Dinuclear Zn2+ complexes in the hydrolysis of the phosphodiester linkage in a diribonucleoside monophosphate diester.

Article abstract of DOI:10.1039/b312301d

Dizinc complexes that were formed from 2:1 mixtures of Zn(NO3)2 and dinucleating ligands TPHP (1), TPmX (2) or TPpX (3) in aqueous solutions efficiently hydrolyzed diribonucleoside monophosphate diesters (NpN) under mild conditions. The dinucleating ligand affected the structure of the aquo-hydroxo-dizinc core, resulting in different characteristics in the catalytic activities towards NpN cleavage. The pH-rate profile of ApA cleavage in the presence of (Zn2+)(2)-1 was sigmoidal, whereas those of (Zn2+)(2)-2 and (Zn2+)(2)-3 were bell-shaped. The pH titration study indicated that (Zn2+)(2)-1 dissociates only one aquo proton (up to pH 12), whereas (Zn2+)(2)-2 dissociates three aquo protons (up to pH 10.7). The observed differences in the pH-rate profile are attributable to the various distributions of the monohydroxo-dizinc species, which are responsible for NpN cleavage. As compared to that using (Zn2+)(2)-1, the NpN cleavage using (Zn2+)(2)-2 showed a greater rate constant, with a higher product ratio of 3'-NMP/2'-NMP. The saturation behaviors of the rate, with regard to the concentration of NpN, were analyzed by Michaelis-Menten type kinetics. Although the binding of (Zn2+)(2)-2 to ApA was weaker than that of (Zn2+)(2)-1, (Zn2+)(2)-2 showed a greater kcat value than (Zn2+)(2)-1, resulting in higher ApA cleavage activity of the former.

Full text of DOI:10.1039/b312301d

View Article Online / Journal Homepage / Table of Contents for this issue
2؉
Dinuclear Zn complexes in the hydrolysis of the phosphodiester
linkage in a diribonucleoside monophosphate diester†
a
b
b
b
Morio Yashiro,* Hideki Kaneiwa, Kenichi Onaka and Makoto Komiyama
a
Department of Applied Chemistry, Faculty of Engineering, Tokyo Polytechnic University,
1583 Iiyama, Atsugi, Kanagawa 243-0297, Japan. E-mail: yashiro@chem.t-kougei.ac.jp;
Fax: ϩ81-46-242-9554; Tel: ϩ81-46-242-9554
Department of Chemistry and Biotechnology, Graduate School of Engineering,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
b
Received 3rd October 2003, Accepted 13th January 2004
First published as an Advance Article on the web 26th January 2004
Dizinc complexes that were formed from 2 : 1 mixtures of Zn(NO ) and dinucleating ligands TPHP (1), TPmX (2)
3
2
or TPpX (3) in aqueous solutions efficiently hydrolyzed diribonucleoside monophosphate diesters (NpN) under
mild conditions. The dinucleating ligand affected the structure of the aquo-hydroxo-dizinc core, resulting in different
characteristics in the catalytic activities towards NpN cleavage. The pH-rate profile of ApA cleavage in the presence
2
ϩ
2ϩ
2ϩ
of (Zn ) -1 was sigmoidal, whereas those of (Zn ) -2 and (Zn ) -3 were bell-shaped. The pH titration study
2
2
2
2ϩ
2ϩ
indicated that (Zn ) -1 dissociates only one aquo proton (up to pH 12), whereas (Zn ) -2 dissociates three aquo
2
2
protons (up to pH 10.7). The observed differences in the pH-rate profile are attributable to the various distributions
2ϩ
of the monohydroxo-dizinc species, which are responsible for NpN cleavage. As compared to that using (Zn ) -1, the
2
2ϩ
NpN cleavage using (Zn ) -2 showed a greater rate constant, with a higher product ratio of 3Ј-NMP/2Ј-NMP. The
2
saturation behaviors of the rate, with regard to the concentration of NpN, were analyzed by Michaelis–Menten type
2
ϩ
2ϩ
2ϩ
kinetics. Although the binding of (Zn ) -2 to ApA was weaker than that of (Zn ) -1, (Zn ) -2 showed a greater k
2
2
2
cat
2ϩ
value than (Zn ) -1, resulting in higher ApA cleavage activity of the former.
2
Introduction
the use of an appropriate linker to join two dipycolylamine
DPA, 4) moieties, which form a fundamental binding unit for
each metal ion. The formation of a stable complex of Zn
(
Recently, non-enzymatic hydrolysis of nucleic acids has
attracted much attention. Since dizinc cores have been found
in the catalytic centers of various phosphatases, it has been
suggested that Zn ions act cooperatively in the enzymatic
reactions. In fact, several synthetic dizinc complexes have
22
2ϩ
1
–14
with the DPA units in an aqueous solution has been shown in
1
5
23
recent studies. By choosing the linker moiety between the two
2
ϩ
DPA units, one can expect to regulate the structure of the dizinc
core, and to elucidate the critical factors for the catalytic prop-
2
–4
been found to hydrolyze ribonucleic acids,
or related
3
erties. Such studies will be useful not only in shedding light on
5,16–18
compounds,
more efficiently than their monozinc analogs.
the mechanism of the metalloenzymes, but also in designing
novel synthetic catalysts. Herein, we present our studies on
several dizinc complexes, and the relationship of their struc-
tures on their abilities to promote RNA cleavage, specifically
We have previously reported that a dizinc complex with a
tetrapyridyl ligand 1 (Fig. 1) can hydrolyze a diribonucleoside
monophosphate diester (NpN) under mild conditions. Tetra-
pyridyl ligands have been widely used as dinucleating ligands
for various transition metal ions (for examples, see refs. 18–21).
A basic concept in designing these dinucleating ligands involves
2
6b,d
NpN, which is the smallest unit of an RNA molecule.
Experimental
Materials
N,N,NЈ,NЈ-Tetrakis[(2-pyridyl)methyl]-2-hydroxy-1,3-diamino-
19
propane (TPHP, 1), bis{N,N-bis[(2-pyridyl)methyl]amino-
20
methyl}-m-benzene (TPmX, 2) and bis{N,N-bis[(2-pyridyl)-
21
methyl]aminomethyl}-p-benzene (TPpX, 3) were prepared
according to reported procedures. 2,2Ј-Dipicolylamine (DPA,
4
) was purchased from Tokyo Kasei and used without further
purifications.
Phosphodiester hydrolysis
In a typical experiment, a solution (1.0 ml) of ApA (0.1 mM),
Zn(NO ) (5.0 mM), 1 (2.5 mM) and HEPES (50 mM) was
3
2
adjusted to pH 7.0, and heated to 50 ЊC. At every 0.5 h, a 40-µl
aliquot of the reaction mixture was removed, quenched by
adding 10% phosphoric acid (10 µl) and analyzed by HPLC
(column, Merck LiChrospher 100 RP-18(e) ODS; eluent, water/
acetonitrile 92 : 8 v/v mixture (pH 5.0) containing disodium
2
ϩ
Fig. 1 Structures of ligands for Zn complexes used in this study, and
2
ϩ
proposed coordination modes of Zn . Remaining coordination sites of
2ϩ
Ϫ
Zn can be occupied with H O, OH or a phosphoester molecule,
2
if present.
hydrogenphosphate (3 mM) and acetic acid (40 mM); flow rate,
Ϫ1
1
.0 ml min ; detection, 260 nm). The reaction was followed up
1
†
Electronic supplementary information (ESI) available: Fig. S1 – H
to ca. 30% conv. The water/acetonitrile ratio of the eluent for
the HPLC analysis was varied depending on ribonucleotides
studied: 92 : 8 or 93.5 : 6.5 for ApA, 99 : 1 for CpC, 96 : 4 for
NMR spectra of the Zn(NO ) ϩ3 mixtures; Fig. S2 – titration data
3
2
and theoretical curves; Fig. 3S – molecular models of Zn 1H and
2
Ϫ2
Zn 2HϪ1. See http://www.rsc.org/suppdata/dt/b3/b312301d/
2
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 4
D a l t o n T r a n s . , 2 0 0 4 , 6 0 5 – 6 1 0
605

View Article Online
GpG and ApU, 98.5 : 1.5 or 100 : 0 for UpU. Identification and
quantification of the products were carried out by comparing
the retention times and the peak areas, respectively, with those
of standard samples. During the entire procedures, great care
was taken in order to avoid contamination that may cause
enzymatic hydrolysis.
Potentiometric titration
To a magnetically stirred solution (5.00 ml) of 1ؒ4HClO (0.025
4
mmol) and Zn(NO ) (0.050 mmol) in a sealed glass vessel, were
3
2
added 50-µl aliquots of 0.1 M NaOH (Nakalai), using a 2.5 ml
syringe (Hamilton Gastight syringe 1002 RN) equipped with a
repeating dispenser (Hamilton PB600-1). The titration was
conducted up to pH 12.13. Since formation of insoluble species
was not observed during the measurements, all data were
used for the analysis, which was carried out using HYPER-
24
ϩ
Ϫ
Ϫ14.02
QUAD2000. The value of K = [H ][OH ] was 10
. Acid
w
1
dissociation constants of the free ligand were determined by a
separate experiment, and were used in the above analysis.
Fig. 2 H NMR spectra (270 MHz) of the Zn(NO ) ϩ1 system at
3 2
rt (pD 7). The concentration of 1 was fixed at 5 mM, and that of
Zn(NO ) was varied. (a) [Zn(NO ) ] = 0 mM, (b) [Zn(NO ) ] = 2.5 mM,
25
3
2
3
2
3 2
Speciation curves were drawn by using HySS.
(
c) [Zn(NO ) ] = 5 mM, (d) [Zn(NO ) ] = 7.5 mM, (e) [Zn(NO ) ] =
0 mM. The region of pyridyl proton signals (left) and that of other
proton signals (right) are shown separately.
2ϩ
2ϩ
3 2 3 2 3 2
Titrations of (Zn ) -2 and (Zn ) -3 were carried out under
2
2
1
similar conditions, with the exception that the solvent was 13%
EtOH, which was determined to be the optimum composition
in dissolving both the free ligand and the corresponding dizinc
complex. Formation of insoluble species was observed for pH
2ϩ
values higher than pH 10.7 and 10.0, for complexes (Zn ) -2
2
2ϩ
and (Zn ) -3, respectively. Accordingly, the calculations were
2
carried out using data obtained below their respective pH
ϩ
Ϫ
Ϫ13.86
Ϫ13.90
values. The value of K = [H ][OH ] was 10
and 10
, for
w
2ϩ
2ϩ
complexes (Zn ) -2 and (Zn ) -3, respectively.
2
2
Measurements
1
H NMR spectra (270 MHz) were obtained using a JEOL
JNM-EX 270 FT-NMR instrument. The pD of the D O
2
solutions of the complexes was adjusted by using D O solu-
2
tions of NaOD and/or DCl. Sodium 3-(trimethylsilyl)-
propionate-2,2,3,3-d was used as the internal standard. The
4
pH values were measured using a TOA HM-30V potentio-
meter.
1
Fig. 3 H NMR spectra (270 MHz) of the Zn(NO ) ϩ2 system at
3
2
rt (pD 6.5). The concentration of 2 was fixed at 2.5 mM, and that
of Zn(NO
.25 mM, (c) [Zn(NO ) ] = 2.5 mM, (d) [Zn(NO ) ] = 3.75 mM,
(e) [Zn(NO
pyridyl and phenyl proton signals (left) and that of other proton signals
right) are shown separately.
Results and discussion
3 2 3 2 3 2
) was varied. (a) [Zn(NO ) ] = 0 mM, (b) [Zn(NO ) ] =
1
3
2
3 2
Formation of dizinc complexes
) ] = 5.0 mM, (f ) [Zn(NO ) ] = 25.0 mM. The region of
3 2 3 2
Dizinc complexes that were formed in aqueous solutions using
(
2
ϩ
2a,19
Zn and tetrapyridyl ligands, such as TPHP (1),
(
TPmX
2) and TPpX (3), were studied using H NMR spectroscopy
in D O at room temperature. The spectra of the Zn(NO ) ϩ 1
2
0
21
1
The spectra of the Zn(NO ) ϩ 2 system is shown in Fig. 3. In
contrast to the simple pattern of the spectrum for Zn(NO ) :
3
2
2
3
2
3 2
system are shown in Fig. 2. While the concentration of 1 was
2 = 2 (Fig. 3(e)), complex patterns were observed for Zn(NO ) :
3 2
held constant (5.0 mM), the concentration of Zn(NO ) was
2 = 0.5, 1 or 1.5 (Fig. 3(b), (c) or (d), respectively). These results
indicated the formation of intermediary complex(es) at
Zn(NO ) : 2 = 0.5–1.5, and the formation of a dizinc complex at
3
2
varied (0, 2.5, 5, 7.5, 10 mM). Depending on the Zn(NO ) :
3
2
1
ratio, the spectra exhibited two sets of signals (Fig. 2, marked
3
2
with ᭜ and ꢀ), which were distinct from the signals of free 1.
The stepwise appearance of the signals, which followed the
increasing Zn(NO ) : 1 ratio, indicated dissimilar structures of
Zn(NO ) : 2 = 2. Further increases in the ratio (Zn(NO ) :
3
2
3
2
2 = 10, Fig. 3(f )) did not result in any additional changes. The
Zn(NO ) ϩ3 system showed a spectral behavior that was simi-
3
2
3 2
the Zn complexes of 1 have different Zn(NO ) : 1 stoichio-
larly dependent on the Zn(NO ) : ligand ratio, as observed for
3
2
3 2
metries. The species marked with ᭜ is dominant when
the Zn(NO ) ϩ 2 system, indicating that a dizinc complex was
3 2
[
ꢀ
Zn(NO ) ] = 5 mM (Zn(NO ) : 1 = 1, Fig. 2(c)), and that with
formed in a likewise manner when Zn(NO ) : 3 = 2 (see the
3
2
3
2
3 2
is dominant when [Zn(NO ) ] = 10 mM (Zn(NO ) : 1 = 2,
Electronic Supplementary Information, Fig. S1†). It is import-
3
2
3 2
1
Fig. 2(e)). In the case for [Zn(NO ) ] = 2.5 mM (Zn(NO ) :
ant to note that the H NMR spectra after storing the solutions
3
2
3 2
1
= 0.5, Fig. 2(b)), the solution was observed as a mixture of
for 30 days at rt were unchanged, and therefore, all the observed
species are the equilibrium products, and are stable under the
given conditions.
free 1 and the species marked with ᭜. On the other hand, the
solution with [Zn(NO ) ] = 7.5 mM (Zn(NO ) : 1 = 1.5,
3
2
3 2
Fig. 2(d)) resulted in a mixture of the ᭜ and ꢀ species. Appar-
ently, the species ᭜ and ꢀ can be assigned as the monozinc and
dizinc complexes, respectively. It is important to note that each
type of complex exhibited distinct signals, and accordingly,
their formation ratio can be directly determined by comparing
the corresponding peak areas.
Based on our observations, it is reasonable to conclude that
an aqueous solution with a 2 : 1 mixture of Zn(NO ) and 1, 2 or
3 will afford a dizinc complex, almost quantitatively. Accord-
3
2
ingly, we prepared these dizinc complexes in situ for the sub-
2ϩ
sequent studies. These complexes are designated as (Zn ) -1,
2
2ϩ
2ϩ
(Zn ) -2 and (Zn ) -3, respectively.
2
2
6
06
D a l t o n T r a n s . , 2 0 0 4 , 6 0 5 – 6 1 0

View Article Online
Table 1 Pseudo-first-order rate constants for NpN cleavage (0.1 mM)
in the presence of zinc complexes in 50 mM HEPES (pH 7.0) at 50 ЊC
Table 2 Results of ribonucleotides cleavage assisted by dizinc com-
a
plexes in 50 mM HEPES (pH 7.0) at 50 ЊC
Ϫ3 Ϫ1
a
Ϫ3 Ϫ1
b
Complex
NpN
kobs/10
h
3Ј-NMP (%)
Complex
Ribonucleotide
kobs/10
h
3Ј-AMP (%)
2
ϩ
b
b
b
2ϩ
(
Zn )2-1
ApA
GpG
CpC
UpU
ApA
GpG
CpC
UpU
ApA
CpC
UpU
ApA
GpG
CpC
UpU
68 ± 15
54 ± 10
59 ± 7
50 ± 4
50 ± 4
37 ± 1
58 ± 3
87 ± 1
86 ± 1
67 ± 1
78 ± 2
86 ± 1
86 ± 1
83 ± 1
68 ± 2
71 ± 2
64 ± 1
67 ± 1
(Zn )2-1
ApA
ApA(2Ј,5Ј)
ApU
68 ± 15
21 ± 1
13 ± 2
29 ± 5
14 ± 1
2900 ± 400
66 ± 5
185 ± 40
17 ± 2
116 ± 10
95 ± 10
11 ± 2
9500 ± 1000
126 ± 10
50 ± 4
50 ± 3
50 ± 6
53 ± 1
50 ± 1
50 ± 6
57 ± 3
87 ± 1
86 ± 1
86 ± 1
86 ± 1
85 ± 1
87 ± 1
77 ± 1
28 ± 4
ApC
2
ϩ
(
Zn )2-2
185 ± 40
159 ± 45
114 ± 15
43 ± 7
120 ± 45
31 ± 4
21 ± 3
12 ± 2
13 ± 1
9 ± 2
ApC(2Ј,5Ј)
2Ј,3Ј-cAMP
UpA
ApA
ApA(2Ј,5Ј)
ApU
2
ϩ
(Zn )2-2
2
ϩ
(
(
Zn )2-3
ApC
2
ϩ
c
Zn )-4
ApC(2Ј,5Ј)
2Ј,3Ј-cAMP
UpA
5 ± 1
b
a
b
[
ribonucleotide]0 = 0.1 mM, [complex]0 = 2.5 mM. [3Ј-AMP]/
([3Ј-AMP]ϩ[2Ј-AMP]).
a
[
3Ј-NMP]/([3Ј-NMP]ϩ[2Ј-NMP]). [Zn(NO ) ] = 5.0 mM, [1], [2] or
3] = 2.5 mM. [Zn(NO ) ] = 5.0 mM, [4] = 5.0 mM.
3
2
c
[
3 2
to generate 2Ј,3Ј-cAMP and the nucleoside, which is indicated
by very large kobs values for 2Ј,3Ј-cAMP cleavages compared
with those for ApN scissions (Fig. 4).
Hydrolysis of diribonucleoside monophosphate diesters (NpN) by
dizinc complexes
Phosphoester hydrolysis is known to proceed via the
formation of a five-coordination phophorus intermediate,
which has a trigonal bipyramid geometry, and the subsequent
cleavage of the O–P bond, which occurs when the oxygen is in
The hydrolysis reactions of NpN (N = A, U, G or C) (0.1 mM)
were carried out in the presence of excess amounts of dizinc
2ϩ
2ϩ
2ϩ
complexes (Zn ) -1, (Zn ) -2 or (Zn ) -3 (2.5 mM) in 50 mM
2
2
2
HEPES (pH 7.0) at 50 ЊC. For comparison, the reaction of
1d
the apical position. The preferential formation of 3Ј-AMP
over 2Ј-AMP indicates that the scissile O–P bond in the apical
position is mainly the 2Ј–O–P rather than the 3Ј–O–P bond.
Therefore, between the two possible intermediates, in the pres-
2ϩ
monozinc complex (Zn )-4 (5.0 mM) was also included.
Every reaction of NpN, with the exception of GpG, yielded
mixtures of products as expected for the hydrolysis of the
phosphodiester linkage: a ribonucleoside 3Ј-monophosphate
2ϩ
ence (Zn ) -2, the structure with 2Ј–O in the apical and 3Ј–O
2
(
3Ј-NMP), a ribonucleoside 2Ј-monophosphate (2Ј-NMP), a
in the equatorial positions is preferred over the alternate struc-
ribonucleoside 2Ј,3Ј-cyclicmonophosphate (2Ј,3Ј-cNMP) and a
ture with 2Ј–O in the equatorial and 3Ј–O in the apical posi-
1d,2,26
ribonucleoside.
However, in the case of GpG, considerable
2ϩ
tions. This indicated that (Zn ) -2 interacts stereospecifically
2
amounts of uncharacterized products (12–26% of the total
amount of products) were formed in addition to the expected
with 2Ј,3Ј-cAMP to preferentially promote the 2Ј–O–P bond
scission.
3
Ј-GMP, 2Ј-GMP, 2Ј,3Ј-cGMP and guanosine. This is presum-
2ϩ
On the other hand, for the (Zn ) -2-assisted reactions, the
1d
2
ably due to side reactions related to the guanine moiety.
rate constant for ApA cleavage was about twelve-fold that of
The rate constants for NpN cleavage were estimated by the
increase in the total amounts of the products, i.e. 3Ј- and 2Ј-
NMP, 2Ј,3Ј-cNMP and the ribonucleoside. As shown by the
pseudo-first order rate constants and product ratios (summar-
ized in Table 1), NpN cleavage at 50 ЊC (pH 7.0) was effectively
promoted by the presence of the dizinc complexes. Addi-
tionally, the type of the nucleic acid base was also shown to
moderately affect the dizinc complex cleavage reaction. In con-
2ϩ
ApA(2Ј,5Ј) (Table 2). Analogous comparisons for the (Zn ) -1-
2
assisted reactions showed only a three-fold difference. For
ApA(3Ј,5Ј) cleavage, the initial step is the intramolecular attack
of the 2Ј–OH group of the 5Ј-terminal nucleoside moiety to the
phosphorus atom, whereas for ApA(2Ј,5Ј) cleavage, the initial
step involves the attack of the 3Ј–OH group. It is reasonable to
2ϩ
suggest that (Zn ) -2 promotes the former intramolecular
2
attack more efficiently than the latter. In contrast, such selectiv-
2ϩ
trast to the dizinc complexes, the reactions using (Zn )-4 were
2ϩ
ity was not observed with the use of (Zn ) -1. The results indi-
2ϩ
2ϩ
2
slower. Interestingly, reactions using (Zn ) -2 and (Zn ) -3
2
2
2ϩ
cate that the interaction between (Zn ) -2 and NpN possesses
2
2
resulted in the preferential formation of 3Ј-NMP over 2Ј-NMP,
ϩ
higher stereospecificity than that for (Zn ) -1.
2ϩ
2
while the reaction using (Zn ) -1 did not show such selectivity.
2
Although the selectivity in cleavage rates between ApA(3Ј,5Ј)
and ApA(2Ј,5Ј) and the product selectivity between 3Ј-AMP
and 2Ј-AMP should be determined in different step of the reac-
2ϩ
The Zn -assisted hydrolysis of 2Ј,3Ј-cUMP at pH 5.6 was
reported to yield 65% 3Ј-UMP (in the mixture of 3Ј- and 2Ј-
6a
UMP). Obviously, the observed selectivities in this study is
attributable to the effects of the dizinc complex structures.
In order to clarify the factors that affect the rate of hydrolysis
and the ratio of the products (3Ј-NMP and 2Ј-NMP), hydroly-
sis reactions of various nucleotides involving an adenosine
moiety were conducted. As shown in Table 2, for both reactions
2ϩ
tion, both were higher in the reactions using (Zn ) -2 than
2
2ϩ
2ϩ
those using (Zn ) -1, indicating (Zn ) -2 interacts with both
2
2
ApA and 2Ј,3Ј-cAMP in more stereospecific manner than
2
ϩ
(
Zn ) -1.
2
Kinetic analyses
2ϩ
2ϩ
using (Zn ) -1 or (Zn ) -2, the rate constants were highly
2
2
dependent on the ribonucleotides. However, for the reactions
involving ApA, ApA(2Ј,5Ј), ApU, ApC, ApC(2Ј,5Ј) and 2Ј,3Ј-
cAMP, the ratios between products 3Ј-AMP and 2Ј-AMP were
essentially identical for both dizinc complexes. The results
clearly indicated that these reactions proceed via a common
intermediate, i.e. 2Ј,3Ј-cAMP (Fig. 4), and the selectivity
between products 3Ј-AMP and 2Ј-AMP is determined by the
ring opening step of the 2Ј,3Ј-cAMP intermediate for all reac-
In order to investigate the differences in NpN hydrolysis among
the dizinc complexes in more detail, kinetic analyses were con-
ducted. As shown in the pH profiles of the rate constant for the
zinc-complex-assisted ApA cleavage (Fig. 5), significant differ-
ences were observed among the dizinc complexes. In the case of
2ϩ
(Zn ) -1, the rate constant increased with the increase of pH
2
until pH 8, at which point, the rate constant remained steady. In
2ϩ
contrast, the pH dependence of the rate constant for (Zn ) -2
2
1d
tions. On the other hand, the rate-determining step of the
ApN cleavage is the transfer of the phosphoryl group of ApN
showed the bell-shaped pattern, with a maximum at pH 8. For
2ϩ
(Zn ) -3, although the pH profile of the rate constant for ApA
2
D a l t o n T r a n s . , 2 0 0 4 , 6 0 5 – 6 1 0
607

View Article Online
Fig. 4 Reaction scheme for the ApN cleavage.
2
ϩ
2ϩ
2ϩ
Table 3 Acid dissociation constants (Ϫlog K ) and formation con-
The speciation curves for (Zn ) -1, (Zn ) -2 and (Zn ) -3
2 2 2
a
stants (log β) for dizinc complexes
(
using the parameters listed in Table 3) are shown in Fig. 6.
Clearly, the best correlation was observed between the pH-rate
constant profile (as illustrated in Fig. 5) and the concentration
of the monohydroxo species for each complex system. Thus,
marked differences in the pH-rate constant profile can be attri-
buted to the varying concentration of the monohydroxo dizinc
species.
Complex
Ϫlog Ka0
Ϫlog Ka1
Ϫlog Ka2
Ϫlog Ka3
log β
2
ϩ
(
(
(
Zn )2-1
4.8
7.8
8.0
6.6
9.1
8.4
15.0
14.1
13.1
2
ϩ
Zn )2-2
2.5
10.6
9.8
2
ϩ
Zn )2-3
ϩ
ϩ
Ka0 = [Zn LH ] / [Zn L][H ]; K = [Zn LH ][H ] / [Zn L]; K
=
2
1
2
a1
2
Ϫ1
2
a2
ϩ
ϩ
[
Zn LH ][H ] / [Zn LHϪ1]; Ka3 = [Zn LH ][H ] / [Zn LHϪ2];
2 Ϫ2 2 2 Ϫ3 2
2ϩ 2
β = [Zn L] / [Zn ] [L]. L stands for the ligand, 1, 2 or 3.
2
Fig. 5 pH–rate constant profiles for ApA cleavage assisted by dizinc
complexes. [ApA] = 0.1 mM, [complex] = 2.5 mM, [HEPES] = 50 mM,
0
2ϩ
2ϩ
2ϩ
2ϩ
5
0 ЊC. ᭝: (Zn )2-1, ᭺: (Zn )2-2, ϩ: (Zn )2-3, ᭹: (Zn )-4.
2ϩ
cleavage was similar to that for (Zn ) -2, the rate constants for
2
2ϩ
2ϩ
(
Zn ) -3 were lower than those for (Zn ) -2 throughout the
2
2
pH region examined.
To investigate the relationship between the pH dependences
of these reactions and the structures of the aquo-dizinc
complexes in solution, the acid dissociation constants of
aquo ligands were determined by pH titrations. Best fit of
the titration data could be attained by assuming the dizinc
2ϩ
2ϩ
2ϩ
structures for (Zn ) -1, (Zn ) -2 and (Zn ) -3; moreover,
2
2
2
data analyses indicated that the formation of the monozinc
1
species was negligible, as predicted by H NMR studies
described above. The proton dissociation constants (Ϫlog K ),
along with the formation constants (log β), are summarized in
a
2
ϩ
2ϩ
Fig.
6
Speciation curves for (a) (Zn )2-1, (b) (Zn )2-2 and
2
ϩ
Table 3.
(c) (Zn
) -3.
2
2ϩ
Within the observable pH ranges, dizinc complexes (Zn ) -1,
2
2ϩ
2ϩ
(
Zn ) -2 and (Zn ) -3 exhibited two, three and three proton
The relationships between the concentration of NpN and the
initial rate for dizinc-complex-assisted NpN cleavage in 50 mM
HEPES (pH 7.0) at 50 ЊC are shown in Fig. 7. Data were
collected for the concentration range of NpN up to 10 mM,
with 2.5 mM of the dizinc complex. Apparently, all of the
2
2
2ϩ
dissociations, respectively. For (Zn ) -1, one of the two proton
2
dissociations can be attributed to the OH group of 1, and thus
the other proton can be assigned as dissociating from a co-
2ϩ
ordinated water molecule of (Zn ) -1. In contrast, all three
2
2ϩ
2ϩ
proton dissociations of (Zn ) -2 and those of (Zn ) -3 can be
V –[NpN] plots did not show linearity, but exhibited saturation
behaviors. Since Zn complexes are labile for the ligand
2
2
0
2ϩ
assigned to the coordinated water molecules.
6
08
D a l t o n T r a n s . , 2 0 0 4 , 6 0 5 – 6 1 0

View Article Online
Fig. 7 Effect of concentration of NpN on the initial rate for the
dizinc-complex-assisted NpN cleavage. [dizinc complex] = 2.5 mM,
0
2
ϩ
pH 7.0 (50 mM HEPES), 50 ЊC. ᭺: ApA with (Zn )2-1, ᮀ: ApA with
2ϩ
2ϩ
2ϩ
(
Zn )2-2, ᭝: CpC with (Zn )2-2, ×: UpU with (Zn )2-2.
27
exchange in solution, it should be reasonable to analyze pres-
ent reactions assuming that both association and dissociation
of the phosphoester molecule with the dizinc complexes are fast
enough compared to the observed NpN cleavage. In fact, the
2
ϩ
2ϩ
reactions of ApA by (Zn ) -1, ApA by (Zn ) -2 and CpC by
2
2
2ϩ
(
Zn ) -2 could be described using Michaelis–Menten type
kinetics. In contrast, the reaction of UpU by (Zn ) -2 did not
2
2ϩ
2
obey Michaelis–Menten type kinetics, which is comparable to
2ϩ
the observation that Zn –cyclen can bind to a thymine moiety
27c
in a nucleoside. The kinetic parameters obtained using the
Ϫ1
Fig.
8
Proposed structures for the hydroxo species of dizinc
Eadie–Hofstee plot are K = 4.6 mM and kcat = 0.10 h for
2ϩ
2ϩ
2ϩ
m
complexes. (a) (Zn )2-1, (b) (Zn )2-2 (trihydroxo species), (c) (Zn ) -
2ϩ
Ϫ1
2
the ApA cleavage by (Zn ) -1, K = 12 mM and k = 0.70 h
2
m
cat
2 (monohydroxo species).
2ϩ
for the ApA cleavage by (Zn ) -2 and K = 13 mM and kcat
.63 h for the CpC cleavage by (Zn ) -2. The solid lines
=
2
m
2ϩ
Ϫ1
0
2ϩ
2
(
(
Zn ) -2 (Zn 2H ) can bear both acid (aquo) and base
2 2 Ϫ1
hydroxo) catalysts simultaneously. The acid–base co-catalyst
shown in Fig. 7 are theoretical curves that were drawn using
these parameters. These studies show that the binding of
is typically regarded as an important factor for bimetallic
2
ϩ
2ϩ
(
Zn ) -1 to ApA is greater than those of (Zn ) -2 to ApA or
1,3,15
2
2
catalysts in phosphatases.
Thus, the higher activity of
Zn ) -2 than that of (Zn ) -1 can be reasonably explained by
2ϩ
2ϩ
CpC. The higher activity of (Zn ) -2 than that of (Zn ) -1 is
2ϩ
2ϩ
2
2
(
2
2
due to the larger kcat value of the former.
2ϩ
the fact that the monohydroxo species of (Zn ) -2 has acidic
protons, in contrast to that of (Zn ) -1. A plausible mechan-
ism of action of dizinc complexes is depicted in Fig. 9.
2
2ϩ
2
Roles of the dizinc cores in the NpN cleavage
In the phosphoester hydrolysis of RNA, it is generally accepted
that the metal ions can serve to (i) promote the deprotonation
of 2Ј–OH, (ii) stabilize the five-coordination phosphorus
intermediate and (iii) facilitate the removal of 5Ј–O–R from the
1d,2a,6f
intermediate.
The presence of the dizinc complexes should
enhance one or more of these processes. Moreover, the catalytic
properties of the dizinc complexes should be dependent on
their structure. Significant structural differences between
2
ϩ
2ϩ
(
Zn ) -2 and (Zn ) -1 were revealed using potentiometric
2
2
2ϩ
titration analyses. Because (Zn ) -2 showed three proton dis-
2
sociations due to the aquo-hydroxo equilibrium, the dizinc core
should contain at least three water molecules, as shown in
Fig. 8(b). Accordingly, in the monohydroxo species (Zn 2H ),
2
Ϫ1
the dizinc core should bear one hydroxo ligand and at least two
additional aquo ligands, as shown in Fig. 8(c). In sharp con-
2ϩ
trast, (Zn ) -1 showed only one proton dissociation due to the
2
aquo ligand in the measurable pH region, indicating that the
dizinc core bears only one aquo/hydroxo ligand (Fig. 8(a)). In
2ϩ
the monohydroxo species of (Zn ) -1 (Zn 1H ), the hydroxo
2
2
Ϫ2
ligand should bridge the dizinc core; otherwise, an additional
water molecule would coordinate, and accordingly, another
proton dissociation should appear. Our argument suggests that
2
ϩ
2ϩ
all Zn ions exist in a five-coordination geometry in (Zn ) -1,
(
2
2ϩ
2ϩ
Zn ) -2 and (Zn ) -3. The five-coordination geometry of
2 2
2ϩ
Zn has been found in many enzymes and their model
5b,15,16,18b,28
compounds.
Attempts to isolate single crystals of the
present complexes, in order to confirm the structure by the
X-ray crystallography, have not been successful.
As discussed above, the monohydroxo dizinc species is
responsible for NpN cleavage for each dizinc complex. In
2
ϩ
contrast to that of the monohydroxo species of (Zn ) -1
2
Fig. 9 A proposed mechanism of action of the dizinc complexes in
2
ϩ
2ϩ
(
Zn 1H ), the dizinc core of the monohydroxo species of
ApA cleavage. (a) (Zn )2-1, (b) (Zn )2-2.
2
Ϫ2
D a l t o n T r a n s . , 2 0 0 4 , 6 0 5 – 6 1 0
609

View Article Online
2
ϩ
The differences in the dizinc core structure of (Zn ) -1 and
K. Nurmi, E. Yousefi-Salakdeh, R. Strömberg and H. Lönnberg,
J. Chem. Soc., Perkin Trans. 2, 1999, 1619–1625; (g) S. Kuusela
and H. Lönnberg, J. Chem. Soc., Perkin Trans. 2, 1994, 2301–2306;
2
2ϩ
(
Zn ) -2 may also explain the observed dissimilarity in their
NpN hydrolysis characteristics; (Zn ) -2 showed higher
selectivities in the product ratio and higher dependence on the
structure of the nucleotide than those of (Zn ) -1. During the
reactions, (Zn ) -2 interacted with NpN with higher stereo-
specificity than the interactions between (Zn ) -1 and NpN.
The higher stereospecificity can be attributed to fact that the
dizinc core in (Zn ) -2 is less tightly-linked than that in
2
2ϩ
2
(
h) S. Kuusela, A. Azhayev, A. Guzaev and H. Lönnberg, J. Chem.
Soc., Perkin Trans. 2, 1995, 1197–1202; (i) S. Kuusela and
H. Lönnberg, Nucleosides Nucleotides, 1998, 17, 2417–2427;
(j) I. Zagórowska, S. Kuusela and H. Lönnberg, Nucleic Acids Res.,
1998, 26, 3392–3396; (k) U. Kaukinen, L. Bielecki, S. Mikkola,
R. W. Adamiak and H. Lönnberg, J. Chem. Soc., Perkin Trans. 2,
2001, 1024–1031.
2ϩ
2
2ϩ
2
2ϩ
2
2ϩ
2
7
8
9
(a) B. Linkletter and J. Chin, Angew. Chem., Int. Ed. Engl., 1995, 34,
2
ϩ
(
Zn ) -1.
2
4
72–474; (b) M. Wall, B. Linkletter, D. Williams, A.-M. Lebuis,
R. C. Hynes and J. Chin, J. Am. Chem. Soc., 1999, 121, 4710–4711.
(a) T. Itoh, H. Hisada, T. Sumiya, M. Hosono, Y. Usui and Y. Fujii,
Chem. Commun., 1997, 677–678; (b) T. Itoh, H. Hisada, Y. Usui and
Y. Fujii, Inorg. Chim. Acta, 1998, 283, 51–60.
K. A. Deal, G. Park, J. Shao, N. D. Chasteen, M. W. Brechbiel and
R. P. Planalp, Inorg. Chem., 2001, 40, 4176–4182.
Conclusion
2
ϩ
Dizinc complexes formed from 2 : 1 mixtures of Zn ions and
dinucleating ligands, which have two DPA units, possess signifi-
cantly different characteristics in NpN hydrolysis, depending on
the linkage between the two DPA units. When the linkage
moiety was 2-hydroxy-1,3-diaminopropane, the dizinc core is
1
1
0 R. Ren, P. Yang, W. Zheng and Z. Hua, Inorg. Chem., 2000, 39,
454–5463.
5
1 (a) K. M. Deck, T. A. Tseng and J. N. Burstyn, Inorg. Chem., 2002,
41, 669–677; (b) E. L. Hegg, S. H. Mortimore, C. L. Cheung,
J. E. Huyett, D. R. Powell and J. N. Burstyn, Inorg. Chem., 1999, 38,
Ϫ
bridged by one hydroxo ligand and the R–O group of 1. On
the other hand, when the linkage moiety was m- or p-xylene-
diamine, the dizinc core bears at least three aquo/hydroxo
ligands. These differences between the structures of the dizinc
core resulted in greater activity and higher stereoselectivity of
the latter towards NpN hydrolysis. Since the acid–base co-
catalyst is regarded as an important factor for bimetallic
2
961–2968.
1
1
2 (a) L. A. Jenkins, J. K. Bashkin, J. D. Pennock, J. Florián and
A. Warshel, Inorg. Chem., 1999, 38, 3215–3222; (b) W. C. Putnam
and J. K. Bashkin, Chem. Commun., 2000, 767–768.
3 (a) D. M. Epstein, L. L. Chappell, H. Khalili, R. M. Supkowski,
W. DeW. Horrocks, Jr. and J. R. Morrow, Inorg. Chem., 2000, 39,
2130–2134; (b) K. P. McCue and J. R. Morrow, Inorg. Chem., 1999,
38, 6136–6142.
1
,3,15
catalysts in phosphatases,
the use of dizinc complexes,
such as (Zn ) -2 and (Zn ) -3, can be quite interesting and
2ϩ
2ϩ
2
2
1
1
4 A. Roigk, R. Hettich and H.-J. Schneider, Inorg. Chem., 1998, 37,
promising in molecular design of enzyme mimetics.
7
51–756.
5 (a) D. E. Wilcox, Chem. Rev., 1996, 96, 2435–2458; (b) W. N.
Lipscomb and N. Sträter, Chem. Rev., 1996, 96, 2375–2433;
Acknowledgements
(
c) N. Sträter, W. N. Lipscomb, T. Klabunde and B. Krebs,
The authors thank Professor Takeshi Yamamura of Tokyo
University of Science for helpful suggestions on potentiometric
titration experiments, Mr. Akira Ishikubo for measuring H
Angew. Chem., Int. Ed. Engl., 1996, 35, 2024–2055; (d ) J. A. Cowan,
Chem. Rev., 1998, 98, 1067–1087; (e) R. G. Kuimelis and L. W.
McLaughlin, Chem. Rev., 1998, 98, 1027–1044.
1
1
6 P. Molenveld, W. M. G. Stikvoort, H. Kooijman, A. L. Spek,
J. F. J. Engbersen and D. N. Reinhoudt, J. Org. Chem., 1999, 64,
NMR spectra of the Zn(NO ) ϩ 1 mixtures and conducting
3
2
preliminary experiments, and Professor Kazuhiko Saigo. This
work was partially supported by Tokyo Ohka Foundation for
the Promotion of Science and Technology, and by a Grant-in-
Aid for Scientific Research (no. 12650845) from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
3
896–3906.
17 M. T. B. Luiz, B. Szpoganicz, M. Rizzoto, M. G. Basallote and
A. E. Martell, Inorg. Chim. Acta, 1999, 287, 134–141.
8 (a) N. V. Kaminskaia, C. He and S. J. Lippard, Inorg. Chem., 2000,
1
3
1
9, 3365–3373; (b) C. He and S. J. Lippard, J. Am. Chem. Soc., 2000,
22, 184–185.
1
9 (a) M. Suzuki, T. Sugisawa, H. Senda, H. Oshio and A. Uehara,
Chem. Lett., 1989, 1091–1094; (b) M. Suzuki, T. Sugisawa and
A. Uehara, Bull. Chem. Soc. Jpn., 1990, 63, 1115–1120; (c) Y. Dong,
S. Ménage, B. A. Brennan, T. E. Elgren, H. G. Jang, L. L. Pearce and
L. Que, Jr., J. Am. Chem. Soc., 1993, 115, 1851–1859.
20 Y. Gultneh, B. Ahvazi, A. R. Khan, R. J. Butcher and J. P.
Tuchagues, Inorg. Chem., 1995, 34, 3633–3645.
21 M. Yashiro, Bull. Chem. Soc. Jpn., 2002, 75, 1383–1384.
22 (a) D. W. Gruenwedel, Inorg. Chem., 1968, 7, 495–501; (b) J. K.
Romary, J. D. Barger and J. E. Bunds, Inorg. Chem., 1968, 7, 1142–
1145.
23 S. Maruyama, K. Kikuchi, T. Hirano, Y. Urano and T. Nagano,
J. Am. Chem. Soc., 2002, 124, 10650–10651.
24 P. Gans, A. Sabatini and A. Vacca, Talanta, 1996, 43, 1739–1753.
25 L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini and A. Vacca,
Coord. Chem. Rev., 1999, 184, 311–318.
26 (a) M. Yashiro, M. Higuchi, Y. Washizu and M. Komiyama,
Bull. Chem. Soc. Jpn., 2002, 75, 1843–1844; (b) M. Yashiro,
M. Higuchi, M. Komiyama and Y. Ishii, Bull. Chem. Soc. Jpn., 2003,
76, 1813–1817.
27 F. A. Cotton and G. Wilkinson, Advanced Inorganic Chemistry,
A Comprehensive Text, John Wiley & Sons, Inc., New York,
2nd edn., 1966, ch. 5.
References and notes
1
(a) J. Chin, Curr. Opin. Chem. Biol., 1997, 1, 514–521; (b) E. Kimura
and T. Koike, Adv. Inorg. Chem., 1997, 44, 229–261; (c) B. N.
Trawick, A. T. Daniher and J. K. Bashkin, Chem. Rev., 1998, 98,
9
39–960; (d ) M. Oivanen, S. Kuusela and H. Lönnberg, Chem. Rev.,
998, 98, 961–990; (e) N. H. Williams, B. Takasaki, M. Wall
1
and J. Chin, Acc. Chem. Res., 1999, 32, 485–493; ( f ) E. Kimura,
Curr. Opin. Chem. Biol., 2000, 4, 207–213.
2
(a) M. Yashiro, A. Ishikubo and M. Komiyama, J. Chem. Soc.,
Chem. Commun., 1995, 1793–1794; (b) M. Yashiro, A. Ishikubo and
M. Komiyama, Chem. Commun., 1997, 83–84; (c) S. Matsuda,
A. Ishikubo, A. Kuzuya, M. Yashiro and M. Komiyama,
Angew. Chem., Int. Ed., 1998, 37, 3284–3286.
3
4
5
W. H. Chapman, Jr. and R. Breslow, J. Am. Chem. Soc., 1995, 117,
5
462–5469.
S. Mikkola, Q. Wang, Z. Jori, M. Helkearo and H. Lönnberg,
Acta Chem. Scand., 1999, 53, 453–456.
(a) C. Bazzicalupi, A. Bencini, A. Bianchi, V. Fusi, C. Giorgi,
P. Paoletti, B. Valtancoli and D. Zanchi, Inorg. Chem., 1997, 36,
2
784–2790; (b) C. Bazzicalupi, A. Bencini, E. Berni, A. Bianchi,
V. Fedi, V. Fusi, C. Giorgi, P. Paoletti and B. Valtancoli,
Inorg. Chem., 1999, 38, 4115–4122.
(a) S. Kuusela and H. Lönnberg, J. Phys. Org. Chem., 1992, 5, 803–
28 (a) E. Kimura, Y. Kodama, T. Koike and M. Shiro, J. Am.
Chem. Soc., 1995, 117, 8304–8311; (b) T. Koike, M. Inoue,
E. Kimura and M. Shiro, J. Am. Chem. Soc., 1996, 118, 3091–
3099; (c) M. Shionoya, E. Kimura and M. Shiro, J. Am. Chem. Soc.,
1993, 115, 6730–6737; (d ) E. Kimura, S. Aoki, T. Koike and
M. Shiro, J. Am. Chem. Soc., 1997, 119, 3068–3076; (e) T. Koike,
M. Takashige, E. Kimura, H. Fujioka and M. Shiro, Chem. Eur. J.,
1996, 2, 617–623.
6
8
3
11; (b) S. Kuusela and H. Lönnberg, J. Phys. Org. Chem., 1993, 6,
47–356; (c) S. Kuusela, M. Rantanen and H. Lönnberg, J. Chem.
Soc., Perkin Trans. 2, 1995, 2269–2273; (d ) S. Kuusela and
H. Lönnberg, Nucleosides Nucleotides, 1996, 15, 1669–1678;
(
e) S. Kuusela, A. Guzaev and H. Lönnberg, J. Chem. Soc.,
Perkin Trans. 2, 1996, 1895–1899; ( f ) S. Mikkola, E. Stenman,
6
10
D a l t o n T r a n s . , 2 0 0 4 , 6 0 5 – 6 1 0