Equilibrium, kinetic and HPLC study of the reactions between platinum(II) complexes and DNA constituents in the presence and absence of glutathione

Article abstract of DOI:10.1039/b411168k

The complex formation equilibria of [Pt(SMC)(H₂O) ₂]⁺ and [Pt(terpy)H₂O]²⁺, where SMC = S-methyl-L-cysteine and terpy = 2,2':6',2 -terpyridine, with some biologically relevant ligands such as inosine (INO), inosine-5'-monophosphate (5'-IMP), guanosine-5'-monophosphate (5'-GMP) and glutathione (GSH) were studied. The stoichiometry and stability constants of the complexes formed are reported, and the concentration distribution of the various complex species have been evaluated as a function of pH. Also the kinetics and mechanism of the complex formation reactions were studied as a function of nucleophile concentration and temperature. For the complex [Pt(SMC)(H₂O) ₂]⁺, two consecutive reaction steps, which both depend on the nucleophile concentration, were observed under all conditions. The negative entropies of activation support an associative complex formation mechanism. Reaction of guanosine-5'-monophosphate (5'-GMP) with Pt(II) complexes was carried out in the presence and absence of glutathione (GSH) at neutral pH. The rate constants clearly showed a kinetic preference toward GSH at neutral pH. The reactions were also monitored by HPLC. However, only a small amount of coordinated 5'-GMP was detected in the HPLC trace. The products were isolated and characterized by MALDI-TOF mass spectrometry.

Full text of DOI:10.1039/b411168k

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F U L L P A P E R
Equilibrium, kinetic and HPLC study of the reactions between
platinum(II) complexes and DNA constituents in the presence and
absence of glutathione
a
a
a
b
b
ˇ
Zivadin D. Bugarcˇic´,* Tanja Soldatovic´, Ratomir Jelic´, Berta Alguero´ and Anna Grandas*
a University of Kragujevac, Faculty of Science, Department of Chemistry, R. Domanovic´a 12,
P. O. Box 60, Sr-34000 Kragujevac, Serbia. E-mail: bugi@kg.ac.yu
b Departament de Qu´ımica Orga`nica, Facultat de Qu´ımica, Universitat de Barcelona, Mart´ı i
Franque`s 1-11, 08028 Barcelona, Spain. E-mail: anna.grandas@ub.edu
Received 21st July 2004, Accepted 7th October 2004
First published as an Advance Article on the web 22nd October 2004
The complex formation equilibria of [Pt(SMC)(H2O)2]+ and [Pt(terpy)H2O]2+, where SMC = S-methyl-L-cysteine
and terpy = 2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine, with some biologically relevant ligands such as inosine (INO),
inosine-5ꢀ-monophosphate (5ꢀ-IMP), guanosine-5ꢀ-monophosphate (5ꢀ-GMP) and glutathione (GSH) were studied.
The stoichiometry and stability constants of the complexes formed are reported, and the concentration distribution
of the various complex species have been evaluated as a function of pH. Also the kinetics and mechanism of the
complex formation reactions were studied as a function of nucleophile concentration and temperature. For the
complex [Pt(SMC)(H2O)2]+, two consecutive reaction steps, which both depend on the nucleophile concentration,
were observed under all conditions. The negative entropies of activation support an associative complex formation
mechanism. Reaction of guanosine-5ꢀ-monophosphate (5ꢀ-GMP) with Pt(II) complexes was carried out in the
presence and absence of glutathione (GSH) at neutral pH. The rate constants clearly showed a kinetic preference
toward GSH at neutral pH. The reactions were also monitored by HPLC. However, only a small amount of
coordinated 5ꢀ-GMP was detected in the HPLC trace. The products were isolated and characterized by
MALDI-TOF mass spectrometry.
At present it is not clear how the platinum(II) species reaches
Introduction
DNA, because Pt(II) has a high affinity for sulfur donors as com-
pared with nitrogen donor ligands such as those of the DNA nu-
cleobases. The cysteine–platinum linkage is quite inert,12 whereas
methionine bonded to platinum can be replaced by thiols or
nucleobases.13–14 Studies on the interaction between the drug
carboplatin and sulfur-containing biomolecules have shown that
long-lived Pt–methionine adducts may be important metabolites
in vivo.15 It has been hypothesized that platinum might initially
bind to sulfur-containing nucleophiles, and, in the case of the
methionine adducts, migrate to DNA to form thermodynam-
ically more stable products.16–19 This assumption is supported
by different experiments carried out with models of cisplatin,3, 20
but it is not confirmed by the results obtained in the experiments
using cisplatin.20 In these circumstances, more studies are
required to understand the evolution of the reactions of plat-
inum(II) complexes with naturally occurring compounds, and
how platinum(II)-based anticancer drugs can reach their target.
The mono-functional [PtCl(terpy)]+ and related complexes of
the general type [Pt(terpy)X]2+, terpy is 2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine, are
very useful models for studying the ligand substitution reactions
of square-planar complexes. The kinetics of the substitution
reactions involving several different X ligands have been inves-
tigated.21–29 We have studied the kinetics of the complex-forma-
tion reactions between [Pd(terpy)H2O]2+ and [Pt(terpy)H2O]2+
with L-cysteine, DL-penicillamine, glutathione and thiourea.28,29
Also, we recently published the results of the substitution reac-
tions of the complexes [Pt(terpy)H2O]2+, [Pt(terpy)(cyst-S)]2+
and [Pt(terpy)(guo-N7)]2+, where cyst-S is L-cysteine and guo-
N7 is guanosine, with some biologically relevant ligands
such as inosine, inosine-5ꢀ-monophosphate, guanosine-5ꢀ-mono-
phosphate, L-cysteine, glutathione, thiourea, thiosulfate and
diethyldithiocarbamate in aqueous 0.10 M NaClO4 at pH 2.5
and 6.0 using variable-temperature and -pressure stopped-flow
spectrophotometry.30 The complex, [Pt(SMC)(H2O)2]+ has been
chosen as a bi-functional complex. However, this complex has
higher solubility and it is more reactive than cisplatin.
The anticancer drugs, cis-diamminedichloroplatinum(II), cis-
[PtCl2(NH3)2], cisplatin, or cis-diammine[1,1-cyclobutanedicar-
boxylato]-O,Oꢀ-platinum(II), carboplatin, are widely used for the
treatment of testicular, ovarian, and other forms of cancer.1,2 Al-
though the anti-tumour activity of cisplatin or carboplatin, is as-
cribed to interactions between the complex and DNA,2–4 a large
amount of the platinum reacts with other biomolecules, such as
proteins and enzymes. In fact, already in the blood, where the Pt
drug is administered by injection or infusion, several molecules
are available for kinetic and thermodynamic competition.3,4
Sulfur containing molecules have a high affinity for platinum
and could form very stable bonds. Moreover, the interaction
of Pt complexes with sulfur containing biomolecules has been
associated with negative phenomena and some drawbacks in
the clinic still remain, such as nephrotoxicity, gastrointestinal
toxicity, ototoxicity and neurotoxicity and drug resistance.5–7
Reactions with thiol (SH) groups of protein side chains (e.g.
in metallothionine and glutathione) are thought to trap and
deactivate the drug before it reaches its cellular DNA target to
form 1,2-intrastrand cross-links with guanine bases, the likely
cytotoxic adduct.8 Glutathione, a tripeptide with a sequence c-
glutamylcysteinylglycine (c-Glu–CysH–Gly, GSH) is frequently
the most prevalent intracellular thiol with concentrations up
to 10 mM and it is the most abundant low molecular weight
peptide. GSH has been adapted through evolution to perform
many divers functions. For instance, GSH protects cells from the
toxic effects of reactive oxygen compounds and is an important
component of the system that uses reduced pyridine nucleotide
to provide the cell with its reducing properties. GSH functions in
catalysis, metabolism and transport. It participates in reactions
involving the synthesis of proteins and nucleic acids and in those
that detoxify free radicals and peroxides. The intracellular level
of GSH is much greater than that of cysteine. Also, GSH serves
as a storage and transport form of cysteine moieties. GSH is
synthesized intracellularly and is exported from the cell.9–11
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
D a l t o n T r a n s . , 2 0 0 4 , 3 8 6 9 – 3 8 7 7
3 8 6 9
©

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Table 1 Equilibrium constants for INO, 5ꢀ-IMP, 5ꢀ-GMP, and GSH
complexes with [Pt(SMC)(H2O)2]+ and [Pt(terpy)(H2O)]2+ at 25 ◦C;
0.1 mol dm−3 ionic strength
With the aim of extending our earlier work, we have per-
formed a detailed study of the complex-formation kinetics
of [Pt(SMC)(H2O)2]+ and [Pt(terpy)H2O]2+ where SMC is
S-methyl-L-cysteine, with inosine, inosine-5ꢀ-monophosphate,
guanosine-5ꢀ-monophosphate and glutathione in an aqueous
solution. The evolution of the reaction of [PtCl(terpy)]+ with 5ꢀ-
GMP in the presence and absence of glutathione was monitored
by reversed-phase HPLC and is also described here.
System
p
q
ra
log bb
[Pt(SMC)(H2O)2]+
1
1
2
0
1
1
0
0
0
1
1
0
0
0
1
1
1
0
0
0
0
1
1
1
1
0
0
0
1
1
0
0
0
0
1
1
0
0
0
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
−1
−2
−1
1
0
0
1
2
3
0
1
1
2
3
0
1
2
1
−3.49(0.02)
−12.29(0.03)
−0.06(0.06)
8.68(0.06)
8.23(0.02)
12.20(0.03)
9.14(0.05)
15.25(0.03)
16.33(0.08)
9.61(0.05)
15.87(0.06)
9.59(0.07)
15.73(0.05)
18.06(0.06)
12.38(0.04)
18.80(0.05)
22.27(0.05)
9.73(0.05)
18.06(0.07)
21.58(0.09)
23.68(0.10)
20.48(0.08)
22.33(0.07)
16.63(0.09)
−4.42 (Ref. 21)
9.59(0.07)
15.73(0.05)
18.06(0.06)
15.85(0.03)
21.25(0.05)
9.73(0.05)
18.06(0.07)
21.58(0.09)
23.68(0.10)
28.43(0.09)
24.90(0.06)
INO
Experimental
5ꢀ-IMP
Synthesis of complexes
The complexes [PtCl(terpy)]Cl·2H2O and [PtCl2(SMC)] were
prepared according to a literature method.31–33 Chemical analy-
sis, UV-VIS and 1H NMR spectral data were in good agreement
with those obtained in previous preparations.
5ꢀ-GMP
GSH
Chemicals and solutions
The chloro complex [PtCl(terpy)]Cl·2H2O or [PtCl2(SMC)], was
converted into the aqua analogue in solution by addition of an
equivalent of AgClO4, heating to 40 ◦C for 1 h, and removing the
AgCl precipitate by filtration through a 0.1 lm pore membrane
filter. Great care was taken to ensure that the resulting solution
was free of Ag+ ions and that the chloro complex had been
converted completely into the aqua species. Since it is known
that perchlorate ions do not coordinate to Pt(II) and Pd(II)
in aqueous solution,34 the kinetics of the complex-formation
reactions were studied in perchlorate medium. The ionic strength
of the solutions was adjusted to 0.1 mol dm−3 with NaClO4
(Merck, p.a.). The pH of the solutions was adjusted with HClO4
and NaOH.
Ligand stock solutions were prepared shortly before use by
dissolving the chemicals, glutathione (Fluka, assay > 99%), ino-
sine, inosine-5ꢀ-monophosphate sodium salt hydrate, guanosine-
5ꢀ-monophosphate sodium salt hydrate (Sigma). The other
reagents were of AR quality. Highly purified, deionised water
was used in the preparation of all solutions.
2
3
4
1
2
0
[Pt(terpy)(H2O)]2+
5ꢀ-GMP
−1
1
2
3
1
2
1
2
3
4
GSH
3
2
a p, q and r are the stoichiometric coefficients corresponding to
[Pt(SMC)(H2O)2]+ or [Pt(terpy)H2O]2+, ligand and H+, respectively.
b Standard deviations are given in parentheses.
Instrumentation
Chemical analyses were performed on a Carlo Erba Elemental
Analyzer 1106. UV-VIS spectra were recorded on Shimadzu UV
250 and Hewlett-Packard 8452A diode-array spectrophotome-
ters with thermostatted 1.00 cm quartz Suprasil cells. Poten-
tiometric measurements were performed using a Metrohm 686
titraprocessor equipped with a 665 dosimat. The electrode and
titraprocessor were calibrated with standard buffer solutions
prepared according to NBS specifications.35 The pH meter read-
ings were converted into hydrogen ion concentration by titrating
a standard acid solution (0.01 mol dm−3), the ionic strength of
which was adjusted to 0.1 mol dm−3 with NaClO4, with standard
base (0.10 mol dm−3) at 25 ◦C. The pH is plotted against p[H].
The relationship pH − p[H] = 0.05 was observed. The [OH−]
value was calculated using a pKw value of 13.997.36 Kinetic
measurements were carried out on a Hi-Tech stopped-flow
spectrophotometer. Kinetic data were collected and analyzed
using the OLIS KINFIT (Bogart, GA) set of programs.37
High pressure liquid chromatography (HPLC) was carried
out using a Shimadzu system equipped with two LC-6A pumps,
an SCL-6B controller, an SIL-6B autoinjector and a variable
wavelength UV-Vis detector. Mass spectrometric characteriza-
tion of the isolated compounds was accomplished by matrix-
assisted laser desorption ionization time-of-flight (MALDI-
TOF) analysis using an Applied Biosystems Voyager-DERPTM
instrument (reflector, 337 nm laser, 3 ns pulses).
complex was determined by titrating 2.5 × 10−3 mol dm−3 of the
solution complex with NaOH. The formation constants of the
complexes were determined by titrating solution mixtures of
[Pt(SMC)(H2O)2]+ (2.5 × 10−3 mol dm−3) and the ligand in con-
centration ratios of 1:1 and 1:2 (complex:ligand). The titration
solution mixtures had a volume of 40 ml. The titrations were
carried out at 25 ◦C by circulating thermostatted water through
the double-wall titration vessel and under a slow and constant
stream of N2 through the test solution. The ionic strength was
adjusted to 0.10 mol dm−3 by NaClO4. A 0.10 mol dm−3 NaOH
solution was used as titrant. At the beginning of the titration
the equilibrium was established in a few minutes (5–10), but
when hydrolysis of the complexes has started the equilibrium
required more time (1–2 h). The equilibrium constants for the
species of the general formula MpLqHr (M = [Pt(SMC)(H2O)2]+
or [Pt(terpy)H2O]2+ and L = glutathione, INO, 5ꢀ-IMP and
5ꢀ-GMP) were calculated using the program36 MINIQUAD-
75. The stoichiometry and stability constants of the complexes
formed were determined by testing various possible composition
models. The selected model gave the best statistical fit and
was chemically consistent with the titration data without giving
any systematic drifts in the magnitude of various residuals, as
describedelsewhere.38 TheresultsaresummarizedinTable1. The
species distribution diagrams were obtained using the program39
SPECIES under the experimental conditions employed.
Potentiometric measurements
Kinetics
The acid-dissociation constants of the ligands were determined
by titrating 2.5 × 10−3 mol dm−3 solution of each with standard
NaOH solution. The hydrolysis constants of [Pt(SMC)(H2O)2]+
Spectral changes resulting from mixing platinum(II) complex
and ligand solutions were recorded over the wavelength range
3 8 7 0
D a l t o n T r a n s . , 2 0 0 4 , 3 8 6 9 – 3 8 7 7

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220 to 450 nm to establish a suitable wavelength at which kinetic
measurements could be performed. Reactions were initiated
by mixing equal volumes of the complex and ligand solutions
directly in the stopped-flow instruments and were followed for
at least 8 half-lives. Complex formation was monitored as an
increase in absorbance at 340 nm, under pseudo-first order
conditions with ligand in at least a 10-fold excess, and at pH 2.5
where the complex [Pt(SMC)(H2O)2]+ exists in the aqua form.
Complex formation of the [PtCl(terpy)]+ was studied at pH ca.
6. The ionic strength of the reaction mixtures was kept constant
at 0.1 mol dm−3 with NaClO4. The temperature was controlled
2[Pt(SMC)(H2O)2]+ ꢀ [Pt2(SMC)2(H2O)2(OH)]+ + H+ (3d)
100
20−1
The pKa value of the carboxylic acid group of coordinated
S-methyl-L-cysteine was too low to be determined potentiomet-
rically. The pKa of coordinated water in [Pt(terpy)H2O]2+ is −
4.4221 and pKa1 and pKa2 of [Pt(SMC)(H2O)2]+ are −3.49 and
−8.80. pKa1 corresponds to deprotonation of the coordinated
water molecule trans to the coordinated amino group, Table 1.
pKa2 has a significantly higher value than the corresponding Pt–
diamine complexes. This is due to the strong trans labilization
effect of sulfur on the coordinated water molecule. The l-
hydroxo species (20−1) is assumed to form through dimerization
of the Pt(II) complex via a hydroxo group bound in the
positions trans to the coordinated amino group. A dimer with
a single hydroxo bridge is formed with [Pt(SMC)(H2O)2]+. The
dimerization reaction can be reformulated as:
◦
throughout all kinetic experiments to 0.1 C. The observed
pseudo-first-order rate constants, kobsd, were calculated as the
average of three to seven independent kinetic runs.
HPLC Studies
[PtCl(terpy)]+ was first reacted with GSH or 5ꢀ-GMP, either
in equimolar amounts or in a 1:2 ratio to ensure complete
reaction. The adducts formed were isolated by HPLC using
the same conditions described to monitor the reaction progress
(see below). Reaction with a mixture of the three compounds
in a relative proportion of Pt(II)/GSH/5ꢀ-GMP 1:1:12 was also
carried out. In all cases the mixture of products was incubated in
water at 37 ◦C for up to seven days, and the platinum(II) complex
concentration was 1 or 2 mmol dm−3.
The evolution of the reaction mixtures was monitored by
reversed-phase HPLC, using a Kromasil C18 column (5 lm,
0.4 × 10 cm) and a linear gradient from 0 to 100% of B in
10 min (solvent A: 0.045% trifluoroacetic acid in H2O, solvent
B: 0.036% trifluoroacetic acid in acetonitrile, flow: 1 ml min−1,
detection wavelength: 244 nm). Retention times: Pt–N7 adduct:
5.4 min, [PtCl(terpy)]+: 5.9 min, Pt–S adduct: 6.1 min.
[Pt(SMC)(H O) ]+ + [Pt(SMC)(H2O)(OH)] ꢀ
2
2
10−1
100
[(SMC)(H2O)Pt − OH − Pt(H2O)(SMC)]+ (3e)
20−1
The equilibrium constant (K) for the dimerization reaction
was determined to be log K = 3.43 (= log b20−1 − log b10−1).
The equilibrium constant for the corresponding dimerization of
[Pd(SMC)(H2O)2]+ was previously found to be log K = 4.12.33
The difference may be explained in terms of the difference in
Lewis acidity of Pd(II) and Pt(II).
The species distribution diagram for the hydrolyzed species of
[Pt(SMC)(H2O)2]+ is shown in Fig. 1.
The aliquots separated from the reaction mixture at different
incubation times were kept frozen prior to analysis under
the described conditions. Peaks with the same retention time
between different runs were collected and lyophilized. All the
isolated products were characterized by MALDI-TOF mass
spectrometric analysis, where M is the mass of the adduct formed
by [Pt(terpy)]2+ and a negatively charged ligand.
MALDI-TOF mass spectrometric analysis
Ionization of platinum adducts was carried out in the positive
mode using 2,5-dihydroxybenzoic acid as the matrix. In general,
0.02–0.1 OD units of the platinum adduct were solubilized in
20 lL H2O for characterization assays. 1 lL of the matrix
solution (10 mg in 1 mL of acetonitrile) was added to 1 lL of the
sample solution, and after mixing by withdrawing and expelling
the solution with a pipette several times, 1 lL of the resulting
solution was spotted on the sample plate and dried. Typical
isotopic distribution was observed, and the highest isotopic
peak was taken as [M]+, where M is the mass of the adduct
[Pt(terpy)(L)].
Fig. 1 Distribution of various species as a function of pH in the
[Pt(SMC)(H2O)2]+ system.
The concentration of the l-hydroxo (20−1) and monoxydroxo
(10−1) species increases with increasing pH. However, the dihy-
droxo (10−2) species starts to form at pH 6.2. The main species
present under physiological conditions is the monohydroxo,
which can interact with DNA constituents.
Complex-formation equilibria of [Pt(SMC)(H2O)2]+ with DNA
constituents
Results and discussion
Equilibrium studies
The purines, inosine, inosine-5ꢀ-monophosphate and guanosine-
5ꢀ-monophosphate have two metal ion binding sites, vs. N1 and
N7. The pH-dependent binding of these N-donors has been
reported before.40 The results show that inosine forms 110 and
120 complexes, Table 1. The 110 species is formed in the acidic
pH range and corresponds to the N7 coordinated complex,
whereas the N1 nitrogen is in the protonated form. The stability
constants of the species 110 is, log b110 = 8.23, and for the 120
species is, log b120 = 12.20.
The acid–base equilibrium of [Pt(terpy)H2O]2+ and [Pt(SMC)-
(H2O)2]+ were characterized by fitting the potentiometric titra-
tion data to various acid–base models. The best model, selected
according to the above mentioned method of calculation, was
consistent with the deprotonation of one or two coordinated
water molecules and formation of hydroxo and l-hydroxo
species, as given by eqn. (3).
+
[Pt(terpy)H2O]2+ ꢀ [Pt(terpy)(OH)]+ + H
(3a)
(3b)
(3c)
The distribution diagram for the inosine complex is given in
Fig. 2. From Fig. 2 it can be seen that INO cannot suppress
the hydrolysis, and the hydroxo species are present in solution.
The complex 10−1 is present in the solution in the pH range
between 2 and 10, and the maximum of the concentration is
10−1
100
[Pt(SMC)(H2O)2]+ ꢀ [Pt(SMC)(H2O)(OH)] + H+
100
10−1
[Pt(SMC)(H2O)(OH)] ꢀ [Pt(SMC)(OH)2]− + H+
10−1
10−2
D a l t o n T r a n s . , 2 0 0 4 , 3 8 6 9 – 3 8 7 7
3 8 7 1

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about 5.5 with the concentration a little higher than 20%. The
complex with INO 110 starts to form at pH 1 and reaches its
maximum concentration at pH 5, where the formation of the
complex 120 starts. The highest concentration of this complex
is at about pH 8.5.
Fig. 4 Distribution of various species as a function of pH in the
Pt(SMC)–GMP system.
with maximum of the concentration at pH 5. The complex 110,
started at pH 5 and the maximum of the concentration is at
pH 9. At physiological pH there are two complexes, 111 and
110, with a little higher concentration of 110.
Fig. 2 Distribution of various species as a function of pH in the
The pKa values of the protonated 5ꢀ-GMP complex are 6.42
(log b111 − log b110) and 3.47 (log b112 − log b111). These values
correspond to the PO2-OH and N1H groups, respectively. The
latter assignment is based on the fact that N7 is coordinated to
Pt(II), and as a result N1H is acidified and its pKa value is lowered
significantly. The pKa of protonated 5ꢀ-IMP complex is 6.26.
This value may correspond to an average of the pKa values for
PO2-OH and N1H groups. It should be noted that the 5ꢀ-GMP
complex (110) is significantly more stable than the inosine and 5ꢀ-
IMP complexes, Table 1. This may be plausible due to hydrogen
bonding between the exocyclic amino group of 5ꢀ-GMP and the
carboxylic group of coordinated S-methyl-L-cysteine. This inter-
action will contribute to the stability of the formed complex.
It could be conclude that 5ꢀ-GMP forms highly stable
complexes in comparison with INO and 5ꢀ-IMP, Table 1. The
stability order is: INO < 5ꢀ-IMP < 5ꢀ-GMP.
Pt(SMC)–INO system.
5ꢀ-IMP forms 111 and 110 complexes with log b111 = 15.87
and log b110 = 9.61, respectively. By comparison of the stability
constants of the complex 110, of INO and 5ꢀ-IMP, it can be seen
that the complex with 5ꢀ-IMP is more stable. There is no 120
complex and this could be explained by the steric effect of the
phosphate groups.
From Fig. 3, it can be seen that the complex formation starts
by formation of 111 complex, at pH 1, and the maximum of the
concentration is at about 5. The complex 110 started at pH 4, and
the maximum of the concentration reached at pH 8. Similarly,
as in the case of INO, 5ꢀ-IMP cannot suppress the hydrolysis of
[Pt(SMC)(H2O)2]+. The hydroxo species, 10−1, is present in the
pH range from 2 to 10, and the maximum of the concentration
(about 20%) is at pH 7. At the physiological pH, the complex
110 is present in the highest concentration.
Finally, a comparison of the stability constant values obtained
from potentiometric and kinetic data are made for 5ꢀ-GMP. The
stability constant of the 1:1 complex formed in the acidic pH
range (where N7 is coordinated) obtained from potentiometric
measurements log K1 = 3.47 (log b112 − log b111) is in good
agreement with the values obtained from kinetic investigations,
viz. log K1 = 3.02 ( = k1/k−1). In the case of INO and 5ꢀ-IMP
this comparison could not be done because the complexes 111
and 112, respectively, have not been found.
Complex-formation equilibria of [Pt(SMC)(H2O)2]+ with
glutathione
In the reaction of [Pt(SMC)(H2O)2]+ with glutathione (GSH)
complexes with stoichiometry coefficients of 112, 111 and 110
have been found. The stability constants are: log b112 = 22.33,
log b111 = 20.48 and log b110 = 16.63. (Table 1). From Fig. 5,
the distribution diagram for this equilibrium can be seen. At
acidic medium the predominant species is 112, fully protonated
GSH, with a maximum of concentration at pH 3. The complex
111 is also present in acidic medium, but with a concentration of
about 20%, with a maximum of concentration at pH 2. However,
in acidic medium the dinuclear l-hydroxo complex has been
detected, 20−1, with a very small concentration. The formation
of the complex 110, started at pH 2, and with increasing the
pH, the concentration of this complex increased. The highest
concentration of this complex is at physiological pH.
Fig. 3 Distribution of various species as a function of pH in the
Pt(SMC)–IMP system.
5ꢀ-GMP forms complexes with stoichiometric coefficients of
110, 111 and 112, whereas hydroxo species are not formed at all.
In the complex 112, 5ꢀ-GMP is protonated with two protons at
the phosphate group, while in the case of the complex 110, 5ꢀ-
GMP is deprotonated, see Fig. 4. However, in this case the hydro-
lysis of the aqua complex, [Pt(SMC)(H2O)2]+, is suppressed.
The stability constants of the complexes, 110, 111 and 112,
are: log b110 = 12.38, log b111 = 18.80 and log b112 = 22.27. From
the distribution diagram, Fig. 4, it can be seen that the forma-
tion of the complex 112 started at acidic medium and at pH 6
it disappeared. The complex 111 forms at pH between 2 and 8,
Complex-formation equilibria of [Pt(terpy)H2O]2+ with DNA
constituents
The complex formation of the monofunctional complex,
[Pt(terpy)H2O]2+ with 5ꢀ-GMP has been studied as a model for
3 8 7 2
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Fig. 5 Distribution of various species as a function of pH in the
Pt(SMC)–GSH system.
Fig. 7 Distribution of various species as a function of pH in the
Pt(terpy)–GSH system.
the bifunctional [Pt(SMC)(H2O)2]+. In this case, the complexes
absorbance at suitable wavelengths as a function of time. The
reaction of [Pt(SMC)(H2O)2]+ with the selected nucleophiles
occurs in two subsequent steps. Both reaction steps exhibit linear
dependence of kobsd on the nucleophile concentration (see Fig. 8).
This suggests that both substitution processes are reversible and
proceed according to the reactions given in eqns. (5) and (6). The
observed rate constants for the two reactions can be expressed
as given in eqn. (7).
ꢀ
ꢀ
z+
[Pt(terpy)(5 -GMP)] , with stoichiometry 111 and 112 where 5 -
GMP is protonated with one (111) and two protons (112) have
been found. The stability constants for these complexes are: log
b111 = 15.85 and log b112 = 21.25. From the distribution diagram,
Fig. 6, it can be seen that complex 112 forms at acidic medium
with a maximum concentration at pH 3, while the complex 111
started at pH 3, and the maximum of the concentration is at
pH 6.5. Also, it can be seen that at the same time as the formation
of the 111 complex, the formation of the hydroxo complex 10−1,
[Pt(terpy)OH]+started. With increasing pH the concentration of
this complex increases as well.
[Pt(SMC)(H2O)2]+ + Nu ꢀ [Pt(SMC)(Nu)(H2O)]+
[Pt(SMC)(Nu)(H2O)]+ + Nu ꢀ [Pt(SMC)(Nu)2]+ + H2O
kobsd1 = k−1 + k1[Nu] and kobsd2 = k−2 + k2[Nu]
(5)
(6)
(7)
Values for the rate constants and thermal activation parameters
estimated from the data in Fig. 8 are summarized in Table 2.
The activation parameters DH‡ and DS‡ were calculated
using the Eyring equation. The reaction for the second step
is significantly slower than for the first one. The faster reaction
step with the larger absorption change was attributed to the
substitution of the first coordinated H2O in the trans position
to the S donor atom of S-methyl-L-cysteine, whereas the slower
reaction was assigned to the displacement of the other H2O
molecule. This is due to the strong trans labilization effect of
coordinated sulfur. Inosine (INO), inosine-5ꢀ-monophosphate
(5ꢀ-IMP) and guanosine-5ꢀ-monophosphate (5ꢀ-GMP) can co-
ordinate to metal ions via N1 and N7. Under our experimental
conditions (pH = 2.5) only the N7 position of INO, 5ꢀ-IMP and
5ꢀ-GMP will bind to the central metal atom, since at this pH the
N1 position is protonated.42,43 Binding through the N7 position
in a neutral or weakly acidic medium has been verified.44 On
average, k1 is ca. 102 times faster than k2, whereas k−1 is on
average ca. 10 times faster than k−2 with the result that K1 (=
k1/k−1) is ca. 10 times larger than K2 (= k2/k−2).
Fig. 6 Distribution of various species as a function of pH in the
Pt(terpy)–GMP system.
Complex-formation equilibria of [Pt(terpy)H2O]2+ with
glutathione
It is known that the complex [Pt(terpy)H2O]2+ does not react
with thioethers,23,24,41 so we studied the complex formation with
thiols, GSH. In the present system the protonated complexes
The introduction of an S-amino acid ligand into the Pt(II) or
Pd(II) coordination sphere results in an increase in substitution
reactivity. Such a labilization has clearly been illustrated by an
earlier study.33 The kinetic data clearly show that 5ꢀ-GMP is
more reactive toward [Pt(SMC)(H2O)2]+ than either INO and 5ꢀ-
IMP. The forward and reverse reactions for both reaction steps
(5) and (6) are characterized by significantly negative activation
entropies, which is in line with an associative substitution
mechanism.
113 and 112 have been found, with stability constants: log b113
=
28.43 and log b112 = 24.90. From the distribution diagram,
Fig. 7, it can be seen that the complex 113 (with fully protonated
GSH) is present in acidic medium, up to pH 5.5. The formation
of the complex started at pH <2, while the maximum of the
concentration is at pH 5.5. However, the hydroxo complex
[Pt(terpy)OH]+, 10−1, is present at pH >4. The concentration
of this complex increases with increasing pH.
The substitution behavior of the [Pt(SMC)(H2O)2]+ complex
is very similar to that reported for the related Pd(II) complex,33
only Pd(II) analogs are ca. 103 times faster.
Kinetic studies for the complex formation of [Pt(SMC)(H2O)2]+
with DNA constituents
Kinetic studies for the complex formation of [PtCl(terpy)]+ with
DNA constituents and glutathione
The kinetics of the substitution of coordinated water was
followed spectrophotometrically by following the change in
The results of the substitution reactions of the [Pt(terpy)H2O]2+
complex with INO, 5ꢀ-IMP and 5ꢀ-GMP have been very recently
D a l t o n T r a n s . , 2 0 0 4 , 3 8 6 9 – 3 8 7 7
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Fig. 8 Observed pseudo-first order rate constants, kobsd, for the first and second reactions, as a function of nucleophile concentration and temperature
(a and b); kobsd, as a function of nucleophile concentration at 25 ◦C (c and d).
published.30 The kinetic data clearly show that these ligands
are very good nucleophiles for the [Pt(terpy)H2O]2+ complex,
and that 5ꢀ-GMP is more reactive.30 Also, the kinetics have
been studied for the complex formation of the [Pt(terpy)H2O]2+
with thiols in aqueous 0.10 M perchloric acid.28 GSH is the
strongest nucleophile. Moreover, GSH is considerably more
reactive than expected. From a comparison of the reactivity
of thiols (L-cysteine, DL-penicillamine and glutathione)28 with
INO, 5ꢀ-IMP and 5ꢀ-GMP in reaction with [Pt(terpy)H2O]2+, it
can be concluded that these N-bonding ligands are even better
nucleophiles than the thiols mentioned.30 The preference of these
N-bonding nucleophiles over thiols in acidic solutions needs
to be addressed. It must be borne in mind that the reactions
with thiols have been investigated at pH 1, where all thiols were
protonated. On the other hand, at pH 2.5 the N7 sites of INO,
5ꢀ-IMP and 5ꢀ-GMP are not protonated.
experimental error limits (Fig. 9a, c), illustrating that the solvent
cannot effectively displace the coordinated nucleophile. Thus no
significant solvent or reverse reaction path was observed in the
present systems, such that direct nucleophilic substitution is the
major observed reaction pathway under the selected conditions.
The following rate law can be formulated:
kobsd = k2[nucleophile]
(9)
where k2 is a second order rate constant for the forward
reaction. The rate law indicates that the reactions proceed via
a direct nucleophilic substitution pathway. The second order
rate constants, obtained from linear least-squares analysis of
the kinetic data, Table 2, clearly point to a kinetic preference
of [PtCl(terpy)]+ toward the GSH at pH ca. 6. 5ꢀ-GMP is also
a very good nucleophile for Pt(II) complexes, but at neutral pH
cannot compete with GSH. The second-order rate constant for
GSH is 102 times higher for GSH than for 5ꢀ-GMP. This is also
reflected in the competition reactions utilizing mixtures of GSH
and GMP. Also, proton and Pt-195 NMR data did not show any
N7 coordination of GMP, in spite of its excess, in the presence
of thiols.12
In this work, we have studied kinetics for the complex
formation of the [PtCl(terpy)]+ with 5ꢀ-GMP in the presence
and absence of GSH at pH ca. 6, with a concentration ratio
[PtCl(terpy)]+:GSH:5ꢀ-GMP of 1:2:10. The observed pseudo-
first-order rate constants, kobsd, as a function of the total
concentration of nucleophile are described by eqn. (8).
However, at or near neutral pH, although less than 10% of
thiols are deprotonated, the N-bonding bases cannot compete
with the thiol containing amino acids and peptides.30,12 There-
fore, binding primarily takes place through the sulfur donor
sites. However, for the GSMe system, rapid coordination to
the sulfur atom followed by migration to the N7 site of the
purine was observed.14,18 Similar competition experiments of the
bifunctional platinum complex, cis-dichloro(ethylendiamine)–
platinum(II) and its hydrolyzed forms with a mixture of 5ꢀ-GMP
or dGpG and thioether containing di- and tri-peptides, also
afforded sulfur bound intermediates, followed by the formation
of N7 coordinated guanine products.46,47
kobsd = k1 + k2[nucleophile]
(8)
A least-squares fit of the data according to eqn. (8), resulted
in values for the forward anation rate constants, k2, and the
reverse aquation rate constant, k1.45 The substitution reactions
are characterized by almost zero values for k1 (see Fig. 9). Thus,
the complex-formation reaction for the GSH goes almost to
completion. Linear plots of the observed pseudo-first-order rate
constants kobsd versus the total concentration of the GSH pass
almost through the origin. The intercept is very small within the
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Table 2 Second-order rate constants and activation parameters for the reaction of Pt(II) complexes with nucleophiles.
[Pt(SMC)(H2O)2]+
First reaction
L
k1298/M−1 s−1
k−1298/s−1
DH1‡/ kJmol−1
DS1‡/J K−1mol−1
5ꢀ-GMP
INO
(22.44 0.82)
(15.92 0.27)
(10.51 0.24)
0.021
0.018
0.012
43
—
—
2
−76
—
—
6
5ꢀ-IMP
Second reaction
L
k2298/M−1 s−1
k−2298/s−1
DH2‡/ kJmol−1
DS2‡/J K−1mol−1
5ꢀ-GMP
INO
(0.24 0.02)
(0.17 0.01)
(0.10 0.01)
0.0016
0.0016
0.0012
59
—
—
2
−61
—
—
5
5ꢀ-IMP
[Pt(terpy)(H2O)]2+
L
k1298/M−1 s−1
DH1‡/kJ mol−1
DS1‡/J K−1 mol−1
5ꢀ-GMPa
5ꢀ-IMPa
INOa
(6.18 0.09) × 102
(5.64 0.08) × 102
(4.02 0.07) × 102
(5.8 0.1) × 102
38
42
33
23
1
1
1
1
−67
−54
−87
−116
2
3
3
GSHb
3
[PtCl(terpy)]+
L
k2298/M−1 s−1
DH2‡/kJ mol−1
DS2‡/J K−1 mol−1
5ꢀ-GMP
5ꢀ-IMP
INO
(9.67 0.46) × 10
(7.84 0.17 ) × 10
(9.23 0.22) × 10
(2.18 0.04) × 103
(4.01 0.17) × 103
43
—
—
36
—
1
−64
—
—
3
GSH
2
−60
6
GSHc
—
a Ref. 30. b Ref. 28. c Rate constants at 37 ◦C in the presence of excess of 5ꢀ-GMP.
It is important to note that glutathione has been used as a
protecting agent and is administered before or after cisplatin.1,3
Cisplatin readily reacts with glutathione and as much as 67%
of the administered platinum has been found to coordinate
to glutathione. However, the role of glutathione appears to be
dual: glutathione both deactivates and activates cisplatin.48 The
higher effectiveness of cisplatin has also been demonstrated by
co-administering cisplatin and glutathione in patients. However,
it is not clear whether this increase in effectiveness is due to the
reduced toxicity or due to the modification of the platinum drug
by binding to the metal. Currently there is much interest in the
mechanisms responsible for the development of resistance.
glutathione than with 5ꢀ-GMP, but this did not prevent a small
amount (< 16%) of [Pt(terpy)(5ꢀ-GMP)]+ from being formed at
the very beginning of the process by reaction of [PtCl(terpy)]+
with some of the large excess of 5ꢀ-GMP present in the reaction
medium. The relative proportion of this adduct remained virtu-
ally constant throughout the reaction process, which indicates
that once formed it remains unaltered. The possibility that
[Pt(terpy)(GS)]+ reacted with the excess of 5ꢀ-GMP present in
the reaction mixture to give [Pt(terpy)(5ꢀ-GMP)]+ can be ruled
out, unless glutathione can replace 5ꢀ-GMP from [Pt(terpy)(5ꢀ-
GMP)]+ at the same reaction rate. The identity of the formed
adducts was confirmed by mass spectrometric analysis of the
products isolated from the reaction mixture by HPLC (Fig. 11).
HPLC studies
The progress of the reaction of [PtCl(terpy)]+ with other
compounds over extended periods of time can be monitored
with techniques such as HPLC, which allows aliquots separated
from the reaction mixture at programmed times to be analyzed.
The studied reactions were carried out in water, without any
buffer, since buffer ions (e.g. phosphate) are potential ligands
for Pt(II). The pH of each solution was regularly checked over
the reaction time, and was shown to be kept between 4.5 and 5.5.
Prior to the study of the reaction between [PtCl(terpy)]+
and glutathione and 5ꢀ-GMP, we first reacted the platinum(II)
complex with each of the nucleophiles. The products formed
were isolated by reversed-phase HPLC and characterized by
MALDI-TOF mass spectrometry. As expected, the products
obtained corresponded to the adducts [Pt(terpy)(GS)]+ and
[Pt(terpy)(5ꢀ-GMP)]+ (m/z 734,2 and 789,8, respectively).
The reaction between [PtCl(terpy)]+, glutathione and 5ꢀ-GMP
was then followed by HPLC (Fig. 10). The ratio of the three
compounds in the repeated assays was 1:1:12, respectively.
It was observed that [PtCl(terpy)]+ reacted much faster with
Conclusions
In conclusion, this investigation demonstrated that N-bonding
ligands such as INO, 5ꢀ-IMP and 5ꢀ-GMP have a high affinity
for [Pt(SMC)(H2O)2]+ and [PtCl(terpy)]+, which may have
important biological implications, since the interactions of Pt(II)
with DNA is thought to be responsible for the antitumour
activity of platinum drugs. 5ꢀ-GMP is more reactive toward Pt(II)
complexes than either INO or 5ꢀ-IMP. Also 5ꢀ-GMP forms the
most stable complexes with Pt(II) compared to INO or 5ꢀ-IMP.
However, the preference of Pt(II) to coordinate to GSH was
demonstrated. The reactions of the Pt(II) complexes with a
mixture of GSH and 5ꢀ-GMP yielded a product of Pt–GSH. 5ꢀ-
GMP cannot effectively compete with the thiol to bind platinum
(the second order constants are listed in Table 2). In fact, only
small amounts of coordinated 5ꢀ-GMP were detected in the
HPLC trace. These data clearly support the HPLC results that
platinum does not significantly bind to 5ꢀ-GMP in the presence
of thiol containing small amino acids and peptides.12,14,48
D a l t o n T r a n s . , 2 0 0 4 , 3 8 6 9 – 3 8 7 7
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Fig. 9 Observed pseudo-first order rate constants, kobsd, as a function of nucleophile concentration and temperature (a and b); (c) kobsd as a function
of nucleophile concentration at 37 ◦C for 5ꢀ-GMP, and GSH without 5ꢀ-GMP (ꢀ) and in the presence of excess of 5ꢀ-GMP (ꢁ); (d) kobsd as a function
of nucleophile concentration at 25 ◦C;
ˇ
and PIV2001-2). Also, Z. D. B. thanks the University of
Barcelona for a fellowship.
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