Active species for Ce(IV)-induced hydrolysis of phosphodiester linkage in cAMP and DNA

Article abstract of DOI:10.1080/15257770600684209

The hydrolysis of cyclic adenosine 3′,5′-monophosphate and 2′-deoxythymidylyl(3′-5′)2′-deoxythymidine by Ce(NH ₄)₂(NO₃)₆ was kinetically studied. The rate of hydrolysis was fairly proportional to the concentration of [Ce ₂^IV (OH)₄]⁴⁺, showing that this is the catalytically active species. According to quantum-chemical calculation, the two Ce(IV) ions in this [Ce₂^IV (OH)₄] ⁴⁺ cluster are bridged by two OH residues. Upon the complex formation with H₂PO₄^- (a model compound for the phosphodiesters), these two Ce(IV) ions bind the two oxygen atoms of the substrate and enhance the electrophilicity of the phosphorus atom. The catalytic mechanism of Ce(IV)-induced hydrolysis of phosphodiesters has been proposed on the basis these results. Copyright Taylor & Francis Group, LLC.

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Active Species for Ce(IV)-Induced
Hydrolysis of Phosphodiester Linkage in
cAMP AND DNA
Jun Sumaoka a , Kenichiro Furuki a , Yuki Kojima a , Masahiko Shibata
b , Kimihiko Hirao b , Naoya Takeda c & Makoto Komiyama a
a Research Center for Advanced Science and Technology , The
University of Tokyo , Komaba, Tokyo, Japan
b Department of Applied Chemistry , Graduate School of
Engineering, The University of Tokyo , Hongo, Tokyo, Japan
c Department of Chemistry and Biotechnology , Graduate School of
Engineering, The University of Tokyo , Hongo, Tokyo, Japan
Published online: 07 Feb 2007.
To cite this article: Jun Sumaoka , Kenichiro Furuki , Yuki Kojima , Masahiko Shibata , Kimihiko
Hirao , Naoya Takeda & Makoto Komiyama (2006) Active Species for Ce(IV)-Induced Hydrolysis of
Phosphodiester Linkage in cAMP AND DNA, Nucleosides, Nucleotides and Nucleic Acids, 25:4-6,
523-538, DOI: 10.1080/15257770600684209
To link to this article: http://dx.doi.org/10.1080/15257770600684209
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Nucleosides, Nucleotides, and Nucleic Acids, 25:523–538, 2006
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770600684209
ACTIVE SPECIES FOR Ce(IV)-INDUCED HYDROLYSIS OF
PHOSPHODIESTER LINKAGE IN cAMP AND DNA
Jun Sumaoka, Kenichiro Furuki, and Yuki Kojima
Research Center
2
for Advanced Science and Technology, The University of Tokyo, Komaba,
Tokyo, Japan
Masahiko Shibata and Kimihiko Hirao
Department of Applied
2
Chemistry, Graduate School of Engineering, The University of Tokyo, Hongo,
Tokyo, Japan
Naoya Takeda
Department of Chemistry and Biotechnology, Graduate School
2
of Engineering, The University of Tokyo, Hongo, Tokyo, Japan
Makoto Komiyama Research Center for Advanced Science and Technology,
2
The University of Tokyo, Komaba, Tokyo, Japan
The hydrolysis of cyclic adenosine 3 ꢁ,5 ꢁ-monophosphate and 2 ꢁ-deoxythymidylyl(3 ꢁ-5 ꢁ)2 ꢁ-
2
deoxythymidine by Ce(NH4)2(NO3)6 was kinetically studied. The rate of hydrolysis was fairly pro-
portional to the concentration of [Ce2IV (OH)4]4+, showing that this is the catalytically active species.
According to quantum-chemical calculation, the two Ce(IV) ions in this [Ce2IV (OH)4]4+ cluster are
bridged by two OH residues. Upon the complex formation with H2 PO4− (a model compound for
the phosphodiesters), these two Ce(IV) ions bind the two oxygen atoms of the substrate and enhance
the electrophilicity of the phosphorus atom. The catalytic mechanism of Ce(IV)-induced hydrolysis of
phosphodiesters has been proposed on the basis these results.
Keywords Artificial nuclease; Cerium; DNA cleavage; Enzyme models; Phosphodiester
hydrolysis
Received 29 December 2005; accepted 9 February 2006.
This work was partially supported by the Bio-oriented Technology Research Advancement Institu-
tion. The support by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture,
Sports, Science and Technology, Japan is also acknowledged.
This article is dedicated to Professor Eiko Ohtsuka on the occasion of her 70th birthday.
Current address for Naoya Takeda: Consolidated Research Institute for Advanced Science and Med-
ical Care, Waseda University, Shinjuku-ku, Tokyo 162-0041, Japan.
Address correspondence to Makoto Komiyama, Research Center for Advanced Science and Tech-
nology, The University of Tokyo, Komaba, Tokyo 153-8904, Japan. E-mail: komiyama@mkomi.rcast.
u-tokyo.ac.jp
523

524
J. Sumaoka et al.
INTRODUCTION
Non-enzymatic hydrolysis of biologically important phosphodiesters
such as cyclic adenosine 3ꢁ,5ꢁ-monophosphate (cAMP), DNA, RNA, and
phospholipids has been attracting increasing interest, because of potential
applications to molecular biology, biotechnology, therapy, and others.[1] Su-
perb catalysts for the hydrolysis of RNA have been successfully obtained.[2]
However, other phosphoesters, especially cAMP and DNA, are so stable
that few catalysts can hydrolyze them at reasonable rates under physiolog-
ical conditions (the half-lives of the phosphodiester linkages of cAMP and
DNA in the absence of catalysts are estimated to be 400 thousand[3] and
200 million years,[4] respectively). About 10 years ago, it was found that
Ce(IV) ion is remarkably active for the hydrolysis of them under physio-
logical conditions.[5−10] No concurrent oxidation of cAMP and DNA was
detectable, indicating a strong potential for practical applications. The ac-
celeration accomplished by this metal ion was as large as 1011–1013 fold. Quite
interestingly and importantly, the catalytic activity of Ce(IV) for the hydrol-
ysis of both cAMP and DNA is overwhelmingly greater than those of other
metal ions. Furthermore, both reactions proceed via nucleophillic attack by
external OH−(or its relevant species), which is in contrast with intramolec-
ular attack by 2ꢁ-OH in RNA hydrolysis (transesterification).[5,7] Thus, they
have many features in common. Several spectroscopic studies have been al-
ready made on Ce(IV)/phosphodiester complexes.[11,12] Nevertheless, the
species responsible for the catalysis has not yet been pinned down and the
mechanism of catalysis by Ce(IV) is not completely clear. One of the most
significant obstacles is the fact that the Ce(IV) ions form complicated gel
of metal hydroxide at physiological pH, which has been preventing detailed
analysis of the reactions.
However, we have now found that highly acidic solutions of Ce(IV) are
free from the precipitation of gel and are appropriate for kinetic analysis.
Significantly, these solutions are sufficiently active for the hydrolysis of cAMP
and DNA, and their catalytic activities are similar to those of the metal hy-
droxide gel obtained at neutral pH. Furthermore, the concentrations of all
the Ce(IV)-derived species in these solutions can be calculated by using the
equilibrium constants which are available in the literature.[13] In this arti-
cle, the hydrolysis of cAMP and 2ꢁ-deoxythymidylyl(3ꢁ-5ꢁ)2ꢁ-deoxythymidine
(TpT) in these acidic solutions is kinetically studied in detail. The de-
pendence of hydrolysis rate on the concentration of each of the Ce(IV)-
derived species is analyzed, and the catalytically active species is determined.
The geometric structure and electric properties of this active species, as
well as those of its complex with a phosphodiester, are investigated by
ab initio quantum-chemical calculation. The reaction mechanism for the
Ce(IV)-induced hydrolysis of phosphodiesters is proposed in terms of these
results.

Active Species for Ce(IV)-Induced DNA Hydrolysis
RESULTS AND DISCUSSION
525
Hydrolysis of cAMP and DNA by Ce(NH4)2(NO3)6
in Acidic Solutions
When the pH of Ce(NH4)2(NO3)6 solution was lower than 2.5, there
existed no precipitates of Ce(IV) hydroxide in the solution. The absence
of colloidal particles in these solutions was confirmed by light-scattering
photometry using the instrument which can detect the particles, if any,
˚
of diameter 15 A or larger. The pseudo-first-order rate constant for the
disappearance of cAMP in the presence of Ce(NH4)2(NO3)6 (1 mM ) at
pH 2.0 and 30◦C is 1.5 × 10−3 s−1.[14] This value is smaller than the value
(4.7 × 10−3 s−1) at pH 7.0, but the difference is only 3-fold. The major prod-
uct was adenosine 3ꢁ-monophosphate, exactly as was the case at pH 7.0.[15]
The 3ꢁ-monophosphate, as well as the 5ꢁ-monophosphate, was gradually hy-
drolyzed to adenosine as the final product. No oxidative cleavage of the nu-
cleobase and the ribose was detectable. According to the redox titration de-
scribed in Experimental section, all the Ce(IV) ions in the solutions retained
their tetravalent states throughout the cAMP hydrolysis. No redox reactions
took place in the mixtures. Cyclic 3ꢁ,5ꢁ-monophosphates of guanosine, cy-
tidine, and uridine were also promptly hydrolyzed by Ce(NH4)2(NO3)6 at
pH 2.0.
The pH-rate constant profile for the hydrolysis of the dinucleotide TpT
by Ce(NH4)2(NO3)6 (10 mM) was also flat. The rate constant at pH 2.0 and
50◦C was 1.1 × 10−5 s−1, whereas the value at pH 7.0 was 5.3 × 10−5 s−1. The
product was mostly thymidine (pT and Tp, formed by the hydrolysis of TpT,
were rapidly hydrolyzed and not much accumulated). Thymine and other
oxidation products were not formed under these conditions (only when the
pH was lower than 1.4, thymine was slightly formed due to the oxidation
and thus the present kinetic studies were carried out at pH > 1.4). Essential
features of the hydrolyses of cAMP and TpT by Ce(NH4)2(NO3)6 in acidic
solutions are similar to those at pH 7.[5,7] In the absence of Ce(NH4)2(NO3)6,
neither cAMP nor TpT was hydrolyzed to a detectable extent, as expected
from their stabilities described above.
Kinetic Analysis of the Hydrolysis of cAMP by Ce(NH4)2(NO3)6
The equilibrium constants for the solvolysis of Ce(IV) were previously
reported at 25◦C and [NaClO4] = 3.0 M (see Eqs. (1–7)).[13] Thus the
concentrations of the Ce(IV)-derived species formed in reaction mixtures
([CeIV]4+, [CeIV(OH)]3+, [CeIV(OH)2]2+, [Ce2IV(OH)2]6+, [CeI2V(OH)3]5+,
[Ce2IV(OH)4]4+, and [Ce6IV(OH)12]12+) can be calculated by using Q1,1
,
Q
1,2, Q2,2, Q2,3, Q2,4, and Q6,12 values. Here the Qx,y is defined by
Eq. (1).

526
J. Sumaoka et al.
ꢁ
x CeIV + y H2O ꢀꢀCeIxV(OH)y (4x − y)+ + yH+
ꢀꢀ
ꢁ
(4x − y)+ꢁ
CeIxV(OH)y
[H+]y
Qx,y
=
(1)
[CeIV]x
The present kinetic analysis was achieved under the same conditions where
these Qx,y values were determined.
CeIV + H2O ꢀ [CeIV(OH)]3+ + H+
logQ1,1 = 1.1
logQ1,2 = 0.3
logQ2,2 = 3.6
logQ2,3 = 4.1
logQ2,4 = 3.5
(2)
(3)
(4)
(5)
(6)
ꢀ
ꢁ
CeIV + H2O ꢀ CeIV(OH)2 2+ + 2H+
ꢀ
ꢁ
2CeIV + 2H2O ꢀ Ce2IV(OH)2 6+ + 2H+
ꢀ
ꢁ
2CeIV + 3H2O ꢀ Ce2IV(OH)3 5+ + 3H+
ꢀ
ꢁ
2CeIV + 4H2O ꢀ Ce2IV(OH)4 4+ + 4H+
ꢀ
ꢁ
6CeIV + 12H2O ꢀ CeI6V(OH)12 12+ + 12H+ logQ6,12 = 15.4 (7)
The equilibrium concentrations of these Ce(IV)-derived species un-
der typical reaction conditions (pH 2.0 and [Ce(NH4)2(NO3)6]0 = 1 mM)
are presented in Table 1. The abundance of these species is in
the following order: [CeIV(OH)2]2+ > [Ce2IV(OH)4]4+ ꢂ [CeIV(OH)]3+
>
[CeI2V(OH)3]5+ ꢂ [CeIV(OH)12]12+ ꢂ [CeIV(OH)2]6+ ≈ [CeIV]4+.
The hydrolysis of6cAMP in acidic so2lutions was sufficiently fast and
fair first-order kinetics was always obtained up to high conversion. As
shown by the open circles in Figure 1a, the pseudo-first-order rate con-
stant kobs monotonously increased with increase in the initial concentra-
tion of Ce(NH4)2(NO3)6. Here, the pH was kept constant at 2.0. All the
experimental points fairly fit the solid line (vi), which shows the equilib-
rium concentration of the bimetallic hydroxo-cluster [CeIV(OH)4]4+. The
dotted lines (iv) and (v) referring to the equilibrium c2oncentrations of
TABLE 1 Distribution of CeIV-Derived Species at pH 2.0,
25◦C, and [Ce(NH4)2(NO3)6]0 = 1 mM ([NaClO4] = 3.0 M )
CeIV-derived species
Mole fraction (%)
4+
[CeIV
]
0.0034
4.2
67
0.0035
1.1
28
[CeIV(OH)]3+
[CeIV(OH)2]2+
[CeI2V(OH)2]6+
[CeI2V(OH)3]5+
[CeI2V(OH)4]4+
[CeI6V(OH)12
]
0.1
12+

Active Species for Ce(IV)-Induced DNA Hydrolysis
527
[CeIV(OH)2]6+ and [Ce2IV(OH)3]5+, respectively, have similar shapes as the
line2(vi). These three species are the candidates for the catalytically active
species. On the other hand, the lines (i–iii) for [CeIV]4+, [CeIV(OH)]3+, and
[CeIV(OH)2]2+ were too flat to satisfy the experimental results, and the line
(vii) for [CeI6V(OH)12]12+ was too steep. These four species cannot be the
active species. Note that the shapes of these lines are concretely determined
a priori only by the Qx,y values, pH, and [Ce(NH4)2(NO3)6]0. On the other
hand, the positions of lines are vertically movable by varying the catalytic rate
constants of the corresponding Ce(IV)-derived species.
The pH dependence of the rate constant of hydrolysis is shown by the
open circles in Figure 1b ([Ce(NH4)2(NO3)6]0 is kept constant at 1 mM).
The profile is rather flat from pH 1.4 to 2.1. All the experimental points are
consistent with the shapes of the theoretical lines for the equilibrium concen-
trations of [CeIV(OH)2]2+ (iii), [CeI2V(OH)4]4+ (vi), and [CeI6V(OH)12]12+
(vii). The concentrations of the other four species rapidly decrease with
increasing pH and are inconsistent with the experimental data (the line
(i) and the line (iv) are completely superimposed with each other). Of
the seven Ce(IV)-derived species in the mixtures, only the tetracationic
bimetallic hydroxo-cluster [CeI2V(OH)4]4+ agrees with both of the analyses
in Figures 1a and 1b, which are independent from each other. It is con-
cluded that this species should be responsible for the hydrolysis of cAMP by
Ce(NH4)2(NO3)6.[16]
The assignment of [CeIV(OH)4]4+ to the catalytically active species
for the hydrolysis has been2 further confirmed by Figure 1c. Here, both
[Ce(NH4)2(NO3)6]0 and pH were varied randomly and independently. As
shown by the open circles, the plot of hydrolysis rate vs. the equilibrium con-
centration of [CeI2V(OH)4]4+ fairly fits the solid straight line which passes
through the origin. From its slope, the second-order catalytic rate con-
stant of this dinuclear cluster is evaluated to be 2.7 M −1 −1. However,
s
there is no linear relationship between the hydrolysis rate and the concen-
tration of [CeIV(OH)2]2+ (the closed circles). Although this mononuclear
Ce(IV) species is abundant in the reaction mixtures, it cannot be the active
species.[16] The ternary reaction system involving two of these monomeric
Ce(IV) species (and the substrate) is unfavorable due to notable loss of en-
tropy. This argument is consistent with the following theoretical studies. No
linear relationship was observed between kobs and the concentration of any
other Ce(IV)-derived species.
Kinetic Analysis of the Hydrolysis of TpT by Ce(NH4)2(NO3)6
Similar kinetic analysis was carried out on the hydrolysis of TpT
by Ce(NH4)2(NO3)6 at 25◦C and [NaClO4] = 3.0 M (Figure 2). Only
hydrolytic product (thymidine) was formed (vide ante). In Figure 2a,
[Ce(NH4)2(NO3)6]0 was varied with the pH kept constant, whereas Figure 2b

528
J. Sumaoka et al.
shows the pH dependence of hydrolysis rate ([Ce(NH4)2(NO3)6]0 is kept
constant). In both figures, the experimental points fairly fit the theoretical
line (the solid one) showing the equilibrium concentration of the bimetallic
hydroxo-cluster [CeI2V(OH)4]4+. No other Ce(IV)-derived species satisfy the
experimental results. Consistently, kobs linearly increased with increasing con-
centration of [CeIV(OH)4]4+ when both [Ce(NH4)2(NO3)6]0 and pH were
varied independe2ntly (the open circles in Figure 2c). On the other hand, the
plot of kobs vs. [CeIV(OH)2]2+ never fit a straight line which passes through
the origin (the closed circles). It is strongly indicated that [CeI2V(OH)4]4+ is
also the catalytic species for TpT hydrolysis, as is the case in the hydrolysis of
cAMP.
Quantum-Chemical Studies on the Catalytically Active
[CeI2V(OH)4]4+Cluster and Its Complex with Phosphodiester
According to ab initio calculation, [CeIV(OH)4]4+ is stable when the two
Ce(IV) ions therein are bridged by two hyd2roxide groups. Its optimized struc-
ture is presented in Figure 3a.[17] All of the two Ce(IV) atoms and these two
bridging OH groups (both the oxygen atoms and the hydrogen atoms) are
located in one plane. The whole structure of the cluster has C2 symmetry
with respect to the straight line connecting the two OH groups. The distance
˚
between the two Ce(IV) atoms is 3.848 A. This value is fairly in accord with the
˚
Ce-Ce distance (3.6 A) in the 2:1 Ce(IV) complex of diphenyl phosphate,
which was evaluated by EXAFS study.[11] It is noteworthy that a bimetallic
Ce(IV) cluster of the same composition is quite unstable when there ex-
ists only one OH bridge between the two Ce(IV) atoms and another OH is
bound to either of the two Ce(IV) ions. Upon optimizing the geometry of
this bimetallic cluster by the quantum-chemical calculation, the OH-bridge
was spontaneously cleaved and accordingly the cluster was decomposed to
two [CeIV(OH)2]2+ molecules.
As a model compound of phosphodiesters for the calculation, H2PO−
was employed since the phosphoesters mostly exist as monoanion under th4e
reaction conditions (the pKa value of the phosphodiester linkage of TpT is
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
FIGURE 1 Plots of logarithm of the pseudo-first-order rate constant of cAMP hydrolysis at 25◦C
([NaClO4] = 3.0 M ) vs. (a) the initial concentration of Ce(NH4)2(NO3)6 and (b) the pH. The pH
was kept constant at 2.0 in (a), whereas [Ce(NH4)2(NO3)6]0 was constant at 1 mM in (b). The solid
line (vi) is the theoretical one showing the concentration of the proposed active species [CeI2V(OH)4]4+
(the right-handed ordinate). The dotted lines are the theoretical ones for (i) [Ce]4+; (ii) [CeIV(OH)]3+
;
(iii) [CeIV(OH)2]2+; (iv) [CeI2V(OH)2]6+; (v) [Ce2IV(OH)3]5+, and (vii) [CeI6V(OH)12 12+. All the theo-
]
retical lines were calculated using the equilibrium constants that were determined under the same condi-
tions as the reaction conditions (25◦C and [NaClO4] = 3.0 M).[13] In (c), both [Ce(NH4)2(NO3)6]0 and
pH were varied independently from each other, and the rate constants were plotted vs. the equilibrium
concentrations of [CeI2V(OH)4]4+ (the open circles) and [CeIV(OH)2]2+ (the closed circles).

Active Species for Ce(IV)-Induced DNA Hydrolysis
529

530
J. Sumaoka et al.
FIGURE 2 Plots of logarithm of the pseudo-first-order rate constant of TpT hydrolysis at 25◦C
([NaClO4] = 3.0 M ) vs. (a) the initial concentration of Ce(NH4)2(NO3)6 and (b) the pH. The pH
was kept constant at 2.0 in (a), whereas [Ce(NH4)2(NO3)6]0 was constant at 10 mM in (b). The solid
and the dotted lines are the theoretical lines (see the legend for Figure 1). In (c), the rate constants were
plotted vs. the equilibrium concentrations of [CeI2V(OH)4]4+ (the open circles) and [CeIV(OH)2]2+ (the
closed circles) using the data in (a) and (b).

Active Species for Ce(IV)-Induced DNA Hydrolysis
531
0.4, according to the previous titration by 31P-NMR−).[5a] Figure 3b shows the
optimized structure of the complex between H2PO and the [CeI2V(OH)4]4+
cluster having two bridging OH groups (the structu4re depicted in Figure 3a).
This complex is stable, and the free energy change for its formation, eval-
uated by the theoretical calculation, is −20.31 kcal mol−1. The two oxygen
atoms of H2PO−4 are coordinated to each of the two Ce(IV) atoms, and the
˚
Ce-O distance for both of the coordination is 2.132 A. The charge (+2.57) on
the phosphorus atom in this complex is remarkably greater than the value of
free H2PO− (+1.89), confirming that the electrons on this atom are notably
withdrawn4by the Ce(IV) ions. As the result, the electrophilicity of this reac-
tion center is enhanced. The positive charges of two Ce atoms in the complex
are 2.26 and 2.27. Upon the complex formation, the symmetric structure of
the bimetallic Ce(IV) cluster in Figure 3a is slightly distorted so that the two
Ce(IV) atoms are pushed towards the H2PO−4 and form two coordination
˚
bonds with their two oxygen atoms. However, the Ce-Ce distance (3.727 A)
is almost unchanged during these processes.
FIGURE 3 The optimized structures determined by the ab initio quantum-chemical calculations.
(a) [CeI2V(OH)4]4+ involving two OH bridges, (b) 1:1 complex between H2PO−4 (a model compound of
phosphodiester) and [Ce2IV(OH)4]4+, (c) the ternary complex between H2PO4− and two [CeIV(OH)2]2+
˚
molecules. The bond length and the interatomic distance are in A unit.

532
J. Sumaoka et al.
For the purpose of comparison, the calculation was also carried out on
the 1:2 complex between one H2PO−4 molecule and two [CeIV(OH)2]2+ clus-
ters. In the optimized structure of this ternary system, each of the two oxy-
gen atoms of H2PO−4 is coordinated to either of these two [CeIV(OH)2]2+
˚
molecules (Figure 3c). The Ce-O distances are 2.198 A. The two Ce atoms are
˚
sufficiently far away from each other (the distance is 6.634 A) and thus these
two [CeIV(OH)2]2+ clusters behave as independent species. This 1:2 complex
is quite unstable in comparison with the 1:1 complex between H2PO−4 and the
dinuclear [CeI2V(OH)4]4+ cluster in Figure 3b. The free energy change for
its formation, evaluated by the calculation, is only –11.04 kcal mol−1, which
is far less favorable than the corresponding value (–20.31 kcal mol−1) for the
latter complex. The charge of P atom is +2.50, which is by 0.07 unit smaller
than the value in the complex of the bimetallic cluster [CeIV(OH)4]4+. It is
highly unlikely that this ternary complex is making a signific2ant contribution
to the phosphodiester hydrolysis.
Proposed Mechanism of the Hydrolysis of Phosphodiesters
The present kinetic studies have concluded that [CeI2V(OH)4]4+ is the
active species for the hydrolysis of cAMP and DNA in acidic solutions. Of all
the Ce(IV) ions in the mixtures, only less than half is forming this bimetallic
hydroxo-cluster (see Table 1: [CeIV(OH)2]2+ is the most abundant species).
Even under these conditions, [CeI2V(OH)4]4+ governs the whole reaction.
The catalytic activity of this bimetallic cluster must be sufficiently large,
in comparison with the activities of the other species. Upon the complex
formation of the phosphodiester with this Ce(IV) cluster, its two oxygen
atoms are coordinated to each of the two Ce(IV) ions in the cluster (Figure
3b). This bidentate coordination is consistent with the results of previous
core-level photoelectron spectroscopy[12] on the complex formation between
diphenyl phosphate and Ce(IV). Because of this coordination, the electrons
of phosphodiester are greatly withdrawn by the Ce(IV) cluster. The struc-
ture of [CeI2V(OH)4]4+ is suitable for this complex formatio−n so that the
˚
Ce-Ce distance is little altered on the coordination of H2PO4 (3.848 A →
˚
3.727 A).
On the basis of these results, the mechanism of the phosphodiester hy-
drolysis is proposed as depicted in Figure 4.[18] First, the Ce(IV)-bound hy-
droxide ion attacks the phosphorus atom as the electrophilic center.[19] Since
the phosphodiester residue is strongly activated by its bidentate coordination
to the two Ce(IV) ions, this intramolecular reaction is efficient. The positive
charges in [CeI2V(OH)4]4+ stabilize the negatively charged transition-state
of the hydrolysis. On the breakdown of the pentacoordinated intermediate,
the water bound to the Ce(IV) ions functions as acid catalyst and assists
the removal of leaving group from the phosphorus atom. Notable D2O sol-
vent isotope effect (2.0) observed for this reaction[5a] is probably associated

Active Species for Ce(IV)-Induced DNA Hydrolysis
533
FIGURE 4 Proposed mechanism for the hydrolysis of phosphodiester by bimetallic hydroxo-cluster
[CeI2V(OH)4]4+
.
with the proton-transfer in this process. Consistently, various dinuclear and
trinuclear metal complexes are far more active for phosphoester hydrolysis
than are the corresponding mononuclear complexes.[20,21] Many enzymes
for phosphodiester hydrolysis also involve two or three metal ions at their
active center.[22] The proposed mechanism of catalysis is further supported
by previous core-level photoelectron spectroscopy[12] showing that the 2p
orbital of P atom of diphenyl phosphate in its Ce(IV) complex is far more
stable than those in the complexes of diphenyl phosphate with other metal
ions. Apparently, the Ce(IV) ion withdraws electrons from the phospho-
diester in far greater extent than do the other metal ions, and drastically
enhances the electrophilicity of the P atom.[23] The results of quantum-
chemical calculation in this article provide strong supports for these
arguments.
CONCLUSION
Among various non-enzymatic catalysts hitherto proposed, only Ce(IV)
ion can hydrolyze cAMP and DNA at reasonable rates under physiological
conditions. The bimetallic hydroxo-cluster [CeIV(OH)4]4+ is the catalytically
active species, as shown by the present kinetic2analysis on the hydrolysis in
acidic solutions. Although the concentration of this cluster is not necessar-
ily large, it governs the reaction due to its large catalytic rate constant. It is
proposed that the stable phosphodiester linkages are strongly activated by
the cooperation of two Ce(IV) ions in this cluster. According to quantum-
˚
chemical theoretical studies, the two Ce(IV) ions at around 3.8 A distance
in this dinuclear Ce(IV) cluster bind two non-bridging oxygen atoms of the
phosphodiester linkages, and notably withdraw electrons from this linkage.
The electron-withdrawing activity of Ce(IV) is overwhelmingly greater than
those of the other metal ions. The phosphorus atom in the phosphodi-
ester linkage thus activated is efficiently attacked by the Ce(IV)-bound
hydroxide, resulting in the hydrolytic scission of the linkage. The origin
of remarkable catalysis of Ce(IV) for phosphodiester hydrolysis has been
for the first time quantitatively clarified. When the hydrolyses are carried
out at around pH 7, similar acid-base cooperation should occur in higher

534
J. Sumaoka et al.
aggregates of [CeI2V(OH)4]4+. The results in the present article should be
useful for design of still more effective catalysts for biologically important
phosphodiesters.[24]
EXPERIMENTAL SECTION
Materials
cAMP and TpT were purchased from Sigma. Ce(NH4)2(NO3)6 was ob-
tained from Nacalai. Water was purified with a Millipore purification system
Milli-Q (the specific resistance >18.3 Mꢀ · cm−1) and sterilized immediately
before use. Throughout the experiments, great care was taken to avoid con-
tamination by natural enzymes.
Kinetic Measurements
Typical procedure for the hydrolysis of cAMP and TpT was as follows.
To 1.0 mL of 3.0 M NaClO4 solution, Ce(NH4)2(NO3)6 was added, and
then the pH of mixture was adjusted. The reaction was initiated by adding
10 µl of a stock solution of substrate. The reaction conditions (25◦C and
[NaClO4] = 3.0 M) are the same as those where the equilibrium constants
Qx,y in Eqs. (2)–(7) were determined in ref 13. The initial concentration
of cAMP or TpT was 0.1 mM. At appropriate interval, the reaction mixture
was analyzed by the reversed-phase HPLC (Merck LiChrospher 18(e) ODS
column, 25 cm). The eluent for the analysis of cAMP hydrolysis was 97:3 mix-
ture containing choline chloride as an ion-pair agent, whereas 93:7 water-
acetonitrile mixture was used for TpT hydrolysis. The HPLC peaks were as-
signed by coinjection with authentic samples. The change in pH during the
reactions was less than 0.1 unit. The rate constants for the hydrolysis of cAMP
were determined by pseudo-first-order plots. On the other hand, the hydrol-
ysis of TpT at 25◦C was slow so that the reaction rate was determined from
the initial slope of time-conversion curve. Duplicate or triplicate runs were
made and the differences between the rate constants obtained were within
10%. The absence of contamination by enzymes and others was confirmed
by repeated control experiments.
Light-Scattering Photometry
The aquesous solutions of Ce(NH4)2(NO3)6 (1 mM) at pH 2.0 were an-
alyzed by an Otsuka Electronics dynamic photoscattering FDLS-3000 spec-
trometer. This analyzer can detect, if any, the particles which are greater than
˚
15 A.

Active Species for Ce(IV)-Induced DNA Hydrolysis
535
Determination of the Concentration of Tetravalent Ce Ion
in the Reaction Mixtures
In order to confirm the lack of reduction of Ce(IV) to the trivalent ion
during the hydrolysis of cAMP and TpT, the concentration of Ce(IV) in
the reaction mixtures was directly determined by titration. A given amount
of FeSO4 was added to the mixture, and the resultant solution was back-
titrated with Ce(NH4)4(SO4)4 using o-phenanthroline as an indicator (its
Fe(II) complex is red, while the Fe(III) complex is light purple). At least
duplicate runs were made for each determination. The solutions of FeSO4
and Ce(NH4)4(SO4)4 were prepared immediately before use.
Quantum-Chemical Calculations
The GAUSSIAN 98 program was used,[25] and the geometries of all the
mono- and dinuclear clusters of Ce(IV), free H2PO− and their complexes
were optimized in a gas phase at RHF level. The Gi4bbs free energies were
obtained by the density-functional-theory calculation with B3LYP level. Con-
tribution of the solvation effects was incorporated by the PCM on the rec-
ommended conditions.[26] In the geometry optimization, the basis function
for the P, O, C, and H atoms was 3 − 21 + G∗, while 6 − 31 + G∗ was used in
the Gibbs free energy calculation. The effective core potential methods were
applied to a 46-electron core ([Kr]4d) of the Ce(IV) atom.[27] The 4f, 5s,
and 5p orbitals were included as outer core and calculated by using the basis
set size of DZP.
REFERENCES
1. (a) Copeland, K.D.; Fitzsimons, M.P.; Houser, R.P.; Barton, J.K. DNA hydrolysis and oxidative cleav-
age by metal-binding peptides tethered to rhodium intercalators. Biochemistry 2002, 41, 343–356
and references cited therein. (b) Komiyama, M.; Sumaoka, J.; Kuzuya, A.; Yamamoto, Y. Sequence-
selective artificial ribonucleases. Methods in Enzymology 2001, 341, 455–468. (c) Sreedhara, A.A.;
Cowan, J. Catalytic hydrolysis of DNA by metal ions and complexes. Journal of Biological Inorganic
Chemistry 2001, 6, 337–347. (d) Komiyama, M.; Takeda, N.; Shigekawa, H. Hydrolysis of DNA and
RNA by lanthanide ions: mechanistic studies leading to new applications. Chemical Communications
1999, 1443–1451. (e) Williams, N.H.; Takasaki, B.; Wall, M.; Chin, J. Structure and nuclease activity
of simple dinuclear metal complexes: Quantitative dissection of the role of metal ions. Accounts of
Chemical Research 1999, 32, 485–493. (f) Komiyama, M.; Sumaoka, J. Progress towards synthetic
enzymes for phosphoester hydrolysis. Current Opinion in Chemical Biology 1998, 2, 751–757. (g)
Hegg, E.L.; Burstyn, J.N. Toward the development of metal-based synthetic nucleases and peptidases:
a rationale and progress report in applying the principles of coordination chemistry. Coordination
Chemistry Reviews 1998, 173, 133–165. (h) Trawick, B.N.; Daniher, A.T.; Bashkin, J.K. Inorganic mim-
ics of ribonucleases and ribozymes: From random cleavage to sequence-specific chemistry to catalytic
antisense drugs. Chemical Reviews 1998, 98, 939–960. (i) Oivanen, M.; Kuusela, S.; Lo¨nnberg, H.
Kinetics and mechanisms for the cleavage and isomerization of the phosphodiester bonds of RNA by
Brønsted acids and bases. Chemical Reviews 1998, 98, 961–990. (j) Morrow, J.R.; Iranzo, O. Synthetic
metallonucleases for RNA cleavage. Current Opinion in Chemical Biology 2004, 8, 192–200.
2. Recent reviews: (a) ref. 1b. (b) ref. 1h. (c) ref. 1i. (d) ref. 1j. (e) Perreault, D.M.; Anslyn, E.V. Unifying
the current data on the mechanism of cleavage—Transesterification of RNA. Angewandte Chemie
International Edition 1997, 36, 432–450.

536
J. Sumaoka et al.
3. Chin, J.; Zou, X. Catalytic hydrolysis of cAMP. Canadian Journal of Chemistry 1987, 65, 1882–1884.
4. Chin, J.; Banaszczyk, M.; Jubian, V.; Zou, X. Co(III) complex promoted hydrolysis of phosphate
diesters—comparison in reactivity of rigid cis-diaquotetraazacobalt(III) complexes. Journal of the
American Chemical Society 1989, 111, 186–190.
5. (a) Komiyama,M.; Takeda, N.; Takahashi, Y.; Uchida, H.; Shiiba, T.; Kodama, T.; Yashiro, M. Efficient
and oxygen-independent hydrolysis of single-stranded-dna by cerium(IV) ion. Perkin Transactions
2 1995, 269–274. (b) Komiyama, M.; Shiiba, T.; Kodama, T.; Takeda, N.; Sumaoka, J.; Yashiro, M.
DNA hydrolysis by cerium(IV) does not involve either molecular-oxygen or hydrogen-peroxide.
Chemistry Letters 1994, 1025–1028. (c) Komiyama, M.; Kodama, T.; Takeda, N.; Sumaoka, J.; Shiiba,
T.; Matsumoto, Y.; Yashiro, M. Catalytically active species for CeCl3-induced DNA hydrolysis. Journal
of Biochemistry 1994, 115, 809–810. (d) Takasaki, B.K.; Chin, J. Cleavage of the phosphate diester
backbone of DNA with cerium(III) and molecular-oxygen. Journal of the American Chemical Society
1994, 116, 1121–1122. (e) Ihara, T.; Shimura, H.; Ohmori, K.; Tsuji, H.; Takeuchi, J.; Takagi, M.
Hydrolysis of nucleotides using actinoid metal ion. Chemistry Letters 1996, 687–688. (f) references
cite in ref. 1.
6. Ce(IV) complexes are also active for DNA hydrolysis: (a) Rammo, J.; Hettich, R.; Roigk, A.; Schneider,
H-J. Catalysis of DNA cleavage by lanthanide complexes with nucleophilic or intercalating ligands
and their kinetic characterization. Chemical Communications 1996, 105–107. (b) Hashimoto, S.;
Nakamura, Y. Characterization of lanthanide-mediated DNA cleavage by intercalator-linked hydrox-
amic acids: Comparison with transition systems. Perkin Transactions 1 1996, 2623–2628. (c) Igawa, T.;
Sumaoka, J.; Komiyama, M. Hydrolysis of oligonucleotides by homogeneous Ce(IV)/EDTA complex.
Chemistry Letters 2000, 356–357. (d) Branum, M.E.; Tipton, A.K.; Zhu, S.; Que, L.Jr. Double-strand
hydrolysis of plasmid DNA by dicerium complexes at 37◦C. Journal of the American Chemical Society
2001, 123, 1898–1904. (f) Kovacic, R.T.; Welch, J.T.; Franklin, S. J. Sequence-Selective DNA Cleavage
by a Chimeric Metallopeptide. Journal of the American Chemical Society 2003, 125, 6656–6662.
(g) references cite in ref. 1.
7. Catalysis by Ce(IV) for cAMP hydrolysis: (a) Sumaoka, J.; Yashiro, M.; Komiyama, M. Remarkably
fast hydrolysis of 3ꢁ,5ꢁ-cyclic adenosine-monophosphate by cerium(III) hydroxide cluster. Journal
of the Chemical Society, Chemical Communications 1992, 1707–1708. (b) Sumaoka, J.; Miyama,
S.; Komiyama, M. Enormous acceleration by cerium(IV) for the hydrolysis of nucleoside 3ꢁ,5ꢁ-
cyclic monophosphates at pH 7. Journal of the Chemical Society, Chemical Communications 1994,
1755–1756. (c) Cullis, P.M.; Snip, E. Stereochemical Course of Cerium(IV)-Catalyzed Hydrolysis of
Cyclic Nucleotides. Journal of the American Chemical Society 1999, 121, 6125–6130.
8. Monophosphates are also hydrolyzed by Ce(IV) ion: Miyama, S.; Asanuma, H.; Komiyama, M.
Hydrolysis of phosphomonoesters in nucleotides by cerium(IV) ions. Highly selective hydrolysis
of monoester over diester in concentrated buffers. Perkin Transactions 2 1997, 1685–1688.
9. In place of using Ce(IV) salts, catalytically active species can be formed by oxidizing Ce(III) ions
with molecular oxygen (refs 5b and d). Molecular oxygen is not required for the catalytic process,
and thus the DNA hydrolysis by Ce(IV) salts is efficient even in its absence (ref. 5b).
10. Lanthanide(III) ions hydrolyze the phosphate esters other than cAMP and DNA: (a) Hay, R.W.;
Govan, N. A lanthanum macrocycle catalyzed-hydrolysis of a phosphate trimester. Journal of the
Chemical Society, Chemical Communications 1990, 714–715. (b) Breslow, R.; Zhang, B.L. Cleav-
age of phosphate esters by a cyclodextrin dimer catalyst that binds the substrates together with
La3+ and hydrogen peroxide. Journal of the American Chemical Society 1994, 116, 7893–7894.
(c) Morrow, J.R.; Aures, K.; Epstein, D. Metal-ion promoted attack of an alcohol on a phosphate
diester—modeling the role of metal-ions in RNA self-splicing reactions. Journal of the Chemical So-
ciety, Chemical Communications 1995, 2431–2432. (d) Matsumura, M.; Komiyama, M. Enormously
fast RNA hydrolysis by lanthanide(III) ions under physiological conditions: Eminent candidates
for novel tools of biotechnology. Journal of Biochemistry 1997, 122, 387–394 and references cited
therein.
11. Shigekawa, H.; Ishida, M.; Miyake, K.; Shioda, R.; Iijima, Y.; Imai, T.; Takahashi, H.; Sumaoka, J.;
Komiyama, M. Extended x-ray absorption fine structure study on the cerium(IV)-induced DNA
hydrolysis: Implication to the roles of 4f orbitals in the catalysis. Applied Physics Letters 1999, 74,
460–462.
12. Shigekawa, H.; Ikawa, H.; Yoshizaki, R.; Iijima, Y.; Sumaoka, J.; Komiyama, M. Core level photoelec-
tron spectroscopy on the lanthanide-induced hydrolysis of DNA. Applied Physics Letters 1996, 68,
1433–1435.

Active Species for Ce(IV)-Induced DNA Hydrolysis
537
13. Base, C.F., Jr.; Mesmer, R.E. 1976. The Hydrolysis of Cations, 138–146. John Wiley & Sons, New York.
14. The half-life of cAMP is only 9 min, corresponding to more than 1013-fold acceleration with respect
to non-catalyzed reaction.
15. The ratio of adenosine 3ꢁ-monophosphate to its 5ꢁ-monophosphate was about 10:1. The P-O(5ꢁ)
linkage was preferentially cleaved.
16. These kinetic analyses were performed on the assumption that only one species of Ce(IV) is respon-
sible for the hydrolytic reaction. The possibility that the combination of Ce(IV)-derived species is
active for the reaction can not be completely ruled out. However, all the results presented here point
to the overwhelming contribution of the proposed species to the catalysis.
17. In Figure 3, water molecules are omitted because of clarity.
18. This mechanism is different from that proposed for the catalysis by CeCl3/O2 combination in ref.
5d, although both involve dinuclear Ce(IV) species. In the previously proposed mechanism, the two
Ce(IV) ions in the cluster are bridged by -O-O- group, which is derived from molecular oxygen,
and this group is the nucleophile for the reaction. Thus, molecular oxygen is inevitable for the
catalysis. In the present mechanism, however, the metal-bound hydroxide as the nucleophile comes
from water. Consistently, no molecular oxygen is required for the hydrolysis of cAMP and DNA by
Ce(NH4)2(NO3)6 (see ref. 9).
19. If the reaction were involving nucleophilic attack by external hydroxide ion in the reaction mixtures,
the reaction rate at pH 7 should be as 105 times as large as that at pH 2. Apparently, it is not the case.
20. (a) Yashiro, M.; Ishikubo, A.; Komiyama, M. Preparation and study of dinuclear zinc(II) complex for
the efficient hydrolysis of the phosphodiester linkage in a diribonucleotide. Journal of the Chemical
Society, Chemical Communications 1995, 1793–1794. (b) Young, M.J.; Chin, J. Dinuclear copper(II)
complex that hydrolyzes RNA. Journal of the American Chemical Society 1995, 117, 10577–10578.
(c). Yashiro, M; Ishikubo, A.; Komiyama, M. Efficient and unique cooperation of three zinc(II)
ions in the hydrolysis of diribonucleotides by a trinuclear zinc(II) complex. Chemical Communica-
tions 1997, 83–84. (d) Molenveld, P.; Engbersen, J.F.J.; Reinhoudt, D.N.; Specific RNA dinucleotide
cleavage by a synthetic calix[4]arene-based trinuclear metallo(II)-phosphodiesterase. Angewandte
Chemie International Edition 1999, 38, 3189–3192. (e) Chapman, W.H. Jr.; Breslow, R. Selective hy-
drolysis of phosphate esters, nitrophenyl phosphates and UpU, by dimeric zinc complexes depends
on the spacer length. Journal of the American Chemical Society 1995, 117, 5462–5469. (f) Yamada,
K.; Takahashi, Y.; Yamamura, H.; Araki, S.; Saito, K.; Kawai, M. Phosphodiester bond cleavage medi-
ated by a cyclic β-sheet peptide-based dinuclear zinc(II) complex. Chemical Communications 2000,
1315–1316. (g) Gajda, T.; Kra¨mer, R.; Jancso´, A. Structure, equilibrium and ribonuclease activity
of copper(II) and zinc(II) complexes formed with a dinucleating bis-imidazole ligand. European
Journal of Inorganic Chemistry 2000, 1635–1644. (h) Worm, K.; Chu, F.; Matsumoto, K.; Best, M.D.;
Lynch, V.; Anslyn, E.V. Preorganized bis-zinc phosphodiester cleavage catalysts possessing natural
ligands: A lesson pertinent to bimetallic artificial enzymes. Chemistry—A European Journal 2003,
9, 741–747. (i) Liu, S.; Hamilton, A.D. Rapid and highly base selective RNA cleavage by a dinuclear
Cu(II) complex. Chemical Communications 1999, 587–588. ( j) Iranzo, O.; Elmer, T.; Richard, J.P.;
Morrow, J.R. Cooperativity between metal ions in the cleavage of phosphate diesters and RNA by
dinuclear Zn(II) catalysts. Inorganic Chemistry 2003, 42, 7737–7746. (k) Liu, C.; Wang, M.; Zhang,
T.; Sun, H. DNA hydrolysis promoted by di- and multi-nuclear metal complexes. Coordination Chem-
istry Reviews 2004, 248, 147–168.
21. The RNA hydrolysis by lanthanide(III) ions was also ascribed to the catalysis by bimetallic hydroxo-
clusters: ref. 10d.
22. Stra¨ter, N.; Lipscomb, W.N.; Klabunde, T.; Krebs, B. Two-metal ion catalysis in enzymatic acyl- and
phosphoryl-transfer reactions. Angewandte Chemie International Edition 1996, 35, 2024–2055.
23. The stability of the trivalent state of Ce should be favorable for this electron-removal. Among all the
lanthanide metals, only Ce takes stable tetravalent state in addition to stable trivalent state.
24. Quite recently, site-selective scission of DNA by Ce(IV)/EDTA complex in which monophosphate
groups of oligonucleotide additives recruit the Ce(IV) to the target site was reported (Chen, W.;
Kitamura, Y.; Zhou, J.-M.; Sumaoka, J.; Komiyama, M. Site-selective DNA hydrolysis by combining
Ce(IV)/EDTA with monophosphate-bearing oligonucleotides and enzymatic ligation of the scission
fragments. Journal of the American Chemical Society 2004, 126, 10285–10291). The information
presented here should be useful to understand the mechanism of these artificial restriction enzymes.
25. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Zakrzewski,
V.G.; Montgomery, J.A.; Stratmann, J.C. Burant, S.; Dapprich, J.M.; Millam, A.D.; Daniels, K.N.;

538
J. Sumaoka et al.
Kudin, M.C.; Strain, R.E.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.;
Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.; Petersson, G.A; Ayala, P.Y.; Cui, Q.; Morokuma, K.;
Malick, D.K.; Rabuck, A.D.; Raghavachari, K.; Foresman, J.B.; Cioslowski, J.; Ortiz, J.V.; Baboul, A.G.;
Stefanov, B.B.; Liu, G.; Liashenko, A.; Piskorz, I.; Komaromi, R.; Gomperts, R.L.; Martin, D.J.; Fox, T.;
Keith, M.A.; Al-Laham, P.; Peng, C.Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P.M.W.;
Johnson, B.G.; Chen, W.; Wong, M.W.; Andres, J.L.; Gonzalez, C.; Head-Gordon, M.; Replogle, E.S.;
Pople, J.A. Gaussian 98 (Rev. A.7), Gaussian, Inc., Pittsburgh, PA, 1998.
26. Solvent, water; Model, PCM/UAHF; Icomp = 4; Version, Matrix inversion; Cavity, Pentakisdodec-
ahedra with 60 initial tesserae. Barone, V.; Cossi, M.; Tomasi, J. A new definition of cavities for
the computation of solvation free energies by the polarizable continuum model. The Journal of
Chemical Physics 1997, 107, 3210–3221.
27. Cundari, T.R.; Stevens, W.J. Effective core potential study of transition-metal and lanthanide catalyzed
hydrogen-exchange. The Journal of Chemical Physics 1993, 98, 5555–5565.