Received: 14 July 2017 Revised: 11 September 2017 Accepted: 11 September 2017

DOI: 10.1002/aoc.4128

FULL PAPER

New anthracene based Schiff base ligands appended Cu(II)

complexes: Theoretical study, DNA binding and cleavage
activities

Ammavasai Gubendran1,2 | Gujuluva Gangatharan Vinoth Kumar3 |

Mookkandi Palsamy Kesavan3 | Gurusamy Rajagopal4 | Periakaruppan Athappan1 |
Jegathalaprathaban Rajesh1,3

1
Department of Inorganic Chemistry,
School of Chemistry, Madurai Kamaraj New anthracene based Schiff base ligands L1 and H(L2), their Cu(II) complexes
University, Madurai 625 021, Tamilnadu, [Cu(L1)Cl2] (1) and [Cu(L2)Cl] (2), (where L1 = N1,N2‐bis(anthracene‐9‐methy-
India
2
lene)benzene‐1,2‐diamine, L2 = (2Z,4E)‐4‐(2‐(anthracen‐9‐ylmethyleneamino)
Department of Chemistry, Saraswathi
Narayanan College, Madurai 625 022,
phenylimino)pent‐2‐en‐2‐ol) have been prepared and characterized by elemen-
Tamilnadu, India tal analysis, NMR, FAB‐mass, EPR, FT‐IR, UV–Vis and cyclic voltammetry.
3
Chemistry Research Centre, Mohamed The electronic structures and geometrical parameters of complexes 1 and 2 were
Sathak Engineering College, Kilakarai
analyzed by the theoretical B3LYP/DFT method. The interaction of these com-
623 806, Tamilnadu, India
4
PG and Research Department of
plexes 1 and 2 with CT‐DNA has been explored by using absorption, cyclic
Chemistry, Chikkanna Government Arts voltammetric and CD spectral studies. From the electronic absorption spectral
College, Tiruppur 641 602, Tamilnadu, studies, it was found that the DNA binding constants of complexes 1 and 2 are
India
8.7 × 103 and 7.0 × 104 M−1, respectively. From electrochemical studies, the ratio
Correspondence of DNA binding constants K+/K2+ for 2 has been estimated to be >1. The high
Dr. Jegathlaprathaban Rajesh, Associate
binding constant values, K+/K2+ ratios more than unity and positive shift of
Professor, Chemistry Research Centre,
Mohamed Sathak Engineering College, voltammetric E1/2 value on titration with DNA for complex 2 suggest that they
Killakarai ‐ 623 806, Ramanthapuram bind more avidly with DNA than complex 1. The inability to affect the conforma-
(District), Tamilnadu, India.
tional changes of DNA in the CD spectrum is the definite evidences of electro-
Email: [email protected]
static binding by the complex 1. It can be assumed that it is the bulky
Funding information anthracene unit which sterically inhibits these complexes 1 and 2 from interca-
DST‐SERB, Grant/Award Number: SB/
FT/CS‐130/2012
lation and thereby remains in the groove or electrostatic. The complex 2 hardly
cleaves supercoiled pUC18 plasmid DNA in the presence of hydrogen peroxide.
The results suggest that complex 2 bind to DNA through minor groove binding.

KEYWORDS
characterization, Cu(II) complexes, DNA binding, DNA cleavage, Schiff base ligands, theoretical
studies

1 | INTRODUCTION

The interaction of transition metal complexes with DNA transfer reactions and to find the potential biological
has been extensively studied in order to develop novel and pharmaceutical activity. Their activity depends on
probes of DNA structure, DNA mediated electron the mode and affinity of the binding with DNA.[1–5] In

Appl Organometal Chem. 2017;e4128. wileyonlinelibrary.com/journal/aoc Copyright © 2017 John Wiley & Sons, Ltd. 1 of 11
https://doi.org/10.1002/aoc.4128
2 of 11 GUBENDRAN ET AL.

human blood plasma, the presence of transition metals 2 | EXPERIMENTAL METHODS

reveals their importance in the living organisms as accu-
mulated storage and transport. Transition metals play a 2.1 | Materials
key role in biological systems such as cell division, respi-
Anthracene‐9‐carbaldehyde, 1,2‐diaminobenzene and
ration, nitrogen fixation and photosynthesis.[6] Among
acetylacetone were purchased from Alfa Aesar and used
the variety of ligand system employed in transition metal
as received. Copper(II) chloride (CuCl2. 2H2O), calf thy-
complexes, Schiff bases are an important class having
mus DNA and pUC18 plasmid DNA were obtained from
many applications and many complexes of different
Sigma Aldrich. All reagents and solvents were purchased
Schiff bases have been reported by a number of
from Merk and Loba chemie (India) and used without
authors.[7–12] Chelating Schiff base ligands containing O
further purification.
and N donor atoms show broad biological activity and
are of special interest because of the variety of ways in
which they are bound to metal ions.[13] Some of the
Schiff base metal chelates shown the minor changes in 2.2 | Physical measurements
the structure of the ligands containing hard soft donor UV–Vis spectra of solutions were recorded using
atoms, markedly affects the activity of the compounds.[14] Shimadzu spectrometer 2500 PC series. The FTIR
Schiff base complexes have received much attention as spectra were recorded from KBr pellets in the range
biomimics model compounds.[15] [Fe(salen)] complex is 400–4000 cm−1 on a Perkin‐Elmer spectrometer. NMR
a model for natural iron protein, and hemerythrin has spectra were obtained on a Bruker 300 MHz spectrome-
the ability to bind molecular oxygen reversibly.[16] Schiff ter. A mass analytical procedure was performed using a
base complexes incorporating two different metal ions system consisting of a LC10ADVP pump and a single
are of special interest, as they are similar to those found quadruple mass spectrometer with an electron spray
in living organisms, like enzymes and proteins.[17] A ionization (ESI) source (LCMS‐2010) (Shimadzu, Kyoto,
large number of Schiff bases based metal complexes have Japan). Electron paramagnetic resonance (EPR) spectra
been studied for their interesting and vital properties were obtained using a Varian E‐112 EPR spectrometer.
such as significant DNA binding ability, anticancer CV measurements were carried out on Bio‐Analytical
activity, catalytic activity in the hydrogenation of olefins, System (BAS) model CV‐50 W electrochemical analyser.
photochromic properties and so on.[18–23] Synthesis of
new Schiff bases and their metal complexes are still
pursued in many recent investigations.[24–26] Reddy et al.
2.3 | Synthetic protocols
has synthesized ternary Cu(II) complexes of the type
[Cu(L’)(L”)](ClO4), where L‘is N N‐donor phenanthroline 2.3.1 | N1‐(anthracene‐9‐methylene)ben-
base and L’ is an ancillary Schiff base ligand.[27] Schiff‐ zene‐1,2‐diamine (L)
base complexes have a wide variety of structures, coordi-
An ethanolic solution of anthracene‐9‐carbaldehyde
nating to metal in either mono‐, bi‐ and tri‐dentate
(0.206 g, 1 mmol was added dropwise to a solution of 1,
modes, depending upon the aldehyde and amines.
2‐diaminobenzene (0.108 g, 1 mmol dissolved in 20 ml
Unsymmetrical Schiff‐base ligand complexes have been
of ethanol under constant stirring and the stirring was
suggested as useful biological models in understanding
continued at room temperature for 6 h. After that, on slow
irregular binding of peptides and also as catalysts in some
evaporation of the solvent at room temperature, white
chemical processes.[11]
shining precipitate was obtained. It was filtered, washed
This paper deals with the preparation of new anthra-
with petroleum‐ether and recrystallized from ethanol.
cene derived Schiff base ligands L1 and H(L2) and their
Yield: 88%.
Cu(II) complexes [Cu(L1)Cl2] (1), [Cu(L2)Cl] (2) and
their characterizations using different elemental, spectro-
scopic and analytical tools. In addition to that, the struc-
2.3.2 | N1,N2‐bis(anthracene‐9‐methylene)
tural parameters of complexes 1 and 2 were investigated
benzene‐1,2‐diamine (L1)
by B3LYP/DFT studies. The DNA binding properties of
complexes 1 and 2 have been studied with a view to An ethanolic solution of anthracene‐9‐carbaldehyde
evaluating their pharmaceutical activities. We believed (0.412 g, 2 mmol) was added drop wise into a ethanolic
that the complexes of 1 and 2 having anthracene deriva- solution of 1, 2‐diaminobenzene (0.108 g, 1 mmol) with
tive ligands, which possess planar aromatic moiety, are continuous stirring at room temperature. The resulting
expected to bind strongly with DNA and to show prom- solution was refluxed for 3 h and the solvent was evapo-
ising chemotherapeutic activity. rated. The residue that formed was removed by filtration
GUBENDRAN ET AL. 3 of 11

and dried. The crude product was then recrystallized from (DFT) analysis were executed by the hybrid exchange‐
ethanol. Yield: 82%. correlation function with 6‐31G (d,p) and B3LYP/
LANL2DZ basis set using Gaussain 09 program.[28] Ini-
tially, the optimized geometries of the ligands (L1 and
2.3.3 | (2Z, 4E)‐4‐(2‐(anthracen‐9‐ H(L2)), complex 1 and 2 were obtained by the DFT‐
ylmethyleneamino)phenylimino)pent‐2‐ B3LYP program.
en‐2‐ol (H(L2))
An ethanolic solution of acetylacetone (0.103 ΜL, 1 mmol)
2.5 | DNA binding experiments
was slowly added to the ligand solution L (0.296 g, 1 mmol
in 25 ml ethanol) under continuous stirring. The resulting The DNA binding experiments were carried out using
mixtures was refluxed for 5 h and evaporated. The residue UV–visible absorption studies, cyclic voltammetry and
that formed was removed by filtration and the resulting circular dichroism studies.
solid was then recrystallized from ethanol. Yield: 70%
The synthetic schemes for ligands L, L1 and H(L2) are
2.6 | DNA cleavage study
given in Scheme 1.
The cleavage of pUC18 DNA by complexes 1 and 2, in the
absence and presence of activating agents H2O2 was dem-
2.3.4 | [Cu(L1)Cl2] (1) onstrated using agarose gel electrophoresis in 10%
The ligand L1 (0.484 g, 1 mmol) was dissolved in 60 mL of DMSO–5 mM Tris–HCl–50 mM NaCl buffer at pH 7.2.
absolute ethanol. An aqueous solution of 1 mmol of the
CuCl2.2H2O was added dropwise to the ligand solution
3 | R ESULTS A ND DISCUSSION
with continuous stirring and refluxed for 5 h. The precip-
itate formed was filtered, washed with diethyl ether and
Initially, complexes 1 and 2 were characterized by ele-
recrystallized from ethanol.
mental analysis (Table 1). The elemental analyses result
of the complexes 1 and 2 are in good agreement with their
2.3.5 | [Cu(L2)Cl] (2) molecular formulas and the proposed chemical structure
of the complexes 1 and 2 are depicted in Figure 1.
The complex 2 was prepared using ligand H(L2)
(0.378 g, 1 mmol) by adopting the same synthetic proce- 1
dure as given for complex 1. 3.1 | H and 13C–NMR spectra
The 1H and 13C–NMR spectra of ligand L1 were
recorded in CDCl3, though L and H(L2) were recorded
2.4 | Theoretical B3LYP/DFT studies
in DMSO‐d6. The 1H–NMR in addition to 13C–NMR
To get the electronic structure of the ground state com- spectrum of L and L1 are shown in Figure S1 and S2,
plexes 1 and 2, we carried out Density Functional Theory respectively. In the 1H–NMR spectra, a sharp singlet

SCHEME 1 Schematic representation

for the synthesis of ligands L, L1 and H(L2)
4 of 11 GUBENDRAN ET AL.

TABLE 1 Elemental analysis and electronic spectral data of complexes 1 and 2

Found (Calcd) λmax (nm)

Complex C H N IL LMCT d‐d
1 69.76 (69.85) 3.93 (3.91) 4.44 (4.53) 255 377 915
2 66.15 (66.22) 4.41(4.49) 5.85 (5.94) 256 380 614

3.3 | EPR spectral analysis

EPR spectra of the complexes 1 and 2 were recorded in
DMSO solvent at liquid nitrogen temperature as well as
at room temperature. At room temperature complexes 1
and 2 exhibit single derivative peak with the g values of
FIGURE 1 The proposed chemical structure of the complexes 1 2.131 and 2.163, correspondingly. But, at liquid nitrogen
and 2 temperature (77 K) the EPR spectra of the Cu(II) complex
1 exhibit a set of four well‐resolved peaks in the low field
region and a weaker signal in the high field region, corre-
peak observed at δ, 8.99 (L) and 8.63 (L1) ppm is due sponding to g║and g⊥ respectively (Figure S4). The same
to = CH (azomethine) proton. The signal due to ‐OH pro- trend was observed in complex 2 (Figure S5).
ton of H(L2) is observed at δ, 12.25 ppm and methyl pro- The g║and g⊥ values are 2.245, 2.019 for 1 and 2.318,
tons appear in the range δ, 1.92–2.24 ppm. The aromatic 2.015 for 2, respectively. The trend observed as g║ > g⊥
protons are clustered at δ, (7.29–8.99 ppm). A broad signal > 2.02, for the complexes 1 and 2 is typical of a Cu(II)
at δ, 6.3 in the spectrum of L may be assigned to ‐NH2 pro- ion (d9) in axial symmetry with the unpaired electron
tons. The 13C–NMR spectral data of ligands confirm the present in the dx2‐y2 orbital.[29] It has been reported that
results obtained from the 1H–NMR. The azomethine car- g║values are close to 2.4 for the complexes containing
bon atom (=CH) is observed at 149.78 ppm for L, copper–oxygen bonds and close to 2.3 for complexes con-
149.04 ppm for L1 and 156.02 ppm for H(L2). Aromatic taining copper–nitrogen bonds.[30] The complex 2 has the
carbons are observed in the range δ, 113.46–156.02, value of g║= 2.318 in conformity with the presence of
121.33–130.19 and 127.10–146.01 ppm for ligands L, L1 both Cu–O and Cu–N bonds. The quotient (g║/ A║),
and H(L2) respectively. The 13C–NMR spectrum of which is empirically treated as a measure of the distortion
H(L2) exhibits –CH3, −CH = C‐CH3, and = C‐OH carbon from planarity, has been found to be 186 and 164 cm−1 for
resonance signals respectively at δ, 19.86–24.20, 95.26 and the complexes 1 and 2, respectively indicating distortion
176.16 ppm, correspondingly. As a result, the NMR spec- from a square planar structure to a deformed tetrahedral
tral study well support with chemical structure of the structure.[31]
ligands L, L1 and H(L2).

3.4 | FT‐IR spectra

3.2 | Mass spectra FT‐IR spectra of free ligands (L, L1 and H(L2)), complexes
1 and 2 were recorded using KBr disc and their character-
The FAB‐mass spectra of the complexes 1 and 2 show istic bands are summarized in Table 2. The IR spectra of
molecular ion peak consistent with the expected molecu- ligand L1 and complex 1 are shown in Figure S6. All the
lar weight and fragmentation pattern in accordance with
the molecular formula. The FAB‐mass spectrum of com-
TABLE 2 FT‐IR spectral assignments of ligands (L, L1, H(L2)),
plex 1 shows molecular ion peak at m/z = 620 correspond- complexes 1 and 2 in cm−1
ing to its molecular weight. The other prominent peak at
m/z = 485 may be due to L1. The complex 2 (Figure S3) Compound υ(O‐H) υ(C = N) υ(M‐N) υ(M‐O)
shows a molecular ion peak at m/z = 496 corresponding L ‐ 1618 ‐ ‐
to its molecular weight. The other important peaks at L 1
‐ 1621 ‐ ‐
m/z = 441 is due to loss of ‐Cl and at m/z = 378 corre- H(L ) 2
3460 1624 ‐ ‐
sponds to H(L2). Thus, mass spectral analysis corrobo-
1 ‐ 1606 417 ‐
rates well with the proposed chemical structure of the
2 ‐ 1606 415 560
complexes 1 and 2.
GUBENDRAN ET AL. 5 of 11

ligands show a strong band in the range, 1624–1618 cm−1 TABLE 3 Voltammetric behavior* of complexes 1 and 2 in DMSO
due to azomethine (−C = N) stretching. These bands are Epc Epa ΔEp ipa/
shifted to lower frequencies, 1610–1589 cm−1 in the com- Complex (V) (V) (V) ipc (A) ipa (A) ipc
plexes, indicating coordination of the Schiff bases through 1 0.012 0.143 0.131 1.15 × 10−5 6.12 × 10−6 0.532
the azomethine nitrogen.[32] The enolic υ(OH) band of the
2 0.011 0.169 0.158 1.19 × 10−5 6.23 × 10−6 0.575
ligand H(L2) observed at 3460 cm−1 disappeared upon
complex formation with Cu(II), indicating the coordina- *Measured vs Ag/AgCl with TBAP as supporting electrolyte at 100 mVs−1.

tion of oxygen after deprotonation to the metal atoms.

For complex 1, the stretching vibration of υ(M‐Cl) is
observed below 350 cm−1 and not observed beyond non‐equivalent current intensity at the cathodic and
400 cm−1. The coordination of the ligands L1 and H(L2) anodic peaks (ipa/ipc = 0.532–0.575). The peaks for the
to the metal through azomethine nitrogen and oxygen CuII/I couple for the complexes 1 and 2 were observed in
atom, after deprotonation, is further confirmed by the the potential range, + 0.400 V to – 0.400 V. The difference
appearance of new bands around 431–417 and in peak potentials, ΔEp = 0.131 (1) and 0.158 (2) V,
560–545 cm−1 regions corresponding to υ(M‐N) and exceeds the Nernstian requirement (0.059 V) suggesting
υ(M‐O) vibrational modes respectively.[33] The IR spectral that the complexes are quasi‐reversible for electrochemi-
data are in good agreement with the proposed structure cal redox reactions.
for the complexes 1 and 2. The broad absorption around
3480 cm−1 is due to water molecules present in the
sample.
3.7 | The ground state structures by
theoretical DFT calculation
3.5 | Electronic absorption spectra
Density functional theory (DFT) calculations were used
The electronic spectral data of complexes 1 and 2 asso- to investigate the mode of complex formation; electronic
ciated structural assignments are given in Table 1. The structures as well as geometrical parameters of com-
UV‐ Vis spectra of these complexes 1 and 2 in DMSO plexes 1 and 2, as their crystal structure have not been
exhibit a sharp absorption at 255 (1) & 256 (2) nm obtained. The structure of complexes 1 and 2 in the gas
and a broad absorption at 377 (1) & 380 (2) nm owing phase was determined to be Cu(II) centered complexes
to intra‐ligand and ligand to metal charge transfer with distorted tetrahedral geometry and the optimized
transitions, (LMCT) respectively (Figure S7). The very structures of complexes 1 and 2 are depicted in
broad band's obtained in the region at 915 (1) & 614 Figure 2. These structures of complexes 1 and 2 are well
(2) nm may be attributed to the d–d transition of Cu(II) correlated with the results of spectroscopic and analytical
ions.[34,35] investigations.
Broad and low energy bands are obtained for Cu(II) Further, the Frontier molecular orbital structures of
complexes 1 and 2 at 915 and 885 nm, respectively. It ligands (L1 and H(L2)), complex 1 and 2 are displayed in
has already been reported that a lower energy transition Figure 3 and 4, respectively. In L1, HOMO is spread over
can be observed between 730 and 900 nm, corresponding the whole π‐moiety and LUMO on the anthracene unit.
to the d–d transition.[36] This value is of particular impor- After complexation with Cu(II) (complex 1) HOMO of
tance since it was highly dependent on the geometry of L1 is localized on the part of the π‐moiety and more
the molecule. It is known that the transition from a spread in metal part and the same behavior is observed
square planar structure to a deformed tetrahedral struc- as L1‐LUMO with little contribution of metal ion. Simi-
ture leads to a red shift of absorption in the electronic larly for H(L2), HOMO and LUMO spread over the whole
spectrum.[37] Thus, the complexes 1 and 2, are not per- π‐moiety. Then, HOMO is spread on imine and metal
fectly square planar and the broad as well as low energy parts and LUMO is spread over the whole molecular
bands obtained at longer wavelengths are attributed to frame structure after complex 2 formations. Moreover,
the deformed tetrahedral structure. the calculated HOMO‐LUMO energy gaps of L1 and
H(L2) are 2.82 eV and 2.97 eV, respectively. After com-
plexation with Cu2+, the HOMO‐LUMO energy gaps of
3.6 | Electrochemical behavior
complex 1 and 2 are found to be 1.76 eV and 2.33 eV.
Electrochemical data for the complexes 1 and 2 are sum- These results obviously indicate that the complexation of
marized in Table 3 and their typical cyclic voltammetric ligands L1 and H(L2) with Cu2+, resulting in the disrup-
responses are shown in Figure S8. Complexes 1 and 2 tion of the internal charge transfer and that resulted in
exhibit a quasi‐reversible behavior as indicated by the the changes in electronic properties.
6 of 11 GUBENDRAN ET AL.

the intensity of the LMCT band at (380 nm) markedly

decreased in presence of DNA, 23–34% hypochromism,
with a red shift up to 4 nm. The binding constant of com-
plex 2 has been found to be 7.0 × 104 M−1 and this values
are higher than that obtained for complex 1 (8.7 × 103 M
−1
). The intrinsic binding constant values obtained for 1
and 2 are lower than that of potential intercalator like
ethidium bromide (Kb = 7.0 × 107 M−1)[38], but compara-
ble to those observed for [Cu(phen)2]+ and
[Ru(phen)3]2+.[39]
From the absorption titration results, it could be seen
that the spectral changes for the complexes 1 and 2 are
not uniform. The spectral characteristic of low
hypochromism, paltry red shift and low Kb values
observed for the complex 1 suggest that there is no likeli-
hood of intercalative binding to DNA due to two bulky
anthracene units which strongly prevent stacking the aro-
matic chromophore between the base pairs of the DNA.
The high Kb values and pronounced hypochromism for
complex 2 are possibly due to the presence of only one
anthracene moiety that facilitates groove binding with
the DNA with the base pairs.[40] Taking into account the
extent of hypochromism and binding constant values the
possibility of intercalative binding of DNA can be ruled
out and only groove binding may occur for complexes 1
and 2.

3.8.2 | Electrochemical studies

Electrochemical investigation of drug–DNA interactions
can provide a useful complement to other methods and
yield information about the mechanism of interaction
FIGURE 2 Gas phase B3LYP/DFT optimized structure of and the conformation of adduct.[41] The electrochemical
complexes 1 and 2 behavior of complexes 1 and 2, and their interaction with
CT‐DNA were carried out by CV. The cyclic voltammetric
data for complexes 1 and 2 in the absence and presence of
3.8 | DNA binding and cleavage studies DNA at room temperature in Tris–HCl buffer (pH = 7.1)
is shown in Table 5.
3.8.1 | Absorption spectroscopic titration
The cyclic voltammogram of complex 1 in the absence
Absorption titration experiments were performed by of DNA reveals reduction of CuII to the CuI form at a
maintaining the Cu(II) complexes 1 and 2 concentration cathodic peak potential (Epc) of −0.010 V versus Ag/AgCl.
as constant at 20 μM while varying the concentration of In the absence of DNA, the separation of the anodic and
CT‐DNA in between 0–100 μM. Table 4 summarizes the cathodic peak potentials, ΔEp = 0.093 V for 1 indicates a
spectral changes when the complexes 1 and 2 are titrated quasi‐reversible redox process. The formal potential, E1/2
with DNA. LMCT bands (377 nm (1) – 380 nm (2)) are (or voltammetric E1/2), taken as the average of Epc and
monitored for absorption spectroscopic titration. Addition Epa is 0.037 V in the absence of DNA. The presence of
of increasing amounts of DNA results in hypochromism DNA in the solution at the same concentration of complex
and a moderate bathochromic shift (2–4 nm) of the peak causes a considerable decrease in the voltammetric cur-
at the LMCT band. The absorption spectra of complexes rent. In addition, both the peak potentials, (Epc and Epa)
1 and 2 in the presence of increasing amounts of DNA and E1/2 have shifted to more negative potential/less pos-
are shown in Figure 5. itive as shown in Figure 6.
For complex 1, LMCT absorption (377 nm) shifts up to The cyclic voltammogram of complex 2 in the absence
only 2 nm with 14–18% hypochromism. As for complex 2 of DNA features a quasi‐reversible redox wave with
GUBENDRAN ET AL. 7 of 11

FIGURE 3 Frontier molecular orbitals

of L1 and complex 1 (from left to right)

FIGURE 4 Frontier molecular orbitals

of H(L2) and complex 2 (from left to right)

TABLE 4 Absorption spectral titration data of the complexes 1 and 2 with DNA in Tris–HCl buffer solution

LMCT (λmax/nm) Red

shift Δ Binding
Hypochromism λ constant
Complex Free Bound (%) (nm) Kb (M−1)
1 377 379 14–18 2 8.7 × 103
2 380 384 23–34 4 7.0 × 104
8 of 11 GUBENDRAN ET AL.

FIGURE 6 Cyclic voltammograms of 1 (a) and 2 (b) in the

FIGURE 5 Absorption spectra of complex 1 (a) and 2 (b) in the absence and in presence of DNA in Tris–HCl buffer pH = 7.1.
absence and presence of increasing amounts of DNA (0–240 μM) [complex] = 100 μM. [R = 0, 4 & 8]
in Tris–HCl buffer (pH = 7.1). Arrow mark indicates the absorbance
change upon increasing DNA concentration. [complex] = 20 μM

and voltammetric E1/2 at the same scan rate. After the

addition of DNA, E1/2 is shifted to −0.034 V, ΔEp is
TABLE 5 Voltammetric behavior of complexes 1 and 2 towards
0.080 V and ipa/ipc value is increased to 0.526.
DNA in Tris–HCl buffer solution
The drop in the voltammetric current in the presence
Epc Epa ΔEp ipa/ E1/2 ΔE1/2 K+/ of DNA in complexes 1 and 2 can be attributed to the dif-
Complex R (V) (V) (V) ipc (V) \(V) K2+ fusion of the Cu(II) complex bound to the large, slowly
1 0 −0.010 0.083 0.093 0.972 0.037 diffusing DNA molecule. In the cyclic voltammograms
4 −0.044 0.077 0.121 1.129 0.017 −0.020 0.46 of 1 and 2, at different concentrations of DNA, the peak
8 −0.102 0.051 0.153 1.057 −0.026 −0.063 0.12 currents decreased with increasing concentrations of
2 0 −0.035 0.083 0.118 0.400 0.006
DNA while both the Epc and E’0 shifted to more positive
or less negative potentials. The phenomena of the shift of
4 −0.077 0.074 0.151 0.526 0.017 −0.029 2.10
E’0 and the decrease of peak current implied forming a
8 −0.114 0.051 0.165 0.983 −0.034 −0.080 7.43 new association of DNA with complex. Based on the shift
of formal potentials in the cyclic voltammograms, the
interaction mode of compounds with DNA can be
Epc = −0.035 V at a scan rate of 100 mVs−1 and the mea- inferred.[42] Therefore, in the light of Bard's report,[43]
sured half wave potential E1/2 for the Cu(II)/(I) couple is complex 2 may interact with DNA by groove binding
0.006 V. The ratio of anodic to cathodic peak current is mode in contrast to 1 whose interaction mode is electro-
0.4 which closely resembles that of the criteria of quasi‐ static due to negative shift of E’0 in these cases. For com-
reversibility. Upon addition of increasing amounts of plex 1, K2+ is higher than K+ while the reverse is observed
DNA the cyclic voltammogram of the complex 2 experi- for the complex 2. This suggests that the B form of DNA
enced a less negative shift in cathodic peak potential tends to stabilize the Cu(II) over the Cu(I) state of the
and more positive shift in both the anodic peak potential complex 1 obviously by electrostatic interaction and
GUBENDRAN ET AL. 9 of 11

thereby indicating that the species Cu(II) interacts with positive band along with a minor red shift (Figure 7) of
DNA to a greater extent than Cu(I). In short, the obvious the band maxima is characteristic of groove binding that
shift of peak potentials and the ratio of binding constant stabilizes right‐handed B form of DNA.[45] The CD spec-
values suggest a weak association of the complex 1 (K+/ tral studies suggest that while the DNA binding of com-
K2+ < 1) with DNA than complex 2 (K+/K2+ > 1). plex 1 involves electrostatic mode, complex 2 wraps the
groove of the DNA. These conclusions are also in accor-
dance with those from electronic spectral and cyclic
3.8.3 | Circular dichroism study
voltammetric studies.
The results of CD studies for 1 and 2 are presented in
Figure 7. Pure DNA produced characteristic bands with
3.8.4 | DNA cleavage study by gel
a positive band at 278 nm and a negative band at
electrophoresis
244 nm. These bands were modified when DNA was
allowed to interact with complexes 1 and 2. Based on the above investigation, Complex 2 has potent
In presence of complex 1, the intensity of the negative DNA binding ability than complex 1 and it needs to probe
ellipticity band decreases almost similar to that for the DNA cleavage characteristics of complex 2 for fulfill-
the positive ellipticity band. This suggests that the DNA‐ ing the basic requirements as an anticancer agent.
binding of the complexes do not affect the conformational Figure 8 illustrates the gel electrophoretic separations
changes of DNA. Furthermore, with increasing concen- showing the cleavage of plasmid pUC18 DNA induced by
tration of complex 1, it proved difficult to detect any obvi- the complex 2 under aerobic conditions. With the increase
ous perturbation in the CD spectrum; illustrating the of complex concentration, no amount of the circular
inability of complexes to affect the conformational hetero- supercoiled DNA is converted into nicked DNA via single
geneity of DNA anymore.[44] strand cleavage (lane 4). It reveals that pUC18 DNA
In presence of complex 2 an increase in the molar induced by the complex in the presence of high concen-
ellipticity values of both the positive and negative elliptic- tration of H2O2 results in the insignificantly small conver-
ity bands of the DNA was observed, consistent with a sin- sion of Form I to Form II (lane 6). H2O2 alone is incapable
gle binding mode. The enhancement of the intensity of of cleaving plasmid DNA (lane 3). It can be seen that
neither the complex 2 alone nor incubation with H2O2
without the complex causes any strand scission (lanes 2
and 3). These experiments demonstrate that both the
copper(II) complex and high amount of H2O2 are required

FIGURE 8 Agarose gel electrophoresis diagram showing the

cleavage of SC pUC18 DNA (500 ng) by complex 2 in Tris–HCl/
NaCl buffer (50 mM, pH = 7.2) lane 1, DNA control; lane 2,
FIGURE 7 CD spectra of CT‐ DNA in the absence (i) and Lane1 + 50 μM complex; lane 3, Lane1 + 100 μM H2O2; lane 4, lane
presence (ii) of complex 1 (a) and complex 2 (b); [DNA] = 200 μM, 1 + 100 μM complex; lane 5, lane 1 + 100 μM complex +100 μM
[complex] = 100 μM, (1/R = 0.5) H2O2; Lane6, lane 1+ 100 μM complex +200 μM H2O2
10 of 11 GUBENDRAN ET AL.

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