Copper(II) terpyridine complexes: Effect of substituent on DNA binding and nuclease activity

Article abstract of DOI:10.1002/ejic.200700053

Mononuclear copper(II) terpyridine complexes, [Cu(ttpy)-Cl]Cl (1) and [Cu(itpy)Cl]Cl (2) (ttpy = tolylterpyridine and itpy = imidazolylterpyridine) were synthesized and characterized. The interaction of the complexes with DNA was studied by electronic and CD spectroscopy, viscosity and gel electrophoresis. Absorption titrations, viscosity and CD experiments reveal an intercalative mode of DNA binding for these complexes. The binding constant values for 1 and 2 are (5.6 ± 0.2) × 10⁴ and (1.4 ± 0.2) × 10⁴ M^-1, respectively, and suggest moderate binding of these complexes to DNA. From computational studies, it was found that the aromatic π cloud is more uniformly distributed in the case of tolylterpyridine (complex 1), which possibly leads to better stacking interactions with the DNA bases and hence a higher binding constant value for complex 1. From the gel electrophoresis experiments, it is inferred that both complex 1 and 2 cleave plasmid DNA in the presence of ascorbic acid and the cleavage efficiency of complex 1 is greater than that of complex 2. Wiley-VCH Verlag GmbH & Co. KGaA, 2007.

Full text of DOI:10.1002/ejic.200700053

FULL PAPER
DOI: 10.1002/ejic.200700053
Copper(II) Terpyridine Complexes: Effect of Substituent on DNA Binding and
Nuclease Activity
Varadarajan Uma,[a] Munusamy Elango,[a] and Balachandran Unni Nair*[a]
Keywords: Copper / Tridentate ligands / DNA binding / Nuclease activity
Mononuclear copper(II) terpyridine complexes, [Cu(ttpy)-
Cl]Cl (1) and [Cu(itpy)Cl]Cl (2) (ttpy = tolylterpyridine and
itpy = imidazolylterpyridine) were synthesized and charac-
terized. The interaction of the complexes with DNA was
studied by electronic and CD spectroscopy, viscosity and gel
electrophoresis. Absorption titrations, viscosity and CD ex-
periments reveal an intercalative mode of DNA binding for
these complexes. The binding constant values for 1 and 2
is more uniformly distributed in the case of tolylterpyridine
(complex 1), which possibly leads to better stacking interac-
tions with the DNA bases and hence a higher binding con-
stant value for complex 1. From the gel electrophoresis ex-
periments, it is inferred that both complex 1 and 2 cleave
plasmid DNA in the presence of ascorbic acid and the cleav-
age efficiency of complex 1 is greater than that of complex
2.
are (5.6Ϯ0.2)ϫ104 and (1.4Ϯ0.2)ϫ104
M
–1, respectively, and
suggest moderate binding of these complexes to DNA. From
computational studies, it was found that the aromatic π cloud
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
investigated as potential site-specific DNA modification
agents.[17–18] The other metal that has been most widely
used in these studies is copper; complexes of copper with
different types of ligands have been tested as potential
chemical nucleases.[19–22] Among them, 1,10-phenanthroline
and its derivatives have attracted great attention due to their
high nucleolytic efficiency.[23–26] Studies by Sigman and co-
workers have shown that bis(1,10-phenanthroline)copper(I)
complex efficiently cleaves DNA through the formation of
a sugar hydrogen abstracting active oxygen species.[27–28]
They have also been broadly used as foot printing agents of
both proteins and DNA,[29] probes of the dimensions of
the minor groove of duplex structures,[30] and identifiers of
transcription starting sites.[31]
Our laboratory has been focusing on DNA interaction
studies of bis-chelated copper(II) complexes with various
tridendate terpyridine ligand systems.[32,33] The present
work stems from our interest to explore the DNA binding
properties and nuclease activity of 1:1 copper(II) complex
with these ligand systems and to examine whether they ex-
hibit a significant change in the binding properties as com-
pared to the bis-chelated complexes. In this paper, we report
the synthesis and DNA interaction and cleavage studies of
copper(II) complexes with two terpyridine derivatives,
namely tolyl terpyridine (ttpy), and imidazolyl terpyridine
(itpy) ligands, the structures of which are shown in Fig-
ure 1. The DNA interaction has been investigated using a
host of physical methods like electronic absorption spec-
troscopy, viscosity measurements, electrochemical methods
and circular dichroic studies and the cleavage reactions have
been assayed using agarose gel electrophoresis.
Introduction
Small molecules that bind and cleave double-stranded
DNA under physiological conditions have found wide ap-
plications in the field of medicine, molecular biology and
biotechnology.[1–5] Transition-metal complexes charac-
terized by high stability, structural versatility and unique
spectroscopic and redox properties are exploited largely in
many of these efforts. These metal complexes are capable of
binding to DNA by a multitude of interactions and cleave
DNA by virtue of their intrinsic chemical, electrochemical
and photochemical reactivities.[6–10] In the majority of the
complexes studied, the metal ion usually serves as the redox
centre and the ligand is responsible for DNA recognition.
The modes of recognition are primarily based upon inter-
calation, groove-binding, hydrogen bonding and electro-
static interactions.
Since the success of platinum complexes as anticancer
drugs, studies on the interaction of transition-metal com-
plexes with nucleic acids has been a topic of interest in bio-
chemical and coordination chemical research.[11] Rutheni-
um(II) polypyridyl complexes have attracted comprehensive
attention due to their significance in the development of
new therapeutic agents and novel nucleic acid structural
probes.[12–14] A number of dinuclear iron complexes are
known to exhibit hydrolytic cleavage of duplex DNA.[15–16]
Nickel(II) and cobalt(II) macrocyclic complexes have been
[a] Chemical Laboratory, Central Leather Research Institute,
Adyar, Chennai, 600020 Tamil Nadu, India
Fax: +91-44-2491-1589
E-mail: bunair@clri.info
3484
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Copper(II) Terpyridine Complexes
FULL PAPER
EPR spectrum of complex 1 and the EPR parameters are
provided in Table 1. From the observed values for both
complex 1 and 2, it is clear that gʈ Ͼ gЌ, which indicates
that the unpaired electron predominantly lies in the dx2–y2
orbital.[34] The ratio of gʈ/Aʈ may be considered as a diag-
nostic parameter of the stereochemistry of the complex, and
the range reported for square-planar complexes is 105–
135 G.[35] In the present case, this value is higher than
135 G, indicating a considerable tetrahedral distortion
around the copper site that could be due to the rigidity of
the terpy moiety and the geometrical constraint it imposes
on the central metal ion.
Figure 1. The structures of the ttpy and itpy ligands.
Results and Discussion
Synthesis and Spectral Properties
Complexes 1 and 2 could be obtained in good yields and
sufficiently pure forms by mixing hot methanolic solutions
of copper chloride and the respective terpyridine ligands in
equimolar quantities. The authenticity of the complexes
was ascertained from mass spectral analysis. The ESI mass
spectra of the complexes show base peaks assignable to the
parent ion [Cu·L·Cl]+, where L corresponds to ttpy and itpy
for complexes 1 and 2, respectively.
The electronic spectra of the complexes exhibit intense
intraligand bands in the region 280–350 nm and a broad
ligand field band in the region 550–800 nm (Table 1). The
absorptions in the UV region show a shift to higher wave-
lengths relative to the unsubstituted terpy complex (228 and
324 nm). This shift is due to the lowering of the LUMO
(π*) energy in the ttpy ligand owing to a more extended
conjugation relative to the terpy ligand. In the case of cop-
per(II) complexes, three d–d transitions are possible:
dxz,dyz Ǟdx2–y2, dz2 Ǟdx2–y2, dxy Ǟdx2–y2. However, only a
single broad band is observed for both the copper(II) com-
plexes, which indicates the total sum of all the above transi-
tions. The broadness associated with the d–d bands is gen-
erally taken as an indication of the geometrical distortion
of the complex from perfect planar symmetry.
Figure 2. EPR spectrum of a frozen solution of complex 1 at 77 K
in DMSO (microwave frequency = 9.5 GHz, power = 3 mW, modu-
lation frequency = 100 kHz, modulation amplitude = 3 G).
Cyclic voltammetric studies to establish the redox behav-
iour of the copper(II) complexes reveal a non-Nernstian,
almost irreversible redox process involving the CuII/CuI
couple near –0.2 V for both of the complexes (values pro-
vided in Table 2). The anodic to cathodic peak current ratio
of around 0.2 for both the complexes suggests poor revers-
ibility of the electron transfer process. Figure 3 depicts the
cyclic voltammograms of complexes 1 and 2. The CuII/CuI
redox potential of complex 1 is slightly more negative rela-
tive to 2, which indicates that the +1 oxidation state is more
stabilized in complex 2 than it is in complex 1.
Table 1. Electronic and EPR (taken at 77 K) spectral parameters
for complexes 1 and 2.
Complex
Electronic spectra[a] g values
λ [nm] (ε, –1 cm–1)
A values
Complex 1
287 (18,050)
318 sh (11,800)
690 br (160)
288 (17,765)
338 (22,995
gʈ = 2.36
gЌ = 2.06
Aʈ = 161 G
Complex 2
gʈ = 2.39
gЌ = 2.08
Aʈ = 165 G
350 sh (23,595)
710 br (153)
[a] br = broad, sh = shoulder.
Table 2. Electrochemical behaviour of complexes 1 and 2.[a]
EPR spectra of complexes 1 and 2 (frozen solutions at
77 K) in DMSO reveal an axial nature with four well-re-
solved peaks of low intensity in the low-field region and
one intense peak in the high-field region. No band corre-
sponding to Ms = Ϯ2 was observed in the spectra, ruling
Complexes
E1/2 [V]
∆Ep [mV]
ipa/ipc
CV
DPV
1
2
–0.257
–0.248
101
109
0.25
0.20
–0.223
–0.224
out any copper–copper interaction. Figure 2 shows the [a] Reference electrode: SCE; supporting electrolyte: TBAP.
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V. Uma, M. Elango, B. U. Nair
FULL PAPER
Figure 3. Cyclic voltammograms of complexes 1 and 2 (0.2 m);
scan rate 50 mVs–1; supporting electrolyte, TBAP (100 m), refer-
ence electrode SCE.
Figure 4. Absorption spectra of complex 2 (20 µ) in the absence
(a) and presence (b–f) of increasing concentrations of CT DNA
(40–120 µ).
Absorption Titration Experiments
The interaction of the metal complex with DNA causes
significant changes in the ligand-centred spectral transitions
of the complex and these changes are monitored to calcu-
late the binding constant. In the presence of increasing con-
centrations of DNA, both 1 and 2 were found to exhibit
hypochromism with an almost negligible shift in the ab-
sorption maxima. This decrease in the intensity of the spec-
tral band is typical of an intercalative mode of binding
wherein the interacting molecule is sandwiched between the
DNA base pairs.[36,37] The planarity and extended aroma-
ticity of the terpyridine ligand systems bring about the
stacking of the molecule between the DNA bases. The spec-
tral changes on interaction of complex 2 with DNA are
shown in Figure 4. The binding constant Kb was estimated
to be (5.6Ϯ0.2)ϫ104 and (1.4Ϯ0.2)ϫ104 –1 for com-
plexes 1 and 2, respectively. The binding constant values for
these complexes are of the same order of magnitude as
those observed for the corresponding bis-chelate systems re-
ported in our earlier publications.[32] Also, the mode of
binding remains intercalative in both cases. This suggests
that the major factor influencing the mode and extent of
binding could be the large size of the ligand system. The
planar terpyridine ring appears to dictate the binding char-
acteristics irrespective of the geometry of the complex (four
or six coordinate).
Figure 5. Optimized molecular structures of R1 (toluene) and R2
(imidazole) obtained at the B3LYP/6-31G* level of calculation
(grey spheres represent carbon atoms; black spheres represent ni-
trogen atoms).
were calculated for R1 and R2[39] and the results are pre-
sented in Table 3. Nucleus independent chemical shift
(NICS) criteria proposed by Schleyer[40] is also calculated
at the centre of the rings. All the three parameters, namely,
η, ω and NICS are measures of reactivity and stability.
From the table, it is observed that R1 is less hard and more
reactive than R2. This is also implied in the values of ω;
the ω value of R1 is higher than that of R2, indicating the
higher reactivity of R1. NICS values indicate the degree of
aromaticity: the more negative the NICS value, the more
aromatic the system and higher the stability. From
Table 3, we observe that the NICS value of R2 is greater
than that of R1, indicating the higher reactivity and lesser
stability of R1. Thus, the tolyl-substituted molecule, com-
plex 1, binds to DNA more strongly [(5.6Ϯ0.2)ϫ104 –1]
To further explore the influence of substituents on the
binding constant values, computational studies were carried
out for complexes 1 and 2. As seen from Figure 1, com-
plexes 1 and 2 differ only in their substituents (R group).
Therefore, calculations were carried out by considering only
the tolyl and imidazolyl groups (R1 and R2, respectively)
as representative of 1 and 2. Hydrogen atoms were added
in the R groups to satisfy the valency (optimized molecular
structures shown in Figure 5).
Table 3. Chemical hardness (η), electrophilicity (ω) and nucleus in-
dependent chemical shift [NICS(0)] values of R1 and R2 calculated
by using the B3LYP/6-31G* level of theory.
The structures were optimized at the B3LYP/6-31G*
level of theory by using the Gaussian 98W suite of pro-
grams.[38] Density functional theory (DFT) based descrip-
tors such as chemical hardness (η) and electrophilicity (ω)
Species
η [eV]
ω [eV]
NICS(0) [ppm]
Toluene
Imidazole
3.27
3.51
1.49
0.97
–9.6
–14.4
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Copper(II) Terpyridine Complexes
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relative to the imidazole substituted molecule, complex 2 plexes 1 and 2 on the specific relative viscosity of DNA is
[(1.4Ϯ0.2)ϫ104 –1].
shown in Figure 7. The relative viscosity of DNA increases
In addition to this, the molecular electrostatic potential steadily with an increase in the concentration of the com-
map of R1 and R2 was calculated at the B3LYP/6-31G* plex and this result parallels the pronounced absorbance
level of theory and is presented in Figure 6. The Gaussview hypochromism that was observed for the complexes and
package was used for viewing the map.[41] In the case of R2, further confirms their intercalative mode of binding. How-
the π cloud is substantially influenced by the electronegative ever, the increase in viscosity is less than that observed for
nitrogen atom of the imidazole ring, but in R1, the aromatic proven intercalators.[43] Among the two, the increase in the
π cloud is uniformly distributed above and below the molec- relative viscosity is more pronounced for complex 1 than
ular plane, which could bring about favourable stacking in- for 2, which parallels the trend in DNA binding studies.
teractions with the DNA bases during intercalation (Fig-
ure 6). Hence, intercalation in this case would be compara-
tively more favourable than it is in R2. Thus, computational
studies clearly show that the tolyl substituent makes com-
plex 1 a better intercalator relative to imidazolyl-substituted
complex 2.
Circular Dichroism
Circular dichroic spectral techniques may give us useful
information on how the conformation of the DNA chain is
influenced by the bound complex. The CD spectrum of CT
DNA consists of a positive band at 275 nm that can be due
to base stacking and a negative band at 245 nm that can be
due to helicity and it is also characteristic of DNA in a
right-handed B form.[44] Thus, simple groove binding and
electrostatic interaction of small molecules with DNA show
less or no perturbations on the base stacking and helicity
bands, whereas intercalation enhances the intensities of
both the bands stabilizing the right-handed B conformation
of CT DNA as observed for classical intercalators.[45] The
CD spectrum of CT DNA was monitored in the presence
of increasing amounts of complexes 1 and 2; the changes
observed in the case of complex 2 are shown in Figure 8.
The positive band showed an enhancement in the molar
ellipticity with a slight redshift of the band maxima whereas
the negative band showed a decrease in the intensity when
the complex concentration was progressively increased.
Similar changes were observed in the CD spectrum of DNA
in the case of complex 2. These observations are supportive
of the intercalative mode of binding of these complexes,
where in the stacking of the complex molecules between the
base pairs of DNA leads to an enhancement in the positive
band and the partial unwinding of the helix is reflected in
the decreased intensity of the negative band. It is also evi-
Figure 6. Total electron density map of R1 and R2 obtained at
0.003 au surface.
Viscosity Experiments
Measurement of DNA viscosity is regarded as the least
ambiguous and the most critical test of the binding mode
of DNA in solution and affords stronger arguments for an
intercalative DNA binding mode. The viscosity of DNA is
enhanced in a complete or partial intercalative mode of
binding where an increase in the length of the polynucleo-
tide occurs as a result of the destacking of the base pairs.[42]
A partial nonclassical mode of binding could bend or kink
the DNA helix, reduce its effective length and thereby its
viscosity. The effect of increasing concentrations of com-
Figure 8. CD spectra of CT DNA (200 µ) in the absence (ϪϪϪ)
and presence of 20 (·-·-·-·-·-·-) and 40 µ (- - - - - - - -) of complex
1.
Figure 7. Effect of increasing concentration of complexes 1 (square)
and 2 (circle) on the relative viscosity of CT DNA (200 µ).
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V. Uma, M. Elango, B. U. Nair
dent from the CD spectra that binding of the complex does Table 4. Extent of DNA cleavage by complexes 1 and 2.
FULL PAPER
not lead to any significant change in the conformation of
CT DNA.
Complexes
Concentration
of Complex [µ]
%SC
%NC
%L[a]
1
25
50
75
65
14
35
86
85
Gel Electrophoresis Studies
15
To determine the ability of the present copper complexes
to serve as metallonucleases, DNA cleavage studies were
performed on plasmid pBR322 DNA and the results were
monitored by gel electrophoresis. The naturally occurring
super coiled form (Form I), when nicked, gives rise to an
open circular relaxed form (Form II). This upon further
cleavage results in the linear form (Form III). When sub-
jected to gel electrophoresis, Form I shows a relatively faster
migration than Forms II and III. Form II migrates very
slowly owing to its relaxed structure whereas the linear
form shows a migration in between Form I and Form II.
Figure 9 shows the electrophoretic pattern of plasmid DNA
treated with complexes 1 and 2. Control experiments sug-
gest that untreated DNA and DNA incubated with either
complex or ascorbic acid alone does not show any signifi-
cant DNA cleavage (Figure 9, lanes 1–3) but in the presence
of ascorbic acid, the complexes were found to exhibit nucle-
ase activity. In the presence of 25 µ of complex 1 and
75 µ of ascorbic acid, slight cleavage of plasmid DNA oc-
curs as is evident from lane 4 (Figure 9) whereas there is
hardly any cleavage in lane 5 (Figure 9), which represents
the same concentration of the complex and reductant for 2.
On increasing the concentration of the complex to 50 µ
while maintaining the concentration of the reducing agent
at 75 µ, the cleavage was found to be much more efficient,
as is evident from the increase in Form II (Figure 9, lanes 6
and 7). A further increase in the concentration of the com-
plex to 75 µ and ascorbic acid to 150 µ leads to complete
conversion of the supercoiled form to Form II and III (Fig-
ure 9, lanes 8 and 9). From Table 4 it is clear that complex
1 exhibits better nuclease activity than 2.
2
25
50
75
89
53
2
11
47
98
[a] SC = supercoiled; NC = nicked circular; L = linear.
electrophoresis were subjected to densitometric quantifica-
tion and the kinetic parameters were analyzed by assuming
a simple pseudofirst-order process for conversion of Form
I to Form II. The rate constants determined from the plot
of ln(%SC DNA) versus time (shown in Figure 10) were
found to be 4.32ϫ10–4 s–1 for complex 1 and 3.14ϫ10–4 s–1
for complex 2. The higher k value for 1 is clearly indicative
of a better nuclease activity of 1 relative to 2.
Figure 10. Plot of ln(%SC DNA) versus time for a complex con-
centration of 75 µ; complex 1 (᭺), Complex 2 (᭹).
To explain the cleavage efficiency of 1 being greater than
2 it is essential to look at the probable mechanistic pathway
for this process. Because the complexes exhibit nuclease ac-
tivity only in the presence of ascorbic acid, it is clear that
the copper(II) complex in both cases is first reduced to a
copper(I) complex, which then reacts with molecular oxy-
gen to produce reactive oxygen species (ROS). A species
with a more negative redox potential is always a better re-
ducing agent and would reduce molecular oxygen much
more readily thereby causing efficient DNA cleavage. In the
present case, because complex 1 has a more negative redox
potential than 2, the former is a better DNA cleaving agent
Figure 9. Cleavage of pBR322 DNA by complexes 1 and 2 in the
presence of ascorbic acid. DNA (250 ng) was incubated with 1 and
2 for 60 min. in Tris buffer (pH 7.5). Lane 1, DNA control; lane 2,
DNA + 1 (75 µ) alone; lane 3, DNA + ascorbic acid (150 µ);
lane 4, DNA + 1 (25 µ) + ascorbic acid (75 µ); lane 5, DNA +
2 (25 µ) + ascorbic acid (75 µ); lane 6, DNA + 1 (50 µ) +
ascorbic acid (75 µ); lane 7, DNA + 2 (50 µ) + ascorbic acid
(75 µ); lane 8, DNA + 1 (75 µ) + ascorbic acid (150 µ); lane 9,
DNA + 2 (75 µ) + ascorbic acid (150 µ).
To quantify the nuclease activity of the complexes, rate than the latter. Similar observations were also reported by
constants were determined for both complex 1 and 2 by other research groups.[46,47] Moreover, the higher binding
further studying the extent of DNA cleavage in a time de- affinity of complex 1 towards DNA relative to 2, could also
pendent manner. The complex concentration was main- possibly be another reason for the higher cleavage efficiency
tained at 75 µ for both 1 and 2 as maximum DNA damage of complex 1. Further studies are under progress to investi-
occurs at this particular concentration. The results of gel gate the DNA cleavage in detail.
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Copper(II) Terpyridine Complexes
FULL PAPER
electron spray source. Infrared spectra were recorded for the com-
plexes and the ligand by using a Perkin–Elmer RX-1 FTIR spec-
trometer and the samples were prepared by KBr mull sampling
technique. Electronic spectra were recorded with a Perkin–Elmer
Lamda35 double beam spectrophotometer. Circular dichroic spec-
tra were recorded with a J-715 spectropolarimeter (Jasco) at 25 °C
with a 0.1-cm pathlength cuvette. Cyclic voltammetry was per-
formed on CH-Instruments, electrochemical analyzer, CH-620 B.
X-band electron paramagnetic resonance (EPR) spectra at room
temperature and liquid nitrogen temperature were recorded with a
Bruker EMX 6/1 spectrometer and the field calibration was done
by using diphenylpicrylhydrazyl (DPPH).
Conclusions
The present communication reports the synthesis and
characterization of two four-coordinate copper(II) com-
plexes with tridentate terpyridine ligands. On the basis of
electronic and EPR spectral studies, the complexes are sug-
gested to have a four-coordinate geometry with consider-
able tetrahedral distortion in the square plane. The com-
plexes are found to adopt an intercalative mode of binding
with moderate binding strengths. The influence of the sub-
stituents of the terpyridine ligand was discussed on the ba-
sis of computational studies. The tolyl substituent in com-
plex 1 is less hard and more reactive relative to the imid-
azolyl substituent of complex 2. Also, the aromatic π cloud
almost uniformly lies above and below the molecular plane
in complex 1, which leads to its better intercalative binding
with DNA. They exhibit efficient nuclease activity in the
presence of ascorbic acid and the differences in their cleav-
age efficiencies were explained on the basis of spectral and
electrochemical results.
DNA Binding and Cleavage Studies – Absorption Titration Experi-
ments: The electronic spectra of complexes 1 and 2 were monitored
in the absence and presence of DNA in the UV region. Absorption
titration experiments were conducted by maintaining the metal ion
concentration constant (20 µ) and varying the concentration of
DNA (0–150 µ). In the reference cell, a DNA blank was placed
so as to cancel any absorbance due to DNA at the measured wave-
length. From the absorption titration data the binding constant
(Kb) was determined by using the following equation.
[DNA]/(εa – εf) = [DNA]/(εb – εf) + 1/Kb(εb – εf)
where εa, εf and εb corresponds to Aobs/[Cu], the extinction coeffi-
cient for the free copper complex and the extinction coefficient for
the complex in the bound form, respectively. A plot of [DNA]/(εa –
εf) versus [DNA] gives Kb as the ratio of the slope to the intercept.
Density functional theory calculations were performed to explore
the reactivity, stability and electron density parameters of the terpy-
ridine substituents. From the theoretical investigation, the influence
of substituent on the magnitude of binding constant is explained.
Experimental Section
General: 2-Acetyl pyridine, tolualdehyde, p-bromobenzaldehyde
and imidazole 2-carboxaldehyde were obtained from Sigma Ald-
rich, USA and used as received. Tris(hydroxymethyl) methylamine
(Tris) was obtained from SRL Chemicals. Calf Thymus DNA (CT
DNA) was obtained from Fluka Chemicals. All chemicals and rea-
gents used were of analytical grade received from Ranbaxy, Mum-
bai. Stock solution of DNA was prepared by stirring a sample dis-
solved in 10 m (Tris) buffer, (pH 7.2) at 4 °C. The solution was
dialyzed exhaustively against the Tris buffer for 48 h and filtered
through a membrane filter obtained from Sartorius (45 µ). The
filtered DNA solution in the buffer gave a UV absorbance ratio
(A260/A280) of about 1.8, indicating DNA was sufficiently free from
protein.[48] The concentration of DNA was determined by using an
extinction coefficient of 6600 –1 cm–1 at 260 nm.[49]
Viscometric experiments were carried out by using an Ostwald-type
viscometer of 2 mL capacity, thermostatted in a water bath main-
tained at 25 °C. The flow rates for the buffer (10 m), DNA
(200 µ) and DNA in the presence of copper(II) complex at various
concentrations (20–140 µ) were measured with a manually oper-
ated timer at least three times to agree within 0.2 s. The relative
viscosity was calculated according to the relation, η = (t – t0)/t0
where t0 is the flow time of the buffer and t is the observed flow
time for DNA, in the presence and absence of the complex. A plot
of (η/η0)1/3 versus 1/R (R = [DNA]/[Complex]) was constructed
from viscosity measurements.[51]
[Cu(ttpy)Cl]Cl (1): The complex was prepared in good yield from
the reaction of CuCl2·2H2O in methanol with tolyl terpyridine li-
gand, which was prepared by a procedure reported in the litera-
ture.[50] The ligand (0.323 g, 1 mmol) and CuCl2·2H2O (0.170 g,
1 mmol) were dissolved in methanol individually and the solutions
were warmed. The hot solution of the ligand was added slowly with
constant stirring to copper chloride when the colour changed to
intense green. The solution was cooled to room temperature and
the green precipitate of the copper–ttpy complex separated out and
Voltammetric experiments were carried out by using a standard
three-electrode system comprising glassy carbon working electrode
with 3 mm diameter, platinum auxillary electrode and saturated cal-
omel reference electrode (SCE). The samples were prepared in
DMSO, and the solutions were deoxygenated by purging nitrogen
gas for 10 min. prior to the measurements. The experiments were
carried out with a complex concentration of 0.2ϫ10–3  by using
tetrabutylammonium perchlorate (TBAP) as the supporting elec-
trolyte.
was filtered and dried. IR [KBr disc]: ν = 3069, 1619, 1451,
˜
1098 cm–1. MS (ESI): m/z = 421 [M – L·Cl]+. C22H17Cl2CuN3
(456.5): calcd. C 57.83, H 3.72, N 9.20, Cu 13.91; found: C 57.78,
H 3.69, N 9.25, Cu 13.93. The complex is soluble in DMSO and
partially soluble in methanol.
Circular dichroic spectrum of DNA in the absence and presence of
complexes was recorded with a J-715 spectropolarimeter (Jasco) at
25 °C with a 0.1-cm pathlength cuvette. The spectra were recorded
for 200 µ of DNA in the absence and presence of 5–40 µ of the
copper(II) complex in the region 220–300 nm.
[Cu(itpy)Cl]Cl (2): The complex was prepared by a similar pro-
cedure to that of complex 1, by taking CuCl2·2H2O (0.170 g) and
itpy (0.299 g) in the mole ratio 1:1; the ligand was prepared by a
similar procedure as that of ttpy. IR (KBr disc): ν = 3068, 1616,
˜
1450, 794 cm–1. MS (ESI): m/z = 397 [M – L·Cl]+. C18H13Cl2CuN5
(432.5): calcd. C 49.94, H 3.00, N 16.18, Cu 14.68; found C 49.87,
H 2.94, N 16.09, Cu 14.60. The solubility of the complex is similar
to that of 1.
Cleavage of plasmid DNA was monitored by using agarose gel elec-
trophoresis. Supercoiled pBR322 DNA (180 ng) in tris(hydroxyme-
thyl)methylamine (Tris) buffer was treated with copper(II) complex
(25–75 µ) and ascorbic acid (75–150 µ). The samples were incu-
bated for 60 min at 35 °C. A second set of experiments was per-
formed by maintaining the complex concentration at 75 µ and
Physical Measurements: ESI mass spectra were recorded with a
Finnigan LCQ Advantage mass spectrometer equipped with an
Eur. J. Inorg. Chem. 2007, 3484–3490
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
3489

V. Uma, M. Elango, B. U. Nair
FULL PAPER
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Published Online: June 8, 2007
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Eur. J. Inorg. Chem. 2007, 3484–3490