A synthetic model for the inhibition of glutathione peroxidase by antiarthritic gold compounds

Article abstract of DOI:10.1021/ic8019183

In this paper, inhibition of the glutathione peroxidase activity of two synthetic organoselenium compounds, bis- [2-(N,N-dimethylamino)benzyl]diselenide (5) and bis[2-(N,N-dimethylamino)benzyl]selenide (9), by gold(I) thioglucose (1), chloro(triethylphosp

Full text of DOI:10.1021/ic8019183

Inorg. Chem. 2009, 48, 2449-2455
A Synthetic Model for the Inhibition of Glutathione Peroxidase by
Antiarthritic Gold Compounds
Krishna P. Bhabak and Govindasamy Mugesh*
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012, India
Received October 8, 2008
In this paper, inhibition of the glutathione peroxidase activity of two synthetic organoselenium compounds, bis-
[2-(N,N-dimethylamino)benzyl]diselenide (5) and bis[2-(N,N-dimethylamino)benzyl]selenide (9), by gold(I) thioglucose
(1), chloro(triethylphosphine)gold(I), chloro(trimethylphosphine)gold(I), and chloro(triphenylphosphine)gold(I) is
described. The inhibition is found to be competitive with respect to a peroxide (H2O2) substrate and noncompetitive
with respect to a thiol (PhSH) cosubstrate. The diselenide 5 reacts with PhSH to produce the corresponding selenol
(6), which upon treatment with 1 equiv of gold(I) chlorides produces the corresponding gold selenolate complexes
11-13. However, the addition of 1 equiv of selenol 6 to complexes 11-13 leads to the formation of bis-selenolate
complex 14 by ligand displacement reactions involving the elimination of phosphine ligands. The phosphine ligands
eliminated from these reactions are further converted to the corresponding phosphine oxides (R3PdO) and selenides
(R3PdSe). In addition to the replacement of the phosphine ligand by selenol 6, an interchange between two
different phosphine ligands is also observed. For example, the reaction of complex 11 having a trimethylphosphine
ligand with triphenylphosphine produces complex 13 by phosphine interchange reactions via the formation of
intermediates 15 and 16. The reactivity of selenol 6 toward gold(I) phosphines is found to be similar to that of
selenocysteine.
Introduction
Gold compounds such as gold thiomalate (myochrisine,
GTM), gold thioglucose (1, solganol, GTG), and auranofin
(2, AUR) have been employed as therapeutic agents for
rheumatoid arthritis (RA) for many years (Figure 1).1 On
the basis of various effects of gold compounds in a number
of different model systems, several pathways have been
proposed to explain the mechanisms of action of gold
compounds in RA,2 but none has been generally accepted
as the mechanism by which gold compounds alter the course
of RA. Recent studies show that the gold drugs GTG and
AUR effectively inhibit certain selenoenzymes such as
Figure 1. Chemical structures of some therapeutic gold(I) compounds.
glutathione peroxidase (GPx),3 type I iodothyronine deio-
dinase (ID-1),4 and thioredoxin reductase (TrxR),5 indicating
that gold compounds may exert some of their pharmacologi-
cal effects by inhibiting one or more selenoenzymes.
*
To whom correspondence should be addressed. E-mail:
mugesh@ipc.iisc.ernet.in.
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Chem. ReV. 1999, 99, 2589–2600. (d) Tiekink, E. R. T. Crit. ReV.
Oncol./Hematol. 2002, 42, 225–248.
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Lipsky, P. E. J. Clin. InVest. 1992, 89, 1839–1848, and references
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10.1021/ic8019183 CCC: $40.75  2009 American Chemical Society
Inorganic Chemistry, Vol. 48, No. 6, 2009 2449
Published on Web 02/11/2009

Bhabak and Mugesh
However, recent evidence suggests that the gold compounds
such as 3 and 4 that inhibit selenoenzymes can also inhibit
GR by reacting with the active-site cysteine residues.8
Therefore, the indirect measurement of the GPx activity by
using the GR-GSSG coupled assay may lead to a complica-
tion in the mechanism of the inhibition of GPx by gold
compounds.
To understand the effect of gold compounds on the GPx
activity and to avoid the complication in the GR-GSSG
coupled assay, we have used bis[2-(N,N-dimethylamino)ben-
zyl]diselenide (5), which has been shown to mimic GPx by
reducing H2O2 in the presence of an aromatic thiol such as
benzenethiol (PhSH).9 By using PhSH instead of GSH, the
GPx activity can be directly measured by following the
formation of diphenyl disulfide (PhSSPh).10 The reduction
of the -Se-Se- bond in compound 5 by thiols leads to the
formation of the corresponding selenol (6), which is respon-
sible for the GPx-like catalytic activity of 5.9 The formation
of selenol 6 was further confirmed from the reaction of
selenol 6 with iodoacetic acid, which produces the corre-
sponding monoselenide as the only product (Figures S22 and
23 in the Supporting Information). If the inhibition of native
GPx by gold(I) compounds occurs through the formation of
a gold selenolate complex as shown in Figure 2, the catalytic
activity of GPx mimic 5 should also be inhibited by gold(I)
complexes. In this paper, we describe inhibition of the GPx-
like antioxidant activity of the diselenide 5 and the corre-
sponding monoselenide 9 by GTG and related gold(I)
compounds. We also describe that inhibition of the GPx
activity by gold(I) compounds is due to the interaction
between the selenol moiety and gold(I) compounds to form
gold selenolate complexes.
Figure 2. Proposed GPx catalytic mechanism and inhibition by AUR.
GPx is an antioxidant selenoenzyme that protects various
organisms from oxidative damage by catalyzing the reduction
of hydrogen peroxide and other organic peroxides with the
help of glutathione (GSH) as the reducing agent.6 The active
site of GPx includes a selenocysteine residue, which under-
goes a series of oxidation and reduction reactions during the
enzyme catalytic cycle (Figure 2). The reaction of the selenol
moiety (E-SeH) at the active site of GPx with H2O2 produces
the selenenic acid (E-SeOH) intermediate, which upon
reaction with GSH produces the corresponding selenenyl
sulfide (E-Se-SG). The attack of a second 1 equiv of GSH
at the -Se-S- linkage eliminates glutathione disulfide
(GSSG) and regenerates E-SeH.6c,7 In both in vivo and in
vitro systems, GSSG produced during the catalytic cycle is
reduced back to GSH by the NADPH-dependent glutathione
reductase (GR) (Figure 2). Therefore, the GPx activity is
generally measured indirectly by following the GR-catalyzed
reduction of GSSG.7
Experimental Section
It has been reported that the gold drugs GTG (1) and AUR
(2) inhibit the GPx activity probably by reacting with E-SeH
of the enzyme to form a stable gold selenolate complex.3
Furthermore, it has been postulated that gold compounds
must undergo ligand displacement reactions with GSH to
produce the gold-GSH complex [Au(SG)2]-, which reacts
with E-SeH to produce the corresponding E-Se-Au-SG
complex (Figure 2).3c Similar to measurement of the GPx
catalytic activity, the inhibition of GPx by gold compounds
is routinely followed by a GR-GSSG coupled assay.3a,c
General Procedure. Compounds 511a and 911b were synthesized
by following literature methods. 1H (400 MHz), 13C (100.5 MHz),
31P (161.9 MHz), and 77Se (76.3 MHz) NMR spectra were obtained
on a Bruker 400 MHz NMR spectrometer. Chemical shifts are cited
with respect to SiMe4 (1H and 13C) as internal standards and H3PO4
(31P) and Me2Se (77Se) as external standards. Mass spectral studies
were carried out on a Bruker Daltonics Esquire 3000plus mass
spectrometer with electrospray ionization mass spectrometry (ESI-
MS) mode analysis.
Synthesis of 6. To a solution of compound 5 (20.0 mg, 0.047
mmol) in CDCl3in an NMR tube was added dithiothreitol (DTT;
(4) Berry, M. J.; Kieffer, J. D.; Harney, J. W.; Larsen, P. R. J. Biol. Chem.
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Requir. Interact. 1998, 194–198. (b) Gromer, S.; Arscott, L. D.;
Williams, C. H., Jr.; Schirmer, R. H.; Becker, K. J. Biol. Chem. 1998,
273, 20096–20101.
(6) (a) Flohe´, L.; Gu¨nzler, E. A.; Schock, H. H. FEBS Lett. 1973, 32,
132–134. (b) Rotruck, J. T.; Pope, A. L.; Ganther, H. E.; Swanson,
A. B.; Hafeman, D. G.; Hoekstra, W. G. Science 1973, 179, 588–
590. (c) Mugesh, G.; Singh, H. B. Chem. Soc. ReV. 2000, 29, 347–
357.
(8) (a) Deponte, M.; Urig, S.; Arscott, L. D.; Fritz-Wolf, K.; Re´au, R.;
Herold-Mende, C.; Koncarevic, S.; Meyer, M.; Davioud-Charvet, E.;
Ballon, D. P.; Williams, C. H., Jr.; Becker, K. J. Biol. Chem. 2005,
280, 20625–20637. (b) Urig, S.; Fritz-Wolf, K.; Re´au, R.; Herold-
Mende, C.; To´th, K.; Davioud-Charvet, E.; Becker, K. Angew. Chem.,
Int. Ed. 2006, 45, 1881–1886.
(9) (a) Iwaoka, M.; Tomoda, S. J. Am. Chem. Soc. 1994, 116, 2557–
2561. (b) Mugesh, G.; Panda, A.; Singh, H. B.; Punekar, N. S.;
Butcher, R. J. J. Am. Chem. Soc. 2001, 123, 839–850. (c) Bhabak,
K. P.; Mugesh, G. Chem.sEur. J. 2008, 14, 8640–8651.
(7) (a) Wilson, S. R.; Zucker, P. A.; Huang, R.-R. C.; Spector, A. J. Am.
Chem. Soc. 1989, 111, 5936–5939. (b) Mugesh, G.; du Mont, W.-W.
Chem.sEur. J. 2001, 7, 1365–1370. (c) Jacob, C.; Giles, G. I.; Giles,
N. M.; Sies, H. Angew. Chem., Int. Ed. 2003, 42, 4742–4758. (d) Roy,
G.; Sarma, B. K.; Phadnis, P. P.; Mugesh, G. J. Chem. Sci. 2005,
117, 287–303. (e) Prabhakar, R.; Vreven, T.; Morokuma, K.; Musaev,
D. G. Biochemistry 2005, 44, 11864–11871. (f) Prabhakar, R.;
Morokuma, K.; Musaev, D. G. Biochemistry 2006, 45, 6967–6977.
(10) (a) Back, T. G.; Moussa, Z. J. Am. Chem. Soc. 2002, 124, 12104–
12105. (b) Back, T. G.; Moussa, Z.; Parvez, M. Angew. Chem., Int.
Ed. 2004, 43, 1268–1270. (c) Sarma, B. K.; Mugesh, G. J. Am. Chem.
Soc. 2005, 127, 11477–11485. (d) Bhabak, K. P.; Mugesh, G.
Chem.sEur. J. 2007, 13, 4594–4601.
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2450 Inorganic Chemistry, Vol. 48, No. 6, 2009

Inhibition of GPx by Antiarthritic Gold Compounds
11.0 mg, 0.070 mmol), and the solution was shaken well for uniform
mixing. The formation of the selenol 6 was monitored by the
disappearance of the peak for compound 5 (426 ppm) and
the appearance of a new peak at 38 ppm in 77Se NMR. Because of
the rapid aerial oxidation of selenol 6 to the corresponding
diselenide 5, it could not be characterized by other techniques.
However, the treatment of iodoacetic acid (2 equiv) to selenol 6
produces corresponding monoselenide, which was further character-
ized by 77Se NMR and mass spectrometric techniques. For the
monoselenide compound: 77Se NMR (ppm, CDCl3): δ 246. ESI-
MS. Calcd for C11H15NO2Se [(M + H)+]: m/z 274.0346. Found:
m/z 274.0341 (Figures S22 and S23 in the Supporting Information).
Synthesis of 10. To a solution of compound 9 (10 mg, 0.028
mmol) in a mixture of chloroform and methanol (4:1) was added
H2O2 (4.0 µL, 0.034 mmol), and the resulting solution was stirred
for 30 min at room temperature. The solvent was removed under
vacuum to obtain 10 as a white amorphous solid in quantitative
yield. 1H NMR (CDCl3, ppm): δ 2.07 (s, 12H), 3.36-3.40 (d, 2H,
J ) 13.6 Hz), 3.50-3.53 (d, 2H, J ) 13.6 Hz), 7.16-7.17 (d, 2H,
J ) 4.4 Hz), 7.28-7.30 (t, 4H, J ) 4.4 Hz), 7.72-7.73 (d, 2H, J
) 3.6 Hz). 13C NMR (CDCl3, ppm): δ 43.6, 62.0, 127.5, 127.7,
127.8, 129.1, 137.9, 141.7. 77Se NMR (CDCl3, ppm): δ 857. ESI-
MS. Calcd for C18H24N2OSe [(M + H)+]: m/z 365.1132. Found:
m/z 365.1125.
General Synthesis of Compounds 11-13. To the selenol 6,
generated from the corresponding diselenide 5, was added a
stoichiometric amount of R3PAuCl (R ) Me, Et, and Ph) in CDCl3
in an NMR tube. The yellow solution of selenol turned almost
colorless immediately upon the addition of gold(I) chlorides. The
formation of phosphinegold(I) selenolate species (11-13) was
characterized by 31P and 77Se NMR spectroscopy and mass
spectrometric techniques. Compound 11. 31P NMR (CDCl3, ppm):
δ -0.62. 77Se NMR (CDCl3, ppm): δ 87. ESI-MS. Calcd [(M +
H)+]: m/z 488.0321. Found: m/z 487.8654. Compound 12. 31P NMR
(CDCl3, ppm): δ 38.31. 77Se NMR (CDCl3, ppm): δ 76. ESI-MS.
Calcd [(M + H)+]: m/z 530.0790. Found: m/z 529.9380. Compound
13. 31P NMR (CDCl3, ppm): δ 37.50. 77Se NMR (CDCl3, ppm): δ
74. ESI-MS. Calcd [(M + H)+]: m/z 674.0790. Found: m/z
673.9761.
HPLC Assay. In this assay, we employed a mixture containing
a 1:2 molar ratio of PhSH and peroxide in methanol at room
temperature as our model system. Runs with and without catalyst
were carried out under the same conditions. In inhibition experi-
ments, increasing concentrations of the inhibitor were added to the
reaction mixture along with the catalyst. Periodically, aliquots were
injected into the reverse-phase C18 (Atlantis, 4.6 × 250 mm, 5 µm)
column and eluted with methanol and water (9:1), and the
concentrations of the product diphenyl disulfide (PhSSPh) were
determined at 254 nm using pure PhSSPh as an external standard.
The amount of disulfide formed during the course of the reaction
was calculated from the calibration plot for the standard (PhSSPh).
Inhibition of the GPx Activity by Gold Drugs. The gold(I)
compounds, such as GTG, and R3PAuCl (R ) Me, Et, and Ph)
were used as inhibitors of GPx mimics. Inhibition experiments of
the GPx-like activities of test compounds by gold(I) drugs were
performed in the presence of an aromatic thiol (PhSH) and different
hydroperoxides [hydrogen peroxide (H2O2), cumene hydroperoxide
(Cum-OOH), and tert-butyl hydroperoxide (t-BuOOH)] using the
reverse-phase HPLC method. Each reaction rate for the reduction
of peroxide was measured at least three times and was corrected
for the background reaction between peroxide and thiol. The rate
for the reduction of peroxides by selenium compounds in the
absence of gold(I) compounds (inhibitors) was considered as 100%
(control activity), and inhibition of the GPx activity in the presence
of various concentrations of gold(I) compounds (inhibitors) was
expressed as a percentage of the control activity. The IC50 values
represent the concentrations of gold(I) compounds required to inhibit
50% of the catalytic activity. These values were calculated by
plotting the percent control activity with respect to the concentration
of gold(I) compounds. The data points were plotted as a sigmoidal
curve fitting using Origin 6.1 software.
Results and Discussion
Mechanism of Inhibition of the GPx Activity of 5 by
Gold(I) Compounds. To understand the mechanism by
which GTG and other gold(I) compounds inhibit the GPx
activity, we have studied the effect of GTG, Et3PAuCl,
Me3PAuCl, and Ph3PAuCl on the reduction of peroxides by
compound 5 in the presence of PhSH. The advantage of using
PhSH as the thiol cofactor is that the activity of 5 can be
followed directly by measuring the rate of formation of
PhSSPh.10 Because there is no GR or NADPH involved,
inhibition of the GPx activity by gold(I) compounds must
be due to the reaction between selenol (6) and gold(I)
compounds. The inhibition experiments were carried out by
using H2O2, t-BuOOH, and Cum-OOH as substrates and
PhSH as a cosubstrate. Interestingly, the trialkyl- or triph-
enylphosphinegold compounds inhibited the GPx activity
much stronger than GTG, supporting the assumption that the
higher inhibitory effect of AUR as compared with GTG is
due to the presence of a triethylphosphine moiety in AUR,
which may stabilize the Au-Se bond (Table 1). In general,
the gold(I) chlorides exhibited higher activity (lower IC50
values) as compared to GTG in all three peroxide assays
(H2O2, t-BuOOH, and Cum-OOH) employed in the present
study, indicating that gold(I) chlorides are better than GTG
as inhibitors in the presence of PhSH.
Furthermore, inhibition of the GPx activity of compound
6 is due to a direct interaction between the selenol moiety
and gold(I) complexes because the thiol cosubstrate (PhSH)
present in the assay does not react with the gold(I) complexes
(Figures S17-S19 in the Supporting Information). Therefore,
inhibition of the GPx activity by gold(I) complexes is
expected to be competitive with respect to peroxide. On the
basis of the inhibition of GPx by AUR (Figure 2), the selenol
6 can react either with the peroxide (H2O2) to produce the
corresponding selenenic acid 7 or with GTG to produce the
corresponding gold selenolate complex 8 (Figure 3). To
understand the nature of inhibition of the GPx activity, we
have performed detailed kinetic experiments at different
concentrations of GTG by varying the concentrations of the
Table 1. Comparison of IC50 Values for Inhibition of the GPx Activity
of 5 by Some Gold(I) Compounds
IC50 values (µM)a
gold(I) compounds
H2O2
t-BuOOH
Cum-OOH
GTG
46.9 ( 1.8
16.8 ( 0.1
18.2 ( 1.0
13.7 ( 0.3
40.6 ( 0.3
14.3 ( 0.5
17.3 ( 0.7
13.7 ( 0.6
43.2 ( 0.5
14.7 ( 0.2
19.4 ( 0.3
12.4 ( 0.5
Et3PAuCl
Me3PAuCl
Ph3PAuCl
a The concentration required to inhibit 50% of the catalytic activity. Assay
conditions: inhibitor (variable), compound 5 (2.5 µM), PhSH (5 mM), and
peroxide (10 mM) in MeOH at 23 °C.
Inorganic Chemistry, Vol. 48, No. 6, 2009 2451

Bhabak and Mugesh
Figure 3. Two possible modes of reaction for the selenol 6 in the presence
of H2O2 and GTG.
Figure 5. Lineweaver-Burk plots obtained for inhibition of the GPx
activity catalyzed by compound 5 in the presence of PhSH at various
concentrations of GTG. [GTG]: (a) 30 µM; (b) 50 µM; (c) 70 µM; (d) 90
µM. For each concentration of GTG, the cosubstrate (PhSH) concentration
was varied, while keeping the concentration of H2O2 constant.
Figure 4. Lineweaver-Burk plots obtained for inhibition of the GPx
activity catalyzed by 5 in the presence PhSH at various concentrations of
GTG. [GTG]: (a) 50 µM; (b) 60 µM; (c) 70 µM; (d) 90 µM; (e) 110 µM.
For each concentration of GTG, the substrate (H2O2) concentration was
varied, while keeping the concentration of PhSH constant.
Figure 6. Reactivity of selenol 6 toward GTG and PhSH.
substrate (H2O2) and thiol cosubstrate for each GTG con-
centration. The initial rates (V0) were calculated for each
concentration of GTG and H2O2 at a fixed concentration of
PhSH. The Lineweaver-Burk plots obtained by plotting 1/V0
versus 1/[H2O2] show linear lines for different concentrations
of GTG, and all of these straight lines intersect the ordinate
with almost identical Vmax values (Figure 4), suggesting that
inhibition of the GPx activity of 5 by GTG is competitive
with respect to the substrate, H2O2.
Figure 7. Mechanism for the catalytic reduction of H2O2 by the monose-
lenide 9 in the presence of PhSH. The formation of PhSSPh was followed
by HPLC techniques, and oxidation-reduction reactions at the selenium
center were confirmed by 77Se NMR spectroscopy.
Although inhibition of the GPx activity of 6 by GTG is
competitive with respect to the peroxide substrates, the
comparable IC50 values obtained for different peroxides
(Table 1) indicate that the nature of peroxide has little effect
on the inhibition. This is in agreement with our previous
observations that the nature of peroxide does not affect the
GPx-like activity of ebselen and related compounds.10d
We carried out further kinetic experiments with different
concentrations of PhSH and GTG at a fixed concentration
of H2O2 to find out the effect of the thiol concentration on
the inhibition. In contrast to the effect of H2O2 on the
inhibition, the Lineweaver-Burk plots obtained from these
experiments show parallel lines, which do not intersect at a
common point (Figure 5), indicating that inhibition of the
GPx activity of 5 by GTG is noncompetitive with respect to
the thiol cosubstrate. Although selenol 6 reacts with the
inhibitor (GTG) to produce the corresponding gold selenolate
complex (8), it does not directly react with the thiol (PhSH)
present in the assay system. Therefore, any variation in the
concentration of the thiol cosubstrate does not affect the
reactivity of selenol 6 toward GTG (Figure 6). This is also
reflected in the noncompetitive nature of the inhibition of
compound 5 by GTG with respect to PhSH. Furthermore,
GTG and other gold(I) compounds do not react with other
catalytic intermediates such as selenenic acid or selenenyl
sulfide in the redox cycle. However, the inactivity of the
electrophilic gold compounds toward selenenic acid or
selenenyl sulfide is not surprising because the selenium atoms
in these intermediates are also electrophilic in nature and an
attack of gold at selenium in these intermediates is not a
favored process.
Effect of Gold(I) Compounds on the GPx-like Activ-
ity of Diaryl Monoselenide 9. The observations that
inhibition of the GPx activity of 5 is due to the reaction of
selenol 6 with gold(I) compounds are further supported by
experiments with the monoselenide 9, which does not
produce any selenol species during the catalytic cycle.
Although the GPx activity of compound 9 (18.4 µM·min-1)
at 250 µM concentration was found to be lower than that of
the diselenide 5 (46.0 µM · min-1) at 2.5 µM concentration,
the activity of 9 was sufficient to follow the inhibition by
Et3PAuCl. It should be noted that compound 9 exhibits its
GPx-like activity via oxidation of the selenium center, as
shown in Figure 7. The oxidation of 9 by H2O2 produced
the corresponding selenoxide 10, which could be conve-
niently reduced back to the monoselenide 9 by thiols. The
formation of selenoxide 10 was confirmed by isolation and
spectral characterization of the product from the reaction of
compound 9 with hydrogen peroxide. The 77Se NMR
experiments indicated that there was no selenol produced
during the entire process.
2452 Inorganic Chemistry, Vol. 48, No. 6, 2009

Inhibition of GPx by Antiarthritic Gold Compounds
in identifying the products formed in the reactions. The mass
spectrum of the product obtained by treating 6 with
Ph3PAuCl showed a peak at 674.0 ppm with the expected
isotopic pattern, which can be ascribed to the gold(I)
selenolate complex (13). Similarly, the formation of com-
pounds 11 and 12 was also confirmed by NMR (31P and 77Se)
and mass spectrometric techniques. The reaction of N-Boc-
L-selenocysteine methyl ester13 with Me3PAuCl produced the
corresponding gold selenolate complex (Figures S50-S54
in the Supporting Information), indicating that the reactivity
of 6 toward gold(I) complexes is similar to that of the
selenocysteine moiety in GPx.
Coffer et al. have previously shown that the reaction of
serum albumin (Alb) with AUR produces a gold thiolate
complex. They have also shown that thiols can displace either
the albumin or the triethylphosphine moiety from the
protein-gold complex, AlbSAuPEt3.14 Shaw et al. have
reported an interesting observation that the reaction of Alb
with the triisopropylphosphine analogue of AUR eliminates
triisopropylphosphine, which, in turn, reacts with the
protein-disulfide bond to produce the corresponding phos-
phine oxide and sulfide.14b Although such displacement
reactions have not been reported for gold selenolate com-
plexes of selenoenzymes,15 the presence of thiols may affect
the stability of gold selenolates. Therefore, we have studied
the interactions of complexes 11-13 with thiols such as
PhSH and DTT. Although compounds 11-13 are reasonably
stable in solution, the 31P and 77Se NMR spectroscopic
experiments suggest that some ligand displacement reactions
take place in the presence of thiols.15 When the reaction of
selenol 6 (produced in situ from the reaction of the diselenide
5 with DTT) with Ph3PAuCl was followed by 31P NMR
spectroscopy, the signal for Ph3PAuCl (33.3 ppm) disap-
peared completely upon addition of the selenol 6. The
Figure 8. Effect of Et3PAuCl on the GPx activity of compounds 5 and 9.
The amount of inhibition is expressed as a percent of the control activity
for compounds 5 (a) and 9 (b) in the absence of Et3PAuCl. The initial rates
(V0) for the reduction of H2O2 by 5 (2.5 µM) and 9 (250 µM) in the absence
of added inhibitor were 46.0 and 18.4 µM·min-1, respectively.
Interestingly, while the GPx activity of compound 5 was
completely abolished at 50-60 µM Et3PAuCl, the activity
of the monoselenide 9 was almost unaffected by treatment
with Et3PAuCl up to a concentration of 60 µM (Figure 8).
The reactivity of the monoselenide 9 toward gold compounds
was further confirmed by 77Se and 31P NMR experiments.
For example, the 77Se NMR signal observed at 342 ppm for
compound 9 was unaffected upon the addition of chloro-
(trimethylphosphine)gold(I). Similarly, the 31P NMR signal
observed for chloro(trimethylphosphine)gold(I) at -10.10
ppm was unchanged in the presence of compound 9 (Figures
S14 and S49 in the Supporting Information). These experi-
ments suggest that the monoselenide 9 does not react with
gold(I) chlorides during the inhibition assay. Therefore,
inhibition of the GPx activity of selenium compounds by
gold(I) drugs can be ascribed entirely to the reaction between
selenol and gold compounds to afford the corresponding gold
selenolate species. The complete inactivation of 5 by
Et3PAuCl indicates that the high GPx activity of this
compound is clearly due to the conversion of diselenide 5
to the corresponding selenol (6) and the activation of the
selenol moiety by the tertiary amino substituent. Therefore,
the gold-based inhibition can also be used to understand the
role of selenol in the GPx-like catalytic mechanism.
Ligand-Exchange Reactions in Gold Selenolate Com-
plexes. Our further studies on the reactivity of selenol 6
toward GTG and R3PAuCl (R ) Me, Et, or Ph) reveal that
the gold(I) chlorides are much more reactive than GTG
toward the selenol moiety. The reaction of 6 with GTG did
not give any isolable product. This reaction afforded a pale-
yellow insoluble material, which could not be characterized.
On the other hand, the reactions of 6 with R3PAuCl afforded
the corresponding gold(I) selenolates (Figure 9), which were
identified by 31P and 77Se NMR spectroscopy and mass
spectrometric techniques (these data are included in the
Supporting Information). For example, free Ph3PAuCl ex-
hibited a 31P NMR signal at 33.3 ppm, which was shifted
slightly downfield (37.5 ppm, in CDCl3) for the gold(I)
selenolate (13).12 Similarly, the 77Se NMR signal observed
at 37 ppm for the selenol 6 showed considerable downfield
shift (74 ppm, in CDCl3) upon coordination with a gold(I)
compound. Mass spectrometry was also found to be useful
(12) The 31P NMR chemical shifts observed for trialkyl/arylphosphinegold
selenolate complexes 11-13 are comparable with that of the triph-
enylphosphinegold(I) complex (36.8 ppm) derived from o-carborane.
Canales, S.; Crespo, O.; Gimeno, M. C.; Jones, P. G.; Laguna, A.;
Romero, P. Dalton Trans. 2003, 4525–4528.
(13) N-Boc-L-selenocysteine methyl ester was synthesized by the following
literature method. (a) Bhat, R. G.; Porhiel, E.; Saravanan, V.;
Chandrasekaran, S. Tetrahedron Lett. 2003, 44, 5251–5253. (b)
Phadnis, P. P.; Mugesh, G. Org. Biomol. Chem. 2005, 3, 2476–2481.
(14) (a) Coffer, M. T.; Shaw, C. F., III.; Hormann, A. L.; Mirabelli, C. K.;
Crooke, S. T. J. Inorg. Biochem. 1987, 30, 177–187. (b) Shaw, C. F.,
III.; Isab, A. A.; Hoeschele, J. D.; Starich, M.; Locke, J.; Schulteis,
P.; Xiao, J. J. Am. Chem. Soc. 1994, 116, 2254–2260. (c) Roberts,
J. R.; Xiao, J.; Schliesman, B.; Parsons, D. J.; Shaw, C. F., III. Inorg.
Chem. 1996, 35, 424–433.
(15) In the presence of GSH, the ligand displacement reactions may take
3c
-
place to produce gold glutathione complex Au(SG)2
.
It has been
reported that thiourea can replace both the triethylphosphine (PEt3)
and thioglucose (SATg-) moieties from AUR. The eliminated trieth-
ylphosphine may be converted to the corresponding triethylphosphine
oxide.14a A similar ligand displacement reaction was observed when
DTT was added to the gold selenolate complex. However, when PhSH
was present, the phosphine displacement reaction was found to be
extremely slow. For example, the 31P NMR signal observed for
Ph3PAuCl at 33.3 ppm in CDCl3 was unchanged upon the addition of
even a large excess (10 equiv) of PhSH. Similar results were obtained
when the experiments were carried out in MeOH. Furthermore, the
phosphine ligand displacement by PhSH was not observed after
the formation of gold selenolate complexes (11-13), suggesting that
the gold selenolate complexes having a Se-Au-P moiety can be
obtained in the presence of thiols. Ahhmad, S.; Isab, A. A. J. Inorg.
Biochem. 2002, 88, 44–52.
Inorganic Chemistry, Vol. 48, No. 6, 2009 2453

Bhabak and Mugesh
Figure 9. Reduction of diselenide 5 to produce selenol 6 and the reaction of 6 with R3PAuCl (R ) Me, Et, or Ph).
products obtained from this reaction exhibited a major peak
at 37.5 ppm along with two minor peaks at 35.4 and 31.1
ppm. The major peak at 37.5 ppm can be assigned to the
expected gold selenolate complex 13, which is further
confirmed by 77Se NMR and mass spectral analysis (Sup-
Figure 10. Reaction of gold selenolate 11 with selenol 6. The trimeth-
ylphosphine eliminated from this reaction is converted to trimethylphosphine
oxide and the corresponding selenide.
porting Information). The peak at 31.1 ppm indicates the
formation of triphenylphosphine oxide (Ph3PdO) during the
reaction. This is due to the spontaneous oxidation of Ph3P
produced by a ligand displacement reaction. A control
experiment involving the reaction of Ph3PAuCl with DTT
indicates that DTT does not replace the triphenylphosphine
in Ph3PAuCl, but the ligand displacement takes place only
after formation of the gold selenolate complex (13). Interest-
ingly, the third product obtained from this reaction was
identified as triphenylphosphine selenide (Ph3PdSe), which
exhibited a peak at 35.4 ppm with the expected 31P-77Se
coupling (JP-Se ) 727 Hz). The intensity of the signal at
35.4 ppm was increased upon the addition of an excess
amount of DTT, indicating that the ligand-exchange reaction
confirmed by mass spectrometric techniques. The peak
corresponding to the eliminated trimethylphosphine was not
observed because the trimethylphosphine produced in this
reaction was further converted to trimethylphosphine selenide
and a trace amount of trimethylphosphine oxide (Figure 10).
This indicates that inhibition of the GPx activity of compound
5 by R3PAuCl is due to the formation of gold selenolate
complexes 11-13 or the bis-selenolate complex 14. How-
ever, the nature of the species formed in this reaction depends
on the ratio of selenol to R3PAuCl concentration. Therefore,
during the inhibition experiments, at a lower concentration
of R3PAuCl, the bis-selenolate complex 14 is expected to
be the predominant species because the reaction of 0.5 equiv
of Me3PAuCl with 1 equiv of selenol 6 produces 14 as the
major product, whereas the formation of gold selenolate
complexes 11-13 may dominate at higher concentrations
of gold(I) chlorides. This is in agreement with the forma-
tion of a linear -S-Au-S- coordination during the
inhibition of human glutathione reductase by compound 3.8b
The reaction of trimethylphosphine complex 11 with Ph3P
produced the corresponding triphenylphosphine complex
(13), indicating that the ligand displacement reactions can
occur with different phosphines. In this phosphine-exchange
reaction, two complexes 11 and 13 do not appear to be in
equilibrium because the PMe3 eliminated in this reaction is
further oxidized to produce Me3PdO and Me3PdSe along
with formation of the triphenylphosphine derivatives
(Ph3PdO and Ph3PdSe). The 31P and 77Se NMR spectro-
scopic experiments indicate that the phosphine-exchange
reaction in complex 11 may take place via the formation of
a trimethylphosphine-triphenylphosphine intermediate (15).
This compound undergoes a further phosphine-exchange
reaction in the presence of an excess amount of PPh3
(5 equiv) to produce another intermediate (16) having
enhances formation of the Ph3PdSe species. A series of 31
P
and 77Se NMR experiments indicate that the attack of DTT
at the gold center leads to the elimination of Ph3P. The rapid
reaction of Ph3P with elemental selenium produces Ph3PdSe.
The elemental selenium present in the system is produced
during the reductive cleavage of diselenide 5 by DTT to
generate the active selenol 6. Because the complexation
reactions were performed with the in situ generated selenol
6 and gold chlorides, a minor amount of the eliminated
trialkyl/arylphosphines by ligand displacement reactions
reacts with the elemental selenium to produce trialkyl/
arylphosphine selenide. The formation of triphenylphoshine
selenide was confirmed independently by treating triph-
enylphosphine with elemental selenium under identical
experimental conditions (Figures S59 and S60 in the Sup-
porting Information). Similarly, compounds 11 and 12
undergo ligand displacement reactions to produce trimeth-
ylphosphine oxide and triethylphosphine oxide, respectively.
Furthermore, the formation of trimethyl- and triethylphos-
phine selenide was also observed in these reactions.
Although DTT enhances ligand displacement reactions
(Figure S26 in the Supporting Information), the stability of
the gold selenolate complex is unaffected by PhSH. Interest-
ingly, when complex 11 was treated with selenol 6, the 77Se
NMR signal at 87 ppm for complex 11 disappeared com-
pletely to produce a new signal at 57 ppm, which can be
ascribed to the formation of complex 14 (Figure 10).16 The
formation of compound 14 in this reaction was further
+
Au(PPh3)2 as the counterion. The intermediate 16 is then
dissociated to produce the gold(I) selenolate complex 13
(Figure 11).
When 0.5 equiv of triphenylphosphine was added to
compound 11, both the starting material (11) and the
triphenylphosphine derivative (13) were detected by 31P
NMR and mass spectral studies (Figures S35-S37 in the
Supporting Information). The addition of an excess amount
of triphenylphosphine (5 equiv) to the reaction mixture led
to the complete conversion of 11 to 13 with an increase in
(16) (a) Jones, P. G.; Tho¨ne, C. Inorg. Chim. Acta 1991, 181, 291–294.
(b) Baker, M. V.; Barnard, P. J.; Berners-Price, S. J.; Brayshaw, S. K.;
Hickey, J. L.; Skelton, B. W.; White, A. H. Dalton Trans. 2006, 3708–
3715. (c) Hickey, J. M.; Ruhayel, R. A.; Barnerd, P. J.; Baker, M. V.;
Berners-Price, S. J.; Filipovska, A. J. Am. Chem. Soc. 2008, 130,
12570–12571.
2454 Inorganic Chemistry, Vol. 48, No. 6, 2009

Inhibition of GPx by Antiarthritic Gold Compounds
Figure 11. Phosphine-exchange reaction in complex 11 and the formation of complex 13.
the intensity of the peak for Me3PdO. The formation of 15
as an intermediate during the conversion of 11 to 13 can be
rationalized from the line broadening in the 31P NMR signals
and the appearance of a new signal at 90 ppm in the 77Se
NMR spectrum. Although three-coordinate gold(I) phosphine
complexes are known,17 the mass spectral data indicate
the formation of [R3PAuPR3]+ during the ligand-excha-
nge reaction (Figure 11). This is in agreement with previous
reports that the formation of two-coordinate [Ph3PAuPPh3]+
species can be isolated in the reaction between Ph3PAuCl
and Ph3P in the presence of sterically bulky counterions
gold selenolate compound. Therefore, inhibition of the GPx
activity by gold compounds can be used as a probe for
identifying the involvement of a selenol in the catalytic
mechanism of synthetic selenium compounds. The gold
selenoate complexes having phosphine ligands undergo
ligand displacement reactions to produce the bis-selenolate
complex in the presence of selenols. In addition to the for-
mation of trialkyl- and triphenylphosphine oxides, these
ligand-exchange reactions lead to the formation of the
corresponding phosphine selenides. Although gold(I) com-
pounds inhibit the GPx activity in vivo, more research is
warranted to determine whether gold(I) inhibition of GPx
has any negative effect on the antioxidant defense mecha-
nism.
18
-
-
-
(PF6 , BF4 , and NO3 ).
Conclusions
In summary, inhibition of the GPx activity by antiarthritic
gold drugs has been studied by utilizing synthetic selenium
compounds as enzyme mimics. The competitive nature of
inhibition of the GPx activity of diaryl diselenide 5 by gold(I)
drugs with respect to the peroxides and the absence of any
such inhibition in the case of diaryl monoselenide 9 support
the assumption that gold(I) compounds inhibit the GPx
activity by reacting with the active selenol moiety to form a
Acknowledgment. This study was supported by the
Department of Science and Technology (DST), New Delhi,
India. We are grateful to the Alexander von Humboldt
Foundation, Bonn, Germany, for the donation of an auto-
mated flash chromatography system. G.M. acknowledges the
DST for a Ramanna Fellowship, and K.P.B. thanks the
Council of Scientific and Industrial Research (CSIR), New
Delhi, India, for a research fellowship.
(17) Bowmaker, G. A.; Dyason, J. C.; Healy, P. C.; Engelhardt, L. M.;
Pakawatchai, C.; White, A. H. J. Chem. Soc., Dalton Trans. 1987,
1089–1097.
(18) (a) Guy, J. J.; Jones, P. G.; Sheldrick, G. M. Acta Crystallogr. 1976,
B32, 1937–1938. (b) Mays, M. J.; Vergnano, P. A. J. Chem. Soc.,
Dalton Trans. 1979, 1112–1115. (c) Staples, R. J.; King, C.; Khan,
M. N. I.; Winpenny, R. E. P.; Fackler, J. P., Jr. Acta Crystallogr.
1993, C49, 472–475. (d) Wang, J.-C. Acta Crystallogr. 1996, C52,
611–613.
Supporting Information Available: Selected tables and all of
the inhibition plots and 31P and 77Se NMR and mass spectra for
the reactions of the thiols and selenols with gold(I) compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
IC8019183
Inorganic Chemistry, Vol. 48, No. 6, 2009 2455