DNA interaction studies of d9 and d10 metal complexes having Schiff base and polypyridyl ligands

Article abstract of DOI:10.1080/00958972.2012.690037

Four mononuclear complexes of Cu(II) and Zn(II) having a Schiff-base ligand and 1,10-phenanthroline/2,2'-bipyridine have been synthesized and characterized. From electronic spectral and magnetic susceptibility data, octahedral geometry has been proposed for the complexes. DNA-binding behaviors of these complexes are studied by absorption titration, electrochemical, and viscosity methods. The results indicate that the complexes bind to calf thymus DNA in an intercalative mode. The gel electrophoresis results reveal that 1 cleaves pBR322 DNA effectively. The ligand and its complexes have been screened for their antimicrobial activities against a few microorganisms. The data exhibit that they are better antimicrobial agents than the ligand.

Full text of DOI:10.1080/00958972.2012.690037

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DNA interaction studies of d9 and d10
metal complexes having Schiff base and
polypyridyl ligands
Krishnan Pothiraj a , Thanasekaran Baskaran a & Natarajan Raman
a
a Research Department of Chemistry , VHNSN College ,
Virudhunagar-626001 , India
Accepted author version posted online: 03 May 2012.Published
online: 16 May 2012.
To cite this article: Krishnan Pothiraj , Thanasekaran Baskaran & Natarajan Raman (2012) DNA
interaction studies of d9 and d10 metal complexes having Schiff base and polypyridyl ligands,
Journal of Coordination Chemistry, 65:12, 2110-2126, DOI: 10.1080/00958972.2012.690037
To link to this article: http://dx.doi.org/10.1080/00958972.2012.690037
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Journal of Coordination Chemistry
Vol. 65, No. 12, 20 June 2012, 2110–2126
DNA interaction studies of d9 and d10 metal complexes
having Schiff base and polypyridyl ligands
KRISHNAN POTHIRAJ, THANASEKARAN BASKARAN and
NATARAJAN RAMAN*
Research Department of Chemistry, VHNSN College, Virudhunagar-626001, India
(Received 12 December 2011; in final form 20 March 2012)
Four mononuclear complexes of Cu(II) and Zn(II) having a Schiff-base ligand and 1,10-
phenanthroline/2,20-bipyridine have been synthesized and characterized. From electronic
spectral and magnetic susceptibility data, octahedral geometry has been proposed for the
complexes. DNA-binding behaviors of these complexes are studied by absorption titration,
electrochemical, and viscosity methods. The results indicate that the complexes bind to
calf thymus DNA in an intercalative mode. The gel electrophoresis results reveal that 1 cleaves
pBR322 DNA effectively. The ligand and its complexes have been screened for their
antimicrobial activities against a few microorganisms. The data exhibit that they are better
antimicrobial agents than the ligand.
Keywords: Schiff base; DNA-Binding; DNA Cleavage; Antimicrobial activity
1. Introduction
Transition metal complexes such as ruthenium, rhodium, cobalt, nickel, copper, zinc,
and lanthanum with nitrogen containing heterocyclic ligands [1, 2] have shown binding
and cleavage activities with DNA [3, 4]. Planar condensed aromatic ligands such as
1,10-phenanthroline, bipyridine, and their derivatives (whose donor properties are
similar to purine and pyrimidine bases) in antitumor (DNA-binding) agents have drawn
attention to their promising properties [5, 6].
These metal complexes are also useful for design and development of synthetic
restriction enzymes, new drugs, DNA foot printing agents etc., because of their
potential to bind DNA and to cleave the duplex [7–10]. The ligands and metal are of
paramount importance in interaction of complexes with DNA, understanding of which
would help in design of new drugs and development of new selective, efficient DNA
recognition, and cleaving agents [11].
Copper(II) complexes play a significant role either as naturally occurring biological
systems or as pharmacological agents [12, 13]. Copper plays an important role in
hematopoiesis, the central nervous system, and bone tissue [14, 15]. Moreover, due to
the ability of copper(II) complexes to cleave DNA, many find possible medical uses
*Corresponding author. Email: drn_raman@yahoo.co.in
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ꢀ 2012 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2012.690037

DNA interactions with Cu and Zn
2111
in the treatment of diseases including cancer [16]. Among the various copper complexes
so far investigated, those containing phenanthroline and its derivatives have attracted
attention for their various biological activities such as antimycobacterial and
antimicrobial activities [17, 18]. We reported herein the synthesis and characterization
of Schiff base with polypyridyl copper(II) and zinc(II) complexes. The DNA-binding
properties of these compounds have been studied by electronic absorption spectra,
electrochemical, and viscosity measurements. The plasmid DNA cleavage of the
complex is also established. The bioefficacy of these complexes are examined against
the growth of the several microorganisms.
2. Experimental
2.1. Chemicals
2-Methylacetoacetanilide was obtained from Sigma Aldrich, Bangalore (India); 1,10-
phenanthroline monohydrate (phen), 2,20 bipyridyl (bpy), o-aminophenol, CuCl2 ꢀ
2H2O, and ZnCl2 were obtained from Merck. All other chemicals and solvents (AR
grade) were purchased from commercial sources and used without purification. Doubly
distilled water was used to prepare the buffer solutions. Supercoiled plasmid (SC)
pBR322 DNA and Tris-HCl buffer were purchased from Bangalore Genei (India).
Calf thymus DNA (ct-DNA), agarose (molecular biology grade), and ethidium bromide
(EB) were obtained from Sigma (USA). The spectroscopic titration was carried out in
aerated buffer (5 mmol Lꢁ1 Tris-HCl/50 mmol Lꢁ1 NaCl, pH 7.2) at room temperature.
2.2. Synthesis of Schiff base (HL)
The Schiff base has been synthesized by refluxing hot ethanol solution (25 mL) of
o-aminophenol (1.08 g, 0.01 mol), and hot ethanol solution (40 mL) of 2-methylacetoa-
cetanilide (1.91 g, 0.01 mol) for 4 h. The precipitate formed after evaporation was
filtered, washed with cold ethanol, recrystallized from hot ethanol, and dried in air.
C17H18N2O2: Yield (%): 79.78, orange red, m.p.: 86ꢂC, M. Wt: 282.34, Anal. Calcd
1
(%): C, 72.32; H, 6.43; N, 9.92. Found (%): C, 72.25; H, 6.38; N, 9.89; H NMR
(DMSO-d6, ꢀ/ppm): 2.05 (s, 3H, aliphatic–CH3), 2.48 (s, 3H, aromatic–CH3), 3.35 (s,
2H, C–CH2–C), 6.82–7.90 (m, 8H, aromatic–H), 8.95 (s, 1H, CONH), 10.54 (s, 1H,
phenolic–OH); IR (KBr, ꢁ/cm–1): 3375 ꢁ(OH), 3238 ꢁ(NH), 3052 ꢁ(Ar–CH), 2980, 2920,
ꢁ(aliphatic–CH), 1670 ꢁ(CONH), 1612 ꢁ(CN), 1546 ꢁ(Ar–CC), 1265 ꢁ(C–O). UV-Vis
(ꢂmax/nm) (DMF): 242, 273, 335.
2.3. Synthesis of metal complexes (1–4)
To a hot suspension of HL (0.282 g, 0.001 mol) in 15 mL ethanol, solution of copper(II)
chloride dihydrate (0.170 g, 0.001 mol) or zinc(II) chloride (0.136 g, 0.001 mol) in 20 mL
ethanol was added followed by the addition of 1,10-phenanthroline (0.180 g, 0.001 mol)/
2,20-bipyridine (0.156 g, 0.001 mol). The mixture was refluxed with stirring for 2 h and
cooled to room temperature. The precipitated complexes were filtered off, washed with

2112
K. Pothiraj et al.
ethanol, and dried in a desiccator over anhydrous calcium chloride under vacuum. The
dried complexes were subjected to elemental and spectroscopic analysis.
[Cu(L)(phen)Cl] (1): Yield (%): 67.25, dark green, m.p.: 162ꢂC, M. Wt: 559.54,
Anal. Calcd for C29H25N4O2CuCl (%): Cu, 11.33; C, 62.13; H, 4.49; N, 9.99; Cl, 6.32.
Found (%): Cu, 11.29; C, 62.14; H, 4.45; N, 9.97; Cl, 6.30; IR (KBr, ꢁ/cmꢁ1): 3237
ꢁ(NH), 3048 ꢁ(Ar–CH), 2984, 2920, ꢁ(aliphatic–CH), 1658 ꢁ(CONH), 1585 ꢁ(CN), 1549
ꢁ(Ar–CC), 1317 ꢁ(C–O), 559 ꢁ(Cu–O), 426 ꢁ(Cu–N); UV-Vis (ꢂmax nmꢁ1) (DMF): 252,
279, 431, 656; molar conductance (DMF): 30.43 Ohmꢁ1 cm2 molꢁ1; ꢃeff (B.M): 1.78.
[Cu(L)(bpy)Cl] (2): Yield (%): 68.12, light green, m.p.: 154ꢂC, M. Wt: 535.52,
Anal. Calcd for C27H25N4O2CuCl (%): Cu, 11.84; C, 60.44; H, 4.69; N, 10.44; Cl, 6.60.
Found (%): Cu, 11.82; C, 60.41; H, 4.67; N, 10.42; Cl, 6.57; IR (KBr, ꢁ/cmꢁ1): 3239
ꢁ(NH), 3051 ꢁ(Ar–CH), 2982, 2918, ꢁ(aliphatic–CH), 1657 ꢁ(CONH), 1600 ꢁ(CN), 1548
ꢁ(Ar–CC), 1325 ꢁ(C–O), 555 ꢁ(Cu–O), 423 ꢁ(Cu–N); UV-Vis (ꢂmax nmꢁ1) (DMF): 248,
275, 435, 638; molar conductance (DMF): 28.25 Ohmꢁ1 cm2 molꢁ1; ꢃeff (B.M): 1.76.
[Zn(L)(phen)Cl] (3): Yield (%): 65.56, yellow, m.p.: 151ꢂC, M. Wt: 561.39,
Anal. Calcd for C29H25N4O2ZnCl (%): Zn, 11.62; C, 61.93; H, 4.48; N, 9.96; Cl,
1
6.30. Found (%): Zn, 11.59; C, 61.91; H, 4.44; N, 9.94; Cl, 6.28; H NMR (DMSO-d6,
ꢀ/ppm): 2.06 (s, 3H, aliphatic–CH3), 2.49 (s, 3H, aromatic–CH3), 3.36 (s, 2H, C–CH2–
C), 6.84–8.61 (m, 16H, aromatic–H), 8.98 (s, 1H, CONH). IR (KBr, ꢁ/cmꢁ1): 3238
ꢁ(NH), 3050 ꢁ(Ar–CH), 2981, 2925 ꢁ(aliphatic–CH), 1654 ꢁ(CONH), 1597 ꢁ(CN), 1551
ꢁ(Ar–CC), 1320 ꢁ(C–O), 565 ꢁ(Zn–O), 435 ꢁ(Zn–N); UV-Vis (ꢂmax nmꢁ1) (DMF):
250, 282, 437; molar conductance (DMF): 29.58 Ohmꢁ1 cm2 molꢁ1; ꢃeff (B.M):
diamagnetic.
[Zn(L)(bpy)Cl] (4): Yield (%): 67.74, yellow, m.p.: 145ꢂC, M. Wt: 537.37, Anal. Calcd
for C27H25N4O2ZnCl (%): Zn, 12.14; C, 60.23; H, 4.68; N, 10.40; Cl, 6.58. Found (%):
1
Zn, 12.12; C, 60.20; H, 4.64; N, 10.38; Cl, 6.56; H NMR (DMSO-d6, ꢀ/ppm): 2.05
(s, 3H, aliphatic–CH3), 2.48 (s, 3H, aromatic–CH3), 3.36 (s, 2H, C–CH2–C), 6.83–8.48
(m, 16H, aromatic–H), 8.96 (s, 1H, CONH). IR (KBr, ꢁ/cmꢁ1): 3238 ꢁ(NH), 3050
ꢁ(Ar–CH), 2980, 2923 ꢁ(aliphatic–CH), 1656 ꢁ(CONH), 1588 ꢁ(CN), 1553 1455
ꢁ(Ar–CC), 1323 ꢁ(C–O), 561 ꢁ(Zn–O), 430 ꢁ(Zn–N); UV-Vis (ꢂmax nmꢁ1) (DMF):
247, 278, 440; molar conductance (DMF): 26.47 Ohmꢁ1 cm2 molꢁ1; ꢃeff (B.M):
diamagnetic.
2.4. Analytical methods
Metal contents of the complexes were analyzed by EDTA titration [19] after
decomposing the organic matter with a mixture of HClO4, H2SO4, and HNO3
(1 : 1.5 : 2.5). Elemental analyses were carried out on an Elementar Vario EL III, Carlo
Erba 1108 elemental analyzer at the Sophisticated Analytical Instrument Facility
(SAIF), Central Drug Research Institute, Lucknow. Molar conductance of
10ꢁ3 mol Lꢁ1 solutions of the complexes in DMF was measured at room temperature
with a Deep Vision Model-601 digital direct reading deluxe conductivity meter.
Magnetic susceptibility measurements were carried out by employing the Gouy method
at room temperature on a powder sample of the complex; CuSO4 ꢀ 5H2O was used as
calibrant. Infrared spectra (IR) (4000–400 cmꢁ1 KBr discs) of the samples were
recorded on an FT-IR Affinity-1 spectrophotometer (Shimadzu). Electronic absor-
bance spectra from 200 to 1100 nm were recorded on a UV-1601 spectrophotometer

DNA interactions with Cu and Zn
2113
(Shimadzu). Proton nuclear magnetic resonance spectra of HL and its zinc complexes
(300 MHz) were recorded on a Bruker Avance DRX-300 FT-NMR spectrometer using
DMSO-d6 as solvent. Tetramethylsilane was used as internal standard. Chemical shifts
are reported in ꢀ scale. EPR spectra of complexes in solid state at 300 K and frozen
DMSO at 77 K were recorded on a Varian E-112 spectrometer at X-band using TCNE
as marker with 100 kHz modulation frequency and 9.1 GHz microwave frequency.
Mass spectrometry experiments were performed on a JEOL-AccuTOF JMS-T100LC
mass spectrometer equipped with a custom-made electrospray interface (ESI).
2.5. DNA interaction study
2.5.1. Electronic absorption titration. All experiments involving interaction of 1–4 with
DNA were carried out in 5 mmol Lꢁ1 Tris-HCl buffer at pH 7.2, containing 50 mmol Lꢁ1
NaCl. The DNA concentration per nucleotide was determined by absorption spectros-
copy using the molar absorption coefficient (6600 (mol L
Absorption titration experiment was performed by maintaining the complex concen-
tration at 10 mmol Lꢁ1 and gradually increasing the concentration (20–100 mmol Lꢁ1
)
ꢁ1 ꢁ1 cmꢁ1) at 260 nm [20].
)
of nucleic acid. The complexes were dissolved in mixed DMF and Tris-HCl buffer. The
reference cuvette also included this mixed solvent. Absorption spectra of each complex
solution were scanned from 200 to 500 nm and then equal amount of ct-DNA was
added to complex solution and the reference cuvette. After each cuvette was incubated
at room temperature for 10 min, absorption spectra were recorded for the same
wavelength range. The changes in the metal complex concentration because of adding
DNA solution to each cuvette were negligible at the end of each titration. For 1–4, the
intrinsic binding constant (Kb) was determined from spectral titration data using the
following equation [21]:
½DNAꢃ=ð"a ꢁ "fÞ ¼ ½DNAꢃ=ð"b ꢁ "fÞ þ 1=Kbð"b ꢁ "fÞ,
where [DNA] is the concentration of DNA in base pairs, the apparent absorption
coefficients "a, "f, and "b correspond to Aobsd/[complex], the extinction coefficient of the
free complex, and the extinction coefficient of the complex when fully bound to DNA,
respectively. Kb is the equilibrium binding constant in (mol Lꢁ1 ꢁ1
) .
2.5.2. Cyclic voltammetric experiments. Cyclic voltammetry studies were performed
on a CH instrument electrochemical analyzer in a 15 mL three-electrode electrolytic cell.
The working electrode was glassy carbon, platinum wire electrode was used as the
counter electrode and an Ag/AgCl electrode saturated with KCl was used as reference
electrode. Cyclic voltammograms of the complexes were recorded in 1 : 2 DMF : buffer
solution at ꢁ ¼ 0.1 Vsꢁ1, where the buffer solution was the supporting electrolyte.
Oxygen was removed by purging the solution with nitrogen which had been previously
saturated with solvent vapors. All electrochemical measurements were performed
at 27ꢂC.
2.5.3. Viscosity measurements. Viscosity measurements were carried out using an
Ubbelodhe viscometer maintained at 37ꢂC in a thermostatic bath. DNA samples

2114
K. Pothiraj et al.
approximately 200 base pairs in average length were prepared by sonicating in order
to minimize complexities arising from DNA flexibility [22]. Flow time was measured
with a digital stopwatch, and each sample was measured three times, and an average
flow time was calculated. Data were presented as (ꢄ/ꢄ0)1/3 versus binding ratio
(R ¼ [complex]/[DNA]) [23], where ꢄ is the viscosity of DNA in the presence of complex
and ꢄ0 is the viscosity of DNA alone.
2.5.4. DNA cleavage experiments. The DNA cleavage activity of newly synthesized
complex 1 was studied by agarose gel electrophoresis. Supercoiled plasmid pBR322
DNA (0.3 mg), dissolved in 5 mmol Lꢁ1 Tris-HCl/50 mmol Lꢁ1 NaCl buffer (pH 7.2),
was treated with all the complexes. The mixtures were incubated at 37ꢂC for 1 h and
then mixed with the loading buffer (2 mL) containing 25% bromophenol blue, 0.25%
xylene cyanol, and 30% glycerol. Each sample (5 mL) was loaded into 1% agarose
gel containing 1.0 mg mLꢁ1 EB. Electrophoresis was undertaken for 2 h at 50 V in
Tris-acetate-EDTA (TAE) buffer (pH 8.0). The DNA cleavage with added reductant or
oxidant was monitored as in the case of cleavage experiment without added reductant
or oxidant using agarose gel electrophoresis. The reaction was also monitored upon
addition of various radical inhibitors, such as sodium azide (NaN3), DMSO, superoxide
dismutase (SOD), hydrogen peroxide (H2O2), and 3-mercaptopropionic acid (MPA),
glutathione (GSH), ascorbic acid (Asc). The samples were incubated for 2 h at 37ꢂC.
The gel was stained with EB for 5 min after electrophoresis and then photographed
under UV light. All experiments were carried out in triplicate under the same
conditions.
2.6. Antimicrobial studies
The disc diffusion method was used to evaluate antimicrobial activities of HL and 1–4
[24]. Antimicrobial activities were assayed on different microorganisms, Staphylococcus
aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger,
Rhizopus stolonifer, Candida albicans, and Rhizoctonia bataicola by the disc diffusion
method. A lawn of microorganism was prepared by pipetting and evenly spreading
100 mL of inoculum into nutrient agar set in Petri discs. Paper discs were impregnated
with stock solution of the samples and dried under sterile conditions. Then the dried
discs were laid on the previously inoculated agar surface. The plates were incubated at
37ꢂC. After 24 h, the activities of the complexes were observed. The experiments were
repeated three times and the results were expressed in average values. A 10 mL spore
suspension (105–106 spore mLꢁ1) of each of the investigated organisms was added to
a sterile agar medium just before solidification, then poured into sterile Petri discs
(9 cm in diameter), and left to solidify. The bacteria were subcultured in agar medium.
The Petri discs were incubated for 24 h at 37ꢂC. Standard antibacterial drug
(Ciprofloxacin) was also screened under similar conditions for comparison. The fungi
were subcultured in potato dextrose agar medium. Standard antifungal drug (flucon-
azole) was used for comparison. The Petri discs were incubated for 48 h at 37ꢂC.
To understand quantitatively the antimicrobial activities of compounds their
minimum inhibitory concentrations (MIC) were evaluated by the microdilution broth
method [25]. Sample solutions were added to broth for different concentrations.
Samples measuring of each microbial suspension were added to the serial dilution of the

DNA interactions with Cu and Zn
2115
test substances. The inoculated test tubes were incubated at 37ꢂC under aerobic
conditions. The MIC was defined as the lowest antimicrobial concentration of the test
samples. Solvent control test was performed to study the effect of 10% DMSO on the
growth of microorganism and it did not inhibit growth. All tests were performed
in duplicate and MICs were constant.
3. Results and discussion
Four mixed-ligand complexes [M(L)(phen/bpy)Cl] (where M is Cu(II) and Zn(II), phen
is 1,10-phenanthroline, and bpy is 2,20-bipyridine) have been prepared by reaction of
metal salts and Schiff base with phen/bpy. They have been characterized by elemental
1
analysis, IR, UV-Visible, H NMR, ESI-MS, and EPR spectroscopic methods.
3.1. Molar conductance measurements
From molar conductance measurements, the complexes are non-electrolytic nature
in 10ꢁ3 mol Lꢁ1DMF solution, implying the presence of chloride in the coordination
sphere of the complexes.
3.2. Electronic absorption spectra and magnetic susceptibility
The electronic absorption spectra of HL and 1–4 were recorded in DMF. The Schiff-
base ligand has ring ꢅ ! ꢅ* bands at 243 and 273 nm and a band at 335 nm due
to n ! ꢅ* transition associated with the azomethine linkage. These bands show a
bathochromic shift due to coordination of azomethine [26]. The moderately intense
broad bands for 3 and 4 at 437–440 nm are assigned to metal-to-ligand charge transfer
(MLCT) transition. The Zn(II) complexes show no appreciable absorption above
450 nm in DMF solution, in accord with the d10 electronic configuration of Zn(II).
At room temperature, the magnetic moments of the Zn(II) complexes are zero and they
are diamagnetic.
The electronic absorption spectra of 1 and 2 show a broad band centered at 656 nm
2
and 638 nm, respectively, corresponding to 2Eg ! T2g transition, consistent with
octahedral geometry around Cu(II). Bands at 431 nm for 1 and 435 nm for 2 are
assigned to MLCT transfer and the remaining bands at 252 nm, 279 nm for 1 and at
248 nm, 275 nm for 2 are due to ꢅ ! ꢅ* transitions of coordinated phen and bpy ligand,
respectively. The magnetic moment of the complexes (1.78 B.M. for 1 and 1.76 B.M.
for 2) at room temperature indicates monomeric Cu(II) complexes.
3.3. IR spectral studies
IR spectra of the complexes are compared with the free ligand to determine the
coordination sites involved in chelation. HL shows a strong band at 1612 cmꢁ1
,
characteristic of the azomethine. Coordination of the Schiff base to metal through
nitrogen is expected to reduce electron density in the azomethine. The band due to

2116
K. Pothiraj et al.
azomethine nitrogen ꢁ(C¼N) shows a modest decrease in frequency for the complexes
appearing around 1585–1600 cmꢁ1, consistent with coordination of the azomethine
nitrogen [27]. The amide stretch, ꢁ(CONH) of 2-methylacetoacetanilide of HL at
1670 cmꢁ1, shifts to 1654–1658 cmꢁ1 upon coordination, indicating coordination of
metal ion via amide carbonyl oxygen. There is an upward shift in the stretching
frequency of ꢁ(C–O) in the complexes from 1265 cmꢁ1 to 1317–1325 cmꢁ1, further
supported by the disappearance of ꢁ(O–H) at 3375 cmꢁ1 for all the complexes,
indicating deprotonation of phenolic proton. The band at 3238 cmꢁ1 (ꢁ(NH)) does not
change for ligand and complexes. Ring stretching frequencies (ꢁ(C¼C) and ꢁ(C¼N)) at
1506, 1420 cmꢁ1 for free phen and 1528, 1455 cmꢁ1 for free bpy shift to higher
frequencies upon complexation (1514, 1423 cmꢁ1 for 1, 1548, 1467 cmꢁ1 for 2, 1517,
1425 cmꢁ1 for 3, and 1545, 1462 cmꢁ1 for 4), indicating coordination of the heterocyclic
nitrogen. Absorptions at 555–565 and 423–435 cmꢁ1 are ascribed to the formation
of M–O and M–N bonds, respectively [28], further supporting coordination of the
azomethine nitrogen and phenolic oxygen.
3.4. 1H NMR spectral studies
1H NMR spectra of HL and 3 and 4 were recorded in DMSO-d6. All the protons were
in their expected regions. Observed signals for HL at 10.54, 8.95, and 6.82–7.90 ppm
are attributed to phenolic OH, amide NH, and aromatic protons, respectively.
Methylene protons at 3.35 ppm and methyl protons at 2.04 ppm for aliphatic (C–CH3)
and 2.48 ppm for aromatic (C–CH3) are practically unaffected by complexation. In 3
and 4, disappearance of phenolic OH proton (ꢀ ¼ 10.54 ppm) is consistent with
deprotonation and coordinating with zinc. Aromatic protons of 3 and 4 are at 6.84–8.61
and 6.83–8.48 ppm, respectively, and the signal of the NH (ꢀ ¼ 8.95 ppm) is slightly
changed, confirming the suggested structures of the complexes. The conclusions drawn
from these studies lend further support to the mode of bonding discussed in IR spectra.
3.5. Mass spectra of Schiff base and 1 and 2
The ESI mass spectra of HL and its polypyridyl complexes 1 and 2 were carried out.
The mass spectrum of HL showed a molecular ion peak at m/z ¼ 282 [M]þ and 283
(M þ 1), which is equivalent to its molecular weight. Complexes 1 and 2 showed
characteristic molecular ion and 37Cl isotopic peaks at m/z 560[M]þ, 562(M þ 2) and
536[M]þ, 538(M þ 2), respectively. The mass spectra of Schiff-base ligand and its
polypyridyl complexes 1 and 2 are in agreement with the proposed structure (scheme 1).
3.6. EPR spectra of 1 and 2
X-band EPR spectra of 1 and 2 have been recorded at room temperature and DMSO
solution at 77 K using 100 kHz field modulation; g-factors are quoted relative to the
standard marker TCNE (g ¼ 2.00277). EPR spectral assignments of 1 and 2 (77 K)
along with the spin Hamiltonian and orbital reduction parameters are summarized
in tables 1 and 2.

DNA interactions with Cu and Zn
2117
Scheme 1. Synthetic route of Schiff base and its complexes.

2118
K. Pothiraj et al.
Table 1. The spin Hamiltonian parameters of 1 and 2 in DMSO at 77 K.
g-tensor
Hyperfine constant ꢄ 10ꢁ4 (cmꢁ1
)
Complex
g
g
?
gav
A
A
Aav
?
1
2
2.27
2.23
2.12
2.09
2.17
2.13
130
123
105
85
133.33
118.33
Table 2. The bonding parameters of 1 and 2 in DMSO at 77 K.
Complex
ꢆ2
ꢇ2
ꢈ2
K
K
K
G
?
1
2
0.72
0.65
0.88
0.82
0.95
0.93
0.61
0.52
0.74
0.63
0.32
0.29
2.279
2.60
EPR spectra of 1 and 2 in the polycrystalline state at 300 K show only one broad
signal at giso ¼ 2.15 and 2.12, respectively. Such isotropic spectra consisting of only one
broad signal and hence only one g value which is due to polar broadening and enhanced
spin lattice relaxation. Unfortunately, these spectra give no information on the
electronic ground state of Cu(II) present in the complexes.
EPR spectra of 1 and 2 in DMSO at 77 K show well-resolved four hyperfine lines
giving g|| 4 g 4 2.00277, corresponding to the presence of an unpaired electron in the
?
dx2ꢁy2 orbital. This reveals that the complexes are monomeric octahedral copper(II)
species. The geometric parameter, G ¼ (g|| ꢁ 2.00277)/(g ꢁ 2.00277) values are less than
?
4.0, consistent with a copper(II) center in dx2ꢁy2 ground state having small exchange
coupling [29].
EPR parameters g||, g?, A||, A (Cu) and the energies of d–d transitions were used to
?
evaluate bonding parameters ꢆ2, ꢇ2, and ꢈ2 which may be regarded as measures of the
covalency of the in-plane ꢉ-bonds, in-plane ꢅ-bonds, and out-of-plane ꢅ-bonds,
respectively [30]. For a copper(II) complex, g|| is sensitive to indicate covalence.
The g|| values for copper(II) complexes less than 2.3 indicate significant covalent
bonding [31, 32].
In 1 and 2, the value of in-plane ꢉ-bonding parameter ꢆ2 was estimated following
Maki and McGarvey. These complexes have K|| 5 K?, which suggests stronger in-plane
ꢅ-bonding [33, 34]. The values of the bonding parameters ꢆ2 (0.65–0.72), ꢇ2 (0.82–0.88),
and ꢈ2 (0.93–0.95) 5 1.0 (value of 1.0 for 100% ionic character) indicate significant
in-plane ꢅ-bonding and in-plane ꢉ-bonding while the axial bond is more polar. The
Fermi contact hyperfine interaction term K ¼ Aiso/Pꢇ2 þ (gav ꢁ 2.00277)/ꢇ2 is a
measure of the contribution of the ‘s’ electrons to the hyperfine interaction, found to
be 0.29–0.32. The calculated empirical factor f ¼ g||/A|| values (175 cm for 1 and 181 cm
for 2) and index of tetragonal distortion are higher than the range 105–135 supporting
high distortion from octahedral geometry.
3.7. DNA-binding and cleavage properties
DNA is the primary pharmacological target of many antitumor compounds [35] and
interactions of metal complexes with DNA have been a subject of importance for

DNA interactions with Cu and Zn
2119
Table 3. Electronic absorption spectral properties of 1–4 with ct-DNA.
ꢂmax (nm)
Complex
Free
Bound
Dꢂ (nm)
Hypochromicity (%)
Kb ꢄ 104 ((mol Lꢁ1 –1
) )
1
2
3
4
431.0
435.0
437.5
440.4
435.5
438.0
440.0
442.3
4.5
3.0
2.5
1.9
12.53
14.50
34.47
26.36
5.72
1.55
2.06
0.98
development of effective chemotherapeutic agents. For this reason, before investigating
the nuclease activity of the compound, we carried out absorption titration, viscosity
measurements, and cyclic voltammetry to examine the complexes mode of, and
propensity for, binding to ct-DNA.
3.7.1. Absorption titration study of complex-DNA interaction. The binding ability
of the complexes with ct-DNA can be characterized by measuring their effects on
absorption spectra. Complex binding with DNA through intercalation usually result
in hypochromism and bathochromism due to the strong stacking interaction between
an aromatic chromophore and the base pairs of DNA [36]. In the present investigation,
the interaction of the bpy- and phen-based complexes (1–4) in DMF solutions (10%)
with ct-DNA have been investigated. Absorption titration experiments of the complexes
in buffer were performed by using a fixed concentration of complexes to which
increments of DNA stock solution were added. The binding of the complexes to duplex
DNA led to a decrease in absorption intensities with a small red shift in the UV-Vis
absorption spectra [37]. To compare quantitatively the affinity of the four complexes
toward DNA, the binding constant (Kb) of the complexes to ct-DNA was determined
by monitoring the changes of absorbance at 431 nm (for 1), 435 nm (for 2), 437.5 nm
(for 3), and 440.4 nm (for 4) with increasing concentration of DNA. The binding
constants (Kb) of complexes are 5.72 ꢄ 104, 1.55 ꢄ 104, 2.06 ꢄ 104, and 0.98 ꢄ 104
(mol L )
ꢁ1 ꢁ1, respectively (table 3). These Kb values are lower than those observed for
classical intercalators (e.g. EB-DNA, ꢅ106 (mol L
)
ꢁ1 ꢁ1) [38], but have the same level
as those of some well-established intercalation agents (ꢅ104) [39–42]. Better binding
affinity of phen complexes to ct-DNA than bpy complexes may be due to the extended
aromaticity and coplanarity of the phenanthroline ring system for better stacking
between the base pairs of DNA [43]. These spectral characteristics suggest that all four
complexes interact with DNA, likely through intercalation. The absorption titration of
1 is shown in figure 1.
3.7.2. Cyclic voltammetric study of complex-DNA interaction. Redox behaviors of 1–4
in the absence and presence of ct-DNA were studied in DMF : buffer (1 : 2) by cyclic
voltammetry at room temperature from 1.0 to ꢁ1.2 V. Cyclic voltammetry is an
important electroanalytical technique for studying interaction of metal complexes with
biomolecules due to the similarity between various chemical and biological redox
processes [44, 45].
Cyclic voltammograms (CV) of 1 and 2 exhibit two quasi-reversible redox waves
corresponding to Cu(III)/Cu(II) and Cu(II)/Cu(I) couple with Epc and Epa values

2120
K. Pothiraj et al.
Figure 1. Absorption spectrum of 1 in Tris-HCl buffer upon addition of ct-DNA. [complex] ¼ 10 mmol Lꢁ1
,
[DNA] ¼ (20–100) mmol Lꢁ1. The arrow shows the absorbance changing upon increasing DNA concentration.
ꢁ0.048, ꢁ0.445 V and ꢁ0.069, ꢁ0.101 V for 1 and ꢁ0.122, ꢁ0.561 V and ꢁ0.105,
ꢁ0.089 V for 2 at 0.1 V sꢁ1 scan rate. The ratio of anodic to cathodic peak currents
Ipa/Ipc was less than 1.0. The formal electrode potentials E1/2 and DEp for Cu(III)/
Cu(II) and Cu(II)/Cu(I) couple (difference in cathodic Epc and anodic Epa peak
potentials) were 0.010, ꢁ0.274 V and 0.117, 0.347 V for 1 and ꢁ0.017, ꢁ0.650 V and
0.227, 0.472 V for 2. The DEp values are larger than the Nernstian values observed for
a one-electron transfer redox couple [46]. At constant parameters, the addition of
ct-DNA shifts E1/2, DEp values for the above-mentioned two redox couples to 0.006,
ꢁ0.243 V and 0.184, 0.306 V for 1 and ꢁ0.084, ꢁ0.632 V and 0.212, 0.242 V for 2
(figure 2). The ratios of Ipa/Ipc were also less than unity for ct-DNA bound complexes.
The shift in potentials and decrease in current ratio suggest binding of 1 and 2 to
ct-DNA [47].
The CV of 3 and 4 in DMF : buffer (1 : 2) solution at a scan rate 0.1 V sꢁ1 features
a quasi-reversible redox wave Zn(II)/Zn(I) with E1/2(ꢁ0.510), DEp(0.725) for 3 and
E1/2(ꢁ0.375), DEp(0.378) for 4 [48]. Upon addition of ct-DNA under the same
conditions, complexes experience a shift in E1/2(ꢁ0.503), DEp(0.656) for 3 and
E1/2(ꢁ0.362), DEp(0.323) for 4 (table 4). The ratio of anodic to cathodic peak currents
(Ipa/Ipc) in the presence and absence of DNA with zinc complexes was less than unity.
The shift in E1/2 for complexes on binding to DNA suggests that both M(II) and M(I)
forms bind but to different extents. Analogous to the treatment of the association of
small molecules with micelles and DNA [49, 50], the ratio of the equilibrium constants,
KR/KO for binding of the M(II) and M(I) forms of complexes to DNA can be estimated
from the net shift in E1/2. For a Nernstian electron transfer in a system in which both
the oxidized and reduced forms associate with a third species such as DNA in solution
can be applied. Here, Mnþ-DNA represents metal complexes bound to DNA with nþ
charge on the metal center. Thus for one-electron transfer,
Eꢂb ꢁ Eꢂf ¼ 0:0591 logðKR=KOÞ,

DNA interactions with Cu and Zn
2121
Figure 2. Cyclic voltammogram of 2 in the absence and presence of increasing amounts of ct-DNA at room
temperature in DMF : Tris-HCl buffer (1 : 2) mixture (pH 7.2) (scan rate ¼ 0.1 V sꢁ1). The arrow shows the
current changing upon increasing DNA concentration.
Table 4. Electrochemical behavior of 1–4 in the absence and presence of ct-DNA.
Epc (V)
Free Bound
Epa (V)
DEp (V)
E1/2 (V)
Free Bound KR/KO
0.010
Complex Redox couple
Free
Bound Free Bound
1
2
Cu(III)/Cu(II) ꢁ0.048 ꢁ0.089
Cu(II)/Cu(I)
0.069
0.095 0.117 0.184
0.006
0.20
7.45
ꢁ0.448 ꢁ0.396 ꢁ0.101 ꢁ0.090 0.347 0.306 ꢁ0.274 ꢁ0.243
0.105
Cu(III)/Cu(II) ꢁ0.122 ꢁ0.148
0.064 0.227 0.212 ꢁ0.017 ꢁ0.084
0.36
3.61
Cu(II)/Cu(I)
Zn(II)/Zn(I)
Zn(II)/Zn(I)
ꢁ0.561 ꢁ0.528 ꢁ0.089 ꢁ0.104 0.474 0.424 ꢁ0.650 ꢁ0.632
ꢁ0.873 ꢁ0.831 ꢁ0.148 ꢁ0.175 0.725 0.656 ꢁ0.510 ꢁ0.503
ꢁ0.564 ꢁ0.524 ꢁ0.186 ꢁ0.201 0.378 0.323 ꢁ0.375 ꢁ0.362
3
4
5.06
4.33
where Eꢂf and Eꢂb are the formal potentials of the M(II)/M(I) couple in the free and fully
bound forms, respectively. KR and KO are the corresponding binding constants for the
M2þ and Mþ species to DNA. The binding ratios of the phen-complexes are higher
than the bpy-complexes. These values suggest that the interaction of metal complexes
with DNA tends to stabilize the M(II) over M(I) (table 4). Based on the above CV data,
all the complexes bind to DNA by both intercalation and electrostatic interaction.
3.7.3. Viscosity measurements. Viscosity is a useful technique for exploring the
binding mode of complexes with ct-DNA, considered as least ambiguous and most
critical test of binding in solution. A classical intercalator causes a significant increase
in the viscosity of DNA solution due to increase in overall DNA contour length [51].
A partial or non-classical intercalation of metal complexes reduces the DNA
viscosity [52]. The effect of phen and bpy complexes on the viscosity of ct-DNA is

2122
K. Pothiraj et al.
Figure 3. Change in relative specific viscosity of ct-DNA (10 ꢄ 10ꢁ5 mol Lꢁ1) on addition of 1 (g), 2 (^),
3 (m), and 4 (f) in Tris-HCl buffer (pH 7.2) at 37ꢂC, R ¼ [complex]/[DNA].
Figure 4. Agarose gel electrophoresis pattern showing cleavage of pBR322 DNA (0.3 mg) by 1. Lane 1,
DNA; Lane 2, DNA þ 1 (20 mmol Lꢁ1); Lane 3, DNA þ 1 (40 mmol Lꢁ1); Lane 4, DNA þ 1 (60 mmol Lꢁ1);
Lane 5, DNA þ 1 (80 mmol Lꢁ1); Lane 6, DNA þ 1 (100 mmol Lꢁ1).
shown in figure 3. The plots show relative viscosity increases with increase in
concentration of complexes. The observed order of DNA-binding in viscosity
measurements is 1 4 3 4 2 4 4, rationalized by intercalation.
3.7.4. DNA cleavage activity of 1. Since 1 shows highest binding tendency with
ct-DNA, the cleavage activity has been evaluated for 1. The nuclease activity of 1 has
been studied using supercoiled pBR322 DNA as substrate. To discover the DNA
cleavage ability, supercoiled pBR322 DNA was incubated with different concentrations
of complex in aqueous buffer solution (5 mmol Lꢁ1 Tris-HCl/50 mmol Lꢁ1 NaCl, pH
7.2) without addition of any reductant or oxidant. The DNA-cleaving ability of 1 was
demonstrated initially by a plasmid relaxation assay, in which conversion of supercoiled
form (Form I, SC) to nicked circular (Form II, NC) or linear open circular (Form III,
LC) DNA was monitored. As observed from figure 4, there is a gradual decrease in the
amount of Form I with a simultaneous increase in Form II (Lanes 2 and 3) on
increasing the concentration of the complex. A notable cleavage feature observed for 1
is the appearance of Form III before the disappearance of Form I (Lanes 3 and 4).

DNA interactions with Cu and Zn
2123
Figure 5. Agarose gel electrophoresis pattern for cleavage of pBR322 DNA (0.3 mg) by 1 (40 mmol Lꢁ1
)
in the presence of different activating agents at 37ꢂC after incubation for 2 h. Lane 1, DNA control;
Lane 2, DNA þ 1 þ GSH (40 mmol Lꢁ1); Lane 3, DNA þ 1 þ Asc (40 mmol Lꢁ1); Lane 4, DNA þ 1 þ MPA
(40 mmol Lꢁ1); Lane 5, DNA þ 1 þ H2O2 (40 mmol Lꢁ1).
This indicates that 1 is capable of direct strand scission and is better suited for
therapeutic applications, while many complexes are only able to cleave a single strand
successively [53].
The cleavage efficiency of copper(II) is usually dependent on activators. Therefore,
to understand the cleavage mechanism, a lower concentration (40 mmol Lꢁ1) of 1 was
allowed to stay in the presence of activators, namely H2O2 and MPA, GSH, Asc; 1
showed significant increase in the cleavage of DNA in the order H2O2 4 MPA 4
Asc 4 GSH (figure 5).
Experiments were carried out in the presence of several reactive oxygen trappers since
dioxygen or redox reagents are believed to be a major cause of DNA damage. To
investigate this mechanistic pathway of DNA cleavage, 1 (80 mmol Lꢁ1) was allowed to
interact with DNA separately with DMSO (Lane 2) and ethanol (Lane 3) as a hydroxyl
radical scavenger under identical conditions; cleavage was not significant as compared
to DNA cleavage without them. Therefore, it was considered that diffusible (ꢆOH)
hydroxyl radicals are not responsible for DNA cleavage. However, with other oxygen
trappers like sodium azide (NaN3) as singlet oxygen scavenger, the cleavage was
significant, revealing the involvement of 1O2 in DNA cleavage (Lane 4). The
1
involvement of O2 was further supported by significant enhancement of the cleavage
activity in D2O, where lifetime of 1O2 is longer than in water [54]. In D2O, a linear form
also appeared, indicating increase in cleavage (Lane 5). In the presence of superoxide
dismutase (SOD), a facile superoxide anion radical (Oꢆ2ꢁ) quencher, cleavage was
observed similar to that observed in the presence of complex only (Lane 6), revealing
that superoxide anion is not the active species in cleavage of DNA as depicted in
figure 6. Hence, the cleavage pattern supports the oxidative cleavage pathway.
3.8. Antimicrobial effect
In vitro testing of antibacterial and antifungal activities of the ligand and 1–4 are shown
in table 5. For comparison, MIC values of ciprofloxacin and fluconazole are also listed
in table 5. The ligand and complexes showed different degrees of antimicrobial
activities. In antibacterial studies, 1 has good activity against S. aureus and E. coli
(MIC ¼ 3.125 mg mLꢁ1) compared to other complexes and also other tested microor-
ganisms. Complexes 2, 3, and 4 have lowest antibacterial activity against P. aeruginosa
(MIC ¼ 25 mg mLꢁ1) and B. subtilis (MIC ¼ 25–50 mg mLꢁ1), respectively. In antifungal

2124
K. Pothiraj et al.
Figure 6. Agarose gel electrophoresis pattern showing cleavage of pBR322 DNA (0.3 mg) by 1 (80 mmol Lꢁ1
)
in the presence of standard radical scavengers at 37ꢂC after incubation for 2 h. Lane 1, DNA control; Lane 2,
DNA þ 1 þ DMSO (15 Units); Lane 3, DNA þ 1 þ ethanol (15 Units); Lane 4, DNA þ 1 þ D2O (70 %); Lane
5, DNA þ 1 þ NaN3 (0.4 mmol Lꢁ1); Lane 6, DNA þ 1 þ SOD (15 Units), respectively.
Table 5. The in vitro antimicrobial activity of HL and 1–4 evaluated by MIC.
Antibacterial activity
Antifungal activity
Complex
S. aureus B. subtilis E. coli P. aeruginosa A. niger R. bataicola R. stolonifer C. albicans
HL
1
2
3
4
100
3.125
12.5
6.25
12.5
200
12.5
6.25
25
50
100
3.125
6.25
6.25
200
6.25
25
12.5
25
100
6.25
12.5
3.125
12.5
100
12.5
25
12.5
6.25
100
6.25
12.5
6.25
25
200
25
50
12.5
50
12.5
Ciprofloxacin
Fluconazole
0.78
–
1.56
–
0.78
–
1.56
–
–
0.78
–
1.56
–
1.56
–
1.56
studies, 3 has greatest activity against A. niger (MIC ¼ 3.125 mg mLꢁ1), while 2 and 4
have moderate activity against C. albicans (MIC ¼ 50 mg mLꢁ1). All compounds have
low to moderate activity compared to ciprofloxacin and fluconazole. Comparison to the
previously reported 2-methylacetoacetanilide Schiff base and copper and
zinc complexes [55, 56], the present complexes are better, perhaps due to phen/bpy
and chloride inside the coordination sphere. In general, the enhancement of
metal complexes in the activity can be explained on the basis of Tweedy’s chelation
theory [57].
4. Conclusion
Synthesis and spectral characterization of Schiff-base polypyridyl copper and zinc
complexes are reported in this work. The Schiff base is tridentate and phen/bpy are
bidentate. Complexes 1–4 adopt octahedral geometry around the metal. DNA-binding
studies reveal that 1 has more intercalative binding than 2–4. Complex 1 shows
significant increase in cleavage of pBR322 DNA and the order of cleavage in different
activators is H2O2 4 MPA 4 Asc 4 GSH. Complex 1 cleaves pBR322 DNA effectively
via oxidative pathway. The antimicrobial activities reveal that ligand and complexes
have antimicrobial activity, depending on the microbial species tested. None of the
present compounds are as effective against the tested microorganisms as currently used
drugs.

DNA interactions with Cu and Zn
2125
Acknowledgments
The authors express their heartfelt thanks to the College Managing Board, Principal,
and Head of the Department of Chemistry, VHNSN College, for providing necessary
research facilities. SAIF, IIT Bombay and CDRI, Lucknow, are gratefully acknowl-
edged for providing instrumental facilities.
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