Allylic nitro compounds as nitrite donors

Article abstract of DOI:10.1021/ja066011v

Allylic nitro compounds were synthesized and evaluated as organic sources of nitrite and nitric oxide. Unactivated allylic nitro compounds do not spontaneously release nitrite and nucleophile promoted nitrite release is slow. However, 2-(nitromethyl)-cycl

Full text of DOI:10.1021/ja066011v

Published on Web 11/24/2006
Allylic Nitro Compounds as Nitrite Donors
Harinath Chakrapani, Michael J. Gorczynski, and S. Bruce King*
Contribution from the Department of Chemistry, Wake Forest UniVersity, Winston-Salem, North
Carolina 27109
Received August 23, 2006; E-mail: kingsb@wfu.edu
Abstract: Allylic nitro compounds were synthesized and evaluated as organic sources of nitrite and nitric
oxide. Unactivated allylic nitro compounds do not spontaneously release nitrite and nucleophile promoted
nitrite release is slow. However, 2-(nitromethyl)-cyclohex-1-ene-3-one spontaneously dissociates in buffer
-
5
-1
(
pH ) 7.4) to release nitrite with a kobs ) 1.6 × 10
s . In the presence of L-cysteine, this compound
rapidly yields nitrite and reacts with hemoglobin similarly to sodium nitrite. Structural modifications and the
nature and amount of nucleophile modulate the rate of nitrite release. In the presence of L-cysteine and
ascorbic acid, this compound forms nitric oxide. Together, these results reveal a new structural architecture
for the tunable liberation of nitrite and nitric oxide from organic compounds.
Introduction
For example, respiring mitochondria use ubiquinol to reduce
nitrite, while xanthine oxidoreductases, which are structurally
Nitrite has long been considered a physiologically inert
byproduct of nitric oxide (NO) oxidation. However, recent
studies suggest that nitrite plays a crucial role in NO-mediated
hypoxic vasodilation, cytoprotection from cardiac and liver
ischemia-reperfusion injury, and gene expression.1-5 The re-
quirement of oxygen for normal functioning of nitric oxide
synthase renders this enzyme ineffective at low oxygen tension,
and nitrite may assume a role as an important hypoxic source
of NO. Deoxyhemoglobin (deoxyHb) reacts with nitrite to
generate iron nitrosyl hemoglobin (HbFe(II)-NO) and meth-
similar to bacterial nitrate and nitrite reductases, catalyze nitrite
reduction to nitric oxide in the presence of NADH.17 Mild
biochemical reductants such as ascorbate, 1,2- and 1,4-dihy-
droxyphenols, and R-tocopherol mediate nitric oxide formation
18
from nitrite. Physiological sources of nitrite include the normal
diet, which contains sizable quantities (0.7-181 mg/day) and
spontaneous auto-oxidation of endogenously produced nitric
1
9
oxide (minor). Furthermore, the intriguing biochemistry of
nitrate has emerged, where the body actively concentrates this
simple anion for its conversion to nitrite (via commensal bacteria
emoglobin (metHb, HbFe(III)), essentially functioning as a
20
competent reductant of nitrite to nitric oxide.6
-10
in the mouth) and ultimately NO in the stomach. Hence, nitrite
represents a major intravascular storage pool of NO and plays
a pivotal role in nitric oxide biology.
Hemoglobin
(Hb) may also react with low concentrations of nitrite to generate
S-nitrosothiols, another possible mechanism for the maintenance
of normal vascular tone.1
1,12
Nitrite has been proposed as a potential therapeutic in the
treatment of various diseases, including neonatal pulmonary
hypertension, subarachnoid hemorrhage associated with vasos-
pasm, and sickle cell disease underscoring the importance of
Additionally, numerous hypoxic
reduction pathways exist for the conversion of nitrite to NO
mediated by several heme- and thiol-containing enzymes.
1
3-16
2
1,22
(
1) Gladwin, M. T. Nat. Chem. Biol. 2005, 1, 308-314.
exogenous sources of nitrite.
The use of inorganic nitrites
(
2) Dejam, A.; Hunter, C. J.; Schecheter, A. N.; Gladwin, M. T. Blood Cells
Mol. Dis. 2004, 32, 423-429.
precludes both controlled delivery and regulation of nitrite
bioavailability, and these drawbacks may limit its utility as a
therapy. We propose the use of allylic nitro compounds as
organic sources of nitrite as an initial strategy to solve the
(
3) Bryan, N. S.; Fernandez, B. O.; Bauer, S. M.; Garcia-Saura, M. F.; Milsom,
A. B.; Rassaf, T.; Maloney, R. E.; Bharti, A.; Rodriguez, J.; Feelisch, M.
Nat. Chem. Biol. 2005, 1, 290-297.
(
4) Webb, A.; Bond, R.; Mclean, P.; Uppal, R.; Benjamin, N.; Ahluwalia, A.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 13683-13688.
5) Duranski, M.; Greer, J.; Dejam, A.; Jaganmohan, S.; Hogg, N.; Langston,
W.; Patel, R.; Yet, S.; Wang, X.; Kevil, C.; Gladwin, M.; Lefer, D. J.
Clin. InVest. 2005, 115, 1232-1240.
(
(14) Benjamin, N.; O’Driscoll, F.; Dougall, H.; Duncan, C.; Smith, L.; Golden,
M.; Mckenzie, H. Nature 1994, 370, 25-26.
(15) Li, H.; Samouilov, A.; Liu, X.; Zweier, J. L. J. Biol. Chem. 2004, 279,
16939-16946.
(
6) Kim-Shapiro, D. B.; Gladwin, M. T.; Patel, R. P.; Hogg, N. J. Inorg.
Biochem. 2005, 99, 237-246.
(16) Kozlov, A. V.; Staniek, K.; Nohl, H. FEBS Lett. 1999, 454, 127-130.
(17) Gangolli, S. D.; Van, Der, Brandt, P. A.; Feron, V. J.; Janowsky, C.;
Koeman, J. H.; Speijers, G. J.; Spiegelhalder, B.; Walker, R.; Wisnok, J.
S. Eur. J. Pharmacol. 1994, 292, 1-38.
(
(
(
7) Cosby, K. Nat. Med. 2003, 12, 1498-1505.
8) Brooks, J. Proc. Royal Soc. B 1937, 123, 368-382.
9) Doyle, M. P.; Pickering, R. A.; Deweert, T. M.; Hoekstra, J. W.; Pater, D.
J. Biol. Chem. 1981, 256, 12393-12398.
(18) Bartsch, H.; Ohshima, H.; Pignatelli, B. Mutat. Res. 1988, 202, 307-324.
(19) Sies, H.; Sharov, V. S.; Klotz, L. O.; Briviba, K. J. Biol. Chem. 1997, 272,
27812-27817.
(
10) Huang, Z.; Shiva, S.; Kim-Shapiro, D.; Patel, R.; Ringwood, L.; Irby, C.;
Huang, K.; Ho, C.; Hogg, N.; Schechter, A.; Gladwin, M. J. Clin. InVest.
2
005, 115, 2099-2107.
(20) Lundberg, J. O.; Weitzberg, E.; Cole, J. A.; Benjamin, N. Nat. ReV.
Microbiol. 2004, 2, 593-602.
(
(
(
11) Nagababu, E.; Ramasamy, S.; Rifkind, J. M. Nitric Oxide Biol. Chem. 2006,
5, 20-29.
12) Angelo, M.; Singel, D. J.; Stamler, J. S. Proc. Natl. Acad. Sci. U.S.A. 2006,
03, 8366-8371.
13) Lundberg, J. O.; Weitzberg, E.; Lundberg, J. M.; Alving, K. Gut 1994, 35,
543-1546.
1
(21) Pluta, R. M.; Dejam, A.; Grimes, G.; Gladwin, M. T.; Oldfield, E. H. J.
Am. Med. Assoc. 2005, 293, 1477-1484.
1
(22) Hunter, C. J.; Dejam, A.; Blood, A. B.; Shields, H.; Kim-Shapiro, D. B.;
Machado, R. F.; Tarekegn, S.; Mulla, N.; Hopper, A. O.; Schecheter, A.
N.; Power, G. G.; Gladwin, M. T. Nat. Med. 2004, 10, 1122-1127.
1
1
6332 J. AM. CHEM. SOC. 2006, 128, 16332-16337
9
10.1021/ja066011v CCC: $33.50 © 2006 American Chemical Society

Allylic Nitro Compounds as Nitrite Donors
A R T I C L E S
Scheme 1
for several days failed to result in nitric oxide or nitrite formation
as judged by chemiluminescence spectroscopy suggesting that
DMSO does not promote nitrite release. Addition of nucleo-
philes (1 equiv) to solutions of 1-6 in DMSO yielded nitrite.
Specifically, nucleophile-assisted nitrite release from 1 followed
the order thiolate > thioacetate [dmt] azide > iodide > no
nucleophile (Figure 1a). By use of phenyl thiolate as a model
nucleophile, nitrite release from various allylic nitro compounds
was examined (Figure 1b). Increasing steric bulk on the C-nitro
carbon retarded nitrite release as evidenced by the diminished
yields of nitrite from 4 and 5 compared to 1. Ring size did not
affect nitrite release as revealed by comparable nitrite yields
from 1, 2, and 3. A vicinal aromatic ring promoted nitrite release
as indicated by greater amounts of nitrite released by 6 when
treated with thiolate than the rest of the substrates tested. These
trends in reactivity remain consistent over several days sug-
gesting substitution of the nitro group as the major operative
pathway for nitrite release (Figure 1b). Preliminary kinetic
studies support a second-order nitrite release process (Supporting
Information).
Scheme 2. Nucleophilic Substitution of Allylic Nitro Compounds
Chart 1. Some Allylic Nitro Compounds as Candidate Nitrite
Donors
aforementioned problems. The development of organic donors
will support our understanding of the underlying mechanisms
of nitrite/nitric oxide biology. Furthermore, such compounds
may form the basis of novel therapeutic agents and could
provide access to versatile NO-based therapeutics including
direct reduction to NO (path a, Scheme 1), conversion to
S-nitrosothiols (path b, Scheme 1), and formation of iron
nitrosylated heme proteins (path c, Scheme 1).
Organic nitro compounds present an attractive approach to
access a range of nitrite release profiles through simple
nucleophilic displacement of the nitro group. In the presence
of soft nucleophiles, allylic nitro compounds undergo nucleo-
philic substitution of the nitro group, presumably accompanied
by nitrite release (Scheme 2).
Allylic nitro compounds undergo regioselective nucleophilic
substitution to directly form nitrite but not nitric oxide.
reactivity pattern of nitrite release may be modified by altering
the nature and amount of nucleophile or the substitution around
the carbon bearing the nitro group. We wish to describe results
at developing allylic nitro compounds as sources of nitrite and
nitric oxide through such substitution and reduction pathways,
preliminary mechanistic studies, and the examination of the
reactions of these compounds with hemoglobin.
The poor aqueous solubility and slow nitrite release profiles
of the allylic nitro compounds 1-6 limit their utility as nitrite
donors (the most efficient source of nitrite was 6, which in the
presence of phenyl thiolate yielded 40% of nitrite over 4 days).
However, these studies show that allylic nitro compounds can
release nitrite and provide insight on potential structural
modifications that could accelerate nitrite release. A suitably
placed electron-withdrawing group may enhance the ability to
release nitrite by making the carbon bearing the nitro group
more susceptible to nucleophilic attack. R-Nitroalkyl enones
undergo regioselective substitution by soft nucleophiles under
25
mild conditions. For example, 2-(nitromethyl)-cyclohex-1-ene-
-one (7) reacts with sodium phenyl thiolate in DMF to give
-(phenylthiomethyl)-cyclohex-1-ene-3-one in 70% yield (Scheme
23,24
3
2
3
23-25
The
2
5
). The allylic nitro compound 7 and 2-(nitromethyl)-cyclo-
hept-1-ene-3-one (8) were synthesized by literature methods
2
5
from 1 and 3, respectively (Scheme 3). The structural
resemblance of these nitrite donor candidates to the NO donor
FK 409, an allylic nitro compound that spontaneously dissociates
to form both nitric oxide and nitrite in buffer, is noteworthy
2
6-30
(Chart 2).
Allylic nitro compound 7 dissolves in nitrite-free milliQ water
Results and Discussion
and DMSO (e6%). Negligible amounts of nitrite were detected
from this solution after several hours at 25 °C. However,
spontaneous nitrite release from 7 occurred in a buffered
solution. Incubation of 7 in 0.1 M, pH 7.4 phosphate buffer
Allylic nitro compounds 1, 2, and 3 were prepared from the
corresponding ketones and nitromethane as previously de-
2
4
scribed. By consideration of direct nucleophilic substitution
SN2) as a potential operating mechanism of nitrite release, the
(PB) produced 16% of nitrite after 4 h and 51% after 30 h at
(
2
5 °C (Figure 2a). Under the same conditions, 8 gave lower
rate and amount of nitrite formed over time should decrease as
the size of the R-substituent increases. In order to test this idea,
compounds 4 and 5 with a methyl and ethyl group as geminal
substituents on the C-nitro group were synthesized.24 Compound
yields of nitrite, possibly due to the greater conformational
flexibility (and poor π-orbital overlap) of the seven-membered
ring leading to reduced activation of the carbon bearing the nitro
group toward nucleophilic substitution by the conjugated ketone.
Under pseudo-first-order conditions, the time course of nitrite
release from 7 in PB (0.1 M, pH 7.4) showed a predictable
6
was also prepared as a prototype to examine effects of
24
electronic perturbation on nitrite release (Chart 1).
Because of the poor aqueous solubility of compounds 1-6,
preliminary investigations were conducted in DMSO. Incubation
of allylic nitro compounds (1-6, 0.1 M in DMSO) at 37 °C
(26) Ohtsuka, M.; Hombo, T.; Esumi, K. CardioVasc. Drug ReV. 1994, 12, 2-15.
(
27) Yoshida, K.; Yasuhiro, K. Curr. Med. Chem. 2005, 5, 675-685.
28) Shinichi, F.; Yoshimi, H.; Yasuko, K.; Mie, N.; Mitsuko, O.; Shigetaka,
N.; Kazuhiro, M.; Masayuki, K.; Yasuhiro, K. J. Pharmacol. Exp. Ther.
1997, 282, 236-242.
(
(
23) Tamura, R.; Kamimura, A.; Ono, N. Synthesis 1991, 423-434.
(
24) Ono, N.; Hamamoto, I.; Yanai, T.; Kaji, A. J. Chem. Soc. Chem. Commun.
(29) Decout, J. L.; Roy, B.; Fontecave, M.; Muller, J. C.; Williams, P. H.;
Loyaux, D. Bioorg. Med. Chem. Lett. 1995, 5, 973-978.
(30) Fukuyama, S.; Kita, Y.; Hirasawa, Y.; Sato, A.; Morokoshi, N.; Koda, S.;
Yasuda, T.; Oka, S.; Sakurai, H. Free Rad. Res. 1995, 23, 443-452.
1
985, 523-524.
(25) Tamura, R.; Katayama, H.; Watabe, K.; Suzuki, H. Tetrahedron 1990, 46,
7
557-7568.
J. AM. CHEM. SOC.
9
VOL. 128, NO. 50, 2006 16333

A R T I C L E S
Chakrapani et al.
Figure 1. (a) Nitrite release from 1 (0.1 M) treated with various nucleophiles (1 equiv) at 37 °C in DMSO. (b) Time course of nitrite release from 0.1 M
solutions of 1-6 treated with 1 equiv PhSNa in DMSO at 37 °C.
Scheme 3. Nucleophilic Displacement of Nitrite from 7 by PhSNa and Synthesis of 7, 8, from 1, 3
Chart 2. Structure of FK 409
cysteine (1 equiv) to 7 at pH 7.0 and 8.7 resulted in quantitative
yields of nitrite in 5 min (Figure 3b). Similar experiments at
pH 4.6 gave only 1% nitrite after 5 min indicating a role for
the thiolate ion (Figure 3b). During the same time period,
treatment of 7 with other amino acids in pH 7.4 PB gave
considerably lower yields of nitrite (<10%) revealing the
importance of the thiol group as a nucleophile in nitrite
formation. However, over 1 h, incubation of 7 with these amino
acids produced significant amounts of nitrite compared to buffer
suggesting potential roles of the carboxylate or amino groups
in nitrite formation (Figure 3c).
pattern of nitrite release with an observed rate of nitrite release
-
5
-1
of 1.6 × 10
s
(Figure 2b). Nitrite yields from 7 in Tris
buffer were similar to those in PB of comparable molarity and
pH (data not shown). These results confirm the ability of allylic
nitro compounds to release nitrite in a buffered solution.
Nitrite release is sensitive to pH and hydroxide ion concentra-
tion. Incubation of 7 in PB (0.1 M) of varying pH showed
increased yields of nitrite as a function of pH as judged by
chemiluminescence detection. Increased basicity led to greater
yields of nitrite until neutral pH, after which point the yield of
nitrite remained nearly constant (Figure 2c). In milliQ water,
only 3% of nitrite was observed after 30 h showing that nitrite
release depends on both pH and ionic strength. The addition of
Independent treatment of 7 with 1 equiv of N-acetyl cysteine,
L-methionine, and N-acetyl-L-methionine further reveals the
requirement of a thiol/thiolate nucleophile for nitrite release.
Addition of N-acetyl cysteine and L-cysteine to 7 gives
comparable and nearly quantitative nitrite in 5 min and suggests
the dominance of the thiol group over the amino group (Figure
3d). Treatment of 7 with L-methionine gives diminished amounts
of nitrite indicating that the thiol group is essential for efficient
nitrite release (Figure 3d). Finally, the lack of participation of
the carboxylate group in nucleophilic displacement was
inferred by the observation of negligible amounts of nitrite
from the reaction of 7 with N-acetyl methionine (Figure 3d).
These results clearly indicate that nitrite release from these
compounds can be controlled by the relative amount and type
of nucleophile present and provide a useful tool for controlled
nitrite release.
7
to milliQ water did not alter the pH of the solution. Varying
the buffer molarity at constant pH provided further evidence
for the importance of ionic strength (and hydroxide ion in the
buffer-mediated decomposition) for nitrite release from 7 (Figure
2
d). Both chemiluminescence and electron paramagnetic reso-
nance analysis (data not shown) failed to provide evidence of
nitric oxide formation from 7 under these conditions demon-
strating that 7 is a selective source of nitrite.
Nitrite release from allylic nitro compounds also depended
on the presence and the amount of added nucleophile. Treatment
of 7 with one equivalent of phenyl thiolate or thioacetate in
milliQ water afforded nearly quantitative yields of nitrite in 30
min, but under the same conditions, the weaker nucleophiles
phenyl sulfinate and azide gave diminished nitrite yields (Figure
The release of nitrite from 7 occurs through substitution of
the nitro group by a suitable nucleophile (Scheme 4). The
products of regioselective nitrite displacement of 7 by various
nucleophiles have been isolated and characterized previously.25
In our hands, the exclusive formation of 2-(azidomethyl)-
cyclohex-1-ene-3-one, the product of nucleophilic displacement
of nitrite from 7 by sodium azide was observed (Supporting
Information). Similarly, NMR studies of the incubation of 7
3a). The biologically relevant nucleophile, L-cysteine, facilitated
nitrite release from 7 as a function of pH and generated nitrite
under neutral and basic buffered solutions. Addition of L-
16334 J. AM. CHEM. SOC.
9
VOL. 128, NO. 50, 2006

Allylic Nitro Compounds as Nitrite Donors
A R T I C L E S
Figure 2. (a) Comparison of nitrite release from 7 and 8 in 0.1 M pH 7.4 buffer. (b) Time course of nitrite release from 7 in 0.1 M pH 7.4 phosphate buffer.
c) Comparison of nitrite release from 7 in 0.1 M PB of varying pH. (d) Comparison of nitrite release from 7 in PB of varying ionic strength at neutral pH.
(
Figure 3. (a) Nitrite release from 7 in milliQ water upon treatment with various nucleophiles. (b) Effect of L-cysteine on nitrite release from 7 in milliQ
water and 0.1 M PB of varying pH (c) Effect of added amino acid on nitrite release from 7 in 0.1 M pH 7.4 PB. (d) Effect of added protected amino acid
on nitrite release from 7 in 0.1 M pH 7.4 PB. *Nitrite measurements done after 5 min.
Scheme 4. Proposed SN2-Type Mechanism of Nitrite Release
from 7
requirement of a suitable nucleophile for nitrite formation.
Previous work demonstrated that radical transfer processes
(
SRN1) in nucleophilic substitution of allylic nitro compounds
2
5
were not important. While the observation of the dependence
of nitrite release rate on the size of the geminal substituent of
the C-nitro group in allylic nitro compounds favors direct nitrite
displacement (SN2), pathways that consist of two sequential
allylic substitutions (SN2′) cannot be excluded.
with L-cysteine in pD 7.4 PB also support this claim (Supporting
Information).31 The thiol group of L-cysteine is the dominant
nucleophilic species but a role for the amino group cannot be
completely excluded. The absence of nitrite formation from 7
and L-cysteine in milliQ water or at pH 4.6 indicates the
Nitrite generated from the allylic nitro compound 7 reacted
(
31) (a) Hamilton, D. S.; Zhang, X.; Dang, Z.; Hubatsch, I.; Mannervik, B.;
Houk, K. N.; Ganem, B.; Creighton, D. J. J. Am. Chem. Soc. 2003, 125,
1
5049-15058. (b) Joseph, E.; Eiseman, J. L.; Hamilton, D. S.; Wang, H.;
Tak, H.; Ding, Z.; Ganem, B.; Creighton, D. J. J. Med Chem. 2003, 46,
194-196.
J. AM. CHEM. SOC.
9
VOL. 128, NO. 50, 2006 16335

A R T I C L E S
Chakrapani et al.
acidified-cysteine formed a red solution with a characteristic
spectral pattern for S-nitrosocysteine (λmax ) 542 nm).37
Chemiluminescence assay also independently confirmed the
formation of S-nitrosothiol. Again, these results support thiol-
mediated nitrite release from 7 and reveal the ability of allylic
nitro compounds to S-nitrosate a thiol in a manner similar to
inorganic nitrite.
Finally and perhaps most importantly in terms of biology,
these allylic nitro compounds act as nitric oxide prodrugs under
mild reducing conditions, as would be expected of a nitrite
donor. Ascorbic acid comprises an important component of
gastric juice and is a vital component in the physiological nitric
oxide generation from nitrite. Several reports employ nitrite and
ascorbic acid mixtures as masked NO sources for therapeutic
3
8-39
purposes.
For example, mixtures of sodium nitrite and
40
ascorbic acid blocked the growth of Streptococus mutans. The
reaction of ascorbic acid with 7 in pH 7.0 PB yielded nitric
oxide, as detected by chemiluminescence headspace analysis
(
≈ 0.1%). At pH 7.0, in the presence of L-cysteine, even higher
amounts of NO (≈ 3%) formed in the reaction headspace after
0 min corroborating nitrite formation and confirming that 7 is
1
a source of nitric oxide. Together, these results demonstrate that
allylic nitro compounds, such as 7, can specifically act as a
selective source of nitrite (in the presence of thiol) or nitric oxide
(in the presence of thiol and ascorbic acid). This compartmen-
talized reactivity makes these compounds mechanistically unique
nitrite donors.
Figure 4. Spectral features of (a) deoxyHb treated with 7 (0.1 M) in buffer
(b) oxyHb treated with 7 (0.1 M) in buffer.
Summary
with heme proteins, thiols, and reductants in a similar fashion
as sodium nitrite, giving further evidence of 7’s ability to act
as a nitrite donor. For example, under the careful exclusion of
oxygen, the reaction of nitrite with deoxyhemoglobin produces
HbFe(II)-NO and methemoglobin in an approximately 1:1
Allylic nitro compounds dissociate in buffer to release nitrite
with vastly different rate profiles that depend on the nature and
relative amount of nucleophile and by the steric and electronic
perturbations of the carbon framework. The addition of an
electron-withdrawing group enhances nitrite release, particularly
in the presence of sulfur nucleophiles. Allylic nitro compound
3
2
ratio. A similar spectral pattern was observed when 7 (100
mM) reacted with deoxyHb (∼3.3 mM) in buffer over 8 h
7
behaved as a stable precursor to both nitrite and nitric oxide.
(Figure 4a). The fact that only 16% of nitrite was formed from
This compound released nitrite in buffer and the addition of
L-cysteine increases the rate of nitrite release. Compound 7 also
reacted with hemoglobin in a similar fashion to sodium nitrite
and S-nitrosated thiols under acidic conditions. Reaction of 7
with ascorbic acid in the presence of L-cysteine generates nitric
oxide showing that this three-component mixture represents a
unique system for nitric oxide formation. These findings reveal
allylic nitro compounds as a new group of nitrite/nitric oxide
sources that have tunable release profiles making them poten-
tially useful in the development of therapeutic agents.
the spontaneous dissociation of 7 in buffer after 16 h reconciled
the observed incomplete reaction of deoxyhemoglobin under
these conditions. OxyHb also reacted with sodium nitrite to form
9
methemoglobin with characteristic spectral features. Reaction
of oxyHb (3.47 mM) with 7 (100 mM) in pH 7.4 PB (0.1 M)
produced these characteristic gradual decreases in absorbances
at 540 and 576 nm with concomitant increases in absorbances
at 630 and 496 nm over 4 h (Figure 4b). This spectral behavior
mimicked the sodium nitrite mediated transformation of Hb to
metHb as reported by Doyle and co-workers.9 Again, the
addition of L-cysteine to the reaction of 7 with deoxyHb and
oxyHb greatly enhanced the rate of reaction further illustrating
Experimental Section
NMR spectra were recorded on a 300-MHz Bruker NMR spectrom-
eter. Nitromethane, nitroethane, 1-nitropropane, cyclopentanone, cy-
clohexanone, cycloheptanone, 1-phenylpropanone, sodium nitrite,
sodium phenyl thiolate, L-cysteine‚HCl, L-methione, L-proline, l-alanine,
L-serine, N-acetyl-L-cysteine, N-acetyl-L-methionine, sodium phenyl
sulfinate, potassium thioacetate, sodium azide, sodium hydrogen
the ability of thiols to induce nitrite release from these allylic
nitro compounds. (Supporting Information).33-35
Under acidic conditions, nitrite reacts with thiols to form
S-nitrosothiols, which have been postulated as important physi-
ological storage forms of nitric oxide.36 Reaction of 7 with
L-cysteine (1 equiv) in pH 7.4 PB followed by treatment with
(
37) Wink, D. A.; Kim, S.; Coffin, D.; Cook, J. C.; Vodovotz, Y.; Christodoulou,
D.; Jourd’heuil, D.; Grisham, M. B. Methods Enzymol. 1999, 301, 201-
211.
(
32) Huang, K. T.; Keszler, A.; Patel, N.; Patel, R. P.; Gladwin, M. T.; Kim-
Shapiro, D. B.; Hogg, N. J. Biol. Chem. 2005, 280, 31126-31131.
(33) Suschek, C. V.; Schewe, T.; Sies, H.; Kroncke, K. D. Biol. Chem. 2006,
(38) Carlsson, S.; Weitzberg, E.; Wiklund, P.; Lundberg, J. O. Antimicrob. Agents
Chemother. 2005, 49, 2352-2355.
(39) Carlsson, S.; Govoni, M.; Wiklund, P.; Weitzberg, E.; Lundberg, J. O.
Antimicrob. Agents Chemother. 2003, 47, 3713-3718.
(40) Radcliffe, C. E.; Lamb, R.; Blinkhorn, A. S.; Drucker, D. B. J. Dent. 2003,
31, 367-370.
3
87, 499-506.
(
34) Weller, R. Exp. Derm. 2003, 28, 511-514.
(
35) Ormerod, A. D.; White, M. I.; Shah, S. A. A.; Benjamin, N. Brit. J.
Dermatol. 1999, 141, 1051-1053.
36) Williams, D. L. H. Acc. Chem. Res. 1999, 32, 869-876.
(
16336 J. AM. CHEM. SOC.
9
VOL. 128, NO. 50, 2006

Allylic Nitro Compounds as Nitrite Donors
A R T I C L E S
phosphate, sodium dihydrogen phosphate, 3-chloroperoxybenzoic acid,
cuvette with a rubber septum until the absorption spectrum showed
the complete removal of oxygen.
2
,2′-dimethyl ethylenediamine, triethylamine, pyridinium chlorochro-
mate, and ascorbic acid were purchased from Sigma-Aldrich and used
as received. Hexanes, ethyl acetate, methylene chloride, acetonitrile,
dimethyl sulfoxide, and ethanol were purchased from Sigma-Aldrich
and used as received. Compounds 1-8 were prepared by known
Absorption Spectroscopy. Hemoglobin solutions (3.47 mM in
heme) in 0.1 M PB (pH 7.4) were treated with 7 or 8 (100 mM) and
placed in a cuvette under anaerobic conditions. Readings were taken
every 30 min for 12 h on a Cary 100 UV-vis spectrometer at 25 °C.
Similar experiments were performed in the presence of L-cysteine (1
eq.) with spectra being recorded every 1 min for 90 min. Similar
experiments were performed with oxyhemoglobin. Mixtures of L-
cysteine with oxyHb and deoxyHb over the reported time periods
provided controls.
2
4-26
methods, and spectrally characterized before use.
Plots were
generated and analyzed using the curve-fitting program of Origin 5.0
2
and the R values of linear fits are reported.
Nitrite Analysis. Test compounds were weighed into vials (3 mL),
sealed with rubber septa, and deoxygenated with argon for 20 min.
Compounds 1-6 were dissolved in DMSO (100 mM), while com-
pounds 7 and 8 were dissolved in milliQ water or buffer (various pH
or molarity) containing ∼5% DMSO to give final concentrations of
S-Nitrosothiol Formation. A solution of L-cysteine (50 µL, 0.13
M in pH 7.0 PB) and 7 (50 µL, 0.12 M in DMSO) were diluted in a
spectrophotometric vial was treated with and 100 µL aqueous solution
of L-cysteine (1 M in milliQ water) acidified with 2-3 drops of dilute
HCl. The resulting solution rapidly turned red, and the UV-visible
spectrum was recorded. Aliquots (50 µL) of this solution were injected
into the NOA reaction chamber containing L-cysteine (3 M) and CuCl
5
-10 mM. The samples were incubated at 37 (1-6) or 25 °C (7 and
8
) and aliquots (50 µL) of the reaction solutions were periodically
injected into the reaction chamber of a Sievers 280 Nitric Oxide
Analyzer (NOA). For nitrite analysis, the reaction chamber contained
1
% w/v potassium iodide in glacial acetic acid, a solution that reduces
(100 µM) in pH 7.0 PB. This system converts S-nitrosothiols to NO.
nitrite to NO. For experiments involving nucleophile additives, equimo-
lar solutions of the nucleophile (in milliQ water or buffer) and the allylic
nitro compound (in DMSO) were mixed and then diluted with the
milliQ water or buffer of suitable molarity and pH to give a final
concentration of 5-10 mM. For example, independent solutions of
L-cysteine (23 mg, 0.13 mmol) in pH 7.4 phosphate buffer (1 mL) and
Ascorbic Acid Experiments. A solution of ascorbic acid (100 µL,
2
M in pH 7.4 PB) was added to a solution of 7 (50 µL, 2 M in DMSO)
at 25 °C and diluted with 400 µL pH 7.4 PB. After 1 h, an aliquot
250 µL) of the headspace was injected into the reaction chamber
containing only milliQ water to determine NO release. The experiment
with L-cysteine was conducted in a similar fashion, except that a solution
of 50 µL of L-cysteine (2M in pH 7.4 PB) was added before the ascorbic
acid and headspace NO was recorded after 10 min. Controls using
sodium nitrite and ascorbic acid give similar results of nitric oxide
release by chemiluminesence.
(
7
(21 mg, 0.13 mmol) in DMSO (1 mL) were prepared. Aliquots (50
µL) of both these solutions were placed in a vial and diluted with pH
7
.4 phosphate buffer (900 µL) to give a final concentration of 7 mM.
Nitric Oxide Analysis. Nitric oxide analyses were carried out using
the same experimental procedure as nitrite analysis except that aliquots
250 µL) of the reaction headspace and solution were injected into the
(
Acknowledgment. This work was supported by the National
Institutes of Health (HL 62198, SBK). 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).
reaction chamber that only contained milliQ water (2 mL). For these
experiments, solutions of the allylic nitro compounds and nucleophile
were generally 20 mM.
(
(
Hemoglobin Preparation. Normal adult hemoglobin was prepared
4
1
as described earlier. The hemoglobin sample was pelleted in liquid
nitrogen and stored at -80 °C. Hemoglobin samples used for
experiments were thawed and used within 24 h. Thawed hemoglobin
samples were further centrifuged to remove any precipitated or
denatured protein. The absence of denatured protein was confirmed
by absorption spectroscopy. Deoxyhemoglobin was formed by gently
flowing argon over a solution of oxyHb in argon-saturated buffer in a
Supporting Information Available: Kinetic analysis of nitrite
release from 1-6, UV-vis spectrum of the reaction of 7 with
deoxyHb and Hb in the presence of L-cysteine, NMR spectra
of the reaction of 7 with sodium azide and L-cysteine, and the
complete list of authors for refs 1 and 7. This material is
available free of charge via the Internet at http://pubs.acs.org.
(
41) Huang, J.; Kim-Shapiro, D. B.; King, S. B. J. Med. Chem. 2004, 47, 3495-
3
501.
JA066011V
J. AM. CHEM. SOC.
9
VOL. 128, NO. 50, 2006 16337