Reductive phosphine-mediated ligation of nitroxyl (HNO)

Article abstract of DOI:10.1021/ol900914s

Nltroxyl (HNO) demonstrates a unique chemical and biological profile compared to nitric oxide (NO). Phosphorus NMR studies reveal that HNO reacts with triarylphosphines to give the corresponding phosphine oxide and aza-ylide. In the presence of a properly situated electrophilic ester, the aza-ylide undergoes a Staudinger ligation to yield an amide with the nitrogen atom being derived from HNO. These results define new HNO reactivity and provide the basis of new HNO detection methods.

Full text of DOI:10.1021/ol900914s

ORGANIC
LETTERS
2009
Vol. 11, No. 13
2719-2721
Reductive Phosphine-Mediated Ligation
of Nitroxyl (HNO)
Julie A. Reisz, Erika B. Klorig, Marcus W. Wright, and S. Bruce King*
Department of Chemistry, Salem Hall, Box 7486, Wake Forest UniVersity,
Winston-Salem, North Carolina 27109
kingsb@wfu.edu
Received April 27, 2009
ABSTRACT
Nitroxyl (HNO) demonstrates a unique chemical and biological profile compared to nitric oxide (NO). Phosphorus NMR studies reveal that
HNO reacts with triarylphosphines to give the corresponding phosphine oxide and aza-ylide. In the presence of a properly situated electrophilic
ester, the aza-ylide undergoes a Staudinger ligation to yield an amide with the nitrogen atom being derived from HNO. These results define
new HNO reactivity and provide the basis of new HNO detection methods.
Recent studies demonstrate the distinct biological and
chemical properties of nitroxyl (HNO), the reduced/
protonated form of the well-known signaling agent nitric
oxide (NO).1-3 Biologically, HNO increases cardiac con-
tractility in both normal and failing canine hearts through
mechanisms that include increased sarcoplasmic reticulum
calcium release and uptake and increased calcium dependent
force development in cardiac tissue.4-7 These properties
indicate the potential of HNO as a new therapy for congestive
heart failure, a condition that causes more than 50 000 deaths
each year in the United States.8 Chemically, HNO represents
the simplest nitroso compound (X-NdO, where X ) H)
and reacts as an electrophile with nucleophiles to yield
addition products (Scheme 1).2,3 Similar to C-nitroso com-
Scheme 1. HNO Reactions
(1) Irvine, J. C.; Ritchie, R. H.; Favaloro, J. L.; Andrews, K. L.; Widdop,
R. E.; Kemp-Harper, B. K. Trends Pharmacol. Sci. 2008, 29, 601–608
(2) Fukuto, J. M.; Bartberger, M. D.; Dutton, A. S.; Paolocci, N.; Wink,
D. A.; Houk, K. N. Chem. Res. Toxicol. 2005, 18, 790–801
.
.
(3) Miranda, K. M. Coord. Chem. ReV. 2005, 249, 433–455
.
pounds, nitroxyl dimerizes to form unstable hyponitrous acid,
which dehydrates to nitrous oxide (N2O, Scheme 1).2,3
Nitroxyl dimerization complicates the study of its biology
and chemistry as it requires: 1) HNO generation from donor
molecules and 2) HNO detection through trapping with metal
complexes, thiols or itself to form N2O.2,3 The previously
reported reactions of organic phosphines with C and S-nitroso
compounds to form phosphine oxides and aza-ylides suggest
a similar reactivity with HNO (if viewed as a simplified
nitroso compound).9-12 Here we report the first reaction of
(4) 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
(5) Paolocci, N.; Katori, T.; Champion, H. C.; St John, M. E.; Miranda,
.
K. M.; Fukuto, J. M.; Wink, D. A.; Kass, D. A. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 5537–5542
.
(6) 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. Circ. Res. 2007, 100, 96–104
(7) Dai, T. Y.; Tian, Y.; Tocchetti, C. G.; Katori, T.; Murphy, A. M.;
Kass, D. A.; Paolocci, N.; Gao, W. D. J. Physiol. 2007, 580, 951–960
(8) Feelisch, M Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4978–4980.
.
.
10.1021/ol900914s CCC: $40.75
Published on Web 06/03/2009
 2009 American Chemical Society

HNO with organic phosphines to produce the corresponding
phosphine oxide and aza-ylide (Scheme 1). We show that
in the presence of an electrophilic trap this aza-ylide
intermediate reacts further to produce a stable and unique
HNO-derived amide product (Scheme 4).
31Phosphorus NMR experiments using authentic syn-
thetic standards provide evidence for aza-ylide formation
from the reaction of HNO with an organic phosphine.
Mixture of Angeli’s salt (Na2N2O3), the most common
HNO donor (t1/2 ) 2.8 min, 37 °C, pH 8.13-4.40),13 with
triphenylphosphine in dioxane/water (2:1) produces a
mixture of triphenylphosphine oxide (δ ) 31 ppm) and
the triphenylphosphine-derived aza-ylide (1, δ ) 26 ppm)
as judged by 31P NMR spectroscopy (Scheme 2 and
Figure 1.
31P NMR spectrum of the reaction of Angeli’s salt (0.04
mmol) with TXPTS (0.04 mmol) in 1:5 D2O/Tris buffer over time.
Resonances correspond to phosphine (-29.7 ppm), aza-ylide 2 (34.5
ppm), and phosphine oxide (39.8 ppm) and are referenced to 85%
H3PO4 (0.0 ppm).
Scheme 2. Reaction of HNO and Triarylphosphines
nitrous oxide formation (>90%) during the aqueous decom-
position of Angeli’s salt indicating reactivity with HNO.
Mixture of TXPTS and Angeli’s salt in buffer in the presence
of glutathione (1 equiv) also forms aza-ylide (1) and the
phosphine oxide of TXPTS suggesting that TXPTS reacts
with HNO faster than glutathione.
Furthermore, the generation of HNO from 4-bromo-N-
hydroxybenzenesulfonamide (3) under basic conditions in
the presence of triphenylphosphine yields 1 and triph-
enylphosphine oxide (Scheme 2).15 Using 15N-labeled 4-bro-
mo-N-hydroxybenzenesulfonamide results in 15N labeled-1
confirming the source of the nitrogen atom (1JN-P ) 31 Hz).16
Under these conditions, 1 also hydrolyzes to triphenylphos-
phine oxide and control experiments indicate that triph-
enylphosphine does not directly react with 3. In general, these
experiments show that HNO, regardless of the source, reacts
with organic phosphines to yield an aza-ylide.
The previously described reaction of S-nitrosothiols (an-
other group of nitroso compounds, X-NdO, where X )
-SR) with triarylphosphines to give the phosphine oxide
and an aza-ylide provides insight to a possible mechanism
for aza-ylide formation in this process.10,11 Reaction of the
phosphine with HNO could yield a product either through
P-addition at N (4) or oxygen (5, Scheme 3). Each of these
Supporting Information). Liquid chromatography/mass
spectrometry further confirms the identity of these prod-
ucts. Under these conditions aza-ylide (1), an intermediate
in the Staudinger reduction of azides to amines14 hydro-
lyzes to triphenylphosphine oxide as expected. 31P NMR
experiments show the formation of 1 and triphenylphos-
phine oxide, the expected byproduct of the reaction, within
5 min and the formation of no other phosphorus containing
products. Control experiments show that triphenylphos-
phine does not directly react with Angeli’s salt or nitrite,
a byproduct of Angeli’s salt decomposition to HNO.
31P NMR experiments also reveal the reaction of Angeli’s
salt with the water-soluble phosphine tris(4,6-dimethyl-3-
sulfonatophenyl)phosphine trisodium salt hydrate (TXPTS)
in Tris buffer (100 mM, pH 7.6) produces two new
phosphorus containing products in a 1:1 ratio (Figure 1).
Comparison to authentic standards confirms the formation
of the same aza-ylide derived from TXPTS (2, Scheme 2)
and the corresponding phosphine oxide. Under these condi-
tions, aza-ylide (2) demonstrates relative stability compared
to 1. 31P NMR experiments indicate the presence of 2 along
with increasing amounts of TXPTS phosphine oxide in this
reaction mixture even after 6 days. Structural differences in
the aromatic portion of the phosphines, especially the electron
donating o and p methyl groups, likely influences the
hydrolytic stability of aza-ylides 1 and 2. TXPTS quenches
Scheme 3. Proposed Mechanism of Aza-Ylide Formation
(9) Sidky, M. M.; Soliman, F. M.; Shabana, R. Egyptian J. Chem. 1980,
21, 29–35.
initial addition products could exist as a three-membered ring
species (6, Scheme 3). Addition of a second phosphine to 6
(or to 4 or 5) would give the corresponding aza-ylide and
phosphine oxide in equal proportions.
(10) Haake, M. Tetrahedron Lett. 1972, 13, 3405–3408.
(11) Wang, H.; Xian, M. Angew. Chem., Int. Ed. 2008, 47, 6598–6601.
(12) Zhang, J; Wang, H.; Xian, M. J. Am. Chem. Soc. 2009, 131, 3854–
3855.
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Org. Lett., Vol. 11, No. 13, 2009

The strong evidence of aza-ylide formation provided by
the preceding NMR studies suggests that a phosphine capable
of undergoing the widely used Staudinger ligation should
effectively provide a means for trapping HNO as the
corresponding amide.17 Treatment of the methyl ester (7)
with Angeli’s salt in a mixture of acetonitrile/Tris buffer (100
mM, pH ) 7.0) at room temperature gives a 1:1 mixture of
the benzamide phosphine oxide (8, δ ) 34.4 ppm) and
methyl ester phosphine oxide (9, δ ) 33.3 ppm, Figure 2,
Scheme 4. Phosphorus Mediated Nitroxyl Ligation
phosphine oxide. These products differ from the reaction of
triphenylphosphine with nitric oxide, which yields triph-
enylphosphine oxide and nitrous oxide.19,18 The inherent
reactivity of the aza-ylide allows ligation to a modified
phosphine reagent producing a stable HNO-unique amide
product. Develoment of these compounds as new HNO
detection methods will require further investigation of their
differential reactivity with NO and selective reaction com-
pared to other biomolecules. The lack of specific HNO
detection methods has impeded the understanding of HNO
in biological systems. In summary, these experiments docu-
ment the reactivity of HNO with triarylphosphines and
provide insight into a new method for distinguishing this
important biologically active nitrogen oxide.
Figure 2.
31P NMR spectrum of the reaction of Angeli’s salt (0.03
mmol) with 7 (0.02 mmol) in 3:1 CD3CN/Tris buffer at 3 h.
Resonances correspond to 8 (34.4 ppm) and 9 (33.3 ppm) and are
referenced to 85% H3PO4 (0.0 ppm).
Acknowledgment. This work was supported by the
National Institutes of Health (HL 62198 and R21 087891).
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).
Scheme 4) as judged by 31P NMR spectroscopy. Liquid
chromatography/mass spectrometry further confirms the
identity of these products. Incubation of 7 with Angeli’s salt
on a preparative scale results in recovery of 8 in 60% yield
(based on the theoretical amount of 8 formed in a 1:1 ratio
from starting material) following chromatography. 31P NMR
experiments show the reaction to be complete within 20 min
with the formation of no other phosphorus containing
products (including the aza-ylide, Figure 2). Control experi-
ments also indicate that 7 does not directly react with
Angeli’s salt or nitrite. Reaction of 7 with 15N-3 produces
15N-7 clearly demonstrating the amide nitrogen derives from
3 and presumably HNO. These results suggest the initial
formation of aza-ylide (10, Scheme 4) by the reaction of
HNO and 7 through a similar mechanism as Scheme 3.
Intramolecular reaction of the aza-ylide group of 10 with
the adjacent ester group generates the amide (8, Scheme 4).
These studies demonstrate that HNO reacts rapidly with
organic phosphines to give the corresponding aza-ylide and
Supporting Information Available: Experimental details
for the synthesis and characterization, including 1H, 13C and
31P NMR spectra, of compounds 1-3 and 7-8. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL900914S
(13) Hughes, M. N.; Cammack, R. Methods Enzymol. 1999, 301, 279–
287.
(14) Ko¨hn, M.; Breinbauer, R. Angew. Chem., Int. Ed. 2004, 43, 3106–
3116.
(15) Toscano, J. P.; Brookfield, F. A.; Cohen, A. D.; Courtney, S. M.;
Frost, L. M.; Kalish, V. J. PCT/US2007/006710, 2007.
(16) Carbajo, R. J.; Lopez-Ortiz, F. J. Magn. Reson. 2001, 148, 165–
168.
(17) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007–2010.
(18) Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc.
2006, 128, 8820–8828.
(19) Lim, M. D.; Lorkovic, I. M.; Ford, P. C. Inorg. Chem. 2002, 41,
1026–1028.
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