Theoretical calculations and reaction analysis on the interaction of pentavalent thioarsenicals with biorelevant thiol compounds

Article abstract of DOI:10.1021/tx700346z

To obtain a rational understanding of the extraordinary interaction of pentavalent thioarsenicals with biorelevant thiol compounds, we carried out ab initio calculations on related arsenic compounds and discussed the correlation between the distribution of observed arsenic species in actual reaction systems and the corresponding calculated reaction enthalpies. Previously, it was considered that pentavalent arsenicals do not form thiol conjugates. However, the dimethylmonothioarsinic acid-glutathione conjugate (DMMTA^V-GSH) was recently reported as the first stable conjugate of a pentavalent arsenical with a thiol compound. We carried out detailed analysis of the DMMTA ^V-GSH formation reaction and demonstrated that this conjugate could be formed nonenzymatically under weakly acidic conditions. On the basis of the ab initio calculations, this conjugation was an exothermic reaction (ΔH = -4.85 kcal/mol) and gave the minimum energy point during the reaction sequence of DMMTA^V with a thiol compound. However, in the case of dimethylarsinic acid (DMA^V), a corresponding oxo acid to DMMTA ^V, conjugation with a thiol compound is an endothermic reaction (ΔH = +0.06 kcal/mol). The minimum energy point of the reaction sequence of DMA^V with a thiol compound was the formation of a trivalent dimethylarsinous acid (DMA^III)-GSH conjugate. Because the formation of arsenic-sulfur bonds is one of the major mechanisms for arsenic toxicity, these energetic results could account for the extraordinary behaviors and toxicities of thioarsenicals in vivo and in vitro in comparison with those of the corresponding oxo acids.

Full text of DOI:10.1021/tx700346z

550
Chem. Res. Toxicol. 2008, 21, 550–553
Theoretical Calculations and Reaction Analysis on the Interaction of
Pentavalent Thioarsenicals with Biorelevant Thiol Compounds
Noriyuki Suzuki,† Hua Naranmandura,† Seishiro Hirano,‡ and Kazuo T. Suzuki*,†
Graduate School of Pharmaceutical Sciences, Chiba UniVersity, Chuo, Chiba 260-8675, Japan, and Research
Center for EnVironmental Risk, National Institute for EnVironmental Studies, Tsukuba 305-8506, Japan
ReceiVed September 22, 2007
To obtain a rational understanding of the extraordinary interaction of pentavalent thioarsenicals with
biorelevant thiol compounds, we carried out ab initio calculations on related arsenic compounds and
discussed the correlation between the distribution of observed arsenic species in actual reaction systems
and the corresponding calculated reaction enthalpies. Previously, it was considered that pentavalent
arsenicals do not form thiol conjugates. However, the dimethylmonothioarsinic acid-glutathione conjugate
(DMMTAV-GSH) was recentry reported as the first stable conjugate of a pentavalent arsenical with a
thiol compound. We carried out detailed analysis of the DMMTAV-GSH formation reaction and
demonstrated that this conjugate could be formed nonenzymatically under weakly acidic conditions. On
the basis of the ab initio calculations, this conjugation was an exothermic reaction (∆H ) -4.85 kcal/
mol) and gave the minimum energy point during the reaction sequence of DMMTAV with a thiol
compound. However, in the case of dimethylarsinic acid (DMAV), a corresponding oxo acid to DMMTAV,
conjugation with a thiol compound is an endothermic reaction (∆H ) +0.06 kcal/mol). The minimum
energy point of the reaction sequence of DMAV with a thiol compound was the formation of a trivalent
dimethylarsinous acid (DMAIII)-GSH conjugate. Because the formation of arsenic-sulfur bonds is one
of the major mechanisms for arsenic toxicity, these energetic results could account for the extraordinary
behaviors and toxicities of thioarsenicals in ViVo and in Vitro in comparison with those of the corresponding
oxo acids.
Introduction
Arsenic is one of the most important pollutants of concern
occurring in the environment, and human health risks such as
hyperkeratosis, pigmentation and blackfoot disease (1, 2), and
lung, skin, and urinary bladder cancers (3–5) are increasing in
populations affected by arsenic-contaminated drinking water.
Although the mechanisms underlying arsenic-induced diseases
and cancers are not precisely understood, the binding of
arsenicals to biomolecules is considered to be one of the major
toxic mechanisms. Trivalent arsenicals are known to have high
affinity for the sulfhydryl groups of biomolecules, such as
glutathione (GSH1) and lipoic acid, and the cysteinyl residues
of many enzymes (1, 2, 6). Because the sulfhydryl groups of
enzymes and cofactors play important roles in many biological
processes, the formation of arsenic(III)-sulfur bonds results in
various harmful effects. However, it is said that pentavalent
arsenicals do not directly bind to sulfhydryl groups. In order to
bind to sulfhydryl groups, the reduction of pentavalent arsenicals
by bioreductants or reduction systems in ViVo into more toxic
trivalent arsenicals is thought to be necessary.
Figure 1. Dimethylarsenicals and dimethylthioarsenicals that have been
previously reported.
sulfhydryl group of GSH (7). They found an unknown arsenic
compound during the study of the metabolism of inorganic and
methylated arsenicals in cabbage (Brassica oleracea) exposed
to dimethylarsinic acid (DMAV), and the unkown compound
was identified as the dimethylmonothioarsinic acid-GSH
conjugate (DMMTAV-GSH, Figure 1) by using HPLC-elec-
trospray mass spectrometry (HPLC-ES MS) and HPLC-
inductively coupled argon plasma mass spectrometry (HPLC-
ICP MS). In addition, they reported that simply mixing
DMMTAV and GSH in water resulted in the formation of the
DMMTAV-GSH complex in the same manner as trivalent
arsenicals.
It is known that such sulfur-containing pentavalent arsenicals
as DMMTAV and dimethyldithioarsinic acid (DMDTAV) are
formed in the metabolic pathway of arsenic in mammals (8–11)
and that these two thioarsenicals showed distinct behaviors and
toxicities in ViVo and in Vitro in comparison with those of the
corresponding oxo acids (12–14). Both Naranmandura et al. and
However, very recently, Raab et al. reported for the first time
that sulfide-activated pentavalent arsenic could also bind to the
* To whom correspondence should be addressed. Dr. Kazuo T. Suzuki,
Professor, Graduate School of Pharmaceutical Sciences, Chiba University,
Chuo, Chiba 260-8675, Japan. Tel: +81-43-226-2865. Fax: +81-43-226-
2865. E-mail: ktsuzuki@p.chiba-u.ac.jp.
† Chiba University.
‡ National Institute for Environmental Studies.
1 Abbreviations: iAsIII, arsenite; DMAIII, dimethylarsinous acid; DMAV,
dimethylarsinic acid; GSH, glutathione; DMMTAV, dimethylmonothioars-
inic acid; DMDTAV, dimethyldithioarsinic acid; ICP MS, inductively
coupled argon plasma mass spectrometry; ES MS, electrospray mass
spectrometry; RHF, restricted Hartree-Fock.
10.1021/tx700346z CCC: $40.75
2008 American Chemical Society
Published on Web 01/08/2008

PentaValent Thioarsenic-GSH Conjugate
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 551
Raml. et al. reported that DMMTAV showed considerably higher
cytotoxicity than nonthiolated DMAV. Therefore, the interaction
of pentavalent thioarsenicals with biorelevant thiol compounds
is important from the viewpoint of their toxicity mechanisms,
if the similar complexes could be formed in mammal tissues.
Here, we report the results of detailed analysis of the
DMMTAV-GSH formation reaction and clarify why this
conjugate could be stably isolated, in contrast to DMAV, which,
instead of forming a conjugate with GSH, is reduced to DMAIII
by GSH.
Experimental Procedures
Synthesis of 34S-DMMTAV. Na234S was synthesized by reduc-
ing elemental [34S]-enriched sulfur (17 mg (0.5 mmol), 99.90%-
enriched, powder) with metallic Na (46 mg (2.0 mmol), lump) in
THF at 80 °C under Ar atmosphere for 12 h. 34S-DMMTAV was
obtained by reacting DMAV and Na234S according to the procedure
in our previous report (15). Chemical and isotopic purities were
confirmed by HPLC-ICP MS.
HPLC-ICP MS and HPLC-ESI MS Analysis. The separation
and detection conditions for arsenic compounds were similar to
those described previously (12, 15, 16). Briefly, arsenic species were
separated on a polymer-based multimode column (Shodex Asahipak
GS-220 HQ, 300 mm × 7.6 mm i.d., Showa Denko, Tokyo, Japan)
eluted with 50 mM ammonium acetate buffer (pH 6.5 at 25 °C) at
a flow rate of 0.6 mL/min. Arsenic in the eluate was monitored
with ICP MS (HP4500; Yokogawa Analytical Systems, Hachiouji,
Japan) and ES MS (LCMS-2010EV series; Shimadzu, Kyoto,
Japan).
Reaction Conditions of DMMTAV and GSH. DMMTAV (133
µM, 1 mL) was mixed with GSH (1.33 mM) and incubated at 37
°C for 12 h at various pH in sodium phosphate buffer (0.1 M, pH
1.5 to 9.0), and then the products were subjected to HPLC-ICP
MS and HPLC-ESI MS analyses.
Ab Initio Calculations. All ab initio calculations were carried
out using the restricted Hartree–Fock (RHF) theory with the
GAMESS quantum calculation package (17). We employed the
6-31G(d,p) basis set and the isodensity polarized continuum method
(IPCM) (18) as an aqueous solvent model for geometry optimiza-
tions and subsequent energy calculations. Reaction enthalpies at
37 °C were obtained by thermodynamic parameters calculated at
37 °C.
Results and Discussion
We previously reported that free DMMTAV and DMDTAV,
but not their GSH conjugates, were detected in mammalian
tissues including liver, in which the concentration of cytosolic
GSH is relatively high (8). Meanwhile, Rabb et al. reported
that simply mixing DMMTAV and GSH in water resulted in
the formation of the DMMTAV-GSH (7). Therefore, we
examined the pH dependence of the reaction. First, in order to
confirm the formation of the DMMTAV-GSH, a reaction
mixture containing DMMTAV and GSH incubated in phosphate
buffer (pH 3) was immediately subjected to HPLC-ICP MS and
HPLC-ESI MS analysis on a multimode GS-220 HQ column.
In this experiment, we prepared DMMTAV labeled with 34S
(34S-DMMTAV) to discriminate pentavalent and trivalent GSH
complexes. The pentavalent complex is considered to possess
34S originating from 34S-DMMTAV, while the trivalent complex
is not because the origin of the sulfur atom of the trivalent GSH
complex is GSH. As shown in Figure 2a, m/z ) 75 (75As), and
the reaction mixture contained a novel arsenic species (peak 2)
distinct from the original DMMTAV (peak 3). DMMTAV
contained only 34S (Figure 2a, m/z ) 48 (32S16O) and 50
(34S16O)), and the novel arsenic species (peak 2) also contained
34S at an equimolar ratio to As, while GSH (peak 1) contained
Figure 2. (a) HPLC-ICP MS and HPLC-ESI MS chromatograms of
the reaction mixture of 34S-DMMTAV and GSH. Detection methods
and monitoring mass numbers are shown in each chromatogram. Peak
1, GSH; peak 2, 34S-DMMTAV-GSH; peak 3, 34S-DMMTAV. (b) ESI
MS spectrum of peak 2.
32S and 34S in the ratio of natural abundance (94.9% and 4.3%,
respectively). For the interference-free detection of S, the signals
of S were detected as that of SO+ by introducing O2 gas into
the mass spectrometer (19, 20). This result shows that this novel
arsenic species contains pentavalent thioarsenic. Peak 2 also
showed a signal at m/z ) 446 in the ESI MS spectrum that
agreed with [M + H]+ of 34S-DMMTAV-32S-GSH (Figure 2a

552 Chem. Res. Toxicol., Vol. 21, No. 2, 2008
Suzuki et al.
Figure 4. Potential energy diagrams for the reaction of pentavalent
arsenicals with methanethiol. The proposed sequential reactions are
presented in a, and enthalpy changes (∆H, kcal/mol) of each step are
presented in b (DMAV) and c (DMMTAV).
weakly acidic conditions, but under neutral or basic conditions,
hydrolysis of DMMTAV and subsequent reduction is predomi-
nant. In fact, in ViVo, free DMDTAV and DMAIII conjugated
with proteins were detected as the two major arsenic species in
rat liver and kidneys exposed to DMMTAV (12).
Previously, it was considered that pentavalent arsenicals do
not form thiol conjugates. To gain a rational understanding of
the abnormality of DMMTAV, we carried out ab initio calcula-
tions of DMAV, DMMTAV, and related compounds. We
assumed a reaction system involving pentavalent arsenicals and
methanethiol (methanethiol was employed instead of GSH to
shorten the calculation time) as shown in Figure 4a, according
to the reduction mechanism proposed by Martin et al. (22). This
proposed mechanism begins with a nucleophilic attack on
pentavalent arsenicals by the first thiol to give a pentavalent
arsenic-SCH3. The second thiol then attacks this complex to
produce the trivalent arsenicals and CH3SSCH3 via the
(CH3)2As(XH)(SCH3)2 intermediate. We calculated the enthalpy
change (∆H) at 37 °C of each step in aqueous solvent by using
the RHF/6-31G(d,p) basis set and the IPCM method (18).
Energetic results of the reaction of DMAV and DMMTAV with
methanethiol are given in Figures 4b and c, respectively. In these
calculation methods, it is hard to consider the pH effects that
we discussed in Figure 3. Nevertheless, we consider that
calculations of enthalpy changes are helpful to understand the
diference of reactivity between two arsenic species with thiol
compounds.
Figure 3. (a) pH dependence of the formation of DMMTAV-GSH
and the hydrolysis of DMMTAV. (b and c) Proposed schemes of the
reaction under acidic or basic conditions.
and b). In this experiment, the formation of DMMTAV-GSH
was confirmed, as reported by Rabb et al. (7).
Next, we analyzed the products of the reaction of DMMTAV
(133 µM, 10 µg As/mL) with GSH (1.33 mM, 10 equivalents
to arsenic), which were incubated at 37 °C for 12 h at various
pH in sodium phosphate buffer (0.1 M, pH 1.5 to 9.0). The
analytical methods for other arsenic compounds except
DMMTAV-GSH were reported in our previous work (15). As
shown in Figure 3a, the formation of DMMTAV-GSH was
promoted under weakly acidic conditions because the dehydra-
tion reaction from the intermediate illustrated in Figure 3b could
be accelerated under acidic conditions. Since the pKa value of
the thiol group of GSH (8.65) (21) is lower than those of simple
alkanethiols (ca. 10), the nucleophilicity of GSH is considered
to be sufficient for this reaction under weakly acidic conditions.
Under strongly acidic conditions, the formation of
DMMTAV-GSH was inhibited in response to the defect of the
nucleophilicity of GSH. In contrast, under neutral or basic
conditions, DMAIII and DMDTAV were formed instead of
DMMTAV-GSH. This phenomenon could be explained as
follows. In the presence of the OH- ion, an equilibrium could
be assumed in which DMMTAV was hydrolyzed to DMAV, and
another DMMTAV molecule reacted with the liberated sulfide
ion to afford DMDTAV (Figure 3c). However, as DMMTAV is
stable even under basic conditions in the absence of a reducing
agent, it is considered to be favorable in this equilibrium.
However, DMAV formed by hydrolysis was immediately
reduced to DMAIII in the presence of GSH; therefore, DMAIII
and DMDTAV were accumulated in the reaction system. These
results show that DMMTAV-GSH is produced rapidly under
Figure 4b indicates that the reduction of DMAV with
methanethiol is indeed an endothermic reaction, while the
subsequent complexation of DMAIII with methanethiol is an
exothermic one (∆H ) -9.42 kcal/mol) and works as a driving
force for this series of reactions. Enthalpy change in the
formation of DMAV-SCH3 indicates a slightly endothermic
reaction, and this DMAV-SCH3 immediately reacts with
another CH3SH molecule. This is why DMAV-thiol conjugates
could not be isolated in the presence of excess thiol compounds.
However, as shown in Figure 4c, the enthalpy change in the
formation of DMMTAV-SCH3 indicates an exothermic reaction
(∆H ) -4.85 kcal/mol), while the reaction with the next CH3SH
molecule is a highly endothermic one (∆H ) +19.66 kcal/mol).
As a result, DMMTAV-SCH3 gives the minimum energy point
during the reaction sequence. This potential energy dia-
gram provides a rational understanding for the stability of

PentaValent Thioarsenic-GSH Conjugate
Chem. Res. Toxicol., Vol. 21, No. 2, 2008 553
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Figure 5. Potential energy diagrams for the CH3SSH elimination
reaction. The proposed reaction is presented in a, and an enthalpy
change (∆H, kcal/mol) of this reaction is presented in b.
DMMTAV-SCH3. Although another reaction pathway could
be assumed in which CH3SSH is eliminated from the
(CH3)2As(SH)(OH)(SCH3) intermediate to directly afford DMAI
-
II (Figure 5a), this pathway is also highly endothermic (∆H )
+26.87 kcal/mol, Figure 5a) and considered to be not favorable.
The results presented in this work could be the key to
understanding the physiological behavior of thioarsenicals. For
example, it is surmised that DMMTAV is hydrolyzed into
DMAV and undergoes further reduction, rather than forming
complexes with thiol groups under physiological conditions in
mammalian cells, as previously reported (12). However, this
result also demonstrates the possibility of the formation of the
DMMTAV-thiol conjugate in any organisms via a nonenzy-
matic reaction in acidic organelles, or via some type of
enzymatic reaction.
Acknowledgment. We acknowledge the Grant-in-Aid from
the Ministry of Education, Culture, Science, Sports and Tech-
nology, Japan (No. 16209004 and No. 17790098).
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