Investigation of DNA binding mechanism, photoinduced cleavage activity, electrochemical properties and biological functions of mixed ligand copper(II) complexes with benzimidazole derivatives: Synthesis and spectral characterization

Article abstract of DOI:10.3109/14756366.2011.592646

The benzimidazole derivative Schiff bases and their copper(II) (Cu(II)) mixed-polypyridyl complexes (14) have been synthesized and characterized by the spectral and analytical techniques. DNA binding/cleavage studies indicate a stronger binding capability for the complex 4 which is confirmed by the absorbance, viscometric and gel-electrophoresis studies. The photocleavage of plasmid pBR322 DNA reveals that hydroxyl radical (OH^?) and singlet oxygen (¹O2) are likely to be the reactive species. Analysis of the growth activity shows that the antimicrobial effect of these Schiff bases on Gram-negative bacteria is higher than that on Gram-positive. Furthermore, the complexes having nitro group show an increased antimicrobial effect.

Full text of DOI:10.3109/14756366.2011.592646

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(3): 380–389
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.592646
ReseaRch aRtIcle
Investigation of DNA binding mechanism, photoinduced
cleavage activity, electrochemical properties and
biological functions of mixed ligand copper(II) complexes
with benzimidazole derivatives: synthesis and spectral
characterization
Natarajan Raman and Abraham Selvan
Research Department of Chemistry, VHNSN College, Virudhunagar, India
abstract
The benzimidazole derivative Schiff bases and their copper(II) (Cu(II)) mixed-polypyridyl complexes (1–4) have
been synthesized and characterized by the spectral and analytical techniques. DNA binding/cleavage studies
indicate a stronger binding capability for the complex 4 which is confirmed by the absorbance, viscometric and gel-
electrophoresis studies. The photocleavage of plasmid pBR322 DNA reveals that hydroxyl radical (OH•) and singlet
oxygen (1O2) are likely to be the reactive species. Analysis of the growth activity shows that the antimicrobial effect
of these Schiff bases on Gram-negative bacteria is higher than that on Gram-positive. Furthermore, the complexes
having nitro group show an increased antimicrobial effect.
Keywords: Copper(II) complex, polypyridyl ligand, DNA interaction, antimicrobial study
Introduction
as imidazole, thiazole, benzimidazole, benzoxazole and
benzothiazole, have been reported2. It has been also sug-
e studies of Copper(II) (Cu(II)) complexes have been
gested that the azomethine linkage is responsible for the
biological activities of Schiff bases such as, antitumor,
antibacterial, antifungal and herbicidal activities3.
widely explored for the versatility of their coordina-
tion geometries, exquisite colors, technical application
dependent molecular structures, spectroscopic proper-
ties and biochemical significance. Octahedral Cu(II)
complexes of ligands containing mixed donor atoms
have been studied extensively due to their potential
applications as molecular materials1. Fused imidazole
derivatives have occupied a prominent place in medici-
nal chemistry because of their significant properties as
therapeutics in clinical applications. us, benzimida-
zole is being explored in the pharmaceutical industries
and the substituted benzimidazole derivatives have also
been found in the diverse therapeutic applications. In
particular, it has been an important pharmacophore
and privileged structure in medicinal chemistry. Several
platinum complexes with N-heterocyclic ligands, such
Transition metal complexes that cleave DNA under
physiological condition are of current interest in the
development of artificial nucleases. Most studies of
nucleic acid cleavage by the small molecules have
been focused on oxidative cleavage and photocleavage.
However, these oxidative cleavage agents require the
addition of an external agent (light, oxidative and/or
reductive agent) to initiate cleavage. e DNA fragments
generated by the hydrolytic cleavage can be enzymati-
cally ligated and endlabeled. Small metal complexes that
promote the hydrolytic cleavage of DNA could be useful
not only in molecular biology and drug design, but also
in elucidating the precise role of metal ions in enzyme
Address for Correspondence: Research Department of Chemistry, VHNSN College, Virudhunagar-626 001, India. Tel.: +091-092451-65958;
Fax: +091–4562-281338. E-mail: drn_raman@yahoo.co.in
(Received 02 April 2011; revised 17 May 2011; accepted 17 May 2011)
380

Investigation of DNA binding mechanism 381
catalysis. One of the most important DNA-related activ-
ity of the transition metal complexes is that, some of the
complexes show the ability to cleave DNA. Very recently,
Cu(II) complexes have been reported to be active in DNA
strand scission4.
a Shimadzu Model 1601 UV-Visible Spectrophotometer.
Cyclic voltammetric measurements were performed at
room temperature on a CHI620C electrochemical ana-
lyzer in freshly distilled DMF solution. e 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. FTIR spectra were
obtained on a Perkin–Elmer Paragon 1000 FTIR spectro-
photometer equipped with KBr discs, using the diffuse
reflectance technique (4000–400 cm−1). Microanalyses
were performed on a Perkin-Elmer 240 elemental ana-
lyzer. 1H NMR spectra were recorded on Bruker (300 MHz)
spectrometer. Mass spectrometry experiments were per-
formed on a JEOL-AccuTOF JMS-T100LC mass spectrom-
eter equipped with a custom-made electrospray interface
(ESI). X-ray diffraction experiments were carried out on
XPERT-PRO diffractometer system. Copper Kα1 line, with
wavelength of 1.5406 Å generated with a setting of 30 mA
and40 kVwiththeelectrodes, wasusedfordiffraction. e
slit width setting was 91 mm. e diffracting angle (2θ)
was scanned from 10.0251 to 79.9251 continuously with
a rate of 2° per minute. Scanning Electron Micrography
study was performed on SEM; JEOL JSM Model 6360 was
used for morphological evaluation. Room temperature
magnetic susceptibility measurements were carried out
on a modified Gouy-type magnetic balance, Hertz SG8-
5HJ. e molar conductivity was measured for (10−3 M)
DMF solution using a conductometer model 601/602.
Studies on the interaction of transition metal com-
plexes with DNA continue to attract the attention of
researchers due to their importance in design and devel-
opment of synthetic restriction enzymes, chemothera-
peutic drugs and DNA foot printing agents5. Binding of
small molecules with DNA has been studied extensively
since DNA is the material of inherence and controls the
structure and function of cells. In this respect, copper(II)
complexes have attracted a great deal of attention due to
their strong DNA-binding and potential anticancer6.
e present work stems from our interest to develop
the chemistry of copper complexes of benzimidazole
derivative based ligands. us, the available literature
encouraged us to synthesize and characterize the Cu(II)
metal complexes [containing 1,10-phenanthroline(phen)
and 2,2′-bipyridine(pby)] with newly synthesized Schiff
bases derived from N,N-bis(benzimidazolyl)oxalate
hydrochloride and 4-substituted anilines. e electron
transfer mechanism is investigated by the aid of cyclic
voltammetry. is paper mainly focuses on exploring
the DNA-binding affinities of the above Cu(II) complexes
using electronic absorption, cyclic voltammetry and vis-
cosity measurements. eir ability to induce the cleav-
age of pBR322 DNA in understanding the recognition
of DNA by small ligands or metal complexes is crucial
for the development of drugs targeted at DNA. We hope
the results will be of high value in further understanding
of DNA binding, the efficiency of DNA recognition and
cleavage by Cu(II) complexes as well as laying the foun-
dation for the rational design of new photoprobes and
photonucleases of DNA.
Synthesis and spectral characterization
Preparation of N,N-bis(benzimidazolyl)oxalate
hydrochloride (L1)
e synthesis route for the ligand L1 is shown in Scheme 1.
Benzimidazole (1.1814 g, 0.01 mol) was dissolved in EtOH
(50 mL) and to this solution was added diethyl oxalate
(1.4615 g, 0.01 mol) in a 2:1 molar ratio. e solution was
refluxed for ca. 1 h, and then conc. HCl (6 mL) was added
dropwise with constant stirring. e cream crystalline
product formed was filtered off under vacuum, washed
thoroughly with hexane and dried in vacuo.
experimental protocols
Chemistry
All the chemicals used were of analytical grade. e sol-
vents were used after purification by the standard method
described in the literature7. Benzimidazole, diethylox-
alate,4-nitroaniline,4-methoxyaniline,1,10-phenanthro-
line, 2,2′-bipyridine and Cu(II) chloride salt were used as
supplied for the preparation of complexes. Calf thymus
DNA (CT DNA) was obtained from the Sino-American
Biotechnology Company. Tris-HCl buffer (5 mM Tris-
HCl, 50 mM NaCl, pH-7.2, Tris=Tris(hydroxymethyl)
aminomethane) solution was prepared by using deion-
ized double distilled water. e concentration of CT
DNA was measured by its known extinction coefficient at
Yield 85% M.F: C16H10N4O2, M.Wt: 290.28, Anal. Calcd.
(%) C(66.24 %), H(3.56 %), N(19.31 %); found (%) C(66.19
1
%), H(3.47 %), N(19.28 %); H NMR (300 MHz, CDCl3) δ,
ppm: 6.78–7.56 (ArH), IR(KBr) γ(cm−1): 1699 (m)(C=O),
1394 (s) (C=N,bz), 1344(w) (C–N,bz), 1448(s) (Ph-C=C),
1529(s) (Ph-C-C), 2872(w) (Ph-C-H).
a
b
Preparation of Schiff base ligands L2 and L2
a
b
e Schiff bases L2 and L2 were synthesized by reflux-
ing ethanolic solutions of L1 (2.9028 g, 0.01 mol) with
4-methoxyaniline (2.463 g, 0.02 mol) or 4-nitroaniline
(2.7624 g, 0.02 mol) in 20 mL ethanolic solution in a 1:2
molar ratio with a constant stirring. en the mixture was
refluxed for 3–4 h. A solid mass separated was collected
and washed by ethanol. Crystallization was done with
ethanol and then dried over CaCl2.
260 nm (6,600 M−1 cm−1)8. e absorbance at 260 nm (A260
)
and at 280 nm (A280) for CT DNA was measured to ensure
its purity. e ratio A260/A280 was found to be 1.84, indicat-
ing that CT DNA was satisfactorily free from protein9.
e spectroscopic titration was carried out in buffer
at room temperature. UV-Vis spectra were recorded on
a
L2 : black powder, yield 65%, M.F: C30H24N6O2, M.Wt:
500.55, Anal. Calcd. (%) C(71.83 %), H(4.87 %), N(16.81
© 2012 Informa UK, Ltd.

382 N. Raman and A. Selvan
%); found (%) C(71.97 %), H(4.81 %), N(16.78 %); 1H
NMR (300 MHz, CDCl3) δ, ppm: 3.42(S, 3H, O-CH3), 6.72-
7.21(m, Ph); IR(KBr) γ(cm−1): 1595 (s) (C=N), 1307(s)
(O-CH3), 1446(m) (Ph-C=C), 1521(s) (Ph-C-C), 2868(w)
(Ph-C-H); UV-Vis (DMF) [nm(frequency,cm−1) (transi-
tion)]; 393(25,445) (intra ligand charge transfer) (ILCT),
267(37,453) (ILCT), 243(41,152) (ILCT), 229(43,668)
(ILCT).
[CuL2 (phen)2]Cl2 (4): green powder, yield 75%, M.F:
b
[CuC52H34N12O4]Cl2, M.Wt: 1025.37, Anal. Calcd. (%)
C(60.92 %), H(3.41 %), N(16.43 %); Cu(6.21 %) found (%)
C(60.89 %), H(3.33 %), N(16.39 %), Cu(6.17 %); IR(KBr)
γ(cm−1): 1516(s) (C=N), 1629(m) (C-NO2), 1425(m)
(Ph-C=C), 1485(s) (Ph-C-C), 2868(w) (Ph-C-H), 414(s)
(M-N); UV-Vis (DMF) [nm(frequency,cm−1)(transition)];
967(10,341) (2B1g→2B2g)(Oh), 735(13,605) (2B1g→2A1g) (Oh),
385(25,974) (ILCT), 293 (34,130) (ILCT).
b
L2 :greenishyellowpowder,yield58%,M.F:C28H18N8O4,
M.Wt: 530.50, Anal. Calcd. (%) C(63.41%), H(3.47%),
N(21.17%);found(%)C(63.37%),H(3.40%),N(21.12%);1H
NMR (300 MHz, CDCl3) δ, ppm: 6.68–7.34(m,Ph); IR(KBr)
γ (cm−1): 1599(s) (C=N), 1631(m) (C-NO2), 1471(w)
(Ph-C=C), 1504(m) (Ph-C-C), 2827(m) (Ph-C-H); UV-Vis
(DMF) [nm(frequency,cm−1) (transition)]; 410(24,390)
(ILCT), 243(41,152) (ILCT), 229(43,668) (ILCT).
Synthesis of Schiff base Cu(II) complexes
All the new Cu(II) complexes were prepared by the fol-
lowing general procedure described in Scheme 1. To
an ethanolic solution of the appropriate Schiff base
(0.001 mol), copper chloride salt (0.001 mol) in ethanol
(15 mL) was added and kept stirring for 30 min. To the
above stirring solution, about 0.002 mol of bpy/phen in
the ethanolic solution was added and refluxed for 2–3 h.
e resultant solid product was washed and recrystal-
lized with ethanol.
a
[CuL2 (bpy)2]Cl2 (1): black powder, yield 78%, M.F:
[CuC50H40N10O2]Cl2, M.Wt: 947.38, Anal. Calcd. (%)
C(63.43 %), H(4.34 %), N(14.83 %), Cu(6.72 %); found (%)
C(63.39 %), H(4.23 %), N(14.79 %), Cu(6.68 %); IR(KBr)
γ (cm−1): 1546(s) (C=N), 1311(s) (O-CH3), 1429(w)
(Ph-C=C), 1516(s) (Ph-C-C), 2874(s) (Ph-C-H), 406(s)
(M-N); UV-Vis (DMF) [nm(frequency,cm−1)(transition)];
955(10,471) (2B1g→2B2g) Octahedral (Oh), 743(13,459)
(2B1g→2A1g) (Oh), 531(18,832) (ILCT), 385 (25,974) (ILCT),
303 (33,003) (ILCT).
a
[CuL2 (phen)2]Cl2 (2): black powder, yield 72%,
M.F: [CuC54H40N10O2]Cl2, M.Wt: 995.43, Anal. Calcd. (%)
C(65.15 %), H(4.07 %), N(14.12 %), Cu(6.38 %); found (%)
C(65.12 %), H(4.01 %), N(14.03 %), Cu(6.35 %); IR(KBr)
γ(cm−1): 1558(m) (C=N), 1303(s) (O-CH3), 1425(s)
(Ph-C=C), 1511(s) (Ph-C-C), 2870(w) (Ph-C-H), 408(s)
(M-N); UV-Vis (DMF) [nm(frequency,cm−1)(transition)];
940(10,638) (2B1g→2B2g)(Oh), 738(13,550) (2B1g→2A1g) (Oh),
522(19,157) (ILCT), 462 (21,645) (ILCT), 291 (34,364)
(ILCT).
b
[CuL2 (bpy)2]Cl2 (3): green powder, yield 68%, M.F:
[CuC48H34N12O4]Cl2, M.Wt: 977.32, Anal. Calcd. (%)
C(59.06 %), H(3.56 %), N(17.21 %), Cu(6.54 %); found (%)
C(58.96 %), H(3.48 %), N(17.18 %), Cu(6.48 %); IR(KBr)
γ(cm−1): 1511(w) (C=N), 1620(m) (C-NO2), 1442(s)
(Ph-C=C), 1481(s) (Ph-C-C), 2868(m) (Ph-C-H), 409(s)
(M-N); UV-Vis (DMF) [nm(frequency,cm−1)(transition)];
965(10,363) (2B1g→2B2g)(Oh), 740(13,514) (2B1g→2A1g) (Oh),
387(25,840) (ILCT), 321 (31,153) (ILCT), 281 (35,587)
(ILCT).
Scheme 1. Synthetic route for the preparation of copper
complexes.
Journal of Enzyme Inhibition and Medicinal Chemistry

Investigation of DNA binding mechanism 383
that of the ligands clearly indicates the formation of a
metal complex in each case.
Biological study
e methods of DNA binding, photoactivated cleavage
experiments and pharmacology studies are discussed in
supplementary material.
e electronic spectra of the 1-4 chelates consist of a
band centered at around 967-735 nm, which is consistent
with an octahedral configuration. On the basis of elec-
tronic spectra, the position of the bands and their weak
intensity have assigned them to d-d transitions, 2B1g→2B2g
Results and discussion
2
and B1g→2A1g. is supports the distorted octahedral
All complexes were soluble in organic solvents like DMF
and DMSO. e synthesized copper complexes were
characterized by elemental analyses, IR, UV–Vis, 1H
NMR, mass spectroscopy, magnetic susceptibility mea-
surement, XRD and SEM analysis.
Cu(II) complex which is usual in the d9 case14. ese
complexes have magnetic moment values in the range
1.96-1.84 BM, which is higher than the spin-only value
(1.73 BM) expected for one unpaired electron and offers
possibility of an octahedral geometry observed for Cu(II)
complexes15.
FTIR studies and mode of bonding
e data of the IR spectra of Schiff base ligands L2 , L2
a
b
Molar conductivity
and their copper complexes are listed in experimental
section. e IR spectra of the complexes are compared
with the free ligands in order to determine the coordina-
tion sites that may involved in chelation. L2a and L2b show
a strong band at 1595 and 1599 cm−1 respectively due
to the imine group vibrations. After complexation, this
band is shifted to a lower frequency, in the range 1511–
1558 cm−1, supporting the binding of the imine nitrogen
with the metal ions10. Furthermore, no bands for free
carbonyls are observed, which indicates that complete
condensation has occurred. e presence of sharp band
in the region 406–414 in the spectra of all the complexes
assigned to ν(M-N) mode further supports to the involve-
ment of nitrogen atom in coordination11. e IR values,
ν(C–H) 860 cm−1 and 735 cm−1 observed for phenanthro-
line are red shifted to 850 cm−1 and 721 cm−1. ese shifts
can be explained by the fact that each of the two nitrogen
atoms of phenanthroline ligands donates a pair of elec-
trons to the central metal forming a coordinate covalent
bond12.
With a view to study the electrolytic nature of the mono-
nuclear metal complexes, their molar conductivities
were measured in DMF (dimethylformamide) for 10−3
M solutions at 25 2°C. e molar conductivity values of
the copper complexes (1–4) are in the range of 52.4–78.3
Ω−1 cm2 mol−1 which indicate the ionic nature of these
complexes. ese high values indicate that mononuclear
metal complexes are electrolytes16 due to presence of
counter ions in the proposed structure of the mononu-
clear metal complexes (Scheme 1).
EPR studies
e EPR spectral assignments of the Cu(II) metal com-
plexes are discussed in supplementary material.
ESI-mass studies
e electron impact mass spectra of L2 ligand and its
b
complex 3 were recorded and investigated at 70 eV
of electron energy. e molecular weights of all the
complexes were established from the molecular ion
peaks observed in the corresponding ESI mass spec-
a
erefore, from the IR spectra, it is concluded that L2
and L2b behave as bidentate ligands with NN donor sites
and bind to the metal ions through two azomethine N
and four nitrogens through two pby/phen as co-ligands.
b
tra. e ESI mass spectrum of L2 Schiff base shows a
molecular ion peak m/z at 530 which is equivalent to
its molecular weight and also exhibits two additional
peaks m/z at 531 and 532 corresponding to (M + 1) and
(M + 2) peaks, respectively. e ESI mass spectrum of
3 shows a molecular ion (M+) peak at m/z = 906, and
also exhibits two additional peaks m/z at 907 and 908,
which are corresponding to (M+1) and (M+2) peaks
respectively. e different competitive fragmentation
pathways of the complex 3 gave important peaks at
m/z 594, 531, 237 (M+2), 412 and 410 (M-2) due to the
fragments [CuC28H18N8O4]+., [C28H18N8O4]+., [C14H11N4]+,
[C21H12N6O4]+. and [C21H10N6O4]+., respectively. Most of
the complexes showed additional peaks corresponding
to the fragments formed due to the loss of the metal ion.
So, it is reasonable to conclude from the assignment
of the fragments of the four studied complexes exist
in a monomeric form. e molecular ion peaks shown
by these complexes are in good agreement with the
structure suggested by elemental analysis, spectral and
magnetic studies.
Magnetic susceptibility and UV–Vis studies
Magnetic susceptibility measurements provide sufficient
data to characterize the structure of the metal complexes.
e formation of the metal complexes was also con-
firmed by electronic spectra. e electronic absorption
a
spectra of the Schiff base ligands L2 , L2b and their copper
complexes were recorded in DMF, over the 200–1100 nm
a
b
range. e L2 and L2 ligands showed strong absorp-
tion bands in the ultraviolet region (263–408 nm), that
could be attributed respectively to the π→π* and n→
π* transitions in the benzene ring or azomethine (–C=N)
groups for free ligand. All the copper complexes showed
the intense electronic bands observed in the spectral
range 550–1000 nm are assigned to the d–d band, which
is in agreement with six-coordinate geometry. e strong
band observed in the range 280–550 nm is associated with
the imine transition in all the metal complexes13. us, a
comparison of the absorption data of the complexes with
© 2012 Informa UK, Ltd.

384 N. Raman and A. Selvan
well-characterized intercalation. is difference of DNA
binding model between Cu(II) complexes 1–4 should be
caused by the different nature of substituents present in
Schiff base ligands of these complexes. For complexes 1
and2, duetothebulkymethoxygrouplocatedinL2a ligand
may restrict from planarity, these complexes could not
completely intercalate into DNA base pairs. e partial
intercalation may act as a “wedge” to pry apart one side
X-ray diffraction and SEM analysis
e structure and surface morphology study of the com-
pounds has been discussed in supplementary file.
Biological study results
Viscosity studies
Viscosity titration measurements were carried out to
clarify the interaction modes between the investigated
compounds and CT DNA. e classic intercalation model
involves the insertion of a planar molecule between DNA
base pairs, which results in a decrease in the DNA helical
twist and lengthening of the DNA, the molecule will be in
close proximity to the DNA base pairs as well. In contrast,
groove-face or electrostatic interactions typically cause a
bend (or kink) in DNA helix reducing its effective length
and thereby its viscosity.
of a base pair stack, as observed for the Δ-[Ru(phen)3]2 + 17
,
but not fully separate the stack as required by the classi-
cal intercalation model. is would cause a static bend
or kink in the helix and decreases the viscosity of DNA.
e complexes 3 and 4, involve an intercalation mode.
is is probably related to the molecular structure of the
complex. In complexes 3 and 4, the bpy/phen ligands
b
and nitro group present in L2 ligand moiety, in which
complex 4 is somewhat more planarity and completely
intercalated with DNA. However, in bpy–Cu(II) complex
3, the bpy ligands bind DNA weakly through intercalative
mode. So, it is likely that the observed slight increase in
relative viscosity of these complexes is not due to interca-
lative interaction but due to partial and/or non classical
interaction.
e effects of the Cu(II) complexes 1, 2, 3 and 4,
together with ethidium bromide (EB) on the viscosity of
rod-like DNA at 25 2°C are shown in Figure 1. e inter-
calator ethidium bromide (EB) significantly increases
the relative specific viscosity of DNA as expected for
the lengthening of the DNA double helix resultant from
e changed degree of viscosity which follows the
order of EB > 4 > 3 > 2 > 1 may depend on its affinity to
DNA, which is consistent with electronic absorption
spectroscopy studies.
Chemical nuclease activity
e chemical nuclease activities of complexes 1–4 have
been studied using supercoiled pBR322 plasmid DNA
as a substrate in a medium of 50 mM Tris–HCl/NaCl
buffer (pH = 7.2) in the presence of hydrogen peroxide
as a activator under physiological conditions. All the
four complexes 1–4 showed remarkable cleavage. e
structure of the ligand plays an important role in the
cleavage18. When circular plasmid DNA is subjected to
electrophoresis, relatively fast migration will be observed
for the supercoiled form (Form I). If scission occurs on
one strand (nicking), the supercoiled form will relax to
generate a slowly moving open circular form (Form II).
If both strands are cleaved, a linear form (Form III) that
migrates between form I and form II will be generated.
e nuclease efficiency of the Cu(II) complexes is
known to depend on the activator used for initiating
Figure 1. Effect of increasing amounts of EB (*) and in presence of
increasing concentration of complexes of 1 (◆), 2 (▲), 3 (■), 4 (×)
on the relative viscosity of CT-DNA at 25 2°C. [DNA]=1.5mM,
R=[complex]/[DNA] or [EB]/[DNA].
Figure 2. Agarose gel showing cleavage of SC pBR322 DNA (0.2 μg) incubated with 50 μM complexes 1–4 in the presence of 0.25mM H2O2
in Tris–HCl/NaCl buffer (50mM, pH=7.2) at 37°C for 1.5h. Lane 1: DNA control; lane 2: DNA + H2O2; lane 3: DNA + 1; lane 4: DNA + 4; lane
5: DNA + H2O2 + 1; lane 6: DNA + H2O2 + 2; lane 7: DNA + H2O2 + 3; lane 8: DNA + H2O2 + 4; lane 9: DNA + H2O2 + 4 + DMSO; lane 10: DNA
+ H2O2 + 4 + SOD (4 units).
Journal of Enzyme Inhibition and Medicinal Chemistry

Investigation of DNA binding mechanism 385
the DNA cleavage. Several authors have studied the
influence of different activators on the cleavage of DNA
by Cu(II) complexes19. In our work, we have found that
hydrogen peroxide is the best activator for DNA cleav-
age. e results of the gel electrophoresis separations of
plasmid pBR322 DNA by the complexes 1–4 are depicted
in Figure 2. e ‘‘chemical nuclease’’ activity follows the
order: 4 (phen)2 > 3 (bpy)2 > 2 (phen)2 > 1 (bpy)2. Control
experiments using the metal complexes alone do not
show any apparent cleavage of SC DNA. e bpy complex
1 shows moderate cleavage activity due to its inability to
bind to DNA. Complexes 3 and 4 having a DNA inter-
calator nitro group show good chemical nuclease activ-
ity. Control experiments show that the hydroxyl radical
scavenger DMSO inhibits the DNA cleavage suggesting
the possibility of hydroxyl radical and/or ‘‘copper-oxo’’
intermediate as the reactive species20. SOD addition
does not have any apparent effect on the cleavage activ-
and L-Histidine (Lane 6) partly diminishes the nuclease
activity of the two compounds which is indicative of the
involvement of hydroxyl radical and the singlet oxygen
or a singlet oxygen-like entity in the cleavage process.
e inhibitory activity of sodium azide can be ascribed
to the affinity of the azide anion for transition metals23.
e EDTA, a Cu(II)-specific chelating agent that strongly
bind to Cu(II) forming a stable complex, can efficiently
inhibit DNA cleavage, indicating Cu(II) complexes play
the key role in the cleavage. e Cu(II) complexes exam-
ined here may be capable of promoting DNA cleavage
through an oxidative DNA damage pathway in the pres-
ence of various reagents, in which giving active oxygen
species such as hydroxyl radical and singlet oxygen or
singlet oxygen-like entity, probably a copper-peroxide
that cleaves DNA.
Spectroscopic studies on DNA binding
.
ity indicating the non-involvement of O2 in the cleavage
DNA-binding studies are important for the rational
design and construction of new and more efficient drugs
targeted to DNA. In these studies, all the compounds
were dissolved in 1% DMF and 99% Tris–HCl buffer
(5 mM Tris–HCl, 50 mM NaCl, pH 7.2) at a concentration
of 25 μM. All the experiments involving the interaction
of complexes with CT DNA were carried out in Tris–HCl
buffer.
reaction. e mechanism involved in the DNA cleavage
reactions is believed to be similar to that proposed by
Sigman and coworkers for the ‘‘chemical nuclease’’ activ-
ity of bis(phen)copper species21. e different DNA cleav-
age efficiency of these four complexes may be due to the
different binding affinity of the complexes to DNA.
Photo-induced DNA Cleavage
A number of metal polypyridyl complexes have been
shown to exhibit DNA photocleaving ability22. e cleav-
age of plasmid pBR322 DNA by the Cu(II) complexes
can be easily monitored by agarose gel electrophoresis.
Complexes 3 and 4 have been found to bring about pho-
tocleavage of supercoiled plasmid pBR322 DNA when
excited at 365 nm. After the photoexposure, the sample
was incubated for 1 h at 37°C, followed by its addition to
the loading buffer containing 25% bromophenol blue,
0.25% xylene cyanol and 30% glycerol (3 μL). e solu-
tions were finally loaded on 1.5% agarose gel containing
1.0 μg mL−1 (EB). Electrophoresis was carried out in a dark
room for 1.5 h at 80 V in Trisacetate–EDTA (TAE) buffer.
e bands were visualized by UV light and photographed.
Figure S1(A, B) (supplementary material) shows the gel
electrophoresis separation of pBR 322 DNA after incuba-
tion with complexes (3 and 4) and irradiation at 365 nm.
e mechanistic aspects of the DNA cleavage reac-
tions involving complexes 3 and 4 (50 μM) have been
investigated at 365 nm wavelength using various con-
trol experiments (Figure S1, supplementary material).
We investigated the DNA cleavage in the presence of
hydroxyl radical scavengers (DMSO, EtOH), singlet oxy-
gen quenchers (NaN3, L-Histidine), superoxide scaven-
ger (SOD), hydrogen peroxide scavenger (catalase) and
chelating agent (EDTA) under our experimental condi-
tions. From Figure S1, it is inferred that no obvious inhibi-
tions are observed for 3 and 4 complexes in the presence
of SOD (Lane 7) and catalase (Lane 8), the results rule
out the possibility of DNA cleavage by superoxide. e
addition of DMSO (Lane 3), EtOH (lane 4), NaN3 (Lane 5)
Electronic absorption spectra
e binding of complexes 1–4 to DNA helix has been
characterized through absorption spectral titrations, by
following changes in absorbance and shift in wavelength.
Formetallo-intercalators, DNA-bindingisassociatedwith
hypochromism and a red shift in the ILCT bands. Figure
S2, supplementary material) shows Cu(II) complexes
in the absence and presence of CT DNA. Addition of
increasing amounts of CT DNA results in hypochromism
and a red shift. As increasing the concentration of CT
DNA, the MLCT transition band of complexes 1 at
530 nm, 2 at 510 nm, 3 at 382 nm and 4 at 381 nm exhib-
its hypochromism of 6.28%, 6.59%, 19.68% and 24.64%,
and bathochromism of 3.5, 4, 4.5 and 5 nm, respectively.
ese spectral characteristics obviously suggest that
complexes 1–4 interact with DNA most likely through an
intercalative and/or electrostatic mode that involves a
stacking interaction between the aromatic chromophore
and the base pairs of DNA.
In order to further elucidate the binding strength of the
complexes, the intrinsic constants Kb were determined
by monitoring the changes of absorbance in the MLCT
band with increasing concentration of CT DNA. e Kb
values of copper(II) chelates are 3.22 × 104, 3.71 × 104,
3.77 × 104 and 3.80 × 104 for 1, 2, 3 and 4, respectively, a
little difference in DNA-binding affinity of the four Cu(II)
complexes can be understood as a result of the fact that
the complex with nitro moiety Schiff base ligand shows
stronger binding affinity with DNA. is may be due to
the high numbers of planar phen ligand and nature of
substituent moieties present in the complex24 which
© 2012 Informa UK, Ltd.

386 N. Raman and A. Selvan
cooperatively act to increase the overall binding ability of
the Cu(II) complexes with DNA.
the large, slowly diffusing DNA molecule. e decrease
extents of the peak currents observed for the above two
complexes upon addition of CT DNA may indicate that
the DNA binding affinity increases in the order: 1 < 2. e
results parallel to the above spectroscopic and viscosity
data of these two complexes in the presence of DNA.
In the absence of DNA, the cyclic voltammogram for
complex 3 exhibits two quasi-reversible one-electron
reduction processes. Inspection of this voltammogram
indicates that the first reduction wave at −0.936 V and
its corresponding oxidation wave appeared at −0.393V
are related to the Cu(II)/Cu(I) reduction process in the
range −0.2 to −1.1 V. Second redox peak potentials for
the Cu(III)/Cu(II) couple are found at Epc = −0.358 V and
Epa = 0.069 V. e ratio of anodic to cathodic peak cur-
rents Ipa/Ipc was 0.217 and 0.925 V for first and second
redox couples respectively. e formal electrode poten-
tials E1/2, ΔEp (difference in cathodic Epc and anodic
Epa peak potentials) were −0.664 and 0.543 V for Cu(II)/
Cu(I), 0.144 and 0.427 V for Cu(III)/Cu(II) couple, respec-
tively. At constant parameters, the addition of CT-DNA
results the shift in E1/2 = −0.639 V and ΔEp = 0.520 V for
Cu(II)/Cu(I), E1/2 = 0.131 V and ΔEp = 0.414 V respectively
for Cu(III)/Cu(II) (Fig. S3) (supplementary material).
e ratio of Ipa/Ipc is 0.143 and 0.665 for CT-DNA bound
metal complex 3, exhibits first and second redox couples,
respectively. e shift in potentials and decrease in cur-
rent ratio suggest the binding of complex 3 to CT-DNA.
e cyclic voltammetric data of complex 4 in the
absence of DNA (Figure S3 B, supplementary mate-
rial) featured the reduction of Cu(II) → Cu(I) form at a
cathodic peak potential. Epc of −0.985 V, and the reoxi-
dation of the Cu(I) form upon scan reversal occurred
at −0.116 V and one more reduction peak is appeared
at Epc = −0.425 V with no corresponding anodic peak.
is is possibly because the electron-withdrawing anion
makes the complex more positively charged and it favors
the reduction of metal ion. Similarly the electron-donat-
ing groups make the complexes less positively charged
and it favors oxidation to Cu(III). Electropotentials of
Cu(III)/Cu(II) couple is showing sensitivity to the nature
of atoms in ligands moiety. e higher reduction poten-
tial can be attributed due to the greater planarity and
electronic properties those are associated with aromatic
rings.
Redox behavior
e redox behavior of the newly synthesized compound
a
a
b
[CuL2 (pby)2]Cl2, [CuL2 (phen)2]Cl2, [CuL2 (pby)2]Cl2
b
and [CuL2 (phen)2]Cl2 in TBAP-DMF have been investi-
gated using cyclic voltammetry. Cyclic voltammetry was
used to confirm the oxidation levels of the metal centers
in the Cu(II) complexes. e electrochemical properties
of metal complexes, particularly with nitrogen donor
atoms have been studied in order to consider spectral
and structural changes accompanying with electron
transfer.
e cyclic voltammogram between +0.4 and −0.4 V
shows oxidation process Cu(II)/Cu(III) while in the range
of 0 to −1.0 V, it shows the reduction process which may
be assigned to Cu(II)/Cu(I) couple (Table 1).All of them
resemble of a quasi-reversible one-electron transfer pro-
cess. Cyclic voltammetric technique has been employed
to study the interaction of the redox active Cu(II) com-
plexes with DNA in order to further testify the DNA bind-
ing modes assessed from the above spectral and viscosity
studies. Summaries of voltammetric results of 1–4 com-
plexes are given in Table 1.
In the absence of DNA, the cyclic voltammogram
recorded at room temperature shows a quasi-reversible
peak for the Cu(II) → Cu(III) couple at 0.439 V and
0.396 V versus Ag/AgCl with a direct cathodic peak for
Cu(III) → Cu(II) at −0.145 V and −0.332 V for complexes 1
and 2, respectively. On titration with DNA, a decrease in
peak current with very slight potential shift was observed
for complexes 1 and 2, which is consistent with the non-
coordinating intercalative binding of copper complexes
through phen/bpy ligand moiety between the DNA
base pairs as also evidenced by the spectral results. It is
observed from the voltammogram that the ΔEp values
are not much sensitive to the DNA addition, while there
is a considerable decrease in peak current as well as in
the Ipa/Ipc values. e formal potential, E1/2 taken as the
average of Epc and Epa shifts slightly towards the nega-
tive side on binding to DNA suggest that both Cu(II) and
Cu(I) forms bind to DNA at different rates. e drop of
the voltammetric currents in the presence of CT DNA can
be attributed to diffusion of the metal complex bound to
Table 1. Redox couples of the complexes, their potentials and the shift of the potentials in the absence and presence of CT-DNA.
Ipc (A)×10−5
Epc (V) E1/2 (V) △Ep (V)
Bound
Complex Redox couple
Koxd/Kred
1.65
Free
Bound
2.90
Free
Bound
−0.132
−0.779
−0.314
−0.953
−0.338
−0.899
−0.404
−0.935
Free
Bound
0.125
−0.503
0.329
−0.467
0.131
−0.639
—
Free
0.584
0.591
0.728
0.900
0.427
0.543
—
1
2
3
4
Cu(III)/Cu(II)
Cu(II)/Cu(I)
Cu(III)/Cu(II)
Cu(II)/Cu(I)
Cu(III)/Cu(II)
Cu(II)/Cu(I)
Cu(III)/Cu(II)
Cu(II)/Cu(I)
3.00
3.24
0.428
0.638
1.04
2.46
1.15
2.67
−0.145
−0.796
−0.332
−0.983
−0.358
−0.936
−0.425
−0.985
0.147
−0.500
0.364
−0.450
0.144
−0.664
—
0.514
0.552
0.659
0.934
0.414
0.520
—
3.20
1.94
0.409
0.474
0.96
2.01
3.21
2.18
1.51
4.22
0.78
2.26
1.65
−0.434
−0.390
0.869
0.780
7.01
Electrochemical data recorded in volt, at room temperature, E1/2=1/2(Epc + Epa).
Journal of Enzyme Inhibition and Medicinal Chemistry

Investigation of DNA binding mechanism 387
Upon the addition of Cu(II) complex 4 with DNA, the
geometry around the Cu(II) ion in complex is changed
due to the interaction of DNA. erefore, the redox
potential of Cu(II)/Cu(I) also changes. CV results show
that a new reduction peak appears at higher potential
than the original reduction peak which can be attributed
to changes in the geometry around copper center in the
complex through the binding of Cu(II) compartmental
Schiff base complex 4 to DNA. erefore, our CV results
suggest the interaction between DNA and copper com-
plex 4. According to these observations, it seems that the
decrease in peak currents of complex 4 after addition of
DNA are caused by the binding of complex 4 to the bulky,
slowly diffusing DNA molecule.
eshiftinE1/2 withincreasingamountsofDNAindicates
a difference in the binding of Cu(II) and Cu(I) species to
DNA. e net shift in E1/2 can be used to estimate the ratio of
equilibrium constants, K2+/K+ for the binding of the Cu(II)
and Cu(I) forms, respectively, to DNA. For a Nernstian
electron transfer system in which both the oxidized and
reduced forms are associated with a third species such as
DNA in solution can be applied. Here, Cun+-DNA repre-
sents the copper complex bound to DNA with n+ charge
on the metal centre. us for a one electron transfer,
while the anodic potential Epa shifts to negative
values [ΔEpa = −0.022V for 1, ΔEpa = -0.064V for 2,
ΔEpa = −0.014V for 3 and ΔEpa = −0.039V for 4 (Figure
S3 B, supplementary material)] for Cu(II)/Cu(I) redox
couple respectively. ese shifts of the potentials show
that complexes 1–4 can bind to DNA by both intercala-
tion and electrostatic interaction25.
Pharmacology effect
e results of antimicrobial screening of all the newly
synthesized compounds are presented in Table 2 (Figure
S4, supplementary material). Some of them showed
moderate to good activity with MIC values in the range
of 5.53–9.26 μg/mL in DMF. In general, the two Schiff
bases have a moderate antimicrobial effect on both
antibacterial and antifungal strains, possibly due to the
presence of azomethine groups (imines) which have
chelating properties. ese properties may be used in
metal transport across the bacterial membranes or to
attach to the bacterial cells at a specific site from which
it can interfere with their growth. is is in agreement
with Huheey et al.26 who mentioned that antibiotics such
as streptomycin, aspergillic acid, and tetracycline with
chelating properties were able to compete successfully
with metal-binding agents for bacteria while not disturb-
ing the metal processing by the host and thus interfering
with the growth of bacteria.
E°b −E°f = 0.0591 log(K2+/K+ )
where E°f and E°b are the formal potentials of the Cu(II)/
Cu(I) couple in the free and bound forms, respectively.
K2+ and K+ are the corresponding binding constants for
the Cu(II) and Cu(I) species to DNA. For complex 4, K2+
is higher than K+, suggests that the interaction of Cu(II)
complexes with DNA tends to stabilize the Cu(II) over the
Cu(I) state.
It can be observed that complexes 1-4 exhibit the
same electrochemical behaviour upon addition of CT
DNA. For increasing amounts of CT DNA, the cathodic
potential Epc shows a positive shift (ΔEpc = +0.013 V
for 1, ΔEpc = +0.018 V for 2, ΔEpc = +0.020 V for 3 and
ΔEpc = +0.021 V for 4) while the anodic potential Epa
shifts to more negative values [ΔEpa = −0.057 V for 1
(Figure S3A, supplementary material), ΔEpa = -0.051 V
for 2, ΔEpa = −0.007 V for 3 for Cu(III)/Cu(II) redox
couple], the cathodic potential Epc shows a posi-
tive shift (ΔEpc = +0.017 V for 1, ΔEpc = +0.030 V for
2, ΔEpc = +0.037 V for 3 and ΔEpc = +0.050V for 4)
b
It was demonstrated that the L2 Schiff base showed
a higher effect on E. coli than S. aureus and this differ-
ence could be due to the Gram-status. It is known that
the membrane of gram-negative bacteria is surrounded
by an outer membrane containing lipopolysaccharides.
e newly synthesized Schiff bases seem to be able to
combine with the lipophilic layer in order to enhance the
membrane permeability of the gram-negative bacteria.
e lipid membrane surrounding the cell favors the pas-
sage of only lipid soluble materials; thus the lipophilic-
ity is an important factor that controls the antimicrobial
activity. Also the increase in lipophilicity enhances the
penetration of Schiff bases into the lipid membranes and
thus restricts further growth of the organism. is could
be explained by the charge transfer interaction between
the Schiff base molecules and the lipopolysaccharide
molecules which lead to the loss of permeability barrier
activity of the membrane.
Table 2. Antimicrobial activities of Cu(II) complexes evaluated by MIC (minimum inhibitory concentration, μg mL−1).
Antibacterial activity Antifungal activity
R. stolonifer C. albicans R. bataticola
Complex
S. aureus
21.34
15.19
7.03
B. subtilis
22.06
17.43
8.11
E. coli
19.57
14.28
6.69
6.78
5.53
5.74
2.54
—
P. aeruginosa
23.45
21.46
8.15
A. niger
25.03
17.54
9.26
a
L2
21.08
19.42
8.54
7.12
6.47
5.92
—
24.34
16.21
7.06
7.63
7.54
6.12
—
22.12
19.38
8.41
8.19
8.24
8.16
—
b
L2
1
2
6.11
7.23
6.41
8.31
3
6.23
7.12
6.74
8.27
4
5.82
6.94
6.23
7.75
Streptomycin
Nystatin
3.25
2.87
3.02
—
—
—
—
2.82
3.31
2.45
2.96
© 2012 Informa UK, Ltd.

388 N. Raman and A. Selvan
e results of anti-bacterial screening reveal that
among all the compounds screened, compounds L2 , L2
reactive species, because DMSO, EtOH, L-Histidine and
azide ions are obviously inhibiting the cleavage activity.
We have performed a comparative study for the influ-
ence of methoxyl and nitro moiety present in the ligands
on DNA cleavage activity. Anti-biogram studies witness
the prominent activity of copper complexes compared to
the activity of ligands.
a
b
and 1-4 showed moderate anti-bacterial activity while
compounds 3 and 4 displayed good anti-bacterial activ-
ity when compared with streptomycin used as standard.
is finding agrees with Azam et al.27 who mentioned
that compounds containing methoxy groups at different
positions of the aromatic ring showed less inhibition on
microbial growth while compounds having halogens,
hydroxyl and nitro groups showed a good inhibitory
effect.
In case of antifungal activity, the Schiff bases and their
Cu(II) complexes were found to be highly active. All the
metal complexes possess higher antifungal activity than
the Schiff bases. Complexes 1 and 2 have more antifungal
active against C. albicans and R. bataticola, respectively
than 3, but less active than 4. is higher antimicrobial
activity of the metal complexes compared to Schiff bases
may be due to the change in structure due to coordina-
tion and chelating tends to make metal complexes to act
as more powerful and potent bactereostatic agents, thus
inhibiting the growth of the microorganisms. Moreover,
coordination reduces the polarity of the metal ion mainly
because of the partial sharing of its positive charge with
the donor groups within the chelate ring system formed
during the coordination. is process, in turn, increases
the lipophilic nature of the central metal atom, which
favors its permeation more efficiently through the lipid
layer of the microorganism, thus destroying them more
aggressively.
acknowledgments
e authors express their sincere thanks to the College
Managing Board, Principal and Head of the Department
of Chemistry, VHNSN College for providing neces-
sary research facilities. Instrumental facilities pro-
vided by Sophisticated Analytical Instrument Facility
(SAIF), IIT Bombay and CDRI, Lucknow are gratefully
acknowledged.
Declaration of interest
e authors report no conflicts of interest. e authors
alone are responsible for the content and writing of this
article.
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