New insights into the palladium-mediated selective hydrolysis of the His18-Thr19 peptide bond in cytochrome c: 1H NMR and density functional theory investigation for model compounds

Article abstract of DOI:10.1039/b211882c

The peptides, CH₃CO-Met-His-GlyH, CH₃CO-CysMe-His-GlyH, CH₃CO-CysMe-His-Gly-OEt and its imidazole derivatives, N^τ-benzyl, N^τ-tosyl, N-benzyl-N^π-phenacyl, have been synthesized and used as model compounds for the mechanistic study of the selective cleavage of cytochrome c promoted by Pd(II) complexes. The peptide bond cleavage of these substrates by cis-[Pd(en)(solvent)₂]²⁺ (solvent: D₂O or CD₃OD) was monitored by ¹H NMR spectroscopy. The results showed that the methionine-containing tripeptide differs from the S-methylcysteine-containing tripeptides in the mode of coordination to Pd(II). The former coordinates to Pd(II) through a sulfur atom, an amide nitrogen of methionine and an N^π atom of imidazole, forming a polycyclic chelate, and is resistant to hydrolysis. The latter, as a model compound for cleavage of the His18-Thr19 bond in cytochrome c with Pd(II) complexes, coordinates to Pd(II) via a sulfur atom, an amide nitrogen and a carbonyl oxygen of histidine to form a polycyclic chelate in which the His-Gly peptide bond is cleaved. Kinetic studies showed that protonation of the N^π atom of imidazole in the S-methylcysteine-containing tripeptides is one of the key factors in controlling the cleavage of the His-Gly bond. In order to obtain theoretical guidance on the cleavage reaction, the geometries of a representative N^π protonated tripeptide cation of CH₃CO-CysMe-His-GlyNMe and its Pd(II) complex with and without ancillary water molecules are optimized at the B3LYP density functional theory level using 3-21G, 6-31G(d) and LanL2DZ basis sets. Based on the experimental and theoretical results obtained from the model compounds, a mechanism is proposed for the first time to explain the nature of selective cleavage of the His18-Thr19 bond in cytochrome c promoted by Pd(II) complexes. Coordination of Pd(II) to the carbonyl oxygen of histidine and hydrogen bond formed between the C=O and ancillary dimer water weaken and polarize the C=O double bond of histidine, giving rise to cleavage of the peptide bond.

Full text of DOI:10.1039/b211882c

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New insights into the palladium-mediated selective hydrolysis of the
1
His18–Thr19 peptide bond in cytochrome c: H NMR and density
functional theory investigation for model compounds
Xiao-juan Sun, Lin Zhang, Yu Zhang, Gao-sheng Yang, Zi-jian Guo and Long-gen Zhu*
State Key Laboratory of Coordination Chemistry, Coordination Chemistry Institute, Nanjing
University, Nanjing 210093, P.R. China. E-mail: Zhulg@public1.ptt.js.cn;
Fax: +86 25 331 4502
Received (in London, UK) 29th November 2002, Accepted 23rd February 2003
First published as an Advance Article on the web 26th March 2003
The peptides, CH3CO-Met-His-GlyH, CH3CO-CysMe-His-GlyH, CH3CO-CysMe-His-Gly-OEt and its
imidazole derivatives, Nt-benzyl, Nt-tosyl, Nt-benzyl-Np-phenacyl, have been synthesized and used as model
compounds for the mechanistic study of the selective cleavage of cytochrome c promoted by Pd(II) complexes.
The peptide bond cleavage of these substrates by cis-[Pd(en)(solvent)2]2+ (solvent: D2O or CD3OD) was
monitored by 1H NMR spectroscopy. The results showed that the methionine-containing tripeptide differs from
the S-methylcysteine-containing tripeptides in the mode of coordination to Pd(II). The former coordinates to
Pd(II) through a sulfur atom, an amide nitrogen of methionine and an Np atom of imidazole, forming a
polycyclic chelate, and is resistant to hydrolysis. The latter, as a model compound for cleavage of the His18–
Thr19 bond in cytochrome c with Pd(II) complexes, coordinates to Pd(II) via a sulfur atom, an amide nitrogen
and a carbonyl oxygen of histidine to form a polycyclic chelate in which the His–Gly peptide bond is cleaved.
Kinetic studies showed that protonation of the Np atom of imidazole in the S-methylcysteine-containing
tripeptides is one of the key factors in controlling the cleavage of the His–Gly bond. In order to obtain
theoretical guidance on the cleavage reaction, the geometries of a representative Np protonated tripeptide
cation of CH3CO-CysMe-His-GlyNMe and its Pd(II) complex with and without ancillary water molecules are
optimized at the B3LYP density functional theory level using 3-21G, 6-31G(d) and LanL2DZ basis sets. Based
on the experimental and theoretical results obtained from the model compounds, a mechanism is proposed for
the first time to explain the nature of selective cleavage of the His18–Thr19 bond in cytochrome c promoted by
Pd(II) complexes. Coordination of Pd(II) to the carbonyl oxygen of histidine and hydrogen bond formed
=
=
between the C O and ancillary dimer water weaken and polarize the C O double bond of histidine, giving rise
to cleavage of the peptide bond.
Selective hydrolysis of peptides and proteins is one of the most
important reactions in both chemical and biochemical pro-
cesses. Over the past decade, a variety of metal complexes have
been developed for directly hydrolytic cleavage of peptide
bonds in peptides1–22 and proteins.23–34 Simple palladium(II)
and platinum(II) complexes were found to promote effectively
the hydrolytic cleavage of peptides containing methio-
nine,6,7,11,12,20–22 cysteine,5,8,15 histidine9,10,14,20–22 or trypto-
phan residues19 through coordination of the side chains. The
use of Pd(II) complexes was also extended to site-specific clea-
vage of proteins such as horse heart cytochrome c,27,31 horse
myoglobin,30 oxidized insulin B chains16,17 and albumin from
cow, pig and chicken eggs.33 However, up to now, little is
known about the factors governing the site specificity of the
cleavage reactions.
In cytochrome c, there are two cysteine residues (Cys14 and
Cys17) and two methionine residues (Met65 and Met80) as
well as three histidine residues (His18, His26 and His33), which
are the potential anchoring sites for Pd(II) complexes. How-
ever, only the His18–Thr19 peptide bond was hydrolyzed
under the experimental condition described in the litera-
ture,27,31 though additional cleavage sites were more recently
observed and identified using a 10-fold excess of Pd(II) over
the protein and a higher incubation temperature.21 These
results indicated that the His18–Thr19 peptide bond is more
susceptible to cleavage, compared to other sites of cleavage.
Previous studies on hydrolysis of CH3CO-Cys-His-AlaH and
CH3CO-CysMe-His-GlyH with Pd(II) complexes showed that
only the His–Ala and His–Gly bonds were cleaved and clea-
vage of Cys–His and CysMe–His was not observed. These
findings further corroborate findings with cytochrome c by
showing the special reactivity of the peptide bond in the
-Cys-His-X- sequence. Alkylation of the sulfur atom does not
affect the regioselectivity of cleavage.27 In order to explain
the selectivity of cytochrome c cleavage, therefore, the short tri-
peptide CH3CO-CysMe-His-GlyH and its derivatives, in which
CysMe is used to mimic the vinylCys in cytochrome c, are cho-
sen as models for mechanistic studies of cytochrome c cleavage.
To prove the necessity of the -Cys-His- sequence for clea-
vage of the peptide bond at the C-terminal of histidine, in this
report, we also prepared CH3CO-Met-His-GlyH. The interac-
tion of the tripeptide with the palladium(II) complex explained
the importance of the cysteine residue in the cytochrome c
cleavage. On the other hand, three imidazole derivatives of
CH3CO-CysMe-His-GlyOEt were synthesized and their kine-
tics of cleavage with the palladium(II) complex were measured
in order to test the hypothesis that protonation of imidazole
facilitates the hydrolytic cleavage. The geometries of a Np-pro-
tonated tripeptide cation of CH3CO-CysMe-His-GlyNMe,
and its Pd(II) complex in the presence and the absence of ancil-
lary water molecules are optimized by B3LYP density func-
tional methods with 3-21G, 6-31G(d) and LanL2DZ basis
818
New J. Chem., 2003, 27, 818–822
DOI: 10.1039/b211882c
This journal is # The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2003

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sets. The interesting results obtained are very helpful for
exploiting the nature of site-specific cleavage of the His18–
Thr19 peptide bond in the cytochrome c with Pd(II) complexes.
was then evaporated under vacuum. The residue was triturated
with ether. The resulting pale solid was dissolved in 10 mL
of THF and 1 equiv of triethylamine was added. A THF solu-
tion containing N-CH3CO-S-methylcysteine-N-hydroxysucci-
nimide ester, which was prepared from 0.44 g (3.0 mmoL) of
CH3CO-CysMe and 0.35 g (3.0 mmoL) of N-hydroxysuccini-
mide, was added to the above solution. The mixture was stir-
red overnight at room temperature. After removing the
solvent under vacuum, the residue was dissolved in 50 mL of
chloroform and sequentially washed with saturated NaCl solu-
tion, 5% acetic acid, 5% NaHCO3 , and saturated NaCl solu-
tion. The chloroform was dried over sodium sulfate and
evaporated to dryness. A slight excess of solid AgNO3 (0.22
g, 1.3 mmol) was added to 20 mL aqueous solution of the resi-
due. After vigorous stirring for 15 min, the white AgCl preci-
pitate was removed by filtration. The filtrate was saturated
with KNO3 and extracted three times with a total of 25 mL
chloroform. The chloroform solution was evaporated to dry-
ness. The residue was recrystallized from chloroform and
ether. The total yield was 52%. The molecular mass observed
was 609; the calculated value for C31H38N5O6S is 608.7.
Experimental
Materials
The deuterium solvents were obtained from Aldrich. The
N-terminal-protected amino acids and EDC (1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride) were
obtained from Sigma. The terminal amino group in each
amino acid or peptide was acetylated. The complex cis-
[Pd(en)Cl2] was prepared by the published procedure35 and
converted to cis-[Pd(en)(CD3OD)2]2+ or cis-[Pd(en)(D2O)2]2+
by treatment with 2 equiv. of anhydrous AgBF4 in CD3OD
or in D2O.6,7 Other common chemicals were of reagent grade.
Measurements
Proton NMR spectra at 500 MHz of D2O or CD3OD solutions,
using sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DDS) as
an internal reference, were recorded with a Bruker AM-500
spectrometer. The sample temperature was kept at 40 ꢀ 0.5 ꢁC.
The pH was measured with an Orion 901 instrument and a
phoenix Ag–AgCl reference electrode. The uncorrected values
in D2O solution and nominal values in CD3OD solution are
designated pH*. The molecular masses of the synthesized pep-
tides were measured by VG-ZAB-HS mass spectrometer.
Cleavage study of peptide bond
cis-[Pd(en)(solvent)2]2+ solution (solvent ¼ CD3OD or D2O)
was freshly prepared to minimize formation of polymeric com-
plexes. Equimolar amounts of substrate and of the Pd(II) com-
plex, both dissolved in CD3OD or D2O, were mixed rapidly in
an NMR tube. The solution was 20 mM in each and the total
volume was 600 mL. Because the pH difference was always less
than 0.10, pH* was measured only at the end of the experi-
ments. Acquisition of the 1H NMR spectra started immedi-
ately after mixing, and 16 scans were collected for each
spectrum. The cleaved product, free glycine or free glycine
ethyl ester, was monitored by enhancement of a singlet at
3.87 or 3.82 ppm, with decrease of the multiplet at 4.06 or
4.01 ppm of the glycyl residue in the substrate. At the end of
the experiments, no new resonance appeared upon addition
of the authentic glycine or glycine ethyl ester to the reaction
mixture. The error in integration of these resonances was esti-
mated at ꢀ5%. First-order logarithmic plots of substrate con-
centration or product concentration versus time were linear for
a period of 3 half-lives. Typical plots consisted of 10–20 points
and correlation coefficients were 0.990–0.999.
Model compounds
CH3CO-Met-His-GlyH. This was prepared in solution
following the synthetic route: Na-Cbz-Nt-Cbz-histidine ! Na-
Cbz-Nt-Cbz-histidylglycine ethyl ester ! Na-H-Nt-Cbz-histi-
dylglycine ethyl ester ! acetyl-methionyl-histidyl-glycine.36,37
The molecular mass observed was m/z 386; calculated value
for C15H23N5O5S is 385.4.
CH3CO-CysMe-His-Gly-OEt. This was prepared in solution
as follows. Na-Boc-Nt-tosyl-histidine ! Na-Boc-Nt-tosyl-histi-
dylglycine ethyl ester ! Na-H-Nt-tosyl-histidylglycine ethyl
ester ! acetyl-S-methylcysteinyl-histidylglycine. The mole-
cular mass calculated for C16H25N5O5S is 399.5; the observed
value was 400.
Optimization of conformations
CH3CO-CysMe-His(Ns-tosyl)-Gly-OEt. This was also
synthesized following the same procedure as that of CH3CO-
CysMe-His-GlyH. Molecular mass calculated for C23H31N5-
O7S2 is 553.6,: the observed value was 554.
The structures of the Np-protonated tripeptide cation of
CH3CO-CysMe-His-GlyNHCH3 and its Pd(II) chelates invol-
ving a thioether, an amide nitrogen and a carbonyl oxygen
of histidine, and one aqua ligand, in the presence and the
absence of ancillary water molecules, were optimized in two
steps—first optimized by the HyperChem v. 6.0 software
employing the MM+ method of molecular mechanics with a
constraint of a hydrogen bond formed between the protonated
Np atom of histidine and the carbonyl oxygen of glycine, then
optimized by the density functional B3LYP theory in the
Gaussian 98 program package40 without any constraint using
the 6-31G(d) basis set for the tripeptide cation, the LanL2DZ
basis set for its Pd(II) chelates with and without the ancillary
water molecules, and the 3-21G basis set for all compounds
above-mentioned. The RMS gradient was less than 0.01 kcal
CH3CO-CysMe-His(Ns-benzyl)-Gly-OEt. This was prepared
following the same procedure as CH3CO-CysMe-His(Nt-tosyl)-
Gly-OEt except that Na-Boc-Nt-tosyl-histidine was replaced
by Na-Boc-Nt-benzyl-histidine. The molecular mass calcu-
lated for C23H31N5O5S is 489.6; the observed value was 490.
CH3CO-CysMe-His(Ns-benzyl-Np-phenacyl)-Gly-OEt imi-
dazolium nitrate. N-CH3CO-S-methylcysteine-N-hydroxy-
succinimide ester was prepared by the standard ethyl
chloroformate method in anhydrous tetrahydrofuran
(THF).38 After filtering triethylamine hydrochloride, the THF
solution was directly used for further coupling reaction with-
out purification. Na-Boc-Nt-benzyl-histidylglycine ethyl ester
was synthesized by the EDC procedure.36 Phenacylation of Np
of imidazole was followed by the method of Fletcher et al.39
The preceding dipeptide ester (0.86 g, 2 mmol) and phenacyl
bromide (0.4 g, 2 mmol) were dissolved in 20 mL of THF
and stirred at room temperature for 4 days. The solvent was
removed under reduced pressure. The residue was dissolved
in 5 mL of trifluoroacetic acid (TFA) for 45 min. The TFA
ꢂ1
molꢂ1 for the MM+ method and 10ꢂ8 atomic units of
˚
A
convergence for B3LYP method.
Results and discussion
Interaction of CH3CO-Met-His-Glyh with cis-[Pd(en)(D2O)2]2+
in D2O
When mixing CH3CO-Met-His-GlyH with cis-[Pd(en)-
(D2O)2]2+ in a weak acid solution of pH* ꢃ1.5, the thioether
New J. Chem., 2003, 27, 818–822
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group at the side chain of methionine rapidly reacts with the
Pd(II) complex, accompanied by a prominent migration of
peptides and cytochrome c,27,31 the imidazole nitrogen in the
complex was detached from Pd(II) and was protonated
(pKa ¼ 5.6), which was replaced by an aqua ligand.
1
the CH3S H resonance downfield to 2.48 ppm. At the same
time, ethylenediamine in the complex is detached from Pd(II)
by acidic action and by the strong trans effect of the thioether
Alcoholysis of CH3CO-CysMe-His-Gly-OEt and its imidazole
derivatives in methanol
2+
group. Free enH2 was readily detected by the growth of a
sharp 1H NMR singlet at 3.37 ppm. During the reaction, a
new singlet resonance at 2.06 ppm was enhanced with the con-
comitant decrease of the acetyl group CH3 1H NMR resonance
of the free peptide at 2.02 ppm. It implied that the new reso-
nance was associated with the acetyl group. After the reaction
was complete, a small amount of acetic acid was added to the
reaction mixture, whereupon a new resonance at 2.08 ppm
appeared. It was evident that the resonance at 2.06 ppm did
not belong to free acetic acid. When NaI was added to the
solution, PdI2 precipitated immediately.6 After removing the
precipitate, no new resonance that was different from the ori-
ginal tripeptide was observed. All these results indicate that
the Pd(II) anchors to the side chain of methionine, followed
by the coordination of the deprotonated amide nitrogen of
methionine, with migration of the acetyl group CH3 resonance
to 2.06 ppm. This coordination reaction was capable of pro-
ceeding to completion, as judged by 1H NMR spectra. The
observed rate constant, kobsd , for the reaction was 21.6 ꢀ
3 ꢄ 10ꢂ3 minꢂ1 at 40 ꢁC and pH* ¼ 1.53. In addition, it was
found that the imidazole of His also coordinated to Pd(II)
via the Np atom, though it is protonated in the free tripeptide
(pKa ¼ 6.5). The H2 and H5 resonances of imidazole moved
from 8.65 and 7.33 to 8.10 and 7.10 ppm upon coordination.
The coordination of Pd(II) to the Nt atom of imidazole was
not observed in the NMR spectra. Although the detailed struc-
Because the imidazole derivatives of CH3CO-CysMe-His-Gly-
OEt are soluble in methanol, rather than in water, the kinetic
studies of Pd(II) complex-mediated cleavage of peptide bond
for these substrates were carried out in methanol solution,
instead of water, for comparison. As shown in Table 1, the
groups linked to the imidazole remarkably affected the alcoho-
lysis of these substrates. CH3CO-CysMe-His-Gly-OEt was
alcoholized to give glycine ethyl ester. When a benzyl group
(an electron-donating group) is linked to the Nt atom of imida-
zole, it would be expected to enhance the basicity of the imida-
zole. As a result, the resonances of H2 and H5 of imidazole
moved toward high field in CD3OD solution of pH* 1.1, from
8.96 and 7.51 ppm for CH3CO-CysMe-His-Gly-OEt to 8.88
and 7.35 ppm for the benzyl group-substituted derivative. Cor-
responding to this, CH3CO-CysMe-His(Nt-benzyl)-Gly-OEt
was alcoholized to give free glycine ethyl ester with kobsd
¼
4.0 ꢄ 10ꢂ3 minꢂ1, which was slightly faster than that of
CH3CO-CysMe-His-Gly-OEt. When tosyl (tolylsulfonyl)
group, a well known strong electron-withdrawing group, was
connected to the Nt atom of imidazole, it was anticipated to
greatly reduce the basicity of the imidazole. Its 1H NMR reso-
nances of H2 and H5 of imidazole shifted downfield and
appeared at 9.41 and 7.79 ppm at pH* 1.1. Only trace amounts
of glycine ethyl ester were detected after 24 h of incubation at
40 ꢁC. No glycine ethyl ester was detected for the substrate of
CH3CO-CysMe-His(Nt-benzyl-Np-phenacyl)-Gly-OEt imida-
zolium nitrate, in which both Nt and Np atoms of imidazole
were blocked. Therefore, it is concluded that the ability to pro-
tonate the Np atom of imidazole is one of the key factors for
controlling the Pd(II) promoted alcoholysis reaction of these
substrates. It is interested to note that the site of cleavage in
alcoholysis of these substrates and hydrolysis of CH3CO-
CysMe-His-GlyH is the same as that in hydrolysis of cyto-
chrome c, promoted by Pd(II) complexes. Therefore, the short
tripeptide CH3CO-CysMe-His-GlyH and its imidazole deriva-
tives can be taken as model compounds for mechanistic studies
of cytochrome c cleavage. The protonation of the Np atom of
histidine is required to stabilize the conformation of the tripep-
tide upon hydrogen bond formation between the protonated
Np atom of histidine and the carbonyl oxygen of glycine (vide
infra).
1
ture is unclear as yet, the H NMR spectra confirmed that the
Pd(II) can be coordinated by the sulfur atom and the amide
nitrogen of methionine, and the Np atom of histidine. It should
be emphasized that hydrolytic cleavage of the peptide bond
does not occur in this case.
Hydrolysis of CH3CO-CysMe-His-GlyH in D2O
CH3CO-CysMe-His-GlyH differs apparently from CH3CO-
Met-His-GlyH in coordination to Pd(II) complexes. When
the S-methylcysteine containing peptide was mixed with cis-
1
[Pd(en)(D2O)2]2+ in a molar ratio of 1:1, the H NMR signal
of SCH3 moved downfield from 2.14 to 2.45 ppm, and ethylene-
diamine was rapidly released, however, the 1H NMR reso-
nances of CH3 of the acetyl group, and of H2 and H5 of
imidazole were unaffected. The hydrolysis of the His–Gly
peptide bond was observed by 1H NMR monitoring, with
observed rate constant kobsd ¼ 4.4 ꢄ 10ꢂ3 minꢂ1 at 40 ꢁC.
The rate of hydrolysis is slightly faster than that of alcoholysis
determined in methanol, as Table 1 shows. Electrospray mass
spectrometry (ESMS) also detected the complex [Pd(S,N,N,O-
CH3CO-CysMe-His-GlyH)] in which Pd(II) was coordinated
by the S atom of CysMe, the amide nitrogen and the carbonyl
oxygen of histidine, and the Np atom of imidazole.41 The coor-
dination of Pd(II) to the carbonyl oxygen of histidine in the
complex may be associated with cleavage of the His–Gly bond.
In acidic solution (pH ꢃ 1.0) used for the hydrolysis studies of
Conformational optimization of Np atom-protonated CH3CO-
CysMe-His-GlyNCH3 and its Pd(II) complexes
Np atom-protonated CH3CO-CysMe-His-GlyNCH3. In our
previous study,42 the conformation of the tripeptide cation
was optimized by combination of the three-dimensional energy
surfaces of AcCysMe and Np protonated AcHisGly cation
using the AM1 semiempirical method. The most stable confor-
mer of the tripeptide cation in that calculation exhibits a typi-
cal hydrogen bond formed between the protonated Np atom of
imidazole and the carbonyl oxygen of glycine, and reveals that
the sulfur, amide nitrogen and carbonyl oxygen atoms of his-
tidine are located on the same side which makes their coordi-
nation to Pd(II) more favorable with minor perturbation of
the conformation. In order to gain further insights into the
two important results, the geometry of the Np protonated tri-
peptide was optimized at the B3LYP/3-21G and B3LYP/6-
31G(d) levels. The selected parameters of geometry and
charges on atoms are listed in Table 2. As shown in Fig. 1
and Table 2, all these features obtained by the AM1 method
are kept in the optimized conformation, though the optimized
Table 1 Kinetic data of cis-[Pd(en)(CD3OD)2]2+-promoted cleavage
of His–Gly bond in CH3CO-CysMe-His-Gly-OEt and its imidazole
derivatives at 40 ꢁC and pH* 1.1 ꢀ 0.1
Substrate
kobsd/minꢂ1
CH3CO-CysMe-His-Gly-OEt
2.3 ꢄ 10ꢂ3
4.0 ꢄ 10ꢂ3
very slow
CH3CO-CysMe-His(Bzl)-Gly-OEt
CH3CO-CysMe-His(Tos)-Gly-OEt
CH3CO-CysMe-His(Bzl, phenacyl)-Gly-OEt
not detected
820
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Table 2 Select parameters of geometries and charges on atoms in the
optimized conformersa b
1
2
3
Pd–S(Cys)
Pd–O(H2O)
Pd–Na(His)
Pd–O(His)
2.426 (2.393)
2.134 (2.134)
1.978 (1.997)
2.110 (2.084)
1.284 (1.292)
1.337 (1.346)
92.20 (88.46)
91.26 (96.59)
80.54 (81.96)
2.422 (2.398)
2.079 (2.081)
1.992 (1.999)
2.158 (2.145)
1.290 (1.296)
1.333 (1.345)
90.84 (89.66)
90.94 (94.50)
78.10 (79.92)
Fig. 2 The conformation of the Pd(II) complex, which contains Np-
protonated CH3CO-CysMe-His-GlyNCH3 cation and one aqua
ligand, is optimized at the B3LYP/LanL2DZ level.
=
C
C–N(Gly)
O(His)
1.243 (1.226)
1.368 (1.364)
S–Pd–O(H2O)
S–Pd–O(His)
Na(His)–Pd–O(His)
O(H2O)–Pd–O(His)
Np(His)–Hꢅ ꢅ ꢅO(Gly)
the Pd–N distance is similar. As shown in Table 2, the coordi-
nation of Pd(II) to the carbonyl oxygen of histidine lengthens
96.71 (93.00) 100.29 (95.88)
2.626 (2.722)
2.612 (2.628)
172.0 (175.8)
2.615 (2.630)
176.0 (177.9)
2.618 (2.736)
158.0 (157.7)
Np(His)–Hꢅ ꢅ ꢅO(Gly) 171.6 (174.6)
=
the C O bond but shortens the C–N bond distance due to p
electron donation from nitrogen to carbon. In other words,
O(H2O)–Hꢅ ꢅ ꢅO(His)
=
the C O bond, rather than the C–N bond, is weakened upon
O(H2O)–Hꢅ ꢅ ꢅO(His)
Charge on atom:
=
C(C O of His)
coordination. The positive charge on the carbonyl carbon is
also increased by means of the coordination, from +0.6934 e
in conformer 1 to +0.7640 e in conformer 2. The increased
+0.693
ꢂ0.492
ꢂ0.695
+0.764
ꢂ0.489
ꢂ0.689
+0.778
ꢂ0.533
ꢂ0.679
=
O(C O of His)
N(C–N of Gly)
=
positive charge of this carbon atom and the weakened C O
double bond are favorable for nucleophilic attack by solvent
water to form a tetrahedral carbon intermediate, followed by
breaking of the C–N bond.
a
The conformers from 1 to 3 represent the conformations given in
b
Figs. 1 to 3. The numbers without parentheses are calculated at
the B3LYP/3-21G level and the numbers in parentheses are calculated
at the B3LYP/6-31G(d) level for conformer 1 and at the B3LYP/
LanL2DZ level for conformers 2 and 3.
Water-assisted cleavage of the His–Gly bond. Two mechan-
isms2,21,22 have been proposed for interpretation of the hydro-
lytic cleavage of the peptide bond by transition metal
complexes. A transition-metal ion either binds to amide oxy-
gen of the scissile peptide bond, resulting in breaking of the
peptide bond by external attack of solvent water molecules,
or delivers an aqua ligand to the scissile amide carbon, then
cleaving it. Although the two mechanisms are kinetically dis-
tinguishable, our experimental and theoretical results reported
here corroborate the external attack mechanism. Because
methanol is more nucleophilic than water for external attack
on carbonyl carbon,11 it would be anticipated that the alcoho-
lysis of the CysMe-containing tripeptide would be faster than
the hydrolysis. However, as indicated above, in fact, the
hydrolysis is slightly faster than the alcoholysis. This implies
that water molecules may assist the cleavage reaction besides
external attack by solvent water molecules. On the other hand,
since almost all Pd(II)-mediated hydrolysis of peptides and pro-
teins is carried out in bulk water, the effect of the water on the
cleavage should be properly considered. In our case, one and
two water molecules are involved in geometry optimization.
As far as only one molecule of water is concerned, the water
molecule is close to the aqua ligand upon hydrogen bonding.
As Fig. 3 shows, only the dimer water molecules involved will
assist the cleavage of the His–Gly bond. The hydrogen bond
formed between the dimer water and the carbonyl oxygen of
geometry may be a local minimum, and the bond lengths and
hydrogen bonds calculated at the B3LYP/3-21G and B3LYP/
6-31G(d) levels are similar.
Pd(II) complex of the tripeptide cation with one aqua ligand.
Based on the above-mentioned experimental and theoretical
studies, the initial structure was constructed as follows for geo-
metry optimization by MM+ molecular mechanics. The Pd(II)
was coordinated by the sulfur atom of S-methylcysteine, the
amide nitrogen and the carbonyl oxygen of histidine, and an
aqua ligand, with a hydrogen bond formed between the proto-
nated Np atom of imidazole and the carbonyl oxygen of
glycine, or the carbonyl oxygen of histidine, or both. The dif-
ferent initial structures were then optimized at the B3LYP/
LanL2DZ level without any constraint. A unique conforma-
tion was finally obtained as shown in Fig. 2. For comparison
of parameters of geometries and charges on atoms between
the free tripeptide cation and its Pd(II) complex, the conformer
2 was also optimized at the B3LYP/3-21G level. The bond
lengths calculated by using these two basis sets are very simi-
lar, but some deviations of bond angles are evident. The para-
meters of geometry and charges on atoms for conformer 2 are
given in Table 2. As Fig. 2 shows, Pd(II) is coordinated in a
square-planar configuration and a typical hydrogen bond
between the protonated Np atom of imidazole and the carbo-
nyl oxygen of glycine is kept, similar to the free tripeptide
cation. Compared with X-ray crystallographic analysis
=
His causes the C O and C–N distances to be further length-
˚
ened and shortened respectively, from 1.284 A to 1.290 A
˚
˚
and from 1.337 A to 1.333 A,. The charges on the carbonyl
˚
results,43 the calculated Pd–S distance is 0.16 A longer and
˚
Fig. 3 The conformation of Pd(II) complex containing the tripeptide
cation, one aqua ligand and dimer water molecules is optimized at the
B3LYP/LanL2DZ level. This conformation is associated with water-
assisted cleavage of the His–Gly bond.
Fig. 1 The conformation of Np-protonated CH3CO-CysMe-His-
GlyNCH3 , optimized at the B3LYP/6-31G(d) level.
New J. Chem., 2003, 27, 818–822
821

View Article Online
15 X. Luo, Y. Song, S. Zhou and L. Zhu, Prog. Nat. Sci., 1999,
9, 909.
16 X. Luo, W. He, Y. Zhang, Z. Guo and L. Zhu, Chem. Lett., 2000,
1030.
17 X. Luo, W. He, Y. Zhang, Z. Guo and L. Zhu, Chin. J. Chem.,
2000, 18, 855.
18 M. Hahn, M. Kleine and W. S. Sheldrick, J. Biol. Inorg. Chem.,
2001, 6, 556.
carbon and oxygen are further increased respectively from
+0.7640 e to +0.7780 e and from ꢂ0.4889 e to ꢂ0.5325 e.
Although the variation of these values is small, it is indicated
=
again that the C O double bond is further weakened and
polarized. Therefore, the dimer water assists the cleavage of
the His–Gly bond. These results are consistent with the fact
that the hydrolysis of the His–Gly bond is slightly faster than
the alcoholysis.
´
19 N. V. Kaminskaia and N. M. Kostic, Inorg. Chem., 2001, 40,
2368.
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Conclusion
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23 T. M. Rana and C. F. Meares, J. Am. Chem. Soc., 1990, 112,
The results obtained satisfactorily explain the necessity of both
the -Cys-His- sequence and the hydrogen bond formed
=
between the N atom of histidine and C O of glycine for clea-
´
´
p
2457.
24 T. M. Rana and C. F. Meares, J. Am. Chem. Soc., 1991, 113,
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chemistry, 1995, 34, 11 311.
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27 L. Zhu, L. Qin, T. N. Parac and N. M. Kostic, J. Am. Chem. Soc.,
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28 E. L. Hegg and J. N. Burstyn, J. Am. Chem. Soc., 1995, 117,
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29 G. Allen and R. O. Campbell, Int. J. Peptide Protein Res., 1996,
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vage of cytochrome c and its model compounds. The hydrogen
bond stabilizes the conformation of the tripeptide cation in
which the sulfur atom of the cysteine residue, the amide nitro-
gen (upon deprotonation) and the oxygen of histidine can
readily coordinate to Pd(II). This type of coordination does
not occur in the methionine-containing peptide of CH3CO-
Met-His-GlyH, because of formation of a labile seven-mem-
bered chelate ring. Although the carbonyl oxygen of histidine
is a weak ligand, its coordination to Pd(II) is facilitated by the
chelate effect. This type of coordination increases the positive
=
chage on the carbonyl carbon and weakens the C O double
bond, rather than the C–N bond. The cleavage of the C–N
bond is probably caused by nucleophilic attact of solvent
water, resulting in a tetrahedral carbon intermediate. The
assistance of the ancillary dimer water futher weakens and
=
polarizes the C O double bond to make the cleavage of the
His–Gly bond more feasible.
´
30 L. Zhu, R. Bakhtiar and N. M. Kostic, J. Biol. Inorg. Chem.,
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33 L. Zhu and N. M. Kostic´, Inorg. Chim. Acta, 2002, 339, 104.
34 L. Zhang, Y. Mei, Y. Zhang, S. Li, X. Sun and L. Zhu, Inorg.
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35 H. Hohmann and R. Van Eldik, Inorg. Chim. Acta, 1990, 174, 87.
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 20271027).
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