Role of Hydrogen Bonding by Thiones in Protecting Biomolecules from Copper(I)-Mediated Oxidative Damage

Article abstract of DOI:10.1021/acs.inorgchem.8b03212

The sulfur-containing antioxidant molecule ergothioneine with an ability to protect metalloenzymes from reactive oxygen species (ROS) has attracted significant interest in both chemistry and biology. Herein, we demonstrated the importance of hydrogen bonding in S-oxygenation reactions between various thiones and H₂O₂ and its significance in protecting the metal ion from H₂O₂-mediated oxidation. Among all imidazole- and benzimidazole-based thiones (1-10), Im^MeS^H (2) showed the highest reactivity toward H₂O₂ - almost 10 and 75 times more reactive than N,N′-disubstituted Im^MeS^Me (5) and Bz^MeS^Me (10), respectively. Moreover, metal-bound Im^MeS^H (2) of [TpmCu(2)]⁺ (13) was found to be 51 and 1571 times more reactive toward H₂O₂ than the metal-bound Im^MeS^Me (5) of [TpmCu(5)]⁺ (16), and Bz^MeS^Me (10) of [TpmCu(10)]⁺ (21), respectively. The electron-donating N-Me substituent and the free N-H group at the imidazole ring played a very crucial role in the high reactivity of Im^MeS^H toward H₂O₂. The initial adduct formation between Im^MeS^H and H₂O₂ (Im^MeS^H·H₂O₂) was highly facilitated (-23.28 kcal mol^-1) due to the presence of a free N-H group, which leads to its faster oxygenation than N,N′-disubstituted Im^MeS^Me (5) or Bz^MeS^Me (10). As a result, Im^MeS^H (2) showed a promising effect in protecting the metal ion from H₂O₂-mediated oxidation. It protected biomolecules from Cu(I)-mediated oxidative damage of through coordination to the Cu(I) center of [TpmCu(CH₃CN)]⁺ (11), whereas metal-bound Im^MeS^Me or Bz^MeS^Me failed to protect biomolecules under identical reaction conditions.

Full text of DOI:10.1021/acs.inorgchem.8b03212

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Role of Hydrogen Bonding by Thiones in Protecting Biomolecules
from Copper(I)-Mediated Oxidative Damage
Rakesh Kumar Rai,§ Ashish Chalana,§ Ramesh Karri,§ Ranajit Das,§ Binayak Kumar,‡
and Gouriprasanna Roy*,§
‡
§Department of Chemistry and Department of Life Sciences, School of Natural Sciences, Shiv Nadar University, NH91, Dadri,
Gautam Buddha Nagar, Uttar Pradesh 201314, India
S
* Supporting Information
ABSTRACT: The sulfur-containing antioxidant molecule ergothio-
neine with an ability to protect metalloenzymes from reactive oxygen
species (ROS) has attracted significant interest in both chemistry and
biology. Herein, we demonstrated the importance of hydrogen bonding
in S-oxygenation reactions between various thiones and H2O2 and its
significance in protecting the metal ion from H2O2-mediated oxidation.
Among all imidazole- and benzimidazole-based thiones (1−10),
ImMeSH (2) showed the highest reactivity toward H2O2almost 10
and 75 times more reactive than N,N′-disubstituted ImMeSMe (5) and
BzMeSMe (10), respectively. Moreover, metal-bound ImMeSH (2) of
[TpmCu(2)]+ (13) was found to be 51 and 1571 times more reactive
toward H2O2 than the metal-bound ImMeSMe (5) of [TpmCu(5)]+
(16), and BzMeSMe (10) of [TpmCu(10)]+ (21), respectively. The
electron-donating N−Me substituent and the free N−H group at the imidazole ring played a very crucial role in the high
reactivity of ImMeSH toward H2O2. The initial adduct formation between ImMeSH and H2O2 (ImMeSH·H2O2) was highly
facilitated (−23.28 kcal mol−1) due to the presence of a free N−H group, which leads to its faster oxygenation than N,N′-
disubstituted ImMeSMe (5) or BzMeSMe (10). As a result, ImMeSH (2) showed a promising effect in protecting the metal ion from
H2O2-mediated oxidation. It protected biomolecules from Cu(I)-mediated oxidative damage of through coordination to the
Cu(I) center of [TpmCu(CH3CN)]+ (11), whereas metal-bound ImMeSMe or BzMeSMe failed to protect biomolecules under
identical reaction conditions.
thioneine exists in two tautomeric forms (thione and thiol
form), mostly in the thione form at physiological pH.17
Although this molecule was discovered more than 100 years
ago, its physiological function has yet to be fully established.
Recently, Brumaghim and co-workers have demonstrated, in
their pioneer work, that the N,N′-disubstituted imidazole-
based thione/selone can prevent the Cu(I)-mediated oxidative
damage of biomolecules by directly binding to the redox active
Cu(I) center and thereby not allowing H2O2 to interact with
the metal ion.18 For instance, ImMeSMe and its selenium
analogue (ImMeSeMe) were reported to show their antioxidant
activities by coordinating to the Cu(I) center of tris(3,5-
dimethylpyrazolyl)-methane (Tpm) Cu(I) complex, [TpmCu-
(CH3CN)]+ (11), through the lone pair of S and Se atoms,
which led to the formation of the corresponding redox inactive
[TpmCu(thione)]+ and [TpmCu(selone)]+ complexes.18,19
Interestingly, because of the presence of a lone pair at the S
or Se center of Cu(I)-bound thione or selone of [TpmCu-
(thione)]+ or [TpmCu(selone)]+, the approaching H2O2 may
readily interact with the metal-bound thione or selone, instead
INTRODUCTION
■
Reactive oxygen species (ROS) are highly toxic and involved in
oxidative damage to biomolecules.1−7 Hydrogen peroxide
(H2O2), a prime member of ROS, is continuously produced in
vivo due to various physiological functions.8 On average,
intracellular and extracellular H2O2 concentrations roughly can
vary from 0.001−0.1 μM and 0.1−10 μM, respectively,
depending upon the normal and oxidative stress conditions.9,10
Moreover, H2O2 is known to produce highly reactive hydroxyl
radical (HO•) in reacting with the free Cu(I) ion via a Fenton-
type reaction, which leads to the oxidative damage of DNA and
protein.8,11 Excess Cu has been implicated in the progression
of neurodegenerative disorders including Alzheimer’s, Parkin-
son’s, and cardiovascular diseases.1,12−14
The sulfur-containing small molecules such as GSH and L-
ergothioneine have evolved to protect cells from Cu-induced
oxidative stress. L-Ergothioneine is mainly synthesized in fungi
and bacteria. Humans acquire this unusual amino acid through
consumption of an ergothioneine-rich diet, primarily by the
consumption of edible mushrooms, black beans, red meat, and
oats.15 In human blood, ergothioneine is estimated to be
present in the range of 1−4 mg/100 mL (ca. 46−183 μM).16
Unlike the thiol (-SH)-containing antioxidant GSH, ergo-
Received: November 16, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.8b03212
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
damage of DNA (IC50 = 1.55 mM, IC50 value is the
concentration required to inhibit 50% of the DNA damage).20
In a separate experiment, Zhu et al. reported that
ergothioneine can prevent Cu(I)-mediated oxidative damage
of DNA and protein in a dose-dependent manner (0.1−1.0
mM), possibly through coordination to the redox active Cu(I)
center.21 Structurally ergothioneine is quite different from
N,N′-dimethyl substituted thione ImMeSMe as it does not have
any substituent at the N atom of the imidazole ring, and,
therefore, it is pertinent to understand the effect of N-
substituents at the five-membered heterocyclic ring on the
reactivity of metal-bound thiones toward H2O2 and their
ability to protect biomolecules from Cu(I)-mediated oxidative
damage. Does a substituent at the N atom of the heterocycle
play any significant role in the reactivity of thiones toward
H2O2? Does ImMeSH, having N−Me and free N−H groups,
have a similar efficacy in protection from Cu(I)-mediated
oxidative damage than N,N′-disubstituted ImMeSMe or
BzMeSMe? Thus, to address these relevant unanswered
questions, we employed various imidazole- (1−5) and
benzimidazole-based (6−10) thiones with different substitu-
ents at the N atom of the heterocycle and the corresponding
Cu(I) complexes 12−21, Figure 1, in our study. Herein we
report a detailed investigation of the reactivity of various free
and Cu(I)-bound thiones toward H2O2 and their ability to
protect biomolecules from Cu(I)-mediated oxidative damage.
Figure 1. Chemical structures of ergothioneine, imidazole, and
benzimidazole-based thiones and their corresponding Cu(I) com-
plexes.
of the Cu(I) ion which is buried inside the complex (Figure 2).
Unfortunately, unlike its Se analogue, the reactivity of ImMeSMe
RESULTS AND DISCUSSION
■
To understand the role of N-substituents at the five-membered
heterocyclic ring of imidazole- and benzimidazole-based
thiones on the reactivity of thiones toward H2O2 and their
ability to inhibit the generation of Cu(I)-mediated HO•
radicals, we synthesized various derivatives of imidazole-
based (1−5) and benzimidazole-based (6−10) thiones by
following the literature procedure.22,23 Thione-bound Cu(I)
complexes (12−21) were obtained by treating 11, [TpmCu-
(CH3CN)]+, with appropriate thione ligands in dichloro-
methane. Detailed synthetic procedures are mentioned in the
Supporting Information. Thiones and 1:1 thione-bound Cu(I)
complexes were characterized thoroughly by NMR, mass
spectrometry, and single crystal X-ray diffraction (SC-XRD).
Reactivity of Thiones toward H2O2. Imidazole-based
thiones 1−5 showed a strong absorbance band at 265 nm due
to the n → π* transition of the C−S moiety. The S-
oxygenation of thiones 1−5 (10 mM) in the presence of 3
equiv of H2O2 was monitored at 37 °C by following the
decrease in absorbance at 265 nm (i.e., the desulfurization of
Figure 2. A representational figure showing how metal-bound thione
protects Cu(I) from H2O2.
toward H2O2 was reported to be extremely slow, and, thus, it
may allow H2O2 to interact with the Cu(I) ion, which leads to
the generated HO• radical via a Fenton-type reaction. As a
result, ImMeSMe poorly inhibits the Cu(I)-mediated oxidative
Figure 3. Desulfurization of imidazole-based thiones 1−5 (a) and benzimidazole-based thiones 6−10 (b) in the presence of 3 equiv of H2O2 at 37
°C.
B
DOI: 10.1021/acs.inorgchem.8b03212
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
thiones), as shown in Figure 3 and Figure S5a. Kinetic studies
of thione oxygenation by H2O2 have revealed that the time
required for the 50% cleavage of C−S bond (t1/2) of N−Me-
substituted thione ImMeSH was almost 3.3 times less than the
time required for ImHSH, which did not have any N-substituent
at the five-membered ring. This observation suggests that the
electron-donating N-Me group probably enhances the
reactivity of ImMeSH toward H2O2.
However, if the electron-donating N-Me group is the only
reason for its higher reactivity toward H2O2, it is expected
therefore that the N,N′-dimethylated ImMeSMe will show higher
reactivity than N-methylated ImMeSH. However, we found that
the t1/2 value for ImMeSMe was 10.7 times higher than the t1/2
value obtained for ImMeSH at 37 °C. This result indicates that
the ImMeSH is more reactive toward H2O2 than ImMeSMe, and
possibly the free N−H group of ImMeSH is playing a significant
role here. The reactivity sequence of imidazole-based thiones is
ImMeSH (2) > ImBnSH (3) > ImHSH (1) > ImCOOMeSH (4) >
ImMeSMe (5), Table 1, which suggests that moving from the
electron-donating N−Me group to the electron-withdrawing
group like −Bn or −CH2CO2Me has reduced the reactivity of
thiones.
H2O2 (3 equiv) and thiones were identified by mass
spectrometry. The end product [MeImH]+ (imidazolium salt),
obtained in the reaction of ImMeSH and H2O2 (3 equiv), was
isolated and characterized thoroughly. LS/MS analysis
confirmed the formation of oxidative intermediate products
such as sulfenic acid ImMeSOH (m/z 131.0287 for [M + H]+)
and sulfonic acid ImMeSO3H (m/z 163.0180 for [M + H]+) or
BzMeSOH (m/z 181.0418 for [M + H]+) and BzMeSO3H (m/z
213.0334 for [M + H]+) from the reaction mixture of ImMeSH/
H2O2 or BzMeSH/H2O2 (Figure 4a,b). This is in good
Table 1. t1/2 Values for the Degradation of C−S Bond of
Thiones Mediated by H2O2 at 37 °C and Charge on the S
Atom of Thiones
a
compd
t1/2 (min)
charge on S
ImHSH (1)
15.54 2.92
4.67 1.73
9.73 1.65
19.33 1.70
49.89 1.50
20.30 2.64
9.6 1.9
14.95 0.61
205.8 2.21
352.29 3.12
−0.277
−0.288
−0.292
−0.283
−0.295
−0.226
−0.235
−0.239
−0.230
−0.241
ImMeSH (2)
ImBnSH (3)
ImCOOMeSH (4)
ImMeSMe (5)
BzHSH (6)
BzMeSH (7)
BzBnSH (8)
BzCOOMeSH (9)
BzMeSMe (10)
a
All experiments were performed at room temperature (37 °C) in
CH3CN, ([Compd] = 10 mM).
Figure 4. (a) Proposed mechanism of C−S bond cleavage of ImMeSH
or BzMeSH by H2O2 (3 equiv) via the formation of sulfenic
(ImMeSOH/BzMeSOH), sulfinic (ImMeSO2H/BzMeSO2H), and sul-
fonic (ImMeSO3H/BzMeSO3H) acid intermediates. ΔG values are in
kcal mol−1. (b) HRMS of intermediates. (c) ORTEP images of end
products MeBzH·HSO4, BnBzH·HSO4, and MeBzMe·HSO4.
We next studied the reactivity of various benzimidazole-
based thiones 6−10 toward H2O2 by following a similar
procedure as mentioned above in the case of thiones 1−5,
except the desulfurization of thiones 6−10 was monitored at
λmax of 310 nm instead of 265 nm. Interestingly, the
benzimidazole thiones (6−10) were found to be less reactive
toward H2O2 in comparison to their imidazole counterparts
(1−5), Figure 3. However, the reactivity sequence of
benzimidazole-based thiones is similar to the imidazole-based
thiones, BzMeSH (7) > BzBnSH (8) > BzHSH (6) > BzCOOMeSH
(9) > BzMeSMe (10). BzMeSMe is ca. 75 and 36 times less
reactive toward H2O2 in comparison to ImMeSH and BzMeSH,
respectively. It should be mentioned that the order of reactivity
of benzimidazole-based thiones toward oxidation mediated by
H2O2 obtained here is slightly different than the order of
reactivity obtained by Doerge et al., BzHSH (6) > BzMeSH (7) >
BzMeSMe (10). The possible reason for this little variation in
reactivity may be due to the use of a different oxidizing reagent,
H2O2, instead of perbenzoic acid in our case.24
agreement with previous oxidation studies.24 A single crystal
obtained from the reaction mixture of BzMeSH/H2O2 in
acetonitrile−dichloromethane (1:1) solution confirmed the
formation of MeBzH·HSO4 (Figure 4c) in which the oxygen
atom of the HSO4− ion formed a strong H-bonding (2.714 Å)
with the N−H group of MeBzH·HSO4 (Figure S7d). Addition-
ally, the OH group of HSO4− formed a second H-bonding with
the oxygen atom of another MeBzH.HSO4 unit leading to the
formation of a chain-like structure. Crystal structures of BnBzH·
HSO4 and MeBzMe·HSO4 are shown in Figure 4c.
Reactivity of Copper(I) Complexes toward H2O2. The
oxidation kinetics of isolated complexes (12−21) in the
presence of H2O2 (3 equiv) was investigated, similar to
thiones, by following the decrease in absorbance (at 265 nm
for 12−16 and at 310 nm for 17−21), Figure 5. The t1/2 value
for [TpmCu(5)]+ (85.56 1.6 min) was found to be 51, 40,
Detection of Intermediates of Oxygenation of
Thiones. The oxidation reactions were monitored by LC/
MS, and the intermediate products obtained in the reactions of
C
DOI: 10.1021/acs.inorgchem.8b03212
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Figure 5. Desulfurization of copper(I) complexes 12−16 (a) and 17−21 (b) in the presence of 3 equiv of H2O2 at 37 °C.
and 26 times more than the t1/2 values obtained for complexes
[TpmCu(2)]+, [TpmCu(3)]+, and [TpmCu(4)]+, respec-
tively, indicating that the metal-bound ImMeSMe (5) reacts
very slowly with the approaching H2O2 than metal-bound
ImMeSH (2), ImBzSH (3), or ImCOOMeSH (4). These
observations strongly suggest that the N−H group of metal-
bound thiones is possibly playing a significant role while
reacting with H2O2, similar to the case of free thiones (vide
supra).
of the reaction between Cu(I) complexes and H2O2 (3 equiv).
Treatment of H2O2 to the solution of [TpmCu(2)]+ in
acetonitrile at room temperature afforded [TpmCu-
(ImMeSOH)]+ (m/z 491.1405), ImMeSO2H (m/z 147.0232),
ImMeSO3H (m/z 163.0180), and [MeImH]+ (m/z 83.0607) in
solution, Figure 6. We also detected HSO4− ion (m/z 96.9646)
in solution by mass spectrometry. Formation of the end
product MeBzH·HSO4 in the reaction of [TpmCu(7)]+ and
H2O2 was confirmed by an X-ray study. On the basis of our
results, we propose a possible pathway of oxidation of ImMeSH
(2)-bound Cu(I) complex [TpmCu(2)]+ by H2O2, as shown
in Figure 6a. Density functional theory (DFT) calculations
show that the oxidation of [TpmCu(2)]+ to [TpmCu-
ImMeSOH)]+ by H2O2 is, indeed, a energetically favorable
(ΔG = −35.34 kcal mol−1) reaction.
Structural Analysis of Copper(I) Thione Complexes.
Single crystals of complexes [TpmCu(4)]+, [TpmCu(7)]+,
[TpmCu(8)]+, [TpmCu(9)]+, and [TpmCu(10)]+ were
obtained through a slow diffusion technique in hexane/
acetonitrile (1:1) solution, and their structures were
determined by X-ray diffraction study, Figure 7. The calculated
four-coordinate geometry index τ4 values of Cu(I) centers of
[TpmCu(4)]+, [TpmCu(7)]+, [TpmCu(8)]+, [TpmCu(9)]+,
and [TpmCu(10)]+ are ranging from 0.67 to 0.72, indicating a
distorted tetrahedral geometry at the Cu(I) center. Upon
coordination to the Cu(I) center through the p-type of S lone
pair (lone pair of thione forms L-type Cu ← S dative covalent
bond), the C−S bond length of metal-bound thiones is
expected to increase. Quantum chemical calculations of all
thiones and their corresponding Cu(I) complexes confirmed
that the C−S bond lengths of metal-bound thiones were,
indeed, increased slightly compared to those in free thiones
(∼0.025−0.042 Å), Table S5.
Reactivity of the imidazole-based thione-bound Cu(I)
complexes toward H2O2 at 37 °C decreases in the order 13
> 14 > 12 > 15 > 16. Likewise, the reactivity of the
benzimidazole-based thione-bound Cu(I) complexes also
decreases in the order 18 > 19 > 17 > 20 > 21. However,
benzimidazole-based thione-bound Cu(I) complexes 17−21
are less reactive in comparison to the corresponding imidazole-
based thione-bound Cu(I) complexes 12−16. Desulfurization
of 21, [TpmCu(10)]+, (t1/2 = 2640 3.4 min) occurred very
slowly at 37 °Cca. 1571 times more slowly than the 13,
[TpmCu(2)]+, (t1/2 = 1.68
1.2 min). Interestingly, the
higher reactivity of the metal-bound thione toward H2O2 is
observed in comparison to the corresponding free thione. This
is very likely due to the elongation of the C−S bond of the
thione upon coordination to the metal center (Table S5, vide
infra), as is evident from the significant upfield shift of the
carbon resonance of C−S in 13C NMR, Table S1.1,25,26
Detection of Intermediates of Oxygenation of Cu(I)-
Complexes. The mass spectral analyses were carried out to
identify the intermediate products produced during the course
Table 2. t1/2 Values for the Degradation of C−S Bond of
Cu(I)-Thione Complexes Mediated by H2O2 at 37 °C and
Charge on the S Atom of Metal-Bound Thiones
Theoretical Insight into the Reaction Pathways. To
further understand the mechanism of S-oxygenation of ImMeSH
(2) or BzMeSH (7) by H2O2, we have performed quantum
chemical calculations (DFT) using the B3LYP/6-31G(d) level
of theory in the gaseous phase. In step 1 of the oxidation of
ImMeSH or BzMeSH by H2O2 involved nucleophilic attack of a
negatively charged S atom of thione (HOMO) to the
antibonding orbital of H2O2 (LUMO). It resulted in the
elongation of the O−O bond of H2O2 and yielded the
oxidation product sulfenic acid ImMeSOH·H2O or BzMeSOH·
H2O via the transition state [ImMeSH - ImMeSOH)]‡ or
[BzMeSH - BzMeSOH)]‡, as shown in Scheme 1. This
transformation was also accompanied by the elimination of
water after abstraction of the N−H proton. Interestingly, the
very first step of the oxidation reaction between ImMeSH (2) or
a
compd
t1/2 (min)
charge on S
[TpmCu(1)]+ (12)
[TpmCu(2)]+ (13)
[TpmCu(3)]+ (14)
[TpmCu(4)]+ (15)
[TpmCu(5)]+ (16)
[TpmCu(6)]+ (17)
[TpmCu(7)]+ (18)
[TpmCu(8)]+ (19)
[TpmCu(9)]+ (20)
[TpmCu(10)]+ (21)
2.75 1.8
1.68 1.2
2.16 2.2
3.2 1.8
85.56 1.6
10.46 0.7
4.42 1.6
8.89 0.4
19.51 0.7
2640 3.4
−0.272
−0.279
−0.291
−0.265
−0.221
−0.240
−0.252
−0.256
−0.231
−0.189
a
All experiments were performed at room temperature (37 °C) in
CH3CN, ([Compd] = 10 mM).
D
DOI: 10.1021/acs.inorgchem.8b03212
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Figure 6. (a) The representative pathway of oxidation of copper(I) complex [TpmCu(2)]+ in the presence of H2O2. HRMS of
[TpmCu(ImMeSOH)]+ is included on the right side. (b) HRMS data of [TpmCu(2)]+ and other intermediate products obtained in the
reaction of [TpmCu(2)]+ and H2O2.
Figure 7. Molecular structures (50% probability density ellipsoids) of [TpmCu(4)]+ (a), [TpmCu(7)]+ (b), [TpmCu(8)]+ (c), [TpmCu(9)]+
(d), and [TpmCu(10)]+ (e).
BzMeSH (7) and H2O2 was the formation of a 1:1 ImMeSH-
H2O2 or BzMeSH-H2O2 adduct, which was greatly stabilized by
intermolecular H-bondings between the H atom of the N−H
group of N-substituted ImMeSH (2) or BzMeSH (7) and H2O2.
The adduct ImMeSH.H2O2 formation was thermodynamically
facilitated (−23.28 kcal mol−1) due to the presence of the
strong N−H···O H-bonding (2.86 Å) between the H atom of
the N−H group of ImMeSH with the O atom of H2O2 and a
relatively weak S···H−O interaction between the S atom of
ImMeSH with the H atom of H2O2, Scheme 1, Figures 8 and
S42. Likewise, in the second step (step 2), the sulfenic acid
(ImMeSOH) further interacted with H2O2 to form a 1:1
ImMeSOH·H2O2 adduct (−25.8 kcal mol−1), which was again
stabilized by two intermolecular H-bondings, namely, N−H···
O (1.84 Å) and O···H−O (1.82 Å). The ImMeSOH·H2O2
adduct yielded a further oxidized product sulfinic acid
ImMeSO2H.H2O (−70.73 kcal mol−1) via transition state
[ImMeSOH - ImMeSO2H]‡. In a similar fashion, in step 3,
sulfinic acid again oxidized, by one more equivalent of H2O2,
to sulfonic acid ImMeSO3H.H2O (−82.87 kcal mol−1) via the
ImMeSO2H·H2O2 adduct. Finally, the unstable sulfonic acid
underwent C−S bond cleavage through a [ImMeSO3H·H2O]#
transition state which led to the formation of the final end
product MeImH.HSO4 (−87.11 kcal mol−1).
E
DOI: 10.1021/acs.inorgchem.8b03212
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Scheme 1. Proposed Mechanism of Degradation of C−S Bond of ImMeSH or BzMeSH by in the Presence of 3 equiv of H2O2
Figure 8. Schematic representation of the potential energy surface (PES) of the reaction of ImMeSH (2) and BzMeSH (7) with H2O2. The energy
values are scaled for the Δ(E + ZPE) values of all the species. Hydrogen bonding distances: d(N−O) = 2.86 Å (ImMeSH·H2O2), d(N−O) = 1.84 Å
(ImMeSOH·H2O2), d(N−O) = 2.88 Å (BzMeSH·H2O2), d(N−O) = 1.82 Å (BzMeSOH·H2O2).
Formation of sulfenic acid ImMeSOH·H2O (−59.51 kcal
mol−1) from ImMeSH (step 1) was 4.85 kcal mol−1
thermodynamically more favorable compared to the formation
of BzMeSOH·H2O (−54.66 kcal mol−1) from BzMeSH, as shown
in Table 3 and Figure 8. This is because the sulfur center of
ImMeSH is more nucleophilic in nature and possesses a large
negative charge (−0.288) compared to the sulfur center of
BzMeSH (−0.235), Table 1. However, the relative energies for
step 2 (i.e., the formation of sulfinic acid from sulfenic acid)
and step 3 (i.e., the formation of sulfonic acid from sulfinic
acid) of benzimidazole-based thione were almost comparable
with the relative energies for step 2 and step 3 of imidazole-
F
DOI: 10.1021/acs.inorgchem.8b03212
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Table 3. Optimized Energy of the Reactants, Products, and Transition States of the Thione Compounds in the Presence of
a
H2O2
B3LYP/6-31g(d)
(Eh)
relative energy
B3LYP/6-31g(d)
(Eh)
relative energy
ImMeSH (2)
(kcal mol−1
)
BzMeSH (7)
(kcal mol−1
)
ImMeSH·H2O2 (2·H2O2)
[2-2a]#
−815.2892932
−815.2474668
−815.3470265
−890.4697636
−890.3876830
−890.5413689
−965.6480714
−23.28
2.96
−59.51
−25.8
25.71
−70.73
−14.4
BzMeSH·H2O2 (7·H2O2)
[7-7a]#
−968.9458258
−968.9012214
−968.9974128
−1044.1195717
−1044.0336805
−1044.1914768
−1119.2977288
−22.29
5.69
−54.66
−26.03
25.91
−71.15
−14.04
ImMeSOH·H2O (2a·H2O)
ImMeSOH·H2O2 (2a·H2O2)
[2a-2b]#
BzMeSOH·H2O (7a·H2O)
BzMeSOH·H2O2 (7a·H2O2)
[7a-7b]#
ImMeSO2H·H2O (2b·H2O)
BzMeSO2H·H2O (7b·H2O)
ImMeSO2H·H2O2 (2b·
BzMeSO2H·H2O2 (7b·
H2O2)
[2b-2c]#
H2O2)
[7b-7c]#
−965.5779924
−965.7571894
−965.6591250
−965.7639423
29.57
−82.87
−21.34
−87.11
−1119.2266936
−1119.4060536
−1119.3017721
−1119.4147539
30.53
−82.02
−16.58
−87.48
ImMeSO3H·H2O (2c·H2O)
[2c·H2O]#
BzMeSO3H·H2O (7c·H2O)
[7c.H2O]#
MeImH·HSO4 (2d·HSO4)
MeBzH·HSO4 (7d·HSO4)
a
[2-2a]# - [ImMeSH - ImMeSOH]# , [2a-2b]# - [ImMeSOH - ImMeSO2H]#, [2b-2c]# - [ImMeSO2H - ImMeSO3H]#, [2c·H2O]# - [ImMeSO3H·H2O]#
[7-7a]# - [BzMeSH - BzMeSOH]#, [7a-7b]# - BzMeSOH - BzMeSO2H]# [7b-7c]# - [BzMeSO2H - BzMeSO3H]# [7c·H2O]# - [BzMeSO3H·H2O]#.
based thione−only 0.42 (for step 2) and 0.85 kcal mol−1 (for
step 3) less favorable compared to imidazole counterpart. Here
it should be noted that the activation energy barriers obtained
for the formation of the corresponding sulfenic, sulfinic, and
sulfonic acids from imidazole-based thione are higher than
those values reported for the endogenous thiol cysteine by
Bayse.27 Significantly, because of the absence of the N−H
group, we have noticed a slow oxidation of ImMeSMe (5) or
BzMeSMe (10) by H2O2, confirming the importance of the free
N−H group in the oxidation mechanism. A similar observation
was also noticed by Mugesh and co-worker in the case of the
oxidation of imidazole-based thiones by peroxynitrite (PN).28
Additionally, recent theoretical study by Bayse and co-workers
has suggested that the presence of both free N−H and N−Me
groups at the five-membered heterocycle of imidazole-based
thiones/selones increases the stability of the Fe(II) complexes
such as [Fe(OH2)5(thione)] and [Fe(OH2)4(thione)2].20b
DFT calculations of thiones (1−10) and their correspond-
ing Cu(I) complexes (12−21) show that the highest-occupied
molecular orbital (HOMO) of ImMeSH (2) is mostly localized
on the sulfur center (like a p-type lone pair localized on S),
Figures 9, S44, and S45.18 Similarly, the HOMO of ImMeSH-
perpendicular to the Cu−S bond, is further destabilized in
thione-bound Cu(I) complex.20a The HOMO of [TpmCu-
(2)]+ is, indeed, 0.84 eV higher in energy than the HOMO of
2. As a result, the energy gap between the HOMO of
[TpmCu(2)]+ and LUMO of H2O2, {ΔE(complex-H2O2)}, is
smaller than the energy gap between the HOMO of 2 and
LUMO of H2O2, {ΔE(thione-H2O2)}, Table 4, causing metal-
Table 4. Energy Gaps between the LUMO of H2O2 and
HOMO of Thiones (1−10) or HOMO Complexes (12−
a
21)
LUMO−HOMO
LUMO−HOMO
b
c
compd
(eV)
complex
(eV)
ImHSH (1)
5.71
5.61
5.64
5.67
5.54
6.03
5.95
5.97
5.99
5.89
[TpmCu(1)]+
(12)
4.79
4.77
4.78
4.91
4.94
4.88
4.85
4.87
5.07
5.50
ImMeSH (2)
ImBnSH (3)
[TpmCu(2)]+
(13)
[TpmCu(3)]+
(14)
ImCOOMeSH
(4)
[TpmCu(4)]+
(15)
ImMeSMe (5)
BzHSH (6)
BzMeSH (7)
BzBnSH(8)
[TpmCu(5)]+
(16)
[TpmCu(6)]+
(17)
[TpmCu(7)]+
(18)
[TpmCu(8)]+
(19)
BzCOOMeSH
[TpmCu(9)]+
(20)
(9)
BzMeSMe (10)
[TpmCu(10)]+
(21)
a
Geometry optimization was performed at B3LYP/6-31G(d) level of
b
c
theory. LUMO of H2O2 and HOMO of thiones (1−10). LUMO of
H2O2 and HOMO of Cu(I)-complexes (12−21).
Figure 9. HOMOs of ImMeSH (2) and [TpmCu(2)]+.
bound thione to be more susceptible to oxidation in
comparison to the free thione. This is in good agreement
with our experimental results. In fact, the HOMOs of all Cu(I)
complexes are found to be higher in energy than the HOMOs
of the corresponding thiones. ΔE(complex-H2O2) values for
imidazole-based Cu(I) complexes are in the order 16 > 15 >
12 > 14 > 13, and the same values for benzimidazole-based
Cu(I) complexes are in the order 21 > 20 > 17 > 19 > 18,
bound Cu(I) complex [TpmCu(2)]+ (13) is largely localized
on the sulfur center, rather than on metal. In fact, the p-type
lone pair of ImMeSH located perpendicular to the Cu−S bond is
the major contributor to this π-type antibonding HOMO.
Brumaghim and co-workers, in their previous work, have
reported that the upon coordination to the metal center the
lone pair of free thione (i.e., the HOMO of thione), which is
G
DOI: 10.1021/acs.inorgchem.8b03212
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
which are precisely the reverse order of their reactivity toward
H2O2.
Likewise, 18 and 21 produced 74% and 12%, respectively,
lower amounts of HO• radicals in comparison to 11,
suggesting that the ImMeSH (2) is more effective than BzMeSH
(7) in protecting Cu(I).
Role of the Thiones in Preventing ROS Generation
via Cu-Mediated Process. Our experimental results strongly
suggest that the metal-bound ImMeSH (2) of [TpmCu(2)]+
(13) and BzMeSH (7) of [TpmCu(7)]+ (18) is more reactive
toward H2O2 compared to metal-bound ImMeSMe (5) of
[TpmCu(5)]+ (16) and BzMeSMe (10) of [TpmCu(10)]+
(21), and, thus, complexes 13 and 18 are expected to produce
lower amounts of HO• radicals in comparison to complexes 16
and 21. To investigate this we have measured the HO• radical-
mediated degradation of methyl orange dye (MO) by
[TpmCu(CH3CN)]+ (11), in the presence of H2O2 at 21
°C, in which the Cu(I) ion is more exposed to H2O2 and, thus,
expected to produce more amounts of HO• radicals, Figure
10a. The amount of HO• radical production, therefore, can be
In order to reconfirm that the Cu(I)-bound ImMeSH (2) or
•
BzMeSH (7) produces lower amount of OH radicals in the
reaction of H2O2 in comparison to [TpmCu(CH3CN)]+ (11),
under identical reaction conditions, the nonfluorescent
terephthalic acid was employed as fluorescence probe, which
is known to react with the HO• radical to form highly
fluorescent 2-hydroxy terephthalic acid with excitation and
emission wavelengths of 315 and 425 nm, respectively.30
Complexes [TpmCu(2)]+ (13) and [TpmCu(5)]+ (16)
produced 88% and 39%, respectively, lower amounts of HO•
radicals in comparison to 11, whereas complexes [TpmCu-
(7)]+ (18) and [TpmCu(10)]+ (21) produced 79% and 15%,
respectively, lower amount of HO• radical in comparison to 11
(Figure 10b). These results again confirmed that the metal-
bound ImMeSH (2) is the most effective among other thiones in
protecting Cu(I) from H2O2.
The protein carbonylation assay was performed to
investigate the effect of 11, 13, 16, 18, and 21 on the
oxidation of protein in the presence H2O2 (Figure 10c). The
extent of oxidative damage of protein (BSA, 1 mg/mL) by
HO• radicals produced in the reaction between Cu(I)
complexes (100 μM) and H2O2 (1 mM) was measured
spectrophotometrically in quantifying the amount of protein
carbonyl formation by following the literature procedure.31
The absorbance band at 370 nm was followed to quantify the
cabonylation of protein.21 The maximum amount of protein
carbonylation was observed in the case of complex 11 (24.57
0.6 nmol/mg of protein, considered as 100%), whereas the
minimum amount of protein carbonylation was observed by
[TpmCu(2)]+ (13) (5.01
0.68 nmol/mg of protein, 20%
only), suggesting that the metal-bound ImMeSH (2) is the most
effective in protecting protein from Cu(I)-mediated oxidative
damage. The metal-bound ImMeSMe (5) of 16 was very slow in
reacting with H2O2 and thus allowed H2O2 to react with the
metal ion, Cu(I), which led to the formation of HO• radicals
and caused oxidative damage to protein. The amounts of
protein carbonylation by 18 and 21 followed a similar trend, as
shown in Figure 10c,d.
Finally we performed the DNA gel electrophoresis experi-
ment to investigate the effect of 11, 13, 16, 18, and 21 on
protecting DNA from Cu(I)-mediated oxidative damage in the
presence of H2O2 (Figure 10e), by following the previous work
by Brumaghim and co-workers.20,32 We have observed ca.
Figure 10. (a) Degradation of methyl orange (a), oxidation of
terephthalic acid (b), and BSA protein carbonylation (c) by Cu(I)
complexes 11, 13, 16, 18, and 21 in the presence of H2O2. (d)
Quantitative data showing the amounts of protein carbonylation by
Cu(I) complexes. (e) Agarose gel showing a reduction in oxidative
DNA damage. Lanes: (1) MW 1 kb ladder; (2) DNA; (3) DNA +
H2O2; (4) 11 (25 μM) + DNA + H2O2; (5) 11 (50 μM) + DNA +
H2O2; (6) 13 (25 μM) + DNA + H2O2; (7) 13 (50 μM) + DNA +
H2O2; (8) 18 (25 μM) + DNA + H2O2; (9) 18 (50 μM) + DNA +
H2O2; (10) 16 (25 μM) + DNA + H2O2; (11) 16 (50 μM) + DNA +
H2O2; (12) 21 (25 μM) + DNA + H2O2; (13) 21 (50 μM) + DNA +
H2O2.
93.03% DNA damage (6.96
3.03% supercoiled DNA and
93.03 3.03% nicked DNA) by complex 11 (50 μM) in the
presence of H2O2 (50 μM). On contrast, we noticed only ca.
10.70% DNA damage (89.30 1.45% supercoiled DNA and
10.70 1.45% nicked DNA) by complex 13 (50 μM), under
identical reaction conditions, suggesting that 13 produced a
lower amount of HO• radicals in comparison to 11. The
detailed experimental procedure and the percentage of DNA
inhibition by 11, 13, 16, 18, and 21 are mentioned in the
Experimental Section and in Table S8. The protein carbon-
ylation and DNA assays were performed in phosphate buffer,
and thus, the stability of the Cu(I) complexes (13, 16, 18 ,and
21) in aqueous phase was also examined by LC/MS as shown
in Figure S19, which suggests that these Cu(I) complexes are
stable in water for a few days. It has been reported that
ImMeSMe is more effective in inhibiting the Fe(II)-mediated
determined indirectly by monitoring the decreased concen-
trations of MO spectrophotometrically at λmax of 417 nm, as
reported in the literature.29 The amounts of MO degradation
by 11 in the presence of H2O2 were considered as 100% and
compared with the amounts of MO degradation obtained by
other thione-based Cu(I)-complexes, [TpmCu(2)]+ (13),
[TpmCu(5)]+ (16), [TpmCu(7)]+ (18), and [TpmCu(10)]+
(21), under identical reaction conditions (see Experimental
Section for more details). As expected, complexes 13 and 16
produced 88% and 36%, respectively, lower amounts of HO•
radicals compared to 11, showing that Cu(I)-bound ImMeSH
(2) of 13 is more effective in protecting the metal ion from
H2O2 compared to the Cu(I)-bound ImMeSMe (5) of 16.
H
DOI: 10.1021/acs.inorgchem.8b03212
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
mL, and the radical minimization efficiency was measured by UV−
visible spectroscopy at λmax = 417 nm.29 The UV−visible spectra for
the compounds are shown in Figure S46. The results shown are an
average of three trials, and the standard deviations are shown by error
bars in Figure 10a. The related data are given in Table S6.
DNA damage than Cu(I)-mediated, and thus, it is very
important to study the effect of these imidazole- and
benzimidazole-based thiones on preventing the other metal-
mediated, say Fe(II), oxidative damage of biomolecules.20
In conclusion, here we demonstrated the importance of
hydrogen bonding in the reactions between imidazole- or
benzimidazole-based thiones with H2O2, and its effect in
protecting the metal ion Cu(I) from H2O2-mediated oxidation.
Metal-bound ImMeSH (2) of [TpmCu(2)]+ (13) showed
remarkably high reactivity toward H2O2. [TpmCu(2)]+ (13) is
51- and 1571-fold more reactive toward H2O2 compared to
[TpmCu(5)]+ (16) and [TpmCu(10)]+ (21), respectively.
DFT calculations revealed that the initial adduct (ImMeSH·
H2O2) formation between ImMeSH (2) and H2O2 is
thermodynamically facilitated by 23.28 kcal mol−1 through
strong H-bonding between the N−H group of ImMeSH (2) and
the O atom of H2O2. Because of its high reactivity, ImMeSH (2)
showed an excellent ability to protect Cu(I) by acting as a
sacrificial antioxidant. Finally, we showed that the metal-bound
ImMeSH (2) of [TpmCu(2)]+ has an excellent ability to protect
DNA and protein from Cu(I)-mediated oxidative damage
through coordination to the Cu(I) center of [TpmCu-
(CH3CN)]+, whereas metal-bound N,N′-methyl substituted
ImMeSMe (5) or BzMeSMe (10) failed to protect Cu(I) from
H2O2-mediated oxidation. We believe that this work will help
in understanding the protective effect of ergothioneine
derivatives to metalloenzymes from reactive oxygen species,
particularly H2O2, and help in designing a better synthetic
thione-based antioxidant molecule.
Hydroxyl Radical (•OH) Detection Using Terephthalic Acid.
The •OH radical was generated by using a classical Fenton-type
reaction (Cu-complex/H2O2). Copper complexes (1 mM), 0.50 mM
terephthalic acid, and 50 mM H2O2 were added and mixed well in
acetonitrile solution. The amount of •OH radical scavenging was
detected by measuring the intensity of the characteristic peak of 2-
hydroxyterephthalic acid. The excitation wavelength was set at 315
nm, and the fluorescence spectra of the samples were collected at an
emission of 425 nm.30 The results shown are an average of three trials,
and the standard deviations are shown by error bars in Figure 10b.
The related data are given in Table S7.
DNA Damage Experiment. The DNA cleavage study was
performed using the previously reported method with little
modification.20,32 The pET15b plasmid of 578 bp size was used as
a model for the experiment. The conversion of covalently closed
circular double-strand supercoiled pET15b DNA (form I) to a relaxed
open circular form (form II) and linear form (III) was used to
investigate DNA strand breakage. The efficiency of the copper
complexes in minimizing the DNA damage was compared. Cu(I)
complexes (25 μM and 50 μM), plasmid DNA (pET15b, 578 bp),
and phosphate buffer (0.1 M, pH 7.4) were initially combined. The
reaction mixture was incubated for 5 min prior to H2O2 (50 μM)
addition. The agarose gel (0.8% agarose) was dissolved in 1× TAE
buffer and heated until boiling. As the boiling solution comes to 60
°C, ethidium bromide (EtBr, 20 μL in 250 mL agarose solution) was
added and poured into a gel casting apparatus. Gel was transferred
into an agarose gel running apparatus, and loading dye (6×, 3 μL) was
added into wells of the agarose gel. Gel was run at constant voltage
(90 V) for 60 min. Images were taken in the exposure of UV-light by
using gel-documentation instrument. Ethidium stains supercoiled
DNA less efficiently than nicked DNA, so supercoiled DNA band
intensities were multiplied by 1.24 prior to comparison.32a,34,35 The
percent of DNA damage inhibition was determined using the formula
1 − [%N/%B]*100, where %N = percentage of nicked DNA in the
thione containing Cu (I) complex (lanes 6−13) and %B = percentage
of nicked DNA in the complex 11 (lane 5). All percentages are
corrected for residual nicked DNA (nicked DNA observed in lane 2)
prior to calculation. Results are the average of at least three trials, and
standard deviations are indicated in Table S8.
Protein Carbonyl Assay. Protein carbonyl formation was studied
in the presence of Cu(I) complexes by following the literature
procedure.31 In general, protein BSA (1 mg/mL), Cu(I) complexes
(0.1 mM), H2O2 (1 mM) was mixed well and incubated for 1 h in 0.1
M phosphate buffer at 21 °C. After 1 h, 0.5 mL of 10 mM DNPH
(DNPH prepared in 2 N HCl) was added in the mixture. The
reaction mixture was further incubated at room temperature for 1 h,
followed by the addition of 0.5 mL of 20% trichloroacetic acid. The
sample was further incubated at ice for 20 min and centrifuged at
12 000 rpm for 10 min. The protein pellet was washed three times
with 3 mL of an ethanol/ethyl acetate mixture (1:1, v/v) and
dissolved in 1 mL of 6 M guanidine (pH 2.3). The peak absorbance at
370 nm was measured to quantitate protein carbonyls. Data were
expressed as nanomoles of carbonyl groups per milligram of protein.
The experiment was repeated three times, and the standard deviation
was shown as error bars in Figure 10c. The data are given in Figure
10d.
EXPERIMENTAL SECTION
■
General Experimental. Imidazole, benzimidazole, and tereph-
thalic acid were obtained from Alfa Aesar. Tetrakis(acetonitrile)-
copper(I)tetrafluoroborate, sulfur powder and H2O2 (30 wt % in
H2O) were obtained from Sigma-Aldrich, and other chemicals were
obtained from local companies. Tris(pyrazolyl)methane was prepared
by following the literature procedure.33 All the experiments were
carried out under anhydrous and anaerobic conditions using standard
Schlenk techniques for the synthesis. H (400 MHz) and 13C (100
1
MHz) NMR spectra were obtained on a Bruker Advance 400 NMR
spectrometer using the solvent as an internal standard for 1H and 13C.
Chemical shifts (1H, 13C) are cited with respect to tetramethylsilane
(TMS). Synthesis of the Cu(I) complexes [TpmCu(1)]+ (12),
[TpmCu(2)]+ (13), [TpmCu(3)]+ (14), [TpmCu(4)]+ (15),
[TpmCu(5)]+ (16), [TpmCu(6)]+ (17), [TpmCu(7)]+ (18),
[TpmCu(8)]+ (19), [TpmCu(9)]+ (20), and [TpmCu(10)]+ (21)
are mentioned in the Supporting Information.
Procedure for Kinetic Studies. The cleavage of the C−S bond
of thiones and their corresponding Cu-complexes in the presence of
H2O2 at 37 °C was followed by UV−visible spectroscopy (Figures S5
and S6). In general, H2O2 was added to a solution of thione (0.01 M)
in CH3CN, in a 1:3 molar ratio, and was stirred continuously. An
aliquot of 20 μL was taken from the reaction mixture at various time
intervals and was further diluted for recording UV spectra. The
decrease in the absorbance band at 265 nm (compounds 1−5 and
complexes 12−16) and 310 nm (compounds 6−10 and complexes
17−21) in the presence of H2O2 was monitored. The initial value at 0
min was considered as 100% of thione (0.01 M). The obtained results
are an average of three trials, and the standard deviations are given in
Table 1 (compounds) and Table 2 (complexes) as well as shown by
error bars in Figure 3 (compounds 1−10) and Figure 5 (complexes
(12−21).
X-ray Crystal Analysis. Crystal structures of compounds were
determined by measuring X-ray diffraction data on D8 Venture
Bruker AXS single crystal X-ray diffractometer equipped with CMOS
PHOTON 100 detector having monochromatised microfocus sources
(Mo−Kα = 0.71073 Å). Single crystals of complex 15
(CCDC1854405), complex 18 (CCDC1854408), complex 19
(CCDC1854409), complex 20 (CCDC1854406), complex 21
(CCDC1854407), compound MeBzH·HSO4 (CCDC1854410), com-
pound BnBzH·HSO4 (CCDC1854412), compound MeBzMe·HSO4
Hydroxyl Radical (•OH) Detection using Methyl Orange.
The solution containing MO (150 μM), H2O2 (37.5 mM), and
complex (4.5 mM) was stirred at 37 °C for 3 min in acetonitrile
solution. A total of 400 μL of the reaction mixture was diluted to 2
I
DOI: 10.1021/acs.inorgchem.8b03212
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(CCDC1854411), compound 4 (CCDC1867238), compound 8
(CCDC1866261), and compound 9 (CCDC1866260) suitable for X-
ray diffraction studies were obtained from a slow evaporation process
using various solvents. All the crystal data were collected at room
temperature. Structures were solved using SHELX program
implemented in APEX3.36−40 The non-H atoms were located in
successive difference Fourier syntheses and refined with anisotropic
thermal parameters. All the hydrogen atoms were placed at the
calculated positions and refined using a riding model with appropriate
HFIX commands. The program Mercury was used for molecular
packing analysis.41 In addition, the anisotropic displacement
parameters for disordered fluorine atoms were fixed using EADP
restraint.42
Computational Details. All the calculations were carried out
using the B3LYP level of theory as implemented in the Gaussian 09
package.43 The 6-31G(d) basis set was used for all atoms (except Cu),
whereas the Stuttgart−Dresden basis set (SDD) was used for the Cu
atom with the respective relativistic effective core potential.44
Frequency calculations of all the optimized structures were performed
to ensure that the optimized structures were the local energy minima
structure without any imaginary frequencies. The NBO Version 3.1
program implemented in Gaussian 09 was used to perform NPA.45
electrochemical comparison of novel iron (II) complexes with thione
and selone ligands. Dalton Trans. 2016, 45, 4697−4711.
(2) Shi, H.; Hudson, L. G.; Liu, K. J. Oxidative stress and apoptosis
in metal ion-induce carcinogenesis. Free Radical Biol. Med. 2004, 37,
582−593.
(3) Jomova, K.; Valko, M. Importance of iron chelation in free
radical-induced oxidative stress and human disease. Curr. Pharm. Des.
2011, 17, 3460−3473.
(4) Jomova, K.; Vondrakova, D.; Lawson, M.; Valko, M. Metals,
oxidative stress and neurodegenerative disorders. Mol. Cell. Biochem.
2010, 345, 91−104.
(5) Valko, M.; Izakovic, M.; Mazur, M.; Rhodes, C. J.; Telser, J. Role
of oxygen radicals in DNA damage and cancer incidence. Mol. Cell.
Biochem. 2004, 266, 37−56.
(6) Haber, A.; Aviram, M.; Gross, Z. Variables that influence cellular
uptake and cytotoxic/cytoprotective effects of macrocyclic iron
complexes. Inorg. Chem. 2012, 51, 28−30.
(7) Valko, M. M. H. C. M.; Morris, H.; Cronin, M. T. D. Metals,
toxicity and oxidative stress. Curr. Med. Chem. 2005, 12, 1161−1208.
(8) Brewer, T. F.; Garcia, F. J.; Onak, C. S.; Carroll, K. S.; Chang, C.
J. Chemical approaches to discovery and study of sources and targets
of hydrogen peroxide redox signaling through NADPH oxidase
proteins. Annu. Rev. Biochem. 2015, 84, 765−790.
ASSOCIATED CONTENT
* Supporting Information
(9) Sies, H. Hydrogen peroxide as a central redox signaling molecule
in physiological oxidative stress: oxidative eustress. Redox Biol. 2017,
11, 613−619.
(10) Stone, J. R.; Yang, S. Hydrogen peroxide: a signaling messenger.
Antioxid. Redox Signaling 2006, 8, 243−270.
(11) Stohs, S. J.; Bagchi, D. Oxidative mechanisms in the toxicity of
metal ions. Free Radic. Free Radical Biol. Med. 1995, 18, 321−336.
(12) (a) De Flora, S.; Izzotti, A. Mutagenesis and cardiovascular
diseases: molecular mechanisms, risk factors, and protective factors.
Mutat. Res., Fundam. Mol. Mech. Mutagen. 2007, 621, 5−17.
(b) Evans, M. D.; Dizdaroglu, M.; Cooke, M. S. OxidativeDNA
damage and disease: induction, repair and significance. Mutat. Res.,
Rev. Mutat. Res. 2004, 567, 1−61.
(13) (a) Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T.; Mazur,
M.; Telser, J. Free radicals and antioxidants in normal physiological
functions and human disease. Int. J. Biochem. Cell Biol. 2007, 39, 44−
84.
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b03212.
Synthesis methods; HR-ESIMS analysis of compounds;
1H, 13C of copper complexes; DFT calculations;
optimized geometries and coordinates of the optimized
structures (PDF)
Accession Codes
CCDC 1854405−1854412, 1866260−1866261, and 1867238
contain the supplementary crystallographic data for this paper.
These data can be obtained free of charge via www.ccdc.ca-
m.ac.uk/data_request/cif, or by emailing data_request@ccdc.
cam.ac.uk, or by contacting The Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:
+44 1223 336033.
(14) (a) Que, E. L.; Domaille, D. W.; Chang, C. J. Metals in
neurobiology: probing their chemistry and biology with molecular
imaging. Chem. Rev. 2008, 108, 1517−1549. (b) Gaggelli, E.;
Kozlowski, H.; Valensin, D.; Valensin, G. Copper homeostasis and
neurodegenerative disorders (Alzheimer’s, prion, and Parkinson’s
diseases and amyotrophic lateral sclerosis). Chem. Rev. 2006, 106,
1995−2044.
AUTHOR INFORMATION
Corresponding Author
*E-mail: Gouriprasanna.Roy@snu.edu.in.
■
̈
(15) Ey, J.; Schomig, E.; Taubert, D. Dietary sources and antioxidant
ORCID
effects of ergothioneine. J. Agric. Food Chem. 2007, 55, 6466−6474.
(16) (a) Moncaster, J. A.; Walsh, D. T.; Gentleman, S. M.; Jen, L. S.;
Aruoma, O. I. Ergothioneine treatment protects neurons against N-
methyl-D-aspartate excitotoxicity in an in vivo rat retinal model.
Neurosci. Lett. 2002, 328, 55−59. (b) Rahman, I.; Gilmour, P. S.;
Jimenez, L. A.; Biswas, S. K.; Antonicelli, F.; Aruoma, O. I.
Ergothioneine inhibits oxidative stress-and TNF-α-induced NF-κB
activation and interleukin-8 release in alveolar epithelial cells. Biochem.
Biophys. Res. Commun. 2003, 302, 860−864.
Gouriprasanna Roy: 0000-0002-0047-3575
Author Contributions
R.K.R. carried out most of the experiments mentioned in this
paper. R.K.R., R.D., and R.K. have solved crystal structures.
R.K.R., A.C., and B.K. performed DNA and PL experiments.
All authors approved the final version of the manuscript for
submission.
Funding
(17) (a) Servillo, L.; D’Onofrio, N.; Balestrieri, M. L. Ergothioneine
antioxidant function: from chemistry to cardiovascular therapeutic
potential. J. Cardiovasc. Pharmacol. 2017, 69, 183−191. (b) Franzoni,
F.; Colognato, R.; Galetta, F.; Laurenza, I.; Barsotti, M.; Di Stefano,
R.; Bocchetti, R.; Regoli, F.; Carpi, A.; Balbarini, A.; Migliore, L.;
Santoro, G. An in vitro study on the free radical scavenging capacity of
ergothioneine: comparison with reduced glutathione, uric acid and
trolox. Biomed. Pharmacother. 2006, 60, 453−457.
This work is supported by Shiv Nadar University (SNU), Shiv
Nadar Foundation (SNF), and Indo French Centre for the
promotion of advanced research (IFCPAR/CEFIPRA, Project
Number. 5605-1).
Notes
The authors declare no competing financial interest.
(18) Kimani, M. M.; Bayse, C. A.; Stadelman, B. S.; Brumaghim, J. L.
Oxidation of biologically relevant chalcogenones and their Cu (I)
complexes: insight into selenium and sulfur antioxidant activity. Inorg.
Chem. 2013, 52, 11685−11687.
REFERENCES
■
(1) Stadelman, B. S.; Kimani, M. M.; Bayse, C. A.; McMillen, C. D.;
Brumaghim, J. L. Synthesis, characterization, DFT calculations, and
J
DOI: 10.1021/acs.inorgchem.8b03212
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(19) Kimani, M. M.; Brumaghim, J. L.; VanDerveer, D. Probing the
antioxidant action of selenium and sulfur using Cu (I)-chalcogenone
tris (pyrazolyl) methane and-borate complexes. Inorg. Chem. 2010, 49,
9200−9211.
(20) (a) Zimmerman, M. T.; Bayse, C. A.; Ramoutar, R. R.;
Brumaghim, J. L. Sulfur and selenium antioxidants: Challenging
radical scavenging mechanisms and developing structure−activity
relationships based on metal binding. J. Inorg. Biochem. 2015, 145,
30−40. (b) Dreab, A.; Brewer, M. I.; Bayse, C. A. DFT modeling of
the prevention of Fe (II)-mediated redox damage by imidazole-based
thiones and selones. J. Inorg. Biochem. 2019, 193, 9−14.
(b) Battin, E. E.; Brumaghim, J. L. Metal specificity in DNA damage
prevention by sulfur antioxidants. J. Inorg. Biochem. 2008, 102, 2036−
2042.
(33) Hallett, A. J.; Anderson, K. M.; Connelly, N. G.; Haddow, M. F.
Bonding modes, structures and fluxionality in rhodium and iridium
tris (3, 5-dimethylpyrazolyl) methane diene complexes. Dalton Trans.
2009, No. 21, 4181−4189.
(34) Lloyd, R. S.; Haidle, C. W.; Robberson, D. L. Bleomycin-
specific fragmentation of double-stranded DNA. Biochemistry 1978,
17, 1890−1896.
(35) Hertzberg, R. P.; Dervan, P. B. Cleavage of double helical DNA
by methidium-propyl-EDTA-iron (II). J. Am. Chem. Soc. 1982, 104,
313−315.
(36) Crystallographic data of all the crystal structures mentioned in
this paper (CCDC 1854405, CCDC 1854406, CCDC 1854407,
CCDC 1854408, CCDC 1854409, CCDC 1854410, CCDC 1854411
and CCDC 1854412) can be obtained free of charge from The
Cambridge Crystallographic Data Centre (CCDC) via www.ccdc.
cam.ac.uk/data_request/cif.
(37) Bruker Support APEX3, SAINT, and SADABS: Software for
data reduction, absorption correction and structure solution; Bruker
AXS Inc.: Madison, WI, USA, 2015; http://www.brukersupport.com/
.
(21) Zhu, B. Z.; Mao, L.; Fan, R. M.; Zhu, J. G.; Zhang, Y. N.; Wang,
J.; Kalyanaraman, B.; Frei, B. Ergothioneine prevents copper-induced
oxidative damage to DNA and protein by forming a redox-inactive
ergothioneine-copper complex. Chem. Res. Toxicol. 2011, 24, 30−34.
(22) Doerge, D. R.; Cooray, N. M. Synthesis of N-substituted
benzimidazole-2-thiones. Synth. Commun. 1991, 21, 1789−1795.
(23) Banerjee, M.; Karri, R.; Chalana, A.; Das, R.; Rai, R. K.; Rawat,
K. S.; Pathak, B.; Roy, G. Protection of Endogenous Thiols against
Methylmercury with Benzimidazole-Based Thione by Unusual
Ligand-Exchange Reactions. Chem. - Eur. J. 2017, 23, 5696−5707.
(24) Doerge, D. R.; Decker, C. J.; Takazawa, R. S. Chemical and
enzymic oxidation of benzimidazoline-2-thiones: a dichotomy in the
mechanism of peroxidase inhibition. Biochemistry 1993, 32, 58−65.
(25) Isab, A. A.; Perzanowski, H. P. 1H, 13C and 199Hg NMR Studies
of the Complexation of HgCl2 by Imidazolidine-2-Thione and its
Derivatives. J. Coord. Chem. 1990, 21, 247−252.
(38) Sheldrick, G. M. SHELXTL Version 2014/7: Programs for the
Determination of Small and Macromolecular Crystal Structures by Single
̈
Crystal X-ray and Neutron Diffraction; University of Gottingen,
̈
Gottingen, Germany, 2014; http://shelx.uni-ac.gwdg.de/SHELX/
index.php.
(26) (a) Meyer, S.; Demeshko, S.; Dechert, S.; Meyer, F. Synthesis,
(39) Sheldrick, G. M. A short history of SHELX. Acta Crystallogr.,
Sect. A: Found. Crystallogr. 2008, 64, 112−122.
(40) Farrugia, L. J. ORTEP-3 for Windows - a version of ORTEP-III
with a Graphical User Interface (GUI). J. Appl. Crystallogr. 1997, 30,
565.
(41) Macrae, C. F.; Bruno, I. J.; Chisholm, J. A.; Edgington, P. R.;
McCabe, P.; Pidcock, E.; Rodriguez-Monge, L.; Taylor, R.; Streek, J.
V.; Wood, P. A. Mercury CSD 2.0 - New Features for the
Visualization and Investigation of Crystal Structures. J. Appl.
Crystallogr. 2008, 41, 466−470.
(42) Muller, P.; Herbst-Irmer, R.; Spek, A. L.; Schneider, T. R.;
Sawaya, M. R. Crystal Structure Refinement; Oxford University Press:
Oxford, U.K., 2006.
(43) Frisch, M. J., et al. Gaussian 09, Revision B.01; Gaussian Inc.:
Wallingford, CT, 2009. The full citation is given in the Supporting
Information.
(44) Figgen, D.; Rauhut, G.; Dolg, M.; Stoll, H. Energy-consistent
pseudopotentials for group 11 and 12 atoms: adjustment to multi-
configuration Dirac−Hartree−Fock data. Chem. Phys. 2005, 311,
227−244.
(45) Glendening, E. D.; Reed, A. E.; Carpenter, J. E.; Weinhold, F.
NBO, version 3.1; University of Wisconsin: Madison, WI.
̈
structure and Mossbauer characterization of polymeric iron(II)
complexes with bidentate thiourea ligands. Inorg. Chim. Acta 2010,
363, 3088−3092. (b) Isaac, J. A.; Mansour, A. T.; David, R.; Kochem,
́
A.; Philouze, C.; Demeshko, S.; Meyer, F.; Reglier, M.; Simaan, A. J.;
Caldarelli, S.; Yemloul, M.; Jamet, H.; Thibon-Pourret, A.; Belle, C.
Tetranuclear and dinuclear phenoxido bridged copper complexes
based on unsymmetrical thiosemicarbazone ligands. Dalton Trans.
2018, 47, 9665−9676.
(27) Bayse, C. A. Transition states for cysteine redox processes
modeled by DFT and solvent-assisted proton exchange. Org. Biomol.
Chem. 2011, 9, 4748−4751.
(28) Bhabak, K. P.; Mugesh, G. Antithyroid Drugs and their
Analogues Protect Against Peroxynitrite-Mediated Protein Tyrosine
NitrationA Mechanistic Study. Chem. - Eur. J. 2010, 16, 1175−
1185.
(29) (a) Shi, L. L.; Zheng, T. R.; Li, M.; Qian, L. L.; Li, B. L.; Li, H.
Y. A series of five-coordinated copper coordination polymers for
efficient degradation of organic dyes under visible light irradiation.
RSC Adv. 2017, 7, 23432−23443. (b) Wen, T.; Zhang, D. X.; Zhang,
J. Two-dimensional copper (I) coordination polymer materials as
photocatalysts for the degradation of organic dyes. Inorg. Chem. 2013,
52, 12−14. (c) Zhao, S.; Zhang, Y. Q.; Zheng, T. R.; Shi, L. L.; Li, B.
L.; Zhang, Y. Synthesis, structure and photocatalytic properties of an
unusual tetranuclear copper (II) coordination polymer. Inorg. Chem.
Commun. 2016, 73, 134−137.
(30) (a) Han, L. J.; Kong, Y. J.; Yan, T. J.; Fan, L. T.; Zhang, Q.;
Zhao, H. J.; Zheng, H. G. A new five-coordinated copper compound
for efficient degradation of methyl orange and Congo red in the
absence of UV−visible radiation. Dalton Trans. 2016, 45, 18566−
18571. (b) Nishikiori, H.; Sato, T.; Kubota, S.; Tanaka, N.; Shimizu,
Y.; Fujii, T. Preparation of Cu-doped TiO2 via refluxing of alkoxide
solution and its photocatalytic properties. Res. Chem. Intermed. 2012,
38, 595−613.
(31) Kocha, T.; Yamaguchi, M.; Ohtaki, H.; Fukuda, T.; Aoyagi, T.
Hydrogen peroxide-mediated degradation of protein: different
oxidation modes of copper-and iron-dependent hydroxyl radicals on
the degradation of albumin. Biochim. Biophys. Acta, Protein Struct. Mol.
Enzymol. 1997, 1337, 319−326.
(32) (a) Battin, E. E.; Zimmerman, M. T.; Ramoutar, R. R.; Quarles,
C. E.; Brumaghim, J. L. Preventing metal-mediated oxidative DNA
damage with selenium compounds. Metallomics 2011, 3, 503−512.
K
DOI: 10.1021/acs.inorgchem.8b03212
Inorg. Chem. XXXX, XXX, XXX−XXX