart failure remains one
of the most lethal diseases in the United States with more than
50 000 deaths each year, which exceeds the deaths from all
forms of cancer combined.17 New work shows that nitroxyl
donors enhance cardiac inotropy and increase plasma calcitonin
gene-related peptide (CGRP) levels but do not affect plasma
levels of cyclic guanylate monophosphate (cGMP). These results
suggest that nitroxyl may influence cardiovascular function
through a different mechanistic pathway than nitric oxide.18,19
potentials (NO/3NO- ) -0.8((0.2) V and NO/1NO-
)
(6) Pino, R. Z.; Feelisch, M. Biochem. Biophys. Res. Commun. 1994, 201, 54-
62.
-7((0.2) V), make reduction of NO to 3NO- and 1NO-
extremely unfavorable.4 HNO and NO- both react with O2, NO,
thiols, and metal ions with HNO also undergoing rapid dimer-
ization.3,5-13 The chemistry and biochemistry of HNO/NO- have
recently been extensively reviewed.14-16
(7) Wong, P. S.; Hyun, J.; Fukuto, J. M.; Shirota, F. N.; DeMaster, E. G.;
Shoeman, D. W.; Nagasawa, H. T. Biochemistry 1998, 37, 5362-5371.
(8) Cook, N. M.; Shinyashiki, M.; Jackson, M. I.; Leal, F. A.; Fukuto, J. M.
Arch. Biochem. Biophysics. 1998, 410, 89-95.
(9) Hughes, N.; Nicklin, H. G. J. Chem. Soc. A 1971, 164-171.
(10) Bazylinski, D. A.; Hollocher, T. C. J. Am. Chem. Soc. 1985, 107, 7982-
7986.
(11) Bazylinski, D. A.; Goretski, J.; Hollocher, T. C. J. Am. Chem. Soc. 1985,
107, 7986-7989.
† Wake Forest University.
(12) Sulc, F.; Immoos, C. E.; Pervitsky, D.; Farmer, P. J. J. Am. Chem. Soc.
2004, 126, 1096-1101.
‡ University of AlabamasBirmingham.
(1) Miranda, K. M.; Paolocci, N.; Katori, T.; Thomas, D. D.; Ford, E.;
Bartberger, M. D.; Espey, M. G.; Kass, A. G.; Feelisch, M.; Fukuto, J. M.;
Wink, D. A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9196-9201.
(2) Bartberger, M. D.; Fukuto, J. M.; Houk, K. N. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 2194-2198.
(13) Doyle, M. P.; Mahapatro, S. N.; Broene, R. D.; Guy, J. K. J. Am. Chem.
Soc. 1988, 110, 593-599.
(14) Miranda, K. M. Coord. Chem. ReV. 2005, 249, 433-455.
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Pharmacol. Toxicol. 2005, 45, 335-355.
(3) Shafirovich, V.; Lymar, S. V. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 7340-
7345.
(16) Fukuto, J. M.; Bartberger, M. D.; Dutton, A. S.; Paolocci, N.; Wink, D.
A.; Houk, K. N. Chem. Res. Toxicol. 2005, 18, 790-801.
(17) Feelisch, M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4978-4980.
(18) Paolocci, N.; Katori, T.; Champion, H. C.; John, M. E. S.; Miranda, K.
M.; Fukuto, J. M.; Wink, D. A.; Kass, D. A. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 5537-5542.
(4) Bartberger, M. D.; Liu, W.; Ford, E.; Miranda, K. M.; Switzer, C.; Fukuto,
J. M.; Farmer, P. J.; Wink, D. A.; Houk, K. N. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 10958-10963.
(5) Hughes, M. N. Biochim. Biophys. Acta 1999, 1411, 263-272.
9
10.1021/ja062365a CCC: $33.50 © 2006 American Chemical Society
J. AM. CHEM. SOC. 2006, 128, 9687-9692
9687

A R T I C L E S
Scheme 1
Sha et al.
Scheme 2
Also, nitroxyl donors precondition cardiac tissue significantly
improving post-ischemic contractility (compared to NO donors)
with no adverse effect on systemic blood pressure.18-20 Nitroxyl
donors appear to be unique entities for the protection and
stimulation of the cardiovascular system during heart failure.
Due to its inherent reactivity, HNO must be generated in situ
from donor compounds with the most common being Angeli’s
salt (Na2N2O3) and Piloty’s acid (PhSO2NHOH). However, the
structural simplicity of Angeli’s salt does not allow extensive
variation for the modification of its release properties. Angeli’s
salt also releases nitrite, which possesses its own biological
profile.21,22 Nitroxyl release from Piloty’s acid occurs only under
basic conditions and decomposition of Piloty’s acid under
physiological conditions may release NO..23 Acyl nitroso
compounds react with nucleophiles to yield nitroxyl but these
highly reactive intermediates must be generated in situ.24 Given
the renewed interest in HNO chemistry and the discovery of
the unique biological properties of HNO, new and mechanisti-
cally unique HNO donors are sorely needed.
Acyloxy nitroso compounds (Scheme 1) have been reported
by several research groups.25-34 Although Rehse and Herpel
showed these compounds inhibit platelet aggregation and
thrombus formation (indicative of NO release), they generate
only small amounts (<1%) of NO and HNO (judged by N2O
formation) under neutral conditions.32 We propose that hydroly-
sis of the acyl portion of these molecules will generate an
unstable intermediate that decomposes to HNO and the corre-
sponding ketone (Scheme 1). Here, we report the synthesis and
structural characterization of various acyloxy nitroso compounds
that release HNO upon hydrolysis.
Table 1. Decomposition of 1-Nitrosocyclohexyl Acetate to NO and
N2O under Different Solvent Conditions
NO formation
(% yield)
N2O formation
(% yield)
solvent conditionsa
none
0.5%
0.4%
0.4-0.5%
0.4%
0%
28%
24%
58%
phosphate buffer, pH 7.4
MeOH + phosphate buffer, pH 7.4
0.1 M NaOH + MeOH
a Room temperature, 2 h incubation
Table 2. Quenching of N2O Formation from 1-Nitrosocyclohexyl
Acetate by Glutathione and Tetraphenylporphyrin Iron(III) Chloride.
N2O formation (% yield)
conditions
1 h
2 h
3 h
overnight
0.1 M NaOH + MeOH
+ 2 equiv glutathione
+ 2 equiv [FeIIITPP]Cl
46%
0%
14%
56%
0%
13%
58%
0%
8%
54%
0%
11%
667 nm and IR absorption for the carbonyl (νCdO ) 1750 cm-1
and nitroso groups (νNdO ) 1561 cm-1).
)
Table 1 summarizes chemiluminescence and gas chromato-
graphic assays that respectively reveal the production of NO
and HNO from 1 under different solvent conditions. A neat
sample of 1 decomposes to a small amount of NO and no N2O
after 2 h. In phosphate buffer, 1 releases a similar amount of
NO, but the yield of N2O, evidence for the intermediacy of
HNO, significantly increases. As N2O arises from the dimer-
ization of 2 HNO molecules, the reported yields are based on
the percentage of the theoretical yield of N2O formation (50%
based on 1). Decomposition of 1 in buffer/methanol solu-
tions (1:1) gives similar results. Under basic conditions, the
amount of NO from 1 remains the same but the yield of N2O
further increases suggesting that 1 gives HNO under basic
conditions. Chemiluminescence experiments reveal only small
amounts (3-4%) of nitrite formation under these conditions.
Extraction of the reaction mixture yields cyclohexanone in
60% yield whose structure was confirmed by 13C NMR
spectroscopy.
Results
Preparation and Evaluation of 1-Nitrosocyclohexyl Ace-
tate as an HNO Donor. Oxidation of cyclohexanone oxime
with lead tetraacetate (LTA) gives 1-nitrosocyclohexyl acetate
in 52% yield (1; Scheme 2).25,26,31 1-Nitrosocyclohexyl acetate
(1) is a bright blue liquid with strong UV-vis absorption at
(19) 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. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10463-10468.
(20) Pagliaro, P. Life Sci. 2003, 73, 2137-2149.
GC competition experiments in the presence of thiols and
ferric iron support the existence of nitroxyl under these con-
ditions. The reaction of these species with nitroxyl should
compete with dimerization and result in a decrease in the yield
of N2O. The addition of glutathione (2 equiv) to the decomposi-
tion of 1 under basic conditions completely suppresses N2O
formation (Table 2).1,15 Likewise, the addition of tetraphen-
ylporphyrin iron (III) chloride ([FeIIITPP]Cl) to the reaction
system significantly decreases the yield of N2O (Table 2).
(21) Gladwin, M. T.; Schechter, A. N.; Kim-Shapiro, D. B.; Patel, R. P.; Hogg,
N.; Shiva, S.; Cannon, R. O., III.; Kelm, M.; Wink, D. A.; Espey, M. G.;
Oldfield, E. H.; Pluta, R. M.; Freeman, B. A.; Lancaster, J. R., Jr.; Feelisch,
M.; Lundberg, J. O. Nat. Chem. Biol. 2005, 1 (6), 308-314.
(22) Ehman, D. L.; Sawyer, D. T. J. Electroanal. Chem. 1968, 16, 541-549.
(23) Fukuto, J. M.; Chiang, K.; Hszieh, R.; Wong, P.; Chaudhuri, G. J. Pharm.
Exp. Ther. 1992, 263, 546-551.
(24) King, S. B.; Nagasawa, H. T. Methods Enzymol. 1998, 301, 211-220.
(25) Lown, J. W. J. Chem. Soc. B 1966, 441-446.
(26) Kropf, V. H.; Lambeck, R. Liebigs Ann. Chem. 1966, 700, 1-17.
(27) Gubelt, G. B.; Warkentin, J. Can. J. Chem. 1969, 47, 3983-3987.
(28) Shafiullah, D.; Ali, H. Synthesis 1979, 124-126.
Monitoring the disappearance of the strong absorption at 667
nm using UV-vis spectroscopy provides information regarding
the kinetics of decomposition of 1. 1-Nitrosocyclohexyl acetate
(1) demonstrates relative stability in neutral buffer (Figure 1).
Incubation in basic solution results in the apparent exponential
decrease of 1 over time with a more rapid decrease in more
concentrated base (Figure 1). From these decompositions, half-
(29) Kotali, A.; Papageorgiou, V. P. J. Chem. Soc., Perkin Trans. 1 1985, 2083-
2086.
(30) Calvet, G.; Dussaussois, M.; Blanchard, N.; Kouklovsky, C. Org. Lett. 2004,
6 (14), 2449-2451.
(31) Zhutov, E. V.; Skornyakov, Y. V.; Proskurina, M. V.; Zefirov, N. S. Russ.
J. Org. Chem. 2003, 39 (11), 1672-1673.
(32) Rehse, K.; Herpel, M. Arch. Pharm. Med. Chem. 1998, 331, 104-110.
(33) Daineko, V. I.; Proskurnina, M. V.; Skornyakov, Y. V.; Trofimov, B. A.;
Zefirov, N. S. Russ. J. Org. Chem. 2002, 38 (10), 1431-1433.
(34) White, H. E.; Considine, J. W. J. Am. Chem. Soc. 1958, 80, 626-630.
9
9688 J. AM. CHEM. SOC. VOL. 128, NO. 30, 2006

Hydrolysis Yields Nitroxyl
A R T I C L E S
Figure 3. X-ray crystallographic structures of 2.
Scheme 4
Figure 1. Kinetics of the decomposition of 1 in phosphate buffer (100
mM, pH ) 7.4) and basic conditions at room temperature as measured by
UV-vis spectroscopy at 667 nm.
Table 3. Decomposition of 1-Nitrosocyclohexyl Trifluoroacetate to
N2O under Different Solvent Conditions
N2O formation
solvent conditions
1 h
2 h
3 h
overnight
MeOH
phosphate buffer, pH 7.4
MeOH + buffer
+ 2 equiv glutathione
+ 2 equiv K2Ni(CN)4)‚H2O
0%
21%
52%
0%
0%
34%
72%
0%
0%
50%
74%
0%
0%
56%
NDa
0%
3%
18%
24%
8%
a ND ) no data
Preparation and Evaluation of 1-Nitrosocyclohexyl 4-Ni-
trobenzoate and 2-Nitrosopropan-2-yl 4-Nitrobenzoate as
HNO Donors. Oxidation of cyclohexanone oxime or acetone
oxime with LTA in the presence of 4-nitrobenzoic acid (10
equiv) gives the 4-nitrobenzoic acid derived nitroso compounds
1-nitrosocyclohexyl 4-nitrobenzoate (2) and 2-nitrosopropan-
2-yl 4-nitrobenzoate (3) as blue solids in 20-30% yield after
recrystallization (Scheme 3).26,32 Similar to 1, GC and EPR
assays reveal the ability of 2 and 3 to release HNO, especially
under basic conditions (Supporting Information). Recrystalli-
zation of 2 provides material suitable for X-ray crystallographic
analysis. These studies confirm the structure of the reaction
product and show that the NdO groups of these compounds
exist in a “nitroxyl-like” bent configuration (C-N-O bond
angle ) 114.2° for 2) with a NdO bond length of 1.183 Å
(Figure 3). In addition, these studies show that these compounds
do not predominately exist as nitroso dimers in the solid state.
Preparation and Evaluation of 1-Nitrosocyclohexyl Tri-
fluoroacetate as an HNO Donor. Oxidation of cyclohexanone
oxime with LTA in the presence of trifluoroacetic acid (10
equiv) in methylene chloride yields 1-nitrosocyclohexyl tri-
fluoroacetate (4) as a bright blue liquid (Scheme 4).33
Figure 2. EPR spectrum of a mixture of hemin and 1 in 1:1 MeOH:1 M
NaOH at 110 K.
Scheme 3
lives of 0.8, 1.7, and 8 min were estimated for each condition
and provide rough estimates for the stability of 1. A further
and more rigorous kinetic examination of this process is
underway.
Incubation of 1 with methemoglobin in phosphate buffer (pH
7.4) fails to produce the expected ferrous nitrosyl hemoglobin
complex as determined by both UV/vis and EPR spectros-
copy.1,15 Under basic conditions, methemoglobin is not a suitable
HNO trap. Incubation of 1 (50 mM) with hemin (50 µM), a
nonprotein porphoryrin ferric iron complex soluble in both basic
aqueous solutions and methanol, in 1:1 methanol:1 M NaOH
yields the ferrous nitrosyl complex as judged by EPR spectros-
copy (Figure 2). The conversion of this ferric heme group to a
ferrous nitrosyl complex provides further evidence for the release
of HNO from the decomposition of 1.
Gas chromatographic assays show that 4 is stable in methanol
but rapidly decomposes in buffer to yield N2O (Table 3).
Incubation of 4 in buffer/methanol (1:1) produces an even larger
amount of N2O (Table 3). Competition experiments show that
the addition of glutathione completely suppresses N2O formation
and the addition of potassium tetracyano-nicklate(II) hydrate
(K2Ni(CN)4)‚H2O) (2 equiv) significantly decreases N2O forma-
tion (Table 3).
Absorption measurements show the characteristic Soret
absorbance of methemoglobin (15 µM in phosphate buffer) at
407 nm shifts to 416 nm upon the addition of 4 (45 mM) in
9
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A R T I C L E S
Sha et al.
Scheme 5
oxime with lead tetraacetate yields 1-nitrosocyclohexyl acetate
(1).25,26,31 Lead tetraacetate oxidation of oximes in the presence
of excess carboxylic acid gives the acyloxy nitroso compounds
(2-4) similar to previous reports.32 In these reactions, the excess
carboxylic acid presumably exchanges with the acetate groups
of lead tetraacetate to form new lead tetraacyl complexes. This
protocol should allow for the preparation of numerous acyloxy
nitroso compounds based on different oxime and carboxylic acid
structures. The bright blue color of these compounds and their
infrared, proton and carbon NMR spectra indicate they exist as
nitroso monomers and not dimers. The first X-ray crystal-
lographic studies of these compounds confirm their monomeric
structures and reveal the nitroso groups possess a “nitroxyl-
like” bent configuration with a normal NdO bond length. The
bond the C-N-O bond angle and the NdO appear similar to
other organic C-nitroso compounds whose structures have been
Figure 4. Absorbance spectrum of methemoglobin (solid line), methemo-
globin, and 4 (long dashed line).
Figure 5. EPR spectrum of a mixture of methemoglobin and 4 at 110 K.
35,36
determined crystallographically
Previous work shows that 1-nitrosocyclohexyl 4-nitrobenzoate
releases only very small amounts (<1%) of NO or HNO under
neutral conditions (phosphate buffer, ethanol, or mixtures of
both).32 NO release from these compounds requires homolytic
C-N bond cleavage and chemiluminescence analysis reveals
little NO (or nitrite) release from 1 under neutral or basic
conditions (Table 1) indicating that homolytic cleavage to give
NO does not constitute a predominant decomposition pathway
for 1 (Table 1 and Scheme 5).
Under conditions that favor ester hydrolysis, 1 acts as a
competent HNO donor. Hydrolysis of these compounds forms
a nitroso-hydroxy species (the adduct of HNO with a ketone)
that decomposes to HNO and the ketone. Kinetic analysis of
the hydrolysis of 1 clearly shows the base-dependent decrease
in absorbance (attributed to the nitroso group) supporting such
a mechanism. Also, examination of the reaction products from
the basic decomposition of 1 reveals the formation of cyclo-
hexanone, which provides further support for this pathway. Early
work shows the treatment of 2-nitroso-2-benzoyloxypropane
with 10 M KOH yields N2O, evidence for HNO, and acetone
and also supports this idea.32 Based in Scheme 5, modification
of the acyl group of the acyloxy nitroso compounds gives 2-4,
which should hydrolyze and release HNO faster than 1.
Although 2 and 3 still require basic hydrolysis to release HNO,
4 rapidly hydrolyzes in buffer to yield HNO at room temperature
as judged by N2O formation and the identification of ferrous
nitrosyl complexes. These results further strengthen a hydrolytic
pathway for the release of HNO from acyloxy nitroso com-
Figure 6. Dose dependent relaxation of pre-constricted rat aortic rings by
Angeli’s salt, 1 and 4.
methanol, which indicates the formation of a ferrous nitrosyl
complex (Figure 4). Similarly, EPR spectroscopy shows the
typical three-line EPR signal of a ferrous nitrosyl complex after
the incubation of 4 with methemoglobin (Figure 5). These
UV-vis and EPR studies provide strong evidence for the
intermediacy of HNO in these reactions.
Biological Results
Figure 6 shows the ability of compounds 1 and 4 to vasodilate
pre-constricted rat aortic rings in a dose-dependent fashion.
Estimated EC50’s (effective concentration to elicit 50% response)
were approximately 10 000 nM for 1 and 885 nM for 4. As a
control, the known HNO donor, Angeli’s salt, demonstrates a
similar profile with an approximate EC50 of 82 nM.
Discussion
(35) Ferguson, G.; Fritchie, C. J.; Robertson, J. M.; Sim, G. A. J. Chem. Soc.
1961, 1976-1987.
This work demonstrates the ability of acyloxy nitroso
compounds to act as HNO donors. Oxidation of cyclohexanone
(36) Felber, H.; Kresze, G.; Prewo, R.; Vasella, A. HelV. Chim. Acta 1986, 69,
1137-1146.
9
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Hydrolysis Yields Nitroxyl
A R T I C L E S
pounds and also suggest the possibility of biological HNO
release through the action of esterases. The ability of 4 to release
HNO at neutral pH clearly separates these compounds from
Piloty’s acid as HNO donors.
class of HNO donors that may provide an alternative to Angeli’s
salt and Piloty’s acid.
Experimental Section
General. Cyclohexanone oxime, acetone oxime, lead tetraacetate,
methemoglobin, hemin, and glutathione were purchased from Sigma
Chemical Company, St. Louis, MO.
Direct HNO detection remains a difficult analytical problem
and alternative complimentary detection methods strongly imply
the intermediacy of HNO during the hydrolysis of 1-4. Gas
chromatographic analysis of the reaction headspace of these
reaction mixtures demonstrates the formation of N2O and
provides evidence for the intermediacy of HNO as nitroxyl
dimerizes to hyponitrous acid that dehydrates to give nitrous
oxide (N2O).37,38 Addition of glutathione or oxidative metal
complexes to these hydrolyses either abolishes or greatly
diminishes N2O formation. Given that nitroxyl rapidly reacts
with thiols to form disulfides and hydroxylamine or sulfinamides
and reduces oxidative metal complexes, the addition of these
species would be expected to decrease N2O formation by
competing with HNO dimerization.39,40 The results from these
trapping experiments further lend evidence for the intermediacy
of HNO in these reactions. Alternative support for HNO
formation from 1-4 comes from the ability of these compounds
to reductively nitrosylate ferric heme groups yielding the rela-
tively stable ferrous nitrosyl complexes as judged by UV/vis
and EPR spectroscopy. Obviously, the development of direct
and sensitive methods of HNO detection remains an important
concern in the field.
1-Nitrosocyclohexyl Acetate (1). A solution of cyclohexanone
oxime (5.46 g, 48.25 mmol) in methylene chloride (50 mL) was added
dropwise with stirring to a solution of LTA (21.39 g, 48.25 mmol) in
methylene chloride (100 mL) at 0 °C. A blue color gradually appeared
with the addition of the oxime. After 1 h at 0 °C, the reaction mixture
was warmed to room temperature. After 2 h at room temperature, water
(30 mL) was added, and the organic layer was extracted with water (2
× 30 mL) and saturated sodium bicarbonate solution (2 × 30 mL).
The organic layer was dried over Na2SO4, the solvent evaporated, and
the residue purified by column chromatography to give 1 as a bright
blue liquid (52% yield): Rf ) 0.68 (pentane/ethyl acetate ) 20:1);
UV/vis (MeOH): λmax ) 667 nm, ꢀ ) 20.7 M-1 cm-1. IR (KBr):
V ) 1750 cm-1 (CdO), 1561 cm-1 (NdO); 1H NMR (300 MHz,
benzene-d6) δ 1.1-1.9 (m, 13H); 13C NMR (300 MHz, benzene-d6) δ
21.0 (CH2), 22.0 (2CH2), 25.1 (2CH2), 29.7 (CH3), 123.8 (O-C-N),
168.5 (CdO); Anal. Calcd. for C8H13NO3: C, 56.11; H, 7.67; N, 8.18.
Found: C, 56.46; H, 7.73; N, 7.82.
1-Nitrosocyclohexyl-4-Nitrobenzoate (2). A solution of cyclohex-
anone oxime (2.66 g, 23.51 mmol) in methylene chloride (50 mL) was
added dropwise with stirring to a solution of LTA (10.42 g, 23.51 mmol)
and 4-nitrobenzoic acid (39.29 g, 235.1 mmol) in methylene chloride
(300 mL) at 0 °C. A blue color gradually appeared with the addition
of the oxime. After 1 h at 0 °C, the reaction mixture was warmed to
room temperature. After 3 h at room temperature, water (50 mL) was
added, and the organic layer was extracted with water (2 × 50 mL)
and 3% sodium bicarbonate solution (2 × 50 mL). The organic layer
was dried over Na2SO4, the solvent was evaporated, and the residue
was recrystalized in diethyl ether/petroleum ether (1:1) in a -5 °C
freezer to give 2 as bright blue crystals (20-25% yield): Rf ) 0.55
(pentane/ethyl acetate ) 20:1); UV-vis (MeOH): λmax ) 666 nm; IR
(KBr): νCdO ) 1732 cm-1, νNdO ) 1562 cm-1, νNO2 ) 1530 cm-1; 1H
NMR (300 MHz, Benzene-d6) δ 1.17-2.04 (m, 10H), 7.38-7.98 (d,
4H); 13C NMR (300 MHz, Benzene-d6) δ 22.1 (2CH2), 25.0 (CH2),
29.7 (2CH2), 123.8 (2Ph-CH), 125.3 (O-C-N), 131.1 (2Ph-CH), 135.3
(Ph-C), 151.1 (Ph-C), 162.6 (CdO).
Compounds 1 and 4 also relax pre-constricted rat aortic rings
demonstrating their ability to elicit biological effects. These
compounds behave similarly to the known HNO donor Angeli’s
salt in this assay reinforcing the ability of these compounds to
act as HNO donors.41 The lower potency of 1 and 4 compared
to Angeli’s salt may be related to (1) lower solubility and (2)
slower HNO release rates (it should be noted that 4 is more
potent than 1). Although relaxation of vascular tissue generally
reflects a response elicited by an NO donor, the observed effects
remain consistent with Angeli’s salt, a known HNO donor.41
These results may indicate the conversion of donor-derived
HNO to NO in this system.
2-Nitrosopropan-2-yl 4-Nitrobenzoate (3). A solution of acetone
oxime (3.11 g, 42.58 mmol) in methylene chloride (50 mL) was added
dropwise with stirring to a solution of LTA (18.88 g, 42.58 mmol)
and 4-nitrobenzoic acid (71.16 g, 425.8 mmol) in methylene chloride
(300 mL) at 0 °C. A blue color gradually appeared with the addition
of the oxime. After 1 h at 0 °C, the reaction mixture was warmed to
room temperature. After 3 h at room temperature, water (50 mL) was
added, and the organic layer was extracted with water (2 × 50 mL)
and 3% sodium bicarbonate solution (2 × 50 mL). The organic layer
was dried over Na2SO4, the solvent was evaporated, and the residue
was recrystalized in diethyl ether/petroleum ether (1:1) in a -5 °C
freezer to give 3 as bright blue crystals (25-30% yield): Rf ) 0.5
(pentane/ethyl acetate ) 20:1); UV-vis (MeOH): λmax ) 659 nm; IR
(KBr): νCdO ) 1738 cm-1, νNdO ) 1563 cm-1, νNO2 ) 1529 cm-1; 1H
NMR (300 MHz, CDCl3) δ 1.69 (s, 6H), 8.31-8.48 (d, 4H); 13C NMR
(300 MHz, CDCl3) δ 21.1 (2CH3), 122.6 (O-C-N), 124.0 (2Ph-CH),
131.4 (2Ph-CH), 135.6 (Ph-C), 151.2 (Ph-C), 162.9 (CdO).
Summary
The recent interest in HNO chemistry, biochemistry, and
biology highlights the need for new and mechanistically unique
HNO donors. Oxidation of oximes in the presence of carboxylic
acids yields acyloxy nitroso compounds that exist as blue
monomeric C-nitroso compounds. Hydrolysis of these com-
pounds release HNO as judged by gas chromatographic
identification of nitrous oxide and the spectroscopic identifica-
tion of ferrous nitrosyl complexes. The modular synthetic
approach allows for alteration of the structure of the acyl group
to control the rate of hydrolysis. Compounds 1 and 4 also relax
a pre-constricted rat aortic ring similar to Angeli’s salt. These
results reveal that acyloxy nitroso compounds represent a new
1-Nitrosocyclohexyl Trifluoroacetate (4). A solution of cyclohex-
anone oxime (4.88 g, 43.16 mmol) in methylene chloride (50 mL) was
added dropwise with stirring to a solution of LTA (19.14 g, 43.16 mmol)
and trifluoroacetic acid (49.22 g, 431.6 mmol) in methylene chloride
(200 mL) at 0 °C. A blue color gradually appeared with the addition
of the oxime. After 1 h at 0 °C, the reaction mixture was warmed to
room temperature. After 3 h at room temperature, water (50 mL) was
(37) Smith, P. A. S.; Hein, G. E. J. Am. Chem. Soc. 1960, 82, 5731-5740.
(38) Kohout, F. C.; Lampe, F. W. J. Am. Chem. Soc. 1965, 87, 5795-5796.
(39) Shen, B.; English, A. M. Biochemistry 2005, 44, 14030-14044.
(40) Donzelli, S.; Espey, M. G.; Thomas, D. D.; Mancardi, D.; Tocchetti, C.
G.; Ridnour, L. A.; Paolocci, N.; King, S. B.; Miranda, K. M.; Lazzarino,
G.; Fukuto, J. M.; Wink, D. A. Free Radical Biol. Med. 2006, 40 (6), 1056-
1066.
(41) Fukuto, J. M.; Chiang, K.; Hszieh, R.; Wong, P.; Chaudhuri, G. J.
Pharmacol. Exp. Ther. 1992, 263 (2), 546-551.
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A R T I C L E S
Sha et al.
added, and the organic layer was extracted with water (2 × 50 mL).
The organic layer was dried over Na2SO4, the solvent was evaporated,
and the residue was purified by column chromatography to give 4 as
a bright blue liquid (30% yield): Rf ) 0.57 (pentane); UV-vis
(MeOH): λmax ) 648 nm; IR (KBr): νCdO ) 1792 cm-1, νNdO ) 1570
cm-1; 1H NMR (300 MHz, Benzene-d6) δ 1.1-1.9 (m, 10H); 13C NMR
(300 MHz, benzene-d6) δ 19.9 (2CH2), 22.9 (CH2), 27.4 (2CH2), 113.8
(q, J ) 286.2 Hz), 127.2 (O-C-N), 153.7 (q, J ) 42.5 Hz); 19F NMR
(300 MHz, benzene-d6) δ -75.7 (CF3).
Chemiluminescence NO Assay. 1-Nitrosocyclohexyl acetate (1) (0.1
mmol) or a solution of 1 (0.1 mmol) in solvent (2 mL) was placed in
a 25 mL flask sealed with a rubber septa. Sample aliquots (10 µL) of
the reaction headspace were injected over time into the reaction vessel
of a Sievers 280 nitric oxide analyzer chemiluminescence detector. This
apparatus directly detects NO and the concentrations of NO were
determined based on a standard curves.
Gas Chromatographic Detection of Nitrous Oxide. A solution of
substrate (0.1 mmol) in solvent (2 mL) was placed in a 25 mL flask
sealed with a rubber septa under an argon atomosphere. In some cases,
glutathione or Fe(III) were added to the solution. Aliquots of the reaction
headspace (250 µL) were injected onto a 6890 Hewlett-Packard gas
chromatograph equipped with a thermal conductivity detector, a 6 ft.
× 1/8 in. Porapak Q column at an operating oven temperature of 50
°C (injector and detector 150 °C) with a flow rate of 16.67 mL/min
(He, carrier gas). The retention time of nitrous oxide was 2.9 min and
identical to known samples of N2O (Aldrich). The yields were calculated
based upon a standard curve prepared by injecting known amounts of
these gases.
UV-vis Assay for the Decomposition of 1. A solution of sodium
hydroxide (0.05 M, 1 mL) was added to a solution of 1 (0.1 mmol,
0.05 M) in methanol (1 mL) at room temperature in a cuvette.
UV-vis measurements were taken every 0.74 min at a wavelength of
667 nm on a Cary 100 Bio UV-visible spectrophotometer (Varian,
Walnut Creek, CA). Similar experiments were performed using 0.1
and 0.2 M sodium hydroxide with UV-vis measurements being taken
every 0.84 min (0.1 M NaOH) and every 1 min for 0.2 M NaOH.
EPR Assay of Ferrous Nitrosyl Complex Formation from the
Reactions of 1 with Hemin. A solution of 1 (50 mM) in methanol
was added to a solution of hemin (50 µM) in 1 M NaOH at room
temperature in a 25 mL flask. Samples (2 mL) for EPR studies were
withdrawn every 2 min for 10 min and then at 15 and 20 min and
transferred to an EPR tube and frozen in liquid nitrogen (77 K). EPR
spectra were taken on a Bruker ER200D spectrometer at 110 K using
10 mW microwave power, 5.0 G modulation amplitude, and 9.3 GHz
microwave frequency.
and Z ) 4 {FW ) 278.26, dcalcd ) 1.391 g cm-3; µa(Mo KRj) ) 0.108
mm-1}. A total of 11378 reflections having 2Θ(Mo KRj) < 54.98° were
collected on a Bruker SMART APEX CCD area detector system using
graphite-monochromated Mo KRj radiation (λ ) 0.71073 Å). Of these
11378 reflections, 3032 were unique (Rint ) 0.089). The structure was
solved using “Direct Methods” techniques with the Bruker AXS
SHELXTL-PC software package. The final anisotropic full-matrix least-
squares refinement on F2 with 181 variables converged at R1 ) 6.31%,
for the 2306 observed data and wR2 ) 17.7% for all data. The
goodness-of-fit was 1.042. Hydrogen atoms were included in the
structure factor calculations as idealized isotropic atoms “riding” on
their respective carbon atoms. Crystallographic software references
include: SMART V5.628 “Program for Data Collection on Area
Detectors” BRUKER AXS Inc., 5465 East Cheryl Parkway, Madison,
WI 53711-5373; SAINT V6.36 “Program for Reduction of Area
Detector Data” BRUKER AXS Inc., 5465 East Cheryl Parkway,
Madison, WI 53711-5373; and SHELXTL-PC V6.12, BRUKER AXS
Inc., 5465 East Cheryl Parkway, Madison, WI 53711-5373.
Vasorelaxation of Rat Aortic Rings. Isometric tension in isolated
rat thoracic aortic ring segments were measured as described previ-
ously.42 Upon sacrifice, the aorta was excised and cleansed of fat and
adhering tissue. Vessels were then cut into individual ring segments
(2-3 mm in width) and suspended from a force-displacement transducer
in a tissue bath (Radnoti). Ring segments were bathed at 37 °C in a
bicarbonate-buffered, Krebs-Henseleit (K-H) solution of the following
composition (mM): NaCl 118; KCl 4.6; NaHCO3 27.2; KH2PO4 1.2;
MgSO4 1.2; CaCl2 1.75; Na2EDTA 0.03; and glucose 11.1 and perfused
continuously with 21% O2/5% CO2/74% N2. A passive load of 2 g
was applied to all ring segments and maintained at this level throughout
the experiments. At the beginning of each experiment, indomethacin-
treated ring segments were depolarized with KCl (70 mM) to determine
the maximal contractile capacity of the vessel. Rings were then washed
extensively and allowed to equilibrate. For subsequent experiments,
vessels were submaximally contracted (50% of KCl response) with
phenylephrine (PE, 3 × 10-8 - 10-7 M), and L-NMMA, 0.1 mM,
was also added to inhibit eNOS and endogenous NO production. After
tension development reached a plateau, HNO donors were added
cumulatively to the vessel bath and effects on tension monitored. Real
time data were collected, downloaded to an IBM PC, and analyzed
using commercially available software. Stock solutions of HNO donors
were prepared immediately (<15 min) before addition to vessel bioassay
chambers and stored in the dark at 4 °C.
Acknowledgment. This work was supported by the National
Institutes of Health (HL 62198, S.B.K., HL70146, R.P.P.). The
NMR spectrometers used in this work were purchased with
partial support from NSF (CHE-9708077) and the North
Carolina Biotechnology Center (9703-IDG-1007). The authors
thank Dr. Jinming Huang for assistance with EPR measure-
ments.
UV-vis and EPR Assay of Ferrous Nitrosyl Complex from the
Reaction of 4 with Methemoglobin. A solution of 4 (45 mM) in
methanol was added to a solution of methemoglobin (15 µM) in
phosphate buffer (100 mM, pH 7.4) at room temperature in a cuvette.
UV-vis measurements were taken every 10 min for 4 h on a Cary
100 Bio UV-visible spectrophotometer (Varian, Walnut Creek, CA).
Similarly, a solution of 4 (45 mM) in methanol was added to a solution
of methemoglobin (15 µM) in phosphate buffer (100 mM, pH 7.4) at
room temperature in a 25 mL flask. Samples (2 mL) for EPR studies
were withdrawn every 2 min for 10 min and then at 15 and 20 min
and transferred to an EPR tube and frozen in liquid nitrogen (77 K).
EPR spectra were taken on a Bruker ER200D spectrometer at 110 K
using 10 mW microwave power, 5.0 G modulation amplitude, and 9.3
GHz microwave frequency.
Supporting Information Available: Experimental details and
results for the gas chromatographic and electron paramagnetic
resonance analysis for the hydrolytic decomposition of 2 and
1
3. Copies of the H and 13C NMR spectra of 1-4 are also
included. Crystallographic Information Files (CIF) for 2 are
included. This material is available free of charge via the Internet
at http://pubs.acs.org.
JA062365A
X-ray Crystallograpy of 2. A blue single crystal of C13H14N2O5
(dimensions 0.25 × 0.16 × 0.13 mm3) was, at 193(2) K, mono-
(42) Crawford, J. H.; Huang, J.; Isbell, T. S.; Shiva, S.; Chacko, B. K.; Schechter,
A.; Darley-Usmar, V. M.; Kerby, J. D.; Lang, J. D., Jr.; Krauss, D.; Ho,
C.; Gladwin, M. T.; Patel, R. P. Blood 2006, 107, 566-575.
5
clinic, space group P2(1)/c-C2h (No. 14) with a ) 6.210(3) Å, b )
11.183(5) Å, c ) 19.130(8) Å, â ) 90.723(7)°, V ) 1328.5(10) Å3,
9
9692 J. AM. CHEM. SOC. VOL. 128, NO. 30, 2006