Hydrolysis of the amide bond in N-acetylated l-methionylglycine catalyzed by various platinum(II) complexes under physiologically relevant conditions

Article abstract of DOI:10.1016/j.poly.2010.12.039

The hydrolytic reactions between various Pt(II) complexes of the type [Pt(L)Cl₂] and [Pt(L)(CBDCA-O,O′] (L is ethylenediamine, en; (±)-trans-1,2-diaminocyclohexane, dach; (±)-1,2-propylenediamine, 1,2-pn and CBDCA is the 1,1-cyclobutanedicarboxylic anion) and the N-acetylated l-methionylglycine dipeptide (MeCOMet-Gly) were studied by ¹H NMR spectroscopy. 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. Under these experimental conditions, a very slow cleavage of the Met-Gly amide bond was observed and this hydrolytic reaction proceeds through the intermediate [Pt(L)(H₂O)(MeCOMet-Gly-S)]⁺ complex. In general, it can be concluded that faster hydrolytic cleavage of the MeCOMet-Gly dipeptide was observed in the reaction with the chloride complex than with corresponding CBDCA Pt(II) complexes. The steric effects of the Pt(II) complex on the hydrolytic cleavage of the amide bond in the MeCOMet-Gly dipeptide were also investigated by ¹H NMR spectroscopy. It was found that the rate of hydrolysis decreases as the steric bulk of the CBDCA and chlorido Pt(II) complexes increase (en > 1,2-pn > dach). These results contribute to a better understanding of the toxic side effects of Pt(II) antitumor drugs and should be taken into consideration when designing new potential Pt(II) antitumor drugs with preferably low toxic side effects.

Full text of DOI:10.1016/j.poly.2010.12.039

Polyhedron 30 (2011) 947–952
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Hydrolysis of the amide bond in N-acetylated L-methionylglycine catalyzed
by various platinum(II) complexes under physiologically relevant conditions
⇑
ˇ
´
ˇ
´
Marija D. Zivkovic, Darko P. Ašanin, Snezana Rajkovic, 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
The hydrolytic reactions between various 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 the 1,1-cyclobutanedicarboxylic anion) and the N-acetylated L-methionylglycine dipeptide
Article history:
Received 26 October 2010
Accepted 23 December 2010
Available online 6 January 2011
(MeCOMet–Gly) were studied by 1H NMR spectroscopy. All reactions were realized at 37 °C with equimo-
lar amounts of the Pt(II) complex and the dipeptide at pH 7.40 in 50 mM phosphate buffer in D2O. Under
these experimental conditions, a very slow cleavage of the Met–Gly amide bond was observed and this
hydrolytic reaction proceeds through the intermediate [Pt(L)(H2O)(MeCOMet–Gly–S)]+ complex. In gen-
eral, it can be concluded that faster hydrolytic cleavage of the MeCOMet–Gly dipeptide was observed in
the reaction with the chloride complex than with corresponding CBDCA Pt(II) complexes. The steric
effects of the Pt(II) complex on the hydrolytic cleavage of the amide bond in the MeCOMet–Gly dipeptide
were also investigated by 1H NMR spectroscopy. It was found that the rate of hydrolysis decreases as the
steric bulk of the CBDCA and chlorido Pt(II) complexes increase (en > 1,2-pn > dach). These results con-
tribute to a better understanding of the toxic side effects of Pt(II) antitumor drugs and should be taken
into consideration when designing new potential Pt(II) antitumor drugs with preferably low toxic side
effects.
Keywords:
Platinum(II) complexes
L
-Methionylglycine
Proton NMR spectroscopy
Hydrolysis
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
catalyst, on this hydrolytic reaction has been extensively investi-
gated in the past two decades. Up until now, most of these inves-
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]. Particularly, 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 plati-
num drugs and toxic side effects, such as nephrotoxicity [5]. More-
over, interest in the study of the interactions of platinum(II) [6–8]
and palladium(II) [7–21] complexes with methionine- and histi-
dine-containing peptides and proteins also became of cardinal
importance after the discovery that their aqua complexes can be
promising reagents for the hydrolytic cleavage of the above-
mentioned peptides. In general, it was shown that platinum(II)
and palladium(II) aqua complexes bind to the heteroatom in the
side chain of methionine [6–13] or histidine [7,8,14–22] and pro-
mote 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 effects of the substrate or
tigations were performed in strongly acidic media, whereas only a
few reports concerning this hydrolytic reaction between methio-
nine- and histidine-containing peptides and platinum(II) antitu-
mor complexes under physiological relevant conditions have
been reported [23–27].
This paper reports on the 1H NMR investigations of the hydroly-
sis reactions between various Pt(II) complexes of the type [Pt(L)Cl2]
and [Pt(L)(CBDCA-O,O0] (where
L is bidentate coordinated
ethylenediamine, en; ( )-trans-1,2-diaminocyclohexane, dach or
( )-1,2-propylenediamine, 1,2-pn; and CBDCA is the 1,1-cyclob-
utanedicarboxylic anion) and the N-acetylated L-methionylglycine
dipeptide (MeCOMet–Gly) under physiological conditions of pH
and temperature (pH 7.40 and 37 °C). The observed rates of the
peptide hydrolysis are explained in terms of the leaving ligand
(chlorido or CBDCA) and the steric hindrance of the chelating
diamine ligand (L) on the Pt(II) complex.
2. Experimental
2.1. Materials
⇑
The distilled water was demineralized and purified to a resis-
tance great than 10 MX cm. The compounds D2O, DNO3, NaOD,
Corresponding author. Tel.: +381 34 300 251; fax: +381 34 335 040.
E-mail address: djuran@kg.ac.rs (M.I. Djuran).
0277-5387/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2010.12.039

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M.D. Zivkovic et al. / Polyhedron 30 (2011) 947–952
948
ethylenediamine (en), ( )-trans-1,2-diaminocyclohexane, (dach),
( )-1,2-propylenediamine (1,2-pn) and K2[PtCl4] were obtained
from the Aldrich Chemical Co. All common chemicals were of re-
2.3.2. NMR (1H and 13C) characterization (DMSO-d6, 250 MHz)
[Pt(en)(CBDCA-O,O0)]: 1H NMR, d (ppm): 2.23 (s, 4H, 2CH2 from
en), 5.55 (s, 4H, 2NH2), 1.64 (m, 2H,
c-CH2 from CBDCA), 2.65 (t,
agent grade. The dipeptide
L
-methionylglycine was obtained from
4H,
a
-CH2 and b-CH2 from CBDCA); 13C NMR, d (ppm): 47.84
the Sigma Chemical Co. The terminal amino group in this peptide
was acetylated by standard methods [9].
(CH2 from en), 14.99 (C3 from CBDCA), 30.19 (C2 and C4 from
CBDCA), 55.47 (C1 from CBDCA), 177.23 (C@O). [Pt(1,2-pn)
(CBDCA-O,O0)]: 1H NMR, d (ppm): 1.11 (d, 3H, CH3 from 1,2-pn),
2.12–2.56 (m, 3H, CH and CH2 from 1,2-pn), 5.22–6.03 (m, 4H,
2.2. Syntheses of [Pt(L)Cl2]-type complexes (L is en, dach or 1,2-pn)
2NH2), 1.65 (m, 2H,
c-CH2 from CBDCA), 2.65 (t, 4H, a-CH2 and
b-CH2 from CBDCA); 13C NMR, d (ppm): 15.82 (CH3 from 1,2-pn),
53.49 (CH2 from 1,2-pn), 56.41 (CH from 1,2-pn), 15.07 (C3 from
CBDCA), 30.10 and 30.38 (C2 and C4 from CBDCA), 56.20 (C1 from
CBDCA), 177.42 (C@O). [Pt(dach)(CBDCA-O,O0)]: 1H NMR, d (ppm):
1.11 (m, 4H, 2CH2, C4 and C5 from dach), 1.62 (m, 4H, 2CH2, C3 and
C6 from dach), 2.03 (m, 2H, 2CH, C1 and C2 from dach), 4.98–5.71
Platinum(II) complexes of the type [Pt(L)Cl2] were synthesized
according to procedures published in the literature [28–30].
K2PtCl4 was dissolved in water and mixed with an equimolar
amount of the diamine ligand. The pH of the solution was adjusted
to ca. 3 by addition of 1 M HCl and the mixture was stirred at 80 °C
for 2 h. All the complexes were crystallized from water at room
temperature. The pure complexes were obtained by recrystalliza-
tion from a small amount of water. The yield was between 80%
and 90%.
(m, 4H, 2NH2), 1.65 (m, 2H, c-CH2 from CBDCA), 2.68 (t, 4H, a-CH2
and b-CH2 from CBDCA); 13C NMR, d (ppm): 24.07 (C4 and C5 from
dach), 31.49 (C3 and C6 from dach), 62.06 (C1 and C2 from dach),
15.04 (C3 from CBDCA), 30.26 (C2 and C4 from CBDCA), 55.47 (C1
from CBDCA), 177.34 (C@O). These data are in accordance with
those reported previously for the corresponding Pt(II) complexes
[33,34].
2.2.1. Elemental microanalyses
Anal. Calc. for [Pt(en)Cl2] = C2H8N2Cl2Pt (FW = 326.08): C, 7.37;
H, 2.47; N, 8.59. Found: C, 7.48; H, 2.43; N, 8.56%. Anal. Calc. for
[Pt(1,2-pn)Cl2] = C3H10N2Cl2Pt (FW = 340.11): C, 10.59; H, 2.96; N,
8.24. Found: C, 11.15; H, 3.17; N, 8.05%. Anal. Calc. for
[Pt(dach)Cl2] = C6H14N2Cl2Pt (FW = 380.17): C, 18.96; H, 3.71; N,
7.37. Found: C, 19,20; H, 3.49; N, 7.46%.
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. NMR spectra of Pt(II) complexes
2.2.2. NMR (1H and 13C) characterization (DMSO-d6, 250 MHz)
[Pt(en)Cl2]: 1H NMR, d (ppm): 2.24 (s, 4H, 2CH2), 5.30 (s, 4H,
2NH2); 13C NMR, d (ppm): 48.76 (CH2). [Pt(1,2-pn)Cl2]: 1H NMR,
d (ppm): 1.11 (d, 3H, CH3), 2.10–2.58 (m, 3H, CH and CH2), 5.12–
5.39 (m, 4H, 2NH2); 13C NMR, d (ppm): 15.71 (CH3), 52.49 (CH2),
55.42 (CH). [Pt(dach)Cl2]: 1H NMR, d(ppm): 1.06 (m, 4H, 2CH2 from
C4 and C5), 1.63 (m, 4H, 2CH2 from C3 and C6), 2.07 (m, 2H, 2CH
from C1 and C2), 4.98–5.71 (m, 4H, 2NH2); 13C NMR, d (ppm):
24.08 (C4 and C5), 31.27 (C3 and C6), 62.66 (C1 and C2). These data
are in accordance with those reported previously for the corre-
sponding Pt(II) complexes [31].
were taken on
a Bruker AC 250 spectrometer operating at
250 MHz (proton) and 62.9 MHz (carbon), using standard Bruker
software; tetramethylsilane (TMS, d 0.00 ppm) was used as a refer-
ence for 1H NMR spectra, whereas the central carbon line of deu-
terated dimethylsulfoxide (DMSO-d6) was set at 39.5 ppm for
carbon-13 NMR spectra.
The reactions of MeCOMet–Gly with the platinum(II) complexes
in 50 mM phosphate buffer at pH 7.40 in D2O were followed by 1H
NMR spectroscopy using a Varian Gemini 2000 spectrometer
(200 MHz). Sodium trimethylsilylpropane-3-sulfonate (TSP) was
used as an internal reference. The 1H NMR spectra were acquired
using the WATERGATE sequence for water suppression. Typical
acquisition conditions were as follows: 90° pulses, 24 000 data
number points, 4 s acquisition time, 1 s relaxation delay, collection
of 16–128 transients and final digital resolution of 0.18 Hz per
point. All the NMR spectra were processed using the Varian VNMR
software (version 6.1, revision C). The chemical shifts are reported
in ppm. Equimolar amounts of the platinum(II) complex and the
dipeptide were mixed in an 5 mm NMR tube and spectra were re-
corded at appropriate time intervals. The final solution was 10 mM
in each reactant. All reactions were performed at 37 °C. Elemental
microanalyses for carbon, hydrogen and nitrogen were performed
by the Faculty of Chemistry of Warsaw University of Technology
and by the Microanalytical Laboratory, Faculty of Chemistry,
University of Belgrade.
2.3. Syntheses of [Pt(L)(CBDCA-O,O0)]-type complexes (L is en, 1,2-pn
or dach)
The chlorido complexes [Pt(en)Cl2], [Pt(1,2-pn)Cl2] and
[Pt(dach)Cl2] were converted into the corresponding diaqua com-
plexes by treatment with 1.95 equiv. of AgNO3 at pH 2.0, according
to a published method [32]. In each case, the formed solid AgCl was
removed by filtration in the dark. To the clear solutions of the aqua
complexes, equimolar amounts of CBDCA (anion of 1,1-cyclobutan-
edicarboxylic acid) and two equivalents of NaOH were added. The
mixture was stirred at 60 °C for 3 h and all complexes were crystal-
lized from water by cooling in a refrigerator. After filtration of the
solid complex, an equivalent volume of ethanol was added to the
filtrate and an additional amount of the CBDCA Pt(II) complex crys-
tallized after cooling in a refrigerator for two days. The yield was
between 50% and 60%.
3. Results and discussion
Hydrolytic reactions between various 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-propylenedi-
amine, 1,2-pn; and CBDCA is the 1,1-cyclobutanedicarboxylic anion)
2.3.1. Elemental microanalyses
Anal. Calc. for [Pt(en)(CBDCA-O,O0)] = C8H14N2O4Pt (FW =
397.29): C, 24.19; H, 3.55; N, 7.05. Found: C, 24.28; H, 3.47; N,
7.04%. Anal. Calc. for [Pt(1,2-pn)(CBDCA-O,O0)] = C9H16N2O4Pt
(FW = 411.31): C, 26.28; H, 3.92; N, 6.81. Found: C, 25.31; H,
and the N-acetylated L-methionylglycine dipeptide (MeCOMet–Gly)
were studied by 1H NMR spectroscopy. All reactions were performed
with equimolar amounts of the platinum(II) complex and the dipep-
tide at pH 7.40 in 50 mM phosphate buffer in D2O and at 37 °C. The
studied platinum(II) complexes are shown in Fig. 1. The different
4.11; N, 6.95%. Anal. Calc. for [Pt(dach)(CBDCA-O,O0)] = C12H20
-
N2O4Pt (FW = 451.38): C, 31.93; H, 4.47; N, 6.21. Found: C, 30.70;
H, 4.53; N, 6.13%.

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M.D. Zivkovic et al. / Polyhedron 30 (2011) 947–952
NH2
NH2
Cl
Cl
chelate diamine ligands (L) in these complexes are inert to substitu-
tion and are expected to remain bound to the platinum(II) atom dur-
ing the reactions with the dipeptide. In all the investigated reactions,
a very slow selective cleavage of the Met–Gly amide bond in the
MeCOMet–Gly dipeptide was observed. The influence of the leaving
ligand (chlorido and CBDCA) and the steric effects of the chelate
diamine ligand (L) on this hydrolytic reaction was investigated in
relation to the toxic side effects of platinum(II) antitumor drugs.
NH2
NH2
CH3
Cl
Cl
NH2
NH2
Cl
Cl
Pt
Pt
Pt
[Pt(en)Cl2]
[Pt(dach)Cl2]
[Pt(1,2-pn)Cl2]
O
O
O
O
O
NH2
NH2
NH2
O
NH2
NH2
CH3
O
O
Pt
Pt
Pt
O
O
NH2
O
O
3.1. Hydrolytic reactions of the [Pt(L)Cl2]- and [Pt(L)(CBDCA-O,O0)]-
type complexes with MeCOMet–Gly
[Pt(dach)(CBDCA-O,O,)]
[Pt(1,2-pn)(CBDCA-O,O,)]
[Pt(en)(CBDCA-O,O,)]
Fig. 1. Platinum(II) complexes used in the reaction with N-acetylated -methio-
nylglycine (MeCOMet–Gly) as catalytic reagents for the hydrolytic cleavage of this
peptide.
L
The reaction scheme of [Pt(L)Cl2]- and [Pt(L)(CBDCA-O,O0)]-type
complexes with the MeCOMet–Gly dipeptide is given in Fig. 2.
O
H3C
NH
O
NH
O
O
S
MeCOMet-Gly
H3C
pH 7.40 in phosphate buffer
t = 37 oC
,
+
[Pt(L)(CBDCA-O,O )]
[Pt(L)Cl2]
+
O
(L = en, 1,2-pn or dach)
O
O
H3C
O
O
NH
H3C
NH
NH
NH
O
O
O
O
O
Cl
O
O
S
S
Pt
H3C
Pt
H3C
NH2
H2N
NH2
H2N
(L)
(L)
[Pt(L)Cl(MeCOMet-Gly-S)]
[Pt(L)(CBDCA-O)(MeCOMet-Gly-S)]
- Cl ; + H2O
CBDCA;+ H2O
O
O
OH2
NH
O
O
H3C
+
+
H3C
NH
O
NH
NH
O
O
O
OH2
OH2
S
S
NH2
(L)
Pt
H2N
Pt
H3C
H3C
NH2
H2N
(L)
internal delivery
external attack
Hidrolitically active [Pt(L)(H2O)(MeCOMet-Gly-S)]+ complex
O
NH2
(L)
NH2
+
OH2
S
H2N
O
O
+
O
O
Pt
Gly
CH3
NH
CH3
[Pt(L)(H2O)(MeCOMet-S)]+
Fig. 2. The reaction scheme of the hydrolytic reaction of the MeCOMet–Gly dipeptide with [Pt(L)Cl2]- and [Pt(L)(CBDCA-O,O0)]-type complexes (L is en, 1,2-pn and dach). The
corresponding Pt(II) complex and dipeptide were mixed in a 1:1 molar ratio and all reactions performed at pH 7.40 and at 37 °C in 50 mM phosphate buffer in D2O solvent.

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M.D. Zivkovic et al. / Polyhedron 30 (2011) 947–952
950
When an equimolar amount of the corresponding Pt(II) complex
was incubated with MeCOMet–Gly under the above-mentioned
conditions, the major product observed in solution after 4 h of
reaction was an intermediate platinum(II)–peptide complex,
[Pt(L)Cl(MeCOMet–Gly–S)] for [Pt(L)Cl2]- and [Pt(L)(CBDCA-
O)(MeCOMet–Gly–S)]ꢀ for [Pt(L)(CBDCA-O,O0)]-type complexes.
The formation of these intermediate products was fast and could
be followed by the simultaneous decline of the proton NMR reso-
nance at 2.11 ppm, arising from the S-methyl protons of free
MeCOMet–Gly, and the growth of the resonance at 2.39–
2.56 ppm, corresponding to the S-methyl protons of the dipeptide
coordinated to Pt(II) through the sulfur atom. The chemical shift of
this resonance is dependent on the type of Pt(II) complex. More-
over, the formation of the ring-opened [Pt(L)(CBDCA-O)(MeCO-
Met–Gly–S)]ꢀ complex could be observed by the simultaneous
opened [Pt(NH3)2(CBDCA-O)(L-Met–S)]ꢀ intermediate product
was formed which has a half-life for Met–S,N ring closure of 28 h
at 310 K [36]. The detection of a Pt(II) complex containing the
ring-opened CBDCA ligand and a monodentate thioether ligand
in the urine of mice treated with the carboplatin drug was also re-
ported [37]. The intermediate [Pt(L)Cl(MeCOMet–Gly–S)] and
[Pt(L)(CBDCA-O)(MeCOMet–Gly–S)]ꢀ complexes are hydrolytically
inactive and after replacement of the chlorido and monodentate
coordinated CBDCA ligand in these complexes by water molecules,
they were converted into the hydrolytically active Pt(II)–peptide
[Pt(L)(H2O)(MeCOMet–Gly–S)]+ complex. It was shown that a coor-
dinated aqua ligand in Pd(II) and Pt(II) complexes plays an impor-
tant role in promoting the regioselective cleavage of the amide
bond in histidine- and methionine-containing peptides [14–
16,18–22]. These findings can be explained in terms of two possi-
ble limiting mechanisms, both presented in Fig. 2. Either the labile
aqua ligand is displaced by the methionine carbonyl oxygen, thus
activating the carbonyl group to external attack by a water mole-
cule, or will itself be delivered to the scissile amide group (internal
delivery) with resulting cleavage. In the present investigated reac-
tions between [Pt(L)Cl2]- and [Pt(L)(CBDCA-O,O0)]-type complexes
with the MeCOMet–Gly dipeptide under the above mentioned
experimental conditions, the hydrolysis of the [Pt(L)Cl(MeCO-
Met–Gly–S)] and [Pt(L)(CBDCA-O)(MeCOMet–Gly–S)]ꢀ complexes
is the rate determining step for the hydrolytic cleavage of the
Met–Gly amide bond. The replacement of the chlorido and CBDCA
ligand by water molecules could not be followed by 1H NMR spec-
decline of the triplet at 2.87 ppm for the H and Hb methylene
a
protons of the bidentate coordinated CBDCA ligand in the
[Pt(L)(CBDCA-O,O0)] complex, and the growth of a new multiplet
at 2.32 ppm for these protons due to the monodentate bound
CBDCA ligand in the ring-opened Pt(II) intermediate complex.
The multiplets for the H methylene protons for the bidentate
c
and monodentate coordinated CBDCA ligand were overlapped
and the changes in their intensities could not be quantified in
the present study. The formation of the ring-opened carboplatin
adducts containing monodentate thioethers were also observed
in the reactions of the anticancer drug carboplatin with a variety
of sulfur-containing amino acids [35,36]. It was found that carbo-
platin reacts very slowly with thiol-type ligands and that sulfur-
bridged species containing four-membered Pt2S2 rings represent
the major products in these reactions. However, it was shown that
the reactions of this Pt(II) antitumor drug with thioether ligands
are much more rapid and the kinetics for the initial stages of the
troscopy. The triplet at 2.50 ppm for the H and Hß methylene pro-
a
tons and the multiplet at 1.97 ppm for the H methylene protons of
c
free CBDCA were overlapped with signals for the H methylene
c
protons and with methyl protons of the acetyl group of the methi-
onine residue, respectively. Hydrolysis of the [Pt(L)Cl(MeCOMet–
Gly–S)] and [Pt(L)(CBDCA-O)(MeCOMet–Gly–S)]ꢀ complexes and
hydrolytic cleavage of the Met–Gly amide bond, upon formation
reaction with
L
-methionine were determined (k = 2.7 ꢁ 10ꢀ3
Mꢀ1 sꢀ1 at pH 7.0). During this reaction, the very stable ring-
Fig. 3. Parts of the 1H NMR spectra during the reactions of [Pt(1,2-pn)Cl2] (a) and [Pt(1,2-pn)(CBDCA-O,O0)] (b) with MeCOMet–Gly. The corresponding Pt(II) complex and the
dipeptide were mixed in a 1:1 molar ratio and all reactions were performed at pH 7.40 and at 37 °C in 50 mM phosphate buffer in D2O with TSP as the internal standard. For
these reactions, the resonances are indicated as follows: (d) GlyCH2 at 3.76 ppm of the MeCOMet–Gly dipeptide and (j) GlyCH2 at 3.55 ppm of the free glycine.

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M.D. Zivkovic et al. / Polyhedron 30 (2011) 947–952
Table 1
of the corresponding hydrolytically active aqua species, are the two
consequential reactions; see Fig. 2. In accordance with this, the
cleavage of the Met–Gly amide bond, as the overall hydrolytic pro-
cess in reactions of [Pt(L)Cl2]- and [Pt(L)(CBDCA-O,O0)]-type com-
plexes with MeCOMet–Gly dipeptide, was followed by 1H NMR
spectroscopy. The resonance at 3.75–3.78 ppm corresponding to
the glycine protons of the non-hydrolyzed peptide decreased while
that at 3.55 ppm for free glycine increased; see Fig. 3. The second
hydrolytic product in this reaction was the [Pt(L)(H2O)(MeCO-
Met–S)]+ complex, containing the N-acetylated amino acid methi-
onine, which is monodentately coordinated through the sulfur
atom for Pt(II). Indeed, the same complex is formed upon mixing
equimolar amounts of the corresponding [Pt(L)(H2O)2]2+ complex
Percentage (%) hydrolyzed amide bond in MeCOMet–Gly dipeptide during its reaction
with [Pt(L)Cl2]- and [Pt(L)(CBDCA-O,O0)]-type complexes after 2 and 7 days at pH 7.40
in 50 mM phosphate buffer and at 37 °C.
Platinum(II) complex
Hydrolyzed Met–Gly amide bond (%)
After 2 days
After 7 days
[Pt(en)Cl2]
24.0
8.6
20.0
8.0
7.5
0
90.0
60.4
60.0
32.0
55.0
25.4
[Pt(en)(CBDCA-O,O0)]
[Pt(1,2-pn)Cl2]
[Pt(1,2-pn)(CBDCA-O,O0)]
[Pt(dach)Cl2]
[Pt(dach)(CBDCA-O,O0)]
for the [Pt(L)Cl2] complexes. A very slow cleavage of the amide
bond was also observed in the reactions between the anticancer
drug cisplatin, cis-[Pt(NH3)2Cl2], and different N-acetylated methi-
onine-containing peptides if these reactions were performed in
neutral or weakly alkaline solutions (7.0 6 pH 6 9.5) [24–27].
with N-acetylated L-methionine, MeCOMet. Upon addition of gly-
cine to the reaction mixture, its resonance was enhanced. The
amounts of the non-hydrolyzed peptide and the hydrolysis prod-
ucts were determined from the known initial concentration of
MeCOMet–Gly and from the integrated resonance of the free gly-
cine. The catalytic abilities of the [Pt(L)Cl2]- and [Pt(L)(CBDCA-
O,O0)]-type complexes were determined by measuring the amount
of hydrolyzed MeCOMet–Gly with time under the same experi-
mental conditions. The concentrations of the free and the hydro-
lyzed peptide were determined every 4 h and the reaction was
followed for 7 days. The time dependence of the hydrolytic cleav-
age of the Met–Gly amide bond in the reactions between [Pt(L)Cl2]-
and [Pt(L)(CBDCA-O,O0)]-type complexes and the MeCOMet–Gly
dipeptide is given in Fig. 4. These complexes differ in the chelate
diamine ligand, which contributes to the steric bulk of the corre-
sponding Pt(II) complex. In accordance with this, it was found that
in all the investigated chlorido and CBDCA Pt(II) complexes, the
rate of peptide hydrolysis decreased in the following order:
en > 1,2-pn > dach (Fig. 4 and Table 1). Furthermore, the rates of
this hydrolytic reaction between [Pt(L)Cl2] and [Pt(L)(CBDCA-
O,O0)] containing the same chelate diamine ligand (L) were com-
pared. In general, it was found that the [Pt(L)Cl2] complexes are
much better hydrolytic agents than the corresponding
[Pt(L)(CBDCA-O,O0)] complexes; see Fig. 4 and Table 1. Differences
in the reactivity between the [Pt(L)(CBDCA-O,O0)] and [Pt(L)Cl2]
complexes can be attributed to the different stability of their inter-
mediate products, [Pt(L)Cl(MeCOMet–Gly–S)] and [Pt(L)(CBDCA-
O)(MeCOMet–Gly–S)]ꢀ, formed in the first stage of the reaction
with the MeCOMet–Gly dipeptide. The very stable intermediate
[Pt(L)(CBDCA-O)(MeCOMet–Gly–S)]ꢀ complex and the slow
replacement of its monodentate coordinated CBDCA ligand by a
water molecule are the main contributing factors for the lower
hydrolytic activity of [Pt(L)(CBDCA-O,O0)] with respect to those
4. Conclusions
Selective hydrolytic cleavage of the MeCOMet–Gly dipeptide in
the reactions with [Pt(L)Cl2]- and [Pt(L)(CBDCA-O,O0]-type com-
plexes occurred under physiological conditions (pH 7.40 and
37 °C). The cleavage of the Met–Gly amide bond in this peptide is
very slow and proceeds through the intermediate [Pt(L)(H2O)
(MeCOMet–Gly–S)]+ complex. The formation of the aqua complex
from the corresponding [Pt(L)Cl(MeCOMet–Gly–S)] and [Pt(L)
(CBDCA-O)(MeCOMet–Gly–S)]ꢀ complexes is the rate determining
step for the hydrolytic cleavage of the MeCOMet–Gly dipeptide.
The higher stability of the initially formed [Pt(L)(CBDCA-O)
(MeCOMet–Gly–S)]ꢀ complex with respect to the analog [Pt(L)Cl
(MeCOMet–Gly–S)] intermediate product is in accordance with
the slow replacement of its CBDCA ligand by a water molecule, fi-
nally resulting in the formation of the hydrolytically active
[Pt(L)(H2O)(MeCOMet–Gly–S)]+ complex. The present findings that
Pt(II) complexes can cleave methionine-containing peptides under
physiological relevant conditions of pH and temperature can have
an importance for a better understanding of the toxic side effects of
Pt(II) antitumor drugs. In this paper, it was demonstrated that
modification of the Pt(II) complex by the introduction of a
sterically hindered diamine ligand, inhibition of the hydrolytic
cleavage of the amide bond involving the carboxylic group of
methionine can be achieved. This latest results should also be
taken into consideration when designing new potential Pt(II)
antitumor drugs with low toxic side effects.
100
(a)
(b)
1
1
2
3
60
20
2
3
8
day(s)
day(s)
1
3
5
7
1
3
5
7
Fig. 4. The time dependence of the hydrolytic cleavage of the Met–Gly amide bond in the MeCOMet–Gly dipeptide with different [Pt(L)Cl2] (a) and [Pt(L)(CBDCA-O,O0)] (b)
complexes (L is ethylenediamine, en (1); ( )-1,2-propylenediamine, 1,2-pn (2); ( )-trans-1,2-diaminocyclohexane, dach (3); and CBDCA is the 1,1-cyclobutanedicarboxylic
anion). The corresponding Pt(II) complex and the dipeptide were mixed in a 1:1 molar ratio and all reactions were performed at pH 7.40 and at 37 °C in 50 mM phosphate
buffer in D2O as solvent.

ˇ
´
952
M.D. Zivkovic et al. / Polyhedron 30 (2011) 947–952
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Acknowledgment
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This work was funded in part by the Ministry of Science and
Technological Development of the Republic of Serbia (Project No.
172036).
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