Selective hydrolysis of the unactivated peptide bond in N-acetylated L-histidylglycine catalyzed by various palladium(II) complexes: Dependence of the hydrolysis rate on the steric bulk of the catalyst

Article abstract of DOI:10.1016/S0277-5387(00)00342-9

Hydrolytic reactions between various palladium(II) complexes of the type cis-[Pd(L)(H₂O)₂]²⁺ in which L is a chelating diamine (ethylenediamine, en; 1,2-propylenediamine, 1,2-pn; N-methylethylenediamine, Meen; isobutylenediamine, ibn and N,N,N',N'-tetramethylethylenediamine, Me₄en) or S,N-coordinated amino acid (S-methyl L-cysteine, MeS-L-HCys and L-methionine, L-HMet) and N-acetylated L-histidylglycine (MeCO-His-Gly), were studied by ¹H NMR spectroscopy. The reactions were carried out in the pH range 2.0-2.5 and at 60°C. In all these reactions, a palladium(II) complex bound to a histidine residue effects the regioselective cleavage of the amide bond involving the carboxylic group of histidine. We found that the rate of hydrolysis decreases as the steric bulk of the palladium(II) complex increases (en > 1,2-pn > Meen > MeS-L-HCys > ibn > L-HMet > Me₄en). The observed rates of hydrolytic reaction are discussed in terms of steric hindrance of the chelating diamine or sulfur-containing amino acid on the palladium(II) complexes. This study is an important step in the development of new palladium(II) complexes as artificial metallopeptidases. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0277-5387(00)00342-9

www.elsevier.nl/locate/poly
Polyhedron 19 (2000) 959–963
Selective hydrolysis of the unactivated peptide bond in N-acetylated
L-histidylglycine catalyzed by various palladium(II) complexes:
dependence of the hydrolysis rate on the steric bulk of the catalyst
*
Milosˇ I. Djuran , Snezˇana U. Milinkovic´
´
Department of Chemistry, Faculty of Science, University of Kragujevac, R. Domanovica 12, P.O. Box 60, 34000 Kragujevac, Yugoslavia
Received 10 December 1999; accepted 24 February 2000
Abstract
Hydrolytic reactions between various palladium(II) complexes of the type cis-[Pd(L)(H2O)2]2q in which L is a chelating diamine
(ethylenediamine, en; 1,2-propylenediamine, 1,2-pn; N-methylethylenediamine, Meen; isobutylenediamine, ibn and N,N,N9,N9-tetramethyl-
ethylenediamine, Me4en) or S,N-coordinated amino acid (S-methyl
L
L-cysteine, MeS-L-HCys and L-methionine, L-HMet) and N-acetylated
-histidylglycine (MeCO-His-Gly), were studied by 1H NMR spectroscopy. The reactions were carried out in the pH range 2.0–2.5 and at
608C. In all these reactions, a palladium(II) complex bound to a histidine residue effects the regioselective cleavage of the amide bond
involving the carboxylic group of histidine. We found that the rate of hydrolysis decreases as the steric bulk of the palladium(II) complex
increases (en)1,2-pn)Meen)MeS-L-HCys)ibn)L-HMet)Me4en). The observed rates of hydrolytic reaction are discussed in terms of
steric hindrance of the chelating diamine or sulfur-containing amino acid on the palladium(II) complexes. This study is an important step in
the development of new palladium(II) complexes as artificial metallopeptidases.
Keywords: Palladium(II) complexes; Catalysts; Peptides; Hydrolysis
q2000 Elsevier Science Ltd All rights reserved.
1. Introduction
ment and the additional chemicals required for cleavage limit
´
the applicability of this method. Recently, Kostic and
coworkers have shown that platinum(II) [13] and palla-
dium(II) [14–21] aqua complexes can be promising rea-
gents for the hydrolytic cleavage of peptides and proteins.
These complexes bind to the heteroatom in the side chain of
methionine [13–17] or histidine[18–21]andpromotecleav-
age of the amide bond involving the carboxylic group of this
anchoring amino acid. Palladium(II) complexes, ascleavage
agents, differ from the well known complexes of other tran-
sition metals because their attachment to substrates is
achieved simply by mixing, the hydrolysis reactions have
half-lives as short as 15 min, and the complexes are easily
removed from the cleaved substrates.
The consistent regioselectivity in the cleavage of methio-
nine or histidine containing peptides promoted by palla-
dium(II) aqua complexes and the mechanism of this
hydrolytic reaction are not completely understood yet. A bet-
ter knowledge of the coordination chemistry of histidine-
containing peptides with palladium(II) complexes is
necessary for understanding the regioselectivity of peptide
and protein cleavage promoted by such complexes. This
paper reports a 1H NMR investigation of the hydrolysis reac-
Many biological processes involve hydrolysis of peptides
and proteins, but relatively little is known about the mecha-
nism of this reaction. The extreme inertness of the amide
bond makes this reaction interesting from a chemical point
of view. Several proteolytic enzymes are used for cleavage
[1], but application of enzymes is limited by their rather
narrow requirements for temperature and pH.
Transition-metal complexes hold promise as new reagents
for protein cleavage by various mechanisms. Several
cobalt(III) complexes coordinate to peptides and facilitate
hydrolysis, but they cleave only the N-terminal amino acid
[2]. However, practical applicationsusuallyrequirecleavage
of internal amide bonds. Studies with some iron-EDTA com-
plexes that are covalently attached to amino acid side chains
revealed some mechanistic features of these hydrolytic reac-
tions, but they did not result in practical methods for analyt-
ical biochemistry [3–12]. Although in some cases the
cleavage was fast, the synthetic work involved in the attach-
* Corresponding author. Tel.: q381-34-335-039; fax: q381-34-335-040;
e-mail: djuran@uis0.uis.kg.ac.yu or djuran@knez.uis.kg.ac.yu
0277-5387/00/$ - see front matter q2000 Elsevier Science Ltd All rights reserved.
PII S0277-5387(00)00342-9
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M.I. Djuran, S.U. Milinkovic´ / Polyhedron 19 (2000) 959–963
tion of a model histidine-containing peptide, N-acetylated
-histidylglycine, catalyzedbyaseriesofpalladium(II)com-
2.4. pH Measurements
L
plex cations of the type cis-[Pd(L)(H2O)2]2q in which L is
a chelating diamine ligand, which differ in the number of
methyl groups at the nitrogen or carbon atom, or S,N-coor-
dinated amino acid. The observed rates of reaction are
explained in terms of steric hindrance of the chelating ligand
on the palladium(II) complexes.
All pH measurements were made at 298 K. The pH meter
(Iskra MA 5704) was calibrated with Fischer certifiedbuffer
solutions of pH 4.00 and 7.00. The results were not corrected
for the deuterium isotope effect. Proton NMR spectra of D2O
solutions containing TSP (3-trimethylsilylpropane-1-sulfo-
nate) as internal reference were recorded withVarianGemini
200 spectrometer.
2. Experimental
3. Results and discussion
2.1. Materials
In the present study hydrolytic reactions between various
complexes of the type cis-[Pd(L)(H2O)2]2q (L is ethyle-
nediamine, en; 1,2-propylenediamine, 1,2-pn;N-methylethy-
Distilled water was demineralized and purified to a resis-
tance greater than 10 MV cm. The compounds D2O, DNO3,
NaOD, and K2[PdCl4] were obtained from AldrichChemical
Co. All common chemicals were of reagent grade. Dipeptide
lenediamine, Meen; S-methyl
isobutylenediamine, ibn; -methionine,
tetramethylethylenediamine, Me4en) and N-acetylated
-histidylglycine, MeCO-His-Gly, were studied by 1H NMR
L
-cysteine, MeS-L-HCys;
L
L-HMet; N,N,N9,N9-
L
L-histidylglycine, amino acids, S-methyl-L-cysteine and L-
spectroscopy. The palladium(II) complexes are as shown in
Fig. 1. The different chelate ligands (L) in these complexes
are inert to substitution and are expected to remain bound to
the palladium(II) atom during the reactions with the amino
acids and peptides. The course of the hydrolytic reaction at
pH 2.0 is shown in Scheme 1. As was shown in previous
studies [18–20,26], acidic solutions are needed to suppress
the formation of hydroxo-bridged oligomeric palladium(II)
complexes, which are catalytically inactive.
methionine and all diamines were obtained from Sigma
Chemical Co. The terminal amino group in His-Gly was
acetylated by standard methods [14].
2.2. Synthesis of palladium(II) complexes
The palladium(II) complexes of the type cis-[Pd(L)Cl2]
(L is ethylenediamine, en; N-methylethylenediamine, Meen;
1,2-propylenediamine, 1,2-pn; S-methyl
L-cysteine, MeS-L-
HCys; -methionine, -HMet; N,N,N9,N9-tetramethylethyle-
L
L
nediamine, Me4en; isobutylenediamine, ibn) were synthe-
sized according to the procedures published in the literature
[22–24]. K2PdCl4 was dissolved in water and mixed with an
equimolar amount of diamine or amino acid. The pH of the
solution was adjusted to ca. 3 by addition of 1 M HCl and the
mixture was stirred at 808C for 2 h. All complexes were
crystallized from water at room temperature. The pure com-
plexes were obtained by recrystallization from a small
amount of water and cooling. The experimental results of the
elemental analysis for C, H and N parameters for all palla-
dium(II) complexesareinaccordancewiththeoreticalvalues
calculated for cis-[Pd(L)Cl2] complexes. The chloro com-
plexes were converted into the corresponding diaqua com-
plexes by treatment with 2 equiv. of AgNO3 at pH 2.0
according to a published method [25]. In each case, the solid
AgCl was removed by filtration in the dark, and fresh stock
solution of the aqua complex wasused infurtherexperiments.
2.3. Reactions of peptide with palladium(II) complexes
Reactions of MeCO-His-Gly with palladium(II) com-
plexes were followed by 1H NMR spectroscopy. Equimolar
amounts of the palladium(II) complex and the peptide were
mixed in an NMR tube. The final solution was 10 mM in each
reactant. ThepH wasvariedintherange2.0–2.5. Allreactions
were carried out at 608C.
Fig. 1. Palladium(II) complexes used in the reaction with MeCO-His-Gly.
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M.I. Djuran, S.U. Milinkovic´ / Polyhedron 19 (2000) 959–963
961
Scheme 1.
3.1. Binding of the catalysts to the substrate
peptide nitrogen atom was not detected in solution even after
the reaction mixture stayed for 5 days at 608C. This is in
accordance with the fact that two methyl groups on the ibn
ligand cause steric crowding on palladium(II) and do not
allow its interaction with the peptide nitrogen atom. Mixing
The previous study with dipeptide MeCO-Gly-His andcis-
[Pd(en)(H2O)2]2q showed that at pH-3 five palla-
dium(II)-peptide products formed [18–20]. These
complexes were distinguished on the basis of the chemical
shifts of the two imidazole protons, H-2 and H-5. The two
major complexes are linkage isomers of each other with uni-
dentate coordination of palladium(II) via the N-3 or N-1
atom to the imidazole ring. The three minor complexes con-
tain more than one palladium(II) atom per dipeptide or
involve more than one donor atom in the dipeptide. The
experiments with MeCO-His-Gly selectively methylated at
the N-1 or N-3 atom of imidazole and cis-[Pd(en)-
(H2O)2]2q showed that only the palladium(II) complex
with monodentate coordination to the N-3 atom of imidazole
can effect the cleavage of the amide bond involving the car-
boxylic group of histidine (Fig. 2); none of the four other
modes of coordination is effective [19]. The cleavage of the
amide bond is regioselective and the reaction is completed in
less than 72 h. Also, the experiments with different histidine-
containing peptides and different palladium(II) complexes
showed that only unidentate coordination of the peptide via
the N-3 atom of the imidazole to the Pd(II) ion effectshydro-
lytic cleavage of the amide bond. This was explained through
the fact that this coordination mode permits the necessary
close approach of the palladium(II) ion and of its aqua ligand
to the scissile peptide bond [18,20].
of cis-[Pd(L)(H2O)2]2q, where L is MeS-
L-HCys or L-
HMet, with an equimolar amount of MeCO-His-Gly under
the same experimental conditions resulted in a spontaneous
formation of three NMR-detectable complexes. In the two
complexes peptide is a unidentate ligand coordinated via the
N-3 or N-1 atom of the imidazole ring to palladium(II). In
the third palladium(II)-peptide complex bidentate coordi-
nation of the peptide occurs via the N-3 atom of imidazole
and the deprotonated peptide nitrogen atom of the terminal
amino group of histidine.
3.2. Regioselective cleavage of the peptide
The reactions between palladium(II) complexes of the
type cis-[Pd(L)(H2O)2]2q and MeCO-His-Gly were car-
ried out at 608C and at 2.0-pH-2.5. The obtained results
compared with those for cis-[Pd(en)(H2O)2]2q [18–20].
In all these reactions only the cleavage of the amide bond
involving the carboxylic group of histidine was observed; see
Scheme 1. In the reactionbetweenthepalladium(II)complex
and MeCO-His-Gly, the 1H NMR resonance at d 3.96 ppm
in the peptide decreased, while that at d;3.80 ppm for free
glycine increased. Upon addition of glycine to the reaction
mixture its resonance is enhanced. The concentrations of the
peptide and the hydrolysis products were determined from
the known initial concentration of MeCO-His-Gly and from
integrated resonance of the free glycine. Some oftheliberated
glycine reacts with the catalyst to form a small amount of the
bis(bidentate) complex cis-[Pd(L)(Gly-N,O)]q, easily
detected by 1H NMR spectroscopy by the resonance at d 3.52
ppm. Indeed, the same complex is formed upon mixing of
equimolar amounts of cis-[Pd(L)(H2O)2]2q and glycine.
When an equimolar amount of cis-[Pd(L)(H2O)2]2q
,
where L is Meen, Me4en, or 1,2-pn, was incubated with
MeCO-His-Gly at 608C and 2.0-pH-2.5, five NMR-
detectable complexes formed. The complexes were distin-
guished on the basis of the chemical shifts of imidazole
protons. These complexes are identical with those for the
reaction of this peptide with cis-[Pd(en)(H2O)2]2q [18–
20]. In the reaction between cis-[Pd(ibn)(H2O)2]2q and
MeCO-His-Gly at 2.0-pH-2.5, only four complexes
formed. The complex with bidentate coordination of peptide
via the N-3 atom of the imidazole ring and deprotonated
3.3. Steric effects of the catalyst
The seven Pd(II) complexes in Fig. 1 differ in the chelate
ligand. The palladium(II) complexes cis-[Pd(Meen)-
(H2O)2]2q
,
cis-[Pd(Me4en)(H2O)2]2q
,
cis-[Pd(1,2-
pn)(H2O)2]2q and cis-[Pd(ibn)(H2O)2]2q in relation to
cis-[Pd(en)(H2O)2]2q have a bidentate ligand with differ-
ent numbers of methyl groups at the nitrogen or carbon atom.
These methyl groups contribute to the steric bulk of the pal-
ladium(II) complex. From Fig. 3 it can be concluded that the
Fig. 2. Catalytically active form of the palladium(II)-peptide complex.
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M.I. Djuran, S.U. Milinkovic´ / Polyhedron 19 (2000) 959–963
Fig. 4. Dependence of the hydrolysis rate on the steric bulk of the palla-
dium(II). The reactions were carried out for 48 h at 2.0-pH-2.5 and at
608C.
Fig. 3. Time dependence of hydrolytic reaction of MeCO-His-Gly with
different palladium(II) complexes at 2.0-pH-2.5 and at 608C.
rate of hydrolysis decreases as the steric bulk of the palla-
dium(II) complex increases (en)1,2-pn)Meen)MeS-
L-
HCys)ibn) -HMet)Me4en). The reaction between
L
cis-[Pd(en)(H2O)2]2q and MeCO-His-Gly after 48 h at
608C is almost two times faster in comparison with cis-
[Pd(Me4en)(H2O)2]2q, or one and a half times faster in
relation to cis-[Pd(ibn)(H2O)2]2q. On comparing the
ligand 1,2-pn with Meen, or Me4en with ibn, we found that
the former are more sterically demanding because the methyl
groups bound directly to the nitrogen atoms are closer to the
metal center, hindering the approach of a nucleophile. It is
very illustrative to compare the rate of reactivity of MeCO-
His-Gly with en, 1,2-pn and ibn, or this peptide with en, Meen
and Me4en palladium(II) complexes, see Fig. 4. It is obvious
that increasing the number of methyl groups at the nitrogen
or carbon atom of the chelate ligand decreases the amount of
hydrolyzed His-Gly peptide bond. For comparison, parallel
work has been done with MeCO-His-Gly and cis-[Pd(MeS-
Scheme 2.
peptide bond promoted by palladium(II) [18–20], see
Scheme 2. In the mechanism involving external attack by
water, the palladium(II) atom, as a Lewis acid, polarizes the
carbonyl group in the scissile peptide bond. In the mechanism
involving internal attack by water, the palladium(II) atom
delivers an aqua ligand to the scissile bond. As can be seen,
both mechanisms require a close approach of the pendant
catalyst to the adjacent peptide bond, and in both cases the
steric bulk of the leaving amino acid as well as palladium(II)
complex hinder this crucial interaction.
L
-HCys-S,N)(H2O)2]2q
(H2O)2]2q. These two complexes differ in the ring size of
the chelate ligand. The MeS- -HCys-palladium(II)complex
with five-membered chelate ring is more reactive than
and
cis-[Pd(L-HMet-S,N)-
Acknowledgements
L
L
-
This work was funded in part by the Ministry of Science
and Technology of the Republic of Serbia (grant 02E35) and
by the Ministry of Science and Progress in Technology of the
FR Yugoslavia (grant OSI-048/1-93). We are grateful to
HMet-palladium(II) with six-membered ring in the square-
planar plane. This inhibition of the hydrolytic reaction can be
attributed to the steric bulk of the palladium(II) complex. A
similar trend to our results has emerged in a previous study
of cis-[Pd(en)(H2O)2]2q and MeCO-His-X peptides con-
taining different C-terminal amino acids (X is Gly, Ala, Ser,
Thr, Leu, Phe, Tyr) [18]. It was found that the rate constant
of the hydrolytic reaction decreases smoothly as the volume
of the leaving amino acid increases.
´
Professor Nenad M. Kostic (Department of Chemistry, Iowa
State University, Ames, IA, USA) and his coworkers for
providing K2PdCl4 and the peptides. We thank also B.
1
´
Mojsilovic for assistance with recording the H NMRspectra.
Summing up together our results with different palla-
dium(II) complexes and previous results with different pep-
tides [18], we can say that inhibition of the hydrolytic
reaction can be attributed to the steric bulk of the palla-
dium(II) complex as well as to the steric bulk of the leaving
amino acid. These findings can be explained in terms of two
possible limiting mechanisms for hydrolytic cleavage of the
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