Novel iron phenanthroline-based photosensitizers for antimicrobial PDT: synthesis, DNA binding and photo-induced DNA cleavage activity

Article abstract of DOI:10.1007/s00044-017-1831-z

The chemistry of Fe(II) complexes showing efficient light-induced DNA cleavage activity, binding propensity to calf thymus DNA and antibacterial photodynamic therapy has been summarized in this article. Complexes of formulation [Fe(mqt)(B)₂](PF₆)(1)–(3), where mqt is 2-thiol-4-methylquinoline and B is N,N-donor heterocyclic base, viz. 1,10-phenanthroline, dipyrido[3,2-d:2′,3′-f]quinoxaline and dipyrido[3,2-a:2′,3′-c]phenazine have been prepared and characterized. The DNA-binding behaviors of these three complexes were explored by absorption spectra, viscosity measurements and thermal denaturation studies. The DNA binding constants for complexes (1), (2) and (3) were determined to be 1.9 × 10³, 3.4 × 10⁴ and 8.1 × 10⁴ M^?1, respectively. The experimental results suggest these complexes interact with DNA through groove-binding mode. The complexes show significant photocleavage of supercoiled DNA proceeds via a type-II process forming singlet oxygen as the reactive species. Antimicrobial photodynamic therapy was studied using photodynamic antimicrobial chemotherapy assay against Escherichia coli and all complexes exhibited significant reduction in bacterial growth on photoirradiation.

Full text of DOI:10.1007/s00044-017-1831-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2017) 26:1160–1169
DOI 10.1007/s00044-017-1831-z
ORIGINAL RESEARCH
Novel iron phenanthroline-based photosensitizers for
antimicrobial PDT: synthesis, DNA binding and photo-induced
DNA cleavage activity
Chittanahalli N. Sudhamani1 Halehatty S. Bhojya Naik1
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Kalligundi R. Sangeetha Gowda1 Manju Giridhar1 Dugganna Girija1
Pasupanetti N. Prashanth Kumar1
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Received: 29 April 2015 / Accepted: 1 March 2017 / Published online: 11 March 2017
© Springer Science+Business Media New York 2017
Abstract The chemistry of Fe(II) complexes showing
efficient light-induced DNA cleavage activity, binding
propensity to calf thymus DNA and antibacterial photo-
dynamic therapy has been summarized in this article.
Complexes of formulation [Fe(mqt)(B)2](PF6)(1)–(3),
where mqt is 2-thiol-4-methylquinoline and B is N,N-donor
heterocyclic base, viz. 1,10-phenanthroline, dipyrido[3,2-
d:2′,3′-f]quinoxaline and dipyrido[3,2-a:2′,3′-c]phenazine
have been prepared and characterized. The DNA-binding
behaviors of these three complexes were explored by
absorption spectra, viscosity measurements and thermal
denaturation studies. The DNA binding constants for
complexes (1), (2) and (3) were determined to be 1.9 × 103,
3.4 × 104 and 8.1 × 104 M−1, respectively. The experi-
mental results suggest these complexes interact with DNA
through groove-binding mode. The complexes show sig-
nificant photocleavage of supercoiled DNA proceeds via a
type-II process forming singlet oxygen as the reactive
species. Antimicrobial photodynamic therapy was studied
using photodynamic antimicrobial chemotherapy assay
against Escherichia coli and all complexes exhibited sig-
nificant reduction in bacterial growth on photoirradiation.
Introduction
Copious biological research have demonstrated that DNA is
the prime intracellular target of anticancer drugs due to the
interaction between drugs and DNA, leading to DNA
damage in cancer cells, thus blocking the division of cancer
cells and resulting in cell death (Jiao et al. 2005; Prakash
Naik et al. 2009a; Bera et al. 2008). Accordingly, the DNA
interaction patterns based on the study of the molecules that
bind to nucleic acids is one of the most significant para-
meters in the screening design for new drugs. Transition
metal complexes having tunable coordination environments
with versatile spectral and electrochemical properties offer
an immense scope of design for species that are suitable for
DNA binding and cleavage activities (Metcalfe and Thomas
2003; Anbu et al. 2009; Prakash Naik et al. 2009b).
Compounds which are able to cleave DNA upon light
irradiation are used in the photodynamic therapy of cancers
(PDT) (Canti et al. 2002; De Rosa and Crutchley 2002).
Photoactive pro-drugs capable of discharging a cytotoxic
drug only at the irradiated site can increase specificity and
thereby minimize toxicity to the surrounding healthy cells.
In the presence of transition metal ions, the Fenton reaction
is a significant pathway for providing oxygen radical spe-
cies. These reactive oxygen species induce different kinds
of damage to DNA. Of which, Fe2+ ions are one such
transition metal ions that induce the DNA cleavage. Since
iron is a very important metal in nature, it is part of the
active center of many enzymes. It can produce highly
reactive radicals, which attack biomolecules such as lipids,
DNA or proteins, and cause damage (Lawrence et al. 2006;
P. Nagababu and Satyanarayana 2007; Peng et al. 2007;
Tan et al. 2007; Richardson et al. 2006).
●
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Keywords Binding propensity Calf thymus DNA
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Groove-binding Thermal denaturation studies
Photocleavage
* Halehatty S. Bhojya Naik
hsb_naik@rediffmail.com
1
Department of Studies and Research in Industrial Chemistry,
School of Chemical Sciences, Kuvempu University,
Shankaraghatta 577 451, India
Due to consequence of antibiotic resistance, researchers
are behind of developing new antibacterial agents.

Med Chem Res (2017) 26:1160–1169
1161
Photodynamic antimicrobial chemotherapy (PACT), a
newly developed therapeutic option that employed photo-
sensitive molecules and visible light to induce an oxidative
damage to microbial pathogens (Hamblin and Hasan 2004;
Wainwright 2004), signifies an alternate antibacterial, anti-
fungal and antiviral treatment against drug resistant organ-
isms (Wainwright 1998).
Melting points were determined in open capillaries and
were uncorrected. Elemental analyses were performed by
Perkin–Elmer (Waltham, MA, USA) Model 240-C CHN
analyzer. The electronic spectra of the complexes were
measured using Shimadzu spectrometer model UV-1650
PC double beam spectrophotometer. The molar con-
ductivities of the complexes in dimethylformamide
(DMF) solution (10−3 M) at room temperature were carried
out by using an Equip-Tronic (India) Digital conductivity
meter model No. EQ-660A. Magnetic susceptibilities of the
solid complexes were determined by employing Gouy
balance at room temperature (28 2 °C). Viscosity mea-
surements were carried out on semimicrodilution capillary
viscometer at room temperature. Infrared (IR) spectra were
recorded in the range of 4000–250 cm−1 using KBr pellets
The use of mixed ligand complexes permits the devel-
opment of increased selectivity, sensitivity, and has great
significance in the field of biological chemistry. Investiga-
tion regarding 1,10-phenanthroline (phen 1) mixed-ligand
chelate systems would offer for an understanding of the
driving forces that led to the formation of such mixed ligand
complexes (Raman and Sobha 2010; Jakupec et al. 2003).
Complexes with ligands containing nitrogenated aromatic
rings deserve an enormous significance, as the complex
with phen 1 proved its ability to break DNA chains (Prab-
hakara et al. 2007a; Sudhamani et al. 2009; Prabhakara and
Bhojya Naik 2008a). In fact, several metal complexes of
sulfur–nitrogen chelating agents have been shown to dis-
play confirmed cytotoxic activities. Moreover, Fe(II) com-
plexes with sulphur and nitrogen containing ligands are the
subject of intensive biological evaluation in the search for
less toxic and more selective anticancer therapies (Keskio-
glu et al. 2008; Tullius 1989).
1
on Shimadzu (Kyoto, Japan) FTIR-8400. H-NMR spectra
were recorded on a Bruker FT NMR spectrometer (300
MHz) at 25 °C in dimethyl sulfoxide-d6 (DMSO-d6) with
Tetramethylsilane as the internal reference. A monochro-
matic 12 W UV lamp of 365 nm was used as a light source
for photonuclease activity.
Synthesis and characterization
Herein, we have designed structurally simple and stable
mono nuclear Fe(II)-based complexes having DNA binder
phenanthroline bases and a photoactive ligand containing a
sulfide moiety to achieve photoneuclease activity. In this
article, we present the synthesis, DNA binding and photo-
induced DNA cleavage activity, and the antimicrobial PDT
of [Fe(mqt)(B)2](PF6)(1)–(3), where 2-thiol-4-methylquino-
line (mqt) acts as a photosensitizer and planar phenanthro-
line bases (B), viz. phen 1, dipyridoquinoxaline (dpq in 2)
and dipyridophenazine (dppz in 3) are DNA binders.
Synthesis of 2-thiol-4-methylquinoline
The initial compounds 2-hydroxy-4-methylquinoline and 2-
chloro-4-methylquinoline were synthesized according to the
method reported earlier (Bhojya Naik et al. 2006). An
ethanolic solution of 2-chloro-4-methylquinoline was mixed
with sodium sulfide in a 1:3 ratio and refluxed for about 4 h
in the presence of a catalytic amount of HCl. The comple-
tion of the reaction was monitored by thin-layer chroma-
tography (ethyl acetate:carbon tetrachloride 8:2). The
reaction mixture was poured into ice cold water and neu-
tralized with sodium carbonate. The product was filtered
and recrystallized.
Experimental
Anal. calc. for C10H9NS: yield 74%, C, 68.51; H, 5.20;
N, 7.99. Found: C, 68.57; H, 5.12; N, 8.01; UV–Visible.
λmax (nm): 281, 380, IR (KBr) cm−1: 1653 ν(C=N), 2480 ν
Materials and methods
1
All reagents and solvents required were of Analytical
Reagents grade, purchased from Himedia (India). The sol-
vents were purified by distillation. The ammonium ferrous
sulfate, phen 1 and tris-HCl were purchased from Merck
(India). Calf thymus (CT) DNA and supercoiled (SC)
pUC19 DNA was procured from Bangalore Genie (India)
and Agarose (molecular biology grade) ethidium bromide
were purchased from Himedia (India). tris-HCl buffer
solution used for binding was prepared using deionized
double-distilled water. tris-borate ethylenediamine tetra-
acetate (TBE) electrolyte buffer was utilized for DNA
cleavage study.
(C–SH); HNMR(δ ppm): 7.1–8.09 (m, ArH, 5H), 2.4 (s,
3H, CH3), 13.4 (s, SH), 13C NMR (DMSO-d6) δ: 19.2
(CH3),116.3(CH), 124.2, 126.3, 129.2, 145.2, 146.5, 180.6
(–C=N), M.W. 175.26.
Synthesis of dpq and dppz ligand
The dpq ligand (dipyrido[3,2-d:2′,3′-f]quinoxaline) and
dppz ligand dipyrido[3,2-a:2′,3′-c] phenazine were prepared
by reported procedures (Yamada et al. 1992; Collins et al.
1998).

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Med Chem Res (2017) 26:1160–1169
Scheme 1 Structures of Fe(II)
complexes
Synthesis of complexes [Fe(mqt)(B)2](PF6)(1)–(3)
1626, 847, 478, 410. UV–Visible. λmax (nm): 278, 356, 438,
562. µeff = 5.71 B.M. ΩM = 145 mhos cm2 mol−1
mqt: 2-thiol-4-methylquinoline and B = phen, 1; dpq, 2;
dppz, 3. (Scheme 1).
The complexes are soluble in DMF, dimethyl sulfoxide
(DMSO) and buffer solution. The complexes are suffi-
ciently stable in the solid and as well as solution phases.
All the complexes were synthesized by following the
similar general procedure. A methanolic solution of
ammonium ferrous sulfate (1 mmol, 0.404 g) and mqt
(1 mmol, 0.175 g) was stirred for 30 min. To the above
reaction mixture, 2 mmol of 10 ml methanolic solution of
the heterocyclic base was added and the stirring was further
continued for 15 min. This solution was treated with a
methanolic solution of NH4PF6 to precipitate as red solid,
which was isolated by filtration. It was washed with cold
methanol followed by diethyl ether and finally dried.
[Fe(mqt)(phen)2]PF6(1). Yield 76%, C, Anal. calc. for
C34H24F6FeN5 PS(%): C, 52.52; H, 3.29; N, 9.52. found
(%): C, 52.55; H, 3.27; N, 9.51. IR (KBr) cm−1: 2504,
1619, 842, 466, 384. UV–visible. λmax (nm): 264, 319, 418,
521. µeff = 5.68 B.M. ΩM = 139 mhos cm2 mol−1
[Fe(mqt)(dpq)2]PF6(2). Yield 68%, Anal. calc. for
C38H24F6FeN9PS (%): C, 54.36; H, 2.88; N, 15.02. found
(%): C, 54.35; H, 2.86; N, 15.05. IR (KBr) cm−1: 2511,
1624, 846, 470, 393. UV–Visible. λmax (nm): 262, 368, 435,
546. µeff = 5.72 B.M. ΩM = 128 mhos cm2 mol−1
[Fe(mqt)(dppz)2]PF6(3). Yield 65%, Anal. calc. for
C46H28F6FeN9 PS(%): C, 58.80; H, 3.00; N, 13.42. found
(%): C, 58.78; H, 2.98; N, 13.46. IR (KBr) cm−1: 2516,
DNA binding experiments
Absorption spectroscopic studies
The concentration of CT DNA was calculated by using its
known extinction coefficient at 260 nm (6600 M−1 cm−1
)
(Pradeepa et al. 2013a). The absorbance at 260 nm and 280
nm for CT-DNA was measured to check the purity level.
The ratio A260/A280 was found to be 1.84, indicating that
CT-DNA was satisfactorily free from protein (Vinay Kumar
et al. 2012a; Prabhakara and Bhojya Naik 2008b). DNA
binding experiments were performed in tris-HCl/NaCl
buffer (5 mM tris (hydroxymethyl) aminomethane, pH 7.2,
50 mM NaCl) solution of the complexes.
Absorption titration experiments were carried out by
varying the DNA concentration (0–100 μM) and maintain-
ing the complex concentration constant (0.5 μM). Absorp-
tion spectra were recorded after each successive addition of
DNA and equilibration for 5 min to allow the complexes to
bind to the CT-DNA. For Fe(II) complexes, the observed
data were then interpreted by using Eq. 1, to obtain the

Med Chem Res (2017) 26:1160–1169
1163
intrinsic binding constant, Kb:
convert the SC DNA to nicked circular (NC) form and
Linear (LC) form.
The inhibition reactions were carried out in the presence
of different additives (NaN3, 100 µM; DMSO, 4 µL; D2O
14 µL) at 365 nm to understand the mechanistic pathways
involved in the photocleavage reactions. These reactions
were performed by adding reagents to SC DNA prior to
addition of the complex before photoirradiation (Vinay
Kumar et al. 2012b; Pradeepa et al. 2013b).
½DNAŠ=ðεa À εfÞ ¼ ½DNAŠ=ðεb À εfÞ þ 1=Kbðεb À εfÞ
ð1Þ
where εa, εf and εb are the apparent, free and bound metal
complex extinction coefficients, respectively. A plot of
[DNA]/(εb−εf) vs. [DNA] gave a slope of 1/(εb−εf) and an
intercept equal to 1/Kb(εb−εf), where Kb is the ratio of the
slope to the y intercept.
Bacterial strains and growth conditions
Viscosity measurements
Escherichia coli ATCC-25922, was grown aerobically
overnight in Luria–Bertani (LB) broth (1% tryptone, 0.5%
yeast extract, 0.5% NaCl w/v) at 37 °C.
To understand the nature of DNA binding of the mixed
ligand metal complexes, the viscosity measurements were
carried out on CT-DNA by varying the concentration of the
added complexes. Flow times were measured using a digital
stopwatch, at least three times and were accepted if they
agreed within 0.1 s. Data were presented as (η/ηo) vs bind-
ing ratio, where η is the viscosity of DNA in the presence of
complex and ηo is the viscosity of DNA alone in 5 mM ltris-
HCl buffer medium (Sudhamani et al. 2014).
PACT assay
The PACT assay was carried out according to the procedure
reported elsewhere (Banfi et al. 2006). Bacterial cultures
were diluted in 15 ml of fresh LB broth to attain a cell
density of 108 cfu ml−1 and after the addition of the 25 and
50 µM concentrations of the iron complexes under investi-
gation, incubated in foil-covered tubes to stay away from
accidental exposure to the light. DMSO in low concentra-
tions (the range from 1 to 8%), was necessary for the pre-
paration of aqueous solution of the iron complexes. After
incubation in the dark, cells were harvested by centrifuga-
tion, washed with 0.1 M phosphate buffered saline (pH 7.4)
and resuspended in the same volume of phosphate buffered
Thermal denaturation
Thermal denaturation experiments were carried out by
monitoring the absorption of CT-DNA (50 µM) at 260 nm at
various temperatures in the presence (5–10 μM) and the
absence of each complex. The melting temperature (Tm), at
which 50% of double-stranded DNA becomes single-
stranded (absorption increase was noticed in the curve
width (σT) and temperature range between 10 and 90%)
occurred and was calculated as reported (Sangeetha Gowda
et al. 2013; Sudhamani et al. 2012a).
saline. The flasks were positioned under
a 500 W
halogen–tungsten lamp at a distance of 20 cm. During light
exposure, cell suspensions were kept under gentle magnetic
stirring. Control cultures, with absence of iron complexes,
were exposed to the light for the same period as
photosensitizer-treated cultures. Aliquots of undiluted,
serially diluted treated and control cultures were plated on
LB agar medium. The percentage of survivors was calcu-
lated according to the equation N1/N0 × 100, where N0
signifies the number of cfu ml−1 in samples before the
irradiation and N1 is the number of cfu ml−1 after light
exposure.
DNA photocleavage experiments
The photocleavage of SC pUC19 DNA was monitored by
using agarose gel electrophoresis. SC pUC19 DNA (0.5 μg)
in ltris-HCl buffer (50 mM) with 50 mM NaCl (pH 7.2) was
treated with metal complex (50 μM), followed by dilution
with ltris-HCl buffer to a total volume of 20 μl. For DNA
photo-nuclease activity, the reactions were carried out under
illuminated conditions using UV light at 365 nm (12 W).
Then after photoexposure of samples, these samples were
incubated for 1 h at 37 °C. A loading buffer containing 25%
bromophenol blue, 0.25% xylene cyanol and 30% glycerol
was added, and electrophoresis was performed at 50 V for
3 h in TBE buffer using 0.8% agarose gel containing
1.0 μg ml−1 ethidium bromide. Bands were visualized using
UV light and photographed. The cleavage efficiency was
measured by determining the ability of the complex to
Results and Discussion
Characterization of metal complexes
The ligand and complexes utilized in this work have been
characterized by elemental analysis, UV/visible, IR and
magnetic susceptibility are summarized in the experimental
section. The formula of the complexes are [Fe(mqt)(phen)2]
(PF6)(1), [Fe(mqt)(dpq)2](PF6)(2) and [Fe(mqt)(dppz)2]

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Med Chem Res (2017) 26:1160–1169
(PF6)(3), respectively. These new complexes were soluble
in DMF, DMSO and in buffer (pH 7.2) solution, and stable
in the solid as well as in a solution phase. The molar con-
ductance of the complexes were in the range of 128–145
ohm−1 cm2 mol−1, demonstrating their 1:1 electrolytic nat-
ure (Mithun Roy et al. 2007).
include binding to minor groove, major groove,
sugar–phosphate backbone and intercalation between the
base pairs. The matching of the shape of the complexes and
the local DNA-binding sites has an impressive effect on the
binding mode (Zhang et al. 2001; Oleksi et al. 2006).
The UV–visible spectra of the ligands and its complexes
were recorded in DMF at 10−3 M concentration. The elec-
tronic spectrum for complexes shows a low intensity bands
Absorption spectral features of DNA binding
The mode and tendency of binding of the complexes to CT-
DNA are studied by different techniques like absorption
spectral technique, DNA thermal denaturation and viscosity
measurements.
5
ascribed to the d–d transition, arising from a T2g→5Eg
transition, which may be consistent with a high spin octa-
hedral geometry around Fe(II) ion. The complexes also
exhibit charge transfer band near 435 nm, which could be
assigned to the sulphur to Fe(II) charge transfer Ligand to
Metal Charge Transfer band (Chaudhary and Singh 2006;
Lobana et al. 1985).
The effective magnetic moment values (5.68–5.72 μB)
were slightly higher than the spin only value that may be
best described in terms of orbital contribution expected in a
high spin state of an octahedral Fe(II) ion (Chaudhary et al.
2002a).
The absorption spectra of iron complexes in absence and
presence of CT-DNA are given in Fig. 1. With increasing
CT-DNA concentration, the hypochromism is found to
increase with a red shift in the ultraviolet (UV) band of the
complexes. The complexes can bind to the DNA in various
binding mode on the basis of their structure, charge and
type of ligands. All complexes show significant hypochro-
misity of 12, 18.1 and 24.6% and minor bathochromic shift
of the spectral band of ~2–3 nm for complexes (1)–(3),
respectively (Fig. 1a, b), signifying mainly groove-binding
propensity of the complexes to the double-stranded DNA.
The intrinsic binding constant (Kb) for complexes (1),(2)
In IR spectra of the complexes, the characteristic fre-
quencies exhibit considerable changes as compared with
those of the parent ligand. The spectrum of the ligand mqt
shows a strong band at 2480 cm−1 assignable to –SH group.
The absence of this band in the spectra of the complexes
indicated the deprotonation of the –SH group on com-
plexation. The complexes (1), (2) and (3) clearly exhibited
strong band ν(C=N) in the range of 1619–1626 cm−1, which
shows a shift to the lower frequencies by 27–34 cm−1 in
comparison with ligand; metal chelates also show some new
bands in the region ν(466–478) and ν(384–410) cm−1, which
are due to formation of M–N and M–S bands, respectively
(Chaudhary and Singh 2002b; Anacona and Jofre 2008). In
addition, the IR spectrum of the PF6 salt of each complex
showed a strong band in the region 842–847 cm−1, ascrib-
able to the counter anion (Lamour et al. 1999).
and (3) were 1.9 × 103, 3.4 × 104 and 8.1 × 104 M−1
,
respectively. The dpq and dppz complexes demonstrated a
considerably higher DNA binding propensity in comparison
to their phen analog probably due to the presence of qui-
noxaline and phenazine rings of extended planar structure,
which could undergo π-stacking interactions with the DNA
bases (Nair et al. 1998).
Viscosity measurements
Viscosity measurement is a consistent method for assigning
the mode of binding of complexes to DNA. In the absence
of crystallographic structural data, hydrodynamic measure-
ment that are sensitive to increase in DNA length are con-
sidered as the least ambiguous and the most critical tests of
binding model in solution. This method is based on the fact
that, unwinding and elongation of the DNA double helix
occuring upon intercalation leads to the significant increase
in the viscosity of DNA. In contrast, complexes binding to
DNA grooves result in minor variation or no variation in the
viscosity of the DNA solution (Chetana et al. 2009).
In this current study, the DNA concentration was kept
constant and that of the metal complexes was varied. The
obtained data were presented as relative viscosity (η/η0)
(where η and η0 are the specific viscosities of DNA in the
presence and absence of the complexes) are plotted against
[Fe]/[DNA]. The effects of all complexes on the viscosity of
DNA are depicted in Fig. 2. The viscosity of DNA increases
slightly with the increasing concentration of complexes.
The 1H NMR spectra of ligand mqt was done in DMSO-
1
d6. The H NMR spectral data are reported along with the
1
possible assignments in the experimental section. The H
NMR spectra of ligand mqt exhibits multiplet signals in the
range δ(7.1–8.09) attributed to aromatic protons. The singlet
at δ 2.4 is assigned to methyl protons and another singlet at
δ 13.4 for the SH proton. The 1H NMR spectra of the
complexes cannot be obtained due to interference in their
paramagnetic properties.
DNA binding studies
The transition metal-based complexes can bind to DNA via
covalent and/or non-covalent interactions. Generally,
covalent bindings include coordination to the DNA base,
sugar and phosphate moieties. Non-covalent bindings

Med Chem Res (2017) 26:1160–1169
1165
Fig. 1 a Absorption spectral traces showing the decrease in absorption
intensity on gradual addition of CT-DNA (0–100 μM) to the solution
of Iron complexes (0.5 μM) in tris-HCl buffer. Inset shows the plot of
[DNA]/εa−εf vs [DNA] for the titration of complexes (1) with CT-
DNA. b Absorption spectral traces showing the decrease in absorption
intensity on gradual addition of CT-DNA (0–100 μM) to the solution
of Iron complexes (0.5 μM) in tris-HCl buffer. Inset shows the plot of
[DNA]/εa−εf vs [DNA] for the titration of complexes (3) with CT-
DNA
0.920
breaking of hydrogen bonding between the bases and gen-
erates a hyperchromic effect on the absorption spectra of
DNA base pairs around 260 nm. The thermal melting tem-
perature (Tm) is the temperature at which half of the DNA
strands are in the double helical state and half of that are in
the random coil. An increase in the thermal melting tem-
perature (Tm) indicates an interaction between DNA and the
metal complex (An et al. 2006).
In the present case, Tm values were determined by
monitoring the absorbance of DNA at 260 nm as a function
of temperature. A moderate positive shift in the DNA
melting temperature (ΔTm) ranging from 1.1 to 3.2 °C was
observed on the addition of the complexes (1)–(3) to DNA
(Fig. 3). This moderate increase in Tm was comparable to
that observed for the other groove binders (Fu et al. 2003).
The results of thermal denaturation experiments presented
here are consistent with the absorption spectral profiles and
viscosity measurements.
Complex (1)
0.915
0.910
0.905
0.900
0.895
0.890
0.885
Complex (2)
Complex (3)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Complex/[DNA]
Fig. 2 Plot of relative viscosity vs [Complex]/[DNA]. Effect of
Fe(II) complexes on the viscosity of CT-DNA at 25 °C. Complex =
0–100 μM, [DNA] = 50 μM
So these complexes do not elongate DNA helix length. This
viscosity result suggests the groove-binding nature of the
complexes (Wu et al. 2005).
Photo-induced DNA cleavage activity
The DNA cleavage reactions generally proceed via oxi-
dative and hydrolytic pathways. The oxidative cleavage
results in the nucleobase oxidation and/or degradation of
sugar moiety by abstraction of sugar hydrogen atom(s),
while the hydrolytic cleavage of DNA involves hydrolysis
Thermal denaturation study
DNA-melting is the process by which double-stranded
DNA gradually dissociates into single strands through the

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Med Chem Res (2017) 26:1160–1169
CT-DNA
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
Complex (1)
Complex (2)
Complex (3)
Fig. 4 Gel electrophoresis diagram showing the photocleavage of SC
pUC19 DNA (0.5 μg) by complex (1) (50 μM) 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 µM); lane 4, DNA + (1) +
DMSO (4 µL); lane 5, DNA + (1) + D2O (14 µL)
40 45 50 55 60 65 70 75 80 85
Temperatureo
C
Fig. 3 Thermal denaturation of CT-DNA in the absence and presence
of Fe(II) complexes. [DNA] = 50 μM, [complex] = 10 μM, buffer:
phosphate
Fig. 5 Gel electrophoresis diagram showing the photocleavage of SC
pUC19 DNA (0.5 μg) by complex (2) (50 μM) and other additives at
365 nm for an exposure time of 1 h. lane 1, DNA control; lane 2, DNA
+ (2); lane 3, DNA + (2) + NaN3 (100 µM); lane 4, DNA + (2) +
DMSO (4 µL); lane 5, DNA + (2) + D2O (14 µL)
of the phosphodiester bond. The oxidative cleavage can
occur by chemical or photochemical means. The source of
light is used in photo-induced oxidative DNA cleavage
instead of any external additives like reducing or oxidiz-
ing agents that are necessary for observing “chemical
nuclease” activity and it has advantages in therapeutic
applications.
The photocleavage activity of SC pUC 19 DNA was
performed by gel electrophoresis to study the potential of
the Fe(II) complexes to act as metallonucleases. The SC
DNA is subjected to electrophoresis, relatively fast
migration will be observed for DNA of closed circular
confirmation (Form I). If one strand is cleaved, the SC
DNA will relax to generate a slower moving NC form
(Form II). If both strands are cleaved, a linear confirma-
tion (Form III) that migrates between Form I and Form II
will be generated (Sudhamani et al. 2012b). The photo-
induced DNA cleavage activity of the complexes has been
carried out in the presence of UV light (365 nm, 12 W)
using a 50 µM concentration of the complexes and SC
pUC19 DNA (0.5 µg) reagent. Figs. 4–6 illustrate the gel
electrophoretic results of the interaction of complexes
with pUC19 DNA. It has been observed that complexes
(2) and (3) cleave DNA effectively with the formation of
both nicked-circular and linear DNA in comparison to
their phen analog, which showed the formation of only
NC form under similar experimental conditions. The
cleavage efficiency follows the order (3) > (2) > (1). The
different DNA-cleavage efficiency of these three com-
plexes was due to the different binding affinity of the
complexes to DNA.
Fig. 6 Gel electrophoresis diagram showing the photocleavage of SC
pUC19 DNA (0.5 μg) by complex (3) (50 μM) and other additives at
365 nm for an exposure time of 1 h. lane 1, DNA control; lane 2, DNA
+ (3); lane 3, DNA + (3) + NaN3 (100 µM); lane 4, DNA + (3) +
DMSO (4 µL); lane 5, DNA + (3) + D2O (14 µL)
Scheme 2 Mechanistic pathways for the cleavage of DNA on
photoirradiation
quencher like sodium azide inhibits the cleavage reaction
of the Fe(II) complexes as seen in line 3 indicated gen-
eration of 1O2, which plays a key role in photo cleavage of
pUC 19 DNA. An enhancement of photo-cleavage of
DNA is observed in D2O (lane 5) in which 1O2 has longer
1
life time that further supports formation of O2 (Prabha-
kara et al. 2007b). Hydroxyl radical scavengers DMSO
did not show significant inhibition in DNA cleavage (lane
4). These results clearly point out a type-II process in
which the photoexcited complexes activate molecular
oxygen from its stable triplet to cytotoxic singlet state
(Scheme 2) (Fig. 7).
The mechanism involved in the DNA cleavage upon
photoirradiation was explored in the presence of various
additives in UV light at 360 nm. A singlet oxygen

Med Chem Res (2017) 26:1160–1169
1167
Fig. 8 CFU reduction (%) in E. coli after the treatments
Fig. 7 Cleavage of SC pUC19 DNA (50 μM) 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)
complexes upto 50 µg ml−1. E. coli show an apparent dose-
dependent lethality when irradiated after the exposure to the
different complexes at 25 and 50 µM concentrations. By
counting colony-forming unit (CFU) of E. coli remaining in
the control, complexes (1), (2) and (3) alone and photo-
irriadiated complexes at different concentrations were made
to determine the effectiveness of PDT. The results of PDT on
E. coli in terms of CFU counts (%) after the treatment can be
seen in Fig. 8. The % survival statistical analysis for E. coli
revealed a nonsignificant reduction of CFU counts between
the control and complexes of 8.1, 10.4, 13.8% for complexes
(1), (2) and (3), respectively. However, a significant reduc-
tion of CFU counts by 58.8, 64.2, 72.5% for 25 µM con-
centration and 70.1, 74.6, 80.8 for 50 µM concentration was
observed for the photoirradiated complexes (1), (2) and (3),
respectively when compared with the control group.
In the control group, a significantly higher (p < 0.06)
bacterial growth (108 CFU ml−1) was exhibited, while the
complexes alone presented no statistically significant (p >
0.06) bacteria reduction, indicating that the complexes alone
seem not to significantly reduce the bacterial viability.
However, all the photo-irradiated complexes were sensitive
to E. coli for the PACT assay, presenting a significantly low
bacterial growth. There was a significant reduction in the
CFU counts (p < 0.06) compared with the initial numbers
recorded in both control and complexes.
PACT assay
The iron complexes (1), (2) and (3) were examined for
their ability to kill E. coli on photoirradiation by a
tungsten–halogen 500 W lamp. The effect of cell viability
on irradiation of visible light emitted by a 500 W
halogen–tungsten lamp was probed to obtain reliable data
about the efficiency of the photodynamic treatment on E.
coli. When E. coli cultures (108 cfu ml−1), in the absence of
complexes, were illuminated for 60 min, no considerable
decrease in the number of viable cells was observed. Thus,
irradiation time was maintained for 60 min in all
PACT assays. To standardize the time of incubation
with the complexes before light exposure, suspensions
(108 cfu ml−1) of the bacteria were incubated with the
complexes in the dark for 30, 60 and 90 min, washed once
in phosphate buffered saline 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 0.053, 0.052, 0.054% for complex (1), 0.074, 0.075,
0.072% for complex (2) and 0.052, 0.053, 0.054% 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 complexes against the above mentioned bacteria. So, for
all photoinactivation experiments of iron complexes under
study, a 30 min incubation period was maintained.
Conclusions
The protocol for photodynamic treatment of E. coli tested
was then further standardized evaluating the effects of
Three novel iron(II) complexes having N,S donor of mqtH
and N, N-donor heterocyclic base(B) are synthesized and

1168
Med Chem Res (2017) 26:1160–1169
Chaudhary A, Jaroli DP, Singh RV (2002a) Antimicrobial, antifertility
and antiinflammatory approach to tetradentate macrocyclic com-
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structurally characterized. The planarity and extended con-
jugation of the phenanthroline bases have a profound effect
on the DNA binding and cleavage activity of the com-
plexes. Binding study shows a minor groove-binding nature
of the iron(II) complexes. The significant results include our
observation of biologically important photoinduced DNA
double-strand breaks leading to linear DNA formation due
to the photosensitizing effect of both dpq and dppz.
1
Mechanistic studies reveal direct involvement of O2 in the
photo-cleavage process. Pathways involving singlet oxygen
in the DNA photocleavage reactions are proposed from the
observation of the complete inhibition of the cleavage in
presence of NaN3 and enhancement of cleavage in D2O.
The sulphur containing ligands with their longer triplet state
lifetimes are suitable for activation of molecular oxygen.
We are making further efforts to understand the DNA
cleavage ability of iron complexes in longer wavelength
(PDT window). These results may throw light towards the
design and development of iron-based complexes with
sulfur containing ligands for PDT applications and as
alternatives to the PDT anticancer drug.
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Acknowledgements This work has been supported by providing
Post Doctoral Fellowship for women (2009–10 & 2010–11) from the
University Grants Commission, New Delhi.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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