Protection of Endogenous Thiols against Methylmercury with Benzimidazole-Based Thione by Unusual Ligand-Exchange Reactions

Article abstract of DOI:10.1002/chem.201605238

Organomercurials, such as methylmercury (MeHg⁺), are among the most toxic materials to humans. Apart from inhibiting proteins, MeHg⁺ exerts its cytotoxicity through strong binding with endogenous thiols cysteine (CysH) and glutathione (GSH) to form MeHgCys and MeHgSG complexes. Herein, it is reported that the N,N-disubstituted benzimidazole-based thione 1 containing a N?CH₂CH₂OH substituent converts MeHgCys and MeHgSG complexes to less toxic water-soluble HgS nanoparticles (NPs) and releases the corresponding free thiols CysH and GSH from MeHgCys and MeHgSG, respectively, in solution by unusual ligand-exchange reactions in phosphate buffer at 37 °C. However, the corresponding N-substituted benzimidazole-based thione 7 and N,N-disubstituted imidazole-based thione 3, in spite of containing a N?CH₂CH₂OH substituent, failed to convert MeHgX (X=Cys, and SG) to HgS NPs under identical reaction conditions, which suggests that not only the N?CH₂CH₂OH moiety but the benzimidazole ring and N,N-disubstitution in 1, which leads to the generation of a partial positive charge at the C2 atom of the benzimidazole ring in 1:1 MeHg-conjugated complex of 1, are crucial to convert MeHgX to HgS NPs under physiologically relevant conditions.

Full text of DOI:10.1002/chem.201605238

A Journal of
Accepted Article
Title: Protection of Endogenous Thiols Against Methylmercury by
Benzimidazole-based Thione via Unusual Ligand Exchange
Reactions
Authors: Gouriprasanna Roy, Mainak Banerjee, Ramesh Karri, Ashish
Chalana, Ranajit Das, Rakesh Kumar Rai, kuber Singh
Rawat, and Biswarup Pathak
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Protection of Endogenous Thiols Against Methylmercury by
Benzimidazole-based Thione via Unusual Ligand Exchange
Reactions**
Mainak Banerjee,[a] Ramesh Karri,[a] Ashish Chalana,[a] Ranajit Das,[a] Rakesh Kumar Rai,[a] Kuber
Singh Rawat,[b] Biswarup Pathak[b] and Gouriprasanna Roy[a]*
Abstract: Organomercurials, such as methylmercury (MeHg+), are
among the most toxic materials to humans. Apart from inhibiting
concentration of GSH, the most abundant intracellular thiol
containing tripeptide, by forming methylmercury-GSH complex
proteins, MeHg+ exerts its cytotoxicity through strong binding with
(MeHgSG) and, as a result it increases the production of
endogenous thiols cysteine (CysH) and glutathione (GSH) to form
reactive oxygen species (ROS) in various tissues including in
MeHgCys and MeHgSG complexes. We report for the first time that
brain leading to DNA damage, protein oxidation, lipid
the N,N-disubstituted benzimidazole-based thione
1 having N-
peroxidation and cell death.[2b,3]
CH2CH2OH substituent exhibits remarkable effect in converting
MeHgCys and MeHgSG complexes to less toxic water-soluble HgS
nanoparticles (NPs) and releases the corresponding free thiol CysH
and GSH from MeHgCys and MeHgSG respectively in solution via
unusual ligand exchange reaction in phosphate buffer at 37 °C.
Whereas the corresponding N-substituted benzimidazole-based
thione 7 and N,N-disubstituted imidazole-based thione 3, in spite of
having N-CH2CH2OH substituent, failed to convert MeHgX (X = Cys,
and SG) to HgS NPs under identical reaction conditions, suggesting
that not only N-CH2CH2OH moiety but the benzimidazole ring and
N,N-disubstitution in 1, which leads to the generation of partial
positive charge at the C2 atom of benzimidazole ring in 1:1 MeHg-
conjugated complex of 1, are crucial to convert MeHgX to HgS NPs
at physiologically relevant conditions.
On the other hand, several microorganisms have been
reported as being MeHg+ tolerant due to their ability to convert
toxic MeHg+ to harmless and insoluble metacinnabar (HgS), by
producing H2S.[6,7] It has also been reported that the H2S-
producing enzyme cystathionine -synthase in mammalian cells
reduces MeHg+ cytotoxicity through the production of (MeHg)2S,
which has little cytotoxicity compared with MeHg+.[8] The
unstable (MeHg)2S gradually decomposes to Me2Hg, CH4, and
HgS.[6,9] Therefore, development of a synthetic molecule which
can convert these highly toxic MeHg+ conjugates, MeHgCys and
MeHgSG, to HgS nanoparticles (NPs) and releases free thiols
CysH and GSH at physiologically relevant conditions has great
significance. Recently we have reported that the N-
methylimidazole-based thione
3 has an ability to convert
MeHgOH to insoluble black HgS NPs at 37 C.[10] However,
unfortunately, compound 3 failed to convert MeHgCys or
MeHgSG to HgS NPs at physiological temperature (37 C).
Introduction
Methylmercury (MeHg+) is a highly neurotoxic environmental
contaminant found in mercury-polluted wetlands, coastal
sediments, and mining areas worldwide.[1] High toxicity of MeHg+
is mainly due to its strong affinity towards endogenous thiols
such as L-cysteine (L-CysH) and glutathione (GSH) and thiol or
selenol containing proteins found in the biological system.[2,3]
The toxicity of MeHg+ varies according to its chemical form and
exposure amount.[4] MeHg+ binds with cysteine residue to form
MeHgCys complex which can easily cross the cellular
membranes through a transporter, called LAT1, and thereby it
acts as a driving force in transporting toxic MeHg+ into various
tissues.[5] Moreover, MeHg+ is known to decrease the
Figure 1. Chemical Structures of imidazole and benzimidazole-based thiones
and selone.
Herein we describe that the presence of N-CH2CH2OH moiety in
compound 1 makes it an excellent molecule with remarkable
ability in converting highly toxic RHgCys and RHgSG (R = Me,
p-C6H4CO2H (Ar)) to less toxic water-soluble HgS NPs under
physiologically relevant conditions via efficient desulfurization
process. Moreover, we observed the release of free thiols (CysH
or GSH) upon addition of compound 1 or 2 into the solution of
RHgCys or RHgSG. Some of the free thiols, released during the
degradation process, utilized to produce water-soluble HgS or
HgSe NPs through binding at the surface of the corresponding
insoluble HgS or HgSe NPs. Surprisingly, compound 7, in spite
of having N-CH2CH2OH substituent, failed to convert RHgX (X =
Cys, and SG) to HgS NPs under identical reaction conditions. In
addition, we show that the replacement of imidazole moiety with
a benzimidazole moiety remarkably alters the reactivity of 1
towards the detoxification of various other organomercurials
[a]
[a]
[**]
M. Banerjee, R. Karri, A. Chalana, R. Das, R. K. Rai, Dr. G. Roy*
Department of Chemistry, School of Natural Sciences, Shiv Nadar
University, NH91, Dadri, Gautam Buddha Nagar, UP 201314, India
E-mail: gouriprasanna.roy@snu.edu.in
K. S. Rawat, Dr. B. Pathak
Discipline of Chemistry and Discipline of Metallurgy Engineering and
Materials Science, Indian Institute of Technology (IIT) Indore,
Indore, MP 453552, India
This work is supported by SNU, IIT Indore, SERB (SB/S1/IC-
44/2013) and CSIR [01(2723)/13/EMR(II)], Govt. of India. We thank
to Prof. Rupamanjari Ghosh (VC, SNU) for continuous support and
encouragement. MB carried out most of the works. RK, AC, MB, RD
and RKR synthesized compounds. RK performed NMR studies. GR,
MB, KSR and BP performed DFT calculations of Scheme 1.
Supporting information for this article is available on the WWW
under http://dx.doi.org/.
[#]
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RHgX (where R = Me, Et, and Ar; X = Cl, and OH) under
identical reaction conditions.
Results and Discussion
The N,N-disubstituted thiones (1, 4, and 5) and selone 2 which
were required for this study were synthesized by in situ
generation of reactive carbenes from benzimidazolium salts.
Whereas N-substituted thiones 7 and 8[11] were synthesized after
protecting the soft sulfur nucleophilic centre of 6 with trityl
chloride followed by N-substitution of benzimidazole ring
(Supporting Information). When ArHgCys (30 mM, m/z
444.0199) was treated with compound 1 in 1:2 molar ratio in
phosphate buffer (pH 9), the HPLC chromatogram showed the
formation of 1:1 ArHg-conjugated complex 9 (m/z 531.0677) via
ligand exchange reaction, which is in equilibrium with the
starting material ArHgCys in solution (9:ArHgCys = 3:1), as
shown in Figure 2a and 2b. However, to our surprise, the above
colorless reaction solution gradually turned black over time
(initial rate = 21.9 ± 6.4 x10-5 Mh-1) compared to the control
reaction (without 1) at 37 °C. The complex 9 was unstable in
solution at 37 °C and, therefore, degraded gradually over time to
form water-soluble black HgS NPs, 11 and Ar2Hg. Interestingly,
to maintain the equilibrium between ArHgCys and 9 in solution,
free thione 1 present in the reaction mixture further reacted with
ArHgCys to produce more amounts of 9, which was
subsequently degraded to HgS NPs over time. As a result, the
concentrations of ArHgCys and 9 were decreased over time as
the reaction was allowed to proceed, Figure 2b. The black HgS
NPs (9 mg) were isolated by centrifugation and characterized by
TEM, SEM, EDX, and IR. FT-IR spectra and EDX analysis
showed that some amounts of cysteine and 11 formed in the
reaction are bound to HgS NPs (Figures S6-S8, and S11,
Supporting Information). The FT-IR spectrum of isolated HgS
NPs showed the presence of amide and C=O bands. Ar2Hg and
11 were detected by NMR and mass spectrometry. Ar2Hg
showed 199Hg NMR peak at -824 ppm. The unbound ketone 11
(yield 32%) which remained free in solution was isolated by
column chromatography and characterized by various
techniques including single crystal X-ray studies (Figure 2g).
The amount of free cysteine (17% of ArHgCys) in solution (not
bound to HgS NPs) was determined by Ellman’s reagent after
15 days (Supporting Information, Figure S9). Similarly, the
treatment of MeHgCys with 1 (1:2 molar ratio) under identical
condition (at pH 9) yielded the formation of water-soluble black
HgS NPs (7 mg), Me2Hg and 11 (29% free 11) after 15 days.
Me2Hg was detected by 199Hg NMR spectroscopy of peak value
at -113 ppm (Figure 2f and Supporting Information).[12] The HgS
NPs obtained in the degradation of 9 and 10 were spherical
shaped with an average size of ca. 4 and 7 nm respectively. The
above reactions were also performed in water in the presence of
weak base, instead of in phosphate buffer of pH 9. Complexes 9
and 10 were unstable in the presence of weak base NaHCO3 or
K2HPO4 (1:2 molar ratio) and gradually degraded into water-
soluble black HgS NPs.
Figure 2. (a) Synthetic scheme for the degradation of RHgCys by 1. (b) Graph
showing the degradation of 9 and ArHgCys over time ( = 254 nm) in the
reaction of ArHgCys/1. (c) Mass spectra of 9 and Ar2Hg. (d) Reaction vials
showing the formation of black water-soluble HgS NPs in the reaction of
ArHgCys/1 in PBS buffer at 37 C. The 199Hg NMR spectra of 10 (e) and
Me2Hg (proton coupled) (f). (g) ORTEP diagram of 11. EDX (h) and TEM (i)
images of HgS NPs obtained in the reaction of ArHgCys/1. (j) Synthetic
scheme and graph showing equilibrium between ArHgCys and 12 over time (
= 254 nm).
On the contrary, when ArHgCys (30 mM) was treated with the
corresponding imidazole-based thione 3 (1:2 molar ratio), the
formation of 12 was observed almost exclusively (12:ArHgCys =
97:3), which was highly stable at 37 °C and the formation of HgS
NPs was not observed under identical reaction conditions,
Figure 2j. Surprisingly, not only with CysH, we found that
compound 1 successfully compete with another endogenous
thiol containing tripeptide GSH for RHg+ (R = Me, Ar) and
convert them into HgS NPs in phosphate buffer (pH 9) at 37 °C
(Figure 3). The treatment of ArHgSG (30 mM, m/z 630.0868)
with 1 in 1:2 molar ratio in phosphate buffer (pH 9), afforded the
formation of 1:1 ArHg-conjugated complex 13 (m/z 531.0677)
via ligand exchange reaction, which is in equilibrium with the
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starting material ArHgSG in solution. However, the above
colorless reaction solution gradually turned black over time
compared to the control reaction (without 1) at 37 °C. Careful
analysis of the reaction products confirmed the formation of
water-soluble HgS NPs (12 mg), 11 (yield of free 11: 32%) and
Ar2Hg. Similarly, the treatment of MeHgSG with compound 1
yielded the formation of water-soluble HgS NPs (11 mg), 11,
Me2Hg, and free GSH (Supporting Information).
initial rate of degradation of the corresponding sulfur analogue 9
(vide supra). Significantly, in all these ligand exchange reactions
with compound 2, mentioned in Figure 3f, we have noticed the
formation of more amounts of free thiols CysH (~ 25%) and GSH
(35%) from RHgX (X = Cys, and GS) in solution compared with
the reactions between RHgX and compound 1. The amounts of
free CysH and GSH were also detected by mass spectrometry
(Figure 3d and 3e). EDX analysis along with FT-IR spectrum of
Hg(SSe) NPs confirmed the presence of S element (which was
either from CysH or GSH) in the NPs (Figure 3h, Supporting
Information). During the conversion of RHgCys or RHgSG to
Hg(SSe) NPs some amounts of thiol molecules attached with
NPs to make them water-soluble and some amounts of thiol
molecules remained free in the solution. The presence of Hg–
Se–S species was indeed found in plasma of rabbits treated with
sodium selenite and HgCl2 where GSH molecules were attached
at the surface of the HgSe core.[13] The presence of Hg(S0.3Se0.7
)
species was also detected in the liver of striped dolphin.[14]
Studies suggest that the selenoprotein P (SeIP) plays an
important role in the detoxification of mercury in plasma through
solubilizing (HgSe)n polymer in bloodstream of rabbits and rats
by forming soluble {(HgSe)n}-SeIP complex which helps to clear
mercury from plasma.[15]
Figure 3. (a) Scheme for the degradation of ArHgSG by 1. (b) Reaction vials
and TEM image showing the formation of HgS NPs in the reaction of
ArHgSG/1. (c) Reaction vials and TEM image showing the formation of HgSSe
NPs in the reaction of ArHgSG/2. Graph showing the production of free GSH
in the reaction of ArHgSG/1 (d) and ArHgSG/2 (e). (f) Synthetic scheme for
degradation of RHgX by 2. (g) Graph showing the production of free cysteine
in the reaction of MeHgCys/2. (h) EDX image of Hg(SSe) NPs obtained in the
reaction of MeHgCys/2.
On the other hand, unlike the case of 1, the treatment of
RHgCys or RHgSG (30 mM) with its selenium analogue 2 in 1:1
molar ratio leads to the formation of the corresponding 1:1 RHg-
conjugated complexes 14-17, which are highly unstable in
solution and degraded very rapidly within few hours in
phosphate buffer (pH 9) at 37 °C to produce water-soluble black
HgSSe NPs (16 mg from ArHgCys, 12 mg from MeHgCys, 19
mg from ArHgSG, and 14 mg from MeHgSG). The initial rate of
degradation of 14 (4.5 ± 1 x10-3 Mh-1) is ~ 20 fold higher than the
Figure 4. a) Synthetic scheme for the degradation of RHgCl by 1 in the
presence of KOH in 1:1 molar ratio in water-acetonitrile solution at 37 °C. b)
HPLC chromatograms showing the degradation of 18 ( = 275 nm) in the
presence of KOH at 37 °C. Reaction vials showing the formation of black HgS
NPs over time in the reaction of ArHgCl/1/KOH (c) and MeHgCl/1/KOH (d) in
water-acetonitrile at 37 °C. (e) Chemical structures of 21-23.
In order to understand the reason behind the degradation of
RHgCys by 1 and the nature of 1:1 RHg-conjugated complexes
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complete degradation of 18–20 (15 mM) was remarkably fast
compared to the time required for the degradation of the
corresponding imidazole-based complexes 21–23 under
identical reaction conditions (Table 1).[10] For instance, the
complete degradation of 18 (15 mM) to HgS in the presence of
KOH at 37 °C was achieved within an hour (t50 = 25 min, initial
rate = 18.1 ± 1.3 x 10-3 M h-1), compared to 9 days for 21 under
identical reaction conditions (t50 = 110 h, initial rate = 5.1 ± 8.4 x
10-5 M h-1). The initial rate of degradation of 18 is almost 350 fold
faster than the initial rate of degradation of 21, indicating that the
replacement of imidazole ring by benzimidazole dramatically
increased the rate of degradation of various organomercurials.
Interestingly, our experimental observation showed that the 1:1
RHg-conjugated complexes 18–20 degraded even in the
presence of weak bases (NaHCO3, K2HPO4, NaOAc and
imidazole) and non-nucleophilic bases like NEt3, N,N-
diisopropylethylamine (DIEA) to produce black insoluble HgS
NPs (Table 1). However, as expected, the initial rate of
degradation of 20 in the presence of NaHCO3 (initial rate = 15.2
± 0.8 x 10-5 Mh-1) or K2HPO4 (initial rate = 10.3 ± 0.4 x 10-5 Mh-1)
was slower compared to the initial rate of degradation of 20 in
the presence of strong base KOH (initial rate = 13.2 ± 0.4 x 10-3
Mh-1). This is in sharp contrast with the nature of corresponding
imidazole-based complexes 21-23 (15 mM), which are highly
stable even in the presence of weak bases (NaHCO3, K2HPO4,
NaOAc, and imidazole) and non-nucleophilic bases (NEt3, DIEA)
at 37 C and the formation of HgS NPs was not observed under
identical reaction conditions. These observations clearly showed
that the benzimidazole moiety plays crucial role in the
degradation of 1:1 RHg-conjugated complexes (9, 10, 13, and
18-20).
(9, 10 and 13), we have synthesized several stable 1:1 RHg-
conjugated complexes 18–20, stabilized with Cl- counter anion
(Figure 4a). Complexes 18–20 were highly stable in water-
acetonitrile (1:1) solution at 37 °C. However, upon addition of
KOH in stoichiometric amount, 18–20 (15 mM) degraded very
rapidly, within an hour, into insoluble black HgS NPs, R2Hg and
11. The gradual degradation of 18 and subsequently formation
of the corresponding ketone 11 was monitored by HPLC, as
shown in Figure 4b. Likewise, complexes 19 and 20 were also
highly stable in water-acetonitrile (1:1) solution at 37 °C and the
formation of black precipitate of HgS NPs was not observed for
10 days in the absence of any base (Figure 5a). Upon addition
of KOH (1:1 molar ratio), however, both 19 and 20 degraded
very rapidly to produce black precipitate of HgS NPs. The HgS
NPs which were insoluble in water and other organic solvents
were characterized thoroughly by TEM and EDX analysis.
Figure 5. (a) Graph showing the degradation of complexes 18-20 (15 mM) in
the presence or absence of KOH at 37 °C. TEM images of HgS NPs obtained
in the reactions of 18/KOH (c), 19/KOH (d) and 20/KOH (e).
Figure 6. Scheme for the formation of 24-27.
Moreover, to understand the role of -OH group in 1 in the
Table 1. Initial rates of degradation of 1:1 RHg-conjugated complexes (15
degradation process, we treated
4 and 5 with MeHgCl
mM) in the presence of various bases at 37 °C.
SR NO.
1
2
3
4
5
6
7
8
9
10
11
12
Reactions
18/KOH
19/KOH
t50
Initial Rate (Mh-1)
18.1 ± 1.3 x 10-3
25.9 ± 4.3 x 10-3
13.2 ± 0.4 x 10-3
20.1 ± 2.7 x 10-5
15.2 ± 0.8 x 10-5
8.5 ± 0.6 x 10-5
10.3 ± 0.4 x 10-5
12.4 ± 0.3 x 10-3
9.0 ± 1.7 x 10-3
17.0 ± 0.4 x 10-3
5.1 ± 8.4 x 10-5
12.4 ± 7.2 x 10-5
separately. Compounds 4 and 5 failed to convert MeHgCl to
HgS NPs at 37 °C under identical reaction conditions, indicating
that the -OH group in 1 plays a crucial role in the desulfurization
process i.e. the cleavage of C–S bond in 1:1 RHg-conjugated
complexes 9, 10, 13, and 18-20. Furthermore, treatment of N-
substituted thione 7 (or 8) with MeHgCl (1:1 molar ratio) in
water-acetonitrile (1:1) solution resulted in the formation of
complex 24 (or 25), Figure 6. However, upon addition of KOH
afforded the deprotonated neutral complex 26 (or 27) which was
highly stable at 37 °C, and did not result in the formation of HgS
NPs even after 15 days of stirring, indicating that the N-
CH2CH2OH moiety alone in 7 was not sufficient to convert
MeHgCl to HgS NPs even in the presence of strong base and
indicating the role of N,N-disubstitution in the benzimidazole ring
of 1 in the desulfurization process. The N,N-disubstitution in 1
25 min
15 min
33 min
43 h
58 h
66 h
20/KOH
19/NaHCO3
20/NaHCO3
19/K2HPO4
20/K2HPO4
19/Et3N
76 h
22 min
48 min
25 min
110h
84h
20/Et3N
20/KOHa
21/KOH
23/KOHa
aConcentrations of 20 and 23 were 20 mM.
The HgS NPs obtained in the degradation of 18–20 were in
cubic shaped and monodisperse with an average size of ca. 4.8,
23 and 74 nm respectively (Figure 5b-d). The time required for
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leads to the generation of partial positive charge at the C2 atom
of benzimidazole ring in 1:1 RHg-conjugated complexes (9, 10,
13, and 18-20) even under basic condition (Figure 4a and Figure
S41, Supporting Information), which is crucial for the cleavage of
C–S bond through the neighboring group participation of the OH
group of N-CH2CH2OH moiety.
opposite side (Figure 8 and 9). Both OH groups and Cl atoms in
one molecular unit formed equally strong intermolecular H-
bonding with Cl atoms and OH groups respectively present in
two different molecular units located on either side leading to the
formation
a
linear chain-like structure; d(O1H•••Cl1)
:
2.38Å;
d(O1H•••Cl1)
:
2.38Å. As result of this equally strong
a
intermolecular H-bonding both the oxygen atoms (O1 and O1)
in 28 are equally far away from the plane of the benzimidazole
ring. The distance between C2 atom in benzimidazole ring and
O1 atom is 3.967Å, as shown in Figure 9. The two Hg–Cl bonds
in 28 are equal in length (2.511Å), Figure 8. X-ray crystal
structure analysis, thus, confirmed that the OH groups in 28 are
not involved in any kind of Hg–O bond formation and rather,
more importantly, they are located close to the C2 atoms of
benzimidazole rings.
Figure 7. Chemical structure of 28.
Figure 9. Crystal structure of 28, drawn in Mercury 3.8 software, showing
intermolecular H-bonding (a) and the orientation of the OH groups (b, c). For
clarity, only the H-bonding participating atoms of other molecular units are
shown.
Figure 8. ORTEP diagram of 28.
Single crystal X-ray diffraction analysis provided very useful
information on the orientation of the OH group in compound 1
and in its Hg-conjugated complex 28 (Figure 7). In the process
of crystallization of 1:1 RHg-conjugated complexes (18-20) in
water-acetonitrile (1:1) solution we noticed the formation of
unexpected complex 28, as a by-product (yield ~ 5%), through
cleavage of Hg–C bond after prolong standing (~ 15 days) which
was characterized by single crystal X-ray diffraction technique.
In general, organomercurials of the type R–Hg–X are
remarkably inert toward Hg–C cleavage under physiological
conditions. However, in the presence of thiones or selones the
protolytic cleavage of Hg–C bonds of RHgX is well known in the
literature.[16] On the other hand, complex 28 could also be
synthesized independently by treatment of 1 with HgCl2 (1:1
molar ratio) in water-acetonitrile solution. Synthetic procedures
of complex 28 are mentioned in the experimental section.
ORTEP diagram of complex 28 is shown in Figure 8. The crystal
packing of 28 displays very interesting feature on the orientation
of the OH group in N-CH2CH2OH moiety. Two benzimidazole
rings in 28 are oriented in trans-fashion to each other, allowing
one set of OH group and Cl atom (O1H group and Cl1 atom) to
face in one side and another set of OH group and Cl atom (O1H
group and Cl1 atom) of the same molecular unit to face on the
Figure 10. (a) The resonance structures of benzimidazole-based thiones. (b)
ORTEP diagrams of compounds 1, 5, and 7.
Experimental results along with the DFT calculations provided
useful information on the nature of benzimidazole-based thiones
(1, 4, 5, 7 and 8). The C–S bond length in N,N-disubstituted (1, 4,
and 5) and N-substituted (7 and 8) thiones are found to be in the
range of 1.648 to 1.684, indicating a shorter bond length
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compared to a C−S single bond (1.761 Å)[17] but a significantly
longer when compared with bond length of C–S double bond of
1H-indene-2(3H)-thione which is devoid of any N-atom in the
five member ring (C=S: 1.625 Å, Figure S42, Supporting
Information). As a consequence, the calculated bond orders of
these thiones (1, 4, 5, 7 and 8) are typically in the range of 1.46-
1.50, between the C–S single and double bond. This clearly
indicates that they mainly exist in zwitterionic form with large
negative charge on the sulfur atom and the distributed positive
charge on the five member heterocyclic ring (Figure 10, Table
2).[18] The C–S bond length and the negative charge on the S-
atom in 1 and its methyl derivative 5 are very similar and yet
they showed completely different behavior towards the
degradation of organomercurials (RHg+) under identical reaction
conditions, once again indicating the role of –OH group in the
degradation process. On the other hand, in spite of having large
negative charge on the S atom (-0.327), the imidazole-based
thione 3 was not able to degrade MeHgCys under identical
reaction conditions indicating that the C–S bond length and the
nucleophilicity of S atom of these two compounds (1 and 3)
cannot be suitably used to explain the high reactivity of 1
towards the degradation of various organomercurials.
of 30 is to some extent (1.6 kcal/mol) more favorable than the
degradation of 29, which is in agreement with experimental
results (Table 1).
Scheme 1. (a) The representative pathway for the degradation of RHgCl by 1
in basic condition. (b) The reaction free energies (G, kcal/mol) of step 2 for 3.
Table 2. The C–S bond lengths, bond orders and the charge on S-atoms of
thiones (1, 3, 4, 5, 7, and 8).
Compds
(C=S) bond
length (Å) (Exp.)
1.6822(29)
(C=S) bond
order (Calc.)a
1.466
Charge on S
(au)a
1
3
4
5
7
8
-0.286
1.6898(17)
1.423
-0.327
1.648(8), 1.660(7)
1.681(8)
1.50, 1.492
1.466
-0.251, -0.260
-0.287
1.6805(19)
1.684(2)b
1.489
-0.262
1.493
-0.257
aThe natural bond orbitals (NBO) analysis was performed at B3LYP/6-
311++G(2d,p) level of theory. The geometry of the compounds was obtained
from the crystal structure of thiones.[19] bref 20.
The possible pathway of degradation of RHgCl by 1 is
mentioned in Scheme 1. The treatment of RHgCl with 1 guided
the formation of 1:1 RHg-conjugated cationic complexes with Cl-
ion as a counter anion (complex-I). However, the addition of
KOH in to the complex-I produced the corresponding 1:1 RHg-
conjugated cationic complexes with OH- ion as a counter anion
(complex-II) which further reacted with another molecule of
RHgOH (in alkaline medium) to produce the corresponding
ketone 11 and insoluble HgS NPs via the formation of unstable
intermediate (RHg)2S.[6,8,9,21] We have indeed observed that the
addition of one more equivalent of Ar1HgOH ([Ar1HgOH]/[1] = 2;
Figure 11. Synthetic scheme for the formation of unusual products 33-35 in
the reaction of 19 in the presence of NEt3, NaOMe, and AcONa respectively.
ORTEP diagrams of 33 and 35 are shown in the right side of the figure.
where Ar1
= p-C6H4CO2Na) has increased the rate of
degradation of 1:1 Ar1Hg-conjugated complex (Figure S23,
Supporting Information). DFT calculations showed that the
degradation of benzimidazole-based 1:1 RHg-conjugated
complexes is energetically more favorable compared to the
degradation of the corresponding imidazole-based 1:1 RHg-
conjugated complexes (step 2, Scheme 1). The degradation of
29 and 30 is 6.5 and 7.0 kcal/mol more favorable compared to
the degradation of 31 and 32 respectively, (G = GB - GI).
Interestingly, DFT calculations also showed that the degradation
While performing the degradation of 1:1 EtHg-conjugated
complex 19 in the presence of other bases such as NEt3,
NaOMe, and AcONa under various reaction conditions, we
noticed the formation of unusual compounds 33-35 as side-
products. Addition of NEt3 into the solution of 19 in
dichloromethane afforded the formation of 33 (yield = 18%)
along with 11. Compound 33 was isolated from the above
reaction mixture and characterized thoroughly by various
techniques including X-ray crystallography (Figure 11). On the
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10.1002/chem.201605238
Chemistry - A European Journal
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other hand, when 19 was treated with NaOMe in methanol, in
1:2.5 molar ratio, we isolated both 11 and its methyl derivative
34 (yield = 15%) from the reaction mixture. Similarly, the
treatment of 19 with sodium acetate (NaOAc) in 1:2 molar ratio
in water-acetonitrile (1:1) solution yielded the formation of 35
along with other products. Compound 35 was obtained in high
yield (32%) when the same reaction was performed in
acetonitrile under anhydrous condition instead of in water-
acetonitrile.
water-soluble HgS NPs at physiologically relevant conditions.
Moreover, during this ligand exchange reaction between
MeHgCys or MeHgSG with 1 we observed the release of free
thiol CysH or GSH into the solution. On the other hand, the N-
substituted benzimidazole-based thione
7 and the N,N-
disubstituted imidazole-based thione 3, in spite of having N-
CH2CH2OH substituent, failed to convert MeHgCys and
MeHgSG to HgS NPS under identical reaction conditions. We
also showed that the introduction of benzimidazole ring in place
of imidazole ring remarkably alters the reactivity of compound 1
towards the degradation of MeHg+ conjugates. Compound 1 can
convert MeHgX (X = Cl, Cys, GS) to HgS NPs at 37 °C in the
presence of weak bases (NaHCO3, K2HPO4, NaOAc and
imidazole). Whereas compound 7 was not able to convert
MeHgX to HgS NPs even in the presence of strong base KOH
under identical reaction conditions, indicating that not only N-
CH2CH2OH substituent but also the N,N-disubstitution in 1 is
crucial to detoxify various toxic organomercurials. The OH group
of N-CH2CH2OH moiety in 1 facilitates the cleavage of C–S bond
of 1:1 RHg-conjugated complexes through neighboring group
participation. This method could possibly be useful to reverse
the inhibition of sulfhydryl group(s), present in endogenous thiols
and proteins, by methylmercury and may help in clearance of
mercury by forming it water-soluble HgS species in biological
system.
Experimental Section
Figure 12. (a) LCMS chromatograms (EIC) and the corresponding mass
spectra of 36. b) Optimized geometry of 36 showing C-O bond lengths in the
molecule. c) Possible pathways of formation of compounds 11 and 33-35 from
36 in the presence of various nucleophiles.
Synthesis of 1-(2-hydroxyethyl)-3-methyl-1H-benzo[d]imidazole-
2(3H)-thione (1). A mixture of N-methyl benzimidazole (0.5 g, 3.78
mmol) and chloroethanol (0.3 g, 3.78 mmol) was heated at 90 C for 4
days. After 4 days of stirring, white solid (0.66 g) was taken in anhydrous
methanol (50 mL) and treated with elemental sulfur (0.12 g, 3.78 mmol)
and anhydrous potassium carbonate (0.52 g, 3.78 mmol). The reaction
mixture was allowed to reflux for 20 h and the solution was then filtered in
hot condition through celite. Filtrate was evaporated under reduced
pressure to yield yellow crude which was purified by column
chromatography packed with silica. The desired product was obtained as
In order to understand the mechanism of formation of unusual
compounds 33-35 we have treated EtHgCl with 1 in MeOH at
low temperature (10C) in anhydrous condition under inert
atmosphere followed by the addition of NaOMe, which yielded
the formation of
a cyclic intermediate 36, through the
neighboring group participation of the hydroxy group present in
N-CH2CH2OH moiety (Figure 12a).[22] The formation of unstable
intermediate 36 was detected by mass spectrometry. However,
in the presence of various nucleophiles, the intermediate 36
gradually converted into other stable products 11 and 33-35,
proceed through pathway A which was thermodynamically more
favorable than pathway B, as shown in Figure 12c. Upon
addition of KOH (aqueous solution) into the solution of 36
produced 11 which is 32.6 kcal/mol thermodynamically more
favorable than 37. In a similar fashion, in the presence of other
nucleophiles (MeO-, Cl-, and AcO-), the intermediate 36
converted into stable products 33-35, which are 35.5, 33.7, and
40.4 kcal/mol thermodynamically more favorable than
compounds 38-40 respectively. Here it is worthwhile to mention
that the degradation of RHgCl to HgS NPs by 1 in the presence
of KOH was also observed at low temperature (10C).
1
a white crystalline solid. Yield: 0.5 g (64%). H NMR (CD3OD) δ (ppm) =
3.75 (s, 3H), 3.92 (t, J = 5.60 Hz, 2H), 4.43 (t, J = 5.60 Hz, 2H), 7.23-7.30
(m, 2H), 7.31-7.32 (m, 1H), 7.33-7.42 (m, 1H), 13C NMR (CD3OD) δ
(ppm) = 31.4, 48.0, 60.5, 110.1, 110.9, 124.0, 124.1, 133.72, 134.0,
170.6. HR-ESIMS (m/z): calcd for [M+H]+ C10H12N2SO = 209.0743,
found: 209.0753.
Synthesis of 1-(2-hydroxyethyl)-3-methyl-1H-benzo[d]imidazole-
2(3H)-selenone (2). Compound 2 was synthesized following the above
procedure of 1 using Se powder. In brief, benzimidzolium salt was
treated with selenium powder (0.30 g, 3.78 mmol), anhydrous potassium
carbonate (0.52 g, 3.78 mmol) in dry methanol (50ml) and reflux for 20 h.
The solution was then filtered in hot condition through celite. Filtrate was
evaporated under reduced pressure to yield yellow crude which was
purified by column chromatography packed with silica. The desired
1
product was obtained as a white crystalline solid. Yield: 0.56 g (58%). H
NMR (DMSO-d6) δ (ppm) = 3.79 (t, J = 5.60 Hz, 2H), 3.81 (s, 3H), 4.90 (t,
J = 5.60 Hz, 2H), 7.26-7.30 (m, 2H), 7.51-7.58 (m, 2H), 13C NMR
(DMSO-d6) δ (ppm) = 33.3, 49.0, 59.1, 110.3, 111.2, 123.4, 123.5, 133.3,
133.9, 165.6. 77Se NMR (DMSO-d6) δ (ppm) = 47 HR-ESIMS (m/z): calcd
for [M+H]+ C10H12N2SeO = 257.0188, found: 257.0186.
In conclusion, we have shown that N,N-disubstituted
benzimidazole-based thione 1 having N- CH2CH2OH substituent
can effectively compete with endogenous thiols cysteine and
glutathione (tripeptide) for MeHg+ and efficiently convert it into
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10.1002/chem.201605238
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Synthesis of 1,3-dimethyl-1H-benzo[d]imidazole-2(3H)-thione (4). To
a solution of N-methyl benzimidazole (0.9 g, 6.80 mmol) in ethyl acetate,
methyl iodide (0.67 mL, 10.15 mmol) was added dropwise at 0°C.
Reaction mixture was stirred for 4 h to produce the corresponding salt as
a white solid (0.75 g, 5.06 mmol) which was isolated after the reaction
and, then, dissolved in dry methanol (50 mL). The sulfur powder (0.24 g,
7.60 mmol) and anhydrous potassium carbonate (1.05 g, 7.60 mmol) was
added to the above methanol solution and the reaction mixture was
allowed to reflux for 20 h. The solution was then filtered in hot condition
through celite. Filtrate was evaporated under reduced pressure to yield
yellow crude which was purified by column chromatography packed with
silica ethyl acetate/hexane as mobile phase (0.3%).The desired product
was obtained as a white crystalline solid. Yield: 0.76 g (63%). 1H NMR
(CD3OD) δ (ppm) = 3.74 (s, 6H), 7.24-7.29 (m, 2H), 7.30-7.33 (m, 2H),
13C NMR (CD3OD) δ (ppm) = 31.4, 110.2, 124.1, 133.6, 170.9. HR-
ESIMS (m/z): calcd for [M+H]+ C9H10N2S = 179.0637, found: 179.0644.
Synthesis
of
1-(2-hydroxyethyl)-3-methyl-1H-benzo[d]imidazol-
2(3H)-one (11). Procedure 1: A mixture of 1 (9.4 mg, 45 µmol) and 4-
(hydroxymercuri)benzoic acid sodium salt (16.2 mg, 45 µmol, Ar1HgOH,
Ar1 = p-C6H4CO2Na) was stirred in 3 mL of water/acetonitrile solution
(1:1) for 2 h at 37°C. After 2h, the black HgS nanoparticles were washed
several times with acetonitrile followed by with water and characterized
by TEM, SEM and EDX analysis. The supernatant part was evaporated
under vacuum and the compound 11 was extracted with chloroform and
purified by column chromatography, yield: 3.5 mg (40%).
Procedure 2:
A mixture of compound 1 (9.4 mg, 45 µmol) and
methylmercury chloride (11.3 mg, 45 µmol) or ethylmercury chloride
(11.9 mg, 45 µmol) or 4-chloromercuribenzoic acid (ArHgCl, Ar = p-
C6H4CO2H) (16.0 mg, 45 µmol) was stirred in 3 mL of water/acetonitrile
mixture (1:1) for 1 h at 37°C. After 1 h, aqueous KOH (45 µmol) was
added to the above reaction mixture and was continued to stir for another
2 h. The black HgS nanoparticles were washed several times with
acetonitrile followed by with water and characterized by several
techniques as mentioned above. Supernatant part was extracted with
organic solvent as mentioned above and compound 11 was purified by
column chromatography, yield = 3.2 mg (37%). Characterization data of
11: 1H NMR (CDCl3) δ (ppm) = 3.37 (s, 3H), 3.95 (t, J = 5.20 Hz, 2H),
4.03 (t, J = 5.20 Hz, 2H), 6.96-7.06 (m, 3H), 7.06-7.12 (m, 1H), 13C NMR
(CDCl3) δ (ppm) = 27.2, 44.4, 61.4, 107.7, 107.9, 121.5, 121.5, 129.7,
130.1, 155.2. HR-ESIMS (m/z): calcd for [M+H]+ C10H12N2O2 = 193.0972,
Found : 193.0974. calcd for [M+Na]+ = 215.0791, found: 215.0789.
Synthesis of 1-(2-methoxyethyl)-3-methyl-1H-benzo[d]imidazole-
2(3H)-thione (5). 1-methylbenzimidazole (0.5 g, 3.78 mmol) and 2-
chloroethylmethylether (0.35 g, 3.78 mmol) was heated at 80 C. After 4
days the white salt (0.66 g) was taken in anhydrous methanol (50 mL)
and treated with elemental sulfur (0.12 g, 3.78 mmol), anhydrous
potassium carbonate (0.52 g, 3.78 mmol). The reaction mixture was
allowed to reflux for 20 h. The solution was then filtered in hot condition
through celite. Filtrate was evaporated under reduced pressure to yield
yellow crude which was purified by column chromatography packed with
silica ethyl acetate/hexane as mobile phase (50 %). The desired product
was obtained as a white crystalline solid. Yield: 0.52 g (63%). 1H NMR
(CDCl3) δ (ppm) = 3.30 (s, 3H), 3.77 (s, 3H), 3.78 (t, J = 5.60 Hz, 2H),
4.49-4.52 (t, J = 5.60 Hz, 2H), 7.15-7.18 (m, 1H), 7.21-7.23 (m, 2H),
7.30-7.33 (m, 1H), 13C NMR (CDCl3) δ (ppm) = 31.2, 45.1, 59.1, 70.3,
108.8, 110.1, 122.9, 123.0, 132.4, 132.8, 169.5. HR-ESIMS (m/z): calcd
for [M+H]+ C11H14N2OS = 223.0900, found: 223.0906.
General procedure for the synthesis of 1:1 RHg-conjugated
complexes (18-20):
Synthesis of 18: A mixture of compound 1 (9.4 mg, 45 μmol) and 4-
chloromercuribenzoic acid (16.1 mg, 45 μmol; ArHgCl, Ar = p-C6H4CO2H)
was stirred in 3 ml of water/ acetonitrile mixture (1:1) for 1 h. Solvent was
evaporated completely under vacuum to obtain 18 as yellow solid. 1H
NMR of 18 (DMSO-d6) δ (ppm) = 3.71 (s, 3H), 3.74 (t, J = 5.6 Hz, 2H),
4.33 (t, J = 5.6 Hz, 2H), 7.23-7.92 (m, 8H), 13C NMR (CD3OD) δ (ppm)
=30.8, 46.8, 58.3, 109.2, 110.1, 122.5, 122.6, 128.0, 128.4, 129.9, 131.8,
132.3, 136.8, 138.1, 158.0, 167.4, 168.5. 199Hg NMR (DMSO-d6) δ (ppm)
-1186. HR-ESIMS (m/z): calcd for [M]+ C17H17HgN2OS = 531.0661,
found: 531.0674.
Synthesis of 1-(2-hydroxyethyl)-1H-benzo[d]imidazole-2(3H)-thione
(7). For the synthesis compound 7, the nucleophilic sulfur center of 6 is
protected with trityl group following the literature procedure and used for
next step.
To the dispersed solution
of 2-(tritylthio)-1H-
benzo[d]imidazole (1 g, 2.54 mmol) in dry acetone (25 ml), potassium
hydroxide (0.71 g, 1.27 mmol) was added and stirred for 5 min, then 2-
bromoethonal (0.38 g, 3.08 mmol) was added dropwise at 0°C and
stirred for 8 h. After 8 h the mixture was poured into 25 ml of water and
extracted with ethyl acetate (2×45ml) and washed with brine and dried
over Na2SO4 and filtered. Filtrate was evaporated under reduced
pressure to yield solid which was dissolved in 5% acetic acid in methanol
and reflux for 30 min. The solution was concentrated and washed with
NaHCO3 solution and the expected compound was extracted with ethyl
acetate. Organic layer was concentrated under reduced pressure to yield
crude which was purified by column chromatography packed with silica
and ethyl acetate/hexane as mobile phase (35%). The desired compound
Synthesis of 19: A mixture of compound 1 (9.4 mg, 45 μmol) and EtHgCl
(11.9 mg, 45 μmol) was stirred in 3 ml of water/ acetonitrile mixture (1:1)
for 1 h. Solvent was evaporated completely under reduced pressure to
yield 19 as white solid. 1H NMR 19 (CD3OD) δ (ppm) = 1.33 (t, J =7.9Hz,
3H), 1.84 (q, J =7.9Hz, 2H), 3.80 (s, 3H), 3.93 (t, J = 5.7 Hz, 2H), 4.47 (t,
J = 5.7 Hz, 2H), 7.28-7.31 (m, 2H) , 7.37-7.48 (m, 2H), 13C NMR
(CD3OD) δ (ppm) = 14.0, 22.7, 31.6, 48.2, 60.4, 110.4, 111.2, 124.3,
124.3, 133.73, 133.9,169.3. 199Hg NMR (DMSO-d6) δ (ppm) -1009. HR-
ESIMS (m/z): calcd for [M]+ C12H17HgN2OS = 439.0762, found: 439.0702.
º
was obtained as a white crystalline solid upon cooling at 4 C. Yield: 0.29
g (59%). 1H NMR of 7 (CD3OD) δ (ppm) = 3.93 (t, J = 5.60 Hz, 2H), 4.40
(t, J = 5.60 Hz, 2H), 7.19-7.23 (m, 3H), 7.37-7.39 (m, 1H). 13C NMR
(CD3OD) δ (ppm) = 47.3, 60.6, 110.7, 111.1, 123.6, 124.1, 132.2, 135.1,
169.7. HR-ESIMS (m/z): calcd for [M+H]+C9H10N2OS= 195.0587, found:
195.0588.
Synthesis of 20: A mixture of compound 1 (9.4 mg, 45 μmol) and MeHgCl
(11.3 mg, 45 μmol) was stirred in 3 ml of water/ acetonitrile mixture (1:1)
for 1 h. Solvent was evaporated completely under vacuum to obtain 20
1
as white solid. H NMR 20 (CD3OD) δ (ppm) = 0.91(s, 3H), 3.79 (s, 3H),
3.93 (t, J = 5.7 Hz, 2H), 4.47 (t, J = 5.7 Hz, 2H), 7.28-7.30 (m, 2H) , 7.36-
7.47 (m, 2H); 13C NMR (CD3OD) δ (ppm) = 3.73,31.58, 48.17, 60.49,
110.37, 111.18, 124.28, 124.31, 133.71, 134.0,169.60. 199Hg NMR
(DMSO-d6) δ(ppm) -848. HR-ESIMS (m/z): calcd for [M]+ C11H15HgN2OS
= 425.0605, found: 425.061.
Synthesis of 1-methyl-1H-benzo[d]imidazole-2(3H)-thione (8).
Compound 8 is synthesized following literature procedure.[10] 1H NMR of
8: (DMSO d6) δ (ppm) = 3.65 (s, 3H), 7.18-7.19 (m, 3H), 7.35-7.36 (m,
1H), 13C NMR (DMSO d6) δ (ppm) = 30.0, 109.4, 109.5, 122.1, 122.8,
130.6, 133.1, 168.5. HR-ESIMS (m/z): calcd for [M+H]+ C8H8N2S=
165.0480, found: 165.0485.
Synthesis of 28: Method 1: Compound 28 was obtained as a by-product
in the form of white crystalline material from a reaction solution of EtHgCl
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10.1002/chem.201605238
Chemistry - A European Journal
FULL PAPER
(11.9 mg, 0.045 mmol) and 1 (9.4 mg, 0.045 mmol) in 1:1 water
acetonitrile medium after slow evaporation (after 15 to 20 days). Yield: ~
5%. X-ray quality crystals of 28 were isolated from mother liquor and the
structure was determined by measuring X-ray diffraction data on D8
Venture Bruker AXS single crystal X-ray diffractometer.
X-Ray crystal analysis.
The crystallization techniques of compounds 1, 4, 5, 7, 8, 11, 28, 33 and
35 are mentioned in the supporting information. Crystal structures of
these 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
Method 2: Compound 28 was also obtained by mixing compound 1 (25
mg, 0.12 mmol) with HgCl2 (16.2 mg, 0.06 mmol) in acetonitrile-
dichloromethane mixture. The resultant solution is mixed well and kept at
60 °C. Over the time growth of white colour cubic crystals were observed.
The supernatant is filtered and the solid precipitate was dried under
reduced pressure to obtain 28 as white crystalline solid, yield = 43 mg
(52%). 1H NMR of 28 (DMSO-d6) δ (ppm) = 3.80 (t, J = 5.58 Hz, 2H), 3.94
(s, 3H), 4.54 (t, J = 5.60 Hz, 2H), 7.47-7.50 (m, 2H), 7.76-7.80 (m, 2H),
13C NMR (DMSO-d6) δ (ppm) = 32.4, 48.3, 58.8, 111.5, 112.2, 124.7,
124.8, 131.8, 132.0, 158.7; 199Hg NMR (DMSO-d6) δ (ppm) -1231.
sources (Mo-Kα
= 0.71073 Å). All the crystal data were collected at
room temperature. The structure was solved using SHELX program
implemented in APEX3.[23-26] 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.[27] Crystal data for compounds 1, 4, 5, 7, 8, 11, 28, 33 and 35
are mentioned below.[28]
Synthesis of 33 and 34: A mixture of compound 1 (0.2 g, 0.96 mmol) and
ethylmercuric chloride (0.31 g, 1.17 mmol) was stirred in 10 mL of freshly
prepared dry methanol under N2 atmosphere for 1h at 37°C to ensure
complete complexation and then NaOMe (0.10g, 1.95 mmol) or triethyl
amine (1.95 mmol) was added and left to stir for 2 hours to ensure
complete degradation. The supernatant part was separately evaporated
under vacuum and the compound was extracted with chloroform. The
organic layer was thoroughly washed with water two more times and then
separated. The solvent was removed completely under vacuum to give
pale-yellow liquid as a crude product which was purified by column
chromatography using ethyl acetate: petroleum ether to yield a white
solid of ketone 33 (yield = 37 mg (18%), which was crystalized from
isopropanol, whereas compound 34, yield = 29 mg (15%) is obtained as
light yellow oil.
Crystal data for compound 1 (CCDC-1483028): C10H12N2OS; Fw: 208.28;
Crystal system: Monoclinic; Space group: P21/n; a: 7.0736 (5) Å; b:
16.2384 (11) Å; c: 9.0772(6) Å; α = 90.00 β = 96.072 (3) γ = 90.00º; V:
1036.79(12) Å3; Dcalc: 1.334 Mg m-3; Z: 4; µ (Mo K) (mm-1): 0.280;
Reflections collected/unique: 15259/2118; Parameters: 130; Rint: 0.0738;
R (all data)b: R1 = 0.091; wR2 = 0.188; R (observed data)a: R1 = 0.066;
wR2 = 0.171; Goodness-of-fit on F2: 1.045; min and max (eÅ-3): -0.269
and 0.436.
Crystal data for compound
2 (CCDC-1496005): C10H12N2OSe; Fw:
255.18; Crystal system: Monoclinic; Space group: P21/C; a: 7.0710 (3) Å;
b: 8.9908 (4) Å; c: 16.7148(7) Å; α = 90.00 β = 94.577 (2) γ = 90.00º; V:
1053.25(2) Å3; Dcalc: 1.61 Mg m-3; Z: 4; µ (Mo K) (mm-1): 3.534;
Reflections collected/unique: 19266/2157; Parameters: 130; Rint: 0.0529;
R (all data)a: R1 = 0.053; wR2 = 0.133; R (observed data)b: R1 = 0.042;
wR2 = 0.120; Goodness-of-fit on F2: 0.886; min and max (eÅ-3): -0.783
and 0.965.
1H NMR of 33 (CDCl3) δ (ppm) =3.36 (s, 3H), 3.75 (t, J = 6.4 Hz, 2H),
4.15 (t, J = 4.8 Hz, 2H), 6.91-7.06 (m, 4H), 13C NMR (CDCl3) δ (ppm) =
27.2, 41.5, 43.1, 107.7, 107.8, 121.5,121.6, 129.3, 130.1, 154.3 HR-
ESIMS (m/z): calcd for [M+H]+ C10H11ClN2O
211.0650.
= 211.0633, found:
Crystal data for compound 4 (CCDC-1483029): C9H10N2S; Fw: 178.25;
Crystal system: Monoclinic; Space group: P21/c; a: 7.377 (2) Å; b: 16.322
(4) Å; c: 15.325 (4) Å; α = 90.00 β = 90.582 (9) γ = 90.00º; V: 1845.2 (8)
Å3; Dcalc: 1.283 Mg m-3; Z: 8; µ (Mo K) (mm-1): 0.295; Reflections
collected/unique: 31131/3806; Parameters: 222; Rint: 0.0587; R (all
1H NMR of 34 (CDCl3) δ (ppm) =3.33 (s, 3H), 3.42 (s, 3H), 3.69 (t, J = 4.8
Hz, 2H), 4.06 (t, J = 5.2 Hz, 2H), 6.95-7.26 (m, 4H), 13C NMR (CDCl3) δ
(ppm) = 27.2, 41.3, 59.0, 70.7, 107.4, 108.3, 121.2, 121.3, 129.9, 130.1,
154.6. HR-ESIMS (m/z): calcd for [M+H]+ C11H14N2O2 = 207.1128, found:
207.1142.
data)a: R1 = 0.266; wR2 = 0.375; R (observed data)b: R1 = 0.134; wR2
=
0.305; Goodness-of-fit on F2: 1.036; min and max (eÅ-3): -0.450 and
0.379.
Synthesis of 35: A mixture of compound 1 (0.2 g, 0.96 mmol) and
ethylmercuric chloride (0.31 g, 1.17 mmol) was stirred in 10 mL 1:1 of dry
ACN for 1h at 37°C to ensure complete complexation and then sodium
acetate (0.16g, 1.95 mmol) was added and left to stir for 3 days to ensure
complete degradation. The supernatant part was separately evaporated
under vacuum and the compound was extracted with chloroform. The
organic layer was thoroughly washed with water two more times and then
separated. The solvent was removed completely under vacuum to give
pale-yellow liquid as a crude product which was purified by column
chromatography using ethyl acetate/petroleum ether to yield a white solid
compound of 37, yield = 72 mg (32%), which was crystallized from
dichloromethane.
Crystal data for compound 5 (CCDC-1483030): C11H14N2OS; Fw: 222.30;
Crystal system: Orthorhombic; Space group: Pna21; a: 7.2516 (18) Å; b:
15.035 (4) Å; c: 10.510 (3) Å; α =β = γ = 90.00º; V: 1145.9 (5) Å3; Dcalc
1.289 Mg m-3; Z: 8; (Mo K) (mm-1): 0.258; Reflections
collected/unique: 2039/1397; Parameters: 139; Rint: 0.0752; R (all data)a:
:
µ
R1 = 0.1489; wR2 = 0.1316; R (observed data)b: R1 = 0.0548; wR2
=
0.1011; Goodness-of-fit on F2: 0.938; min and max (eÅ-3): -0.186 and
0.209.
Crystal data for compound 7 (CCDC-1483021): C9H10N2OS; Fw: 194.25;
Crystal system: Triclinic; Space group: P-1; a: 7.3434 (5) Å; b: 7.9920 (6)
Å; c: 9.2454 (6) Å; α = 68.908 (2) β = 72.248 (2) γ = 82.675º (2); V:
482.05 (6) Å3; Dcalc: 1.338 Mg m-3; Z: 2; µ (Mo K) (mm-1): 0.296;
Reflections collected/unique: 14457/1960; Parameters: 124; Rint: 0.0477;
1H NMR of 35 (CDCl3) δ (ppm) =1.98 (s, 3H ), 3.42 (s, 3H ), 4.14 (t, J =
5.6 Hz, 2H), 4.36 (t, J = 5.6 Hz, 2H), 6.96-7.09 (m, 4H), 13C NMR
(CDCl3) δ (ppm) = 20.9 , 27.2 , 40.1 , 62.2, 107.6, 107.7, 121.4, 121.5,
129.6, 130.2, 154.4, 170.9. HR-ESIMS (m/z): calcd for [M+H]+
C12H14N2O3 = 235.1077, found: 235.1068.
R (observed data)a: R1 = 0.0477; wR2 = 0.1277; R (all data)b: R1
=
0.0357; wR2 = 0.1106; Goodness-of-fit on F2: 0.695; min and max (eÅ-
3): -0.188 and 0.230.
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Crystal data for compound 11 (CCDC-1483025): C10H12N2O2; Fw: 192.22;
Crystal system: Orthorhombic; Space group: Pbca; a: 7.1994 (4) Å; b:
16.2551 (10) Å; c: 16.4466 (8) Å; α =β = γ = 90.00º; V: 1924.70 (18) Å3;
NBO Version 3.1 program implemented in Gaussian 03 was used to
perform NPA60 for thiones and selone. To calculate the free energies for
Figure12, optimization of 11, 33-35, and 37-40 were performed in
gaseous phase at B3LYP level of theory using 6-311++G(2d,p) basis
set.[32-34] The B3LYP method and 6-311++G(2d,p) basis set were used
for main group based compounds (without Hg). The optimized
geometries with co-ordinates are given in Supporting Information.
Dcalc
collected/unique: 16300/1959; Parameters: 130; Rint: 0.0575;
: µ
1.327 Mg m-3; Z: 8; (Mo K) (mm-1): 0.094; Reflections
R
(observed data)a: R1 = 0.0539; wR2 = 0.1294; R (all data)b: R1 = 0.0912;
wR2 = 0.148; Goodness-of-fit on F2: 1.036; min and max (eÅ-3): -0.191
and 0.228.
Crystal data for compound 28 (CCDC-1483031): C20H24Cl2HgN4O2S2; Fw:
688.07; Crystal system: Monoclinic; Space group: P2/c; a: 9.5007 (5) Å; b:
8.6511 (4) Å; c: 14.8130 (6) Å; α = 90.00 β = 95.463 (3) γ = 90.00º; V:
1211.97 (10) Å3; Dcalc: 1.885 Mg m-3; Z: 2; µ (Mo K) (mm-1): 6.536;
Reflections collected/unique: 5095/2342; Parameters: 144; Rint: 0.0395;
R (all data)a: R1 = 0.040; wR2 = 0.107; R (observed data)b: R1 = 0.028;
wR2 = 0.094; Goodness-of-fit on F2: 0.759; min and max (eÅ-3): -0.430
and 0.599.
Acknowledgements
This work is supported by SNU, IIT Indore, SERB (SB/S1/IC-44/2013)
and CSIR [01(2723)/13/EMR(II)], Govt. of India. We thank to Prof.
Rupamanjari Ghosh (VC) for continuous support encouragement. MB, RK, AC,
RD and RKR thank to SNU for fellowship. We thank to AIRF, JNU for TEM
and SEM facilities.
Keywords: detoxification• methylmercury • sulphur • selenium • MeHgCys
Crystal data for compound 33 (CCDC-1483013): C10H11ClN2O; Fw:
210.66; Crystal system: Monoclinic; Space group: P21/c; a: 8.603 (4) Å; b:
4.480 (2) Å; c: 26.119 (12) Å; α = 90.00 β = 91.916 (12) γ = 90.00º; V:
1006.1 (8) Å3; Dcalc: 1.391 Mg m-3; Z: 4; µ (Mo K) (mm-1): 0.347;
Reflections collected/unique: 7499/1986; Parameters: 129; Rint: 0.0897;
R (all data)a: R1 = 0.173; wR2 = 0.2850; R (observed data)b: R1 = 0.1020;
wR2 = 0.2570; Goodness-of-fit on F2: 1.097; min and max (eÅ-3): -
0.337 and 0.440.
CAUTION! Organomercurials are highly toxic to humans, and thus appropriate
safety precautions must be taken in handling these toxic chemicals.
Author Contributions
GPR planned the research work, designed experiments, and written MS. MB
carried out most of the works (kinetic studies, crystal structure determination,
characterization of nanoparticles and isolation several intermediates, synthesis
and DFT calculations). RK, AC, MB, RD and RKR have synthesized all
compounds reported here. RK performed NMR studies. GPR, MB, KSR and
BP performed DFT calculations of Scheme 1. GPR and MB analyzed and
interpreted data. All authors approved the final version of the manuscript for
submission.
Crystal data for compound 35 (CCDC-1483026): C12H14N2O3; Fw: 234.25;
Crystal system: Monoclinic; Space group: P21/c; a: 9.3632(5) Å; b:
16.3177 (9) Å; c: 8.2183 (4) Å; α = 90.00 β = 106.696 (2) γ = 90.00º; V:
1202.43 (11) Å3; Dcalc: 1.43 Mg m-3; Z: 4; µ (Mo K) (mm-1): 0.094;
Reflections collected/unique: 23452/2466; Parameters: 157; Rint: 0.047;
R (observed data)a: R1 = 0.044; wR2 = 0.105; R (all data)b: R1 = 0.063;
wR2 = 0.114; Goodness-of-fit on F2: 1.037; min and max (eÅ-3): -0.151
and 0.175.
Funding Sources
This work is supported by SNU, IIT Indore, SERB (SB/S1/IC-44/2013), CSIR
[01(2723)/13/EMR(II)], Govt. of India.
General procedure for the degradation of RHgCys or RHgSG by 1
and 2. Compound 1 (37.5 mg, 180 µmol) or 2 (22.9 mg, 90 µmol) was
added to the solution of RHgCys or RHgSG (R = Ar/Me, 90 µmol) in 3 ml
of PBS buffer (pH 9) and stirred the reaction mixture at 37°C. The clear
solution was gradually turned black after 120 min in case of 2 and 6 h in
case of 1. An aliquot of 5 l was taken from the above reaction mixture at
various times and then diluted to further for HPLC and LCMS analysis.
Mobile phase: water/acetonitrile gradient. After completion of reaction,
the black water-soluble HgS (or water-soluble Hg(SSe)) nanoparticles
were isolated by centrifugation and washed intensively with water and
followed by with acetonitrile. HgS and HgSe powder were dried
completely over vacuum and characterized thoroughly by various
techniques such as SEM, TEM and EDX and IR analysis (Supporting
Information).
Notes
The authors declare no competing financial interest
Keywords: detoxification• methylmercury • sulphur • selenium • MeHgCys
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Mainak Banerjee, Ramesh
Karri, Ashish Chalana, Ranajit
Das, Rakesh Kumar Rai, Kuber
Singh Rawat, Biswarup Pathak
and Gouriprasanna Roy*
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Title: Protection of Endogenous
Thiols Against Methylmercury by
Benzimidazole-based Thione via
Unusual Ligand Exchange Reactions
MeHg+ exerts its cytotoxicity through strong binding with endogenous thiols such as CysH and GSH (tripeptide) to form MeHgCys and
MeHgSG complexes. Herein we show that the N,N-disubstituted benzimidazole-based thione 1 having N-CH2CH2OH substituent exhibits
remarkable effect in converting MeHgCys and MeHgSG to water-soluble HgS NPs and releases the corresponding free thiol (CysH or GSH)
via unusual ligand exchange reactions in phosphate buffer at 37 °C, whereas N-substituted benzimidazole based thione 7 and N,N-
disubstituted imidazole-based thione 3, in spite of having N-CH2CH2OH substituent, failed to convert MeHgX (X = Cys and SG) to HgS NPs
under identical reaction conditions.
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