Synthesis and kinetics of decomposition of some novel S-nitrosothiols

Article abstract of DOI:10.1139/v99-026

The S-nitrosothiols 2-acetamido-2-deoxy-S-nitroso-1-thio-β-D-glucopyranose 3,4,6-triacetate (GPSNO) and S-nitroso-N-carbamyl-D,L-penicillamine (SNCP) were synthesized by S-nitrosation of the corresponding thiols, isolated, and fully characterized. The nitrosothiol (TGSNO) from 1-thioglycerol was obtained as a red gelatinous liquid, which decomposed rapidly at room temperature and so was not characterized. The kinetics of decomposition of GPSNO showed that there is a surprisingly large thermal pathway overlaid with a Cu²⁺/RS^- catalyzed reaction. The results strongly suggest that the product disulfide complexes Cu²⁺ (for which there is some spectral evidence), leading to incomplete conversion by that route. Ascorbate also acts as a Cu²⁺ reductant. Another S-nitroso sugar, S-nitroso-1-thio-β-D-glucose (SNTG), behaved very similarly from solutions generated and used in situ. The decomposition of TGSNO shows induction periods suggesting that slow initial generation of Cu⁺ (the true catalyst) is taking place. There appears to be also a significant alternative pathway (analogous to that found for GPSNO), where the rate appears to be independent of [Cu²⁺], but very unusually this pathway is effectively halted by addition of EDTA either at the start of the reaction or at a later time. Reaction schemes are put forward to account for these unusual reaction characteristics.

Full text of DOI:10.1139/v99-026

550
Synthesis and kinetics of decomposition of
some novel S-nitrosothiols
Andrew P. Munro and D. Lyn H. Williams
Abstract: The S-nitrosothiols 2-acetamido-2-deoxy-S-nitroso-1-thio-β-D-glucopyranose 3,4,6-triacetate (GPSNO) and
S-nitroso-N-carbamyl-D,L-penicillamine (SNCP) were synthesized by S-nitrosation of the corresponding thiols, isolated,
and fully characterized. The nitrosothiol (TGSNO) from 1-thioglycerol was obtained as a red gelatinous liquid, which
decomposed rapidly at room temperature and so was not characterized. The kinetics of decomposition of GPSNO
showed that there is a surprisingly large thermal pathway overlaid with a Cu2+/RS– catalyzed reaction. The results
strongly suggest that the product disulfide complexes Cu2+ (for which there is some spectral evidence), leading to
incomplete conversion by that route. Ascorbate also acts as a Cu2+ reductant. Another S-nitroso sugar, S-nitroso-1-thio-
β-D-glucose (SNTG), behaved very similarly from solutions generated and used in situ. The decomposition of TGSNO
shows induction periods suggesting that slow initial generation of Cu+ (the true catalyst) is taking place. There appears
to be also a significant alternative pathway (analogous to that found for GPSNO), where the rate appears to be
independent of [Cu2+], but very unusually this pathway is effectively halted by addition of EDTA either at the start of
the reaction or at a later time. Reaction schemes are put forward to account for these unusual reaction characteristics.
Key words: S-nitrosothiols, nitric oxide, ascorbate, copper catalysis.
Résumé : On a synthétisé les S-nitrosothiols, 3,4,6-triacétane de 2-acétamido-2-désoxy-S-nitroso-1-thio-β-D-glucopyra-
nose (GPSNO) et S-nitroso-N-carbamyl-D,L-pénicillamine (SNCP), en procédant à la S-nitrosation des thiols correspon-
dants, on les a isolés et caractérisés complètement. Le nitrosothiol (TGSNO) obtenu à partir du 1-thioglycérol est un
liquide gélatineux rouge qui se décompose rapidement à la température ambiante et il n’a pas été caractérisé. La ciné-
tique de décomposition du GPSNO montre qu’il existe une voie réactionnelle thermique importante qui est recouverte
par une réaction catalysée par Cu2+/RS–. Les résultats suggèrent fortement que le disulfure formé se complexe avec le
Cu2+ (quelques données spectrales supportent ces hypothèses); cette interaction provoque une conversion incomplète
par cette voie. L’ion ascorbate agit aussi comme réducteur du Cu2+. Un autre S-nitrososucre généré en solution et uti-
lisé in situ, le S-nitroso-1-thio-β-D-glucose (SNTG), se comporte d’une façon très semblable. La décomposition du
TGSNO présente des périodes d’induction qui suggèrent une génération initiale lente du Cu+ (le vrai catalyseur). Il
semble exister une voie de réaction alternative importante (analogue à celle observée pour le GPSNO) dans laquelle la
vitesse semble indépendante de [Cu2+]; il est toutefois surprenant de réaliser que cette voie est effectivement inter-
rompue par l’addition d’EDTA soit au début de la réaction soit plus tard. Des schémas réactionnels sont proposés pour
expliquer ces caractéristiques réactionnelles inusitées.
Mots clés : S-nitrosothiols, oxyde nitrique, ascorbate, catalyse par le cuivre.
[Traduit par la Rédaction] Munro and Williams 556
Introduction
the storage and transport of NO in the body. S-Nitrosoglu-
tathione GSNO (1), S-nitrosoproteins (2), and the S-nitroso
derivative of hemoglobin (3) have been identified as natu-
rally occurring compounds, and there are literature hypothe-
ses regarding their role in the storage and transport of NO
around the body (2, 4). A recent suggestion that the inter-
conversion between the Fe-NO and side chain S-NO forms
in nitrosated hemoglobin, in some way controls blood pres-
sure (3), has attracted much interest. The biological (5) and
chemical (6) properties of S-nitrosothiols have recently been
reviewed.
Formerly, it was believed that most RSNO compounds
(with a few exceptions, notably GSNO and S-nitroso-N-acetyl
penicillamine, SNAP) were too unstable in the pure state to
allow their isolation and characterization, but in recent years
an increasing number have been shown to be stable at room
temperatures, although others decompose during extraction.
In general, the corresponding O-nitroso compounds, the
alkyl nitrites, are much more widely stable and of course
One aspect of the continuing intense and widespread in-
terest in the “nitric oxide story” concerns the chemistry of
S-nitrosothiols, RSNO. Not only are these compounds under
active consideration as possible nitric oxide donors for med-
ical therapeutic use, but also currently there is much interest
in establishing the part played by them in vivo, possibly in
Received February 16, 1998.
This paper is dedicated to Jerry Kresge in recognition of his
many achievements in chemistry.
A.P. Munro and D.L.H. Williams.1 Department of
Chemistry, Durham University, South Road, Durham
DH1 3LE, U.K.
1Author to whom correspondence may be addressed.
Telephone: (44) 191 374 3125. Fax: (44) 191 384 4737.
e-mail: D.L.H.Williams@durham.ac.uk
Can. J. Chem. 77: 550–556 (1999)
© 1999 NRC Canada

Munro and Williams
551
well known. Fairly dilute (1 × 10–3 to 1 × 10–4 M) solutions
of all RSNO are easily generated from thiols and nitrous
acid in an equilibrium process, which lies very much on the
side of RSNO, and are usually sufficiently stable (even for
those examples which cannot be obtained in the pure state)
to allow experiments to be successfully carried out in situ.
For most RSNO compounds that have been studied, the
thermal decomposition reaction is rather slow at room tem-
perature, with half lives of many tens of hours. However, we
have shown (7, 8) that in solution, in phosphate buffer at
pH 7.4, RSNO species decompose giving NO and RSSR, in
a copper-catalyzed process. The effective reagent is Cu+,
generated in situ by thiolate reduction of Cu2+ (eqs. [1]–[3]),
which in many cases occurs when both RS– and Cu2+ are
present only at the trace impurity level. In contrast to the
corresponding photochemical reaction, there is no EPR evi-
dence for intermediate thiyl radical formation. There is a
large reactivity range that results from the presence or other-
wise of suitable substituents (e.g., -NH2) which will allow
bidentate binding by Cu+ to occur. If the reactions are car-
In this paper we describe the synthesis and characteriza-
tion of a S-nitrosothiol derived from a thio sugar and exam-
ine its decomposition together with that of the S-nitroso
derivative of 1-thioglycerol (prepared in solution) to nitric
oxide, both of which turn out to show some unusual fea-
tures.
Experimental section
Materials
An aqueous solution of sodium nitrite (0.11 g in 1.65 cm3)
was added dropwise over 2 min with vigorous stirring to a
solution of a commercial sample of 2-acetamido-2-deoxy-1-
thio-β-D-glucopyranose 3,4,6-triacetate (0.30 g in 1:1 metha-
nol: 1 M HCl, 3.3 cm3, and containing concentrated sulfuric
acid 0.17 cm3). The mixture became pale red almost imme-
diately, was stirred for 1 h, and the precipitate collected by
filtration. The pink solid was washed with ice-cold water
and air-dried for 24 h. (65% yield). The solid was stable at
room temperature but decomposed on heating, so no melting
point could be recorded. Elemental analysis calcd. for
C14H20N2O9S: C 42.9, H 5.15, N 7.1%; found: C 43.0, H
5.03, N, 6.6%. The UV-visible spectrum showed λmax (H2O)
343 and 557 nm, ε 450 and 17 dm3 mol–1 cm–1 respectively.
The infrared spectrum included bands at 1572 cm–1 (NO
–
ried out aerobically then the ultimate fate of NO is NO2
after initial oxidation of NO, although at very low NO con-
centrations, such as those encountered in vivo, oxidation of
NO by oxygen is very slow and probably will not compete
with other NO reactions.
[1]
[2]
[3]
[4]
2Cu2+ + 2RS᎐ = 2Cu+ + RSSR
1
stretch) and 654 cm–1 (CS stretch). The complex H NMR
RSNO + Cu+ = RS– + Cu2+ + NO
spectrum was consistent with the expected structure 1.
᎐
4NO + 4OH᎐ + O2 = 4NO2 + 2H2O
Attempts to prepare the S-nitroso derivative of 1-thiogly-
cerol (3-mercapto-1,2-propanediol) TGSNO (structure 2) re-
sulted in the formation of a red gelatinous liquid, which
decomposed rapidly at room temperature, but which could
be kept for a short while below 4° in the dark. It was, how-
ever, too unstable to purify and characterize. However, a so-
lution prepared from equimolar quantities of 1-thioglycerol
and sodium nitrite gave a UV-visible spectrum with λmax 333
and 544 nm, ε 881 and 21 dm3 mol–1 cm–1, respectively.
Similarly, the S-nitroso derivative of 1-thio-β-D-glucose
SNTG (structure 4) was too unstable to isolate, but we ob-
tained the characteristic UV-visible spectrum with λmax 342
and 557 nm with ε 436 and 14 dm3 mol–1 cm–1, respectively.
These spectral characteristics are very similar to those found
for GPSNO.
RSNO + R′SH = R′SNO + RSH
Added thiols can produce a variety of effects. If the thiol
is structurally different from that from which RSNO is de-
rived, then transnitrosation (eq. [4]) can occur, leading to the
formation of a new R′SNO (9). If the added thiol is the same
as that from which RSNO is derived, then at low concentra-
tion of added thiol, catalysis can occur (increasing the rate
of Cu+ formation), whereas at higher concentrations, thiol
complexation of Cu2+ can occur, often resulting in the ap-
pearance of substantial induction periods, during which Cu+
is being generated from very low concentrations of free Cu2+
(8, 10). The relative importance of each reaction (reduction
and complexation) is governed by the thiol concentration
and structure. At very much higher added thiol concentra-
tions, a quite different reaction or series of reactions occurs,
leading principally to ammonia formation, together with
some nitrous oxide (11, 12).
Recently, we have shown that ascorbate ion can bring
about two reactions with S-nitrosothiols (13): (i) at low as-
corbate concentration, the copper catalyzed reaction is domi-
nant in which ascorbate takes over the role of the thiolate
and acts as a reducing agent for Cu2+ (this reaction can be
completely halted by EDTA addition), and (ii) at much
higher ascorbate concentration, a different reaction occurs,
unaffected by the presence of Cu2+ or EDTA, and which
leads to NO and thiol (and not diulfide) formation; under
these conditions, all the evidence suggests that ascorbate
acts as a nucleophile and undergoes electrophilic nitrosation
in the same way as do nitrous acid and alkyl nitrites — the
former being a very well known reaction.
The same synthetic procedure gave a 54% yield of the
S-nitroso derivative SNCP (structure 3) of N-carbamyl
penicillamine (provided as a gift by the former Wellcome
company). This was a stable green solid with red reflections,
very similar in appearence to the closely related and very
well known S-nitroso-N-acetyl penicillamine (SNAP). Ele-
mental analysis calcd. for C6H11N3O4S: C 32.6, H 4.98, N
19.0%; found: C 32.3, H 4.86, N 18.4%. The UV-visible
spectrum showed λmax (H2O) 340 and 590 nm, ε 853 and
20 dm3 mol–1 cm–1. The infrared spectrum included bands at
1
1494 cm–1 (NO stretch) and 661 cm–1 (CS stretch). The H
NMR spectrum was in accord with the expected structure
and included the characteristic (14) downfield shift of the
proton signals in the vicinity of the sulfur atom upon
nitrosation.
All other materials were commercial samples of the high-
est available purity grade.
© 1999 NRC Canada

552
Can. J. Chem. Vol. 77, 1999
CH3OCOCH2
CH3OCO
SNO
OH
O
SNO
OH
NHCOCH3
CH3OCO
1 (GPSNO)
2 (TGSNO)
HOCH2
SNO
O
HO
SNO
HO2C
NHCONH2
OH
HO
3 (SNCP)
4 (SNTG)
Results
Fig. 1. Absorbance–time plots for the decomposition of GPSNO
(1 × 10–3 M) in the presence of Cu2+: (a) no added Cu2+;
(b) [Cu2+] 0.1 × 10–5 M; (c) [Cu2+] 0.5 × 10–5 M;
(d) [Cu2+] 1.0 × 10–5 M; (e) 5.0 × 10–5 M.
2-Acetamido-2-deoxy-S-nitroso-1-thio-β-D-glucopyranose
3,4,6-triacetate (GPSNO), 1
We have prepared GPSNO by direct S-nitrosation of the
corresponding thiol, using a modification of the method used
by Field et al. (15) for the first synthesis of SNAP. It was
characterized by satisfactory elemental analyses and by the
observation of the NO and CS stretching frequencies in the
infrared spectrum and also by observation of the characteris-
tic UV-visible absorbances at 343 and 557 nm. The pink
solid is quite stable at room temperature. Analysis of an
aqueous solution of GPSNO by Ellman’s method (16)
showed that the thiol level resulting from the equilibration
of GPSNO in solution was 1.9% when the total GPSNO
concentration was -5 × 10–3 mol dm–3.
0.4
0.3
0.2
(a)
(b)
(c)
0.1
0
(d)
(e)
0
100
200
300
400
500
600
700
In solution at pH 7.4, the decomposition of GPSNO
showed the rather unusual absorbance–time plots shown in
Fig. 1 for a series of solutions containing different concen-
trations of added Cu2+. To separate any metal ion catalyzed
process from any other reaction, we carried out two experi-
ments with and without the addition of the general metal ion
chelator EDTA, and these traces are given in Fig. 2. The
faster reacting component of the reaction (which is Cu2+
catalyzed) is completely attenuated, leaving the other com-
ponent unchanged. Addition of the specific Cu+ chelator
neocuproine generated the same pattern of behaviour (not
shown).
The effect of ascorbate in low concentration was also in-
vestigated, and the results are presented graphically in
Fig. 3. Clearly ascorbate catalyzes the reaction, and also re-
sults in the quantitative decomposition of GPSNO observed
now as a single process.
time / min
Spectral measurements
These were all carried out in aqueous phosphate buffer,
pH 7.4, at 25° in a conventional spectrophotometer operat-
ing in the time-drive mode at around 340 nm or in the
spectral scan mode. Typical concentrations of GPSNO were
-1 × 10–3 mol dm–3. Solutions of TGSNO and SNTG were
prepared at the same concentration by reaction of equimolar
amounts of 1-thioglycerol or 1-thio-β-D-glucose and sodium
nitrite in mildly acidic solution, immediately prior to investi-
gations of the decomposition characteristics at pH 7.4.
Nitric oxide and nitrite ion analysis
Nitric oxide was determined in the reaction solutions, un-
der nitrogen, using a commercial World-Precision ISO-NO
specific electrode, calibrated with standard ascorbic acid-
sodium nitrite solutions. Nitrite ion was determined by a
modification of the Griess method (8).
Repeat scans of the decomposition (shown in Fig. 4) at
high concentration of added Cu2+ (5 × 10–5 mol dm–3) showed
the clear development of a small absorbance at around
280 nm, which can be interpreted as the Cu2+ complex of the
© 1999 NRC Canada

Munro and Williams
553
Fig. 2. Absorbance–time plots for the decomposition of GPSNO
(1 × 10–3 M) in the presence of Cu2+ (1 × 10–5 M) (a) without
EDTA, (b) with EDTA (1 × 10–3 M).
Fig. 4. Repeat scans (4 min intervals) for the decomposition of
GPSNO (1 × 10–3 M) with added Cu2+ (5 × 10–5 M).
Absorbance increases at 280 nm and decreases at 350 nm.
0.45
0.4
0.3
0.2
0.1
0
0.4
0.35
(b)
0.3
(a)
0.25
0.2
220
290
360
430
500
0
50
100
150
200
nm
time / min
GPSNO in that it appears that there are two reactions: one
catalyzed by added [Cu2+] and the other not. In contrast,
however, to the results for GPSNO, the spectral traces of the
Cu2+-dependent reaction show evidence of induction periods
which are shorter as the [Cu2+] and the concentration of the
added 1-thioglycerol (not shown) is increased. In marked
contrast to the behaviour of GPSNO, however, the effect of
added metal ion chelator EDTA to solutions of TGSNO
(Fig. 6) progressively halted both components of the reac-
tion (the Cu2+-dependent and Cu2+-independent pathways),
leaving a virtually stable solution of TGSNO over at least
12 h. Addition of neocuproine produced the same effect.
When EDTA was added at various time points after the start
of the reaction, decomposition was immediately halted at
those time points. The NO-probe gave a value of -45% for
NO production and the nitrite analysis, and when the reac-
tion was carried out aerobically, gave a figure of 97% of the
theoretical maximum.
Fig. 3. Absorbance–time plots for the decomposition of GPSNO
(1 × 10–3 M) in the presence of Cu2+ (1 × 10–5 M) with (a) no
ascorbate, (b) ascorbate (5.0 × 10–5 M), (c) ascorbate (1.0 × 10–4 M).
0.5
0.4
0.3
(a)
0.2
(b)
0.1
(c)
0
0
100
200
300
400
time / min
disulfide derived from GPSNO. When the reaction was car-
ried out anaerobically, NO was detected using the NO–elec-
trode system in the solution at -30%. The same experiment
carried out aerobically gave a value of 51% of the theoreti-
cal maximum of nitrite anion.
Ascorbic acid, added in low concentration (1 × 10–5 to 1 ×
10–4 M) also catalyzed the decomposition of TGSNO and
resulted in quantitative reaction, just as in the case of
GPSNO. At much higher ascorbate concentration (0.04–0.24
M) and in the presence of EDTA, the reaction was fully first
order in TGSNO concentration, and there was an excellent
linear relationship between the observed first-order rate con-
stant and ascorbate concentration, yielding a value of 1.59 ×
10–3 M–1 s–1 for the second-order rate constant. Under these
There was a very rapid reaction between GPSNO and sul-
fite ion at pH 7.4 in the presence of added EDTA, yielding a
second-order rate constant of 8630 M–1 s–1. No nitric oxide
or nitrite ion were detected in these reaction products.
Attempts to isolate the S-nitroso derivative of 1-thio-β-D-
glucose were unsuccessful, even though it was clear from
the development of a red colour that S-nitrosation had oc-
curred. The decomposition of solutions generated from the
thiol and nitrous acid were examined spectrophotometrically
at pH 7.4 and showed very similar behaviour to that shown
in Fig. 1 for GPSNO. In the same way, the addition of
EDTA completely suppressed the Cu2+-catalyzed reaction
leaving a significant thermal reaction.
–
conditions NO (or NO2 if aerobic) is formed, together with
the thiol product.
Repeat scans (not shown) of the reaction carried out in the
presence of added Cu2+ (5 × 10–5 M) clearly showed the
disappearence of the absorbance at around 340 nm, and
there was some evidence of the development of a small
absorbance in the 260 nm region, which disappeared during
the course of the reaction.
S-Nitroso 1-thioglycerol (TGSNO), 2
S-Nitroso-N-carbamyl penicillamine (SNCP), 3
All attempts to effect the nitrosation of 1-thioglycerol (3-
mercapto-1,2-propanediol) using the known procedures gen-
erated an unstable red gelatinous liquid. It is almost certain
that this is the S-nitroso derivative of 1-thioglycerol and so-
lutions prepared from equimolar amounts of 1-thioglycerol
and nitrous acid gave the expected UV-visible spectrum so
characteristic of S-nitrosothiols.
The S-nitroso derivative of N-carbamyl penicillamine
(SNCP) was synthesized, fully characterized, and shown to
be a stable solid at room temperature. In aqueous solution,
1.5% free thiol was determined by Ellman’s method, reflect-
ing again the equilibration in solution. The thiol was a gift
from the former Wellcome company, and the quantity of
SNCP synthesized did not allow enough material for the
study of its decomposition characteristics. However, some
rate measurements were carried out on the decomposition of
The decomposition behaviour is shown in Fig. 5 and re-
veals a similar pattern of behaviour to that found earlier for
© 1999 NRC Canada

554
Can. J. Chem. Vol. 77, 1999
Fig. 5. Absorbance–time plots for the decomposition of TGSNO
(1 × 10–3 M) with (a) no added Cu2+, (b) Cu2+ (0.5 × 10–5 M),
(c) Cu2 (1 × 10–5 M).
Fig. 6. Absorbance–time plots for the decomposition of TGSNO
(1 × 10–3 M) with added Cu2+ (1 × 10–5 M) (a) without EDTA,
(b) with EDTA (1 × 10–5 M), (c) with EDTA (2 × 10–5 M),
(d) with EDTA (5 × 10–5 M).
0.9
(d)
(c)
0.8
(a)
0.6
0.6
(b)
0.3
0.4
(b)
(c)
0.2
0
(a)
0
100
200
300
0
time / min
0
100 200 300 400 500 600 700
time / min
SNCP generated and used in situ from equimolar quantities
of the thiol and nitrous acid. Good first-order plots were ob-
tained for experiments with added Cu2+ in the range 0.3–1.0
× 10–5 M, and the measured first-order rate constants were
linearly dependent on [Cu2+].
tion of GPSNO (-5 × 10–3 M), which is a fairly typical
figure, and which is high enough to effect Cu2+ reduction.
We estimate the first-order rate constant (assuming the de-
composition to be a first-order process) for the decomposi-
tion of GPSNO by the thermal route to be -3 × 10–5 s–1, i.e.,
with a half life of about 6–7 h. This compares with a value
for the half life for the thermal decomposition of S-nitroso
cysteine (one of the more unstable nitrosothiols in the pure
state) of -55 h at the same temperature (8). Other related
structures e.g., SNAP, behave similarly.
Discussion
GPSNO (1)
The successful synthesis of GPSNO and and of SNCP to-
gether with the demonstration of their apparent indefinite
stability in the solid state has added further examples to the
small list of stable S-nitrosothiols. It is likely that it, along
with all RSNO species tested, will show the general charac-
teristics of vasodilation and inhibition of platelet aggregation
in vivo. Testing is underway. It is possible that this sugar de-
rivative may have significant advantages (e.g., of solubility
in water) over SNAP and GSNO for in vivo administration
in order to effect these properties. A related sugar derivative,
which does not have the stability of GPSNO in the pure
state, has been shown (17) to have vasodilatory properties,
including when the application is made transdermally to hu-
man cutaneous vascular smooth muscle.
The copper-catalyzed part of the reaction shows an inter-
esting feature in that reaction without added Cu2+ appears to
cease after only less than 30% conversion. As the added
Cu2+ increased so does the % conversion, until at 5 × 10–5
M
added Cu2+ reaction is essentially quantitative and is so
rapid as to effectively swamp the thermal reaction. We have
not observed this pattern of behaviour with other S-nitro-
sothiols. Incomplete conversion, however, has been noted
earlier in the glutathione (GSH)/Cu2+ promoted decomposi-
tion of GSNO, in the presence of added oxidized
glutathione, GSSG (19), which was interpreted in terms of
complexation of Cu2+ by the added GSSG. For the GSNO
reaction, there was spectroscopic evidence of such a com-
plex with a shoulder at 250 nm and at higher concentration,
a peak of low extinction at 620 nm, characteristic of a Cu2+
complex, for which a 1:1 structure has been proposed (20).
Here with GPSNO decomposition we find (Fig. 4) the devel-
opment of a small absorbance at around 280 nm which could
reasonably be ascribed to the formation of the Cu2+ complex
of the disulfide.
The characteristics of in vitro decomposition and NO re-
lease of GPSNO are somewhat unusual compared with those
of other S-nitrosothiols, particularly those derived from
cysteine and related compunds. Figure 1 shows the pattern.
It is clear that there are two reactions here, an initial faster
reaction which is Cu2+-catalyzed (and which does not go to
completion unless the [Cu2+] is relatively high –5 × 10–5
M
in this case), and a slower reaction which is unaffected by
the [Cu2+], shows no signs of stopping at incomplete conver-
sion, and which appears to be a thermal reaction. This inter-
pretation is supported by the evidence in Fig. 2 which shows
the disappearance of the copper-promoted reaction when
EDTA is present (7), leaving the thermal reaction unaffected.
The same result was obtained (not shown) when the specific
Cu+ chelator neocuproine was added, at the same concentra-
tion, instead of EDTA, confirming our earlier suggestions
that in these copper ion catalyzed reactions, the true catalyst
is Cu+ (8), generated by thiolate ion reduction of Cu2+.
Thiolate ion is likely to be present in all RSNO solutions be-
cause of the reversibility of the S-nitrosation process (18). In
this case we have measured a thiol level of -2% in a solu-
The derivative from 1-thio-β-D-glucose (generated in situ)
behaved in a very similar manner, suggesting that the basic
sugar unit provides sites which are available in the product
disulfides for coordination of Cu2+, and which allows a sig-
nificantly larger rate of thermal decomposition of S-nitroso
sugars generally when compared with simpler cysteine de-
rivatives.
We detected nitric oxide in the decomposition reaction
when it was carried out anaerobically. With relatively long
reaction times it is extremely difficult to determine NO
quantitatively, because of its reactivity, particularly towards
traces of oxygen. Under these circumstances a measured
yield of -30% NO is not unusual. A little more difficult to
explain is the relatively low yield of nitrite anion (51%)
© 1999 NRC Canada

Munro and Williams
555
determined when the reaction was carried out aerobically.
We have no obvious explanation at this stage.
not immediately obvious what is going on here. There are
clearly two reactions taking place, one catalyzed by “free”
Cu2+ and the other not, yet both are stopped by EDTA addi-
tion. A possible explanation is that the slower reaction is a
copper-promoted reaction, but the effective catalyst, Cu+, is
obtained by thiolate reduction of the RSSR–Cu2+ complex,
which would be expected to be a slower process than the
thiolate reduction of free Cu2+, and which would lead to
complete, rather than partial decomposition of TGSNO. Fur-
ther work is needed to clarify this point.
When ascorbic acid was added at 5 × 10–5 and 1 × 10–4
M, reaction proceeds to completion, implying that Cu2+
bound to the disulfide product is accessible for reduction by
ascorbate but not by thiolate.
As part of another study which is looking at reactions in-
volving nucleophilic attack at the nitroso nitrogen atom,2 we
discovered that sulfite ion is generally very reactive towards
S-nitrosothiols, bringing about complete decomposition in
seconds or fractions of seconds in most cases. The reaction
forms the thiol and not the disulfide product, and does not
generate nitric oxide (or nitrite ion in the presence of air),
and by analogy with the corresponding reaction of nitrous
acid (21), probably forms hydroxylamine disulfonate.
GPSNO shows the same characteristics in reaction with
sulfite ion with a second-order rate consant of 8630 M–1 s–1,
which means that for reaction of GPSNO (1 × 10–3 M) and
sulfite (2 × 10–2 M), the half life of reaction is only 4 × 10–3 s.
Since reaction is so rapid and thiol products are readily
analysed by the Ellman procedure, this reaction could be the
basis for a quantitative analytical procedure for the determi-
nation of S-nitrosothiols.
Some evidence of RSSR–Cu2+ complex formation is evi-
dent from the repeat scan experiments (not shown), but the
complex does not appear to be as stable as that generated
from GPSNO. Ascorbate also acts as a reducing agent at low
concentration, resulting in complete decomposition of
TGSNO, implying that, as for GPSNO, Cu2+ bound to the
disulfide product is accessible for reduction by ascorbate.
At higher concentrations of addded ascorbate, the role of
ascorbate changes from that of a reducing agent to that of a
nucleophile, which undergoes electrophilic nitrosation by
–
TGSNO, resulting in the formation of NO (becoming NO2
if aerobic) and the thiol 1-thioglycerol (cf. the reaction of
nitrous acid with ascorbic acid (22)). Its reactivity is com-
parable to that of SNAP and S-nitroso-N-acetylcysteine.4 A
similar reaction has been reported (13) for the reaction of
TGSNO with 1-thioglycerol at high [1-thioglycerol], where
RS– now acts as a nucleophile, leading to ammonia forma-
tion.
TGSNO (2)
Unfortunately, it appears that TGSNO is not sufficiently
stable at room temperature to enable a complete character-
ization and to allow standard solutions to be prepared from
it. The red liquid, which could be kept at 0° for some weeks,
is almost certainly the S-nitroso compound, and the UV-
visible spectrum supports this. TGSNO was chosen for this
study because preliminary in vivo testing experiments with
solutions generated and used in situ had shown some un-
usual results which could result in some beneficial therapeu-
tic properties.3
The decomposition features shown in Fig. 5 are in some
ways similar to those found for GPSNO, in that there appear
to be two reactions, one copper ion catalyzed and the other
not. The copper-catalyzed reaction, however, does show in-
duction periods, not evident in the reactions of GPSNO. In-
duction periods of this kind have been observed on a number
of previous occasions (8, 10), and have been interpreted as
the relatively slow initial formation of Cu+ (from Cu2+ and
RS–) with the regeneration of both reactants (eq. [2]). As ex-
pected, the induction period is reduced as the [Cu2+] is in-
creased. It is also effectively removed by addition of
thioglycerol (RS–), not shown. Nitrite ion was formed quan-
titatively when reactions were carried out aerobically, and a
substantial amount of NO (45%) was measured when oxy-
gen was excluded.
SNCP (3)
Because of a shortage of material, we were unable to
carry out a full kinetic analysis on the decomposition of
SNCP. However, we found good first-order behaviour over a
range of [Cu2+] and obtained a value of 780 dm3 mol–1 s–1
for the second-order rate constant k2 (defined by rate =
k2[RSNO][Cu2+]), which compares with a value of 20 dm3
mol–1 s–1 for the closely structurally related SNAP. It is
tempting to account for the reactivity difference in terms of
the relative abilities of the N-COCH3 and N-CONH2 groups
to bind Cu+ in an intermediate leading to NO formation, but
this does not take account of the relative thiolate concentra-
tions in both cases, which can dramatically affect reactivity
in the ability to generate Cu+ from Cu2+ (10).
Conclusions
We have prepared and characterized two new stable S-
nitrosothiols, GPSNO and SNCP, and have examined the de-
composition in aqueous buffer (pH 7.4) of GPSNO, TGSNO,
and SNTG (the latter two generated and used in situ). All re-
actants decomposed by two pathways, (i) GPSNO by a sig-
nificant thermal pathway in parallel with a Cu2+-catalyzed
reaction, which tends to stop at incomplete conversion, as
the Cu2+ catalyst is tied up by complexation with the disul-
fide product, and (ii) TGSNO by a Cu2+ pathway, which also
stops at incomplete conversion probably for the same rea-
son, together with a component which may arise by copper
One major difference between the reactions of TGSNO
and GPSNO is shown in Fig. 6, where it can be seen that the
addition of EDTA effectively stops both reactions. Reaction
can also be halted when the EDTA is added during the
course of the reaction, rather than at the beginning. The non-
Cu2+-catalyzed reaction cannot therefore, for TGSNO, be a
thermal reaction, if it is halted by the addition of EDTA. It is
2 A.P. Munro and D.L.H. Williams. To be published.
3 A.R. Butler. Personnal communication.
4 A.J. Holmes and D.L.H. Williams. To be published.
© 1999 NRC Canada

556
Can. J. Chem. Vol. 77, 1999
catalysis where the true catalyst Cu+ is generated from the
product disulfide–Cu2+ complex. Both compounds showed
the “normal” reactivity with relatively high concentrations
of reactive nucleophiles such as ascorbate and sulfite.
8. S.C. Askew, D.J. Barnett, J. McAninly, and D.L.H. Williams.
J. Chem. Soc. Perkin Trans. 2, 741 (1995).
9. D.J. Barnett, A. Rios, and D.L.H. Williams. J. Chem. Soc.
Perkin Trans. 2, 1279 (1995).
10. A.P. Dicks, P.H. Beloso, and D.L.H. Williams. J. Chem. Soc.
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11. S.P. Singh, J.S. Wishnock, M. Keshive, W.M. Deen, and S.R.
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Acknowledgement
We thank the EPSRC for a research studentship awarded
to A.P.M.
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