Nucleophilic reactivity of thiolate, hydroxide, and phenolate ions toward a model O2-arylated diazeniumdiolate prodrug in aqueous and cationic surfactant media

Article abstract of DOI:10.1002/poc.1607

The kinetics of aromatic nucleophilic substitution of the nitric oxide-generating diazeniumdiolate ion, DEA/NO, by thiols (L-glutathione, L-cysteine, DL-homocysteine, 1-propanethiol, 2-mercaptoethanol, and sodium thioglycolate) from the prodrug, DNP-DEA/NO, has been examined in aqueous solution and in solutions of cationic DOTAP vesicles. Second-order rate constants in buffered aqueous solutions (k_RS-=3.48-30.9M^-1 s^-1; 30°C) gave a linear Bronsted plot (β_nuc=0.414±0.068) consistent with the rate-limiting S_NAr nucleophilic attack by thiolate ions. Cationic DOTAP vesicles catalyze the thiolysis reactions with rate enhancements between 11 and 486-fold in Tris-HCl buffered solutions at pH 7.4. The maximum rate increase was obtained with thioglycolate ion. Thiolysis data are compared to data for nucleophilic displacement by phenolate (k_PhO-=0.114M^-1 s^-1) and hydroxide (k_OH-=1.82×10^-2M^-1 s ^-1, 37°C) ions. The base hydrolysis reaction is accelerated by CTAB micelles and DODAC vesicles, with the vesicles being ca 3-fold more effective as catalysts. Analysis of the data using pseudo-phase ion-exchange (PIE) formalism implies that the rate enhancement of the thiolysis and base hydrolysis reactions is primarily due to reactant concentration in the surfactant pseudo-phase. Copyright

Full text of DOI:10.1002/poc.1607

Research Article
Received: 13 February 2009,
Revised: 8 June 2009,
Accepted: 2 July 2009,
Published online in Wiley InterScience: 9 September 2009
(www.interscience.wiley.com) DOI 10.1002/poc.1607
Nucleophilic reactivity of thiolate, hydroxide,
and phenolate ions toward a model
O2-arylated diazeniumdiolate prodrug in
aqueous and cationic surfactant media
Matthew S. Ninga, Stacy E. Pricea, Jackie Taa and Keith M. Daviesa
*
The kinetics of aromatic nucleophilic substitution of the nitric oxide-generating diazeniumdiolate ion, DEA/NO, by
thiols (L-glutathione, L-cysteine, DL-homocysteine, 1-propanethiol, 2-mercaptoethanol, and sodium thioglycolate)
from the prodrug, DNP-DEA/NO, has been examined in aqueous solution and in solutions of cationic DOTAP vesicles.
Second-order rate constants in buffered aqueous solutions (kRS- ¼ 3.48–30.9 MS1 sS1; 30 -C) gave a linear Brønsted
plot (bnuc ¼ 0.414 W 0.068) consistent with the rate-limiting SNAr nucleophilic attack by thiolate ions. Cationic DOTAP
vesicles catalyze the thiolysis reactions with rate enhancements between 11 and 486-fold in Tris-HCl buffered
solutions at pH 7.4. The maximum rate increase was obtained with thioglycolate ion. Thiolysis data are compared to
data for nucleophilic displacement by phenolate (kPhO- ¼ 0.114 MS1 sS1) and hydroxide (kOH- ¼ 1.82 T 10S2 MS1 sS1
,
37 -C) ions. The base hydrolysis reaction is accelerated by CTAB micelles and DODAC vesicles, with the vesicles being ca
3-fold more effective as catalysts. Analysis of the data using pseudo-phase ion-exchange (PIE) formalism implies that
the rate enhancement of the thiolysis and base hydrolysis reactions is primarily due to reactant concentration in the
surfactant pseudo-phase. Copyright ß 2009 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: catalysis; Brønsted plots; diazeniumdiolates; nitric oxide; surfactant vesicles
On account of interest in the activation of 1 in different
INTRODUCTION
microenvironments, we have also examined the effect of cationic
CTAB micelles and DODAC and DOTAP vesicles on thiolysis and
base hydrolysis reactions of 1 and have compared the effective-
ness of the surfactant aggregates as catalytic media for the
reactions. To assess factors influencing the reactions at the
aqueous interfaces of the micellar and vesicle systems, we have
employed pseudo-phase ion-exchange (PIE) formalism, with
consideration of ion-exchange between the OHꢀ and RSꢀ
nucleophiles and the bound halide counter ions of the surfactant
head groups in the interfacial region.
Diazeniumdiolates (NONOates) are widely used nitric oxide
generating agents in biomedical research studies, both in ionic
forms, R1R2NN(O)NOꢀ, that undergo spontaneous acid-catalyzed
dissociation at physiological pH and as O2-protected prodrugs,
R1R2NN(O)NOR, that are stable until activated for NO release by
enzymes at targeted biological sites.[1–4] Prodrug strategies
involving O2-arylated diazeniumdiolates, in particular, have been
of recent interest because of theirsuccess in delivering NO to acute
myeloid leukemia cells on activation by glutathione/glutathione
S-transferase[5–7] and this process is believed to be responsible for
the anticancer properties that they display. Key to the effectiveness
ofO2-arylateddiazeniumdiolatesasprodrugsistheirstabilityunder
physiological conditions until nucleophilic attack by glutathione
frees the diazeniumdiolate ion, resulting in its spontaneous
hydrolysis and release of NO. To better understand the reactivity
of diazeniumdiolates of the O2-arylated class, we have studied
reactions of a model diazeniumdiolate prodrug, O2-(2,4-dinitro-
phenyl) 1-(N,N-diethylamino)diazen-1-ium-1, 2-diolate (DNP-DEA/
NO, 1) with different thiols (RSH) in buffered aqueous solutions and
have compared the nucleophilicity of thiol anions toward 1 with
that of hydroxide and phenolate ions.
EXPERIMENTAL
Materials
O2-(2,4-Dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-dio-
late (DNP-DEA/NO, 1) was synthesized by reaction of DEA/NO
(sodium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate) with
2,4-dinitrofluorobenzene (Acros Organics) in dimethylsulfoxide.[8]
The structure was confirmed using UV-Vis (lmax ¼ 304 nm,
*
Correspondence to: K. M. Davies, Department of Chemistry and Biochemistry,
George Mason University, 4400 University Drive, Fairfax, VA 22030, U.S.A.
E-mail: kdavies@gmu.edu
a
M. S. Ning, S. E. Price, J. Ta, K. M. Davies
Department of Chemistry and Biochemistry, George Mason University,
Fairfax, VA 22030, USA
J. Phys. Org. Chem. 2010, 23 220–226
Copyright ß 2009 John Wiley & Sons, Ltd.

MODEL O2-ARYLATED DIAZENIUMDIOLATE PRODRUG REACTIVITY
e ¼ 14.2 mMꢀ1 cmꢀ1) and by 1H NMR in CDCl3 (300 MHz,
Bruker). DOTAP (1,2-dioleoyl-3-trimethylammonium-propane)
was obtained as a chloroform solution from Avanti Polar Lipids
(Alabaster, Alabama). L-glutathione, L-cysteine, DL-homocysteine,
1-propanethiol, 2-mercaptoethanol, cetyltrimethylammonium
bromide (CTAB), and dimethyldioctadecylammonium bromide
(DODAB) were obtained from Sigma-Aldrich. D-penicillamine and
sodium thioglycolate were obtained from Acros. All the materials
were used as received.
Corporation). Base hydrolysis rate constants were measured by
monitoring the decrease in absorbance of the diazeniumdiolate
chromophore at 305 nm. For runs with short half-lives requiring
more rapid mixing, reaction was initiated by delivering 1.0 ml
aliquots of a previously thermostated buffer to 10 ml of
diazeniumdiolate substrate in the thermostated cell.
Measurement of thiol association constants
Values for the vesicle association constants, KRSH, for glutathione
and thioglycolic acid were determined spectrophotometrically
from changes in thiol absorbance (A) at 210 nm in the presence of
variable amounts of sonicated DOTAP, using reported pro-
cedures.[10] Solutions containing variable amounts of DOTAP (0–
6.0 ꢃ 10ꢀ3 M) in 0.050 M Tris-HCl buffer were thermostated,
blanked, and treated with the same amount of thiol. Spectral
scans (200–250 nm) showed the thiol absorbance decreasing
progressively with increase in DOTAP concentration and the
thiol absorption maximum shifting to longer wavelengths.
Measured absorbance values were fitted to the equation
A ¼ (Aw þ AvesKRSH[DOTAP])/(1 þ KRSH[DOTAP]), where Aw and Aves
are the absorbance values for thiol in water and in the vesicle
pseudo-phase, respectively. KRSH values were obtained from a
nonlinear regression fit of the data to the above equation using
SigmaPlot (Systat Software Inc., 2007) (refer to Supporting
Information; Table E and Figure A).
Preparation of vesicle solutions
Unilamellar DOTAP vesicles were prepared from chloroform
solutions of the lipid by removing the solvent on a rotorvap under
a N2 flux, followed by hydration in buffer and bath sonication, as
described in detail elsewhere.[9] The size of the DOTAP vesicles,
prepared as above in 0.050 M Tris-HCl buffer, has been previously
determined by dynamic light scattering. Their mean diameter
(38.8 ꢁ 0.20 nm) showed no significant change over the time
period (20–90 min) that they were examined.[9] Dimethyldiocta-
decylammonium chloride (DODAC) was prepared by passing a
methanol–chloroform (70:30, v/v) solution of the bromide salt,
DODAB, through a Dowex 1X8-100 anion-exchange resin,
followed by evaporation of the solvent and recystallization of
the solid obtained twice from acetone–water (95:5, v/v). DODAC
vesicles (ca. 10 mM) were prepared in a thermostated cell at 55 8C
by probe sonication with an Ace Glass sonicator (Model GEX 750-
5B) operated at 20 kHz for 5 s on/5 s off for a 1 h period. Total
sonication time was 30 min. Titanium particles were removed
from the resulting solution by passing the solution through a
PTFE 0.20 mm filter (Fisher). All vesicle solutions were prepared
daily prior to their use in rate studies and used in kinetic runs
immediately after preparation.
RESULTS AND DISCUSSION
Effect of pH on the thiolysis of 1 in buffered aqueous media
The nucleophilicity of different thiols (RSH) toward 1 in aqueous
media was determined by measuring the thiolysis rate constants
as a function of pH in 0.050 M Tris-HCl or glycine-NaOH buffered
aqueous solutions at 30 8C. With the concentration of the thiol in
20–50-fold excess of the substrate, pseudo-first-order rate
constants increased with increase in the pH, consistent with
nucleophilic attack by the thiolate anion, RSꢀ. The dependence
of the measured first-order rate constant on pH is described by
Eqns (1)–(3) and individual values for the second-order rate
Rate measurements
Rate constants for the thiolysis reactions were measured
spectrophotometrically by following the formation of the S-
(2,4-dinitrophenyl)-conjugates at ꢂ350 nm, using a Hewlett
Packard 8453 Diode Array UV-visible spectrophotometer. Stock
solutions of 1 were prepared in DMSO and added to aqueous
buffer to obtain substrate concentrations in kinetic runs (0.50–
1.0 ꢃ 10ꢀ4 M) that contained 0.5–1% DMSO. All thiol stock
solutions (0.15–0.40 M) were prepared immediately before use in
the kinetic experiments and were stored in capped vials with a
small head-space. Series of rate measurements were typically
completed within 30–60 min and replicate rate measurements,
routinely made at the beginning and end of this period, showed
no indication of variability due to thiol oxidation. Data obtained in
duplicate sets of experiments, carried out on different days with
freshly prepared thiol solutions, also showed good reproduci-
bility. During the preparation of glutathione stock solutions,
additional NaOH was needed to bring glutathione solutions to pH
7.4. The 1-propanethiol stock solution was prepared in
acetonitrile. For liposome experiments, variable amounts of
0.050 M Tris buffer (pH 7.42) and 0.010 M DOTAP vesicle solution
in 0.050 M Tris buffer were mixed in a 1 ml cuvette and
thermostated before the reaction was initiated by the addition of
consecutive 10 ml aliquots of thiol and substrate. For buffer
experiments, thiol stock solutions were prepared in a Tris-HCl or
glycine/NaOH buffer. pH values were checked against standard
buffer solutions using an Orion 350 pH meter (Thermo Electron
ꢀ
kRS Ka½RSHꢄT
kobs
¼
(1)
(2)
½Hþꢄ þ Ka
ꢀ
¼ kRS athiol½RSHꢄT
where
Ka
1
athiol
¼
¼
(3)
½Hþꢄ þ Ka
1 þ 10ðpKaꢀpHÞ
ꢀ
The constant, kRS , and the apparent Ka of the thiol under
the experimental conditions were obtained from plots of
[RSH]T/kobs versus [Hþ] (Fig. 1). The excellent linearity of the
reciprocal plots and the close agreement between the measured
and literature pKa values[11–13] is consistent with the reaction
involving nucleophilic attack by the thiol anion in each case.
When the nucleophilicities of the anions were compared through
a Brønsted plot of log kRS- against pKa, the nucleophilicity
increased with increase in the basicity of the thiol and (with
the exception of penicillamine) was described by a linear
relation log kRS- ¼ bnuc ꢅ pKRSH þ C, where bnuc ¼ 0.414 ꢁ 0.068
and C ¼ ꢀ2.83 ꢁ 0.60 (Fig. 2). The finding is consistent with
the thiolysis reactions proceeding through rate-limiting SNAr
J. Phys. Org. Chem. 2010, 23 220–226
Copyright ß 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/poc

M. S. NING ET AL.
In contrast to the kRS- values, ko2 is negatively correlated with
the pKa of the thiols (Fig. 2), and although thiols with higher pKa
values are intrinsically better nucleophiles, their reactivity with
1 at pH 7.4 is offset by their lower tendency to generate thiolate
anions, making the cellular thiols, glutathione, cysteine, and
homocysteine, the most effective in activating 1 for NO release at
physiological pH. Data are summarized in Table 1. The much
smaller kRS- value obtained with penicillamine and its failure to
follow the Brønsted reactivity pattern established for the other
thiols may be tied to steric constraints arising from the methyl
groups adjacent to the sulfhydryl group of penicillamine and the
fact that its thiol group is the only one attached to a tertiary
carbon. Similar irregular rate behavior by penicillamine in
Brønsted plots has been reported for the thiol-mediated
decomposition of S-nitrosothiols.[16]
Figure 1. Plot of [GSH]/kobs versus [Hþ] for the reaction of glutathione
with 1 in 0.050 M Tris-HCl buffer (pH 8.5–9.7) at 30 8C
Nucleophilic displacement by phenolate and
hydroxide ions
The rate of nucleophilic displacement of the diazeniumdiolate
ion, DEA/NO, by thiol anions has been compared to that obtained
by phenolate and hydroxide ions. A value for the second-order
rate constant for nucleophilic attack by the phenolate ion was
determined from rate measurements carried out in glycine/NaOH
buffer (pH 9.3–10.8) through a plot of [phenol]/kobs versus [Hþ].
Data were obtained again under pseudo-first-order conditions
with [phenol] ¼ 5.0 mM and [1] ¼ 1 ꢃ 10ꢀ4 M. The slope and
intercept of the plot yielded a value of 0.114 Mꢀ1 sꢀ1 for kPhO- and
a pKa of 10.0 for phenol, consistent with the phenolate ion, as the
effective nucleophile in the reaction.
The second-order rate constant for the base hydrolysis of 1 was
obtained from reactions carried out with [NaOH] ¼ 2.0–10.0 mM.
Nucleophilic displacement of the diazeniumdiolate ion by
hydroxide occurred readily in the basic solution with the
formation of the diazeniumdiolate and dinitrophenolate ions.
nucleophilic attack of the thiolate anion on the ipso carbon of the
O2-protected prodrug in each case.[14] Diazeniumdiolate ions,
R2N[N(O)NO]ꢀ, have been shown to be good leaving groups in
SNAr reactions with a displacement rate by hydroxide between
that of chloride and fluoride ions,[8] and the O2-oxygen of the
diazeniumdiolate ion (pKa ꢆ 4.5)[15] is less basic than that of the
thiols employed in the study. Spectral evidence of a solvent
stabilized intermediate Meisenheimer complex has been
reported for the reaction of 1 with sodium methoxide in
dimethylsulfoxide.[8]
The thiol-mediated reactivity of 1 at physiological pH is also of
interest. Here, reaction rates are determined not only by the
inherent strengths of the thiolate ions as nucleophiles, but also by
their relative concentrations in the reaction solutions.
A
comparison was obtained through values of the second-order
rate constant ko2 for reaction at pH 7.4 that were estimated using
the following equation:
ꢀ
kRS
1 þ 10ðpKaꢀpHÞ
k2o ¼ kRS a ¼
(4)
ꢀ
Table 1. Dependence of nucleophilic rate constants (kRS-)
and apparent second-order rate constants at pH 7.4 (ko2) on the
pKa of the thiol for the thiolysis of 1 at 30 8C
Thiol
kRS- (Mꢀ1 sꢀ1
)
ko2 (Mꢀ1 sꢀ1
)
pKa
L-Cysteinea
3.48
5.62
10.4
22.7
28.4
30.9
0.18
0.431
0.215
0.292
0.0638
0.0284
0.0245
0.0329
8.25
8.80
8.94
9.95
10.4
10.5
8.05
DL-Homocysteinea
L-Glutathionea
2-Mercaptoethanolb
Sodium thioglycolateb
1-Propanethiolb
D-Penicillaminea
a 0.050 M Tris-HCl buffer.
b 0.050 M glycine/NaOH buffer. [Thiol] ¼ 1.5–5.0 mM. [1] ¼
Figure 2. Dependence of second-order rate constant for thiolysis of 1 in
ꢀ
aqueous solution on thiol pKa. (O) k2 ¼ kRS : log kRS- ¼ 0.414 pKRS- - 2.83;
0.10 mM.
R2 ¼ 0.959. (&) k2 ¼ ko2: log k2o ¼ ꢀ0.566 pKa þ 0.0466; R2 ¼ 0.973
www.interscience.wiley.com/journal/poc
Copyright ß 2009 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2010, 23 220–226

MODEL O2-ARYLATED DIAZENIUMDIOLATE PRODRUG REACTIVITY
Our data established a first-order dependence on hydroxide
ion, kobs ¼ kOH- [OHꢀ], and a second-order rate constant, kOH
hydrophobic association must predominate for 1-propanethiol
and 2-mercaptoethanol, while the relatively modest catalysis
found with the two amino acids cysteine and homocysteine is
consistent with the weaker vesicle interactions expected for
those two zwitterions.
-
¼ 1.82 ꢃ 10ꢀ2 ꢁ 1.04 ꢃ 10ꢀ3 Mꢀ1 sꢀ1 at 37 8C, in good agreement
with the value, 7.4 ꢃ 10ꢀ3 Mꢀ1 sꢀ1
, previously determined
at 25 8C by HPLC.[8] The second-order rate constants for
nucleophilic attack by phenolate (kPhO- ¼ 0.114 Mꢀ1 sꢀ1) and
hydroxide (kOH- ¼ 1.82 ꢃ 10ꢀ2 Mꢀ1 sꢀ1) ions compare to the
values, kRS- ¼ 3.48–30.9 Mꢀ1 sꢀ1 obtained in the thiolysis reac-
tions. The kPhO- and kOH- values are consistent with the slower
rates typically found with oxygen nucleophiles in SNAr reactions
compared to those of sulfur anions.
Apart from differences in thiol binding, additional contri-
butions to the catalysis are also expected to arise from enhanced
ionization of the thiol groups at the cationic surface and possibly
from changes in the intrinsic nucleophilic reactivity in the vesicle
pseudo-phase. A rate enhancement of over a million-fold has
been observed for p-nitrophenyl octanoate thiolysis by 1-
heptanethiol in cationic DODAC vesicles.[18] We applied the
same PIE model (Eqn (5)), developed for reactions between a
neutral substrate and a nucleophilic anion derived from a weak
acid,[19] to the relatively modest catalysis that we observed for the
thiolysis of 1 by glutathione, the thiol of relevance to the
enzymatic activation of O2-arylated prodrugs in vivo, and
thioglycolate, the thiol that exhibited the greatest catalysis in
our DOTAP studies.
Thiolysis of 1 in cationic DOTAP vesicle media
The effect of cationic DOTAP liposomes on the thiolysis of 1 was
examined in 0.050 M Tris-HCl buffer at pH 7.42 with
[RSH]T ¼ 5.0 mM and [DOTAP] ¼ 0.10–10.0 mM. kobs versus
[DOTAP] rate profiles obtained were typical of bimolecular
reactions catalyzed by vesicle interfaces, with pseudo-first-order
rate constants increasing to a maximum at ca 4 mM lipid. A
measure of the liposome catalysis is provided by catalytic factors,
kcalc ¼ ½RSHꢄTKa
ꢀ
ꢁ
2
3
5
½Clꢀꢄb
½Clꢀꢄf
k2v
V
k2o þ
KSKRS=Cl
k
cat, the ratio of the maximum rate constant in the buffered
4
ꢃ
liposome solution (kobs) to that in the buffer solution alone (kw).
kw values were estimated for the conditions of the DOTAP
experiments (pH 7.42, [thiol] ¼ 5.0 mM) using kw ¼ ko2[thiol].
Values for kcat were found to depend on the thiol employed,
ranging from 486 for sodium thioglycolate to 11 for homo-
cysteine. Data are summarized in Table 2.
ð1 þ KRSH½DOTAPꢄÞð½Hþꢄw þ KapÞð1 þ KS½DOTAPꢄÞ
(5)
In Eqn (5), ko2 and k2v are the second-order rate constants for
thiolysis in the aqueous and vesicle phases, respectively, KRS/Cl
and Kap are the selectivity constants for thiolate-chloride ion-
exchange and the apparent thiol acidity constant at the cationic
vesicle interface, and KRSH is the vesicle association constant for
the protonated thiol. We used values of 0.20 for a, the degree of
counter-ion dissociation at the vesicle surface, 0.58 for V, the
effective volume of the vesicle pseudo-phase,[19] and 710 Mꢀ1 for
Ks, obtained from the binding of 1 to DPPC and DOPE vesicles.[20]
The values of 263 and 693 Mꢀ1, respectively, that we determined
for glutathione and thioglycolic acid association with DOTAP
vesicles were used for KRSH. These compare to values of 1200 Mꢀ1
reported for 1-heptanethiol association with DODAC vesicles[21]
and 150 Mꢀ1 for benzyl thiol association with CTAB micelles.[22]
[Clꢀ]b and [Clꢀ]f, the concentrations of bound and free chloride
ions in the Tris-HCl buffered DOTAP vesicle solutions were
calculated using Eqns (6) and (7), where [RSꢀ]b and [OHꢀ]b are the
concentrations of vesicle-bound thiolate[23]
Assuming that the liposome catalysis is predominantly due to
the concentration of the reactants in the vesicle pseudo-phase, as
has been established for the vast majority of micellar and vesicle-
catalyzed organic reactions, the differences in kcat values in our
study must arise in large part from differences in thiol association
with DOTAP vesicles. Although such associations will involve
differing degrees of electrostatic and hydrophobic interactions,
electrostatic binding is expected to dominate at the cationic
vesicle interface. The most pronounced catalysis that was
observed with sodium thioglycolate is presumably due to
strong electrostatic interaction between thioglycolate’s car-
boxylic acid group (pKa ¼ 3.68)[17] and the cationic DOTAP
surface, bringing the small thioglycolate ion in close proximity to
the diazeniumdiolate substrate in the vesicle bilayer. Similar
considerations apply to the bulkier glutathione molecule, with its
two carboxylate groups (pKa ¼ 2.12, 3.59)[17] effectively orienting
the thiol for reaction at the vesicle surface. In contrast,
½Clꢀꢄb ¼ ð1 ꢀ aÞ½DOTAPꢄ ꢀ ½RSꢀꢄb ꢀ ½OHꢀꢄb
½Clꢀꢄf ¼ a½DOTAPꢄ þ ½RSꢀꢄb þ ½OHꢀꢄb þ ½Clꢀꢄbuffer
(6)
(7)
and hydroxide[24] ions. Since Kap is expected to vary as a function
of [DOTAP], we used values for Kap calculated by Eqn (8).[19] We
have previously observed a lowering of the pKa of
Table 2. Rate data for thiolysis of 1 in 0.050 M Tris/HCl buffer
and in Tris-buffered DOTAP vesicle media at pH 7.42 and 30 8C
ꢂ
ꢃ
1 þ KRS=Cl½Clꢀꢄb=½Clꢀꢄf
1 þ KRSH½DOTAPꢄ
kmax (sꢀ1
)
kw (sꢀ1
)
kcat ¼ kmax/kw
Kap ¼ Ka
(8)
Sodium thioglycolate
1-Propanethiol
L-glutathione
2-Mercaptoethanol
L-cysteine
0.0690
0.0175
0.122
0.0207
0.0250
0.0120
0.000142
0.000123
0.00146
0.000319
0.00215
0.00108
486
143
84
65
12
glutathione by 0.5 units in the presence of DOTAP vesicles.[20]
To analyze our data, the equations defining [RSꢀ]b, [OHꢀ]b,
[Clꢀ]f, [Clꢀ]b, Kap, and kcalc, were linked in an Excel spreadsheet
(MS Office, 2007) and values of KGS/Cl and k2v were left as
adjustable parameters and systematically varied to achieve the
closest fit between kobs and curves generated by kcalc for [DOTAP]
between 0 and 0.010 M. Fig. 3 show the best fits obtained for the
DL-homocysteine
11
[Thiol] ¼ 5.0 mM. [1] ¼ 0.10 mM. [DOTAP] ¼ 1.0–10.0 mM.
J. Phys. Org. Chem. 2010, 23 220–226
Copyright ß 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/poc

M. S. NING ET AL.
Figure 4. Plots of kobs versus [Dn] for hydrolysis of 1 in 0.010 M (D) and
0.0050 M (*) NaOH solution at 37 8C. The solid lines are calculated with
Eqn (9).
Figure 3. Plot of kobs versus [DOTAP] for reaction of 1 with glutathione
(&) and sodium thioglycolate (*) in 0.050 M Tris-HCl buffer at pH 7.4 and
30 8C. Solid line (kcalc) is calculated with Eqn (5) using best-fit values
indicated in the text
thiolysis of 1 by glutathione and thioglycolate that we obtained
using KGS/Cl ¼ 10 and 45, respectively. The corresponding k2v/k2o
values were 1.2 and 1.6. Although we recognize that such k2v/k2o
ratios are subject to some uncertainty due to the compensatory
nature of the values used for KGS/Cl and k2v, we believe that they
are indicative of only a small increase in the intrinsic reactivity of
the substrates in DOTAP media. Such changes compare to a k2v/k2o
ratio of 38 and a rate enhancement in excess of 106 that were
reported for the thiolysis of p-nitrophenyl octanoate by
heptanethiol in cationic DODAC vesicle media.[18,21] This clearly
indicates that the electrostatic binding of thiols at the DOTAP
vesicle interface is not as effective in modifying the intrinsic
reactivity of our substrates as is the hydrophobic solubilization of
p-nitrophenyl octanoate and heptanethiol in the tetraalkylam-
momium DODAC bilayer. In contrast to the DODAC vesicles, the
polarity of the acetate bridges in the dioleoyl chains of DOTAP
would appear to inhibit penetration of the thiol reactants into the
hydrophobic core of the DOTAP vesicles to an extent that is
apparently necessary to achieve the local concentration of
reactants and the greater catalysis obtained with DODAC.
constant observed in the surfactant solutions and kw was that for
base hydrolysis in the absence of surfactant. In a 5.0 mM NaOH
solution, kmax/kw was 290 for DODAC vesicles compared to 93 for
CTAB micelles.
Quantitative analysis of rate-surfactant profiles in sodium
hydroxide solutions was made using the PIE formalism developed
by Quina and Chaimovich[25] with explicit consideration of ion-
exchange between the bound halide counter ions, Xꢀ, of the
surfactant head groups and OHꢀ ions in the interfacial region.
Although the PIE model has been shown to have limitations at
high base concentrations, it has found wide application in
treating kinetic data for base-hydrolysis reactions catalyzed by
surfactant micelles and vesicles.[10,24–30] The kinetic data
obtained for the hydrolysis of 1 in NaOH solutions in CTAB
and DODAC solutions were fitted to the following equation:
½Xꢀꢄm
ꢄ
ꢅ
½OHꢀꢄT
KsKOH=X
þ k2w
k2m
V
½Xꢀꢄw
kcalc
¼
(9)
ð1 þ KsDnÞð1 þ KOH=X½Xꢄm=½XꢄwÞ
where KOH/X is the ion-exchange constant between the hydroxide
and the halide at the micellar or vesicle surface, k2m is the second-
order rate constant in the surfactant phase, k2w is the second-
order rate constant in the aqueous phase, and [Dn] and V are the
Catalysis of base hydrolysis by CTAB micelles and DODAC
vesicles
Our examination of the hydrolysis of 1 in NaOH solutions was
extended to microenvironments provided by the cationic
interfaces of both CTAB micelles and DODAC vesicles. Reactions
were conducted at NaOH concentrations between 2.0 and
10.0 mM in large excess of the substrate (1 ꢃ 10ꢀ4 M). When the
base hydrolysis reaction was examined in CTAB solutions as a
function of added surfactant, rate profiles typical of micellar-
catalyzed bimolecular reactions were observed (Fig. 4) with the
measured first-order rate constant increasing with added [CTAB]
to a maximum at ca 3 mM surfactant before decreasing sharply at
higher surfactant concentrations. The acceleration of pseudo-
first-order rate constants was also noted in the presence of the
bilayer vesicles provided by the synthetic surfactant DODAC, with
greater rate enhancements being apparent with DODAC vesicles
than with CTAB micelles. The rate profiles obtained in NaOH
solutions are compared in Figs 4 and 5. A comparison of the
catalytic efficiency of the two surfactant media was obtained
through the ratio, kmax/kw, where kmax was the maximum rate
Figure 5. Plots of kobs versus [DODAC] for hydrolysis of 1 at 37 8C in
0.010 M NaOH (*), 0.005 M NaOH (^), and 0.002 M NaOH (&). The solid
lines are calculated with Eqn (9)
www.interscience.wiley.com/journal/poc
Copyright ß 2009 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2010, 23 220–226

MODEL O2-ARYLATED DIAZENIUMDIOLATE PRODRUG REACTIVITY
Table 3. Comparison of rate and equilibrium parameters for micelle- and vesicle-catalyzed hydrolysis of 1 in NaOH solutions at 378C
Surfactant
[NaOH], M
k2m/k2w
Ks (Mꢀ1
)
kmax/kw
k2m,2v (Mꢀ1 sꢀ1
)
CTAB
CTAB
DODAC
DODAC
DODAC
0.010
0.0050
0.010
0.0050
0.0020
1.43
1.21
0.63
0.46
0.25
350
340
730
770
756
76
97
250
279
334
2.10 ꢃ 10ꢀ2
2.20 ꢃ 10ꢀ2
1.1 ꢃ 10ꢀ2
8.0 ꢃ 10ꢀ3
4.0 ꢃ 10ꢀ3
concentration and effective volume of the micellized surfactant.
The concentrations of the bromide ions (CTAB) or chloride ions
(DODAC) in the aqueous and surfactant pseudo-phases, [Xꢀ]w
and [Xꢀ]m, respectively, were calculated using the ion-exchange
equations described in detail elsewhere.[23–25] The value of a
(0.20), the degree of counter-ion dissociation at both the micelle
and vesicle surface was assumed to be independent of the
surfactant concentration, and cmc values of 5.0 ꢃ 10ꢀ4 and
so, this implies a similar reaction behavior at the outer and inner
vesicle interfaces.
CONCLUSIONS
The linear Brønsted plot obtained for the thiolysis reactions is
consistent with the rate-limiting SNAr nucleophilic attack of the
thiolate anion on the ipso carbon of the O2-protected prodrug.
Although weaker nucleophiles, the cellular thiols employed in the
study are the most effective in activating 1 for NO release at
physiological pH. The intrinsic nucleophilic reactivity of gluta-
thione, the active thiol for the thiolysis of 1 in vivo, is 570 times
that of hydroxide ion. Also, at physiological pH and typical
intracellular concentrations (1.0–10.0 mM), free glutathione is
estimated to react with DNP-DEA/NO a million times more rapidly
than hydroxide ion, even without the participation of the
enzyme. Vesicle-mediated catalysis of the thiolysis and base
hydrolysis reactions is mostly due to substrate concentration in
the surfactant pseudo-phase, with the smaller size of OHꢀ and
thioglycolate ions giving them a rate advantage for catalysis by
cationic vesicle interfaces.
6 ꢃ 10ꢀ6 M were used for CTAB and DODAC. In treating our data,
[26]
we used V ¼ 0.37 Mꢀ1 for CTAB micelles
and 0.58 Mꢀ1 for
DODAC vesicles,[25] and values of 0.08[24,25] and 1.6[10] for KOH/Br
and KOH/Cl, respectively. With these assumptions, rate-surfactant
profiles were fitted to Eqn (9) to provide best fit values for k2m and
k2v. The agreement between the experimental data and the
theoretical line calculated with Eqn (9) is shown in Figs 4 and 5,
and the obtained rate and equilibrium parameters are
summarized in Table 3.
The result that the larger DODAC vesicles are more effective
than CTAB micelles in enhancing base hydrolysis rates has been
observed previously for other CTAB and DODAC-catalyzed
reactions.[10,26] Such size-dependent differences in catalytic
efficiency for alkylammonium micelles and vesicles have
generally been attributed to differences in substrate binding
to the surfactant aggregates, with the increased hydrophobicity
and fluidity of the vesicle bilayers[31] making them more effective
in concentrating hydroxide ions at the interface.[21] Although in
our study, both CTAB micelles and DODAC vesicles accelerate the
rate of alkaline hydrolysis of 1, the increased reaction rate is not
reflected in values calculated for the intrinsic second-order rate
constants, k2v and k2m, for reaction in the two pseudo-phases. The
value obtained for k2v is lower than k2w and those of k2m and k2w
are more comparable in magnitude. Such findings have been
previously observed in other reactions catalyzed by micelles and
vesicles, where a reduction in the intrinsic reactivity is more than
compensated by an increase in the local concentration of the
reactants at the interface. The lowering of the intrinsic reactivity
in the presence of DODAC vesicles and CTAB micelles, compared
to that in water is generally attributed to differences in the
polarity of the media, particularly the lower dielectric at the
surface of the surfactant aggregates and the diminished
hydrogen bonding ability of the DODAC interface compared
to that in CTAB micelles.[10,21,25]
Acknowledgements
Support for this work from the National Institutes of Health (grant
number R15-HL078750-01) is gratefully acknowledged.
REFERENCES
[1] L. K. Keefer, R. W. Nims, K. M. Davies, D. A. Wink, Meth. Enzymol. 1996,
268, 281–293.
[2] L. K. Keefer, Annu. Rev. Pharmacol. Toxicol. 2003, 43, 587–607.
[3] C. A. Valdez, J. E. Saavedra, B. M. Showalter, K. M. Davies, T. C. Wilde, M.
L. Citro, J. J. Barchi, J. R. Deschamps, D. Parrish, S. El-Gayar, U.
Schleicher, C. Bogdan, L. K. Keefer, J. Med. Chem. 2008, 51, 3961–3970.
[4] B. M. Showalter, M. M. Reynolds, C. A. Valdez, J. E. Saavedra, K. M.
Davies, J. R. Klose, G. N. Chmurny, M. L. Citro, J. J. Barchi, S. I. Merz, M. E.
Meyerhoff, L. K. Keefer, J. Am. Chem. Soc. 2005, 127, 14188–14189.
[5] P. J. Shami, J. E. Saavedra, L. Y. Wang, C. L. Bonifant, B. A. Diwan, S. V.
Singh, Y. Gu, S. D. Fox, G. Buzard, M. L. Citro, D. J. Waterhouse, K. M.
Davies, L. K. Keefer, Mol. Canc. Therm. 2003, 2, 409–417.
[6] P. J. Shami, J. E. Saavedra, C. L. Bonifant, J. Chu, V. Udupi, S. Malaviya, B.
I. Carr, S. Kar, M. Wang, L. Jia, X. Ji, L. K. Keefer, J. Med. Chem. 2006, 49,
4356–4366.
Although the vesicle bilayers of DODAC and DOTAP provide
both internal and external surfaces as potential reaction sites, we
have found no evidence of biphasic kinetic behavior resulting
from differences in reactivity at the inner and outer interfaces or
rate limiting permeation of reactants through the membrane.
This may suggest that the rate of transmembrane equilibration of
reactants is fast, compared to the vesicle catalyzed reaction and, if
[7] H. Chakrapani, R. C. Kalathur, A. E. Maciag, M. L. Citro, X. Ji, L. K. Keefer,
J. E. Saavedra, Bioorg. Med. Chem. 2008, 16, 9764–9771.
[8] J. E. Saavedra, A. Srinivasan, C. L. Bonifant, J. Chu, A. P. Shanklin, J. L.
Flippen-Anderson, W. G. Rice, J. A. Turpin, K. M. Davies, L. K. Keefer, J.
Org. Chem. 2001, 66, 3090–3098.
J. Phys. Org. Chem. 2010, 23 220–226
Copyright ß 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/poc

M. S. NING ET AL.
[9] B. T. Dinh, S. E. Price, A. Majul, M. El-Hajj, V. Morozov, J. A. Hrabie, K. M.
Davies, Nitric Oxide, 2008, 18, 113–121.
[21] M. K. Kawamuro, H. Chaimovich, E. B. Abuin, E. A. Lissi, I. M. Cuccovia,
J. Phys. Chem. 1991, 95, 1458–1463.
´
´
[10] P. Herves, J. R. Leis, J. C. Mejuto, J. Perez-Juste, Langmuir 1997, 13,
[22] V. R. Correia, I. M. Cuccovia, H. Chaimovich, J. Phys. Org. Chem. 1993, 6,
7–14.
[23] E. R. Triboni, M. J. Politi, I. M. Cuccovia, H. Chaimovich, P. B. Filho, J.
Phys. Org. Chem. 2003, 16, 311–317.
6633–6637.
[11] S.-E. W. Huskey, W. P. Huskey, A. Y. H. Lu, J. Am. Chem. Soc. 1991, 113,
2283–2290.
[12] S.-S. Tang, G.-G. Chang, Biochem. J. 1995, 309, 347–353.
[13] Lange’s Handbook of Chemistry and Physics, 13th edn, (Ed.: J. A. Dean),
McGraw-Hill, New York, 1985.
[14] D. J. Hupe, W. P. Jencks, J. Am. Chem. Soc. 1977, 99, 451–464.
[15] K. M. Davies, D. A. Wink, J. E. Saavedra, L. K. Keefer, J. Am. Chem. Soc.
2001, 123, 5473–5481.
[16] T.-M. Hu, T.-C. Chou, AAPS J. 2006, 8, 485–E492.
[17] I. H. Pitman, I. J. Morris, Aust. J. Chem. 1979, 32, 1567–1573.
[18] I. M. Cuccovia, F. H. Quina, H. Chaimovich, Tetrahedron 1982, 38, 917–
990.
[19] H. Chaimovich, J. B. S. Bonilha, D. Zanette, I. M. Cuccovia, in:
Surfactants in Solution, Vol. 2, (Eds.: K. L. Mittal, B. Linderman), Plenum
Press, New York, 1984. pp. 1121–1138.
[24] F. H. Quina, M. J. Politi, I. M. Cuccovia, E. Baumgarten, S. M. Martins-
Franchetti, H. Chalmovich, J. Phys. Chem. 1980, 84, 361–365.
[25] F. H. Quina, H. Chaimovich, J. Phys. Chem. 1979, 83, 1844–1850.
[26] J. H. Fendler, W. L. Hinze, J. Am. Chem. Soc. 1981, 103, 5439–
5447.
´
[27] L. Garc´ıa-Rio, P. Herves, J. R. Leis, J. C. Mejuto, J. Perez-Juste, J. Phys.
Org. Chem. 1998, 11, 584–588.
[28] M. D. Nascimento, M. A. Lezdcano, F. Nome, J. Phys. Chem. 1992, 96,
5537–5540.
[29] M. V. Scarpa, P. S. Araujo, S. Schreier, A. Sesso, G. O. Anselmo, H.
Chaimovich, I. M. Cuccovia, Langmuir 2000, 16, 993–999.
[30] T. J. Broxton, J. R. Christie, R. P.-T. Chung, J. Org. Chem. 1988, 53, 3081–
3084.
[20] B. T. Dinh, M. N. Zanbrakji, K. Dove, S. E. Price, M. Peterson, K. M.
Davies, Nitric Oxide 2008, 19, 326–332.
[31] T. Kunitake, Y. Okahata, R. Ando, S. Shinkai, S. Hirakawa, J. Am. Chem.
Soc. 1980, 102, 7877–7881.
www.interscience.wiley.com/journal/poc
Copyright ß 2009 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2010, 23 220–226