Design of a mononuclear copper(II)-phenanthroline complex: Catechol oxidation, DNA cleavage and antitumor properties

Article abstract of DOI:10.1016/j.poly.2015.12.055

A mononuclear copper(II) complex [Cu(phen)(OH₂)₂(NO₃)](NO₃) (1) [phen = 1,10-phenanthroline] has been synthesized and structurally characterized by different spectroscopic characterization methods including sing

Full text of DOI:10.1016/j.poly.2015.12.055

Accepted Manuscript
Design of a mononuclear copper(II)-phenanthroline complex: Catechol oxida-
tion, DNA cleavage and antitumor properties
Dhananjay Dey, Subrata Das, Hare Ram Yadav, Anandan Ranjani, Loganathan
Gyathri, Sanjay Roy, Partha Sarathi Guin, Dharumadurai Dhanasekaran,
Angshuman Roy Choudhury, Mohammad Abdulkader Akbarsha, Bhaskar
Biswas
PII:
S0277-5387(15)00805-0
DOI:
http://dx.doi.org/10.1016/j.poly.2015.12.055
POLY 11754
Reference:
Polyhedron
To appear in:
Received Date:
Accepted Date:
9 July 2015
28 December 2015
Please cite this article as: D. Dey, S. Das, H.R. Yadav, A. Ranjani, L. Gyathri, S. Roy, P.S. Guin, D. Dhanasekaran,
A.R. Choudhury, M.A. Akbarsha, B. Biswas, Design of a mononuclear copper(II)-phenanthroline complex:
Catechol oxidation, DNA cleavage and antitumor properties, Polyhedron (2016), doi: http://dx.doi.org/10.1016/
j.poly.2015.12.055
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Design of a mononuclear copper(II)-phenanthroline complex:
Catechol oxidation, DNA cleavage and antitumor properties
Dhananjay Dey,[a] Subrata Das,[a] Hare Ram Yadav,[b] Anandan Ranjani,[c]
Loganathan Gyathri,[c] Sanjay Roy,[d] Partha Sarathi Guin,[d] Dharumadurai
Dhanasekaran,[c] Angshuman Roy Choudhury,[b] Mohammad Abdulkader
Akbarsha,[e] and Bhaskar Biswas,[a]*
aDepartment of Chemistry, Raghunathpur College, Purulia-723 133, India
bDepartment of Chemical Sciences, Indian Institute of Science Education and Research,
Mohali, Mohali 140 306, India
cDepartment of Microbiology, Bharathidasan University, Tiruchirappalli 620 024, India
dDepartment of Chemistry, Shibpur Dinobundhoo Institution (College), Howrah- 711 102,
India
eMahatma Gandhi-Doerenkamp Center, Bharathidasan University, Tiruchirappalli 620 024,
India; Department of Food Science and Nutrition, College of Food and Agriculture, King
Saud University, Riyadh, Kingdom of Saudi Arabia.
___________________________________________________________________________
Abstract
A mononuclear copper(II) complex [Cu(phen)(OH2)2(NO3)](NO3) (1) [phen = 1,10-
phenanthroline] has been synthesized and structurally characterized by different spectroscopic
characterization methods including single crystal X-ray diffraction study. X-ray crystal
structure analysis shows that 1 adopts square pyramidal geometry with CuN2O3 chromophore
and the molecule crystallises in P21/n space group. 1 has been evaluated as model system for
the catechol oxidase enzyme by using 3,5-di-tert-butylcatechol (DTBC) as the substrate in
methanol medium, revealing that 1 exhibits greater catalytic activity with Kcat value 3.91×103
hour-1 compared to a few copper(II) complexes of the same class. Electrochemical analysis
suggests that the mononuclar Cu(II) complex mimics the catechol oxidase enzyme in
methanolic medium through radical pathway. The complex cleaves the double strand of pBR
322 plasmid DNA in a concentration-dependent manner so as for the DNA to change from
super coiled form to both nicked circular and linear forms. The complex is cytotoxic to the
human hepatocarcinoma cell HepG2, as revealed in MTT assay. AO/EB and Hoechst 33528
fluorescent staining techniques were used to find the mode of cell death. Interestingly, AO/EB
and Hoechst stained cells observed at IC50 concentration shows that 1 brings about
conformational change on DNA to induce apoptosis which would be the basis underlying its
cytotoxic property.

2
Keywords: Copper(II); Crystal structure; Catecholase activity; DNA Cleavage; Antitumor
activity
___________________________________________________________________________
*To whom correspondence should be addressed. Fax/Tel: +91 3251 255235; E-mail:
mr.bbiswas@rediffmail.com / icbbiswas@gmail.com (B. Biswas)
1. Introduction
Synthesis of potential metal-based drugs, which may achieve wider spectrum of activity,
enabling the administration of a lower dose, attack on different types of tumor cells, overcome
drug resistance problems, and exhibit better selectivity and lower toxicity than cis-platin have
received considerable curiosity [1-3]. So, there is substantial attention focused on the design
of new metal-based anticancer drugs that exhibit enhanced selectivity and novel modes of
DNA interaction like non-covalent interactions that mimic the mode of interaction of
biomolecules [4-7]. Sigman and co-workers [8] developed the first chemical nuclease,
bis(1,10-phenanthroline)copper(I) complex, that effectively cleaves DNA by binding the
minor groove in the presence of hydrogen peroxide. Very recently, certain mixed ligand
copper(II) complexes, which strongly bind and cleave DNA, have been shown to exhibit
prominent anticancer activities and regulate apoptosis [9-11]. Therefore, designing suitable
copper complexes for DNA binding and cleavage under physiological conditions is of
remarkable importance in taking into account the advantages of processes that produce
fragments similar to those formed by restriction enzymes [12-15].
On the other hand, investigation on the synthetic analogues of catechol oxidase has emerged
as a field of modern research in bioinorganic chemistry to gain insight into functional
mechanisms of catechol oxidase and elucidate different internal and external parameters like
pH, nature of model substrates and solvents, the flexibility of the coordinating ligands, and the
coordination environment of the copper centers which may influence the activity [16-21]. As
the active site of catechol oxidase consists of a bridged-dicopper(II) moiety, several dinuclear
copper(II) complexes derived from various chelating ligands have been widely studied for this
purpose [22-26]. In addition to the dicopper(II) systems, a few mononuclear copper(II)
complexes [27-29] and even a few copper(II) clusters and polymers have been found as active
catalysts [30]. All these facts reflect that explorations of the possibility of exhibiting
catecholase activity by new types of compounds are very much demanding, and our research
group is engaged in this endeavor. In the present work, we report a mononuclear copper(II)-

3
phen complex of square pyramidal geometry with potential catecholase activity. Spectroscopic
investigation and electrochemical analysis suggest rapid conversion of catechol to quinone via
Cu(I)-superoxide intermediates with hydrogen peroxide as by product. The mononuclear
copper(II) complex effectively cleaves DNA under physiological conditions. It is cytotoxic to
a human hepatocarcinoma cell line (HepG2) and the mechanism of cell death is essentially
apoptosis.
2. Materials and methods
2.1. Chemicals, solvents and starting materials
High purity 1,10-phenanthroline (E. Merck, India), copper(II) nitrate trihydrate (E. Merck,
India), 3,5-di-tert-butylcatechol (Sigma-Aldrich, USA) and all reagents were purchased from
the respective outlets and used as received.
2.2. Physical measurements
Infrared spectrum (KBr) was recorded with
a
FTIR-8400S SHIMADZU
spectrophotometer in the range 400–3600 cm–1. 1H NMR spectrum in DMSO-d6 was obtained
on a Bruker Avance 300 MHz spectrometer at 25°C and was recorded at 299.948 MHz.
Chemical shifts are reported with reference to SiMe4. Ground state absorption was measured
in a JASCO V-730 UV-vis spectrophotometer. Electrospray ionization (ESI) mass spectrum
was recorded using a Q-tof-micro quadruple mass spectrometer. The pH value of the solutions
was measured in Systronics pH meter at room temperature. Elemental analyses were
performed on a Perkin Elmer 2400 CHN microanalyzer.
2.3. Synthesis of [Cu(phen)(OH2)2(NO3)](NO3) (1)
A methanolic solution (10 mL) of phen (0.180 g, 1 mM) was added drop-wise to an aqueous
solution (15 mL) of Cu(NO3)2.3H2O (0.305 g, 1 mM). The blue solution was filtered and the
supernatant liquid was kept in air for slow evaporation. Yield: 0.448 g (69% based on metal
salt). Anal. Calc. for C12H12N4O8Cu (1): C, 35.69; H, 3.00; N, 13.88. Found: C, 35.80; H,
2.95; N, 13.95%. IR (KBr, cm-1): 3393 (vOH),1626, 1604 (vC=N), 1384 (vNO3); UV-Vis (λmax
,
nm): 278, 302, 695.
2.4. Crystal structure determination and refinement
Single crystal X-ray diffraction data were collected using a Rigaku XtaLABmini
diffractometer equipped with Mercury375R (2x2 bin mode) CCD detector. The data were
collected with graphite monochromated Mo-Kα radiation (λ=0.71073 Å) at 298(2) K using ω

4
scans. The data were reduced using Crystal Clear suite, and the space group determination
was done using Olex2. The structure was resolved by direct method and refined by full-matrix
least-squares procedures using the SHELXL-97 software package using OLEX2 suite [31,
32].
2.5. Catalytic oxidation of 3,5-DTBC
In order to examine the catecholase activity of the complex, a 10−4M solution of 1 in
methanol solvent was treated with 100 equiv. of 3,5-di-tert-butylcatechol (3,5-DTBC) under
aerobic conditions at room temperature. Absorbance vs. wavelength (wavelength scans) of
these solutions were recorded at a regular time interval of 5 min in the wavelength range 300–
500 nm.
The kinetics of oxidation of 3,5-DTBC were determined by the method of initial rates and
involved monitoring the growth of the quinone band at 400 nm as a function of time [33].
Kinetic experiment was performed with 1 (at a constant concentration of 1×10-4 M) and 3,5-
DTBC (varying the concentration from 1 × 10-3 M to 1 × 10-2 M) in methanol adopting UV-
Vis spectrophotometer. The experimental procedure involved the preparation of stock
solutions of the mononuclear Cu(II) complex and the substrate 3,5-DTBC at higher
concentrations in methanol medium. 2 mL of 3,5-DTBC at appropriate concentration,
obtained by accurate dilution from the stock solution, was taken into the UV-Vis quartz cell
and kept for a while inside the cell holder which was attached with a thermostat to keep the
temperature at 25 °C. Then, 0.04 mL of stock solution of the complex was added to it to
achieve the ultimate concentration of the complex as 1×10-4 M. The formation of 3,5-DTBQ
was monitored with time at a wavelength of 400 nm. The initial rate method was applied to
determine the rate constant value for each concentration of the substrate and each experiment
was repeated thrice.
2.6. Detection of presence of hydrogen peroxide in the catalytic oxidation of 3,5-
DTBC
To detect the formation of hydrogen peroxide during the catalytic reaction, we followed a
reported method [34]. Reaction mixtures were prepared as in the kinetic experiments. During
the course of the oxidation reaction, the solution was acidified with H2SO4 to pH 2 to stop
further oxidation after a certain time and an equal volume of water was added. The formed
quinone was extracted three times with dichloromethane. To the aqueous layer were added 1
mL of a 10% solution of KI and three drops of a 3% solution of ammonium molybdate. The

5
formation of I3− could be monitored spectrophotometrically because of the development of the
characteristic I3− band (λmax= 353 nm) (Fig. S6).
2.7. Electrochemical analysis
The electrochemical analysis of 1 in 50% methanol in the presence of 0.1 M NaF as an
supporting electrolyte was performed with Ag/AgCl, and saturated KCl, respectively, at the
scan rate of 20 mVs-1. The number of electrons involved in the reduction was confirmed by
the chronocoulometry study. A plot of cathodic peak current (Ipc) with the square root of the
scan rate (
was studied using the following equation (1) to check whether the reduction is
purely diffusion controlled or not and also to detect the presence of any adsorbed species on to
the electrode surface.
Ipc = (2.69 ×105) n3/2 DO1/2 A C v1/2
…(1)
To find the catalytic role of copper complex, a mixture containing one part of copper
complex and 100 parts of 3,5-DTBC was prepared and incubated for 1 minute which was then
followed by cyclic voltammetry without purging of argon gas.
2.8. DNA cleavage studies
Cleavage of DNA was monitored by agarose gel electrophoresis technique, with the
complex [Cu(phen)(OH2)2(NO3)](NO3) (1) at two different concentrations (25 and 50µg per
mL). pBR 322 DNA was incubated with the complex for one hour at 37 °C. The reaction was
also monitored in the presence of the radical activator (H2O2) and an inhibitor (DMSO). The
inhibition reaction was carried out by the addition of the inhibitor reagent prior to the addition
of the complex and the activator. After incubation 2 µL of bromophenol blue dye solution was
mixed with the reaction mixture. The latter was then loaded carefully onto the electrophoresis
chamber wells in the agarose gel when control DNA was loaded onto a separate well. TAE
buffer was used as the running buffer and the electrophoresis was conducted at a constant
current of 50 V per well for 30 min. The bands were observed in the gel documentation
system and photographed to find the extent of DNA cleavage.
2.9. Anticancer activity of the [Cu(phen)(OH2)2(NO3)](NO3) (1)
2.9.1. Cell culture
The HepG2 human hepatocarcinoma cell was obtained from the National Center for
Cell Science (NCCS), Pune, India. The cells were cultured in DMEM medium (Sigma–
Aldrich, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (Gibco) and 100

6
U/mL of penicillin and 100 µg/mL of streptomycin as antibiotics (Gibco), in 96 well culture
plates, at 37 °C, in a humidified atmosphere of 5% CO2, in a CO2 incubator (Forma, Thermo
Scientific, USA). All experiments were performed using cells from passage 15 or less.
2.9.2. Cytotoxicity assay (MTT assay)
The complex, in the concentration range from 50 to 500 µM in DMSO, was added to
the wells 24 h after seeding of 5 × 103 cells per well in 200 µL of fresh culture medium.
DMSO solution was used as the solvent control. A miniaturized viability assay using 3-(4,5-
di-methylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was carried out according
to the method described by Mosmann [35]. After 24 and 48 h, 20 µL of MTT solution [5
mg/mL in phosphate-buffered saline (PBS)] was added to each well and the plates were
wrapped with aluminum foil and incubated for 4 h at 37 °C. The purple formazan product was
dissolved by addition of 100 µL of DMSO to each well. The absorbance was monitored at 570
nm (measurement) and 630 nm (reference) using a 96-well plate reader (Bio-Rad, Hercules,
CA, USA). Data were collected for three replicates each and used to calculate the mean. The
percentage inhibition was calculated, from this data, using the formula:
[Mean OD of untreated cells (control) - Mean OD of treated cells (treated)] × 100
=
Mean absorbance of untreated cells (control)
From these data, the IC50 (the concentration at which the complex killed 50% of the cells at
the respective durations of treatment) for 24 and 48 h treatment were arrived at.
2.9.3. Acridine orange (AO) and ethidium bromide (EB) staining
Acridine orange and ethidium bromide staining was performed as described by Spector
et al. [36]. The cell suspension of each sample containing 5 × 105 cells, was treated with 25 µL
of AO and EB solution (3.8 µM of AO and 2.5 µM of EB in PBS) and examined in a
fluorescent microscope (Carl Zeiss, Jena, Germany) using an UV filter (450–490 nm). Three
hundred cells per sample were counted in triplicate for each dose point. The cells were scored
as viable, apoptotic or necrotic as judged by the staining, nuclear morphology and membrane
integrity [36], and the percentages of apoptotic and necrotic cells were then calculated.
Morphological changes were also observed and photographed.
2.9.4. Hoechst 33528 staining
The HepG2 cells were cultured in 6-well plates and treated with the 24 h IC50 of the
complex. After incubation, the treated and untreated cells were harvested and stained with

7
Hoechst 33258 (1 mg/mL, aqueous) for 5 min at room temperature. A drop of cell suspension
was placed on a glass slide, and covered with a cover slip to reduce light diffraction. At
random 300 cells, in duplicate, were observed at ×400 in a fluorescent microscope (Carl Zeiss,
Jena, Germany) fitted with a 377–355 nm filter, and the percentage of cells reflecting
pathological changes was calculated.
3. Results and discussion
3.1. Synthesis and formulation
The mononuclear Cu(II) complex was prepared out of reaction among copper(II) nitrate
trihydrate and 1,10-phenanthroline in water medium. The coordination geometry of 1 was
determined by mainly single crystal X-ray diffraction study along with different spectroscopic
and analytical techniques. The blue crystals suitable for X-ray data collection were obtained
by slow evaporation of resultant reaction mixture. The different formulations were confirmed
by elemental analysis, IR, UV-vis, mass spectral analysis, thermogravimetric analysis and
crystallographic structure analysis of the compound.
+
OH2
H2O
OH2
N
N
Cu(NO3)2
+
Cu
N
N
ONO2
Scheme 1. Procedure for preparation of the copper(II) complex (1)
3.2. Crystal structure
The X-ray structural determination of compound 1 shows that the mononuclear cationic
copper(II) complex adopts distorted square pyramidal geometry around Cu(II) in the space
group P21/n. An ORTEP view of 1 with an atom labelling scheme is shown in Figure 1. The
coordination geometry around copper(II) is described as distorted square pyramidal as evident
from the value of the trigonal index, τ = 0.26 ; [τ = |β−α|/60, where α=90.0° and β=106.132°; τ
is 1 for a perfect trigonal bipyramidal geometry and is zero for a perfect square pyramidal
geometry [37]. The coordination geometry around the Cu centre consists of equatorial plane
which is formed by nitrato oxygen (O1), pyridine nitrogens (N1/N2) and the oxygen atom
(O5) from water and the axial position is occupied by oxygen atom of water molecule (O4).
The charge of cationic unit is counterbalanced by anionic charge of nitrate molecule. The

8
crystal packing shows an extended H-bonding scheme (Fig. S1). In fact, uncoordinated
oxygen atoms of the nitrate molecule act as hosts to form H-bonds towards C-H/O-H bonds,
and lead to one dimensional (1D) chain arrangement (Fig. S1). From supramolecular
perception it is seen that oxygen atoms (O6,O7,O8) of counteranionic nitrate molecule hold
diagonally two mononuclear copper(II) units through H-bonding interactions O···H-O
(O8···H4A, 2.10 A°, O6···H5B, 2.66 A°, O7···H5B, 1.86 A°) between axial water (O4H2) and
equatorial water (O5H2) while each of the mononuclear units in a layer extends via O3···H8-
C8 (2.44 A°). The H-bonding interactions help to form self-assembled 1D chain along b axis.
The crystallographic structural parameters of 1 are listed in Table 1. Selected bond lengths and
angles are presented in Table S1, and the relevant hydrogen bonding parameters are
summarized in Table S2.
3.3. IR and electronic spectra of [Cu(phen)(OH2)2(NO3)](NO3) (1)
A moderately strong, sharp peak due to a C=N stretching vibration, at 1626 cm−1 for the
complex shows the presence of imine group in 1. In complex 1, there is a single band at 3393
cm−1, indicating the presence of O-H stretching vibration of water. The IR spectrum shows
close splitting of the band centered at 1384 cm−1 due to different coordination motifs of nitrate
stretching [38,39]. The bands at 1273, 1421 and 1491 cm−1 and a single band at 1384 cm−1 for
complex 1, corroborate the presence of coordinated and ionic nitrate respectively (Figure S2).
The electronic spectrum of the compound is recorded in methanolic solution at room
temperature. In the higher-energy region of the optical spectrum, 1 shows characteristic bands
at 272 and 302 nm which are assignable to π-π* transition of the C=N chromophore in
phenanthroline while a low intensity broad band centered at 695 nm is observed in the visible
region (Fig. S3), suggesting the square pyramidal geometry around copper centre [40]. The
structural integrity in solution state for the copper(II) complex has been determined by ESI
mass spectral analysis. The ESI mass spectral study of 1 in methanol medium exhibits the
molecular ion peak, at m/z 277.9484 as [Cu(phen)(H2O)2]2+-H+]+ (Fig. S4). The release of
coordinated nitrate ion from the primary zone of coordination is also evident from the ESI-MS
spectrum of 1 and further consolidates the solution instability.

9
3.4. Catechol oxidase activity
3,5-Di-tert-butylcatechol (3,5-DTBC) is the most widely used substrate for the study of
potential catecholase activity of biomimicking coordination compounds, primarily for the
following reasons: (i) its low reduction potential makes it easy to oxidize, (ii) the bulky tert-
butyl substituents prevent further overoxidation reactions such as ring-opening, and (iii) the
oxidation product, 3,5-di-tert-butylquinone (3,5-DTBQ), is highly stable with a characteristic
absorption band maxima at 401 nm in pure methanol.
Scheme 2. Catalytic oxidation of 3,5-DTBC to 3,5-DTBQ in air-saturated methanol solvent
As a model of the catechol oxidase enzyme we have taken one mononuclear copper(II)
complex and examined its efficiency in oxidation of 3,5-DTBC to 3,5-DTBQ. The catalytic
activity of complex 1 was pursued by treating 1×10−4 M solution of the complex with 1×10−2
M of 3,5-DTBC. The reaction was monitored for 1 h after addition of 3,5-DTBC to the
methanolic solution of the complex (Fig. 2). Upon addition of 3,5-DTBC, a new band is
generated at 400 nm and with times to go the band exhibits significant increment of the
absorbance at 400 nm. Since it is well established that 3,5-DTBQ shows band maxima at 400
nm in pure methanol,[41] the experiment unequivocally proves oxidation of 3,5- DTBC to
3,5-DTBQ catalyzed by 1. 3,5-DTBQ was purified by column chromatography and isolated in
high yield (73.7% for 1).
Kinetic studies were performed to understand the extent of the catalytic efficiency of 1.
The kinetics of oxidation of 3,5-DTBC were determined by the method of initial rates and
involved monitoring the growth of the quinone band at 400 nm as a function of time. The
average rate constant values for 1 shows that the rate is first order at low concentrations of the
3,5-DTBC substrate but zero order at its higher concentrations. Figure 3 represents the
dependency of initial rate on the concentration of catechol for complex 1 in methanol medium.
The rates of reaction obtained for various 3,5-DTBC concentrations were fitted to the
Michaelis–Menten equation (Fig. 3) and linearized by means of Lineweaver–Burk plot to

10
determine various enzymatic kinetic parameters (Km, Vmax and Kcat) as in Table 2 for the
copper(II) compound. Comparing the turnover rate between our copper(II) complex and the
reported ones by some renowned scientists it is found that ranging from 4.1 to 23.5 h-1 have
been found which are comparable to those reported by Krebs et al. and Neves et al. [16,22]
and to an order of hundred times more than the complexes (2) reported by Panja et al., Hundal
et al. [47,48] but are significantly lower than those reported by Krebs et al., and Monzani et al.
[24,25,41].
3.5. Mechanistic insight of catecholase activity via electrochemical analysis of 1
The cyclic voltammogram (Fig. 4a) of 1 in 50% methanol in the presence of 0.1 M NaF as
the supporting electrolyte shows one-electron reduction peak (Ia) with the corresponding
oxidation peak (Ib) at -150 mV and + 80 mV vs. Ag/AgCl, saturated KCl, respectively, at the
scan rate of 20 mVs-1. A plot of cathodic peak current (Ipc) with the square root of the scan
rate (
(Fig. 4b) shows a linear relationship passing through the origin which clearly
indicates that this reduction is purely diffusion controlled (diffusion coefficient for reduction =
1.82×10-5 cm2s-1) and there is no adsorption on to the electrode surface. Comparing the
experimental results of the present copper complex with that of earlier results [42] one can say
that the reduction of the present complex is due to the formation of Cu(I)-semiquinone species
in solution. In the complex the negative value of the reduction potential (-150 mV) suggests
that the reduction is quite difficult which is due the fact that the ligands increase the electron
density significantly on central Cu(II). This is the reason why the reduced species becomes
unstable and is oxidised at a positive potential (i.e., + 80 mV). This means that the reduced
species easily oxidises to the original complex (1). In addition there is a sharp reduction peak
(II) in the cyclic voltammogram of the copper complex (Fig. 4a). A plot of cathodic peak
current (Ipc) of this peak with the scan rate (v) shows a linear relationship which clearly
indicates that this reduction is solely due to the adsorption of the compound onto the electrode
surface but not due to bulk electrolysis.
To find the catalytic role of copper complex, electrochemical investigation of a mixture
containing one part of copper complex and 100 parts of 3,5-DTBC shows a reduction and an
oxidation peak at -235 and +210 mV respectively (Fig. 5). In the cyclic voltammogram of the
mixture 3,5-DTBC and copper complex an oxidation hump nearly at +50 mV was observed
which is definitely due to the oxidation of Cu(I)-semiquinone generated in the catalytic
pathways. The decrease in the intensity of the reduction peak of Cu(II) in the cyclic

11
voltammogram of 3,5-DTBC in the presence of copper complex is indicative of the reduction
of Cu(II) to Cu(I) during the course of electro-catalytic effect of the complex.
3.6. Mechanistic inferences
Despite the clarification of the active sites of catechol oxidases being known from its
crystal structure the catalytic mechanism is still a much debated issue. Krebs et al. [43] and
Solomon et al. [44] have suggested largely accepted bio-mimetic mechanism of catechol
oxidase enzyme involving different coordination nature of catechol to the copper centers.
Previously, Chung-Yuan etal. [45] investigated a biomimetic study of a dicopper-
phenanthroline complex encapsulated in silica towards catechol oxidase and showed that
conversion of hydroxo-bridged dicopper(II) complexes into µ-peroxo dicopper(II) complexes
in presence of molecular oxygen, and produced quinone with water as byproduct . To
establish the pathway for the catalytic reaction, we initially detected the presence of hydrogen
peroxide following a reported procedure [34] and found the band at 353 nm which is due to
−
existence of I3 (see experimental section) using a 1×10−2 M concentration of catechol with a
catalyst concentration of 1×10−4 M for 1 (Fig. S5). To obtain a mechanistic inference of the
catecholase activity by 1 and to investigate possible complex−substrate intermediates, we
recorded ESI-MS spectrum for a 1:100 mixture of the complex and 3,5-DTBC within 10 min
of mixing in a methanol solvent (Fig. S6). The spectrum of 1 shows a base peak at m/z 243.9
(100%), which can be assigned to the quinone sodium aggregate [3,5-DTBQ-Na+]+. Other
characteristic peaks at m/z 464.38 indicate the presence of the bidentate catecholate adduct B
as [Cu(phen)(3,5-DTBC)]+-H+]+. Another important peak at m/z 501.0 possibly corroborates
the formation of species C as [Cu(phen)(O2)(3,5-DTBC)-Na+]+. Alternatively, molecular
oxygen exists in triplet state with two electrons occupying two degenerate molecular orbitals
and as a diradical, it can directly react with the Cu(I)-radical species in a termination (radical-
radical) reaction. This reaction pathway may be an alternative to the formation of species C.
From electrochemical analysis and mass spectrometric investigation it is evident that in
solution there is existence of Cu(I) species, semiquinone species, catecholate adduct and
oxygen bound species in the catalytic cycle of the reaction. Now, we are at the stage to
suggest a plausible pathway in which at the first stage active catalyst (B) is generated by the
reaction of 3,5-DTBC with one equivalent of complex (Scheme 3). The catecholate-bound
species exists in equilibrium with copper(I)-semiquinone adduct (B) which generates the
superoxide intermediate (C) with aerial oxygen in which copper(I) center in B undergoes an

12
oxidation to a copper(II) center in C (Scheme 3). In the catalytic cycle, next step involves the
elimination of quinone as major product with hydrogen peroxide as byproduct with
concomitant origination of [Cu(phen)(S)2] (A); where S = H2O.
OH2
N
CuII
A
N
OH2
HO
But
OH
But
MeOH
But
HO
HO
S
But
O
O
N
N
B
u
t
N
N
But
N
N
CuII
CuI
CuII
O
O
S
O
But
But
B
O
A
O
O
N
O
But
CuII
O
2H2O
N
But
+
H2O2
O
O
But
But
C
Scheme 3 Proposed catalytic cycle for the oxidation of 3,5-DTBC by the copper(II) complex
3.7. DNA cleavage studies
.
The irradiation of pBR322 plasmid DNA in the presence of the copper complex in a
concentration (µg/mL) dependent manner was studied so as to determine the efficiency with
which it sensitizes DNA cleavage. The conversion of circular supercoiled DNA to its nicked
circular form was monitored. Though there was conversion of supercoiled form (Form I) to
linear form (Form III) at both the concentrations, cleavage increased with the increasing
concentration of the complex (50 µg) (Fig 6b,c). The cleavage activity was also observed
using a cleavage activator (H2O2) and an inhibitor (DMSO). To find the involvement of
oxygen species in the damage to the DNA, the hydroxyl radical scavenger DMSO was used
along with the complex and the hydroxyl radical (OH·) (Fig 6d), which resulted in significant
inhibition of DNA cleavage confirming the involvement of diffusible hydroxyl radical as the

13
reactive species causing strand scission. The nuclease activity of the complex increased in the
presence of the activator along with the complex (Fig 6e). Sigman et al.,[46,47] proved that
copper(I) complex of 1,10-phenanthroline (OP), [(OP)2-CuI]+, with H2O2 as a co-reactant, is a
chemical nuclease that nicks DNA by either diffusing to the minor groove where it is oxidized
by H2O2 or binds to minor groove where it gets reduced before reacting with H2O2 and the
phenomenon is sensitive to the conformation of the nucleic acid and cleavage of DNA. For
our molecule, the major reason behind this is that the hydroxyl radical’s reactions with the
deoxyribose sugar backbone are initiated by hydrogen abstraction from a deoxyribose carbon,
and the predominant consequence is eventual strand breakage and base release. The DNA
cleavage experiment clearly reveals that the addition of copper(II)-phen complex to the DNA
led to the mild transition from Form I (Supercoiled) to Form III (Linear). But the addition of
the activator led to the conversion of Form I to Form II (Nicked circular) which was absent
with the addition of the hydroxyl radical scavenger.
3.8. Anticancer activity of [Cu(phen)(OH2)2(NO3)](NO3) (1)
3.8.1. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay
Since the present mononuclear copper(II) complex has the ability to cleave DNA in the
presence and absence of a reductant, and since DNA cleavage is considered important for a
drug to act as an anticancer agent [48,49], the cytotoxic effect of the complex, dissolved in
DMSO, was investigated against a human hepatocarcinoma cell line (HepG2) adopting MTT
assay. The IC50 values were obtained by plotting the cell viability against the concentration of
the complex (Fig. 7). The results revealed that the IC50 at 48 h (60 0.2 µM) is lower than at 24
h (80 0.1 µM) clearly indicating that the complex exhibits cytotoxicity against HepG2 in a
dose- and duration-dependent manner. Thus, the cytotoxicity exhibited by the complex is
consistent with its strong binding with DNA, and its efficiency in cleaving DNA in the
absence of an external agent is responsible for its potency to induce cell death.
3.8.2. Fluorescent staining for apoptosis
Apoptotic cell death is known as characterized by different cellular changes such as cell
shrinkage, nuclear condensation, DNA fragmentation, membrane blebbing and formation of
apoptotic bodies. These apoptotic characteristics as produced in HepG2 human
hepatocarcinoma cell by the [Cu(phen)(OH2)2(NO3)](NO3) (1) complex were analyzed
adopting AO/EB staining. In this staining method, the fluorescence pattern depends on the

14
viability and membrane integrity of the cells. In general, dead cells only are permeable to
ethidium bromide and fluoresce orange-red, whereas live cells are permeable to acridine
orange only and thus fluoresce green. The cytological changes which were observed in the
treated cells are classified into four types based on the fluorescence emission and
morphological features of chromatin condensation in the AO/EB stained nuclei: i) viable cells,
possessing highly organized nuclei, fluoresce green; ii) early apoptotic cells, which show
nuclear condensation, emit orange-green fluorescence; iii) in late apoptotic cells, with highly
condensed or fragmented chromatin, the nuclei fluoresce orange to red; and iv) cells
undergoing necrosis fluoresce orange to red, with no indication of chromatin fragmentation.
All these morphological changes were observed after the treatment of cancer cells with the
complex. Figure 8 indicates the apoptotic morphologies induced by the complex at 24 h IC50.
Since the copper complex is a cationic molecule consisting of a planar hetero-polyaromatic
phenanthroline ligand and two aqua molecules at Cu(II) centre, it has the natural ability to
bind with DNA through heterogeneous base pair with ease. The replacement of labile aqua
molecules by DNA base pair or intercalation of phenanthroline ring through base pair may
very effectively cause apoptosis of the affected cells. Figure 9 shows the efficacy of the
complex at its 24 h IC50 to induce apoptosis in close to 50% of cells as revealed in the AO/EB
assay. The IC50 values and morphological changes consolidate the potential anticancer activity
of the molecule. However, further studies are needed in this direction to confirm the mode of
cell death induced by the complex.
3.8.3. Hoechst staining
Morphological changes in the nucleus and chromatin were revealed by Hoechst 33528
staining method. The cells treated with 24 h IC50 of the complex showed changes in the
morphology of the nuclei. In the control cells the nuclei were round with intact chromatin
while after treatment with the complex changes such as chromatin marginalization,
condensation and fragmentation were observed (Figure 8), which are characteristic of
apoptosis. It is interesting that close to 50% of treated cells exhibited nuclear morphologies
indicating apoptosis (Fig. 10).
4. Conclusion
We have synthesized and characterized a mononuclear copper(II)-phenanthroline complex of
the type [Cu(phen)(OH2)2(NO3)](NO3) which can act as potential catalyst for the oxidation of

15
catechols to quinones, mimicking the catecholase activity of catechol oxidase with high
turnover number, Kcat (h-1) value as 3.91×103. The DNA cleavage experiment clearly reveals
that the addition of 1 led to the partial transition from Form I (Supercoiled) to Form III
(Linear). But the addition of an activator led to the conversion of Form I to Form II (Nicked
circular) which was not the case following addition of the hydroxyl radical scavenger.
Induction of apoptosis in the human hepatocarcinoma cell line (HepG2) was assessed in
relation to the changes in cell viability, cellular morphology and nuclear cytology, which
shows the efficacy of 1 to induce apoptosis in time and dose-dependent manner. Interestingly,
the observed IC50 values reveal that 1 effects conformational change on DNA strongly.
Moreover, detailed experimentation related to DNA interaction, cleavage and cytotoxicity on
this complex will help to expound clinically relevant information for developing possible new
anticancer drugs.
Supplementary data
Supplementary crystallographic data are available free of charge from The Director,
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44-1223-336033; E-mail:
deposit@ccdc.cam.ac.uk or www: http://www.ccdc.cam.ac.uk) upon request, quoting
deposition number CCDC 1061531.
Acknowledgments
DD acknowledges fellowship conferred upon him by the Department of Science and
Technology (DST), New Delhi. The work was supported financially by DST, India, under the
FAST TRACK SCHEME for YOUNG SCIENTIST (No. SB/FT/CS-088/2013dtd.
21/05/2014). The single crystal X-ray facility of the Department of Chemical Sciences, IISER,
Mohali, is acknowledged. HRY thanks CSIR for the fellowship to him. MAA thanks
Doerenkamp-Zbinden Foundation, Switzerland, for the fund to establish the in vitro lab at
MGDC.
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19
Table 1. Crystallographic refinement parameters of [Cu(phen)(H2O)2(NO3)](NO3) (1)
Crystal parameters
Empirical formula
Formula weight
Temperature
1
C20H20CoN5Cl2O5
540.21
293 K
Wavelength
0.71073 Å
Crystal system
Space group
Triclinic
P-1
Unit cell dimensions
a = 7.0995(3) Å α= 91.829(4)o
b = 12.2760(6) Å ß= 91.508(4)o
c = 13.1897(7) Å ᵞ = 99.633(4)°
3
Volume
Z
1132.16(10) Å
2
3
Density (calculated)
1.573 Mg/m
-1
Absorption coefficient
F(000)
1.036 mm
544
Reflections collected
Independent reflections
R(int)
28913
6566
0.021
Completeness to theta
99.6 %
2
Goodness-of-fit on F
1.058
Final R indices [I>2sigma(I)]
R indices (all data)
R1 = 0.0786, wR2 = 0.2835
R1 = 0.0623, wR2 = 0.1488
-3
0.610 and -0.426 e. Å
Largest diff. peak and hole
Table 2. Kinetic parameters for the oxidation of 3,5-DTBC catalyzed by 1 in methanol
Complex
Vmax (M s-1)
1.08×10-3
1.31×10-7
1.145×10-7
Km (M)
Kcat ( h-1)
3.91×103
2.35×101
4.1
Ref
Present
50
2.7×10-3
2.27×10-3
2.12×10-3
1*
2
2
51
*Std. Error for Vmax (MS-1) = 6.40×10-6; Std. Error for Km (M)= 3.71×10-4

20
Fig 1. An ORTEP diagram of [Cu(phen)(OH2)2(NO3)](NO3) (1) with atom numbering
scheme and 30% probability ellipsoids.
3,5-DTBQ
0.04
0.02
0.00
300
400
500
600
Wavelength (nm)
Fig 2. UV-vis spectral change of complex at a regular interval of 5 min with 3,5-DTBC.

21
0.00010
0.00008
0.00006
0.00004
0.00002
0.00000
35000
30000
25000
20000
15000
10000
0
200
400
600
800
1000
1/[Substrate]
0.000
0.002
0.004
0.006
0.008
0.010
[Substrate] (M)
Fig. 3 Plot of rate vs. [substrate] in presence of 1 in MeOH; inset: Lineweaver-Burk plot
Fig. 4(a): Cyclic voltammogram of 1 in 50% methanol. Scan rate: 20 mVs-1. [1] = 10-3 M,
[NaF] = 0.1 M, T = 298.15 K; (b): Plot of cathodic peak current (Ipc) vs. the square root
of the scan rate (v1/2) for the reduction of 1.

22
Fig 5. Cyclic voltammogram of 1 and 3,5-DTBC mixture in 50% methanol. Scan rate: 20
mVs-1. [1] = 10-3 M, [3,5-DTBC], = 0.1 M, [NaF] = 0.1 M, T = 298.15 K.
Fig 6. DNA cleavage activities of 1 [Lane a: Control pBR 322 DNA, Lane b: pBR 322 DNA +
25 µg of 1, Lane c: pBR 322 DNA + 50 µg of 1, Lane d: pBR 322 DNA + 25 µg of 1+ 5 µl
DMSO + 5 µl H2O2 , Lane 5: pBR 322 DNA + 25 µg of 1+ 5 µl H2O2]

23
Fig 7. MTT assay of [Cu(phen)(OH2)2(NO3)](NO3) (1) in HepG2 cells treated for for 24 h and
48 h
Fig 8. Representative morphological changes observed in HepG2 cells after 24 h incubation
with [Cu(phen)(OH2)2(NO3)](NO3) (1) as revealed in AO/EB staining (left) and Hoechst
staining (right)

24
Fig 9. The relative percentage of morphological changes in HepG2 cells after 24 h incubation
with [Cu(phen)(OH2)2(NO3)](NO3) (1) as revealed in AO/EB staining
Fig 10. The relative percentage of morphological changes in HepG2 cells after 24 h incubation
with [Cu(phen)(OH2)2(NO3)](NO3) (1) as revealed in Hoechst staining.

25
Design of a mononuclear copper(II)-phenanthroline complex:
Catechol oxidation, DNA cleavage and antitumor properties
Dhananjay Dey,[a] Subrata Das,[a] Hare Ram Yadav,[b] Anandan Ranjani,[c] Loganathan
Gyathri,[c] Sanjay Roy,[d] Partha Sarathi Guin,[d] Dharumadurai Dhanasekaran,[c] Angshuman
Roy Choudhury,[b] Mohammad Abdulkadhar Akbarsha,[e] and Bhaskar Biswas,[a]*
G R A P H I C A L A B S T R A C T
________________________________________________________________________
A mononuclear copper(II) complex [Cu(phen)(OH2)2(NO3)](NO3) (1) has been evaluated as
model system for the catechol oxidase enzyme by using 3,5-di-tert-butylcatechol (DTBC) as
the substrate in methanol medium, revealing that 1 exhibits greater catalytic activity with Kcat
value 3.91×103 hour-1. The complex cleaves the double strand of pBR 322 plasmid DNA in a
concentration-dependent manner and is cytotoxic to the human hepatocarcinoma cell HepG2.
________________________________________________________________________________________________

26
Design of a mononuclear copper(II)-phenanthroline complex:
Catechol oxidation, DNA cleavage and antitumor properties
Dhananjay Dey,[a] Subrata Das,[a] Hare Ram Yadav,[b] Anandan Ranjani,[c] Loganathan
Gyathri,[c] Sanjay Roy,[d] Partha Sarathi Guin,[d] Dharumadurai Dhanasekaran,[c] Angshuman
Roy Choudhury,[b] Mohammad Abdulkadhar Akbarsha,[e] and Bhaskar Biswas,[a]*
P I C T O G R A M
________________________________________________________________________
________________________________________________________________________________________________