DNA binding, prominent photonuclease activity and antibacterial PDT of cobalt(II) complexes of phenanthroline based photosensitizers

Article abstract of DOI:10.1080/15257770.2018.1508691

The chemistry of Co(II) complexes showing efficient light induced DNA cleavage activity, binding propensity to calf thymus DNA and antibacterial PDT is summarized in this article. Complexes of formulation [Co(mqt)(B)₂]ClO₄ 1–3 where mqt is 4-methylquinoline-2-thiol and B is N,N-donor heterocyclic base, viz. 1,10-phenanthroline (phen 1), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq 2) and dipyrido[3,2-a:2′,3′-c]phenazine (dppz 3) 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 1.6 × 10³ M^?1, 1.1 × 10⁴ M^?1 and 6.4 × 10⁴ M^?1 respectively. The experimental results suggest that these complexes interact with DNA through groove binding mode. The complexes show significant photocleavage of supercoiled (SC) DNA proceeds via a type-II process forming singlet oxygen as the reactive species. Antimicrobial photodynamic therapy was studied using photodynamic antimicrobial chemotherapy (PACT) assay against E. coli and all complexes exhibited significant reduction in bacterial growth on photoirradiation.

Full text of DOI:10.1080/15257770.2018.1508691

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
DNA binding, prominent photonuclease activity
and antibacterial PDT of cobalt(II) complexes of
phenanthroline based photosensitizers
Chittanahalli N. Sudhamani, Halehatty S. Bhojya Naik, Kalligundi R.
Sangeetha Gowda, Dugganna Girija & Manju Giridhar
To cite this article: Chittanahalli N. Sudhamani, Halehatty S. Bhojya Naik, Kalligundi R. Sangeetha
Gowda, Dugganna Girija & Manju Giridhar (2018): DNA binding, prominent photonuclease
activity and antibacterial PDT of cobalt(II) complexes of phenanthroline based photosensitizers,
Nucleosides, Nucleotides and Nucleic Acids, DOI: 10.1080/15257770.2018.1508691
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2018.1508691
DNA binding, prominent photonuclease activity and
antibacterial PDT of cobalt(II) complexes of
phenanthroline based photosensitizers
Chittanahalli N. Sudhamani, Halehatty S. Bhojya Naik, Kalligundi R. Sangeetha
Gowda, Dugganna Girija, and Manju Giridhar
Department of Studies and Research in Industrial Chemistry, School of Chemical Sciences,
Kuvempu University, Shankaraghatta, India
ABSTRACT
ARTICLE HISTORY
Received 15 September 2015
Accepted 31 July 2018
The chemistry of Co(II) complexes showing efficient light
induced DNA cleavage activity, binding propensity to calf thy-
mus DNA and antibacterial PDT is summarized in this article.
Complexes of formulation [Co(mqt)(B)2]ClO4 1–3 where mqt is
4-methylquinoline-2-thiol and B is N,N-donor heterocyclic
base, viz. 1,10-phenanthroline (phen 1), dipyrido[3,2-d:20,30-
f]quinoxaline (dpq 2) and dipyrido[3,2-a:20,30-c]phenazine
(dppz 3) 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 com-
KEYWORDS
Groove binding; singlet
oxygen; photocleavage;
photoirradiation
plexes 1, 2 and 3 were determined to be 1.6 ꢀ 103 Mꢁ1
,
1.1 ꢀ 104 Mꢁ1 and 6.4 ꢀ 104 Mꢁ1 respectively. The experimental
results suggest that these complexes interact with DNA
through groove binding mode. The complexes show signifi-
cant photocleavage of supercoiled (SC) DNA proceeds via a
type-II process forming singlet oxygen as the reactive species.
Antimicrobial photodynamic therapy was studied using photo-
dynamic antimicrobial chemotherapy (PACT) assay against E.
coli and all complexes exhibited significant reduction in bac-
terial growth on photoirradiation.
Introduction
Exploration of the interaction of DNA with small molecules is basic work
for the new types of pharmaceutical molecules that are capable of binding
and cleaving DNA, which is related to their utility in the design and devel-
opment of new drugs. The interactions of metal complexes with DNA rep-
resent a significant area of research which creates a center of attention
from both inorganic chemists and biochemists.[1–4] In this regard, the
mixed-ligand metal complexes have been found to be primarily useful
CONTACT Halehatty S. Bhojya Naik
hsb_naik@rediffmail.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/15257770.2018.1508691.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.
ß 2018 Taylor & Francis Group, LLC

2
C. N. SUDHAMANI ET AL.
because of their potential to bind DNA via a multitude of interactions and
to cleave the duplex by virtue of their intrinsic chemical, electrochemical
and photochemical reactivities.[5–9] Recently, it has been revealed that
organic moieties having nitrogen and sulfur atom that plays a vital role in
view of the wide spectrum of medicinal applications. In fact, several metal
complexes containing sulfur-nitrogen chelating agent have been shown to
cytotoxic activity.
Photodynamic therapy (PDT) has materialized in recent years as a non-
invasive therapeutic modality for the cure of various infections by bacteria,
fungi and viruses, in addition to variety of cancers.[10–13] This therapy is
characterized as an oxygen dependent photochemical reaction that occurs
upon light–mediated activation of a photosensitizing compound leading to
the generation of cytotoxic reactive oxygen species, predominantly singlet
oxygen.[14] Owing to increase of resistance to antibiotic drugs, researchers
are searching for new antibacterial agents. In this direction, photodynamic
antimicrobial chemotherapy (PACT) is an alternate antibacterial, antifungal
and antiviral treatment against drug-resistant organisms.[15]
Currently, the study of cobalt complexes is of immense interest since
cobalt complexes have similar interesting characteristics and DNA cleaving
properties as ruthenium complexes, but have not received as much atten-
tion as the ruthenium (II) systems.[16] Cobalt metal is present in the active
sites of several important classes of metalloproteins and these complexes
have attained greater importance because of their application as potential
hypoxia-activated prodrugs.[17–21] Co(II) ion has been used as significant
spectroscopic probe for proteins that contain an N, S ligated metal center.
Redox-active agents like cobalt polypyridyl complexes that damage DNA in
vitro might confirm apoptotic activities in living systems by inducing oxi-
dative stress and/or damage of DNA. So the complexes may cleave DNA
with producing reactive oxygen species in vitro, and could be prospective
anticancer drugs by killing cancer cells.[22–26]
The current work stems from our interest to design and develop redox
active metal complexes that are capable to bind and cleave DNA photolyti-
cally. We have designed stable mononuclear cobalt(II) based complexes
having DNA binder phenanthroline bases and a photoactive ligand having
a sulfide moiety to achieve DNA photocleavage activity and perhaps
become a photoactive component for PACT.
Experimental
Material and methods
All reagents and solvents required were of AR grade, procured from
Himedia (India) Laboratories Pvt. Ltd. The solvents were purified by

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
distillation. The [Co(H2O)6](NO3)2, 1,10-phenanthroline and Tris-HCl were
purchased from Merck (India). Calf thymus (CT) DNA and supercoiled
(SC) pUC19 DNA were purchased from Bangalore Genie (India) and
Agarose (molecular biology grade) ethidium bromide were purchased from
Himedia (India). D2O was purchased from Sigma Aldrich. Tris-HCl buffer
solution used for binding was prepared using deionized double-distilled
water. Tris-borate ethylenediamine tetraacetate (TBE) electrolyte buffer uti-
lized for DNA cleavage study.
The electronic spectra of the complexes were acquired by Shimadzu spec-
trometer model UV-1650 PC double beam spectrophotometer. 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 molar conductivities of the complexes in
dimethylformamide (DMF) solution (10ꢁ3 M) at room temperature were
carried out by an Equip-Tronic (India) Digital conductivity meter model
No. EQ-660A. Magnetic susceptibilities of the solid complexes were deter-
mined by employing Gouy balance at room temperature (28 2 ꢂC).
Viscosity measurements were carried out on semimicrodilution capillary
viscometer at room temperature. Infrared (IR) spectra were recorded in the
range of 4000–250 cmꢁ1 using KBr pellets on Shimadzu (Kyoto, Japan)
1
FTIR-8400. H-NMR spectra were obtained using a Bruker FT NMR spec-
trometer (300 MHz) at 25 ꢂC in DMSO-d6 with TMS as the internal refer-
ence. A monochromatic 12 W UV lamp of 365 nm was used as light source
for photonuclease activity.
Synthesis of methylquinoline-2-thiol
The initial compounds 2-hydroxy-4-methylquinoline and 2-chloro-4-meth-
ylquinoline were prepared by reported procedures.[27] An ethanolic solution
of 2-chloro-4-methylquinoline was mixed with sodium sulfide in a 1:3 ratio
and refluxed for about 4 hours in the presence of a catalytic amount of
HCl. The completion of the reaction was monitored by TLC (ethyl acetate:
carbon tetrachloride 8:2). The reaction mixture was poured into ice cold
water and neutralized, the product was filtered and recrystallized.
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. kmax (nm): 281, 380, IR (KBr) cmꢁ1:
1
1653 ꢀ(C ¼ N), 2480 ꢀ(C-SH) ; HNMR(d ppm): 7.1-8.09 (m, ArH, 5H),
2.4 (s, 3H, CH3), 13.4 (s, SH), M.P. 196 ꢂC, M.W. 175.26.
The dpq ligand (dipyrido[3,2-d:20,30-f]quinoxaline) and dppz ligand
dipyrido[3,2-a:20,30-c] phenazine were synthesized according to the method
reported earlier.[28,29]

4
C. N. SUDHAMANI ET AL.
Synthesis of complexes
[Co(mqt)(B)2]ClO4 1-3 where mqt is 4-methylquinoline-2-thiol and B is N,N-
donor heterocyclic base, 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)
A general procedure was adopted for the preparation of all the com-
plexes. A methanolic solution of [Co(H2O)6](NO3)2 (1 mmol, 0.30 g) and
mqt (1 mmol, 0.175 g) was stirred for 1 hour. To the above reaction mix-
ture, 1 mmol of 10 ml methanolic solution of the heterocyclic base was
added. The resulting solution on refluxing for 30 min to afford red solu-
tion. This solution was treated with a methanolic solution of NaClO4
(0.18 g, 1.5 mmol) to precipitate a brick red soild. The solid was isolated by
filtration and washed with cold methanol followed by diethyl ether and
finally dried in a vacuum desiccator over P2O5.
[Co(mqt)(phen)2]ClO4 (1). Yield 74%, Anal. Calc. for C34H24ClCoN5O4S (%):
C, 58.92; H, 3.49; N, 10.11. found (%): C, 58.89; H, 3.51; N, 10.11. IR
(KBr) cmꢁ1: 2935 s, 1596 s, 1086 vs(ClO4), 466 m, 384 m. UV-Visible. kmax
(nm): 272, 311, 409, 518. meff ¼ 5.12 B.M. XM ¼ 76 mhos cm2 molꢁ1.
M.P. 391 ꢂC.
[Co(mqt)(dpq)2]ClO4 (2). Yield 70%, Anal. Calc. for C38H24ClCoN9O4S (%):
C, 57.26; H, 3.03; N, 15.81. found (%): C, 57.27; H, 2.98; N, 15.83. IR (KBr)
cmꢁ1: 2910 s, 1600 s, 1091 vs(ClO4), 481 m, 398 m. UV-Visible. kmax(nm):
268, 348, 452, 546. meff ¼ 4.8 B.M. XM ¼ 68 mhos cm2 molꢁ1 . M.P. 426 ꢂC.
[Co(mqt)(dppz)2]ClO4 (3). Yield 68%, Anal. Calc. for C46H28ClCoN9O4S (%):
C, 61.58; H, 3.15; N, 14.05. found (%): C, 61.55; H, 3.17; N, 14.05. IR
(KBr) cmꢁ1: 2894 s, 1596 s, 1082 vs(ClO4), 481 m, 406 m. (m, medium; s,
strong; vs, very strong; w, weak) UV-Visible. kmax (nm): 276, 366, 455, 576.
meff ¼ 4.91B.M. XM ¼ 81 mhos cm2 molꢁ1. M.P. 441 ꢂC.
Caution! Perchlorate salts are potentially explosive and only small
quantity was handled with care.
DNA binding experiments
Absorption spectroscopic studies
The concentration of CT-DNA per nucleotide [C(p)] was measured using
its known extinction coefficient at 260 nm (6600 Mꢁ1 cmꢁ1).[30] The absorb-
ance at 260 nm (A260) and 280 nm (A280) for CT-DNA were measured in
order to check the purity level. The ratio A260/A280 was found to be 1.8 to
1.9, representing that CT-DNA was satisfactorily free from protein. Buffer
(5 mM tris (hydroxymethyl) aminomethane, pH 7.2, 50 mM NaCl) was
used for the absorption experiments.[31]

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
UV ꢁ Visible absorption titration experiments were performed by varying
DNA concentration (0 to 100 lM) and maintaining the metal-complex con-
centration constant (0.5 lM). 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 Co(II) complexes, the observed
data were then interpreted by using Equation (1), in order to obtain the
intrinsic binding constant, Kb:
½
ꢃ
½
ꢃ
DNA = ea ꢁ ef ¼ DNA = eb ꢁ ef þ 1=Kb eb ꢁ ef
(1)
ð
Þ
ð
Þ
ð
Þ
Where ea, ef, and eb are the apparent, free and bound metal complex
extinction coefficients respectively. A plot of [DNA]/(ebꢁef) versus [DNA]
gave a slope of 1/(ebꢁef) and a y intercept equal to 1/Kb(ebꢁef), where Kb
is the ratio of the slope to the y intercept.[32]
Viscosity measurements
To understand the nature of DNA binding of the mixed ligand metal com-
plexes, 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. Relative specific viscosity was calculated according to Cohen
and Eisenberg Plots of (g/go)1/3 (g and go are the relative specific viscos-
ities of DNA in the presence and absence of the drug) vs. [drug]/[DNA]
were constructed. Data were presented as (g/go) vs binding ratio, where g
is the viscosity of DNA in the presence of complex and go is the viscosity
of DNA alone in 5 mM Tris–HCl buffer medium.[33]
Thermal denaturation
DNA thermal denaturation studies were carried out by monitoring the
absorption intensity of the CT DNA (50 mM) at 260 nm varying the tem-
perature from 45 to 85 ꢂC, both in the absence and presence of the com-
plexes in 1 mM phosphate buffer (pH 7.0).[34]
DNA photocleavage experiments
The degree of photoinduced cleavage of supercoiled (SC) pUC19 DNA by
the complexes was analyzed by monitoring the conversion of supercoiled
DNA (Form I) to nicked DNA (Form II) and linear DNA (Form III) using
an agarose gel electrophoresis technique. Each pUC19 DNA sample (0.5 lg)
was in 50 mM Tris-HCl buffer (pH, 7.2; ionic strength, 25 mM) containing
50 mM NaCl in the presence of the complex (50 lM), followed by dilution
with Tris-HCl buffer to a total volume of 20 lL. For DNA photonuclease
activity, the reactions were carried out under illuminated conditions using

6
C. N. SUDHAMANI ET AL.
UV light at 365 nm (12 W) for 1 h. After photoexposure of samples under
UV light, these samples were incubated for 1 h at 37 ꢂC followed by add-
ition to the loading buffer containing 25% bromophenol blue, 0.25% xylene
cyanol and 30% glycerol and the solution was finally loaded on 0.8% agar-
ose gel containing 1.0 lg/ml ethidium bromide. Electrophoresis was carried
out in a dark chamber for 3 h at 50 V in Tris-borate buffer (45 lmol/L
Tris-borate, 1 mmol/L EDTA) TBE buffer. The data were documented using
a UVITEC Gel Documentation center.
The inhibition reactions were carried out in the presence of different
additives (NaN3, 100 mM; DMSO, 4 mL; D2O 14 mL) at 365 nm for mechan-
istic insights of the photo-induced DNA cleavage reactions.These reactions
were done by adding reagents to SC DNA prior to addition of the complex
before photoirradiation.[35,36]
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 ꢂC.
PACT assay
The PACT assay was performed according to the procedure reported else-
where.[37] 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 25 mM and
50 mM concentrations of the cobalt complexes under investigation, incu-
bated in foil-covered tubes to avoid accidental exposure to the light. 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 phosphate buffer saline. The flasks were placed under a 500 W halogen–-
tungsten lamp at a distance of 20 cm. During light exposure, cell suspen-
sions were maintained under gentle magnetic stirring. Control cultures,
with no cobalt complexes added, were exposed to the light for the same
period as photosensitizer treated cultures. Aliquots of undiluted and serially
diluted treated and control cultures were plated on LB agar medium. The
percentage of survivors was determined according to the equation N1/
N0 ꢀ 100, where N0 represents the number of cfu/ml in samples before the
irradiation and N1 is the number of cfu/mL after light exposure.
Results and discussion
Characterization of metal complexes
The ligand and complexes employed in this work have been characterized by
elemental analysis, UV-visible, IR and magnetic susceptibility which are
summarized in the experimental section. The formula of the complexes are

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
[Co(mqt)(phen)2]ClO4 1, [Co(mqt)(dpq)2]ClO4 2, [Co(mqt)(dppz)2]ClO4 3
respectively. These novel 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 conductance values of the complexes in DMF solutions fall in the
range 68–81 ohmꢁ1 cm2 molꢁ1, demonstrating their electrolytic nature.
The IR spectra of the ligands were compared with those of the metal
complexes in order to confirm the binding mode of the ligands to the cor-
responding metal ion in the complexes. The IR spectrum of the free ligand
mqt exhibits a strong band at 2480 cmꢁ1, due to the –SH group. The
absence of this band in the spectra of the complexes indicated the deproto-
nation of the –SH group on complexation. The band due to ꢀ(C ¼ N) in
the range of 1596–1600 cmꢁ1 suffers a negative shift by 53–57 cmꢁ1 from
free ligands suggesting the involvement of the quinoline nitrogen in the
coordination. In addition, these complexes show new bands in the region
466–481 cmꢁ1 and 384–406 cmꢁ1 which are due to the formation of M-N
and M-S bands respectively.[38–40] All complexes display the characteristic
infrared band for the perchlorate anion in the region 1082–1091 cmꢁ1.[41]
1
1
The H NMR spectra of ligand mqt was done in DMSO-d6. The H
NMR spectral data are reported along with the possible assignments in the
1
experimental section. The H NMR spectra of ligand mqt exhibits multiplet
signals in the range d(7.1–8.09) attributed to aromatic protons. The singlet
[39]
at d 2.4 is assigned to methyl protons
and another singlet at d 13.4 for
the SH proton.[42] The H NMR spectra of the complexes cannot be
1
obtained due to interference in their paramagnetic properties.
The electronic spectra of Co(II) complexes show absorption bands in the
ꢄ
region of 272–366 nm that are attributed to intraligand p–p transition and
in the region of 409–455 nm assigned for n–p . On the other hand complexes
ꢄ
show two absorption bands at 518–576 nm region, which were assignable to
4T1g(F)! A2g(F) and T1g(F) ! T2g(F) transitions in an octahedral environ-
ment.[43,44] The magnetic susceptibility values were found to be 4.8, 4.91 and
5.12 B.M for complexes 1, 2 and 3, respectively. This is comparable with that
of 4.3–5.2 B.M expected for six coordinated octahedral Co(II) complexes,
indicative of octahedral geometry for the present Co(II) complexes.[45,46]
Based on the analytical data and physicochemical properties, the follow-
ing structure was proposed for the complexes (Figure 1).
4
4
4
DNA binding studies
Absorption spectral features of DNA binding
To establish the DNA binding propensity of metal complexes, different
techniques like absorption spectral technique, thermal denaturation and
viscometric titration experiments have been widely employed.

8
C. N. SUDHAMANI ET AL.
Figure 1. Structures of Co(II) complexes.
Electronic absorption spectroscopy is the foremost technique for the
assessment of the mode of interaction of metal complexes with CT-DNA.
The spectroscopic titrations of complexes in solutions with CT-DNA were
carried out and absorption spectra of the complexes in the absence and pres-
ence of calf thymus DNA are illustrated in Figure 2a and b. The successive
addition of DNA led to increasing of hypochromism in the absorption inten-
sities with a small red shift. The complexes can bind to the DNA in different
binding mode on the basis of their structure, charge and type of ligands. The
percentage hypochromism of all the complexes were found to be 10.2, 16.6
and 21.4% and a minor bathochromic shift of the spectral band of ꢅ2–3 nm
for complexes 1–3 was observed respectively, signifying mainly the groove-
binding propensity of the complexes to the double-stranded DNA.[47]
In order to compare quantitatively the binding strength of these com-
plexes, the intrinsic binding constant Kb was determined by monitoring the
changes in absorbance of the complexes with increasing concentration of
CT-DNA. The observed intrinsic binding constants Kb of complexes 1, 2 and
3 were 1.6 ꢀ 103 Mꢁ1, 1.1.ꢀ104 Mꢁ1 and 6.4 ꢀ 104 Mꢁ1, respectively. The
dpq and dppz complexes demonstrate a considerably higher DNA binding
propensity in comparison to their phen analog probably due to the presence
of quinoxaline and phenazine rings of extended planar structure.[48]
Viscosity measurements
Optical photophysical titration normally offers necessary information, but
not satisfactory evidence for the binding mode of the complexes with

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
Figure 2. Absorption spectra of cobalt complex in Tris–HCl buffer upon addition of DNA.
[Complex] ¼ 0.5 lM, [DNA] ¼ (0–100 lM). Arrow shows the absorbance changing upon increase
of DNA concentration. The inner plot of [DNA]/eaꢁef vs [DNA] for the titration of DNA with
complex. (a) complex 1 and (b) complex 2.
DNA. The viscosity studies provide strong evidence for intercalation or
groove binding. A classical intercalation model demands increase in the
DNA helix length as base pairs are separated to accommodate the binding
ligand, leading to an increase of DNA viscosity. In contrast, complexes
binding to DNA grooves result in minor variation or no variation in the

10
C. N. SUDHAMANI ET AL.
viscosity of the DNA solution.[49,50] To throw further light on the DNA
binding mode, the viscometric titration experiments were carried out on
CT DNA by varying the concentration of added complexes 1, 2 and 3. In
this present study, the DNA concentration was kept constant and that of
the metal complexes was varied. The obtained data were presented as rela-
tive viscosity (g/g0) (where g and g0 are the specific viscosities of DNA in
the presence and absence of the complexes) are plotted against [Co]/
[DNA]. The effects of all complexes on the viscosity of DNA were depicted
in a Supplementary file (Figure S1). The viscosity of DNA increases slightly
by increasing the concentration of complexes. So these complexes do not
elongate DNA helix length. These viscosity results suggested the groove-
binding nature of the complexes[51] and are consistent with the results of
the electronic absorption spectroscopic study.
Thermal denaturation study
The DNA melting experiments were performed to investigate the effect of
DNA duplex stability due to binding of the complexes. Duplex DNA at its
melting temperature unwinds to give single strand DNA, thus increasing its
absorbance at 260 nm. Thus, the helix to coil transition temperature can be
determined by monitoring the absorbance of DNA at 260 nm as a function
of temperature. The thermal melting temperature (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 tempera-
ture indicates an interaction between DNA and the metal complex.
In the current case, the thermal denaturation profile of DNA in the
absence and presence of Co(II) complexes is provided in Supplementary
file (Figure S2). A change in the DNA melting temperature was observed
on addition of the complexes to CT ꢁ DNA and their absorbance moni-
tored at 260 nm. A moderate positive shift in the DNA melting temperature
(DTm) ranging from 1.3 to 2.8 was observed on the addition of the com-
plexes 1–3 to DNA. This moderate increase in Tm value emphasizes that
these complexes were comparable with the other groove binders.[52–54] The
results of thermal denaturation experiments presented here are consistent
with the absorption spectral profiles and viscosity measurements.[55]
Photo-induced DNA cleavage activity
The Photoneuclease activity of supercoiled pUC 19 DNA was executed by
gel electrophoresis to study the potential of the Co(II) complexes as metal-
lonucleases. DNA cleavage was analyzed by monitoring the conversion of
supercoiled DNA (Form I) to nicked DNA (Form II) and linear DNA
(Form III). The photo-induced DNA cleavage activity of the complexes has

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
Figure 3. Gel electrophoresis diagram showing the photocleavage of SC pUC19 DNA (0.5 lg)
by complex 1 (50lM) and other additives at 365 nm for an exposure time of 1 h. lane 1, DNA
control; lane 2, DNA þ 1; lane 3, DNAþ 11NaN3 (100 mM); lane 4, DNAþ 11 DMSO (4 mL); lane
5, DNAþ11 D2O (14mL).
Figure 4. Gel electrophoresis diagram showing the photocleavage of SC pUC19 DNA (0.5 lg)
by complex 2 (50 lM) and other additives at 365 nm for an exposure time of 1 h. lane 1, DNA
control; lane 2, DNA þ 2; lane 3, DNAþ 21NaN3 (100 mM); lane 4, DNAþ 21 DMSO (4 mL); lane
5, DNAþ 21 D2O (14mL).
Figure 5. Gel electrophoresis diagram showing the photocleavage of SC pUC19 DNA (0.5 lg)
by complex 3 (50 lM) and other additives at 365 nm for an exposure time of 1 h. lane 1, DNA
control; lane 2, DNA þ 3; lane 3, DNAþ (3)1NaN3 (100 mM); lane 4, DNAþ 31 DMSO (4 mL);
lane 5, DNAþ 31 D2O (14mL).
been performed in the presence of UV light (365 nm, 12 W) using a 50 mM
concentration of the complexes and super-coiled pUC19 DNA (0.5 mg)
reagent. Figures 3–5 illustrate the gel electrophoretic consequences of the
interaction of complexes with pUC19 DNA. The nucleolytic efficiencies of
the complexes 2 and 3 are better than complex 1 with the formation of
both nicked-circular and linear DNA in comparison to their phen analog,
which showed the formation of only nicked circular (NC) form under simi-
lar experimental conditions. The chemical environment and their geometric
structures may also affect the nucleolytic efficiency of cobalt complexes.[56]
Therefore, the different DNA-cleavage efficiency of these three complexes
was due to the different binding affinity of the complexes to DNA and fol-
lows the order 3 > 2> 1.
The mechanistic aspects of the DNA cleavage reactions of all the com-
plexes were explored in the presence of various additives in UV light at
360 nm (Supplementary file Figure S3). A singlet oxygen quencher like

12
C. N. SUDHAMANI ET AL.
Scheme 1. Mechanistic pathways for the cleavage of DNA on photoirradiation.
sodium azide inhibits the cleavage reaction of the Co(II) complexes as seen
in lane 3 indicated generation of 1O2 plays a key role in photo cleavage of
pUC 19 DNA. Hydroxyl radical scavengers DMSO did not show any
inhibitory effect, thus excluding the possibility of a type I pathway forming
hydroxyl radical (ꢁOH) (lane 4). This can be further confirmed by testing
the cleavage of supercoiled (SC) pUC19 DNA in the presence of D2O. Due
to the longer lifetime of singlet oxygen in D2O the expected strand scission
would be great in D2O.[57,58] The formation of singlet oxygen is addition-
ally supported by the enhancement of the percentage of SC DNA cleavage
in D2O (lane 5). The reason for the enhanced cleavage ability of these com-
1
plexes may be accredited to the generation O2 during the photocleavage
process Scheme 1.
PACT assay
The cobalt 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)
alone, in the absence of complexes were illuminated for 60 min, no consid-
erable decrease in the number of viable cells was observed. Thus, irradi-
ation time was maintained for 60 min in all PACT assays. To standardize
the time of incubation with the complexes before light exposure, bacterial
suspensions (108 cfu/mL) were incubated with the complexes in dark for
30, 60 and 90 min, washed once in phosphate buffer saline and then irradi-
ated with light for the period previously determined. After the light treat-
ment the percentage of survivors for E. coli were calculated to be 0.064%,
0.062%, 0.063% for complex 1, 0.075%, 0.072%, 0.074% for complex 2 and
0.062%, 0.064%, 0.066% for complex 3. Consequently, results suggested
that, increasing the time of the contact between the complexes and the bac-
terial cells did not improve the performance of the complexes against the
above mentioned bacteria. So, for all photoinactivation experiments of
cobalt complexes under study a 30 min incubation period was maintained.
The protocol for photodynamic treatment of E.coli tested was then fur-
ther standardized evaluating the effects of complexes upto 50 mg/ml. E.coli
exhibited apparent dose-dependent lethality when irradiated after the
exposure to the different complexes at 25 mM and 50 mM concentrations.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
By counting colony forming unit (CFU) of E.coli remaining in the control,
complexes (1, 2 and 3) alone and photoirriadiated complexes at different
concentrations were carried out to determine the effectiveness of PDT[37]
.
The results of PDT on E.coli in terms of CFU counts (%) after treatment
can be seen in a supplementary file (Figure S4). The % survival statistical
analysis for E.coli revealed a nonsignificant reduction of CFU counts
between the control and complexes of 8.4%, 11.6% and 14.1% for com-
plexes 1, 2 and 3, respectively. However, a significant reduction of CFU
counts by 58.8%, 64.2%, 72.5% for 25 mM concentration and 70.1%, 74.6%,
80.8% for 50 mM concentration were observed for the photoirradiated com-
plexes 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) was exhibited, while the complexes alone presented no stat-
istically significant (p > 0.06) bacteria reduction, indicating that the com-
plexes alone seems not to significantly reduce 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 signifi-
cant reduction in the CFU counts (p < 0.06) compared with the initial
numbers recorded in both control and complexes.
Conclusions
From the studies detailed herein, we have synthesized the cobalt(II) com-
plexes of type [Co(mqt)(B)2]ClO4 as a novel class of DNA photocleavers.
The planarity and extended conjugation of the phenanthroline bases have a
strong effect on the DNA binding and cleavage activity of the complexes.
Binding studies confirmed a minor groove-binding nature of the cobalt(II)
complexes. The complexes having phenanthroline bases displayed good
binding propensity to the calf thymus DNA giving an order 3 (dppz) > 2
(dpq) > 1 (phen). The complexes also show efficient photo-induced DNA
cleavage activity on irradiation with a monochromatic UV light of 365 nm
and cleavage efficiency follows the order 3 (dppz) > 2 (dpq) > 1 (phen).
Mechanistic studies revealed direct involvement of 1O2 in the photo-
cleavage process. Sulphur containing ligands with their longer triplet state
lifetimes are suitable for activation of molecular oxygen. This work has
opened up new area of research involving Co(II) complexes for photocleav-
age of DNA with an objective for future PDT applications.
Acknowledgement
This work has been supported by providing Post Doctoral Fellowships for women
(2009–10 & 2010–11) from the University Grants Commission, New Delhi.

14
C. N. SUDHAMANI ET AL.
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