Synthesis, DNA interactions and antibacterial PDT of Cu(II) complexes of phenanthroline based photosensitizers via singlet oxygen generation

Article abstract of DOI:10.1016/j.saa.2014.11.085

Cu(II) complexes [Cu(mqt)(B)H₂O]ClO₄(1-3) of 2-thiol 4-methylquinoline and phenanthroline bases (B), viz 1,10-phenanthroline (phen in 1), Dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq in 2) and Dipyrido[3,2-a:2′,3′-c]phenazine (dppz in 3) have been prepared and characterized by elemental analysis, IR, UV-Vis, magnetic moment values, EPR spectra and conductivity measurements. The spectral data reveal that all the complexes exhibit square-pyramidal geometry. The DNA-binding behaviors of the three complexes were investigated by absorption spectra, viscosity measurements and thermal denaturation studies. The DNA binding constants for complexes (1), (2) and (3) were determined to 2.2 × 10³, 1.3 × 10⁴ and 8.6 × 10⁴ M^-1 respectively. The experimental results suggest that these complexes interact with DNA through groove-binding mode. The photo induced cleavage studies shows that the complexes possess photonuclease property against pUC19 DNA under UV-Visible irradiation via a mechanistic pathway involving formation of singlet oxygen as the reactive species. Antimicrobial photodynamic therapy was studied using photodynamic antimicrobial chemotherapy (PACT) assay against Escherichia coli and all complexes exhibited significant reduction in bacterial growth on photoirradiation.

Full text of DOI:10.1016/j.saa.2014.11.085

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 138 (2015) 780–788
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, DNA interactions and antibacterial PDT of Cu(II) complexes
of phenanthroline based photosensitizers via singlet oxygen generation
⇑
C.N. Sudhamani, H.S. Bhojya Naik , K.R. Sangeetha Gowda, M. Giridhar, D. Girija, P.N. Prashanth Kumar
Department of Studies and Research in Industrial Chemistry, School of Chemical Sciences, Kuvempu University, Shankaraghatta 577 451, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
Synthesis of biologically active Cu(II)
mixed ligand metal complexes and
their characterization.
ꢀ
ꢀ
All the complexes exhibited efficient
DNA groove-binding propensity.
The complexes were able to cleave
pUC19 DNA effectively via singlet
oxygen generation.
ꢀ
Screening of newly synthesized
complexes as an antibacterial
photosensitizer.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2014
Received in revised form 8 November 2014
Accepted 20 November 2014
Available online 11 December 2014
Cu(II) complexes [Cu(mqt)(B)H2O]ClO4(1–3) of 2-thiol 4-methylquinoline and phenanthroline bases (B),
viz 1,10-phenanthroline (phen in 1), Dipyrido[3,2-d:20,30-f]quinoxaline (dpq in 2) and Dipyrido[3,2-
a:20,30-c]phenazine (dppz in 3) have been prepared and characterized by elemental analysis, IR, UV–
Vis, magnetic moment values, EPR spectra and conductivity measurements. The spectral data reveal that
all the complexes exhibit square-pyramidal geometry. The DNA-binding behaviors of the three com-
plexes were investigated by absorption spectra, viscosity measurements and thermal denaturation stud-
ies. The DNA binding constants for complexes (1), (2) and (3) were determined to 2.2 ꢁ 103, 1.3 ꢁ 104 and
8.6 ꢁ 104 Mꢂ1 respectively. The experimental results suggest that these complexes interact with DNA
through groove-binding mode. The photo induced cleavage studies shows that the complexes possess
photonuclease property against pUC19 DNA under UV–Visible irradiation via a mechanistic pathway
involving formation of singlet oxygen as the reactive species. Antimicrobial photodynamic therapy was
studied using photodynamic antimicrobial chemotherapy (PACT) assay against Escherichia coli and all
complexes exhibited significant reduction in bacterial growth on photoirradiation.
Keywords:
Groove-binding
Metal complexes
Cleavage
Photonuclease activity
PACT assay
Ó 2014 Elsevier B.V. All rights reserved.
Introduction
plexes that DNA on photoirradiation for their potential use in
PDT because of the noninvasive nature of this therapeutic process
Exploration of the interaction between transition metal com-
plexes and DNA has fascinated many benefits due to their signifi-
cance in cancer therapy and molecular biology [1–4]. In recent
years, there is a growing importance in the design of metal com-
requiring photoactivation of the drug at the tumor cells, leaving
other healthy cells unexposed to light [5–7]. Hence, to a great
extent attention has been targeted on the design of novel metal-
based complexes which can bind and cleave DNA.
The universal rise in the rates of antibiotic resistance of bacteria
emphasizes the need for alternate antibacterial agents. A promis-
ing approach to destroy antibiotic-resistant bacteria uses light in
⇑
Corresponding author. Fax: +91 8282 256255.
E-mail address: hsb_naik@rediffmail.com (H.S. Bhojya Naik).
http://dx.doi.org/10.1016/j.saa.2014.11.085
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

C.N. Sudhamani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 138 (2015) 780–788
781
combination with a photosensitizer to persuade a phototoxic reac-
tion. This photodynamic approach could be a possible alternative
to general medications in treating localized infections, thus avoid-
ing the development of microbial resistance to general drugs. Eas-
ily reachable oral or skin infections could be good candidates for
PACT treatment [8–12].
IR spectra were recorded in 4000–250 cmꢂ1 region using KBr pel-
lets on Shimadzu (Kyoto, Japan) FTIR-8400S spectrophotometer.
1H-NMR spectra were recorded on a Bruker FT NMR spectrometer
(300 MHz) at 25 °C in DMSO-d6 with TMS as the internal reference.
Electron paramagnetic resonance (EPR) spectra was measured
using Bruker BioSpin GmbH.
Photosensitizing uniqueness has become predominantly rele-
vant due to their potential applications in photodynamic antimi-
crobial chemotherapy (PACT), which takes up photosensitizers
and visible or ultraviolet light. The basic principles are the interac-
tion between light and photoactive drugs, forms reactive oxygen
species (ROS) produced through either electron transfer (type I)
or energy transfer (type II) reactions [13]. These ROS will react with
many cellular components that will persuade oxidative processes
leading to cell death [14–16]. Copper is a bioessential element in
all living systems and has been found to be involved in mixed
ligand complex formation in a number of biological processes
[17]. Copper complexes containing polypyridine ligands and their
derivatives are of great significance since they exhibit various bio-
logical activities such as antitumor [18], anti-candida [19], antibac-
terial [20] and antimicrobial [21,22] activities, etc. The presence of
both the copper(II) center and photoactive ligand has been found
to be essential for light induced DNA cleavage activity [23,24].
Complexes with ligands containing nitrogenated aromatic rings
have deserved an immense importance since the complex with
1,10-phenanthroline proved its ability to break DNA chains [25–
28]. Dhar et al. reported [29,30] that the sulfur-containing ligands
act as photosensitizers and the photosensitizing effect is greater
when the sulfur is bound to the metal ion. The literature reveals
to thio or thione moieties are known to show efficient intersystem
crossing to the triplet state on photo-irradiation [31–33].
Viscosity measurements were carried out on semimicro
dilution capillary viscometer. Thermal denaturation studies were
carried out with a Perkin–Elmer model 554 with a Shimazdu
UV–Vis recording spectrophotometer coupled to a temperature
controller (Model TCC-240A) using quartz cuvettes of 10 mm
light-path. A monochromatic 12 W UV lamp of 365 nm was used
as light source for photonuclease activity.
Synthesis and characterization
Synthesis of ligand (methylquinoline-2-thiol)
The starting compounds 2-hydroxy-4-methylquinoline and 2-
chloro-4-methylquinoline were synthesized according to the
method reported earlier [34]. Ethanolic solution of above synthe-
sized 2-chloro-4-methyl quinoline was mixed with sodium sulfide
in 1:3 ratios and refluxed for about 4 h in the presence of catalytic
amount of HCl. The completion of the reaction was monitored by
TLC eluting the phase ethyl acetate: carbon tetrachloride (8:2).
The reaction mixture was poured into ice cold water and neutral-
ized; finally product was filtered and recrystallized.
Anal. Calc. for C10H9NS: C, 68.51; H, 5.20; N, 7.99. Found: C,
68.57; H, 5.12; N, 8.01; UV–Visible. kmax (nm): 281, 380, IR (KBr)
cmꢂ1: 1653
m(C@N), 2480 m
(C–SH); 1H NMR (d ppm): 7.1–8.09
(m, ArH, 5H), 2.4 (s, 3H, CH3), 13.4 (s, SH), M.W. 175.26.
So, we aimed to synthesis engineered bichromophoric photo-
sensitizers (photoactive drugs) competent to mediate intramolecu-
lar energy and/or charge transfer through DNA double helix which
results in improved efficiency toward bacterial cells.
Herein, we present the synthesis, DNA binding and photoin-
duced DNA cleavage activity of [Cu(mqt)(B)H2O] (1)–(3), where
2-thiol 4-methylquinoline (mqt) acts as a photosensitizer and pla-
nar phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1),
dipyridoquinoxaline (dpq in 2) and dipyridophenazine (dppz in 3)
are DNA binders. In this study, [Cu(mqt)(B)2H2O] has emerged to
be a promising molecule as a photoactive component for PACT.
Synthesis of ligands (dpq and dppz)
The dpq ligand (dipyrido[3,2-d:20,30-f]quinoxaline) and dppz
ligand dipyrido[3,2-a:20,30-c] phenazine were prepared by reported
procedure [35,36].
Synthesis of complexes [Cu(mqt)(B)H2O]ClO4 (1)–(3)
mqt: 2-thiol 4-methylquinoline and B = phen, 1; dpq, 2, dppz,3.
All the present complexes were synthesized by following the
same general procedure. Mixture of methanolic solution of
Cu(ClO4)2ꢃ6H2O (1 mmol, 0.37 g) and solution of the mqt (1 mmol,
0.175 g) was stirred for 30 min. To the above reaction mixture,
1 mmol of methanolic solution (10 ml) of B was added and the stir-
ring was continued for 30 min more. Then obtained precipitate was
separated by filtration.
Material and methods
Chemicals and instrumentations
[Cu(mqt)(phen)H2O]ClO4 (1). Yield 76%, C, Anal. Calc. for C22H18
ClCuN3O5S (%): 49.35; H, 3.39; N, 7.85. Found (%): C, 49.37; H,
3.40; N, 7.83. IR (KBr) cmꢂ1: 3456 br, 3050m, 2925s, 1600s, 1091
vs (ClO4), 1633 vs 993m, 755s, 485w, 386m (br, broad; m, medium;
s, strong; vs very strong; w, weak). UV–Visible. kmax (nm): 276,
All chemicals used for the synthesis were of analytical grade
and procured from HiMedia Laboratories Pvt., Ltd. All the solvents
were purified by distillation and used. The Cu(ClO4)2ꢃ6H2O was
purchased from Merck (India) and used as received without further
purification. D2O purchased by Sigma Aldrich. Calf thymus
(ds)DNA and Super coiled (SC) pUC19 DNA were purchased from
Bangalore Genie (India), Agarose (molecular biology grade) ethi-
dium bromide were purchased from Himedia (India). Tris–HCl buf-
fer solution used for binding was prepared using deionised double
distilled water. Tris–borate ethylenediaminetetraacetate (TBE)
electrolyte buffer used for DNA cleavage study. Elemental analysis
was done on Perkin-Elmer Model 240-C CHN analyzer. Conductiv-
ity measurements were determined in DMF (10ꢂ3 M) using an
Equip-Tronic Digital conductivity meter model No. EQ-660A. Mag-
netic measurements were carried out by the Gouy method at room
temperature (28 2 °C) using Hg[Co(SCN)4] as calibrant. The elec-
tronic spectra of the complexes were measured using Shimadzu
spectrometer model UV-1650 PC double beam spectrophotometer.
320, 400, 520. l
eff = 1.81 B.M. XM = 70 mhos cm2 molꢂ1
[Cu(mqt)(dpq)H2O]ClO4 (2). Yield 72%, Anal. Calc. for C24H18ClCuN5
O5S (%): C, 49.07; H, 3.09; N, 11.92. Found (%): C, 49.02; H, 3.13; N,
11.90. IR (KBr) cmꢂ1: 3489 br, 3052m, 2924s, 1606s, 1084 vs
(ClO4), 1626 vs 989m, 776s, 478m, 392m. UV–Visible. kmax (nm):
265, 370, 452, 602. l
eff = 1.76 B.M. XM = 58 mhos cm2 molꢂ1
[Cu(mqt)(dppz)H2O]ClO4 (3). Yield 67%, Anal. Calc. for C28H20
ClCuN5O5S (%): C, 52.75; H, 3.16; N, 10.98. Found (%): C, 52.69;
H, 3.21; N, 10.89. IR (KBr) cmꢂ1: 3471 br, 3052m, 2925s, 1602s,
1087 vs (ClO4), 1618 vs 989w,738s, 470 w, 398w. UV–Visible. kmax
(nm): 279, 356, 450, 628, l
eff = 1.81 B.M. XM = 67 mhos cm2 molꢂ1

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C.N. Sudhamani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 138 (2015) 780–788
Solubility and stability
bromide. Electrophoresis was carried out in a dark chamber for
3 h at 50 V in Tris–borate buffer (45 mol/L Tris–borate, 1 mol/L
EDTA) TBE buffer. Bands were visualized using UV light and photo-
graphed. The cleavage efficiency was measured by determining the
ability of the complex to convert the supercoiled (SC) DNA to
nicked circular (NC) form and linear (LC) form.
The complexes are soluble in dimethylformamide (DMF),
dimethyl sulfoxide (DMSO) and buffer solution. The complexes
are sufficiently stable in the solid and solution phases.
Caution! Perchlorate salts are potentially explosive and only
small quantity was handled with care.
l
l
The inhibition reactions were carried out in the presence of dif-
ferent additives (NaN3, 100 lM; DMSO, 4 lL; D2O 14 lL) at 365 nm
DNA binding experiments
for mechanistic insights of the photo-induced DNA cleavage reac-
tions. These reactions were done by adding reagents to SC DNA
prior to addition of the complex before photoirradiation [42,43].
Absorption spectroscopic studies
DNA binding experiments were executed in Tris–HCl/NaCl buf-
fer (5 mM tris (hydroxymethyl) aminomethane, pH 7.2, 50 mM
NaCl) solution of the complexes. The concentration of CT-DNA (in
the base pairs) was determined using its known extinction coeffi-
cient at 260 nm (6600 Mꢂ1 cmꢂ1) [37]. The absorbance at 260 nm
and 280 nm for CT-DNA was measured in order to check the purity
level. The ratio A260/A280 was found to be 1.8–1.9, signifying that
CT-DNA was adequately free from protein [38,39].
Bacterial strains and growth conditions
Escherichia coli ATCC- 25922, was grown aerobically overnight
in LB broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl w/v) at
37 °C.
UV–Visible absorption titration experiments were performed
PACT assay
by varying the DNA concentration (0–100
lM) and maintaining
PACT assay was carried out according to the reported procedure
elsewhere [44]. Bacterial cultures were diluted in 15 ml of fresh LB
broth to obtain a cell density of 108 cfu/ml and after the addition of
the metal-complex concentration constant (0.5
l
M). Absorption
spectra were recorded after each successive addition of DNA and
equilibration for 5 min to allowing the complexes to bind to the
CT DNA. For Cu(II) complexes, the observed data were then inter-
preted by using Eq. (1), to obtain the intrinsic binding constant, Kb:
the 25 lM and 50 lM concentrations of the copper complexes
under investigation, incubated in foil-covered tubes to avoid acci-
dental exposure to the light. DMSO in low concentrations (the
range from 1% to 8%), was necessary for the preparation of aqueous
solution of the copper complexes. After incubation in the dark, cells
were harvested by centrifugation, washed with 0.1 M phosphate
buffer saline (pH 7.4) and resuspended in the same volume of
PBS. The flasks were placed under a 500 W halogen–tungsten lamp
at a distance of 20 cm. During light exposure, cell suspensions were
maintained under gentle magnetic stirring. Control cultures, with
no copper complexes added, were exposed to the light for the same
period of PS treated cultures. Aliquots of undiluted and serially
diluted treated and control cultures were plated on LB agar med-
ium. The percentage of survivors was calculated according to the
equation N1/N0 ꢁ 100, where N0 represents the number of cfu/ml
in samples before the irradiation and N1 the number of cfu/ml after
light exposure.
DNA
DNA
1
¼
þ
ð1Þ
ð
ea
ꢂ
ef
Þ
ð
eb
ꢂ
ef
Þ
Kbðeb
ꢂ
ef
Þ
where ea, ef, and eb are the apparent, free and bound metal complex
extinction coefficients respectively. A plot of [DNA]/(eb f) vs
[DNA] gave a slope of 1/(eb f) and a y intercept equal to 1/Kb
f), where Kb is the ratio of the slope to the y intercept [23].
ꢂ
e
ꢂ
e
(eb
ꢂ
e
Viscosity measurements
Viscosity measurements were performed using semi-micro
dilution capillary viscometer at room temperature and flow time
was measured with a digital stopwatch. Each experiment was car-
rying out three times and an average flow time was calculated. The
data was presented as (g|go) vs binding ratio, where g is the viscos-
ity of DNA in the presence of complex and go is the viscosity of
DNA alone in 5 mM Tris–HCl buffer medium [40].
Results and discussion
Thermal denaturation
Characterization of metal complexes
Melting studies were carried out by monitoring the absorption
intensity of CT DNA (50
lM) at 260 nm various temperatures in the
The elemental analysis data, UV–Visible, IR and magnetic sus-
ceptibility of the new complexes are summarized in the experi-
presence (5–10 M) and absence of each complex. As such, the
l
melting temperature (Tm), at which 50% of double-stranded DNA
becomes single-stranded (absorption increase was noticed in the
curve width (rT) and temperature range between 10% and 90%)
occurred and was calculated as reported [41].
mental
section.
The
formula
of
the
complexes
[Cu(mqt)(phen)(H2O)]ClO4 (1), [Cu(mqt)(dpq)(H2O)]ClO4 (2) and
[Cu(mqt)(dppz)(H2O)]ClO4 (3), respectively. These new complexes
are soluble in DMF, DMSO and in buffer (pH 7.2) solution and sta-
ble in the solid as well as in a solution phase. The molar conduc-
tance values of the complexes in DMF solutions fall in the range
58–70 ohmꢂ1 cm2 molꢂ1, demonstrating their electrolytic nature.
The complexes are one-electron paramagnetic at room tempera-
ture corresponding to d9 electronic configuration for copper(II)
center.
The complexes exhibit a prominent broad d–d band in the range
of 520–628 nm for complexes (1)–(3) in DMF. In addition, strong
bands in the UV region assigned to ligand to metal charge transfer
(LMCT). The electronic spectra of the complexes are in very good
agreement with the previously reported square pyramidal geome-
try of the complexes Cu(II) complexes [45–47]. The dpq and dppz
complexes display a moderately strong band near 440 nm possibly
due to n–p⁄ transition involving the quinoxaline or phenazine moi-
eties as this band is not observed in the phen complexes
DNA photocleavage experiments
The extent of photoinduced cleavage of supercoiled (SC) pUC19
DNA by the complexes was studied by agarose gel electrophoresis.
Each pUC19 DNA sample (0.5
7.2; ionic strength, 25 mM) containing 50 mM NaCl in the presence
of the complex (50 M) followed by dilution with Tris–HCl buffer
to a total volume of 20 L. For DNA photo-neuclease activity, the
lg) in 50 mM Tris–HCl buffer (pH,
l
l
reactions were carried out under illuminated conditions using UV
light at 365 nm (12 W). After photoexposure of samples under
UV light, these samples were incubated for 1 h at 37 °C followed
by addition to the loading buffer containing 25% bromophenol
blue, 0.25% xylene cyanol and 30% glycerol and the solution was
finally loaded on 0.8% agarose gel containing 1.0 lg/ml ethidium

C.N. Sudhamani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 138 (2015) 780–788
783
In IR spectra of the complexes, the characteristic frequencies
exhibit significant changes as compared with those of the parent
ligand. The spectrum of the ligand mqt shows a strong band at
2480 cmꢂ1 assignable to –SH group. The absence of this band in
the spectra of the complexes indicating the deprotonation of the
–SH group on complexation. The complexes (1), (2) and (3) clearly
concentration, the hypochromism is found to increase with a red
shift in the UV band of the complexes. All complexes show minor
bathochromic shift of the spectral band of ꢄ2–3 nm with signifi-
cant hypochromisity of 12%, 19.8% and 28.6% for complexes (1)–
(3) respectively (Fig. 2a and b), suggesting mainly groove-binding
propensity of the complexes to the double-stranded DNA. The
intrinsic binding constant (Kb) values are in the range of
2.2 ꢁ 103, 1.3 ꢁ 104 and 8.6 ꢁ 104 Mꢂ1 follows the order
(3) > (2) > (1). The dpq and dppz complexes prove significantly
higher DNA binding propensity in comparison to their phen ana-
logue possibly due to the presence of quinoxaline and phenazine
exhibited strong band
m
(C@N) in the range of 1618–1633 cmꢂ1
which shows a shifting to the lower frequencies by 20–35 cmꢂ1
in compared with ligand and metal chelates also show some new
bands in the region 485–479 cmꢂ1 and 386–402 cmꢂ1 which are
due to formation of M–N and M–S bands respectively. All the com-
plexes exhibit a board band at 3400–3500 cmꢂ1 attributable to
rings of extended planar structure which could undergo
interactions with the DNA bases [50].
p-stacking
water molecules followed by appearance of rocking qr (HOH) fre-
quency at 723–790 cmꢂ1, assignable to coordinated water mole-
cules. Besides, complexes are display characteristic infrared band
The complex (3) having a dppz ligand with its extended fused
aromatic rings show significant binding propensity whereas the
complex (1) which show less binding affinity to the double-
stranded CT-DNA. The evaluated Kb values and the results are sum-
marized in the Table 1.
for the perchlorate anion at 1090 cmꢂ1
.
Complexes (1)–(3) are one-electron paramagnetic having leff
value of ꢄ1.8 BM. The complexes display axial EPR spectra giving
g
and g\ values of ꢄ2.2 (F = 162 G) and ꢄ2.0, respectively. The
k
k
EPR spectral features are characteristic of an essentially square-
Viscosity measurements
pyramidal geometry [48,49].
Based on the analytical data and physicochemical properties,
the following structure is proposed for the complexes (See Fig. 1).
The hydrodynamic measurements that are sensitive to length
change (viscosity and sedimentation) are regarded as the least
ambiguous and the most critical test of a binding in solution, in
the absence of crystallographic structural data [51]. This method
is based on the well-known fact that, unwinding and elongation
of the DNA double helix occur upon intercalation leads to the sig-
nificant increase in the viscosity of DNA [52]. In contrast, com-
plexes binding to DNA grooves result in minor variation or no
variation in the viscosity of the DNA solution.
The viscosity measurements were carried out on CT-DNA by
varying the concentration of added complex. The effect of Cu(II)
complexes on the viscosity of DNA is depicted in Fig. 3. A marginal
increase of the relative viscosity is observed on the addition of the
present complexes to the DNA solution, suggesting the groove-
binding nature of the complexes.
DNA binding studies
Absorption spectral features of DNA binding
The mode and propensity of binding of the complexes to calf
thymus (CT) DNA are studied by different techniques like absorp-
tion spectral technique, DNA thermal denaturation and viscosity
measurements.
The absorption spectral technique is used to evaluate the intrin-
sic binding constants (Kb) of the complexes to CT DNA by monitor-
ing the absorption intensity of the charge transfer spectral band of
the complexes near 300 nm attributed to the LMCT transition
involving the heterocyclic base and the metal ion with increasing
concentration of CT DNA keeping the complex concentration as
constant. The extent of the hypochromism gives the strength of
the interaction of complex to DNA. On increasing the CT-DNA
Thermal denaturation study
Thermal behavior of DNA in the presence of metal complexes
can give insight into their conformational changes when
Fig. 1. Structures of Cu(II) complexes.

784
C.N. Sudhamani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 138 (2015) 780–788
Fig. 2. Absorption spectra of copper complex in Tris–HCl buffer upon addition of DNA. [Complex] = 0.5
l
M, [DNA] = (0–100
lM). Arrow shows the absorbance changing upon
increase of DNA concentration. The inner plot of [DNA]/ea
ꢂ ef vs [DNA] for the titration of DNA with complex. (a) Complex (1) and (b) complex (2).
is defined as the temperature where half of the total base pairs is
bounded, is usually introduced [53].
Table 1
Effect of CT-DNA on absorbance and binding constant of the Cu(II) complexes.
A moderate positive shift in the DNA melting temperature
Complex
D
k (nm)
H (%)
Kb (Mꢂ1
)
(
D
Tm) ranging from 1.4 to 3.6 is observed on the addition of the
(1)
(2)
(3)
2
2
3
12.0
19.8
28.6
2.2 ꢁ 103
1.3 ꢁ 104
8.6 ꢁ 104
complexes (1)–(3) to DNA (Fig. 4). The low value of Tm primarily
suggest the groove-binding nature of the complexes to DNA in
preference over an intercalative mode of binding to DNA that nor-
D
mally gives significantly high positive
DTm values [54].
temperature is raised and information about the interaction
strength of the complexes with DNA. The double- stranded DNA
tends to gradually dissociate to single strands on increase in the
solution temperature and generates a hyperchromic effect on the
absorption spectra of DNA bases (kmax = 260 nm). In order to
identify this transition process, the melting temperature Tm, which
Photo-induced DNA cleavage activity
The cleavage of supercoiled pUC 19 DNA was monitored by gel
electrophoresis to examine the capability of the present complexes

C.N. Sudhamani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 138 (2015) 780–788
785
Fig. 3. Plot of relative viscosity vs [Complex]/[DNA]. Effect of Cu(II) complexes on the viscosity of CT-DNA at 25 °C. Complex = 0–100 lM, [DNA] = 50 lM.
Fig. 4. Thermal denaturation of CT-DNA in the absence and presence of Cu(II) complexes. [DNA] = 50 lM, [complex] = 10 lM, buffer: phosphate.
(1)–(3) to serve as metallonucleases. The photo-induced DNA
cleavage experiments have been carried out in UV (365 nm,
age efficiency follows the order (3) > (2) > (1). The reason for differ-
ence in the cleavage activity also depends on the DNA binding
capabilities.
12 W) using 50
pUC19 DNA (0.5
l
M
concentration of the complexes and SC
lg) reagent. The dpq and dppz complexes show
Mechanistic aspects of the photoinduced DNA cleavage reac-
tions are investigated in the presence of various additives in UV
light (Figs. 5 and 6). Addition of singlet oxygen quencher like
sodium azide to complexes (1)–(3) inhibits the cleavage reaction,
that signifying 1O2 generation plays a key role in photo cleavage
of pUC 19 DNA. While the hydroxyl radical scavenger DMSO has
no apparent effect on the cleavage process, thus excluding the pos-
sibility of a type-I pathway forming hydroxyl radical (ÅOH). The for-
mation of singlet oxygen is further supported by the presence of
D2O. An enhancement of photocleavage of DNA is observed in
D2O solvent in which 1O2 has longer lifetime so induce more strand
scissions [57,58]. Data from control experiments unequivocally
suggest the involvement of singlet oxygen in a type II process in
more significant cleavage activity with the formation of both
nicked-circular and linear DNA in comparison to their phen ana-
logue showing formation of only NC form under similar experi-
mental conditions (Fig. 5). The DNA binder heterocyclic bases
dpq and dppz have photoactivatable quinoxaline and phenazine
moieties with conjugated C@N bonds that could generate photoex-
cited 3(n–p⁄) or 3( p⁄) state(s) effecting DNA cleavage following
p–
an oxidative pathway in addition to the photoactive sulfur of the
mqtH ligand show a significant photosensitization effect, enhanc-
ing the cleavage activity of complexes (2) and (3) to a greater
extent along with the formation of linear DNA by attacking two
complementary DNA strands at close proximity [55,56]. The cleav-

786
C.N. Sudhamani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 138 (2015) 780–788
Fig. 5. Gel electrophoresis diagram showing the photocleavage of SC pUC19 DNA (0.5
l
g) by complex (1), (2) and (3) (50
L); lane 5, DNA+(1)+D2O (14
L); lane 11, DNA control; lane 12, DNA+(3); lane 13, DNA+(3)+NaN3
l
M) and other additives at 365 nm for an exposure
time of 1 h. lane 1, DNA control; lane 2, DNA+(1); lane 3, DNA+(1)+NaN3 (100 M); lane 4, DNA+(1)+DMSO (4
l
l
l
L);. lane 6, DNA control; lane 7,
DNA+(2); lane 8, DNA+(2)+NaN3 (100
l
l
M); lane 9, DNA+(2)+DMSO (4
L); lane 15, DNA+(3)+D2O (14
l
l
L); lane 10, DNA+(2)+D2O (14 l
L).
(100 M); lane 14, DNA+(3)+DMSO (4
l
Fig. 6. Cleavage of SC pUC19 DNA (50 lM) by complexes (1), (2) and (3) in the presence of various additives upon photo irradiation at 365 nm in 50 mM Tris–HCl/NaCl buffer
(pH 7.2).
Scheme 1. Mechanistic pathways for the cleavage of DNA on photoirradiation.
the metal assisted photo-excitation process involving ligand n–p⁄
and
p⁄ transitions (Scheme 1).
tial decrease in the number of viable cells was observed. Thus, irra-
diation time was maintained for 60 min in all PACT assays. To
standardise the time of incubation with the complexes before light
exposure, suspensions (108 cfu/ml) of the bacteria was incubated
with the complexes in the dark for 30, 60 and 90 min, washed once
in PBS and then irradiated with light for the period previously
determined. The percentage of survivors for E. coli after the light
treatment were found to be 0.043%, 0.041%, 0.044% for complex
(1), 0.068%, 0.067%, 0.065% for complex (2) and 0.043%, 0.042%,
0.044% for complex (3). Consequently results suggested that,
increasing the time of the contact between the complexes and
the bacterial cells did not improve the performance of the
p
–
PACT assay
The novel copper complexes (1), (2) and (3) were tested for
their ability to kill E. coli on photoirradiation by a tungsten–halo-
gen 500 W lamp. The effect of cell viability on irradiation of visible
light emitted by a 500 W halogen–tungsten lamp was investigated
to obtain dependable data about the efficiency of the photody-
namic treatment on E. coli. When E. coli cultures (108 cfu/ml) alone,
in absence of complexes, were illuminated for 60 min, no substan-

C.N. Sudhamani et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 138 (2015) 780–788
787
Fig. 7. CFU reduction (%) in E. coli after treatments.
complexes against the above mentioned bacteria. So, for all
photoinactivation experiments of copper complexes under study
30 min incubation period was maintained.
The protocol for photodynamic treatment of E. coli tested was
then further standardized evaluating the effects of complexes up
due to the photosensitizing effect of both dpq and dppz. Mechanis-
tic studies reveal direct involvement of 1O2 in the photo-cleavage
process. Pathways involving singlet oxygen in the DNA photoclea-
vage reactions are proposed from the observation of the complete
inhibition of the cleavage in presence of NaN3 and enhancement of
cleavage in D2O. Sulfur containing ligands with their longer triplet
state lifetimes are suitable for activation of molecular oxygen. We
are making further efforts to understand the DNA clevage ability of
copper complexes in longer wavelength (PDT window). These
results may through light toward the design and development of
copper-based complexes with sulfur containing ligands for PDT
applications and as alternatives to the PDT anticancer drug.
to 50
when irradiated after the exposure to the different complexes at
25 M and 50 M concentrations. The DMSO used in low concen-
lg/ml. E. coli showed a apparent dose-dependent lethality
l
l
trations for the preparation of aqueous solution of the copper com-
plexes also did not alter cell viability. By counting colony forming
unit (CFU) of E. coli remaining in the control, complexes (1), (2) and
(3) alone and photoirradiated complexes at 25 lM and 50 lM con-
centrations was made to determine effectiveness of PDT. The
results of PDT on E. coli in terms of CFU counts (%) after treatment
can be seen in Fig. 7. The% survival statistical analysis for E. coli.
revealed a nonsignificant reduction of CFU counts between the
control and Complexes 7.6%, 9.3%, 12.8% of complexes (1), (2)
and (3) respectively. However, a significant reduction of CFU
Acknowledgment
This work has been supported by providing Post Doctoral Fel-
lowship for women (2009–10 & 2010–11) from the University
Grants Commission, New Delhi.
counts by 56.8%, 62.3%, 72.4% for 25
lM concentration and 71.3%,
76.8%, 82.3% for 50 M concentration was observed for the phot-
oirradiated complexes (1), (2) and (3) respectively when compared
l
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