Kinetics and thermodynamics of oxidation mediated reaction in L-cysteine and its methyl and ethyl esters in dimethyl sulfoxide-d6 by NMR spectroscopy

Article abstract of DOI:10.1016/j.molstruc.2016.11.038

L-Cysteine (L-Cys), L-Cysteine methyl ester (L-CysME) or L-Cysteine ethyl ester (L-CysEE), when dissolved in dimethyl sulfoxide, undergoes an oxidation process. This process is slow enough and leads to nuclear magnetic resonance (NMR) spectral changes that could be monitored in real time. The oxidation mediated transition is modeled as a pseudo-first order kinetics and the thermodynamic parameters are estimated using the Eyring's formulation. L-Cysteine and their esters are often used as biological models due to the remarkable thiol group that can be found in different oxidation states. This oxidation mediated transition is due to the combination of thiol oxidation to a disulfide followed by solvent-induced effects may be relevant in designing cysteine-based molecular models.

Full text of DOI:10.1016/j.molstruc.2016.11.038

Journal of Molecular Structure 1131 (2017) 196e200
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Kinetics and thermodynamics of oxidation mediated reaction in -
L
cysteine and its methyl and ethyl esters in dimethyl sulfoxide-d6 by
NMR spectroscopy
,
, b, *
Ryan J. Dougherty a 1, Jaideep Singh a, V.V. Krishnan a
a Department of Chemistry, California State University, Fresno, CA, 93740, USA
b Department of Pathology and Laboratory Medicine, School of Medicine, University of California, Davis, CA, 95616, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
L
-Cysteine (L-Cys), L-Cysteine methyl ester (L-CysME) or L-Cysteine ethyl ester (L-CysEE), when dissolved
Received 17 August 2016
Received in revised form
8 November 2016
Accepted 13 November 2016
Available online 14 November 2016
in dimethyl sulfoxide, undergoes an oxidation process. This process is slow enough and leads to nuclear
magnetic resonance (NMR) spectral changes that could be monitored in real time. The oxidation
mediated transition is modeled as a pseudo-first order kinetics and the thermodynamic parameters are
estimated using the Eyring's formulation. L-Cysteine and their esters are often used as biological models
due to the remarkable thiol group that can be found in different oxidation states. This oxidation mediated
transition is due to the combination of thiol oxidation to a disulfide followed by solvent-induced effects
may be relevant in designing cysteine-based molecular models.
Keywords:
NMR
1H
© 2016 Elsevier B.V. All rights reserved.
13C
Oxidation
Kinetics
Solvent effect
L-cysteine.
L-Cysteine methyl ester (L-
CysME) and
Short title
L-Cysteine ethyl ester (L-CysEE)
1. Introduction
reduced toxicity) [1]. Furthermore, the thiol group of cysteine takes
part in a variety of biochemical reactions such as the formation of a
L
-Cysteine (L-Cys) is one of the natural sulfur-containing amino
weak hydrogen bond at receptor sites and thus to initiate or
mediate biological responses. L-CysME or L-CysEE is also used as a
biomimetic ligand for metal complexes that contain Ni or Zn. These
metal complexes serve as models for structural motifs such as zinc
fingers [5,6].
acids. With the capacity to form disulfide bridges, it has an integral
role in protein structure. L-Cys and their derivatives are often used
as biological models due to the remarkable thiol group that can be
found in different oxidation states, and thus dictating the biological
function of the proteins. For example, the functional derivative of L-
In previous reports, it has been shown that thiols can be directly
oxidized by DMSO to a corresponding disulfide [2,7e13]. When L-
Cys and their ester derivatives were dissolved in DMSO, we
observed a similar oxidation event. However, to our surprise, we
also observed what we believe to be a change in the resulting di-
sulfide, that is predominantly due to the oxidation of thiol along
with other solvent-mediated interactions. This observed oxidation
mediated change was slow enough to be monitored in real time
using NMR spectroscopy. This resulting change is much slower than
the characteristic time of the typical NMR experiment and leads to
significant changes in the NMR spectrum that could be measured as
a function of time. The data fit a pseudo-first order process with
respect to L-Cys at the temperature range studied. This agrees quite
Cys, L-Cysteine ethyl ester (L-CysEE), has many applications in
chemistry, biochemistry, food sciences, and biomedical research
[1e4]. L-CysEE has been used in the chemical synthesis of the
majority of nonsteroidal anti-inflammatory drugs (NSAIDs) due to
the presence of the SH functional group (to confer antioxidant
properties) and the presence of the natural amino acid scaffold (for
* Corresponding author. Department of Chemistry, California State University,
Fresno, CA, 93740, USA.
E-mail
(V.V. Krishnan).
addresses:
krish@csufresno.edu,
vvkrishnan@ucdavis.edu
1
Currently at Department of Chemistry, University of California, Davis, USA.
http://dx.doi.org/10.1016/j.molstruc.2016.11.038
0022-2860/© 2016 Elsevier B.V. All rights reserved.

R.J. Dougherty et al. / Journal of Molecular Structure 1131 (2017) 196e200
197
nicely with the rate law proposed by Wallace and Mahon and a
detailed derivation of that rate law can be found in their seminal
study [13]. The enthalpic and entropic contributions to the reaction
were then estimated via Erying Plot. Further evidence of an
oxidatively induced change was observed from chemical shift
changes in the 13C NMR data. The observed changes and the derived
thermodynamic parameters are similar between all the three
molecules studied.
The simplified rate equation (Eq. (2)) can be used to describe the
kinetics by virtue of the fact that the total concentration remains
the same, and that are no other any side reactions during the
measurement time of the kinetics.
The concentration of reactant and product were calculated ac-
cording to
½Aꢁ
½Cꢁ ¼ ½Cꢁ0
½Sꢁ
(3)
2. Materials and methods
where [A] is the area of the NMR signal of the reactant, [S] is the
total area of the NMR signals from the product, and [C]0 is the initial
concentration of the reactant at each temperature. Solution to
Equation (3) is given by
2.1. Materials
Commercially, L-Cys and their ester derivatives are readily
ꢀ
ꢁ
available as hydrochloride salts.
L-Cysteine, L-Cysteine methyl ester,
½Cꢁ ¼ ½Cꢁ0eꢃkt or ½Cꢁ ¼ 1 ꢃ ½Cꢁ0eꢃkt
(4)
and -Cysteine ethyl ester hydrochloride were purchased from
L
Sigma-Aldrich (98% purity) and used with no further modification.
Deuterated dimethyl sulfoxide spiked with 0.05% by volume with
tetramethylsilane (TMS) was used as a solvent and internal refer-
ence (Sigma-Aldrich).
With [C] defined in Eq. (3), a linear fit between the estimated
transition rates (natural logarithm scale) vs. inverse of temperature
in K (1000/T) was used to estimate the activation energy of the
Eyring equation given by
2.2. NMR measurements
ꢂ
ꢃ
ꢂ
ꢃꢂ ꢃ
kh
kBT
ꢃ
D
Hz
1
T
D
Sz
ln
¼
þ
R
(5)
All the 1H and 13C NMR experiments were performed in a 400-
MHz (1H resonance frequency) VNMRS spectrometer (Varian-Agi-
lent) and using the one-NMR probe. Measurement of the exact
sample temperatures in the NMR probe was calibrated using the
chemical shift changes in neat methanol standard (CH3OH) [14].
The probe temperature was set between 30 and 55 ꢀC in the
spectrometer, and the calibrated sample temperature was used in
the calculations. Each sample was prepared by freshly dissolving in
DMSO-d6 to a concentration of ~243 mM just before the start of the
NMR experiment. The probe was tuned and the pulse width was
calibrated at each temperature using the experimental sample. The
approximate delay from the time the sample is prepared to start the
collection of the first transient was 10 min. One-dimensional ex-
periments were performed in an arrayed fashion. All the 1D NMR
experiments were performed at a pulse angle that corresponds to
Ernst angle (~70ꢀ) [15]. Each one-dimensional experiment was
collected over 8 transients, with an acquisition time per FID (free
induction decay) of 2.043 s, and a relaxation recycling delay of 4 s.
The total time for each experiment in the array was 48.34 s. The
array continuously collected until the reaction was almost
completed. The reaction completion time ranges from 2 h at high
temperature to >16 h at low temperatures. All the spectra were
processed using Mestrenova (Mestrelab Research, Santiago de
Compostela, Spain).
R
where h is Planks constant (6.62697 ꢂ 10ꢃ34 J$s), kB is Boltzmann
constant (1.3807 ꢂ 10ꢃ23 J/K), R is the gas constant (8.314 J/
(K$mol)), and the T is sample temperature in Kelvins. The enthalpy
D
Hz was calculated by multiplying the negative slope by R and the
entropy
3. Results
The oxidation mediated change ensuring the formation of the
D
Sz was calculated by multiplying the intercept value by R.
disulfide adduct is modeled as a pseudo-first order reaction with
respect to L-Cys. Fig. 1 highlights the effect of the oxidation medi-
ated changes on L-Cys (Fig. 1a), L-CysME (Fig. 1b) and L-CysEE
(Fig. 1c) on the chemical shifts of the 1H
b protons. The spectra
plotted in blue are at the beginning of the experiment while the
ones plotted in red are after the reaction at 30 ꢀC. The chemical
shifts of the 1H and 13C nuclei at t z 0 and at the end of reaction are
listed in Table 1. Supporting information shows a representative 1H
spectrum along with the chemical shift assignments (Fig. S1) and
the observed chemical shift changes in the 13C spectra (Fig. S2) in
the case of L-CysEE. The major chemical shift changes occur at the
b
-position for all the molecules. The 1H
0.3 ppm while the 13C
resonances shift ~13 ppm for all the mol-
ecules (Fig. S2). The oxidation mediated chemical shift changes in
the 13C
are in the range of 2.7e2.9 ppm for all the molecules.
b protons shift downfield by
b
2.3. Reaction kinetics
a
Remaining nuclei show no significant changes in the chemical
shifts, except the N1 protons, become much narrower and slightly
shielded with an integration that yields approximately three pro-
tons (Fig. S1). This trend continues at all the temperatures studied.
The oxidation mediated change is modeled as a pseudo-first
order reaction process. Ignoring the reverse reaction, the differen-
tial rate law can be written as
The
being the
b
-protons show the AB part of the ABX spin system with X
proton. The chemical shifts of the -protons are shiel-
d½Cꢁ
a
b
¼ k0½Cꢁ½DMSOꢁ
(1)
dt
ded (blue to red spectrum in Fig. 1) and the NMR spectra do not
show the thiol protons (Fig. S1).
where [C] is considered representative of either one of the three
molecules studied; -Cysteine. -Cysteine methyl ester (L-CysME)
and
The chemical shifts of the b-protons between the initial and final
L
L
forms of L-CysEE are well resolved and therefore the area under the
curve of each of them is used to quantify the reactants and prod-
ucts. Fig. 2 shows the change in the concentration of the reactants
and products with time for each temperature in the case of L-CysEE.
The corresponding fit to the pseudo-first order process (Eq [4]) is
also shown as continuous curves. The pseudo-first order rate con-
stants, k, estimated from the exponential decay of the reactants
L-Cysteine ethyl ester (L-CysEE). Upon rearranging Eq. (1), and
defining a pseudo first order rate constant k ¼ k0 ꢂ ½DMSOꢁ, giving
rise to the simplified rate equation,
d½Cꢁ
¼ k½Cꢁ
(2)
dt

198
R.J. Dougherty et al. / Journal of Molecular Structure 1131 (2017) 196e200
Fig. 1. Chemical shift changes due to the oxidation mediated changes in f L-Cysteine (a), L-Cysteine Methyl Ester (b) and L-Cysteine Ethyl Ester (c). Sub-spectra of the b-protons of the
reactant (blue) and that of the product (red) obtained in dissolving the DMSO-d6. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
Table 1
Changes in the NMR parameters in the oxidation process.a
Molecule
L-Cys
Nucleus
1H (ppm)
t~0
13C (ppm)
t~0
t » 1 m
t » 1 m
N1
a
b
C1
N1
a
8.6
4.2
3.1
8.8
4.2
3.3
53.9
24.2
169.0
51.3
37.4
169.2
L-CysME
L-CysEE
8.9
4.3
3.0
3.8
9.0
4.3
3.4
3.8
54.0
24.1
52.9
168.1
51.1
36.8
52.9
168.3
b
ε1
C1
N1
a
8.8
4.3
3.0
4.2
1.2
8.9
4.3
3.4
4.2
1.3
53.9
24.2
61.9
13.9
169.6
51.1
36.9
62.1
13.9
167.8
b
ε1
ε2
C1
a
Chemical shift changes > 0.1 ppm are marked with bold font.
(Fig. 2b) and that of the increase of the product to maximum
(Fig. 2a) estimated, are listed Table 2 for each molecule. The rate of
the reaction follows a typical pseudo-first order process. The in-
crease in temperature increased the rate of the reaction:
1.67e2.08 m m
M/min at 30 ꢀC to 14.6e17.5 M/min at 55 ꢀC (Table 2).
The estimated half-life of the reaction (t ¼ ln (2)/k) ranges from
½
330 to 415 min (30 ꢀC) to 39e45 min (55 ꢀC).
Table 2 also lists Eyring analysis of the pseudo-first-order reac-
tion kinetics. Linear fit was used to determine the activated
enthalpy (
mol ¼ ꢃslope ꢂ 1000/R, and
D D D
Hz) and entropy ( Sz) of the reaction process. ( Hz kJ/
D
Sz J/(mol.K) ¼ (Y-intercept ꢃ ln (kB/
h)) ꢂ R, where R, kB, and h are gas, Boltzmann and Planks' con-
stants, respectively in SI units). As an example in the case of L-
CysEE, the following thermodynamic parameters are estimated
(average of rate constants from both the fits):
D
Hz ¼ 389.6 6 kJ/
mol and
reaction of these molecules is
D
Sz ¼ ꢃ40.07 2 J/(mol.K). The average free energy of the
D
Gz ¼ 402 20 kJ/(mol). The ther-
Fig. 2. First order kinetics of the reaction process of L-CysEE at various temperatures.
The reduction of the reactant ([CysEE-H] þ) measured directly by NMR (b) for various
sample temperatures and the corresponding increase of the ionized product ([CysEE])
(a). The experimental measurements are shown as symbols (symbols are not resolved
due to high sampling rate) and the non-linear least square fit to the respective
experimental data are shown as dashed lines. Various colors of the data points and
dashed lines correspond to the spectrometer set temperature: 30 ꢀC (black), 35 ꢀC
(red), 40 ꢀC (green), 45 ꢀC (yellow), 50 ꢀC (blue), and 55 ꢀC (pink). (For interpretation of
the references to colour in this figure legend, the reader is referred to the web version
of this article.)
modynamic estimates clearly show a free energy of activation
barrier that is large enough to suggest the formation covalent bond,
such as the SeS bond due to the oxidation process. One of the
drawbacks of Eyring plot is that Y-intercept (used to determine
is normally a large extrapolation from the experimental data to
D
Sz)

R.J. Dougherty et al. / Journal of Molecular Structure 1131 (2017) 196e200
199
Table 2
Kinetic and thermodynamic parameters of the oxidation.a
Temperature
L-Cys
k (
M/min)b
L-CysME
k (
M/min)b
L-CysEE
k (
M/min)b
m
k (
m
M/min)c
m
k (
m
M/min)c
m
k (m
M/min)c
30 ꢀC
35 ꢀC
40 ꢀC
45 ꢀC
50 ꢀC
55 ꢀC
1.67 0.01
2.74 0.01
3.89 0.01
6.19 0.03
9.68 0.04
14.6 0.04
1.69 0.01
3.48 0.01
4.3 0.02
6.95 0.02
11.2 0.11
15.22 0.15
1.99 0.02
2.84 0.01
4.73 0.01
7.41 0.02
9.4 0.02
2.31 0.02
3.75 0.01
5.61 0.01
8.15 0.02
8.5 0.02
2.08 01
2.19 0.01
3.4 0.01
5.73 0.02
8.21 0.03
11.83 0.05
17.22 0.07
3.33 0.01
5.45 0.02
8.13 0.03
11.71 0.05
16.72 0.06
16.89 0.57
17.62 0.6
Thermodynamic parameters
D
D
D
Hz (kJ/mol)
Sz (J/mol K)
Gz (kJ/mol K)
405.3 30
ꢃ37.5
416.2 55
403.2 25
346.6 36
ꢃ49.5 10
385.0 57
395.2 20
387.3 10
392.0 10
1
ꢃ37.6
4
ꢃ38.3
4
ꢃ40.5
2
ꢃ39.6
2
402.3 20
a
b
c
LCys: L-Cysteine, L-CysME: L-Cysteine methyl ester, L-CysEE: L-Cysteine ethyl ester.
k measures using the decay of the reactant and.
k measured using the growth of the product.
high temperatures. In our case, the adjusted R2 values of the linear
is related to increase in the absorbance at 310 nm, suggesting the
formation of SeS bond (Fig. S3), which further suggests oxidation
mediated change [19]. With chemical shifts highly sensitive to the
local environment, the oxidation induced change is the only
possible explanation that can accommodate the observed chemical
shift differences between the reactant and product in all the three
molecules studied. The kinetic events measured in this study on
fresh samples of L-Cys and its ester derivatives suggest that the
reaction process is mediated by the oxidation of the thiol group
[20]. More importantly, the estimation of the free energy (~400 kJ/
molK) from variable temperature kinetic measurements (Table 2),
is large enough such that it establishes the formation of a covalent
bond associated with oxidation of the thiol group.
fit are 0.99 and lead to reliable estimates D
Sz.
4. Discussion
The rate of oxidation mediated change in L-Cys and its ester
derivatives by DMSO was measured using quantitative NMR spec-
troscopy. The spectral features of the initial and final forms are
clearly distinguishable in the one-dimensional NMR spectra at
400 MHz (Fig. 1 and Table 1). The decay of the reactant to form the
oxidized product follows a pseudo-first-order reaction kinetics
(Fig. 2). This suggests an oxidation process that takes place
following the formation of the disulfide adduct. The rate of con-
version increases with temperature with a half-life of the reaction
close to 5 h at 30 ꢀC to 45 min at 55 ꢀC (Table 2). Eyring's activated
complex approach is used to determine the enthalpy and entropy of
the reaction kinetics (Table 2).
5. Conclusions
L-Cysteine and its esters are excellent model compounds to
In spite of the importance of L-Cysteine and its ester derivatives,
study fundamental molecular reactions such as ionization and
oxidation. Many of the molecular chemistry involving cysteine and
related compounds are well explored in the literature and are
routinely discussed in advanced bioorganic and biophysical
chemistry texts. In spite of such a vast knowledge in the area,
systematic investigation of molecular reactions using NMR spec-
troscopy allowed the measurement of the rate of the reaction
process. The ability to measure the reaction kinetics in real time in
such systems further opens up possibilities to explore these re-
actions and in particular the kinetics. The polarity and the aprotic
nature of the solvent slow the reaction kinetics and thus opening
up newer possibilities to study fundamental molecular mecha-
nisms related to the oxidation process.
structural and dynamics studies of these molecules are limited,
particularly in solvents such as DMSO. In solvents such as water, the
reaction is much faster than in DMSO and NMR-based methods are
incapable of measuring the rates of the reaction. Typically, solvents
will affect the reaction rates depending on whether the substrate is
positively or negatively charged, or electrically neutral [16]. In a
study by Walters and Leyden's on molecules
L
-cysteine,
-cysteine noted significant changes in
and protons [17]. In the case of
L-cysteine
methyl ester and S-methyl-
L
the chemical shifts of both
a
b
L-
Cysteine, the chemical shift changes over a pH range measured (pH:
1e13) the and protons exhibited a shift of 1.3 and 0.31 ppm,
respectively. Such shifts are in the order of 0.85 ppm ( ) and
0.38 ppm ( ) for -cysteine methyl ester and 0.98 ppm ( ) and
0.43 ppm ( ) for S-Methyl- -cysteine. The chemical shift changes in
a
b
a
b
L
a
b
L
the present study are in the order of 0.32 ppm (Fig. 1 and Table 1).
One of the earliest and detailed studies of cysteine and related
compounds including L-CysEE was presented by Benesch and
Benesch [18]. According to this study, in comparison with cysteine
when the carboxyl group is modified, the acidity of the ammonium
proton is increased more from the electrostatic contributions than
the inductive effects. As a result, the acidity of the ammonium
proton becomes greater than that of the thiol proton. This is an
important and differentiable characteristic of L-CysME or L-CysEE
Acknowledgements
RJD thanks, Dr. Golden for many discussions. The authors thank
Dr. S. Attar for insightful comments on the reaction involving the
thiol group and Candice Courtney for critical reading of the
manuscript. RJD was supported in part by a graduate fellowship
from National Science Foundation (NSF Award # 1059994). This
work was in part supported by a RIMI (Research Infrastructure for
Minority Institutions) Grant (P20 MD 002732).
in comparison with unmodified L-cysteine. L-CysME and L-CysEE
have functional groups with relatively acidic protons, and each
proton has a relatively similar pKa value. The large change in the
chemical shifts of the proton and carbon nuclei at the Cb position,
that is closest to the thiol group (Fig. S1) suggests oxidation. We
have noticed the sample color (sealed tube) changes with time and
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.molstruc.2016.11.038.

200
R.J. Dougherty et al. / Journal of Molecular Structure 1131 (2017) 196e200
[9] O.G. Lowe, Oxidation of thiols and disulfides to sulfonic acids by dimethyl
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