S-Nitrosocaptopril formation in aqueous acid and basic medium. A vasodilator and angiotensin converting enzyme inhibitor

Article abstract of DOI:10.1039/c1ob05747b

The reaction of S-nitrosocaptopril (NOcap) formation was studied in both aqueous acid and basic medium. Captopril (cap) reacts rapidly with nitrous acid in strong acid medium to give the stable - in the timescale of the experiments - NOcap. The kinetic study of the reaction involving the use of stopped-flow, shows that at low sodium nitrite (nit) concentration, the reaction is first-order in both [nit], [H⁺], and is strongly catalysed by Cl ^- or Br^- (= X^-): rate = (k₃ + k ₄[X^-])[H⁺][nit][cap]. In aqueous buffered solution of acetic acid-acetate the reaction rate is much slower and the decomposition of NOcap was observed; however, the rate of NOcap decay is more than 30-fold slower than its formation. In aqueous basic medium of carbonate-hydrogen carbonate buffer, as well as in alkaline medium, the kinetics of the nitroso group (NO) transfer from tert-butyl nitrite (tBN) to cap was studied using either conventional or stopped flow methods. In mild basic medium, the NOcap decomposes. The NOcap formation is first-order in both tBN and cap concentrations, and the reaction rate increases with pH until to, approximately, pH 11.5, above which value it becomes pH independent or even invariable with the [OH^-]. Kinetic results show that the thiolate ion of cap is the reactive species. In fact, the presence of anionic micelles of sodium dodecyl sulfate (SDS) inhibits the reaction due to the separation of the reagents; whereas, cationic micelles of tetradecyltrimethylammonium bromide (TTABr) catalyse the reaction at low surfactant concentration due to reagents concentration in the small volume of the micelle. The rate equation is: rate = k_f K_SH[cap][tBN]/(K_SH + [H⁺]). The rate of NOcap decomposition in mild basic medium is first-order in both [cap] and [NOcap], and decreases on increasing pH; but, in alkaline medium the NOcap is stable within the timescale of the experiments. Based on the results, the NOcap decomposition yields the disulfide compound that is formed in the nucleophilic attack of the -SH group of cap to the sulfur electrophilic center of NOcap, -S-NO. The resulting rate equation is: rate = k_d[H ⁺][cap][NOcap]/(K_SH + [H⁺]).

Full text of DOI:10.1039/c1ob05747b

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PAPER
S-Nitrosocaptopril formation in aqueous acid and basic medium.
A vasodilator and angiotensin converting enzyme inhibitor†
a
b
Alexia Sexto and Emilia Iglesias*
Received 12th May 2011, Accepted 12th July 2011
DOI: 10.1039/c1ob05747b
The reaction of S-nitrosocaptopril (NOcap) formation was studied in both aqueous acid and basic
medium. Captopril (cap) reacts rapidly with nitrous acid in strong acid medium to give the stable—in
the timescale of the experiments—NOcap. The kinetic study of the reaction involving the use of
stopped-flow, shows that at low sodium nitrite (nit) concentration, the reaction is first-order in both
+
-
-
-
-
+
[
nit], [H ], and is strongly catalysed by Cl or Br (= X ): rate = (k
3
+ k [X ])[H ][nit][cap]. In aqueous
4
buffered solution of acetic acid–acetate the reaction rate is much slower and the decomposition of
NOcap was observed; however, the rate of NOcap decay is more than 30-fold slower than its formation.
In aqueous basic medium of carbonate–hydrogen carbonate buffer, as well as in alkaline medium, the
kinetics of the nitroso group (NO) transfer from tert-butyl nitrite (tBN) to cap was studied using either
conventional or stopped flow methods. In mild basic medium, the NOcap decomposes. The NOcap
formation is first-order in both tBN and cap concentrations, and the reaction rate increases with pH
until to, approximately, pH 11.5, above which value it becomes pH independent or even invariable with
-
the [OH ]. Kinetic results show that the thiolate ion of cap is the reactive species. In fact, the presence of
anionic micelles of sodium dodecyl sulfate (SDS) inhibits the reaction due to the separation of the
reagents; whereas, cationic micelles of tetradecyltrimethylammonium bromide (TTABr) catalyse the
reaction at low surfactant concentration due to reagents concentration in the small volume of the
+
micelle. The rate equation is: rate = k
f
K
SH[cap][tBN]/(KSH + [H ]). The rate of NOcap decomposition
in mild basic medium is first-order in both [cap] and [NOcap], and decreases on increasing pH; but, in
alkaline medium the NOcap is stable within the timescale of the experiments. Based on the results, the
NOcap decomposition yields the disulfide compound that is formed in the nucleophilic attack of the
–
SH group of cap to the sulfur electrophilic center of NOcap, –S–N O. The resulting rate equation is:
+
+
rate = k [H ][cap][NOcap]/(KSH + [H ]).
d
Introduction
The process does not alter the ability of cap to inhibit ACE and
provides an additional mechanism by which NOcap can affect
vascular relaxation. Both properties in the same molecule make
it potentially a pharmacologically very interesting NO-donating
drug.
Activation of guanylate cyclase and inhibition of angiotensin
converting enzyme (ACE) are the two major mechanisms for
1,2
vasodilation. NO is a biological transmitter that mediates in
3–5
vasodilation and in angiogenesis. The interactions of NO with
sulfhydryl-containing molecules and enzymes, the S-nitrosation
reactions (also referred to as S-nitrosylation), affect protein
function and determine the many effects of NO in vivo. The
nitrosation of the thiol group of captopril (cap) results in the
It is of particular interest to optimize the various experimental
conditions of NOcap formation, as well as to investigate its
stability, since the decomposition of NOcap releases the biological
significant species (NO and cap) to the in vivo system. In addition,
S-nitrosothiols can also modify protein structure and function
by inducing mixed disulfides and intramolecular disulfide in
the direct attack of the thiol/thiolate group of enzyme-lysine
6–8
9,10
formation of S-nitrosocaptopril (NOcap).
11
to the S-nitrosothiol. In this sense, Wang et al. studied the
effect of S-nitrosothiols on the cellular regulatory mechanism of
papain inactivation. Since many disease states such as muscular
dystrophy, inflammation, and rheumatoid arthritis are associated
with elevated proteolytic activity of cysteine proteases, much
attention has been paid to the rational design and synthesis
a
Department of Biolog ´ı a Celular y Molecular, Facultad de Ciencias,
Universidad de La Coru n˜ a, 15071 La Coru n˜ a, SPAIN
b
Department of Qu ´ı mica F ´ı sica e E. Q. I, Facultad de Ciencias, Universidad
de La Coru n˜ a, 15071 La Coru n˜ a, SPAIN. E-mail: emilia.iglesias@udc.es;
Fax: +34 981 167065; Tel: +34 981 167000
†
Electronic supplementary information (ESI) available: Potentiometric
12,13
data. Details of biexponential fitting. See DOI: 10.1039/c1ob05747b
of selective inhibitors of these types of enzymes.
Papain is
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Org. Biomol. Chem., 2011, 9, 7207–7216 | 7207

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one of the most studied plant cysteine proteases that shares
many features with physiologically important mammalian cysteine
proteases. The active site of papain and others cysteine proteases
glass electrode and calibrated using commercial buffers of pH
4.01, 7.02, and 9.26 (Crison). The reported [buffer] refers to the
total buffer concentration. In strong acid (HClO ) and alkaline
4
+
-
(
enzymes capable of hydrolysing peptide bonds), contain an
(NaOH) media the acidity is given as [H ] or [OH ], respectively.
Kinetics of fast reactions were studied on a Bio-Logic SFM-
20 stopped-flow system interfaced with a computer and operated
by a Bio-Kine32 software (V4.51, 2009), noting the increasing
absorbance at 325 nm due to the S-nitrosocaptopril.
14
essential cysteine sulfhydryl and histidine imidazole unit. Wang’s
results suggest that inactivation of papain by S-nitrosothiols could
be due to a direct attack of the reactive thiolate of cysteine unit
25
(Cys ) in the enzyme active site, on the sulfur of S-nitrosothiols
11
to form a mixed disulfide between the inactivator and papain.
The UV–vis spectra and kinetics of slow reactions (t1/2>60 s)
were recorded with a Kontron-Uvikon double beam spectropho-
tometer fitted with thermostatted multicell holders.
The aim of this work is to explore the best experimental
conditions of NOcap formation in vitro and its stability as a
function of the acidity, with the goal of exploring the mechanism
of enzyme inactivation by NOcap. For that, we investigated the
NOcap formation in both acid and basic medium. In acid medium,
sodium nitrite (nit) was used as the NO-precursor, whereas the
NO-transfer from tert-butylnitrite (tBN) to cap has been applied
in the formation of NOcap in basic medium. The reaction
has also been studied in aqueous micellar solutions of both
anionic micelles of sodium dodecyl sulfate (SDS) and of cationic
micelles of tetradecyltrimethylammonium bromide (TTABr). Our
results clearly support the hypothesis of Wang on the basis of a
mechanism that yields disulfide bonds in the interaction of –SH
and –SNO groups at pH values close to that of the imidazolium
Methods
Experiments were performed under pseudo-first order conditions
with either cap or nit as the limiting reagent concentration;
experiments carried out in basic medium, with tBN being the
NO-transfer, were performed with an excess of [cap] over [tBN].
Measurements of absorbance (A) versus time (t) fit perfectly the
first-order integrated rate equation, A
unless otherwise indicated. The non-linear regression analysis of
A–t data gives k , A , and A as optimizable parameters, with
being the pseudo-first order rate constant and A, A , and A
t
= A
•
+ (A
o
- A
•
)·exp(-k
o
t),
o
o
•
k
o
o
•
14
being the absorbance values at times t, zero, and at the end of the
reaction.
(
pK ~8.5) of the active site of cysteine proteases. The results
a
in micelles show the effective interaction of the cap anion with
the cationic micelles of TTABr; this feature is consistent with
the presence of imidazolium cation on the active site of cysteine
proteases such as papain.
◦
All experiments were carried out at 25 C and the ionic strength
has not been adjusted because the effect was negligible (at least at
I <0.1 M).
Products characterization
Experimental
Spectroscopic characteristics of reaction product coincide with
those observed for NOcap that has been prepared and charac-
terized by Loscalzo. Decomposition of thionitrites, RSNO, in
Materials
9
Captopril or N-[(S)-3-mercapto-2-methyl propionyl)-L-proline of
the higher available purity was obtained from Sigma and was
used as received. t-Butylnitrite was synthesized by treating the
corresponding alcohol with sodium nitrite in aqueous sulfuric
acid, purified by fractional distillation under reduced pressure
and stored at low temperature over molecular sieves to prevent
neutral aqueous solutions to give the corresponding disulfide and
initially nitric oxide with quantitative yields is well documented in
the literature.
6,16
Results and discussion
15
hydrolysis. All other reagents were of the maximum commercially
available purity and were used as supplied.
1
. Potentiometric titration
Stock solution of t-butylnitrite was prepared daily in dioxane. A
small volume of this solution (<100 mL) was dissolved in weakly
basic medium (~2 mM carbonate buffer or sodium hydroxide)
in which it is stable during the required time. The percentage of
dioxane in the final reaction mixture never exceeded 1% by volume.
The rest of the solutions were prepared with water that was firstly
deionised and subsequently twice distilled (the first distillation
over potassium permanganate).
The captopril molecule is a mercapto-proline derivative that
contains two acid ionisable groups: the carboxylic group of the
proline residue and the thiol group of the propionyl moiety. Within
the interval of acidity used in the present study, both acid groups
of cap can be ionized. Because of that, the potentiometric titration
has been performed from solutions that contain 8.8 mM of cap
at ionic strength 0.10 M (NaCl) and titrating with a standardized
sodium hydroxide solution in order to remove the two protons
of the molecule and to obtain the macroscopic pKOH and pKSH
(Scheme S1 of ESI†). Both equilibrium constants were calculated
form the potentiometric titration data, which are shown in Fig.
The addition of a small concentration (20 mM) of EDTA (which
would complex the possible trace quantities of metal ions such as
+2
+
Cu /Cu that catalyse the S-nitroso compounds’ decomposition)
to the reaction sample did not affect the kinetic results.
17
S1 of ESI,† by means of the Hyperquad 2003 program as
pKOH = 3.52 ± 0.02 and pKSH = 10.00 ± 0.01. The first value is
attributed to the carboxylic group and the second one, to the thiol
group. In this sense, the aqueous solution of 8.8 mM of cap at
0.10 M (NaCl) ionic strength has a pH = 2.82 in the absence of
Techniques
The pH was controlled using buffer solutions of acetic acid–
acetate or carbonate–hydrogen carbonate and was measured with
a Crison 2001 pH-meter equipped with a GK2401B combined
18
titrant; then, using the approach of Nyasulu et al. that easily
and rapidly determines K and K of uncharged substrates, one
a
b
7
208 | Org. Biomol. Chem., 2011, 9, 7207–7216
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calculates pKOH = 3.50, which is in perfect agreement with the value
calculated from the acid–base titration curve. For comparison
purposes, the microscopic pK corresponding to the ionization
of –COOH group in, for instance, cysteine varies from 2.00 to
either in aqueous perchloric acid or acetic acid–acetate buffer
(0.25–0.50 M, pH 3.6 to 5.2), or even in alkaline medium ([OH ] =
0.035 and 0.10 M).
-
To carry out the kinetic experiments, the concentration of the
limiting reagent must not exceed 2 mM and the reaction progress
was recorded at 325 nm. To check the reaction stoichiometry, the
absorbance at 325 nm was measured at the end of the reaction for
different [cap]/[nitrite] ratios; the results depicted in Fig. 2 show
that the maximum value was obtained at equal concentration of
both reagents, which indicates a 1 : 1 cap : nitrite stoichiometry in
the nitrosation reaction.
4
.99, whereas the microscopic pK of ionization of –SH group in
the same compound varies between 7.44 and 9.87 and the pK of
a
19
propane thiol is reported as 10.65.
2
.
Characteristics of nitrosocaptopril formation reaction
Equimolar concentrations (14 mM) of cap and nit were mixed
and allowed to react in strong acid medium (HClO
4
0.060 M)
◦
at 25 C. The reaction mixture turns red–brown instantaneously,
due to the NOcap formation. The UV–visible spectrum, recorded
between 200–650 nm, does not change with time, and shows strong
absorption below 450 nm and a much less intense absorption band
-
1
-1
centered at 547 nm (e = 20 M cm ). The longer absorption band
is attributed to the forbidden n
N
→p* transition and determines
the color of NOcap. In order to properly observe the band at lower
wavelength, the solution should be diluted at least 5-fold; then, the
characteristic band of S-nitroso compounds centered at 332 nm
-
1
-1
(
e = 1020 M cm for NOcap), attributed to the allowed n
O
→p*
transition, is observed. There is a third strong absorption band
below 260 nm, attributed to the p→p*, which cannot be used to
follow the reaction due to the overlap with cap spectrum, whose
aqueous solutions show strong absorption at wavelengths smaller
than 250 nm, approximately, Fig. 1.
Fig. 2 Stoichiometric plot of the reaction of nit and cap in aqueous
perchloric acid 0.050 M showing the variation of the absorbance readings
at the end of the reaction for [cap] = [nit] = 2 mM mixed at different
concentration ratios.
In strong acid medium, the cap-nitrosation reaction is too fast
to follow it by conventional methods; therefore, the stopped-flow
technique has been used. By contrast, in mild acid medium of
acetic acid–acetate buffer the reaction was followed by conven-
tional methods registering the increase absorbance also at 325 nm;
Fig. 3(a) shows the reaction spectra in acetic acid–acetate buffer
under conditions of [nit] < [cap]. The reaction spectra in aqueous
basic medium of NOcap formation in the NO-transfer reaction
from tert-butyl nitrite to cap are shown in Fig. 3(b). Notice the
-
higher absorption of NOcap than either HNO
2
/NO
2
system or
tBN, and the clear isosbestic point drawn at 277 nm in the reaction
of cap + tBN → NOcap + tBOH (t-butanol).
+
Fig. 1 UV–visible spectra of [1] cap 2.9 mM at [H ] = 0.05 M; [2] NOcap
formed in the reaction sample of [nit] = 1.0 mM, [cap] = 2.9 mM, and
3
. NO-transfer in strong acid medium
+
[
[
H ] = 0.050 M; and [3] NOcap formed in the reaction sample of [cap] =
nit] = 0.0 12 M and [H ] = 0.060 M.
+
The NOcap formation was studied in aqueous perchloric acid
using the stopped-flow method. Several parameters that affect the
reaction rate have been analysed including [nit], [H ], and [X ]
+
-
The NO-transfer to cap from S-nitroso-N-acetyl-D,L-
10
-
-
-
penicillamie (SNAP) has been studied in aqueous basic medium.
(with X = Cl or Br ).
+
The closeness of the maximum wavelength absorption of SNAP
and NOcap and the resulting overlapping of the spectra compli-
cates the kinetic study, which had been followed at 547 nm, using
high reagent concentrations. The use of tBN as the NO-donor
results in a non-complicated system from the spectral point of
view.
When no excess nitrite is present in the reaction mixture, the
generated NOcap is stable for longer. In this respect, the reaction
followed during 15 h did not show appreciable decomposition,
At constant [H ] = 0.025 M the influence of [nit] was investigated
in the range (3–30) mM with cap as the limiting reagent (0.7–
0.07) mM. In every experiment the A versus t fits perfectly the
first-order integrated rate equation with c < 0.002. The pseudo-
first order rate constant, k , increases with [nit] according to eqn
(1), Fig. 4(a). The second-order term in [nit] will be significant at
high nitrite concentrations. The non-linear regression analysis of
k
k
2
o
-
1
3
-1
o
against [nit] to eqn (1) gives k
= (442.5 ± 0.3) mol dm s (cc = 0.999 ).
1
= (56.1 ± 0.8) mol dm s and
-
2
6
-1
2
7
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Fig. 3 (a) Reaction spectrum recorded at 5 min interval (scans 1 to 8)
for the NO-transfer to cap 1.76 mM in aqueous acetic acid solutions
Fig. 4 (a) Variation of k
o
as a function of [nit] for the reaction of NOcap
+
(
[buffer] = 0.55 M, pH 4.1) of [nit] = 1.0 mM; ( ◊ ◊ ◊ ) no nitrite; (---) at
the end of the reaction. (b) Reaction spectrum recorded at 2 min interval
scans 3 to 9) for the NO-transfer to cap 1.6 mM from tBN 1.1 mM in
carbonate–hydrogen carbonate buffer ([buffer] = 0.066 M, pH 10.2); scans
and 2 show the spectrum of cap and tBN, respectively, under the same
formation in aqueous perchloric acid solutions at [H ] = 0.025 M; (b) Plot
+
of k
o
against [H ] (HClO
4
) obtained in the NOcap formation in aqueous
(
solutions of (᭹) [cap] = 0.45 mM and [nit] = 8.5 mM and (᭢) [cap] =
6.0 mM and [nit] = 0.45 mM, at 25 C.
◦
1
+
The variation of k
o
as a function of [H ], depicted in Fig. 4(b),
experimental conditions of the reaction, and scan 10 corresponds to the
spectrum at the end of the reaction.
describes good straight lines with zero-intercept.
Under the experimental conditions of the results of Fig. 4(b), the
contribution of the second-order term in [nit] of eqn (1) is much
smaller than the first-order term (i.e, under the less favourable
2
k
o
= k
1
[nit] + k
2
[nit]
(1)
conditions of [nit] > [cap] it represents 6.7%), therefore, it can be
+
Aqueous acid solutions of nit produce two nitrosating agents:
stated that rate = k
3
[nit][cap][H ]. The least-squares fit of k
o
vs.
+
8,20
+
+
-1
3
-1
the nitrosyl ion (NO ) and dinitrogen trioxide (N
2
O
3
). NO is
[H ] gives the slope value of (21.1 ± 0.2) mol dm s when [nit] =
-
1 3
-1
formed by the protonation and subsequent dehydration of nitrous
8.5 mM and of (15.1 ± 0.2) mol dm s when [cap] = 6.0 mM.
+
+
-7
-1
-2
acid (HNO
2
+ H ꢀ NO OH
2
, KNO = 3 ¥ 10 M ); the formation
ꢀ N
= 3 ¥ 10 M ); then, it is kinetically detected at high [nit].
These results afford comparable values of k = 2482 and 2517 mol
dm s , respectively, to the experiments performed with cap or nit
3
6
-1
of N implies two nitrous acid molecules (2HNO
2
O
3
2
2
O
3
,
-
3
-1
K
N O
2 3
as the limiting reagent.
-
-
Therefore, the first-order term in [nit] is due to cap nitrosation
The effect of Cl and Br was investigated at [cap] = 0.45 mM,
+ +
+
+
-
by NO (NO OH
nitrosation step by N
, it can be concluded that at [nit] <~15 mM the nitrosation of
2
), whilst the second-order term results from the
[nit] = 9.0 mM and [H ] = 0.010 M (for Cl ) and [H ] = 0.014 M
-
-
2
O
3
. Considering the relative values of k and
1
(for Br ). In either case, the reaction is strongly catalysed by Cl or
Br , although the effect of bromide is higher than that of chloride.
-
k
2
+
2
-
-
-
-
cap goes mainly through NO , i.e. k
1
[nit] > 8.5k
2
[nit] . Therefore,
The plot of k
o
vs. [X ] (X means Cl or Br ) is a straight line with
in order to facilitate the quantitative treatment of the results, low
non-zero intercept, but, in order to highlight the different effect
+
[
[
nit] was used in the following experiments.
The influence of [H ] was studied by working with either [cap] <
nit] or [cap] > [nit]. Under any of the two experimental situations,
of both halide ions, Fig. 5 shows the variation of k
as a function of [X ]. The least-squares fit of the data yields the
o
/([nit][H ])
+
-
intercept (= k
50) mol dm s and k
experiments with Cl and k
(155.6 ± 0.9)¥10 mol dm s for the experiments with Br .
3
6
) and slope (= k
4
) values of eqn (2) as k
3
= (2445 ±
-
2
1
3
-3
9
-1
the A–t data fits perfectly the first-order integrated equation, i.e.,
first-order with respect to [nit] in the first set of experiments and
first-order with respect to [cap] in the second set of experiments.
4
= (44.3 ± 0.5)¥10 mol dm s for the
-
-2
6
1
3
= (2720 ± 70) mol dm s and k =
4
3
-3
9
-1
-
7
210 | Org. Biomol. Chem., 2011, 9, 7207–7216
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Table 1 Experimental conditions used in the NOcap formation in aqueous perchloric acid from nitrous acid, values of the kinetic rate constants
determined experimentally, k
i
(i = 1,2,3, or 4), and their correspondence with the rate constants of the reaction mechanism of Scheme 1
+
XNO/M-1 s-1
[
nit]/mM
[cap]/mM
[H ]/M
k
exp
equivalence
value
K
equilibrium
k
k
a
+
56.1 M-1 s-1
442.5 M
2482 M
2517 M
2445 M
2720 M
NO = 3 ¥ 10 M-1
-7
9
3
3
8
0
9
9
9
9
–30
–30
.5
0.7–0.07
variable
0.45
6.0
0.45
0.45
0.45
0.45
0.025
0.025
variable
variable
0.10
0.14
0.10
k
k
k
1
2
3
k
NO
K
NO[H ]
K
K 2
NO = 7.5 ¥ 10
a
-2 -1
-3
-1
5
k 2
N
O3 K 2
N
O3
s
N
O3 = 3 ¥ 10
M
M
k 2
N
O3 = 1.5 ¥ 10
a
-2 -1
-7
-1
9
9
k
NO
K
NO
s
K
NO = 3 ¥ 10
k
k
k
k
k
k
NO = 8.5 ¥ 10
NO = 8.4 ¥ 10
a
-2 -1
.45
s
b
-2 -1
9
.0
.0
.0
s
NO = 8.15 ¥ 10
c
-2 -1
9
s
NO = 9.0 ¥ 10
b
-3 -1
ClNO = 1.1 ¥ 10 M-2
BrNO = 0.051 M
-3
7
k
k
4
4
k
k
ClNO
BrNO
K
K
ClNO
44.3 M
s
K
K
ClNO = 3.9 ¥ 10
BrNO = 3.1 ¥ 10
c
155.6 M s-1
-3
-2
6
.0
0.14
BrNO
a
No added Cl or Br ; variable [Cl ]; variable [Br-]
-
-
b
-
c
Scheme 1 Equilibrium and reaction steps that occur in the NOcap
formation in aqueous acid solutions of sodium nitrite.
+
+
Fig. 5 Variation of k
o
not dependent on [H ] and [nit]-i.e. k
o
/([H ][nit])-
[
nit] used in the experiments of Fig. 4(b), the second-order term
on [nit] is negligible (the nitrosation step by N is distinguished
experimentally by the absence of acid catalysis, apart from the
second order dependence upon [HNO ]).
-
-
as a function of (᭹) [Cl ] and (᭡) [Br ] obtained in the NOcap formation
in aqueous perchloric acid solutions of sodium nitrite.
2
O
3
2
−
+
⎢
⎥ ⎢
)
⎥
H [nit]
ko =
(
k + k X
3
4
(2)
⎢
⎥ ⎢
⎥
⎦
−
⎥ ⎢
⎦ ⎣
+
⎣
⎦ ⎣
⎡
⎤ ⎡
)
⎤
H
rate =
(
kNOKNO + kXNOKXNO
X
[ ]
nit][cap +
⎥
⎦
⎢
⎣
(
3)
2
This rate equation suggests a reaction mechanism with two
slow reaction steps due to nitrosation by both NO and XNO.
A common feature of many nitrosation reactions is the ability of
halide ions and other nucleophiles to act as catalysts in acidic
solution. This is due to the equilibrium displacement towards
kN KN
O O
2 3 2
[nit] [cap
]
3
+
The comparison of the experimental rate constants, k
i
i = 1, . . . ,
4
, with the corresponding expression of eqn (3), allows the values
8,20
of the bimolecular rate constants, kNO, kN2O3 , and kXNO reported in
Table 1, along with the experimental conditions.
+
-
the nitrosating agent NOX (HNO
2
+ H + X ꢀ XNO, KXNO),
when compared with the equilibrium constant of NO formation
+
As is often found in nitrosation studies, the most reactive agent
+
-
3
-2
-2
with thiol group of cap is the NO . The mean value of k = 8.3 ¥
1
constant. N O is the less reactive species and ClNO transfers
(
K
NOX = 1.1 ¥ 10 and 5.1 ¥ 10 M for X = Cl and Br, respectively).
NO
9
-1
3
-1
0
mol dm
s
corresponds to a diffusion controlling rate
The rate constants for the nitroso group transfer from NOX
8
,20,21
follow the order NOCl > NOBr,
demonstrating that the
2
3
the nitrosyl group 10-fold faster than BrNO, due to the greater
electronegativity of Cl than Br.
catalytic effect is due to the thermodynamics of the formation
of the nitrosating agent.
The overall observed experimental facts can be easily interpreted
according to a reaction mechanism in which the thiol group of cap
4.
NO-transfer in mild acid medium
(
but not thiolate) reacts in a rate limiting step with the nitrosating
S-nitrosocaptopril formation has also been studied in aqueous
-
-
-
-
agent XNO, where X = H
reaction mechanism is shown in Scheme 1
2
O, NO
2
, Cl or Br . The corresponding
solutions of acetic acid–acetate. Nitrous acid is a weak acid (pK =
a
◦
22
3.15 at 25 C). Measurements at pH >~2.5 require the correction
-
In strong acid medium the stoichiometric [nit] is in the form
due to NO
2
formation to be made. That is, the overall nitrite
◦
22
of HNO
2
, due to the pK
a
= 3.14 at 25 C of this weak acid.
concentration is the sum of nitrite ion and nitrous acid; which
+
+
Then, the rate eqn derived from Scheme 1 is that of eqn (3), which
implies that [HNO
2
] = [nit][H ]/(K
a
+ [H ]). Therefore, the first
predicts each one of the observed experimental facts, of which, it
observed feature is the reduction of the reaction rate as the pH
+
-
is convenient to remark the fact that the straight line of k
o
vs. [H ]
increases, as can be see in Fig. 6(a). The presence of either Cl or
-
plot with zero-intercept indicates that, firstly, the thiol group (and
not the thiolate) is the reactive form of cap, and secondly, at the low
Br enhances the reaction rate, due to the increase of the effective
nitrosating agent, XNO, Fig. 6(b).
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Table 2 Values of the observed rate constant, k
o
, measured in the NOcap
formation in aqueous acetic acid–acetate solutions of nitrite as a function
of total buffer concentration
[buffer]/M
k
o
/10- s-1
3
[buffer]/M
k
o
/10-3 s-1
pH 4.0
pH 4.5
[
cap] = 0.80 mM
o
[nit] = 10 mM
[cap]
o
= 0.80 mM
[nit] = 10 mM
0.15
0.20
0.25
0.30
0.45
0.60
0.70
0.80
0.95
1.41
1.42
1.45
1.51
1.59
1.65
1.77
1.78
1.79
0.10
0.15
0.20
0.25
0.30
0.45
0.55
0.65
0.70
0.364
0.377
0.381
0.391
0.410
0.435
0.452
0.467
0.475
N-acetyl-D,L-penicillamine (SNAP) has recently been studied in
10
aqueous basic medium of phosphate buffer. The closeness of
the maximum wavelength absorption of SNAP and NOcap and
the resulting overlapping of the spectra complicates the kinetic
study, which had to be carried out in the visible spectral region by
working with high reagent concentrations. The use of tBN as the
NO-donor results in a very much simpler system, at least from the
experimental point of view.
Therefore, in this study tBN was used in the NO-transfer
reaction to cap. The reaction was studied in aqueous solutions
of carbonate–bicarbonate buffer (pH range in between 9 to 11,
approximately) and in alkaline medium (NaOH) by noting the
increase in absorbance at 325 nm due to NOcap formation, and
with tBN being the limiting reagent ([cap] ≥ 10[tBN]).
The absorbance–time (A–t) profiles suggest the existence of a
rapid reaction (due to NOcap formation) followed by a much
slower one (due to NOcap decomposition). The occurrence
of both reactions is clearly observed at low pH (solutions of
Fig. 6 (a) Effect of pH on k
o
for NOcap formation in 0.31 M acetic
acid–acetate buffer at [nit] = 12.5 mM, [cap] = 0.80 mM and (᭹) no added
-
-2
HCO
3
/CO ); nevertheless, as the pH is increasing—i.e. in
3
-
-
Br (᭡) [Br ] = 0.075 M; (b) variation of k
o
for NOcap formation reaction
alkaline medium—only the reaction due to NOcap formation was
observed. Fig. 7 shows representative A–t plots of both reactions.
in 0.25 M acetic acid–acetate buffer at [nit] = 12.5 M as a function of (᭹)
-
-
[
Cl ] at pH 4.05 and of (᭡) [Br ] at pH 3.90.
The reaction rate is slightly affected by the total buffer concen-
tration, as the data in Table 2 show, which may be attributed to
the ionic strength effect. In this sense, at pH >4 more than 75%
of the stoichiometric cap concentration is in the ionic form, i.e.
-
the carboxylic group—pK
1
= 3.52—is ionized: –COO , and the
-
-
+
nitrosating agent in the absence of Cl or Br is the NO .
Finally, as distinct from the reaction in strong acid medium,
the NOcap is not stable under these experimental conditions.
However, the decay rate is very slow, e.g. at pH 4.26 the observed
-
5
-1
rate constant has been measured as 4.1 ¥ 10 s , which is almost
2
0-fold lower than the rate of NOcap formation.
. NO-transfer in basic medium
5
Aqueous basic solutions of nitrite salts do not generate nitrosating
agents. One alternative is the use of alkyl nitrites, RONO, which
are among the few nitrosating agents for basic media. In acid
medium, alkyl nitrites such as tBN hydrolyse very fast to the parent
Fig. 7 Plot of absorbance readings at 325 nm against time for the reaction
of cap 12.7mM and tBN 1.1 mM in aqueous basic medium of 0.085 M
carbonate–bicarbonate buffer of pH 10.1, A–t data fits perfectly to A = A
t) + C t), the plot contains 1000 experimental points
•
+
C
1
·exp(-k
1
2
·exp(-k
2
15
alcohol and nitrous acid; by contrast, their basic hydrolysis is a
and the solid line (undistinguishable) of the fit process passes thorough
every point; the inset shows the same plot for the reaction of 12.7 mM cap
and 1.1 mM tBN in alkaline medium of [OH ] = 0.14 M, but the A–t data
23
very slow process and can be used as NO-donors under these
24,25
-
experimental conditions
and in basic medium the nitrite ion is
not a nitrosating agent. The NO-transfer to cap from S-nitroso-
fits perfectly to A = A
•
- (A
o
- A
•
)·exp(-k
1
t).
7
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The influence of [cap] was analysed at pH 10.1, [buffer] = 0.085
M; NaOH solution was used to adjust the pH on increasing [cap]
because of the ionization of the acid group of proline moiety
and thiol. The A–t data profiles were fitted to the biexponential
To further elucidate the process, the effect of the pH was studied
in aqueous solutions of 0.15 M carbonate–bicarbonate buffer and
[cap] = 12.7 mM. At approximately pH <11, the A–t data profiles
were fit to the double exponential equation, whilst above pH 11,
or in alkaline medium, no decomposition of NOcap was detected,
at least within the same time scale.
equation A = A
•
+ C
1
·exp(-k
, C , k
In order to obtain a proper convergence of the
fitting process, the input values of C , and k were estimated in
1
t) + C
2
·exp(-k
2
t) by non-linear
being the optimized
regression analysis, with A
•
1
1
, C , and k
2
2
26,27
parameters.
The k values increase with pH by describing a sigmoid curve,
1
1
1
Fig. 9(a). This experimental fact suggests the occurrence of an acid
ionization equilibrium with the conjugate base being the reactive
species; in other words, the ionization of the -SH group of cap
to thiolate takes place and it is the nucleophilic attack of thiolate
that provokes the NO-transfer. Scheme 2(a) illustrates the reaction
steps.
a pre-fitting of only the increasing portion of the overall A–t
curve, i.e. the reaction due to NOcap formation. The reported
values correspond to that obtained in the fit of A–t data to
the biexponential equation, with correlation coefficients >0.9999.
Both rate constants, k
1
and k , increase proportionally to [cap],
2
-
1
see Fig. 8, according to the equations k
1
= (0.991 ± 0.009) M
-
1
-2
-1 -1
s ·[cap] and k
than 30-fold k
2
= (2.70 ± 0.03) ¥ 10
M
s ·[cap], but k
1
is more
2
.
Fig. 9 Variation of the rate constants (a) k
fer from tBN to cap, and (b) k , determined for NOcap decomposition,
as a function of pH; experimental conditions [cap] = 12.7 mM, [tBN] =
.11 mM, [buffer] = 0.15 M (carbonate–bicarbonate), above pH~11.5 only
1
, determined in the NO-trans-
2
1
NaOH has been used.
Fig. 8 (a) Variation of the pseudo-first order rate constant, k
function of [cap] for the NOcap formation, at [buffer] = 0.085 M of pH 10.1
carbonate–bicarbonate buffer); (b) values of the rate constant of NOcap
decomposition, k , measured as a function of [cap] in 0.085 M total [buffer]
1
, as a
Taking into account that the stoichiometric cap concentration
(
-
-2
is [cap]
o
= [capH ] + [cap ] and the acid ionization constant of
2
thiol, KSH, the eqn (4) can be obtained.
pH 10.1 (carbonate–bicarbonate buffer).
kf KSH
[
cap
+
⎢
⎣
]
rate = k1
[
tBN
]
with k1
=
(4)
The pseudo-first order rate constant k
1
corresponds to the
KSH + ⎡H ⎤
⎥
⎦
reaction between tBN and the thiol/thiolate group of cap, whereas
is due to the decomposition reaction of NOcap and increases
also with [cap], that is, the excess cap participates in the NOcap
decomposition reaction.
k
2
This eqn predicts that the graph of the reciprocal plot of k
against [H ] should result in a straight line, as experimentally
was obtained, Fig. 10(a). Least-squares fitting of the data gives:
1
+
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-
Table 3 Values of k determined in the NO-transfer for tBN to cap as a function of [OH ] and the limit value of the bimolecular rate constant k for the
1
f
27
reaction of tBN and the thiolate group of cap, along with literature values of k
f
with different thiols
thiol
-
/10-2 s-1
/M-1 s-1
k
f
/M-1 s-1
[
OH ]/M
k
1
k
f
28
0.073
0.092
0.110
0.137
0.165
0.202
0.257
2.67
2.71
2.85
2.81
2.65
2.66
2.68
2.10
2.13
2.24
2.20
2.10
2.10
2.10
limit value of k
cysteine
cysteine-methyl ester
cysteine-ethyl-ester
N-acetyl-cysteine
glutathione
f
for tBN and the thiol
1.7
1.6
1.5
1.8
1.8
4.9
thioglycol acid
Scheme 2 Proposed equilibrium and reaction steps that occur in basic
medium (a) in the NO-transfer reaction, k , from tert-butyl nitrite and
captopril and (b) in the S-nitrosocaptopril decomposition, k , to give the
f
d
disulfide compound in the reaction with the thiol group of captopril.
The numbers on S, H, N, and O atoms refers to the corresponding
electronegativity.
intercept = 38 ± 1 s and slope = (3.91 ± 0.045)¥10 M-1 s (cc =
1
1
0
k
.999
2
), from which values can be determined pKSH = 10.01 and
-
1
-1
f
= 2.07 M s for the bimolecular reaction between tBN and
the thiolate group of cap. The kinetic pKSH matches the value
determined by potentiometry and also compares quite well with
10
the published value, pKSH = 9.80. On the other hand, the value
of k is in good agreement with either the values obtained in
f
28
the nitrosation of other thiols by tert-butylnitrite, see Table 3,
or with the limit value directly determined in alkaline medium,
that is, in conditions of no pH effect, and reported also in the
Table 3.
+
Fig. 10 (a) Reciprocal plot of k
1
against [H ] to show the linear behaviour
By contrast, the observed rate constant of NOcap decom-
+
+
according to eqn (4); (b) linear-correlation of [H ]/k
predicts eqn (5).
2
against [H ] that
position, k
2
, decreases as the pH increases, and, at pH >11
no decomposition was observed; meanwhile, the k
maximum value and remains invariable on increasing [OH ] as
1
reaches its
-
+
can be seen from data in Table 3. On the other hand, k
2
increases
k
d
[H ][cap]
k2 =
(5)
+
with [cap] at pH 10.1. These experimental facts suggest a decay
reaction of NOcap with the participation of the thiol group of
a second cap molecule, to give the disulfide compound, Scheme
KSH +[H ]
+
+
According to this equation the plot of [H ]/k
2
versus [H ] should
2
(b). Considering the electronegativity values of S, H, N, and O
result in a straight line, as can be see in Fig. 10(b); in addition, the
ratio of intercept/slope of the line equals KSH. Least-squares fit of
atoms (shown in Scheme 2), the S-atom of the thiol group is a
nucleophilic center capable of attack on the electrophilic S-atom
of thionitrite group to give the disulfide compound, RSSR.
-
1
-7
the linear plot gave: intercept = KSH(k [cap]) = (1.28 ± 0.06) ¥ 10
d
-
1
2
M s and slope = (k [cap]) = (8.15 ± 0.25) ¥ 10 s. From the quotient
d
-
The rate eqn will be rate = k
d
[capH ][NOcap], with k
2
=
of both values one determines pKSH = 9.8, in good agreement with
-
-
-2
k
d
[capH ]. Considering that [cap] = [capH ] + [cap ] and the acid
the published value and the experimentally determined pKSH, and
-
1
3
-1
ionization equilibrium of thiol group, KSH, one easily arrives to
k
d
= 0.097 mol dm s .
eqn (5) that relates the experimental k
cap concentration and [H ].
2
with the stoichiometric
To further test our proposal of the NO-transfer from tBN to
cap-thiolate group, the reaction has been carried out in aqueous
+
7
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-
1
micellar solutions of both the cationic surfactant tetrade-
cyltrimethylammonium bromide (TTABr) and the anionic surfac-
tant sodium dodecylsulfate (SDS). The kinetics have been followed
the binding constants of tBN as K
s
= 15 and 32 M respectively
to SDS and TTABr micelles. Under the experimental conditions
-
2
used, all cap is in the form of thiolate ions, i.e. cap (Scheme
2). Therefore, the inhibition observed in the presence of SDS
micelles is due to the reactants’ separation: tBN associates to SDS
micelles, whilst cap is excluded from the micellar interphase by
electrostatic repulsions, Scheme 3; therefore, only reaction in the
water pseudophase is possible.
-
with the stopped-flow equipment at [OH ] = 0.065 M and [cap] =
0
.014 M and [tBN] = 1.2 mM. The experimental data of A–t fits
2
-2
perfectly the first-order integrate rate equation with c < 0.001.
The variation of the observed rate constant, i.e. k
-
1
1
/s , with the
surfactant concentration can be seen in Fig. 11 for the case of
either SDS (a) or TTABr (b).
Scheme 3 Equilibrium and reaction steps that occur in the NO-transfer
reaction form tert-butyl nitrite (tBN) to captopril (cap) in alkaline medium
in the presence of (a) SDS micelles and of (b) TTABr micelles.
Considering that total tBN concentration is the sum of the con-
centrations in both aqueous and micellar pseudophases, [tBN] =
[
tBN]
the eqn (6) is derived. In this eqn, k
constant in the water pseudophase, and as defined previously, k
w
+ [tBN]
m
, and the distribution equilibrium constant, K ,
s
w
1
is the pseudo-first order rate
f
is the bimolecular rate constant between tBN and thiolate ions of
cap, and cmc represents the critical micellar concentration of SDS
(~6.5 mM in the present conditions).
29,30
Fig. 11 Influence of (a) SDS micelles and (b) TTABr micelles on the
rate constant k
1
measured in the NO-transfer from tBN to cap in alkaline
-
w
medium, [OH ] = 0.065 M, at [cap] = 14.3 mM and [tBN] = 1.2 mM. The
k1
w
k1
=
with k1 = kf
[
cap
]
(6)
solid line fits eqn (6); for parameters, see text.
1
+ Ks
(
[
SDS
]
-cmc
)
It can be noted that anionic micelles of SDS inhibit the
reaction throughout all the concentration range, whereas cationic
micelles strongly catalyse the reaction at [TTABr] <0.020 M,
approximately; a further increase of surfactant concentration
The non-linear regression analysis of experimental k
1
- [SDS]
-
1
-1
w
-2 -1
data yields: k
f
= 1.93 M
s
(k
1
= (2.74 ± 0.02) ¥ 10 s ) and
-
1
K
s
= (25.0 ± 0.4) M taking cmc = 6.5 mM. The k
perfectly with those directly determined and listed in Table 3. The
value is higher than that determined in the acid hydrolysis of
f
value agrees
results in a decrease of k
1
values. The observed k
1
–[TTABr] profile
K
s
29
is typically found when reactive anions (captopril thiolate ions in
tBN; however, the concordance of the results is acceptable if one
takes into account that, firstly, in the published datum the sample
-
the present case) exchange with the surfactant counterions (Br in
+
the present case).
solutions contain 1% dioxane and secondly, the presence of H in
t-Butylnitrite is a hydrophobic neutral compound that binds
the micellar interphase modifies its polarity.
By contrast, cationic micelles of TTABr catalyse the NO transfer
reaction due to reagent concentration in the small volume of the
29
specifically to either cationic or anionic micelles. The magnitude
of the interaction depends on the micelle hydrophobicity, i.e. on
the number of C-atoms in the hydrocarbon-chain of the surfactant.
For the present case, previous studies in this lab have determined
-
2
micelles: tBN binds to TTABr micelles and cap ions exchange
-
with the micellar counterions, Br , according to the equilibrium:
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cap-
the data requires the knowledge of K
of reaction in both water and micellar interphases, having similar
reactivities, explains the observed increase in k at low surfactant
due to low [Br ]) and the subsequent
2
+ 2Br-
ꢀ cap
-2
+ 2Br-
, K
. The quantitative treatment of
. In any case, the possibility
2 L. Ignarro and F. Murad, Nitric Oxide: Biochemistry, Molecular
Biology, and Therapeutic Implications, Academic Press, Inc, San Diego,
CA, 1995.
w
m
m
w
I
I
3
R. Butler and D. L. H. Williams, Chem. Soc. Rev., 1993, 22, 233–
241.
1
-
2
-
concentration (high [cap ]
m
4 S. H. Snyder, Nature, 1994, 372, 504–505.
5 R. Trouillon, D.-K. Kang, S.-I. Chang and D. O’Hare, Chem. Commun.,
-
decreasing as the Br ions, introduced with the surfactant, displace
the cap- from the micellar interphase in the exchange equilibrium
process, Scheme 3(b).
2
011, 47, 3421–3423.
2
6
(a) D. L. H. Williams, Chem. Soc. Rev., 1985, 14,
171–196; (b) D. L. H. Williams, Chem. Commun., 1996, 1085–
1
091.
7
8
D. L. H. Williams, Acc. Chem. Res., 1999, 32, 869–876.
D. L. H. Williams, “Nitrosation Reactions and the Chemistry of Nitric
Oxide”, Elsevier R. V., Amsterdam, 2004.
Conclusions
S-nitrosocaptopril is really formed in aqueous acid solutions of
sodium nitrite or in aqueous basic solutions of t-butylnitrite. In
either strong acid or alkaline medium the NOcap is stable, whereas
in mild acid or basic medium it decomposes in the timescale
of the experiments. The thiol is the reactive group towards the
9 J. Loscalzo, D. Smick, N. Andon and J. Cooke, J. Pharmacol. Exp.
Ther., 1989, 249, 726–729.
1
1
0 D. V. Aquart and T. P. Dasgupta, Org. Biomol. Chem., 2005, 3, 1640–
1
646.
1 M. Xian, X. Chen, Z. Liu, K. Wang and P. G. Wang, J. Biol. Chem.,
2000, 275, 20467.
-
-
12 H.-H. Otto and T. Schirmeister, Chem. Rev., 1997, 97, 133–
71.
nitrosating agents for the acid medium, XNO (X = H
2
O, NO ,
2
1
-
-
Cl , Br , . . . ), whereas the nucleophilic attack of the thiolate
ion to the nitroso group of tBN provokes the transnitrosation
reaction in basic medium. In this sense, the thionitrites’ (RSNO)
behaviour is quite different from that of alkyl nitrites (RONO).
The lower electronegativity of sulfur atoms than that of oxygen
1
3 R. E. Babine and S. L. Bender, Chem. Rev., 1997, 97, 1359–
1472.
14 J. W. Keillor and R. S. Brown, J. Am. Chem. Soc., 1992, 114, 7983–
7
989.
1
5 E. Iglesias, L. Garc ´ı a-Rio, R. Leis, M. E. Pe n˜ a and D. L. H. Williams,
J. Chem. Soc., Perkin Trans. 2, 1992, 1673–1679 and references therein.
(
or nitrogen) atoms makes the S-atom of RSNO an electrophilic
16 B. Roy, A. Moulinet and M. Fontecave, J. Org. Chem., 1994, 59, 7019–
7
026.
7 (a) P. Gans, A. Sabatini and A. Vacca, Talanta, 1996, 43, 1739–1753;
b) E. Iglesias, I. Brandariz, C. Jim e´ nez and R. G. Soengas, Metallomics,
011, 3, 521–528.
18 F. Nyasulu, M. Moehring, P. Arthasery and R. Barlag, J. Chem. Educ.,
center. Then, the RSNO decomposes to the disulfide compound;
whereas alkyl nitrites do not give the peroxide derivative. The
present results showed that NOcap decomposition occurs under
1
(
2
pH conditions similar to the imidazolium pK of the active site
a
2
011, 88, 640–642.
of cysteine proteases to yield the disulfide compound (RSSR) in
the attack of the thiol group of a cap molecule to the thionitrite
group of NOcap. Extrapolating to the in vivo system, if the thiol
group would correspond to a cysteine molecule in, e.g. papaine,
the RSSR formation changes the protein structure. This fact
affects the protein function and, at the same time, the NO releases
affect vascular relaxation. The results in cationic micelles justified
the cationic character of the active site of cysteine proteases.
In summary, the present results are in line with many papers
proposing that the biological effects of nitrosothiols are due to
their tendency to liberate NO or to modify other functional
cysteine containing proteins through S-transnitrosation.
1
9 R. Stewart, “The proton applications to organic chemistry”, Academic
Press, Orlando, 1985, p. 206-213 and 45-47.
20 D. L. H. Williams, Nitrosation, Cambridge University Press, 1988, and
references therein.
1 D. L. H. Williams, Adv. Phys. Org. Chem., 1983, 19, 381–428 and
references therein.
2 J. Tummavuori and P. Lumme, Acta Chem. Scand., 1968, 22, 2003–
2011.
2
2
2
2
3 S. Oae, N. Asai and K. Fujimore, J. Chem. Soc., Perkin Trans. 2, 1978,
5
71–577.
4 (a) E. Iglesias and A. Fern a´ ndez, J. Chem. Soc., Perkin Trans. 2, 1998,
1691–1969; (b) E. Iglesias, J. Am. Chem. Soc., 1998, 120, 13057–13069.
25 E. Iglesias and J. Casado, Int. Rev. Phys. Chem., 2002, 21, 37–74 and
references therein.
2
6 J. H. Espenson, Chemical Kinetics and Reaction Mechanisms, 2nd ed.,
995, McGraw-Hill, p. 70-76.
7 See ESI† for the meaning of C
8 H. M. S. Patel and D. L. H. Williams, J. Chem. Soc., Perkin Trans. 2,
990, 37–42.
9 L. Garc ´ı a-Rio, E. Iglesias, J. R. Leis and M. E. Pe n˜ a, Langmuir, 1993,
, 1263–1268.
0 (a) N. M. van Os, J. R. Haak and L. A. M. Rupert, Physico-Chemical
properties of Selected Anionic, Cationic and Nonionic Surfactants,
Elsevier Sicence Publishing, Amsterdan, 1993; (b) A. Dom ´ı nguez, A.
Fern a´ ndez, E. Iglesias and L. Montenegro, J. Chem. Educ., 1997, 74,
1227–1231.
1
2
2
1
2
and C .
Acknowledgements
1
This work was funded by the Ministerio de Educaci o´ n y Ciencia
of Spain (Project CTQ2008-04429/BQU).
2
3
9
Notes and references
1
J. P. Cooke, N. Andon and J. Loscalzo, J. Pharmacol. Exp. Ther., 1989,
49, 730–734.
2
7
216 | Org. Biomol. Chem., 2011, 9, 7207–7216
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