Kinetics and mechanism of reactions of the drug tiopronin with platinum(IV) complexes

Article abstract of DOI:10.1016/j.jinorgbio.2013.04.003

Tiopronin, a synthetic thiol-containing drug being used in treatments of cystinuria and certain types of rare arthritis, is also a hepatoprotective and a detoxifying agent. Many analytical methods have been developed based on its redox chemistry with metal ions/complexes, but the kinetic and mechanistic aspects are poorly understood. In this work, the oxidation of tiopronin by cisplatin prodrug and a model compound, cis-[Pt(NH₃) ₂Cl₄] and trans-[PtCl₂(CN)₄] ^{2 -}, was investigated. The oxidation kinetics was followed by a stopped-flow spectrophotometer over a wide pH range under the pseudo first-order conditions of [Tiopronin][Pt(IV)]. Time-resolved spectra were also recorded for both Pt(IV) complexes, enabling to establish an overall second-order rate law: - d[Pt(IV)] / dt = k′[Tiopronin][Pt(IV)], where k′ pertains to observed second-order rate constants. Under the kinetic conditions, tiopronin was oxidized to form the tiopronin-disulfide exclusively as identified by mass spectrometry. A reaction mechanism was proposed, involving parallel reductions of the Pt(IV) complexes by the three protolytic tiopronin species as rate-determining steps. The rate constants for the rate-determining steps were derived. The fully deprotonated tiopronin is about 4 × 10⁴ more reactive than its corresponding thiol form for both Pt(IV) complexes; the huge reactivity difference orchestrates closely with the fact that the nucleophilicity of thiolate is much higher than the corresponding thiol. Hence, the attack of the sulfur atom in thiol/thiolate of tiopronin on the axially-coordinated chloride in the Pt(IV) complexes is nucleophilic in nature in the rate-determining steps, resulting in a bridge formation and a subsequent bridged electron-transfer.

Full text of DOI:10.1016/j.jinorgbio.2013.04.003

Journal of Inorganic Biochemistry 125 (2013) 9–15
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Kinetics and mechanism of reactions of the drug tiopronin with
platinum(IV) complexes
Shuying Huo a,b, Hongmei Shi , Dongzhi Liu ⁎, Shigang Shen ⁎, Jiong Zhang ,
b,1
a,
b,
b
b
b,
Changying Song , Tiesheng Shi ⁎
a
School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China
Key Laboratory of Medicinal Chemistry and Molecular Diagnostics, The Ministry of Education and College of Chemistry and Environmental Science, Hebei University, Baoding 071002,
b
Hebei Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Tiopronin, a synthetic thiol-containing drug being used in treatments of cystinuria and certain types of rare
arthritis, is also a hepatoprotective and a detoxifying agent. Many analytical methods have been developed
based on its redox chemistry with metal ions/complexes, but the kinetic and mechanistic aspects are poorly under-
Received 21 December 2012
Received in revised form 28 February 2013
Accepted 11 April 2013
stood. In this work, the oxidation of tiopronin by cisplatin prodrug and a model compound, cis-[Pt(NH
3
)
2
Cl
4
] and
Available online 19 April 2013
2
−
2 4
trans-[PtCl (CN) ]
, was investigated. The oxidation kinetics was followed by a stopped-flow spectrophotometer
over a wide pH range under the pseudo first-order conditions of [Tiopronin]≫[Pt(IV)]. Time-resolved
spectra were also recorded for both Pt(IV) complexes, enabling to establish an overall second-order
rate law: −d[Pt(IV)] / dt = k′[Tiopronin][Pt(IV)], where k′ pertains to observed second-order rate constants.
Under the kinetic conditions, tiopronin was oxidized to form the tiopronin-disulfide exclusively as identified by
mass spectrometry. A reaction mechanism was proposed, involving parallel reductions of the Pt(IV) complexes
by the three protolytic tiopronin species as rate-determining steps. The rate constants for the rate-determining
steps were derived. The fully deprotonated tiopronin is about 4 × 104 more reactive than its corresponding
thiol form for both Pt(IV) complexes; the huge reactivity difference orchestrates closely with the fact that the
nucleophilicity of thiolate is much higher than the corresponding thiol. Hence, the attack of the sulfur atom in
thiol/thiolate of tiopronin on the axially-coordinated chloride in the Pt(IV) complexes is nucleophilic in nature
in the rate-determining steps, resulting in a bridge formation and a subsequent bridged electron-transfer.
Keywords:
Tiopronin
Pt(IV) complex
Oxidation kinetics
Mechanism
©
2013 Elsevier Inc. All rights reserved.
1
. Introduction
and chemiluminescent approaches [19–22]. Moreover, some of the
analytical methods are based on the oxidation chemistry of tiopronin
by metal ions/complexes involving iron(III) [17,18], thallium(III) [19],
and cesium(IV) [20,21]. Kinetically or pharmacokinetically, studies on
tiopronin are, in contrast, very scarce [23–26]. As a consequence, we
have carried out a systematic investigation on kinetics and mechanisms
of oxidation of tiopronin by some important metal ions/complexes; in
this work we report the oxidation of tiopronin by two platinum(IV) com-
Tiopronin (also called thiola, N-(mercaptopropionyl)glycine, a race-
mic mixture around the single chiral center, cf. the structure in Fig. 1) is
a synthetic thiol-containing drug, which has been used to treat cystinuria
[1,2] and certain types of rare arthritis [3,4]. It has also been used as a
hepatoprotective agent [5,6], and a detoxifying agent. For instance,
tiopronin was found to have a function of protecting the nephtotoxicity
and ototoxicity caused by cisplatin [7–9]. In addition, tiopronin protected
gold clusters/nanoparticles have been found to possess some very attrac-
tive properties [10–12].
A number of analytical methods have been developed for the
determinations of tiopronin in various kinds of samples due to its
widely therapeutic applications as pointed out above; these methods
encompass chromatographic [13–15], spectrophotometric [16–18],
2
−
plexes: cis-[Pt(NH
Fig. 1).
3
)
2
Cl
4
] and trans-[PtCl
2
(CN)
4
]
(structures shown in
Currently, there is a strong interest in the designs and syntheses of
new type of platinum(IV) anticancer drugs and meanwhile, new drug
delivery methods for some Pt(IV) anticancer prodrugs are vigorously
pursued in order to find a new generation of platinum based anticancer
3 2 4
drugs [27–37]. cis-[Pt(NH ) Cl ] is the prodrug of cisplatin due to its
readiness of reduction and to the formation of cisplatin [27–31].
2
−
2 4
trans-[PtCl (CN) ]
has been used as a model compound for the
⁎
Corresponding authors. Tel.: +86 3125079525; fax: +86 312 5079525.
E-mail addresses: dzliu@tju.edu.cn (D. Liu), shensg@hbu.edu.cn (S. Shen),
platinum(IV) anticancer drugs such as ormaplatin (cf. Fig. 1) as it has
several characters very suitable to the kinetic studies [38–40]. In this
work, the oxidations of tiopronin by the two Pt(IV) complexes are char-
acterized kinetically over a wide pH range and a detailed mechanistic
rock@hbu.edu.cn (T. Shi).
1
Present address: School of Public Health, Hebei Medical University, Shijiazhuang
0
50017, Hebei Province, PR China.
0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2013.04.003

10
S. Huo et al. / Journal of Inorganic Biochemistry 125 (2013) 9–15
_
2
Cl
Pt
Cl
Cl
Pt
H N
Cl
Cl
3
CN
CN
Cl
Cl
NC
NC
NH2
NH2
Pt
H N
3
Cl
Cl
Cl
2-
cis-[Pt(NH ) Cl ]
trans-[PtCl (CN) ]
2 4
3
2
4
Ormaplatin
O
NH
NH
HOOC
S
HOOC
SH
S
COOH
NH
O
O
Tiopronin
Tiopronin-disulfide (fully protonated form)
Fig. 1. Structures of cis-[Pt(NH
3
)
2
Cl
4 2
], trans-[PtCl (CN)
4
]2−, ormaplatin, tiopronin (a racemic mixture at the chiral center), and tiopronin-disulfide.
picture is delineated. Moreover, the oxidation products are well charac-
terized by mass spectrometry.
0.90 M NaClO , 0.09 M NaCl and 0.01 M HCl. The stock solution of
4
cis-[Pt(NH Cl ] was used daily in fresh. Solutions of platinum(IV)
4
)
3 2
complexes and tiopronin were prepared, respectively, by adding an
appropriate amount of the Pt(IV) stock solution and of tiopronin to
the buffer. Those solutions were flushed for 10 min with nitrogen before
loading on the stopped-flow machine and were only used for a couple of
hours. Reactions were initiated by mixing equal volumes of platinum(IV)
and tiopronin solutions directly in the stopped-flow machine and were
followed under pseudo-first-order conditions with tiopronin being at
least 10-fold excess.
2
. Materials and methods
2
.1. Materials
2 4 2 3 2 4
Tiopronin, L-glutathione, K [Pt(CN) ]·3H O and cis-[Pt(NH ) Cl ]
were obtained from Sigma-Aldrich (St. Louis, MO). Acetic acid, sodium
acetate, sodium dihydrogenphosphate, disodium hydrogenphosphate,
sodium carbonate, and sodium bicarbonate, sodium perchlorate, and
perchloric acid were, all in analytical grade, purchased either from Fisher
Scientific (in Beijing, China) or from Alfa Aesar (in Tianjin, China) and
were used for preparations of buffer solutions without further purifica-
tion. The synthesis of K
method reported in a previous work [41]; the UV-Visible (UV-vis) spec-
tra of the solutions prepared from K [PtCl (CN) ] are in excellent agree-
ment with that reported earlier for trans-[PtCl
distilled water was used to prepare all the solutions.
2.4. Time-resolved spectra
UV–vis spectra were recorded with a TU-1900 spectrophotometer
(Beijing Puxi, Inc., Beijing, China) using 1.00 cm quartz cells. The
spectrophotometer was equipped with a cell compartment which
could be thermostated by circulation of water from a thermostate
(BG-chiller E10, Beijing Biotech Inc., Beijing). Time-resolved spectra
were recorded on the above spectrophotometer for the reaction between
2 2 4
[PtCl (CN) ] was carried out according to the
2
2
4
2
−
2
4
(CN) ]
[42]. Doubly
cis-[Pt(NH
For the reaction between trans-[PtCl
3 2 4
) Cl ] and tiopronin in an acetic acid/acetate buffer of pH 3.68.
2
−
2
.2. Buffer solutions
2
(CN)
4
]
and tiopronin, the time-
resolved spectra were recorded on the stopped-flow spectrometer in
The following combinations (with concentrations of 0.2–0.3 M)
were prepared to cover a wide pH range: acetic acid/sodium acetate,
NaH PO /Na HPO and NaHCO /Na CO and Na HPO /Na PO . All
0.020 M perchloric acid solutions.
2
4
2
4
3
2
3
2
4
3
4
2.5. Mass spectrometric analysis
the buffers, which contained 2 mM EDTA and 0.10 M NaCl, were ad-
justed to 1.00 M ionic strength with sodium perchlorate. The role of
EDTA was to eliminate the possible catalytic effect of traces of metal
ions such as Cu(II) and Fe(III) [43–45] during the thiol autooxidation
processes, while the addition of NaCl is to suppress the hydrolysis of
the platinum(IV) complexes. The pH values of buffer solutions were
measured with an Accumet Basic AB15 Plus pH meter equipped with
an Accumet AccutupH® combination pH electrode (Fisher Scientific,
Pittsburgh, PA). Standard buffers of pH 4.00, 7.00 and 10.00, also from
Fisher Scientific, were used to calibrate the electrode just before the
pH measurements.
Mass spectra were recorded on an Agilent 1200/6310 ion trap mass
spectrometer with electrospray ionization (ESI); both positive and nega-
tive ionization modes were employed. For each of the Pt(IV) complexes, a
reaction mixture containing 8 mM tiopronin, 1 mM Pt(IV) and 20 mM
HCl was subjected to the mass analysis. In addition, mass spectra were
also recorded for pure tiopronin in dilute HCl; they were used as a refer-
ence for mass analysis.
3. Results and discussions
3.1. Time-resolved spectra and kinetic data
2
.3. Kinetic experiments
(CN)
4
]2
−
Time-resolved spectra for the reaction between trans-[PtCl
2
An Applied Photophysics SX-20 stopped-flow spectrometer
Applied Photophysics Ltd., Leatherhead, U.K.) was used for kinetic
and tiopronin under a set of reaction conditions are displayed in Fig. 2.
The large increase in the absorption peak around 255 nm can be assigned
(
2
−
runs and for recording time-resolved spectra. Stock solutions of 1.0 mM
to the formation of the reaction product of [Pt(CN)
4
]
[42]. Moreover,
2
−
3 2 4 2 4
cis-[Pt(NH ) Cl ] and trans-[PtCl (CN) ]
were prepared by dissolving
two clear isosbestic points at 241.1 nm and 285.7 nm (an inset in Fig. 2
appropriate amount of the Pt(IV) complexes in solutions containing
shows an enlarged scale at one isosbestic point) are found from the figure.

S. Huo et al. / Journal of Inorganic Biochemistry 125 (2013) 9–15
11
These isosbestic points suggest that the reduction process is the transfor-
2
−
mation between the two absorbing species trans-[PtCl
2
(CN)
, while the absorption variation from the tiopronin oxidation
does not make a significant contribution to the overall spectral changes
4
]
and
2
−
4
[Pt(CN) ]
2
−
displayed in Fig. 2. Reduction of trans-[PtCl
at 255 nm. By use of pseudo first-order conditions ([tiopronin] ≥ 10∙
Pt(IV)] and constant pHs controlled by buffers), the kinetic traces at
2
(CN)
4
]
was thus followed
[
2
55 nm could be simulated very well by single exponentials, indicating
2
−
2 4
that the reduction is indeed first-order in [PtCl (CN) ]. Pseudo first-
order rate constants kobsd, derived from the simulations, are reported as
the average values from 5 to 7 runs; standard deviations are usually
much less than 5%.
Time-resolved spectra for the reaction between cis-[Pt(NH
and tiopronin in an acetic acid/acetate buffer are shown in Fig. 3A.
The absorbance decrease was simulated by Eq. (1), where A , A
and A stand for absorbances at time t, zero and
3 2 4
) Cl ]
t
0
,
∞
A ¼ ðA –A Þ expð−kobsdtÞ þ A∞
ð1Þ
t
0
∞
infinity, respectively. The simulations around two major bands gave
rise to very good fittings (shown in Fig. 3B) and further, the values
of kobsd obtained from the fittings at different wavelengths were in
excellent agreement within the experimental errors. These attributes
manifest that the absorbance decrease in Fig. 3 corresponds to the re-
duction of cis-[Pt(NH ) Cl ] without other complications. Hence, the
3 2 4
reduction kinetics was followed around 230 nm, and similarly, the
obtained kobsd values were from 3 to 5 duplicate runs. Values of
k
obsd as functions of [Tiopronin] and pH at 25.0 °C and ionic strength
of 1.00 M are summarized in Supporting Tables S1 and S2 in the sup-
plementary materials.
3
.2. Rate law
The influences of varying [Tiopronin] on the reduction rates were
studied in different buffer solutions covering a wide range of pH.
Values of kobsd as a function of [Tiopronin] at several pHs are shown
in Figs. 4 and 5. Clearly, all the plots of kobsd versus [Tiopronin] are
of straight lines with no significant intercepts, demonstrating that
the reductions are also first-order in [Tiopronin]. Therefore, the
Fig. 3. (A) Time-resolved spectra for reaction between cis-[Pt(NH
under the reaction conditions: [Pt(IV)] = 0.05 mM, [Tiopronin] = 1.00 mM, [Cl ] =
3
)
2
Cl
4
] and tiopronin
−
0
.10 M, pH = 3.68 acetic acid/acetate buffer, μ = 1.0 M and 25.0 °C. The first spec-
trum was obtained ca. 10 s after reaction and time between scans was 38 s. (B) Kinetic
traces at 227 nm and 270 nm for reaction between cis-[Pt(NH Cl ] and tiopronin; the
3
)
2
4
data points are from the absorbance readings in Fig. 3A. The solid curves were obtained
by fitting Eq. (1) the experimental data by use of a nonlinear least-squares method.
reductions can be generally described by an overall second-order
rate law (2), where k′ denotes observed second-order rate constants.
h
i
h
i
2
–
2–
d PtðCNÞ
¼
=dt ¼ k
PtCl ðCNÞ
4
obsd
2
4
h
i
2
–
k′½Tioproninꢀ PtCl ðCNÞ
ð2aÞ
2
4
or
ꢀ
ꢁ
ꢀ
ꢁ
−
d PtðNH Þ Cl =dt ¼ k
PtðNH Þ Cl
3
2
4 ꢀ
obsd
ꢁ
3
2
4
¼
k′½Tioproninꢀ PtðNH Þ Cl
ð2bÞ
ð3Þ
3
2
4
and
kobsd ¼ k′½Tioproninꢀ:
]2 and tiopronin under
−
Fig. 2. Time-resolved spectra for reaction between trans-[PtCl
2
(CN)
4
+
Values of k′ as a function of pH are listed in Table 1; they were calcu-
lated from the linear plots of kobsd versus [Tiopronin].
the reaction conditions: [Pt(IV)] = 0.05 mM, [Tiopronin] = 1.00 mM, [H ] = 0.020 M,
Cl ] = 0.10 M, μ = 1.0 M and 25.0 °C. The time between scans was 17 s.
−
[

12
S. Huo et al. / Journal of Inorganic Biochemistry 125 (2013) 9–15
Fig. 5. kobsd as a function of [Tiopronin] for oxidation of tiopronin by cis-[Pt(NH
at 25.0 °C and ionic strength of 1.0 M.
3 2 4
) Cl ]
Fig. 4. Pseudo first-order rate constants, kobsd, as a function of [Tiopronin] for oxidation
2
−
of tiopronin by trans-[PtCl
2
(CN)
4
]
at 25.0 °C and ionic strength of 1.0 M.
3.4. Reaction mechanism
3
.3. Reaction products
The mass spectra for pure tiopronin, for the reaction mixtures of
As discussed above, the time-resolved spectra shown in Figs. 2 and 3
suggest that the reductions of the two Pt(IV) complexes follow a simple
process. This coincides with the nature of platinum(IV) complexes,
namely, substitution inert as characterized by rather slow substitution
reactions. The present reduction reactions are fast in general and in
2
−
tiopronin with trans-[PtCl
2
(CN)
4
]
3 2 4
and with cis-[Pt(NH ) Cl ] are given
in supporting Figs. S1–S3 in supplementary materials. Assignments of
the major peaks are also provided in each of the figures. Under the condi-
tions used which are similar to those employed in the kinetic experi-
ments, tiopronin was found to be oxidized almost exclusively to form
tiopronin-disulfide (structure also shown in Fig. 1) by both the Pt(IV)
complexes; some other conceivable products from deeper oxidations of
Table 1
Observed second-order rate constants k′ as a function of pH at 25.0 °C and 1.0 M ionic
strength.
2
−
tiopronin were not noticeable. Previously, trans-[PtCl
2
(CN)
4
]
was uti-
k′/M−1 s−1
Pt(IV) Complex
trans-[PtCl (CN)
pH
lized, as a very efficient reagent, for formation of intramolecular disulfides
in dithiol-containing peptides [46,47] and small dithiol compound [40]. In
this work, we found that formation of intermolecular disulfide bond by
]2
–
1.98
22.6 ± 0.7
73.6 ± 2.5
2
4
2
3
.51
.10
250 ±
2
2
−
trans-[PtCl
2
(CN)
4
]
was also efficient under the kinetic conditions used.
3.47
3.91
571 ± 20
(1.14 ± 0.01) × 103
(1.93 ± 0.02) × 103
The mass spectra do not, however, provide any meaningful infor-
mation about the platinum complexes after reaction; this is ascribed
to their much lower sensitivity compared to tiopronin disulfide. On
the other hand, the UV–vis spectra assisted us to clarify this issue.
The UV–vis spectra for 0.08 mM [Pt(CN)
reaction mixture of 0.08 mM trans-[PtCl
4
4
5
5
.17
.50
.12
.53
(4.28 ± 0.03) × 103
(1.58 ± 0.02) × 104
(3.97 ± 0.04) × 104
2
−
5
4
]
in 5 mM HCl and for a
6.22
(1.73 ± 0.02) × 10
2
−
(6.9 ± 0.1) × 105
6
7
.82
.24
2
(CN)
4
]
with 0.30 mM
(1.5 ± 0.1) × 106
3.24 ± 0.05
9.3 ± 0.1
tiopronin in 5 mM HCl are shown in the supporting Fig. S4. Virtually,
the two spectra give rise to two identical absorption peaks around
cis-[Pt(NH
)
3 2
Cl
4
]
3.27
.61
3
2
2
55 nm and 279 nm, while the minor spectral difference in the region
25–235 nm is caused by the tiopronin background contribution.
4.51
5.29
50.1 ± 0.4
304 ±
(4.39 ± 0.07) × 10
(2.32 ± 0.03) × 104
4
3
6.53
Therefore, it can be safely concluded that the reduced product of
trans-[PtCl
2
−
2−
7.38
2 4
(CN) ]
is [Pt(CN)
4
]
.

S. Huo et al. / Journal of Inorganic Biochemistry 125 (2013) 9–15
13
contrast; it is therefore anticipated that formation of short-lived Pt(IV)
species through substitution by the tiopronin has no time to occur.
The second-order pH-dependent rate constants k′ listed in Table 1 dis-
play a similar trend for the Pt(IV) complexes, increasing several orders of
magnitude when the reaction media are changed from acidic solutions to
slightly basic ones. This trend clearly indicates that the thiolate form of
tiopronin is much more reactive than its thiol forms. By taking the three
protolytic forms of tiopronin into account, a reaction mechanism is
proposed and depicted in Scheme 1. In this mechanism, all the protolytic
species of tiopronin reduce the Pt(IV) complexes in parallel relying
on the solution pH. In addition, all the reactions described by k
1
− k
3
are rate-determining steps, generating transient chlorothiol and
sulfenylchloride species (cf. more discussions on the rate determining
steps below). From the product analysis, it can be concluded that the
chlorothiol and sulfenylchloride species are rapidly and mainly trapped
intermolecularly by the excess thiol/thiolate forms of tiopronin, leading
to formation of tiopronin-disulfide.
3
.5. Calculation of rate constants
According to Scheme 1, Eq. (4) was derived as the rate law, where
denotes to
Fig. 6. Second-order rate constants k′ as a function of pH at 25.0 °C and ionic strength
2
−
of 1.0 M for oxidation of tiopronin by trans-[PtCl
2
(CN)
4
]
(data points). The solid
a
H
curve was obtained by fitting Eq. (5) the experimental data by use of a weighed
nonlinear least-squares method.
−
d½PtðIVÞꢀ=dt ¼ d½PtðIIÞꢀ=dt
2
k a þ k K a þ k Ka1K
1
H
2
a1
H
3
a2
¼
½PtðIVÞꢀ½Tioproninꢀ
ð4Þ
aH2 þ K a þ K Ka2
Eq. (6) was also used to simulate the data in Fig. 6, giving rise to
a1
H
a1
exactly the same values of k
that the k -term in Eq. (5) has indeed a negligible contribution. In the
case of cis-[Pt(NH Cl ], Eq. (6) was directly employed to simulate
2 3
and k when Eq. (5) was used, confirming
1
the proton activity, corresponding to the pH measurements. When
Eq. (4) is compared to Eq. (3), Eq. (5) can be obtained:
)
3 2
4
the k′–pH data, and the simulation also rendered a good fitting shown
in Fig. 7. The derived rate constants for the rate-determining steps are
summarized in Table 2.
2
′
k1aH þ k2Ka1aH þ k3Ka1Ka2
k ¼
ð5Þ
aH2 þ K a þ K
K
a1 a2
a1
H
3.6. Rate-determining steps
The k′–pH data in Table 1 were analyzed by Eq. (5) with k
1
, k
2
and
3
k as adjustable parameters whereas pKa1 = 3.42 and pKa2 = 8.33
determined at 25.0 °C by other researchers were utilized as fixed
It has been interpreted that the reductive elimination of the
trans-dichoro-platinum(IV) complexes takes place through a chloride-
bridged electron-transfer process [38–40,49–53]. By analog, the rate-
determining steps in Scheme 1 proceed conceivably through a similar
transition state where a bridge is formed between the axially coordinated
chloride of the Pt(IV) complex and the sulfur atom from the thiol/thiolate
group of tiopronin [38–40,49–53]. It follows by the collapse of the bridge,
values [48]. A weighted nonlinear least-squares analysis in the case
2
−
of trans-[PtCl
2
(CN)
4
]
resulted in a good fitting shown in Fig. 6; mean-
−
1
−1
while, the fitting affords k
1
= 0.11 ± 0.79 M
s
, k
. Apparently, k
has no meaning because it is indeterminate within the data collected.
2
= 630 ±
−
1
−1
7
−1 −1
6
M
s
, and k
3
= (2.45 ± 0.02) × 10 M
s
1
1
Eq. (5) is reduced to Eq. (6) when the k -term is neglected:
+
resulting in a Cl transfer from the Pt(IV) complexes to tiopronin species
(
to form chlorothiol and/or sulfenylchloride); concurrent to the collapse
′
k2Ka1aH þ k3Ka1Ka2
k ¼
ð6Þ
of the bridge, the Pt(IV) complexes are acquiring two electrons in
one-step from the bridged chloride (inner-sphere electron transfer),
2
a
þ K a þ K
K
a1 a2
H
a1
H
NH
HOOC
k1
NH
HOOC
+
SH
_
RSH
_
SHCl
O
Pt(II) + Cl
O
_
2
H+ + Cl
_
Ka1
n
Cl
Pt
Cl
+
RSH H + Cl
_
NH
_
OOC
k2
NH
OOC
RSSR
+
+
SH
_
SHCl
O
Pt(II) + Cl
O
Ka2
NH
H+ + Cl
_
_
k3
_
NH
OOC
OOC
_
RSH
_
SCl
S
Pt(II) + Cl
O
O
_
2
For n =2, Pt(IV) = trans-[PtCl (CN) ] ; For n = 0, Pt(IV) = cis-[Pt(NH ) Cl ]
2
4
3 2
4
RSH denotes any of the three protolytic species of tiopronin indicated above
Scheme 1. Suggested reaction mechanism.

14
S. Huo et al. / Journal of Inorganic Biochemistry 125 (2013) 9–15
Table 3
3 2 4
Observed second-order rate constants k′ for reduction of cis-[Pt(NH ) Cl ] and satraplatin
at 25.0 °C and 1.0 M ionic strength.
Pt(IV) Complex
cis-[Pt(NH Cl
Reductant
pH
k′/M−1s−1
)
3 2
4
]
Ascorbate
Glutathione
Tiopronin
Ascorbate
Glutathione
7.16
7.36
7.38
7.12
675a
4
b
1.31 × 10
2.32 × 104
c
−
2 d
Satraplatin
5.08 × 10
NA
NA, not available.
a
Ref [53].
b
Measured in this work, cf. Fig. S5 in the supporting material.
This work.
Ref. [52].
c
d
Tiopronin was reported to have a nephroprotective property for
cisplatin [7,8] while glutathione has not. This differentiation may stem
from their interactions with cisplatin in some different ways. It is thus
reasonable that tiopronin may also have a nephroprotective effect for
Pt(IV) prodrugs of cisplatin although tiopronin and glutathione reduce
Fig. 7. Second-order rate constants k′ as a function of pH at 25.0 °C and ionic strength
of 1.0 M for oxidation of tiopronin by cis-[Pt(NH Cl ] (data points). The solid curve
3 2 4
cis-[Pt(NH ) Cl ] with reaction rates differing not too much each other.
3
)
2
4
was obtained by fitting Eq. (6) the experimental data by use of a weighed nonlinear
least-squares method.
4. Conclusions
and are being reduced to their Pt(II) counterparts. For the two Pt(IV) com-
We have carried out a careful and detailed study on the oxidations
of tiopronin by cisplatin prodrug cis-[Pt(NH Cl ] and a model complex
in a wide pH range. A rate law is established through
4
plexes, the ratios of k
3
/k
2
are similar and about 4 × 10 , highlighting the
3
)
2
4
2
−
huge reactivity difference between thiolate form of tiopronin and its
corresponding thiol form. This huge reactivity difference coincides
with the fact that the nucleophilicity of thiolate is much higher than
the corresponding thiol observed by other researchers [54,55]. In
other words, the chloride bridged electron-transfer process resembles,
in nature, to a nucleophilic substitution.
2 4
trans-[PtCl (CN) ]
the stopped-flow spectrometric and kinetic measurements. The mass
spectrometric analysis of the oxidations products, together with the rate
law, enables us to suggest a convincing reaction mechanism and to gain
some insights into the oxidative degradation of tiopronin. It appears to
be the first detailed kinetic and mechanistic study for the oxidation of
tiopronin by metal ions/complexes, although some of the oxidation
processes have been widely used in determinations of tiopronin in vari-
ous samples. Since cis-[Pt(NH ) Cl ] is rapidly reduced to cisplatin by
3
.7. Biological/biomedical relevance
3
2
4
tiopronin and by other thiol-containing compounds, it is anticipated
that tiopronin may also protect the nephtotoxicity and ototoxicity caused
by Pt(IV) prodrugs of cisplatin [7–9].
A series of Pt(IV) anticancer active compounds including ormaplatin
and some recently developed ones [56,57] have a configuration similar
to that of cis-[Pt(NH Cl ]; reduction of this class of Pt(IV) agents by
)
3 2
4
tiopronin will be expected to follow the reaction mechanism delineated
above. Observed second-order rate constants k′ for reductions of
Acknowledgments
cis-[Pt(NH
3
)
2
Cl
4
] by ascorbate and glutathione at biological relevant
Financial support of this work by a grant from the Natural Science
Foundation of Hebei University (2012ZD01) is gratefully acknowledged.
pH are listed in Table 3; all these reductions proceed via the inner-
sphere electron transfer mode. Reduction of the orally active Pt(IV)
drug satraplatin by ascorbate is about 10,000 times slower than that
Appendix A. Supplementary data
3 2 4
of cis-[Pt(NH ) Cl ] (Table 3). Due to the poor bridging effect of the
coordinated acetate in satraplatin, the much slower reduction was
believed to take place via an outer-sphere electron transfer [52]. On
the other hand, reduction of satraplatin by glutathione appeared to be
more complex, not following the simple second-order kinetics [58].
Ascorbic acid or glutathione or both are often supposed to be responsi-
ble for in vivo reductions of Pt(IV) prodrugs, but large biomolecules may
also play a big role for these reductions as pointed out by Gibson [29].
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2013.04.003.
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