Reactivity of sulfur nucleophiles with N-methyl-N-nitroso-p- toluenesulfonamide

Article abstract of DOI:10.1039/b316200a

The transfer of the nitroso group from N-methyl-N-nitroso-p- toluenesulfonamide (MNTS) to cysteine (CYS) and 2-aminoethanethiol (AET) has been studied in a pH range between pH = 7 and pH = 13. Kinetic results clearly indicate that both nucleophiles react through the corresponding thiolate to give the corresponding nitrosothiol. The existence of two (AET) or three (CYS) macroscopic acidity constants has been kinctically evidenced and the nitrosation rates of the corresponding bases have been identified. Nitrosation rate constants of the different species present in the reaction medium have been determined and a Bronsted-type plot has been established giving a β_nuc value ? 0.08 clearly different from the values of β_nuc ? 0.7 obtained in the nitrosation of primary and secondary amines by MNTS. The low β_nuc value has been attributed to the need for previous desolvation of the nucleophile.

Full text of DOI:10.1039/b316200a

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Reactivity of sulfur nucleophiles with N-methyl-N-nitroso-
p-toluenesulfonamide
C. Adam,† L. García-Río* and J. R. Leis
Departamento de Química Física, Facultad de Química, Universidad de Santiago, 15782,
Santiago, Spain. E-mail: qflgr3cn@usc.es
Received 12th December 2003, Accepted 23rd February 2004
First published as an Advance Article on the web 17th March 2004
The transfer of the nitroso group from N-methyl-N-nitroso-p-toluenesulfonamide (MNTS) to cysteine (CYS) and
2-aminoethanethiol (AET) has been studied in a pH range between pH = 7 and pH = 13. Kinetic results clearly
indicate that both nucleophiles react through the corresponding thiolate to give the corresponding nitrosothiol.
The existence of two (AET) or three (CYS) macroscopic acidity constants has been kinetically evidenced and the
nitrosation rates of the corresponding bases have been identified. Nitrosation rate constants of the different species
present in the reaction medium have been determined and a Brønsted-type plot has been established giving a βnuc
value ≅ 0.08 clearly different from the values of βnuc ≅ 0.7 obtained in the nitrosation of primary and secondary
amines by MNTS. The low βnuc value has been attributed to the need for previous desolvation of the nucleophile.
diol has been observed to react almost exclusively at the nitroso
Introduction
group of the MNTS. The possibility that a nucleophile may
react simultaneously through both electrophilic centers of the
MNTS has also been confirmed when studying its reaction with
the hypochlorite ion (90% of reaction through the sulfonyl
group) or with acetohydroxamic acid.8
Nitroso compounds have attracted considerable interest mainly
due to their important biological relevance. A wide variety of
structurally related compounds possessing the N-nitroso-N-
alkyl functionality have demonstrated a cancer chemothera-
peutic potential.1 As is well documented, the basic hydrolysis
of N-methyl-N-nitroso-p-toluenesulfonamide (MNTS) yields
diazomethane,2 a powerful alkylating agent3 of DNA. The
possible carcinogenic effect of these N-nitroso compounds can
be even larger when considering the possibility that they may
transfer the nitroso group to other nucleophilic substrates by a
transnitrosation process.4
Another type of nitroso compounds are the S-nitrosothiols11
(henceforth called RSNO), which have attracted growing inter-
est since ca. 1990 due to a spectacular series of discoveries
related to the synthesis in vivo of nitric oxide and its effects on a
wide range of physiological functions.12 In spite of the fact that
the reactivity of the MNTS with different nucleophilic agents
has been studied, there is, however, little information on its
behavior when the nucleophilic atom is a sulfur atom. We have
found in the literature only one mechanistic study of the
reactivity of MNTS with cysteine.13 In this study, the MNTS is
found to transfer its nitroso group to the sulfur atom of the
cysteine giving the corresponding S-nitrosothiol.
The chemical and biological activity of MNTS becomes
more complex due to the existence of two reactive centers: the
nitrogen atom of the nitroso group and the sulfur atom of the
sulfonyl group (Scheme 1). Experimental results obtained in our
laboratory show that different types of nucleophiles react at
either one or the other electrophilic center. Soft nucleophiles5
react exclusively at the nitroso group. Amines,6 carbanions7 and
The acid–base behavior of the cysteine allows us to establish
the existence of two possible nucleophilic species: Hcys(؊) and
Cys(2؊) whose macroscopic pKa are 8.21 and 10.4 respectively
(see Scheme 2). The existence of these two nucleophilic forms
should be apparent from its behavior toward the nitroso group.
However, the results obtained when studying its reaction with
MNTS13 indicate that the reaction rate increases as the pH of
the medium increases, but ignoring that this should reach
a limiting value for values of pH > 11. The absence of the
limiting value should be a consequence of the high ethanol
concentration in the reaction media (≅ 25%). At high pH values
the presence of ethanol yields CH3CH2OϪ and a new decom-
position pathway for MNTS. The existence of this new
decomposition pathway (its importance increases on increasing
the basicity of the medium) is contrary to the tendency of the
cysteine nitrosation reaction rate to reach a limiting value. An
anomalous behavior has also been observed when studying the
reactivity of cysteine with the nitroso group of alkyl nitrites.14
The results obtained show that the only reactive species is
Hcys(؊), noting that the rate constant reaches a limiting value
for pH > 10.
other nucleophiles such as:8 SCNϪ; IϪ; S2O3 and SO3 must
be included in this category. Hard nucleophiles such as the
hydroxyl ion9 or very basic alkoxides react through the sulfonyl
2Ϫ
2Ϫ
group. There is
a continuous gradation between these
behaviors, in such a way that when the basicity of the alcohol
decreases the percentage of reaction on the nitroso group
increases.10 As an extreme case, the hexafluoroacetone derived
These results contrast with the parallelism existing between
basicity and nucleophilic character, widely reported in the
literature and observed in particular in nitroso group transfer
reactions.15 In order to provide insight into the causes of this
anomaly, we have carried out the present study which includes
the reaction of MNTS with cysteine (CYS) and 2-amino-
Scheme 1
† Permanent address: Departamento de Química, Area de Química
Orgánica, Facultad de Ingeniería Química, Universidad Nacional del
Litoral, 3000 Santa Fe, Argentina.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
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Scheme 2
ethanethiol (AET) in a pH range between pH = 7 and pH = 13.
The results obtained show that, unlike those reported in the
literature, both the cysteine and the AET react with the MNTS
through the two microscopic forms possessing a negative charge
on the sulfur atom, with the reaction rate leveling off for high
pH values.
were conducted in aqueous solution at 25 ЊC using a number of
buffer solutions to cover the pH range 7–12. Good first order
behavior was found throughout, following every run to at least
90% reaction. Each run was repeated at least five times and the
average value of the pseudo-first-order rate constant, kobs, used
in the subsequent analysis was obtained. Standard error of kobs
is < 3%.
Experimental section
Results
All reactants used (MNTS, CYS and AET) were from Aldrich,
of the highest purity commercially available and used without
further purification. Kinetic experiments were performed with a
large excess of nucleophile over MNTS, [MNTS] = 1 × 10Ϫ4 M.
The concentrations of the nucleophiles used were typically in
the range 1.00 × 10Ϫ2 to 0.25 M. Due to the low solubility of
MNTS in water, its solutions were prepared in an acetonitrile/
water mixture, in such a way that the percentage of acetonitrile
in the reaction mixture was always < 1% (v/v). The pH was
controlled by buffer solutions of the nucleophile itself. The pH
was measured with a Radiometer 82 pH-meter equipped with
GK2401C or GK2401B (for pH > 11) combined glass
electrodes.
When MNTS is placed in a CYS or AET buffer solution two
kinetic processes are observed. Both kinetic processes take
place on very different time scales. A fast process, occurring
within a few seconds and characterized by an increase in
the absorbance at λ = 330 nm due to the formation of the
corresponding thionitrite, is observed. The obtained variation
in absorbance is consistent with the quantitative formation of
the corresponding S–NO compound and therefore in agree-
ment with an attack of the sulfur nucleophile on the nitroso
group of the MNTS. The second kinetic process occurs over a
period of hours and results in the disappearance of the absorp-
tion band centered at 330 nm corresponding to the thionitrite.17
The influence of the cysteine concentration on kobs in its reac-
tion with MNTS has been studied. The results shown in Fig. 1
indicate the existence of a clear linear dependence with a zero
intercept between kobs and [CYS]. The absence of intercept is
indicative of the fact that the basic hydrolysis of MNTS does
not take place on time scales competing with the transfer of the
nitroso group to the sulfur atom of the cysteine.9 The results
illustrated in Figs. 1 and 2 (influence of the AET concentration
on kobs in its nitrosation by MNTS) satisfy equation 1, where
Values of cysteine microscopic ionization constants have
been obtained from the literature (Scheme 2). The corre-
sponding AET values (Scheme 3) have been experimentally
obtained using the method described by Clement and Hartz.16
kapp is an apparent bimolecular rate constant since it includes
2
the different acid–base equilibrium constants of the nucleophile
Scheme 3
Kinetic experiments were performed in an Applied Photo-
physics MX17 stopped-flow spectrophotometer monitoring the
increasing absorbance at λ = 320 nm (CYS) and λ = 330 nm
(AET) due to the formation of thionitrites. All experiments
Fig. 1 Influence of the cysteine concentration on kobs in its reaction
with MNTS at 25 ЊC. (᭺) pH = 7.47; (᭹) pH = 7.94 and (ᮀ) pH = 8.63.
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sponding increase in kapp. For pH ≅ 9.5 all the cysteine should
2
be in the form of Hcys(؊), and the rate constant should be
pH-independent, however because of the third pKa of cysteine
the structure Hcys(؊) is in equilibrium with Cys(2؊) and the
rate constant will continue increasing with the pH. Such an
increase in the rate constant will reach a limiting value when all
the cysteine in solution is in the structure Cys(2؊), which
happens for pH larger than 12. Subsequently, the rate constant
kapp increases again with the pH until it reaches a limiting value
2
for pH > 12.
Discussion
The experimental results can be quantitatively explained by a
reaction mechanism involving nitroso group transfer from
MNTS to the forms of the cysteine Hcys(؊) and Cys(2؊)
(Scheme 4).
Fig. 2 Influence of the 2-aminoethanethiol (AET) concentration on
kobs in its reaction with MNTS at 25 ЊC. (᭺) pH = 7.44; (᭹) pH = 8.19
and (ᮀ) pH = 8.56.
From Scheme 4, the following expression (equation 2) for the
apparent rate constant kapp can be obtained:
and, [Nucleophile] represents the total concentration of CYS or
AET. As can be seen in Figs. 1 and 2, the slopes of the plots of
kobs versus the concentration of CYS or AET increase with the
2
pH of the medium or, according to equation 1, kapp increases
(2)
2
with the pH.
kobs = kapp[Nucleophile]T
(1)
2
Figures 3 and 4 show the influence of the pH on kapp. As
where the equilibrium constants refer to the processes depicted
in Scheme 2 and k1 and k2 are the rate constants corresponding
to the nitrosation of Hcys(؊) and Cys(2؊) respectively as
shown in Scheme 4.
2
can be observed, the kapp values increase with decreasing [Hϩ]
2
reaching a first leveling off for pH values ≅ 9.5. That leveling off
is not very clear because of the third and second pKa values of
cysteine and AET respectively. Increasing the pH increases the
concentration of Hcys(؊) in the cysteine solution, with a corre-
The experimental results nicely fit equation 2 as is made clear
by the curve plotted in Fig. 3. The value corresponding to the
first ionization, pK1 = 1.87, has been kept constant for the fitting
process. From the fit of the kinetic results to equation 2,
the macroscopic ionization constants can be obtained for the
second and third process: pK2 = 8.33 and pK3 = 10.24. These
values compare well with those reported in the literature (see
Scheme 2). From the kinetic analysis, the rate constants k1 =
11.4 MϪ1 sϪ1 and k2 = 19.2 MϪ1 sϪ1 have also been obtained.
These rate constants indicate that Cys(2؊) has a higher
reactivity toward the nitroso group than Hcys(؊), as might be
expected from its higher basicity.
The experimental behavior observed for the nitroso group
transfer from MNTS to AET can be quantitatively explained
on the basis of the existence of two simultaneous reaction
pathways: reaction of SNH and SN species (see Scheme 3).
These two reaction pathways allow us to obtain the following
expression (equation 3) for kapp
:
2
Fig. 3 Influence of pH on kapp for the transfer of the nitroso group
2
from MNTS to cysteine at 25 ЊC.
(3)
where the equilibrium constants refer to the processes shown in
Scheme 3 and the rate constants k1 and k2 refer to the nitros-
ation of SNH and SN respectively.
The analysis of kinetic data yielded values for the macro-
scopic acidity constants: pK1 = 8.46 and pK2 = 10.95, which
are in very good agreement with literature values. Likewise,
rate constants k1 = 13.0 MϪ1 sϪ1 and k2 = 18.8 MϪ1 sϪ1 have
been obtained. As had been observed in the cysteine nitros-
ation, the rate constants increase as the corresponding basicity
of the nucleophile increases.
Structure–reactivity correlations
Fig. 4 Influence of pH on kapp for the transfer of the nitroso group
The values obtained for the nitroso group transfer from MNTS
to sulfur nucleophiles show that these are more effective than
2
from MNTS to 2-aminoethanethiol (AET) at 25 ЊC.
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Scheme 4
the corresponding nitrogen or carbon nucleophiles of similar
(4)
basicity. In particular, the rate of nitrosation of SNH and
Hcys(؊) by MNTS is approximately 2000 times higher than
that of morpholine, a nitrogen nucleophile of the same basicity.
This higher strength of the sulfur nucleophiles is well
documented in the literature as evidenced by the different
values in Ritchie’s Nϩ scale.18
The variation of the nucleophilic reactivity of SN/SNH and
Cys(2؊)/Hcys(؊) with the basicity of the nucleophile shows a
behavior clearly different from that of the nitrogen or carbon
nucleophiles. The rate constant increases slightly by 75% by
increasing the basicity of the nucleophile approximately 200
times. The same increase in basicity brings about a rise of 104%
in the rate constant of the nitrosation of amines or carbanions
by MNTS. Such a difference of behavior is made clear when
establishing a Brønsted correlation giving values of βnuc ≅ 0.7
for the nitrosation of primary and secondary amines by
MNTS,15 whereas the value obtained for the nitrosation of the
sulfur nucleophiles studied is βnuc ≅ 0.08.
In view of the fact that the higher the basicity of RSϪ, the
more difficult the desolvation is, βd < 0 can be expected. Thus, if
βnuc is low, βnuc may be dominated by βd and be close to zero
or even negative. The values reported in the literature where
βnuc values are quite low for thiolate ion addition to a variety
of electrophiles are very common.23 These low βnuc values are
indicative of a transition state with little bond formation.
Acknowledgements
Financial support from Ministerio de Ciencia y Tecnología
(Project BQU2002-01184) and from Xunta de Galicia
(PGIDT00PXI20907PR and PGIDT03-PXIC20905PN) is
gratefully acknowledged. C. Adam thanks the University of
Santiago for a postdoctoral fellowship.
This change in the sensitivity of the reaction to the basic
strength of the nucleophile is unusual although not without
precedent and it has traditionally been regarded as a con-
sequence of the effect of desolvation on the reaction rate or on
reactivity–structure correlations. In fact, there are many cases
where the rate of certain nucleophilic attacks has been found
to decrease as the basicity of the nucleophile is increased,
leading to negative Brønsted exponents. This behavior has been
observed for some phosphoryl transfer reactions to amines,19
and for reactions of highly reactive carbocations with amines20
and for reactions of thiolate ions with Fischer carbene com-
plexes.21 In the same way, values of the Brønsted exponent close
to zero have been found for reactions of diphenylketene with
amines.22 Studies carried out by Jencks19 indicate that these
anomalous Brønsted exponents result from a requirement for
partial desolvation of the nucleophile prior to the reaction. The
desolvation is usually considered to be a pre-equilibrium that
occurs in a separate step, in such a way that a two-step model
like that illustrated in Scheme 5 can be adopted for a nucleo-
philic attack:
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Scheme 5
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