1H NMR study of the reactions between carboplatin analogues [Pt(en)(Me-mal-O,O′)] and [Pt(en)(Me2-mal-O,O′)] and various methionine- and histidine-containing peptides under physiologically relevant conditions

Article abstract of DOI:10.1016/j.ica.2012.11.004

¹H NMR spectroscopy was applied to the study the reactions of [Pt(en)(Me-mal-O,O′)] and [Pt(en)(Me₂-mal-O,O′)] complexes (en is ethylenediamine, Me-mal and Me₂-mal are bidentate coordinated anions of 2-methylmalonic and 2,2-dimethylmalonic acids, respectively) with N-acetylated Ac-l-Met-Gly and Ac-l-Met-l-His-type peptides (Ac-l-Met-l-His, Ac-l-Met-Gly-l-His-GlyNH₂ and Ac-l-Met-Gly-Gly-l- His-Gly). The use of Me-mal and Me₂-mal Pt(II) complexes in the above reactions allows convenient monitoring of their biscarboxylate group via methyl peaks by ¹H NMR measurements. All reactions were realized at 37°C with equimolar amounts of the Pt(II) complex and the dipeptide at pH 7.40 in 50 mM phosphate buffer in D₂O. In all these reactions the ring-opened Me-mal and Me₂-mal Pt(II) adducts as an intermediate products were detected in solution for more than 48 h. We found that during this time in the reaction with Ac-l-Met-Gly these monodentate bound malonate ligands have been replaced by water molecule leading to the formation of the corresponding aqua Pt(II)-peptide complex which further promotes the regioselective cleavage of the peptide. However, in the reaction with Ac-l-Met-l-His-type peptides a selective intramolecular replacement of these malonate anions by the N3 imidazole nitrogen atom from histidine residue was occurred. This replacement reaction leads to the formation of the S,N3-macrochelate Pt(II)-peptide complex which was shown as very stable and hydrolytically inactive for more than two weeks.

Full text of DOI:10.1016/j.ica.2012.11.004

Inorganica Chimica Acta 395 (2013) 245–251
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Inorganica Chimica Acta
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1H NMR study of the reactions between carboplatin analogues [Pt(en)(Me-mal-O,O0)]
and [Pt(en)(Me2-mal-O,O0)] and various methionine- and histidine-containing
peptides under physiologically relevant conditions
⇑
ˇ
ˇ
´
´
Snezana Rajkovic, Darko P. Ašanin, Marija D. Zivkovic, Miloš I. Djuran
Department of Chemistry, Faculty of Science, University of Kragujevac, R. Domanovica 12, P.O. Box 60, 34000 Kragujevac, Serbia
a r t i c l e i n f o
a b s t r a c t
1H NMR spectroscopy was applied to the study the reactions of [Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-
mal-O,O0)] complexes (en is ethylenediamine, Me-mal and Me2-mal are bidentate coordinated anions of
Article history:
Received 1 August 2012
Received in revised form 30 October 2012
Accepted 9 November 2012
Available online 20 November 2012
2-methylmalonic and 2,2-dimethylmalonic acids, respectively) with N-acetylated Ac-
L
-Met-Gly and Ac-
L-
Met- -His-type peptides (Ac- -Met- -His, Ac- -Met-Gly- -His-GlyNH2 and Ac- -Met-Gly-Gly-L
L
L
L
L
L
L
-His-Gly).
The use of Me-mal and Me2-mal Pt(II) complexes in the above reactions allows convenient monitoring
of their biscarboxylate group via methyl peaks by 1H NMR measurements. All reactions were realized
at 37 °C with equimolar amounts of the Pt(II) complex and the dipeptide at pH 7.40 in 50 mM phosphate
buffer in D2O. In all these reactions the ring-opened Me-mal and Me2-mal Pt(II) adducts as an interme-
diate products were detected in solution for more than 48 h. We found that during this time in the reac-
Keywords:
Proton NMR spectroscopy
Malonate Pt(II) complexes
L
-Methionine- and
L-histidine-containing
tion with Ac-
molecule leading to the formation of the corresponding aqua Pt(II)–peptide complex which further pro-
motes the regioselective cleavage of the peptide. However, in the reaction with Ac- -Met- -His-type pep-
L-Met-Gly these monodentate bound malonate ligands have been replaced by water
peptides
Ring-opened Pt(II)–peptide complexes
L
L
tides a selective intramolecular replacement of these malonate anions by the N3 imidazole nitrogen atom
from histidine residue was occurred. This replacement reaction leads to the formation of the S,N3-macr-
ochelate Pt(II)–peptide complex which was shown as very stable and hydrolytically inactive for more
than two weeks.
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
peptides. In general, it was shown that these complexes bind to
the heteroatom in the side chain of methionine [9–16] or histidine
Recent years have witnessed an increasing interest in the study
of the interactions of platinum(II) and palladium(II) complexes
with sulfur- and histidine-containing peptides and proteins [1–
4]. This interest was induced through the facts that interactions
of platinum(II) complexes with methionine or cysteine residues
in peptides and proteins are thought to be responsible for a variety
of biological effects, such as deactivation of platinum(II) antitumor
complexes, development of cellular resistance to platinum drugs
and to toxic side effects, such as nephrotoxicity [5]. The presence
of histidine has also been established in a large number of enzyme
active centers [6] and the histidyl residue is probably the most
important metal-binding site in biological systems [7,8]. Moreover,
interest in the study of the interactions of platinum(II) [9–11] and
palladium(II) [10–24] complexes with methionine- and histidine-
containing peptides and proteins also become of cardinal impor-
tance after the discovery that their aqua complexes can be promis-
ing reagents for the hydrolytic cleavage of the above-mentioned
[10,11,17–25] and promote cleavage of the amide bond involving
the carboxylic group of the anchoring amino acid. The influence
of different factors, such as pH, temperature, solvent and steric ef-
fects of the substrate or catalyst, on this hydrolytic reaction has
been extensively investigated in the past two decades. Up to
now, most of these investigations were performed in strong acidic
media, whereas only a few reports concerning this hydrolytic reac-
tion between methionine- and histidine-containing peptides and
platinum(II) antitumor complexes under physiologically relevant
conditions have been reported [26–30]. The findings that Pt(II)
complexes can cleave peptides and proteins under physiologically
relevant conditions of the pH and temperature can have impor-
tance for a better understanding of the toxic side effects of Pt(II)
antitumor drugs. Our recent studies of the reactions between var-
ious Pt(II) complexes of the type [Pt(L)Cl2] and [Pt(L)(cbdca-O,O0] (L
is ethylenediamine, en; ( )-trans-1,2-diaminocyclohexane, dach;
( )-1,2-propylenediamine, 1,2-pn and cbdca is cyclobutane-1,1-
dicarboxylate) and Ac-L-Met-Gly dipeptide showed that hydrolysis
of the Met-Gly amide bond in this peptide occurred under physio-
logical conditions of pH and temperature (pH 7.40 and 37 °C) [31].
⇑
Corresponding author. Tel.: +381 34 300 251; fax: +381 34 335 040.
E-mail address: djuran@kg.ac.rs (M.I. Djuran).
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.11.004

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S. Rajkovic et al. / Inorganica Chimica Acta 395 (2013) 245–251
246
The cleavage of this peptide was much slower in the reaction with
[Pt(L)(cbdca-O,O0] than with [Pt(L)Cl2] complexes. Difference in the
catalytic abilities between these two type of Pt(II) complexes was
attributed to the different stability of their intermediate
AgNO3 at pH 2.00, according to a published method [41]. The solid
AgCl was removed by filtration in the dark. To the clear solution of
the [Pt(en)(H2O)2]2+ complex, equimolar amounts of the solid Me-
H2mal (or Me2-H2mal) acid and two equivalents of NaHCO3 were
added. The mixture was stirred at 60 °C for 3 h. All the complexes
were crystallized from water by cooling in a refrigerator. The pure
complexes were obtained by recrystallization from small amount
of water. The yield was between 50% and 60%. Anal. Calc. for
[Pt(en)(Me-mal-O,O0)] = C6H12N2O4Pt (FW = 371.25): C, 19.41; H,
3.26; N, 7.55. Found: C, 19.50; H, 3.22; N, 7.48%. Anal. Calc. for
[Pt(en)(Me2-mal-O,O0)] = C7H14N2O4Pt (FW = 385.28): C, 21.82; H,
3.66; N, 7.27. Found: C, 21.54; H, 3.42; N, 7.43%.
[Pt(L)(cbdca-O)(Ac-
complexes, respectively. The higher stability of the initially formed
[Pt(L)(cbdca-O)(Ac-
-Met-Gly-S)]ꢀ complex with respect to the
analogue [Pt(L)Cl(Ac- -Met-Gly-S)] intermediate product is in
L L-Met-Gly-S)]
-Met-Gly-S)]ꢀ and [Pt(L)Cl(Ac-
L
L
accordance with the slow replacement of its cbdca ligand by a
water molecule, finally resulting in the formation of the hydrolyt-
ically active [Pt(L)(H2O)(Ac-
are in accordance with those previously reported that very stable
ring-opened [Pt(NH3)2(cbdca-O)( -HMet-S)] complex was detected
during the reaction of carboplatin with -HMet and that similar
L
-Met-Gly-S)]+ complex. These findings
L
L
2.3. NMR (1H and 13C) characterization (D2O, 200 MHz)
ring-opened species was also found in the urine of animals treated
with carboplatin (carboplatin, [Pt(NH3)2(cbdca-O,O0)], is a widely
used second generation anticancer drug) [32]. Malonate complexes
of Pt(II)-am(m)ine exhibited antitumor activity without the neph-
rotoxic effects of cisplatin, cis-[Pt(NH3)2Cl2] [33,34].
[Pt(en)(Me-mal-O,O0)]: 1H NMR, d (ppm) 2.57 (s, 4H, 2CH2 from
en), 4.16 (q, H, a-CH from Me-mal), 1.37 (d, 3H, CH3 from Me-mal);
13C NMR, d (ppm) 16.54 (CH3 from Me-mal), 50.79 (CH2 from en),
53.68 (CH from Me-mal), 183.30 (C@O from Me-mal).
[Pt(en)(Me2-mal-O,O0)]: 1H NMR, d (ppm) 2.59 (s, 4H, 2CH2 from
en), 1.77 (s, 6H, 2CH3 from Me2-mal); 13C NMR, d (ppm) 27.87 (CH3
from Me2-mal), 50.68 (CH2 from en), 55.32 (C from Me2-mal),
185.63 (C@O from Me2-mal).
We report here reactions of peptides with methionine (Ac-
Met-Gly) and both methionine and histidine in the side chains
(Ac- -Met- -His, Ac- -Met-Gly- -His-GlyNH2 and Ac- -Met-Gly-
Gly-
-His-Gly) with carboplatin analogues, [Pt(en)(Me-mal-O,O0)]
L-
L
L
L
L
L
L
and [Pt(en)(Me2-mal-O,O0)]. Our use of 2-methylmalonate (Me-
mal) and 2,2-dimethylmalonate (Me2-mal) in the place of cyclobu-
tane-1,1-dicarboxylate (cbdca) allows convenient monitoring the
biscarboxylate group via methyl peaks by 1H NMR measurements.
Also, [Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-mal-O,O0)] complexes
showed better solubility in comparison with analogue [Pt(L)
(cbdca-O,O0)] (L is en, dach and 1,2-pn).
These data are in accordance with those reported previously for
the corresponding Pt(II) complexes [39,42].
2.4. Measurements
All pH measurements were realized at ambient temperature
using an Iskra MA 5704 pH meter calibrated with Fischer certified
buffer solutions of pH 4.00 and 7.00. The results were not corrected
for the deuterium isotope effect.
2. Experimental
The reactions of Ac-
L
-Met-Gly, Ac-
L-Met-L-His, Ac-L-Met-Gly-L-
2.1. Materials
His-GlyNH2 and Ac- -Met-Gly-Gly-
L
L
-His-Gly with the platinum(II)
complexes in 50 mM phosphate buffer at pH 7.40 in D2O were fol-
lowed by 1H NMR spectroscopy using a Varian Gemini 2000 spec-
trometer (200 MHz). Sodium trimethylsilylpropane-3-sulfonate
(TSP) was used as an internal reference. The final solution was
10 mM in each reactant. All reactions were performed at 37 °C.
Equimolar amounts of the platinum(II) complex and the peptide
were mixed in an 5 mm NMR tube and the rate constants were
determined from the proton NMR data recorded at appropriate
time intervals. The formation of the ring-opened complexes 1a
and 1b (Fig. 1) in the reactions of [Pt(en)(Me-mal-O,O0)] and
Distilled water was demineralized and purified to a resistance
greater than 10 M
(en), malonic acid (H2mal), 2-methylmalonic acid (Me-H2mal),
2,2-dimethylmalonic acid (Me2-H2mal) and K2[PtCl4] were ob-
tained from the Aldrich Chemical Co. All common chemicals were
X
cmꢀ1. The compounds D2O, ethylenediamine
of reagent grade. The dipeptide
was obtained from the Sigma Chemical Co. The dipeptide
nyl- -histidine ( -Met- -His) and pentapeptide -methionylglycyl-
glycyl- -histidylglycine ( -Met-Gly-Gly- -His-Gly) were obtained
from the Bachem A.G. The tetrapeptide N-acetylated- -methionyl-
glycyl- -histidylglycineamide (Ac- -Met-Gly- -His-GlyNH2) was
L-methionylglycine (
L-Met-Gly)
L-methio-
L
L
L
L
L
L
L
L
[Pt(en)(Me2-mal-O,O0)] complexes with Ac-
Met- -His dipeptides, respectively, was fitted to a second-order
L-Met-Gly and Ac-L-
L
L
L
L
synthesized by manual solid phase peptide synthesis using
Fmoc-chemistry [35,36]. The peptide was purified using semi-pre-
parative RP-HPLC, and analyzed by analytical HPLC and electro-
spray ionization mass spectrometry. The terminal amino group in
this peptide was acetylated by a standard method [12]. The
[Pt(en)Cl2] complex was synthesized according to a procedure pub-
lished in the literature [37–39]. The purity of the complex was
checked by elemental microanalyses. Anal. Calc. for [Pt(en)Cl2] =
C2H8N2Cl2Pt (FW = 326.08): C, 7.37; H, 2.47; N, 8.59. Found: C,
7.32; H, 2.50; N, 8.52%.
process [43] by plotting x/ao(ao ꢀ x) against t (ao is the initial con-
centration of the Pt(II) complex and x is the concentration of the
corresponding ring-opened complex at time t). The concentrations
of [Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-mal-O,O0)] complexes
were determined by integration of the resonances for the CH3 pro-
tons of bidentatly coordinated Me-mal at 1.37 and Me2-mal at
1.77 ppm and those for these protons at 1.24 for Me-mal and
1.27 ppm for Me2-mal both coordinated in monotopic fashion of
the corresponding ring-opened Pt(II)–peptide complex.
The deatachment of Me-mal and Me2-mal ligands from the
ring-opened Pt(II)–peptide complexes 1a and 1b leading to the for-
mation of the aqua Pt(II)–peptide complex 2 and macrochelate
complex 4 (Fig. 1), respectively, was fitted to a first-order process
[43] by plotting ln(ao/at) against t (ao is the concentration of the
corresponding ring-opened Pt(II)–peptide complex and at is the
concentration of this complex at time t). The concentrations of
the ring-opened complexes 1a and 1b were determined by integra-
tion of the resonances for the CH3 protons of the Me-mal at 1.24
and Me2-mal at 1.27 ppm coordinated in monotopic fashion to
2.2. Syntheses of [Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-mal)]
complexes
The [Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-mal-O,O0)] com-
plexes were synthesized by modification of the procedure pub-
lished in the literature [40].
The chlorido complex [Pt(en)Cl2] was converted into the corre-
sponding diaqua complex by treatment with 1.95 equivalents of

´
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S. Rajkovic et al. / Inorganica Chimica Acta 395 (2013) 245–251
O
H2
N
O
H2
N
O
O
O
O
CH3
CH3
H
Pt
Pt
or
CH3
N
N
O
H2
O
H2
[Pt(en)(Me-mal-O,O`)]
[Pt(en)(Me -mal-O,O`)]
2
o
pH = 7.4; t = 37
C
Ac-L-Met-L-His
Ac-L-Met-Gly
H
CH3
H
CH3
O-
N 3
1NH
O-
H2
N
H2
N
O
O
O
O
O
O
O
Pt
O
Pt
N
O-
O-
N
S
S
H2
H2
H3C
N
H3C
N
H
H
O
O
HN
HN
H3C
O
O
H3C
1a
1b
-
-
[Pt(en)(Me-mal-O)(Ac-L-Met-Gly-S)]
[Pt(en)(Me-mal-O)(Ac-L-Met-L-His-S)]
O
O
-O
H
-O
H
-O
CH3
-O
CH3
O
O
O
H
N
O
O-
H3C
N
H
H3C
NH
O
O
O
H
N
OH2
O-
O
S
Pt NH2
H3C
S
H2N
2
H3C
H2N
3
N
1NH
Pt
NH2
+
[Pt(en)(H O)(Ac-L-Met-Gly-S)]
2
4
+
O
H
[Pt(en)(Ac-L-Met-L-His-S,N )]
3
H3C
N
O-
O
O
OH2
+
H2N
O-
S
Pt NH2
Gly
H3C
H2N
3
+
[Pt(en)(H O)(Ac-L-Met-S)]
2
Fig. 1. Different pathways of the reaction of the Ac-L-Met-Gly and Ac-L
-Met-His-type peptides with [Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-mal-O,O0)] complexes. The
corresponding Pt(II) complex and peptide were mixed in a 1:1 M ratio and all reactions performed at pH 7.40 and at 37 °C in 50 mM phosphate buffer in D2O.
the platinum(II) and those for these protons of the free Me-mal and
Me2-mal ligands at 1.31 and 1.34 ppm, respectively.
lar amounts of the platinum(II) complex and peptide at the pH 7.40
in 50 mM phosphate buffer in D2O and at 37 °C. Under these exper-
imental conditions ethylenediamine ligand remains bound to the
Pt(II) [33], while bidentate coordinated Me-mal and Me2-mal li-
gands in the platinum(II) complexes undergo substitution during
their reactions with peptides. In the reactions of [Pt(en)(Me-mal-
3. Results and discussion
The reactions of [Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-mal-
O,O0)] complexes (en is ethylenediamine, Me-mal and Me2-mal
are bidentate coordinated anions of 2-methylmalonic and 2,2-dim-
O,O0)] and [Pt(en)(Me2-mal-O,O0)] complexes with Ac-
L-Met-Gly
very slow hydrolytic cleavage of the Met-Gly amide bond was ob-
served, while in the reactions between these Pt(II) complexes and
ethylmalonic acids, respectively, with N-acetylated Ac-
and Ac- -Met- -His-type peptides (Ac- -Met- -His, Ac- -Met-Gly-
-His-GlyNH2 and Ac- -Met-Gly-Gly- -His-Gly) were studied by
1H NMR spectroscopy. All reactions were performed with equimo-
L
-Met-Gly
peptides containing both L-methionine and L-histidine amino acids
L
L
L
L
L
in the side chains no hydrolysis of these peptides was observed and
only macrochelate platinum(II)–peptide complexes formed as a fi-
nal products. In all these reactions an intermediate Pt(II)–peptide
L
L
L

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S. Rajkovic et al. / Inorganica Chimica Acta 395 (2013) 245–251
248
Table 1
Characteristic proton NMR chemical shifts for the reactions of [Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-mal-O,O0)] with Ac-L-Met-Gly dipeptide at pH 7.40 in 50 mM phosphate
buffer in D2O and at 37 °C.
Reactants/products
Characteristic 1H NMR chemical shifts (d, ppm; J, Hz)a
a-CH-mal
CH3-mal
(CH3)2-mal
CH3-Met
Gly-CH2
Ac-
L
-Met-Gly
2.11(s)
3.78 (s)
[Pt(en)(Me-mal-O,O0)]
4.16 (q, J = 6.92)
3.29 (q, J = 7.02)
1.37 (d, J = 6.96)
1.24 (d, J = 7.14)
[Pt(en)(Me2-mal-O,O0)]
[Pt(en)(Me-mal-O)(Ac-
[Pt(en)(Me2-mal-O)(Ac-
[Pt(en)(H2O)(Ac-
Free Me-mal
Free Me2-mal
Free Gly
1.77 (s)
1.27 (s)
L
-Met-Gly-S)]ꢀ (1a)
-Met-Gly-S)]ꢀ (1a)
2.38 (s)
2.38 (s)
2.38 (s)
3.78 (s)
3.78 (s)
3.78 (s)
L
L
-Met-Gly-S)]+ (2)
1.31(d)
1.34 (s)
3.56 (s)
a
In all complexes the multiplet for methylene protons of en is centered at 2.70 ppm.
Table 2
Observed rate constants for the reactions of [Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-
mal-O,O0)] complexes with Ac-L-Met-Gly and Ac-L-Met-L-His dipeptides. All rate
constants were obtained from 1H NMR measurements at pH 7.40 in 50 mM
phospahate buffer in D2O and at 37 °C.
Reactions
Second-order rate
constants for
First-order rate
constants for
formation of the ring- conversion of 1a into
opened complexes 1a 2 (Ac- -Met-Gly) and
L
(Ac-
L
-Met-Gly) and
1b into 4 (Ac-
L-Met-L-
1b (Ac-L-Met-L-
His)106 k/sꢀ1
His)103k2/Mꢀ1 sꢀ1
[Pt(en)(Me-mal-O,O0)]
(26.20 0.62)
(3.02 0.02)
(1.45 0.02)
(5.22 0.03)
(5.98 0.03)
+ Ac-
[Pt(en)(Me2-mal-O,O0)]
+ Ac- -Met-Gly
[Pt(en)(Me-mal-O,O0)]
+ Ac- -Met- -His
[Pt(en)(Me2-mal-O,O0)] +
Ac- -Met- -His
L-Met-Gly
(4.05 0.04)
(11.95 0.31)
(6.19 0.02)
L
L
L
L
L
Fig. 2. Parts of the 1H NMR spectra recorded during the reaction of [Pt(en)(Me2-
mal-O,O0)] with an equimolar amount of Ac-
-Met-Gly dipeptide as a function of
products containing ring-opened Me-mal and Me2-mal rings were
detected in solution.
L
time at pH 7.40 and at 37 °C in 50 mM phosphate buffer in D2O with TSP as the
internal standard. The resonances assigned as (j), (N) and (d) correspond to the
methyl malonate protons of the [Pt(en)(Me2-mal-O)(Ac-
mal and [Pt(en)(Me2-mal-O,O0)], respectively.
L
-Met-Gly-S)]ꢀ, free Me2-
3.1. Reactions of Pt(II) complexes with Ac-L-Met-Gly
The schematic presentation of the reactions of Ac-L-Met-Gly
of Ac-L
-Met-Gly with [Pt(en)(Me2-mal-O,O0)] was observed in 1H
dipeptide with two platinum(II) complexes, [Pt(en)(Me-mal-O,O0)]
and [Pt(en)(Me2-mal-O,O0)], is given in Fig. 1. When an equimolar
amount of the Pt(II) complex was incubated with Ac-L-Met-Gly dipep-
NMR spectrum from difference in the chemical shifts of the singlet
for the methyl Me2-mal protons at 1.77 ppm for [Pt(en)(Me2-mal-
O,O0)] and that for these protons at 1.27 ppm due to the complex 1a
(Fig. 1 and Table 1). However, in the present study the formation of
the ring-opened complex in the reaction of [Pt(en)(mal-O,O0)] (mal
tide under the above-mentioned experimental conditions, the first
product observed in solution after 15 min of the reaction was an
intermediate complex 1a (Fig. 1). The formation of this intermedi-
ate product containing monodentate coordinated Me-mal (or
Me2-mal) ligand was evidenced in the 1H NMR spectrum by the
simultaneous decline of the resonances at 2.11 ppm, arising from
is bidentate coordinated anion of malonic acid) with Ac-
L
-Met-Gly
-CH2
could not be followed by 1H NMR spectroscopy based on the
a
protons of malonate anion because of the rapid exchange of its
methylene protons with deuterium from D2O solvent. Also, this ex-
change occurred when pure malonic acid was dissolved in D2O. The
coordination of malonic acid to Pt(II) complex additionally acti-
vates H/D exchange [44,45].
the S-methyl protons of free Ac-L-Met-Gly, and growth of the
resonance at 2.38 ppm, corresponding to these protons for the
dipeptide coordinated to Pt(II) through the sulfur atom (Table 1).
Additionally, formation of the intermediate complex 1a for the
L
-Met-Gly dipeptide with [Pt(en)(Me-mal-O,O0)]
The formation of the ring-opened adducts 1a in the above
investigated reactions was followed during time and second-order
reaction of Ac-
was followed from difference in the chemical shifts of the reso-
nances for -CH at 4.16 (q, J = 6.92) and CH3 malonate protons at
rate constant for the reaction of [Pt(en)(Me-mal-O,O0)] with Ac-
L-
a
Met-Gly, k2 = (26.20 0.62) ꢁ 10ꢀ3 Mꢀ1 sꢀ1, was six times lager
from that for the reaction of this peptide with analogue
[Pt(en)(Me2-mal-O,O0)] complex, k2 = (4.05 0.04) ꢁ 10ꢀ3 Mꢀ1 sꢀ1
(Table 2).
1.37 ppm (d, J = 6.96 Hz) for [Pt(en)(Me-mal-O,O0)] and those for
these protons of 1a at 3.29 (q, J = 7.02) and 1.24 ppm (d, J =
7.14 Hz), respectively (Fig. 1 and Table 1). These chemical shifts
are in accordance with those previously reported for the ring-
opened Me-mal-O adducts containing monodentate coordinated
thioethers in the reactions of the [Pt(en)(Me-mal-O,O0)] complex
It was found that complex 1a under the above mentioned
experimental conditions was converted into the hydrolytically ac-
tive complex 2 (Fig. 1). This conversion proceeds through the
with Ac-L-Met and Met-Gly [33]. Formation of 1a in the reaction

´
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S. Rajkovic et al. / Inorganica Chimica Acta 395 (2013) 245–251
[Pt(en)(Me-mal-O)(Ac-L-Met-Gly-S)]- (1a)
(a)
(b)
[Pt(en)(Me2-mal-O)(Ac-L-Met-Gly-S)]- (1a)
free Me2-mal
free Me-mal
[Pt(en)(Me-mal-O,O’)]
[Pt(en)(Me2-mal-O,O’)]
Fig. 3. Time dependence of relative percentages of Me-mal and Me2-mal species (based on the CH3 malonate protons) for the reactions of [Pt(en)(Me-mal-O,O0)] (a) and
[Pt(en)(Me2-mal-O,O0)] (b) complexes with an equimolar amount of Ac-
-Met-Gly dipeptide at pH 7.40 in 50 mM phosphate buffer in D2O and at 37 °C.
L
detachment of the monodentate coordinated Me-mal and Me2-mal
ligands from Pt(II) and their replacement by water molecules. The
conversion of 1a into 2 is evident in the 1H NMR spectrum by the
simultaneous decline of the doublet at 1.24 and singlet at 1.27 ppm
due to the methyl protons of monodentate coordinated Me-mal
and Me2-mal ligands, respectively, and the growth of these reso-
nances at 1.31 and 1.34 ppm for the free Me-mal and Me2-mal li-
gands, respectively (Table 1). The parts of 1H NMR spectra
measured during time for the reaction between [Pt(en)(Me2-mal-
following order: [Pt(en)(cbdca-O,O0)] < [Pt(en)(Me2-mal-O,O0)] <
[Pt(en)(Me-mal-O,O0)] < [Pt(en)(mal-O,O0)] < [Pt(en)Cl2]. This find-
ing can be attributed to the steric influence of the leaving ligands
on the catalytic properties of the Pt(II) complexes indicating that
cbdca ligand is more sterically demanding in comparison with
bidentate malonate-type or monodentate chlorido ligands. This is
clearly demonstrated in the reaction of Ac-
with three malonate Pt(II) complexes which differ in the number
of the methyl groups attached on the -malonate carbon atom.
L-Met-Gly dipeptide
a
O,O0)] and Ac-
L-Met-Gly are presented in Fig. 2. The plots illustrat-
The rate of the cleavage of this dipeptide was decreased in the fol-
lowing order: mal > Me-mal > Me2-mal (see inserted chart in
Fig. 4). This undoubtedly confirms that inhibition of the hydrolytic
ing the changes in concentration [%] of 1a and free Me-mal and
Me2-mal ligands during 48 h are presented in Fig. 3. From this fig-
ure it can be seen that concentration of 1a was increased in the
first 5 h for the Me-mal and 15 h for the Me2-mal ring opened com-
plex. After this time the concentrations of these ring-opened ad-
ducts were decreased finally being about 25% after 48 h. The
first-order rate constants for the conversion of 1a into 2 were
determined by 1H NMR measurements, k = (3.02 0.02) ꢁ 10ꢀ6 sꢀ1
for Me-mal and k = (1.45 0.02) ꢁ 10ꢀ6 sꢀ1 for Me2-mal (see Sec-
tion 2 and Table 2).
mal
Me-mal
Me2-mal
We found that complex 2, obtained after displacement of mono-
dentate coordinated Me-mal and Me2-mal ligands from complex
1a by water molecule, promote the regioselective cleavage of the
Met-Gly amide bond in the Ac-L-Met-Gly dipeptide (Fig. 1). This
hydrolytic reaction is very slow and it could be followed success-
fully by 1H NMR spectroscopy. The resonance at 3.78 ppm corre-
sponding to the glycine protons of the non-hydrolyzed peptide
decreased while that at 3.56 ppm for free glycine increased (Ta-
ble 1). Upon addition of glycine to the reaction mixture, its reso-
nance was enhanced. The amount of hydrolyzed Met-Gly amide
bond in the complex 2 was determined by integration of the reso-
nance for the glycine protons in the non-hydrolyzed peptide and
that for the free glycine. In this study we compared the catalytic
[Pt(en)Cl2]
[Pt(en)( mal-O,O’)]
[Pt(en)(Me-mal-O,O’)]
[Pt(en)(Me2-mal-O,O’)]
[Pt(en)(cbdca-O,O’)]
abilities in the cleavage of the Ac-L-Met-Gly dipeptide for Pt(II)
complexes with malonate-type ligands, [Pt(en)(mal-O,O0)],
[Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-mal-O,O0)], with those for
previously investigated [Pt(en)Cl2] and [Pt(en)(cbdca-O,O0)]
complexes [31] (Fig. 4). From this figure in can be concluded that
the catalytic abilities of these Pt(II) complexes increase in the
Fig. 4. The time dependence of the hydrolytic cleavage of the Met-Gly amide bond
in the Ac-L-Met-Gly dipeptide with different Pt(II) complexes. All reactions were
realized at 37 °C with equimolar amounts of the Pt(II) complex and the dipeptide at
pH 7.40 in 50 mM phosphate buffer. The inserted figure shows the hydrolysis of the
Met-Gly bond in the presence of three malonato–Pt(II)-type complexes during
2 days.

´
S. Rajkovic et al. / Inorganica Chimica Acta 395 (2013) 245–251
250
Table 3
Characteristic 1H NMR chemical shifts (d, ppm) for the Ac-L-Met-L-His-type peptides and corresponding macrochelate Pt(II)-peptide complexes 4 formed in the reaction with
[Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-mal-O,O0)] complexes at pH 7.40 and at 37 °C in D2O. The chemical shifts of monodentate coordinated Me-mal and Me2-mal ligands of the
ring-opened complexes 1b are identical with those for complex 1a shown in Table 1.
Peptide/macrochelate Pt(II)-peptide complex 4
Imidazole protons
C2H
S-CH3
Gly1CH2
Gly2CH2
Gly3CH2
C5H
Ac-
[Pt(en)(Ac-
Ac- -Met-Gly1-
[Pt(en)(Ac- -Met-Gly-
Ac- -Met-Gly1-Gly2- -His-Gly3
[Pt(en)(Ac- -Met-Gly-Gly-
-His-Gly-S,N3)]+
L
-Met-
L
-His
-Met-
-His-Gly2-NH2
-His-Gly-NH2-S,N3)]+
8.44
8.13
8.31
8.09
8.61
8.07 7.09
7.20
7.08
7.19
7.02, 6.97
7.34
2.10
2.58,2.60
2.11
2.57
2.11
L
L
-His-S,N3)]+
L
L
3.93
3.98
4.04
4.00
3.93
3.93
3.96
3.94
L
L
L
L
3.80
3.81
L
L
7.09
2.59
reaction was effected by the methyl group in the leaving malonate
ligand. The same conclusion can be drawn from our kinetic data
presented in Table 2. From this table we can see that replacement
of monodentate coordinated Me2-mal ligand by water molecule in
the ring-opened complex 1a (Fig. 1) was two times slower in re-
spect to the corresponding Me-mal complex, both resulting in
the formation of the hydrolytically active complex 2. Difference
in the replacement of these two ligands can be correlated with dif-
ferent stability of their ring-opened complexes.
resonances at 1.24 and at 1.27 ppm for these protons in the
ring-opened [Pt(en)(Me-mal-O)(Ac- -Met- -His-S)] and [Pt(en)(Me2-
mal-O)(Ac- -Met- -His-S)] complexes, respectively, had been
L
L
L
L
appeared. The concentrations of the starting Pt(II) complex and
corresponding ring-opened species were determined during time
by integration of the above-mentioned resonances. The second-order
rate constants for the reactions of [Pt(en)(Me-mal-O,O0)] and
[Pt(en)(Me2-mal-O,O0)] complexes with Ac-
L-Met-L-His are similar
with those for the reactions between these Pt(II) complexes and
Ac-
L
-Met-Gly dipetide, k2 = (11.95 0.31) ꢁ 10ꢀ3 Mꢀ1sꢀ1 for Me-mal
and k2 = (6.19 0.02) ꢁ 10ꢀ3 Mꢀ1 sꢀ1 for Me2-mal (Table 2).
3.2. Reactions of Pt(II) complexes with Ac-L-Met-L-His-type peptides
The second step of the reaction between Pt(II) complexes and
In the second part of this study we used 1H NMR spectroscopy
to investigate the reactions of [Pt(en)(Me-mal-O,O0)] and
[Pt(en)(Me2-mal-O,O0)] complexes with peptides containing both
Ac-L-Met-His-type peptides is conversion of 1b into 4 (Fig. 1). This
conversion proceeds through the intramolecular replacement of
monodentate coordinated Me-mal and Me2-mal ligands from 1b
with the N3 imidazole nitrogen atom of the histidine residue.
The conversion of 1b into 4 is evident in the 1H NMR spectrum
by the simultaneous decline of the resonances due to the methyl
protons of the Me-mal and Me2-mal coordinated in monotopic
fashion to the Pt(II) and growth of those for these protons of free
Me-mal and Me2-mal ligands. The chemical shifts for these reso-
nances are almost identical with those observed for the conversion
L
-methionine and
namely Ac- -Met-
Gly-Gly- -His-Gly (Ac-
molar amount of Pt(II) complex was incubated with the corre-
sponding Ac- -Met- -His-type peptide under the above
L
-histidine amino acids in the side chains,
-His, Ac- -Met-Gly- -His-GlyNH2 and Ac- -Met-
-Met- -His-type peptides). When an equi-
L
L
L
L
L
L
L
L
L
L
mentioned experimental conditions only one Pt(II)–peptide com-
plex in these reactions was observed in solution as a final product
after 48 h (see Fig. 1, complex 4). As it was shown in Fig. 1 complex
4 has bidentate coordinated peptide via the N3 atom of the imid-
azole ring and methionine sulfur atom. The complex 4 was showed
as very stable product during time and no hydrolysis of amide
bonds in the above investigated peptides had been observed dur-
ing two weeks. The formation of this macrochelate complex pro-
ceeds in two steps. The first step of this reaction is monodentate
of 1a into
2
in the reaction of [Pt(en)(Me-mal-O,O0)] and
-Met-Gly dipeptide
[Pt(en)(Me2-mal-O,O0)] complexes with Ac-
L
(Table 1). Additionally, the conversion of 1b into 4 can be followed
in the 1H NMR spectrum from difference in the chemical shifts of
the C2H and C5H imidazole protons for the free and N3-bound his-
tidine side chain to Pt(II) (Table 3). From Table 3 it can be seen that
the C2H and C5H resonances are upfield shifted after peptide coor-
dination through the N3 nitrogen atom of the imidazole to Pt(II).
coordination of Ac-L-Met-L-His-type peptide to the Pt(II) through
The higher field chemical shifts for the C2H (
Dd = 0.22–0.31 ppm)
the methionine sulfur atom. This reaction yields to the formation
of the ring-opened complex 1b which was evidenced in the 1H
NMR spectrum from difference in the chemical shifts of the methyl
protons for the free at 2.10–2.11 and these protons for the Pt(II)–
sulfur bound peptide at 2.57–2.60 ppm after 15 min (Table 3).
These chemical shifts are in accordance with those previously re-
ported for the reactions of Pt(II) complexes with different methio-
nine-containing peptides [10,30]. The monodentate coordination
with respect to the C5H proton ( d = 0.12–0.17 ppm) can be attrib-
D
uted to the fact that this proton is closer to the N3 binding center
[46–48]. The first-order rate constants for conversion of 1b into 4,
k = (5.22 0.03) ꢁ 10ꢀ6 sꢀ1 for Me-mal and k = (5.98 0.03) ꢁ
10ꢀ6 sꢀ1 for Me2-mal, are lager from those for the conversion of
1a into 2, k = (3.02 0.02) ꢁ 10ꢀ6 sꢀ1 for Me-mal and k =
(1.45 0.02) ꢁ 10ꢀ6 sꢀ1 for Me2-mal (Table 2). This can be attrib-
uted to the fact that N3 nitrogen atom of imidazole ring from
of Ac-L-Met-L
-His-type peptides to the [Pt(en)(Me-mal-O,O0)] and
Ac-
in the reaction with Ac-
same S,N3-coordination mode of the Pt(II) occurred in the reactions
with Ac- -Met- -His, Ac- -Met-Gly- -His-GlyNH2 and Ac- -Met-
Gly-Gly- -His-Gly peptides. Also, no influence of Gly residue (one
L
-Met-
L
-His dipeptide is better nuclephile than water molecule
[Pt(en)(Me2-mal-O,O0)] caused the opening of the malonate rings
in these complexes. This ring-opening process can be followed in
the 1H NMR spectrum from difference in the chemical shifts of
the methyl malonate protons for bidentate and those for these pro-
tons of monodentate bound Me-mal and Me2-mal ligands. These
chemical shifts are almost identical with those for the reactions
of [Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-mal-O,O0)] complexes
L-Met-Gly dipeptide. We found that the
L
L
L
L
L
L
or two) inserted between Met and His anchoring amino acids on
the formation rate of complex 4 was observed.
with Ac-L-Met-Gly dipeptide (see previous section and Table 1).
The second-order rate constants for the formation of 1b were
determined from 1H NMR measurements (Table 2). The doublet
at 1.37 and singlet at 1.77 ppm due to the methyl malonate pro-
tons of [Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-mal-O,O0)] com-
plexes, respectively, were decreased during time and new
4. Conclusions
From the present investigation, it can be stated that the reaction
of [Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-mal-O,O0)] complexes
with N-acetylated Ac-L-Met-Gly and Ac-L-Met-L-His-type peptides

´
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S. Rajkovic et al. / Inorganica Chimica Acta 395 (2013) 245–251
[12] L. Zhu, N.M. Kostic´, Inorg. Chem. 31 (1992) 3994.
under physiological conditions (pH 7.40 and 37 °C) primarily pro-
´
[13] L. Zhu, N.M. Kostic, J. Am. Chem. Soc. 115 (1993) 4566.
ceeds with the formation of the ring-opened malonate adducts
containing monodentate methionine bound peptide to the Pt(II).
These ring-opened species are present in solution for more than
48 h and during that time the monodentate coordinated malonate
ligand (Me-mal or Me2-mal) has been slowly replaced intermolec-
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L
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L
L
[20] X. Chen, L. Zhu, H. Yan, X. You, N.M. Kostic´, J. Chem. Soc., Dalton Trans. (1996)
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water molecules leads to the formation of the hydrolytically active
aqua Pt(II)–peptide complex which further promotes slow hydro-
lysis of the Met-Gly amide bond in the Ac-
However, intramolecular replacement of these malonate anions
by the histidine residue of Ac- -Met- -His-type peptides leads to
the formation of very stable and hydrolytically inactive macroche-
late Pt(II)–peptide complex. The previous results confirming that
the ring-opened carboplatin adducts containing monodentate thio-
ethers were also observed in the reactions of the anticancer drug
2653.
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carboplatin with
a variety of sulfur-containing amino acids
ˇ
´
´
[49,50] together with those for the presently investigated reactions
of carboplatin analogues [Pt(en)(Me-mal-O,O0)] and [Pt(en)(Me2-
mal-O,O0)] with methionine-containing peptides contribute to the
current hypothesis that the ring-opened adducts of chelated dicar-
boxylate platinum anticancer complexes with methionine deriva-
tives could play important role in their mechanism of action.
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Acknowledgements
This work was funded in part by the Ministry of Education, Sci-
ence and Technological Development of the Republic of Serbia
(Project No. 172036).
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