Synthesis, characterization and DNA-binding characteristics of Ru(II) molecular light switch complexes

Article abstract of DOI:10.1007/s13738-012-0150-z

A series of four polypyridyl Ru(II) complexes such as [Ru(L) ₄(PIP)]²⁺ and [Ru(L)₄PPIP]²⁺ where L is 4-amino pyridine and Pyridine (PIP = 2-phenylimidazo[4,5-f] [1, 10] phenanthroline), (PPIP = 2-(40-phenoxy-phenyl) imidazo[4,5-][1, 10]phenanthroline) have been synthesized and characterized by elemental analysis, physicochemical methods such as UV-vis, IR and NMR spectroscopic techniques. The DNA-binding behavior of these complexes was investigated by electronic absorption titrations, fluorescence spectroscopy, viscosity measurements and salt-dependent studies. The experimental results indicate that all these complexes can bind to DNA through an intercalation mode, the DNA-binding affinities of these complexes follow the order [Ru(4-APy) ₄(PPIP)]²⁺(1)[[Ru(Py)₄PPIP]²⁺(2) < [Ru(4-APy)₄(PIP)]²⁺(3)[[Ru(Py)₄PIP] ²⁺(4). Noticeably, these complexes have been found to be efficient photosensitisers for strand scissions in plasmid DNA. Further, all four complexes screened for their antimicrobial activity indicate that the complexes show appreciable activity against Escherichia coli and Neurospora Crassa. In addition, in the presence of Co²⁺, the emission of DNA-[Ru(L ₄)PPIP/PIP]²⁺ can be quenched and recovered by the addition of EDTA, which exhibited the DNA "light switch" properties. Iranian Chemical Society 2012.

Full text of DOI:10.1007/s13738-012-0150-z

J IRAN CHEM SOC (2012) 9:671–680
DOI 10.1007/s13738-012-0150-z
ORIGINAL PAPER
Synthesis, characterization and DNA-binding characteristics
of Ru(II) molecular light switch complexes
•
C. Shobha Devi Penumaka Nagababu
•
•
M. Shilpa Y. Praveen Kumar M. Rajender Reddy
•
•
•
Nazar Md Gabra S. Satyanarayana
Received: 8 October 2011 / Accepted: 14 January 2012 / Published online: 22 August 2012
Ó Iranian Chemical Society 2012
Abstract A series of four polypyridyl Ru(II) complexes
such as [Ru(L)4(PIP)]2? and [Ru(L)4PPIP]2? where L is
4-amino pyridine and Pyridine (PIP = 2-phenylimidazo[4,5-f]
[1, 10] phenanthroline), (PPIP = 2-(40-phenoxy-phenyl)
imidazo[4,5-][1, 10]phenanthroline) have been synthesized
and characterized by elemental analysis, physicochemical
methods such as UV–vis, IR and NMR spectroscopic tech-
niques. The DNA-binding behavior of these complexes was
investigated by electronic absorption titrations, fluorescence
spectroscopy, viscosity measurements and salt-dependent
studies. The experimental results indicate that all these
complexes can bind to DNA through an intercalation mode,
the DNA-binding affinities of these complexes follow the
order [Ru(4-APy)4(PPIP)]2?(1) [ [Ru(Py)4PPIP]2?(2) [
[Ru(4-APy)4(PIP)]2?(3) [ [Ru(Py)4PIP]2?(4). Noticeably,
these complexes have been found to be efficient photosen-
sitisers for strand scissions in plasmid DNA. Further, all four
complexes screened for their antimicrobial activity indicate
that the complexes show appreciable activity against Esche-
richia coli and Neurospora Crassa. In addition, in the pres-
ence of Co2?, the emission of DNA-[Ru(L4)PPIP/PIP]2? can
be quenched and recovered by the addition of EDTA, which
exhibited the DNA ‘‘light switch’’ properties.
Abbreviations
PIP
[2-Phenylimidazo [4,5-f] [1, 10]
phenanthroline]
[2-(40-Phenoxy-phenyl) imidazo[4,5-f][1, 10]
PPIP
phenanthroline]
Py
Pyridine
4-APy
Bpy
4-Aminopyridine
2,20-Bipyridine
Phen
Dmb
Dip
1,10-Phenanthroline
4,40-Dimethyl-2,20-bipyridine
4,40-Diphenyl-2,20-bipyridine
Ethidium bromide
EB
DMSO
TMS
EDTA
Dimethylsulphoxide
Tetramethylsilane
Ethylenediaminetetraacetic acid
CT DNA Calf thymus DNA
Introduction
There has been great deal of interest on Ru(II) polypyridyl
complexes owing to their wide range of applications in
various fields such as photophysics [1], photochemistry,
supramolecular chemistry [2] and biochemistry [3, 4].
They also play a crucial role in biological processes such as
respiration, photosynthesis and oxidative DNA cleavage
[5]. From the literature study, it is evident that many Ru(II)
polypyridyl complexes with their planar aromatic ligands
can intercalate through DNA base pairs. These small
molecules binding to DNA are very important in the
development of new chemotherapeutic reagents [6–9], base
pair mismatches and DNA molecular probes. Because of
their feasibility in the synthesis, strong luminescent
Keywords Ru(II)complexes ꢀ Calf-thymus DNA ꢀ
Emission quenching ꢀ Light switch effect ꢀ Photocleavage
C. Shobha Devi ꢀ M. Shilpa ꢀ Y. Praveen Kumar ꢀ
M. Rajender Reddy ꢀ N. M. Gabra ꢀ S. Satyanarayana (&)
Department of Chemistry, Osmania University,
Hyderabad 500 007, Andhra Pradesh, India
e-mail: ssnsirasani@gmail.com
P. Nagababu
Institute of Chemistry, Academia Sinica,
Taipei, Taiwan
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J IRAN CHEM SOC (2012) 9:671–680
characteristics and DNA-binding affinity, Ru(II) interca-
lators became targets for many researchers [10, 11].
DNA may have potential application as a template or as
an element in DNA-based nanocircuits and molecular
electronics including photo electronics. A requirement for
the latter is a means to switch the photoactive molecule on
and off; otherwise, irradiation always results in charge
transport. This requirement can be fulfilled by Ru(II)
polypyridyl complexes, since these complexes can also
function as ‘‘molecular light switches’’ in aqueous solution,
exhibiting weak luminescence in the absence of DNA and
strong luminescence upon addition of DNA. Most of the
studies of DNA binding with metal complexes have
emphasized on intercalative than auxiliary ligands. How-
ever, Ji et al. [12–15] have studied role of auxiliary ligands
in polypyridyl Ru(II) complexes for their interaction with
DNA. The auxiliary ligand of polypyridyl ruthenium(II)
complexes plays a key role in the spectral properties and
interaction with DNA [16]. But the intercalating ligand in
the complex is the polypyridyl ligand not the ancillary
ligand.
Tris HCl, 50 mM NaCl, pH 7.2). The DNA had a ratio of
UV absorbance at 260 and 280 nm of about 1.8–1.9:1,
indicating that the DNA was sufficiently free of protein
[19]. The concentration of DNA per nucleotide was
determined by spectroscopy using a molar absorption
coefficient of 6,600 M-1 cm-1 (260 nm) [20].
Synthesis and characterization
1,10-Phenanthroline-5,6-dione [21], PIP[PIP = 2-pheny-
limidazo[4,5-f] [1, 10] phenanthroline], PPIP[PPIP =
2-(40-phenoxy-phenyl) imidazo[4,5-][1, 10]phenanthroline]
[22] and [Ru(L)4Cl2]2? [23] were synthesized according to
the literature procedure. Synthetic strategies of these
ligands and their Ru(II) complexes are shown in Fig. 1.
Synthesis of compound 1
Tetra 4-aminopyridine, 2-(40-phenoxy-phenyl) imidazo[4,
5-f][1, 10]phenanthroline ruthenium(II) perclorate was syn-
thesized by mixing [Ru(4-APy)4Cl2]ꢀ2H2O (0.5 mM) and
PPIP (0.5 mM) in 25 mL ethanol and 15 mL water. This
mixture was refluxed for 8 h under nitrogen atmosphere to
give a clear and red solution. After cooling, the solution was
treated with a saturated aqueous solution of sodium perchlo-
rate to give red precipitate. The red solid was collected and
washed with 2 mL of ice cooled ethanol, then it was dried in
vacuo. Yield: 70 %. Anal. calcd. for (%) C45H44N12O11Cl2Ru
C, 49.1; H, 4.03; N, 15.27. Found (%): C, 49.15; H, 4.1; N,
15.5. IR (KBr) 3,445 (broad) (N–H), 1,654 (C=N), 1,540
(C=C), 550 cm-1 (Ru–N). 1H-NMR (DMSO-d6, 400 MHz):
d9.1 (m, 2H, H1, H3), d8.3 (m, 2H, H16, H17), d8.1 (d, 1H, H2),
d7.8 (m, 2H, H8, H9), d7.5 (t, 1H, H14), d7.3 (t, 1H, H13), d7.1
(t, 1H, H12), d3.5 (NH2), d2.1 (NH).
In most of the compounds described so far, the Ru(II)
metal center is bound to bidentate aromatic and polyaro-
matic compounds such as bpy, phen, dmb and dip [17, 18].
Less attention has been paid to compounds containing
monodentate ligands. Particularly, Ru(II)-PIP/PPIP com-
plexes with four pyridines/aminopyridines have not been
reported yet. In this contribution, we report synthesis and
characterization of four ruthenium polypyridyl complexes
[Ru(4-APy)4(PPIP)]2?
,
[Ru(Py)4PPIP]2?
,
[Ru(4-APy)4
(PIP)]2? and [Ru(Py)4PIP]2?. Their interactions with DNA
were investigated by electronic absorption, emission
quenching studies, viscosity and salt-dependent studies.
Effects of light switching on and off were also studied.
These complexes can intercalate into DNA base pairs and
cleave the pBR 322 DNA with high activity upon irradia-
tion. We have also tested all four complexes for their
antimicrobial activity.
Synthesis of compound 2
Tetra 4-pyridine, 2-(40-phenoxy-phenyl) imidazo[4,5-f][1,
10] phenanthroline ruthenium(II) perchlorate was
obtained by a similar procedure to that described above,
[Ru(Py)4Cl2]ꢀ2H2O (0.5 mM) was used in place of [Ru(4-
APy)4Cl2]ꢀ2H2O. Yield: 65 %. Anal. calcd. for (%)
C45H40N8O11Cl2Ru C, 51.93; H, 3.87; N, 10.77. Found (%):
C, 52.2; H, 3.92; N, 11.05. IR (KBr) 3,447 (N–H), 1,652
(C=N), 1,560 (C=C), 549 cm-1 (Ru–N). 1H-NMR (DMSO-
d6, 400 MHz): d9.4 (d, 1H, H1), d8.6 (d, 1H, H16), d8.5 (t, 1H,
H2), d8.45 (d, 1H, H3), d8.4 (t, 1H, H18), d8.3 (t, 1H, H17),
d8.22 (d, 1H, H8), d8.2 (m, 2H, H9,12), d8.0 (d, 1H, H13), d7.8
(d, 1H, H14), d2.2 (NH). 13C[1H]-NMR (DMSO-d6,
400 MHz): d157.8 (1C, C1), d156.7 (1C, C15), d154.2 (1C,
C5), d150.0 (1C, C16), d136.9 (1C, C4), d136.0 (1C, C6),
d130.5 (1C, C3), d129.0 (1C, C17), d128.0 (1C, C18), d125.0
Experimental
Materials
All reagents and solvents were purchased commercially
and were used as received unless otherwise noted. RuCl3,
1,10-Phenanthroline monohydrate and pyridines were
purchased from Merck, calf thymus (CT) DNA was pur-
chased from Aldrich and supercoiled pBR 322 DNA was
obtained from Fermentas life sciences. Double distilled
water was used for preparing various buffers. Interaction of
the complexes with DNA was studied in tris buffer (5 mM
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J IRAN CHEM SOC (2012) 9:671–680
673
Fig. 1 Synthetic routs for ligand and 1, 2, 3 and 4 complexes
(1C, C2), d124.6 (2C, C8,9), d124.0 (1C, C10), d123.0 (2C,
C2), d128.8 (1C, C3), d126.9 (1C, C4), d110.9 (2C, C5,14),
d109.5 (1C, C13), d108.9 (2C, C6,7), d108.0 (1C, C10),
d107.0(2C, C9,8).
C7,14), d120.4 (1C, C11), d119.7 (1C, C13), d119.0 (1C, C12).
Synthesis of compound 3
Synthesis of compound 4
Tetra 4-aminopyridine, 2-phenylimidazo[4,5-f] [1, 10]
phenanthroline ruthenium(II) perchlorate complex was
obtained by a similar procedure to that described above;
PIP (0.5 mM) was used in place of PPIP. Yield: 75 %.
Anal. calcd. for (%) C39H41N12O10Cl2Ru C, 46.42; H,
4.06; N, 16.66. Found (%): C, 46.56; H, 4.1; N, 16.78. IR
(KBr) 3,373 (broad) (N–H), 1,636 (C=N), 1,516 (C=C),
Tetra pyridine, 2-phenylimidazo[4,5-f] [1, 10] phenan-
throline] ruthenium(II) perchlorate complex was obtained
by
a similar procedure to that described above,
[Ru(Py)4Cl2]ꢀ2H2O (0.5 mM) was used in place of [Ru(4-
APy)4Cl2]ꢀ2H2O. Yield: 75 %. Anal. calcd. for (%)
C39H36N8O10Cl2Ru C, 49.36; H, 3.9; N, 11.84. Found (%):
C, 49.38; H, 4.051; N, 11.479. IR (KBr) 3,368 (broad)
(N–H), 1,650 (C=N), 1,560 (C=C), 549 cm-1 (Ru–N)
1H-NMR (DMSO-d6, 400 MHz): d8.4 (m, 2H, H1, H11),
d8.0 (m, 3H, H2, H7, H12), d7.6 (m, 2H, H9, H13), d6.7 (d,
1H, H3), d6.6 (d, 1H, H8), d2.5 (NH).
1
550 cm-1 (Ru–N) H-NMR (DMSO-d6, 400 MHz): d9.1
(d, 1H, H12), d8.10 (m, 2H, H1, H13), d7.9 (m, 2H, H2, H9),
d7.7 (d, 1H, H3), d7.6 (d, 1H, H10), d7.5 (d, 1H, H8),
d3.10(NH2), d2.1 (NH). 13C [1H]-NMR (DMSO-d6,
400 MHz): d156.1 (1C, C1), d144.7 (2C, C11,12), d129 (1C,
123

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Physical measurements
Results and discussion
UV–visible spectra were recorded with an Elico SL159
spectrophotometer. IR spectra were recorded on KBr
Electronic absorption titration
1
disks on a Perkin-Elmer FT-IR-1605 spectrometer. H and
The application of electronic absorption spectroscopy in
DNA-binding studies of Ru-polypyridyl complexes is one
of the most useful techniques [24–26]. A complex binding
to DNA through intercalation usually results in hypochro-
mism and bathochromism, due to the intercalation mode
involving a strong p–p* stacking interaction between an
aromatic chromophore and the base pairs of DNA [22]. It is
generally accepted that the extent of the hypochromism in
the UV–visible band is consistent with the strength of in-
tercalative interaction [27–30]. The absorption spectra of
complexes 1–4 in the absence and presence of CT-DNA (at
constant concentration of complex, i.e., 10–15 lM) are
given in Fig. 2. As the concentration of DNA increases,
absorption bands of the complexes display clear hypo-
chromism. An insignificant red shift in the MLCT band of
the complexes was observed. The hypochromism
(H % = 100x (Afree - Abound)/Afree) of the MLCT bands at
469, 466, 471 and 475 of complexes 1–4 was determined to
be about 11.5, 9.41, 16.2 and 12.4 %, respectively. To
compare the DNA-binding affinities of all four complexes,
quantitatively, their intrinsic binding constant Kb to DNA
was obtained by monitoring the changes of the MLCT
absorbance at their respective wavelengths, according to
the following Eq. [31].
13C NMR spectra were recorded on a Bruker 400 MHz
spectrometer with DMSO as solvent at RT and TMS as
the internal standard. Microanalyses (C, H, N) were car-
ried out with a Perkin-Elmer 240 elemental analyzer.
Absorption titration experiment was performed at room
temperature to determine the binding affinity between the
DNA and complex. A fixed ruthenium concentration
3.0 ml (20 lM) was taken in a cuvette to which incre-
ments of the DNA stock solution were added. DNA
solution (concentration 30–200 lM per nucleotide) was
added to each cuvette reference and sample to eliminate
the absorbance of DNA itself. Solutions were mixed for
3 min and absorption spectra were recorded. Titrations
were continued until there was no further change in the
spectra was noted. Fluorescence measurements were
performed on a Hitachi F-2500 spectrofluorimeter. All
measurements were made at 25 °C using a thermostated
cuvette holder. Emission titration experiments were per-
formed at a fixed metal complex concentration (10 lM) to
which increments of a DNA solution (10–150 lM) were
added. Viscosity experiments were carried out on Ostwald
viscometer immersed in thermostated water bath main-
tained at 30 0.1 °C. Flow time was measured with a
digital stopwatch and every sample was measured three
times and an average flow time was calculated. Data were
presented as (g/go)1/3 versus the concentration of [Ru(II)]/
[DNA] [36], where g is the viscosity of DNA in the
presence of complexes and go is the viscosity of DNA
alone. For gel electrophoresis experiments, supercoiled
pBR 322 DNA(0.1 lg/lL) was treated with 20–80 lM of
Ru(II) complexes and the mixtures were irradiated at
365 nm for 1 h. The samples were then analyzed by
0.8 % agarose gel electrophoresis (Tris–Acetic acid-
EDTA buffer, pH 8.0) at 50 V for 2 h. The gel was
stained with 1 lg ethidium bromide and photographed
under UV light. The antibacterial activity of the com-
plexes was studied against Escherichia coli and Neuros-
pora crassa. Each of the Ru(II) complex was dissolved in
DMSO at a concentration of 1 mg mL-1. Paper disks of
Whatman filter paper no. 1 were sterilized in an auto-
clave. The paper disks saturated with 10 lL of the Ru(II)
complex were placed in the Petri dishes containing LB
(Luria Bertini) agar media inoculated with E. coli and N.
crassa separately. The Petri dishes were incubated at
37 °C and the inhibition zones were recorded after 24 h
of incubation. The results were also compared with
standard antibacterial drug Ampicillin at the same
concentration.
½DNAꢁ=ðea ꢂ efÞ ¼ ½DNAꢁ=ðeb ꢂ efÞ þ 1=Kbðeb ꢂ efÞ
where [DNA] is the concentration of DNA in nucleotides,
ea,ef and eb are the extinction coefficient for the free Ru(II)
complex, extinction coefficient of complex in the presence
of DNA, and the extinction coefficient of the Ru(II) com-
plex in the fully bound form, respectively. Intrinsic binding
constant Kb of complexes is given in Table 1. The Kb
values of complexes are in the order of 1 [ 2 [ 3 [ 4.
This suggests that there is a mode of binding that involves a
striking interaction between the complex and the base pairs
of DNA. As PPIP possesses a greater planar area and
extended p system, it penetrates more deeply into the DNA
base pairs. Therefore, binding constants for 1, 2 complexes
are more than that of 3 and 4. The difference in binding
strengths of complexes 1 and 2; 3 and 4 is probably caused
by the difference in auxiliary ligands. The four additional
NH2 groups in complexes 1 and 3 may exert some addi-
tional interactions (may be H-bonding) with DNA base
pairs and hence have higher binding constants. The Kb
values are more than those reported earlier for their
bidentate ligands [Ru(bpy)2PPIP] (4.3 9 104) [22]. This
difference is due to the difference in the ancillary ligands.
When these complexes are bound to DNA, there is no
conformational change observed except a change in the
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J IRAN CHEM SOC (2012) 9:671–680
675
7.8E-10
6.8E-10
5.8E-10
4.8E-10
3.8E-10
Fig. 2 Absorption spectra of
[Ru(4-APy)4(PPIP)] (1),
[Ru(Py)4PPIP] (2), [Ru(4-
APy)4(PIP)] (3) and
1.25E-09
1.05E-09
8.5E-10
6.5E-10
4.5E-10
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
2.0
1.5
1.0
0.5
0.0
0.000001 0.000006 0.000011
[DNA]
0.000001
0.000006
[Ru(Py)4PIP](4) in Tris–HCl
buffer upon addition of CT-
DNA in absence (top) and
presence of CT-DNA (lower)
the [complex] = 10–15 lM.
[DNA] = 0–120 lM. Inset
plots of [DNA]/(ea- ef) versus
[DNA] for the titration of DNA
with Ru(II) complexes. Arrow
shows change in absorption
with increasing DNA
[DNA]
350
400
450
500
550
600
300
400
500
600
Wavelength (nm)
Wavelength (nm)
7.5E-10
6.5E-10
5.5E-10
4.5E-10
concentration
1.00
0.75
0.50
1.00
0.75
0.50
0.25
0.000001
0.000006
[DNA]
0.000011
7.5E-10
6.5E-10
5.5E-10
4.5E-10
3.5E-10
1E-06 3E-06 5E-06 7E-06 9E-06
[DNA]
350
400
450
500
550
600
350
400
450
500
550
Wavelength (nm)
Wavelenth nm
absorption intensity. The DNA is still in B form without
conformational change.
I0=I ¼ 1 þ Ksv½Qꢁ
where I0 and I are the fluorescence intensities in the
absence and presence of quencher, respectively, Q is the
concentration of the quencher, Ksv is a linear Stern–Volmer
quenching constant. In the quenching plot of I0/I versus
[Q], Ksv is given by the slope. Figure 4 shows the ferro-
cyanide quenching plots for complexes in the absence,
presence and excess of DNA. All four complexes show
linear Stern–Volmer plots. The Ksv values for all four
complexes are given in Table 1. The quenching studies
indicate that the DNA-binding abilities of the complexes
follow the order: 1 [ 2 [ 3 [ 4. These Stern–Volmer
plots are linear and complex is cation and quencher is
anion, a type of fluorophore–quencher complex (F–Q) is
formed. It could be static quenching.
Emission studies
All four complexes show emission in Tris buffer at ambient
temperature [32]. The excitation peaks appeared at 467,
462, 470 and 464 nm and emission peaks at 588, 598, 591
and 599 nm for 1, 2, 3 and 4 complexes, respectively.
Upon addition of CT-DNA, the emission intensities
increase further than the complex alone as shown in Fig. 3.
This implies that complexes can strongly interact with
DNA. Binding data were cast into the scatchard plot of r/Cf
versus r, where r is the binding ratio (Cb/[DNA]) and Cf is
the free ligand concentration. Non linear least square fitting
of the data yield K and n, the n exclusion parameter is
3.2( 0.3) bp. The binding constants calculated are also
comparable with UV absorption data. The K (emission
binding constant) values are given in Table 1.
Emission quenching experiments using [Fe(CN)6]4- as
quencher further support the above proposal. As illustrated
in Fig. 4 in the absence of DNA, complexes were effi-
ciently quenched by [Fe(CN)6]4- resulting in two linear
Stern–Volmer plots. In the presence of DNA, quenching
was small, because of the highly negatively charged
[Fe(CN)6]4- would be repelled by the negative charge of
the DNA phosphate backbone which would hinder the
quenching of the emission of the bound complexes. The
Stern–Volmer quenching constant Ksv can be determined
using Stern–Volmer equation [33].
Light switch on off effect
From Fig. 6, it can be seen that after binding to DNA
(switch on), the emission of DNA-[Ru(APy)4(PPIP)] can
be quenched by Co2? ion, thus turning the light switch
off [34, 35]. The addition of 0.03 mM Co2? to 0.01 mM
of complex bound to 0.2 mM DNA results in the loss of
luminescence due to the formation of Co2?-[Ru(A-
Py)4(PPIP)] (heterometallic complex). In order to further
provide additional evidence for the quenching which
originated in the formation of heterometallic complex, the
emission spectra of [Ru(APy)4(PPIP)] without DNA in the
absence and presence of Co2? are measured. Similar
quenching of luminescence was observed. This observation
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Table 1 Absorption peaks,
absorption, emission and
quenching binding constants of
Ru(II)complexes
Complex
Absorption
kmax (nm)
Absorption binding
constant Kb (M-1
(K) Emission
binding constant
(Ksv
Only Comp ? DNA
Comp 1:50 1:200
)
[Ru(4-APy)4(PPIP)]
[Ru(Py)4PPIP]
469, 362, 329
466, 359, 326
461, 360, 307
471, 361, 315
2.01 9 105
1.21 9 105
7.5 9 104
6.16 9 104
2.12 9 105
1.375 9 105
7.23 9 104
6.5 9 104
219, 142, 30
152, 79, 10
208, 134, 20
128, 81, 15
[Ru(4-APy)4(PIP)]
[Ru(Py)4PIP]
(1)
(2)
(3)
(4)
Fig. 3 Emission spectra of [Ru(4-APy)4(PPIP)](1), [Ru(Py)4PPIP](2) [Ru(4-APy)4(PIP)](3), [Ru(Py)4PIP](4) in Tris–HCl buffer at 25 °C upon
addition of CT-DNA, [Ru] = 20 lM, [DNA] = 0–120 lM. The arrow shows the increase in intensity upon increasing CT-DNA concentrations
Viscosity measurements
is an indication of the formation of non-luminescent species,
Co2?-[Ru(APy)4(PPIP)]. The emission of DNA-intercalated
complex (light switch on) can be quenched by transition
metal ions, thus turning the light switch off, these are clearly
demonstrated in Fig. 5. While adding EDTA into the (buffer)
system containing Co2?-[Ru(APy)4(PPIP)] or ([Ru(APy)4
(PPIP)(Co)]4?), the emission intensity of the complex is
recovered again (light switch on). This indicates that het-
erometallic complex Co2?-[Ru(APy)4(PPIP)] becomes free
again. Inthisexperiment, adding0.03 mMofCo2? decreased
the intensity of [Ru(APy)4(PPIP)] and on adding equimolar
EDTA 0.03 mM the luminescence was recovered. Similar
observations were obtained for all the four complexes.
Though photo physical studies are quite useful in deter-
mining binding constants of metal complexes to DNA [36,
37], to further elucidate the binding mode of the present
complexes, viscosity measurements were carried out. A
classical intercalation model demands that the DNA helix
lengthens as base pairs are separated to accommodate the
binding ligand hence leads to an increase in the viscosity of
DNA [38]. In contrast, a partial intercalation could bend
the DNA helix and reduce its effective length and con-
comitantly its viscosity [39, 40]. The changes in relative
viscosity of rod like CT-DNA in the presence of complexes
123

J IRAN CHEM SOC (2012) 9:671–680
677
1.35
1.3
(1)
(2)
a
b
1.5
a
b
1.4
1.3
1.2
1.1
1
1.25
1.2
1.15
1.1
c
1.05
1
c
0.9
0.0003
0.95
0.0003
0.0008
0.0013
0.0018
0.0023
0.0008
0.0013
0.0018
K4[Fe(CN)6] mM/L
K4[Fe(CN)6] mM/L
(3) 1.4
(4) 1.3
1.25
1.2
a
b
1.35
a
b
c
1.3
1.25
1.2
1.15
1.1
1.15
1.1
1.05
1
1.05
1
c
0.95
0.9
0.95
0.9
0.0003
0.0003
0.0008
0.0013
0.0018
0.0023
0.0008
0.0013
0.0018
K4[Fe(CN)6] mM/L
K4[Fe(CN)6] mM/L
Fig. 4 Emission quenching of [Ru(4-APy)4(PPIP)](1), [Ru(Py)4PPIP] (2), [Ru(4-APy)4(PIP)] (3) & [Ru(Py)4PIP] (4) with K4[Fe(CN)6] in the
absence (a) and presence (b) [Ru] = 20 lm, and excess of DNA (c)
affinity to DNA, follows the order EB [ 1 [ 2 [ 3 [ 4
(Fig. 7). These results also suggest that all four complexes
intercalate between the base pairs of DNA and parallel the
result obtained by absorption, fluorescence and quenching
measurements. Based on the binding data and the viscosity
experiment, we conclude that these four complexes bind to
DNA by intercalation. But they do not intercalate as
strongly as proven intercalators [36].
Salt-dependent studies
Fig. 5 Schematic representation of molecular light switch properties
of [Ru(4-APy)4(PPIP)] in presence of DNA (1) (switch on), in
presence of Co(II) (switch off) (2) (heterometallic complex) and
Figure 8 shows the salt dependence of 1 and 3 complexes
recovered luminescence upon addition of EDTA (3)
binding to DNA and comparative data for proven interca-
lators. The clear dependence of the binding constants for
1, 2, 3 and 4 are shown in Fig. 7. Ethidium bromide, a
well-known DNA intercalator, increases the relative vis-
cosity strongly by lengthening the DNA double helix
through intercalation. As seen from Fig. 7 upon increase in
the amounts of complexes 1 and 2, the relative viscosity of
the DNA increases steadily similar to the behavior of
ethidium bromide, whereas in complexes 3 and 4 the
increase in viscosity is comparatively less. The increased
degree of viscosity, which may depend on the biding
these complexes upon Na? binding to DNA may be ana-
lyzed by polyelectrolyte theory [41]. From Record theory,
slope of the lines in Fig. 8 provide an estimate of Zw,
where Z is the charge on the complex and w is the fraction
of counter ions associated with each DNA phosphate
(w = 0.88 for double-stranded B-form DNA). The data in
Fig. 8 show that the slopes of the lines are greater than 1,
being -1.31 and -1.236 for 1 and 3 complexes, respec-
tively. These values are less than the theoretically expected
123

678
J IRAN CHEM SOC (2012) 9:671–680
44
42
40
38
36
34
50
Fig. 6 Luminescence changes
of 0.01 mM [Ru(PPIP)(APy)4]
in the presence of 0.2 mM of
DNA (a), in the absence of
0.2 mM DNA (b) and (1),
presence of 0.03 mM Co2? (2)
and addition of 0.03 mM of
EDTA (3)
(a)
(b)
40
3
30
1
3
1
20
10
0
1
2
3
1
2
3
2
2
Wavelength
Wavelength
A
2.6
2.1
1.6
1.1
0.6
B
C
D
E
0.013
0.033
0.053
0.073
0.093
0.113
[Ru]/[DNA]
Fig. 8 Salt dependence of the equilibrium binding constants for
DNA-binding ligands. Data are given for [Ru(4-APy)4(PPIP)] (a) and
[Ru(4-APy)4(PIP)] (b). Slopes are -1.315 and -1.216, respectively
Fig. 7 Effect of increasing amount of ethidium bromide (a) [Ru(4-
APy)4(PPIP)] (b), [Ru(Py)4PPIP] (c), [Ru(4-APy)4(PIP)] (d) and
[Ru(Py)4PIP] (e) on relative viscosity of CT-DNA at 30 0.1 °C.
The total concentration of DNA is 0.25 mM
circular DNA was subjected to electrophoresis, relatively
fast migration was observed for the supercoiled form (form
I). As scission occurs on one strand, the supercoiled DNA
generated electrophoretically slower-moving open circular
form (form II). When both strands are cleaved, a linear
form (III) was generated that migrated between forms I and
II [44, 45]. With increasing concentrations of the com-
plexes (lanes b–e), the amount of form I DNA diminished
gradually, whereas form II increased. Further, the unirra-
diated control solution confirmed that the complexes did
cause photosensitized cleavage. Under comparable exper-
imental conditions, all complexes showed photocleavage
activity.
value of Zw = 2 9 0.88 = 1.76. Such lower values could
arise from coupled anion release (from the ligand) or from
changes in ligand or DNA hydration upon binding. By
increasing the Na? concentrations [42], the relative binding
affinities of the complexes decreased similar to that of
proven intercalators [43] like ethidium bromide.
Photocleavage of plasmid DNA by Ru(II) complexes
The potential of the present complexes to cleave DNA was
studied by gel electrophoresis using supercoiled pBR 322
DNA. There has been considerable interest in DNA
endonucleolytic cleavage reactions that are activated by
metal ions. The delivery of high concentration of metal ion
to the helix, in locally generating singlet oxygen (1O2) or
hydroxyl radicals ꢀOH yields an efficient DNA cleavage
reaction. Figure 9 shows gel electrophoresis separation of
pBR 322 DNA after incubation with complexes and irra-
diation at 365 nm. No DNA cleavage was observed for
control in which complex was absent (lane a). When this
Antimicrobial activity
Anticancer, antimicrobial activity and toxicological studies
have been reported by our group [17, 18] for various Ru(II)
complexes with polypyridyl ligands. Thus, antimicrobial
activity was attempted for these complexes. The antibac-
terial activity was found in all four complexes. The anti-
microbial activities of these compounds were determined
123

J IRAN CHEM SOC (2012) 9:671–680
679
1
Form-II
Form-I
Table 2 Antibacterial activity of Ru(II) complexes at 20 lg/ml
Bacterial species
E. coli
Fungal species
N. crassa
2
3
Form-II
Form-I
DMSO
Nil
12
Nil
[Ru(4-APy)4(PPIP)]
[Ru(Py)4PPIP]
[Ru(4-APy)4(PIP)]
[Ru(Py)4PIP]
Ampicillin
9
10
8
Form-II
Form-III
Form-I
19
15
6
1
18–22
13–15
4
Form-II
Form-I
Values of zone of inhibition (mm, including the diameter of the disk)
conformation. All the complexes show effects of light
being switched on and switched off.
a
c
b
d
e
Fig. 9 Photo cleavage of pBR 322 DNA in the presence of [Ru(4-
APy)4(PPIP)] (1), [Ru(Py)4PPIP] (2), [Ru(4-APy)4(PIP)] (3) and
[Ru(Py)4PIP] (4) complexes, after irradiation with UV light at
365 nm. Lane a control plasmid DNA (untreated pBR 322), lanes b–e
addition of complexes 20, 40, 60 and 80 lM
Acknowledgments Council of Scientific and Industrial Research
(CSIR), New Delhi, India. is greatfully acknowledged for the finan-
cial support in the form of Junior Research Fellow to C. Shobha Devi.
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disk method [46]. The antimicrobial activity data (Table 2)
indicate that the complexes show appreciable activity
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