Reaction of Nitroxyl (HNO) with Hydrogen Sulfide and Hydropersulfides

Article abstract of DOI:10.1021/acs.joc.0c02412

Nitroxyl (HNO) has gained a considerable amount of attention because of its promising pharmacological effects. The biochemical mechanisms of HNO activity are associated with the modification of regulatory thiol proteins. Recently, several studies have suggested that hydropersulfides (RSSH), presumed signaling products of hydrogen sulfide (H2S)-mediated thiol (RSH) modification, are additional potential targets of HNO. However, the interaction of HNO with reactive sulfur species beyond thiols remains relatively unexplored. Herein, we present characterization of HNO reactivity with H2S and RSSH. The reaction of H2S with HNO leads to the formation of hydrogen polysulfides and sulfur (S8), suggesting a potential role in sulfane sulfur homeostasis. Furthermore, we show that hydropersulfides are more efficient traps for HNO than their thiol counterparts. The reaction of HNO with RSSH at varied stoichiometries has been examined with the observed production of various dialkylpolysulfides (RSSnSR) and other nitrogen-containing dialkylpolysulfide species (RSS-NH-SnR). We do not observe evidence of sulfenylsulfinamide (RS-S(O)-NH2) formation, a pathway expected by analogy with the known reactivity of HNO with thiol.

Full text of DOI:10.1021/acs.joc.0c02412

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Article
Reaction of Nitroxyl (HNO) with Hydrogen Sulfide and
Hydropersulfides
Jessica Zarenkiewicz,† Vinayak S. Khodade,† and John P. Toscano*
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ABSTRACT: Nitroxyl (HNO) has gained a considerable amount of attention because
of its promising pharmacological effects. The biochemical mechanisms of HNO activity
are associated with the modification of regulatory thiol proteins. Recently, several studies
have suggested that hydropersulfides (RSSH), presumed signaling products of hydrogen
sulfide (H2S)-mediated thiol (RSH) modification, are additional potential targets of
HNO. However, the interaction of HNO with reactive sulfur species beyond thiols
remains relatively unexplored. Herein, we present characterization of HNO reactivity
with H2S and RSSH. The reaction of H2S with HNO leads to the formation of hydrogen polysulfides and sulfur (S8), suggesting a
potential role in sulfane sulfur homeostasis. Furthermore, we show that hydropersulfides are more efficient traps for HNO than their
thiol counterparts. The reaction of HNO with RSSH at varied stoichiometries has been examined with the observed production of
various dialkylpolysulfides (RSSnSR) and other nitrogen-containing dialkylpolysulfide species (RSS−NH−SnR). We do not observe
evidence of sulfenylsulfinamide (RS−S(O)−NH2) formation, a pathway expected by analogy with the known reactivity of HNO
with thiol.
disulfide (RSSR) and hydroxylamine (NH2OH) (Scheme 1,
Path B). HNO-mediated oxidation of protein thiols to
disulfides is considered a biologically reversible modification
because disulfides are readily reduced to thiols in the presence
of biological reductants. However, oxidation to RS(O)NH2
represents a modification more difficult to reverse in a
biological setting.12
In the last decade, hydrogen sulfide (H2S) has emerged as a
cell signaling molecule along with NO and carbon monoxide.13
In mammals, H2S is produced enzymatically mainly via three
enzymes: cystathionine γ-lyase (CSE), cystathionine β-
synthase (CBS), and 3-mercaptopyruvate sulfurtransferase.14,15
H2S is capable of influencing a myriad of physiological
functions.16−19 In aqueous solution, H2S (pKa = 6.98) is in
equilibrium with its deprotonated HS− form,20 which
predominates under physiological conditions (pH 7.4).
Despite the involvement of H2S in various physiological
processes, the biochemical mechanisms by which it elicits
different responses remain largely unknown. Oxidative post-
translational modification of protein cysteine residues to
hydropersulfides (RSSH) has been proposed as a significant
pathway for H2S-induced biological effects.21 Indeed, recently
it has been postulated that at least a part of the biological
1. INTRODUCTION
Nitroxyl (HNO), the one-electron reduced and protonated
form of nitric oxide (NO), is a potential therapeutic for several
conditions, including heart failure, alcoholism, vascular
dysfunction, and cancer.1−4 HNO shows a chemical and
biological profile distinct from that of NO. One of the
important chemical properties of HNO is its electrophilicity
toward soft nucleophiles like thiols (RSH).5 The chemical
biology of HNO indicates that thiols and related species are
likely targets for HNO-mediated biological activity.6−8 It has
been reported that the reaction of HNO with thiols is relatively
fast (k = 2 to 20 × 106 M−1 s−1) and thermodynamically
favorable.9,10 The reaction of HNO with thiols proceeds via
initial attack of the thiol sulfur atom on the electrophilic
nitrogen of HNO, giving a short-lived N-hydroxysulfenamide
(RS−NH−OH) intermediate (Scheme 1).6,7,11 At low thiol
concentrations, this intermediate rearranges to a sulfinamide
(RS(O)NH2), presumably via dehydration of the protonated
N-hydroxysulfenamide intermediate to form an alkyliminosul-
fonium intermediate followed by reaction with water (Scheme
1, Path A). However, at high thiol concentrations, the N-
hydroxysulfenamide intermediate reacts with thiol to produce a
Scheme 1. Reaction of HNO with Thiols
Received: October 11, 2020
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activities of H2S is attributed to the generation of RSSH rather
than H2S itself.21−23 Recent advances in analytical methods
revealed the prevalent nature of small molecule and protein
RSSH in biological systems. For example, Ida and co-workers
reported that mammalian tissues contain >100 μM of
glutathione hydropersulfide (GSSH).24 Likewise, significant
levels of cysteine hydropersulfide (Cys-SSH) are also present
in cells.24 Furthermore, cysteine residues in a variety of
proteins/enzymes have been reported to be modified to the
corresponding hydropersulfide. For example, 10−25% of
proteins in mouse liver lysate are persulfidated under
physiological conditions.21 Three enzymes, CSE, CBS, and
cysteinyl-tRNA synthetases, have been reported to catalyze the
formation of Cys-SSH.24,25 Interestingly, RSSH display distinct
chemical properties that may be relevant to their biology. For
example, RSSH are more acidic than the corresponding RSH.
Everett and co-workers have estimated the pKa of 2-[(3-
aminopropyl)amino]ethane hydropersulfide to be 6.2, which is
1.6 units lower than the corresponding thiol.26 The pKa of
cysteine hydropersulfide was computationally estimated to be
∼4 units lower than that of the cysteine.27 More recently,
Alvarez and co-workers reported the pKa of GSSH to be 5.45,
which is 3.49 units lower than GSH.28 These results indicate
that a higher ratio of RSS−/RSSH compared with RS−/RSH
under physiological conditions. Additionally, RSSH have
greater reducing potential than the corresponding RSH.29−31
Also, RSSH are more nucleophilic than the corresponding
RSH,27,28,30 presumably because of the alpha effect.
dissolved in aqueous solution using a semipermeable
membrane that allows the dissolved gases, but not water, to
enter a mass spectrometer.35−37 When AS (500 μM) is
incubated with H2S (100 μM) in pH 7.4 phosphate-buffered
saline (PBS) under anaerobic conditions, a reduction in the
signal corresponding to the HNO dimerization product N2O
(m/z = 44) is observed (Figure 1a). Consistent with this
observation, a reduction in the signal corresponding to H2S
(m/z = 34) is also observed (Figure 1b), confirming that H2S
reacts with HNO.
Figure 1. MIMS signals observed at (a) m/z = 44 corresponding to
N2O+ and (b) m/z = 34 corresponding to H2S+ during incubation of
AS (500 μM) and H2S (100 μM) alone or together in argon-purged
PBS (pH 7.4, 100 mM) containing the metal chelator DTPA (100
μM) at 37 °C.
The ability of HNO to target thiols and thiol-containing
proteins makes it likely that small molecule and protein
hydropersulfides are additional potential targets for HNO.
While the reaction of HNO and various thiols has been well
characterized, the reaction between HNO and other reactive
sulfur species such as H2S and RSSH remains relatively
unexplored. Being highly thiophilic, HNO is expected to react
with H2S as well as with RSSH. Indeed, it has been proposed
that the specificity of HNO signaling may be a function of the
presence of cysteine hydropersulfide residue in proteins.8 This
suggestion is consistent with the idea that HNO may react
preferentially with RSSH because RSSH have enhanced
nucleophilicity and reducing capability. Indeed, Fukuto and
co-workers have demonstrated the interaction between RSSH
and HNO,32 although the chemical details of this reaction
remain to be further elucidated. Herein, we present character-
ization of HNO reactivity toward H2S and RSSH along with a
comparison of this reactivity with RSH.
To verify the reaction between HNO and H2S, we
independently analyzed the yield of N2O by gas chromatog-
raphy (GC) headspace analysis. For comparison, analogous
experiments were conducted with N-acetylcysteine methyl
ester (pKa = 7.28) because of its similar pKa to H2S (pKa =
6.98).20 As the thiolate anion is the active species in this
reaction, thiol concentrations were adjusted such that equal
amounts of thiolate anion were present in solution. Incubations
of AS (200 μM) with varying concentrations of H2S show a
marked decrease in N2O production relative to AS only
(Figure 2, black bars). Similarly, addition of N-acetylcysteine
methyl ester to buffer solutions containing AS also results in a
decrease in N2O production (Figure 2, red bars). Quantifica-
tion of the N2O yields reveals that H2S is a more efficient trap
2. RESULTS AND DISCUSSION
2.1. Reactivity of HNO with H2S. Because of HNO’s
inherent reactivity, it must be generated in situ. We used
Angeli’s salt (AS, Na2N2O3) as a HNO donor.33 AS
decomposes spontaneously under physiological conditions via
protonation of the dianion to produce equimolar amounts of
nitrite and HNO with a half-life of about 2.4 min at 37 °C.34 In
the absence of chemical traps, HNO rapidly dimerizes (k = 8 ×
106 M−1 s−1) and dehydrates to form nitrous oxide (N2O).33
However, addition of a chemical trap such as thiol competes
with HNO dimerization leading to a reduction in N2O yield.
The inorganic salt, sodium sulfide (Na2S), was used as the
source of H2S.
Figure 2. GC-determined relative yields of N2O in the presence of
increasing concentrations of HNO trap, H2S (black bars), or N-
acetylcysteine methyl ester (red bars). Incubations were performed
with 0, 50, and 100 μM HS− or RS− and 200 μM AS in phosphate
buffer (pH 7.4, 100 mM) with DTPA (100 μM) at 37 °C for 3 h.
Initially, we examined the reaction of HNO with H2S by
membrane inlet mass spectrometry (MIMS). This technique
can monitor the relative amounts of small hydrophobic gases
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for HNO compared with N-acetylcysteine methyl ester. In
addition, a comparison between H2S and thiophenol (PhSH)
reactivity with HNO reveals that H2S is likewise a better trap
for HNO compared with PhSH (Supporting Information,
Figure S1).
After confirming HNO trapping by H2S, we examined the
mechanism of this reaction. Based on previous reports on the
reaction of HNO with thiols, we expected that the HNO
reaction with H2S should proceed via the intermediacy of N-
mercaptohydroxylamine 1 (HSNHOH), and depending on the
relative concentrations of H2S, this intermediate would either
produce hydrogen disulfide (H2S2) and NH2OH (Scheme 2,
Figure 3. (a) Analysis of polysulfides generated from the H2S reaction
with HNO. AS (25 μM) was incubated with Na2S (100 μM) in pH
7.4 ammonium bicarbonate buffer (25 mM) containing DTPA (100
μM) at 37 °C. After 15 min, an aliquot of the reaction mixture was
withdrawn and incubated with HPE-IAM (1 mM) for 15 min. The
asterisk indicates the presence of impurity in the commercial HPE-
IAM sample. HS-HPE-AM coelutes with bis-SS-HPE-AM under these
conditions. (b) A comparison of sulfur species (e.g., H2S, H2S2, and
H2S3) measured by detection of the trapped HPE-AM species from
H2S alone vs H2S reaction with HNO.
Scheme 2. Proposed Mechanism of HNO Reaction with H2S
(Supporting Information, Figure S7), confirming NH2OH
generation under these conditions.
We then analyzed the reaction of H2S with excess HNO,
analogous to previously studied thiol−HNO reactions, which
primarily result in sulfinamide formation.4 Incubation of H2S
with excess AS (4 equiv) results in a purple solution that turns
colorless within 5 min with concomitant formation of a white
precipitate. We speculate that the white solid formed under
these conditions is S8. To explore this possibility, we analyzed
for S8 by a triphenylphosphine (PPh3)-based 31P NMR assay.
PPh3 is known to react with S8 to form triphenylphosphine
sulfide (SPPh3), which can be detected by 31P NMR
spectroscopy.40 We extracted the white precipitate formed
during the reaction of H2S with excess HNO in CDCl3 and
incubated with PPh3. 31P NMR analysis of this mixture shows a
new peak at 43.3 ppm (Figure 4b), indicating S8 generation
under these conditions. The same species is also generated
from the reaction of authentic S8 with PPh3 (Figure 4a). Based
on a calibration curve generated from the reaction of known
Path A) or sulfinamide (Scheme 2, Path B). In addition, the N-
mercaptohydroxylamine intermediate might also undergo
dehydration to yield thionitrosyl hydride (HNS) (Scheme 2,
Path C).
First, we analyzed the products of this reaction under
conditions of excess H2S. We anticipated that if the N-
mercaptohydroxylamine intermediate 1 reacts further with
H2S, we should observe H2S2 and NH2OH (Scheme 2, Path
A). We examined H2S2 generation by trapping with β-(4-
hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) (Scheme 3).
Scheme 3. Polysulfide Trapping with HPE-IAM to Produce
Bis-(S)n-HPE-AM
HPE-IAM was chosen because it is a soft electrophile and has
been shown to be relatively resistant toward electrophile-
mediated decomposition of longer chain polysulfides,38,39 if
they are formed. As expected, ultraperformance liquid
chromatography−mass spectrometry (UPLC−MS) analysis
of H2S incubation with HPE-IAM shows thioether bis-S-
HPE-AM formation as a major product (Figure 3a, bottom
trace). However, analogous analysis of H2S (4 equiv)
incubation with HNO shows a significant increase in bis-SS-
HPE-AM formation with concomitant decrease in bis-S-HPE-
AM (Figure 3b), consistent with H2S2 generation. In addition,
a small amount of bis-SSS-HPE-AM is also observed,
presumably produced by disproportionation of H2S2 to H2S3
and H2S. Interestingly, no longer-chain polysulfides are
observed under these conditions. We also examined NH2OH
formation, another anticipated product of HNO reaction with
excess H2S (Scheme 2, path A), by derivatization with 4-
cyanobenzaldehyde. High-performance liquid chromatography
(HPLC) analysis shows 4-cyanobenzaldehyde oxime formation
Figure 4. Selected region of the 31P NMR spectra following
incubation of (a) authentic S8, and (b) the white precipitate produced
during the reaction of H2S (5 mM) with HNO (20 mM) extracted in
CDCl3 with PPh3 (−5.3 ppm). SPPh3 is observed at 43.3 ppm. The
peak at −17.7 ppm corresponds to triphenyl phosphate (O
P(OPh)3), which is used as an internal standard.
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amounts of commercially available elemental sulfur (S8) with
PPh3, we determined the S8 yield to be approximately 79% of
the sulfur introduced into the reaction mixture. To verify S8
generation, the white solid was independently analyzed by
electron ionization (EI)-MS. As anticipated, a new peak with
m/z = 257.8 (expected m/z = 257.5) is observed (Supporting
Information, Figure S8), confirming S8 generation.
points to 8:1 at the 25 min mark (Supporting Information,
Figure S3). These results suggest another contributor to the
m/z = 28 signal, which we attribute to N2. The variation in the
44:28 ratio over the course of the experiment suggests that N2
is generated at early times and decreases as the experiment
progresses.
We independently examined the H2S reaction with excess
HNO using 2-bromo-Piloty’s acid (2-BrPA). In aqueous
solution, 2-BrPA produces HNO and 2-bromophenylsulfinic
acid as a byproduct.38 Incubation of H2S with 2-BrPA (4
equiv) in pH 7.4 PBS again shows the formation of S8 and N2
(Supporting Information, Figures S4 and S9). This result
confirms that nitrite, a byproduct of AS decomposition, is not
involved in S8 and N2 formation during the H2S reaction with
excess HNO.
Two pathways can be envisioned for S8 generation under
conditions of H2S reaction with excess HNO (Scheme 4). As
Scheme 4. Proposed Mechanism of S8 Generation from H2S
Reaction with Excess HNO
Alternatively, S8 can also be produced by disproportionation
of the N-mercaptohydroxlamine intermediate 1 followed by
intramolecular cyclization as shown in Scheme 4, eq 3.
However, HPE-IAM trapping studies at early time points,
before the formation of the white precipitate, show that no
longer-chain polysulfides are formed (Supporting Information,
Figure S15), indicating that the proposed mechanism in
Scheme 4, eq 3 is likely not operative under these conditions.
Furthermore, intermediate 1 could also undergo homolytic
cleavage to produce hydrosulfide radicals (HS•), which can
lead to higher order sulfur species in a process catalyzed by
either trace metals or residual oxygen present in solution.43
However, we do not observe changes in the S8 yield from the
HNO reaction with H2S under aerobic versus anaerobic
conditions (Supporting Information, Figure S10). In addition,
the presence or lack of a metal chelator diethylenetriamine-
pentaacetic acid (DTPA) in solution also does not influence
the final products of the reaction, indicating that HS• is likely
not involved in this reaction.
Based on the significant S8 formation observed under
conditions of excess HNO, it appears that sulfinamide
formation is not a major pathway under these conditions;
however, more studies are required. In addition, as suggested
in Scheme 2, Path C, the N-mercaptohydroxylamine
intermediate 1 could undergo dehydration to yield HNS. If
formed, HNS may undergo further reaction, similar to HNO,
yielding N2S and H2S. However, MIMS analysis shows no
evidence of HNS or N2S formation under conditions of either
excess HNO or H2S (Supporting Information, Figure S5),
indicating that this reaction pathway is presumably not
operative. Taken together, our results indicate that H2S reacts
with HNO to produce either short-chain hydrogen polysulfides
(H2Sn) or S8 depending on their relative concentrations. With
excess H2S, H2Sn formation is favored (Scheme 2, Path A). In
contrast, S8 is produced under conditions of excess HNO
(Scheme 4, eqs 1 and 2).
shown in Scheme 4, eq 2, reaction of the initial N-
mercaptohydroxlamine intermediate 1 with a second equiv-
alent of HNO would lead to the formation of intermediate 2.
This pathway is supported by our GC data (Figure 2) in which
H2S incubation with 4 equiv of HNO shows nearly 2 equiv of
HNO trapping, supporting that intermediate 1 reacts with a
second equivalent of HNO to produce intermediate 2. We
propose that intermediate 2 can undergo a sulfur extrusion
reaction to generate S0 and dihydroxyhydrazine, which then
decomposes to produce nitrogen (N2) and water.41 To test
this hypothesis, we analyzed for N2 generation using MIMS.
Incubation of H2S under conditions of excess HNO shows an
increase in the m/z = 28 signal (Figure 5), indicating N2
Figure 5. MIMS signals observed at m/z = 44 corresponding to N2O+
and m/z = 28 corresponding to a combination of N2O fragmentation
to N2+ and N2 generated from the reaction of H2S with excess HNO.
2.2. Reactivity of HNO with Hydropersulfides. The
propensity of RSSH to undergo decomposition under aqueous
conditions precludes convenient and direct accessibility of
RSSH for chemical and biological studies. Hence, donor
molecules capable of releasing RSSH in situ are needed. We
utilized our recently developed alkylamine-substituted perthio-
carbamate 3a as a primary alkyl RSSH donor, and 3b as a
tertiary alkyl RSSH donor (Scheme 5).44 At physiological pH,
these precursors release RSSH and 1,3-dimethyl-2-imidazoli-
dinone (4) as a byproduct (Scheme 5, eq 1). In the absence of
trapping agents, RSSH reacts with the precursor producing
dialkyltrisulfide (S3) and a thiocarbamate intermediate
generation. However, the detection of N2 is complicated by
N2O (m/z = 44) fragmentation, which also generates an m/z =
28 signal (Supporting Information, Figure S2). Hence, we
compared the ratio of 44:28 signals from AS alone with that for
AS + H2S. The 44:28 ratio for N2O alone produced from AS
decomposition was found to be 9.5:1 (Supporting Information,
Figure S3). This result is consistent with the NIST reported a
44:28 ratio of 9.25:1 for the EI mass spectrum of N2O.42 In
contrast, MIMS monitoring of the H2S reaction with excess
HNO results in a 44:28 ratio that varies from 2:1 at early time
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their pKa. We assumed a pKa difference of 1.5 units for this
study. With this assumption, the hydropersulfide is 98%
deprotonated at physiological pH. After correcting for
differences in pKa, we find that RSSH (3a and 3b) are better
traps for HNO than their thiol counterparts (5a and 5b)
(Figure 7). Additionally, 3b exhibited better HNO-trapping
Scheme 5. RSSH Release from Precursors 3a and 3b
(Scheme 4, eq 2), which rapidly decomposes to release
carbonyl sulfide (COS) (Scheme 5, eq 3). In addition, RSSH
also undergoes disproportionation reactions to produce
dialkylpolysulfides (Scheme 5, eqs 4 and 5).44
Initially, the reaction of HNO with RSSH was examined by
MIMS. Incubation of AS (100 μM) with 3a (25 μM) in PBS
(pH 7.4) shows a reduction in the signal corresponding to
N2O (m/z = 44) (Figure 6a), indicating that RSSH reacts with
Figure 7. GC-determined relative yield of N2O in the presence of
increasing concentrations of 3a (black bars), 3b (red bars), N-
acetylcysteine methyl ester (5a, blue bars), and N-acetylpenicillamine
methyl ester (5b, green bars). Incubations were performed with 0, 50,
100, and 200 μM RSS− or RS− and 200 μM AS in PBS (pH 7.4, 100
mM) with DTPA (100 μM) at 37 °C for 3 h.
efficiency than 3a, likely because of competitive RSSH trapping
by the sterically accessible disulfide bond in the precursor 3a
itself. We note that even if the pKa of RSSH is 4 units lower
than the corresponding thiol, the RSSH anion concentration
would be 99.9% (rather than 98%) under the conditions of our
experiments, a negligible impact on the RSSH concentrations
employed.
Next, we studied the mechanism of this reaction. Based on
the known thiol−HNO reaction, a variety of outcomes for the
reaction between RSSH and HNO are possible. The initial
reaction of HNO with RSSH is expected to proceed through
an N-hydroxy-perthiosulfenamide (RSS−NH−OH) intermedi-
ate 6 (Scheme 6). As with thiols, we anticipate that the
concentration of RSSH relative to that of HNO will play an
important role in the nature of the observed products.
Initially, 3a decomposition was examined in the absence of
HNO by UPLC−MS. Incubation of 3a in pH 7.4 buffer shows
dialkyldisulfide (labelled as S2) generation as a major product
(Figure 8a, bottom trace). In addition, a small amount of
dialkyltrisulfide, dialkyltetrasulfide, dialkylpentasulfide, and
Figure 6. MIMS signals observed at (a) m/z = 44 corresponding to
N2O+ during incubation of either AS (100 μM) alone or with RSSH
precursor 3a (25 μM), and (b) m/z = 60 corresponding to COS+
during incubation of either RSSH precursor 3a (25 μM) alone or with
AS (100 μM) in argon-purged PBS (pH 7.4, 100 mM) containing
DTPA (100 μM) at 37 °C.
HNO. In addition, a reduction in the signal attributed to COS
(m/z = 60) is also observed (Figure 6b), also supporting that
RSSH reacts with HNO, and as a result it is less available to
react with precursor 3a itself to produce dialkyltrisulfide (S3)
and thiocarbamate-derived COS (Scheme 5, eqs 2 and 3).
Analogous experiments with precursor 3b also show HNO
trapping (Supporting Information, Figure S6), albeit better
than 3a presumably because of the sterically hindered disulfide
bond in precursor 3b inhibiting its reaction with released
RSSH.
Scheme 6. Proposed Mechanism of HNO Reaction with
RSSH
The ability of RSSH to react with HNO was independently
analyzed by GC headspace analysis. A thiol comparison was
used to examine the nucleophilicity of RSSH. We began by
determining the pKas of N-acetylcysteine methyl ester (5a, pKa
= 7.28) and N-acetylpenicillamine methyl ester (5b, pKa =
7.02). Because of the unstable nature of RSSH in the
deprotonated state, there is not a reliable way to determine
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These results suggest that intermediate 6 undergoes dehy-
dration to produce the intermediate 7 (Scheme 6, Path C),
which can be trapped either by water to produce RS−S(O)−
NH2 or by RSSH to produce RSS−NH−SSR. The lack of RS−
S(O)−NH2 formation does not exclude its formation because
it can react further with RSSH under these conditions to
produce S3.
We also examined the HNO reaction with the tertiary alkyl
RSSH precursor 3b. UPLC−MS analysis of 3b incubation in
the absence of HNO shows a peak at 5.67 min corresponding
to RSSH as well as polysulfides (RSSnSR, n = 1−4) and N-
acetylpenicillamine methyl ester (5b) (Figure 9a, bottom
Figure 8. (a) UPLC−MS chromatograms of primary alkyl hydro-
persulfide precursor 3a (50 μM) incubated without and with
increasing concentrations of AS (50, 200, 400, and 800 μM) in
ammonium bicarbonate buffer (pH 7.4, 25 mM) containing DTPA
(100 μM) at 37 °C for 15 min, followed by quenching in 1% formic
acid. RSSH-derived symmetrical dialkyl polysulfide, labeled as S2 to S6
(RSSnSR, n = 0−4) formation is evident. A peak at 3.52 min
attributed to the byproduct 4 is also observed. A small peak at 5.4 min
corresponding to RSS−NH−SSR coelutes with S4. (b) Comparison of
RSSH-derived symmetrical dialkyl polysulfide, labelled as S2 to S6
(RSSnSR, n = 0−4) from the 3a and 3a + AS reaction mixtures.
dialkylhexasulfide (labeled as S3, S4, S5, and S6, respectively)
formation is also observed, presumably via RSSH disproportio-
nation reactions (Scheme 5, eqs 4 and 5). In contrast, when 3a
is incubated with AS (1 equiv), a slight decrease in S2 with a
concomitant increase in S3 is observed (Figure 8b).
Furthermore, as the ratio of AS to 3a is increased, an increase
in the relative amount of S3 with a concomitant decrease in S2
is observed. These results suggest that RSSH indeed reacts
with HNO to produce intermediate 6, which reacts further
with RSSH producing S3 and N-mercaptohydroxylamine
intermediate 1 (Scheme 6, Path A). The intermediate 1
might react further with RSSH to produce RSSSH. Consistent
with this observation, we also observe reduced levels of S4, S5,
and S6 with increasing concentrations of AS, demonstrating
that RSSH traps HNO, and thus it is less available to undergo
disproportionation reactions to produce longer-chain poly-
sulfides. Alternatively, RSSH can also react with the external
sulfur of intermediate 6 to produce S4 and NH2OH (Scheme 6,
Path B). However, a lack of major change in the S4
concentration with increasing concentration of HNO suggests
that Path B is likely not operative under these conditions,
presumably because of the sterically accessible internal sulfur
atom of intermediate 6 being available to react with RSSH to
produce S3. We anticipated that the intermediate 6 might
rearrange to sulfenylsulfinamide (RS−S(O)−NH2) under
conditions of excess HNO (Scheme 6, Path C). However,
UPLC−MS analysis shows no evidence of its formation.
Instead, a minor new peak at 5.4 min with m/z = 454.0199 is
observed (Supporting Information, Figure S22). We assign this
new peak to RSS−NH−SSR (Figure 8a, labeled as S2NHS2).
Figure 9. (a) UPLC−MS chromatograms of tertiary alkyl hydro-
persulfide precursor 3b (50 μM) incubated without and with
increasing concentrations of AS in ammonium bicarbonate buffer
(pH 7.4, 25 mM) containing DTPA (100 μM) at 37 °C for 15 min,
followed by quenching in 1% formic acid. RSSH-derived symmetrical
dialkylpolysulfide, labelled as S3 to S6 (RSSnSR, n = 1−4), formation is
evident. A peak at 3.52 min attributed to the byproduct 4 is also
observed. The asterisk indicates an unknown product. (b)
Comparison of RSSH-derived symmetrical dialkylpolysulfide, labeled
as S3 to S6 (RSSnSR, n = 1−4) and RSS−NH−SnR (n = 1−3) from 3b
and 3b + AS reaction mixtures.
trace). This result indicates that RSSH undergoes dispropor-
tionation reactions and its presence is likely because of
equilibrium reactions with polysulfides.44 We then examined
the RSSH reaction with HNO under conditions of excess
RSSH. When 4 equiv of 3b is incubated with AS, the peak
attributed to RSSH disappears and the level of S3 increases
(Figure 9b, red bars). In addition, small peaks at 6.14, 6.43,
and 6.74 min that we ascribe to RSS−NH−SR, RSS−NH−
SSR, and RSS−NH−SSSR, respectively (Figure 9a and
Supporting Information, Figures S33−S35, labeled as S2NHS,
S2NHS2, and S2NHS3) are observed, presumably formed by
the reaction of intermediate 7 with RSH, RSSH, and RSSSH,
respectively. We then examined the 3b reaction with AS at
equimolar concentrations. UPLC−MS analysis shows the
reduced level of S3 and increased levels of S4 and RSS−
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NH−SnR (n = 1−3) (Figure 9b, blue bars). Furthermore, the
3b reaction was also checked under conditions of excess HNO
and we observe reduced levels of polysulfides (S3, S4, and S5)
and RSS−NH−SnR (n = 1−3). In addition, two new minor
peaks at 2.9 and 3.3 min with m/z = 205.643 were observed
(Figure 9a, top trace and Supporting Information Figures S30
and S31). We assign these peaks to N-(2-hydroxy-5,5-
dimethyl-3-oxoisothiazolidin-4-yl)acetamide isomers 9, pre-
sumably formed by the intramolecular cyclization of
intermediate 8 as shown in Scheme 7. Intermediate 8 can be
S41). As expected, UPLC−MS analysis shows a significant
reduction in RSS-HPE-AM adduct formation during the 3b
reaction with HNO compared with 3b alone, confirming that
RSSH indeed reacts with HNO. However, no evidence of
inorganic polysulfide formation is observed, suggesting that
RSSH reacts efficiently with HNO and as a result is less
available to undergo disproportionation reactions to produce
inorganic polysulfides. Taken together, these results indicate
that ONSS− is likely not formed under these conditions.
3. CONCLUSIONS
Scheme 7. Proposed Mechanism of 9 Formation from the
3b Reaction with Excess HNO
Reactive sulfur species (RSS) and reactive nitrogen species
play diverse and critical roles in cellular signaling and the
fundamental chemistry of these species as well as their
generation and consumption are critical for understanding
their participation in signaling mechanisms. In this work, we
first studied the reaction of HNO with H2S. Our results
indicate that H2S also reacts with HNO to produce either
hydrogen polysulfides (H2Sn) or S8 depending on their relative
concentrations. H2Sn represents an emerging class of RSS
whose presence and potential roles in biological systems are
only recently beginning to be appreciated.46 In addition,
comparison of the reactivity of thiol with analogous RSSH
shows that RSSH are more potent traps for HNO. These
results indicate the specificity of HNO signaling may be a
function of reaction with RSSH. Furthermore, HNO reaction
with small molecule RSSH produces various RSSnSR and
RSS−NH−SnR species with no evidence of RS−S(O)−NH2
under the conditions studied.
obtained by the HNO reaction with thiol 5b, produced either
by RSSH exchange reactions with RS−Sn−SR or HNO-
induced decomposition of intermediate 6 (Scheme 7, Path B).
To verify this observation, 5b was independently incubated
with excess HNO and we observe HNO-mediated thiol
oxidation to disulfide as a major product (Supporting
Information, Figure S36). In addition, similar peaks at 2.9
and 3.3 min were evident in the case of the reaction of 5b with
HNO, suggesting that cyclization product 9 is likely formed
during the 3b reaction with excess HNO.
We predicted that the concentration of RSSH relative to that
of HNO would have an important role in the nature of the
observed products. However, all conditions studied here
indicate that HNO-induced modification of RSSH results in
the formation of various dialkylpolysulfides. Interestingly,
several unique species such as RSS−NH−SnR are also
observed.
Recently, Pluth and coworkers reported that RSSH react
with nitrite to produce NO via inorganic polysulfides and a
perthionitrite (ONSS−) intermediate.45 UV−vis analysis of the
ONSS− intermediate obtained from the reaction of adamantyl
persulfide with tetrabutylammonium nitrite in THF exhibits an
absorbance at 446 nm. Decomposition of ONSS− subsequently
leads to the formation of NO and inorganic polysulfide. To test
if a similar reaction pathway is operative during the reaction of
3b with AS (which produces nitrite as byproduct), we analyzed
this reaction by UV−vis spectroscopy. Incubation of 3b with
AS in pH 7.4 PBS at 37 °C shows no absorption band between
400 and 450 nm (Supporting Information, Figure S43),
indicating that ONSS− is likely not produced under these
conditions. We also examined the 3b reaction with sodium
nitrite and UV−vis analysis shows no absorption band between
400 and 450 nm (Supporting Information, Figure S44). We
then analyzed NO generation from the reaction of 3b with AS,
and nitrite by GC headspace analysis. We see no evidence of
NO generation (Supporting Information, Figure S46),
indicating that RSS− is likely not reacting with nitrite under
these conditions. We also examined the RS(S)nH, and
HS(S)nH generation during the reaction of 3b with HNO by
trapping with HPE-IAM (Supporting Information, Figure
4. EXPERIMENTAL PROCEDURES
4.1. General Methods. All chemicals were purchased from
commercial sources and used as received unless stated otherwise.
NMR spectra were obtained on a 400 MHz FT-NMR spectrometer.
All chemical shifts of spectra were reported in parts per million (ppm)
relative to tetramethylsilane (δ = 0). The pH measurements were
performed using a Fisher Scientific Accumet AB15 pH-meter.
Ultraviolet−visible (UV−vis) absorption spectra were obtained
using a diode-array spectrophotometer. GC analysis was performed
on an instrument equipped with an electron capture detector and
Restek column (ShinCarbon ST 80/100, 2m, 1/8″ OD). HPLC was
performed on Agilent Technologies 1100 Series, attached with a C-18
column (Hichrom, 5 μm, 4.6 × 150 mm). High-resolution mass
spectra were obtained from a Waters Acquity Q-ToF MS/MS
instrument. UPLC−MS analysis was carried out with a Waters
Acquity/Xevo-G2 UPLC−MS system equipped with ACQUITY
UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm). The mass signals
for products of polysulfides trapping with HPE-IAM and dialkylpo-
lysulfides were obtained via deconvolution using MassLynx 4.1
software. EI-MS spectra were acquired using a VG-70S Magnetic
Sector Mass Spectrometer. To ensure that equal amounts of anion
(HS− and RS− or RSS− and RS−) are present in solution during the
reaction of sulfur nucleophiles with HNO, we calculated the
concentration of anion using the pKa of H2S and thiol/RSSH of
interest to determine the percentage of anion present at pH 7.4; pH =
pKa + log(A−)/(HA)
4.2. Synthesis and Characterization. The HNO donors,
Angeli’s salt,47 and 2-bromo-N-hydroxybenzenesulfonamide (2-
BrPA)48 were prepared as previously described. Hydropersulfide
precursors (3a and 3b), N-acetyl-penicillamine methyl ester (5b), and
4-cyanobenzaldehyde oxime were synthesized as previously reported,
and analytical characterization data were consistent with the reported
values.44,49
4.3. Analysis of HNO Reaction with H2S and RSSH by MIMS.
MIMS was carried out using a Hiden HPR-40 system containing a 20
mL sample cell and a membrane selective for detecting gases dissolved
G
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in aqueous solution. The sample cell was filled with 20 mL of PBS
(pH 7.4, 100 mM) containing DTPA (100 μM) and purged with
argon for at least 30 min prior to analysis. A stock solution of AS was
prepared in 10 mM NaOH. A preweighed solid sample of Na2S was
dissolved in PBS to obtain the desired concentration of H2S in
solution. Hydropersulfide precursor and thiol stock solutions were
prepared in DMSO. These stock solutions were purged with nitrogen
for 10 min and used shortly after preparation. Aliquots (200 μL) of
these solutions were injected into the sample cell using a gastight
syringe and masses of interest were monitored with continuous
sampling in positive ion mode.
4.4. GC Headspace Analysis of HNO Reaction with H2S and
RSSH. Hydropersulfide precursor and thiol stock solutions were
prepared in DMSO. In order to compare the inherent nucleophilicity
of the hydropersulfide compared to its thiol counterpart, the
concentrations of hydropersulfide and thiol were corrected to have
the same amount of anion present in solution. As the pKa values of
hydropersulfides are not known, it was assumed to be 1.5 units lower
than the determined thiol pKa. In a 15 mL vial sealed with rubber
septum, 5 mL PBS (pH 7.4, 100 mM) containing DTPA (100 μM)
was purged with argon for 25 min. These vials were placed in a heated
cell block, which was held at 37 °C. The Na2S or RSSH precursor or
thiol and AS solutions were added to each vial to obtain 5 mL total
volume, and the resulting solutions were incubated for 2 h at 37 °C.
Headspace gas samples (60 μL) were injected into Agilent 8860 GC
attached with Restek column (ShinCarbon ST 80/100, 2m, 1/8″
OD) to analyze N2O. These experiments were carried out in triplicate
for each concentration of interest and three injections were performed
for each vial.
The reaction mixture was then extracted with CDCl3 (1.5 mL × 3).
To this, 500 μL of PPh3 (50 mM, stock solution prepared in CDCl3)
was added and the resulting solution was incubated overnight at rt in
a sealed vial. An internal standard triphenyl phosphate (1 mM) was
added to the reaction mixture and analyzed using 31P NMR
spectrometry. A calibration curve was generated by reacting known
amounts of commercially available sulfur (S8) with equimolar
amounts of PPh3 along with 1 mM OP(OPh)3 as an internal
standard. 31P NMR spectra were acquired in CDCl3 on a Bruker
AVANCE I 400 MHz UltraShield NMR spectrometer.
4.8. Analysis of S8 by EI-MS. As a second method of
confirmation, the white precipitate produced from the reaction of
H2S with HNO (4 equiv) was analyzed by EI-MS. In a 20 mL
scintillation vial, a reaction mixture was prepared with a final
concentration of 2 mM Na2S and 8 mM AS giving a final reaction
volume of 10 mL in pH 7.4 PBS (100 mM) containing DTPA (100
μM). The reaction was allowed to proceed until completion
(approximately 2−3 h) and was then extracted with chloroform
(1.5 mL × 3). The solvent was evaporated under vacuum to yield a
white solid that was analyzed by EI-MS.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c02412.
■
sı
UV−vis spectra, HPLC traces, GC chromatograms,
MIMS analysis, UPLC−MS traces, mass spectra, and 31
NMR spectra (PDF)
P
4.5. Analysis of Polysulfides by UPLC−MS. Polysulfides
generated from the reaction of H2S with HNO were analyzed by
trapping with HPE-IAM by UPLC−MS. The reaction was performed
in a 20 mL scintillation vial with a total reaction volume of 3 mL. H2S
(1 equiv) was incubated with various concentrations of AS (0.25 or 4
equiv) in freshly prepared ammonium bicarbonate buffer (pH 7.4, 50
mM) containing DTPA (100 μM). A 200 μL aliquot was taken from
the reaction mixture at specified times and added to a solution of
HPE-IAM (10 equiv) in ammonium bicarbonate buffer and incubated
for 30 min. The samples were then loaded into vials in an autosampler
maintained at 4 °C and analyzed using UPLC−MS as follows: mobile
phase: 0−1 min 90% water + 0% ACN + 10% formic acid (0.1%); 1−
7.5 min gradient up to 10% water + 80% ACN + 10% formic acid
(0.1%); 7.5−8.4 min 10% water + 80% ACN + 10% formic acid
(0.1%); 8.4−8.5 min gradient up to 90% water + 0% ACN + 10%
formic acid (0.1%); and 8.5−10 min 90% water + 0% ACN + 10%
formic acid (0.1%). Flow rate = 0.3 mL min−1. Similarly, various
RSSnSR and RSS−NH−SnR species produced from the reaction of
RSSH with HNO were analyzed by UPLC−MS. The mass signals for
bis-(S)n-HPE-AM, RS(S)nSR, and RSS−NH−(S)nR were obtained
via deconvolution using MassLynx 4.1 software.
AUTHOR INFORMATION
Corresponding Author
■
John P. Toscano − Department of Chemistry, Johns Hopkins
University, Baltimore, Maryland 21218, United States;
orcid.org/0000-0002-4277-3533; Email: jtoscano@
jhu.edu
Authors
Jessica Zarenkiewicz − Department of Chemistry, Johns
Hopkins University, Baltimore, Maryland 21218, United
States
Vinayak S. Khodade − Department of Chemistry, Johns
Hopkins University, Baltimore, Maryland 21218, United
States; orcid.org/0000-0003-2406-5856
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c02412
4.6. Hydroxylamine Analysis. An HPLC-based assay has been
used for the detection of hydroxylamine (NH2OH) by derivatization
with vanillin.50 We used this assay with slight modification. Briefly,
H2S (400 μM) was incubated with AS (100 μM) in pH 7.4 PBS (100
mM, 2 mL total volume) containing DTPA (100 μM) for 30 min at
37 °C. This mixture was then incubated with 4-cyanobenzaldehyde (1
mM) in pH 5.5 sodium acetate buffer (100 mM) for 2 h at 37 °C to
convert NH2OH to 4-cyanobenzaldehyde oxime. The resulting
mixture was analyzed by Agilent high-performance liquid chromatog-
raphy (HPLC). HPLC methodmobile phase A: water, and mobile
phase B: ACN, flow rate: 1 mL/min, run time: 24 min, and the
gradient elution method: 10 to 25% B from 0 to 18 min, 25 to 90% B
from 18 to 24 min. Detection wavelengths: 254 and 268 nm. Column:
Hichrom C-18 reversed phase column (150 mm × 4.6 mm, 5 μm).
4.7. Analysis of S8 Using a Triphenylphosphine-31P NMR-
Based Assay. A white precipitate formed in the reaction of H2S with
HNO was analyzed using a triphenylphosphine (PPh3)-based 31P
NMR assay. In a 20 mL scintillation vial, Na2S (5 mM) was incubated
with AS (20 mM) in ammonium bicarbonate buffer (pH 7.4, 50 mM)
containing DTPA (100 μM) (final volume 5 mL) for 2 h at 37 °C.
Author Contributions
†J.Z. and V.S.K. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We gratefully acknowledge the National Science Foundation
(CHE-1900285) for generous support for this research.
■
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