Structure and DNA cleavage properties of two copper(ii) complexes of the pyridine-pyrazole-containing ligands mbpzbpy and Hmpzbpya

Article abstract of DOI:10.1039/b704390b

The DNA-cleavage properties of the two copper(ii) complexes, [Cu(mbpzbpy)Br₂](H₂O)_2.5 (1) and [Cu(mpzbpya)Cl](CH₃OH) (2), obtained from the ligands 6,6′-bis(3,5-dimethyl-N-pyrazolmethyl)-2,2′-bipyridine) (mbpzbpy) and 6′-(3,5-dimethyl-N-pyrazolmethyl)-2,2′-bipyridine-6-carboxylic acid) (Hmpzbpya), respectively, are reported. Upon coordination to Cu ^II chloride in methanol, one arm of the ligand mbpzbpy is hydrolyzed to form mpzbpya. Under the same experimental conditions, the reaction of mbpzbpy with CuBr₂ does not lead to ligand hydrolysis. The ligand mpzbpya is coordinated to a copper(ii) ion generating a CuN₃OCl chromophore, resulting in a distorted square-pyramidal environment, whereas with the N ₄ mbpzbpy ligand, the Cu^II ion is four-coordinated in a distorted square planar geometry. Both complexes promote the oxidative DNA cleavage of X174 phage DNA in the absence of reductant. The oxidative nature of the DNA cleavage reaction has been confirmed by religation and cell-transformation experiments. Studies using standard radical scavengers suggest the involvement of hydroxyl radicals in the oxidative cleavage of DNA. Although both compounds do convert form I (supercoiled) DNA to form II (nicked, relaxed form), only complex 1 is able to produce small amounts of form III (linearized DNA). This observation may be explained either by the attack of the copper(ii) complexes to only one single strand of DNA, or by a single cleavage event. Statistical analysis of relative DNA quantities present after the treatment with both copper(ii) complexes supports a random mode of DNA cleavage. This journal is The Royal Society of Chemistry.

Full text of DOI:10.1039/b704390b

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PAPER
www.rsc.org/dalton | Dalton Transactions
Structure and DNA cleavage properties of two copper(II) complexes of the
pyridine-pyrazole-containing ligands mbpzbpy and Hmpzbpya†‡
a
a
a
a
a
Palanisamy Uma Maheswari, Kristian Lappalainen, Michael Sfregola, Sharief Barends, Patrick Gamez,
b
b
a
a
Urho Turpeinen, Ilpo Mutikainen, Gilles P. van Wezel and Jan Reedijk*
Received 22nd March 2007, Accepted 21st May 2007
First published as an Advance Article on the web 5th July 2007
DOI: 10.1039/b704390b
The DNA-cleavage properties of the two copper(II) complexes, [Cu(mbpzbpy)Br
2
](H O)2.5 (1) and
2
ꢀ
[
2
Cu(mpzbpya)Cl](CH
3
OH) (2), obtained from the ligands 6,6 -bis(3,5-dimethyl-N-pyrazolmethyl)-
ꢀ
ꢀ
ꢀ
,2 -bipyridine) (mbpzbpy) and 6 -(3,5-dimethyl-N–pyrazolmethyl)-2,2 -bipyridine-6-carboxylic acid)
II
(
Hmpzbpya), respectively, are reported. Upon coordination to Cu chloride in methanol, one arm of
the ligand mbpzbpy is hydrolyzed to form mpzbpya. Under the same experimental conditions, the
reaction of mbpzbpy with CuBr does not lead to ligand hydrolysis. The ligand mpzbpya is coordinated
to a copper(II) ion generating a CuN
environment, whereas with the N
2
3
OCl chromophore, resulting in a distorted square-pyramidal
mbpzbpy ligand, the Cu ion is four-coordinated in a distorted
II
4
square planar geometry. Both complexes promote the oxidative DNA cleavage of φX174 phage DNA in
the absence of reductant. The oxidative nature of the DNA cleavage reaction has been confirmed by
religation and cell-transformation experiments. Studies using standard radical scavengers suggest the
involvement of hydroxyl radicals in the oxidative cleavage of DNA. Although both compounds do
convert form I (supercoiled) DNA to form II (nicked, relaxed form), only complex 1 is able to produce
small amounts of form III (linearized DNA). This observation may be explained either by the attack of
the copper(II) complexes to only one single strand of DNA, or by a single cleavage event. Statistical
analysis of relative DNA quantities present after the treatment with both copper(II) complexes supports
a random mode of DNA cleavage.
Introduction
promising coordination compounds typically contain a DNA-
binding moiety that targets either the major groove or the minor
The development of artificial chemical nucleases is essential in
groove, and may additionally act as intercalator, thereby increasing
1
–3
the field of biotechnology and drug design. The ability to
accomplish site-specific DNA cleavage will undoubtedly allow the
development of new chemotherapeutic agents and antimicrobial
19,20
the DNA-targeting ability of the metal complex.
However, this
procedure does not accurately characterize the in vivo activity of a
chemical nuclease. The induced site-specificity is a difficult task in
the design of DNA-cleaving agents. Most strategies used are based
on the linkage of a DNA-recognition element to the chemical
nuclease. Such DNA targeting can be achieved with either an
antisense RNA, or with a highly specific DNA-binding protein,
such as a zinc-finger motif.
4–6
drugs. In addition, artificial nucleases will provide important
7
,8
new tools for DNA manipulation to molecular biologists.
For example, a copper complex of 1,10-phenanthroline is used
in DNA-footprinting experiments, which are important for the
detailed study of DNA–protein interactions. Transition-metal
complexes are well suited for application as artificial nucleases,
because of their diverse structural features, and the possibility to
tune their redox potential through the choice of proper ligands.
Also, ligands can be designed to incorporate biological functional
groups with a suitable metal ion to mimic the active sites found in
9
The interaction of transition metals, like Mn, Fe, Cu, with
dioxygen (in the presence of a reductant) often generates reactive
1
0–17
21
oxygen species that ultimately may cleave DNA. The cleavage
mechanism can be either oxidative at the sugar or at the nucle-
obase, or hydrolytic at the phosphodiester backbone of DNA. The
18
metalloproteins.
single- or double-strand oxidative DNA cleavage by redox-active
The efficiency of the DNA cleavage can be enhanced through the
increase of the affinity of the metal complex for DNA. Successful,
2−
metal complexes like [Fe(edta)] or Cu(1,10-phenanthroline)
2
2
Cl ,
is initiated by the production of reactive oxygen species, like
the hydroxyl radical or singlet oxygen through a Fenton-type
a
Leiden Institute of Chemistry, Leiden University, P.O. Box, 9502, 2300, RA
22–25
mechanism.
These free radicals abstract the most accessible
Leiden, The Netherlands. E-mail: reedijk@chem.leidenuniv.nl; Fax: +31
and exposed sugar hydrogens and initiate the oxidative cleavage,
leading to DNA-cleavage products. In fact the site of hydrogen
atom abstraction from the DNA-sugar depends on the active
metallonuclease used. This class of metallonucleases has potential
applications in site-specific recognition of DNA and as DNA
footprinting agents, however, their use in living cells (in-vivo) is
limited as their activity towards DNA cleavage is only observed
7
15274671
b
University of Helsinki, Department of Chemistry, Laboratory of Inorganic
Chemistry, Helsinki, 00014, Finland
†
CCDC reference numbers 637327 & 637328. For crystallographic data
in CIF or other electronic format see DOI: 10.1039/b704390b
Electronic supplementary information (ESI) available: Fig. S1–S4 show-
‡
ing the hydrogen bonding and packing style for the complexes 1 and 2. See
DOI: 10.1039/b704390b
3
676 | Dalton Trans., 2007, 3676–3683
This journal is © The Royal Society of Chemistry 2007

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in the presence of excess of external reductants, such as sodium
ascorbate or mercaptopropionic acid and dioxygen. The real
challenge is the search for simple, synthetic metal complexes that
bind and cleave DNA by a self-activating mechanism, also under
in-vivo conditions.
Iron bleomycin was the first reported natural product to
cleave DNA in an oxidative pathway, and its importance was
soon recognized because of its novel and broad spectrum an-
26–28
titumor properties.
The nuclease activity of this compound
is self-activated through coordination to iron(II), in the pres-
29,30
ence of molecular dioxygen.
The copper(II) adducts of 4-
methoxypyrrolic marine natural products were also reported to
cleave DNA, in the absence of any reductant, suggesting the
II
involvement of hydroxyl radicals. These Cu compounds have also
Scheme 1 Reaction pathway to synthesize the ligand mbpzbpy.
shown promising antitumor properties against breast-, colon- and
30–35
liver-cancer cells by facilitating apoptosis.
More recently, the
with HBr and NaNO . The resulting 2-bromopicoline is pu-
2
complex [Fe(N Py)(CH CN)](ClO was reported as a synthetic
4
3
4
)
2
rified by distillation (yield = 77%). Next, the coupling reac-
model of ‘activated bleomycin’, able to cleave DNA in the absence
of reductant, owing to the formation of transient low-spin Fe(III)–
tion of 2-bromopicoline catalysed by wet Pd/C produces, after
ꢀ
ꢀ
ꢀ
nine days, 6,6 -dimethyl-2,2 -bipyridine with a yield of 50%. 6,6 -
14
ꢀ
OOH species.
Dimethyl-2,2 -bipyridine is subsequently brominated with N-
We recently reported the discovery of a copper(II) complex,
prepared from Hpyramol, that catalytically cleaves target DNA in
the absence of reductant, by attacking multiple positions of the
bromosuccinimide (NBS) in refluxing CCl to yield the dibromide
4
ꢀ
ꢀ
derivative 6,6 -bis(bromomethyl)-2,2 -bipyridine (yield = 21%).
The final step is the reaction between the dibromide derivative
and the sodium salt of the 3,5-dimethylpyrazole, giving the ligand
mbpzbpy (Fig. 1a) in good yields (77–94%); details of the reaction
conditions are given in the Experimental section.
13
nucleotide. It was suggested that the DNA cleavage reaction is
oxidative, through a non-diffusible radical mechanism, because
radical scavengers do not significantly inhibit the DNA cleavage
13
reaction. The above copper complex also shows interesting
cytotoxic properties in selected cancer cell lines, comparable to
the antitumor drug cisplatin. This copper complex is self-activated
by the dehydrogenation of the precursor Hpyramol ligand upon
coordination to the metal ion, i.e. the dehydrogenation of the
ligand Hpyramol to Hpyrimol has now been observed with
II
III
II
II 36,37
transition metals like Cu , Fe , Mn and Zn .
The oxidative
DNA-cleavage mechanism appears to be purely ligand-based, as
38
also the redox-inactive, zinc complex also shows DNA cleavage.
In the present paper the synthesis and structures of two new,
related copper(II) complexes, namely [Cu(mbpzbpy)Br ](H
complex 1) and [Cu(mpzbpya)Cl](MeOH) (complex 2), are
Fig. 1 Schematic representations of the ligands mbpzbpy (a) and
Hmpzbpya (b).
2
2
O)2.5
(
Preparation of the copper complexes
described. Both complexes are soluble in water and cleave DNA
without the presence of any added reductant. This is a valuable fea-
ture for an application as chemotherapeutic agents in anticancer
treatments. DNA cleavage studies with various concentrations of
the complexes have been performed on φX174 phage DNA. DNA
cleavage investigations have also been carried out in the presence of
different additives including selected radical scavengers, to clarify
the mechanism of action of each complex. Religation and cell-
transformation experiments have been performed to determine
the mode of DNA cleavage, i.e. hydrolytic, or oxidative. All studies
suggest the involvement of hydroxyl radical species which generate
DNA scissions via an oxidative mechanism.
The two complexes are prepared from CuBr
the ligand mbpzbpy [2-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)-6-
6-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridine-2-yl)pyridine]
in absolute methanol. Starting from CuCl O, the ligand
·6H
mbpzbpy is modified to the partially hydrolysed ligand Hmpzbpya
6-(−((3,5-dimethyl-1H-pyrazol-1-yl)methyl)pyridine-2-yl)pyri-
dine-2-carboxylic acid] (Fig. 1b) yielding complex 2. Starting
from CuBr the ligand has been found not to degrade under
2
and CuCl
2
·6H
2
O and
(
2
2
[
2
comparable conditions, yielding complex 1.
Crystal structure descriptions
[
Cu(mbpzbpy)Br
2
](H
2
O)2.5 (1). The reaction of the ligand
in absolute methanol yields dark blue
Results and discussion
mbpzbpy with CuBr
2
crystals of 1 after one week. 1 crystallises in the C2/c monoclinic
space group. An ORTEP perspective view of complex 1 is depicted
in Fig. 2. Selected bond lengths and angles are given in Tables 1
Synthesis of the ‘mbpzbpy’ ligand
The tetradentate ligand ‘mbpzbpy’ is synthesized from the
ꢀ
ꢀ
II
commercially available compound 6,6 -dimethyl-2,2 -bipyridine
and 2, respectively. The Cu ion is in a distorted square-planar
ꢀ
ꢀ
(
Scheme 1). 6,6 -Dimethyl-2,2 -bipyridine can also be prepared
coordination environment formed by the N ligand. The distortion
4
from 2-aminopicoline, which is first brominated by reaction
most likely arises from the small bite angle of the bipyridine moiety.
This journal is © The Royal Society of Chemistry 2007
Dalton Trans., 2007, 3676–3683 | 3677

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Table 1 Selected bond lengths ( A˚ ) for complexes 1 and 2
yields light-green plate crystals of 2 after two weeks, the crystal
structure reveals that the original mbpzbpy ligand had been
converted to Hmpzbpya (Fig. 1). 2 crystallises in the P-1 triclinic
space group. An ORTEP perspective view of 2 is shown in Fig. 3.
Selected bond lengths and angles are given in Tables 1 and 2,
respectively. The copper(II) ion is pentacoordinated in a highly
distorted square-pyramidal environment (the s factor amounts to
Complex 1
Complex 2
Cu1–N21
Cu1–N21b
Cu1–N11
Cu1–N11b
Cu1–Br
1.992(2)
1.992(2)
2.010(2)
2.010(2)
3.245(1)
Cu1–N11
Cu1–N31
Cu1–O18
Cu1–N21
Cu1–Cl1
1.932(4)
2.011(4)
2.018(3)
2.043(4)
2.4384(14)
39
0
.392) with a N OCl donor set. The equatorial plane is defined
3
by the two pyridine nitrogen atoms N11 and N21, the pyrazole
nitrogen atom N31, and by the O18 atom from the carboxylate
donor (Fig. 3). The apical position is occupied by the chloride
anion Cl1. The Cu–N, Cu–O, Cu–Cl distances are in the expected
◦
Table 2 Selected bond angles ( ) for complexes 1 and 2
Complex 1
Complex 2
40
ranges (Table 1). The basal angles varying from 78.97(16) to
N21–Cu1–N21b
N21b–Cu1–N11
N21–Cu1–N11
N11–Cu1–N11b
98.87(12)
93.06(8)
93.06(8)
80.95(11)
N11–Cu1–N31
N11–Cu1–O18
N31–Cu1–O18
N11–Cu1–N21
N31–Cu1–N21
N11–Cu1–Cl1
N31–Cu1–Cl1
O18–Cu1–Cl1
N21–Cu1–Cl1
148.15(17)
80.98(15)
102.51(15)
78.97(16)
91.43(16)
111.50(12)
99.42(12)
98.34(11)
94.66(12)
◦
102.51(15) (Table 2) reflect the significant distortion of the square-
pyramid, stemming from the methylene group linking the pyrazole
moiety to the bipyridine. Indeed, while N11, N21, and O18 are in
the same plane, the pyrazole nitrogen N31 is out of this plane
(
Fig. S3‡). The crystal packing of 2 shows that the oxygen atom
O19 from the carboxylate group is involved in a strong hydrogen-
bonding interaction with a lattice methanol molecule (the O1–
˚
H1A · · · O19 distance is 2.715 A, Fig. S4‡).
Fig. 3 ORTEP drawing (30% probability level) of [Cu(mpzbpya)-
Cl](MeOH) (2). Hydrogen atoms and lattice MeOH has been left out
for clarity.
Fig. 2 ORTEP drawing (30% probability level) of [Cu(mbpzbpy)-
Br ](H O)2.5 (1). Hydrogen atoms and lattice water have been omitted
2
2
for clarity. Symmetry operation codes: a: −x, y, 1/2−z; b: 1−x, y, 1/2−z;
Copper complexes from pyrazole ligands are known to mediate
c: 1/2−x, 1/2−y; d: 1/2 + x, 1/2−y, −1/2 + z.
41
oxidation reactions; therefore, the [Cu(mbpzbpy)Cl ] compound
2
As a result, the two pyrazole rings deviate from the plane of
the bipyridine unit to minimize the steric interactions between
the methyl substituents. Thus, the Cu–N distances involving the
is believed to first oxidize one methylene group of the ligand
in MeOH, yielding ultimately the carboxylic acid product (Hm-
pzbpya) after further oxidation (see Scheme 2).
˚
bipyridine unit are longer (Cu1–N11 = 2.010(2) A, Cu1–N11b =
˚
˚
2
.010(2) A) than the pyrazole ones (Cu1–N21 = 1.992(2) A, Cu1–
˚
N21b = 1.992(2) A). The equatorial N–Cu–N angles, varying from
◦
8
0.95(11) to 98.87(12) , reveal the strong geometric distortion
induced by the bipyridine unit, and by the steric hindrance between
two methyl groups of the pyrazole groups. The crystal packing
of 1 shows that the bromide counter ions are interacting with the
copper center, at semi-coordinating distances of the axial positions
˚
of the N
4
plane (Cu · · · Br separation of 3.245 A, Fig. S1‡). In fact,
these bromide anions are also involved in a hydrogen bonding
network with lattice water molecules (Fig. S2‡). Each bromide
thus exhibits close contact with three water molecules (the O–
Scheme 2 Proposed pathway for the formation of Hmpzbpya.
˚
DNA cleavage properties
H · · · Br distances range from 3.369 to 3.453 A).
[
Cu(mpzbpya)Cl](CH
3
OH) (2). The room temperature reac-
·6H O in absolute methanol
Investigations of the DNA-cleaving abilities of both complexes
1 and 2 revealed cleavage activities in the absence of reductant,
tion of the ligand mbpzbpy with CuCl
2
2
3
678 | Dalton Trans., 2007, 3676–3683
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Even though complex 1 did result in the formation of some
linear DNA (Fig. 4A, form III), it cannot be assigned as direct
double-strand cleavage as explained below. The ratio of double-
strand cuts to single-strand cuts was about 0.033 : 1 at a complex
concentration of 220 lM. The number of double-strand breaks per
molecule of DNA expected from completely random single-strand
42
breaks is given by the Freifelder–Trumbo equation. The amount
of double-strand breaks depends on the maximum separation in
bases between cuts on complementary strands that can produce a
linear molecule and the number of phosphodiester bonds in the
plasmid. Using this statistical model, one would expect only one
dsDNA break per about 100 ss DNA breaks. In the present study,
the occurrence of approximately 0.033 double-strand breaks and
Fig. 4 Agarose gel electrophoresis of φX174 DNA treated with increasing
concentrations of complex 1 (A) or complex 2 (B). Lanes 1–11 represent
the DNA cleavage using 20–220 lM of complex 1 or complex 2 (in 20 lM
increments) with 20 lM (in base pairs) of φX174 DNA. Incubation time
was 2 h at 37 C for all reactions. Lane 12 is the DNA control without
the copper complexes. Small amounts of form III are visible only for
2.37single-strandbreaks per molecule is determined, and therefore
◦
the ratio is around 72. The slightly lower value (72) compared to
the expected one (100) indicates that a random cleavage path to
the formation of linear DNA indeed is most likely taking place.
In contrast, complex 2, where the original ligand is partially
hydrolysed, does not show any sign of linearized DNA (form
III) at any stage, thus ruling out the possibility of direct double-
stranded cleavage. Thus complex 2 accomplished the conversion
of form I (supercoiled) to only form II DNA (nicked or single-
strand cut); even at a concentration as high as 220 lM, the
highest concentration tested, still 23% of the initial form I of DNA
remained intact in the reaction mixture (Table 4). The inability of
complex 2 to produce linear DNA (form III) may be due to its
reduced cleavage efficiency, as evidenced by the fact that in the
presence of 220 lM of complex 2 (Fig. 4B, lane 11) still less DNA
is cleaved than in the case of complex 1 at 100 lM (Fig. 4A, lane 5).
Conversely, both the complexes 1 and 2 cleave supercoiled DNA
complex 1.
◦
after a reaction time of 2 h with φX174 DNA at 37 C (Fig. 4A,
B). The reaction of complex 1 with φX174 DNA (20 lM, in base
pairs) resulted in significant amounts of form II, and in more
than stoichiometric amounts (around 1–10 fold). About 90% of
the supercoiled DNA (form I) was nicked to produce relaxed or
open circle DNA (form II), when incubated with 80 lM complex
1
(Fig. 4A). Although this efficient DNA cleavage was promising,
only a very small proportion of the DNA was linearized by
complex 1 (Fig. 4A). Double-stranded DNA cleavage cannot be
easily repaired by the host DNA repair system and is therefore
much more desirable from the perspective of anti-cancer drug
development for any DNA-cleaving transition metal complex.
Table 3 Crystal data and structure refinement for [Cu(mbpzbpy)Br
2
](H
2
O)2.5 (1) and [Cu(mpzbpya)Cl](CH OH) (2)
3
Coordination compound
1
2
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C
640.87
173 K
0.71073
Monoclinic
C2/c
a = 18.391(3)
b = 15.522(3)
c = 9.1220(18)
a = 90.00
b = 104.03(3)
c = 90.00
2526.3(8)
4
1.685
4.061
1288
Blue, block
0.30 × 0.30 × 0.20
3.10–27.53
−23 23, −20 20, −11 11
18 757
2893
2328
22
H
29Br
2
CuN
6
O
2.50
18 4 3
C H19ClCuN O
438.36
173 K
0.71073
Triclinic
P-1
a = 7.9130(16)
b = 11.833(2)
c = 11.894(2)
a = 115.45(3)
b = 102.53(3)
c = 102.35(3)
920.2(3)
◦
Unit cell dimensions/ A˚ ,
Volume/ A˚ 3
Z
Density (calculated)
Absorption coefficient
F(000)
Crystal color, morphology
Crystal size
Theta range for data collection
Index range h,k,l
Reflections collected
Independent reflections
Observed reflections
Data/restraints/parameters
2
1.582
1.359
450
Green, plate
0.18 × 0.18 × 0.02
2.81–27.50
−10 10, −15 15, −15 15
15 782
4221
3101
4221, 12, 245
0.965
0.0670
0.0922
1.230, −1.277
2893, 0, 161
1.048
0.0287
0.0454
0.457, −1.202
2
Goodness-of-fit on F
Final R/wR
R (all data)
2
[I > 2r(I)]
Largest diff. peak and hole
This journal is © The Royal Society of Chemistry 2007
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Table 4 Relative percentages of different forms of DNA after cleavage by
the copper complexes 1 and 2
could efficiently religate the DNA that was linearized using the
restriction enzyme BamHI, and subsequently religated using T4
DNA ligase as illustrated by the high transformation efficiency
of 94%, which is comparable to untreated control DNA (100%
by default). Fig. 6 indicates low transformation efficiency for the
DNA cleaved by complex 1 in comparison to complex 2. As the
undigested, initial form I of DNA is the only DNA giving rise to
colonies in the cell-transformation experiments, the DNA cleavage
is suggested to be oxidative, with probable alterations in the sugar
and/or base units.
Form I (%)
Form II (%)
Form III (%)
3.1
Complex 1 100 lM
Complex 2 220 lM
0.9
23
96
77
a
—
a
Complex 2 shows no formation of form III, even at the highest
concentration of 220 lM.
very efficiently to form III (linearized DNA) in the presence of the
reducing agent ascorbic acid at a low concentration of 5 lM.
Evidence for an oxidative pathway for the cleavage of DNA has
been obtained through religation and transformation of pUC19
plasmid cleavage products using complexes 1 and 2, containing
form I and form II DNA. All ligation reactions contained the
same total amount of DNA, but with different form I : form II
ratios. Complex 2 at a concentration of 220 lM converted about
Further evidence for an oxidative mechanism of the DNA
cleavage is achieved by additional DNA-cleavage reactions per-
formed in the presence of standard radical scavengers, such
as the enzyme superoxide dismutase (SOD), the minor groove-
specific antibiotic distamycin, and by using the chemicals dimethyl
sulfoxide (DMSO) and sodium azide (NaN ) (Fig. 7 and 8). The
3
action of complex 2 is inhibited by DMSO, suggesting the potential
involvement of hydroxyl radical intermediates in the oxidative
DNA cleavage. The action of complex 1 is also inhibited in the
presence of DMSO and, to a much lesser extent, by sodium azide.
These results suggest the participation of hydroxyl radicals and
singlet oxygen in the reaction. Interestingly, the DNA cleavage
is seriously enhanced in the presence of SOD, sodium chloride,
or in dark conditions (Fig. 8). Moreover, the DNA cleavage is
also enhanced under anaerobic conditions, showing that cleavage
7
5% of the DNA, while more than 90% of the initial DNA was
converted by a 80 lM solution of complex 1 (Fig. 5). As shown in
Fig. 6, religation of the chemical cleavage experiments (obtained
by the incubation of both complexes 1 and 2 with pUC19 plasmid)
essentially gave lower transformation efficiencies as compared to
control pUC19 plasmid (untreated with BamHI and T4 DNA
ligase), and comparable to the digests not treated with T4 DNA
ligase. Expectedly, in contrast to DNA cleaved chemically, we
may occur in an O independent manner, just as observed in
2
prokaryotic systems. The enhanced oxidative DNA cleavage in the
presence of SOD indicates that superoxide anions are not involved.
However, the in situ production of H
2
2
O and its reaction with
SOD ultimately may produce reactive oxygen species. The DNA
cleavage is inhibited for both the complexes in the presence of
distamycin, indicative of competitive binding interactions between
the copper complexes and distamycin in the minor groove of DNA
(Fig. 7, lane 5 for 1, and Fig. 8, lane 5 for 2). This observation
Fig. 5 Analysis of the cleavage reaction by determination of the religation
efficiency. Lane 1, pUC19 DNA cleavage products treated with complex
1
, 100 lM; Lane 2, 20 lM DNA + complex 2, 220 lM; Lane 3, pUC19
DNA digested with BamHI (expectedly showing only linear DNA (form
III)); Lane 4, untreated pUC19 DNA (20 lM; control).
Fig. 7 Agarose gel electrophoresis of the oxidative cleavage reaction of
φX174 supercoiled phage DNA (20 lM) with complex 1 (100 lM) after
◦
2
h of incubation at 37 C in phosphate buffer (pH 7.2) (Lane 11). DNA
was incubated with: Lane 1, no additives (control); Lane 2, 200 lM NaN
Lane 3, 0.5 U of superoxide dismutase, Lane 4, DMSO, Lane 5, 100 lM
distamycin, Lane 6, D O; Lane 7, under argon, Lane 8, in the dark, Lane
, 350 lM NaCl, Lane 10, 20 lM ascorbic acid.
3
;
2
9
Fig. 6 Analysis of the cleavage reaction by determination of the religation
efficiency. Samples refer to pUC19 plasmid DNA treated with: 1, Complex
1
(100 lM); 2, Complex 2 (220 lM); 3, Restriction enzyme BamHI,
4
, untreated (transformation control). Samples were incubated with (+)
Fig. 8 Agarose gel electrophoresis of the oxidative cleavage reaction of
(20 lM) φX174 supercoiled phage DNA with complex 2 (200 lM) after
2 h of incubation at 37 C in phosphate buffer (pH 7.2) (Lane 11). DNA
or without (−) DNA ligase prior to transformation to E. coli JM109.
CFU, colony forming units. BamHI-digested DNA was almost completely
religated by the T4 DNA ligase, while very little religation was observed for
DNA cleaved with either Complex 1 or Complex 2, suggesting the cleavage
was oxidative. Error bars deduced from three independent experiments are
shown.
◦
was incubated with: Lane 1, no additives (control); Lane 2, 200 lM NaN
Lane 3, 0.5 U of superoxide dismutase, Lane 4, DMSO, Lane 5, 100 lM
distamycin, Lane 6, D O; Lane 7, under argon, Lane 8, under dark, Lane
3
;
2
9, 350 lM NaCl, Lane 10, 20 lM ascorbic acid.
3
680 | Dalton Trans., 2007, 3676–3683
This journal is © The Royal Society of Chemistry 2007

View Article Online
strongly indicates that the complexes 1 and 2 preferentially bind
DNA in the minor groove, rather than in the major groove.
As stated above, the oxidative DNA cleavage properties of
complexes 1 (form I to form II) and 2 are explicitly different.
This difference can be explained (i) by a charge effect and/or
transformation experiments have shown that the DNA cleavage
reaction is oxidative rather than hydrolytic.
Experimental
(
ii) a structural geometry effect. Complex 1 which has a 2+ charge
General
most likely experiences a better interaction with the negative DNA
helix (as the two bromide anions are only at semi-coordinating
positions in the crystal lattice) than the neutral complex 2 (as
the chlorido and the carboxylato ligands are coordinated to the
metal centre). The second aspect is the slightly distorted square-
planar environment of complex 1 with easily dissociated bromide
anions. As a result, the cation part of 1 is expected to interact
more efficiently with the DNA double helix, via static interactions
All chemicals were used as obtained without further purification.
Elemental analyses (C, H, N) were carried out on a Perkin-
Elmer 2400 series II analyzer. FTIR spectra were recorded with a
Perkin-Elmer Paragon 1000 FTIR spectrophotometer, equipped
with a Golden Gate ATR device, using the reflectance technique
−
1
(4000–300 cm ). The ligand field spectra of the compounds in
solution were recorded in the 200–1100 nm range with a Cary
1
5
0 spectrophotometer. H NMR spectra were recorded using a
(
such as intercalation or electrostatic contacts) than the distorted
DPX 300 Bruker (300 MHz) instrument. Chemical shifts are
reported in ppm (parts per million) relative to the solvent peak. X-
band electron paramagnetic resonance (EPR) measurements were
performed at 77 K in the solid state on a Jeol RE2x electron spin
resonance spectrometer, using DPPH (g = 2.0036) as a standard.
square-pyramidal neutral complex 2.
It has been demonstrated that the oxidative cleavage of DNA
in the absence of a reductant is possible with copper(II) complexes
through an effective activation of molecular oxygen, generating
9
reactive oxygen species. The favourable Cu(II) to Cu(I) redox
potential is then coupled with a self-hydrogen abstraction from
the DNA molecule (most probably from the sugar moieties). The
occurrence of this process instigates a single DNA cleavage event,
through a Fenton mechanism. This DNA cleavage may become
catalytic if the ligands coordinated to the Cu ion facilitate the
Syntheses
Synthesis of the ligand mbpzbpy and its precursors
2-Bromopicoline. 10 g (92 mmol) of 2-aminopicoline were
38
Cu(II)/Cu(I) cycle. In the present case, complex 1 is more effective
than complex 2 to undergo a single cleavage event, however,
both compounds fail to cleave DNA in a catalytic manner.
So the distinct cleavage efficiencies observed for the present
complexes can be rationalized by their significantly different
coordination geometries. Indeed, it has been shown that the
coordination geometry plays an important role in Cu(II)/Cu(I)
dissolved in 50 mL of HBr (47%) at RT. Then, the solution
◦
was cooled to −20 C, and 13.3 mL (259 mmol) of cooled
bromine were added dropwise over a period of 30 min, maintaining
◦
the temperature at −20 C. The resulting paste was stirred for
9
0 min at this temperature. Next, a solution of 17 g (246 mmol)
of sodium nitrite in water (25 mL) was added dropwise. After
◦
that, the reaction mixture was allowed to warm to 15 C for a
43
redox processes. Complex 1 which is square-planar, can more
period of 1 h, and stirred for an additional 45 min. The mixture
2
2
easily accommodate an electron in its co-planar d(x −y ) orbital
compared to complex 2. Accordingly, 1 can easier proceed to
◦
was cooled to −20 C, and treated with cooled aqueous NaOH
44
(
67 g, 100 mL). During the addition, the temperature was kept
1
0
a d configuration and thus to a tetrahedral geometry, which
is likely to occur with a Fenton-type mechanism. Complex 2
exhibits a distorted square-pyramidal geometry with a weak axial
r bond (chlorido ligand) and a strong r bond in the basal
◦
to −10 C. Subsequently, the mixture was allowed to warm to
room temperature, and stirred for 1 h. The solution was extracted
with ethyl acetate, dried over sodium sulfate, and filtered. The
solvent was evaporated under reduced pressure to give a crude oil,
which was distilled in vacuo, yielding a colourless liquid (48–50 C,
1
45
plane (carboxylato moiety). As a result, the accommodation
◦
2
2
of an electron in the co-planar dx −y orbital is unlikely to
happen due to steric as well as electronic repulsive forces. Thus,
complex 2 with a CuN O chromophore and a distorted square
pyramidal geometry is less efficient in DNA cleavage than complex
, which exhibits a CuN core with a distorted square-planar
1
mmHg), namely 2-bromopicoline. Yield: 12.3 g (77%). H-NMR
45
(CDCl
3
, 300 MHz) d (ppm) 2.54 (s, 3H, CH –C(6)), 7.10 (d, 1H,
3
3
H–C(5)), 7.29 (d, 1H, H–C(3)), 7.43 (t, 1H, H–C(4)).
ꢀ
ꢀ
1
4
6,6 -Dimethyl-2,2 -bipyridine. A mixture of 2-bromopicoline
(4.2 g, 24.4 mmol), sodium formate (2.1 g, 30.8 mmol), 10%
wet Pd/C (0.15 g), benzyltriethylammonium chloride (0.7 g,
geometry.
3.1 mmol), NaOH (84 mg, 2.1 mmol) and 10 mL of water was
refluxed for 9 days. Additional amounts of sodium formate (0.1 g)
and Pd/C catalyst (0.01 g) were added each day. After 9 days,
the mixture was filtered. The water phase was extracted with
dichloromethane, and the black solid material was washed with
dichloromethane. The combined organic phase was dried over
magnesium sulfate, filtered, and the solvent was evaporated under
reduced pressure. The brown crude product was recrystallized
Concluding remarks
Even though both copper(II) complexes are soluble in water and
cleave φX174 DNA, under more than stoichiometric conditions
without the presence of any reductant, both complexes fail to pro-
duce catalytic double-strand DNA cleavage. The DNA cleavage
studies with various concentrations of the complexes performed
on φX174 phage DNA and in the presence of different additives
including selected radical scavengers, indicate the mechanism of
DNA cleavage to be oxidative with the involvement of reactive
oxygen species, such as hydroxyl radicals. Religation and cell-
ꢀ
ꢀ
twice from pentane and hexane. 6,6 -Dimethyl-2,2 -bipyridine was
1
isolated as a slightly brown powder (yield = 1.1 g, 50%). H-NMR
ꢀ
(CDCl
3
, 300 MHz) d 2.63 (s, 6H, CH
3
–C(6,6 )), 7.15 (d, 2H, H–
ꢀ
ꢀ
ꢀ
C(5,5 )), 7.68 (t, 2H, H–C(3,3 )), 8.18 (d, 2H, H–C(4,4 )) ppm.
This journal is © The Royal Society of Chemistry 2007
Dalton Trans., 2007, 3676–3683 | 3681

View Article Online
ꢀ
ꢀ
ꢀ
6
,6 -Bis(bromomethyl)-2,2 -bipyridine. A mixture of 6,6 -di-
a broad signal without anisotropy centred around a g value of
2.124. Frozen solution EPR in a MeOH glass, shows an axially
ꢀ
methyl-2,2 -bipyridine (2.76 g, 15.5 mmol) and N-bromosuccini-
mide (5.10 g, 28.6 mmol) in 150 mL of CCl
4
was refluxed for
resolved spectrum with gꢁ = 2.22 and Aꢁ = 175 G, with g
⊥
as
3
0 min, and 30 mg of benzoyl peroxide were subsequently added.
2.054.
The resulting mixture was refluxed overnight, and the resulting
succinimide by-product was filtered off. The solution was cooled
X-Ray crystal structure determinations. Crystallographic data
and refinement details are given in Table 3. A crystal was selected
for the X-ray measurements and mounted to the glass fiber using
◦
to 0 C, and the ensuing precipitate was filtered and washed with
ꢀ
ꢀ
MeOH to give 0.8 g of 6,6 -bis(bromomethyl)-2,2 -bipyridine. The
CCl solution was evaporated under reduced pressure yielding a
slightly yellow powder. This powder was washed with a solution
of MeOH and CH Cl (2 : 98). The insoluble part gives another
46
the oil drop method and data were collected at 173 K on a
Nonius Kappa CCD diffractometer (Mo Ka radiation, graphite
monochromator, k = 0.71073 nm). The intensity data were cor-
rected for Lorentz and polarization effects, and for absorption. The
4
2
2
1
crop of product 0.3 g (total yield = 1.1 g, 21%). H-NMR (CDCl
3
,
47
48
49
programs COLLECT, SHELXS-97, SHELXL-97 were used
for data reduction, structure solution and structure refinement,
respectively. The non-hydrogen atoms were refined anisotropically.
The H atoms were introduced in calculated positions and refined
with fixed geometry with respect to their carrier atoms. Rotation
about the exocyclic C–C bonds was allowed in the refinements
of the riding methyl H atoms at C26 and C27 in compound
1. Rotation about the exocyclic C–C bonds was allowed in the
refinements of the riding methyl H atoms at C36 and C37 in
compound 2.
3
00 MHz) d 4.63 (s, 4H, CH
2
–Br), 7.47 (dd, J = 7.7; 0.8 Hz,
ꢀ
ꢀ
2
H, H–C(5,5 )), 7.82 (t, J = 7.8 Hz, 2H, H–C(4,4 )), 8.39 (dd,
ꢀ
J = 7.9; 0.7 Hz, 2H, H-C(3,3 )) ppm. The reaction also gives
other brominated side products which can be separated by column
chromatography.
ꢀ
ꢀ
6
,6 -Bis(3,5-dimethyl-N-pyrazolmethyl)-2,2 -bipyridine (mbpz-
ꢀ
bpy). The compound mbpzbpy was synthesized from 6,6 -bis-
bromomethyl)-2,2 -bipyridine (1.0 g, 2.92 mmol), 3,5-dimethyl-
ꢀ
(
pyrazole (0.62 g, 6.5 mmol) and NaH (0.15 g, 6.5 mmol). The
product was obtained as a white microcrystalline powder. (Yield =
1
DNA cleavage studies. Cleavage experiments were performed
using φX174 supercoiled DNA purchased from Invitrogen Life
Technologies. Typical reactions were carried out using 20 lM
DNA (base pairs) in 10 mM phosphate buffer, and incubated
0
.66 g, 94%). H-NMR (CDCl
3
, 300 MHz) d 2.24 and 2.26 (s, 6H,
CH
3
–pyrazol), 5.40 (s, 4H, CH
2
–py), 5.89 (s, 2H, CH–pyrazol),
ꢀ
6
.83 (d, J = 7.6 Hz, 2H, H–C(5,5 )), 7.72 (t, J = 7.8 Hz, 2H,
ꢀ
ꢀ
13
H–C(4,4 )), 8.27 (d, J = 7.7 Hz, 2H, H–C(3,3 )) ppm, C-NMR
◦
at 37 C for 2 h. Reactions with various concentrations of copper
(CDCl , 300 MHz) d 11.2; 13.5; 54.5; 105.7; 119.7; 121.0; 137.9;
3
+
complex, from 20 lM to 220 lM, were performed. The reactions
were stopped by the addition of loading buffer (bromophenol
blue, xylene cyanol, and 25% ficoll). The cleavage reactions were
analyzed by agarose gel electrophoresis. The reaction samples were
loaded on 0.8% agarose gel containing ethidium bromide, and were
run at 80 mV for 60–90 min in a TBE buffer. After washing with
de-ionized water, the gels were documented using a BioRad Gel
Doc 1000 apparatus. Additional reactions were performed in the
presence of several additives. Thus, standard radical scavengers
139.8; 148.0; 155.4 and 156.8 ppm, MS (m/z) 372 (M , 100); IR
−
1
1572, 1550, 1425, 785, 780, 602 cm .
Synthesis of the copper complexes
Cu(mbpzbpy)Br ](H O)2.5 (1). 0.268 mmol of ligand (0.1 g)
[
2
2
were dissolved in warm absolute methanol (10 mL). Then, a
solution of 0.268 mmol of CuBr (0.059 g) in methanol was added
2
dropwise to the ligand solution under stirring. The solution was
stirred for 10 min and filtered. The filtrate was left in air for the
slow evaporation of the solvent. Dark blue crystals of 1, suitable
for X-ray diffraction, were obtained after one week (Yield = 18%).
such as NaN
(100 lM), and D
3
(100 lM), SOD (0.5 units), DMSO, distamycin
O were used. The reactions were also carried out
2
in the presence of ascorbic acid (20 lM), excess sodium chloride
(350 lM), and under anaerobic and dark conditions.
−
1
IR (neat, cm ): 2360, 1608, 1578, 1554, 1471, 1427, 1395, 1296,
1
032, 861, 792, 691. UV-Vis (neat, nm): 472 (vide infra). Anal. calcd
CuN 2.50: C, 41.23; H, 4.56; N, 13.11%. Found: C,
1.20; H, 4.46; N, 13.11%. The powder EPR spectrum exhibits a
broad signal with a small anisotropy, centred around a g value of
.077. Frozen solution EPR in a MeOH glass, shows a nice axially
resolved spectrum with gꢁ = 2.22 and Aꢁ = 178 G, with g as
.048.
Religation studies. The cleavage of pUC19 plasmid DNA
were carried out essentially as described above. The cleavage of
DNA achieved by the copper complexes was further analysed by
gel electrophoresis. Additionally, positive and negative controls
were carried out using undigested pUC19 and pUC19 treated
with the restriction enzyme BamHI. The reactions mixtures were
purified using a QIAGEN PCR purification kit, and the accurate
DNA concentrations were measured using a NanoDrop ND-1000
spectrophotometer. 50 ng of sample DNA were religated in a
solution of 2 lL of 10X ligation buffer with 1 unit of T4 DNA
ligase in a total volume of 20 lL. Negative controls without ligase
were also included. The reactions were incubated overnight at
for C22
4
H
29Br
2
6
O
2
⊥
2
[
Cu(mpzbpya)Cl](CH
were dissolved in warm absolute methanol (10 mL). Then, a
solution of 0.268 mmol of CuCl O (0.046 g) in methanol
was added dropwise to the ligand solution under stirring. The
solution was stirred for 10 min and filtered. The filtrate was left
in air for the slow evaporation of the solvent. Light green plates
of 2, suitable for X-ray diffraction, were obtained after two weeks
3
OH) (2). 0.268 mmol of ligand (0.1 g)
2
·6H
2
◦
16 C. These reactions were then used to transform 200 lL of
competent DH5a cells. The cells were heat-shocked for 2 min in
−
1
◦
◦
(
1
Yield = 32%). IR (neat, cm ): 3353, 2360, 1601, 1575, 1552, 1432,
289, 1054, 792, 648. UV-Vis (neat, nm): 495 (vide infra). Anal.
calcd for C18 : C, 49.34; H, 4.37; N, 12.78%. Found:
C, 49.31; H, 4.36; N, 12.70%. The powder EPR spectrum exhibits
a 42 C water bath, and then incubated at 37 C for 20 min,
following the addition of 800 lL of medium. These samples were
then plated on LB-agar containing carbenicillin at three different
concentrations, i.e. 10, 100, and 900 lL. The plates were incubated
H
19ClCuN
4
O
3
3
682 | Dalton Trans., 2007, 3676–3683
This journal is © The Royal Society of Chemistry 2007

View Article Online
◦
overnight at 37 C. All ligation reactions performed were carried
25 M. X. Li and N. Q. Li, Chem. J. Chin. Univ., 2001, 22, 1230–
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232.
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2
6 A. Decker, M. S. Chow, J. N. Kemsley, N. Lehnert and E. I. Solomon,
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Dalton Trans., 2007, 3676–3683 | 3683