Reactivity of kiteplatin with S-donor biomolecules and nucleotides

Article abstract of DOI:10.1039/c4dt01474j

Kiteplatin, (cis-1,4-DACH)dichloridoplatinum(ii), contains an isomeric form of the carrier ligand present in the successful antitumor drug oxaliplatin and has been recently found to be very active against oxaliplatin-resistant colon cancers, confirming that, by changing the nature of the amine ligand, it is possible to obtain platinum drugs that are not cross-resistant to those already in clinical use. Apart from interaction with DNA, another factor that can affect the activity of platinum drugs is their metabolic fate in the cellular environment. Therefore, kiteplatin has been reacted with S-donor biomolecules, such as glutathione, cysteine, and methionine. The investigation has further confirmed the different reactivity of methionine as compared to cysteine-containing peptides and has unraveled the possibility of cis-1,4-DACH to become mono-coordinated with one free end (a situation never seen for isomeric 1,2-DACH ligands) and to labilize cis ligands as a consequence of its large steric hindrance. The reaction of kiteplatin-GSH adducts with 5′-GMP has also shown how the reaction products can be different depending upon the aerobic or anaerobic reaction conditions used. This journal is the Partner Organisations 2014.

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Reactivity of kiteplatin with S-donor biomolecules
and nucleotides†
Cite this: Dalton Trans., 2014, 43,
12851
Emanuele Petruzzella,a,b Nicola Margiotta,b Giovanni Natileb and
James D. Hoeschele*a
Kiteplatin, (cis-1,4-DACH)dichloridoplatinum(II), contains an isomeric form of the carrier ligand present in
the successful antitumor drug oxaliplatin and has been recently found to be very active against oxaliplatin-
resistant colon cancers, confirming that, by changing the nature of the amine ligand, it is possible to
obtain platinum drugs that are not cross-resistant to those already in clinical use. Apart from interaction
with DNA, another factor that can affect the activity of platinum drugs is their metabolic fate in the cellular
environment. Therefore, kiteplatin has been reacted with S-donor biomolecules, such as glutathione,
cysteine, and methionine. The investigation has further confirmed the different reactivity of methionine as
compared to cysteine-containing peptides and has unraveled the possibility of cis-1,4-DACH to become
mono-coordinated with one free end (a situation never seen for isomeric 1,2-DACH ligands) and to
labilize cis ligands as a consequence of its large steric hindrance. The reaction of kiteplatin–GSH adducts
with 5’-GMP has also shown how the reaction products can be different depending upon the aerobic or
anaerobic reaction conditions used.
Received 19th May 2014,
Accepted 11th July 2014
DOI: 10.1039/c4dt01474j
www.rsc.org/dalton
nature of the amine ligand, it is possible to obtain complexes
that are active toward cisplatin- and oxaliplatin-resistant cell
Introduction
Cisplatin (cis-diamminedichloridoplatinum(II)) (Fig. 1) is one lines. Moreover, since nuclear DNA is the putative target for
of the most potent anticancer drugs known and is widely used this class of drugs, several mechanistic and structural studies
in the treatment of solid tumors, such as testicular, ovarian, of adducts of different platinum complexes with DNA have
lung and bladder cancers.1–4 Despite its efficacy, cisplatin has been carried out.18,19 Another factor that can affect the activity
several limitations due to its toxicity, acquired resistance and of platinum complexes in different cell lines, is the metabolic
low activity against a variety of cancers.5–7 In order to overcome fate of the drugs, that, once in the cellular environment, can
these limitations, second and third generation Pt(II) com- interact with several biological substrates, particularly S-donor
pounds have been developed. Unlike carboplatin, a second biomolecules20 such as glutathione and proteins containing
generation drug that is cross-resistant to cisplatin, oxaliplatin, methionines and cysteines (Fig. 1).21 The reaction with these
(1R,2R-DACH)(oxalato-O,O′)platinum(II) (DACH = diaminocyclo- platinophiles reduces the percentage of platinum reaching
hexane),8 a third generation compound, is active against colo- DNA,22 the total of which is estimated to be only about 1%.23
rectal cancer (Fig. 1).9 However, resistance limitations have
Glutathione, GSH, is a tripeptide containing a central
been observed for oxaliplatin.10 Kiteplatin ((cis-1,4-DACH) L-cysteine attached by peptidic bonds to the γ carboxylic group
dichloridoplatinum(II)),11 a compound containing an isomeric of L-glutamate and to the amino group of L-glycine. GSH acts
form of the carrier ligand of oxaliplatin, has been recently as a biological anti-oxidant24 and is present in concentrations
investigated as a promising drug candidate against oxaliplatin- of 0.5–10 mM in most cells.25 High levels of intracellular gluta-
resistant colon cancers.12–16
thione have been correlated with low cisplatin activity26 and
It has been extensively demonstrated17 that the nature of high intracellular levels of glutathione have been found in cis-
the amine carrier ligand(s) has a strong influence on the platin-resistant cells.27,28 Nevertheless, co-administration of
activity of platinum complexes. Therefore, by changing the cisplatin and glutathione (as a chemoprotectant) does not
appear to decrease the antitumor activity of cisplatin.29 It has
been hypothesized that Pt-GS adducts could act as a drug
aChemistry Department, Eastern Michigan University, Ypsilanti, MI 48197, USA.
reservoir from which platinum could bind to DNA in a sub-
E-mail: jhoesche@emich.edu; Tel: +1 7344876681
bDipartimento di Chimica, Università degli Studi di Bari Aldo Moro, Via E. Orabona 4,
sequent step in exerting its antitumor activity.30 Studies of the
interaction of platinum complexes with DNA in the presence
70125 Bari, Italy
†Electronic supplementary information (ESI) available. See DOI: 10.1039/c4dt01474j of S-containing molecules indicate that, at physiological pH,
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Fig. 1 Structures of antitumor Pt complexes (cisplatin, oxaliplatin, kiteplatin) and labelled structures of S-donor biomolecules (glutathione, cysteine,
methionine).
the affinity of platinum for S-donors is greater than that for amines, such as cisplatin, where the dimeric [{Pt(NH3)2(L-cys-
N-donors (such as a nucleobase).31,32
μ-S)}2]2+ adduct was the major product, and [Pt(NH3)2(L-cys-S)2]
The reaction between GSH and diamineplatinum(II) com- and [Pt(NH3)2(L-cys-N,S)]+ were the less abundant products.34,35
plexes, such as [PtCl2(en)] (en = ethylenediamine), oxaliplatin In contrast, reaction with L-methionine, met, can lead to
or its dichlorido analogue ((1R,2R-DACH)dichloridoplatinum(II)), displacement of the carrier ligand(s) forming the [Pt(L-met-
led to the formation of two different types of adducts.25,33 S,N)2]2+ adduct.35 In the case of complexes with diamine
The first adduct was a dimer formed by two glutathione resi- ligands, as in oxaliplatin, it was found that methionine dis-
dues bridging two diamineplatinum units (A in Fig. 2). The places only the labile ligands without displacement of the
second adduct was a macrochelate formed by two diamine- diamine (forming [Pt(1R,2R-DACH)(Met-S,N)]+ in the case of
platinum units bridged by a single glutathione residue (B in oxaliplatin).36,39
Fig. 2). Sadler hypothesized that the latter macrochelate
adduct could be a reactive species.
It is clear, from the above considerations, that a detailed
investigation of the interaction between a potentially new Pt
Dimeric A-type adducts were shown to be formed also by antitumor drug, such as kiteplatin, and S-donor biomolecules
Pt(II) complexes with monoamine ligands, such as cisplatin,34,35 is a necessary step to understand the metabolic fate of this
together with the [Pt(GS)2] adduct, resulting from the displace- drug candidate and to learn to what extent it is able to over-
ment of both labile and inert ligands.21,36 Also, proteins con- come the deactivating action of platinophiles.
taining L-methionine, and L-cysteine can act as platinophiles.35
Oxaliplatin biotransformation products containing mono-
Experimental section
Materials and methods
cysteine and mono-methionine have been found in rat
kidney,37 confirming the role of these amino acids in the
metabolism of Pt drugs.
Commercial reagent grade chemicals and solvents were used
The reaction with L-cysteine, cys, does not lead to displace-
as received without further purification.
ment of the carrier ligand(s) even in the case of monodentate
NMR spectra were recorded using Jeol ECX 400 MHz and
Bruker Avance DPX 300 MHz instruments at 25 °C. NMR
spectra were recorded on samples of concentration of ca.
1
5 mM, 32 scans were performed for H 1D NMR experiments
and 256 iterations were performed for 1H 2D COSY NMR experi-
ments. 1H NMR experiments were referenced to the residual
HOD peak (1H), positioned at 4.78 ppm.40 NMR data were pro-
cessed with Mestre-C software.
ESI-MS analyses were performed using a Waters LCT
Premier mass spectrometer. Samples were prepared by dissol-
ving the solid compounds in water with 0.1% formic acid,
obtaining a final concentration of ca. 10 μM, and then injected
with a flow rate of 0.2 mL min−1 of 50/50 water–acetonitrile
Fig. 2 Structure of dimeric (A) and macrochelate (B) adducts obtained
by reaction of diamineplatinum(II) complexes with glutathione.
mixture into the mass spectrometer ion source.
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Reaction mixtures were fractionated using a Thermo Scien-
tific Dionex ICS-5000 station, equipped with a reverse-phase
column (Hypersil GOLD 250 × 4.6 mm, 5 μm, 175 Å) and a UV
detector at 220 nm, and eluting with water for 5 min followed
by a 0–30% acetonitrile gradient over 25 min with 0.1% tri-
fluoroacetic acid (TFA) as an ion-pairing agent.
A Thermo Scientific Orion 4 star pH meter, equipped with a
Thermo Scientific Orion 8192BNUWP combination electrode
standardized with buffers at pH 4.01, 7.00 and 10.01, was used
for pH measurements.
Kiteplatin was prepared using a method derived from that
of Khokhar,41 and developed in our laboratories.42
Reaction between kiteplatin and S–platinophiles. Kiteplatin
(5.7 mg, 15.0 μmol) was dissolved in water (14.3 mL) and then
treated with the stoichiometric amount (1 : 1 ratio) of the
selected platinophile (GSH, cysteine, or methionine) dissolved
in water (0.70 mL) and adjusted to pH 7 with NaOH. The final
solution (1.0 mM in both Pt complex and platinophile) was
kept at 37 °C for 24 h.
Fig. 3 HPLC chromatograms of the reaction between kiteplatin and
GSH. (a) Kiteplatin solution; (b) soon after addition of GSH; (c) after 3 h
at 37 °C; (d) after 24 h at 37 °C.
Reaction between kiteplatin, glutathione and 5′-guanosine
monophosphate (GMP). Kiteplatin (5.7 mg, 15.0 μmol) was
dissolved in water (13.0 mL H2O and 1.5 mL D2O), and treated
with a stoichiometric amount (1 : 1 ratio) of GSH dissolved in
water (0.50 mL) and adjusted to pH 7 with NaOH. The final
solution (1.0 mM in both Pt complex and GSH) was kept at
37 °C for 24 h under magnetic stirring and then treated with 2
equivalents of GMP (12.2 mg, 30.0 μmol). The reaction course
was monitored by 1H NMR spectroscopy.
Reaction between kiteplatin, glutathione and 5′-guanosine
monophosphate (GMP) under N2 atmosphere. This experi-
ment was performed under the same conditions used for the
previous one, except for bubbling N2 gas into solvents and
solutions for 2 minutes in order to remove dissolved oxygen.
with the pure reagent. After 3 h of reaction at 37 °C, a new
peak with a retention time of ca. 15 min (I) and corresponding
to the major reaction product was observed (Fig. 3c). Peak I
was collected, frozen, and lyophilized for subsequent mass-
spectrometry analysis. ESI-MS analysis of I revealed the pres-
ence of a doubly-charged cationic peak at m/z = 615.1 ([M]2+),
corresponding to the molecular formula [C32H60N10O12S2Pt2]2+,
as confirmed by isotopic pattern distribution (Fig. S1 in ESI†).
This peak can be assigned to the dimeric adduct [{Pt(cis-
1,4-DACH)(GS)}2] (GS denotes deprotonated GSH) with two
S-atoms bridging the platinum units (Fig. 4). This assignment
was confirmed by a negative peak detected at m/z = 1227.1,
Results and discussion
Kiteplatin and glutathione
The reaction between kiteplatin and glutathione (GSH) has
previously been investigated by means of biophysical tech-
niques.14 The results showed that, compared to cisplatin, kite-
platin reacts with GSH at a similar or slightly higher rate.
Despite this high reactivity, the efficacy of kiteplatin toward
A2780cisR tumor cells, whose resistance to cisplatin appears also
to be dependent upon an elevated levels of GSH, remains good.
In the present work, we have monitored the reaction of
kiteplatin with glutathione by HPLC, performed at 37 °C
(Fig. 3). In the course of the reaction the pH decreased from 7
to a value between 4 and 5. Nevertheless, we preferred not to
use a buffer, such as phosphate, since it could interfere with
platinum complexation.25
Pure kiteplatin showed two peaks eluting after ca. 10 and
11 min, assigned to the monoaqua and the dichlorido species,
respectively (Fig. 3a).43 After addition of GSH to the kiteplatin
solution, a new peak, having a retention time of ca. 9 min, was
Fig. 4 Structure of kiteplatin–glutathione adduct I (a) and possible
observed (Fig. 3b); this peak was assigned to GSH by comparison equilibrium between syn and anti conformers of adduct I (b).
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Table 1 1H NMR chemical shifts (ppm) for kiteplatin–glutathione adducts I, II and III in D2O
Glutathione
Glutamate
Cysteine
Glycine
cis-1,4-DACH
NH2
Adduct
NH
Hα
Hβ
Hγ
NH Hα
Hβ
NH
Hα
CH
CH2
Kiteplatina
GSHb
GSSG
4.91
3.19
1.74–1.73
—
—
3.78 2.13
3.84 2.18
2.48
2.55
—
—
4.53 2.88
4.77 3.30, 3.00
—
—
3.93
3.99
I
II
—
3.76 1.68
2.18
—
—
4.76 3.71
5.29 2.08
—
4.04
—
3.38
3.67, 3.57,
3.31, 3.06
1.40–0.78
1.45–0.80
6.91, 5.59 2.88 1.93, 1.60 2.67, 2.11
8.97 3.93 6.41, 5.90,
5.10, 4.49
III
—
4.09 2.34 3.30 2.52
—
5.72 2.61
7.82 3.95 5.69, 5.34, 5.11, 3.70, 3.52, 3.17 1.89–1.48
4.67, 4.28
a From ref. 42. b From ref. 36.
whose molecular formula, [C32H57N10O12S2Pt2]−, is consistent corresponding to [C23H42N7O8Pt2S]− ([M − 2H + HCOO]−), as
with [M − 3H]−, as confirmed by isotopic distribution pattern confirmed by comparison of experimental and theoretical iso-
(Fig. S1 in ESI†).
topic pattern distributions (Fig. S3 in ESI†). Formic acid was
Adduct I was characterized also by 1H 1D and 2D COSY added to the solution used for ESI-MS analysis for improving
NMR (Fig. S2 in ESI† and Table 1). Besides the main signals, volatility.
other minor signals were detected. This finding suggests the
Positive ESI-MS analysis of III showed the same mass peak
presence of an equilibrium between two conformers, syn and reported for II as a major peak, and also a minor doubly-
anti, in solution, with one conformer largely favoured over the charged cationic peak at m/z = 493.0, consistent with an
other (Fig. 4).33,36
adduct of formula [C22H43N7Na2O7Pt2S]2+, as confirmed by the
The HPLC chromatogram recorded after 24 h reaction isotopic distribution pattern (Fig. S4 in ESI†), and assigned to
between kiteplatin and GSH at 37 °C showed a new peak [M + H2O + 2Na − 2H]2+. These data led us to conclude that
having a retention time of ca. 12 min which was assigned to adducts II and III have a molecular composition similar to the
the oxidized form of glutathione44 (GSSG) by comparison with macrochelate reported by Sadler,25,33 i.e., two Pt(cis-1,4-DACH)
the chromatogram of pure GSSG recorded in the same con- moieties and one molecule of glutathione, with the addition,
ditions (data not shown). The chromatogram also showed the in the case of III, of a water molecule.
1
presence of a major peak with a retention time of ca. 14 min
Both II and III were characterized also by H NMR spectro-
(peak II in Fig. 3d). The fraction containing peak II was col- scopy (Fig. S5 in ESI†) and the assignments are reported in
lected and lyophilized. Once redissolved in water, this product Table 1.
was not stable and was partially transformed into another
Fig. 6 reports the glutamate region of the 1H 2D COSY
peak having a retention time of 15.5 min (peak III in Fig. 5). spectra of II and III, where the largest differences between the
This latter peak was present also in the HPLC chromatogram two adducts are observed. Glutamate Hα shifts downfield from
taken after 24 h at 37 °C, (III in Fig. 3d). Peak III was collected 2.88 to 4.09 ppm on passing from II to III. Moreover, in II the
and lyophilized. Also compound III was not stable in water CβH2 methylene protons are magnetically non-equivalent and
where it was partially transformed into II. These data indicate are observed at 1.60 and 1.93 ppm. In contrast, in III the Hβ
that II and III are in equilibrium.
methylene protons are magnetically equivalent and are found
ESI-MS analysis of II showed a doubly-charged cation at 2.34 ppm. Finally, while in II glutamate aminic protons were
at m/z
= 461.5, consistent with the molecular formula observed at 6.91 and 5.59 ppm, no aminic protons were
[C22H43N7O6Pt2S]2+ ([M]2+), as also confirmed by the isotopic observed in III. The slow exchange with deuterium in the case
pattern distribution. Negative ESI-MS showed a peak at m/z = 966.2 of II could be due to coordination of the aminic group to a
metal center, while the fast exchange with deuterium of the
solvent in the case of III is indicative of uncomplexed aminic
group.
All these data are in accord with compound II having a
molecular arrangement similar to the macrochelate reported
by Sadler for other diamine complexes of Pt(II) (Fig. 2).25,33
Coordination of the aminic group of glutamate to platinum
and consequent formation of a 9-membered chelate ring can
fully explain not only the slow exchange rate of the aminic
protons of glutamate, but also the remarkable diastereotopic
splitting of the aminic and β-methylene protons of glutamate
Fig. 5 HPLC chromatogram of adduct II redissolved in water and gen-
erating also adduct III.
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Fig. 6 Comparison of 1H 2D COSY NMR spectra of II (a) and III (b) recorded in D2O, with assignments of glutamate protons.
as a consequence of hindered rotation of the C–C and C–N react with chloridoamine–Pt(II) compounds leading to the
bonds within the macrochelate. In contrast, in the case of III formation of the same products formed in the case of GSH.
the glutamate aminic group would be uncomplexed and one This was explained with the possibility of chloridoamine–
platinum core would complete the coordination shell by Pt(II) complexes to catalyze the disproportionation of GSSG
binding a water molecule. Thus, in the case of III, the set of into GSH and GSO2H (2 GSSG + 2 H2O → 3 GSH + GSO2H).45
chemical shifts is consistent with a glutathione coordinated Reaction of kiteplatin with GSSG (molar ratio 1 : 0.67 so to
only by the S and N atoms of cysteine.21
allow for formation of one GSH per platinum after dispropor-
Attempts to shift the equilibrium between II and III by tionation) led to formation of the same adducts obtained
changing the pH (by addition of TFA or NaOH) were unsuc- in the direct reaction between kiteplatin and GSH (Fig. S6
cessful. Sadler reported that the oxaliplatin–glutathione macro- in ESI†).
chelate was stable in the pH range 2.8–8.7.25 In the case of
Reaction of kiteplatin–glutathione adducts with 5′-GMP. As
kiteplatin, the corresponding macrochelate II is quite reactive, already reported, reaction of Pt(II) complexes with S-donor bio-
and the aminic group of glutamate can be easily displaced by a molecules is thought to cause deactivation of Pt(II)-based
water molecule, leading to the formation of III (Fig. 7).
drugs, although the co-administration of cisplatin and gluta-
The possibility that the 9-membered macrochelate ring of II thione (the latter as chemoprotectant) does not appear to
can easily open up, making a platinum coordination site avail- influence the efficacy of the drug in terms of therapeutic
able for coordination of other nucleophiles, such as water or a outcome.29
guanine base of DNA, could render compound II not an end
product, but a drug reservoir able to interact, in a subsequent adducts could act as a drug reservoir in tumor cells.
step, with biomolecules and exert an anticancer effect. In order to assess if one of the adducts formed by kiteplatin
It has also been hypothesized that platinum–glutathione
We also investigated the reaction between kiteplatin and and a S-containing biomolecule can react with nucleic acids,
oxidized glutathione. It has been reported that GSSG can we have carried out a preliminary NMR investigation using
5′-GMP as a DNA model.
Therefore, in one experiment kiteplatin was allowed to react
with glutathione for 24 h at 37 °C (a time sufficient for the
reaction to be completed) and then treated with 5′-GMP (2
1
equivalents). The reaction was monitored by H NMR focusing
our attention on H8 and H1′ signals of 5′-GMP (Fig. 8).30
The spectrum recorded immediately after addition of
5′-GMP shows the signals of the free nucleotide: a sharp
singlet at 8.16 ppm assigned to H8, a broad signal at 6.35 ppm
assigned to the NH2 group and a doublet at 5.91 ppm
assigned to H1′ of the ribose (Fig. 8a). The pH of the solution,
initially acidic, shifted to neutral values after addition
Fig. 7 Equilibrium between adducts II (left) and III (right).
of GMP.
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equilibrium in favour of the formation of the kiteplatin-
(GMP)2 adduct. When the reaction is carried out under a N2
atmosphere, the oxidation process cannot take place, and thus
the glutathione remains in its reduced form that has higher
affinity for the Pt(II) center than for GMP.
Kiteplatin and cysteine
Reaction between kiteplatin and cysteine led to the formation
of a major product that was isolated by HPLC and character-
ized by ESI-MS and NMR spectroscopy.
ESI-MS analysis showed a doubly-charged positive peak at
m/z
=
386.57, assignable to the molecular formula
[C15H34ClN5O2Pt2S]2+ (M2+). This is consistent with a mole-
cular arrangement formed by two [Pt(cis-1,4-DACH)] moieties,
a chloride and a deprotonated cysteine, as confirmed by
the isotopic distribution pattern. A second peak was detected
at m/z = 772.14, corresponding to the molecular formula
[C15H33ClN5O2Pt2S]+ and consistent with [M − H]+, as also con-
firmed by the isotopic distribution pattern (Fig. S7 in ESI†).
The isolated adduct was characterized by NMR (Fig. S8 in
ESI†) and the proton chemical shifts are reported in Table 2.
Two signals (at 3.77 and 3.51 ppm) were assigned to the
methynic protons of coordinated DACH. These two signals cor-
relate with the multiplet centered at 1.87 ppm, that was
assigned to the methylenic protons of DACH.
A signal detected at 3.71 ppm was assigned to the α protons
of cysteine. It correlates with a multiplet centered at 3.12 ppm,
that was assigned to the β protons of cysteine.
The presence of two different signals for the methynic
protons of DACH suggests that the two aminic groups have
different ligands in the trans position.
All the reported data are consistent with a molecular
arrangement having two (cis-1,4-DACH)Pt(II) units bridged by a
single S-atom of cysteine. One Pt(II) unit could complete its
coordination shell by coordinating the aminic group of
cysteine, while the second Pt(II) center could complete its
coordination shell by coordinating a chlorido ligand. The
overall formula is sketched in Fig. 9 (compound IV). For the
first platinum, the overall arrangement would be similar to
that reported for the cysteine–cisplatin adduct [Pt(NH3)2(L-cys-
N,S)]+,34 while for the second platinum the arrangement
corresponds to the substitution of the first chloride by the
incoming cysteine nucleophile.
Fig. 8 1H NMR spectra of the reaction between kiteplatin–GSH adducts
and GMP: (a) soon after addition of GMP to a solution of kiteplatin and
glutathione kept for 1 d at 37 °C, (b) after 1 day at 37 °C, (c) after 6 days,
(d) after 10 days, (e) after 13 days.
After 1 day at 37 °C, only a very weak signal was detected at
8.51 ppm. Most likely this signal belongs to H8 of a mono-
adduct between Pt and GMP (Fig. 8b).38 After 6 days at 37 °C,
the signal at 8.51 ppm remained very weak and a new weak,
more shielded, signal was detected at 7.98 ppm (Fig. 8c). The
latter signal grew with time and, after 10 days reaction, its
intensity was comparable to that of unreacted GMP (Fig. 8d).
After 13 days, no free GMP remained. The singlet detected at
7.98 ppm belongs to the H8 protons of GMP in the bis-adduct
[Pt(cis-1,4-DACH)(5′-GMP)2]2+, as shown in a previous work.42
The latter compound is also characterized by NH2 (6.31 ppm)
and H1′ GMP protons (5.68 ppm) slightly shifted to higher
field with respect to free GMP (Fig. 8e).
This experiment proves that kiteplatin already bound to a
S-containing biomolecule is still able to react with 5′-GMP at
non-acidic pH values. We were surprised by the extremely low
reactivity observed in the first 6 days (spectra a–c in Fig. 8) as
opposed to the quite high reactivity observed in the following
4 days and hypothesized that handling and shaking of the
NMR sample could have favoured GSH oxidation by air. There-
fore we repeated the experiment under anaerobic conditions
(flushing with N2). In the latter conditions, no reaction was
observed even after a prolonged reaction time. Therefore we
can argue that the displacement of glutathione by GMP is
triggered by oxygen which, by oxidizing GSH, shifts the
Compound IV has strong relationship with the glutathione-
adducts II and III previously described. In all cases, one
cysteine–sulfur bridges two (cis-1,4-DACH)Pt(II) units, and the
Table 2 1H NMR chemical shifts (ppm) for adducts of kiteplatin with cysteine and methionine in D2O
Amino acid
cis-1,4-DACH
Adduct
Hα
Hβ
Hγ
CH3
NH2
CH
CH2
Cysteine
Methionine
IV
V
4.00
3.87
3.71
3.61, 3.33
3.15–3.02
2.24–2.09
3.12
2.65
2.14
—
3.77, 3.51
1.87
1.91, 1.60
2.73, 2.54, 2.24
2.10, 2.09, 2.00, 1.97
5.54–4.99
3.41, 3.12, 2.69, 2.51
1.46–0.76
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Fig. 9 Sketch of kiteplatin–cysteine adduct (IV).
N atom of cysteine coordinates to one platinum unit forming a
5-membered ring. Unlike compound II, in which the aminic
group of glutamate coordinates to the second Pt(II) unit, in the
case of III and IV the coordination shell of the second plati-
num is completed by an extra ligand (a water molecule and a
chlorido ligand, respectively).
Fig. 10 Portion of 1H NMR spectrum recorded on a sample of kiteplatin–
methionine adduct in D2O.
Both Pt centers retain their cis-1,4-DACH ligand. This fact
could allow kiteplatin to retain its antitumor activity after
interacting with cysteine or cysteine containing peptides, since
the intact (cis-1,4-DACH)Pt(II) moiety could still be able to
coordinate to DNA and exert a pharmacological effect.
Kiteplatin and methionine
The interaction between cisplatin and methionine has been
considered a possible cause of nephrotoxicity observed in
patients treated with cisplatin.46 Therefore, it was of interest to
investigate the reaction of kiteplatin with methionine.
Fig. 11 Equilibrium between trans and cis isomers of the kiteplatin–
methionine adduct (V).
Reaction between kiteplatin and methionine led to the for-
mation of a major adduct that was isolated by HPLC and
characterized by ESI-MS and 1H NMR spectroscopy.
possible configurations at the S atom (R or S) and the two con-
figurations are made inequivalent by the asymmetry of the α
carbon center. This would explain the observation of four
methyl signals. A similar multiplicity has been observed for
the [Pt(L-Methionine-S,N)2]2+ adduct.35
Notably, the formation of a similar adduct, with only one
coordinated ammine and a chelated methionine was hypo-
thesized in the reaction of the aqua species of cisplatin with
methionine.47
The 7-member chelate ring of cis-1,4-DACH is strained, as
evidenced by the NH2–Pt–NH2 angle of ca. 97°;11,42 hence, it
can easily open up with the diamine becoming mono-
coordinated. It is rather surprising that the diamine is not
completely displaced by a second methionine molecule as
observed in the case of cisplatin46 and carboplatin.35 It is possi-
ble that an additional interaction between the free end of the
diamine and the metal core stabilizes this molecular arrange-
ment (anchimeric assistance).
ESI-MS showed a cationic peak at m/z = 493.95, corres-
ponding to the molecular formula, [C11H25ClN3O2PtS]+ (M+),
as also confirmed by the isotopic distribution pattern. An
anionic peak was also detected at m/z = 527.94, corresponding
to the molecular formula [C11H24Cl2N3O2PtS]− ([M + Cl − H]−),
as also confirmed by the isotopic distribution pattern (Fig. S9
in ESI†). These data are consistent with a reaction product
having the formula [PtCl(cis-1,4-DACH)(met)]+ (compound V).
This product was characterized also by NMR (Fig. S10 in
ESI†) and the proton chemical shifts are reported in Table 2.
1
The H NMR spectrum showed four signals assignable to CH3
groups of coordinated methionine (Fig. 10). Since a mono-
coordinated, S-bonded methionine would give only one methyl
signal, we deduce that, most likely, methionine coordinates to
the Pt center through the S and N atoms, forming a 6-member
ring and, possibly, two different isomers are present. If the
methionine is chelated, we must deduce that one end of the
diamine has been displaced leaving room for one chloride
to coordinate. Moreover, being the cis-1,4-DACH mono-
coordinated with a chloride in cis position, there is the possi-
Conclusion
bility to have two isomers with the methionine sulfur trans to The investigation has shown that kiteplatin, like other plati-
nitrogen or to chloride (Fig. 11). Each isomer would have two num compounds with diamines, reacts with glutathione
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Dalton Transactions
forming a dimeric sulfur-bridged adduct containing two plati-
num units and a GSH moiety. Specific for kiteplatin is the
equilibrium between two forms of the macrochelate, one with
the glutamate aminic group coordinated to platinum (II) and
the other with uncoordinated glutamate aminic group and a
water molecule completing the coordination shell of one plati-
num (III). Such a dissociation equilibrium has not been
observed in the case of the analogous species formed by other
platinum substrates,25,33 and could be a consequence of the
steric hindrance of cis-1,4-DACH which can destabilize cis
ligands.
The reaction of kiteplatin with cysteine led to the formation
of an adduct having again two Pt(II)(cis-1,4-DACH) cores
bridged by a S atom. The overall coordination of the amino
acid is similar to that of GSH in compound III with the only
difference that a chloride has replaced the coordinated water
molecule.
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