New regioselectivity in the cleavage of histidine-containing peptides by palladium(II) complexes studied by kinetic experiments and molecular dynamics simulations

Article abstract of DOI:10.1021/ja982369i

Palladium(II) complexes promote hydrolytic cleavage of amide bonds in N- acetylhistidylglycine (AcHis-Gly), N-acetylhistidine (AcHis), and their derivatives methylated at the N-1 or N-3 atom of imidazole. Methylation controls coordination of imidazole to palladium(II) and allows stereochemical analysis of the reactions. The complex [PdCl₄]^2- regioselectively cleaves the amide bond involving the carboxylic group of histidine, the bond His- Gly; the rate constants of cleavage are virtually the same when the peptides coordinate to palladium(II) via the N-1 and the N-3 atom. The complex [Pd(H₂O)₄]²⁺ cleaves, at comparable rates, the amide bonds involving both the carboxylic (His-Gly) and the amino (AcHis) groups of histidine in the acetylated dipeptide. This unprecedented reactivity is examined by theoretical calculations in which molecular dynamics and solution of Poisson- Boltzmann equation are combined in a new way. When the Pd(H₂O)₃²⁺ group is attached to the N-1 atom, both scissile bonds can be cleaved by internal delivery of aqua ligands. When the Pd(H₂O)₃²⁺ group is attached to the N- 3 atom, both scissile bonds can be cleaved by internal delivery of aqua ligands and by external attack of water; in some conformers the two modes of cleavage may be combined in the reaction mechanism. In both N-1 and N-3 linkage isomers internal delivery seems to be assisted by weak hydrogen bonding. The rate constants for cleavage by [Pd(H₂O)₄]²⁺ are approximately 10 times greater than those for cleavage by [PdCl₄]^2-. This difference is explained semiquantitatively by consideration of the aquation equilibria involving [PdCl₄]^2-. This study shows that kinetics and regioselectivity of peptide cleavage may be controlled simply by choosing ligands in palladium(II) complexes. This is another step in our development of simple metal complexes as artificial metallopeptidases.

Full text of DOI:10.1021/ja982369i

J. Am. Chem. Soc. 1999, 121, 3127-3135
3127
New Regioselectivity in the Cleavage of Histidine-Containing
Peptides by Palladium(II) Complexes Studied by Kinetic Experiments
and Molecular Dynamics Simulations
†
‡
,†
Tatjana N. Parac, G. Matthias Ullmann, and Nenad M. Kosti c´ *
Contribution from the Department of Chemistry, Iowa State UniVersity, Ames, Iowa 50011, and Freie
UniVersit a¨ t Berlin, Institute f u¨ r Kristallographie, Takustrasse 6, 14195 Berlin, Germany
ReceiVed July 6, 1998
Abstract: Palladium(II) complexes promote hydrolytic cleavage of amide bonds in N-acetylhistidylglycine
(AcHis-Gly), N-acetylhistidine (AcHis), and their derivatives methylated at the N-1 or N-3 atom of imidazole.
Methylation controls coordination of imidazole to palladium(II) and allows stereochemical analysis of the
reactions. The complex [PdCl4]2 regioselectively cleaves the amide bond involving the carboxylic group of
histidine, the bond His-Gly; the rate constants of cleavage are virtually the same when the peptides coordinate
to palladium(II) via the N-1 and the N-3 atom. The complex [Pd(H2O)4]2+ cleaves, at comparable rates, the
amide bonds involving both the carboxylic (His-Gly) and the amino (AcHis) groups of histidine in the acetylated
dipeptide. This unprecedented reactivity is examined by theoretical calculations in which molecular dynamics
-
2
+
and solution of Poisson-Boltzmann equation are combined in a new way. When the Pd(H2O)3 group is
attached to the N-1 atom, both scissile bonds can be cleaved by internal delivery of aqua ligands. When the
Pd(H2O)32 group is attached to the N-3 atom, both scissile bonds can be cleaved by internal delivery of aqua
ligands and by external attack of water; in some conformers the two modes of cleavage may be combined in
the reaction mechanism. In both N-1 and N-3 linkage isomers internal delivery seems to be assisted by weak
+
2
+
hydrogen bonding. The rate constants for cleavage by [Pd(H2O)4] are approximately 10 times greater than
2-
those for cleavage by [PdCl4] . This difference is explained semiquantitatively by consideration of the aquation
2-
equilibria involving [PdCl4] . This study shows that kinetics and regioselectivity of peptide cleavage may be
controlled simply by choosing ligands in palladium(II) complexes. This is another step in our development of
simple metal complexes as artificial metallopeptidases.
Introduction
reagents can be attached to the substrate by a covalent
1
5-19,23,24,27-31
tether
or simply by spontaneous coordination of
Selective cleavage of proteins has long been one of the most
important procedures in analytical biochemistry. The amide
bond, however, is extremely unreactive. Uncatalyzed hydrolysis
of peptides by water at pH around 7 occurs with the half-lives
32-51
amino acid side chains to the metal atom.
Transition-metal
(
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of 250-600 years. Even the nonselective hydrolysis, under
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temperature of the dipeptide Gly-Gly are 2 days in 1.0 M NaOH
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under reflux overnight. Only several proteolytic enzymes are
(
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1
(
2
commonly used in determinations of amino acid sequence.
Moreover, these enzymes require narrow ranges of temperature
and pH.
Transition-metal complexes have emerged as promising
reagents for cleavage of peptides and proteins.3-51 These new
(
(
1
(
†
Iowa State University.
Freie Universit a¨ t Berlin.
‡
(
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1) Radzicka, A.; Wolfenden, R. J. Am. Chem. Soc. 1996, 118, 6105.
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(
1
0.1021/ja982369i CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/19/1999

3128 J. Am. Chem. Soc., Vol. 121, No. 13, 1999
Parac et al.
complexes are well suited for the traditional task of sequence
determination and for new challenges, such as preparation of
semisynthetic proteins, structural mapping and functional analy-
sis of protein domains, analysis of protein interactions with other
proteins and with nucleic acids, elucidation of protein folding,
analysis of nonnative states, and development of new drugs.
Because of their small size, transition-metal complexes can be
applied in structural studies that require relatively high “resolu-
tion”, at the level of functional groups and chemical bonds in
proteins.
Experimental Procedures
2 4
Chemicals. The deuterium-containing compounds D O, DClO , and
NaOD and the salts K [PdCl ] and AgClO monoxydrate (99.999%
2 4 4
pure) were obtained from Aldrich Chemical Co. All other chemicals
were of reagent grade. Dipeptide His-Gly and amino acids 1-Me-His
and 3-Me-His were obtained from Sigma Chemical Co. The terminal
amino group in each of these substrates was acetylated by a published
procedure.53 Dipeptides 1-Me-AcHis-Gly and 3-Me-AcHis-Gly were
synthesized from 1-Me-AcHis and 3-Me-AcHis by a standard solid-
state method, and their purity was checked by HPLC; this was done
by the staff of the Protein Facility. The complex [Pd(H
prepared by a published method.
Measurements. Proton NMR spectra of solutions in D
as an internal reference, were recorded with Bruker DRX 400 and
Varian Unity 500 spectrometers. Temperature was kept within (0.5
°C. The pH was measured with Fisher 925 instrument and a Phoenix
Ag/AgCl reference electrode. The pD values were calculated by the
standard formula:54 pD ) pH + 0.41. Ultraviolet-visible spectra were
recorded with a Perkin-Elmer λ-18 spectrophotometer.
2
+
2
O)
4
]
was
Studies in our laboratory showed that palladium(II) complexes
spontaneously bind to the side chains of methionine and histidine
residues and effect hydrolytic cleavage of short peptides with
36
2
O, with DSS
35-51
half-lives that range from several hours to several minutes.
Because palladium(II) complexes are diamagnetic, kinetic
1
experiments are easily done by H NMR spectroscopy. Because
the cleavage reactions occur with turnover,4
3,48,50
the simple
palladium(II) complexes that catalyze them can be considered
primitive artificial peptidases. Our successes in selective cleav-
Study of Hydrolysis. Because all the solutions were made in D
all the aqua ligands actually were D O. For simplicity and consistency
with our previous publications, however, we show aqua ligands as H O.
For the cleavage of peptides with K [PdCl ], 400 µL of a 100 mM
solution of K [PdCl ] in D O, 100 µL of a 100 mM solution of peptide
in D O, 50 µL of a 100 mM solution of DSS in D O, and 150 µL of
D O were mixed in the NMR tube. For the cleavage of peptides with
2
O,
age of cytochrome c,38 myoglobin, and other proteins bode
well for the general applicability of these new reagents in
biochemistry and allied disciplines. Before palladium(II) com-
plexes can become widely accepted, the basis for their selectivity
must be explained in studies with peptides.
51
52
2
2
2
4
2
4
2
2
2
2
2+
[
Pd(H
was mixed with 500 µL of a 100 mM solution of the peptide or the
histidine derivative in D O, in the Eppendorf vial. The pH was adjusted
to 1.0 (i.e., pD to 1.4) with 2.0 M DClO and was measured at the
2 4 2
O) ] , 2.00 mL of a 100 mM solution of this complex in D O
The present study shows, for the first time, that peptide bonds
2
-
can be cleaved with commercially available salts of [PdCl4]
2
anion; no chemical derivatization or modification of this simple
complex is required. Aquation of this complex, however, causes
4
beginning and at the end of each experiment. The difference was always
less than 0.10. Samples were kept at 40 °C for different periods of
time. The reactions were followed by H NMR spectroscopy, and the
spectra were very similar to those that we published before.
2+
a remarkable difference in reactivity; the complex [Pd(H2O)4]
cleaves peptides more rapidly and with different regioselectivity
than its chloro precursor does. We combine experimental
kinetics and theoretical molecular dynamics to study how simple
substitution of ancillary ligands affects hydrolytic cleavage of
peptides by palladium(II) complexes and to determine the
stereochemical basis for the regioselectivity of cleavage.
1
40,43
The
error in integration of the resonances was estimated at (5%. In all the
2
+
experiments with [Pd(H
[PdCl ]
4
2
O)
, the reactions were quenched by adding 400 µL of a 200
mM solution of sodium diethyldithiocarbamate (Naddtc) in D O to 200-
µL aliquots of the reaction mixture. The insoluble [Pd(ddtc) ] was
4
] , and in the early experiments with
2-
2
2
1
(27) Heyduk, E.; Heyduk, T. Biochemistry 1994, 33, 9643.
removed by centrifugation, and H NMR spectra of the clear supernatant
(
28) Ghaim, J. B.; Greiner, D. P.; Meares, C. F.; Gennis, R. B.
were recorded. Free glycine and free acetic acid were identified by
their H NMR chemical shifts and by enhancement of the corresponding
Biochemistry 1995, 34, 11311.
29) Greiner, D. P.; Hughes, K. A.; Gunasekera, A. H.; Meares, C. F.
Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 71.
30) Miyake, R.; Murrakami, K.; Owens, J. T.; Greiner, D. P.; Ozoline,
O. N.; Ishihama, A.; Meares, C. F. Biochemistry, 1998, 37, 1344.
1
(
resonance upon addition of these compounds to the reaction mixture.
Molecular Dynamics Simulation. To sample the conformational
space of the Pd(II)-peptide complex, we performed a Langevin
molecular dynamics simulation in a vacuum (dielectric constant of 1.0),
over a period of 3.00 ns, with an integration step of 1.00 fs; the energies
were saved after each 10 fs, for further analysis. The system was
coupled to a heat bath at 1000 K, and the friction coefficient â was set
(
(
31) Rana, T. M. AdV. Inorg. Biochem. 1993, 10, 177.
(32) Yashiro, M.; Takarada, T.; Miyama, S.; Komiyama, M. J. Chem.
Soc., Chem. Commun. 1994, 1757.
33) Allen, G.; Campbell, R. O. Int. J. Peptide Protein Res. 1996, 48,
65.
(
2
-
1
55
to 2.0 ps . In addition to the original set of CHARMM parameters,
the improper torsion centered at the C atom of histidine was constrained
to keep this atom trigonal and therefore keep the C -C bond coplanar
(
(
(
(
(
34) Hegg, E. L.; Burstyn, J. N. J. Am. Chem. Soc. 1995, 117, 7015.
35) Burgeson, I. E.; Kosti c´ , N. M. Inorg. Chem. 1991, 30, 4299.
36) Zhu, L.; Kosti c´ , N. M. Inorg. Chem. 1992, 31, 3994.
37) Zhu, L.; Kosti c´ , N. M. J. Am. Chem. Soc. 1993, 115, 4566.
38) Zhu, L.; Qin, L.; Parac, T. N.; Kosti c´ , N. M. J. Am. Chem. Soc.
γ
â
γ
with the imidazole ring, as it must be. The palladium(II)-ligand torsion
angles were left unconstrained. The parameters that are not included
in the CHARMM set are given in Supporting Information, Table S1.
The bonded energy parameters for the coordinated water were taken
from the SPCE model for water.56 Atomic charges in the palladium(II)
complex were calculated by the extended H u¨ ckel method and are given
in Supporting Information, Table S2. Nonbonded energies for all pairs
of atoms were calculated without any cutoffs. The C-terminus of the
peptide was reasonably assumed to be protonated.
1
994, 116, 5218.
(39) Zhu, L.; Kosti c´ , N. M. Inorg. Chim. Acta 1994, 217, 21.
(40) Parac, T. N.; Kosti c´ , N. M. J. Am. Chem. Soc. 1996, 118, 51.
(41) Korneeva, E. N.; Ovchinnikov, M. V.; Kosti c´ , N. M. Inorg. Chim.
Acta 1996, 243, 9.
42) Chen, X.; Zhu, L.; Yan, H.; You, X.; Kosti c´ , N. M. J. Chem. Soc.,
Dalton Trans. 1996, 2653.
(
(
(
43) Parac, T. N.; Kosti c´ , N. M. J. Am. Chem. Soc. 1996, 118, 5946.
44) Chen, X.; Zhu, L.; Yan, H.; Kosti c´ , N. M.; You, X. Chin. Sci. Bull.
The molecular dynamics trajectory was used to find conformations
with the interatomic distances that are comparable to, or smaller than,
the sum of the van der Waals radii of the two atoms involved. The
1
996, 41, 390.
(
(
45) Chen, X.; Zhu, L.; Kosti c´ , N. M. Chin. Chem. Lett. 1996, 7, 127.
46) Milinkovi c´ , S. U.; Parac, T. N.; Djuran, M. I.; Kosti c´ , N. M. J.
Chem. Soc., Dalton Trans. 1997, 2771.
(
(
(
(
(
47) Parac, T. N.; Kosti c´ , N. M. J. Serb. Chem. Soc. 1997, 62, 847.
48) Chen, X.; Zhu, L.; Kosti c´ , N. M. J. Biol. Inorg. Chem. 1998, 3, 1.
49) Parac, T. N.; Kosti c´ , N. M. Inorg. Chem. 1998, 37, 2141.
50) Karet, G. B.; Kosti c´ , N. M. Inorg. Chem. 1998, 37, 1021.
51) Zhu, L.; Bakhtiar, R.; Kosti c´ , N. M. J. Biol. Inorg. Chem. 1998, 3,
(53) Wheeler, G. P.; Ingersol, A. W. J. Am. Chem. Soc. 1951, 73, 4604.
(54) Covington, A. K.; Paabo, M.; Robinson, R. A.; Bates, R. G. Anal.
Chem. 1968, 40, 700.
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Swaminathan, S.; Karplus, M. J. Comput. Chem. 1983, 4, 187.
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1987, 91, 6269.
3
83.
52) Milovi c´ , N. M.; Kosti c´ , N. M. Unpublished results.
(

Peptide CleaVage by Palladium(II)
J. Am. Chem. Soc., Vol. 121, No. 13, 1999 3129
pertinent distances are two: first, from the oxygen atom of an aqua
ligand on palladium(II) to the carbon atom of an amide bond; and
second, from the palladium(II) ion to the oxygen atom of an amide
bond. The energy of each of these conformations was minimized in
three steps. First, all non-hydrogen atoms were fixed in their position
obtained by MD simulation, as mentioned above; only the hydrogen
atoms were used in the minimization. In the second step, the
non-hydrogen atoms were gently constrained in their position by a
harmonic potential with the force constant of only 1.0 kcal mol-1. In
the final step of energy minimization all constraints were removed,
and all degrees of freedom of the molecule were allowed to vary. This
procedure is very likely to achieve the goalsfind local energy minima
close to the conformations with relatively small O-C and Pd-O
distances, which were found in the trajectories.
Scheme 1
-
1
is common to all of them. The parameter b was set at 5.0 cal mol
-
2 59
A
.
C
Coulumbic energy (G ) arises from pairwise interactions among all
Energetics of Various Conformations. In the molecules of interest
the atoms and can be calculated according to eq 5, in which i and j run
2+
a Pd(H
ring of AcHis-Gly. At least three factors contribute to the change in
the energy of different conformations, ∆G ; see eq 1. Changes in
2
O)
3
group is attached to the N-1 or N-3 atom in the imidazole
1
G (ꢀ ) ) /
(q q /ꢀ r )
(5)
C
s
2
∑
i
j
s ij
T
j*i
∆
G ) ∆∆G + ∆G + ∆GC
(1)
T
R
NE
over all the atoms. In eqs 2, 3, and 5, ꢀ
m
was set at 80.0 for water, and
ꢀ
s
was set at 4.0 for the peptide interior. In eqs 2 and 3, ionic strength
solvation energy come from electrostatic, ∆G
R
, and nonelectrostatic,
was set at zero, and a Stern layer of 4.0 Å was used. In the finite-
difference algorithm for numerically solving the Poisson-Boltzmann
equation, an 81 × 81 × 81 grid with a 0.5-Å spacing was used.
The Thermodynamic Cycle. Numerical solution of the Poisson-
Boltzmann equation requires discrete redistribution of charge on the
grid. The artifact resulting from this redistribution is termed grid energy.
It precludes direct calculation of the change of the conformational
∆
G
NE, interactions. There are also changes in Coulombic electrostatic
energy, ∆G . The G term is energy calculated with the Coulomb law,
using a single dielectric constant for a uniform medium. The ∆G term,
which is calculated with the Poisson-Boltzmann equation, includes
but contains an additional term if more than one dielectric medium
C
C
R
∆G
C
is present. In calculations of solvation energy with a thermodynamic
cycle, the Coulombic terms cancel out in the difference; see the next
subsection. We neglect contributions to the change of the conforma-
tional energy that arise from the bonded-energy terms. The three terms
in eq 1 can be estimated under various approximations.
Electrostatic contribution to the solvation energy (∆G
polarization of the medium by the charges in the (H O)
complex. This contribution can be calculated by solving numerically
energy ∆G
can be avoided by calculating the other three steps in the thermodynamic
cycle.61 We calculated the energy required to transfer the (H
O) Pd-
T
, along the thick horizontal line in Scheme 1. This artifact
2
3
peptide complex in an arbitrary reference conformation from a medium
with the dielectric constant of water to a medium with the dielectric
constant of the peptide (the vertical on the left) and the energy required
to transfer the complex in a different conformation in the same direction
(the vertical on the right). The grid energies canceled out in the
“vertical” calculations, and we obtained reliable quantities for the two
R
) arises from
2
3
Pd-peptide
5
7-59
the Poisson-Boltzmann equation.
molecule with the dielectric constant ꢀ
dielectric constant ꢀ to a medium with the dielectric constant ꢀ
given in eq 2. The energy of a molecule within its reaction field is
defined in eq 3. In it, q are the atomic charges in the molecule, and φ
The energy required to bring a
from a medium with the
is
s
“vertical” processes in Scheme 1. Because in the calculation of ∆G
the dielectric constants of the solute and of the medium are equal (ꢀ
C
m
s
s
,
ꢀ
s
in the upper portion of Scheme 1), this term was calculated with the
Coulomb law and a single dielectric constant. Estimation of ∆GNE
completed the cycle and allowed an estimation of ∆G . These
i
i
T
∆
G ) G (ꢀ ,ꢀ ) - G (ꢀ ,ꢀ )
(2)
(3)
R
R
s
m
R
s
s
6
2
calculations were done with the program package MEAD 1.1.3.
1
G (ꢀ ,ꢀ ) ) /
q φ (ꢀ ,ꢀ )
i i s i
R
s
i
2
∑
Results and Discussion
is the electrostatic potential at the position of the charge i inside a
dielectric cavity with a dielectric constant ꢀ in a medium with a
dielectric constant ꢀ such that ꢀ is ꢀ or ꢀ . The cavity is defined by
the water-accessible surface of the molecule. The potential φ can be
obtained by solving numerically the Poisson-Boltzmann equation or
its linearized form.
Nonelectrostatic contribution to the solvation energy (GNE) is caused
by van der Waals interactions between the solute and the solvent and
also by creation or enlargement of the cavity for the solute against the
solvent pressure. This, the second term in eq 1, can be estimated by
the empirical eq 4, in which ∆A is the change in the water-accessible
s
Substrates for Hydrolysis. In all the substrates the terminal
amino group was acetylated, so that it cannot be the first one
to coordinate to palladium(II). In four of the substrates the N-1
or N-3 atom of the imidazole ring was methylated, to enforce
palladium(II) binding to the other nitrogen atom. The six
substrates are AcHis, AcHis-Gly, 1-Me-AcHis, 1-Me-AcHis-
Gly, 3-Me-AcHis, and 3-Me-AcHis-Gly. See Chart 1. For the
sake of brevity, the amide bonds involving the amino and
carboxylic groups of histidine, respectively, are designated “left”
and “right.” (In biochemical parlance, these bonds are designated
as lying “upstream” and “downstream” from the histidine
residue.) The “left” amide bond exists in all the substrates; the
i
i
m
s
i
∆GNE ) a + b∆A
(4)
surface during a conformational transition. The water-accessible surface
“
right” one exists only in the dipeptide and its two methyl
(A) is defined by rolling a sphere with a diameter of 1.4 Å over the
derivatives.
(
H
2
O)
3
Pd-peptide molecule. The parameters a and b are empirically
Palladium(II) Complexes. Although the reactions were run
in D2O and therefore the aqua complexes contain D2O, we write
H2O for the sake of clarity and consistency with our previous
5
9,60
fitted.
Since we are interested in relative, not absolute, energies of
various conformations, we justifiably neglected the parameter a, which
2
+
(
(
57) Warwicker, J.; Watson, H. C. J. Mol. Biol. 1982, 186, 671.
58) Klapper, I.; Fine, R.; Sharp, K. A.; Honig, B. H. Proteins 1986, 1,
publications. The complex [Pd(H2O)4] was made by the
4
7.
(
(
(61) Ullmann, G. M.; Knapp, E.-W.; Kosti c´ , N. M. J. Am. Chem. Soc.
1997, 119, 42.
(62) You, T.; Bashford, D. B. Biophys. J. 1995, 69, 1721.
59) Sitkoff, D.; Sharp, K. A.; Honig, B. J. Phys. Chem. 1994, 98, 1978.
60) Ben-Naim, A. Curr. Opin. Struct. Biol. 1994, 4, 264.

3130 J. Am. Chem. Soc., Vol. 121, No. 13, 1999
Parac et al.
Chart 1
Figure 1. Hydrolytic cleavage at pD 1.4 and 60 °C of the histidine-
2
glycine bond in the peptide AcHis-Gly. The solution in D O was
2 4
initially 32 mM in AcHis-Gly and 128 mM in K [PdCl ].
Chart 2
aquation reaction in acidic aqueous solution, as shown in eq 6.
2-
+
[
PdCl4] + 4Ag + 4H O f
2
2+
[
Pd(H O) ] + 4AgCl(s) (6)
2 4
The ultraviolet absorption maximum occurs at 390 nm, as
reported previously.36 Control experiments showed that AgClO4
in the absence of palladium(II) complexes does not promote
cleavage of peptides.36 The aqua complex was freshly prepared
before the kinetic experiments and kept at pD 1.4, to suppress
formation of hydroxo-bridged polynuclear complexes.
Regioslective Cleavage of AcHis-Gly Catalyzed by
2
-
2-
[
PdCl4] . Upon mixing AcHis-Gly with [PdCl4] , three
complexes detectable by NMR spectroscopy formed rapidly.
The known modes of coordination, shown in Chart 2, were
defined on the basis of the characteristic chemical shifts of the
imidazole H-2 and H-5 protons.40 The amide proton can be
displaced by the palladium(II) ion, as in the complex designated
, even at pD < 2.0.6
3-69
The unspecified ligands on the
2
-
palladium(II) ion are Cl anions, at least initially. (Their
aquation on standing will be discussed below.) The major
species is the complex designated 3.
As Figure 1 shows, in the presence of [PdCl4]2 glycine was
completely released from the dipeptide AcHis-Gly. Two
derivatives of AcHis-Gly, containing a methyl group at either
nitrogen atom in the imidazole ring, also undergo this cleavage
-
1
reaction. It was followed by the decline of the H NMR
detected by NMR spectroscopy even after 1 week. Clearly, only
the “right”, not the “left”, amide bond was cleaved. This
regioselectivity was further examined in the reaction of [PdCl4]2
with AcHis, which lacks the “right” amide bond. Although the
complexes 1 and 3 formed immediately upon mixing of the
reactants, the “left” amide bond was not cleaved to a detectable
resonance for the glycyl residue in the coordinated dipeptide
and the growth of the resonance for the free glycine. The decline
and growth occur with the same rate constant, which is listed
in Table 1. Under the same conditions free acetic acid was not
-
(
(
(
(
(
63) Sigel, H.; Martin, R. B. Chem. ReV. 1982, 82, 385.
64) Sovago, L.; Martin, R. B. J. Inorg. Nucl. Chem. 1981, 43, 425.
65) Menabue, L.; Saladini, M.; Sola, M. Inorg. Chem. 1990, 29, 1293.
66) Wilson, E. W., Jr.; Martin, R. B. Inorg. Chem. 1971, 10, 1197.
67) Kasselauri, S.; Garoufis, A.; Hadjiliadis, M.; Hadjiliadis, N. Coord.
2-
extent. As Table 1 shows, [PdCl4] did not promote cleavage
of methylated AcHis, either. This unreactivity confirms that the
-
PdCl3 group bound to either nitrogen atom in the histidine
Chem. ReV. 1990, 104, 1.
side chain does not promote cleavage of the “left” amide bond.
(
(
68) Wilson, E. W., Jr.; Martin, R. B. Inorg. Chem. 1970, 9, 528.
69) Laussac, J. P.; Haran, R.; Hadjiliadis, N. C. R. Acad. Sci. Pans Ser.
That the substrate is consumed at the same rate at which the
products are formed indicates that in the cleavage reaction
II 1985, 300, 137.

Peptide CleaVage by Palladium(II)
J. Am. Chem. Soc., Vol. 121, No. 13, 1999 3131
Table 1. Rate Constants (104kobs, in min-1)a for the Hydrolysis of
forms complex 3. Cleavage of the “right” amide bond is easily
followed by H NMR spectroscopy. The singlets at 3.89 and
Histidine-Containing Substrates Catalyzed by Two Palladium(II)
1
b
Complexes, at pD 1.4 and 40 °C
3
.48 ppm correspond to free glycine and to the complex cis-
2 4
Pd(H O)
]2
+
+
[
[Pd(N,O-Gly)(H2O)2] , in which glycine is coordinated via its
substratec
[PdCl
]2- “right”
5.0
“left”
“right”
49
amino and carboxylate groups. This mode of glycine chelation
4
7
0
2+
is well known. Indeed, an equimolar mixture of [Pd(H2O)4]
AcHis-Gly
AcHis
-Me-AcHis-Gly
-Me-AcHis
-Me-AcHis-Gly
-Me-AcHis
29
21
22
20
11
10
1
and glycine showed the same singlet in the H NMR spectrum.
1
1
3
3
5.2
4.1
43
49
1
Since the H NMR resonance of the acetyl group in AcHis-
Gly and its methyl derivatives is broadened upon addition of
Pd(H2O)4] , this resonance overlaps with that of free acetic
2
+
[
acid, and cleavage of the “left” amide bond could not be
observed directly. Fortunately, upon removal of the palladium-
a
4
To obtain kobs, divide the number by 10 . Estimated error is (10%.
Blank spaces correspond to reactions that cannot occur with a given
b
(II) species as the insoluble complex [Pd(ddtc)2], acetic acid, a
c
substrate. The “left” and “right” amide bonds are shown in Chart 1.
product of the cleavage of the “left” amide bond, was easily
detected and quantitated. As Table 1 shows, the “right” bond
is cleaved more rapidly than the “left” one. The respective yields
of cleavage are 100% and 85% with AcHis-Gly and 100%
and 75% with 3-Me-AcHis-Gly. Partial oligomerization of [Pd-
1
intermediates do not accumulate. Indeed, H NMR spectra did
not contain any resonances attributable to intermediates.
The substrate 1-Me-AcHis-Gly forms complexes 1 and 2,
which are interconverted by (de)protonation of the “left” amide
nitrogen atom. Our previous study showed that the chelate
complex 2 is inactive in hydrolysis.43 In it the “left” amide bond
is stabilized by the nitrogen coordination to palladium(II),
whereas the “right” amide bond is remote from the palladium-
2
+
(
H2O)4] into polynuclear, hydroxo-bridged complexes may
be slow enough not to affect the faster cleavage, but it may
compete somewhat with the slower one. Moreover, 3-Me-AcHis
7
1
tends to form polynuclear complexes.
AcHis and its methyl derivatives lack the “right” amide bond
(
II) that is firmly held on the “left” side. Complex 3, however,
2
+
-
when they are treated with [Pd(H2O)4] , acetic acid and
histidine (or its methyl derivative) are formed. Indeed, this aqua
complex promotes the cleavage of the “left” amide bond. In
is flexible enough to allow the approach of the PdCl3 group
to the “right” amide bond but, judging from Table 1, not to the
“
left” amide bond. Because the substrate 3-Me-AcHis-Gly
2+
this respect, it differs from cis-[Pd(en)(H2O)2] , which cleaves
only the “right” amide bond in AcHis-aa dipeptides, in which
aa designates glycine and several other amino acids.40
forms only the complex 3, this linkage isomer must be
responsible for the substrate cleavage.
In cleavage reactions promoted by cis-[Pd(en)(H2O)2] , the
2
+
Table 1 shows several interesting results. First, both amide
bonds are cleaved in AcHis-Gly and also in both methyl
derivatives of it. EVidently, attachment of the Pd(H2O)32 group
to either nitrogen atom in the imidazole ring results in cleaVage
of both amide bonds in the dipeptide. Second, the adjacent
numbers in the “left” column form three pairs. Within each pair
the numbers are nearly the same. Clearly, the rate constant for
the cleavage of the one amide bond is only slightly affected by
the presence (and cleavage) of the other. Cleavage of the “right”
bond is 1.7 to 4.5 times faster than cleavage of the “left” bond.
This difference in the rate constants exceeds the margins of error,
and we consider it real. But the difference is too small to be
explained with certainty. The main purpose of this study is to
explain in stereochemical terms our qualitative finding that
unspecified ligands in Chart 2 are ethylenediamine and H2O.
as in the present one, only the “right”
amide bond was cleaved. In the previous case, however, the
In this previous case,4
0,43
+
linkage isomer 3 was at least 50 times more reactive than the
linkage isomer 1,43 whereas in the present case the two isomers
are nearly identical in reactivity, as Table 1 shows. The ancillary
ligandssbidentate ethylenediamine versus two unidentate Cl-
ionssapparently exert decisive influence on the cleavage
reaction.
Catalytic Turnover. One equivalent of the [PdCl4]2- com-
plex cleaves as many as five equivalents of AcHis-Gly. This
is a modest turnover number, but it proves the principle of
catalysis. There are two “connected” catalytic cycles in Scheme
2
, for the linkage isomers 1 and 3. All the species shown in
2
+
1
attachment of the Pd(H O)
3
group to either nitrogen atom in
Scheme 2 were detected by H NMR spectroscopy. So was the
unreactive complex 2, which yielded the active complex 1 upon
protonation; this process and free chloride ions are omitted from
Scheme 2, for the sake of clarity.
2
the imidazole ring results in cleavage of both amide bonds in
the peptide.
Mechanism of Peptide Hydrolysis Promoted by Transi-
tion-Metal Complexes. Transition-metal complexes bind to a
nucleophilic side chainsin this study, the imidazole ringsand
effect cleavage of a proximate peptide bond by two limiting
Turnover is assisted by the relative lability of the palladium-
(II)-imidazole bond and by the acidic solvent. Because hydro-
nium ions displace palladium(II) from AcHis, the cleavage
reaction is not inhibited by this product of cleavage. The
palladium(II) complex detaches from AcHis and attaches to the
AcHis-Gly molecule to be cleaved next.
7,8
mechanisms; see Chart 3. In one mechanism, the metal cation
acts as a Lewis acid, interacts with the amide oxygen atom,
further polarizes the carbonyl group, and makes the carbon atom
more electrophilic and therefore susceptible to external attack
by water from the solvent. This mechanism requires close
approach of the amide oxygen atom and the metal cation in an
orientation favorable for a bonding interaction. In the case of
the square-planar palladium(II) complex, axial approach of the
oxygen atom allows partial donation of the lone pair of electrons
Cleavage of AcHis-Gly Promoted by [Pd(H2O)4]2+.
Because the acidic solvent protonates the imidazole ring in the
peptide and lowers its affinity for palladium(II), the [Pd-
+
(
H2O)4]2 complex was always added in a 4-fold molar excess
over the dipeptide, to enhance coordination. Thorough control
experiments showed that in the acidic solution of pD 1.4, in
the absence of palladium(II) reagents, the peptide is not cleaved
to a significant degree.
The reaction of interest is shown in Scheme 3. Dipeptide
AcHis-Gly forms all three complexes shown in Chart 2; 1-Me-
AcHis-Gly forms complexes 1 and 2; and 3-Me-AcHis-Gly
2
into the vacant 4dz orbital.
(
70) Appleton, T. G.; Bailey, A. J.; Bedgood, D. R., Jr.; Hall, J. R. Inorg.
Chem. 1994, 33, 217.
71) Remelli, M.; Munerato, C.; Pulidon, F. J. Chem. Soc., Dalton Trans.
1994, 2050.
(

3132 J. Am. Chem. Soc., Vol. 121, No. 13, 1999
Parac et al.
Scheme 2
In the other mechanism, the metal aqua complex internally
delivers one of its ligands to the scissile amide bond. If the
aqua complex is sufficiently acidic to be deprotonated, cleavage
is enhanced because hydroxo ligand is more nucleophilic than
aqua ligand. Because the pKa value of the aqua ligand bound
to palladium(II) is by several units greater than the pH of our
reaction mixture, the concentration of the palladium(II) hydroxo
species must be very low. In our case, the cleavage reaction
may be assisted by the proximity of the aqua ligand to the amide
carbon atom. This is the main requirement for the second
mechanism.
and the solution of Poisson-Boltzmann equation (for treatment
of the solvation effects) have not been combined in previous
studies. We did the calculations with complexes designated 1
and 3 in Chart 2 containing water as the three unspecified
ligands in the coordination sphere. The simulated peptide
molecules, like the actual chemicals used in the experiments,
contain hydrogen. Even though the actual ligands are D2O, the
difference in isotopes has no effect on our calculations and on
their relevance to the experiments. Atomic mass affects
vibrational frequencies, which we do not calculate. In classical
mechanics and statistical thermodynamics, energy of the system
does not depend on the mass (if gravity is negelected). Energies
of the complexes are calculated by using the thermodynamic
cycle in Scheme 1 and are shown in Table 2; a similar cycle
was successfully used in our recent study of metalloprotein
complexes. Because the two linkage isomers are different
compounds, their energies cannot be compared. Consequently,
differences between the rate constants in the same column in
Table 1 cannot be explained quantitatively. But the regioselec-
tivity of peptide cleavage is explained below. The calculations
provided stereochemical information that would be difficult or
impossible to obtain by experimental methods.
Also possible is a combination of these two limiting mech-
anisms, in which the metal ion simultaneously polarizes the
amide bond and delivers a nucleophilic ligand to that same
8
bond. None of the mechanisms in Chart 3 requires breaking
6
1
of a bond between a hydrogen atom and another atom.
Consequently, primary isotope effect H/D is not expected. The
isotope effect of the solvent D2O, if it exists, would change the
kinetics so little that it would be undetectable in our experiments.
Since the aforementioned mechanisms are indistinguishable
by kinetic methods, we applied molecular-dynamics calculations
in several steps to analyze possible interactions between the
Pd(H2O)32 group bound to the imidazole ring and the adjacent
amide bonds in AcHis-Gly. The composite molecule is
relatively large but still amenable to thorough calculations
without debilitating assumptions and constraints. Fortunately,
this molecule was also amenable to kinetic investigations
discussed above. Because the theoretical and the experimental
parts of this study were done with the same system, the analyses
of conformations in the next three subsections explain the kinetic
results well.
+
We examined the trajectories obtained by the simulations and
sought conformers in which the atoms of interest approach each
other. We took the sum of van der Waals radii as the contact
7
2,73
distance between two atoms:
3.6 Å between oxygen of an
aqua ligand and amide carbon (for the internal-delivery mech-
anism) and 3.2 Å between palladium(II) and amide oxygen (for
the external-attack mechanism). We also considered the spatial
orientation of the groups that potentially are involved in the
(
(
72) Bondi, A. J. Phys. Chem. 1964, 68, 441.
73) Mayo, S. L.; Olafson, B. D.; Goddard, W. A., III J. Phys. Chem.
Molecular-Dynamics Calculations. To our knowledge, high-
temperature molecular dynamics (for conformation sampling)
1990, 94, 8897.

Peptide CleaVage by Palladium(II)
J. Am. Chem. Soc., Vol. 121, No. 13, 1999 3133
Scheme 3
-
1
Table 2. Energies (in kcal‚mol ) for Selected Conformers of the
Conformations when the N-1 Atom Is Coordinated to the
Pd(H2O)3 Group. The results of simulations for this linkage
2
+
Dipeptide AcHis-Gly Bearing a Pd(H
Atom (the upper four rows) or at the N-3 Atom (the lower four
rows) of the Imidazole Ring
2
O)
3
Group at the N-1
2+
isomer, designated 1, are given in the upper half of Tables 2
II
and 3; see also Figure 2. Judging by the Pd -O distances that
solvation
are never shorter than 3.5 Å, this interaction, and consequently
the external attack, is unlikely.
conformer
total ∆G
T
∆∆G
R
∆GNE
Coulombic ∆G
C
A1
B1
C1
D1
A3
B3
C3
D3
-724.54
-722.72
-725.87
-723.55
-718.53
-722.58
-722.50
-728.12
-124.06
-123.32
-123.99
-117.87
-107.30
-113.23
-108.78
-115.43
2.81
2.86
2.95
2.83
2.60
2.68
2.64
2.65
-603.29
-602.26
-604.83
-608.52
-613.82
-612.03
-616.37
-616.33
In conformer A1 an aqua ligand sits at 3.3 Å from the carbon
atom of the “left” amide bond, whereas in conformer B1 an
aqua ligand sits at 3.6 Å from the carbon atom of the “right”
amide bond. In both cases the spatial orientation of the oxygen
lone pair and the C-N antibonding orbital of the amide group
is favorable for cleavage by internal delivery. Interestingly, in
both of these conformers the oxygen atom of the scissile amide
bond and the oxygen atom of the aqua ligand poised to cleave
it sit at distances that suggests O-H-O hydrogen bonding: 2.7
Å in A1 and 2.6 Å in B1. Although weak, these hydrogen bonds
may further polarize the amide bonds and render them more
electrophilic and therefore more reactive toward the internally
delivered aqua ligand. Conformers A1 and B1 have similar total
energies and therefore similar probabilities of existence. The
similarity of the rate constants (in Table 1) for cleavage of the
two amide bonds may be attributed to these probabilities.
cleavage. The dihedral angle between the planes of the amide
bond and of the aqua ligand is relevant to internal delivery,
whereas the angle formed by the amide carbonyl group and the
palladium(II) ion is relevant to external attack. The structures
having minimal energies in their respective simulations may
not correspond to the respective global minima in solution,
because the simulations were performed in a vacuum. But
contributions of solvation were included in the calculation of
the conformational energy via the thermodynamic cycle, as
explained above. Each conformational family (a set of similar
conformers) is designated with a capital letter. In each family
we highlight the conformer having the shortest interatomic
distances that are relevant to the mechanisms of cleavage shown
in Chart 3. Eight such conformers, four for each of the two
linkage isomers, are included in Tables 2 and 3. Six of them
are shown in Figure 2.
Conformations When the N-3 Atom Is Coordinated to the
2
+
Pd(H2O)3 Group. The results of simulations for this linkage
isomer, designated 3, are given in the lower half of Tables 2
and 3; see also Figure 2. In an interesting family, represented
by conformer A3, one aqua ligand is proximate to the “left”
amide bond, another aqua ligand is proximate to the “right”
amide bond, and the palladium(II) ion is proximate to oxygen
atoms in both amide bonds! Moreover, the aqua ligands are

3134 J. Am. Chem. Soc., Vol. 121, No. 13, 1999
Parac et al.
Table 3. Interatomic Distances (in Å) between the Amide Carbon Atom and the Oxygen Atom of the Aqua Ligand and between the Amide
Oxygen Atom and the Palladium Atom; Dihedral Angles (in deg) between the Plane of the Amide Bond and the Plane of the Aqua Ligand;
and Angles (in deg) Formed by the Amide Carbonyl Group and the Palladium(II) Ion for Selected Conformers of the Dipeptide AcHis-Gly
2
+
a
Bearing a Pd(H
2
O)
3
Group at the N-1 Atom (the upper four rows) or at the N-3 Atom (the lower four rows) of the Imidazole Ring
distance dihedral angle angle
left right
conformer
C
left-OH
2
C
right-OH
2
Oleft-Pd
O
right-Pd
left
right
A1
B1
C1
D1
A3
B3
C3
D3
3.3
4.4
3.7
4.3
3.2
4.0
3.7
4.4
5.8
3.6
6.1
3.8
3.4
3.2
3.9
3.6
4.1
4.3
3.5
4.8
3.1
4.2
3.0
2.9
6.6
4.2
7.6
3.8
3.1
3.0
3.0
3.0
52
-129
175
142
105
-173
141
-135
-79
-128
-34
-100
-73
-88
126
60
10
19
26
30
53
71
76
23
35
64
10
42
26
27
30
122
a
The “left” and “right” amide groups are shown in Chart 1.
Chart 3
Figure 2. Selected conformers of the dipeptide AcHis-Gly bearing a
2+
Pd(H
2
O)
3
group at the N-1 atom (the upper two pictures) or at the
N-3 atom (the lower four pictures) of the imidazole ring. These
conformers were identified by thorough molecular-dynamics simula-
tions, as described in the text.
nearly perpendicular to the respective amide planes, and the
amide oxygen atoms sit above the palladium(II) coordination
plane. Naturally, the oxygen atoms from both amide groups
cannot simultaneously occupy the axial or nearly axial positions.
This family is well suited to cleavage of both amide bonds by
the mechanism that combines internal delivery of aqua ligands
and external attack by water from the solvent.
In the conformational family represented by D3, conditions
exist for the cleavage of the “right” amide group by a combined
mechanism and of the “left” amide group by external attack;
again, the oxygen atom of the “left” amide bond sits in an axial
position above the palladium(II) ion.
Interestingly, in all of the conformers discussed in this
subsection, the oxygen atoms of an aqua ligand and of the
proximate amide group are separated by a distance consistent
with (weak) hydrogen bonding. These interactions may activate
the amide group by both electronic and steric effectssby
polarizing this group and by holding it in the orientation
favorable for nucleophilic cleavage.
In the conformational family represented by B3, an aqua
ligand approaches the carbon atom of the “right” amide bond
at the dihedral angle favorable for internal delivery, and the
palladium(II) ion approaches the oxygen atom of the same bond.
Conditions exist for cleavage of the “right” amide bond by the
combined mechanism.
In the conformational family represented by C3, the “left”
amide bond may be cleaved by the combined mechanism;
because the amide oxygen atom sits in an axial position with
respect to the palladium(II) ion, external attack may be an
important component in this mechanism. In this family, the
Clearly, the conformational space of the linkage isomer 3 is
rich in conformers suitable for hydrolytic cleavage of both amide
groups in the dipeptide. This finding explains the similarity of
corresponding rate constants from the last two columns in Table
1.
“
right” amide bond may be cleaved by the external attack.

Peptide CleaVage by Palladium(II)
J. Am. Chem. Soc., Vol. 121, No. 13, 1999 3135
Importance of the Aqua Ligands on Palladium(II) for
initial concentration of this complex was 75 mM. Although it
attaches to the imidazole ring in the peptide and aquates
simultaneously, we separated these two reactions and applied
equilibrium constants in eq 7 to estimate the composition of
Hydrolysis of the Amide Bond. We can only surmise why the
-
[
PdCl4]2 complex does not cleave the “left” amide bond. Under
reasonable assumption that both amide bonds in the same
peptide are cleaved by the same mechanism, the difference
2
-
the mixture. The solution of [PdCl4] at equilibrium would be
2+
2-
2-
-
between [Pd(H2O)4] , which cleaves both bonds, and [PdCl4] ,
which cleaves only the “right” one, may be due to steric factors.
The former complex is smaller than the latter and presumably
may approach the scissile bonds more closely. This explanation
is consistent with our previous findings that chelate aqua
complexes, which are relatively bulky, cleave only the “right”
amide bond, i.e., the one downstream from the anchoring
methionine or histidine residue in short peptides.3
approximately 37 mM in [PdCl4] , 28 mM in [PdCl3(H2O)] ,
+
8.0 mM in [PdCl2(H2O)2], 1.8 mM in [PdCl(H2O)3] , and 0.20
2+
mM in [Pd(H2O)4] . The imidazole ring of the peptide is much
more likely to displace an aqua than a chloro ligand. Therefore
the total concentration of the complexes bearing both the
imidazole ligand and at least one aqua ligand is approximately
10 mM, a sum of 8.0 + 1.8 + 0.20 mM. If we consider the
requirement that the aqua ligand for internal delivery to the
amide bond needs to be in a cis position to the imidazole ring,
the concentration of the complexes suitable for cleavage
6,37,39,40,43
We can, however, explain the different rates of cleavage by
the two palladium(II) complexes. As Table 1 shows, the
dipeptide is cleaved about 10 times more rapidly by [Pd-
2+
becomes even smaller than 10 mM. Clearly, [Pd(H2O)4] is
more reactive than [PdCl4] in promoting hydrolytic cleavage.
+
2-
2-
(
H2O)4]2 than by [PdCl4] . We examine this finding at two
levels. In a simple analysis, we consider displacement of only
one of the four identical ligands by the imidazole ring (the
The semiquantitative argument in this subsection explains the
approximately 10-fold difference between the rate constants for
these two complexes in Table 1.
2+
peptide). The Pd(H2O)3 group is more effective than the
-
PdCl3 group in both limiting mechanisms for hydrolysiss
Conclusions
external attack and internal delivery. Palladium(II) ion in the
cationic complex is a stronger Lewis acid, and therefore better
able to activate the amide group for external attack, than that
ion in the anionic complex. The aqua complex, obviously, is
This study shows that kinetics and regioselectivity of cleavage
of histidine-containing peptides can be controlled by the choice
of ligands in palladium(II) complexes. Whereas selectivity of
enzymes depends on the structural fit between the active site
and the substrate, selectivity of the new artificial dipeptidases
depends on the proximity of the anchored metal complex to
the scissile amide bonds. This study shows, for the first time,
that stereochemical requirements for efficient cleavage by
palladium(II) complexes can be explained by thorough molec-
ular dynamics calculations. Although the new reagents so far
achieved only modest turnover of substrate, their proven
catalytic ability justifies their designation as artificial peptidases.
better suited than the chloro complex for internal delivery of
water. Because the Pd(H2O)32+ group attached to the imidazole
ring in the dipeptide contains several aqua ligands, conforma-
tions that brings one of them near the scissile amide bond are
likely to exist. Indeed, our molecular-dynamics simulations
confirm this intuitive expectation.
In a more detailed analysis, we take into consideration the
-
aquation of the PdCl3 group attached to the dipeptide. In eq 7
K1
K2
K3
\z
Acknowledgment. This work was funded by the National
Science Foundation, through grant CHE-9404971. G.M.U.
thanks Boehringer Ingelheim Fonds for a fellowship. We thank
Vassiliki-Alexandra Glezakou, Dr. Michael W. Schmidt, Profes-
sor Mark S. Gordon, and Professor Gordon J. Miller for useful
discussions.
2-
-
[
PdCl4]
y\ z [PdCl (H O)] y
\z [PdCl (H O) ] y
3
2
2
K
2
2
4
+
2+
[
PdCl(H O) ] y\ z [Pd(H O) ] (7)
2 3 2 4
the entering aqua ligands and leaving chloro ligands are implied;
showing them explicitly would have needlessly complicated the
equation. The equilibrium constants K1, K2, K3, and K4 are 4.2
Supporting Information Available: Table S1, energy
parameters not included in the CHARMM force field (Ki and
úo), and Table S2, charges used in the molecular dynamics
simulation (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
-
2
-3
-4
-5
74,75
×
10 , 3.8 × 10 , 5.2 × 10 , and 3.3 × 10 at 25 °C.
As expected, every substitution step is less favorable than the
2
-
previous one. In the cleavage experiments with [PdCl4] the
(
(
74) Elding, L. I. Inorg. Chim. Acta 1972, 6, 647.
75) Elding, L. I. Inorg. Chim. Acta 1972, 6, 683.
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