DNA interaction, antimicrobial studies of newly synthesized copper (II) complexes with 2-amino-6-(trifluoromethoxy)benzothiazole Schiff base ligands

Article abstract of DOI:10.1016/j.jphotobiol.2016.10.027

Four novel Schiff base ligands, L¹ (1-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)naphthalen-2-ol, C₁₉H₁₁F₃N₂O₂S), L² (3-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)benzene-1,2-diol, C₁₅H₉F₃N₂O₃S), L³ (2-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)-5-methoxyphenol, C₁₆H₁₁F₃N₂O₃S) and L⁴ (2-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)-4-bromophenol, C₁₅H₈BrF₃N₂O₂S) and their binary copper(II) complexes 1 [Cu(L¹)₂], 2 [Cu(L²)₂], 3 [Cu(L³)₂] and 4 [Cu(L⁴)₂] have been synthesized and characterized by elemental analysis, ¹H NMR, ¹³C NMR, ESI mass, FT-IR, ESR, UV–Visible, magnetic susceptibility, TGA, SEM and powder XRD studies. Based on spectral and analytical data, a square planar geometry is assigned for all Cu(II) complexes. The ligands and their Cu(II) complexes have been screened for antimicrobial activity against bacterial species E. coli, P. aeruginosa, B. amyloliquefaciens and S. aureus and fungal species S. rolfsii and M. phaseolina and it is observed that all Cu(II) complexes are more potent than corresponding ligands. DNA binding (UV absorption, fluorescence and viscosity titrations) and cleavage (oxidative and photo cleavage) studies of Cu(II) complexes have also been investigated against calf thymus DNA (CT-DNA) and supercoiled pBR322 DNA respectively. From the experimental results, it is found that the complexes bound effectively to CT-DNA through an intercalative mode and also cleaved pBR322 DNA in an efficient manner. The DNA binding and cleavage affinities of newly synthesized Cu(II) complexes are in the order of 2 > 1 > 3 > 4.

Full text of DOI:10.1016/j.jphotobiol.2016.10.027

Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 147–156
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
DNA interaction, antimicrobial studies of newly synthesized copper (II)
complexes with 2-amino-6-(trifluoromethoxy)benzothiazole Schiff
base ligands
⁎
Aveli Rambabu, Marri Pradeep Kumar, Somapangu Tejaswi, Narendrula Vamsikrishna, Shivaraj
Department of Chemistry, Osmania University, Hyderabad, Telangana 500007, India
a r t i c l e i n f o
a b s t r a c t
Four novel Schiff base ligands, L1 (1-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)naphthalen-
2-ol, C19H11F3N2O2S), L2 (3-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)benzene-1,2-diol,
C15H9F3N2O3S), L3 (2-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)-5-methoxyphenol,
C16H11F3N2O3S) and L4 (2-((E)-(6-(trifluoromethoxy)benzo[d]thiazol-2-ylimino)methyl)-4-bromophenol,
C15H8BrF3N2O2S) and their binary copper(II) complexes 1 [Cu(L1)2], 2 [Cu(L2)2], 3 [Cu(L3)2] and 4 [Cu(L4)2]
have been synthesized and characterized by elemental analysis, 1H NMR, 13C NMR, ESI mass, FT-IR, ESR, UV–Vis-
ible, magnetic susceptibility, TGA, SEM and powder XRD studies. Based on spectral and analytical data, a square
planar geometry is assigned for all Cu(II) complexes. The ligands and their Cu(II) complexes have been screened
for antimicrobial activity against bacterial species E. coli, P. aeruginosa, B. amyloliquefaciens and S. aureus and fun-
gal species S. rolfsii and M. phaseolina and it is observed that all Cu(II) complexes are more potent than corre-
sponding ligands. DNA binding (UV absorption, fluorescence and viscosity titrations) and cleavage (oxidative
and photo cleavage) studies of Cu(II) complexes have also been investigated against calf thymus DNA (CT-
DNA) and supercoiled pBR322 DNA respectively. From the experimental results, it is found that the complexes
bound effectively to CT-DNA through an intercalative mode and also cleaved pBR322 DNA in an efficient manner.
The DNA binding and cleavage affinities of newly synthesized Cu(II) complexes are in the order of 2 N 1 N 3 N 4.
© 2016 Elsevier B.V. All rights reserved.
Article history:
Received 8 June 2016
Received in revised form 21 September 2016
Accepted 21 October 2016
Available online 24 October 2016
Keywords:
Schiff base
Binary Cu(II) complex
Antimicrobial activity
DNA interaction
1. Introduction
analogues made, it is an urgent requirement to develop new drugs
with minimal side effects and maximal curative potential [9].
DNA is the primary target molecule for most anticancer therapies.
The interaction between DNA and transition metal complexes is a
main fundamental issue in life sciences [1]. Compared to natural DNA
binding nucleases, artificial nucleases are found to have low molecular
weight and less reactive conditions [2,3]. The binding of small molecules
to DNA is extremely useful in understanding the drug-DNA interactions,
and DNA offers many potential binding sites for transition metals in-
cluding the electron rich bases and the major or minor grooves [4].
Metal complexes can interact with DNA through a non-covalent way,
via electrostatic binding, groove binding and intercalative binding [5].
Among these binding modes, intercalation shows special attractions
owing to its various applications in cancer therapy and molecular biolo-
gy [6,7]. Metal complexes that can bind and react with particular DNA
sites under physiological conditions are of considerable interest in the
designing of anticancer agents [8]. Cisplatin is a most effective antican-
cer drug widely used in the treatment of various human carcinomas.
However, the side effects of Cisplatin and its second generation
Palaniandavar et al. pointed out that copper(II) complexes are the best
alternatives to Cisplatin [10]. And a huge number of transition metal
complexes have been used as cleavage agents for DNA, and complexes
can cleave DNA through three types of mechanisms viz., hydrolytic
cleavage [11], oxidative cleavage [12] and photolytic cleavage [13].
Benzothiazole, a bicyclic heterocyclic compound is a unique synthon
in synthetic medicinal chemistry because of its significant pharmacolog-
ical activity [14]. The heterocyclic unit usually involved five or six mem-
bered ring with hetero atoms like N, O and S which are considered as
more polarizable than carbon [15]. Schiff bases and corresponding
metal complexes play a key role in understanding the coordination
chemistry of transition metals [16,17]. The imine (−C_N–) group of
Schiff base provides an opportunity for the stupendous biological activ-
ities such as antibacterial, antifungal, antitumor and herbicidal activities
[18]. The aromatic moiety of Schiff base plays a key role in enhancing
the binding capacity of DNA through stacking interaction and in stabiliz-
ing the DNA double helix [19,20]. Copper is an essential trace element
with relevant oxidation states [21] and many copper(II) complexes
have appeared in the literature with various biological activities. Syn-
thesis, characterization, biological activity and DNA interaction of
⁎
Corresponding author.
E-mail address: shivaraj_sunny@yahoo.co.in (Shivaraj).
http://dx.doi.org/10.1016/j.jphotobiol.2016.10.027
1011-1344/© 2016 Elsevier B.V. All rights reserved.

148
A. Rambabu et al. / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 147–156
Cu(II) complexes of different isoxazole Schiff bases were reported earli-
er from our laboratory [22,23]. In view the above, in the present work,
we focused on design, synthesis, characterization, biological activity
and DNA interactions of Cu(II) complexes derived from novel N, O
donor 2-amino-6-(trifluoromethoxy)benzothiazole Schiff bases.
placed on the surface of the culture impregnated agar plates. The culture
plates were incubated for 24 h at 37 °C. During the incubation, the test
solution was diffused and the growth of the inoculated microorganisms
was affected.
2.4. DNA Binding Studies
2. Experimental
2.4.1. Preparation of Stock Solutions
2.1. Materials and Instrumentation
Using UV-VIS spectrophotometer, the concentration of CT-DNA
stock solution at A260 was determined after 1:30 dilutions of original
DNA(1 mg/ml) sample with 5 mM Tris-HCl/50 mM NaCl buffer at
pH = 7.2. The molar absorptivity value was taken as 6600 M−1 cm−1
[26]. Solution of CT-DNA in 5 mM Tris-HCl/50 mM NaCl gave a ratio of
UV absorption at 260 nm and 280 nm A260/A280 of ca. 1.8–1.9: 1, indicat-
ing that the DNA was sufficiently free of protein [27]. All the stock solu-
tions were stored at 4 °C and were used within four days. The DNA
binding experiments were done in Tris-HCl buffer using stock solution
of compounds (ligands and complexes).
All chemicals and solvents are of analytical reagent grade obtained
from Finar, Merck and Sigma-Aldrich, and solvents such as chloroform,
methanol, petroleum ether and water were purified by standard proce-
dures [24]. Calf thymus DNA (CT-DNA) and supercoiled pBR322 DNA
(Merck made) were purchased from Genei, Bangalore and stored at
4
°C. Product numbers of CT-DNA and pBR322 DNA are
615100680011730 and 611600670501730 respectively. For DNA inter-
action studies, double distilled water was used.
NMR spectra of Schiff bases were recorded on Bruker 400 MHz NMR
instrument using TMS (tetramethylsilane) as standard. ESI mass spec-
trometry was done on VG AUTOSPEC mass spectrometer. Using KBr pel-
lets, IR spectra of the compounds were recorded in the range of 4000–
250 cm−1 on Perkin-Elmer Infrared model 337. Electronic spectral
data were obtained from Shimadzu UV–Vis 1601 spectrophotometer.
Magnetic susceptibilities of Cu(II) complexes were measured on Gouy
balance model 7550 using Hg[Co(NCS)4] as standard. The percentage
composition of C, H, N and S was determined using micro analytical
techniques on Perkin Elmer 240C (USA) elemental analyzer. Copper
content of the complexes was estimated by atomic absorption spectros-
copy after decomposing the complexes with concentrated HNO3 using
GBC Avanta 1.0 AAS. Melting points of all the compounds were deter-
mined on Polmon instrument (Model No. MP-96). ESR spectra were re-
corded at liquid nitrogen temperature using JES-FA200 ESR
spectrometer (JEOL-Japan). Thermal studies were carried out on
Shimadzu TGA-50H instrument in the temperature range of 27–
1000 °C under dynamic nitrogen atmosphere (20 mL min−1) with a
heating rate of 10 °C min−1. Fluorescence study was performed in a
Hitachi F4500 spectrofluorimeter. Viscosity measurements were per-
formed using Ostwald viscometer (Vensil). Powder X-ray diffraction
(XRD) analysis was made with an X'pert Pro diffractometer. The mor-
phology of ligands and complexes was determined by SEM (scanning
electron microscopy, Zeiss evo18).
2.4.2. Absorption Study
DNA binding study through UV–Vis absorption titrations was carried
out for ligands (L1–4) and their binary Cu(II) complexes (1–4) with fixed
compound concentrations (10 μM) while varying the CT-DNA concen-
tration (0–10 μM). Because of low soluble nature of the compounds in
the buffer solution, they were dissolved in DMSO first to get a com-
pound stock solution. While measuring the absorption, steady incre-
ments of CT-DNA were added to both the compound solution and the
reference solution to eliminate the absorbance of CT-DNA itself and
the resulting compound solution was incubated for 5 min before ab-
sorption spectra were recorded.
2.4.3. Fluorescence Study
The interaction pattern between the compounds and CT-DNA was
studied with ethidium bromide (EB) bound CT-DNA solution of 5 mM
Tris-HCl/NaCl buffer (pH 7.2) using fluorescence spectrophotometer.
The emission spectra of EB (12.5 μM) bound CT-DNA (125 μM) were re-
corded in the range of 530–680 nm (350 nm excitation) by the addition
of variable ligand/complex concentrations (0–60 μM). The relative bind-
ing affinity of the ligands and corresponding Cu(II) complexes with CT-
DNA was determined by measuring the quenching constant (K) from
the slopes of straight lines of Stern-Volmer equation [28]:
Io=I ¼ 1 þ Ksv
r
2.2. Synthesis of 2-Amino-6-(trifluoromethoxy)benzothiazole (i)
where, Io and I are the fluorescence (emission band) intensities in the
absence and presence of the quencher, respectively and r = [CuL1–4] /
[DNA].
The starting material (i) was prepared according to method reported
early [25]. And the synthesis procedure of Schiff base ligands and their
binary Cu(II) complexes (Scheme 1) is given in supplementary
information.
2.4.4. Viscosity Study
Viscosity measurements were performed using Ostwald viscometer
2.3. Antimicrobial Study
at 30
0.1 °C by keeping the concentration of CT-DNA constant
(200 μM), and varying the concentration of the complex from 0 to
200 μM. The flow times were measured with a digital stop-watch and
each sample was measured three times to get an average flow time.
To study the effect of DNA binding on the viscosity of the complex, the
results were presented as (η / ηo)1/3 versus [complex]/[DNA], where η
and ηo are the viscosity of CT-DNA solution in the presence and absence
of the complex, respectively. Viscosity values were calculated from the
experimental flow time of CT-DNA containing solutions corrected
from the flow time of buffer alone.
Antimicrobial activity of the ligands (L1–4) and their copper(II) com-
plexes (1–4) was evaluated by paper disc technique using nutrient agar
as the medium. The in vitro antibacterial activity was tested against two
Gram negative bacteria Escherichia coli (E. coli) and Pseudomonas
aeruginosa (P. aeruginosa) and two Gram positive bacteria Bacillus
amyloliquefaciens (B. amyloliquefaciens) and Staphylococcus aureus (S.
aureus), and the antifungal activity was tested against Sclerotium rolfsii
(S. rolfsii) and Macrophomina phaseolina (M. phaseolina). Streptomycin
and Mancozeb were the standard drugs for antibacterial and antifungal,
respectively. The stock solution of each compound was prepared by dis-
solving 5 mg of sample in 10 mL of DMSO to make the concentration of
500 μg/mL. The nutrient agar medium was inoculated with the test or-
ganisms and left for overnight to develop the culture. The sterilized
blank paper discs (6 mm diameter) were soaked in test solution and
2.5. DNA Cleavage Study
DNA cleavage activity of the Cu(II) complexes (1–4) was studied by
agarose gel electrophoresis. Oxidative cleavage (using H2O2) and photo-
lytic cleavage (using UV light) were performed with supercoiled

A. Rambabu et al. / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 147–156
149
Scheme 1. Synthetic route of Schiff bases and their copper(II) complexes.
pBR322 DNA by treating with variable concentrations of Cu(II) com-
3. Results and Discussion
plexes and followed by dilution with Tris-HCl/NaCl buffer(5:50 mM,
pH 7.2). Then mixed samples were incubated for 2 h at 37 °C and follow-
ed by mixing with bromophenol blue (2 μL). Electrophoresis was per-
formed at 70 V for 45 min by loading the mixed samples(10 μL out of
16 μL) in the wells of 1% agarose gel which was placed in TAE (Tris-ac-
etate-EDTA) buffer (pH 8.0) containing tray. The resulting gel was
stained with ethidium bromide before the images were taken under
UV light using Gel documentation.
Schiff bases and their Cu(II) complexes are coloured, highly stable at
room temperature and non-hygroscopic. The ligands are soluble in
methanol, ethanol, chloroform, acetonitrile, DMF and DMSO and the
complexes are soluble in DMF and DMSO, whereas both are insoluble
in water. The analytical data of all synthesized complexes are in good
agreement with calculated for 1:2 stoichiometric ratios of metal to
ligand.

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A. Rambabu et al. / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 147–156
Table 1
Visible, ESR, magnetic susceptibility, TGA, SEM and powder XRD studies.
The elemental analysis, NMR, ESI mass, SEM and powder XRD data are
given in supplementary file.
Characteristic IR absorption frequencies (cm−1) of Schiff bases and their Cu(II) complexes.
Compound
ν(OH)
ν(C_N)
ν(C\\O)
ν(M\\O)
ν(M\\N)
L1
3441
–
3330
–
3441
–
3438
–
1610
1598
1616
1593
1626
1612
1613
1598
1169
1138
1184
1169
1152
1147
1173
1154
–
575
–
575
–
589
–
–
462
–
445
–
439
–
[Cu(L1)2] (1)
L2
3.1.1. FT-IR Spectra
The IR spectra of Schiff bases (L1–4) and respective complexes were
recorded in the region of 4000–250 cm−1 using KBr pellet and the
data is presented in Table 1. The coordination mode of the ligands to-
wards the metal ion has been investigated by comparing the infrared
spectra of the free ligands with corresponding Cu(II) complexes (Fig.
S1). The strong absorption band that is observed in the range 1571–
1585 cm−1 in ligands are assigned to ν(C_N) group in the benzothiazole
ring. This band is overlapped with the aromatic ν(C_C) [29]. These bands
are remained unchanged in respective complexes confirming the
azomethine nitrogen of benzothiazole ring is not participated in coordi-
nation to the metal ion. The ligands exhibited a characteristic strong
band in the region of 1626–1610 cm−1 due to ν(C_N) group of Schiff
base ligands. In complexes, this band is shifted by 12–23 cm−1 to a
lower wave number indicating that the coordination occurred through
azomethine nitrogen atom of Schiff bases [30–33]. A broad band ob-
served in the range of 3330–3441 cm−1 due to phenolic ν(OH) group
of Schiff bases is disappeared in complexes confirming the oxygen of
phenolic –OH group is participated in coordination to the metal ion.
This is further confirmed by the shift of the ν(C\\O) band at 1152–
1184 cm−1 towards lower frequency region to the extent of 5–
31 cm−1 in respective Cu(II) complexes [34]. The coordination of
azomethine nitrogen and phenolic oxygen is further supported by the
appearance of two non-ligand bands in the range of 575–589 cm−1
and 439–462 cm−1 due to ν(Cu\\O) and ν(Cu\\N), respectively [35,36].
[Cu(L2)2] (2)
L3
[Cu(L3)2] (3)
L4
[Cu(L4)2] (4)
581
443
Table 2
Electronic spectral data and magnetic moment values.
Compound
π-π*
n-π*
d-d
μ
eff (BM)
L1
264(38461)
261(38314)
240(38759)
265(37735)
253(39525)
274(36496)
270(37037)
266(37593)
338(24330)
317(31545)
349(28653)
341(29325)
385(25974)
310(32258)
385(25974)
334(29940)
–
–
[Cu(L1)2] (1)
L2
575(17391)
1.77
–
1.74
–
1.82
–
1.78
–
[Cu(L2)2] (2)
L3
592(16891)
–
[Cu(L3)2] (3)
L4
566(17667)
–
585(17094)
[Cu(L4)2] (4)
3.1. Spectral Characterization
All ligands and corresponding Cu(II) complexes have been charac-
terized by elemental analysis, 1H NMR, 13C NMR, ESI mass, FT-IR, UV–
Fig. 1. UV–Visible spectra of ligands and their complexes.

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151
Table 3
Antimicrobial activity results of Schiff bases and their Cu(II) complexes at 500 μg/mL
concentration.
Compound
Bacterium (mm)
Fungi (mm)
S. M.
Gram-positive bacteria
Gram-negative
bacteria
rolfsii phaseolina
B.
S.
E.
P.
amyloliquefaciens aureus coli aeruginosa
L1
L2
L3
L4
1
2
3
4
2
4
2
4
2
6
4
8
3
2
2
6
4
8
6
8
11
–
2
2
3
6
4
6
4
6
10
–
2
3
3
2
4
5
4
4
10
–
4
5
4
2
7
7
9
6
–
14
5
2
5
5
6
7
8
6
Fig. 2. ESR spectrum of complex 1.
Streptomycin 15
Mancozeb
–
15
–
3.1.2. Electronic Spectra and Magnetic Susceptibility
The electronic absorption spectra of ligands (L1–4) and their Cu(II)
complexes (1–4) were recorded in DMSO at room temperature and
the data is shown in Table 2. The absorption spectra of ligands exhibited
two bands at in the range of 240–270 nm and 338–385 nm are assigned
to π-π* and n-π* transitions of phenyl ring and (−C_N–) group, re-
spectively [37,38]. These absorptions are shifted to 261–274 nm and
310–341 nm in complexes which supports the coordination of the li-
gands to Cu(II) ion. Besides this, all the complexes exhibit an additional
and characteristic broad d-d transition band at 575 for (1), 592 for (2),
566 for (3) and 585 nm for (4) in the visible region, due to 2B1g → 2E1g
transition (Fig. 1).
The effective magnetic moments of Cu(II) complexes were calculat-
ed by the equation μeff = 2.828 [χm·T]1/2 and are found to be in the
range of 1.74–1.82 BM, (where ‘χm’ is the molar susceptibility and ‘T’
is the room temperature), which are consistent with the presence of a
single unpaired electron [39]. From the electronic spectral data and
magnetic susceptibility values, a square planar geometry is assigned to
all Cu(II) complexes [40].
which is an indicative of covalent nature of metal-ligand bond [44].
The G values calculated are found to be in the range 4.6280–5.5210,
clearly demonstrating a negligible exchange interactions between the
copper ions according to Hathaway [42].
3.1.4. Thermal Analysis
Thermo gravimetric analysis (TGA) is useful technique to determine
the thermal stability of the metal complexes. The thermal analysis
(TGA) of all Cu(II) complexes was carried out at a heating rate of
10 °C min−1 under nitrogen atmosphere over a temperature range of
ambient temperature to 1000 °C. Thermo grams of complexes (1–4)
are given in Fig. 3. The results obtained are suggested that all complexes
have a similar decomposition process mainly in two steps. All com-
plexes showed high thermal stability up to 200 °C suggesting the ab-
sence of any solvent/water molecules in/out of the coordination
sphere. In first step, the complexes showed a sudden weight loss around
220 °C associated with the partial loss of respective ligands. In second
decomposition step, a gradual weight loss is appeared in the
3.1.3. ESR Spectra
ESR spectra of Cu(II) complexes provide information about the ex-
tent of the delocalization of unpaired electron and the nature of
metal-ligand bond. The ESR spectra of all Cu(II) complexes (1–4) were
recorded in DMSO at liquid nitrogen temperature (77 K) and the spec-
trum of complex 1 is shown in Fig. 2. The parameters g‖, g┴, Δg, gav
and G values have been calculated. The order of g values is found to
be, g‖ N g┴ N ge (2.0023) suggesting the unpaired electron is localized
2 2
in d
orbital with 2B1g which is the ground state of Cu(II) square pla-
x − y
nar complex [41]. The g‖ values for all complexes are found to be b2.3,
Fig. 4. Zone of inhibition (in mm) of all ligands and complexes tested against bacterial
Fig. 3. Thermo grams of complexes, 1–4.
strains.

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temperature range of 420–730 °C may be attributed to the complete de-
composition of the organic part around the metal ion and the further
horizontal line may be due to CuO residue as remaining part.
3.2. Antimicrobial Activity
The ligands and corresponding Cu(II) complexes were screened for
antimicrobial activity against two Gram-positive bacteria, B.
amyloliquefaciens and S. aureus and two Gram-negative bacteria, E. coli
and P. aeruginosa as well as two fungal species such as S. rolfsii and M.
phaseolina by paper disc method. Table 3. shows the inhibition zone
values (mm) of the compounds. From the graphs (Figs. 4 and 5), it is
clear that the zone of inhibition area is somewhat larger for the com-
plexes than free ligands. Such increased activity of the complexes can
be explained on the basis of Overtone's concept [43] and Tweedy's che-
lation theory [44]. Overtone's concept of cell permeability explains that
the lipid membrane that surrounds the cell favours the passage of only
the lipid-soluble materials, which makes the liposolubility is an impor-
tant factor which controls the antimicrobial activity. Moreover, chela-
tion reduces the polarity of the metal ion considerably because of the
partial sharing of its positive charge with the donor groups and also
due to π-electron delocalization on the whole chelating ring. This pro-
cess, in turn, increases the lipophilic nature of the central metal atom,
Fig. 5. Zone of inhibition (in mm) of all ligands and complexes tested against fungal
strains.
Fig. 6. UV–Vis absorption spectra of ligands (L1–4) and complexes (1–4) in the absence (dashed line) and presence (solid lines) of increasing concentrations of CT-DNA in Tris-HCl buffer
(pH 7.2). Conditions: [Ligand] or [Complex] = 10 μM, [DNA] = 0–10 μM. Arrow (↓) shows the absorbance changes upon increasing DNA concentration. Inset: linear plot for the calculation
of the intrinsic binding constant, Kb for DNA binding.

A. Rambabu et al. / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 147–156
153
Fig. 6 (continued).
which favours its permeation more effectively through the lipid layer of
cell membrane [45] thus destroying them more aggressively.
DNA in base pairs, and the ɛa, ɛf and ɛb are apparent absorption coeffi-
cients of the compound in the presence of DNA, free compound and
the fully DNA-bound compound, respectively [50]. Kb (binding con-
stant) is calculated from the slope to intercept ratio. The Kb values for li-
3.3. DNA Binding Studies
gands and complexes are found to be, 4.76
0.02 × 104 M−1 (L1),
3.3.1. UV–Vis Spectroscopic Studies
3.35
0.02 × 104 M−1 (L2), 1.93
0.01 × 104 M−1 (L3), 1.53
0.01 × 104 M−1 (1), 1.59
Electronic absorption spectroscopy is an effective method to scruti-
nize the binding mode of DNA with metal complexes [46,47]. In general,
the binding nature of metal complexes to DNA helix is investigated by
the changes in absorbance and wavelength. Molecules binding with
DNA through an intercalation usually result in the reduction in absor-
bance (hypochromism) and the shift towards higher wavelength (red
shift/bathochromic shift) due to the intercalation involving a strong π-
π* stacking interaction between the aromatic chromophore and the
DNA base pairs [48]. The magnitudes of hypochromism and
bathochromic shift depend on the strength of the intercalative interac-
tion [49]. In the present investigation, the complexes interact with DNA,
most likely through a mode that involves a stacking interaction between
the aromatic naphthalene/phenyl ring and the base pairs of DNA. The
Cu(II) complexes showed a band in the range of 247–252 nm attributed
to the intra ligand π-π* transitions. Upon addition of increasing amounts
of DNA, the intensity of the π-π* transition band decreases by 7–22%
(hypochromism) with a slight red shift of 2–6 nm. The absorption spec-
tra of ligands L1–4 and respective complexes 1–4 in absence and pres-
ence of CT-DNA are shown in Fig. 6.
0.02 × 104 M−1 (L4), 6.99
0.01 × 105 M−1 (2), 2.21
0.02 × 104 M−1 (3) and 1.90
0.01×
104 M−1 (4), respectively. From these Kb values, it is found that the
binding strength of complexes are stronger than the respective ligands,
and among the complexes, the complex 2 shows stronger binding affin-
ity, probably due to strong hydrogen bond of the hydroxyl group on the
ligand of the complex 2 with suitable DNA nucleobases [51]. However
these results indicate that the binding affinity between all the com-
pounds and CT-DNA is less than that of potential intercalators like
ethidium bromide (EB) (Kb = 7 × 107 M−1) [52] and the binding
order of Cu(II) complexes with DNA is as follows, 2 N 1 N 3 N 4.
3.3.2. Fluorescence Study
To get further proof for the mode of binding of ligands (L1–4) and
Cu(II) complexes (1–4) with CT-DNA, the relative binding ability of
the compounds with DNA has been investigated by ethidium bromide
(EB) displacement method. EB is a weak fluorescent compound, in
Tris-HCl buffer, it shows low intrinsic fluorescence/emission intensity
due to quenching by solvent molecules. But the fluorescence intensity
of EB dramatically enhances when it binds to DNA, in an intercalation
mode. This higher fluorescence intensity of EB-DNA can be quenched
by the addition of other DNA binding agents. The DNA binding agents
The intrinsic binding constant Kb could be determined from a plot of
[DNA] / (ɛa − ɛf) versus [DNA] using the equation: [DNA] / (ɛa − ɛf) =
[DNA] / (ɛb − ɛf) + 1/Kb(ɛb − ɛf), where [DNA] is the concentration of

154
A. Rambabu et al. / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 147–156
Fig. 7. Emission spectra of EB bound DNA in the absence and presence of ligands (L1–4) and complexes (1–4). [EB] = 12.5 μM, [DNA] = 125 μM, [Ligand] or [Complex] = 0, 10, 20, 30, 40, 50
and 60 μM, respectively; λex = 350 nm. The arrow ↓ shows the intensity changes on increasing the ligand or complex concentration.
compete with the EB bound DNA, displace the EB from DNA, this leads
to a reduction in the emission intensity of EB-DNA [53]. In the present
work, the emission spectra of EB-DNA in the absence and presence of li-
gands and Cu(II) complexes have been shown in Fig. 7. The fluorescence
intensity of EB-DNA decreases gradually on steady increasing the con-
centration of the compounds. This may be due to the displacement of
the EB molecule from the intercalating site by the compounds. The re-
sults reveal that all the compounds bind with DNA by the intercalation
mode. The relative binding constants of the ligands and their complexes
have been estimated using Stern-Volmer equation and the values are in
the range 1.56 × 104 M−1 (L1), 4.09 × 104 M−1 (L2), 1.45 × 104 M−1
(L3), 1.49 × 103 M−1 (L4), 1.85 × 104 M−1 (1), 9.50 × 104 M−1 (2),
4.48 × 103 M−1 (3) and 2.37 × 103 M−1 (4), respectively.
In the present investigation, the binding of Cu(II) complexes (1–4)
with CT-DNA was elucidated by measuring the relative specific viscosity
of DNA on the addition of varying concentration of complexes. On in-
creasing the complex concentration, an extension occurs in the helix
and hence the viscosity of DNA increases [56]. The effects of all Cu(II)
complexes on the viscosity of DNA at 30 0.1 °C are shown in Fig. 8.
The experimental results clearly showed that all the complexes can
bind to DNA, in an intercalation mode, and also evidenced by UV–Vis
spectroscopic results. The relative viscosity of CT-DNA increased steadi-
ly on the addition of increasing concentration of the copper (II) com-
plexes in the following order, 2 N 1 N 3 N 4.
3.4. DNA Cleavage
3.3.3. Viscosity Study
The cleavage of super coiled pBR322 DNA with Cu(II)complexes was
investigated by agarose gel electrophoresis method at variable concen-
trations using H2O2 and UV light, and 2 h incubation. The results are
shown in Figs. 9 and 10 and Figs. S2 and S3. The ability of the complexes
to cleave DNA was estimated by monitoring the conversion of circular
supercoiled DNA (Form I) to nicked circular (Form II) and linear form
(Form III). The supercoiled form (Form I) moves very fast. If one strand
of supercoiled DNA is cleaved, the supercoils relax to convert into a
slower-moving nicked form (Form II). If both strands are cleaved, a lin-
ear form (Form III) is produced which migrates in between Form I and
Form II [57].
The viscosity study is also an important tool to investigate the mode
of binding of metal complexes to DNA. In the absence of crystallographic
data, hydrodynamic measurements are too sensitive to changes in
length (i.e., viscosity and sedimentation), so these measurements are
proved to be the most critical and least ambiguous test to support a
complex-DNA binding mode [54]. A molecule binds to DNA, in classical
intercalation mode, results in a significant increase in DNA viscosity due
to the lengthening of DNA helix, which is caused by an increase in the
separation of base pairs at interaction sites and an increase in overall
double helix length. In contrast, molecules bind exclusively in DNA
groove by a partial or non-classical intercalation mode, result in less or
no changes in DNA viscosity due to the bending/kinking of DNA helix,
which reduces the effective length of DNA [55].
In the present study the results show that, no cleavage is observed in
DNA control (Lane 1), when there was no complex. By addition of Cu(II)
complex with constant increasing the concentrations of complexes

A. Rambabu et al. / Journal of Photochemistry & Photobiology, B: Biology 165 (2016) 147–156
155
Fig. 7 (continued).
(from Lane 2 to Lane 7 for photo cleavage and Lane 3 to Lane 8 for oxi-
4. Conclusion
dative cleavage), the percentage of supercoiled DNA band (Form I) di-
minished gradually whereas nicked circular DNA (Form II) and linear
DNA (Form III) increased. Further, it is observed that all complexes
(1–4) promote the cleavage of supercoiled pBR322 DNA more
efficiently.
Four novel Schiff bases and respective mononuclear binary Cu(II)
complexes have been synthesized and characterized. From the analyti-
cal and spectral data, it is observed that all the complexes adopted a
square planar geometry with 1:2 (metal:ligand) stoichiometry. The
Cu(II) complexes exhibited good antimicrobial activity against the bac-
terial (E. coli, P. aeruginosa, B. amyloliquefaciens and S. aureus) and fungal
strains (S. rolfsii and M. phaseolina) compared to Schiff base ligands. The
binding interaction of ligands/complexes with calf thymus DNA has
been investigated using absorption titrations, fluorescence quenching
and viscosity measurements. The results obtained revealed that these
Fig. 9. Oxidative cleavage of supercoiled pBR322 DNA(0.2 μg, 33.3 μM) at 37 °C in 5 mM
Tris-HCl/5 mM NaCl buffer by the Cu(II) complexes. Lane 1: DNA control, Lane 2:
DNA + H2O2 (1 mM), Lane 3–8: DNA + H2O2 (1 mM) + complex 1/2, [complex] = 20,
30, 40, 50, 60 and 70 μM, respectively.
Fig. 8. Effect of increasing amounts of EB, and complexes 1, 2, 3 and 4 on the relative
viscosity of CT-DNA at 30 0.1 °C.

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