Characterization of the mechanism of reduction of trans-diamminetetrachloroplatinum(IV) by l-cysteine and dl-homocysteine

Article abstract of DOI:10.1007/s11243-015-9983-5

The interactions between Pt(IV) anticancer prodrugs incorporating two ammines/amines in trans positions in their equatorial planes and some important thiols have not been exploited to date. In this work, the reduction of one such Pt(IV) prodrug, namely trans-[Pt(NH₃)₂Cl₄], by two thiol-containing amino acids l-cysteine (Cys) and dl-homocysteine (Hcy) which are prevalent in human plasma has been characterized by stopped-flow spectroscopic and ESI high-resolution mass spectral methods. The reduction process obeys overall second-order kinetics. The dependencies of the observed second-order rate constants k′ on pH have been established between pH 4.03 and 11.24. Mass spectral analysis indicates that cystine and homocystine are the dominant products for the Cys and Hcy oxidations, respectively. The suggested reaction mechanism involves all possible protolytic species of Cys/Hcy, which attack one of the two apically coordinated chlorides in parallel (all as rate-determining steps), leading to a Cl⁺ transfer to the attacking sulfur atom. The rate expression has been derived, and the rate constants for the rate-determining steps have been calculated. Features of the reduction process are discussed based on the obtained rate constants. The overall kinetic and mechanistic picture enables an in-depth understanding of the reduction process of this type of Pt(IV) anticancer prodrug.

Full text of DOI:10.1007/s11243-015-9983-5

Transition Met Chem (2015) 40:869–875
DOI 10.1007/s11243-015-9983-5
Characterization of the mechanism of reduction of trans-
diamminetetrachloroplatinum(IV) by L-cysteine and DL-
homocysteine
1
1
1
1
1
1
•
Taotao Lu
Sufang Sun
•
Jingran Dong
•
Chunxia Nan
•
Shuying Huo
•
Shigang Shen
1
•
Tiesheng Shi
Received: 20 July 2015 / Accepted: 11 September 2015 / Published online: 6 October 2015
Ó Springer International Publishing Switzerland 2015
Abstract The interactions between Pt(IV) anticancer
prodrugs incorporating two ammines/amines in trans
positions in their equatorial planes and some important
thiols have not been exploited to date. In this work, the
reduction of one such Pt(IV) prodrug, namely trans-
Introduction
Platinum(II)-based anticancer drugs such as cisplatin and
carboplatin, both of which are in worldwide use, play a central
role in cancer chemotherapies [1, 2]. Although the importance
of these drugs needs no further emphasis, both of them have
been found to possess several side effects encompassing
nephrotoxicity, ototoxicity, neurotoxicity, and also cross-re-
sistance [3]. Therefore, the development of a new generation
of platinum anticancer drugs is of great interest in order to
minimize the toxic effects of such drugs and also to broaden
the spectrum of treatable cancers. This process has involved
thedesignandsynthesisofnumerousPt(IV)compounds[2, 4–
9]. Although Pt(IV) anticancer drugs usually have two
ammines/amines in cis positions in their equatorial planes
similar to cisplatin and other Pt(II) drugs [2, 4–6], somePt(IV)
complexes with two ammines/amines in trans positions in
their equatorial planes also show anticancer activities [7–9].
Nevertheless, Pt(IV) complexes mostly have a typical octa-
hedral configuration and their ligand substitution reactions are
generally very slow. As a consequence, some properties of the
Pt(IV) anticancer drugs can be tuned by choosing different
ligands in the apical positions [4–6].
[
Pt(NH ) Cl ], by two thiol-containing amino acids L-cys-
3 2 4
teine (Cys) and DL-homocysteine (Hcy) which are prevalent
in human plasma has been characterized by stopped-flow
spectroscopic and ESI high-resolution mass spectral
methods. The reduction process obeys overall second-order
kinetics. The dependencies of the observed second-order
0
rate constants k on pH have been established between pH
4
.03 and 11.24. Mass spectral analysis indicates that
cystine and homocystine are the dominant products for the
Cys and Hcy oxidations, respectively. The suggested
reaction mechanism involves all possible protolytic species
of Cys/Hcy, which attack one of the two apically coordi-
nated chlorides in parallel (all as rate-determining steps),
?
leading to a Cl transfer to the attacking sulfur atom. The
rate expression has been derived, and the rate constants for
the rate-determining steps have been calculated. Features
of the reduction process are discussed based on the
obtained rate constants. The overall kinetic and mecha-
nistic picture enables an in-depth understanding of the
reduction process of this type of Pt(IV) anticancer prodrug.
Contrary to their inert behaviors toward substitution,
Pt(IV) anticancer drugs can be reduced readily to their
Pt(II) counterparts and they are thus often referred to as
prodrugs [1, 2, 4–9]. Some dominant small molecule
reductants in plasma [10] such as L-cysteine, glutathione,
and ascorbic acid are believed to be responsible for the
reduction of Pt(IV) anticancer prodrugs [11–15]. However,
large molecules like proteins [16–19] and selenium-con-
taining antioxidants [20] might be strong competitors to
these small reductants, at least under some circumstances.
In this work, we report the characterization of the reduc-
tion process of trans-diamminetetrachloroplatinum(IV),
&
&
Shigang Shen
shensg@hbu.edu.cn
Tiesheng Shi
rock@hbu.edu.cn
1
College of Chemistry and Environmental Science, and the
MOE Key Laboratory of Medicinal Chemistry and Molecular
Diagnostics, Hebei University,
Baoding 071002, Hebei Province,
People’s Republic of China
123

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Transition Met Chem (2015) 40:869–875
trans-[Pt(NH ) Cl ], by L-cysteine (Cys) and DL-homocys-
3
were all analytical grade, purchased either from Alfa Aesar
or from Fisher Scientific and were used without further
purification. Standard buffers of pH 4.00, 7.00, and 10.00
for pH meter calibrations were purchased from Fisher
Scientific. Doubly distilled water was employed to prepare
all the solutions.
2
4
teine (Hcy), which are important thiol-containing amino
acids in plasma [10]; Scheme 1 illustrates their structures.
Trans-[Pt(NH ) Cl ] was chosen as a representative for the
3
2
4
class of Pt(IV) anticancer active compounds bearing a trans-
diammine configuration in the equatorial plane [7]. To the
best of our knowledge, the kinetic and mechanistic aspects of
the reduction of Pt(IV) anticancer prodrugs with a trans-
diammine configuration in their equatorial planes by thiol-
containing compounds have not been studied before [11–20].
Buffers
Several buffers with combinations of acetic acid/sodium
acetate, NaH PO /Na HPO , NaHCO /Na CO , and Na
2-
2
4
2
4
3
2
3
HPO /Na PO (concentrations of 0.15–0.25 M) were pre-
4
3
4
Experimental
pared to cover a pH range of 3.22–11.24. All the buffers
which included 2 mM EDTA and 0.10 M NaCl were tuned
to an ionic strength (l) of 1.0 M with sodium perchlorate.
An Accumet Basic AB15 Plus pH meter, equipped with an
AccumetAccutupHÒ combination pH electrode (Fisher
Scientific, Pittsburgh, PA), was employed to determine the
pH values of the buffer solutions; the electrode was cali-
brated using standard buffers of pH 4.00, 7.00, and 10.00
just before the pH determinations. The function of EDTA
in the buffers was to eradicate possible catalytic effects
during thiol autooxidation by traces of metal ions such as
Cu(II) and Fe(III) [21–23], while the purpose of NaCl in
the buffers was to restrain the hydrolysis of trans-
Chemicals
Cys, Hcy, and trans-[Pt(NH ) Cl ] were obtained from
3
2
4
Sigma-Aldrich. Acetic acid, sodium acetate, sodium chlo-
ride, hydrochloric acid, sodium dihydrogen phosphate,
disodium hydrogen phosphate, sodium bicarbonate, sodium
carbonate, trisodium phosphate, and sodium perchlorate
[Pt(NH ) Cl ].
3 2 4
Stock solutions and kinetic measurements
Stock solutions of 1.0 mM trans-[Pt(NH ) Cl ] were pre-
3
2
4
pared by dissolving the required amount of the complex in
a solution containing 0.90 M NaClO , 0.09 M NaCl, and
4
0.01 M HCl; these solutions were only used for several
hours. Stock solutions of Cys/Hcy were prepared by adding
the desired amount of Cys/Hcy to a buffer of specific pH
and were then flushed for 10 min with nitrogen. Solutions
of Pt(IV) and Cys/Hcy for kinetic measurements were
prepared by dilution of these stock solutions with the same
pH buffer and were flushed 10–15 min with nitrogen
before loading into an Applied Photophysics SX-20 stop-
ped-flow spectrometer (Applied Photophysics Ltd,
Leatherhead, UK). Reactions were started by mixing equal
volumes of platinum(IV) and Cys/Hcy solutions directly in
the stopped-flow machine and were followed under pseudo
first-order conditions such that the concentrations of Cys/
Hcy were at least a tenfold excess over those of Pt(IV).
Product analysis
Fig. 1 a A typical kinetic trace for the reaction between trans-
[
Pt(NH
machine under the reaction conditions: [Pt(IV)] = 0.20 mM,
Hcy] = 3.00 mM, pH 5.76 phosphate buffer, 25.0 °C and
l = 1.0 M. b The kinetic trace in a was highlighted between 0 and
s (red dots) and was simulated by Eq. 1 by use of a nonlinear least-
squares routine (blue line). (Color figure online)
3 2 4
) Cl ] and Hcy recorded at 280 nm by the stopped-flow
Product identifications were performed by analysis of high-
resolution mass spectra recorded on an Apex Ultra 7.0T
FT-ICR mass spectrometer (Bruker Daltonik, Germany)
using electrospray ionization (ESI) in positive mode. A
[
2
1
23

Transition Met Chem (2015) 40:869–875
871
fresh solution of 5 mM Hcy/Cys in 10 mM acetic acid and
a reaction mixture containing 5 mM Hcy/Cys and 1 mM
trans-[Pt(NH ) Cl ] in 10 mM acetic acid after reaction for
values are given in Figs. 2 and 3, respectively. Most of the
plots pass through the origin, while a couple of plots show
small intercepts. However, these intercepts are too small to
be significant and are within the experimental errors. Thus,
the reductions of trans-[Pt(NH ) Cl ] by both Cys and Hcy
3
2
4
about 5 min were subjected to mass analysis.
3
2
4
are overall second order, being first order in [Pt(IV)] and in
0
[
Cys]/[Hcy] (as described by Eq. 3), where k represents
Results and discussion
the observed second-order rate constants and [Reduc-
tant] = [Cys] or [Hcy].
Kinetic traces
Â
Values of k have been evaluated from the plots in Figs. 2
Ã
Â
Ã
Àd PtðNH Þ Cl =dt ¼ k
PtðNH Þ Cl
3
2
4
obsd
0
3 2
4
Â
Ã
The electronic spectra of trans-[Pt(NH ) Cl ] solutions in a
3
2
4
¼ k ½ReductantŠ PtðNH Þ Cl
ð3Þ
3
2
4
phosphate buffer of pH 5.76, recorded on a TU-1900
spectrophotometer (Beijing Puxi, Inc, Beijing, China)
using 1.00-cm quartz cells, displayed an absorption band at
ca. 278 nm. A typical kinetic trace followed at 280 nm for
the reaction between trans-[Pt(NH ) Cl ] and Hcy in the
0
and 3 and are summarized in Table 1.
Identification of oxidation product(s)
3
2
4
same buffer is shown in Fig. 1a, where the absorbance of
Hcy is negligible. The kinetic trace is clearly biphasic and
can be interpreted in terms of two consecutive reactions
ESI high-resolution mass spectra for a solution of 5 mM
Hcy in 10 mM acetic acid and for a reaction mixture ini-
tially containing 5 mM Hcy/Cys and 1 mM trans-
[
24]; the rapid initial decrease in absorbance (displayed in
[Pt(NH ) Cl ] in 10 mM acetic acid after a reaction time of
3 2 4
Fig. 1b after expansion) is attributed to the reduction of
5
min are displayed in Fig. 4. Peak assignments are as
?
trans-[Pt(NH ) Cl ] to its Pt(II) counterpart trans-
3
2
4
follows: m/z 136.0427 for HcyÁH ; m/z 158.0247 for
[
Pt(NH ) Cl ]. The relatively slow increase in the absor-
3 2 2
bance is ascribed to the substitution of one of the chloride
ligands in trans-[Pt(NH ) Cl ] by the excess Hcy in the
2
3
2
reaction mixture [24]. These two consecutive reactions
have well-separated timescales. Consequently, the absor-
bance decrease in Fig. 1b can be simulated by Eq. 1, where
A , A , and A pertain to the absorbances at time t, zero,
t
0
?
and infinity, respectively [25].
At ¼ ðA0ÀA1Þ expðÀkobsdtÞ þ A1
ð1Þ
This behavior suggests that the reduction is first order in
Pt(IV)] and the value of the observed first-order rate
[
constant, kobsd, was obtained from the simulation. The
kinetic traces for the reaction of trans-[Pt(NH ) Cl ] with
3
2
4
Cys resembled that of Fig. 1a. In this work, our attention is
paid to the reduction process, whose behavior is described
by Eq. 2;
Â
Ã
Â
Ã
Àd PtðNH3Þ Cl4 =dt ¼ kobsd PtðNH3Þ Cl4
For each concentration of Cys/Hcy, values of k
ð2Þ
2
2
obsd
reported in this work were averages from 3 to 5 duplicate
runs; standard deviations were usually less than 5 %.
Overall second-order kinetics
The effects of varying [Cys] or [Hcy] on the reduction rate
were investigated in different buffer solutions in order to
find the reaction order with respect to thiol. The concen-
trations of [Cys]/[Hcy] were varied between 0.20 and
Fig. 2 Pseudo first-order rate constants, kobsd, as a function of [Cys]
5
.00 mM such that there were no pH changes in the buf-
for reduction of trans-[Pt(NH
3
)
2
Cl
4
]
by Cys at 25.0 °C and
fers. Plots of kobsd versus [Cys] and [Hcy] at different pH
l = 1.0 M and in different pH buffers
123

8
72
Transition Met Chem (2015) 40:869–875
0
Table 1 Observed second-order rate constants k for reduction of
trans-[Pt(NH
and l = 1.0 M
3
)
2
Cl
4
] by Cys and Hcy as a function of pH at 25.0 °C
0
-1 -1
s
Reductant
pH
4.03
k /M
Cys
31.4 ± 0.7
4.50
5.10
5.76
6.29
6.78
7.36
7.40
7.98
8.54
9.04
9.55
9.99
85.8 ± 1.9
305 ± 7
3
(1.46 ± 0.04) 9 10
3
(5.3 ± 0.2) 9 10
4
4
4
5
5
(1.39 ± 0.03) 9 10
(4.15 ± 0.14) 9 10
(4.42 ± 0.08) 9 10
(1.15 ± 0.02) 9 10
(2.04 ± 0.04) 9 10
5
(3.3 ± 0.1) 9 10
5
(4.8 ± 0.1) 9 10
5
(8.0 ± 0.2) 9 10
5
10.36
10.86
11.24
4.03
(7.8 ± 0.2) 9 10
6
(1.08 ± 0.3) 9 10
6
(1.34 ± 0.3) 9 10
Hcy
5.7 ± 0.1
4
5
5
6
6
7
7
7
8
9
9
9
.50
.06
.76
.29
.75
.36
.40
.98
.54
.04
.46
.99
13.7 ± 0.4
Fig. 3 Plots of kobsd versus [Hcy] for reduction of trans-[Pt(NH
3 2-
)
71 ± 2
Cl
4
] by Hcy at 25.0 °C and l = 1.0 M and in different pH buffers
297 ± 9
3
3
4
4
4
5
5
5
(1.26 ± 0.03) 9 10
(2.98 ± 0.08) 9 10
(1.03 ± 0.02) 9 10
(1.07 ± 0.02) 9 10
(3.08 ± 0.07) 9 10
(1.06 ± 0.15) 9 10
(1.46 ± 0.07) 9 10
(1.74 ± 0.06) 9 10
?
?
?
HcyÁNa ; m/z 180.0067 for {Hcy-H ?2Na }; m/z
?
?
2
71.0784 for (2Hcy)ÁH ; m/z 293.0605 for (2Hcy)ÁNa ;
?
and m/z 269.0623 for homocystineÁH . It is clear from the
peak assignments that homocystine is the predominant
oxidation product of Hcy. Similarly, the mass spectra (not
shown) for Cys and for the reaction mixture of Cys with
trans-[Pt(NH ) Cl ] indicate that Cys is mainly oxidized to
3
2
4
cystine. Hence, disulfide bonds are formed in both cases.
5
(4.5 ± 0.1) 9 10
Previously, trans-dichloro-Pt(IV) complexes such as trans-
2?
5
1
1
1
0.36
0.86
1.24
(3.5 ± 0.1) 9 10
2
-
[
PtCl (CN) ] and trans-[PtCl (en) ] have been utilized
2 4 2 2
5
(7.4 ± 0.2) 9 10
for formation of intramolecular disulfide bonds in peptide
synthesis and in protein folding [26–31].
5
(7.9 ± 0.2) 9 10
Reaction mechanism
Hcy participates in the reaction after the rate-determining
step(s). Thus, it is logical to assume that all the protolytic
0
The observed second-order rate constants k in Table 1 (also
species of Cys/Hcy can reduce trans-[Pt(NH ) Cl ] but with
3 2
see the data points in Figs. 5, 6) increase rapidly from pH 4 to
about 9, followed by a more gradual increase from 9 to 11.24.
The increase covers about five orders of magnitude as the pH
is raised from 4.03 to 11.24. These observations suggest that
the thiolate forms of Cys and Hcy possess much higher
reactivities toward trans-[Pt(NH ) Cl ] than their corre-
4
different reactivities. This leads to the reaction mechanism
proposed in Scheme 2, with Reactions 4–7 being rate-de-
termining steps. These rate-determining steps involve
attacks of the sulfur atom in Cys/Hcy at one of the apically
coordinated chlorides in trans-[Pt(NH ) Cl ], resulting in
3 2 4
3
2
4
the formation of transition-state intermediates. A conceiv-
able transition state for Reaction 3 is shown in Scheme 3 as
an example (Scheme 3).
sponding thiol forms [32]. On the one hand, the overall
second-order kinetics established in Eq. 3 suggests that only
one molecule of Cys/Hcy is participating in the rate-deter-
mining step(s). On the other hand, the oxidation products
cystine and homocystine imply that another molecule of Cys/
In the transition state, a Cl–S bond is partially formed
and, simultaneously, the Cl–Pt(IV)–Cl bonds are partially
1
23

Transition Met Chem (2015) 40:869–875
873
Fig. 4 ESI high-resolution mass spectra for 5 mM Hcy in 10 mM acetic acid (top) and for a reaction mixture containing 5 mM Hcy and 1 mM
trans-[Pt(NH Cl ] in 10 mM acetic acid obtained after a reaction time of about 5 min (bottom)
3
)
2
4
0
0
Fig. 5 Observed second-order rate constants k as a function of pH at
5.0 °C and l = 1.0 M for reduction of trans-[Pt(NH Cl ] by Cys
data points). The solid curve was obtained by fitting Eq. 10 to the
Fig. 6 Values of k as a function of pH at 25.0 °C and l = 1.0 M for
reduction of trans-[Pt(NH Cl ] by Hcy (data points). The solid
curve was obtained by fitting Eq. 10 to the experimental data by use
2
(
3
)
2
4
3
)
2
4
experimental data by use of a weighted nonlinear least-squares
method
of a weighted nonlinear least-squares method
SH
Cl
SH
NH3
Cl
CH2
CH2
Cl
Pt
broken. Formation of the Cl–S bond produces a short-lived
?
sulfenyl chloride species 3 via a Cl transfer to the
CH2
NH3
Cl
HOOCCHNH2
L-Cysteine (Cys)
HOOCCHNH2
DL-Homocysteine (Hcy)
attacking sulfur atom [18, 19]. Species 3 will be rapidly
trapped by the excess of Cys/Hcy to form cystine/homo-
cysteine, as shown in Scheme 4 [33, 34].
trans-[Pt(NH ) Cl ]
3
2
4
Scheme 1 Structures of trans-[Pt(NH
3
)
2
Cl
4
], Cys and Hcy
123

8
74
Transition Met Chem (2015) 40:869–875
Table 2 Derived values of rate constants for the rate-determining
steps
+
SH
CH
ClSH
(CH2)n
k1
(
2
)
n
_
+
Pt(II) + Cl (4)
+
HOOCCH-NH3+
-1 -1
s
HOOCCH-NH
Ka1
3
Reductant
Cys
k
m
Value/M
1
a
k
k
k
1
2
3
–
+
SH
ClSH
9.4 ± 0.7
k2
_
(
CH
2
)
n
(CH
2 n
)
+
Pt(II) + Cl (5)
5
Cl
Pt
_
_
_
+
+
(2.8 ± 0.2) 9 10
OOCCH-NH
3
OOCCH-NH
3
6
Cl
NH
NH
3
2
k4
(1.26 ± 0.05) 9 10
a
–
K
a2
+
3
Cl
Hcy
k
_
1
SCl
S
Cl
_
k3
(CH2)n
OOCCH-NH3+
k2
1.3 ± 0.2
(CH
2
)
n
+ Pt(II) + Cl (6)
_
+
5
OOCCH-NH
3
k
k
3
4
(3.3 ± 0.1) 9 10
3
5
K
a3
(9.1 ± 0. 3) 9 10
_
a
SCl
Could not be evaluated based on the kinetic data collected
S
_
k
4
(
CH
2
)
n
+ Pt(II) + Cl (7)
(
CH
2 n
)
_
_
OOCCH-NH
2
OOCCH-NH
2
4
3
H
2
H
2
H
k1a þ k2Ka1a þ k3Ka1Ka2aH þ k4Ka1Ka2Ka3
k0 ¼
ð9Þ
3 2 2
Pt(II) = trans-[Pt(NH ) Cl ]. For Cys, n = 1; for Hcy, n = 2.
3
H
a þ Ka1a þ Ka1Ka2aH þ Ka1Ka2Ka3
Scheme 2 Proposed reaction mechanism
Protolytic constants for Cys at 25.0 °C and l = 1.0 M
have been reported as pKa1 = 1.9, pKa2 = 8.10, and
pKa3 = 10.1 [35], while those for Hcy are pKa1 = 2.22
[36], pKa2 = 8.9, and pKa3 = 10.9 [37] at 25.0 °C and
l = 0.50 M. With these protolytic constants as fixed val-
ues and k - k as tunable parameters, Eq. 9 was utilized
_
_
COO
_
Cl----Pt----Cl----S (CH
2
)
n
CH
+
3
NH
1
4
0
to simulate the k versus pH data in Figs. 5 and 6 by use of
Scheme 3 Proposed transition-state structure
a weighted nonlinear least-squares routine. The simulation
results indicated that k was indeterminate; this could be
1
easily understood since we did not collect any kinetic data
_
SCl
in very acidic media. Neglecting the k term in Eq. 9
1
S
fast
+
NH3+
H N
(
CH2)
OOCCH-NH +
+
3
n
(CH2)n
OOCCH-NH3+
renders Eq. 10;
CH(CH ) -S-S-(CH ) CH
2
n
2 n
_
_
_
_
COO
3
OOC
2
H
k2Ka1a þ k3Ka1Ka2aH þ k4Ka1Ka2Ka3
3
k0 ¼
_
ð10Þ
+
Cl
3
H
2
H
a þ Ka1a þ Ka1Ka2aH þ Ka1Ka2Ka3
Scheme 4 Fast trapping reaction of sulfenyl chloride
0
Simulations of Eq. 10 using the k versus pH data in
Figs. 5 and 6 all generated good fits and provided the
values of k - k listed in Table 2.
2
4
By analog, species 1, 2, and 4 are formed via similar
transition states to that described above and then trapped by
reactions similar to Scheme 4, giving the same end
products.
Features of the reduction process
A few salient features for the reduction process can be
determined from the rate constants as follows. (1) The
reduction of trans-[Pt(NH ) Cl ] by both Cys and Hcy
Rate constants for the rate-determining steps
3
2
4
0
proceeds rapidly at the physiological pH, with values of k
Equation 8 was deduced mathematically as the overall rate
expression based on the reaction scheme;
4
.42 9 10 and 1.07 9 10 M
4
-1 -1
4
7
s
at 25.0 °C and pH
.40 for Cys and Hcy, respectively (Table 1). This points to
Â
Ã
À d PtðNH3Þ Cl4 =dt
2
a very short half-life of trans-[Pt(NH ) Cl ] or similar
3 2 4
3
2
k a þ k K a þ k K K a þ k K K K
trans-dichloro-Pt(IV) anticancer prodrugs [8, 9] in human
plasma. The rapid reduction of trans-[Pt(NH ) Cl ] will
1
H
2
a1
H
3
a1 a2
H
4
a1 a2 a3
¼
ð8Þ
3
H
2
H
a þ K a þ K K a þ K K K
3 2
4
a1
a1 a2
Ã
H
a1 a2 a3
Â
undoubtedly induce oxidative stress, ether in the plasma or
in the cells. Currently, the relation between the oxidative
stress and toxicological aspects of Pt(IV) prodrugs is not
well understood. (2) The thiolate species of Cys/Hcy are
much more reactive toward trans-[Pt(NH ) Cl ] than their
½
ReductantŠ PtðNH3Þ Cl4
2
where a stands for the proton activity, which corresponds
H
exactly to the pH measurements. When Eq. 8 is compared
with Eq. 3, Eq. 9 is obtained.
3
2
4
1
23

Transition Met Chem (2015) 40:869–875
875
corresponding thiol forms, as illustrated by the ratios k /
3
5. Varbanov HP, Jakupec MA, Roller A, Jensen F, Galanski M,
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4
k2 = 3 9 10 and k /k = 1.3 9 10 for Cys and k3/
5
4
2
6
7
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k = 2.5 9 10 and k /k = 7 9 10 for Hcy. These large
5
2
4 2
0
reactivity ratios explain the k versus pH profiles in Figs. 5
and 6; similar ratios have also been noticed in the case of
ormaplatin when a dithiol compound is used as a reductant
8. Martinez A, Lorenzo J, Prieto MJ, de Llorens R, Font-Bandia M,
Avilex FX, Moreno V (2005) ChemBioChem 6:2068
9
. Rakic GM, Grguric-Sipka S, Kaluderovic GN, Bette M, Filipovic
L, Arandelovic S, Radulovic S, Tesic ZLJ (2012) Eur J Med
Chem 55:214
[
19]. (3) Although the oxidation site is the sulfur atom in
Cys and Hcy, the deprotonation of the electron-withdraw-
?
ing group –NH3 can still cause a significant reactivity
10. Turell L, Radi R, Alvarez B (2013) Free Radic Biol Med 65:244
1
1
1
1. Shi T, Berglund J, Elding LI (1996) Inorg Chem 35:3498
2. Lemma K, Shi T, Elding LI (2000) Inorg Chem 39:1728
3. Jovanovic S, Petrovic B, Bugarcic ZD, van Eldik R (2013) Dalton
Trans 42:8890
difference, as evidenced by ratios of k /k 4.5 and 2.8 for
4
3
Cys and Hcy, respectively. This clearly indicates that the
electron density of the sulfur atom is a key factor governing
the reactivity of each particular protolytic species of Cys or
Hcy toward trans-[Pt(NH ) Cl ].
14. Zhang JZ, Wexselblatt E, Hambley TW, Gibson D (2012) Chem
Commun 48:847
1
1
3
2
4
5. Sinisi M, Intini FP, Natile G (2012) Inorg Chem 51:9694
6. Carr JL, Tingle MD, McKeage MJ (2006) Cancer Chemother
Pharmacol 57:483
Conclusion
The reduction of trans-[Pt(NH ) Cl ] by Cys and Hcy, the
17. Nemirovski A, Kasherman Y, Tzaraf Y, Gibson D (2007) J Med
Chem 50:554
1
1
8. Huo S, Shen S, Liu D, Shi T (2012) J Phys Chem B 116:6522
9. Ren Y, Dong J, Shi H, Huo S, Dai T, Shi T (2015) Transit Met
Chem 40:347
3
2
4
major two thiol-containing amino acids in human plasma,
gives cystine and homocysteine, respectively, as the major
products. The reduction process follows overall second-
20. Huo S, Dong J, Shen S, Ren Y, Song C, Xu J, Shi T (2014)
Dalton Trans 43:15328
2
2
1. Taylor JE, Yan JF, Wang J-L (1966) J Am Chem Soc 88:1663
2. Kachur AV, Koch CJ, Biaglow JE (1998) Free Radic Res 28:259
0
order kinetics with pronounced dependencies of k on pH.
The overall picture including the kinetic and mechanistic
aspects enables us to understand the reduction process in
depth and will undoubtedly provide some useful informa-
tion regarding the design and synthesis of similar types of
Pt(IV) anticancer prodrugs.
23. Ehrenberg L, Harms-Ringdahl M, Fedorcsak I, Granath F (1989)
Acta Chem Scand 43:177
2
2
2
4. Dong J, Huo S, Song C, Shen S, Ren Y, Shi T (2014) Transit Met
Chem 39:127
5. Huo S, Dong J, Song C, Xu J, Shen S, Ren Y, Shi T (2014) RSC
Adv 4:7402
6. Shi T, Rabenstein DL (1999) J Org Chem 64:4590
Acknowledgments Financial support of this work by Grants from
the Natural Science Foundation of Hebei Province (B2015201073),
from the You Bo Program of Hebei University (YB201403), and from
the Natural Science Foundation of Hebei University (2014-11) is
gratefully acknowledged.
27. Shi T, Rabenstein DL (2000) J Am Chem Soc 122:6809
28. Shi T, Rabenstein DL (2001) Tetrahedron Lett 42:7203
29. Shi T, Rabesntein DL (2002) Bioorg Med Chem Lett 12:2237
30. Shi T, Spain SM, Rabenstein DL (2006) Angew Chem Int Ed
45:1780
3
1. Narayan M, Welker E, Wanjalla C, Xu C, Scheraga HA (2003)
Biochemistry 42:10783
3
2. Huo S, Shi H, Liu D, Shen S, Zhang J, Song C, Shi T (2013) J
Inorg Biochem 125:9
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