Water soluble acyloxy nitroso compounds: HNO release and reactions with heme and thiol containing proteins

Article abstract of DOI:10.1016/j.jinorgbio.2012.07.023

Nitroxyl (HNO) has gained interest as a potential treatment of congestive heart failure through the ability of the HNO donor, Angeli's salt (AS), to evoke positive inotropic effects in canine cardiac muscle. The release of nitrite during decomposition limits the use of AS requiring other HNO sources. Acyloxy nitroso compounds liberate HNO and small amounts of nitrite upon hydrolysis and the synthesis of the water-soluble 4-nitrosotetrahydro-2H-pyran-4-yl acetate and pivalate allows for pig liver esterase (PLE)-catalysis increasing the rate of decomposition and HNO release. The pivalate derivative does not release HNO, but the addition of PLE catalyzes hydrolysis (t_1/2 = 39 min) and HNO formation (65% after 30 min). In the presence of PLE, this compound converts metmyoglobin (MetMb) to iron nitrosyl Mb and oxyMb to metMb indicating that these compounds only react with heme proteins as HNO donors. The pivalate in the presence and the absence of PLE inhibits aldehyde dehydrogenase (ALDH) with IC₅₀ values of 3.5 and 3.3 μM, respectively, in a time-dependent manner. Reversibility assays reveal reversible inhibition of ALDH in the absence of PLE and partially irreversible inhibition with PLE. Liquid chromatography-mass spectrometry (LC-MS) reveals formation of a disulfide upon incubation of an ALDH peptide without PLE and a mixture of disulfide and sulfinamide in the presence of PLE. A dehydroalanine residue forms upon incubation of this peptide with excess AS. These results identify acyloxy nitroso compounds as unique HNO donors capable of thiol modification through direct electrophilic reaction or HNO release.

Full text of DOI:10.1016/j.jinorgbio.2012.07.023

Journal of Inorganic Biochemistry 118 (2013) 140–147
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Water soluble acyloxy nitroso compounds: HNO release and reactions with heme and
thiol containing proteins
Jenna F. DuMond, Marcus W. Wright, S. Bruce King ⁎
Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 April 2012
Received in revised form 7 July 2012
Accepted 9 July 2012
Available online 6 October 2012
Nitroxyl (HNO) has gained interest as a potential treatment of congestive heart failure through the ability of the
HNO donor, Angeli's salt (AS), to evoke positive inotropic effects in canine cardiac muscle. The release of nitrite
during decomposition limits the use of AS requiring other HNO sources. Acyloxy nitroso compounds
liberate HNO and small amounts of nitrite upon hydrolysis and the synthesis of the water-soluble
4-nitrosotetrahydro-2H-pyran-4-yl acetate and pivalate allows for pig liver esterase (PLE)-catalysis increasing
the rate of decomposition and HNO release. The pivalate derivative does not release HNO, but the addition of
PLE catalyzes hydrolysis (t1/2=39 min) and HNO formation (65% after 30 min). In the presence of PLE, this
compound converts metmyoglobin (MetMb) to iron nitrosyl Mb and oxyMb to metMb indicating that these
compounds only react with heme proteins as HNO donors. The pivalate in the presence and the absence of
PLE inhibits aldehyde dehydrogenase (ALDH) with IC50 values of 3.5 and 3.3 μM, respectively, in a time-
dependent manner. Reversibility assays reveal reversible inhibition of ALDH in the absence of PLE and partially
irreversible inhibition with PLE. Liquid chromatography–mass spectrometry (LC–MS) reveals formation of a
disulfide upon incubation of an ALDH peptide without PLE and a mixture of disulfide and sulfinamide in the
presence of PLE. A dehydroalanine residue forms upon incubation of this peptide with excess AS. These results
identify acyloxy nitroso compounds as unique HNO donors capable of thiol modification through direct
electrophilic reaction or HNO release.
Keywords:
Nitroxyl
Nitric oxide
Thiol protein
Heme proteins
Esterase
Dehydroalanine
©
2012 Elsevier Inc. All rights reserved.
1
. Introduction
Acyloxy nitroso compounds (1–3, Fig. 1) act as HNO donors through
ester hydrolysis to give an unstable intermediate that decomposes to
HNO without generation of nitrite in buffered conditions [21]. Varying
the pH and the R group of the ester of these structures varies their sta-
bility, HNO donor activity and ability to relax pre-constricted rat aorta
[22]. Compounds 1 and 2 only slowly hydrolyze and competitively
react with other nucleophiles (thiolates) without HNO formation, but
3 hydrolyzes to HNO under all conditions [22]. While compounds 1–3
provide information concerning the reactivity and HNO release of
acyloxy nitroso compounds, their lack of water solubility limits their
use in biological systems. Incorporating oxygen into the ring of 1 and
2 gives 4 and 5, and should greatly increase water solubility allowing
for esterase mediated hydrolysis to increase the rate of decomposition
(Fig. 1) [23].1
The increased water solubility of 4–5 and their structural similarity
to C-nitroso compounds, which also react with thiols [24], allow
evaluation with myoglobin (Mb), a heme protein, and aldehyde
dehydrogenase (ALDH), a thiol containing protein, known HNO targets
in the presence of pig liver esterase (PLE) to enhance HNO release
[11,25,26]. Our results show that acyloxy nitroso compounds can act
Nitroxyl (HNO/NO−), is the one electron reduced relative of nitric
oxide (NO), an important biological signaling agent [1–5]. Nitroxyl
has distinct biological properties compared to NO and is a potential
treatment of myocardial reperfusion injury and congestive heart
failure [6–8]. Nitroxyl avidly reacts with heme and thiol containing
proteins and these reactions likely mediate its biological actions
[8–11]. Nitroxyl must be generated from donors due to its fast dimer-
O, k=8×106
M
−1
s
−1
) and a limited
, AS) is the
ization to nitrous oxide (N
number of donors exist [12–14]. Angeli's salt (NaN O
2 3
most widely used HNO donor, but at physiological pH AS forms an
equivalent of nitrite during decomposition [15], Nitrite reacts with
all of the redox forms of hemoglobin and possesses its own biological
profile, which must be accounted for in biological evaluations of HNO
2
activity [15–19]. Other limitations of AS include the fast rate of
decomposition (k=10−4
s
−1
) and difficulty in modifying the
structure [14,15]. The ability of AS to elicit positive inotropic ef-
fects in canine cardiac muscle highlights the need for new HNO
donors as potential new therapies [7,20].
1
⁎
Corresponding author. Tel.: +1 336 758 5774; fax: +1 336 758 4656.
During this work, the following patent appeared describing the preparation of 4
E-mail address: kingsb@wfu.edu (S.B. King).
and 5.
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.07.023

J.F. DuMond et al. / Journal of Inorganic Biochemistry 118 (2013) 140–147
141
Fig. 1. Hydrolytic HNO release from 1–5.
as efficient HNO donors in the presence of PLE, which may be useful in
further probing HNO biology.
UV–vis spectra were taken every 5 min for 6 h at 660 nm. Similar ex-
periments were performed using a solution of PLE (9 U/μmol) in PBS
(
100 mM, pH 7.4).
2
. Experimental
2.4. Gas chromatographic N
2
O analysis [28]
2
.1. General
For headspace analysis, a solution of 4 or 5 (0.08 μmol) in anhy-
All chemicals were purchased from Sigma Aldrich Chemical Compa-
drous DMF (8 μL) followed by PBS (10 mM, pH 7.4, 0.8 mL) was
injected into a 10 mL round bottom flask sealed with a rubber septum
and flushed with inert gas. To some samples PLE (9 U/μmol) and GSH
ny and used as received. AS and 15N-AS were prepared as described,
and stored dry at −20 °C [27]. Analytical TLC was performed on silica
gel plates with C-4 Spectroline 254 indicator. Visualization was accom-
plished with UV light. Solvents for extraction and purification were of
(
1.60 μmol) were added, the sample was incubated at 37 °C, and at
desired timepoints headspace aliquots (100 μL) were injected via a
gas-tight syringe onto a 7890 A Agilent Technologies gas chromato-
graph equipped with a micro-electron capture detector and a
0 m×0.32 m (25 μm) HP-MOLSIV capillary column. The oven was
operated at 200 °C for the duration of the run (4.5 min). The inlet
was held at 250 °C and run in split mode (split ratio 1:1) with a
technical grade and used as received. Liquid chromatography–mass
spectrometry (LC–MS) solvents were HPLC grade. 1H NMR and
13
C
NMR spectra were recorded using a Bruker Avance 300 MHz NMR
spectrometer. Chemical shifts are given in ppm (δ); multiplicities are
indicated by s (singlet), d (doublet), t (triplet), q (quartet). Yeast
ALDH (E.C. 1.2.1.5) was purchased from Sigma Aldrich Chemical Com-
pany. UV–vis spectrometry was performed on a Cary 100 Bio UV–
3
total flow (N
The micro electron capture detector (μECD) was held at 325 °C with
a makeup flow (N ) of 5 mL/min. The retention time of nitrous
2
as carrier gas) of 4 mL/min and a pressure of 37.9 psi.
visible (UV–vis) spectrophotometer (Varian, Walnut Creek, CA). IC50
s
2
were obtained by fitting concentration-dependent percent of control
data non-linear curve fitting using sigmoidal curve with standard
slope algorithm using GraphPad Prism software. ALDH peptide
A286-R307 (AIDWIAAGIFYLNSGQNCTANSR) was purchased from
Peptide 2.0 (Chantilly, VA) with >98% purity. High-resolution mass
spectra were obtained using a Thermo Scientific LTQ Orbitrap mass
spectrometer equipped with a heated electrospray ionization source
operated in positive ion mode.
oxide was 3.4 min, and yields were calculated based on a standard
curve for nitrous oxide (Matheson Tri-Gas).
−
2.5. Chemiluminescence NO, NO2 assay [21]
A solution of 4 or 5 (0.08 μmol) in anhydrous DMF (8 μL) followed
by PBS (10 mM, pH 7.4, 0.8 mL) was injected into a 10 mL round
bottom flask sealed with a rubber septum and flushed with inert
gas. To some samples PLE (9 U/μmol) was added and the sample
2
2
.2. Synthetic procedures
was incubated at 37 °C, and at desired timepoints, aliquots (200 μL
−
headspace, NO or 5 μL reaction mixture, NO
2
) were injected into
.2.1. 4-Nitrosotetrahydro-2H-pyran-4-yl Acetate (4)
Ultraviolet–visible (UV–vis) (PBS) λmax =660 nm, ε=20.7 M−
1
the reaction vessel of a Sievers 280 NOA chemiluminescence detector.
This apparatus detects nitrite by reduction of nitrite to NO, followed
by chemiluminescence NO detection (the reaction vessel contained
cm−
1
,
1
H NMR (300 MHz, benzene-d
.7 (s, 3H), 1.9 (ddd, J=5.0, 11.5 Hz, 2H), 3.3 (td, J=2.7, 11.6 Hz, 2H),
.6 (ddd, J=4.6, 11.8 Hz, 2H), 13C NMR (75 MHz, benzene-d
) δ 21.8,
6
) δ 1.5 (dd, J=2.5, 14.0 Hz 2H),
1
3
3
1
% NaI in glacial acetic acid). The yields were calculated based on a
6
standard curve for nitrite.
1.3, 64.2, 120.2, 167.6.
2
.2.2. 4-Nitrosotetrahydro-2H-pyran-4-yl Pivalate (5) [22]
2
2
.6. UV–vis assay of Mb with 5
−
1
cm−1, 1
H
UV–vis (DMF:PBS, 1:29) λmax =660 nm, ε=20.7 M
NMR (300 MHz, benzene-d ) δ 1.2 (s, 9H), 1.5 (dd, J=2.5, 14.0 Hz,
2
3
2
6
.6.1. UV–vis assay of ferrous nitrosyl complex from the reaction of 5
H), 1.9 (ddd, J=5.0, 11.5 Hz, 2 H), 3.3 (td, J=2.4, 11.5 Hz, 2H),
with metmyoglobin (metMb)
.6 (ddd, J=4.8, 11.7 Hz, 2H), 13C NMR (75 MHz, benzene-d
6
) δ
Under degassed conditions, a solution of 5 (2.58 μmol) in anhy-
drous DMF (25.8 μL) was added to a solution of metMb (0.129 μmol)
in PBS (100 mM, pH 7.4, 1.47 mL) at 37 °C in a sealed cuvette. In the
reactions that contained PLE (9 U/μmol, 85 U/mL in PBS, 100 mM, pH
8.3, 31.1, 40.2, 64.3, 119.6, 175.0.
2
.3. UV–vis spectroscopy assay for the decomposition of 4 and 5 [21]
7
.4), it was added directly before UV–vis measurements began.
A solution of phosphate buffered saline (PBS, 100 mM, pH 7.4)
UV–vis spectra were taken every 1.0 min for 4 h, and the disappear-
ance of metMb was monitored by the decrease in absorbance at 502
and 630 nm, while the appearance of Mb–NO was monitored by the
increase in absorbance at 542 and 583 nm [26].
was added to a solution of 4 (5 μmol) in PBS (100 mM, pH 7.4,
00 μL) or 5 (5 μmol) in anhydrous dimethyl formamide (DMF)
50 μL) to give a total volume of 1.5 mL at 37 °C in a sealed cuvette.
1
(

142
J.F. DuMond et al. / Journal of Inorganic Biochemistry 118 (2013) 140–147
2
.6.2. UV–vis assay of metmyoglobin (metMb) from the reaction of 5
with oxymyoglobin (MbO
A solution of 5 (84 nmol) in anhydrous DMF (1 μL) was added to a
solution of MbO (42 nmol) in PBS (100 mM, pH 7.4, 999 μL) at 37 °C
in a sealed cuvette. In some reactions, PLE (9 U/μmol, 85 U/mL in PBS,
00 mM, pH 7.4) was added directly before UV–vis measurements
began. UV–vis spectra were taken every 2.0 min for 4 h, and the
disappearance of MbO was monitored by the decrease in absorbance
observed in these experiments. N-Ethylmaleimide (NEM) thiol trapping
of the unreacted peptide was performed and analyzed as above.
2
)
2
3. Results and discussion
1
3.1. Synthesis
2
Substitution of the cyclohexane ring of 1–2 with an oxygen-
containing tetrahydropyran ring gives new acyloxy nitroso com-
pounds (4–5) that can be prepared using previously reported meth-
odology (Fig. 2) [21,22]. Condensation of tetrahydropyran-4-one
with hydroxylamine hydrochloride results in dihydro-2H-pyran-
at 540 and 580 nm, while the appearance of metMb was monitored
by the increase in absorbance at 502 and 630 nm.
2
2
.7. Inhibition studies
4(3H)-one oxime and lead (IV) tetraacetate oxidation in the absence
.7.1. In vitro inhibition of yeast ALDH [24]
and presence of 5 equivalents of 2, 2-dimethylpropanoic acid gives
Yeast ALDH (0.1 U) was preincubated for 10–120 min with the in-
hibitor (AS dissolved in 10 mM NaOH, nitrosobenzene (NB) in DMSO,
in DMF in the presence or absence of PLE (9 U/μmol, 85 U/mL,
00 mM PBS, pH 7.5)) at 37 °C in the primary reaction mixture
100 mM PBS, pH 7.5) to give a total volume of 0.1 mL. After
0–120 min, 20 μL of the primary mixture was added to a cuvette
4-nitrosotetrahydro-2H-pyran-4-yl acetate and pivalate (4 and 5, Fig. 2).
1
13
H and C NMR spectroscopy confirm the structure of the oxime as well
as 4 and 5, which exist as bright blue oils. Compounds (4–5) demonstrate
significant water solubility (1.7 mM in buffer (compound 4) or a 3% DMF/
buffer mixture (compound 5)) presumably due to the increased polarity
and hydrogen bonding interactions of the tetrahydropyran ring oxygen
atom with water.
5
1
(
1
that contained the secondary mixture (90 mM potassium phosphate
+
buffer, pH 8.0, 1.0 mM EDTA, 0.5 mM NAD and 30% glycerol) to
+
give a final volume of 1.0 mL. The reduction of NAD to NADH was
3.2. Decomposition kinetics
activated by the addition of benzaldehyde (0.6 μmol), and the activity
of ALDH was followed by the increase in absorbance at 340 nm over
A hydrolytic mechanism provides a reasonable explanation for the
observed decomposition rates and HNO formation from 4 and 5
(Fig. 3) [21,22]. Similar to 1–3, HNO release from 4 and 5 requires
ester hydrolysis to generate an unstable α-hydroxy nitroso intermedi-
ate that collapses to cyclohexanone and HNO (Fig. 3) [21]. The
improved aqueous solubility of 4 and 5 allows the addition of pig
liver esterase (PLE) as a catalyst for hydrolysis (Fig. 3). Monitoring
the disappearance of the absorption at 660 nm using ultraviolet–visible
(UV–vis) spectroscopy provides kinetic information regarding the de-
composition of 4 and 5 in the presence and absence of PLE. Compound
4 decomposes under buffered conditions with a rate constant of k=
3
0 min Measurements were taken in triplicate and IC50 values were
calculated.
2
.7.2. In vitro reactivation of inhibited yeast ALDH [25]
The experimental conditions were repeated as in the inhibition
assay with each inhibitor at a concentration of 10 times above the
calculated IC50. DTT (10 mM) was added to the primary mixture
after the initial 1–10 min of incubation with the respective inhibitor
at 37 °C. After the addition of DTT, the reaction mixture sat for
3
min at room temperature and an aliquot (20 μL) of this reaction
−
5
−1
mixture was added to the cuvette and the assay was completed as
above to measure ALDH activity.
3.0×10
s
(t1/2=6.4 h, compared to t1/2=14.8 h for 1) and the
addition of PLE increases decomposition with an observed rate con-
−
4
−1
stant of k=5.0×10
s
(t1/2=23 min, under these conditions
2
.8. LC–MS studies
which include 9 U of PLE per μmol of 4). Decomposition of 4 in a
completely aqueous environment compared to the aqueous/organic
mixture required to dissolve 1 likely enhances hydrolysis. Incubation
of 5 in a 3% mixture of DMF:PBS (100 mM, pH 7.4) at 37 °C shows
very slow decomposition over time with a rate constant of k=
2
.8.1. LC–MS: reactivity of ALDH peptide A286-R307 with nitrosobenzene
(
NB), AS, and 5 in the presence and absence of PLE
ALDH peptide A286-R307 (10 μM in 100 mM NH
cubated with NB (10 eq., anhydrous DMF), AS (10, 1000 eq., stock solu-
4 3
CO buffer) was in-
−
6
−1
6.0×10
s
(t1/2=32 h, compared to t1/2=37.8 h for 2, Supple-
15
tion in 100 mM NaOH), N AS (100 eq., stock solution in 100 mM
mental Data). Under the same reaction conditions, addition of PLE
NaOH), and 5 (10 eq., stock solution in anhydrous DMF) in the presence
increases the decomposition of 5 50-fold with an observed rate
−
4
−1
or absence of PLE (9 U/μmol, stock solution in 100 mM NH
4
CO
3
buffer)
constant of k=3.0×10
s
(t1/2=39 min, under these conditions
for 1 h or 3.5 h (5×t1/2 of 5+PLE) at room temperature (unless noted
at 37 °C), and then analyzed by LC–MS on a Thermo Scientific LTQ
Orbitrap XL. Separations were achieved using a Thermo Scientific Hypersil
GOLD C18 column (150×2.1 mm, 1.9 μm). During LC, solvents A (0.1%
which include 9 U of PLE per μmol of 5, Fig. 3). As expected, the im-
proved water solubility of 5 compared to 2 does not affect hydrolysis
of the stable pivalate ester.
−
formic acid in H
2
O) and B (0.1% formic acid in MeOH) were held at a
3.3. Gas chromatographic and chemiluminescence N
2
O and NO
2
analysis
flow rate of 400 μL/min. Conditioning the column involved transitioning
from acetonitrile to 95% A and 5% B over 15 min, and held for 5 min. LC
analysis consisted of injections (10 μL) using a gradient elution of 5 to
Nitrous oxide (N
2
O), the dimerization and dehydration product of
HNO, provides evidence for HNO intermediacy during the decomposi-
9
1
5% B over 10 min, followed by lowering to 5% B over 10 s, and held for
min. After each analysis two blanks were run. Electrospray ionization
tion of 4 and 5 (Fig. 3) [13]. Fig. 4 shows that the decomposition of 4
in the buffer generates N
matography (GC), but the decomposition of 5 does not generate N
over time. The addition of PLE (9 U/μmol) to 5 results in N O (65%)
2
O (17%, 30 min) as determined by gas chro-
(ESI) parameters consisted of a spray voltage of 4 kV, capillary voltage
2
O
of 40 V, and a capillary temperature of 325 °C. Positive mode high resolu-
tion mass spectra were collected with 30,000 Hz resolution from 200 to
2
after 30 min, which does not increase over time and suggests other
reactions of HNO compete with dimerization under these conditions.
2
000 m/z. Extracted masses with an error of +/−5 ppm were found to
+2 +2
be 1186.5573 [M(thiol)+2H] , 1202.0613 [M(sulfinamide)+2H]
,
Addition of glutathione (GSH) traps HNO and quenches N
tion, providing further evidence for the presence of HNO (data not
shown). Addition of PLE to 4 also increases the amount of N O formed
(Fig. 4) and the amount of N O formation again corresponds to the
observed decomposition rates in the presence of PLE. These results
2
O forma-
+
2
1192.0491 [M(dehydroalanine(dhA))+2Na] , 1240.0791 [M(N-phenyl
+
2
+4
sulfinamide)+2H] , and 1185.5501 [M(disulfide (RSSR))+4H]
In addition, the presence of extracted masses for [M(sulfinic
acid(SO
.
2
2
−
+2
2−
+2
2
))+2H]
and [M(sulfonic acid(SO
3
))+2H]
was not

J.F. DuMond et al. / Journal of Inorganic Biochemistry 118 (2013) 140–147
143
Fig. 2. Synthesis of 4–5.
strongly suggest that PLE, a serine-based esterase that accepts large
hydrophobic substrates catalyzes hydrolysis and HNO release from
(84 μM) and PLE to a solution of MbO
2
(42 μM) show the disappear-
ance of the peaks at 543 and 582 nm with the appearance of peaks at
503 and 633 nm. These results reveal the conversion of oxyMb to
metMb and further support HNO formation (Fig. 5) [26]. Kinetic analy-
these compounds (Fig. 3) [29,30].
−
Fig. 4 also shows NO
2
formation, as monitored by chemilumines-
−
4
−1
cence, from the decomposition of 4 and 5 in the presence and absence
sis of these processes yields rate constants of =3.0×10
s
(t1/2=
of PLE. In the absence of PLE, decomposition of 4 or 5 only yields small
39 min), which coincide with the rate of PLE-mediated decomposition
of 5 supporting HNO formation through PLE catalyzed hydrolysis and
decomposition of an α-hydroxy nitroso intermediate (Fig. 3 and Sup-
−
amounts of NO
Data for 5). In the presence of PLE, the amount of NO
over time and 4 yields (17%, 4 h, conditions that give 56% N
under the same conditions 5 produces 10% NO
2
(4–8%, Fig. 4) and no nitric oxide (Supplemental
−
2
increases
O), and
2
2
plemental Data). Incubation of 5 with metMb or MbO in the absence
−
2
after 4 h (five-times
of PLE results in no change in the absorption spectra indicating that
the nitroso groups of these acyloxy nitroso compounds do not react
with these iron-heme proteins (Supplemental Data). The low level of
nitrite, which reacts with both metMb and MbO , generated from the
2
esterase catalyzed hydrolysis makes 5 a potentially valuable alternative
the half-life of decomposition under these conditions), an amount far
less than the one equivalent generated by Angeli's salt. Nitrite forma-
tion appears linked to the presence of the esterase as little nitrite
forms in the absence of PLE and the amount of nitrite is proportional
to the amount of PLE added (data not shown). Nucleophilic addition
of the serine residue of the esterase to the electrophilic nitroso
group of 4 or 5 (rather than the ester carbonyl) would yield an addi-
tion product that may decompose to alternative products including
nitrite [29,30]. Alternatively, the aerobic oxidation of HNO forms
nitrite as the final nitrogen-containing product and presents another
path for nitrite formation in these reactions (Fig. 3) [31–33]. These
results identify 5 as a water-soluble and switchable HNO-donor that
does not hydrolyze (or release HNO), and likely behaves as a
C-nitroso compound until esterase catalyzed hydrolysis rapidly re-
leases HNO. This information generally segregates the acyloxy nitroso
compounds (1–5) into three broad groups regarding kinetic decom-
position (and HNO release) at neutral pH with 1 and 4 being slow
HNO donors, 2 and 5 (in the absence of PLE) do not release HNO
and 3 and 5 (in the presence of PLE) being rapid HNO donors similar
to AS [22].
HNO source for studying reactions with iron-heme proteins.
3
3
.5. Inhibition studies
.5.1. In vitro inhibition of yeast ALDH
Nitrosobenzene (NB), a C-nitroso compound which cannot hydro-
lytically release HNO and Angeli's salt-derived HNO (the simplest
nitroso compound) both inhibit the thiol-containing enzyme ALDH
(
reported IC50 values of 2.5 and 1.8 μM, respectively) [24,25]. ALDH
inhibition arises from a direct reaction of these nitroso compounds
with the active site thiol (C302) resulting in inhibition characterized
by both an irreversible and reversible (by DTT) component resulting
from sulfinamide and disulfide modifications [25]. Previous work de-
fines the reactivity of acyloxy compounds (1–3) with small molecule
thiols and while the trifluoroacetate derivative (3) hydrolyzes to form
HNO under all conditions (like AS), 1–2 directly react with thiolates
(
similar to C-nitroso compounds) to give the corresponding disulfide
3
.4. Reaction of 5 with MetMb and MbO
2
and oxime [22]. Based on this precedent, acyloxy compound 5 should
inhibit ALDH both in the presence and absence of PLE through differ-
ent mechanisms. Nitrosobenzene and AS inhibit ALDH in a concen-
tration dependent manner with IC50 values of 0.2 and 2.1 μM,
respectively, and 5 inhibits ALDH (IC50 values of 3.5 and 3.3 μM
in the absence and presence of PLE, respectively, Supplemental
Data) with similar potency to NB and AS. The inhibition of yeast
ALDH by NB, AS, and 5 with or without PLE, (at concentrations
of inhibitor one-half [IC50]) increases with increased incubation
time suggesting a reactive process resulting in a covalent enzyme
modification (Fig. 6). For NB and AS, the majority of the inhibition
occurs within the first 10 min of incubation while inhibition with
The solubility and HNO release properties of 4 and 5 allows evalua-
tion of their interactions with biological targets. Based on the drastic
differences in the kinetics of 5 in the absence and presence of PLE,
further experiments focused on the reactions of 5 with heme and
thiol containing proteins. UV–vis measurements of the incubation
with 5 in the presence of PLE with metMb (86 μM) in PBS reveal the
disappearance of the peaks at 503 and 632 nm with the appearance
of peaks at 542 and 581 nm. These results show the conversion of
metMb to the ferrous nitrosyl complex (Fig. 5), and support HNO
formation from 5 [26]. Similar measurements upon addition of 5
Fig. 3. Decomposition of 4–5 in the presence of PLE.

144
J.F. DuMond et al. / Journal of Inorganic Biochemistry 118 (2013) 140–147
Fig. 6. Effects of incubation time on ALDH inhibition of various C-nitroso compounds or
HNO donors at one-half [IC50]. Control indicates enzyme and substrate incubated at 10
and 120 min.
Fig. 4. Yields of N
and chemiluminescence analysis.
O and NO−
from 4 and 5 as a function of PLE as determined by GC
2 2
5
(with and without PLE) progresses over 2 h (Fig. 6). Experiments with
inactivated by 5 alone with DTT restores 63% of enzyme activity
suggesting a reducible disulfide as the predominant modification and
suggests that 5 inhibits ALDH via different mechanisms from both NB
and AS (Fig. 7). Treatment of ALDH inactivated by 5 in the presence of
PLE with DTT restores 43% of the enzyme activity, similar to AS, indicat-
ing a mixture of reversible (reducible) and non-reducible modifications,
further supporting esterase catalyzed hydrolysis of 5 and HNO forma-
tion (Fig. 7). Increasing the incubation time of 5 in the presence of PLE
to five times the half-life of decomposition does not change DTT
NB and AS at concentrations ten-fold the IC50, show complete inhibition
of the enzyme within 10 min (Supplemental Data).
3
.5.2. In vitro reactivation of ALDH activity
Treatment of ALDH inactivated by NB with dithiothreitol (DTT)
restores 15% of the enzyme activity revealing
a non-reducible
modification and reactivation of AS-inactivated ALDH returns 46% of
the enzyme activity suggesting a mixture of reversible and irreversible
modifications, as previously reported (Fig. 7) [25]. Treatment of ALDH
reactivation of the enzyme (Supplemental Data). Previous work
−
shows that NO
2
from AS does not inhibit ALDH indicating inhibition
by 5 with PLE in these experiments occurs via HNO [25]. Overall, these
results indicate that 5 in the presence of PLE releases HNO and inhibits
ALDH in a similar fashion to HNO generated from AS. Nitrosobenzene
inhibition arises from the formation of a non-reducible enzyme
modification while inhibition with 5 alone likely generates a reducible
disulfide revealing that while acyloxy nitroso compounds demonstrate
electrophilic behavior similar to C-nitroso compounds, the reactions of
acyloxy nitroso and C-nitroso compounds yield different products.
3.6. LC–MS analysis of the reaction of HNO donors with the ALDH peptide
A285-R307
High resolution LC–MS experiments provide further molecular
insight to the mechanism of ALDH inhibition by nitrosobenzene, AS
Fig. 5. UV–vis assay of the reaction of 5 with Mb. A) Incubation of metMb (86 μM,
100 mM PBS, pH 7.4, 37 °C) with 5 (1.7 mM) and PLE (9 U/μmol). B) Incubation of
Fig. 7. DTT reactivation of ALDH activity after incubation with C-nitroso compounds or
MbO (42 μM, 100 mM PBS, pH 7.4, 37 °C) with 5 (84 μM) and PLE (9 U/μmol).
2
HNO donors (ten-times [IC50]).

J.F. DuMond et al. / Journal of Inorganic Biochemistry 118 (2013) 140–147
145
and 5 in the absence and presence of PLE. Initial experiments using full
length intact ALDH followed by tryptic digestion fail to yield clear results
regarding the products of these reactions and necessitate the use of
ALDH A285-R307 peptide (AIDWIAAGIFYLNSGQNCTANSR), which con-
tains the active site thiol (C302), as a simple model. As this peptide only
contains a single cysteine, intramolecular disulfides that play a role in re-
versible HNO-mediated ALDH inhibition cannot form and results from
the reactions of these compounds and this peptide provide structural
rather than functional information [25]. Such MS experiments show
the expected mass for the unreacted peptide and the addition of
N-ethyl maleimide confirms the presence of a thiol (Table 1). Treatment
of the ALDH peptide A285-R307 with NB results in the formation
of a new peak with a m/z of 1240.0791, which has been assigned
the N-phenyl sulfinamide structure (Table 1 and Supplemental
Data section, which contains detailed MS data for each incuba-
tion). Reaction of the peptide with 5 only forms the disulfide
derived from two peptides (m/z=1185.5496, Table 1). These
modifications, formed from electrophilic but non-HNO releasing
compounds, explain (within the limitations of this model peptide)
the observed inhibition and reactivation results with the full pro-
tein as DTT would reduce the disulfide returning activity but not
the N-phenyl sulfinamide.
Table 1
LC–MS analysis of the reaction of C-nitroso compounds and HNO donors and ALDH peptide A285-R307.
Reagent
Retention time (min)
6.98
m/z
Product (s)
——
1186.5573 [M+2H]+2
Unreacted peptide
NB
7.17
7.54
1240.0791 [M(N-phenyl sulfinamide+2H]+2
N-phenyl sulfinamide
5
1185.5496 [M(RSSR)+4H]+4
RSSR
AS (10 eq.)
6.82
1192.0491 [M(dhA)+2Na]+2
dhA-major
6
.94
1202.0620 [M(sulfinamide+2H]+2
Sulfinamide-minor
AS (1000 eq.)
6.89
6.91
7.52
1202.0613 [M(sulfinamide+2H]+2
1202.5616 [M(15Nsulfinamide+2H]+2
1185.5514 [M(RSSR)+4H]+4
Sulfinamide
15N-sulfinamide
RSSR
15N-AS (1000 eq.)
5
+PLE
6
.92
1202.0618 [M(sulfinamide+2H]+2
Sulfinamide

146
J.F. DuMond et al. / Journal of Inorganic Biochemistry 118 (2013) 140–147
As expected for the reaction of a thiol with excess HNO [31,34,35], in-
cubation with AS (1000 eq.) gave a peak with a m/z=1202.0613, which
peptide thiol/thiolate on an electrophilic nitroso compound. Pathway
A shows the addition of this thiolate (more reactive form) to the
nitroso group of NB or HNO, generated from AS or the PLE-mediated
hydrolysis of 5, to yield an initial N-hydroxysulfenamide that re-
arranges to the corresponding sulfinamide through dehydration to a
sulfenium ion intermediate and rehydration at sulfur [31,34]. Numerous
nucleophiles add to NB and previous work shows the formation of
N-substituted sulfinamides upon reaction of thiols with nitrosobenzene
[41,42]. The addition of thiols to HNO yields a similar intermediate that
reacts with another thiol to give disulfides or rearranges to the
sulfinamide [31,34], which generally resist DTT reduction resulting in
enzyme inactivation [25]. Pathway B shows thiolate addition to the
acyloxy nitroso compound (5) to give a product that apparently does
not dehydrate but reacts with excess thiol to yield disulfide, which
would be reduced by DTT, and the corresponding oxime. This reactivity
mirrors previous work that shows disulfide and oxime formation from
the reaction of 1 and 2 with various thiolates [22]. The structural differ-
ence in the initial addition products of Pathways A and B, with B bearing
a suitable carboxylate leaving group that favors decomposition through
disulfide formation rather than dehydration, appears to influence this
difference in reactivity. Based on these findings, the mixture of disulfide
and sulfinamide products observed during the reaction of the ALDH
peptide with 5 in the presence of PLE likely forms from a mixture of
Pathways A and B, with A yielding the sulfinamide via HNO reactions
and B yielding disulfide via direct reactions with 5. Indeed, incubation
of the ALDH peptide with 5 and PLE for longer times at 37 °C (rather
than room temperature) to ensure full HNO release yields sulfinamide,
as the major product suggesting an increased contribution from
Pathway A (HNO release). Acyloxy nitroso compounds thus act
in two separate ways: 1) as electrophiles directly reacting with
protein thiol residues to give disulfides (not N-substituted sulfinamides)
or 2) sources of electrophilic HNO and both pathways result in thiol
modification leading to enzyme inhibition. Acyloxy nitroso compounds
demonstrate a distinct direct reactivity with thiols yielding only disul-
fide products compared to both C-nitroso compounds and HNO allowing
these compounds to be considered as “organic nitroxyls” that con-
vert thiols to disulfides. Dehydroalanine formation likely results
from the elimination of a sulfur–nitrogen intermediate or the
final sulfinamide (Pathway A). Direct amination of cysteine resi-
corresponds to a sulfinamide as the only product (Table 1) and use of
1
5
15
N-AS yields the N-substituted sulfinamide (Table 1) [36]. Interesting-
ly, LC–MS analysis of the reaction of ALDH peptide A285-R307 with AS
(
(
10 eq.) does not give the expected disulfide but yields a major product
based on ion count) with a m/z=1192.0491, which corresponds to the
loss of HSONH , suggesting dehydroalanine (dhA) containing peptide
2
formation (Table 1). A minor product with a m/z=1202.0620 that cor-
responds to a sulfinamide also forms, and no evidence of sulfinic or sul-
fonic acids was detected (Table 1). Addition of L-cysteine to this reaction
mixture followed by LC–MS gives an L-cysteine addition product further
confirming the structure and revealing that dehydroalanine formation
occurs before mass spectrometry. MS2 experiments confirm the se-
quence of these peptides and site of modification (Supplemental
Data). No precedent currently exists for dehydroalanine formation
from the reaction of HNO with cysteine, although treatment of
cysteine containing proteins with the aminating reagent, O-
mesitylenesulfonylhydroxylamine, rapidly forms dehydroalanine
[
37–40]. Whether this modification occurs in full length protein,
or why it does not form at higher HNO concentrations remains un-
known at this time, but such an HNO-mediated transformation war-
rants deeper investigation. Sulfinamide and dehydroalanine formation
from reaction of AS with the full protein explains the observed irrevers-
ible portion of inhibition while disulfide formation (possible in the full
protein) explains the reversible part.
Reaction of the peptide with 5 in the presence of PLE at room
temperature results in the formation of the major product (m/z=
185.5514) corresponding to the disulfide derived from two peptides,
as well as a minor product with the m/z=1202.0618, indicating the
presence of sulfinamide (Table 1). Based on the above results, the di-
sulfide appears to arise from the direct reaction of the peptide with 5
while the sulfinamide would arise from the reaction with HNO gener-
ated through the PLE-mediated hydrolysis of 5. Incubation at 37 °C for
longer times (3.5 h) to assure complete HNO release (and increase the
amount of HNO) from 5 yields sulfinamide as the major product
1
(
Table 1), as expected if sulfinamide only arises from the reaction
with HNO. No evidence of dhA product formation was observed. The
formation of these products explains the observed reactivation results
with the full length protein and 5 appears to inhibit ALDH in the pres-
ence of PLE by two mechanisms: 1) the direct reaction of 5 with ALDH
to yield a disulfide and 2) the PLE-catalyzed hydrolysis of 5 to give
HNO, which reacts with the protein to give the sulfinamide.
dues yields dehydroalanine and the loss of HOSNH
2
from the
sulfinamide finds some precedent in sulfoxide eliminations to
yield alkenes [37–40,43]. Regardless of the mechanism, the forma-
tion of dehydroalanine from the reaction of HNO and thiol-
containing peptides indicates new HNO chemistry that may be
exploited for HNO detection.
Fig. 8 outlines mechanisms for the formation of the observed
modifications that include the direct nucleophilic attack of the ALDH
Fig. 8. Mechanisms for the formation of the observed modifications of the ALDH peptide thiol when reacted with C-nitroso compounds or HNO donors.

J.F. DuMond et al. / Journal of Inorganic Biochemistry 118 (2013) 140–147
147
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