Selective and an efficient oxidative conversion of glutathione to glutathione disulfide with N-bromosuccinimide in aqueous acidic solution: Kinetic and mechanistic chemistry

Article abstract of DOI:10.1080/17415993.2010.499943

Oxidative conversion of thiols to disulfides is an important chemical transformation in organic synthesis. A tripeptide, glutathione (GSH), composed of glutamate, cysteine and glycine, has been found to be the most abundant low molecular weight thiol in most biological systems. Its importance in mammal systems is believed to be related to its functions in oxidative metabolism and detoxification. It is noted that despite the importance of this substrate, less information is available in the literature on the oxidation of this substrate viewed from its kinetic and mechanistic studies. N-Bromosuccinimide (NBS) is a mild and selective oxidant for many organic compounds, and hence, it has been used as an oxidant for the present redox system. Consequently, the kinetics of oxidation of GSH with NBS in aqueous HClO4 medium has been investigated at 283K. The reaction rate exhibits first-order dependence on [NBS]o and fractional-order dependence each on [GSH]o and [H+]. The effect of added succinimide, ionic strength and dielectric constant of the medium on the rate of the reaction has been studied. The solvent isotope effect was studied using D2O. The reaction was studied at different temperatures and thermodynamic parameters have been computed. Glutathione disulfide is characterized as the oxidation product of GSH. The protonated species RN+HBr (here R = (CH2O)2-) of the NBS is assumed to be the reactive oxidizing species. The reaction constants involved in the mechanism were evaluated. The observed results have been explained by a plausible mechanism, and the related rate law has been deduced.

Full text of DOI:10.1080/17415993.2010.499943

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Selective and an efficient oxidative
conversion of glutathione to
glutathione disulfide with N-
bromosuccinimide in aqueous acidic
solution: kinetic and mechanistic
chemistry
Ramalingaiah a , K. N. Vinod b , Puttaswamy b , Nirmala Vaz c & R.
V. Jagadeesh d
a Department of Chemistry, P.E.S. College of Science (Affiliated to
University of Mysore), Mandya, 571401, India
b Department of Chemistry, Bangalore University, Central College
Campus, Bangalore, 560 001, India
c Department of Chemistry, Jyoti Nivas College (Autonomous),
Hosour Road, Bangalore, 560 095, India
d Leibniz Institute for Catalysis, University of Rostock, Albert
Einstein Street 29a, D-18059, Rostock, Germany
Published online: 28 Sep 2010.
To cite this article: Ramalingaiah , K. N. Vinod , Puttaswamy , Nirmala Vaz & R. V. Jagadeesh
(2010): Selective and an efficient oxidative conversion of glutathione to glutathione disulfide with
N-bromosuccinimide in aqueous acidic solution: kinetic and mechanistic chemistry, Journal of Sulfur
Chemistry, 31:5, 405-414
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Journal of Sulfur Chemistry
Vol. 31, No. 5, October 2010, 405–414
Selective and an efficient oxidative conversion of glutathione to
glutathione disulfide with N-bromosuccinimide in aqueous
acidic solution: kinetic and mechanistic chemistry
Ramalingaiaha*, K.N. Vinodb, Puttaswamyb, Nirmala Vazc and R.V. Jagadeeshd
aDepartment of Chemistry, P.E.S. College of Science (Affiliated to University of Mysore), Mandya 571401,
India; bDepartment of Chemistry, Bangalore University, Central College Campus, Bangalore 560 001,
c
India; Department of Chemistry, Jyoti Nivas College (Autonomous), Hosour Road, Bangalore 560 095,
India; dLeibniz Institute for Catalysis, University of Rostock, Albert Einstein Street 29a, D-18059, Rostock,
Germany
(Received 24 April 2010; final version received 07 June 2010)
Oxidative conversion of thiols to disulfides is an important chemical transformation in organic synthesis.
A tripeptide, glutathione (GSH), composed of glutamate, cysteine and glycine, has been found to be the
most abundant low molecular weight thiol in most biological systems. Its importance in mammal systems
is believed to be related to its functions in oxidative metabolism and detoxification. It is noted that despite
the importance of this substrate, less information is available in the literature on the oxidation of this
substrate viewed from its kinetic and mechanistic studies. N-Bromosuccinimide (NBS) is a mild and
selective oxidant for many organic compounds, and hence, it has been used as an oxidant for the present
redox system. Consequently, the kinetics of oxidation of GSH with NBS in aqueous HClO4 medium has
been investigated at 283 K. The reaction rate exhibits first-order dependence on [NBS]o and fractional-
order dependence each on [GSH]o and [H+]. The effect of added succinimide, ionic strength and dielectric
constant of the medium on the rate of the reaction has been studied. The solvent isotope effect was studied
using D2O. The reaction was studied at different temperatures and thermodynamic parameters have been
computed. Glutathione disulfide is characterized as the oxidation product of GSH. The protonated species
RN+HBr (here R = (CH2O)2−) of the NBS is assumed to be the reactive oxidizing species. The reaction
constants involved in the mechanism were evaluated. The observed results have been explained by a
plausible mechanism, and the related rate law has been deduced.
Keywords: glutathione; N-bromosuccinimide; oxidation; kinetics; acid medium
1. Introduction
The thiol group is generally oxidized to the corresponding disulfide, although instances of oxi-
dation behind disulfide stage are reported depending on the reaction conditions and nature of
the reactants (1). The disulfides are important intermediates for the preparation of sulfinyl and
sulfenyl compounds and also have industrial applications (2). The mechanistic chemistry of the
*Corresponding authors. Email: rlingh_chem@yahoo.com or pswamy_chem@yahoo.com
ISSN 1741-5993 print/ISSN 1741-6000 online
© 2010 Taylor & Francis
DOI: 10.1080/17415993.2010.499943
http://www.informaworld.com

406 Ramalingaiah et al.
oxidative conversion of thiols to disulfides has received considerable attention in understanding
the reaction pathways of a particular redox system (3). This kind of chemical transformation is an
important reaction because the disulfide bridges are important for the three-dimensional structure
of proteins. Glutathione (GSH) (γ -glutamyl-cysteinyl-glycine) is a tripeptide and its importance
in mammal systems is believed to be related to its functions in oxidative metabolism and detoxifi-
cation (4). Controlled oxidative conversion of GSH to glutathione disulfide (GSSH) forms a very
important synthetic route in biochemical reactions. In the literature, many reports are available
on the oxidation of GSH with various reagents (4–7). However, most of these methods are com-
plex in nature and not economical. And also, information on the kinetic and mechanistic aspects
of these redox reactions are lacking. By keeping this in view, in the present research, we have
developed a systematic route for the formation of GSSH from GSH which has significant advan-
tages such as: simple with short reaction time, cost-effectiveness, the use of relatively non-toxic
reagents, ease of isolation of products and the ability to be scaled up for industrial operations.
The study has been extended to explore the mechanistic aspects of this oxidation through kinetic
investigations.
N-halosuccinimides are important group of N-halo reagents and relatively stable compounds
when compared with other N-halo reagents (8). Depending on the reaction conditions, these
reagents behave as a source of both halonium ion as well as a nitrogen anion (9). As a result,
these reagents react with a wide range of functional groups leading to an array of molecular
transformation (8, 9). The important member of this class is N-bromosuccinimide (NBS). NBS
is a potent oxidant and it has been used in the quantitative estimation as well as oxidation-
kinetic study of diverse group of substrates (9–12). As a consequence, in the present kinetic
study, NBS has been used as an oxidant in order to establish optimum conditions for the for-
mation of GSSH and also to examine the mechanistic behavior of this reagent toward GSH
oxidation.
In the light of the above facts and in continuation of our research work (2) on the oxidation of
some thiols, the present paper reports a systematic investigation on the kinetics and mechanism
of oxidation of GSH with NBS in perchloric acid medium. The very objective of the present work
is to develop optimum conditions for the smooth oxidative conversion of GSH to GSSH through
kinetic and mechanistic investigations.
2. Results and discussion
Preliminary kinetic experiments revealed that the reactions between NBS and GSH were too
rapid to be measured in a neutral or alkaline medium at an ambient temperature. To investigate
the kinetic and mechanistic aspects of this redox system, detailed kinetic experiments were carried
out at 283 K in perchloric acid medium and optimum conditions for the quite oxidation of GSH
with NBS were established. The kinetics of oxidation of GSH with NBS was investigated at
several different initial concentrations of the reactants in HClO4 medium.
2.1. Effect of varying reactant concentrations on the rate of the reaction
With the substrate in excess, at constant [GSH]o, [HClO4] and temperature, plots of log [NBS]
versus time were linear (R2 > 0.9839), indicating a first-order dependence of rate on [NBS]o.
Further, the values of kꢀ were unaltered with the variation in [NBS]o (Table 1), confirming the
first-order dependence on [NBS]o. Under the same experimental conditions, the reaction rate
increased in [GSH]o (Table 1) and a plot of log kꢀ versus log [GSH]o was linear (R2 = 0.9921)
with a slope of 0.71, indicating a fractional-order dependence of rate on [GSH]o.

Journal of Sulfur Chemistry 407
Table 1. Effect of varying concentrations of NBS, GSH and HClO4
on the rate of reaction at 283 K.
104 [NBS]o
103 [GSH]o
103 [HClO4]
(mol dm−3
)
(mol dm−3
)
(mol dm−3
)
104kꢀ (s−1
)
6.0
7.0
8.0
9.0
10.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
1.0
3.0
5.0
8.0
12.0
8.0
8.0
8.0
8.0
8.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
1.0
2.0
5.0
10.0
18.0
2.14
2.08
2.15
2.20
2.18
0.51
1.10
1.62
2.15
3.34
0.87
1.36
2.15
3.08
4.28
2.2. Effect of varying perchloric acid concentration on the rate of the reaction
Values of kꢀ increased with the increase in [HClO4] (Table 1) and a plot of log kꢀ versus log [HClO4]
was linear (R2 = 0.9991) with a slope of 0.55, indicating a fractional-order dependence on
[HClO4].
2.3. Effect of added succinimide concentration on the rate of the reaction
Addition of the reduction product of NBS, succinimide (4.0 × 10−4 mol dm−3), had no effect on
the rate signifying that it is not involved in the pre-equilibrium with the oxidant.
2.4. Effect of ionic strength of the medium on the rate of the reaction
The effect of ionic strength on the reaction rate was studied in the presence of 0.20 mol dm−3
KClO4 solution. It was observed that the ionic strength had no significant effect on the rate of
the reaction. This suggests the involvement of non-ionic species in the rate-determining step
(rds). Consequently, no attempts were made to fix the ionic strength of the medium constant for
kinetic runs.
2.5. Effect of varying dielectric constant of the medium on the rate of the reaction
In order to find the nature of reactive species, the dielectric constant (D) of the medium was
varied by adding MeOH (0–20% v/v) to the reaction mixture by keeping all other experimental
conditions constant. The reaction rates (104 kꢀ s−1) at 76.73, 74.50, 72.37, 67.48 and 62.71 of D
were found to be 2.15, 2.42, 2.89, 3.40 and 4.15, respectively. A plot of log kꢀ versus 1/D was
linear (R2 = 0.9959) with a positive slope indicating an ion–dipole type of interaction. Blank
experiments showed that there was no oxidation of methanol by NBS during the experimental
period. Values of D were taken from the literature (13).

408 Ramalingaiah et al.
Table 2. Effect of varying temperature on the rate
of reaction and thermodynamic parameters for the
oxidation of GSH by NBS in acid medium.
Temperature (K)
104kꢀ(s−1
)
278
283
288
293
1.42
2.15
3.28
4.02
46.3
Ea (kJ mol−1
)
ꢀH== (kJ mol−1
)
)
43.9 0.01
89.5 0.20
−160 0.13
4.85 0.15
ꢀG (kJ mol−1
ꢀS= (JK−1 mol−1
log A
)
Note: [NBS]o = 8.0 × 10−4 mol dm−3
;
[GSH]o = 8.0×
10−3 mol dm−3; [HClO4] = 5.0 × 10−3 mol dm−3
.
2.6. Effect of solvent isotope on the rate of the reaction
The rate studies in D2O medium showed that the rate of the reaction is faster in heavy water. The
value of kꢀ in D2Owas3.32 × 10−4 s−1 while the corresponding value in H2Owas2.15 × 10−4 s−1
leading to a solvent isotope effect kꢀ(H2O)/kꢀ(D2O) = 0.65.
2.7. Effect of varying temperature on the rate of the reaction
The reaction was studied at different temperatures (278–293 K) and from the linear Arrhenius
plot of log kꢀ versus 1/T (R2 = 0.9908), values of activation energy and other thermodynamic
parameters (ꢀH =, ꢀS=, ꢀG= and log A) were deduced.All these data were tabulated in Table 2.
2.8. Test for free radicals
The addition of reaction mixture to aqueous acrylamide solution did not initiate polymerization
showing the absence of free radical species. The proper control experiments were also performed
under similar experimental conditions without the oxidant.
2.9. Reactive species of NBS
NBS (RNBr here R = (CH2O)2−) acts as a mild oxidant in both acidic and alkaline media (8, 9).
In general, NBS undergoes a two electron change in its reactions. As a consequence, this reagent
reacts with a wide range of functional groups affecting an array of molecular transformations (8,
9). Depending on the pH of the medium, NBS furnishes different types of equilibria in acid and
alkaline solutions (14, 15) as shown below:
+
RNBr + H+
RNBr + H2O
HOBr + OH−
RNBr + H+
RN HBr
(1)
(2)
(3)
(4)
(5)
(6)
−−→
←−−
−−→
←−−
RNH + HOBr
−
−−→
←−−
OBr + H2O
+
−−→
←−−
RNH + Br
+
+
−−→
←−−
Br + H2O
H2O Br
RNBr + OH−
RNH + OBr .
−
−−→
←−−

Journal of Sulfur Chemistry 409
Therefore, the possible oxidizing species in the acid medium are NBS itself (RNBr), protonated
NBS (RN+HBr) and Br+, and in alkaline NBS solution, they are NBS, HOBr and OBr−.
In present investigations, all the kinetic runs were carried out in the presence of mercuric
acetate (5.0 × 10−3 mol dm−3) in order to avoid any possible bromine oxidation. It was observed
that addition of mercuric acetate had no significant effect on the rate of the reaction. This indicates
that it is not involved in NBS oxidation and functions only as a bromide ion trapping agent (16). It
also suppresses completely the oxidation by Br2, which would have been formed by the interaction
of HBr and NBS as
RNBr + HBr −→ RNH + Br2.
(7)
Mercuric acetate exists as HgBr24− or unionized HgBr2 and thus ensures that the oxidation takes
place through NBS. Further, the acceleration of the rate by an increase in the concentration of
H+ ions and insignificant effect of added succinimide (RNH) on the reaction rate assumes that
RN+HBr is the most probable oxidizing species in the present case.
2.10. Reaction scheme
Inviewoftheabovekineticobservations, Scheme 1isproposedtoexplainthemechanismofoxida-
tion of GSH with NBS in perchloric acid medium. An initial equilibrium involves the protonation
of NBS forming the active oxidizing species RN+HBr as shown in Step (i) of Scheme 1. In the
next fast pre-equilibrium step (Step (ii)), the lone pair of electrons of sulfur atom of GSH attacks
the positive bromine of the RN+HBr species forming an intermediate Complex-I. This Complex-
I, in a slow and rds (Step (iii)), disproportionates to form another intermediate Complex-II with
the elimination of succinimide. In the next step (Step (iv)), the intermediate Complex-II interacts
with another mole of substrate, with the elimination of bromide, to yield the final product, GSSH.
2.11. Rate law
Based on Scheme 1, the rate law for the above redox system can be derived as follows:
The total effective concentration of NBS is [NBS]t, then
[NBS]t = [RN–Br] + [RN+HBr] + [Complex-I].
From Step (i) of Scheme 1,
(8)
(9)
[RN+HBr]
[RN+HBr]
K1[H+]
K1 =
or [RNBr] =
.
[RNBr][H+]
From Step (ii) of Scheme 1,
[Complex-I]
[Complex-I]
K2[GSH]
K2 =
or [RN+HBr] =
.
(10)
[RN+HBr][GSH]
By substituting for [RN+HBr] from Equation (10) into Equation (9) one obtains,
[Complex-I]
[RNBr] =
.
(11)
K1K2[GSH][H+]
By substituting for [RN+HBr] and [RNBr] from Equations (10) and (11), respectively, into
Equation (8) and solving for [Complex-I], we get
K1K2[NBS]t[GSH][H+]
[Complex-I] =
.
(12)
1 + K1[H+]K1K2[GSH][H+]

410 Ramalingaiah et al.
O
O
K1
+
N
H
(i)
H+
N
Br
+
Br
(fast)
O
(RNH+Br)
O
(RNBr)
..
S
H
O
O
H
HOOC
N
COOH
+
N
H
N
O
+
(ii)
H
Br
+
N
O
NH2
H
O
Br
(RNH+Br)
(GSH)
+
O
K2
(fast)
S
H
O
H
HOOC
O
N
COOH
N
+
N
H
H
O
NH2
Br
(Complex-I)
+
S
H
O
O
k3
(slow and rds)
H
(iii)
HOOC
N
COOH
N
H
Br
O
NH2
O
S
(Complex-I)
O
H
HOOC
N
H
+
N
COOH
N
H
O
O
NH2
(Complex-II)
Br
S
..
S
H
O
O
H
HOOC
H
N
COOH
(iv)
HOOC
+
N
COOH
N
N
H
H
O
NH2
NH2
O
NH2
(GSH)
-Br-
(Complex-II)
O
H
N
HOOC
N
H
COOH
O
S
S
O
H
HOOC
NH2
N
COOH
N
H
O
(Glutathione disulfide)
Scheme 1. A general mechanistic scheme for the oxidation of GSH by NBS in acid medium.
From slow/rds (Step (iii)) of Scheme 1,
−d[NBS]t
Rate =
= k3[Complex-I].
(13)
dt
By substituting for [Complex-I] from Equation (12) into Equation (13), the following rate law
(Equation (14)) is obtained:
K1K2K3[NBS]t[GSH][H+]
Rate =
.
(14)
1 + K1[H+]K1K2[GSH][H+]

Journal of Sulfur Chemistry 411
Since rate = kꢀ [NBS]t, Equation (14) can be transformed into Equation (15)
K1K2K3[GSH][H+]
kꢀ =
.
(15)
1 + K1[H+]K1K2[GSH][H+]
Equation(15)mayberearrangedintoEquations(16)and(17), andtheyaresuitableforverification.
1
kꢀ
1
1
1
1
=
=
+
+
(16)
(17)
K1K2K3[GSH][H+] k3K2[GSH] k2
ꢀ
ꢁ
1
1
1
1
+
+
.
kꢀ
[GSH] K1K2k3[H+] k3K2
k2
Double reciprocal plots of 1/kꢀ versus 1/[GSH] and 1/kꢀ versus 1/[H+] were found to be linear
(Figure 1; R2 > 0.9912). From the intercepts and slopes of such plots, values of protonation
constant K1, equilibrium constant K2 and decomposition constant k3 could be derived as 10.50 ×
104 dm3 mol−1, 0.45 × 103 dm3 mol−1 and 3.44 × 10−4 s−1, respectively. Further, the proposed
mechanism and derived rate law are supported by the following experimental facts.
2.12. Effect of solvent isotope
As expected for an H+ catalyzed reaction, the reaction rate is increased in the D2O medium. It is
well known that D3O+ is a stronger acid than H3O+ by a factor of 2–3, a solvent isotope of this
magnitude is to be expected (17, 18). The observed solvent isotope effect of kꢀ(H2O)/kꢀ(D2O) <
1 conform to the concept of solvent isotope effect. However, the magnitude of increase in rate
in D2O is small which can be attributed to the fractional-order dependence of rate on [H+]. The
increase in rate in D2O medium also implies a fast equilibrium hydrogen ion transfer (Step (i) of
Scheme 1).
Figure 1. Double reciprocal plots of 1/kꢀ versus 1/[GSH] and 1/kꢀ versus 1/[H+]. Experimental conditions are as in
Table 1.

412 Ramalingaiah et al.
2.13. Effect of dielectric constant
The influence of solvent on the rate and frequency factors of reactions in solutions can be explained
on the basis of the theory of absolute reaction rate (19). The effect of varying solvent composition
on the reaction rate has been described in several publications (19–22). For the limiting case of
zero angle of approach between two dipoles or an ion–dipole system, Amis (19) has shown that
a plot of log kꢀ versus 1/D yields a straight line, with a negative slope for a negative ion–dipole
or dipole–dipole interaction, and positive slope results for a positive ion–dipole interaction. The
positive dielectric effect observed in the present study supports the interaction between a positive
ion and a dipole in the reaction sequence (Scheme 1).
2.14. Thermodynamic parameters
The proposed mechanism is also supported by the moderate value of energy of activation. The
positive values of free energy of activation and enthalpy of activation signify that the transition
state is highly solvated. The high negative entropy of activation reflects that the transition state is
more rigid than initial state with lesser degrees of freedom. Further, the change in ionic strength of
the medium did not alter the rate, indicating that the non-involvement of ionic species in the rds.
The reduction product of NBS, succinimide, does not influence the rate, showing that it does not
participate in pre-equilibrium. All these experimental observations provide additional evidence
for the proposed mechanism and the derived rate law.
3. Conclusion
The kinetics of oxidation of GSH by NBS in the acid medium obeys the rate law–d[NBS]/dt =
0.71
k[NBS]o[GSH]o [HClO4]0.55. Optimum conditions for the smooth oxidative conversion of GSH
to GSSH were established. Thermodynamic parameters and reaction constants have been deduced.
Protonated NBS has been postulated as the reactive oxidizing species. A general mechanism
consistent with the observed kinetics has been proposed and discussed.
4. Experimental
4.1. Materials
An aqueous solution of NBS (Merck) was prepared afresh each time and its strength was ascer-
tained by the iodometric method and preserved in brown bottles to prevent its photochemical
deterioration. Fresh aqueous solution of GSH (Aldrich, 98%) was prepared whenever required.
Mercuric acetate (E. Merck) was used. Solvent isotope studies were made with D2O (99.4%)
supplied by the Bhabha Atomic Research Centre, Mumbai, India. All other reagents used were of
AnalaR grade. Double-distilled water was used throughout the course of the reaction. Regression
analysis of experimental data was carried out using Microsoft Excel, from which the regression
coefficient (R2) was calculated.
4.2. Kinetic procedure
All the kinetic runs were performed under pseudo-first-order conditions by maintaining the excess
of [GSH]o over [NBS]o, and the procedure followed is similar to that reported earlier (2). The

Journal of Sulfur Chemistry 413
course of the reaction was studied for at least two half-lives. The pseudo-first-order rate constants,
kꢀ, calculated from linear plots of log [NBS] versus time, were reproducible within 3–5%.
4.3. Stoichiometry
The stoichiometry of the reaction was determined by equilibrating varying ratios of [GSH] and
[NBS] in presence of 5.0 × 10−3 mol dm−3 HClO4 at 283 K for 48 h under kinetic conditions.
Iodometric determination of unreacted NBS showed that the oxidation of GSH involves a single
electron change with NBS, which is stoichiometrically represented as
O
N
SH
O
H+
HOOC
NH
COOH
+
Br
2
NH
O
O
NH 2
O
NH 2
COOH
NH
(18)
HOOC
NH
O
O
S
S
+
N
H
+
HBr
O
O
HOOC
NH
COOH
NH
NH 2
O
4.4. Characterization of products
The products in the GSH–NBS reaction mixture were extracted several times with diethyl ether.
The combined ether extract was evaporated and subjected to column chromatography on silica gel
(60–120 mesh) using gradient elusions (chloroform). After initial separation, the products were
further purified by recrystallization. The oxidation product of GSH is GSSH. The presence of
disulfide in the reaction products was detected by paper chromatography with PhOH saturated with
H2O as a solvent and ninhydrin as a spray reagent (Rf = 0.088). GSSH was further confirmed by
gas chromatography–mass spectroscopy (GC–MS). The mass spectrum showing an M+ parent ion
peak at 613 amu clearly confirms GSSH. Other peaks observed in the spectrum can be interpreted
in accordance with the observed structure. The reduction product of NBS, succinimide, was
identified by spot test (23). It was also observed that there was no further oxidation of these
products under the present reaction conditions.
Acknowledgement
One of the authors (Ramalingaiah) is grateful to the Principal and Management of PES Trust, Mandya, for encouragement.
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