Oxidative cleavage of DNA by transition metal complexes: Synthesis, spectral characterization and DNA interactions of copper(II) complexes with quinquedentate Schiff base ligands

Article abstract of DOI:10.14233/ajchem.2019.21558

Designing of dinucleating ligands, with an additional donor atom that can bridge two metals in a more or less fixed geometry has rapidly developed in recent years. Part of the interest stems from the fact that the corresponding complexes are often studied

Full text of DOI:10.14233/ajchem.2019.21558

Asian Journal of Chemistry; Vol. 31, No. 1 (2019), 148-156
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2019.21558
Oxidative Cleavage of DNA by Transition Metal Complexes: Synthesis,
Spectral Characterization and DNA Interactions of Copper(II)
Complexes with Quinquedentate Schiff Base Ligands
2
M. PRAGATHI1,* and K. HUSSAIN REDDY
1Department of Chemistry, Sri Vishnu Gavimatam Government Degree College, Kalyandurg-515761, India
2Department of Chemistry, Sri Krishnadevaraya University, Anantapuramu-515003, India
*Corresponding author: E-mail: khussainreddy@yahoo.co.in; pragathi.madur@gmail.com
Received: 3 July 2018;
Accepted: 6 September 2018;
Published online: 30 November 2018;
AJC-19174
Designing of dinucleating ligands, with an additional donor atom that can bridge two metals in a more or less fixed geometry has rapidly
developed in recent years. Part of the interest stems from the fact that the corresponding complexes are often studied as enzyme mimics.
Two quinquedentate ligands have been synthesized by condensing salicylaldehyde/o-hydroxy- acetophenone with 2-hydroxy-1,3-
propanediamine. The ligands and their metal complexes are synthesized and characterized by physico-chemical and spectral analysis.
Electrochemical behaviour of the complexes is investigated through cyclic voltammetric studies. E1/2 values are observed at 0.360 and
0.331 V vs. Ag/AgCl for the complexes. The non-equivalent current in cathodic and anodic peaks (ic/ia = 1.224 and 1.065 at 100 mV s-1)
for metal complexes indicate quasi-reversible behaviour. Binding interactions of the dinuclear copper(II) complexes with calf thymus
DNA are investigated using absorption spectrophotometry. Cleavage activities of these complexes are uncovered on a double stranded
pBR plasmid DNA by using gel electrophoresis experiments in different conditions. At micromolar concentration, the ligands exhibit no
significant activity, whereas the metal complexes show significantly enhanced nuclease activity due to the presence of metal ions. Copper
complexes cleave DNA more effectively in the presence of oxidant. This is consistent with the increased production of hydroxyl radicals
by cuprous ions similar to the well known “Fenton reaction”.
Keywords: Oxidative cleavage of DNA, Quinquedentate ligands, Copper(II) complexes, Spectrophotometry, Fenton reaction.
metal ions and their complexes have been of interest for many
years [10-15]. When designing dinuclear metal complexes the
choice of the ligand system is of prime importance. It should
be capable of incorporating two metal ions. Ligands which
afford complexes with the metal ions sharing at least one donor
atom (the so-called compartmental ligands) seem to be appro-
priate for this purpose.
The design of dinucleating ligands, with an additional
donor atom that can bridge two metals in a more or less fixed
geometry has rapidly developed in recent years. Part of the
interest stems from the fact that the corresponding complexes
are often studied as enzyme mimics [16-18].
INTRODUCTION
Interaction of metal complexes with nucleic acids is an
exciting area of research due to their potential use as drugs,
tools for biochemical and biomedical applications in gene
regulation. Considerable efforts are being made to design
sequence-specific DNA cleaving agents that bind DNA at any
desired sequence and cleave DNA efficiently at the binding
site [1-4]. Complexes that are capable of cleaving DNA hydro-
lytically and selectively would be highly desirable as they do
not require any external agents. Several artificial metallo-
nucleases developed to bind to DNA for the cleavage of DNA,
have potential applications as therapeutic agents and as
versatile replacements for nucleases as laboratory tools [5-9].
Binucleating Schiff base ligands are highly inclined to give
homo and hetero polynuclear complexes with many transition
Although metal complexes derived using other quinqueden-
tate ligands have been reported [17-19] in the past. The investi-
gations on DNA binding and cleavage activities of such complexes
are not reported so far. In continuation of our ongoing research
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Vol. 31, No. 1 (2019)
Oxidative Cleavage of DNA by Transition Metal Complexes 149
R
C
R
C
work on metal DNA interactions [20-31], herein we report the
synthesis, characterization and DNA interaction of copper(II)
complexes derived from condensation of salicylaldehyde/
o-hydroxyacetophenone and 2-hydroxy-1,3-propanediamine.
N
N
EXPERIMENTAL
OH
OH
HO
Salicylaldehyde, 2-hydroxyacetophenone and 2-hydroxy-
1,3-propanediamine used in preparation of ligands were of
reagent grade (Sigma-Aldrich) and were used without further
purification. Metal salts used for the synthesis of metal comp-
lexes were of reagent grade (Merck). Solvents used in the present
study were distilled before use. Calf thymus DNA and plasmid
pBR 322 were purchased from Genie Bio Labs, Bangalore,
India. All other chemicals were ofAR grade and used without
further purification.
where, R = H {2-hydroxy-(1,3-diiminopropyl)}bisphenol (HDPH3)
R = CH3 {2-hydroxy-(1,3-diiminopropyl)2,2’-methyl}bisphenol (HDMPH3)
Fig. 1. General structure of quinquedentate ligands
infrared spectra of HDPH3 and HDMPH3 showed the bands at
(cm-1): 3512, 3446; 1633, 1612; 2896, 2925; 1275, 1279 assig-
ned to ν(OH); ν(C=N); ν(aromatic C-H); and ν(C-O) stretching
1
vibrations, respectively. H NMR: HDPH3: δ(13.2) (singlet
2H), δ(8.4) (singlet 1H), δ(6.4-7.2) (multiplet 8H), δ(4.2)
(singlet 2H) and δ(3.9) (multiplet 4H) and δ(3.8) (multiplet
1H) assigned, respectively to phenolic -OH, alcoholic OH,
phenyl H, N=CH-, -CH2- and CH-OH (methine) protons, respec-
tively and HDMPH3: δ(14.1) (singlet 2H), δ(8.2) (singlet 1H),
δ(6.8-7.2) (multiplet 8H), δ(4.4) (singlet 2H) and δ(3.7)
(multiplet 1H), δ(2.4) (doublet 2H) and δ(1.6) (singlet 6H)
assigned, respectively to phenolic -OH, alcoholic OH, phenyl
H, N=CH-, CH-OH(methine), -CH2- and –CH3 protons, respec-
tively. GC-mass spectra of HDPH3 and HDMPH3 show mole-
cular ion peaks at (m/z) 298 and 326, respectively. GC-MS
Spectrum of HDMPH3 is shown in Fig. 2.
Magnetic measurements of all the copper(II) complexes
at 298 K were obtained on a Faraday’s magnetic susceptibility
balance (Sherwood Scientific, Cambridge, UK). High purity
pentahydrated copper sulfate was used as standard. The
conductance measurements at 298 2 K in dry and purified
dimethylformamide were made on CM conductivity cell
(model 162 Elico). Infrared spectra in KBr disc were recorded
in the range 4000-400 cm-1 on a Perkin-Elmer spectrum 100
spectrometer. Electronic spectra were recorded in N,N-dimethyl
formamide on a Perkin-Elmer UV Lamda-50 spectrophotometer.
Elemental analyses were carried out with a Heraeus Vario EL
III Carlo Erba 1108 instrument. Mass spectra of the ligands
were recorded on Jeol GC MATE II GC-Mass spectrometer in
EI+ ionization mode. 1H NMR spectra were recorded at 400.00
MHz on aAvance-400 Bruker spectrometer at CDRI, Lucknow.
ESR spectra were recorded in solid state and in DMF at 298 K
and at liquid nitrogen temperature (L.N.T) on a Varian E-112
spectrometer with 100 KHz field modulation. The g|| and g⊥
values are computed from the spectrum using tetracyanoethylene
(TCNE) free radical as ‘g’. Cyclic voltammetry was performed
with a CH Instruments 660C electrochemical analyzer and a
conventional type electrode, Ag/AgCl reference electrode,
glassy carbon working electrode and platinum counter electrode.
Nitrogen was used as purge gas and all solutions were prepared
in DMF containing 0.1 M concentration in tetrabutylammonium-
hexaflorophosphate (TBAPF6). DNA cleavage activities were
performed on a UVI-tech-UK Xplorer Gel documentation system.
Synthesis of ligands: Ligands were prepared according to
literature methods [19]. The ligand [2-hydroxy-(1,3-diimino-
propyl)]bisphenol (HDPH3) was synthesized by the refluxion
of salicylaldehyde (20 mmol, 2.1 mL) with a methanolic solution
of 1,3-diaminopropan-2-ol (0.9 g, 10 mmol) for 30 min.Whereas
the ligand [2-hydroxy-(1,3-diiminopropyl)2,2’-methyl]bisphenol
(HDMPH3) was synthesized by the reaction of 2-hydroxy-
acetophenone (20 mmol, 2.4 mL) and 1,3-diaminopropan-2-
ol (0.9 g, 10 mmol). The bright yellow crystalline Schiff bases
are separated out on slow evaporation of the solvent. Ligands
were further washed with methanol and dried in vacuoo.
HDPH3:Yield: 82 %; m.p.: 99-101 °C.Anal. (%) Calc. (found):
C-68.32 (68.44); H-5.98 (6.08); N-9.51(9.39); O-16.19 (16.09);
HDMPH3: Yield: 88 %; m.p.: 174-176 °C. Anal. (%) Calc.
(found): C-70.12 (69.92); H-6.68 (6.79); N-8.46 (8.58); O-
14.74 (14.71); Fig. 1 gives the general structure of ligands. The
Synthesis of copper(II) complexes: To a stirring methanolic
solution of copper(II) acetate monohydrate (1.99 g, 10 mmol),
a hot methanolic solution of HDPH3 (5 mmol, 1.49 g)/HDMPH3
(5 mmol, 1.63 g) was added drop-wise with constant stirring.
The contents were stirred magnetically for 3 h. The contents
were then filtered. Shining dark green coloured crystalline
products were obtained on slow evaporation of the solvent.
The complexes were washed with methanol and dried in vacuoo.
[Cu2(CH3COO)(HDP)]: Yield: 62 %; m.p.: 218-220 °C (D).
[Cu2(CH3COO)(HDMP)]: Yield: 58 %; m.p.: 268-270 °C (D).
LC-MS spectra of copper(II) complexes showed molecular
ion peaks corresponding to their calculated molecular weights
(Fig. 3). The peak at m/z = 509.21 (cal. 509.50) represents the
molecular ion peak of the complex Cu2(CH3COO)HDMP. The
LC-MS spectrum of Cu2(CH3COO)HDP shows peak at m/z =
481.98 (cal. 481.45) related to molecular ion peak of the complex
Cu2(CH3COO)HDP. Analytical data of complexes are given
in Table-1.
DNA binding experiments: Binding interactions of the
complexes with DNA were carried out in tris-buffer. Solution
of calf thymus-DNA (CT-DNA) in (0.5mM NaCl/5mM Tris-
HCl; pH = 7.0) buffer gave absorbance ratio at 260 and 280
nm of 1.8 indicating that the DNA was sufficiently free of
proteins. The DNA concentration per nucleotide was deter-
mined by absorption coefficient (6600 dm3 mol-1 cm-1) at 260
nm. Stock solutions stored at 4 °C were used after not more
than 4 days. The electronic spectra of metal complexes were
monitored in the absence and presence of CT-DNA.Absorption
titrations were performed by maintaining the metal complex
concentration 5 × 10-5 M and varying the nucleic acid concen-
tration (0-19 × 10-8 M).Absorption spectra were recorded after
each successive addition of DNA solution. The intrinsic binding

150 Pragathi et al.
Asian J. Chem.
109.0361
100
270.0781
80
124.0606
149.0407
136.0450
60
79.0171
40
253.9307
59.8688
20
95.0137
326.0897
307.0738
236.9008
193.0562
220.9715
173.9797
207.1392
162.0459
316.3365
283.0940
296.1334
100
150
200
m/z
250
300
Fig. 2. GC-MS spectrum of HDMPH3
TABLE-1
PHYSICO-CHEMICAL AND ANALYTICAL DATA OF COPPER(II) COMPLEXES
Molar conductance
(Ω-1 cm-2 mol-1)
Elemental analysis (%): Found (Calcd.)
µeff
(BM)
Colour
(Yield %)
Decomposition
point (°C)
Complex
m.w. *
C
H
N
Cu
Dark green
(62)
Dark green
(58)
481.98
(481.45)
509.21
47.36
(47.40)
49.78
3.81
(3.77)
4.22
5.66
(5.82)
5.74
26.48
(26.40)
24.64
Cu2(CH3COO)HDP
218-220
268-270
8.22
6.34
1.06
1.26
Cu2(CH3COO)HDMP
(509.50)
(49.50)
(4.35)
(5.50)
(24.94)
*Determined using LC-MS.
×105
3.8
xylene cyanol + 30 % glycerol and solutions were loaded on
0.8 % agarose gel containing 100 µg of ethidium bromide.
Electrophoresis was performed at 75 V in TBE buffer until
the bromophenol blue reached to 3/4 of the gel. Bands were
visualized by UV transilluminator and photographed. The
efficiency of DNA cleavage was measured by determining the
ability of the complex to form open circular (OC) or nicked
circular (NC) DNA from its supercoiled (SC) form. The
reactions were carried out under different conditions.
358.0771
3.6
3.4
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
267.1728
333.1624
509.2084
436.2297
200 250 300 350 400 450 500 550 600 650 700 750 800 850
Counts vs. Mass-to-charge (m/z)
RESULTS AND DISCUSSION
Fig. 3. LC-MS spectrum of Cu2(CH3COO) HDMP
The ligands containing two imino groups and three hydroxyl
groups have been synthesized and characterized. Copper(II)
complexes of these ligands are stable at room temperature,
non-hygroscopic, insoluble in water and methanol, but readily
soluble in DMF and DMSO. In spite of several repeated efforts
the complexes could not be isolated as single crystals suitable
for XRD. Hence, the complexes have been characterized based
on the physico-chemical analyses and spectral data. Low molar
constant (Kb) was calculated by using the equation, [DNA]/
(εa-εf) = [DNA]/(εb-εf) + 1/Kb (εb-εf), where [DNA] is the molar
concentration of DNA in base pairs, εa, εb and εf are apparent
extinction coefficient (Aobs/[M]), the extinction coefficient for
the metal (M) complex in the fully bound form and the extinc-
tion coefficient for free metal (M), respectively.
DNA cleavage experiments: The extent of cleavage of
DNA by ligands and their copper(II) complexes was monitored
using agarose gel electrophoresis with pBR 322 DNA. After
incubation for 30 min at 37 °C, the samples were added to the
loading buffer containing 0.25 % bromophenol blue + 0.25 %
-1
conductivity values (< 20 Ω cm-2 mol-1) (Table-1) of present
copper(II) complexes suggest non-electrolytic nature of the
complexes [32]. The magnetic moment values of copper comp-
lexes (1.06 and 1.26 BM) are found to be sub-normal to spin

Vol. 31, No. 1 (2019)
Oxidative Cleavage of DNA by Transition Metal Complexes 151
only value [33-35]. The data suggest the presence of antiferro-
magnetically coupled copper centers in the complexes.
In the electronic spectra of Cu2(CH3COO)HDP and
Cu2(CH3COO)HDMP complexes intense band are observed
at 37037 and 37174 cm-1, respectively, which are attributed to
intra ligand π–π* aromatic ring and imine moiety. The respec-
tive bands at 27027 and 27472 cm-1 are assigned to metal to
ligand charge transfer transition (M→L CT). Typical electronic
spectrum of Cu2(CH3COO)HDP is shown in Fig. 4. The corres-
ponding low intensity broad structured bands at 15600 and
5898 cm-1are assigned to d-d transitions. Since the molar absor-
cm-1 indicates monodentate coordination, whereas difference
smaller than 200 cm-1 indicates bridging coordination mode.
In the complexes, the frequencies of vibration νasy(COO) appears
in the range at 1552 and 1546 cm-1 while those characteristic
of the νsym(COO), appeared at 1409 and 1387 cm-1. A value of
Dν < 200 cm-1, indicates bridging coordination mode of acetate
group [40]. In the IR spectra of all the complexes aromatic
ring vibrations and C=C vibrations are not much affected. The
non-ligand absorption bands occurring in the regions 509-506
cm-1 and 412-406 cm-1 are assigned to ν(M-O) and ν(M-N),
respectively [41].
ESR spectra studies: ESR spectra of copper(II) complexes
were recorded at room temperature and at liquid nitrogen
temperature (LNT) in both solution ((DMF) and solid state.
Typical ESR spectra of Cu2(CH3COO)HDP in powder state at
300 K, at liquid nitrogen temperature, in DMF solution at 300
K and at liquid nitrogen temperature are shown in Fig. 5. The
spin Hamiltonian and orbital reduction parameters of copper
complexes are given in Tables 2(a) and 2(b). The g|| and g⊥ values
are computed from the spectrum using tetracyanoethylene
(TCNE) free radical as ‘g’ marker. It is significant from the
data that the observed g|| values for all these copper complexes
are less than 2.3 suggesting covalent character of the metal-
ligand bonding [39]. The g tensor values of copper(II) comp-
lexes can be used to derive the ground state. In square planar
*
ptivity values (π–π ~ 1700, CT ~ 760, d-d ~ 300 L mol-1 cm-1)
2
are quite high, the bands assigned 2T2 → E electronic transition
in favour of tetrahedral structure.
Infrared spectra of the complexes in KBr disc were recorded
in the range 4000-400 cm-1 on a Perkin-Elmer spectrum 100
spectrometer. The donor sites of ligands have been identified
from infrared spectral studies. The ν(OH) stretching vibrations
are observed in 3512-3446 cm-1 region in ligands. These absorp-
tion bands are absent in complexes suggesting the deprotonation
of all the three –OH groups in complex formation. Thus, the
ligand is triply deprotonated in the complex formation. The
C=N (imine) vibrations are observed at 1633 and 1612 cm-1 in
the IR spectra of ligands. These bands are shifted to lower
wave number in IR spectra of the complexes suggesting the
participation of azomethine nitrogen atom in coordination with
metal atom [36]. Strong bands observed at 1552 and 1546 cm-1
are due to the presence of bridging acetato group [23,37].
Stretching vibrations of free or non-coordinated acetate ion
occurs in the range 1160-1115 cm-1 whereas the terminal acetate
shows vibrational peak in 1060-1040 cm-1 region. The lowering
of the position of the phenolic C-O bands in the complexes
indicates the formation of covalent bond between metal and
oxygen [38]. The spectra of the copper(II) complexes show
bands which could be assigned due to bridging coordination
mode of the acetate anion. The asymmetric (νasy) and the symm-
etric (νsym) stretching vibrations of the acetate and in particular,
their differences, ∆ν = νasy – νsym have been used as empirical
indicators of coordination modes of the acetate group. Accor-
ding to Deacon and Philip [39], a difference larger than 200
2
2
complexes, the unpaired electron lies in the dx -y orbitals giving
2B1g as the ground state with g|| > g⊥ >2.0023, while the unpaired
2
2
electron lies in the dz orbital giving A1g as the ground state
with g⊥ > g|| > 2.0023. From the observed values of complexes
at 300 K and 77 K in solid state spectrum it is clear that g|| > g⊥
> 2.0023 which suggests the fact that the unpaired electron
2
2
lies predominantly in the dx orbital [42]. The gav value for
-y
these complexes is greater than 2 indicating covalent nature
of the metal-ligand bond [43].
In the solid state, similar spectra of these complexes at 77 K
and 300 K indicates that the geometry around copper(II) ion
is unaffected on cooling to liquid nitrogen temperature. In these
conditions the axial symmetry parameter G, which measures
the interaction between copper centres in unit cell is calculated
from the following equation:
0.35
1.8
(B)
0.30
0.25
0.20
0.15
0.10
0.05
0.00
-0.05
1.6
(A)
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
400
500
600
700
800
900
200
300
400
500
600
700
800
900 1000
Wavelength (nm)
Wavelength (nm)
Fig. 4. Electronic spectra of Cu2(CH3COO)HDP (A) in DMF solvent and (B) spectrum of high concentration (1 × 10-3 M)

152 Pragathi et al.
Asian J. Chem.
(A)
(C)
(D)
(B)
2000
3000
4000 2000
3000
4000
Fig. 5. (A) X-band powder ESR spectra of Cu2(CH3COO)HDP at 300K, (B) at LNT, (C) in DMF solution at 300K and (D) at LNT in DMF
solution
TABLE-2(a)
SPIN HAMILTONIAN AND ORBITAL REDUCTION PARAMETERS OF COPPER COMPLEXES IN POWDER STATE
At room temperature
At liquid nitrogen temperature (LNT)
Complex
g||
gav
G
g||
gav
G
G⊥
G⊥
Cu2(CH3COO)HDP
Cu2(CH3COO)HDMP
2.114
2.136
2.077
2.098
2.089
2.110
1.495
1.397
2.117
2.138
2.081
2.101
2.093
2.113
1.457
1.374
TABLE-2(b)
SPIN HAMILTONIAN AND ORBITAL REDUCTION PARAMETERS OF COPPER COMPLEXES IN DMF SOLUTION
A||
A⊥
α2
Complex
g||
g(av)
G
K||
Aav
g||/A||
g⊥
λ
K⊥
(cm-1)
(cm-1)
Cu2(CH3COO)HDP
Cu2(CH3COO)HDMP
2.124
2.148
2.068
2.082
2.086
2.104
1.852
1.828
335
413
0.841
0.837
1.236
1.238
0.0182
0.0190
0.0128
0.0125
0.0146
0.0146
117
113
0.315
0.308
G = [g|| – 2.0023/g⊥ – 2.0023]
parameters (K||, K⊥). The trend g|| > g⊥ > ge (2.0023) observed
for these complexes suggests that the unpaired electron is
The calculated G values are found to be less than 4 for
the copper complexes suggesting that there are considerable
interactions between metal ions in the solid complex [44]. The
broad ESR spectra clearly reveal that there is strong antiferro-
magnetic interaction between two metal ions in the complexes.
This antiferromagnetic coupling occurs due to the quenching
of the spin of electrons of one metal ion by the adjacent metal
ion [37].
ESR spectra were recorded in DMF at room temperature
and liquid nitrogen temperature to obtain more accurate mole-
cular values by giving four hyperfine signals for all the comp-
lexes. The ESR parameters (g||, g⊥, A||, A⊥) of the complexes
and the energies of d-d transitions are used [45-47] to evaluate
spin-orbit coupling constant (λ) and the orbital reduction
2
2
localized in dx -y orbital [48] of the copper(II) ion. For both
copper(II) complexes the lowest g value greater than 2.0023
2
-y
2
is also consistent with a dx ground state. The spin-orbit
coupling constant (λ) value is calculated using the relation,
gav = (g|| + 2 g⊥)1/3and gave = 2(1-2 λ/10 Dq), is less than the
free copper(II) (832 cm-1) which also supports covalent
character of M-L bond. The observed K|| < K⊥ relation in all
the complexes indicates the presence of in plane π-bonding
[49]. The in-plane bonding parameter α values are calculated
using the following relation:
α = _ (A||/0.036) + (g|| 2.0023) + 3/7 (g⊥ _ 2.0023) + 0.04
The empirical factor g||/A|| (cm-1), is an index of tetrahedral
distortion. In these two complexes, g||/A|| falls in the range 113-
2
2
_

Vol. 31, No. 1 (2019)
Oxidative Cleavage of DNA by Transition Metal Complexes 153
117 cm corresponding to a copper(II) center with medium
distortion [50].
Based on molar conductance, magnetic moment, GC-MS,
electronic, FT-IR and ESR spectral data the following general
structure (Fig. 6) is assigned to copper(II) complexes of quinque-
dentate Schiff base ligands.
complexes. The non-equivalent current in cathodic and anodic
peaks (ic/ia = 1.224 and 1.065 at 100 mV s-1) for complexes
indicate quasi-reversible behaviour [51]. The difference ∆Ep
= Epc - Epa in all the complexes exceeds the Nerstian requirement
59/n mV (n = number of electrons involved in oxidation reduc-
tion) which suggests quasi-reversible character associated with
a considerable reorganization of the coordination sphere during
electron transfer [52]. The complexes have large separation
(129 and 154 mv) between anodic and cathodic peaks indica-
ting quasi-reversible character. The E1/2 values of copper comp-
lexes are inversely related to the size of the complex. As the
molecular weight of the complex increases, the E1/2value decreases
[53].
DNA binding studies of copper(II) complexes: The inter-
action of metal complexes with calf-thymus DNA was moni-
tored by UV-visible spectroscopy. The absorption spectra of
complexes were compared in the absence and in the presence
of CT-DNA. In the presence of increasing amounts of DNA,
the spectra of all complexes showed a strong decrease (Hypo-
chromicity) in intensity with shift in absorption maxima towards
lower (blue-shift) wavelengths.
Copper(II) complexes exhibit an intense absorption band
around 352-341 nm which is attributed to metal-ligand charge
transfer (MLCT) transitions.Absorption spectra were recorded
in the range of 250-500 nm. Electronic absorption spectral
data upon addition of CT-DNA and binding constants of these
complexes are given in the Table-4. The change in absorbance
values with increasing amounts of CT-DNA was used to evaluate
the intrinsic binding constant Kb, for the complexes. In the
presence of increasing amounts of CT-DNA, the UV-visible
absorption spectra of copper(II) complexes show bathochromic
shift (blue shift) (λmax: 2-3 nm). It is evident from the table,
that all the complexes bind with DNA with high affinities and,
the estimated binding constants are in the range 1-4 × 104 M-1.
This may be due to the presence of π-stacking of phenyl ring
present in the Schiff base ligand. Typical absorption spectra
of Cu2(CH3COO)HDP in presence and in absence of DNA
are shown in Fig. 8. The binding constants (Kb) for DNA intera-
ction of the complexes have been calculated by using the follo-
wing equation:
CH3
C
O
O
O
O
Cu
Cu
O
C
R
N
N
C
R
where R = H/CH3
Fig. 6. Tentative structure of the acetato bridged dinuclear copper complex
Electrochemical studies:Redox behaviour of the copper(II)
complexes has been investigated by cyclic voltammetry in DMF
using 0.1 M tetrabutylammonium hexaflourophosphate as
supporting electrolyte. The cyclic voltammetric profile of
Cu2(CH3COO)HDEP is given in Fig. 7. The electrochemical
data of the complexes are presented in Table-3.
a
b
[DNA]/(εa-εf) = [DNA]/(εb-εf) + 1/Kb (εb-εf)
From Table-4, it is evident that complex with lower mole-
cular weight i.e., Cu2(CH3COO)HDP shows higher binding
affinity towards DNA (Kb = 4.42 × 104 M-1) rather than the
heavier copper complex Cu2(CH3COO)HDMP (Kb = 1.77 ×
104 M-1).
0.6
0.5
0.4
0.3
0.2
0.1
0
Potential (V)
Fig. 7. Cyclic voltammetric profile of Cu2(CH3COO)HDMP at different
scan rates (a) 100 mV s-1 and (b) 50 mV s-1
DNA cleavage activities of copper(II) complexes: Nuclease
activities of quinquedentate Schiff base ligands and their copper(II)
compexes have been studied by agarose gel electrophoresis
using pBR 322 plasmid DNA in Tris-HCl/NaCl (50 mM/5
mM) buffer (pH 7) in the presence and absence of H2O2 after
Repeated scans at various scan rates suggest the presence of
stable redox species in solution. E1/2 values are observed at 0.360
and 0.331V vs.Ag/AgCl for the complexes Cu2(CH3COO)HDP
and Cu2(CH3COO)HDMP, respectively. It may be inferred that
Cu(II) complexes undergo reduction to their respective Cu(I)
TABLE-3
CYCLIC VOLTAMMETRIC DATA OF COPPER(II) COMPLEXES
log Kca
0.260
0.218
∆Ep (mV)
129
154
-∆G°b
1495
1252
Complex
Redox couple
Epc (V)
0.296
0.254
Epa (V)
0.425
0.408
E1/2
-ic/ia
1.224
1.065
Cu2(CH3COO)HDP
Cu2(CH3COO)HDMP
II/I
II/I
0.360
0.331
a log Kc = 0.434 ZF/RT∆Ep; b ∆G° = -2.303 RT log Kc

154 Pragathi et al.
Asian J. Chem.
TABLE-4
ELECTRONIC ABSORPTION DATA UPON ADDITION OF CT-DNA TO Cu(II) COMPLEXES
λmax (nm)
Complex
m.w.
H (%)
Kb (M-1)
∆λ (nm)
Free
352
344
Bound
350
341
Cu2(CH3COO)HDP
Cu2(CH3COO)HDMP
481
509
2
3
12.17
8.74
4.42 × 104
1.77 × 104
30
25
20
15
10
5
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
0
5
10
[DNA]
15
20
Fig. 9. Agarose gel (0.8 %) showing results of electrophoresis of 1 µL of
pBR 322 Plasmid DNA; 4 µL of Tris-HCl/NaCl (50 mM/5 mM)
buffer (pH-7); 2 µL of complex ligand in DMF (1 × 10-3 M); 11 µL
of sterilized water; 2 µL of H2O2 (total volume 20 µL) were added,
respectively, incubated at 37 °C (30 min); Lane 1: 1 kb DNA Ladder;
Lane 2: DNA control; Lane 3: DNA control + H2O2; Lane 4: HDPH3
(100 µM) + DNA; Lane 5: HDPH3 (100 µM) + DNA+ H2O2; Lane
6: HDMPH3 (100 µM) + DNA; Lane 7: HDMPH3 (100 µM) + DNA
+ H2O2
-0.1
200 250 300 350 400 450 500 550 600 650
Wavelength (nm)
(Fig. 10: lane 7 and 12), whereas the complexing agent EDTA
could not show considerable effect over the DNA cleavage
activity of the complexes.
Fig. 8. Absorption spectra of Cu2(CH3COO)HDP in the absence and in the
presence of increasing concentration of CT-DNA; top most spectrum
is recorded in the absence of DNA; A plot of [DNA]/(εa-εf) versus
[DNA] is shown in the inset
Conclusions
• Physico-chemical and spectral studies suggest that the
copper(II) complexes of quinquedentate ligands are acetato
bridged dinuclear complexes with tetrahedral geometry.
• The complexes have covalent character as suggested
by ESR spectral data.
• The cyclic voltammetric studies suggest that all the
complexes undergo quasi-reversible one electron reduction.
Repeated scans as well as various scan rates show that the comp-
lexes do not undergo any dissociation. The non-equivalent
current intensity of cathodic and anodic peak indicates quasi-
reversible behaviour of these complexes. Comparison of the
E1/2 values of present copper(II) complexes with analogous
nickel(II) complexes reveals that the complexes undergo more
facile redox change which seems to be a requirement to the
DNA cleavage.
0.5 h incubation period at 37 °C [22,28].At micromolar concen-
tration, the ligands exhibit no significant activity in the absence
or in the presence of the oxidant as shown in Fig. 9. But the
copper(II) complexes show enhanced nuclease activity due to
the presence of metal ions. Nuclease activity of complexes was
also investigated in presence of free radical scavenger (DMSO),
chelating agent (EDTA) and reducing agent DTT. Quantification
of the gel afforded data of three forms is presented in Tables 5
and 6. From the data it is clear that Cu2(CH3COO)HDMP has
higher nuclease activity than the complex Cu2(CH3COO)HDP.
In the absence of H2O2 the complexes cleaved supercoiled DNA
(Form 1) into nicked DNA (Form II) (Fig. 10 lane 3 and 8).
From Fig. 9 (lanes 4 & 10) and quantification data, it is evident
that copper complexes cleave DNA more effectively in the
presence of oxidant which may be due to hydroxyl radical
(OH) reaction with DNA. This is consistent with the increased
production of hydroxyl radicals by cuprous ions similar to the
well known Fenton reaction [54]. In presence of DTT (reducing
agent) the cleavage activity of the complexes was further enhanced
• Ligands do not show any binding affinity towards CT-
DNA, but the affinity is greatly enhanced by the incorporation
of metal ion in respective ligands. Copper(II) complexes derived
from tridentate ligands show higher binding affinity which may
TABLE-5
SELECTED SC pBR322 DNA CLEAVAGE DATA OF LIGANDS IN Fig. 9
Percentage of
Lane No.
Reaction condition
Form I
Form II
Form III
2
3
4
5
6
7
DNA
95.17
94.38
94.25
92.92
94.32
91.48
4.83
5.62
5.75
7.08
5.68
8.52
ND
ND
ND
ND
ND
ND
DNA + H2O2 (10 µM)
DNA + HDPH3 (62.5 µM)
DNA + HDPH3 (62.5 µM) + H2O2 (10 µM)
DNA + HDMPH3 (62.5 µM)
DNA + HDMPH3 (62.5 µM) + H2O2 (10 µM)

Vol. 31, No. 1 (2019)
Lane No.
Oxidative Cleavage of DNA by Transition Metal Complexes 155
TABLE-6
SELECTED SC pBR322 DNA CLEAVAGE DATA OF COPPER COMPLEXES IN FIG. 10
Percentage of
Reaction condition
Form I
Form II
Form III
1
2
3
4
5
6
7
8
9
DNA
DNA + H2O2 (10 µM)
DNA + Cu2(CH3COO)HDP(62.5 µM)
97.18
96.33
88.25
32.98
28.32
71.48
22.13
41.79
08.32
09.26
71.23
20.13
2.82
3.67
ND
ND
ND
ND
ND
ND
ND
ND
6.96
1.08
ND
ND
11.75
67.02
71.68
30.52
77.87
58.21
84.72
89.66
28.77
89.87
DNA + Cu2(CH3COO)HDP (62.5 µM) + H2O2 (10 µM)
DNA + Cu2(CH3COO)HDP (62.5 µM) + DMSO (10 µM)
DNA + Cu2(CH3COO)HDP (62.5 µM) + EDTA (10 µM)
DNA + Cu2(CH3COO)HDP (62.5 µM) + DTT (10 µM)
DNA + Cu2(CH3COO)HDMP (62.5 µM)
DNA + Cu2(CH3COO)HDMP (62.5 µM) + H2O2 (10 µM)
DNA + Cu2(CH3COO)HDMP (62.5 µM) + DMSO (10 µM)
DNA + Cu2(CH3COO)HDMP (62.5 µM) + EDTA (10 µM)
DNA + Cu2(CH3COO)HDMP (62.5 µM) + DTT (10 µM)
10
11
12
Fig. 10. Agarose gel (0.8 %) showing results of electrophoresis of 1 µL of pBR 322 Plasmid DNA; 4 µL of Tris-HCl/NaCl (50 mM/5 mM) buffer (pH-7); 2 µL
of complex in DMF(1x10-3M); 11 µL of sterilized water; 2 µL of H2O2 (total volume 20 µL) were added, respectively, incubated at 37 °C (30 min);
Lane 1: DNA control; Lane 2: DNA control + H2O2; Lane 3: Cu2(CH3COO)HDP + DNA; Lane 4: Cu2(CH3COO)HDP + DNA+ H2O2; Lane 5:
Cu2(CH3COO)HDP + DNA + DMSO; Lane 6: Cu2(CH3COO)HDP + DNA + EDTA; Lane 7: Cu2(CH3COO)HDP + DNA+DTT; Lane 8:
Cu2(CH3COO)HDMP + DNA; Lane 9: Cu2(CH3COO)HDMP + DNA+ H2O2; Lane 10: Cu2(CH3COO)HDMP + DNA + DMSO; Lane 11:
Cu2(CH3COO)HDMP + DNA + EDTA; Lane 12: Cu2(CH3COO)HDMP + DNA + DTT
be due to the presence of good leaving group (H2O), whereas
the metal complexes with polymethylene backbones show least
binding affinity towards CT-DNA because of steric hindrance
of bulky ligands. Binding constants of nickel(II) complexes are
found to be less when compared to those of copper(II) complexes.
• Ligands do not show significant nuclease activity, but
their copper(II) complexes show remarkable activity. Copper(II)
complexes show more nuclease activity in presence of an oxi-
dizing agent (H2O2).
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