Synergie effect of two metal centers in catalytic hydrolysis of methionine-containing peptides promoted by dinuclear palladium(II) hexaazacyclooctadecane complex

Article abstract of DOI:10.1039/b500210a

The species obtained by the reaction of [Pd₂([18]aneN ₆)Cl₂](ClO₄)₂ (where [18]aneN ₆ is 1,4,7,10,13,16-hexaazacyclooctadecane) with AgBF₄ have been determined by electrospray ionization mass spectrometry (ESI-MS) to be an equilibrium mixture of three major types of dinuclear Pd(II) complex cations, [Pd₂(-O)([18]aneN₆)]²⁺, [Pd ₂(μ-OH)-([18]aneN₆)]³⁺ and [Pd ₂(H₂O)(OH)([18]aneN₆)]³⁺, in aqueous solution. The hydroxo-group-bridged one, [Pd₂(μ-OH)-([18] aneN₆)]³⁺, is a dominant species, whose crystal structure has been obtained. The crystal structure of [Pd₂(μ-OH)-([18] aneN₆)](ClO₄)₃ shows that each Pd(II) ion in the dinuclear complex is tetra-coordinated by three nitrogen atoms and one hydroxo group bridge in a distorted square configuration. The two Pd(II) ions are 3.09 A apart from each other. The dinuclear Pd(II) complex cations [Pd₂(μ-OH)([18]aneN₆)]³⁺ and [Pd ₂(H₂O)(OH)([18]aneN₆)]³⁺ can efficiently catalyze hydrolysis of the amide bond involving the carbonyl group of methionine in methionine-containing peptides with turnover number of larger than 20. In these hydrolytic reactions, the two Pd(II) ions are synergic; one Pd(II) ion anchors to the side chain of methionine and the other one delivers hydroxo group or aqua ligand to carbonyl carbon of methionine, or acts as a Lewis acid to activate the carbonyl group of methionine, resulting in cleavage of Met-X bond. The binding constant of dinuclear Pd(II) complex cations with AcMet-Gly and AcMet were determined by ¹H NMR titration to be 282±2 M^-1 and 366±4 M^-1, respectively. The relatively low binding constants enable the catalytic cycle and the possible catalytic mechanism is proposed. This is the first artificial mimic of metallopeptidases with two metal active centers. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b500210a

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F U L L P A P E R
Synergic effect of two metal centers in catalytic hydrolysis of
methionine-containing peptides promoted by dinuclear
palladium(II) hexaazacyclooctadecane complex†
a,b
a
a
a
a
a
Gaosheng Yang, Ren Miao, Yizhi Li, Jing Hong, Chuenmei Zhao, Zijian Guo and
a
Longgen Zhu*
a
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 8331 4502; Tel: (+86) 25 8359 7066
b
School of Chemistry and Material Science, Anhui Normal University, Wuhu, 241000,
P. R. China
Received 6th January 2005, Accepted 14th March 2005
First published as an Advance Article on the web 31st March 2005
The species obtained by the reaction of [Pd
octadecane) with AgBF have been determined by electrospray ionization mass spectrometry (ESI-MS) to be an
equilibrium mixture of three major types of dinuclear Pd(II) complex cations, [Pd
2
([18]aneN
6
)Cl
2
](ClO
4
)
2
(where [18]aneN is 1,4,7,10,13,16-hexaazacyclo-
6
4
2
+
2
(l-O)([18]aneN
)] , in aqueous solution. The hydroxo-group-bridged one, [Pd
)] , is a dominant species, whose crystal structure has been obtained. The crystal structure of [Pd
)](ClO shows that each Pd(II) ion in the dinuclear complex is tetra-coordinated by three nitrogen atoms
6
)] , [Pd
2
(l-OH)-
(l-OH)-
(l-OH)-
3
+
3+
([18]aneN
([18]aneN
([18]aneN
6
6
6
)] and [Pd
2
(H
2
O)(OH)([18]aneN
6
2
3
+
2
)
4 3
˚
and one hydroxo group bridge in a distorted square configuration. The two Pd(II) ions are 3.09 A apart from each
3
+
3+
other. The dinuclear Pd(II) complex cations [Pd
2
(l-OH)([18]aneN
6
)] and [Pd
2
(H
2
O)(OH)([18]aneN )] can
6
efficiently catalyze hydrolysis of the amide bond involving the carbonyl group of methionine in methionine-
containing peptides with turnover number of larger than 20. In these hydrolytic reactions, the two Pd(II) ions are
synergic; one Pd(II) ion anchors to the side chain of methionine and the other one delivers hydroxo group or aqua
ligand to carbonyl carbon of methionine, or acts as a Lewis acid to activate the carbonyl group of methionine,
resulting in cleavage of Met–X bond. The binding constant of dinuclear Pd(II) complex cations with AcMet–Gly and
1
−1
−1
AcMet were determined by H NMR titration to be 282 ± 2 M and 366 ± 4 M , respectively. The relatively low
binding constants enable the catalytic cycle and the possible catalytic mechanism is proposed. This is the first
artificial mimic of metallopeptidases with two metal active centers.
Introduction
to dinuclear one, in order to obtain new artificial hydrolytic
cleavage reagents for peptides and proteins with high efficiency
and large catalytic turnover number.
The selective hydrolytic cleavage of peptides and proteins is one
of the most important reactions involved in various biological
In this report, the dinuclear complex [Pd
2
([18]aneN
6
)Cl ]-
2
1
processes. Over the past decade, a variety of metal complexes
26
(
ClO
4
)
2
(where [18]aneN
6
is 1,4,7,10,13,16-hexaazacycloocta-
have been used as “inorganic proteases” to cleave the amide
bonds in peptides and proteins.
and platinum(II)
effectively the hydrolysis of peptides and proteins containing
methionine and histidine residues through coordination to the
side chains. According to the reaction mechanism they are
mononuclear model complexes; only one metal ion participates
in binding to substrate and activation of amide bond in the vicin-
ity of the binding site. However, many natural metallopeptidases
have been discovered to contain a dinuclear metal active center.
Bovine lens leucine aminopeptidase contains two zinc ions, 3.0 A
apart from each other. Aeromonas proteolytica aminopeptidase
contains two zinc ions, with a distance of 3.5 A. Methionine
decane) was selected as a model compound. There were two
advantages of this choice. The first was that this dinuclear
2
–9
10–17
Several palladium(II)
1
6–18
complexes have been found to promote
26
complex is well characterized and has high stability, and only
one site of coordination for each Pd(II) center can be replaced by
side chain of methionine or other donor groups in peptides and
proteins. The second was that the distance of two palladium(II)
˚
˚
ions in this complex is 3.04 A, similar to that of 3–5 A found
23
in natural metallopeptidases. It would be expected that the
synergic effect of two palladiums occurs in the activation of
substrate promoted by the dinuclear palladium(II) complex.
Fortunately, we have found that the hydrolysis of methionine-
containing peptides can be catalyzed by the species obtained
19
˚
20
21
˚
from [Pd
2
([18]aneN
6
)Cl
2
](ClO
4
)
2
treated by AgBF . To the best
4
aminopeptidase from E. coli contains two Co(II) ions, with a
of our knowledge, this is the first true dinuclear metal center
model for synergic hydrolysis of peptides.
22
˚
distance of 2.9 A. Both metal ions cooperatively participate
in substrate binding and activation. Various hydrolytic enzymes
for the cleavage of polynucleotides contain multiple metal ions
at their active sites.
found more effective than mononuclear ones in hydrolysis of
19,23,24
Dinuclear model complexes have been
Results and discussion
25
phosphate ester. Therefore, it is necessary to extend the re-
search of artificial metallopeptidases from mononuclear model
Composition of dinuclear Pd(II) complex in solution
An aqueous solution, obtained from [Pd
2
([18]aneN
6
)Cl
2
](ClO
4
)
2
via removal of AgCl formed by adding AgBF
4
at pH 1.2
†
Electronic supplementary information (ESI) available: Variation of
1
(adjusted by HClO ), was detected by electrospray ionization
4
the H NMR chemical shift of the CH
tio of Pd(II) complex to peptide. See http://www.rsc.org/suppdata/
dt/b5/b500210a/
3
S group with molar ra-
mass spectrometry (ESI-MS). As shown in Fig. 1, four major
peaks appear at m/z 244.0, 287.9, 293.9 and 302.9 with
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
D a l t o n T r a n s . , 2 0 0 5 , 1 6 1 3 – 1 6 1 9
1 6 1 3

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Fig. 1 ESI mass spectrum of an aqueous solution obtained from [Pd
AgBF
2
([18]aneN
6
)Cl
2
](ClO
4
)
2
through removal of AgCl formed by adding
2+
2+
4
30 6 2 4 6 2
. (A) High resolution scans of the four major peaks; (B) Isotope distribution patterns for (a) [C12H N OPd ] , (b) [C12H31BF N OPd ] ,
2+ 2+
(
6 5 2 6 6 2
c) [C12H31ClN O Pd ] , (d) [C12H33ClN O Pd ] calculated by the IsoPro 3.0 program.
Scheme 1 The major species observed by ESI-MS.
2
+
doubly positive charges. The spectrum simulations based on
corresponding composition given in Scheme 1 fit to each of the
higher-resolution spectra very well. On the basis of ESI-MS
measurement, three major types of dinuclear Pd(II) complex
N
6
)(CH
3
OH)] , respectively. The last three complexes formed
under ESI-MS experimental conditions.
2
+
3+
Crystal structure of [Pd
2
(l-OH)([18]aneN
6
4 3
)](ClO )
cations, [Pd
Pd (H O)(OH)([18]aneN
in equilibrium, as shown in Scheme 2. It is worth noting that the
2
(l-O)([18]aneN
6
)] , [Pd
2
(l-OH)([18]aneN
6
)] and
3
+
[
2
2
6
)] , exist in aqueous solution and are
To gain further insight into the structure of the species formed in
solution, the aqueous solution prepared at the same condition
of ESI-MS measurements was crystalized at 4 C, the single
2
+
◦
existence of [Pd
2
(l-O)([18]aneN
6
)] may be favored under
the ESI-MS conditions over that in aqueous solution, since
charge reduction of the multiply charged ion is a common phe-
crystals of [Pd (l-OH)([18]aneN )](ClO ) were obtained and
confirmed by X-ray crystallographic analysis. The structure of
2
6
4 3
27
nomenon in ESI-MS. Judging by the relative ESI-MS intensi-
ties, the dominant species is the hydroxo-group-bridged one, the
crystal structure of which has been obtained. The other minor
peaks appeared at m/z 252.9, 260.3, 268.9, 276.1 are attributed to
[Pd (l-OH)([18]aneN )](ClO ) consists of a dinuclear cation of
2
6
4 3
3
+
[Pd (l-OH)([18]aneN )] and three ordered counterpart anions
2
6
of perchlorate. Fig. 2 shows a drawing of the molecular structure
of the cationic complex. The selected bond lengths and bond
angles and crystallographic data are listed in Table 1. Each
palladium ion in the dinuclear complex is tetra-coordinated by
2
+
2+
[
(
Pd
OH)(OCH
2
(OH)
2
([18]aneN
3
6
)] , [Pd
2
(OH)(OCH
3
)([18]aneN
6
)] , [Pd
2
-
2
+
)([18]aneN
6
)(H O)] , [Pd
2
2
(OH)(OCH
3
)([18]ane-
1
6 1 4
D a l t o n T r a n s . , 2 0 0 5 , 1 6 1 3 – 1 6 1 9

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Scheme 2 An equilibrium among the dinuclear Pd(II) complex cations in aqueous solution.
˚
Pd–N distance of 1.996 to 2.094 A in the cationic complex
˚
are comparable to that of 1.98–2.174 A in [Pd
2
([18]aneN
([20]aneN
6
)-
)-
26
29
Cl
Br
Pd–O distances are also comparable to those in other hydroxo-
group-bridged dinuclear Pd(II) complexes, such as (NBu
2
](ClO
4
)
2
2
,
[Pd
2
([18]aneN
6
)Br
2
]Br
8
2
(H
2
O)
4
,
[Pd
2
6
2
9
30
2
]Br (H
2
O), and [Pd ([24]aneN
2
)](ClO
4
)
4
complexes. The
4
)
2
-
31
[
(
(C
PMe
6
F
3
5
)
2
Pd(l-OH)
2
Pd(C
6
F
5
)
2
]
and [(Me
3
P)
2
Pd(l-OH)
2
Pd-
3
2
)
2
](CF
3
SO ) .
3
2
It is noted that the structure of [Pd
2
(l-OH)([18]aneN
6
4 3
)](ClO )
determined corresponds exactly to the predominant species
observed in ESI-MS measurement in the same preparation
Fig. 2 Drawing of the dinuclear cation of [Pd
6
2
(l-OH)([18]ane-
conditions of pH 1.2. On the basis of the pK value of 3.0
a
N
)](ClO with 30% thermal displacement ellipsoids. All hydrogen
4 3
)
33
for mononuclear Pd(II) aqua complexes, in our experimental
condition of pH ∼ 1, the predominant species would be
aqua-ligand-coordinated, instead of hydroxo-group-bridged or
coordinated. However, in fact, all species observed by ESI-MS
measurements and crystallographic analysis showed hydroxo
bridges or oxygen bridges or hydroxo coordination. This means
that the dinuclear Pd(II) complex has a strong tendency towards
formation of a hydroxo species, even in more acidic solution.
The hydrolytic reaction is very common for the hydrated metal
ions with high oxidation state. Therefore, it is reasonable to
believe that the formation of a hydroxo species of the dinuclear
Pd(II) complex occurring in more acidic solution is probably
due to the high positive charges of the cationic complex. These
hydroxylated species observed are realistic ones present in the
solution.
atoms and three ordered perchlorate anions are omitted for clarity.
◦
Table 1 Selected bond lengths/ A˚ and angles/ for [Pd
(l-OH)([18]-
2
a
6 4 3
aneN )](ClO )
Pd1–O1
Pd1–N1
Pd1–N2
Pd1–N3
O1–Pd1–N1
O1–Pd1–N2
O1–Pd1–N3
N1–Pd1–N2
N1–Pd1–N3
N2–Pd1–N3
2.087(6)
2.085(9)
1.996(8)
2.014(9)
99.0(3)
177.3(3)
91.8(3)
83.7(4)
169.0(4)
85.5(4)
Pd2–O1
Pd2–N4
Pd2–N5
Pd2–N6
O1–Pd2–N4
O1–Pd2–N5
O1–Pd2–N6
N4–Pd2–N5
N4–Pd2–N6
N5–Pd2–N6
Pd1–O1–Pd2
2.076(6)
2.094(9)
2.002(8)
2.041(9)
96.0(3)
177.7(3)
93.3(3)
86.2(3)
170.2(3)
84.6(4)
95.9(3)
a
C
12
H
31Cl
3
N
6
O
13Pd
2
, M = 786.58, crystal system = orthorhombic,
(no. 19), a = 9.5368(7) A˚ , b = 13.202(1) A˚ ,
Interaction of peptides with dinuclear Pd(II) complex cations
space group = P2
1
2
1
2
1
c = 19.890(2) A˚ , U = 2504.2(3) A˚ , T = 293(2) K, Z = 4, l(Mo Ka) =
3
A solution of dinuclear Pd(II) complex obtained by treatment
−
1
1
(
0
.8 mm , No. of reflections collected = 13483, No. of unique data
int) = 2791 (0.051). The final R indices [I > 2r(I)]: R = 0.0472; wR
.1195, R indices (all data): R = 0.1244.
= 0.0608; wR
of AgBF
Cl ](ClO
4
for removal of chloride ions from [Pd
2
([18]aneN )-
6
R
1
2
=
2
4 2
)
was mixed with methionine-containing peptide in a
1
2
molar ratio of 1:1 at pH 1.1 and room temperature for 10 min,
and then measured by ESI-MS. When AcMet–Gly was taken
as an example, besides the peaks at m/z 244.0, 287.9 and 293.9
present in Fig. 1, a new peak at m/z 367.9 separated by 0.5 m/z
appeared and was assigned to [Pd ([18]aneN )(OH)(AcMet–
three nitrogen atoms and one hydroxo group bridge in a distorted
square configuration. The two palladium ions are 3.091(1) A
apart from each other. The angle between the coordination
˚
2
6
2
+
planes of Pd1 (containing Pd1, N1, N2, N3, O1) and Pd2
Gly)–H] based on a high-resolution scan and simulation of
the isotope distribution pattern. The species, as is shown in
Fig. 3, was formed by interaction of AcMet–Gly either with
◦
(
containing Pd2, N4, N5, N6, O1) is 85.7(2) . The conformation
of the cationic complex is twist-boat-like.
3
+
Hydroxide-ion-bridged dinuclear Pd(II) complexes have been
[Pd (l-OH)([18]aneN )] via attack of AcMet–Gly to one of the
2
6
28
known for many years. However, the dinuclear Pd(II) com-
plex of polyazacycloalkane with a bridging hydroxo group
is reported here for the first time. As Table 1 shows, the
two Pd(II) centers, followed by breaking the Pd–OH bond, or
2+
with [Pd (H O)(OH)([18]aneN )] via substitution of the aqua
2
2
6
ligand.
2+
Fig. 3 Assignment for ESI-MS peak at m/z 367.9. (A) High-resolution scan; (B) Isotope distribution patterns for [C21
by the IsoPro 3.0 program.
H
46
N
8
O
5
SPd
2
]
calculated
D a l t o n T r a n s . , 2 0 0 5 , 1 6 1 3 – 1 6 1 9
1 6 1 5

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Attachment of peptides to the dinuclear Pd(II) complex
cations was also observed in H NMR spectra. The initial
coordination of Pd(II) to side chain of methionine-containing
peptides was accompanied by migration of chemical shift of the
Table 2 Catalytic hydrolysis of the Met–X bond in AcMet–X, pro-
1
moted by dinuclear Pd(II) complex cations at pH* ∼ 1.0, and 50 ±
◦
0
.5 C
3
−1
Substrate
Molar ratio, promoter : substrate
1 : 1
10 kobs/h
CH
electronic supplementary information (ESI)†). Furthermore, as
shown in Table S1, the chemical shift of CH S varies with the
molar ratio of the complex to substrate. In H NMR spectrum,
only one signal of the CH S appeared in the range of molar
3
S towards downfield, as summarized in Table S1 (in the
AcMet–Gly
74.9 ± 0.5
65.0 ± 0.5
51.9 ± 0.4
31.7 ± 0.2
17.3 ± 0.1
0.82 ± 0.01
38.1 ± 0.2
33.1 ± 0.2
25.1 ± 0.1
15.9 ± 0.1
3
0
.5 : 1
1
0.2 : 1
0.1 : 1
.05 : 1
3
0
ratio varied indicates a rapid exchange in the NMR time scale
between the free and binding AcMet-X at room temperature. A
detail NMR titration for AcMet–Gly was made, and the result
shown in Fig. 4 confirmed again that AcMet–Gly is coordinated
to the dinuclear Pd(II) complex in 1:1 stoichiometry. According
to the equation in ref. 34 the binding constant, K, was calculated
a
0
: 1
AcMet–Ala
1 : 1
.5 : 1
.2 : 1
0
0
0.1 : 1
0.05 : 1
1 : 1
a
9.2 ± 0.1
−
1
AcMet–Ala–Ser
AcMet–Pro
35.1 ± 0.3
32.6 ± 0.2
23.4 ± 0.2
14.5 ± 0.1
to be 282 ± 2 M , which is relatively low.
0
0
0
.5 : 1
.2 : 1
.1 : 1
a
0
.05 : 1
1 : 1
6.3 ± 0.1
37.0 ± 0.2
32.9 ± 0.3
24.1 ± 0.2
15.0 ± 0.1
0
0
0
.5 : 1
.2 : 1
.1 : 1
a
0
.05 : 1
7.6 ± 0.1
a
Followed for less than three half-lives.
Table 3 Effect of pH on hydrolysis of Met–Gly bond in AcMet–Gly,
◦
promoted by dinuclear Pd(II) complex cations, at 50 ± 0.5 C
3
−1
Molar ratio, promoter : substrate
pH*
10 kobs/h
1
1
1
: 1
: 1
: 1
2.01
1.65
0.98
23.7 ± 0.2
34.6 ± 0.3
74.9 ± 0.5
oligomerization of mononuclear Pd(II)-aqua complexes, caused
11
by hydroxo-bridging. In this study, the acidic solution may
be associated with breaking of hydroxo-bridging in [Pd (l-
)] , favorable for
2
2
Fig. 4 (A) NMR titration curve in D O. (B) Jobs plot.
2
+
3+
O)([18]aneN
6
)] and [Pd
2
(l-OH)([18]aneN
6
binding of substrate to the dinuclear Pd(II) complex.
The effect of temperature on the hydrolytic reaction was also
examined with the substrate AcMet-Gly. The data are given
in Table 4, and the apparent activation enthalpy, DH and
Catalytic hydrolysis of peptides promoted by dinuclear Pd(II)
complex cations
‡
‡
−1
As discussed above, the composition of the dinuclear Pd(II)
complex-anchored by peptide is well characterized (See Fig. 3)
by means of combination of ESI-MS measurement and H
activation entropy, DS are 75.0 ± 3.2 kJ mol and −103.4 ±
−1 −1
9.9 J K mol respectively, calculated according to the Eyring
equation.
1
2
+
NMR titration. The hydrolysis reaction occurred in the complex
As known from previous studies, [Pd(dien)(H
2
O)] and
O)] is inactive for the hydrolysis of amide
bond, due to the absence of extra aqua ligands after its
3
+
1
2+
[
Pd
2
([18]aneN
6
)(OH)(AcMet-Gly)] and was monitored by H
[Pt(dien)(H
2
NMR. The amide bond at carboxyl terminus of methionine was
10–12
12,17
hydrolyzed, as reported previously.
Table 2 gives the observed
binding to substrate.
However, in the complex [Pd ([18]-
2
3
+
rate constants, kobs for different methionine-containing peptides.
As shown in Table 2, the rate constant decreases in some extent
as the molar excess of the dipeptide over the promoter increases.
Although the reaction is not fast enough, it is significant in
comparison with control experiment. At the same experimental
condition, the observed rate constant for dipeptide hydrolysis
in the absence of the dinuclear Pd(II) complex was determined
aneN )(OH)(AcMet–Gly)] , the peptide coordinates to one
6
Pd(II) center, and the other Pd(II) center remains to be coor-
dinated by the hydroxo group or an aqua ligand. Therefore, it is
evident that the amide bond of Met–X in AcMet–X is hydrolyzed
by the two Pd(II) centers.
−
3
−1
to be 0.82 × 10 h , 21 times slower than the lowest one
in the presence of the dinuclear Pd(II) complex. Thereby, the
achievement of the catalysis is important, though the turnover
number of 20 is relatively small.
Table 4 Effect of temperature on hydrolysis of Met–Gly bond in
AcMet–Gly, promoted by dinuclear Pd(II) complex cations, at pH* ∼
1.0
◦
3
−1
The effect of pH was examined with the substrate AcMet–
Gly. As shown in Table 3, the observed rate constant for
hydrolysis of Met–Gly bond increases as the solution is more
acidic. Previous studies reported that the acidic solution can
enhance the hydrolytic rate of amide bond through inhibiting the
Molar ratio, promoter : substrate Temperature/ C 10 kobs/h
1
1
1
:1
:1
:1
40
50
60
27.9 ± 0.1
74.9 ± 0.5
167.5 ± 1.6
1
6 1 6
D a l t o n T r a n s . , 2 0 0 5 , 1 6 1 3 – 1 6 1 9

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Scheme 3 Proposed catalytic cycle for hydrolysis of AcMet–X (herein X = Gly) promoted by dinuclear Pd(II) complex. Three intermediates are
involved in cleavage of Met–Gly bond via internal delivery of a hydroxy (I) or an aqua ligand (II) or Lewis acid activation (III).
The catalytic cycle
crystallization. The crystals obtained has the same structure as
given in Fig. 2, further confirming that the complex of [Pd (l-
2
As mentioned above, AcMet–Gly coordinates to the dinu-
clear Pd(II) complex with a relatively low binding constant,
OH)([18]aneN
6
)](ClO
4
3
) is stable and can be recycle.
and the hydrolytic reaction occurs in the complex [Pd -
2
3
+
(
[18]aneN )(OH)(AcMet–Gly)] with a turnover number of
6
at least 20. This means that these hydrolytic reactions are
efficiently catalyzed by the dinuclear Pd(II) complex. Impor-
tantly, these reactions can be taken as an artificial mimic of
metallopeptidases with two metal active centers, though the
hydrolytic reactions promoted by the model complex are not fast
enough at the moment. A possible catalytic cycle is proposed in
Conclusion
In this report, a well defined dinuclear Pd(II) complex of
[Pd ([18]aneN )Cl ](ClO ) was chosen as a precursor to mimic
2
6
2
4 2
metallopeptidases with two metal active centers. In contrast to
mononuclear Pd(II) complex of [Pd(dien)(H O)] that is inactive
towards hydrolysis of amide bond in peptides, the hydrolysis
of methionine-containing peptides can be catalyzed by the
2
+
2
3
+
Scheme 3. In the catalytic cycle, both [Pd
2
(l-OH)([18]aneN
6
)]
3
+
and [Pd
2
(H
2
O)(OH)([18]aneN
6
)] are able to be coordinated by
3
+
one AcMet–Gly via side chain of methionine residue to form
intermediate complexes. The cleavage of Met–Gly bond occurs
in three possible ways. A hydroxo group or an aqua ligand that
coordinates to another Pd(II) center attacks the carbonyl carbon
of methionine, as shown in Scheme 3(I) and (II). Lewis acid
activation of Met–Gly bond is also involved in the Scheme 3(III).
The release of AcMet from the Pd(II) center is the key step for
completing the catalytic cycle. The binding constant of AcMet
to the dinuclear Pd(II) complex was also determined to be 366 ±
dinuclear Pd(II) complex cations of [Pd (l-OH)([18]aneN )]
2
6
3+
and [Pd (H O)(OH)([18]aneN )] with turnover number of at
2
2
6
3
+
least 20. In the complex of [Pd (OH)([18]aneN )(AcMet-X)] ,
2
6
the amide bond at carboxyl terminus of methionine is cleaved
via synergism of two metal centers and internal delivery or Lewis
acid activation mechanism or both. The low binding ability
of substrates and leaving products to one Pd(II) center of the
dinuclear Pd(II) complex makes the catalytic cycle proceeding
smoothly. Although the catalytic reaction is not fast enough
currently, this finding is promising and provides a clue in the
pursuit of the final goal of artificial metallopeptidases with two
metal active centers.
−
1
4
M . Again, the relatively low binding constant makes the
catalytic cycle possible. After completing the hydrolytic reaction
1
◦
monitored by H NMR, the NMR tube was kept at 4 C for
D a l t o n T r a n s . , 2 0 0 5 , 1 6 1 3 – 1 6 1 9
1 6 1 7

View Article Online
Experimental procedures
CCDC reference number 253854. See http://www.rsc.org/
suppdata/dt/b5/b500210a/ for crystallographic data in CIF
or other electronic format.
Chemicals
Double distilled water was used for preparation of solu-
tions. The D
2
O, anhydrous AgBF
4
, and K
2
[PdCl
4
] were ob-
Kinetics of hydrolysis
tained from Aldrich. 1,4,7,10,13,16-Hexaazacyclooctadecane
(
[18]aneN
6
) was obtained from Fluka Chemie. Methionyl glycine
The concentration of the freshly prepared aqueous solution
of chloride-free dinuclear palladium(II) complex was 20.0 mM
at pH* ca. 1.2. A variable amount of above solution of
palladium(II) complex, 120 lL AcMet–X (75.0 mM, X is Gly,
Ala, Ala–Ser and Pro) and 10.0 lL DSS (100 mM), all as
(
Met–Gly), methionyl alanine (Met–Ala), and methionyl alanyl
serine (Met–Ala–Ser) were obtained from Sigma. The pep-
tides used were acetylated based on a published procedure.
Acetyl methionyl proline (AcMet–Pro) was synthesized by
16
the GL Biochem (Shanghai) Ltd, and its purity was checked
D
2
O solutions, were mixed in an NMR tube. The pH* value
1
by HPLC. H NMR spectrum of the AcMet–Pro in D
2
O
was adjusted with 1.5 M HClO in D O at the beginning,
4
2
showed the following principal d values: 3.92–3.81 and 3.78–
.68 (m, CH N of Pro), 2.12 (s, CH S), 2.01 (s, CH CO).
The complex [Pd ([18]aneN )Cl ](ClO was prepared by a
published procedure, the chloride ions in which were re-
moved by the following procedure. To a solution of 15.0 mg
and was measured again at the end of reaction, the difference
was less than 0.10 and the final value was reported. The
3
2
3
3
total volume was adjusted with D O to 600 lL. The final
2
2
6
2
4
)
2
26
1
concentration of peptide was 15.0 mM. Acquisition of H NMR
spectra began as soon as possible, and 32 scans were taken
at each time. The sample temperatures were kept at 40, 50,
(
0.02 mmol) [Pd
added 0.060 mL of 1.50 M HClO
AgBF . Water was then added to adjust the total volume up
2
([18]aneN
6
)Cl
2
](ClO
4
)
2
in 0.70 mL water were
◦
and 0.100 mL of 0.400 M
or 60 ± 0.5 C. Resonances of the following groups allow
4
clearly monitoring of peptides and hydrolysis products: CH
2
4
to 1.00 mL. This mixture solution was stirred for 12 h at
of Gly, CH
3
of Ala, CH
2
N of Pro and CH of Ala–Ser. The
3
◦
35
C. A white precipitate of AgCl formed was removed by
concentrations of the peptides and of the hydrolysis products
were determined, with an estimated error of ± 5%, from the
known initial concentrations of peptide and from the integrated
resonances of the leaving group and the DSS internal standard.
The hydrolysis products were identified by addition of pure Gly,
Ala, Ala–Ser or Pro to the corresponding reaction mixture,
centrifugation. All the above procedures were performed in
1
the dark. The pH value of the resulting solution was 1.2. H
NMR spectrum of the species prepared in D
following principal d values: 3.66–3.49(m), 3.41–3.19(m) and
.13–2.52(m). The species in H O or D O were freshly prepared
2
O showed the
3
2
2
1
prior to use.
only enhancing the H NMR signals of hydrolysis products,
without new signal appeared. First-order logarithmic plots of
substrate concentration or hydrolysis product concentration
versus time were linear for three half-lives. A few slow reactions
were monitored for less than three half-lives. A typical kinetic
plot was consisted of 10–18 points, and correlation coefficients
were higher than 0.99.
Measurements
1
Routine H NMR spectra in D
2
O with sodium 4,4-dimethyl-
4
-silapentane-1-sulfonate (DSS) as an internal reference, were
recorded with Bruker Avance 300 NMR spectrometers. pH
was measured with an Orion 901 instrument and a Phoenix
Ag–AgCl reference electrode. The uncorrected values in D O
2
Acknowledgements
were designated pH*. Electrospray ionization mass spectra were
recorded using an LCQ electrospray mass spectrometer (ESI-
MS, Finnigan) by loading 1.0 lL solution into the injection
valve of the LCQ unit and then injecting into the mobile phase
solution (50% of aqueous methanol), which was carried through
the electrospray interface into the mass analyzer at a rate of
This work was funded by the National Natural Science Foun-
dation of China (Nos. 20271027 and 20231010). We thank
Yongjiang Liu for the X-ray data collection, and Yuhua Mei
for the ESI-MS analysis.
−
1
2
00 lL min . The voltage employed at the electrospray needles
◦
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D a l t o n T r a n s . , 2 0 0 5 , 1 6 1 3 – 1 6 1 9

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34 a/(dobs − d
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D a l t o n T r a n s . , 2 0 0 5 , 1 6 1 3 – 1 6 1 9
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