Synthesis, characterization, DNA binding properties, fluorescence studies and toxic activity of cobalt(III) and ruthenium(II) polypyridyl complexes

Article abstract of DOI:10.1007/s10895-010-0743-9

The new ligand 4-(isopropylbenzaldehyde)imidazo[4,5-f][1,10]phenanthroline (ippip) and its complexes [Ru(phen)₂(ippip)]²⁺ (1), [Co(phen)₂(ippip)]³⁺ (2), [Ru (bpy)₂(ippip)] ²⁺ (3), [Co(bpy)<

Full text of DOI:10.1007/s10895-010-0743-9

J Fluoresc (2011) 21:563–572
DOI 10.1007/s10895-010-0743-9
ORIGINAL PAPER
Synthesis, Characterization, DNA Binding Properties,
Fluorescence Studies and Toxic Activity of Cobalt(III)
and Ruthenium(II) Polypyridyl Complexes
Penumaka Nagababu & Mynam Shilpa & J. Naveena Lavanya Latha & Ira Bhatnagar &
P. N. B. S. Srinivas & Yata Praveen Kumar & Kotha Laxma Reddy &
Sirasani Satyanarayana
Received: 27 May 2010 /Accepted: 29 September 2010 /Published online: 8 October 2010
#
Springer Science+Business Media, LLC 2010
.
Keywords Cobalt(III) and ruthenium(II) mixed-ligands
Abstract The new ligand 4-(isopropylbenzaldehyde)imi-
dazo[4,5-f ][1,10]phenanthroline (ippip) and its complexes
[Ru(phen)2(ippip)]2+ (1), [Co(phen)2(ippip)]3+ (2), [Ru
(bpy)2(ippip)]2+ (3), [Co(bpy)2(ippip)]3+ (4) (bpy=2,2-
bipyridine) and (phen=1,10-phenanthroline) were synthe-
sized and characterized by ES+-MS, 1H and 13C NMR. The
DNA binding properties of the four complexes were
investigated by different spectrophotometric methods and
viscosity measurements. The results suggest that complexes
bind to calf thymus DNA (CT-DNA) through intercalation.
When irradiated at 365 nm, the complexes promote the
photocleavage of pBR322 DNA, and complex 1 cleaves
DNA more effectively than 2, 3, 4 complexes under
comparable experimental conditions. Furthermore, photo-
cleavage studies reveal that singlet oxygen (1O2) plays a
significant role in the photocleavage.
.
.
DNA-binding Fluorescence studies Photocleavage
.
of plasmid DNA Toxic studies
Introduction
Among the host of DNA-binding and cleaving agents
reported so far, transition metal complexes are of relevance
to the present work. Major interests in this regard concern
the utility of metal complexes in the development of site
specific cleavers, structural/spectroscopic probes, restriction
enzymes, molecular photo switches and, of course, anti-
tumor drugs. Metal complexes have been found to be
particularly useful for the above mentioned purposes
because of their potential to bind DNA through multitude
of interactions and to cleave the duplex by virtue of their
intrinsic chemical, electrochemical and photochemical
reactivities. During the last decade, a number of
transition-metal complexes have been utilized to probe
nucleic acid structure [1, 2], and in the development of
DNA-cleaving agents [3, 4], DNA photo probes [5, 6]
DNA-molecular light switches [7, 8] and so forth. It has
also been well documented that metal complexes can bind
to DNA covalently as well non covalently [6, 9].
On the other hand, Barton and co-workers reported that a
number of metal complexes containing aromatic diimine
ligands [10] could act as photoluminescent metal-based
probes for the study of DNA binding. These complexes
have been reported to bind DNA through π–π* and/or
electrostatic interactions and cause oxidative cleavage of
DNA. Moreover, an oxidative cleavage of DNA on
irradiation with visible light has gained considerable
interest due to its potential application in photodynamic
:
:
:
:
P. Nagababu M. Shilpa Y. P. Kumar K. L. Reddy
S. Satyanarayana (*)
Department of Chemistry, Osmania University,
Hyderabad, Andhra Pradesh, India PIN-500 007
e-mail: ssnsirasani@gmail.com
I. Bhatnagar
CCMB (Centre for Cellular and Molecular Biology),
Hyderabad 500007, India
P. N. B. S. Srinivas
National Institute of Nutrition,
Hyderabad, India
J. N. L. Latha
Department of Biotechnology, Krishna University,
Machilipatnam, India

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J Fluoresc (2011) 21:563–572
therapy of cancer [11, 12]. Recently, several mixed ligand
complexes of ruthenium(II) and cobalt(III) have also been
reported [13–16]. In view of the above, we explore DNA
binding and photo-induced cleavage activity of cobalt and
ruthenium complexes.
60%). Analytical data for RuC46H39N8Cl2O12. ES+-MS cal:
1,060 found: 1,059. IR: 1,479 (C=C), 1,598 (C=N), 721
1
(Co–N (L)), 623 cm−1 (Co–N(ippip)). H NMR (DMSO,
TMS): 9.09 (d, 6 H), 8.74 (d, 6 H), 8.50 (d, 4H), 8.28 (s,
4H), 8.13 (t, 4H), 8.02(t, 2H), 7.99 (d, 2H), 7.53 (d, 1H),
7.35(d, 1H), 3.61 (d, 1H), 1.8 CH3 peak. 13C NMR
(200 MHz, DMSO, 298 K, major peaks) δ: 155.71,
155.38, 151.56, 151.30, 143.61, 143.42, 131.34, 127.32,
127.07, 126.87, 33.44, 23.64.
Experimental
Materials
[Co(phen)2(ippip)](ClO4)3. 4H2O
CT (calf thymus) DNA was purchased from Aldrich. The
synthesis of 1,10 phenanthroline-5, 6-dione was prepared as
per the procedure given in literature [17]. Interactions of the
complexes with DNA were studied in tris buffer (5 mM
Tris-HCl, 5 mM NaCl, pH 7.0). DNA had a ratio of UV
absorbance at 260 and 280 nm of about 1.90 indicating its
purity [18]. The DNA concentration per nucleotide was
determined by absorbance at 260 nm using the molar
A mixture of [Co(phen)2Br2]Br.3H2O (0.531 g,1.0 mmol),
ippip (0.489 g,1.5 mmol) in ethanol (20 cm3) was refluxed
for 4 h to give a yellow solution. After filtration, the
complex was precipitated by addition of a saturated
ethanolic solution of NaClO4. The complex was filtered
and further dried under vacuum before recrystallization
(Me2CO-Et2O). (Yield=75%) Analytical data for
CoC45H38N8Cl3O16. ES+-MS calc:1,128 found:1,126. IR:
1,541 (C=C), 1,430 (C=N), 714 (Co–N (L)), 624 cm−1
−1
absorption coefficient (6,600 M cm−1) [19].
1
Synthesis of Ligands
(Co–N(ippip)). H NMR (DMSO, TMS):δ 9.58(d, 6 H),
9.43(d, 6 H), 8.76 (d, 4H), 8.59 (s, 4H), 8.34 (t, 4H), 7.9(t,
2H), 7.74 (d, 2H), 7.55,(d,1H), 7.20,(d,1H), 3.8(d,1H), 1.6
CH3 peak.
(ippip) =4-(isopropyl benzaldehyde)imidazo[4,5-f ][1, 10]
phenanthroline
Phen-dione (0.53 g, 2.50 mmol), 4-isopropyl benzaldehyde
(0.152 g, 1.2 mmol) and ammonium acetate (3.88 g,
50.00 mmol) and glacial acetic acid (15 mL) were refluxed
together for 4 h as per Steck and Day [20], cooled thereafter
to room temperature and diluted with water. Dropwise
addition of liquor ammonia gave a yellow precipitate which
was collected, washed with water, dried and purified by
recrystallization. Formula: C22H18N4, Anal. Calc. H 5.36, C
78.08, N 16.56 found: H 4.9, C 77.0, N 15.6 (yield=80%)
[Ru(bpy)2ippip)(ClO4)2. 3 H2O
This complex was synthesized as described above complex,
(yield=63%), analytical data for RuC42H34N8Cl2O11. ES+-MS
calc: 1,004 found: 1,002. IR: 1,601 (C=C), 1,448 (C=N), 724
(Co–N (L)), 626 cm−1 (Co–N(ippip)). H NMR ((DMSO,
1
TMS), 300 MHz, TMS):
8.34 (d, 2H), 8.23(t, 2H), 8.16 (d, 2H), 8.10 (t, 3H), 8.0
(d, 2H), 7.971(m, 2H), 7.97(q, 4H), 7.93(d, 2H) 7.85(d,
2H), 7.7471(d, 2H), 7.5(d, 2H), 7.4(d, 1H), 7.34(d, 1H),
3.01(d, 1H), 1.5 CH3 peak. 13C NMR (200 MHz, DMSO,
298 K, major peaks) δ:152.87, 152.69, 150.91, 150.91,
150.165, 147.30, 147.22, 145.37, 136.86, 130.51, 128.13,
127.16, 126.75, 126.40, 126.10, 33.47, 23.77.
1
Analytical data:ES+-MS calculated: 338.15 found: 339. H
NMR (DMSO, TMS): 9.03 (d, 2H), 8.95 (d, 2H), 8.24 (d,
2H), , 7.21 (d, 1H), 7.03 (d, 1H), 6.95 (d, 1H), 2.51(d, 1H),
1.65 CH3 peak.
Synthesis of Complexes
[Co(bpy)2(ippip)](ClO4)3. 2H2O
[Ru(phen)2(ippip)(ClO4)2. 3.5H2O
This complex was synthesized as described above complex,
with a mixture of [Co(bpy)2Br2]Br.3H2O and ippip
(0.564 g, 1.42 mmol), (yield=60%), analytical data for
CoC42H34N8Cl3O14 . ES+-MS cal: 1,042 found: 1,040. IR:
1,560 (C=C), 1,448 (C=N), 718 (Co–N (L)), 622 cm−1
(Co–N(ippip)). 1H NMR, (DMSO, TMS):δ 9.46(d, 2H),
9.44(d, 4H), 9.40(d, 2H), 9.1(m,4H), 8.72(d, 2H), 8.62(t,
4H), 8.42 (t, 2H), 8.15(m, 4H), 7.65(d, 2H), 7.5(d, 1H),
7.38(d, 1H), 3.1(d, 1H), 1.5CH3 peak. (Molecular structure
of complexes Fig. 1).
A mixture of [Ru(phen)2Cl2]·2H2O (0.531 g, 1.0 mmol),
ippip (0.489 g,1.0 mmol) and ethanol (15 mL) was
thoroughly deoxygenated. The purple mixture was heated
for 8 h at 120 °C under N2 atmosphere. When the solution
finally turned red, it was cooled to room temperature and an
equal volume of saturated aqueous sodium perchlorate
solution was added under vigorous stirring. The red solid
was collected and washed with small amounts of water,
ethanol, and diethyl ether, then dried under vacuum, (yield=

J Fluoresc (2011) 21:563–572
565
N
H
N
N
N
N
CH
3
Ru
[Ru(phen) ]Cl
2
2
N
CH
3
N
N
N
H
N
CH3
CH3
N
N
N
CH
3
[Co(phen) Br ]Br
2
2
Co
OHC
N
CH
3
N
O
O
N
CH3
CH3
N
N
N
Gla.AcOH,
NH4OAc, Ref.4h
N
H
N
N
N
H
N
[Ru(bpy) ]Cl
2
2
N
N
CH
N
3
Ru
N
N
CH
N
3
[Co(bpy) Br ]Br
N
2
2
H
N
N
CH
N
3
Co
N
CH
N
3
N
N
Fig. 1 Molecular structure of complexes
Physical Measurements
spectra were recorded. Titration was continued as above till
there was no further change in absorption indicating
saturation of binding. Change in concentration of complex
due to dilution was negligible in view of the small volumes
of DNA solution added. The above procedure was repeated
minimum of three times.
Fluorescence emission titration experiments were per-
formed at a fixed metal complex concentration (10 μM) to
which DNA (1–160 μM) was added in a volume of (10–
100 μL). Excitation wavelength was kept constant, emission
was recorded. Fluorescence emission enhancement was based
on comparison of emission intensity at 558 nm of complexes
in the absence and presence of CT DNA.
Dialysis experiment were conducted at room temperature
with 3 cm3 of CT-DNA(120 μM) sealed in a dialysis bag
and 6 cm3 of complexes(20 μM) outside the bag with the
solution stirring for 24 h.
Viscosity was determined in an Ostwald viscometer at
30.0 0.1° in a thermostated water-bath. CT DNA (∼200
base pairs) was prepared by sonication [21]. Data are
UV-Visible spectra were recorded in an Elico Bio-
spectrophotometer model BL198. IR spectra were obtained
in KBr phase in a Perkin-Elmer FTIR-1605 spectropho-
tometer. NMR spectra were measured with a Varian XL-
400 MHz spectrometer with DMSO as solvent at room
temperature and TMS as the internal standard. ES+-MS
mass spectra were recorded. Microanalyses (C, H, N) were
carried out with a Perkin-Elmer 240 elemental analyzer.
Fluorescence measurements were performed in a HitachiF
4500 spectrofluorimeter.
Spectrophotometric titrations were carried out at room
temperature to determine the binding affinity of complexes
to DNA. For this, the reference cuvette contained tris-buffer
(pH 7.0, 3.0 ml). The complex (30 μm in buffer, 3.0 ml)
was taken in another sample cuvette. Spectra were recorded
in the range of 200–600 nm. During the titration small
aliquots of buffered DNA (1–10 μl, ∼5 to 10 mM in NP)
were added to both solutions mixed for ∼5 min and then the

566
J Fluoresc (2011) 21:563–572
Table 1 Absorption spectrophotometric titration and thermal denaturation of DNA bound to complexes 1–4
Complexes
Excitation peaks
Emission peaks
UV/Vis peaks (nm) Δλ Absorption λmax (nm) TM °C Hypochromism (%)
Free Bound
DNA alone
–
260 nm
–
4
3
3
3
–
366
297
446
307
60
67
66
64
64
1
2
3
4
274, 329, 383 nm 319, 358, 582 nm 298, 366, 446 nm
358, 403, 451 nm 380, 359, 409 nm 297, 349 nm
268, 318, 405 nm 360, 410, 591 nm 256, 288, 446 nm
356, 452, 405 nm 370, 360, 409 nm 362, 302 nm
370
300
449
310
20%
19%
17%
15%
presented as (η/ηο)1/3 vs the concentration of complex
bound to DNA, where ŋ is the viscosity of DNA in the
presence of complex and η0 is the viscosity of DNA alone.
Viscosity values were based on flow time of DNA-
containing solution (t>100 s) corrected for flow time of
buffer alone (to), η=t−tο/ tο [22].
Thermal denaturation studies were carried out with an
Elico Bio-spectrophotometer model BL198, equipped with
temperature-controlling programmer ( 0.1 °C). The absor-
bance at 260 nm was continuously monitored for solutions
of CT-DNA (100 μM) in tris-buffer in the absence and
presence of the complex (10 μM), with temperature
1.20E+02
Fig. 2 Absorption spectra of
complexes 3 and 1 in absence
(red) and presence (lower) of
DNA. [complex]=10 μM;
[DNA]/complex]=0, 5, 10, 15.
Arrow shows the absorbance
changes upon increasing DNA
concentrations. Insert plots of
[DNA]/[εa−εf] vs [DNA] for the
titration of DNA with
0.54
3
1.00E+02
8.00E+01
6.00E+01
4.00E+01
2.00E+01
0.00E+00
0.49
0.44
0.39
0.34
0.29
0.24
0.19
0.14
0.09
0.04
2.05
0.00E 2.00E 4.00E 6.00E 8.00E
+00 +01 +01 +01 +01
[DNA]/105M
1.55
complexes; (•) experimental data
points; solid lines, linear fitting
of the data
1.05
0.55
364
384
404
424
444
464
484
504
0.05
214
264
314
364
414
464
514
1
2.50E-06
2.00E-06
1.50E-06
1.00E-06
5.00E-07
0.00E+00
2.55
2.05
1.55
1.05
0.55
0.00E 2.00E 4.00E 6.00E 8.00E
+00
-04
-04
[DNA]/M
-04
-04
0.75
0.7
0.65
0.6
0.55
350
370
390
410
430
450
470
280
330
380
430
480

J Fluoresc (2011) 21:563–572
567
increments of 1 °C, min−1. Supercoiled pBR322 DNA and
complex mixture was irradiated for 1 h at lmax=365 nm.
All samples were analyzed by electrophoresis for 2.5 h at
40 v/cm on a 0.8% agarose gel in buffer, pH 8.2, gel DNA
was visualized by staining with 1 μg/ml ethidium bromide
and then photographed under UV light.
The cell line HL-60 (human myeloid leukemia) was
obtained from the National Centre for Cellular Sciences
(NCCS), Pune, India. HL-60 cells were cultured in RPMI
1640 media supplemented with 10% (v/v) heat inactivated
fetal bovine serum (FBS), 100 units/ml penicillin and
100 μg/ml streptomycin. HL-60 cell lines were maintained
in culture at 37 °C in an atmosphere of 5% CO2.
600 nm. The bands below 300 nm are attributed to
intraligand (IL) π−π* transitions, the band at 354 nm is
attributed to the π−π* transition and the lowest energy
bands at 446, 448, 349 and 362 nm for complexes 1, 2, 3
and 4 are assigned to the metal-to-ligand charge transfer
(MLCT) transitions (Table 1) by comparison with the
spectra of other polypyridyl Ru(II) complexes [24–27].
The application of electronic absorption spectroscopy in
DNA-binding studies is one of the most useful techniques.
The complex binding with DNA through intercalation
usually results in hypochromism and bathochromism. The
interaction of these complexes with CT-DNA was moni-
tored by the absorption titration method. In the presence of
increasing CT-DNA, both complexes 1, 3 showed a strong
decrease in intensity (hypochromicity) and a small red shift
Fig. 2. The change in the absorbance of the MLCT band
with increasing amounts of CT-DNA was used to derive the
intrinsic binding constants (Kb). The values of Kb derived
for 1, 2, 3, and 4 complexes are 2.1 0.2×105, 1.9 0.2×
105, 8.5 0.2×104 and 7.32 0.2×104 respectively. The
different DNA-binding properties of complexes 1, 2, 3,
and 4 are due to the different ancillary ligand. On going
Spectroscopic Characterization
The IR spectral data for the complexes exhibit bands at
1,458 cm−1 and at 1,578–1,590 cm−1 due to C=C and C=N
vibrations of the ring respectively. Bands were present
around 587 cm−1 and 570 cm−1 corresponding to Co–N
1
(bpy) or Co–N of (phen). In the H-NMR spectra of the
complexes the peaks due to various protons of ligand shifted
downfield compared to the free ligand suggesting complex-
ation. As expected, the signal for the ligand appeared in the
range around 7 to 9.8 ppm and the 6 hydrogens of the
methyl group peaks appeared at 1.5–1.8 ppm
23
4
300
18
13
8
3
200
100
0
1 2 3 4 5 6 7 8
[DNA]/[Co]
Results and Discussion
DNA-binding Studies
The absorption titrations of Ru(II) & Co(III) complexes in
buffer were performed by using a fixed concentration of
ruthenium or cobalt complexes concentration to which
increments of the DNA stock solution(conc 4.5×10−4) were
added. Ruthenium (or) cobalt-DNA solutions were allowed
to incubate for 5 min before the absorption spectra was
recorded. The intrinsic binding constants Kb of complexes
to DNA were derived as follows [23].
525
550
600
650
695
Wavelength[nm]
2
4
3
2
1
0
157
100
1
2 3 4 5 6 7
[DNA]/[Co]
½DNAꢀ=ðea ꢁ ef Þ ¼ ½DNAꢀ=ðeb ꢁ ef Þ þ 1=ðKbðeb ꢁ ef ÞÞ
where [DNA] is the concentration of CT-DNA in base pairs,
the apparent absorption coefficients εa, εf, and εb correspond
to Aobsd/[Ru](or)[Co], the absorbance for the free complex,
and the absorbance for the complex in fully bound form,
respectively. Kb is given by the ratio of slope to intercept.
50
14
374
400
420
440
449
Wavelength[nm]
Fig. 3 Fluorescence emission spectra of complexes 2 and 4 in tris
buffer in the presence of CT DNA, where [cobalt or ruthenium]=
20 μM, [DNA]/Co=0, 5, 10, 15, 20 etc. The arrows indicate the
intensity changes upon increasing concentration. Insert: plots of
relative integrated emission intensity versus [DNA]/[Co]
Absorption Titration
The absorption spectra of 1, 2, 3 and 4 complexes shows
three or two well resolved bands in the range of 200–

568
J Fluoresc (2011) 21:563–572
Fig. 4 Emission quenching
curves of complexes (1), (2),
(3), and (4) in absence of DNA
(a) presence of DNA (1:20) (b),
excess of DNA (1:200 μM) (c)
1
2 18
a
18
16
14
12
10
8
16
14
a
b
12
10
8
b
c
6
4
2
0
6
4
c
2
0
0
0.01
0.02
0.03
0.04
0
0.01
0.02
0.03
0.04
[Fe(CN)6]4-mmol-1
[Fe(CN)6]4-mmol-1
14
3
4
a
16
14
12
10
8
12
10
8
a
b
b
6
6
4
4
c
2
0
c
2
0
0
0.01
0.02
0.03
0.04
0
0.01
0.02
0.03
0.04
[Fe(CN)6]4-mmol-1
[Fe(CN)6]4-mmol-1
from bpy to phen, the plane area and hydrophobicity
increases, and leading to a greater DNA-binding affinity for
complex 1. The intrinsic binding constant Kb of four
complexes with DNA is in the order 1>2>3>4.
increased relative viscosity of DNA. Based on relative
magnitude of viscosity binding affinity of complexes to
DNA follows the order 1>2>3>4 in agreement with the
conclusion that these complexes intercalate between the
base pairs of DNA.
Fluorescence Quenching
Thermal Denaturation
Complexes 1, 2, 3 and 4 exhibit fluorescence in tris buffer
at ambient temperature with λmax at 582, 408, 405 and
409 nm, addition of CT-DNA, the fluorescence emission
intensities of 1, 2, 3 and 4 increased by a factor of 4.2, 3.2,
2.1 and 1.2 respectively. This indicates that the strong
intercalation with DNA and the consequent restriction of
mobility of the complexes at the binding site has the effect
of enhancing, emission intensity Fig. 3. As per the
procedures described [21], fluorescence quenching experi-
ments were performed at ambient temperature with K4[Fe
(CN)6]4- as the quenching agent. From the data for all
complexes shown in Fig. 4, maximal quenching of
fluorescence of DNA bound complex is seen with complex
1 and this effect decreases as one goes from complex 1 to
complex 4.
To examine the effects of complex binding on thermal
denaturation, all complexes were allowed to interact with
DNA according to the conditions detailed and were heated
20
18
16
14
12
10
8
a
b
c
d
6
4
2
0
0
0.01
0.02
0.03
0.04
Viscosity Measurements
[Complex]/[DNA]
Since Intercalation result in enhanced DNA viscosity [21,
22], viscosity of DNA in presence of complexes 1–4, have
been determined as shown in Fig. 5. All complexes lead to
Fig. 5 Effect of increasing amount of complexes on the relative
viscosities of CT DNA at 25 0.1 °C complex -1 (a), complex-2 (b),
complex -3 (c) and complex -4 (d)

J Fluoresc (2011) 21:563–572
569
DNA alone
from room temp up to 80 °C to obtain DNA melting curves
from which Tm values were derived. Free un-complexed
CT DNA used herein had a Tm=60 °C. Tm for each
complex was determined as the difference between Tm of
complex + DNA and Tm of DNA alone. On this basis Tm of
complex increased by 4–7 °C, in the order 1>2>3>4 as
shown by the data of Table 1. Thus the binding of
complexes did enhance the stability of DNA and further-
more, strength of binding was greatest for complex 1,
decreasing in the same order as indicated by other data of
this study. (Table 1).
a
b
c
d
For the gel electrophoresis experiment, Supercoiled
pBR322 DNA (0.1 l g) was treated with the Ru(II) & Co
(III) complexes in 50 mM Tris–HCl, 18 mM NaCl buffer,
and the solution was then irradiated at room temperature
with a UV lamp (365 nm, 10 W). The samples were
analyzed by electrophoresis for 1.5 h at 80 V on a 0.8%
agarose gel in TAE buffer. The gel was stained with lg/ml-1
ethidium bromide and photographed under UV light.
Wavelength (nm)
Fig. 7 Circular dichroism spectra of the dialysate of complexes 1 (a),
2 (b), 3 (c) and 4 (a) after 24 h dialysis against with CT DNA
are cleaved, a linear form (Form III) that migrates between
Form I and Form II will be generated [30]. The cleavage
effect upon irradiation of the plasmid pBR322 DNA in the
presence of different concentrations of complexes 1, 2, 3,
and 4 have been tested and shown in Fig. 6 Lane 1 DNA
alone, lanes 2–6 DNA + complexes with increasing
concentration of complex. Form II increases of and, Form
I decreases gradually as the concentration of complex
increase. The results suggest concentration-dependent
single-strand cleavage of supercoiled Form I to the nicked
Form II. Under comparable experimental conditions,
complex 1 exhibits more effective DNA cleavage activity
than complexes 2, 3, and 4. The different cleaving
efficiency may be ascribed to the different binding affinity
of complexes to DNA.
Photoactivated Cleavage of pBR322 DNA
There is substantial and continuing interest in DNA
endonucleolytic cleavage reactions that are activated by
metal ions [28, 29]. The cleavage reaction on plasmid DNA
(pBR322 DNA) can be monitored by agarose gel electro-
phoresis. When circular plasmid DNA is subjected to
electrophoresis, relatively fast migration will be observed
for the intact supercoil form (Form I). If scission occurs on
one strand (nicking), the supercoil will relax to generate a
slower-moving open circular form (Form II). If both strands
Lane
1
1
2
3
4
5
6
Enantioselective Binding
Form II
Form I
A difference in biological activities between the enantiomers
of an optically active metal complex has been noted in many
examples such as toxicity and drug efficiency [31, 32]. In
order to understand such affects better at the level of
molecular interaction. It is of therefore important to examine
enantiomeric effects on the DNA binding of a metal complex.
According to the insertion model proposed by Barton and co-
workers [22, 33], the Δ enantiomer of the complex, a right-
handed propeller-like structure, will display a greater affinity
than the Λ enantiomer with the right-handed CT-DNA helix,
due to the appropriate steric matching. This discrimination
can be observed via equilibrium dialysis experiments and
provide strong evidence in support of intercalation.
2
3
4
Form II
Form I
Form II
Form I
Form II
Form I
The CD spectrum of CT DNA exhibits a positive band at
274 nm due to base stacking and a negative band at 243 nm
due to the helicity of DNA. In the presence of complex an
increase in the molar eliplicity values of the both positive
and negative band of the CT DNA observed Fig. 7. It can
Fig. 6 Photocleavage of pBR 322 DNA, in absence and in the
presence of complexes 1, 2, 3 and 4 light after 60 min irradiation at
365 nm. Lane 1 control plasmid DNA (untreated pBR 322), lanes 2 to
6 addition of complexes, in amounts of (0.01 M) 5, 10, 20, 30, 40 μl
of complex concentraction

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J Fluoresc (2011) 21:563–572
Fig. 8 Effects of complexes on
the viability of HL-60 cells,
following continuous incubation
for 24 h, with increasing drug
concentration (0.1–500 μM)
1
3
65
60
55
50
45
40
35
30
25
20
15
10
60
55
50
45
40
35
30
25
20
15
0.75
1
1.25 1.5 1.75
2
2.25 2.5
0.75
1
1.25 1.5 1.75
2
2.25
Concentration (µM)
Concentration (µM)
EC ~63.4 µM
50
IC ~61.7 µM
4
50
60
55
50
45
40
35
30
25
20
15
0.75
1
1.25 1.5 1.75
2
2.25 2.5
Concentration (µM)
IC ~89.9 µM
50
be concluded that the interaction of all the complexes
towards CT-DNA follows the same order as the former
experiments. Those significant changes indicate conforma-
tional changes and unwinding of DNA base pairs with
destabilization of the DNA double helix, which is consistent
with DNA intercalation mode suggested above.
plate was incubated at 37 °C in the dark for 4 h. The
formazan crystals were solubilized in DMSO (100 μl/well)
and the reduction of MTT was quantified by absorbance at
570 nm in a spectrophotometer (Spectra MAX Plus;
Molecular Devices; supported by SOFT max PRO 3.0).
Effects of test compounds on cell viability were calculated
using cells treated with DMSO as control. The data were
subjected to linear regression analysis and the regression
lines were plotted for the best straight line fit Fig. 8. The
IC50 (inhibition of cell viability) concentrations were
calculated using the respective regression equation.
Cytotoxicity Studies
Cell proliferation or viability was measured using the MTT
[3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasodium
bromide] assay [34]. Cells were seeded in each well
containing 100 μl medium at a final density of 2×104
cells/well, in 96 well microtiter plates at identical con-
ditions. After overnight incubation, the cells were treated
with different concentrations of testing ligand complexes
(20–100 μg/ml in RPMI 1640 medium and filtered) in a
final volume of 200 μl with five replicates each. After 24 h,
10 μl of MTT (5 mg/ml) was added to each well and the
Observations
The cytotoxic effects of test complexes were investigated
on human leukemia cell lines. After 24 h of treatment, the
number of live cells were measured by MTT assay and IC50
values for each cell line were determined (Table 2). It is
evident from Table 1 that HL-60 cells were sensitive to
Table 2 CD data for the
Complexes
Positive peak
Negative peak
Cytotoxic activity
of test complexes(IC50
values in μM)
interaction of CT DNA and
Cytotoxic data of complex
1 to 4
DNA alone
274 243 nm
273, 242 nm
275, 243 nm
275.2 243 nm
276.2 242 nm
−246 nm
−244 nm
−246.1 nm
−247 nm
−246.8 nm
1
2
3
4
63.4 μM
No activity
61.7 μM
89.9 μM

J Fluoresc (2011) 21:563–572
571
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complex 1 and 3, with an IC50 value of 61.7 and 63.4 μg/ml,
as compared with complex 4. Complex 2 did not show any
activity against the HL-60 cells and the decreasing order of
activity among the test complexes was in the order 1>3>4
Fig. 8. The chemical structure of the mixed ligand
complexes could be a factor for exhibiting differential
anti-proliferative activities on the cancerous cell lines.
Conclusions
Four Ru(II) and Co(III) complexes of extended planar
aromatic ligands have been isolated, characterized and their
interaction with CT DNA examined. Absorption, Emission,
thermal denaturation and viscosity experiments were
performed and the result obtained suggests an intercalative
mode of DNA binding for the four complexes. The planarity
of the ligands plays an important role in dictating DNA
binding affinity. Thus the complex 1 binds to DNA more
strongly than remaining complexes binding, affinity follows
the order 1>2>3>4.The present study demonstrates that the
ancillary ligands with hydrogen bonding potential support the
intercalative interaction of ligands with extended aromatic
rings and enhances the DNA binding affinity.
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Acknowledgements We are grateful to G. Bhanuprakash reddy,
Scientist-G, National Institute of Nutrition, Hyderabad for helping to record
CD spectra and also grateful to DST New Delhi for financial support.
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