Studies on DNA binding, electrochemical activation, DNA photocleavage, and biopotency of N and O donor bidentate ligands with Cu(II), Co(II), and Zn(II)

Article abstract of DOI:10.1080/00958972.2010.551767

Three Schiff-base ligands, namely 2-(3,4-dimethoxybenzylideneamino)phenol (HL¹), 2-(3-hydroxy-4-nitrobenzylidene-amino)phenol (HL²), and 2-(3-methoxy-4-hydroxy-benzylideneamino)phenol (HL³) and their Cu(II), Co(II), and Zn

Full text of DOI:10.1080/00958972.2010.551767

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Studies on DNA binding,
electrochemical activation, DNA
photocleavage, and biopotency of N
and O donor bidentate ligands with
Cu(II), Co(II), and Zn(II)
N. Raman a & A. Selvan a
a Research Department of Chemistry , VHNSN College, Madurai
Kamaraj University , Virudhunagar 626 001, India
Published online: 24 Jan 2011.
To cite this article: N. Raman & A. Selvan (2011) Studies on DNA binding, electrochemical
activation, DNA photocleavage, and biopotency of N and O donor bidentate ligands
with Cu(II), Co(II), and Zn(II), Journal of Coordination Chemistry, 64:3, 534-553, DOI:
10.1080/00958972.2010.551767
To link to this article: http://dx.doi.org/10.1080/00958972.2010.551767
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Journal of Coordination Chemistry
Vol. 64, No. 3, 10 February 2011, 534–553
Studies on DNA binding, electrochemical activation, DNA
photocleavage, and biopotency of N and O donor bidentate
ligands with Cu(II), Co(II), and Zn(II)
N. RAMAN* and A. SELVAN
Research Department of Chemistry, VHNSN College, Madurai Kamaraj University,
Virudhunagar 626 001, India
(Received 15 September 2010; in final form 16 November 2010)
Three Schiff-base ligands, namely 2-(3,4-dimethoxybenzylideneamino)phenol (HL1),
2-(3-hydroxy-4-nitrobenzylidene-amino)phenol
(HL2),
and
2-(3-methoxy-4-hydroxy-
benzylideneamino)phenol (HL3) and their Cu(II), Co(II), and Zn(II) complexes were
synthesized and characterized by spectroanalytical techniques. The UV-Vis and magnetic
moment data revealed square-planar Cu(II) and tetrahedral Co(II) and Zn(II). Conductivity
data showed the non-electrolytic nature of the complexes. Absorption and viscometric titration
as well as cyclic voltammetry studies revealed the complexes as avid binders of calf thymus (CT)
DNA through intercalation. A profound effect of substituents on the Schiff base was observed
on binding of complexes to CT-DNA and in cleavage of supercoiled pBR322 DNA. All the
complexes showed DNA cleavage in the presence of mercaptopropionic acid (MPA) except
HL1 complexes. The cleavage activity increased with longer exposure time of irradiation at
365 nm and higher complex concentration. The cleavage reactions in the presence of MPA
involve hydroxyl radical. The synthesized Schiff bases and their metal complexes were
also tested for antibacterial and antifungal activities. The metal complexes exhibited
higher antibacterial and antifungal activities than the parent ligands and their biopotency
is discussed.
Keywords: Electrochemistry; DNA interaction; DNA photocleavage; Antimicrobial
1. Introduction
Bidentate Schiff bases and their metal complexes have significant importance in
biological processes and constitute an active area of research in modern coordination
chemistry. The high affinity of Schiff bases for chelation to transition metal ions is
utilized in preparing complexes. Compared with research on tridentate, quadridentate,
macrocyclic, and acyclic hydroxy Schiff bases [1], very little attention has been focused
on complexes of bidentate hydroxy Schiff bases [2]. Chelating ligands containing
nitrogen and oxygen donors show broad biological activities and are of special interest
because of their coordination to metals at active sites of metallobiomolecules [3].
Copper participates in transmutation and collagenation whereas other metals
*Corresponding author. Email: drn_ raman@yahoo.co.in
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ꢀ 2011 Taylor & Francis
DOI: 10.1080/00958972.2010.551767

DNA binding and photocleavage
535
participate in natural biological systems [4]. Metal ions bonded to biologically active
compounds may enhance their antibacterial, antifungal, and anticancer activities [5].
Design of molecules capable of targeting specific DNA sites offers possibilities in
developing tools for both pharmaceuticals and biotechnology [6]. Metal complex can
bind to DNA non-covalently, with electrostatic effect, groove binding, and intercala-
tion. Applications of metal complexes require that complexes bind to DNA through
intercalation inducing cellular degradation [7]. The intercalating ability not only
correlates with the planarity of ligand [8], but also relates to coordination geometry of
the metal ion and donor type of the ligand [9]. Metal ion type and valency also play
important roles in deciding the binding extent of complexes to DNA [10]. Copper
complexes are widely used as DNA foot printing, DNA structural probes, potential
anticancer drugs, etc. Changes in intensities of electronic spectra can be used to explain
the nature and strength of stacking interactions between chromophores and DNA base
pairs. There has been considerable interest in DNA photocleavage reactions activated
by metal ions [11].
Herein, we investigate the ligation of Schiff bases derived from 2-aminophenol and
disubstituted benzaldehydes and their complexes of Cu(II), Co(II), and Zn(II). Based
on the literature [12, 13], we have synthesized disubstituted HL1 and HL3 and designed
and synthesized the Schiff base HL2 to discuss the effect of substituents on biological
studies. This article focuses on exploring the DNA-binding affinities of the metal
complexes using electronic absorption, cyclic voltammetry (CV), and viscosity
measurements. Ability to induce cleavage of pBR322 DNA and understand recognition
of DNA by small ligands or metal complexes is crucial for development of drugs
targeted at DNA. Our results on further understanding of DNA binding, efficiency of
DNA recognition, and cleavage by metal(II) complexes lay the foundation for design
of new photoprobes and photonucleases of DNA.
2. Experimental
2.1. General measurements and materials
All reagents were purchased from the commercial sources as reagent grade. Calf thymus
(CT) DNA was obtained from the Sigma Company. All solvents were purified by
regular methods. Tris-HCl buffer (5 mmol Lꢀ1 Tris-HCl, 50 mmol Lꢀ1 NaCl, pH, 7.2,
Tris ¼ Tris(hydroxymethyl)aminomethane) solution was prepared using deionized
double-distilled water. UV-Vis spectra were recorded on a Shimadzu Model 1601
UV-Visible Spectrophotometer. CV measurements were performed at room tempera-
ture on a CHI 620C electrochemical analyzer in freshly distilled DMF. The X-band
EPR spectra of the complexes were recorded on a Jeol RE2X electron spin resonance
spectrometer at RT (300 K) and LNT (77 K) using TCNE as the g-marker.
Thermogravꢀim1 etric analysis (TGA) was carried out in dynamic air at a heating rate
of 10ꢁC min using a Mettler Toledo System. X-ray diffraction (XRD) experiments
were carried out on XPERT-PRO diffractometer system. Cu-Kꢀ1 line, with a
˚
wavelength of 1.5406 A generated with a setting of 30 mA and 40 kV with the
electrodes, was used for diffraction. The slit width setting was 91 mm. The diffracting
angle (2ꢁ) was scanned from 10.0251 to 79.9251 continuously with a rate of 2ꢁ per

536
N. Raman and A. Selvan
minute at 25ꢁC. Scanning Electron Micrography performed on SEM, JEOL JSM
Model 6360, was used for morphological eva1uation. FTIR spectra were obtained on a
Perkin-Elmer Paragon 1000 FTIR spectrophotometer equipped with KBr pellets using
the diffuse reflectance technique (4000–400 cmꢀ1). Microanalyses were performed on
1
a Perkin-Elmer 240 elemental analyzer. H-NMR spectra were recorded on a Bruker
(300 MHz) spectrometer. Mass spectrometry experiments were performed on a JEOL-
AccuTOF JMS-T100LC mass spectrometer equipped with a custom-made electrospray
interface. Room temperature magnetic susceptibility measurements were carried out on
a modified Gouy-type magnetic balance, Hertz SG8-5HJ. The molar conductivity was
measured for (10ꢀ3 mol Lꢀ1) DMF solutions using a conductometer model 601/602.
2.2. DNA binding and cleavage experiments
DNA binding and cleavage experiments were performed at room temperature. The
absorption titrations of Cu(II) and Co(II) complexes in Tris-HCl buffer (5 mmol Lꢀ1
Tris-HCl, 50 mmol Lꢀ1 NaCl, pH, 7.2) were performed by a fixed complex concentra-
tion to which increments of the DNA stock solution were added. Complex solutions
employed were 25 mmol Lꢀ1 in concentration and CT-DNA was added at a ratio of 2 : 1
[DNA] : [complex]. Complex-DNA solutions were allowed to incubate for 10 min before
absorption spectra were recorded. The intrinsic binding constant ratio Kb of Cu(II) and
Co(II) complexes to DNA was calculated using the following equation:
½DNAꢂ=ð"a ꢀ "fÞ ¼ ½DNAꢂ=ð"b ꢀ "fÞ þ 1=½Kbð"b ꢀ "fÞꢂ,
where [DNA] is the concentration of DNA in base pairs, "a, "f, and "b are the apparent,
free, and bound metal complexes extinction coefficients, respectively. In plots of
[DNA]/("a ꢀ "f) versus [DNA], Kb is given by the ratio of slope to the intercept.
CV was carried out at different scan rates in DMF to determine the binding affinity
between DNA and complexes using a three-electrode configuration (glassy carbon
working electrode, Pt counter electrode, and Ag/AgCl reference electrode). All the
samples were purged with nitrogen prior to measurements.
Viscosity measurements were carried out using an Ubbelodhe viscometer maintained
at a constant temperature of 25ꢁC in a thermostated water bath. Flow time was
measured with a digital stopwatch with each sample measured three times and an
average flow time calculated. Data are presented as (ꢂ/ꢂo)1/3 versus binding ratio, where
ꢂ is the viscosity of CT-DNA in the presence of complex and ꢂo is the viscosity of
CT-DNA alone.
For the gel electrophoresis experiments, supercoiled (SC) circular pBR322 DNA
(0.5 mg) was treated with complexes in 50 mmol Lꢀ1 Tris-HCl buffer (pH 7.2) containing
50 mmol Lꢀ1 NaCl in the presence and absence of reducing agent in the dark, then
irradiated at room temperature with a UV-light (365 nm) for 45 min.
Inhibition reactions for the ‘‘chemical nuclease’’ reactions were carried out in the
dark in the presence of reagents like distamycin (100 mmol Lꢀ1) and DMSO (4 mL) prior
to addition of complexes and the reducing agent 3-mercaptopropionic acid (MPA).
Inhibition reactions for the photo-induced DNA cleavage study were carried out at
365 nm using reagents, namely NaN3 (5 mL), ethanol (4 mL), KI (4 mL), DMSO (4 mL),
and histidine (50 mmol Lꢀ1) prior to the addition of the complexes. For the D2O
experiment, this solvent was used for dilution of the sample to 10 mL. The samples after

DNA binding and photocleavage
537
incubation for 1 h at 37ꢁC in a dark chamber were added to the loading buffer
containing 25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol (3 mL) and the
samples were analyzed by electrophoresis for 30 min at 50 V in 50 mmol Lꢀ1 Tris-HCl
buffer (pH 7.2) containing 0.8% agarose gel. The gel was stained with 1 mg (molꢀ1
)
ethidium bromide (EB) and then photographed under UV light.
2.3. Microbial assay
The Schiff-base metal(II) complexes were tested for their effect on certain pathogenic
bacteria and fungi by the disc diffusion method [14]. The complexes were stored dry at
room temperature and dissolved in DMF (1%). Both Gram-positive (Staphylococcus
aureus, Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa)
bacteria were grown in nutrient agar medium and incubated at 37ꢁC for 48 h followed
by frequent subculture to fresh medium and were used as test bacteria. The Aspergillus
niger, Rhizopus stolonifer, Candida albicans, and Rhizoctonia bataticola, used as test
fungi, were grown into dextrose agar medium and incubated at 27ꢁC for 72 h followed
by periodic subculturing to fresh medium. Then the Petri discs were inoculated with a
loop full of bacterial or fungal culture and spread throughout the Petri discs uniformly
with a sterile glass spreader. To each disc, the test samples (10 mg mLꢀ1) and reference
streptomycin (5 mg discꢀ1 for bacteria) or nystatin (10 mg discꢀ1 for fungi) were added
with a sterile micropipette. The plates were then incubated at 35 ꢃ 2ꢁC and 27 ꢃ 1ꢁC for
bacteria and fungi, respectively, and growth of microorganisms was observed. When
no growth of microorganisms was observed in the medium containing the lowest
concentration of test materials, the minimum inhibitory concentration (MIC) of the test
material was defined at this point of dilution. The MIC was measured to be the lowest
concentration after the period of incubation.
2.4. Synthesis and characterization of ligands
The Schiff-base ligands 2-(3,4-dimethoxybenzylideneamino)phenol (HL1) and 2-(3-
methoxy-4-hydroxy-benzylideneamino)phenol (HL3) were synthesized according to
literature procedures [12, 13] whereas another Schiff-base ligand 2-(3-hydroxy-
4-nitrobenzylidene-amino)phenol (HL2) was prepared by condensation of o-hydro-
xyaniline (1.09 g, 0.01 mol) with 3-hydroxy-4-nitrobenzaldehyde (1.67 g, 0.01 mol)
refluxed in ethanol for 3 h, then cooled to room temperature to give a precipitate
which was collected and then washed with ethanol.
HL1: light brown powder, yield 75%, molecular formula: C15H15NO3, molecular
weight: 257.28, m.p.: 98ꢁC, Anal. Calcd C(70.02%), H(5.87%), N(5.44%); found:
C(69.85%), H(5.75%), N(5.42%); 1H-NMR (300 MHz, DMSO-d6) ꢃ: 3.97(S, 3H,
O–CH3), 9.86(s, 1H, OH), 6.78–7.18(m, 4H, Ph), 7.26–7.58(m, 3H, Ph), 8.60(s, 1H,
N¼CH) IR(KBr) ꢄ (cmꢀ1): 1620(s) (HC¼N), 1374(m) (O–CH3), 1328(m) (C–O),
2845(m) (O–H ꢄ ꢄ ꢄ N), 1416(s) (Ph–C¼C), 1515(m) (Ph–C–C), 2936(s) (Ph–C–H) UV-
Vis (DMF) [nm (frequency, cmꢀ1) (transition)]; 308(32,467) (intra ligand charge
transfer) (ILCT), 275(36,363) (ILCT), 268(37,313) (ILCT), 254(39,370) (ILCT).

538
N. Raman and A. Selvan
HL2: yellow powder, yield 82%, molecular formula: C13H10N2O4, molecular weight:
258.13, m.p.: 182ꢁC, Anal. Calcd C(60.46%), H(3.90%), N(10.84%); found:
1
C(60.38%), H(3.91%), N(10.83%); H-NMR (300 MHz, DMSO-d6) ꢃ: 10.57(s, 1H,
OH), 6.73–7.02(m, 4H, Ph), 7.21–7.62(m, 3H, Ph), 8.55(s, 1H, N¼CH) IR(KBr) ꢄ
(cmꢀ1): 1625(s) (HC¼N), 1620(m) (C–NO2), 3381(b) (free OH), 1321(m) (C–O),
2852(w) (O–H ꢄ ꢄ ꢄ N), 1431(m) (Ph–C¼C), 1531(m) (Ph–C–C), 2925(m) (Ph–C–H)
UV-Vis (DMF) [nm (frequency, cmꢀ1)(transition)]; 350(28,571) (ILCT), 265(37,735)
(ILCT), 248(40,323) (ILCT).
HL3: brown powder, yield 78%, molecular formula: C14H13NO3, molecular weight:
243.26, m.p.: 88ꢁC, Anal. Calcd C(69.12%), H(5.38%), N(5.75%); found C(69.13%),
H(5.33%), N(5.73%); 1H-NMR (300 MHz, DMSO-d6) ꢃ: 3.96(s, 3H, O–CH3), 10.13(s,
1H, OH), 6.75–7.12(m, 4H, Ph), 7.25–7.55(m, 3H, Ph), 8.48(s, 1H, N¼CH), IR(KBr) ꢄ
(cmꢀ1): 1615(s) (HC¼N), 1331(s) (O–CH3), 3444(b) (free OH), 1332(m) (C–O), 2849(w)
(O–H ꢄ ꢄ ꢄ N), 1416(m) (Ph–C¼C), 1515(m) (Ph–C–C), 2940(s) (Ph–C–H) UV-Vis
(DMF) [nm (frequency, cmꢀ1)(transition)]; 361(27,701) (ILCT), 265(37,735) (ILCT),
251(39,840) (ILCT).
2.5. Synthesis and characterization of metal complexes
The synthetic route to the complexes is summarized in scheme 1. All the complexes were
synthesized by mixing ethanolic solution of ligands and metal(II) salts in required molar
ratio (1 : 2). The reaction mixture was refluxed on a water bath for 2–3 h and then
concentrated to a small volume on a hot plate at ꢅ50ꢁC. After cooling, the solid
complexes were filtered, recrystallized, washed in ethanol, and dried in vacuum
over CaCl2.
Scheme 1. Synthetic route for preparation of the complexes, where M ¼ Cu(II), Co(II), and Zn(II).

DNA binding and photocleavage
539
1a
CuL2 (1): black, yield 65%, molecular formula: CuC30H28N2O6, molecular weight:
576.10, m.p.: 194ꢁC, Anal. Calcd C(62.54%), H(4.89%), N(4.86%), Cu(11.03%);
found: C(62.51%), H(4.91%), N(4.85%), Cu(11.01%); IR(KBr) ꢅ (cmꢀ1): 1584(s)
(HC¼N), 1370(w) (O–CH3), 1317(m) (C–O), 421(m) (M–N), 557(m) (M–O), 1421(s)
(Ph–C¼C), 1496(m) (Ph–C–C), 2963(s) (Ph–C–H) UV-Vis (DMF) [nm (frequency,
2
cmꢀ1)(transition)(geometry)]; 503(19,880) (2B1g ! A1g)(square-planar; SP), 417
2
(23,980) (2B1g ! E1g) (SP), 268(37,313) (ILCT) magnetic moment, ꢆeff (BM); 1.68.
2b
CuL2 (2): black, yield 73%, molecular formula: CuC26H18N4O8, molecular weight:
577.99, m.p.: 127ꢁC, Anal. Calcd C(54.02%), H(3.14%), N(9.71%), Cu(10.99%);
found: C(53.97%), H(3.12%), N(9.68%), Cu(10.96%); IR(KBr) ꢅ (cmꢀ1): 1585(s)
(HC¼N), 1620(m) (C–NO2), 3375(w) (free OH), 1312(m) (C–O), 417(m) (M–N), 538(s)
(M–O), 1472(m) (Ph–C¼C), 1534(m) (Ph–C–C), 2924(m) (Ph–C–H) UV-Vis (DMF)
2
[nm (frequency, cmꢀ1)(transition)(geometry)]; 508(19,685) (2B1g ! A1g)(SP),
2
435(22,988) (2B1g ! E1g) (SP), 350(28571) (ILCT), 265(37,735) (ILCT) magnetic
moment, ꢆeff (BM); 1.71.
3c
CuL2 (3): brown, yield 68%, molecular formula: CuC28H24N2O6, molecular weight:
548.05, m.p.: 4250ꢁC, Anal. Calcd C(61.36%), H(4.41%), N(5.11%), Cu(11.59%);
found: C(61.29%), H(4.37%), N(4.95%), Cu(10.98%); IR(KBr) ꢅ (cmꢀ1): 1585(m)
(HC¼N), 1328(m) (O–CH3), 3401–3443(b) (free OH), 1321(m) (C–O), 415(m) (M–N),
570(s) (M–O), 1414(m) (Ph–C¼C), 1518(m) (Ph–C–C), 2933(w) (Ph–C–H) UV-Vis
2
(DMF)[nm (frequency, cmꢀ1)(transition)(geometry)]; 535(18,691) (2B1g ! A1g)(SP),
2
416(24,038) (2B1g ! E1g) (SP), 265(37,735) (ILCT), 229(43,668) (ILCT) magnetic
moment, ꢆeff (BM); 1.65.
1a
CoL2 (4): light brown, yield 66%, molecular formula: CoC30H28N2O6, molecular
weight: 571.49, m.p.: 188ꢁC, Anal. Calcd C(63.05%), H(4.93%), N(4.90%), and
Co(10.31%); found: C(62.96%), H(4.91%), N(4.83%), and Co(10.28%); IR(KBr) ꢅ
(cmꢀ1): 1587 (s) (HC¼N), 1363(w) (O–CH3), 1315(m) (C–O), 428(m) (M–N), 565(s)
(M–O), 1428(m) (Ph–C¼C), 1485(m) (Ph–C–C), 2972(s) (Ph–C–H) UV-Vis (DMF) [nm
4
(frequency, cmꢀ1)(transition)(geometry)]; 581(17,211) [4T1(F) ! A2(F)] (tetrahedral),
4
435(22,988) [4T1(F) ! T1(P)] (tetrahedral), 308(32,467) (ILCT), 275(36,363) (ILCT)
magnetic moment, ꢆeff (BM); 4.28.
2b
CoL2 (5): brown, yield 78%, molecular formula: CoC26H18N4O8, molecular weight:
573.38, m.p.: 153ꢁC, Anal. Calcd C(54.46%), H(3.16%), N(9.77%), Co(10.27%);
found: C(54.38%), H(3.12%), N(9.76%), Co(10.26%); IR(KBr) ꢅ (cmꢀ1): 1591(s)
(HC¼N), 1612(m) (C–NO2), 3402(w) (free OH), 1314(m) (C–O), 415(m) (M–N), 525(s)
(M–O), 1468(s) (Ph–C¼C), 1529(m) (Ph–C–C), 2930(m) (Ph–C–H) UV-Vis (DMF) [nm
4
(frequency, cmꢀ1)(transition)(geometry)]; 556(17,985) [4T1(F) ! A2(F)] (tetrahedral),
4
436(22,935) [4T1(F) ! T1(P)] (tetrahedral), 264(37,878) (ILCT), 221(45,248) (ILCT)
magnetic moment, ꢆeff (BM); 4.51.
3c
CoL2 (6): light brown, yield 72%, molecular formula: CoC28H24N2O6, molecular
weight: 543.44, m.p.: 4250ꢁC, Anal. Calcd C(61.88%), H(4.45%), N(5.15%),
Co(10.84%); found: C(61.85%), H(34.38%), N(5.10%), Co(10.82%); IR(KBr) ꢅ
(cmꢀ1): 1582(m) (HC¼N), 1329(m) (O–CH3), 3317(m) (free OH), 1319(m) (C–O),
421(m) (M–N), 578(s) (M–O), 1418(m) (Ph–C¼C), 1521(m) (Ph–C–C), 2933(w) (Ph–
C–H) UV-Vis (DMF) [nm (frequency, cmꢀ1)(transition)(geometry)]; 562(17,793)

540
N. Raman and A. Selvan
4
4
[4T1(F) ! A2(F)] (tetrahedral), 435(22,988) [4T1(F) ! T1(P)] (tetrahedral), 265
(37,735) (ILCT), 251(39,840) (ILCT) magnetic moment, ꢆeff (BM); 4.36.
1a
ZnL2 (7): greenish yellow, yield 62%, molecular formula: ZnC30H28N2O6, molecular
weight: 577.94, m.p.: 204ꢁC, Anal. Calcd C(62.34%), H(4.88%), N(4.84%),
Zn(11.31%); found: C(62.31%), H(4.75%), N(4.78%), Zn(11.28%); 1H-NMR
(300 MHz, DMSO-d6) ꢃ: 3.95(s, 3H, O–CH3), 6.69–7.01(m, 4H, Ph), 7.35–7.61(m,
3H, Ph), 8.62(s, 1H, N¼CH), IR(KBr) ꢅ (cmꢀ1): 1593(s) (HC¼N), 1368(w) (O–CH3),
1312(m) (C–O), 427(m) (M–N), 545(m) (M–O), 1428(s) (Ph–C¼C), 1487(m) (Ph–C–C),
2971(s) (Ph–C–H).
2b
ZnL2 (8): brown, yield 65%, molecular formula: ZnC26H18N4O8, molecular weight:
579.83, m.p.: 148ꢁC, Anal. Calcd C(53.85%), H(3.13%), N(9.66%), Zn(11.27%);
1
found: C(53.79%), H(3.12%), N(9.58%), Zn(11.24%); H-NMR (300 MHz, DMSO-
d6) ꢃ: 3.94(s, 3H, O–CH3), 6.86–7.02(m, 4H, Ph), 7.14–7.55(m, 3H, Ph), 8.56(s, 1H,
N¼CH), IR(KBr) ꢅ (cmꢀ1): 1587(m) (HC¼N), 1618(w) (C–NO2), 3381(m) (free OH),
1311(m) (C–O), 427(m) (M–N), 543(s) (M–O), 1482(s) (Ph–C¼C), 1528(s) (Ph–C–C),
2938(m) (Ph–C–H).
3c
ZnL2 (9): light brown, yield 73%, molecular formula: ZnC28H24N2O6, molecular
weight: 549.88, m.p.: 4250ꢁC, Anal. Calcd C(61.16%), H(4.40%), N(5.09%),
Zn(11.89%); found: C(61.08%), H(4.36%), N(5.03%), Zn(11.87%); 1H-NMR
(300 MHz, DMSO-d6) ꢃ: 3.93(s, 3H, O–CH3), 6.64–6.99(m, 4H, Ph), 7.16–7.35(m,
3H, Ph), 8.43(s, 1H, N¼CH), IR(KBr) ꢅ (cmꢀ1): 1585(m) (HC¼N), 1329(m) (O–CH3),
3381(m) (free OH), 1323(m) (C–O), 432(m) (M–N), 581(s) (M–O), 1419(m) (Ph–C¼C),
1527(m) (Ph–C–C), 2938(w) (Ph–C–H).
3. Results and discussion
3.1. 1H-NMR spectra
1H-NMR spectra of HL1, HL2, and HL3 and their zinc complexes were recorded in
DMSO-d6; data along with assignments are provided in section 2. All proton
resonances of the aromatic region were assigned. The OH signal in the spectrum of
HL1 at ꢃ 9.86 disappeared in its complex spectrum (7), indicating that OH is involved
in complexation with Zn(II).
3.2. Mass spectra
Mass spectra provide a vital clue for elucidating the structure of compounds.
The electronic impact mass spectrum of the HL2 showed a molecular ion (Mþ) peak
at m/z ¼ 258 corresponding to [C13H10N2O4]þ, which confirmed the proposed formula.
The different competitive fragmentation pathways of ligand gave peaks at m/z 58, 79,
91, 93, 99, 103, 139, 151, 165, 213, and 257 which are shown in scheme S1
(Supplementary material). The intensities of these peaks give the stability and
abundance of the fragments. Complex 8 showed a molecular ion peak at m/z 579
with isotopic peaks at 578, 580, 581, and 582 m/z, suggesting monomeric complex.

DNA binding and photocleavage
541
The molecular ion peaks shown by other complexes were also in good agreement with
the structural formula suggested by elemental analysis, spectral, and magnetic studies.
3.3. Electronic and magnetic moment studies
The electronic spectra of ligands and complexes were recorded in DMF from 200 to
1100 nm. The ligands showed absorption of the ꢇ ! ꢇ* and n ! ꢇ* transitions of C¼N
chromophore or charge-transfer transition arising from ꢇ electron interactions between
the metal and ligand, which involves either a metal-to-ligand or ligand-to-metal electron
transfer [15, 16].
The n–ꢇ* bands in the complexes show a slight shift due to donation of electrons to
the metal and hence, coordination of azomethine, with a reduction of intensity. Strong
bands at 229–350 nm are observed in spectra of all Cu(II) complexes. For the Cu(II)
2
2
2
2
complexes there are two spin-allowed transitions, B1g ! A1g and B1g ! E1g,
suggesting SP geometry around Cu(II) [17, 18]. Their magnetic moments support a
distorted SP geometry [19].
Two d–d bands were observed in spectra of the cobalt(II) complexes between 581 and
4
4
4
4
435 nm due to T1(F) ! A2(F) and T1(F) ! T1(P) transitions, in agreement with
assignments made earlier for tetrahedral cobalt(II) complexes [20, 21]. Together with
the magnetic moment, this gives adequate support to a tetrahedral geometry for the
cobalt(II) complexes [22]. High intensity bands at 221–308 nm have been assigned to
charge transfer transitions arising from excitation of an electron from the metal to the
unfilled molecular orbitals derived from the ꢇ* level of the ligands [23].
Zinc(II) complexes are diamagnetic as expected; its geometry is probably tetrahedral
with N, O donor Schiff bases. The electronic spectral data and empirical formula of the
zinc(II) complexes are consistent with a tetrahedral geometry [24].
3.4. Molar conductance measurements
The chelates were dissolved in DMF and their molar conductivities of 10ꢀ3 mol Lꢀ1
solutions at 25 ꢃ 2ꢁC were measured. Complexes 1–9 lie in the range 0.74–3.91 ꢀꢀ1
cmꢀ2 molꢀ1 indicating non-electrolytes [25].
3.5. IR spectra and mode of bonding
IR spectral data of HL1, HL2, and HL3 and their complexes are listed in section 2.
The azomethine ꢅ(C¼N) stretch (in free ligand at 1615–1625 cmꢀ1) shifted to lower
frequencies in complexes, indicating participation of the azomethine nitrogen in
coordination [26]. The stretching frequency at 2845–2852 cmꢀ1 for the ligands showed
the presence of O–H ꢄ ꢄ ꢄ N intramolecular hydrogen bonds [27]. This ꢅ(OH) band
(intramolecular H bonding OH) of the free ligands disappeared on complexation,
indicating coordination through deprotonated phenolic oxygen. Participation of the
OH group was further conErmed by clarifying the effect of chelation on the ꢅ(C–O) and
in-plane bending, ꢃ(OH) [28]. New bands in spectra of the complexes at 525–581 cmꢀ1
were assigned to ꢅ(M–O) stretch, while bands at 415–432 cmꢀ1 to the ꢅ(M–N) mode.
Therefore, the above arguments together with the elemental analyses indicate that the

542
N. Raman and A. Selvan
Table 1. The spin Hamiltonian parameters of Cu(II) complexes in DMSO at
300 K and 77 K.
g-tensor
Hyperfine constant ꢇ 10ꢀ4 (cmꢀ1
)
Complex
g
k
g
?
giso
A
A
?
Aiso
k
1
2
3
2.74
2.76
2.79
2.14
2.17
2.15
2.34
2.37
2.36
70
74
79
260
262
265
196
199
203
ligand is monobasic bidentate in coordination via the azomethine N and deprotonated
phenolic O [29].
3.6. EPR spectra
EPR spectra of the Schiff-base complexes were recorded for powder and solution
samples at RT and LNT, and the results are described in tables 1 and 2. The Cu(II)
complexes at room temperature exhibit isotropic signals with g values (table 1)
characteristic for axial symmetry [30]. The trend, g|| 4 g 4 ge (2.0027) showed that the
?
unpaired electron was localized in the dx2 ꢀ y2 orbital of Cu(II) [31]. Exchange coupling
between two Cu(II) ions can be explained by the Hathaway expression G ¼ (g|| ꢀ 2)/
(g ꢀ 2). When G 4 4.0, interaction between metal centers is negligible in solid
?
complexes (table 2). The observed values indicate that odd electron density was less on 1
than the other two chelates. Hence, delocalization of the metal d-electrons is greater
in 1 than the other two copper complexes.
The EPR parameters g||, g , gav, A||, and A and the energies of the d–d transitions
?
?
were used to evaluate the bonding parameters ꢀ2, ꢈ2, and ꢄ2 which may be regarded as
a measure of covalency of the in-plane ꢉ-bonding, in-plane ꢇ-bonding, and out-of-plane
ꢇ-bonding, respectively. The values of ꢀ2 and ꢈ2 were estimated from the following
expression [32]; lower values of ꢀ2 compared to ꢈ2 indicate that the in-plane ꢉ-bonding
is more covalent than the in-plane ꢇ-bonding.
ꢀ2 ¼ ꢀðA =0:036Þ þ ð g ꢀ 2:0023Þ þ ð3=7Þð g ꢀ 2:0023Þ þ 0:04,
jj
jj
?
ꢈ2 ¼ ð g ꢀ 2:0023ÞE= ꢀ 8ꢊꢀ2:
jj
According to Hathaway [33], K|| ꢆ K ꢆ 0.77 for the pure in-plane ꢉ-bonding and
?
K|| 5 K for the in-plane ꢇ-bonding, while for the out-of-plane ꢇ-bonding K|| 4 K . In
?
?
the Cu(II) complexes, K|| 4 K indicates more significant out-of-plane ꢇ-bonding than
?
the in-plane ꢇ-bonding in metal–ligand ꢇ-bonding. K is dimensionless, as a measure of
the contribution of s electrons to the hyperfine interaction and is generally found to
have a value of 0.30. The K values obtained for all the complexes are in agreement with
those estimated by Assour [34] and Abragam and Pryce [35].
3.7. Thermogravimetric studies
HL1 and 2 were subjected to TGA (figure S1, Supplementary material). The TGA
profiles from 30 to 200ꢁC are probably due to the loss of hydration, which may be

DNA binding and photocleavage
543
Table 2. The bonding parameters of Cu(II) complexes in DMSO
solution.
Complex
ꢀ2
ꢈ2
ꢄ2
K||
K
K ꢇ 10ꢀ4 (cmꢀ1
)
G
?
1
2
3
1.03 2.15 1.60 2.21 1.65
1.07 2.10 1.86 2.25 1.99
1.11 2.00 1.50 2.22 1.66
326
340
340
5.36
4.53
5.34
bound to the hydroxyl group of the ligand by hydrogen bonds. Above 220ꢁC, the
complexes decomposed gradually rather than with sharp decomposition, which may be
due to fragmentation and thermal degradation of the organic moiety. As the
temperature increased, the thermal decomposition of HL1 occurred in one step from
225 to 328ꢁC with 30.23% weight loss (calculated: 31.12%).
The thermogram of 2 indicated two stages of decomposition loss of aldehyde and
oxidative degradation of the remainder to CuO. The first stage of decomposition
started at 230ꢁC with 21.79% (calculated: 22.14%) corresponding to loss of aldehyde.
The second stage of decomposition took place at 409–520ꢁC, consistent with oxidative
decomposition of the complex, leading to the formation of metal oxide. No further
change was observed from 520ꢁC to 800ꢁC. Apart from evaluating the thermal stability
of the metal complex, this study also helped to characterize the complex.
3.8. XRD and morphology study
The XRD and morphology studies of the synthesized compounds and their discussions
are given in ‘‘Supplementary material’’.
3.9. DNA unwinding properties
There is continuing interest in DNA endonucleolytic cleavage reactions activated by
metal ions. Cleavage of plasmid DNA can be monitored by agarose gel electrophoresis.
Irradiation of pBR322 plasmid DNA in the presence of the complexes was studied to
determine the efficiency with which it sensitizes DNA cleavage. This can be achieved by
monitoring the transition from the naturally occurring covalently closed circular form
(Form I) to the open circular relaxed form (Form II). This occurs when one of the
strands of the plasmid is nicked and can be determined by gel electrophoresis of the
plasmid. Extended irradiation results in nicks on both strands of the plasmid, which
eventually results in its opening to the linear form (Form III). When circular plasmid
DNA is subjected to gel electrophoresis, relatively fast migration is observed for the SC
form (Form I). Form II migrates slowly and Form III migrates between Form II and
Form I [36].
Chemical nuclease activity of 1–3 in the presence of 3-MPA (500 mmol Lꢀ1) was
investigated using plasmid SC pBR 322 DNA (30 mmol Lꢀ1, 0.2 mg) in 50 mmol Lꢀ1
Tris-HCl buffer/5 mmol Lꢀ1 NaCl (pH 7.2) as shown in figure S2 (Supplementary
material). The extent of DNA cleavage, observed by agarose gel electrophoresis, gives
the order 2 4 3 4 1. Complex 2 completely cleaved SC-DNA into its nicked circular
form in the presence of 500 mmol Lꢀ1 MPA, while 1 and 3 did not show significant

544
N. Raman and A. Selvan
Scheme 2. Mechanistic pathways proposed for chemical nuclease activity by 1, 2, and 3.
chemical nuclease activity under similar conditions. The bulk of HL1 and HL3
containing the methoxy substituent could reduce the cleavage activity by lowering DNA
binding. Control experiments did not show any cleavage of SC-DNA under similar
reaction conditions. The different DNA cleavage of the three complexes may be due
to different binding affinity to DNA [37]. The DNA cleavage in the presence of MPA
probably proceeds through the hydroxyl radical pathway as proposed by Sigman
(scheme 2) [38].
The change in mobility of Form I DNA band with increase in concentration of
compounds is due to unwinding of the SC Form I DNA. The increase in intensity of the
Form II band with increase in concentration of the compounds is due to partial nicking
of Form I DNA to produce Form II DNA [39]. Figure 1 shows gel electrophoresis
separation of pBR322 DNA after incubation with 2 and 3 with irradiation at 365 nm.
No DNA cleavage was observed for controls in which the complex was absent (lane 1).
With increasing concentration of 2 and 3 (figure 1a), the amount of Form I of pBR322
DNA diminished gradually, whereas Form II increased. No obvious DNA SC of Form
I to nicked Form II happened at 20 and 40 mmol Lꢀ1 (for 3) and 20 mmol Lꢀ1 (for 2),
indicating that these complexes unwind duplex DNA in a concentration-dependent
manner with an activity of DNA cleavage in the order 2 4 3. At 80 mmol Lꢀ1, 2 and 3
promote almost complete conversion of DNA from Form I to Form II. The greater
nuclease activity of 2 may be attributed to its redox nature, i.e., the ease of reduction of
Cu(II) to Cu(I). At the same concentration of complex, with increasing irradiation time
Form II also increased (figure 1b). Similar results have been reported in other cases [40].
Under comparable experimental conditions, 2 exhibited more effective DNA cleavage
than 3.
In order to establish the reactive species responsible for photoactivated cleavage of
plasmid DNA, the following experiments were carried out. Photoactivated cleavage of
pBR322 in the presence of 2 is shown in figure 2. For 2, in the presence of different
hydroxyl radical scavengers, such as KI, DMSO, and ethanol, different degrees of
inhibition in the photo-induced cleavage of the plasmid were observed (figure 2),
indicating that hydroxyl radical (OH) is likely the reactive species [41]. Thus
photoreduction of 2 with concomitant hydroxide oxidation is the important step in
DNA cleavage. DNA incubated with singlet oxygen quenchers histidine, D2O, and
sodium azide were also carried out, and no inhibition was observed, confirming that
singlet oxygen radical was not involved in the cleavage.
3.10. Electrochemical behavior of 1, 4, and 7
Electrochemical properties of the complexes are described in ‘‘Supplementary
material’’.

DNA binding and photocleavage
545
Figure 1. (a) Photoactivated cleavage of pBR322 DNA in the presence of 2 and 3 after 60 min irradiation at
365 nm. Lane 1 and lane 6, DNA alone; lanes 2–5 different concentrations of 2: (2) 20; (3) 40; (4) 60; and (5)
80 mmol Lꢀ1; lanes 7–10, in different concentrations of 3: (7) 20; (8) 40; (9) 60; and (10) 80 mmol Lꢀ1. (b)
Photoactivated cleavage of pBR322 DNA in the presence of 2 and 3 (20 mmol Lꢀ1) after irradiation at 365 nm
for 0, 20, 40, 60, and 80 min (lanes 2–6) for 2 and (lanes 7–11) for 3, Lane 1, DNA alone.
Figure 2. Gel electrophoresis diagram showing the photocleavage of SC pBR 322 DNA by 2 (50 mmol Lꢀ1
)
in the presence of various reagents (30 min) in DMF–Tris buffer medium on irradiation with UV light of
365 nm wavelength: Lane 1, DNA control; lane 2, DNA þ DMSO (4 mL); lane 3, DNA þ ethanol (4 mL);
lane 4, DNA þ KI (4 mL); lane 5, DNA þ 2; lane 6, DNA þ DMSO þ 2; lane 7, DNA þ ethanol þ 2;
lane 8, DNA þ KI þ 2; lane 9, DNA þ NaN3 (5 mL) þ 2; lane 10, DNA þ D2O þ 2; lane 11, DNA þ histidine
(50 mmol Lꢀ1) þ 2.
3.11. DNA-binding experiments
Transition metal complexes bind to DNA via both covalent and/or non-covalent
interactions [42]. In covalent binding, a labile ligand of the complexes is replaced by a
nitrogen base of DNA such as guanine N7. Non-covalent DNA interactions include
intercalative, electrostatic, and groove (surface) binding of metal complexes outside of
the DNA helix. DNA can provide three distinctive binding sites for quinolone metal
complexes, groove binding, electrostatic binding to phosphate group, and intercalation
[43]. This behavior is of importance to the biological role of quinolone antibiotics in
the body [44].
3.11.1. Electronic absorption titration. Electronic spectra are the most common way
to investigate interactions of complexes with DNA. For metallo-intercalators, DNA
binding causes hypochromism and red shift (bathochromism) due to intercalation

546
N. Raman and A. Selvan
Figure 3. Absorption spectra of (a) 1 and (b) 4 in the presence (——) and absence (- - -) of DNA in Tris-HCl
buffer upon addition of CT-DNA [complex] ¼ 25 mmol Lꢀ1. Arrow shows the absorbance changing upon the
increase of DNA concentration.
involving a strong stacking interaction between aromatic chromophore and the base
pairs of DNA. Absorption spectra of 1, 2, and 3 in the absence and presence of CT-
DNA (at [Complex] ¼ 25 mmol Lꢀ1) are illustrated in figure 3(a). Upon increasing
concentration of DNA, the complexes exhibited pronounced hypochromism, along
with a modest bathochromic shift. For 1, the hypochromism in the MLCT band at
437.5 nm reached as high as 16.47% with a red shift of 6 nm; for 2, the MLCT band at
441 nm exhibited hypochromism of about 9.78% with a red shift of 6 nm; for 3, the
MLCT band at 438.5 nm exhibited hypochromism of about 15.69% with a red shift of
5.5 nm. Similar hypochromicity was reported for 4 (figure 3b), 5, and 6 in table 4. These
spectral characteristics suggest that the complexes interact with DNA through an
intercalation mode, which could subsequently stabilize the DNA duplex [45]. After
intercalating the base pairs of DNA, the ꢇ* orbital of the intercalated ligand can couple
with the ꢇ orbital of the base pairs, thus decreasing the ꢇ–ꢇ* transition energy and

DNA binding and photocleavage
547
Table 3. Electrochemical behavior of Cu(II), Co(II), and Zn(II) complexes in the presence of CT-DNA.
ipc (A) ꢇ 10ꢀ5
Epc (V)
Free Bound
E1/2 (V)
DEp (V)
Complex Redox couple Free Bound
Free
Bound Free Bound Koxd/Kred
1
2
3
Cu(II)/Cu(I)
Cu(I)/Cu(0)
Cu(II)/Cu(I)
Cu(I)/Cu(0)
Cu(II)/Cu(I)
Cu(I)/Cu(0)
Co(III)/Co(II) 2.65
Co(II)/Co(I) 3.70
Co(III)/Co(II) 2.53
1.50
1.23
2.14
1.39
1.51
0.72
0.89
0.95
2.04
1.18
1.26
0.62
2.33
2.92
1.81
1.45
0.40
1.64
ꢀ0.763 ꢀ0.773
ꢀ0.241 ꢀ0.255
0.480
0.185
0.478 0.566
0.207 0.852
0.589
0.911
0.636
0.370
0.67
1.03
2.01
0.76
0.32
1.36
0.48
0.05
1.94
2.75
1.59
2.64
ꢀ0.781 ꢀ0.799 ꢀ0.477 ꢀ0.481 0.607
ꢀ0.206 ꢀ0.213 ꢀ0.009 ꢀ0.028 0.394
ꢀ0.755 ꢀ0.784 ꢀ0.424 ꢀ0.409 0.662 ꢀ0.749
ꢀ0.088 ꢀ0.096
ꢀ0.657 ꢀ0.676
0.318
0.272
0.327 0.813
0.259 0.360
0.847
0.390
0.585
0.466
1.111
0.455
0.27
4
5
6
7
8
9
ꢀ1.046 ꢀ1.123 ꢀ0.804 ꢀ0.830 0.484
ꢀ0.808 ꢀ0.825 ꢀ0.562 ꢀ0.592 0.491
Zn(II)/Zn(I)
Zn(II)/Zn(I)
Zn(II)/Zn(I)
2.09
0.76
1.99
ꢀ0.541 ꢀ0.567
0.018
0.344
0.023 1.046
0.363 0.441
0.124
0.136
ꢀ0.963 ꢀ0.938 ꢀ0.748 ꢀ0.803 0.43
Table 4. Absorption spectroscopic properties of Cu(II) and Co(II) complexes
on binding to CT-DNA.
ꢊmax (nm)a
Bathochromism
Dꢊ (nm)b
Hypochromicity
(%)c
Kb ꢇ 104
Complex
Free
Bound
(mol Lꢀ1
)
1
2
3
4
5
6
431.5
435
433
433
434
432
437.5
441
438.5
437.5
440
6.0
6.0
5.5
4.5
6.0
6.5
16.47
9.78
15.69
17.05
8.05
2.56
3.83
3.58
2.53
3.92
3.68
438.5
16.44
aIn DMF.
bAbsorption maximum red shift in the presence of CT-DNA in Tris-HCl buffer, pH – 7.2.
cAbsorbance decrease extent at absorption maximum in the presence of CT-DNA in
Tris-HCl buffer, pH – 7.2.
resulting in the bathochromism [46, 47]. On the other hand, the coupling ꢇ orbital is
partially Elled by electrons, thus decreasing the transition probabilities and concom-
itantly resulting in hypochromism [48].
The intrinsic binding constant Kb of complexes (table 4) can be obtained from the
decay of the absorbance in the MLCT band with increasing concentration of CT-DNA.
In general, a planar extension of the intercalative ligand would increase the strength of
the interaction of the complexes with DNA [49]. The binding constants indicate that 2
and 5 bind more strongly than 1, 3, 4, and 6, as expected, since HL2 possesses greater
planar area and extended ꢇ system than HL1 and HL3, allowing HL2 to penetrate more
deeply into, and stack more strongly with, the base pairs of DNA. The difference could
also be attributed to the different steric effect induced by the substituent groups in the
ligand at 3 and 4 positions.
3.11.2. Electrochemical studies. CV is a sensitive technique to determine changes
in redox behavior of metallic species in the presence of biologically important
molecules [50]. The redox behavior of metallic species is sensitive to the coordination

548
N. Raman and A. Selvan
surrounding the metal center; therefore, metal-based interaction can be detected using
this technique.
The quasi-reversible redox couple for each complex in 1 : 2 (DMF : buffer solution)
has been studied upon addition of CT-DNA; the shifts of the cathodic Epc potentials
are given in table 3.
No new redox peaks appeared after addition of CT-DNA to each complex, but the
current intensity of all the peaks decreased significantly, suggesting the existence of an
interaction between each complex and CT-DNA. The decrease in current intensity and
the ipa/ipc values can be explained in terms of an equilibrium mixture of free and DNA-
bound complex to the electrode surface [51, 52].
The electrochemical behaviors of the Cu(II) complexes in DMF (table 3) show two
redox couples corresponding to Cu(II)/Cu(I) and Cu(I)/Cu(0) in the absence of CT-
DNA. For increasing amounts of CT-DNA, the cathodic potential Epc showed a
negative shift, DEpc ¼ ꢀ10 mV [Cu(II)/Cu(I)] and DEpc ¼ ꢀ14 mV [Cu(I)/Cu(0)] for 1
(figure S3a, Supplementary material), DEpc ¼ ꢀ19 mV [Cu(II)/Cu(I)] and DEpc ¼ ꢀ7 mV
[Cu(I)/Cu(0)] for 2 (figure S3b, Supplementary material), DEpc ¼ ꢀ29 mV [Cu(II)/Cu(I)]
and DEpc ¼ ꢀ8 mV [Cu(I)/Cu(0)] for 3 (figure S3c, Supplementary material), on binding
to DNA suggesting that both Cu(II) and Cu(I) forms bind to DNA at different rates.
These two peaks showed their reduction behavior in 1, 2, and 3. The first one electron
reduction occurs at Cu(II) coordinated to the unsaturated nitrogens and the second at
Cu(II) coordinated to saturated oxygen. In the anodic region, two peaks correspond to
oxidative behavior of Cu(I)/Cu(II) and Cu(0)/Cu(I) (figure S3(a–c), Supplementary
material). These shifts of potentials show that 1, 2, and 3 bind to DNA by intercalation.
The shift in E1/2 with increasing amounts of DNA indicates a difference in the
binding of Cu(II) and Cu(I) to DNA. The net shift in E1/2 can be used to estimate the
ratio of equilibrium constants K2þ/K for binding of Cu(II) and Cu(I), respectively, to
þ
DNA. This is consistent with the unequal values of ipc/ipa which is less than one on
increasing the concentration of DNA.
CV has been employed to study the interaction of the redox active Co(II) complexes
(4, 5, and 6) with DNA. Summaries of voltammetric results of three complexes are given
in table 3. The CV data of 4 and 6 in the absence of DNA featured reduction of þ3 to
the þ2 form at a cathodic peak potential Epc of ꢀ0.657 V for 4 and ꢀ0.808 V for 6
versus Ag/AgCl; the re-oxidation of the þ2 form upon scan reversal occurred at 0.113
and ꢀ0.317 V, respectively. In the absence of DNA, the separation of the anodic and
cathodic peak potentials, DEp ¼ 0.360 V for 4 and 0.491 V for 6, indicate a quasi-
reversible one-electron redox process. The formal potential E1/2, taken as the average
of Epc and Epa, are 0.272 and ꢀ0.562 V, respectively. Complex 4 showed one more
oxidation wave with Epa ¼ 0.089 V in the absence of DNA which can be assigned to the
oxidation of þ1 form as shown in figure S3d (Supplementary material). The presence of
DNA in the solution at the same concentration of these two complexes caused a
considerable decrease in the voltammetric current. In addition, the peak potentials, Epc
and Epa, as well as E1/2 shift to less negative potential. The drop of the voltammetric
currents in the presence of CT-DNA can be attributed to diffusion of the metal complex
bound to the large, slowly diffusing DNA.
CV of 5 exhibited one redox couple corresponding to Co(II)/Co(I) at Epc ¼ ꢀ1.046 V
and Epa ¼ ꢀ0.562 V with DEp ¼ 0.484 V. In the presence of CT-DNA, at the same
concentration of complex, the peak potentials (Epc ¼ ꢀ1.123 V and Epa ¼ ꢀ0.538 V)
were shifted to less negative potential and the redox couple was quasi-reversible with

DNA binding and photocleavage
549
DEp ¼ 0.585 V. Thus, the apparent E1/2 shifted to more negative potentials by
26 mV in the presence of DNA. In the absence of DNA, the CV of 5 did not show a
reduction of þ3 to þ2 in cathodic peak potential. The change in the substituent in the
ligands may affect the oxidation potential of the metal ion, indicating that small
changes in the electronic and structural coordination environment affect the redox
potential of the complexes. The decrease in the peak currents for these complexes
upon addition of CT-DNA indicates that DNA-binding affinity increases in the
order 5 5 4 5 6.
The ligand has a signiEcant effect on E1/2 for all the complexes; electron-withdrawing
groups stabilize cobalt(II) while electron-donating groups favor oxidation to
cobalt(III). Electropotentials of Co(II)/Co(III) couple show sensitivity to the nature
of ligand.
In the absence of DNA, CVs of 7, 8, and 9 show quasi-reversible Zn(II)/Zn(I)
oxidation waves with Epa/Epc at ꢀ0.541/0.505, 0.124/0.565, and ꢀ0.963/ꢀ0.533 V,
respectively, and quasi-reversible Zn(II)/Zn(I) reductive waves at E1/2 ¼ 0.018 V (7),
0.344 V (8), and ꢀ0.748 V (9). Since all the complexes have similar coordination
geometry, the observed trend can be interpreted by the electron-donating effect of the
methoxy. For 7 (figure S3e, Supplementary material), the KZn(II)/KZn(I) value was
calculated to be 2.75, and for 9, 2.64, suggesting that both 7 and 9 complexes of
Zn(I) and Zn(II) interact with DNA almost to the same extent. The complexes 7, 8,
and 9 interact with DNA through intercalation mode with the binding ability to
DNA in the order 7 4 9 4 8. Upon addition of CT-DNA, the voltammetric currents of
7, 8, and 9 decrease from slow diffusion of the complexes bound to the large
DNA molecule.
Differential pulse voltammogram (DPV) experiments were also performed to observe
the changes in the formal potential as well as the current density during the addition of
DNA to solution due to the intercalative interaction of 7, 8, and 9. The reduction
process was monitored at a scan rate of 0.1 Vsꢀ1 (figure S4a and figure S4b,
Supplementary material). DPV also certifies the changes found in CV experiments and
hence zinc complexes with 3,4-disubstituted ligands bind to DNA by intercalation,
with effective insertion of the HL1 and HL3 between the base pairs of the DNA
duplex strand.
3.12. Viscosity measurements
Viscosity measurements are sensitive to length change of DNA and regarded as the least
ambiguous and the most critical tests of binding mode in solution in the absence of
crystallographic structural data. Under appropriate conditions, intercalation of drugs
like EB causes a significant increase in the viscosity of DNA solution due to the increase
in separation of base pairs of intercalation sites increasing DNA length. Molecules
binding exclusively in the DNA grooves cause less pronounced or no change in DNA
solution viscosity [53]. The effect of 1–9 and EB on the viscosity of rod-like DNA is
shown in figure 4. Our results reveal a large increase in the relative viscosity of CT-
DNA, indicating that interaction involves intercalation, with the compound binding
between two adjacent base pairs [54]. The increase in viscosity produced is similar to
that caused by EB. For EB, increasing the ratio of EB to DNA increases relative specific
viscosities of DNA. On increasing the amounts of 1–9, the relative viscosity of DNA

550
N. Raman and A. Selvan
Figure 4. Effect of increasing amounts of EB (˙) and in the presence of increasing concentrations of copper
complexes of 1 (_), 2 (þ), 3 (ꢈ), 4 (\), 5 (*), 6 (ꢀ), 7 (g), 8 (m) and 9 (˙) on the relative viscosity of CT-DNA
at 30ꢁC. [DNA] ¼ 1.5 mmol Lꢀ1, R ¼ [complex]/[DNA] or [EB]/[DNA].
increased steadily, correlating with DNA-intercalating potential, in the order
EB 4 7 4 1 4 9 4 8 4 4 4 3 4 6 4 2 4 5. These complexes bind to DNA through
intercalation, consistent with absorption spectra titration, and reFect the tendency of
each ligand to intercalate into DNA base pairs.
3.13. Antimicrobial activity
Antimicrobial activity of metal complexes generally depends on chelation ability of
the ligand, the nature of nitrogen donor ligands, the total charge of the complex,
the existence and nature of the metal ion, and the nuclearity of the metal center in the
complex [55].
Results shown in table 5 clearly indicate that inhibition is much larger by metal
complexes than free ligand. Higher activity of Cu(II) complexes is probably due to
effective chelation. This activity is quite comparable to the reference drugs streptomy-
cin/nystatin for bacterial/fungal (52.45 mg mLꢀ1) test, respectively. However, the
reduced activities in some cases can be attributed to the inability of the complexes to
form hydrogen bonds with the cell constituents [56–58].
The results in table 5 show that 1, 2, and 3 had the stronger effect against bacteria
and fungi than the other complexes used. Complex 8 exhibited higher antibacterial
effect against E. coli and 5 against A. niger, R. stolonifer, and C. albicans than 1, 2,
and 3. Activity is concentration and substituent dependent as the growth of
inhibition increases with an increase in concentration of the complexes.

DNA binding and photocleavage
551
Table 5. MIC (in mg mLꢀ1) of ligands and their metal complexes against Gram-positive and Gram-negative
microorganisms.
Antibacterial activity
Antifungal activity
Compound
S. aureus B. subtilis E. coli P. putida A. niger R. stolonifer C. albicans R. bataticola
1a
2b
3c
1
2
3
4
5
6
7
32.18
20.19
31.28
11.02
7.53
10.24
17.54
9.37
9.63
13.81
9.57
10.64
1.98
41.27
36.49
35.22
13.14
6.58
11.12
19.31
11.34
11.02
15.26
6.84
21.46
18.76
20.41
9.87
6.03
11.34
16.75
8.49
8.57
17.54
5.69
14.38
1.04
–
42.25
28.41
35.41
9.21
29.48
29.25
41.53
10.43
9.14
9.58
21.47
5.28
12.10
15.20
5.67
16.05
–
1.82
34.16
33.24
38.41
11.87
7.42
9.46
18.73
7.28
7.23
17.31
8.64
12.47
–
36.49
35.48
36.40
9.21
9.76
9.14
18.46
8.12
9.54
15.42
11.05
14.39
–
28.17
28.02
25.15
9.20
5.24
9.34
17.15
10.51
8.68
17.24
9.18
11.34
–
8.15
12.14
16.41
8.10
11.98
16.48
10.95
13.64
1.16
8
9
13.47
2.32
–
Streptomycin
Nystatin
–
–
2.45
2.18
1.96
Complexes with electron-withdrawing groups are more active than complexes
substituted with electron-donating groups of these complexes.
4. Conclusions
Three Schiff bases and nine complexes have been synthesized and characterized. The
experimental data allow assigning SP geometry for Cu(II) complexes and tetrahedral
for Co(II) and Zn(II) complexes. The X-ray powder patterns show that the synthesized
compounds have orthorhombic systems with formation of amorphous compounds.
Spectral titration and viscosity experiments support that the complexes intercalate into
DNA base pairs. When irradiated at 365 nm, the complexes are efficient photocleavers
of plasmid pBR322 DNA. The hydroxyl radical is likely responsible for cleavage of
plasmid pBR322 DNA. Complex 2 promotes cleavage of plasmid pBR322 DNA from
the SC Form I to the open circular Form II upon irradiation. In cyclic voltammograms
of the complexes, quasi-reversible waves attributed to redox couples, characteristic for
each metal complex, have been recorded at potentials expected. Substituents at different
positions on the intercalative ligand cause some interesting differences in the properties
of the resulting complexes. CV studies show that the complexes bind to CT-DNA
by intercalation. A second finding demonstrates the substituent effect of coordinating
intrinsically active ligands to bioactive metals in growth inhibition against
microorganisms.
Acknowledgments
The authors express their sincere thanks to the College Managing Board, Principal, and
Head of the Department of Chemistry, VHNSN College for providing necessary

552
N. Raman and A. Selvan
research facilities. Instrumental facilities provided by Sophisticated Analytical
Instrument Facility (SAIF), IIT Bombay, and CDRI, Lucknow are gratefully
acknowledged.
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