The pH of HNO donation is modulated by ring substituents in Piloty's acid derivatives: Azanone donors at biological pH

Article abstract of DOI:10.1016/j.jinorgbio.2012.10.008

A group of Piloty's acid (N-hydroxybenzenesulfonamide) derivatives were synthesized and fully characterized in order to assess the rates and pH of HNO (azanone, nitroxyl) donation in aqueous media. The derivatives, with electron-withdrawing and -donating substituents include methyl, nitro, fluoro, tri-isopropyl, trifluoromethyl and methoxy groups. The most interesting modulation observed is the change in pH range in which the compounds are able to donate HNO. UV-visible kinetic measurements at different pH values were used to evaluate the decomposition rate of the donors. A novel technique based on electrochemical measurements using a Co-porphyrin sensor was used to assess the release of HNO as a function of pH, by direct measurement of [HNO]. The results were contrasted with DFT calculations in order to understand the electronic effects exerted by the ring substituents, which drastically modify the pH range of donation. For example, while Piloty's acid donates HNO from pH 9.3, the corresponding fluoro derivative starts donating at pH 4.0.

Full text of DOI:10.1016/j.jinorgbio.2012.10.008

Journal of Inorganic Biochemistry 118 (2013) 134–139
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journal homepage: www.elsevier.com/locate/jinorgbio
The pH of HNO donation is modulated by ring substituents in Piloty's acid derivatives:
☆
azanone donors at biological pH
⁎
Kiran Sirsalmath, Sebastián A. Suárez, Damián E. Bikiel, Fabio Doctorovich
Departamento de Química Inorgánica, Analítica y Química Física/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón II,
Buenos Aires (C1428EHA), Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 June 2012
Received in revised form 10 October 2012
Accepted 10 October 2012
Available online 17 October 2012
A group of Piloty's acid (N-hydroxybenzenesulfonamide) derivatives were synthesized and fully characterized in
order to assess the rates and pH of HNO (azanone, nitroxyl) donation in aqueous media. The derivatives, with
electron-withdrawing and -donating substituents include methyl, nitro, fluoro, tri-isopropyl, trifluoromethyl
and methoxy groups. The most interesting modulation observed is the change in pH range in which the compounds
are able to donate HNO. UV–visible kinetic measurements at different pH values were used to evaluate the decom-
position rate of the donors. A novel technique based on electrochemical measurements using a Co-porphyrin sensor
was used to assess the release of HNO as a function of pH, by direct measurement of [HNO]. The results were
contrasted with DFT calculations in order to understand the electronic effects exerted by the ring substituents,
which drastically modify the pH range of donation. For example, while Piloty's acid donates HNO from pH 9.3, the
corresponding fluoro derivative starts donating at pH 4.0.
Keywords:
Ring substituents
Piloty's acid
Azanone donors
Nitroxyl donors
© 2012 Elsevier Inc. All rights reserved.
1. Introduction
to HNO at a rate comparable to Angeli's salt and PA [22–24]. A review
addressing all the previous issues has been recently published [25].
Azanone (nitroxyl, HNO), the one-electron reduction product of
nitric oxide, is a short-lived molecule that has been intensively investi-
gated due to its singular chemical and biological properties [1–3]. From
a physiological perspective, encouraging studies suggest that azanone
can be produced under particular cofactors conditions by NO-producing
proteins, such as the nitric oxide synthases (NOS) [4–6]. Furthermore,
several studies have shown distinct pharmacological effects for nitric
oxide and nitroxyl donors, indicating that the lifetime of HNO in living
tissues is probably much longer than previously assumed. This fact
suggests the coexistence of HNO and NO in certain tissues [7–9]. The
reactivity of nitroxyl points to hemeproteins and thiols as the biologi-
cally relevant targets [10,11].
Preparation of PA was first reported in 1896 [26]. It was firstly
postulated by Angeli [27] that its decomposition occurs via elimina-
tion of azanone from the conjugate anion (Eq. (1)), analogous to the
self-decomposition of AS. This is one of several examples in the
literature of organic chemistry in which HNO elimination is strongly
indicated.
ð1Þ
Azanone can be generated in solution by trioxodinitrate [12]
(Na2N2O3, Angeli's salt, AS), which readily decomposes to form HNO
via oxyhyponitrite anion HN2O3−. [8] In recent years, donors that release
HNO photochemically [13], thermally [14], enzymatically [15], hydro-
lytically [16], or by oxidation [17] have been developed. Another HNO
donor is N-hydroxybenzenesulfonamide (C6H5SO2NHOH, Piloty's acid,
PA), which decomposes above pH 9 producing HNO through a
base-catalyzed deprotonation mechanism, or releases NO upon
oxidation [2,18–21].
Our attention has been drawn to N-hydroxybenzenesulfonamide
decomposition as a source of the intermediate molecule HNO, in con-
tinuation of earlier studies of this reactive species [28]. It was demon-
strated by mass spectrometry that HNO is evolved during pyrolysis of
the sodium salt C6H5SO2NHONa [29]. Exner and Juska [30] have
interpreted the infrared and ultraviolet spectra of benzohydroxamic
acid (C6H5CONHOH) and its alkyl derivatives under acidic, neutral,
and alkaline conditions, showing their dissociable hydrogen atoms
to be nitrogen bound. Infrared spectra of these and other compounds
and their lithium salts in the crystalline state and in dioxane and
chloroform solutions have apparently affirmed this conclusion and
extended it to include PA [31]. Further evidence for N-deprotonation
has been found by solid-state X-ray photoelectron spectroscopy
[32]. The dissociation equilibrium thus inferred (Eq. (2)) the possi-
bility that the intermediate species released in the decomposition
Methanesulfohydroxamic acid (MHSA), the simplest member of
the aliphatic sulfohydroxamic acid series, is also able to decompose
☆
Dedicated to Professor Francis T. Bonner.
Corresponding author.
⁎
E-mail address: doctorovich@qi.fcen.uba.ar (F. Doctorovich).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.10.008

K. Sirsalmath et al. / Journal of Inorganic Biochemistry 118 (2013) 134–139
135
Table 1
reaction (Eq. (1)) could be the triplet molecule NOH rather than the
singlet HNO.
Names, abbreviations and substituents for the PA derivatives used in this work.
Benzenesulfonamide
Abbreviation
R1
H
R2
H
R3
1
N-hydroxy-4-methyl
Me-PA
Me
(or TSHA)
NO2-PA
F-PA
TriIso-PA
OMe-PA
(NO2–CF3)-PA
2
3
4
5
6
N-hydroxy-4-nitro
N-hydroxy-4-Fluoro
N-hydroxy-2,4,6-triisopropyl
N-hydroxy-4-methoxy
N-hydroxy-2-nitro-4-trifluoromethyl
H
H
IsoP
H
H
H
H
IsoP
H
NO2
NO2
F
IsoP
OMe
CF3
ð2Þ
This molecule would yield triplet NO− upon deprotonation,
constituting a second example of the triplet intermediate observed in
the NO–NH2OH reaction [33,34]. Bonner and Ko [21] reported kinetic,
isotopic, and NMR studies on these questions.
2.1. Synthesis
In this work we have studied six PA derivatives with electron-
donating and -withdrawing substituents on the aromatic ring (Fig. 1
and Table 1). They have been characterized structurally and by density
functional theory (DFT) calculations. Their decomposition and HNO
donation capabilities have been studied as a function of pH. The substit-
uents on the aromatic ring change the electronic properties and acidity
of the –SO2NHOH group, and the N–O bond strength, allowing their
decomposition (and HNO donation) to start at a large range of pH values
(−1 to 10), depending on the substituents. Surprisingly enough, five of
six of these donors operate at physiological pH, as it will be shown below.
N-hydroxy-4-methyl benzenesulfonamide (1) [35], N-hydroxy-
4-nitro benzene sulfonamide (2) [35], N-hydroxy-4-fluoro benzene-
sulfonamide (3) [36], N-hydroxy-2,4,6-triisopropyl benzene-
sulfonamide (4) [35], N-hydroxy-4-methoxy benzenesulfonamide (5)
[36], and N-hydroxy-2-nitro-4-trifluoro methyl-benzenesulfonamide (6)
[24], were synthesized according to the procedure published by
Porcheddu et al. [35]. NMR and other relevant analysis are reported
only for the compounds which were poorly described (5) or not
described before (6).
N-hydroxy-4-methoxy benzenesulfonamide (5). 69% yield–98% purity,
mp 160 °C (dec). 1H NMR (MeOD 500 MHz) 7.7 (m, 2 H), 6.9 (m, 2 H),
3.85 (s, 3 H). 13C NMR (MeOD 500 MHz) 129.4 (1C), 126.7 (2 C), 112.9
(2 C), 54 (1 C).
2. Materials and methods
N-hydroxy-2-nitro-4-trifluoromethyl-benzenesulfonamide (6). This
product was prepared according to the literature procedure [35], but
the reaction time was shortened to 1–2 h in order to minimize the
product decomposition. It was isolated as a white solid (yield ca.
70%); mp 212–213 °C (dec.). 1H NMR (DMSO, ppm): 7.51 (br s, 2H),
6.92 (s, 2 H), 4.53 (m, 2H), 2.75 (m, 1H), 1.13 (d, J=6.9 Hz, 6H), 1.07
(d, J=6.8 Hz, 12H). 13C NMR (DMSO, ppm): 147.4, 146.9, 141.8, 121.5,
33.4, 28.2, 24.9, 23.9. Microanalysis did not fit the calculated values due
to the fast spontaneous decomposition of the product.
All reactions were carried out in anaerobic conditions using standard
Schlenk techniques with nitrogen or argon of high purity. Manganese(III)
meso-(tetrakis(4-sulfonato-phenyl)) porphyrinate (MnIIITPPS) was
purchased from Frontier Scientific. Hydroxylamine hydrochloride and
sulfonyl chloride derivatives were purchased from Sigma-Aldrich
Argentina. Magnesium oxide was purchased from Biopack. THF and
methanol were purchased from Aldrich. All reagents were used
without further purification. MilliQ water was employed in the kinetic
experiments. Although solutions of Piloty's acid display substantial
stability at low pH, they are subject to air oxidation under neutral and
alkaline conditions. Hence, the solutions for the kinetic experiments
were prepared fresh under anaerobic conditions and kept refrigerated
until usage.
2.2. Instrumental procedures
All known compounds were characterized by melting point, 1H,
and 13C NMR. The analysis agreed with the previously reported ones
[35–37]. The amount of solvent present in the molecular formula
was determined by 1H NMR spectra and microanalysis. Melting
points were taken on a capillary melting point apparatus. 1H NMR
and 13C NMR spectra were measured in CDCl3 on a Bruker AM 500
spectrometer using tetramethylsilane as internal standard.
2.3. Kinetic measurements
Spontaneous decomposition of the donors was followed at various
pH values. Kinetic data were obtained measuring the UV absorbance
spectra change in a HP 8453 spectrometer using a 1-cm path quartz
cell sealed with a septum ensuring an inert atmosphere. The temper-
ature was controlled using a Lauda RE 207 thermostat at 25 °C. All the
solutions were degassed for 20 min before the experiment using N2
or Ar. The pH was controlled by proper buffer solutions (ca. 0.1 M).
Reactions were unaffected by the irradiation of the sample with the
light source of the spectrometer. Donor solutions were freshly prepared
for each set of measurements and kept on ice and the concentration was
checked before each measurement by UV–vis following its absorbance.
Donors were dissolved in ethanol 96%, and added to the porphyrin solu-
tions to a final ethanol/water ratio smaller than 1%. The kinetic data were
analyzed by a program written in MATLAB2008a, which allows fitting the
experimental data to a particular proposed mechanism. It is composed by
a singular value decomposition (SVD) subroutine which extracts the
Fig. 1. Piloty's acid derivatives studied in this work. See Table 1 for R1, R2 and R3.

136
K. Sirsalmath et al. / Journal of Inorganic Biochemistry 118 (2013) 134–139
Table 2
relevant data, diminishing the noise and a minimization subroutine
which uses the simplex algorithm of Nelder–Mean to adjust the pro-
posed kinetic constants. In addition to the decomposition UV–visible
experiments, kinetic experiments were conducted by 1H NMR in an
AM500 Bruker spectrometer. In all cases only one organic product was
observed by NMR, identified as the corresponding sulfinate by compar-
ison with the reported infrared spectra (see Supplementary Informa-
tion (SI)) [38].
Isolated yields of PA derivatives used in this work.
Product
R
Yield (%)
1
2
3
4
5
6
4-MeC6H4
4-O2NC6H4
4-FC6H4
2,4,6-(i-Pr)3C6H2
4-OMeC6H4
2-NO2-4-CF3C6H4
85
97
96
90
72
ca 70
2.4. Measurements of HNO production as a function of pH
In all cases the current intensity, which corresponds to the electrical
response to HNO generated by decomposition of the donors, was mea-
sured with the electrode described in previous literature [39]. Under
inert atmosphere, 0.015 mmol of donor were added to 15 mL of an
0.1 M KNO3 aqueous solution at the initial pH, which was previously ad-
justed by addition of HCl (aq.) (initial pH=2 for all donors). The pH was
raised slowly by addition of 0.01 M aqueous NaOH up to pH=11.
Current intensity, time and pH were recorded simultaneously with
the setup shown in Figure SI1.
In order to obtain the effect of the pH of the medium over the de-
composition of the compounds, we ran UV–vis kinetics at different pH
values for each compound, at 25 ° C (see Fig. S1). The decomposition
rates were tested at various pH values in independent experiments,
and the approximate pH at which the decomposition started to be ob-
served was determined (as asserted by following the UV–vis and/or
the 1H NMR spectra for 5 h). Table 3 shows the rate constants for the
decomposition of the PA derivatives, which was modeled as a first
order reaction. As can be observed in Table 3, the decomposition rate
is in all cases in the range 10−3 to 10−4 s−1. However, the pH at
which the donors start to decompose shows a broad range (ca. 10 pH
units). This cannot be due to a change in acidity of the –NHOH substit-
uent only, since for example in the case of arenesulfonic acids similar
substituents on the aromatic ring produce a change on the acidity of
the –SO3H group of less than 1 pH unit (−7.18 for p-NO2 versus
−6.57 for p-CH3) [40]. On the other hand, OMe-PA, containing en
electron-donor substituent, decomposes at the same pH than NO2-PA,
which contains an electron-attracting substituent. Therefore, other
factors such as the N–O bond strength of the –SO2–NHOH moiety
and solvation effects should be considered, as it will be discussed
below.
The pH at which the donation of HNO starts was determined in
each case by using an HNO-selective electrode [39], which signal
comprises a current proportional to [HNO] and is completely insensitive
to NO. Starting with a solution of the donor at a pH in which the com-
pound is stable, the pH was raised slowly by addition of diluted
NaOH(aq.), while the pH and current were measured simultaneously.
A large increase in the current indicates that [HNO] is raising, reaching
a plateau when the maximum rate of HNO formation is obtained.
Table 3 shows that the inflexion point of the current vs. pH curve is in
most cases around one pH unit after the pH at which the decomposition
starts to be observed, except for TriIso-PA (Fig. 2, the rest of the curves
are shown in Fig. SI2).
2.5. Single-crystal diffraction studies
All crystal donors were obtained by slow diffusion from acetonitrile
at room temperature and were mounted on a nylon loop. Single-crystal
data were collected on an Oxford Diffraction Gemini E CCD Diffractom-
eter equipped with a sealed tube with Mo KR radiation (λ=0.71073 Å)
at room temperature. For data collection, cell refinement and data
reduction CrysAlis PRO software was used. Absorption correction
was made by multi-scan. The structures were solved using direct
methods with SHELXS-97 and refined by the full-matrix least-
squares technique with SHELXS-97 based on F2. Software used to
prepare material for publication: WinGX publication routines and
ORTEP-3 for Windows. See Table SI1 for experimental data.
2.6. DFT calculations
All gas-phase DFT calculations performed in this work were carried
out using the Gaussian 98 software package. Calculations were carried
out at two different levels of theory. Geometries were fully optimized
at the B3P86 level using 6–311+G(2df,p) for all atoms in order to
obtain comparable geometrical parameters against DRX bonds. Opti-
mizations at the B3LYP level using 6–31 G(d,p) for all atoms using
water (polarizable continuum model—PCM) were used in order to
take into account solvation effects of the energetics of the reactions.
Deprotonation reactions were modeled using an explicit water molecule
as the proton acceptor. The NOH molecule was calculated in the triplet
state, while all the remaining molecules were calculated as singlets.
In some cases suitable crystals for X-ray diffraction experiments
could be obtained. The structures are shown in Fig. 3 and relevant
parameters are listed in Table 4. The S–N bond distances are quite
3. Results and discussion
The selected PA derivatives contain electron attracting (2, 3, 6) and
donating groups (1, 4,5). In the case of 4 there is also an important steric
hindrance around the –SO2–NHOH moiety. All donors were prepared by
following a recently described method (Eq. (3)) [35]. Since some of
these derivatives were not reported with this synthetic procedure,
Table 2 shows the yields obtained for all derivatives, which are very
good. Unfortunately, the derivative 6 decomposes spontaneously in
the course of a few hours even in the solid form.
Table 3
Rate constants for the decomposition of Piloty's acid derivatives.
a
Product
Rate constant (k, s−1
)
Decomposition HNO donation pHc
pHb
PA
(2.44 0.23)×10−4 [21] 9.3 [21]
–
1
2
3
4
5
6
Me-PA
NO2-PA
F-PA
(4.4 0.6)×10−4
(3.6 0.4)×10−4
(1.9 0.3)×10−3
(5.0 0.5)×10−3
(3.5 0.4)×10−4
9
5
3
3
10.0 0.5
5.7 0.8
4.0 0.3
5.4 0.2
5.8 0.7
Not measured
TriIso-PA
OMe-PA
5
(NO2-CF3)-PA (6.3 0.8)×10−4
ca. −1
a
Rate constant at the pH where the HNO electrode current reaches a plateau (Figs. 2
and SI2). In the case of 6, the rate was taken two pH units after the decomposition pH.
b
pH at which decomposition starts to be observed by UV–visible and/or NMR.
Inflexion point for the current vs. pH curves (Figs. 2 and SI2).
c
ð3Þ

K. Sirsalmath et al. / Journal of Inorganic Biochemistry 118 (2013) 134–139
137
Fig. 3. Crystal structure of: a) Me–PA; b) NO2–PA; c) F-PA. Ellipsoids are drawn at 50%
probability level.
O-deprotonation to form the corresponding anion sets the –SO2NHOH
moiety to release HNO while in the case of the N-anion, the breakage
of the S–N bond produces the NOH isomer which eventually can convert
to HNO. The process 3NOH→1HNO is favorable from the energetic point
of view, −15.5 kcal/mol according to our calculations, ca. −20 kcal/mol
by measurements from previous lit. [41], but it has to be taken into
account that this triplet to singlet process is forbidden and could be
relatively slow. Summarizing, taking into account the energetic re-
quirements, in aqueous media O-deprotonation and the subsequent
1HNO release seems more likely to take place than N-deprotonation
and 3NOH release, contrary to what have been suggested before in or-
ganic solvents. [31] Regarding the second deprotonation, the energy re-
lease from the N-anion to yield the dianion is the highest one. From this
point, the breakage to produce NO− is a very favorable process,
suggesting that at extremely high pH values, NO− could be formed di-
rectly from the dianion.
Fig. 2. pH versus HNO current intensity for: a) Me–PA b) F–PA.
similar in all cases. However, the N–O bond tends to be shorter
when electron-attracting groups are present in the ring (–NO2, –F).
This shorter bond would facilitate the formation of HN=O, which
requires a double bond between the N and O atoms. The trend
shown in Table 4 is quite in agreement with the HNO donation pH
values listed in Table 3. However, it has to be taken into account
that the situation predominating in solution could differ from solid-
state measurements.
For the rest of the PA derivatives, all energies discussed above
were also calculated (SI and Table 5). Table 5 presents the calculated
energies for the processes:
O ꢀ deprotonationðΔEOH
Þ
ð4Þ
R ꢀ SO2–NH–OH→½R ꢀ SO2–NH–Oꢁ− þ Hþ
3.1. NH versus OH deprotonation, and electron-withdrawing versus
electron-donating groups
N ꢀ deprotonationðΔENH
Þ
ð5Þ
ð6Þ
R ꢀ SO2–NH–OH→½R ꢀ SO2–N–OHꢁ− þ Hþ
Deprotonation as the first step for PA decomposition has been
suggested initially by Angeli [27]. The –SO2NHOH moiety contains
two proton-like hydrogen atoms, one on the N atom and the other
one on the O atom. One question that arises is which proton loss
(from O–H or N–H) is more relevant for the decomposition pathway.
By means of electronic structure calculations we optimized the structures
for TSHA (Me-PA), its two derived monoanions, and the dianion. The
deprotonation processes are depicted in Scheme 1. The results show
that both deprotonations hold similar energetic requirements, with
N-deprotonation producing a more stable anion than O-deprotonation
by ca. 10 kcal/mol. However, decomposition from the O-anion is more
favorable than from the N-anion by almost 30 kcal/mol. Moreover,
Decomposition from O ꢀ anionðΔEHNO
Þ
½R ꢀ SO2–NH–Oꢁ−→½R ꢀ SO2ꢁ− þ HNO þ Hþ
Decomposition from N ꢀ anionðΔENOH
Þ
ð7Þ
½R ꢀ SO2–N–OHꢁ−→½R–SO2ꢁ− þ NOH þ Hþ
The trends presented in Table 5 can be rationalized in terms of the
electron-donating/withdrawing nature of the substituent. In fact, the
easiest deprotonation occurs for the nitro derivative which can be
assumed to have the most positively charged –NHOH group and
hence the most stable anion. On the other hand, –OMe (an electron-

138
K. Sirsalmath et al. / Journal of Inorganic Biochemistry 118 (2013) 134–139
Table 4
Table 5
Relevant experimental bond distances and angles.
Calculated energies for reactions 1–4 (kcal/mol), and estimated pKa values for com-
pounds 1 to 5.
Compound
dS–N (Å)
dN–O (Å)
bSNO (°)
R
ΔEOH
ΔENH
pKa(NH)
ΔEHNO
ΔENOH
Me-PA
PA
NO2-PA
F-PA
1.659
1.645
1.656
1.649
1.431
1.414
1.407
1.392
110.34
109.67
111.16
108.56
H
56.1
56.6
52.5
55.5
55.0
57.0
(56.7)a
44.4
45.1
40.0
43.9
45.7
45.7
(45.3)a
9.29 [21]
9.8
6.0
8.9
10.3
10.3
−1.0
−0.6
−3.2
−1.3
−3.8
−0.3
(−0.5)a
26.2
26.5
24.8
25.9
21.1
26.6
(26.4)a
1
2
3
4
5
Me
NO2
F
iPr3
OMe
donating group at the para position, but electron-withdrawing by in-
ductive effect at the meta position) destabilizes the anion formed at
the para position and hence its deprotonation is less favorable. The
effects are more important for the electron-withdrawing than for
the donating groups. For example, for the deprotonation of –OH the
difference between PA (“H” substituent) and the strongest electron-
withdrawing nitro group (2) amounts 3.6 kcal/mol, while the difference
with the electron-donating methoxy group (5) is only of 0.9 kcal/mol.
This last difference is reduced if a water molecule H-bound to the –
OMe group is added to the calculations (see Table 5 and Fig SI4), and in
the presence of two water molecules it is reduced even further. This
tendency and the previously shown experimental evidence indicate
that in aqueous solution the electron-attracting inductive effect of
the para –OMe group predominates over the electron-donating
resonance effect. In the same direction, if we analyze the relative
stability of the sulfinate anions produced (Figure SI5 and Table SI4),
while the electron-withdrawing substituents generate more stable
anions, electron donor ones produces the opposite effect. In fact,
there is a clear trend between the relative energy of the sulfinate
anion and the energy of the HNO donation process. TriIso-PA is an
exception, since it is more prone to donate HNO than the sulfinate
stability suggests, probably due to steric constraints (i.e. longer S–N
bond).
a
Calculated with the addition of one water molecule H-bound to 5.
which can be rearranged to yield:
K2 ¼ K1expð−ðΔG2−ΔG1Þ=RTÞ
and according to the proposed approximation
ð9Þ
K2 ¼ K1expðꢀðΔE2−ΔE1Þ=RTÞ
ð10Þ
By using one of the compounds as standard (i.e. PA with pKa=9.29),
the remaining pKa values can be calculated. The pKa values corresponding
to ΔENH are shown in Table 5, and similar results are obtained if the pKa
values corresponding to ΔEOH are calculated. As it can be observed
there is a good agreement between the calculated pKa and the HNO dona-
tion pH (Table 3) in the cases of 1 and 2 (pKa(NH) =9.8 and 6.0 vs. HNO
donation pH=10.0 and 5.7 respectively). However, the HNO donation
pH values of 3, 4 and 5 are ca. 5 pH units smaller than the estimated
pKa values.
All these trends can be also rationalized by using a free-energy
correlation function such as the Hammett equation. Fig. SI3 shows a
plot of the σp Hammet parameters versus the OH and NH deprotonation
energies ΔEOH and ΔENH. The correlation is not far from linearity and in
agreement with the previous comments.
The acidity corresponding to the NH and OH groups can be estimated
by using ΔG°=−RTlnK, assuming that the fundamental differences be-
tween the different compounds are due to the enthalpic contribution.
Given two different donors:
4. Conclusions
Piloty's acid derivatives have been synthesized and the pH values at
which HNO donation starts were determined by measurement of the
released HNO. These values are approximately coincident with the pH
values of decomposition, which cover a broad pH range (ca. −1 to 10).
From the kinetic point of view, the stability of the anions derived
from the donors is similar, since the decomposition rate constants are
all in the range 10−3–10−4. However, F-PA and TriIso-PA, show the
fastest decomposition rates, and therefore a kinetic effect could be
predominating in these cases. In the case of PA, as well as for the donors
ΔG2−ΔG1 ¼ −RTlnK2 þ RTlnK1
ð8Þ
Scheme 1. Mechanisms of HNO/NO– donation from TSHA.

K. Sirsalmath et al. / Journal of Inorganic Biochemistry 118 (2013) 134–139
139
NO2-PA and Me-PA, the pH at which the HNO donation starts seems to
References
be dominated by the thermodynamic acidity constants. However, an-
other predominant effect seems to be “structural.” Table 4 shows a
clear tendency; dN–O is in the order: F-PA (3)bNO2-PA(2)bPAbMe-PA
(1) which follows the same trend than the HNO donation pH. This
could be due to the fact that the shorter this bond is, more facile results
the formation of HN=O. One would expect to see this situation
reflected in the ΔEHNO values of Table 5, which are more favorable for
NO2-PA, F-PA and TriIso-PA. However, these differences are rather
small and the solvent could be playing an important role enhancing
these variations through H-bonding to the N and O atoms of the HNO
moiety. Finally, steric hindrance may be important in the case of
TriIso-PA, and solvation is definitely expected to be important in the
case of OMe-PA through H-bonding to the –OMe group.
Contrary to PA and TSHA, the rest of the donors with electron-
attracting or withdrawing substituents work as HNO donors at physio-
logical pH. All these PA derivatives are easily synthesized in one-step
reactions from the corresponding sulfonyl chlorides with very good
to excellent yields. Since a large variety of sulfonyl chlorides is com-
mercially available, an enormous number of donors derived from PA
working at any pH are available one step away.
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Abbreviations
AS
PA
trioxodinitrate—Na2N2O3—Angeli's salt
N-hydroxybenzenesulfonamide—Piloty's acid
Me-PA (or TSHA) N-hydroxy-4-methylbenzenesulfonamide
NO2-PA N-hydroxy-4-nitro benzenesulfonamide
F-PA
N-hydroxy-4-fluorobenzenesulfonamide
TriIso-PA N-hydroxy-2,4,6-triisopropylbenzenesulfonamide
OMe-PA N-hydroxy-4-methoxybenzenesulfonamide
(NO2–CF3)-PA N-hydroxy-2-nitro-4-trifluoromethyl
benzenesulfonamide
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MnIIITPPS manganese(III) meso-(tetrakis(4-sulfonato-phenyl))
porphyrinate
DFT
density functional theory
PCM
Polarizable Continuum Model
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Acknowledgements
This work was financially supported by UBA (UBACYT W583),
FONCyT (PICT 2010-2649), CONICET (PIP 112-201001-00125) and
PME-2006-01113.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2012.10.008.