Intramolecular hydrogen-bonding activation in cysteines: A new effective radical scavenger

Article abstract of DOI:10.1039/c3cp50743b

The challenge of developing organic molecules with improved antioxidant activities for a competitive marketplace requires, given the great amount of possibilities, much laboratory work. Nowadays, the ability of methodologies based on quantum chemistry to determine the influence of different modifications on a molecule core provides a powerful tool for selecting the most useful derivatives to be synthesized. Here, we report the results of the assessment of antioxidant activity for quaternary amino acids, specifically for cysteine derivatives. The effect of introducing different substituents on the cysteine core is evaluated by using DFT to obtain an adequate structure-antioxidant activity relationship. This theoretical study shows a small panel of targets among which (R)-N-acetyl-2-methylcysteine methyl ester 15 exhibits special features and relevant antioxidant activity. The conformational ¹H NMR study of this synthesized compound indicates the existence of an intramolecular C₇ member ring involving S-H?OC substructure, which is reported for the first time in the literature for this amino acid unit. This unusual conformation seems to be the reason for the high antioxidant capacity experimentally found for this compound.

Full text of DOI:10.1039/c3cp50743b

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Intramolecular hydrogen-bonding activation in
cysteines: a new effective radical scavenger†
Luisa Haya,a In˜aki Osante,b Ana M. Mainar,a Carlos Cativielab and Jose S. Urieta*a
Cite this: Phys. Chem. Chem. Phys., 2013,
15, 9407
The challenge of developing organic molecules with improved antioxidant activities for a competitive
marketplace requires, given the great amount of possibilities, much laboratory work. Nowadays, the
ability of methodologies based on quantum chemistry to determine the influence of different
modifications on a molecule core provides a powerful tool for selecting the most useful derivatives to
be synthesized. Here, we report the results of the assessment of antioxidant activity for quaternary
amino acids, specifically for cysteine derivatives. The effect of introducing different substituents on the
cysteine core is evaluated by using DFT to obtain an adequate structure–antioxidant activity relationship.
This theoretical study shows a small panel of targets among which (R)-N-acetyl-2-methylcysteine methyl
ester 15 exhibits special features and relevant antioxidant activity. The conformational 1H NMR study of
this synthesized compound indicates the existence of an intramolecular C7 member ring involving
S–HÁ Á ÁOQC substructure, which is reported for the first time in the literature for this amino acid unit.
This unusual conformation seems to be the reason for the high antioxidant capacity experimentally
found for this compound.
Received 18th February 2013,
Accepted 17th April 2013
DOI: 10.1039/c3cp50743b
www.rsc.org/pccp
The most common thiol-type antioxidants8 are cysteine,
Introduction
N-acetylcysteine (NAC) and derivatives9 such as g-glutathione
(GSH, g-L-glutamyl-cysteinyl-glycine).10 The importance of their
chemical and biological properties11,12 makes advisable a ratio-
nalised research focused on the cysteine-core. As occurs with
many other substances,13 it can be expected that the modifica-
tion of the original molecule of cysteine would yield even more
multipotent antioxidants. However, there is such a large number
of possible combinations of substituents in different positions of
cysteine that a method for screening a subset of target products
with enhanced antioxidant activity would be very useful in order
to reduce the amount of time that the synthesis and testing of all
of the possibilities would involve.
Therefore, in this paper a methodology based on quantum
calculations is developed to assess ‘‘a priori’’ (in silico) the
antioxidant activity of a set of modified cysteines. Four sub-
stitutions are considered, each corresponding to a different
position in the core molecule of cysteine. The first focuses on
the influence of a protecting group in carboxylic acid (R1)
dealing with either the free acid or the corresponding methyl
ester. The second deals with the effect of introducing an acetyl
protecting group into the amine (R2). In the third one, the effect
of replacing the hydrogen atom in the sulfhydryl group by a
methyl group (R3) is considered. Finally, an alkyl substituent
which would presumably change the rigidity of the cysteine
skeleton is introduced into the Ca-carbon (R4). Table 1 shows
Antioxidant is a term applied in medicine and nutrition to
substances that, in low concentration levels, reduce reactive
oxygen species (ROS) or reactive nitrogen species (RNS) before
they can oxidize other molecules. They also bind heavy metals
and react with other potentially harmful substances. Owing to
these properties, antioxidant activity is very important for
delaying the aging process in biological systems, preserving
cells from damage and preventing the oxidation of food and
pharmaceutical products. There is a wide range of compounds
on the marketplace, either natural or synthetic, that have more
or less pronounced antioxidant activity1–3 comprising several
sulphur derivatives,4,5 among which the thiol-type antioxidants
are probably the most significant for their good electron donor
ability,6 and for their interactions, as nucleophiles, with electro-
philic groups of ROS or RNS species.7
a Group of Applied Thermodynamics and Surfaces (GATHERS), Aragon Institute for
Engineering Research (I3A), Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009,
Zaragoza, Spain. E-mail: urieta@unizar.es; Fax: +34 976 761 202;
Tel: +34 976 761 298
b Amino Acids and Peptides Group, Department of Organic Chemistry,
Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009, Zaragoza, Spain.
E-mail: cativiela@unizar.es; Fax: +34 976 761 210; Tel: +34 976 761210
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3cp50743b
c
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Table 1 Set of cysteines with different modifications considered in this work
between cation radicals and neutral species, and from the
HOMO energy applying Koopmans’ theorem.18
R1
R2
R3
R4
Experimental section
No.
1
2
3
4
5
6
7
8
H
CH3
H
H
H
CH3
H
H
CH3
H
CH3
CH3
H
H
H
H
H
H
CH3
H
H
H
H
H
CH3
H
H
CH3
H
CH3
CH3
H
The radical scavenging activities of cysteines were evaluated
using DPPH (1,1-diphenyl-2-picrilhydrazyl) radical assays,
following the method described by Brand-Williams et al.19 with
several modifications. Solutions with different concentrations
of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid) in methanol (42 ml) were added to 2.5 ml of a 0.03 g lÀ1
DPPH solution in methanol. The decrease in absorbance of the
DPPH radical at 514 nm was measured under agitation in a
thermostated cell at 25 1C for two hours using a Thermo
Scientific evolution 300 UV-Vis spectrophotometer. The exact
initial and final DPPH concentrations in the reaction medium
were calculated from a calibration curve. The results are
reported as EC50, that is, the amount of antioxidant necessary
COCH3
H
H
COCH3
COCH3
H
H
CH3
CH3
CH3
H
9
H
10
11
12
13
14
15
16
COCH3
H
COCH3
COCH3
H
COCH3
COCH3
H
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
to reduce the DPPH concentration to 50%. The EC50 value
20
the complete array of modified cysteines studied in this work.
The results pose the possibility of activating unusual intra-
molecular hydrogen bonding as a strategy to synthesize new
effective radical scavengers from the cysteine core.
obtained for Trolox was 0.23 Æ 0.02 molTrolox/molDPPH
.
The EC50 for cysteines was determined from the Trolox EC50
value. A cysteine solution in water (42 ml) was added to 2.5 ml of
a DPPH solution of 0.03 g lÀ1 in methanol. The decrease in
absorbance was measured under the same working conditions
as those used for Trolox. The difference between the initial and
final DPPH concentrations was normalized to the cysteine
concentration in the reaction mixture. The EC50 value of the
cysteines was obtained by extrapolation of the normalized
concentration to the Trolox EC50. This method was tested with
pure cysteine 1, a compound whose antioxidant ability is
commonly measured using the DPPH method.21–23
Computational methods
All the calculations reported in this work were performed using
the Gaussian 09 program.14 Gas-phase molecular geometries of
all the cysteines in their neutral and radical cation forms were
optimized using Density Functional Theory (DFT) with the
hybrid B3LYP method and the 6-31+G(d,p) basis set. A fre-
quency analysis was carried out to verify the nature of the
minimum states of all molecules. Zero-point energies and
thermal corrections together with entropies were used to con-
vert internal energies to Gibbs energies at 298.15 K. Finally, in
order to obtain consistent energy calculations, single point
calculations were performed at the M06-2X/6-31+G(d,p) level
of the B3LYP/6-31+G(d,p). The effect of different solvents was
calculated using the polarizable continuum solvation model
(PCM).
The procedures for the synthesis, isolation and spectro-
scopic characterization of compounds 2, 5, 6 and 15 are
described in the ESI.†
Results and discussion
In silico prediction of antioxidant activity
In general, to reliably assess the antioxidant activity of a
molecule it would be extremely advantageous to establish a
relationship between that activity and one or several funda-
mental parameters of the molecule that could be calculated or
experimentally determined. As the main factor governing the
antioxidant activity of a molecule is its ability to transfer
electrons, several properties can be chosen that, in principle,
are intimately related to the ease with which an electron is
released. Following previous studies on redox processes,15 the
HOMO energy of the molecule as well as the formal redox
potential, and the first ionization energy for the formation of
the most stable radical cation (on the sulphur atom) have been
selected. Then, the existence of a correlation between the
properties and a suitable experimental parameter accounting
Redox potentials for modified cysteines were calculated
using a thermodynamic cycle,15–17 via eqn (1).
DG(soln)1 = DG(g)1 + DDG(solv)
1
(1)
The Gibbs energy of the one electron oxidation reaction in the
gas-phase, DG(g)1, and the Gibbs energies of solvation, DG(solv)1,
for neutral and radical cation species were calculated following
the procedure described above. The resulting value of the Gibbs
energy in solution, DG(soln)1, is related to the absolute formal
redox potential, Eabs, according to eqn (2).
Eabs = DG(soln)1/nF
(2)
where n is the number of electrons transferred and F is the for the antioxidant activity needs to be checked to prove the
Faraday constant. usefulness of the property for gauging the activity. The experi-
The ionization energies (IE) of the modified cysteines were mental parameter chosen as an indicator of the reducing char-
computed according to the difference in the Gibbs energy state acter is the EC50 determined by means of the DPPH radical assay.
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Table 2 Experimental EC50 and calculated absolute redox potentials (Eabs),
Table 3 Calculated absolute redox potentials in methanol (Eabs), ionization
potentials (IE) and HOMO energies of several cysteines, together with their
estimated EC50 from EHOMO, Eabs and IE regressions
ionization potentials (IE) and HOMO energies for several cysteines
a
No
EC50 (molCys/molDPPH
)
Eabs (V)
IE (eV)
ÀEHOMO (eV)
a
b
c
EC50
(molCys
EC50
(molCys
EC50
(molCys/
1
2
3
5
6
0.42b
0.38
0.38c
0.39
0.36
6.79
6.71
6.69
6.69
6.67
8.81
8.67
8.45
8.65
8.33
8.36
8.27
8.26
8.33
8.18
ÀEHOMO
/
Eabs
/
IE
No
(eV)
molDPPH
)
(V)
molDPPH
)
(eV)
molDPPH)
1
2
3
4
5
6
7
8
8.36
8.27
8.26
7.88
8.33
8.18
7.91
7.86
7.81
7.87
8.23
7.88
8.08
7.78
7.78
8.01
0.41
0.38
0.38
0.25
0.40
0.35
0.26
0.25
0.23
0.25
0.37
0.25
0.32
0.22
0.22
0.30
6.79
6.71
6.69
6.27
6.69
6.67
6.27
6.37
6.27
6.50
6.66
6.57
6.21
6.39
6.45
6.18
0.43
0.39
0.38
0.17
0.38
0.37
0.17
0.22
0.17
0.28
0.36
0.32
0.14
0.23
0.26
0.12
8.81
8.67
8.45
8.19
8.65
8.33
7.91
8.25
8.07
8.26
8.52
8.43
7.86
8.13
8.16
7.78
0.42
0.40
0.37
0.34
0.40
0.35
0.30
0.34
0.32
0.35
0.38
0.37
0.29
0.33
0.33
0.28
a
n = 3, s = Æ0.02 where n and s are the number of replicates and the
b
standard deviation, respectively. Bibliographic EC50 (1) = 0.40 Æ
c
0.02 molNAC/molDPPH
.
EC50 (3) = 0.32 Æ 0.02 molCYS/molDPPH
.
Five cysteines that are commercially available or that can be
easily synthesized (compounds 1, 2, 3, 5 and 6) were chosen to
check the procedure. DFT calculations were used to obtain
either directly HOMO energy or indirectly the absolute formal
redox potential and ionization energy. The EC50 of the cysteines
was also measured. The obtained results are gathered in
Table 2.
Fig. 1 shows that there is a quite good linear correlation
between the EC50 and each fundamental molecular property of
the cysteines. Thus, any of the properties could be used to
gauge the antioxidant activity. These results also indicate that
the molecular properties are related to each other, as would be
expected. In fact, a good correlation had already been obtained
between the formal redox potential and the HOMO energy for a
set of carbamates.15 This is important because, although the
formal redox potential is likely to be the more rigorous indi-
cator from the theoretical point of view, the HOMO energy
entails less computational cost allowing us, nevertheless, to
estimate EC50 values with a very reasonable accuracy.
9
10
11
12
13
14
15
16
a
b
c
Estimated EC50 from ÀEHOMO
.
Estimated EC50 from Eabs. Estimated
EC50 from IE.
than pure cysteine but the improvement is remarkable in
several compounds. These are S-methyl-L-cysteine 4, N-acetyl-
S-methyl-L-cysteine 7, (R)-S-methyl-2-methylcysteine 8, (R)-S-methyl-
L-cysteine methyl ester 9, (R)-N-acetyl-2-methylcysteine 10,
N-acetyl-S-methyl-L-cysteine methyl ester 12, (R)-N-acetyl-S-methyl-
2-methylcysteine 13, (R)-S-methyl-2-methylcysteine methyl ester 14,
(R)-N-acetyl-2-methylcysteine methyl ester 15 (NAMCME) and
(R)-N-acetyl-S-methyl-2-methylcysteine methyl ester 16. Eight
out of ten compounds are S-Me-type and the remaining two
are thiol-type compounds.
It should be indicated that the more stabilized the radical
cation the better are the EC50 values. This is to be expected. As
is well known, antioxidant activity and the mechanism of the
oxidation process are intimately related to the active centre.
Therefore, in the scavenging process S-methyl and thiol-type
compounds yield different types of products through different
pathways. Mechanistically, the oxidation process of thiol com-
pounds yields a thiyl radical that finally leads to the formation
of a disulfide bond.24 However, in vitro antioxidant activity of
S-methylcysteines implies that the molecules can undergo,
depending of the ROS, either one or two-electron oxidations
to give, respectively, radical cations or sulfoxides.25 However,
despite these differences, the processes share a common key
step, which is the base of our theoretical methodology: the loss
of a first electron to yield a high unstable radical cation. In this
way, an effective modification on the cysteine core must imply
the radical cation stabilization. For this reason any electro-
auxiliary group, e.g. a methyl group, attached to the sulphur
atom will promote a logical enhancement of the antioxidant
activity. Then, most of the cysteines with the best antioxidant
activity have the sulphur atom protected, and their activity is
due to the s-electron density of the methyl group directly
attached to the sulphur atom. But this line of argument makes
noticeable the high antioxidant activity predicted for non
Once the correlation between HOMO energy and EC50 was
established, HOMO energies (as well as the absolute redox
potentials and ionization energies) were calculated and the EC50
estimated for all the remaining cysteine derivatives is included in
Table 3. All of the derivatives exhibit better antioxidant activity
Fig. 1 EC50 vs. absolute redox potential, ionization potential and HOMO energy,
respectively.
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Table 4 Geometrical parameters for C7 and C5-membered ring hydrogen bonds
(10 and 15) and non-intramolecular hydrogen bonds (3 and 6) at B3LYP/6-
31+g(d,p)
No Solvent
d(S–HÁ Á ÁOQC)a
F
c
w1
d(N–HÁ Á ÁOQC)a
10 Gas-phase 2.092
Heptane 2.080
Methanol 2.053
À178.0 177.8 À95.7 2.085
À177.7 177.7 À94.9 2.085
À176.4 177.0 À92.5 2.081
À176.3 176.9 À92.4 2.081
À176.2 176.9 À92.2 2.081
DMSO
Water
2.052
2.051
3
Gas-phase 3.696
Heptane 3.794
Methanol 3.907
À107.7 164.5 À66.1 2.520
À104.6 165.4 À66.1 2.618
À107.0 170.2 À65.4 2.783
À107.0 170.6 À65.3 2.790
À107.0 171.0 À65.2 2.796
Fig. 2 Optimized structures obtained by DFT calculations for compounds 15
and 6.
DMSO
Water
3.913
3.917
remarkable because of the intramolecular S–HÁ Á ÁOQC hydrogen
bond. Although the S–HÁ Á ÁOQC hydrogen bond has been
observed in other compounds,27 it is very unusual. In fact, in
the Cambridge Structural Database, out of 1811 Y-S-Z substruc-
tures only 26 show that hydrogen bond and only one of these is
intramolecular.28
Taking into account these results, it can be proposed that
the Ca-methyl group would activate intramolecular hydrogen
bonds in 10 and 15 thus enhancing the stability of the neutral
species and remarkably that of the radical cation, with the
subsequent reduction of the ionization energy. It is likely that
the cooperative effects existing between C7 and C5-membered
15 Gas-phase 2.080
Heptane 2.066
Methanol 2.040
À177.3 178.2 À94.8 2.067
À176.5 178.1 À93.5 2.066
À175.1 177.4 À90.9 2.065
À175.0 177.4 À90.8 2.065
À175.0 177.3 À96.7 2.065
DMSO
Water
2.039
2.039
6
Gas-phase 3.697
Heptane 3.752
Methanol 3.990
À107.9 161.5 À66.1 2.501
À103.8 166.5 À65.6 2.564
À103.4 165.8 À65.8 2.795
À103.6 166.2 À65.7 2.799
À107.2 161.1 À66.1 2.802
DMSO
Water
3.992
3.994
a
Distances (d) are in Å.
S-methylated compounds, thus implying a different strategy of rings would donate to the sulphur atom an amount of electron
the molecules to stabilize the radical cation species. density similar to that provided by a methyl group leading to a
Then, regarding a theoretical answer for the stabilization in similar antioxidant activity.
compounds 10 and 15 the geometrical parameters obtained for
It has also been considered advisable to study the effect of
the minimum structures in the gas phase, listed in Table 4, different solvents on the structure of 10 and 15 to know
suggest the formation of an intramolecular S–HÁ Á ÁOQC (on the whether and to what extent the intramolecular hydrogen bonds
N-acetyl group) hydrogen bond. When comparing pairs of are maintained under the influence of different types of solute–
compounds (10 vs. 3 and 15 vs. 6) in which the quaternary solvent interactions. This is due to two reasons. On the one
methyl group (R4) is, respectively, present or absent, shorter hand, as is well known, molecules in solution can adopt
distances between functional groups are observed in the different conformations depending on the nature of the
presence of the Ca-methyl group. In fact, in compounds 3 and solvent. On the other hand, antioxidant activity is exerted and
6, the carbonyl OQC of the N-acetyl group is oriented away measured in specific media. Therefore, the PCM model, which
from hydrogen (S–H). Thus, the methyl group in Ca causes a provides good results in describing the behaviour in solution of
restructuration of the torsional angles w1, c and f promoting compounds able to form intramolecular hydrogen bonds,29,30
the approach of S–H and OQC of the N-acetyl group which has been used to optimize the molecular geometry of com-
could allow the formation of a S–HÁ Á ÁOQC intramolecular pounds 3, 6, 10 and 15 in several solvents with increasing
hydrogen bond giving a seven-membered ring. At the same polarity, namely, heptane, methanol, DMSO, and water
time the formation of the C7 member ring is accompanied by (Table 4). Compounds 3 and 6 form intermolecular hydrogen
the formation of a second intramolecular C5 member ring if the bonds with the solvents as indicated by the increase of
distances between N–H and OQC on the carboxylic group, distances d(S–HÁ Á ÁOQC) and d(N–HÁ Á ÁOQC) with increased
d(N–HÁ Á ÁOQC), are taken into account. This C5 member ring solvent polarity. But for 10 and 15 the intramolecular distances
does not appear for molecules that are non-methylated in Ca. d(S–HÁ Á ÁOQC) and d(N–HÁ Á ÁOQC) are not affected by the
Besides, the values for the dihedral angles (f,c) = (À177.3, media. This could be indicative of the presence of a strong
178.2) obtained by the DFT calculations in the gas phase are intramolecular hydrogen bond that is not affected by solvent
characteristic of a C5 conformation due to the alkyl side chains nature. This particularity seems to be a result of the so called
attached to the Ca-carbon.26 Fig. 2 shows, as an example, the ‘‘structural effects’’ that do not involve the rupture of the
differences between optimized structures of 6 and 15. In hydrogen bonds. Either water or methanol molecules can form
conclusion, compounds 10 and 15 exhibit a structure consis- three or two-dimensional networks through intermolecular
tent with the existence of two intramolecular hydrogen bonds, hydrogen bonds. These networks will have cavities of different
a structure never previously observed in a cysteine. What is sizes and shapes where 10 or 15 molecules could be inserted.
more, it must also be noticed that these compounds are very This insertion would bring about an increase in the regularity
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of the network by forcing intermolecular hydrogen bonds of the
solvent to stretch in such a way that there are no changes in the
conformation of the solute molecule in a state of infinite
dilution.31–33
Experimental corroboration of the existence of intramolecular
hydrogen bonds in Ca-quaternary cysteines and of their
antioxidant activity
The surprising structures predicted for cysteines 10 and 15 as
well as their good antioxidant activity were worth an experi-
mental verification to test the validity of the method in the
prediction of both EC50 and structures. Compound 10 was
discarded because in non-polar media the carboxylic acids
usually exist as dimers due to their tendency to ‘‘self-associate’’,
and this would interfere with the verification of the existence of
Scheme 1 Synthesis of (R)-N-acetyl-2-methylcysteine methyl ester 15 and
N-acetyl-L-cysteine methyl ester 6.
Fig. 3 Experimental 1H NMR chemical shift in CDCl3 when adding different portions of DMSO-d6 (a) 6 and (b) 15.
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the intramolecular hydrogen bonds. Thus, (R)-N-acetyl-2- Besides, it could also be useful for synthetic oligomer design
methylcysteine methyl ester 15 was synthesized. Scheme 1 by profiting from its steric and dihedral angle constraints
shows the reaction path and the yields of compounds 15 and which could enable modulating their structural features for
6. Then the conformational preferences of their molecules in possible applications involving protein recognition.37,38
1
solution were studied through H NMR.
A 1H NMR titration was performed on compounds 6 and 15
Conclusions
by increasing the polarity of the medium (CDCl3) with the
addition of increasing amounts of DMSO. In this sense, the
1H NMR chemical shift of a proton involved in an intra-
molecular hydrogen bond would be scarcely disturbed if the
polarity of the medium changes.
This paper describes a method devised to estimate antioxidant
activity based on the correlation of HOMO energies, obtained
by DFT calculations, of a subset of cysteines with their respec-
tive experimental EC50. This procedure, combined with the
PCM calculations, has been applied to an array of cysteines
allowing us to discover intramolecular hydrogen bond activa-
tion in cysteines 10 and 15 by introducing a Ca-methyl group.
The experimental structure of (R)-N-acetyl-2-methylcysteine
methyl ester 15 shows two cooperative C7 and C5 member
rings. This conformation had never been previously reported
in a single cysteine and it is responsible for its remarkable
antioxidant activity. Additionally, the experimental EC50 value
of 15 coincides reasonably well with the value predicted using
the method, thus confirming its validity.
From the 1H NMR spectrum, Fig. 3b, the S–H proton of
compound 15 shows no change in chemical shift when increas-
ing the polarity of the medium but at the same time it exhibits
important structural changes. The S–H proton shows different
coupling constants, JSH–CH = 9.6 Hz and JSH–CH = 8.5 Hz, with
a
b
each proton in CH2 (CHaHb) in pure CDCl3 (doublet of doublets)
but it shows the same coupling constant, JSH–CH H = 9.1 Hz, for
a
b
both CHaHb protons when the concentration of DMSO increases
to 60 mL (triplet). That is, CH2 protons are diastereotopic (CHaHb)
in pure CDCl3 suggesting a substantial S–HÁ Á ÁOQC intra-
molecular H-bonding. Different behavior can be observed for
the non-hydrogen bound and non-methylated Ca compound 6
(Fig. 3a).
Acknowledgements
In the case of N–H its shift changed little with the addition
of DMSO-d6 to CDCl3, the values of Dd for the amide N–H
protons for cysteines 15 and 6 are 0.50 and 0.90 ppm, respec-
tively, as can be seen in Fig. 3. The amide N–H proton for the
cysteine 15 was less shifted downfield and agrees with the
values described in the bibliography for quaternary amino
acids.34,35 Then, the possibility of the amide N–H proton in
15 being involved in an intramolecular hydrogen bond
N–HÁ Á ÁOQC with the oxygen of the ester carbonyl group can
be reasonably assumed. Therefore, the 1H NMR study confirms
that the thiol S–H and the amide N–H are involved in coopera-
tive intramolecular hydrogen bonds with the oxygen of the
acetyl group and the oxygen of the ester carbonyl group,
respectively, as was predicted by the DFT calculations.
Finally, the experimental antioxidant activity (EC50) found
for (R)-N-acetyl-2-methylcysteine methyl ester 15 was 0.27 Æ
0.02 molCys/molDPPH. Considering the experimental antioxidant
activity of compound 6, 0.36 Æ 0.02 molCys/molDPPH, and the
conformation found experimental and theoretically for both
cysteines (in the gas-phase and at infinite dilution), the unusual
structure of (R)-N-acetyl-2-methylcysteine methyl ester 15 is
responsible for its effectiveness as a radical scavenger. More-
over, the EC50 value of 15 quite coincides with the extrapolated
value (Table 3) in such a way that it constitutes a strong support
for the validity of the prediction method proposed, especially if
it is considered that the correlation was based on values of EC50
ranging from 0.36 to 0.42, with the extrapolated value being
quite far away from that range.
´
The authors thank the Ministerio de Ciencia e Innovacion-
FEDER (grants CTQ2010-17436, CTQ2009-14629-CO2-02, CTQ2012-
38219-C03-02 and IPT-2011-0860-060000) and the Gobierno de
Aragon-FSE (research groups E40 and E52) for financial support.
The authors thank Juan I. Pardo for helpful discussions.
L.H. thanks UZ for a predoctoral contract and I.O. thanks CSIC
for a postdoctoral contract.
´
Notes and references
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