Di and tetranuclear Cu(II) complexes with simple 2-aminoethylpyridine: Magnetic properties, phosphodiester hydrolysis, DNA binding/cleavage, cytotoxicity and catecholase activity

Article abstract of DOI:10.1016/j.poly.2019.02.015

Di and tetranuclear Cu(II) complexes, [Cu₂(2-AEP)₄(μ-Cl)](ClO₄)₃ (1) and [Cu₄(μ₃-OH)₂(μ₂-OH)₂(2-AEP)₄(μ₂-ClO₄)₂]

Full text of DOI:10.1016/j.poly.2019.02.015

Accepted Manuscript
Di and Tetranuclear Cu(II) Complexes with Simple 2-Aminoethylpyridine:
Magnetic Properties, Phosphodiester Hydrolysis, DNA Binding/Cleavage, Cy-
totoxicity and Catecholase Activity
Popuri Sureshbabu, Qazi Mohammad Junaid, C. Upadhyay, W. Victoria,
Vidhyapriya Pitchavel, Sakthivel Natarajan, S. Sabiah
PII:
S0277-5387(19)30108-1
https://doi.org/10.1016/j.poly.2019.02.015
POLY 13759
DOI:
Reference:
Polyhedron
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 September 2018
29 January 2019
1 February 2019
Please cite this article as: P. Sureshbabu, Q. Mohammad Junaid, C. Upadhyay, W. Victoria, V. Pitchavel, S.
Natarajan, S. Sabiah, Di and Tetranuclear Cu(II) Complexes with Simple 2-Aminoethylpyridine: Magnetic
Properties, Phosphodiester Hydrolysis, DNA Binding/Cleavage, Cytotoxicity and Catecholase Activity,
Polyhedron (2019), doi: https://doi.org/10.1016/j.poly.2019.02.015
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Di and Tetranuclear Cu(II) Complexes with Simple 2-
Aminoethylpyridine: Magnetic Properties, Phosphodiester Hydrolysis,
DNA Binding/Cleavage, Cytotoxicity and Catecholase Activity
Popuri Sureshbabu,a Qazi Mohammad Junaid,a C. Upadhyay,b W. Victoria,a Vidhyapriya
Pitchavel,c Sakthivel Natarajanc and S. Sabiaha*
aDepartment of Chemistry, Pondicherry University, Pondicherry – 605 014, India.
bSchool of Materials Science and Technology, Indian Institute of Technology (BHU) – 221005, India,
cDepartment of Biotechnology, Pondicherry University, Puducherry – 605 014, India.
*Email. sabiahs@gmail.com
Abstract
Di and tetranuclear Cu(II) complexes, [Cu2(2-AEP)4(µ-Cl)](ClO4)3 (1) and [Cu4(µ3-
OH)2(µ2-OH)2(2-AEP)4(µ2-ClO4)2](ClO4)2 (2), with the simple 2-aminoethylpyridine (2-AEP)
ligand have been synthesized and characterized by different spectroscopic and analytical
techniques. The X-ray structure reveals that complex 1 is a dimer with a monochloro bridge
connecting the two copper atoms and complex 2 is a tetramer with µ2 and µ3 hydroxo bridges
connecting the four copper atoms. Both copper centers in complex 1 have a distorted square
pyramidal (sp) geometry, whereas two copper centres show a sp geometry and the other two
copper centres show a distorted octahedral geometry in complex 2. Variable temperature
magnetic susceptibility analysis reveals that complex 1 shows a ferromagnetic interaction with 2J
= +1.73 cm-1, whilst 2 shows predominantly antiferromagnetic interactions between the
copper(II) ions with J1 = +2.98 and J2= -16.91 cm-1. Both complexes 1 and 2 hydrolyze the
phosphodiester BNPP with rate constants k = 9.65 × 10-3 and 1.42 × 10-2 s-1 in CH3CN/H2O,
respectively. These complexes interact with DNA as evidenced by theoretical and experimental
methods. The DNA in silico study suggests that complexes 1 and 2 bind with CT-DNA through
minor groove interactions, which is further confirmed by UV-Vis spectroscopic titrations, CD
measurements, viscosity studies and electrochemical methods. The binding interaction of both
1

complexes with calf thymus DNA shows efficient binding with Kb values of 3.54 × 104 M-1 for 1
and 3.18 × 104 M-1 for 2. Both complexes proficiently cleave plasmid DNA (pBR322) to the
linear form (form III) at 25 µM under oxidative conditions and both exhibit moderate cytotoxic
activity on cervical cancer cell lines (ME-180 and SiHa). In addition, both complexes catalyze
the oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC) to 3,5-di-tert-butylquinone (3,5-DTBQ), as
studied by UV-Vis spectroscopic titrations. The rate of the reaction is 1.47 × 10-3 M s-1 for 1 and
3.45 × 10-3 M s-1 for 2. The present complexes, with the simple 2-AEP ligand, show diverse bio-
inorganic aspects.
Keywords: 2-Aminoethylpyridine, BNPP hydrolysis, DNA binding, tetranuclear copper,
cytotoxicity, catecholase activity.
1. Introduction
Di and multinuclear metal complexes with bridging oxo, hydroxo, chloro and carboxylate
ligands have been comprehensively studied to understand structural as well as magnetic
properties.1 Since several type 3 copper metalloproteins and non-heme iron centers have similar
bridging motifs, di/multinuclear complexes with bridging ligands are considered as bio-inspired
structural motifs.2,3 Especially, a bridging moiety with a nucleophilic nature is suitable for
attacking the phosphorus atom of phosphodiesters.4-6 Hence, the hydrolytic or oxidative cleavage
of phosphoesters by multinuclear copper complexes has gained much attention.7-9 Since DNA
contains phophodiester bonds, these complexes can also be viewed as targets for nucleic acid
chemistry.7-9 In that sense, their interaction and binding of DNA have received greater interest,
partly due to their redox nature, lewis acidity, pharmacological activity and bio-relevance.10-12
Apart from this, di/tetra copper complexes with a bridging motif are well suited for
magnetostructural correlation via super exchange pathways.13 They may show ferromagnetic or
2

antiferromagnetic behavior, depending on the bridging ligand, geometry at the metal center, Cu–
Cu distance and Cu–L(Bridge)–Cu angles.13 Although many complexes have been reported with di
hydroxo bridging ligands,14, 15 mono chloro bridged dimers and tetrahydroxo bridged tetramers
are less studied with regard to magneto-structural chemistry, especially complexes with bidentate
N-donor ligands. Apart from this, bi/multinuclear copper complexes are often explored as
catecholase models to convert catechols to their corresponding ortho-quinones.16-22
By considering these facts, we are interested to synthesize bi and multinuclear copper
complexes with the simple 2-aminoethyl pyridine (2-AEP) ligand, with bridging hydroxyl and
chloride ligands. In this perspective, we have successfully synthesized two homoleptic di and
tertanuclear copper (II) complexes, [Cu2(2-AEP)4(µ-Cl)](ClO4)3 (1) and [Cu4(µ3-OH)2(µ2-
OH)2(2-AEP)4(µ2-ClO4)2](ClO4)2 (2). These complexes show intraferromagnetic (1) and
antiferromagnetic (2) interactions via the bridging ligands. Both complexes are effective towards
phosphodiester hydrolysis, DNA binding/cleavage and catecholase activit, and they show
anticancer activity against ME-180 and SiHa cancer cell lines. The results obtained from these
studies are elaborated in the present paper.
2. Results and discussion
2.1. Synthetic aspects of the copper complexes
In the present study, the simple, cheap and readily available bidentate ligand 2-
aminoethylpyridine (2-AEP) was used for complexation with Cu(II) salts. The reaction of 2-AEP
with copper(II) chloride dihydrate (CuCl2.2H2O) and copper(II) perchlorate hexahydrate
(Cu(ClO4)2.6H2O) in a 1:1 ratio yielded di and tetranuclear copper(II) complexes 1 and 2 in good
yields, as illustrated in Scheme 1. Although the PF6 analogue of 2-AEP is known, its synthesis
was entirely different from that in the present work.23e Complexes 1 and 2 were found to be air
and moisture stable and are soluble in common solvents like DMSO, DMF, CH3CN and H2O.
3

These complexes are partially soluble in MeOH. Both complexes were characterized by
spectroscopic (UV-Vis, FT-IR and EPR) and elemental analyses. Variable temperature magnetic
susceptibility studies were investigated to understand the magnetic nature of these bridged
copper complexes. Structurally characterized dinuclear copper(II) complexes containing a single
chloro bridge with bidentate ligands are scant in the literature. Only nine examples are known
with bidentate ligands, and their magnetic data were not available.23, 43
3+
N
N
NH2
NH2
NH2
CuCl2 . 2H2O
Cl
Cu
Cu
(ClO4)3
NaClO
EtOH,
rt, 2 h
4
H2N
N
N
NH2
N
1
O
, 45%
O
O
Cl
2+
O
H2
N
H
N
Cu(ClO4)2 . 6H2O
O
Cu
Cu
H
O
N
Et3N/EtOH
rt, 2 h
N
(ClO4)2
H2
H2
N
O
H
N
Cu
Cu
N
O
H
O
N
H2
O
O
Cl
O
2
, 70%
Scheme 1. Synthetic pathway for the di and tetranuclear copper complexes 1 and 2.
2.2. Structural description of 1 and 2
2.2.1. Complex 1: Complex 1 was crystallized from an acetonitrile/methanol mixture as blue
crystals in the monoclinic system with the C 2/c space group [Fig. 1a]. The asymmetric unit of
complex 1 consists of one Cu(II) ion, two 2-AEP ligands, one coordinated chloride anion (Cl-)
and two perchlorate anions [Fig. 1b]. There is an inversion centre within the molecule. The two
4

Cu(II) centers are connected by a singly bridging chloride anion to form a dinuclear unit having
the molecular formula [Cu2(2-AEP)4(µ-Cl)](ClO4)3. Both Cu(II) centres are in a distorted square
pyramidal (sp) geometry, with contributions from four nitrogen atoms of the two 2-AEP ligands
and the fifth coordinating site being occupied by the mono bridged chloride anion (Fig. 1(c)).
The geometry on each copper(II) ion could be best described with the geometric parameter 
(trigonality index) whose value suggests a square pyramidal geometry when  is close to zero
and a trigonal bipyramidal geometry when  is close to 1.24 For complex 1,    at both the
Cu1 and Cu2 atoms, and hence the geometry around the metal centers is considered as distorted
square pyramidal. The two copper(II) ions are separated from each other by 5.280 Å, which is
longer than for the PF6 analogue.23e The average Cu-N bond length of 2.028 Å (Table 2) is
comparable with the values reported for other dinuclear Cu(II) amine complexes.25-27 Complex 1
exhibits moderate H-bonding between the hydrogen atoms on the NH2 and aliphatic CH2 groups
and the perchlorate oxygen atoms, which stabilize the structure {C-H6∙∙∙O4 (2.705 Å) and
NH2∙∙∙O3 (1.990 Å); O(4)∙∙∙H(6b)-C(6) = 146.77° and O(3)∙∙∙H(4B)-N(4) = 159.91°}.
5

Fig. 1. (a) Crystal structure of complex 1. (b) Asymmetric unit. Color code: H, C (grey), N
(blue), Cl (green) and Cu (cyan). (c) Distorted square pyramidal (SP) geometry around the
copper(II) ion. (d) Polyhedral view of the SP geometry.
2.2.2. Complex 2: Complex 2 was crystallized from acetonitrile solvent as blue crystals. Single
crystal XRD analysis shows a triclinic system with the P-1 space group. The asymmetric unit of
complex 2 consists of two copper(II) ions, two AEP ligands, two coordinated hydroxyl groups,
one perchlorate ligand and one perchlorate anion [Fig. 2a].
6

Fig. 2. Diamond view. (a) Asymmetric unit of complex 2. Color code: H, C(grey), N (blue), O
(red), Cl (green) and Cu (cyan). (b) Typical distorted SP geometry around the Cu1(II) ion (one
dimer unit). (c) Crystal structure of 2.
Both Cu(II) centers have different spacial arrangements , namely like five coordination at Cu1(II)
and six coordination at Cu2(II) by 2-AEP N donor atoms, four bridging hydroxyls (two µ2-OH
and two µ3-OH) and a bridging perchlorate oxygen atom, resulting in typical distorted square
pyramidal and octahedral geometries [Fig. 2b]. Hence the tetrameric unit has the formula
[Cu4(µ3-OH)2(µ2-OH)2(2-AEP)4(µ2-ClO4)2](ClO4)2 (Fig. 2c). The geometric parameter  = 0.06
7

at the Cu1 atom supports the square pyramidal geometry around the Cu1(II) center in complex 2.
The Cu---Cu distances vary from 2.872 to 6.161 Å. An effective interaction is seen between the
Cu1 and Cu2 atoms, with a separation of 2.872 Å. The average Cu-N bond length of 2.018 Å
(Table 2) is comparable with the values reported for other tetranuclear Cu(II) N-donor
complexes.25-27 Many hydrogen bonds exist between the nitrogen bonded hydrogen atoms of 2-
AEP and the oxygen atoms of the perchlorate anions, which stabilize the structure. Packing
diagrams of complexes 1 and 2 are shown in Fig. 3.
Fig. 3. Illustration of the crystal packing diagrams in two dimensional sheets of (a) complex 1,
viewed along the c-axis, and (b) complex 2, viewed along the b-axis.
8

Table 1. Crystal parameters for 1 and 2
Parameters
CCDC
1
2
1567730
1567729
Molecular formula
Molecular weight
Crystal system
Space group, Z
a (Å)
C28H40Cl4Cu2N8O12 C28H44Cl4Cu4N8O20
949.56
Monoclinic
C 2/c, 4
21.975(8)
12.215(3)
15.288(6)
90
1208.68
Triclinic
P-1, 1
8.846(5)
11.236(7)
12.663(8)
112.305(6)
105.254(5)
95.851(5)
1094.39(13)
293(2)
b (Å)
c (Å)
α (°)
105.847(4)
90
β (°)
γ (°)
V (Å3)
3948.2(2)
293(2)
Temperature (K)
λ (Å)
0.71073
1.597
0.71073
1.834
Dc (Mg/m3)
µ (mm-1)
1.415
2.247
3867
5041
Reflections collected
Reflections used
2921
3593
No.of refined
parameters
276
394
aR1 [1>2σ(I)]
0.1682
0.0759
0.0851
1.0671
bR2w
0.1867
1.034
Goodness of fit
aR1=Σ||Fo| -|Fc||/Σ|Fo|, bwR2=|Σw(|Fo|2-|Fc|2)|/Σ|w|(Fo)2|1/2
9

Table 2. Selected bond lengths (Å) and angles (°) of [Cu2(2-AEP)4(µ-Cl)](ClO4)3 (1) and
[Cu4(µ3-OH)2(µ2-OH)2(2-AEP)4(µ2-ClO4)2](ClO4)2 (2)
Complex 1
Bond lengths (Å)
2.036 Cu1-N4
Bond angles (°)
N1-Cu1-N2 89.41 N3-Cu1-N4
Cu1-N1
Cu1-N2
Cu1-N3
2.011
2.640
5.280
93.10
95.01
109.85
87.68
93.93
2.030 Cu1-Cl
N1-Cu1-N3 177.31 N1-Cu1-Cl
2.036 Cu1- Cu1
N1-Cu1-N4 86.90
N2-Cu1-N3 89.51
N2-Cu1-Cl
N3-Cu1-Cl
N2-Cu1-N4 156.17 N4-Cu1-Cl
Complex 2
Bond lengths (Å)
2.024 Cu1-O5
Bond angles (°)
N1-Cu1-N2 95.32 N3-Cu2-O2
Cu1-N1
Cu1-N2
Cu1-O1
Cu1-O2
Cu2-N3
Cu2-N4
Cu2-O1
Cu2-O2
2.768
2.685
2.873
3.167
3.968
2.873
3.968
6.161
163.00
162.59
92.92
1.989 Cu2-O5
N1-Cu1-O1 172.36 N4-Cu2-O1
1.966 Cu1- Cu2
1.941 Cu1- Cu1ꞌ
2.029 Cu1- Cu2ꞌ
1.970 Cu1ꞌ- Cu2ꞌ
1.964 Cu2- Cu1ꞌ
1.929 Cu2- Cu2ꞌ
N1-Cu1-O2 96.22
N2-Cu1-O1 91.44
N4-Cu2-O2
Cu1-O1-Cu2 93.02
N2-Cu1-O2 166.37 Cu1-O2-Cu2 95.85
N3-Cu2-N4 95.00
N3-Cu2-O1 97.83
O1-Cu1-O2
O1-Cu2-O2
76.64
76.67
3. Spectral characterization
The synthesized copper(II) complexes 1 and 2 were further characterized by UV-Vis, FT-IR,
EPR and cyclic voltammetric techniques.
3.1. UV-Vis spectra
The UV-Vis spectra of complexes 1 and 2 were recorded in acetonitrile at RT in the
spectral window 190-1100 nm and are illustrated in Fig. 4. The absorption spectrum of the
mono-chloro bridged complex 1 displayed a broad lower energy band centered around 735 nm (ε
= 206 M-1cm-1), corresponding to the d-d transition of a Cu(II) ion in a square pyramidal
geometry. Complex 2 displayed a d-d transition around 608 nm (ε = 294 M-1cm-1), similar to
other hydroxo bridged Cu(II) complexes.28, 29
10

Fig. 4. The UV-Vis spectra of the di and tetranuclear copper complexes 1 (5 mM) and 2 (2.5
mM) in acetonitrile.
3.2. Infrared (FT-IR) spectra
The FT-IR spectra of complexes 1 and 2 were recorded at room temperature in the region
4000-400 cm-1. Complexes 1 and 2 exhibited a characteristic band at 3314-3447 cm-1 ascribed to
ν(NH2) coordinated to copper(II) centers.30, 31 The sharp bands around 3107-3214 cm-1 were due to
ν(=CH) stretching vibrations of pyridine groups and those in the range 2917-2924 cm-1 were due to
ν(CH2) vibrations of the aliphatic methylene groups. In addition to these bands, the complexes
displayed strong bands around 1080-1088 and 623-630 cm-1 due to perchlorate group
asymmetric stretching and bending modes respectively. Complex 2 displayed a broad
characteristic envelop at 3482 cm-1 due to bridging OH groups.
3.3. EPR spectra
The solid state EPR spectra of complexes 1 and 2 were recorded with a magnetic field
strength of 2000-4500 G (Fig. 5) at room temperature. The spectra revealed two signals,
indicating tetragonally distorted Cu(II) centers with g||= 2.27 and g⊥= 2.05 for 1, and g||= 2.25
and g⊥= 2.06 for 2. The obtained values match with those of distorted Cu(II) environments in
similar complexes.32
11

Fig. 5. EPR spectra of (a) [Cu2(2-AEP)4(µ-Cl)](ClO4)3 (1), (b) [Cu4(µ3-OH)2(µ2-OH)2(2-
AEP)4(µ2-ClO4)2](ClO4)2 (2), at room temperature, scan range = 2000–4500 G, mod. amplitude
= 5 G and microwave frequency = 9.40715 GHz.
3.4. Magnetic studies
Magneto-structural correlations of di and multinuclear complexes with bridging ligands
often gained attention to understand super exchange pathways. Especially, tetra-copper
complexes with µ-hydroxo systems and bidentate ligands were studied by a few groups.33 The
magneto-structural correlation of complexes with a singly bridging chloride ion and bidentate
ligands is still a matter of investigation. Hence we have carried out a magnetic susceptibility
study on the present complexes.
The magnetic susceptibility studies of complexes 1 and 2 were carried out on crystalline
samples in the temperature range 2-300 K. The magnetic responses are illustrated in Figs. 6 and
7. The magnetic behaviour of both complexes have been analyzed using the modified Bleaney-
Bowers equation (3.4.1),34a,b where g is the magnetic field splitting factor, J is the exchange
parameter with +J and -J indicating ferromagnetic and antiferromagnetic exchange interactions
between two Cu(II) ions, respectively. TIP is the temperature independent paramagnetism
contribution of 1 mole of copper(II) ions. The modified Bleany-Bowers equation used for fitting
the obtained data is given by eq 3.4.1.
12

2NA g2 2
3KB (T )
m 

11/3exp(2J / KBT)

1 TIP

3.4.1

Fig. 6. Plot (a) χmvs T (b) χmTvs T in the range 2-300 K for 1 (inset 1/χmvs T) (the solid line
indicates the theoretical curve from the Bleaney-Bowers equation).
The obtained data for complexes 1 and 2, in the form of molar magnetic susceptibility
(χm) versus temperature (T) and 1/χm versus temperature (T), are shown in Figs. 6 and 7a. From
the strong deviation in the plot, it was proposed that complex 1 displays a dual nature (ferro and
antiferromagnetism) with respect to temperature. The fitting of the obtained data in the equation
(3.4.1) in the higher temperature range for complex 1 showed ferromagnetic-like behaviour (2J =
+1.73 cm-1), whereas the lower temperature range showed antiferromagnetic nature (2J = -13.48
cm-1). At lower temperatures, intermolecular antiferromagnetic interactions or zero field splitting
might have an influence on the structure of the complex.35, 36 However, the positive J value
suggests an intramolecular ferromagnetic exchange interaction between the two copper(II)
centers. The χmT values for 1 at 300 and 50 K are 2.8 and 1.18 cm3 mol−1 K, respectively. A
superexchange interaction in complex 1 is possible through the chloride bridge between the
Cu(II) centers. The exchange interaction between the two copper(II) ions mainly depends on the
13

Cu∙∙∙Cl∙∙∙Cu bridging angle, together with the Cu∙∙∙Cu and Cu∙∙∙Cl distances.37 The magnetic
parameters obtained from the fitting of data using eq 3.4.1 are listed in Table 3 and compared
with available complexes in Table 4. From Table 4, it is clear that only a limited number of
mono chloro bridged dicopper complexes are available with bidentate N-donor ligands.
Unfortunately, the J values are not available for these complexes to bring about any meaningful
magneto structural correlation.
The equations used for fitting the obtained data for tetranuclear complex 2 are given below:34c
imp
exp
(1- )
 complex
=
+
+ TIP
2 2
2N g

{[2 exp(-y) + exp(-x) + 5 exp(x)]
exp
=
kT
/[exp(-2y) + 6 exp(-y) + exp(-2x) + 3 exp(-x) + 5 exp(x)]}
........................(3.4.2)
J2
J1
y
=
and
with
x
=
kT
kT
The magnetic properties of complex 2, in the form of molar magnetic susceptibility (χM)
versus temperature (T) and 1/χm versus temperature (T) plots, are shown in Fig.7. The inset of
Figure 7(a) shows the residual plot of the measured and fitted data. The difference between the
measured and fitted data is ~ 1%, showing the goodness of the fitting. The χmT values for
complex 2 at 300 and 50 K are 1.08 and 1.05 cm3 mol−1 K; J1 = +2.98 and J2= -16.91 cm-1. In
general, the magnetic susceptibility was influenced by several factors, such as the geometry
around the copper center, Cu-O(H)-Cu angle, Cu-O bond distances and Cu-Cu bond lengths.
Among these, the Cu-O(H)-Cu angle plays a major role for spin coupling between copper
centers, with S = ½.37 Hence, ferromagnetism is observed when the Cu−O(H)−Cu angles are
smaller than 97.5° due to accidental orthogonality of the magnetic orbitals and
antiferromagnetism is observed at higher angles, especially in dihydroxo bridged dicopper(II)
complexes38, 39 Ruiz et al., explained the two possible mechanistic pathways for the exchange
14

interaction in bis-µ-hydroxo di and tetranuclear copper(II) complexes: 1) direct interaction
between Cu(II) ions, 2) superexchange interaction propagated through the hydroxyl bridge.40 The
observed Cu1−O1(H)−Cu2 angle of 95.7° and Cu1−O2(H)−Cu2 angle of 92.9°, with a positive
J1 = +2.98 value (Table 3), suggest a ferromagnetic interaction. At the same time, the J2 value of
-16.91 cm-1 suggests the dominance of an antiferromagnetic interaction. The magneto structural
correlation of selected hydroxo bridged tetracopper complexes with bidentate ligands is shown in
Table 5. From the table, it is visible that complex 2 shows a moderate antiferromagnetic
interaction, comparable with the reported complexes.
Fig. 7. Plot of (a) χmvs T (b) χmTvs T in the range 2-300 K for 2 (the solid line indicates the fitted
curve using the Bleaney-Bowers equation).
Table 3. Selected magnetic data for di and tetranuclear bridged copper(II) complexes
exchange
parameter
(cm-1)
TIP
T
(K)
Θ
Impurities
(%)
Complex
g
(cm3
R2
(Kelvin)
/mol)
1
-2.13
2.15
5 x 10–6
2-300
2-300
2J=+1.73
0.005
0.01
0.99
0.99
2
J1 = +2.98
-3.99
2.12
5 x 10–6
J2 = -16.91
15

Fig.8. Magnetization vs applied field curve for (a) 1 and (b) 2. Right bottom inset shows the M-H
plot at 300 K and left top shows the M-H plot at the lowest temperature.
The obtained magnetic susceptibility data for complexes 1 and 2 can also be correlated by
their M-H curves. The M-H behavior of both complexes is distinct, as shown in Fig.8. In the case
of complex 1, the M-H plot at 2 K showed a non-linear ferromagnetic hysteresis curve with an
remanence of 3 x 10-4 emu/g and coercivity of 23 Oe (as shown in the top left inset), whereas we
observed that the M-H plot at 300 K (as shown in the inset) behaved like a complex
antiferromagnetic system, having a non-saturating magnetization, even at an applied field of 5 T.
This further confirmed our assignment of a ferromagnetic to antiferromagnetic like transition in
complex 1. In the case of complex 2, the M-H behavior showed an almost linear pattern at both
the temperatures 300 and 5 K. However, it also had a coercivity of 30 Oe and remanent
magnetization of 7 × 10-3 emu/g. The value of the remanence is almost an order of magnitude
higher than that of complex 1. It may be noted that the J value was relatively high in comparison
with that of complex 1, correlating well with these observations.
16

Fig. 9. The plot of 2J vs.  (Cu-OH-Cu) for related tetranuclear copper bis-μ-hydroxo bridged
complexes with bidentate ligands.
The correlation of 2J vs  for selected tetranuclear copper bis-μ-hydroxo complexes with
bidentate ligands (selected from Table 5) is shown in Fig. 9. The correlation pattern for complex
2 falls on the linear relationship.
Table 4. Comparison of bond angles and J values for dinuclear mono-chloro bridged Cu(II)
complexes with bidentate N-donor ligands.
Complex
Cu-Cl-Cu
angle (°)
Cu…Cu
distance
(Å)
Cu…Cl
distance
(Å)
J value
(cm-1)
Ref
[Cu2(2-AEP)4(µ-Cl)](ClO4)3 (1)
Cu2(phen)2(µ-Cl)(Cl3)
180.00
90.86
127.50
89.92
5.280
3.564
2.640
2.706
+1.73 This work
-
-
-
-
-
-
-
-
-
43(b)
23(a)
23(b)
23(c)
23(d)
23(e)
23(f)
23(g)
23(h)
[Cu2(acec)2(bpy)2(µ-Cl)](ClO4)(H2O)
[Cu2(2,9-tb-phen)2(µ-Cl)(Cl3)](SbF6)
[Cu2(2,5-bamp)2(µ-Cl)(Cl3)]
[Cu2(tmeda)2(µ-Cl)(CO2)](BPh4)
[Cu2(2-aep)4(µ-Cl)](PF6)3
[Cu2(maep)(µ-Cl)(Cl2)]n
[Cu2(PYP)(µ-Cl)(Cl2)]n
[Cu2(aei)(µ-Cl)(Cl2)]n (Cl)(H2O)2
4.500
3.097
3.709
3.643
5.060
4.263
3.785
5.232
2.509
2.196
2.227
2.306
2.530
2.785
2.701
2.856
95.24
102.96
180.00
113.58
98.64
131.79
17

Table 5. Magneto-structural correlation of selected tetranuclear bis-(µ-OH) bridged Cu(II)
complexes with bidentate N-donor ligands
Complex
Cu-O-Cu Cu…Cu
angle (°)
distance distance (cm-1)
(Å) (Å)
Cu…O
J1
Ref
This
Work
[Cu4(2-AEP)4(µ3-OH)2(µ2-OH)2(µ2-ClO4)2](ClO4)2 (2)
95.71(0) 2.872(0) 1.931(0) +2.98
[Cu4(bpy)4(OH)4 (H2O)2](C8HO4)3.6H2O
[Cu4(bpy)4(OH)4 (H2O)2](NO3)2(C7H5O2)2.6H2O
[Cu4(bpy)4(OH)4 (H2O)2](NO3)2(C5H6O4).8H2O
[Cu4(bpy)4(OH)4 (H2O)2](C5H6O4)2.16H2O
[Cu4(dmbpy)2(OH)4(H2O)2](BF4)2 (H2O)4
[Cu4(bpy)4(OH)4(H2O)(BTC)]NO3.8H2O
[Cu4(bpy)4(OH)4](PF6)4
96.30(0) 2.915(0) 1.930(0) +6.79 33a
98.00(0) 2.898(0) 1.919(0) +64.1 33b
99.23(0) 2.918(0) 1.920(0) +20.3 33b
98.74(0) 2.912(0) 1.924(0) +35.4 33b
97.65(0) 2.939(0) 1.973(0) +31.1 33c
99.29(0) 2.907(0) 1.905(0) -21.1 33d
96.60(2) 2.914(0) 1.947(2) +12.0 33e
4. Phosphodiester hydrolysis
Having synthesized the di and tetranuclear copper(II) complexes, we have performed
phosphodiester (PDE) hydrolysis experiments as the complexes possess hydroxo/chloro
nucleophiles which are capable of attacking the electrophilic phosphorus atom of
phosphodiesters. Bis(4-nitrophenyl) phosphate (BNPP) is often used as a substrate to study such
reactions. When BNPP undergoes hydrolysis, the product 4-nitrophenolate (p-NP) can be
detected in basic pH by its characteristic band around λmax = 400 nm in UV-Vis spectra [Fig.
10a].41
Pseudo-first-order rate constants were determined using the initial rate method.42a, 44c
From these preliminary studies, the rate and rate constant of BNPP hydrolysis were calculated
from the slope of a linear plot of [p-NP] vs time (Figs. 10b and 11b). The rate and rate constant
for complex 1 were 8.49 × 10-5 mM s-1 and 9.65 × 10-3 s-1. Similarly, these values for complex 2
were 9.79 × 10-5 mM s-1 and 1.42 × 10-2 s-1, respectively. The present di and tetra-nuclear
complexes showed hydrolytic activity comparable to many reported copper complexes. For
instance, dinuclear copper complexes with tacn ligands bearing alkyl guanidine moieties were
18

reported by Leone Spiccia’s group and showed hydrolytic activity k = 1.35 × 10-6 s-1.8b
R.E.H.M.B. Osória reported dinuclear copper(II) complexes and studied phosphodiester
hydrolysis with rates of the reaction in the range 1.8 – 5.1 × 10-4 s-1.42b Sabiah, et al. reported a
dinuclear copper(II) complex for phosphodiester hydrolysis with reaction rate of 1.5 × 10-5 s-1.8a
Fig. 10. (a) Spectral profile for the hydrolysis of BNPP by complex 1. Spectra were recorded
every 30 min. (b) Plot of concentration vs time.
Fig. 11. (a) Spectral profile for the hydrolysis of BNPP by complex 2. Spectra were recorded
every 30 min. (b) Plot of concentration vs time.
5. DNA interaction studies
5.1. UV-Vis spectrophotometric study
19

Since we were interested in understanding the DNA binding interactions with the di and
tetranuclear complexes 1 and 2, we have carried out interaction studies using UV-Vis, FT-IR,
CD and CV methods. The binding affinity of complexes 1 and 2 with calf thymus DNA (CT-
DNA) was measured by electronic absorption spectroscopy as it is one of the simple, commonly
employed methods to determine the binding nature of metal complexes with CT-DNA. The
titrations for the present complexes were carried out by varying the concentration of CT-DNA
from 0 to 20 µM and fixing the complex concentration constant (1 = 5 mM; 2 = 2.5 mM). The
absorption intensity of complexes 1 and 2 kept decreasing upon each addition of CT-DNA. The
spectral profiles of complexes 1 and 2 in the presence and absence of CT-DNA are shown in
Figs. 12a and 13a. The binding constants were calculated from the equation given below.43
[DNA]/|ɛa-ɛf| = [DNA]/|ɛb-ɛf|+1/Kb|ɛb-ɛf|
where [DNA] represents the concentration of CT-DNA, ɛa, ɛb and ɛf represents the extinction
coefficients of partially bound DNA to the complex, the fully DNA bound complex and the free
complex. The binding constant (Kb) for the complexes was estimated from the plot of [DNA]/ɛa-
ɛf vs [DNA], which yielded a straight line with the ratio of slope/intercept equal to the binding
constant. The calculated binding affinities of the copper(II) complexes 1 and 2 are shown in
Table 6. They show moderate binding affinities with CT-DNA, which were well comparable
with reported values.44
20

Fig. 12. (a) Spectral profile of the dinuclear copper(II) complex 1 (5 mM) with CT-DNA (0-20
µM), (b) binding plot of [DNA]/(ɛa-ɛf) Vs [DNA].
Fig. 13. (a) Spectral profile of the tetranuclear copper(II) complex 2 (2.5 mM) with CT-DNA (0-
20 µM), (b) binding plot of [DNA]/(ɛa-ɛf) Vs [DNA].
Complexes 1 and 2 show similar binding interactions towards CT-DNA as compared to
many copper complexes. For instance, N.M.R. Martins et al. reported dinuclear copper(II)
complexes with an arylhydrazone of ethyl 2-cyanoacetate with binding constants in the range 2.7
– 3.3 × 105 M-1.44a Q.Q. Zhang et al. reported mono chloro bridged copper complexes of
phenanthroline ligands with a binding constant of 4.75 × 104 M-1.44b S. Ramakrishnan et al.
reported mixed-ligand dinuclear copper(II) complexes of a benzamide based ligand with diimine
21

auxiliary ligands, with binding constants in the range 0.5 – 7.6 × 104 M-1.44d M. Jiang et al.
described bridged binuclear copper(II) complexes with an oxamide ligand, having binding
constants 1.73 × 105 M-1 for[Cu2(heae)(pic)2] and 1.92 × 105 M-1 for [Cu2(heae)(Me2phen)2].44e
Binuclear copper(II) complexes were reported by K. Zheng et al. with a binding constant of 8.35
× 105 M-1.44f Mixed ligand copper(II) complexes of bipyridyl/phenanthroline were reported by
F.-H. He et al., showing kb values in the range 1.1 – 1.8 × 104 M-1.44g
Table 6. Rate constants and binding constants for complexes 1 and 2.
Complex
Phosphodiester
Hydrolysis
Rate constant (k) s-1
DNA Binding
Constant (kb M-1)
9.65 × 10-3
1.42 × 10-2
3.54 × 104
3.18 × 104
[Cu2(2-AEP)4(µ-Cl)](ClO4)2 (1)
[Cu4(µ3-OH)2(µ2-OH)2(2-AEP)4(µ2-ClO4)2](ClO4)2 (2)
5.2. FT-IR study
FT-IR spectral studies were performed to understand the binding nature between the metal
complexes with nucleotides of CT-DNA. In the literature, the vibrational bands for free DNA
appear at 1261, 1402 and 1639 cm-1, which correspond to thymidine, cytosine and adenine bases,
respectively.45 The bands at 1087 and 991 cm-1 are assigned to symmetric and asymmetric
stretching modes of phosphate groups respectively. After interaction of the complexes with
cytosine and adenine (CT-DNA) bases, the bands are shifted to 1392 and 1627 cm-1 (1), and
1393 and 1628 cm-1 (2) respectively. These complexes also interacted with the phosphodiester
moieties of DNA, as the phosphate bands shifted to 1119 and 1071 cm-1 (complex 1), and 1117
and 1077 cm-1 (complex 2). The obtained results strongly suggest that these complexes interact
with CT-DNA via an electrostatic/groove binding mode.45
5.3. CD spectrometric study
22

Circular dichroism spectral studies are performed to study the interaction of molecules/metal
complexes with DNA. CT-DNA was dissolved in 10 mM Tris-HCl buffer (pH 7.4) and the CD
spectra of the solution showed characteristic positive and negative peaks around 285 and 240 nm
due to base stacking and helicity, respectively. If any molecule/metal complex interacts with CT-
DNA via a groove/electrostatic interaction, it will exhibit a small or no perturbation on both the
helicity bands and base stacking in the spectra, where as intercalation enhances the intensities of
both the bands, as well as shifting the position of the peaks. Complexes 1 and 2 were incubated
along with CT-DNA at [DNA]:[complex] ratios of 1:0.5 (1) and 1:0.25 (2) ratio, and the spectra
were recorded at ambient temperature in 10 mM buffer solution. The obtained spectra revealed
that there was a small change in the intensities of both the negative and positive bands, but there
was no considerable shift in the peaks (Fig. 14). Hence, the results suggested that both copper
complexes bind with CT-DNA via the groove mode.46
Fig. 14. CD spectral profile of CT-DNA with 1 and 2 in 7.4 pH buffer at room temperature,
where [CT-DNA]:[complex] = 1:0.5 (1) and 1:0.25 (2).
5.4. Viscosity measurements
A viscosity study is a simple, often used and effective method to find the mode of binding
between complexes and CT-DNA.46 Generally this test suggests that when complexes interact
23

with DNA via a classical intercalative mode, there is a significant increment in the viscosity of
CT-DNA. However, groove or electrostatic binding interactions result in very little perturbation
of the DNA viscosity. In order to understand the binding mode, viscosity studies were performed
at physiological temperature with varying concentrations of 1 and 2 (from 0 to 140 µM). The
relative viscosity (R) of CT-DNA does not show any significant changes on increasing the
complex concentration. This result clearly indicates that the complexes do not bind with CT-
DNA by an intercalative mode (Fig. 15). Hence the present di and tetranuclear copper complexes
bind with CT-DNA via electrostatic interactions or a groove binding mode.46c, d
Fig. 15. Effect of increasing amounts of complexes 1 and 2 on the viscosity of CT-DNA.
Measurements were performed in 10 mM Tris–HCl buffer, pH 7.4 and at RT with [CT-DNA]=
100 µM; R = [complex]/[CT-DNA]; relative viscosity (η/η0)1/3 is plotted against R
5.5. Cyclic voltammetric study
Cyclic voltammetric analysis is one of the simple and useful techniques to understand the
binding behavior of redox active copper complexes with CT-DNA.47 The cyclic voltammograms
of the copper complexes 1 and 2 in the absence and presence of CT-DNA exhibited considerable
shifts in the oxidation and reduction potentials, which indicates that the complexes interact with
DNA (Fig. 16, Table 7).
24

The cyclic voltammetric studies of 1 and 2 were performed with 10 mM Tris-HCl buffer
with tetrabutylammonium perchlorate (TBAP) as the supporting electrolyte at pH 7.4 (Fig. 14).
Both complexes displayed a quasi-reversible one-electron cathodic peak Epc (reduction
potential) at -0.51 and -0.54 V due to the Cu(II)/Cu(I) couple and a one step anodic peak Ea
(oxidation potential) at 0.08 and 0.16 V due to the Cu(I)/Cu(II) couple. The obtained potentials
matched those of reported copper(II) complexes.47 M. Jiang et al. reported an Epc value of -0.35
V for N,N’-bis(N-hydroxyethylaminoethyl)oxamide bridged binuclear copper(II) complexes44e
and K. Zheng et al. reported a reduction potential Epc at -0.49 V for dinuclear copper(II)
complexes with N-phenolato-N′-[2-(dimethylamino)ethyl]- oxamide ligands.44f
Fig. 16. Cyclic voltammograms of 1 (1 mM) and 2 (0.5 mM) in 10 mM Tris-HCl buffer (pH =
7.4); potentials are referenced vs Ag/AgCl; [Bu4NClO4] =10 mM; scan rate: 0.05 V s-1.
The ratio of the equilibrium binding constants KR/KO was calculated by using the peak change of
the initial potential (ΔEo). Bard and Carter expressed the interaction with small molecules/metal
complexes using the simple equation as given below.49
ΔE° = Eb° ─Ef° – 0.059 log(KR/KO)
where Ef° and Eb° are the potentials of the free complex and complex bound with DNA,
respectively, and Kox and Kred are binding constants for the oxidised and reduced forms with CT-
25

DNA. The Kred/Kox values were 0.98 (1) and 1.02 (2). The obtained results recommend that both
complexes have comparable binding strengths with DNA through groove/electrostatic binding.25
Table 7. CV data for complexes 1 and 2.
Redox
couple
Ipc (A) × 10-5
Epc (V)
E1/2 (V)
ΔEp (V)
Kred/
Kox
Free Bound Free
Bound Free
-0.21
Bound Free Bound
Cu(II)/Cu(I)
1
2
3.65 2.51
1.78 1.73
-0.50 -0.51
-0.20
-0.19
0.58 0.62
0.68 0.70
0.98
1.02
Cu(II)/Cu(I)
-0.55 -0.54
-0.21
6.1. DNA cleavage studies
The interaction studies of 1 and 2 with supercoiled plasmid DNA (pBR322 DNA) were
performed in the absence and presence of reducing agent (ascorbate) by agarose gel
electrophoresis. To study the capability of these complexes as DNA cleavage agents, DNA was
incubated with different concentrations of 1 and 2 in 50 mM Tris-HCl buffer at pH 7.4 for 2 h
and then subjected to gel electrophoresis. The concentration of complex 1 was varied from 0-50
µM, keeping the DNA concentration (0.4 µg) constant. Interestingly, the electrophorogram
showed (Fig. 17, lanes 4 and 5) that complex 1 oxidatively cleaves SC (supercoiled circular)
DNA to 42% linear DNA (Form III) at 25 µM and 48 % at 50 µM concentration with the
reducing agent (Fig. 17, lanes 4 and 5). Complex 1 does not show any significant DNA cleavage
in the absence of a reducing agent (lanes 6-8).
26

Fig.. 17. (a) Cleavage activity of pBR322 DNA with 1 as determined by gel electrophoresis
analysis. Each lane contained 0.4 µg DNA in buffer (pH=7.4). Lane 1: reference DNA; Lane 2:
DNA + 1 mM ascorbate; lane 3-5: 1 mM ascorbate + 1 (12.5, 25 and 50 µM, respectively); lane
6-8: 1 (12.5, 25 and 50 µM, respectively). (b) Bar chart representing the percentage of degraded
DNA.
27

Fig. 18. (a) Cleavage activity of pBR322 DNA with 2 as determined by gel electrophoresis
analysis. Each lane contained 0.4 µg DNA in buffer (pH=7.4). Lane 1: reference DNA; Lane 2:
DNA + 1 mM ascorbate; lane 3-5: 1 mM ascorbate + 2 (6.25, 12.5 and 25 µM respectively); lane
6-8: 2 (6.25, 12.5 and 25 µM respectively). (b) Bar chart representing the percentage of DNA
forms.
Similarly, we performed DNA cleavage studies for complex 2, the electrophorogram
showed (Fig. 18, lanes 3-5) that complex 2 oxidatively cleaves SC (supercoiled circular) DNA to
30% linear plasmid DNA (Form III) at 6.25 µM, 42 % at 12.5 µM and 49 % at 25 µM
concentration with a reducing agent (Fig. 18, lanes 3-5). In the absence of a reducing agent, 2 did
not cause any significant DNA cleavage (lanes 6-8).
The achieved results were comparable with copper complexes reported by various
groups.50,51 The heteroleptic dinuclear copper(II) complexes of a benzamide based ligand
28

reported by S. Ramakrishnan et al. were able to cleave plasmid DNA to the nicked form at very
high concentrations (100 µM).44d Dinuclear copper(II) complexes with a bis(benzoyl hydrazone)
ligand, demonstrated by R. H. Sabina and his coworkers, were able to cleave plasmid DNA to
Form II (nicked) at 10 µM.50a Dinuclear copper(II) complexes with a biimidazole ligand,
synthesized by Y. Li’s research group, showed cleavage activity at 50 µM concentration,
whereas the dinuclear copper(II) complexes with a naphthalene-sulfonyl-triazole ligand reported
by J. Hernández-Gil et al. exhibited cleavage at 30 µM concentration.51a,b S. Gama and his
coworkers described tridentate pyrazole based dinuclear copper(II) complexes that could cleave
plasmid DNA at 50 µM concentration.51c The synthesis of dinuclear copper(II) complexes with
amidino-O-methylurea ligands was explored by A. Meenongwa et al. and these complexes
showed cleavage to Form II at 50 µM concentration.51d When we compare complexes 1 and 2
with the above examples, both complexes showed good results at very low concentrations (25
µM).
6.2. DNA quenching studies
In order to understand the reactive oxygen species (ROS) responsible for DNA cleavage,
quenching studies were done for complexes 1 and 2 with different scavengers, as shown in Fig.
19. These scavengers were tertiary butanol (tert-BuOH) and dimethylsulfoxide (DMSO) for
hydroxyl radicals, sodium azide (NaN3) for singlet oxygen, catalase for hydrogen peroxide and
superoxide dismutase (SOD) for superoxides. For both compounds 1 and 2, DNA adduct
formation was observed in the presence of catalase, therefore DNA migration was prevented
(Fig. 19). The obtained results showed a strong quenching effect by sodium azide for complex 1
(Fig. 19, Lane 6), whereas SOD showed a weak quenching effect and there was no significant
change with the other scavengers (Fig. 19, Lane 8). In presence of tert-butanol and DMSO,
29