DNA hydrolysis catalyzed by tris(diisopropyl-1,4,7-triazacyclononanes) ethane metal complexes

Article abstract of DOI:10.1002/jccs.200400176

Novel trinuclear coordinated ligands were designed using three of 1,4,7-triazacyclononane moieties with a tribromo linker. These ligands show greater capability and potency in association with various metal ions. Tris(diisopropyl-1,4,7-triazacyclononanes)ethane (2) associated with Cu ²⁺ and Ce⁴⁺ ions shows better reactivity to achieve DNA hydrolysis at 37 °C at pH 8 for 5 hr. Using high resolution of polyacrylamide gel electrophoresis, DNA cleavage reaction in Tris-HCl buffer causes a nicking site at the single-stranded region. Alternatively, the reaction in HEPES buffer shows a random DNA cleavage included the double-stranded region. Hydrolysis of phosphodiester bond mediated by trinuclear metal complex is capable of performing at the physiological temperature 37 °C, but it has a lower reactivity compared to mono- and di-nuclear metal complexes. The result suggests that trinuclear metal complexes have a bulky environment to prevent from going toward the phosphodiester bond. The design of an efficient chemical hydrolyase will be a challenge in order to construct a coordinate ligand with the enhancement of hydrolytic activity and the reduction of the steric environment.

Full text of DOI:10.1002/jccs.200400176

Journal of the Chinese Chemical Society, 2004, 51, 1201-1208
1201
DNA Hydrolysis Catalyzed by
Tris(diisopropyl-1,4,7-triazacyclononanes)ethane Metal Complexes
+
Chien-Chung Cheng* (
), Yan-Chen Huang (
Department of Chemistry, Tamkang University, Taipei, Taiwan 251, R.O.C.
) and Ming-Chan Liu (
)
Novel trinuclear coordinated ligands were designed using three of 1,4,7-triazacyclononane moieties with
a tribromo linker. These ligands show greater capability and potency in association with various metal ions.
2
+
4+
Tris(diisopropyl-1,4,7-triazacyclononanes)ethane (2) associated with Cu and Ce ions shows better reac-
tivity to achieve DNA hydrolysis at 37 °C at pH 8 for 5 hr. Using high resolution of polyacrylamide gel elec-
trophoresis, DNA cleavage reaction in Tris-HCl buffer causes a nicking site at the single-stranded region. Al-
ternatively, the reaction in HEPES buffer shows a random DNA cleavage included the double-stranded re-
gion. Hydrolysis of phosphodiester bond mediated by trinuclear metal complex is capable of performing at
the physiological temperature 37 °C, but it has a lower reactivity compared to mono- and di-nuclear metal
complexes. The result suggests that trinuclear metal complexes have a bulky environment to prevent from go-
ing toward the phosphodiester bond. The design of an efficient chemical hydrolyase will be a challenge in or-
der to construct a coordinate ligand with the enhancement of hydrolytic activity and the reduction of the steric
environment.
Keywords: DNA hydrolysis; Triazacyclononane; Cerium; Copper; Trinuclear metal complex.
INTRODUCTION
Multi-nuclear metal complexes are arousing more and
nuclear metal complexes are believed to further raise the
hydrolytic capability to cleave DNA. Even though trinuclear
copper complex has the lowest reactivity compared to mono-
and bi-nuclear metal complexes, trinuclear copper com-
more interest, as their mimicking of the biological functions
of metalloenzymes is better understood. For instance, ascor-
2
1
plexes show specific hydrolytic cleavage at a DNA junction
1
2
22
bate oxidase and p-methane monooxygenase are capable of
catalyzing the electron transfer reaction to induce chemical
transformation. Its catalytic site consists of an unsymmetrical
tricopper center resolved by a physical method such as X-ray
crystallography or EPR. In the study of DNA hydrolysis,
metal ions play an important role in cleavage of phospho-
between single- and double-stranded regions.
In our laboratory, novel trinuclear ligands using macro-
cycles were designed and synthesized in order to improve the
reactivity and selectivity in DNA hydrolysis. For instance,
macrocyclic compounds such as 1,4,7-triazacyclononanes
(TACN) and cyclen are known to be excellent ligands in asso-
3
-5
17,23
diester bonds. Metal ion as a Lewis acid stabilizes the tran-
ciation with metal ions to perform DNA hydrolysis.
In
6
,7
sition state conformation as well as metal ion is capable of
generating a metal-hydroxide as a nucleophile to attack the
this study, a novel coordinate ligand was designed based on
TACN moiety to prepare a three-macrocyclic moieties within
a molecule. This configuration of ligand is capable of form-
ing a pre-organized structure for grasping three metal ions as
well as to interact with each arm as shown in Fig. 1. The for-
mation of trinuclear metal complexes with metal ions such as
8
-11
phosphodiester bonds
to assist DNA hydrolysis. A com-
plex with plural metal ions may act as a Lewis acid and a nu-
5
,12-15
cleophile
simultaneously to improve the reactivity and
1
6-18
selectivity in DNA hydrolysis.
The use of a coordinate
2
+
2+
2+
3+
3+
4+
ligand is known to promote the hydrolytic strength and to in-
crease the specific selectivity. Indeed, synthetic bimetallic
complexes were found to enhance the cleavage of dou-
Zn , Co , Cu , La , Tb , and Ce was characterized by
electronic absorption and ESI-mass spectroscopy. The ability
of DNA hydrolysis was evaluated by agarose gel electropho-
resis with supercoiled plasmid DNA. The preference of DNA
1
9,20
ble-stranded DNA at 37 °C and pH 8.
Therefore, multi-
th
Dedicated to Professor Sunney I. Chan on the occasion of his 67 birthday and his retirement from professional life.
*
+
Corresponding author. Fax: +886-2-26209924; e-mail: cccheng@mail.tku.edu.tw
Graduate student, Department of Chemistry, National Taiwan University, Taipei, Taiwan, R.O.C.

1
202 J. Chin. Chem. Soc., Vol. 51, No. 5B, 2004
Cheng et al.
sequences for various metal complexes was examined by
high-resolution polyacrylamide gel electrophoresis at differ-
ent conditions such as buffers, pH, and temperatures. As a re-
plexes were found to increase the rate of DNA hydrolysis in
1
8,21,22
physiological condition but with low specificity.
In or-
der to increase the selectivity and reactivity in DNA hydroly-
sis, a novel ligand containing three TACN were designed and
prepared for this purpose.
4
+
sult, a three-macrocyclic ligand associated with Ce ion pro-
vides better capability in hydrolyzing the phosphodiester
bonds in DNA.
Ligand preparation
TACN was prepared by a modification of Richman and
1
2
RESULTS AND DISCUSSIONS
Atkins’ method. TACN has three secondary amino groups
which are believed to be oxidized easily. Thus, alkylation of
the amino groups of TACN was necessary to prevent the
ligand degradation as well as to avoid aggregation of two lig-
ands with one metal ion. Dialkyl TACN was prepared by se-
lective deprotection of two p-toluenesulfonate groups in
tri-tosylated TACN derivatives using 30% HBr in acetic acid
and phenol for 3 days to obtain a white solid in a 90% yield.
Then, diisopropyl TACN derivative (6, dip-TACN) was pre-
pared using the coupling reaction with isopropyl bromide and
Metal complexes are known to enhance DNA hydroly-
sis by acting as Lewis acids to stabilize the transition state en-
ergy. As well, metal complexes also act as nucleophiles to
generate the metal-hydroxide species to attack DNA phos-
2
4
phodiester linkage. The property of the coordinated ligand
in metal complexes is important to tune the extent of Lewis
8
acids and the strength of metal-hydroxide. Macrocyclic
compounds such as 1,4,7-triazacyclononane associated with
2
+
2+
3+
various metal ions such as Co , Cu , and La are known to
dry Na
2
CO
3
in CH
3
CN for 72 hrs and followed by the detosyl-
SO at 120 °C for 18 hr in a 60%
1
2,13
hydrolyze DNA.
The rigidity of macrocyclic compounds
ation by concentrated H
2
4
provide the pre-organized structure to reduce the entropy in
coordination with metal ions. A ligand containing two TACN
moieties with a linker was designed to possess both a Lewis
acid and a metal-hydroxide to reduce the degree of freedom.
As a result, two TACN moieties linked with an ethylene
group show a dramatic enhancement in DNA hydrolysis. Ac-
cording to the model study of DNA polymerase, it was postu-
lated that the increase of the number of the coordinate ligands
might provide greater binding to phosphodiester bonds to re-
duce the transition state energy. Indeed, trinuclear metal com-
yield as shown in Scheme I. A tris-TACN ligand is achieved
by the coupling reaction of dip-TACN (6) with 1,3,5-trisbro-
3 2 3
momethyl species in a CH CN solution of anhydrous Na CO
for 3-5 days. For instance, the formation of tris(diisopropyl-
Scheme I Synthesis of diisopropyl-1,4,7-triazacy-
clononane
CH3
NH2
NH2
o
NHTs
NHTs
in pyridine, 55 C, 30 min
+
+
NH
3
Ts N
TsO
O
S
O
+ H O, stirred overnight
2
(
86%)
Cl
linker
CH3
+
+
triethylamine inCH Cl2
2
HO
OH
2
o
OTs
O
S
O
0 C, O , 12 hr (90%)
2
Cl
M
M
M
+
NaH in DMF,
o
N2, 105 C, 4.5 hr
(
80%)
Ts
Ts
N
N
N
N
N
+
PhOH, in 30% HBr/AcOH
N
N
N
N
N
o
N
N
H
H
90 C , 36 hr, dry tube (83%)
Ts
Ts
4
N
N
N
N
3
N
N
N
+2-bromopropane, Na2CO3
in CH CN, reflux 18 hr, N2
N
N
N
N
(
94%)
3
N
Ts
N
H
N
1
, tdip-b-TACN
2, tdip-e-TACN
in 18 M H SO , (60%)
20 C, 18 hr, dry tube
2
4
N
N
o
N
N
1
Fig. 1. Chemical structures of trinuclear coordinate
ligands containing three macrocyclic moieties.
6
5

DNA Hydrolysis
J. Chin. Chem. Soc., Vol. 51, No. 5B, 2004 1203
1
,4,7-triazacyclononanemethyl)benzene (1, tdip-b-TACN)
electronic absorption spectra. It provides useful information
regarding the time scale for the in situ preparation of metal
complexes prior to reacting with DNA.
ligand was accomplished by reacting with 3.1 equivalent of
dip-TACN and 1 equivalent of 1,3,5-tribromomethyl ben-
zene (7) to generate ligand 1 in Scheme II. Ligand 1 was char-
acterized by NMR spectra and ESI-mass spectroscopy with a
molecular ion at 754.9 (M+1) suggesting the formation of
three TACN moieties within a ligand.
DNA Reactions
In the study of DNA reactions, the relaxation of super-
coiled plasmid DNA was used to examine the hydrolytic re-
activity of trinuclear metal complexes. Ligand 2-Cu complex
was used in DNA reaction due to its tetrahedron-geometry. In
the absence of external oxidant, ligand 2 was reacted with
Scheme II Preparation of three-dimensional ligands
using different tribromo linkers
2
+
various concentrations of Cu ions for time periods of 120-
200 min in order to complete the complexation. The solu-
H
N
1
tion was allowed to react with phage fX-174 DNA at 10 mM
sodium phosphate buffer (pH 8.0) at 37 °C for an additional 5
hr before DNA fragments were analyzed by 1% agarose gel.
As a result, ligand 2-Cu complex is indeed capable of induc-
ing the hydrolytic DNA cleavage at 37 °C. The increase con-
centration of circular DNA (Form II) depends upon the in-
N
N
OH
Br
reflux 24 hr
1
Na CO in CH CN,
2 3 3
RT 48hr, N2
HO
dry tube PBr3
Br
OH
Br
7
H
N
2
+
N
N
crease of the Cu concentration shown in Fig. 2. As well,
DNA hydrolysis reaction is also a time-dependent process.
The longer reaction time with DNA, the more circular DNA
was cleaved by ligand 2-Cu complex. Moreover, reaction
temperature was known to be one of the important factors in
DNA hydrolysis. The result shows an increase of the circular
DNA concentration cleaved by ligand 2-Cu complex along
with the higher reaction temperature (25, 35, 45, and 55 °C)
in Fig. 3.
reflux 24 hr
2
dry tube PBr3
Br
r.t., N2
Na2CO3, in CH3CN
OH
Br
OH
Br
OH
8
9
Ligand 1 containing a linker of 1,3,5-tri-substituted
benzene has less interaction among each substituted arm due
to the flat structure of benzene. In order to increase the inter-
action among the three arms, tribromoethane (9) with a tetra-
hedron-geometry was used as a linker. It was prepared by
2
+
The use of Cu ion in DNA hydrolytic cleavage has the
2
+
+
bromination of pentaglycerin with PBr
3
to give a 40% yield in
possibility of the reduction of Cu to Cu , which is sensitive
Scheme II. Ligand 2 (tdip-e-TACN) was obtained in a similar
manner by coupling dip-TACN and tribromo species to give a
product having a molecular ion peak at 706.7 (M+1) con-
firmed by ESI-mass spectrum.
to the external oxidant such as O
2
and thiol-containing spe-
+
cies such as dithiothreotiol (DTT). The formation of Cu -O
2
species is known to generate the hydroxyl radical inducing
the oxidative DNA cleavage instead of the hydrolytic cleav-
+
age. In order to eliminate the possibility of the Cu interfer-
3
+
3+
Complexation of various metal ions
ence, the inert transitional metal ions such as La , Tb , and
4
+
Due to a lack of the chromophore moiety, ligand 2 has
less interference in the UV absorption measurement ranging
from 600 to 800 nm. Therefore, it will be an advantage to per-
form the binding study with transitional metal ions. For in-
stance, when ligand 2 is titrated stepwise with an aqueous so-
Ce were used to complex with ligand 2 in a different metal-
to-ligand ratio to examine the capability in DNA hydrolysis
4
+
in Fig. 4A. Recently, Ce with EDTA ligand was found to be
2
5
capable of cleaving DNA. As a result, ligand 2-Ce complex
displays a better reactivity in the hydrolytic DNA cleavage at
37 °C for 5 hr. Ligand 2-Ce complex also has more reactivity
lution of CuCl
2
, the UV absorption wavelength at 653 nm
2
+
4+
representing the free Cu ion was shifted to 798 nm upon the
than that of the Ce ion only, as shown in Fig. 4B. Com-
2
+
increasing of the Cu concentration suggesting the forma-
tion of ligand 2-Cu complex. No isosbectic point was ob-
served during the titration. Under similar conditions, various
monly, the metal ion has more accessibility and less steric
hindrance toward phosphodiester bonds.
In order to further examine the selectivity and reactiv-
2
+
2+
4+
32
metal complexes with Ni , Co , and Ce ions were pre-
pared. Furthermore, the time required for the formation of
metal complex was found in a second range monitored by
ity of ligand 2-metal complex in DNA, a P-5¢-end-labeled
hairpin oligonucleotides HIV-27 DNA (5¢-GCA-GAT-CTG-
AGC-CTG-GGA-GCT-CTC-TGC-3¢) was used and ana-

1
204 J. Chin. Chem. Soc., Vol. 51, No. 5B, 2004
Cheng et al.
lyzed by polyacrylamide gel electrophoresis. DNA sequence
was designed based on RNA hairpin from the trans-activa-
tion response element (TAR-RNA) in HIV. HIV-27 DNA
forms a double-stranded DNA structure with a three-base
bulge and a six-base single-stranded loop but it is not neces-
sary to refer to a model study of TAR-RNA. DNA cleavage
reactions were performed with a pre-mixing solution of
ligand 2 and metal ions for a period of time. Then, the solu-
from the position of the Maxam-Gilbert G lane which is gen-
erated from the oxidative damage followed by the piperidine
treatment. It suggests that DNA fragments cleaved by ligand
2-Ce complex belong to the hydrolytic cleavage not the oxi-
dative cleavage based on the different terminal residue.
4
+
Interestingly, the reactivity and selectivity of Ce com-
plex in DNA cleavage was found to be buffer-dependent.
DNA cleavage patterns were dramatically different between
HEPES and Tris-HCl buffers as shown in Fig. 5. When the re-
action was performed in a 10 mM Tris-HCl buffer at pH 8.0,
DNA cleavage showed more intensity at the single-stranded
region in HIV-27 DNA. No sequence preference was found
using HEPES buffer at pH 8.0 under similar conditions. In or-
der to understand the buffer interference, DNA binding study
was used to examine the interaction with DNA, metal com-
plex, and buffers. When the gel retardation assay (shown in
the supplementary data) was conducted in HEPES buffer, a
slow migration band was observed in the presence of ligand
2-Ce complex indicating the formation of high molecular
3
2
tion of metal complex was allowed to react with 5¢- P-la-
beled oligonucleotide (4-5 mM) for 5 hr at 55 °C. Ligand 2-Ce
complex is capable of cleaving DNA; however, no specific
cleavage site was observed in sodium phosphate buffer at pH
8
. The confirmation of DNA hydrolysis induced by metal
complexes is by the migration position of DNA fragments in
high-resolution polyacrylamide gel electrophoresis. Com-
monly, hydrolytic DNA cleavage will form a free hydroxyl
group attached to the 3¢-end terminal and a phosphate group
to the 5¢-end terminal. Using high resolution PAGE, the posi-
tion of DNA bands induced by ligand 2-Ce complex differs
Fig. 2. Time-dependent DNA cleavage induced by ligand 2-Cu complexes at 10 mM sodium phosphate buffer at 37 °C.
FC-174 plasmid DNA (125 ng, 60 mM per nucleotide) was incubated with ligand 2 (20 mM) and CuCl (10, 20, 50,
00, and 200 mM) in 10 mM HEPES buffer (pH 8) for 2, 5 and 16 hrs. Quantification of DNA cleavage was obtained
2
1
from the above figure. The Y-axes represent the relative intensity and the X-axes represent the concentration of metal
ion from 10, 20, 50, 100, and 200 mM, respectively.

DNA Hydrolysis
J. Chin. Chem. Soc., Vol. 51, No. 5B, 2004 1205
bands of DNA-HEPES-metal complex. The increase of metal
complex concentration did not further slow down the migra-
tion position suggesting that the concentration of metal com-
plex at 0.2 mM in HEPE buffer (10 mM) reaches the equilib-
rium binding. However, under the same condition, DNA
binding in Tris buffer did not alter the migration position.
Therefore, the reaction in Tris buffer is capable of specifi-
cally inducing DNA hydrolysis at the single-stranded region
and in HEPES buffer switching to random DNA cleavage in-
cluded the double-stranded region.
complex is greater than that of metal-TACN. It is because the
introduction of the di-isopropyl groups is known to avoid the
formation of the metal-TACN dimmer. As a result, metal-
(dip-TACN) provides more labile sites to associate with DNA
and water molecules resulting in DNA stranded scission.
In summary, novel trinuclear coordinated ligands were
prepared with three macrocyclic moieties and a tribromo
linker. These ligands show greater capability in association
2
+
with various metal ions. Ligand 2 associated with Cu and
4
+
Ce ions shows better reactivity in DNA hydrolysis at 37 °C
at pH 8 for 5 hr. The sequence-specific DNA cleavage in-
duced by ligand 2-Ce complex is found to be a buffer-depen-
dent reaction due to the formation of a metal-buffer-DNA
A number of macrocyclic compounds was examined to
compare the efficiency of multi-nuclear metal complexes in
DNA hydrolysis. Obviously, the reactivity of the macrocyclic
complex in nicking DNA is in a decreasing order: metal ion >
metal-(dip-TACN) > metal-TACN > metal-(tdip-e-TACN)
(
see supplementary data). The use of metal ion shows better
reactivity in DNA hydrolysis. It is rationalized that a free
metal ion has a small volume easily accessible to phospho-
diester bonds in DNA framework. Although a coordinated
ligand is capable of enhancing the hydrolytic force in metal
ion centers, it inevitably increases the steric effect and rigid-
ity of metal complexes to reduce the affinity toward phospho-
diester bonds. The hydrolytic reactivity of metal-(dip-TACN)
Fig. 4. DNA cleavage induced by ligand 2 and various
metal ions in the different metal-ligand ratio.
(
A) FC-174 plasmid DNA (125 ng, 60 mM per
nucleotide) was incubated with ligand 2 (50
mM) and metal ions (50, 100, and 150 mM) in 10
mM HEPES buffer (pH 8) at 55 °C for 15 hr.
Lanes 1, DNA only, lane 2-4, ligand 2 with
2
+
2+
Co ; lane 5, Co ion only (150 mM); lane 6-8,
2
+
2+
ligand 2 with Cu ion; lane 9, Cu ion only
150 mM); lane 10-12, ligand 2 with La ion;
lane 13, La ion only (150 mM); lane 14-16,
ligand 2 with Tb ion; and lane 17, Tb ion
3
+
(
3
+
3
+
3+
only (150 mM). (B) Supercoiled plasmid DNA
Fig. 3. Temperature-dependent DNA cleavage induced
by ligand 2-Cu complexes at 10 mM sodium
phosphate buffer for 5 hr. FC-174 plasmid
DNA (125 ng, 60 mM per nucleotide) was incu-
4
+
cleavage caused by Ce ion. FC-174 plasmid
DNA (125 ng, 60 mM per nucleotide) was incu-
4
+
bated with ligand 2 and Ce ions in 1 mM Tris
buffer (pH 8) at 37 °C for 3 hr. Lane 1, DNA
only; lane 2-4, ligand 2 (50, 100, and 150 mM)
bated with ligand 2 (50 mM) and CuCl
in 10 mM HEPES buffer (pH 8) for 5 hr. Lanes
, 25 °C without metal complex; lanes 2, 25 °C
2
(50 mM)
4
+
4+
complexed with Ce ion (50 mM); lane 5, Ce
ion only (150 mM); lane 6-7, ligand 2 (50 mM)
1
with metal complex; lane 3, 35 °C with metal
complex; lane 4, 45 °C with metal complex; and
lane 5, 55 °C with metal complex.
4
+
complexed with Ce ion (100 and 25 mM); lane
4
+
8
, Ce ion (50 mM) only.

1
206 J. Chin. Chem. Soc., Vol. 51, No. 5B, 2004
Cheng et al.
adduct. The use of different buffers showed the specific se-
lectivity in DNA hydrolysis. Trinuclear metal complexes are
capable of inducing DNA stranded scission; however, they
have lower reactivity compared to that of mono- and di-
the hydrolytic capability and to reduce the steric environment
simultaneously. Thus, the design of a novel ligand to possess
multi-nuclear metal binding sites is a challenge for the devel-
opment of chemical hydrolyases.
1
4
nuclear metal complexes. A similar observation reported by
Rokita’s group was the use of pyridine derivatives in associa-
tion with three metal ions with lower reactivity in DNA hy-
drolysis. The result suggests that trinuclear metal complexes
have the bulky environment to hinder their approach toward
the phosphodiester bonds. But, when cyclen (1,4,7,10-tetra-
azacyclodecane) was used as a module for the formation of
trinuclear metal complex, it showed the best reactivity in
EXPERIMENTAL SECTION
Materials and Methods
All reagents and ACS grade solvents were purchased
from commercial sources and were used without further puri-
fication unless otherwise noted. All reagents and ACS grade
solvents were purchased from commercial sources and were
1
7
DNA cleavage reported by Kimura’s group. Recently, more
multi-nuclear metal complexes show significant contribu-
tions in biological systems such as Cu-cluster in p-methane
monooxygenase and Mn-cluster in oxygen-evolving com-
plex (photosystem II). It is required for a ligand to increase
1
used without further purification unless otherwise noted. H
NMR spectra were determined on a Bruker ACF-300 NMR
2
spectrometer. UV-Vis spectra were obtained in H O, and 10
mM phosphate buffer on a Hewlett-Packard 8453 diode array
spectrometer. l-Phage FC-174 supercoiled plasmid DNA
was purchased from In Vitrogene Technologies (USA). No
purification was needed prior to use. Synthetic oligonucleo-
tides were purchased from Biobasic Inc. (Canada). The syn-
thesis and characterization of tri-tosylated TACN and di-iso-
propyl TACN in Scheme I were prepared as described in the
literature. Compounds 3-7 were prepared by following litera-
2
6-29
ture procedures.
Tris(bromomethyl)ethane (9)
,1,1-Trishydroxyethane (60 g, 0.5 mol) was added
slowly to a phosphorus tribromide (203.6 g, 0.76 mol) in
CH CN at room temperature. The solution was heated to 100
C for 30 min, and then allowed to heat to reflux for an addi-
1
3
°
tional 24 hrs. The mixture solution was cooled to room tem-
perature, quenched with water, and filtrated to obtain a solu-
tion. The solution was extracted twice with benzene. The
combined organic layer was washed with saturated sodium
4
bicarbonate solution, water, brine, and dried over MgSO .
Fig. 5. Buffer-dependent DNA cleavage induced by
The solvent was vaporized to obtain a pale-yellow solid.
2
+
ligand 2-Cu complex. DNA cleavage reac-
1
Yield: 61.2 g (40%). H NMR (CDCl
3
) d 3.48 (s, 6H, -CH
2
Br),
3
2
tions were using a 5¢-end P-isotope labeled
DNA substrate (3 mM) with a pre-mixing solu-
13
1
.27 (s, 3H, -CH
3
); C NMR (DMSO-d
6
), d 39.3, 39.1, 20.7.
4
+
tion of ligand 2 (100 mM) and Ce ion in 10
Tris(diisopropyl-1,4,7-triazacyclononanemethyl)benzene
(1)
mM Tris-HCl buffer or HEPES buffer (pH 8) at
5
5 °C for 16 hrs followed by 0.7 M piperidine
treatment. Cleavage products were analyzed on
a 20% denaturing polyacrylamide gel (7 M
urea). Lanes 1, DNA only; lane 2-5, ligand 2
An anhydrous CH
TACN was added to a CH
methyl)benzene and K CO
under N . The mixture was filtrated and the solvent was va-
porized to obtain a yellow solid. The yellow solid was washed
with CH Cl twice to remove the starting materials and then
3
CN solution of 3.1 eq. diisopropyl
CN solution of 1 eq. tris(1-bromo-
at room temperature for 2 days
3
4
+
2
3
and Ce ion (100, 200, 500, and 1000 mM) in
4
+
2
HEPES buffer; lane 6-9, ligand 2 and Ce ion
100, 200, 500, and 1000 mM) in Tris-HCl
buffer.
(
2
2

DNA Hydrolysis
J. Chin. Chem. Soc., Vol. 51, No. 5B, 2004 1207
purified by column chromatography packed with Al
2
O
3
gel
(Amicon) using ultracentrifuge (6,000 rpm, Beckmann
GS-15R equipped with rotor F0850) at 4 °C for 80 min, fol-
lowed by an additional centrifuge with Milli-Q water (1 mL)
for 60 min. A further dilution to proper radiation intensity
with deionized water was performed prior to reacting with
metal complexes.
eluting with CHCl
amine). Yield: 29%. H NMR (D
.66 (s, 6H, Ar-CH -); 3.04 (m, -CH(CH
CH ); 2.71 (s, 12H, N-CH -); 0.95-1.05 (m, -CH(CH
ESI-MS (m/e) = 754.9 (M+1).
3
:MeOH = 19:1 (containing 1% triethyl-
1
2
O) d 7.11 (s, 3H, Ar-H);
); 2.6-2.9 (m, N-
).
3
2
3 2
)
2
2
3 2
)
Tris(diisopropyl-1,4,7-triazacyclononanemethyl)ethane (2)
An anhydrous CH CN solution of a 3.01 equivalent of
diisopropyl TACN was added to a CH CN solution of 1
equivalent of trisbromomethylethane and dry K CO at 60 °C
and then stirred for 2 days under N . The mixture was filtrated
and the solvent vaporized to obtain a yellow solid. The yel-
DNA Reactions30
3
A total volume 20 mL solution containing the final con-
3
2
3
centration of ~ 8 nCi of 5¢-g- P -labeling oligomer, unlabeled
DNA (3-5 mM), and 10 mM sodium phosphate buffer (pH
6.96) was combined with a desired amount of ligands and
metal ions at a desired temperature for 3-24 hr. The reaction
was quenched by adding sonicated calf thymus DNA (5 mg),
3 M sodium acetate (pH 7, 15 mL), and 95% EtOH (700 mL),
then cooled at -78 °C for 20 min, centrifuged (12,000 rpm) at
4 °C for 6 min, and lyophilized to dryness to obtain a pellet. In
some cases, the reaction mixture was subjected to a piperi-
dine treatment by adding 0.7 M piperidine aqueous solution
(60 mL) and heating at 90 °C for 30 min. After lyophilizing,
2
3
2
low solid was washed with CH
2
Cl
) d 3.42 (s, 6H, C-CH
), 2.72 (t, 12H, N-CH ), 2.56 (t, 12H,
-); 2.44 (s, 12H, N-CH -); 1.23 (s, 3H, C-CH ); 0.87-
.97 (m, 39H, -CH(CH ). ESI-MS (m/e) = 706.7 (M+1).
2
twice to remove the start-
1
ing materials. H NMR (CDCl
3
2
N), 2.83
(
m, 3H, -CH(CH
3
)
2
2
N-CH
2
2
3
0
3 2
)
Hydrolysis of supercoiled DNA examined by agarose gel
electrophoresis
washing with deionized H
2
O, and lyophilizing again to dry-
DNA cleavage was assayed in a volume of 10 mL con-
taining FC-174 supercoiled plasmid DNA (125 ng, ~ 60 mM
nucleotide) and 10 mM sodium phosphate (pH 7). Ligand
ness, the DNA sample was resuspended in a gel-loading
buffer (5 mL) containing 0.25% bromophenol blue, 0.25%
xylene cyanol FF, and 7 M urea. The DNA fragment was ana-
lyzed by 20% denaturing polyacrylamide gel (7 M urea) and
then visualized using Kodak BioMax MR-1 films with inten-
sifying screens. The optical density of DNA fragments was
quantified using an image program from UVP Inc. (GelBase/
(
50-200 mM) and metal ions were added sequentially and the
reaction was incubated at various temperatures for a period of
time. The reaction was quenched with a gel-loading solution
containing bromophenol blue (0.25%), xylene cyanol FF
TM
(
0.25%), and glycerol (50%). Form I (supercoiled) and Form
GelBlot Pro) equipped with a scanner (HP scanner).
II (circular) DNA were separated by agarose gel electropho-
resis (1% agarose, 1x TAE), stained with ethidium bromide
Modified Maxam-Gilbert G Lane
3
1
(
0.1 mg/mL), destained with deionized H
photographed with CCD equipment from Vilber Lourmat
Model 008-SD).
2
O for 20 min, and
A modified Maxam-Gilbert G lane was performed by
the following procedures: a DNA solution containing ~10
3
2
(
2
nCi P-labeling oligomer and deionized H O in a total vol-
ume of 20 mL was cooled at 0-4 °C prior to use, followed by
successive steps of adding DMS (<1 mL) to the DNA solu-
tion, votexing the tube (< 1 sec), immediately adding 2-mer-
captoethanol (10 mL), and votexing for an additional 30 sec to
quench the reaction. After adding sonicated calf thymus
DNA (5 mg) and 3.0 M sodium acetate (pH 7.0, 15 mL), a
DNA solution was precipitated with 95% EtOH (1 mL) and
centrifuged to obtain a DNA pellet which was then treated
with piperidine as mentioned above before loading on gels.
DNA Preparation
Purification of synthetic oligonucleotides was carried
out by gel purification in a 20% denaturing polyacrylamide
gel (7 M urea). The DNA bands were visualized with a UV
lamp (lmax 254 nm) by placing the gel on a TLC F254 plate (20
´
20 cm, Merck) as previously described. The DNA concen-
tration was determined using the extinction coefficient (lmax
60 nm) or molecular weight method (1 O.D. ~ 33 mg and the
average molecular weight of one nucleotide = 330 daltons).
2
3
2
The 5¢- P -end labeling oligomer was prepared using T4
polynucleotide kinase (New England Biolabs) and deoxy-
ACKNOWLEDGMENTS
3
2
adenosine-5¢-[g- P]-triphosphate (DuPont). The excess free
3
2
g- P-ATP was removed by filtration with Centricon-10
Financial support from Academia Sinica, Tamkang

1
208 J. Chin. Chem. Soc., Vol. 51, No. 5B, 2004
Cheng et al.
University, and the National Science Council of the Republic
of China is gratefully acknowledged.
14. Humphreys, K. J.; Karlin, K. D.; Rokita, S. E. J. Am. Chem.
Soc. 2002, 124, 6009.
1
1
1
5. Rossi, L. M.; Neves, A.; Horner, R.; Terenzi, H.; Szpoganicz,
B.; Sugai, J. Inorganica Chimica Acta 2002, 337, 366.
6. Komiyama, M.; Sumaoka, J. Current Opinion in Chemical
Biology 1998, 2, 751.
Received March 23, 2004.
7. Kimura, E.; Aoki, S.; Koike, T.; Shiro, M. J. Am. Chem. Soc.
1
997, 119, 3068.
18. Yashiro, M.; Ishikubo, A.; Komiyama, M. Chem. Commun.
997, 83.
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