Synthesis, characterization, DNA interaction and cleavage activity of new mixed ligand copper(II) complexes with heterocyclic bases

Article abstract of DOI:10.1016/j.poly.2006.08.026

Mixed ligand complexes having the formulae Cu(RPO)₂Py₂, Cu(RPO)₂Im₂ and Cu(DBO)₂Py₂ [RPO = resacetophenone oxime, DBO = 2,4-dihydroxybenzophenone oxime, Py = pyridine and Im = imidazole] have been synthesized and characterized by UV-Vis, IR, ESR, cyclic voltammetry and magnetic susceptibility methods. Absorption studies revealed that each of these octahedral complexes is an avid binder of calf thymus DNA. The apparent binding constants for mixed ligand complexes are in order of 10⁴-10⁵ M^-1. Based on the data obtained in the DNA binding studies a partial intercalative mode of binding is suggested for these complexes. The nucleolytic cleavage activity of the adducts was carried out on double stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment in the presence and absence of oxidant (H₂O₂). All the metal complexes cleaved supercoiled DNA by hydrolytic and oxidative paths. The oxidative path dominates the hydrolytic cleavage. The hydrolytic cleavage of DNA is evidenced from the control experiments showing discernable cleavage inhibition in the presence of the hydroxyl radical inhibitor DMSO or the singlet oxygen quencher azide ion.

Full text of DOI:10.1016/j.poly.2006.08.026

Polyhedron 26 (2007) 572–580
www.elsevier.com/locate/poly
Synthesis, characterization, DNA interaction and cleavage activity
of new mixed ligand copper(II) complexes with heterocyclic bases
a
a,
b
M.S. Surendra Babu , K. Hussain Reddy *, Pitchika G. Krishna
a
Department of Chemistry, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh 515003, India
Department of Zoology, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh 515003, India
b
Received 20 June 2006; accepted 16 August 2006
Available online 9 September 2006
Abstract
Mixed ligand complexes having the formulae Cu(RPO)2Py2, Cu(RPO)2Im2 and Cu(DBO)2Py2 [RPO = resacetophenone oxime,
DBO = 2,4-dihydroxybenzophenone oxime, Py = pyridine and Im = imidazole] have been synthesized and characterized by UV–Vis,
IR, ESR, cyclic voltammetry and magnetic susceptibility methods. Absorption studies revealed that each of these octahedral complexes
is an avid binder of calf thymus DNA. The apparent binding constants for mixed ligand complexes are in order of 104–105 Mꢀ1. Based
on the data obtained in the DNA binding studies a partial intercalative mode of binding is suggested for these complexes. The nucleolytic
cleavage activity of the adducts was carried out on double stranded pBR322 circular plasmid DNA by using a gel electrophoresis exper-
iment in the presence and absence of oxidant (H2O2). All the metal complexes cleaved supercoiled DNA by hydrolytic and oxidative
paths. The oxidative path dominates the hydrolytic cleavage. The hydrolytic cleavage of DNA is evidenced from the control experiments
showing discernable cleavage inhibition in the presence of the hydroxyl radical inhibitor DMSO or the singlet oxygen quencher azide ion.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Mixed ligand copper(II) complexes; Oximes; Aromatic bases; CT DNA; Binding interaction; Hydrolytic cleavage
1. Introduction
light of the above and in continuation of our on going
research work [9,10] on metal–DNA interactions herein
we describe the synthesis characterization, DNA binding
and cleavage activity of copper(II) complexes with resace-
tophenone oxime (2,4-dihydroxyacetophenone oxime)
and 2,4-dihydroxybenzophenone oxime and their pyridine
and imidazole adducts.
Oximes are widely recognized ligands [1–5]. Transition
metal complexes with O-hydroxy aromatic oximes (e.g.
salicylaldoxime (SAO)) have attracted much attention, as
they exist as cis and trans geometrical isomers. Copper
complexes are known to assume trans structures while
cobalt complexes have cis structures. We have investigated
several cobalt complexes with O-hydroxy carbonyl Schiff
bases (tetradentate ligands) containing additional hydroxyl
groups [6]. Such additional groups have been exploited to
anchor the complexes on a polymer support [7].
2. Experimental
2.1. Chemical
Investigation on DNA interactions and cleavage activity
of metal complexes with oximes are quite limited [8]. In
2,4-Dihydroxybenzophenone and resacetophenone were
purchased from Merck, and metals used in the preparation
of the complexes are of reagent grade. The solvents used in
the synthesis of the ligands and metal complexes were dis-
tilled before use. All other chemicals were of AR grade and
were used without further purification. Agarose, used in gel
*
Corresponding author. Tel.: +91 8554 224347/255752; fax: +91 8554
229043.
E-mail address: Khussainreddy@yahoo.co.in (K.H. Reddy).
0277-5387/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2006.08.026

M.S. Surendra Babu et al. / Polyhedron 26 (2007) 572–580
573
electrophoresis, was purchased from Sigma–Aldrich, Calf
thymus DNA and plasmid pBR322 were purchased from
Genie Biolabs, Bangalore, India.
where ea, ef and eb are the apparent free and bound metal
complex extinction coefficients, respectively. A plot of
[DNA]/(ea ꢀ ef) versus [DNA] gave a slope of 1/(eb ꢀ ef)
and a Y-intercept equal to 1/Kb (ea ꢀ ef), Kb is the ratio
of the slope to the Y-intercept.
2.2. Instrumentation
2.4. Assay of nuclease activity
The elemental analyses were performed using a Perkin–
Elmer 2400 CHNS elemental analyzer. Magnetic moments
were determined in the polycrystalline state on a PAR
model-155 vibrating sample magnetometer operating at
field strength of 2–8 kG. High purity Ni metal (saturation
moment 55 emu/g) was used as standard. The molar con-
ductance of the complexes in DMF (10ꢀ3 M) solution
was measured at 28 2 ꢁC with a systronic model 303
direct-reading conductivity bridge. The electronic spectra
were recorded in DMF with a Schimadzu UV-160A spec-
trophotometer. FT-IR spectra were recorded in the range
4000–50 cmꢀ1 with a Bruker IFS 66V in KBr and polyeth-
ylene medium. ESR spectra were recorded on Varian E-122
X-band spectrophotometers at liquid nitrogen temperature
in DMF. The voltammetric measurements were performed
on Bio-Analytical Systems (BAS), CV-27 assembly in con-
junction with an X–Y recorder. Measurements were made
on the degassed (N2 bubbling for 5 min) solution in
DMF (10ꢀ3 M) containing 0.1 M tetraethyl ammonium
perchlorate as the supporting electrolyte. The three-
electrode system consisted of a glassy carbon (working),
platinum (auxiliary) and Ag/AgCl (reference) electrodes.
UV–Vis spectra were recorded on a Tech-Comp UV-8500
instrument.
A DMF solution containing the metal complexes (1 ll
of 1 mM) was taken in a clean eppendroff tube and plasmid
DNA (1 ll of 0.10 lg/ml) was added. The contents were
incubated for 2 h at 37 ꢁC and loaded on 1% agarose gel
after mixing 5 ll of loading buffer (0.25% bromophenol
blue + 25% xylenecyanol + 30% glycerol sterilized dis-
tilled). Electrophoresis was performed at constant voltage
(80 V) until the bromophenol blue reached to 3/4 of the
gel. Then the gel was stained for 10 min by immersing it
in ethidium bromide solution. The gel was then de-stained
for 10 min by keeping it in sterile distilled water and the
plasmid band was visualized by viewing the gel under a
transilluminator and photographed. The efficiency of the
DNA cleavage was measured by determining the ability
of the complex to form open circular (OC) or nicked circu-
lar (NC) DNA from its supercoiled (SC) form by quantita-
tively estimating the intensities of the bands using the
Vilber Lourmat (V 99.01) Gel Documentation System.
The reactions were carried out under hydrolytic conditions.
A control experiment was done in presences of hydroxyl
radical scavenger DMSO and singlet oxygen quencher
azide ion.
3. Preparation
2.3. DNA binding experiments
3.1. Preparation of ligands
A solution of CT DNA in 0.5 mM NaCl/5 mM Tris–
HCl (pH 7.0) gave a ratio of UV absorbance at 260 and
280 nm, A260/A280 of 1.8–1.9, indicating DNA was suffi-
ciently free of protein. A concentrated stock solution of
DNA was prepared in 5 mM Tris–HCl/50 mM NaCl in
water, pH 7.0 and the concentration of CT DNA were
determined by UV absorbance at 260 nm after 1:100 dilu-
tions (in 250 lM in NP). The molar absorption coefficient
was taken as 6600 Mꢀ1 [11]. Stock solutions were stored at
4 ꢁC and were used after no more than four days. Doubly
distilled water was used to prepare the buffer solutions.
Solution were prepared with the appropriate copper com-
plexes (35 lM of a 1.0 mM solution in DMF), CT DNA
(diluted from a 1 mg/ml solution), NaCl (final concentra-
tion 50 mM) and Tris–HCl buffer (pH 7.0, final concentra-
tion 50 mM) and diluted with H2O to a total volume of
1 ml. After equilibration (ca. 10 min), the spectra were
recorded against an analogous blank solution containing
the same concentration of DNA/NaCl and Tris–HCl
buffer.
Resactophenone oxime (RPO) and 2,4-dihydroxybenzo-
phenone oxime (DBO) ligands were prepared by the
reaction of hydroxylammoniumchloride (NH2OH Æ HCl)
with resacetophenone and 2,4-dihydroxybenzophenone,
respectively.
Hydroxylammoniumchloride (0.03 mol) and resaceto-
phenone (0.03 mol) were taken in 50% aqueous methanol
or 2,4-dihydroxybenzophenone (0.03 mol) in benzene sol-
vent. The resulting mixture was acidified with a few drops
of glacial acetic acid and refluxed for 2–3 h. The precipi-
tated compound was filtered, washed with cold water dried
under vacuum and recrystallized from aqueous ethanol.
The infra red spectrum of RPO and DBO shows strong
and broad bands due to hydrogen-bonded phenolic OH at
20-position in the region 3000–2800 cmꢀ1. They also exhibit
two separate OH bands due to oxime OH at 3237,
3137 cmꢀ1 and phenolic OH at 3400, 3350 cmꢀ1, respec-
tively, in addition to ˜C@N stretching at 1598, 1588
cmꢀ1, respectively. The 1H NMR spectra of RPO and
DBO were recorded in DMSO-d6 solvent. RPO shows
signals at 2.22 (s, 3H), 6.42–7.21 (m, 3H), 9.15 (s, 1H)
10.85 (s, 1H) and 11.79 (s, 1H) assigned to –CH3, –C6H3,
The data were then fitted to Eq. (1) to obtain the intrin-
sic binding constant, Kb [12]
½DNAꢁ=ðea ꢀ ef Þ ¼ ½DNAꢁ=ðeb ꢀ ef Þ þ 1=Kbðea ꢀ ef Þ;
ð1Þ

574
M.S. Surendra Babu et al. / Polyhedron 26 (2007) 572–580
DBO (0.05 mol)] in a 1:2 ratio in 50% aqueous ethanolic
medium. The reaction mixture was stirred for 30 min,
and the precipitate formed was filtered, washed with hot
water and cold methanol. The complexes were dried at
110 ꢁC.
The copper(II) complex (1.5 g) of RPO or DBO was
placed in a schlenk tube and dissolved in pyridine (3 ml).
The solution was stirred magnetically for 30 min and n-hex-
ane (25 ml) was added. After standing for 3–4 days, the
dark green product that formed was washed with water
and n-hexane and dried under reduced pressure over CaCl2.
Cuproxime of RPO (0.01 mol) was placed in a 250-ml
round bottom flask. Imidazole (0.05 mol) dissolved in
CH2Cl2 was added to the contents of the flask. The reaction
mixture was refluxed on a water bath for 2 h. On cooling the
dark green precipitate that formed was filtered, washed with
cold hexane and dried under vacuo over CaCl2.
-1.0
-1.4
-1.6
-1.8
-1.2
-0.8
-0.4 -0.6
E/V
Fig. 1. Cyclicvoltametric profile diagram of RPO (------) and DBO (——).
ortho phenolic OH, para phenolic OH and @N–OH
(oxime), respectively. Signals corresponding to –C6H3,
–C6H5, two phenolic OH’s and oxime-OH are, respectively,
observed in the NMR spectrum of DBO at 6.30–6.45 (m,
3H), 7.22–7.80 (m, 5H), 10.09–10.42 broad (2H) and
12.45 (s, 1H) d values, respectively. The mass spectrum of
RPO shows a molecular ion peak at 167 (m/z) correspond-
ing to its molecular weight. The other important peaks at
m/z 150 and 135 are due to loss of –OH and –CH3 from
the molecular ion peak. A peak at 121 corresponds to the
4. Results and discussion
The present ligands contain two functional groups viz.,
oxime and phenolic groups (Fig. 2). The ligands and their
metal complexes (both the cuproximes and their adducts)
are stable at room temperature, are non-hygroscopic, solu-
ble partially in methanol, ethanol and easily soluble in
dimethylformamide. The colour, melting point, analytical
data, molar conductance and magnetic moment data are
summarized in Table 1. Elemental analysis supports the
present composition of the complexes. The molar conduc-
tivity data suggest that the complexes are non-electrolytes.
The magnetic moment values of the complexes indicate the
þ
formation of the HOC6H3NCH3 ion. The mass spectrum
of DBO shows peaks at 229, 213, 183 and 108 correspond-
ing to the molecular ion (M+Å), M+Åꢀ16, HOC6H3N–Ph+Å
OH
and
+., respectively.
O
The presence of only cathodic peaks [ꢀ1.35 and ꢀ1.83
(RPO); ꢀ1.42 and ꢀ1.87 V (DBO)] in cyclicvoltammo-
grams (Fig. 1) of the ligands suggest an irreversible reduc-
tion of the azomethine group. Each peak corresponds to a
one electron reduction. A possible mechanism (Scheme 1)
is proposed for the reduction of these ligands.
R
HO
C=N
OH
3.2. Synthesis of cuproxime complexes and their pyridine and
imidazole adducts
OH
Cuproximes were prepared by mixing copper(II) chlo-
ride (4.3 g, 0.025 mol) and oxime [RPO (0.05 mol) or
Fig. 2. Structure of ligands; where R = CH3, RPO; R = C6H5, DBO.
R
R
-
+
-
H
-e,
C
N
HO
OH
C
NH
OH
HO
OH
OH
+
-
H
-e,
R
CH
NH
OH
HO
OH
Scheme 1. A possible mechanism for reduction of oxime ligands.

M.S. Surendra Babu et al. / Polyhedron 26 (2007) 572–580
575
Table 1
Physical properties of copper complexes of RPO and DBO and their base adducts
S. no. Complexes
Colour
Melting
point (ꢁC)
Elemental analysis
Molar conductance leff (BM)
(Xꢀ1 cm2 molꢀ1
)
Carbon observed Hydrogen observed Nitrogen observed
(calulated)
(calulated)
(calulated)
I
II
III
IV
V
Cu(SAO)2
Cu(RPO)2
Cu(RPO)2Py2 dark green
Cu(RPO)2Im2 bluish green 185–188
Cu(DBO)2
green
green
208–210
270–275
193–196
50.32 (50.27)
48.12 (48.31)
52.15 (52.82)
48.75 (48.94)
52.17 (52.49)
53.48 (53.12)
3.38 (3.5)
7.99 (8.12)
7.12 (7.32)
8.22 (8.39)
11.74 (12.58)
4.62 (4.76)
7.61 (7.65)
5.8
16.8
9.0
7.5
12.2
8.5
2.18
2.83
2.12
2.32
2.34
2.78
4.32 (4.52)
4.48 (4.82)
4.78 (4.74)
3.49 (3.52)
3.58 (3.49)
green
224–228
215–217
VI
Cu(DBO)2Py2 dark green
presence of one unpaired electron. The copper salicyl-
aldoxime complex [Cu(SAO)2] is used for comparative
studies.
the far IR spectra of the metal complexes are assigned to
m(M–N) (265, 271 and 289 cmꢀ1) m(M–O) (576, 589 and
595 cmꢀ1
)
stretching vibrations. Additional bands
The electronic spectral data of the metal complexes are
given in Table 2. The spectral data of the complexes are
dominated by intense intra-ligand (p–p*) and charge trans-
fer (CT) bands. The presence of a single d–d band may be
attributed to the symmetric nature of the ligand field. In the
electronic spectra of the mixed ligand complexes, d–d
bands are observed in the high-energy region (Table 2)
due to Jahn–Teller distortion observed in octahedral
complexes.
observed (1610–1600, 1540–1520, 260–220, 245–230 cmꢀ1
)
in the IR spectra of the mixed ligand complexes are pre-
sumably due to the binding of bases (pyridine/imidazole)
to copper via nitrogen donor atoms, preferably in axial
positions.
Oxime OH stretching vibrations are observed at a lower
frequency in the chelates (3221 and 3128 cmꢀ1), indicating
the involvement of oxime OH in strong hydrogen bonding
which leads to the formation of a stable 5-membered ring
structure. Observance of three evenly distributed bands in
the 620–450 cmꢀ1 region is characteristic of a trans-struc-
ture for the complexes [13]. IR spectral data together with
electronic and magnetic moment data suggest a trans
square planar structure for the cuproximes and a trans
octahedral structure for the mixed ligand copper(II) com-
plexes (Fig. 3a and b).
ESR spectra of all the complexes are recorded in DMF
solvent at liquid nitrogen temperature (LNT). The ESR
spectra of the copper(II) complexes exhibit a set of four
well-resolved peaks in the low field region and a weaker sig-
nal in high field region, corresponding to gi and g^, respec-
tively (Fig. 4). The gi and g^ values are 2.245, 2.019 for
Cu(RPO)2 and 2.218, 2.015 for Cu(DBO)2, respectively.
The trend observed, gi > g^ > 2.02, for the present copper
complexes is typical of a copper(II) (d9) ion in axial
symmetry with the unpaired electron present in the dx2ꢀy2
orbital. The gi value of the parent complexes being less
than 2.3 is indicative of significant covalent bonding
The broad band in the IR region 3000–2800 cmꢀ1 in
RPO and DBO are absent in the corresponding copper che-
lates suggesting the deprotonation of the 20-OH phenolic
group due to formation of a covalent bond between the
copper and phenolic oxygen. Strong bands observed at
1598 and 1588 cmꢀ1 assigned to ˜C@N– stretching vibra-
tion in the ligands are shifted to lower frequency in the
complexes revealing the participation of the azomethine
nitrogen in chelation. The non-ligand bands observed in
Table 2
Electronic spectral data (cmꢀ1) of the copper(II) complexes
Complex
p–p*
Charge transfer (CT)
d–d
I
II
III
IV
V
35710
38460
35710
36360
37470
36410
28570
29850
28250
28960
29760
27760
14950
15625
15380
15150
16390
15880
VI
OH
a
b
OH
O
H
O
H
R
R
B
O
O
C=N
C=N
Cu
Cu
N=C
N=C
O
O
B
R
R
H
O
H
O
HO
HO
Fig. 3. (a) Structure of copper(II) complexes where R = CH3, RPO; R = C6H5, DBO. (b) Structure of copper(II) adducts where R = CH3, RPO;
R = C6H5, DBO; B = pyridine/imidazole.

576
M.S. Surendra Babu et al. / Polyhedron 26 (2007) 572–580
Fig. 4. X-band ESR spectra of the Cu(RPO)2Py2 complex at liquid nitrogen temperature (LNT) in DMF.
[14,15] in the complexes. The orbital reduction parameters
data (Ki > K^) in Table 3 suggest that the present com-
plexes have out-of-plane p-bonding [16].
Massacesi et al. [17,18] reported gi values in the range
2.3–2.4 for complexes containing copper–oxygen bonds
and 2.2–2.3 for complexes containing copper–nitrogen
bonds. The present copper oxime complexes have
gi = 2.2–2.4 in conformity with both Cu–O and Cu–N
bonds in the cuproximes and their adducts.
The redox behavior of these complexes has been investi-
gated by cyclic voltammetry at a glassy carbon electrode.
Table 4 gives the electrochemical data obtained at a glassy
carbon electrode in DMF solution. The cathodic peak cur-
rent function values were found to be independent of the
scan rate. Repeated scans, as well as different scan rates
showed that dissociation does not take place. Fig. 5 shows
the profile of Cu(RPO)2Py2 and Cu(DBO)2Py2 complexes
at 50 mV sꢀ1 scan rates.
The P values for a complex containing N2O2 donor
atoms fall in the range 0.022–0.029 cmꢀ1 [19]. The P val-
ues for the mixed ligand complexes are consistent with
bonding of copper with nitrogen and oxygen donor atoms
[20].
The copper complexes are redox active and show a
cyclic voltammetric response in the potential range of
0.35–0.60 V which is assignable to the reduction peak of
Cu(II)/Cu(I). All these complexes exhibit quasi-reversible
behavior [21] as indicated by the non-equivalent current
Table 3
Spin Hamiltonian and orbital reduction parameters of the copper(II) complexes
Complex
gi
g^
gav
G
Ki
K^
Ai
A^
Aav
a2
P
II
2.245
2.346
2.354
2.218
2.236
2.019
2.015
2.086
2.015
2.069
2.094
2.073
2.175
2.096
2.158
8.078
5.264
4.202
10.212
5.007
0.7657
0.9111
0.9121
0.8161
0.8987
0.4017
0.7945
0.7725
0.1615
0.8028
0.0147
0.0279
0.0214
0.0172
0.0149
0.0075
0.0139
0.0055
0.0055
0.0139
0.0098
0.0187
0.0157
0.0094
0.0143
1.5597
0.7532
0.9758
1.1918
1.3112
0.0261
0.0287
0.0289
0.0296
0.0276
III
IV
V
VI
Table 4
Cyclic voltammetric data of RPO and DBO copper(II) complexesa
ꢀDGꢂc
b
Complex
Redox couple
CV cathodic, Epc
CV anodic, Epa
DEp (mV)
250
250
170
210
200
170
E1/2
ꢀic/ia
logKc
I
II/I
II/I
II/I
II/I
II/I
II/I
0.34
0.35
0.38
0.36
0.38
0.42
0.59
0.60
0.55
0.57
0.58
0.59
0.47
0.47
0.45
0.46
0.48
0.51
1.18
1.28
1.09
1.13
1.29
1.04
178.13
133.11
195.58
158.33
166.25
195.58
8.12 · 105
7.72 · 105
11.35 · 105
9.19 · 105
9.65 · 105
11.35 · 105
II
III
IV
V
VI
a
Recorded in DMF at room temperature with Et4NClO4 as supporting electrolyte, glassy carbon as working electrode, Pt wire as auxiliary electrode
and Ag/AgCl as reference electrode; scan rate 50 mV sꢀ1
.
b
logKc = 0.434ZF/RTDEp.
c
DGꢂ = ꢀ2.303RTlogKc.

M.S. Surendra Babu et al. / Polyhedron 26 (2007) 572–580
577
Table 5
Effect of CT DNA on the absorbance bands and binding constant of the
copper complexes and adducts
Complex
kmax (nm)
Dk (nm)
H (%)
Kb (Mꢀ1
)
Free
Bound
I
341
342.5
274.5
274.2
272.4
284.8
345.2
290.2
324.2
1.5
1.5
1.3
2.1
2.3
12.8
1.3
1.3
ꢀ16.6
ꢀ12.8
+11.8
+12.4
ꢀ15.6
ꢀ23.2
+4.8
2.5 · 104
3.8 · 105
2.6 · 104
2.1 · 105
9.9 · 105
II
III
IV
V
276.0
275.5
274.5
287.1
357.0
291.5
325.5
VI
1.4 · 104
+0.5
hypsochromic shifts (kmax: 1.0 0.5 nm) and hypochro-
mism [hypochromicity: 12.8% for Cu(RPO) and 15.4%
for Cu(DBO)]. While the mixed ligand complexes showed
hypsochromic (kmax: 1.0 0.5 nm) and hyperchromism
[hyperchromicity: +11.8%, +12.4% and +4.8%]. The
change in hypochromic to hyperchromic shift from parent
complex to mixed ligand complexes suggests that the type
of binding mode is different (Fig. 6). This may be attributed
to a change in structure from square planar (parent) to
octahedral nature (adducts). The binding nature in the
mixed ligand complexes is significant due to p-stacking or
a hydrophobic interaction of the aromatic pyridine or
imidazole bases. The binding constants of the parent com-
plexes are sufficiently high, possibly due to their square pla-
nar nature [24]. A strong binding constant for II and V
compared with I may be attributed to the presence of an
extra OH group in complexes II and V, while V has the
highest binding constant, which may be due to an increased
p-stacking interaction facilitated by the presence of extra
phenyl rings [25].
Fig. 5. Cyclic voltametric profile of (a) Cu(RPO)2Py2 (——); (b)
Cu(DBO)2Py2 (------).
intensity of cathodic and anodic peaks (ic/ia = 0.512–
0.322 V). All complexes have a large separation between
the anodic and cathodic peaks (100–250 mV) indicating
the quasi-reversible character. The DGꢂ values of the mixed
ligand complexes are greater than the parent complexes,
showing that these complexes are more stable than their
parent complexes.
The E1/2 values of the adducts and complexes are com-
parable with other complexes showing nuclease activity
[22]. The E1/2 values of the parent complexes and mixed
ligand complexes are high suggesting that the complexes
undergo a more facile redox change which is the require-
ment for DNA cleavage [23].
4.2. Nuclease activity
Gel electrophoresis experiments using pBR322 circular
plasmid DNA were performed with the ligands and their
complexes in the presence and absence of H2O2 as an oxi-
dant. At micro-molar concentrations, for 2 h incubation
periods, the ligands exhibit no significant cleavage activity
in the absence or presence of oxidant (H2O2). The nuclease
activity is greatly enhanced by the incorporation of copper
ion in the respective ligands. The nuclease activity of the
complexes was also investigated in the presence of a free
radical scavenger, dimethylsulfoxide (DMSO) and a singlet
oxygen quencher, azide ion (NaN3).
From Fig. 7a it is evident that the complexes cleave
DNA more efficiently in the presence of an oxidant. This
may be attributed to the formation of hydroxyl free
radicals. The production of a hydroxyl radical due to the
reaction between the metal complex and oxidant may be
explained as shown below [26].
4.1. Electronic absorption titrations
The binding interaction of the copper complexes with
DNA was monitored by UV–Vis spectroscopy. The
absorption spectra of the copper complexes are compared
with and without CT DNA. In the UV region spectra, all
the copper(II) complexes exhibit an intense absorption
band around 280 nm which is attributed to an n–p* or
p–p* transition. With increasing concentration of CT
DNA, the absorption bands of the complexes are affected,
resulting in the obvious tendency of hyperchromism or
hypochromism with a hypsochromic shift (1–4 nm). The
change in absorbance values with increasing amount of
CT DNA were used to evaluate the intrinsic binding con-
stant Kb for the present complexes (Table 5).
In the presence of increasing amounts of CT DNA, the
UV–Vis absorption of Cu(RPO)2 and Cu(DBO)2 showed
(L)Cu2þ + H2O2 ! (L)Cu3þ + OH + OHÅ

578
M.S. Surendra Babu et al. / Polyhedron 26 (2007) 572–580
Fig. 6. Variation in absorption spectra of: (i) Cu(RPO)2, (ii) Cu(RPO)2Py2, and (iii) Cu(RPO)2Im2 in the absence and presence of increasing amounts of
DNA. [CuL] = 35 lM. Arrows show the absorbance changes upon increasing the DNA concentration.
Fig. 7a. Agarose gel (1%) showing the results of electrophoresis of 1 ll of (0.10 lg/ml) pBR 322 plasmid DNA, 2 ll of 0.1 M Tris–HCl (pH 8.0) buffer: 1 ll
(1 M) complex in DMF; 10 ll of sterilized distilled water and 2 ll of 9.0 mM H2O2 were added, incubation at 37 ꢁC (120 min): Lane 1: Marker; lane 2:
DNA (control); lane 3: DNA + H2O2 (control); lane 4: DNA + Cu(RPO)2; lane 5: DNA + Cu(RPO)2 + H2O2; lane 6: DNA + [Cu(RPO)2]Py2; lane 7:
DNA + [Cu(RPO)2]Py2 + H2O2; lane 8: DNA + [Cu(RPO)2]Im2; lane 9: DNA + [Cu(RPO)2]Im2 + H2O2; lane 10: DNA + [Cu(DBO)2]; lane 11:
DNA + [Cu(DBO)2] + H2O2; lane 12: DNA + [Cu(DBO)2]Py2; and lane 13: DNA + [Cu(DBO)2]Py2 + H2O2.
Table 6
Selected SC pBR322 DNA cleavage data of copper complexes in Fig. 7a
Lane no.
Reaction condition
Percentage of form I
Percentage of form II
Percentage of form III
2
3
4
5
6
7
8
9
10
11
12
13
DNA
DNA + H2O2
DNA + Cu(RPO)2
92.5
92.0
79.6
69.9
64.4
ND
51.5
28.5
65.5
20.6
ND
ND
7.5
8.0
ND
ND
3.3
17.1
21.9
34.6
57.2
25.2
50.5
21.3
45.8
73
DNA + Cu(RPO)2 + H2O2
DNA + Cu(RPO)2Py2
DNA + Cu(RPO)2Py2 + H2O2
DNA + Cu(RPO)2Im2
DNA + Cu(RPO)2Im2 + H2O2
DNA + Cu(DBO)2
DNA + Cu(DBO)2 + H2O2
DNA + Cu(DBO)2Py2
DNA + Cu(DBO)2Py2 + H2O2
6.5
ND
39.8
23.3
21.0
13.2
32.4
27
51.4
48.6
ND, not detected.

M.S. Surendra Babu et al. / Polyhedron 26 (2007) 572–580
579
Fig. 7b. Gel electrophoresis diagram for reactions done in the control experiment with an incubation time of 60 min at 37 ꢁC in dark condition with NaN3
(1 mM) and DMSO (4 ll): Lane 1: DNA; lane 2: DNA + DBO; lane 3: DNA + CuCl2; lane 4: DNA + [Cu(DBO)2]Py2; lane 5: DNA + [Cu(DBO)2]-
Py2 + DMSO(4 ll); lane 6: DNA + [Cu(DBO)2]Py2 + NaN3 (1 mM); lane 7: DNA + RPO; lane 8: DNA + [Cu(RPO)2]Py2; lane 9: DNA + [Cu(RPO)2]-
Py2 + DMSO(4 ll); and lane 10: DNA + [Cu(RPO)2]Py2 + NaN3.
These OHÅ free radicals participate in the oxidation of
the deoxyribose moiety, followed by hydrolytic cleavage
of the sugar phosphate backbone [27]. The more pro-
nounced nuclease activity of these adducts in the presence
of oxidant as compared to the parent complexes (Fig. 7a,
lanes 6–9, 12 and 13) may be due to the increased produc-
tion of hydroxyl radicals. Even in the absence of oxidant,
the mixed ligand complexes exhibit significant DNA cleav-
age activity when compared to the parent complexes (e.g.
lanes 4, 6 and 8). This may be due to the ability of the
mixed ligand complexes to associate with DNA, facilitated
by aromatic bases (hydrophobic interaction) leading to fair
nucleolytic cleavage. From Table 6 it is clear that the per-
centage of cleavage activity in the presence of oxidant
(H2O2) follows the order Cu(RPO)2Py > Cu(DBO)2Py >
Cu(RPO)2Im2.
The nuclease activity of the complexes has also been
investigated in the presence of a free radical scavenger
(dimethylsulfoxide) and a singlet oxygen quencher (azide
ions). To this end, standard scavengers of reactive oxygen
intermediates were included in the electrophoresis process
(Fig. 7b). Quantification of the gel afforded the data given
in Table 7. Azide (lanes 6 and 10) inhibits to some extent,
indicating the involvement of a singlet oxygen in the cleav-
age activity. The hydroxyl radical scavenger, dimethylsulf-
oxide (lanes 5 and 9) diminishes the nuclease activity of the
compound, which is indicative of the involvement of the
hydroxyl radical in the cleavage process. Nuclease activity
of the complexes is not completely diminished either in the
presence of free radical scavenger or in the presence of sin-
glet oxygen quencher. This means that some DNA is
cleaved by other than an oxidative mechanism, possibly
by a discernable hydrolytic path.
5. Conclusions
Reports on DNA interactions of copper complexes of
polypyridyl and phenanthroline mixed ligand complexes
are numerous in the literature, those of interactions of
metal oximates and of mixed ligand copper complexes with
oximes and aromatic bases have not been investigated so
far. In this study, we have attempted to unravel the
DNA interactions and cleavage activity of mixed ligand
copper complexes. The increase in binding constants of
copper complexes II and V compared with the Cu(SAO)2
complex (I) may be due to formation of a strong hydrogen
bonding interaction between the coordinated ligands (RPO
and DBO). The mixed ligand complex with imidazole
appears to be a better DNA binding agent than the corre-
sponding pyridine analogue, possibly due to the additional
free nitrogen atom that may facilitate to strengthen the
DNA interaction via hydrogen bonding. Since the com-
plexes are found to cleave DNA even in the presences of
free radical scavenger (DMSO) and singlet oxygen
quencher (NaN3) these complexes may also be regarded
as hydrolytic cleavage agents.
Table 7
DNA cleavage of pBR 322 by the copper(II)–pyridine adducts (1 mM) in
the presence of potential inhibitors (Fig. 7b)
Complex
Percentage of Percentage of Percentage of
form I
form II
form III
DNA
DBO
CuCl2
Cu(DBO)2Py2
Cu(DBO)2Py2 + DMSO 61.5
Cu(DBO)2Py2 + NaN3
RPO
Cu(RPO)2Py2
Cu(RPO)2Py2 + DMSO 79.6
87.6
85.4
84.7
52.3
12.4
14.6
15.3
13.4
7.6
14.9
19.5
29.3
20.4
26.2
ND
ND
ND
34.3
30.9
22.5
ND
ND
ND
ND
62.6
80.5
70.7
Acknowledgment
Financial support received form the University Grant
Commission, New Delhi, India [F12-118/2001] is gratefully
acknowledged.
Cu(RPO)2Py2 + NaN3
73.8
ND, not detected.

580
M.S. Surendra Babu et al. / Polyhedron 26 (2007) 572–580
[14] M.J.M. Campbell, Coord. Chem. Rev. 15 (1975) 277.
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