Reactivity of nitrogen nucleophiles towards S-nitrosopenicillamine

Article abstract of DOI:10.1039/a905901f

We report the results of a kinetic study of the reactions of a number of nitrogen nucleophiles with the nitrosothiol S-nitrosopenicillamine (SPEN). The range of nucleophiles includes primary, secondary and tertiary aliphatic amines, together with hydrazine, hydroxylamine, azide ion, ammonia, semicarbazide, thiomorpholine and S-methylcysteine. Secondary amines form N-nitrosamines quantitatively. As expected, reaction occurs via the free base forms of the nucleophiles and consequently most of the reactions take place readily only at relatively high pH. Experiments were carried out with [nucleophile] ? [RSNO], and for many reactions, plots of the first order rate constant vs. [nucleophile] were linear. For ammonia and the primary amines, however, this plot tended to level off at high [nucleophile] and an explanation is offered involving the reversible formation of an inactive RSNO-amine complex, for which there is spectral evidence, in parallel with the main reaction. For the secondary amines there is a reasonably good Broensted plot with a β value of ～0.2. The much greater reactivities of S-methylcysteine and thiomorpholine, compared to those of primary amines and morpholine respectively are consistent with initial attack at the sulfur atom, followed by an internal rearrangement. Over the whole range of nucleophiles studied there is a reasonable correlation with the Ritchie N⁺ parameter, and not with the Pearson n scale. Comparisons are made with the corresponding reactions of alkyl nitrites and N-methyl-N-nitrosotoluene-p-sulfonamide (MNTS).

Full text of DOI:10.1039/a905901f

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Reactivity of nitrogen nucleophiles towards S-nitrosopenicillamine
Andrew P. Munro and D. Lyn H. Williams*
Department of Chemistry, Durham University, South Road, Durham, UK DH1 3LE
Received (in Cambridge, UK) 21st July 1999, Accepted 20th August 1999
We report the results of a kinetic study of the reactions of a number of nitrogen nucleophiles with the nitrosothiol
S-nitrosopenicillamine (SPEN). The range of nucleophiles includes primary, secondary and tertiary aliphatic amines,
together with hydrazine, hydroxylamine, azide ion, ammonia, semicarbazide, thiomorpholine and S-methylcysteine.
Secondary amines form N-nitrosamines quantitatively. As expected, reaction occurs via the free base forms of the
nucleophiles and consequently most of the reactions take place readily only at relatively high pH. Experiments were
carried out with [nucleophile] ӷ [RSNO], and for many reactions, plots of the first order rate constant vs. [nucleophile]
were linear. For ammonia and the primary amines, however, this plot tended to level off at high [nucleophile] and an
explanation is offered involving the reversible formation of an inactive RSNO–amine complex, for which there is
spectral evidence, in parallel with the main reaction. For the secondary amines there is a reasonably good Brönsted
plot with a β value of ~0.2. The much greater reactivities of S-methylcysteine and thiomorpholine, compared to
those of primary amines and morpholine respectively are consistent with initial attack at the sulfur atom, followed by
an internal rearrangement. Over the whole range of nucleophiles studied there is a reasonable correlation with the
Ritchie Nϩ parameter, and not with the Pearson n scale. Comparisons are made with the corresponding reactions of
alkyl nitrites and N-methyl-N-nitrosotoluene-p-sulfonamide (MNTS).
There is currently much interest in the chemistry of S-nitroso-
thiols (or thionitrites) RSNO, in connection with aspects of the
now well-known1 ‘nitric oxide story’. They are under active
consideration as nitric oxide donors for possible medical use
to make up for any deficiencies in the natural in vivo synthesis
of NO from -arginine, and they are currently believed to be
involved in the storage and transport of NO within the body.2
S-Nitrosoglutathione (GSNO) is presently used medically to
inhibit platelet aggregation during some operations,3 and both
GSNO and S-nitroso-N-acetylpenicillamine (SNAP), are stable
solids which have been, and continue to be, much used in a
range of in vivo and in vitro experiments as NO generators. Less
stable (generally non-isolable in the pure state) RSNOs are
sometimes used for the same purpose, when they are generated
in solution by thiol nitrosation4 and used as such in situ.
Clearly the mechanism of NO release is important. We have
shown5 that apart from the thermal reaction (which is usually
very slow at room temperature) and the photochemical
pathway, reaction can occur in aqueous solution at pH 7.4,
generating the disulfide and initially nitric oxide. This occurs in
a Cuϩ-catalysed process, in which the catalyst is generated from
Cu2ϩ and RSϪ, which are often present in buffered solutions of
RSNOs at trace impurity levels. In aerated aqueous solutions
the nitric oxide is rapidly and quantitatively converted to nitrite
anion, at the concentrations used. There is a large structure–
reactivity dependence, which has been rationalised. It has been
further shown that ascorbate (at low concentration) will also
act as a reducing agent, and that Cuϩ can also be generated, by
thiolate reduction from Cu2ϩ bound to peptides and proteins.6
Another important reaction of RSNOs is that with thiolate
ion (eqn. (1)), which leads to a transnitrosation, forming a new
ion, and that mechanistically the reaction can be regarded as
a nucleophilic substitution at the nitroso nitrogen atom,9 or
alternatively as an electrophilic S-nitrosation by RSNO. At this
stage there is no evidence in favour of (or against) the involve-
ment of a reaction intermediate. Since there is a wide range of
reactivity of RSNOs towards NO generation,10 it is possible
that NO can be stored in vivo as a ‘stable’ RSNO, which could
release NO by first undergoing a transnitrosation to give a more
unstable RЈSNO. It is known for example that S-nitroso-
proteins are much more stable than is S-nitrosocysteine itself to
loss of NO.11
Another example of reactions where RSNOs can act as
electrophilic nitrosating species is to be found in their reactions
with ascorbate. As previously mentioned, it has been demon-
strated that ascorbate can act as a reducing agent (at quite low
concentration, typically 1 × 10Ϫ4 mol dmϪ3) to generate Cuϩ,
which brings about RSNO decomposition. However, in the
presence of EDTA, which cuts out any metal ion promoted
reactions, and at higher concentrations, ascorbate brings
about the decomposition of RSNOs generating NO and the
corresponding thiol12 (eqn. (2)). As yet the exact form of
2RSNO ϩ Ascorbate →
2RSϪ ϩ 2NO ϩ Dehydroascorbic acid ϩ 2Hϩ (2)
the nucleophile has not been properly established, but since the
reactions were carried out at pH 7.4, it is likely that the nucleo-
phile is the monoanion of ascorbic acid. Similarly it has been
shown recently that S-nitrosothiols react with hydrogen per-
oxide (via the anion) generating initially the peroxynitrite
anion, stable at high pH, but which isomerises to nitrate anion
at lower pH.13
We thought it to be of some interest to determine the range
of nucleophiles which would undergo electrophilic nitrosation
by RSNOs, thus establishing the possible generality of the reac-
tion. There are literature reports14 that amines can be nitrosated
in this way, but there has not been a proper mechanistic study.
It is possible that amine nitrosation could occur by RSNO
decomposition (by the copper catalysed route) to give NO,
RSNO ϩ RЈSϪ
RSϪ ϩ RЈSNO
(1)
S-nitrosothiol if the R groups are different. This exchange pro-
cess has been known qualitatively for some time,7 but more
recent work has measured the equilibrium and rate constants
for the process.8 Kinetic studies over a range of pH values have
shown, as expected, that the reactive species is the thiolate
J. Chem. Soc., Perkin Trans. 2, 1999, 1989–1993
This journal is © The Royal Society of Chemistry 1999
1989

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Table 1 Values of kO for the reaction of SPEN with piperidine (0.2
which by oxidation and further reaction with NO would give
N2O3, which could readily nitrosate any amine group present.
Indeed we showed this to be the case10 with the reaction of
SNAP with N-methylaniline, where in the presence of air, the
N-nitrosamine was formed essentially quantitatively, whereas
when oxygen was rigorously excluded, very little N-nitrosamine
was formed. The copper catalysed pathway is readily blocked
by EDTA addition.
Electrophilic nitrosation by other compounds containing the
-NO group such as alkyl nitrites and a range of N–NO com-
pounds is well known, and has significant synthetic advantages
over the more usual nitrous acid nitrosation, in that generally it
can be carried out under basic conditions, and also in a wider
range of solvents. Detailed mechanistic studies of reactions of
alkyl nitrites15 and of N-nitrososulfonamides16 have been pub-
lished, and there are some results on the reactions of N-nitros-
amines.17 We hoped to compare the results of RSNO reactions
with nitrogen nucleophiles with these literature data.
mol dmϪ3) as a function of pH
pH
kO/10Ϫ5 sϪ1
10.58
10.73
10.87
11.00
11.21
11.39
11.56
11.74
11.92
12.02
12.10
5.8
7.2
9.1
10.6
13.9
16.6
19.8
22.3
23.0
24.2
25.3
We found generally that the reactions of amines with RSNO
species were quite slow, so drawing on our experience with reac-
tions of several RSNOs18 with thiolate ion, we elected to work
initially with one substrate, S-nitrosopenicillamine (SPEN) 1.
This compound was significantly more reactive than the other
nitrosothiols used, towards thiolate ion at high [thiolate ion],
when a different reaction, leading principally to ammonia
formation, occurs, but where the rate limiting step is believed to
involve thiolate attack at a nitrosothiol. Although SPEN has
never been isolated in the pure state, it is easy to work with a
solution generated by S-nitrosation of penicillamine, which is
sufficiently stable as a stock solution in mildly acidic solution
for the purpose.
Fig. 1 Plots of kO vs. total amine concentration for the reaction of
SPEN with (a) pyrrolidine at pH 11.57 and (b) piperidine at pH 11.21.
Results and discussion
(a) Reactions of secondary aliphatic amines
A range of secondary aliphatic amines reacted with SPEN
(generated in situ from equimolar quantities of penicillamine
and nitrous acid) in water at pH values near to the pKa of the
protonated amine. Reactions were followed by noting the dis-
appearance of the absorbance at 340 nm due to SPEN. Final
spectral scans all showed the complete disappearance of the 340
nm band and the appearance of absorbances in the 230–240 nm
region consistent with N-nitrosamine formation. Spectral
measurements in this range showed that nitrosamine formation
is essentially quantitative. All kinetic experiments were carried
out with [amine] ӷ [SPEN], and in the presence of EDTA to
prevent SPEN decomposition to NO via a copper-catalysed
reaction. Good first-order kinetics were always found and the
first-order rate constant (kO) was linearly dependent on the
total stoichiometric amine concentration, [Amine]T, as shown
in Fig. 1 for both the reactions of pyrrolidine (a) and piperidine
(b) at a constant pH. This establishes experimentally the rate
eqn. (3). For both of these amines, values of kO were also
Fig. 2 Plot of (kO)Ϫ1 vs. [Hϩ] for the reaction of SPEN with piperidine.
should be related by eqn. (4), where k2 is the bimolecular rate
kO = k2Ka[Amine]T/(Ka ϩ [Hϩ])
(4)
constant for reaction between SPEN and the free base form of
the amine. Plots of (kO)Ϫ1 against [Hϩ] should then be linear
with a positive slope and intercept. A typical plot is shown in
Fig. 2 for the reaction of piperidine. The data for pyrrolidine
gave a similar straight line plot. Values of the pKa of the
protonated form of the amines found from the slopes and inter-
cepts of these lines were determined as 11.15 (piperidine) and
11.55 (pyrrolidine), which agree well with the respective liter-
ature values19 of 11.12 and 11.30. These plots also gave values
of 1.34 × 10Ϫ3 dm3 molϪ1 sϪ1 (piperidine) and 7.00 × 10Ϫ3 dm3
molϪ1 sϪ1 (pyrrolidine) for k2, the bimolecular rate constant for
reaction of the free amine with SPEN.
The reactions of eight secondary amines with SPEN were
followed kinetically. Each individual experiment gave good
first-order behaviour, and plots of kO against [Amine]T were all
linear. Typical experimental plots (for piperidine and pyrrol-
idine) are shown in Fig. 1. The other amines behaved similarly
Rate = k[SPEN][Amine]T = kO[SPEN]
(3)
obtained over a range of pH values in the region of the pKa
value of the protonated amine. The experimental results for
piperidine are given in Table 1. In both cases, plots of kO vs. pH
are S-shaped with an inflexion point in the vicinity of the pKa
value, confirming that reaction occurs via the free base form of
the amine as expected. This means that kO and the acidity
1990
J. Chem. Soc., Perkin Trans. 2, 1999, 1989–1993

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Table 2 Values of k2 from eqn. (4) for the reaction of SPEN with a
range of secondary amines
Table 3 Values of k2 for reaction of SPEN with a range of nitrogen
nucleophiles
Amine
k2/dm3 molϪ1 sϪ1
Nucleophile
k2/dm3 molϪ1 sϪ1
Pyrrolidine
7.2 × 10Ϫ3
Propylamine
Ethylamine
Ethylenediamine
Quinuclidine
Trimethylamine
Hydrazine
7.8 × 10Ϫ4
4.9 × 10Ϫ4
1.8 × 10Ϫ4 a
7.1 × 10Ϫ4
3.6 × 10Ϫ4
3.3 × 10Ϫ2 a
1.1 × 10Ϫ2 a
1.5 × 10Ϫ5
9.9 × 10Ϫ4
4.4 × 10Ϫ5
1.5 × 10Ϫ2
3.8 × 10Ϫ2
7.0 × 10Ϫ3 a
7.9 × 10Ϫ3 b
1.7 × 10Ϫ3
Piperidine
1.4 × 10Ϫ3 a
5.8 × 10Ϫ4
,-Proline
Sarcosine
Piperazine
4-Hydroxyproline
Morpholine
N-Methylaniline
1.3 × 10Ϫ3
Azide ion
3.5 × 10Ϫ4 c
6.1 × 10Ϫ4
Methoxyamine
Hydroxylamine
Semicarbazide
Thiomorpholine
S-Methylcysteine
3.8 × 10Ϫ4
Too slow to measure
a From the reciprocal plot (kO)Ϫ1 vs. [Hϩ]. b From a reaction carried out
anaerobically. c Corrected for the statistical factor.
a Corrected for the statistical factor.
is a reasonable Brönsted plot with a β value of ~0.2. This is
significantly smaller than the value of 0.6 found for the corre-
sponding reactions of secondary amines with MNTS21 suggest-
ing that charge transfer in the transition state of the RSNO
reactions is less extensive, possibly reflecting the powerful
electron withdrawing effect of the sulfonyl group in MNTS.
Less basic amines such as N-methylaniline (NMA) react
much too slowly for a meaningful rate constant to be obtained.
This means that an earlier report of nitrosamine formation10
from NMA and a nitrosothiol, carried out without removal
of all trace amounts of Cu2ϩ ions, was correctly interpreted
in terms of prior NO formation followed by oxidation and
hydrolysis giving nitrous acid which effects N-nitrosation,
rather than as a direct nitrosation reaction by the nitrosothiol.
Fig. 3 Plot of kO vs. [OHϪ] for the alkaline hydrolysis of SPEN.
(b) Reactions of other nitrogen nucleophiles
except that in some cases there was a small positive intercept
at [Amine]T = 0, which will be discussed later. Values of k2
obtained from the slopes and from eqn. (4) are listed in Table 2.
The value for pyrrolidine is duplicated, within the experimental
error, when reaction is carried out anaerobically, demonstrating
that dissolved oxygen plays no part in these reactions.
Primary aliphatic amines behave very much like their secondary
counterparts, particularly at relatively low concentrations of
amine and k2 values were obtained in the same way. Values
obtained are given in Table 3. Again reaction was shown to
occur via the free base form by the linearity of a plot of (kO)Ϫ1
vs. [Hϩ], and satisfactory agreement was found between the
derived and literature pKa values. Reactivity was comparable
with that of the secondary amines of similar basicity. Other
nitrogen nucleophiles (of lower basicity), azide ion, and hydra-
zine reacted much more rapidly than did the amines: hydroxyl-
amine also reacted, but at a rate comparable with those of the
primary amines. Reaction also occurred with tertiary amines
e.g. trimethylamine and quinuclidine, as is the case with nitrous
acid nitrosation,22 but reactions were quite slow and were not
examined in detail.
We did find one significant difference between the behaviour
of the primary and secondary amines, in that for the former the
plots of kO against [Amine]T showed a very pronounced tend-
ency to curvature leading to a levelling off at high [Amine]T,
which was not present for any of the secondary amines studied.
An example is shown in Fig. 4 for the reaction of propylamine
at pH 10.9. All of the primary amines behaved similarly, but
there was no obvious curvature in the plots for the reactions
of azide ion, hydrazine and hydroxylamine. For the primary
amines therefore values of k2 were obtained at lower concen-
trations of amine (below ~0.09 mol dmϪ3) where the inequality
[Amine]T ӷ [SPEN] still was valid. The products were not char-
acterised; it is very likely that a primary aliphatic diazonium ion
is formed, as in the nitrous acid reaction, leading to a range of
products of deamination. There was, as expected, no absorb-
ance formation in the 230–240 nm range corresponding to the
formation of a stable nitrosamine.
Small positive intercepts for the kO against [Amine]T plots are
evident for the slower reacting amines, notably for proline,
piperazine and morpholine, and can reasonably be ascribed to
the alkaline hydrolysis pathway, where the nucleophile is the
hydroxide ion. This explanation was suggested20 to account for
similar small intercepts observed in the nitrosation of amines
brought about in alkaline solution by N-methyl-N-nitroso-
toluene-p-sulfonamide (MNTS). To check this possibility, we
examined the kinetics of the alkaline hydrolysis of SPEN
independently. Over the range of [OHϪ] 0.06–0.20 mol dmϪ3,
first order behaviour occurred and the plot of kO against [OHϪ]
is linear with a very small intercept, which could arise from the
spontaneous thermal decomposition of SPEN (see Fig. 3). The
expected ‘nitrogen’ product in the hydrolysis reaction is the
nitrite anion, which was determined in the final reaction solu-
tion only at ~50% yield. This suggests that in addition to attack
of OHϪ at the nitrogen atom, some reaction also occurs pos-
sibly by way of initial attack at the sulfur atom, which could
lead to nitrous oxide formation, notionally by release of NOϪ.
Reactions of nucleophiles with MNTS show this ambident
feature,21 for while amines and other nitrogen nucleophiles
attack the nitroso-nitrogen atom (leading to amine nitrosation),
the ‘harder’ nucleophiles OHϪ and OEtϪ attack at the sulfur
atom leading to diazomethane formation.
There is an acceptable agreement between the k2 values
obtained for piperidine and pyrrolidine from the variation of kO
with [Amine]T (Table 2) and those obtained from the reciprocal
plot (Fig. 2). Qualitatively there is a clear link between the
reactivity of the secondary amines and their basicity, and there
The curvature of the k2 vs. [Amine]T plot was even more
pronounced in the reaction of ammonia with SPEN, which did
proceed readily (with kO values in the region of 1 × 10Ϫ3 sϪ1),
J. Chem. Soc., Perkin Trans. 2, 1999, 1989–1993
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Fig. 6 Possible structure of a complex between RSNO and a second-
ary amine or ammonia (R = H).
Fig. 4 Plot of kO vs. total amine concentration for the reaction of
SPEN with propylamine.
Fig. 7 Plot of log k2 vs. the Ritchie Nϩ parameter for the reactions of
SPEN with a range of nucleophiles. (1) Semicarbazide, (2) methoxy-
amine, (3) hydroxylamine, (4) morpholine, (5) ethylamine, (6) piper-
azine, (7) piperidine, (8) hydrazine and (9) azide.
the presence of the sulfur atom results in an increased reactivity,
suggesting that reaction occurs initially at sulfur, a more nucleo-
philic site. This contrasts with the results obtained for the reac-
tions of MNTS,16 where the kinetic results indicate that the
nitrogen site is more nucleophilic than the sulfur atom. This
suggests that MNTS and SPEN possess different discriminating
properties. Details of the reaction of S-nitrosothiols with a
range of sulfur nucleophiles will be published shortly,23 when
this point will be discussed further. Comparable ‘additional
reactivity’ has been described for the nitrosation of S-methyl-
cysteine,24 thioproline25 and thiomorpholine26 by nitrous acid
itself, and the results were also interpreted in terms of initial
attack at the sulfur atom followed by internal rearrangement of
the NO group to the amino nitrogen atom, but these results
refer to reaction in an acid medium, where the amino group is
substantially protonated, and so provides no direct comparison
for the relative nucleophilicities of N- and S- sites.
Fig. 5 Spectra of (a) SPEN (2.5 × 10–4 mol dmϪ3) alone and (b) with
added ammonia (0.2 mol dmϪ3) at pH 9.55.
which precluded the determination of a k2 value. Curved plots
of this type could arise if an inactive complex between the
amine and the nitrosothiol is formed rapidly as indicated in
Scheme 1. The general expression for kO from such a scheme is
RSNO ϩ RЈNH2
[RSNOؒRЈNH2] rapid
K
RSNO ϩ RЈNH2 → RSϪ ϩ RЈN2 slow k2
ϩ
Scheme 1
given by eqn. (5), which predicts a constant value for kO at high
kO = k2Ka[RЈNH2]T/(Ka ϩ [Hϩ] ϩ KKa[RЈNH2]T) (5)
Correlation of the kinetic results
[RЈNH2]T if K[RЈNH2]T ӷ 1. Support for this idea comes from
the observation of a significant change in the initial spectrum
of SPEN in the 340 nm range at high [RЈNH2]T. This is shown
in Fig. 5 for the interaction of ammonia with SPEN at pH 9.55.
A possible structure for this inactive complex could be that
shown in Fig. 6, where there are two H-bonding interactions
involving the O- and S-atoms of RSNO in a six-membered ring
structure. This is possible for primary amines and ammonia,
but not for secondary amines, in keeping with the experimental
findings.
Although there is a reasonable correlation between log k2 and
the pKa values for the family of secondary amines, there is no
discernible link when all of the nitrogen nucleophiles are
included. Similarly there is no correlation with the Pearson
nucleophilicity parameter n27 for the nucleophiles in this work
for which values are available (for example both hydrazine and
azide ion are substantially more reactive than are pyrrolidine
and piperidine, even though their n values would predict other-
wise). There is however a reasonable correlation between log k2
and the Ritchie Nϩ parameter28 as shown in Fig. 7. The slope
of the drawn line is 0.8, a little lower than the predicted value of
1.0 and the one point for hydrazine lies well above the line.
These results parallel almost exactly those reported in an
extensive study of nucleophilic reactivity for attack at the nitro-
gen atom of the nitroso group in the nitrososulfonamide
MNTS.16,20,21 Here also there was a correlation with Nϩ but not
with n. This was interpreted using the ideas of Hoz29 and
Shaik30 in terms of a Klopman frontier-controlled reaction31 in
which the transition state has substantial diradical nature.
From the general parallel between the MNTS (and an alkyl
nitrite) and SPEN reactions it appears that direct nitrosation
(c) Ambident nucleophiles
There are two examples in Table 2 where there is evidence that
SPEN effects nitrosation initially at a sulfur site, which is then
followed by an internal rearrangement of the nitroso group to
the nitrogen atom. The rate constant for the reaction with thio-
morpholine is 40 times greater than that for morpholine, and
the value for S-methylcysteine is ~100 times larger than the
values for simple primary amines: a direct comparison with e.g.
alanine is not possible since, unusually, the kinetics for that
reaction were not straightforward. It is quite clear however that
1992
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7 S. Oae, Y. H. Kim, D. Fukushima and K. Shinhama, J. Chem. Soc.,
Perkin Trans. 1, 1978, 913; J. W. Park, Biochem. Biophys. Res.
Commun., 1988, 152, 916.
reactions effected by S-nitrosothiols can be characterised in the
same way.
8 D. J. Meyer, H. Kramer, N. Ozer, B. Coles and B. Ketterer, FEBS
Lett., 1994, 345, 177; D. J. Barnett, J. McAninly and D. L. H.
Williams, J. Chem. Soc., Perkin Trans. 2, 1994, 1131.
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Trans. 2, 1995, 1279.
10 S. C. Askew, D. J. Barnett, J. McAninly and D. L. H. Williams,
J. Chem. Soc., Perkin Trans. 2, 1995, 741.
11 J. S. Stamler, D. I. Simon, J. A. Osborne, M. E. Mullins, O. Jaraki,
T. Michel, D. J. Singel and J. Loscalzo, Proc. Natl. Acad. Sci. USA,
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13 P. J. Coupe and D. L. H. Williams, J. Chem. Soc., Perkin Trans. 2,
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14 S. Oae and K. Shinhama, Org. Prep. Proc. Int., 1983, 15, 165.
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Experimental
SPEN was generated in mildly acid solution by the reaction of
equimolar quantities (around 1 × 10Ϫ2 mol dmϪ3) of sodium
nitrite and penicillamine. This stock solution was stable for
many hours and a fresh batch was made up daily. Reaction
solutions were prepared with [SPEN] typically 1 × 10Ϫ3 mol
dmϪ3 and the nucleophile typically in the range 0.002–0.200 mol
dmϪ3. The pH was adjusted by the addition of perchloric acid
or sodium hydroxide solution, and the amine/amine salt acted
as a buffer. All other materials used were commercial samples
of the highest purity available.
Kinetic measurements were carried out in aqueous solution
containing EDTA (1 × 10–4 mol dmϪ3) at 25 ЊC at 340 nm in a
conventional spectrophotometer following the disappearance
of the absorbance due to the nitrosothiol. Good first order
behaviour was found throughout and rate constants were
obtained using the Enzfitter program and were generally repro-
ducible to 3%. Nitrite analysis was done by the conventional
diazotisation–azo dye procedure, following pH adjustment
to 7.
19 D. D. Perrin, Dissociation Constants of Organic Bases in Aqueous
Solution, Butterworths, London, 1965.
20 A. Castro, J. R. Leis and M. E. Pena, J. Chem. Soc., Perkin Trans. 2,
1989, 1861.
21 J. R. Leis, M. E. Pena and A. M. Rios, J. Chem. Soc., Perkin Trans.
2, 1995, 587.
22 D. L. H. Williams, Nitrosation, Cambridge University Press,
Cambridge, 1988, pp. 98–101 and references therein.
23 A. P. Munro and D. L. H. Williams, unpublished work.
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Acknowledgements
We thank the EPSRC for the award of a research studentship to
APM and Dr M. R. Crampton and Dr S. Amado for construc-
tive discussions.
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