Effect of substituent of terpyridines on the DNA-interaction of polypyridyl ruthenium(II) complexes

Article abstract of DOI:10.1016/j.saa.2011.09.037

An octahedral complexes of ruthenium with 2,9-dimethyl-1,10-phenanthroline (dmphen) and substituted terpyridine have been synthesized. The Ru^II complexes have been characterized by elemental analyses, thermogravimetric analyses, magnetic moment measurements, FT-IR, electronic, ¹H NMR and FAB mass spectra. The binding strength and mode of interaction of the complexes with Herring Sperm DNA has been investigated using absorption titration and viscosity measurement studies. Results suggest that the substituent on terpyridine ligand affects the binding mode and binding ability of the complexes. Effect of time and ionic strength on DNA cleavage ability of complex has also been studied by gel electrophoresis. Results suggest that more than 200 mM concentration of NaCl decreases the cleavage ability of complex.

Full text of DOI:10.1016/j.saa.2011.09.037

Spectrochimica Acta Part A 84 (2011) 243–248
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Effect of substituent of terpyridines on the DNA-interaction of polypyridyl
ruthenium(II) complexes
Mohan N. Patel∗, Deepen S. Gandhi, Pradhuman A. Parmar
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar-388 120, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
An octahedral complexes of ruthenium with 2,9-dimethyl-1,10-phenanthroline (dmphen) and substi-
tuted terpyridine have been synthesized. The RuII complexes have been characterized by elemental
analyses, thermogravimetric analyses, magnetic moment measurements, FT-IR, electronic, 1H NMR and
FAB mass spectra. The binding strength and mode of interaction of the complexes with Herring Sperm
DNA has been investigated using absorption titration and viscosity measurement studies. Results suggest
that the substituent on terpyridine ligand affects the binding mode and binding ability of the complexes.
Effect of time and ionic strength on DNA cleavage ability of complex has also been studied by gel elec-
trophoresis. Results suggest that more than 200 mM concentration of NaCl decreases the cleavage ability
of complex.
Received 28 April 2011
Received in revised form
10 September 2011
Accepted 14 September 2011
Keywords:
Mixed ligand ruthenium(II) complex
Substituted terpyridines
DNA-interaction
© 2011 Elsevier B.V. All rights reserved.
Ionic strength
1. Introduction
still remain unknown. Therefore, extensive studies on structurally
different tridentate ligands are necessary to further evaluate and
Metal complexes binding with nucleic acid are currently inves-
tigated because of their utility as DNA structural probes, DNA
foot printing and specific cleavage agents, and potential anticancer
drug [1]. Studies of the interaction of transition metal complexes
with DNA have been a pet subject of researchers in the field of
bioinorganic chemistry [2]. Transition-metal complexes are well
suited for application as artificial nucleases, because of their diverse
structural features, and the possibility to tune their redox poten-
tial through the choice of proper ligands [3]. Synthetic routes to
2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridines have attracted considerable interest because
these heterocycles are used extensively in both coordination chem-
istry and supramolecular chemistry [4,5].
Dipyridyl complexes of ruthenium have been reported to show
high excited state potential but are not very efficient DNA interca-
lators, while phenanthroline complexes of ruthenium are reported
to bind DNA via intercalation. The ruthenium complexes of ter-
pyridine ligands are more rigid compared to bipyridine ligands,
and have not been investigated systematically. The complexes of
terpyridine moieties have several synthetic and structural advan-
tages over the bipyridine complexes [2]. As far as RuII complexes
with tridentate ligands, the concerned studies have received a lim-
ited degree of attention, because interest in such complexes is
restricted by the absence of room temperature luminescence of
[Ru(tpy)2]2 [6], and their exact mode and extent of DNA-binding
understand the factors that determine the DNA-binding mode and
extent [7]. The octahedral polypyridyl RuII complexes bind to DNA
in three dimensions, but the ancillary ligands can also play an
important role in governing DNA-binding of the complexes. By
varying substitutive group or substituent position in the ancillary
ligand creates some interesting differences in the space config-
uration and the electron density distribution of RuII polypyridyl
complexes, which will result in some differences in spectral prop-
erties and the DNA-binding behaviors of the complexes, and will be
helpful to understand the binding mechanism of RuII polypyridyl
complexes to DNA [8].
In this article, we report the synthesis and characteri-
zation of three complexes: [RuII(4-cptpy)(dmphen)Cl]ClO4
(1),
ttpy)(dmphen)Cl]ClO4 (3), where 4-cptpy = 4ꢀ-(4-chlorophenyl)-
2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine, ptpy = 4ꢀ-phenyl-2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine,
4-ttpy = 4ꢀ-(4-tolyl)-2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine,
dmphen = 2,9-
[RuII(ptpy)(dmphen)Cl]ClO4
(2)
and
[RuII(4-
dimethyl-1,10-phenanthroline. Binding behaviors of the
synthesized complexes towards Herring Sperm DNA have been
investigated using absorption titration and viscosity measurement
methods. Cleavage of pUC19 DNA by the complexes has also been
studied by gel electrophoresis technique.
2. Experimental
2.1. Materials and chemicals
∗
2-Acetyl pyridine, 4-chlorobenzaldehyde, benzaldehyde and
4-methylbenzaldehyde were purchased from Spectrochem
Corresponding author. Tel.: +91 2692226858x221.
E-mail address: jeenen@gmail.com (M.N. Patel).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.09.037

244
M.N. Patel et al. / Spectrochimica Acta Part A 84 (2011) 243–248
4-chlorobenzaldehyde. Yield: 1.26 g, 39%, mp: 151–152 ◦C. 1H NMR
(Mumbai, India). Ruthenium trichloride and sodium perchlo-
rate were purchased from Chemport (Mumbai, India). Luria
Broth, agarose, ethidium bromide (EB), TAE (Tris–Acetyl–EDTA),
bromophenol blue and xylene cyanol FF were purchased from
Himedia, India. Herring Sperm DNA was purchased from Sigma
Chemical Co., India. 2,9-Dimethyl-1,10-phenanthroline was pur-
chased from Loba Chemie (India). Culture of pUC19 bacteria
(MTCC 47) was purchased from Institute of Microbial Technology,
Chandigarh, India.
ꢀ
ꢀ
ꢀꢀ
(CDCl3, 400 MHz) ı/ppm 8.798 (s, 2H, H3 ,5 ), 8.776 (d, 2H, H3,3 ),
ꢀꢀ
ꢀꢀ
8.721 (d, 2H, H6,6 ), 7.924 (dd, 2H, H4,4 ), 7.869 (d, 2H, Hph2,6),
7.404 (dd, 2H, H5,5 ), 7.344 (d, 2H, Hph3,5), 2.453 (s, 3H, CH3). 13C
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
NMR (CDCl3, 100 MHz) ı/ppm 156.26 (C2 ,6 ), 155.75 (C2,2 ), 150.18
ꢀ
ꢀꢀ
ꢀꢀ
(C4 ), 148.98 (C6,6 ), 139.1 (Cph1), 136.92 (C4,4 ), 135.45 (Cph4),
ꢀꢀ
ꢀꢀ
129.66 (Cph3,5), 127.15 (Cph2,6), 123.75 (C5,5 ), 121.42 (C3,3 ), 118.7
ꢀ
ꢀ
(C3 ,5 ), 21.25 (CH3). Anal. Calc. for C22H17N3: C, 81.71; H, 5.30; N,
12.99. Found: C, 81.96; H, 5.13; N, 13.12. UV–Vis (DMSO): ꢁ/nm
(εmax/dm3 mol−1 cm−1) 283.0 (3.5 × 104).
2.2. Physical measurements
2.4. Synthesis of complexes
Infrared spectra were recorded on Fourier transform IR
(FTIR) Shimadzu spectrophotometer as KBr pellets in the range
4000–400 cm−1. The 1H NMR and 13C NMR were recorded on a
Bruker Avance (400 MHz). The fast atomic bombardment mass
spectra (FAB MS) were recorded on Jeol SX 102/Da-600 mass spec-
trometer/data system using Argon/Xenon (6 kV, 10 mA) as the FAB
gas. The accelerating voltage was 10 kV and spectra were recorded
at room temperature. The electronic spectra were recorded on a
UV-160A UV–Vis spectrophotometer, Shimadzu (Japan). TGA was
carried out using a 5000/2960 SDTA, TA instrument (USA) operating
at a heating rate of 10 ◦C per minute in the range of 20–800 ◦C under
N2. C, H and N elemental analyses were performed with a model
240 Perkin Elmer elemental analyzer. The magnetic moments were
measured by Gouy’s method using mercury tetrathiocyanatocobal-
tate(II) as the calibrant (ꢀg = 16.44 × 10−6 cgs units at 20 ◦C), with a
Citizen Balance.
2.4.1. Synthesis of [RuII(4-cptpy)(dmphen)Cl]ClO4 (1)
The [RuIII(4-cptpy)Cl3] (5 mmol) was synthesized by stirring
4-cptpy in ethanol (500 mL) with gentle heating till dissolution fol-
lowed by addition of RuCI3·3H2O (5 mmol) to it and the solution
was refluxed for 3 h with stirring. The mixture was allowed to cool
at room temperature resulting in the formation of brown precipi-
tate. The precipitate was filtered off and was washed with ethanol
and ether followed by drying in air [9]. The complex [RuIII(4-
cptpy)Cl3] (280 mg, 0.5 mmol), 2,9-dimethyl-1,10-phenanthroline
(114 mg, 0.55 mmol), excess LiCl (122 mg, 2.94 mmol) and NEt3
(0.9 mL) were taken in 50 mL ethanol and the mixture was refluxed
for 2 h under a dinitrogen atmosphere (Scheme 1). In this reaction,
NEt3 functions as a reducing agent and facilitates dissociation of
the Ru–Cl Bond. The initial dark brown color of the solution grad-
ually changed to a deep purple. The solvent was then removed
under reduced pressure. The dry mass was dissolved in a min-
imum volume of acetonitrile and an excess saturated aqueous
solution of NaClO4 was added to it. The precipitate was filtered
off and washed with cold ethanol followed by ice-cold water.
The product was dried in vacuum and purified using a silica col-
umn. The complex was eluted by 2:1 CH2Cl2/CH3CN. Yield: 0.279 g,
71%, mol. wt. 788.04. IR (KBr): ꢂ 3066 w,br; 2920 sh; 1603 m,sh;
2.3. Synthesis of ligands
2.3.1. Synthesis of 4ꢀ-(4-chlorophenyl)-2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine
(4-cptpy)
2-Acetylpyridine (2.42 g, 20.0 mmol) was added to 70 mL
ethanolic solution of 4-chlorobenzaldehyde (1.4 g, 10.0 mmol).
KOH pellets (1.4 g, 26 mmol) and aqueous NH3 (30 mL, 25%,
0.425 mol) were added to the solution and was then stirred at room
temperature for 8 h (Scheme 1). An off-white solid was formed
which was collected by filtration and washed with H2O (3× 10 mL)
and ethanol (2× 5 mL). Recrystallization from CHCl3–MeOH gave
white crystalline solid. Yield: 1.48 g, 43%, mp: 168–169 ◦C. 1H NMR
1498 m,sh; 1088 s,sh; 756 s,sh; 626 vs,sh; 492 w,sh cm−1
[dimethyl sulfoxide-d6 (DMSO-d6), 400 MHz] ı/ppm 9.512 (s, 2H,
.
1H NMR
ꢀ ꢀ ꢀꢀ
T3 ,5 , where T = Terpyridine), 9.117 (d, 2H, T6,6 ), 8.491 (d, 1H, P7,
ꢀꢀ
ꢀꢀ
where P = Phenanthroline), 8.402 (d, 2H, T3,3 ), 8.083 (t, 2H, T4,4 ),
7.875 (d, 2H, Tph3,5), 7.864 (s, 2H, P5,6), 7.792–7.775 (m, 2H, P4,8),
ꢀꢀ
7.757 (d, 1H, P3), 7.557 (d, 2H, Tph2,6), 7.295 (t, 2H, T5,5 ), 3.321 (s,
ꢀ
ꢀ
ꢀꢀ
(CDCl3, 400 MHz) ı/ppm 8.753–8.744 (m, 4H, H3,3 ,5 ,3 ), 8.697 (d,
3H, CH3), 2.747 (s, 3H, CH3). Anal. Calc. for C35H26Cl3N5O4Ru: C,
53.34; H, 3.33; N, 8.89%. Found: C, 53.55; H, 3.16; N, 9.05%. FAB
MS: m/z = 789 [M]+, 691 [M–ClO4+H]+, 653 [M–ClO4–Cl]+, 345 [4-
cptpy+2H]+, 209 [dmphen+H]+.
ꢀꢀ
ꢀꢀ
2H, H6,6 ), 7.939–7.859 (m, 4H, H4,4 , Hph2,6), 7.495 (d, 2H, Hph3,5),
7.390 (dd, 2H, H5,5 ). 13C NMR (CDCl3, 100 MHz) ı/ppm 156.24
ꢀꢀ
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
ꢀꢀ
(C2 ,6 ), 155.74 (C2,2 ), 149.24 (C4 ), 148.81 (C6,6 ), 137.4 (C4,4 ),
136.82 (Cph1), 135.3 (Cph4), 129.23 (Cph2,6), 128.65 (Cph3,5), 123.96
(C5,5 ), 121.65 (C3,3 ), 118.83 (C3 ,5 ). Anal. Calc. for C21H14ClN3: C,
73.36; H, 4.10; N, 12.22. Found: C, 73.12; H, 4.24; N, 12.06. UV–Vis
(DMSO): ꢁ/nm (εmax/dm3 mol−1 cm−1) 280.5 (3.1 × 104).
2.4.2. Synthesis of [RuII(ptpy)(dmphen)Cl]ClO4 (2)
ꢀꢀ
ꢀꢀ
ꢀ
ꢀ
The complex was synthesized in a manner identical to that
described for [RuII(4-cptpy)(dmphen)Cl]ClO4, with [RuIII(ptpy)Cl3]
(258 mg, 0.5 mmol) in place of [RuIII(4-cptpy)Cl3]. Yield: 0.233 g,
62%, mol. wt. 753.6. IR (KBr): ꢂ 3072 w,br; 2924 sh; 1596 m,sh;
2.3.2. Synthesis of 4ꢀ-phenyl-2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine (ptpy)
The ligand was prepared by the same method as described
above, but using benzaldehyde (1.06 g, 10 mmol) instead of 4-
chlorobenzaldehyde. Yield: 1.11 g, 36%, mp: 202–204 ◦C. 1H NMR
1496 m,sh; 1083 s,sh; 757 s,sh; 623 vs,sh; 496 w,sh cm−1 1H NMR
.
ꢀ
ꢀ
ꢀꢀ
(DMSO-d6, 400 MHz) ı/ppm 9.481 (s, 2H, T3 ,5 ), 9.119 (d, 2H, T6,6 ),
ꢀꢀ
ꢀꢀ
8.44 (d, 2H, T3,3 ), 8.34 (d, 1H, P7), 8.078 (t, 2H, T4,4 ), 7.864 (s,
2H, P5,6), 7.798–7.778 (m, 2H, P4,8), 7.698 (d, 1H, P3), 7.619 (d, 2H,
ꢀ
ꢀ
ꢀꢀ
(CDCl3, 400 MHz) ı/ppm 8.802 (s, 2H, H3 ,5 ), 8.771 (d, 2H, H3,3 ),
ꢀꢀ
ꢀꢀ
8.72 (d, 2H, H6,6 ), 7.959–7.91 (m, 4H, H4,4 , Hph2,6), 7.556–7.456
ꢀꢀ
Tph2,6), 7.584–7.571 (m, 3H, Tph3,4,5), 7.298 (t, 2H, T5,5 ), 3.354 (s, 3H,
(m, 3H, Hph3,4,5), 7.395 (dd, 2H, H5,5 ). 13C NMR (CDCl3, 100 MHz)
ꢀꢀ
CH3), 2.77 (s, 3H, CH3). Anal. Calc. for C35H27Cl2N5O4Ru: C, 55.78; H,
3.61; N, 9.29%. Found: C, 55.93; H, 3.49; N, 9.42%. FAB MS: m/z = 753
[M]+, 655 [M–ClO4+H]+, 619 [M–ClO4–Cl]+, 311 [ptpy+2H]+, 209
[dmphen+H]+.
ꢀ
ꢀ
ꢀꢀ
ꢀ
ꢀꢀ
ı/ppm 155.88 (C2 ,6 ), 155.55 (C2,2 ), 150.47 (C4 ), 148.78 (C6,6 ),
138.31 (Cph1), 137.32 (C4,4 ), 129.11 (Cph2,6), 127.39 (Cph3,4,5),
123.94 (C5,5 ), 121.6 (C3,3 ), 119.2 (C3 ,5 ). Anal. Calc. for C21H15N3:
C, 81.53; H, 4.89; N, 13.58. Found: C, 81.32; H, 4.71; N, 13.41. UV–Vis
(DMSO): ꢁ/nm (εmax/dm3 mol−1 cm−1) 279.5 (3.7 × 104).
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀ
ꢀ
2.4.3. Synthesis of [RuII(4-ttpy)(dmphen)Cl]ClO4 (3)
The complex was synthesized in
a manner identical to
2.3.3. Synthesis of 4ꢀ-(4-tolyl)-2,2ꢀ:6ꢀ,2ꢀꢀ-terpyridine (4-ttpy)
The ligand was prepared by the same method as described
above but using 4-methylbenzaldehyde (1.2 g, 10 mmol) instead of
that described for [RuII(4-cptpy)(dmphen)Cl]ClO4, with [RuIII(4-
ttpy)Cl3] (265 mg, 0.5 mmol) in place of [RuIII(4-cptpy)Cl3]. Yield:
0.284 g, 74%, mol. wt. 767.62. IR (KBr): ꢂ 3071 w,br; 2925 sh; 1594

M.N. Patel et al. / Spectrochimica Acta Part A 84 (2011) 243–248
245
Scheme 1. Synthesis of terpyridine ligands and ruthenium(II) complexes (1, R Cl; 2, R H; 3, R CH3).
m,sh; 1495 m,sh; 1087 s,sh; 762 s,sh; 628 vs,sh; 487 w,sh cm−1
.
of each sample was measured three times and an average flow
time was calculated. Data were represented graphically as (Á/Á0)1/3
versus concentration ratio ([Complex]/[DNA]) [18], where Á is vis-
cosity of DNA in the presence of complex and Á0 is viscosity of DNA
alone. Viscosity values were calculated from the observed flow time
of DNA-containing solutions (t > 100 s) corrected for the flow time
of buffer alone (t0), Á = t − t0.
1H NMR (DMSO-d6, 400 MHz) ı/ppm 9.473 (s, 2H, T3 ,5 9.124 (d,
ꢀ
ꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
2H, T6,6 ), 8.387 (d, 2H, T3,3 ), 8.353 (d, 1H, P7), 8.068 (t, 2H, T4,4 ),
7.873 (s, 2H, P5,6), 7.795–7.773 (m, 2H, P4,8), 7.705 (d, 1H, P3),
ꢀꢀ
7.584–7.594 (d, 2H, Tph3,5), 7.554 (d, 2H, Tph2,6), 7.284 (t, 2H, T5,5 ),
3.387 (s, 3H, CH3), 2.747 (s, 3H, CH3), 2.525 (s, 3H, CH3). Anal. Calc.
for C36H29Cl2N5O4Ru: C, 56.33; H, 3.81; N, 9.12%. Found: C, 56.51;
H, 3.67; N, 9.26%. FAB MS: m/z = 767 [M]+, 669 [M−ClO4+H]+, 633
[M−ClO4−Cl]+, 325 [ptpy+2H]+, 209 [dmphen+H]+.
2.7. Gel electrophoresis
Caution: Perchlorate salts of metal complexes with organic
ligands are potentially explosive. Only small amounts of mate-
rial should be prepared, and these should be handled with care
[8,10–12].
Cleavage of pUC19 DNA (100 ␮g/mL) by RuII complexes
(200 ␮M) was measured by the conversion of supercoiled pUC19
DNA to open circular and linear. Gel electrophoresis of pUC19 DNA
was carried out in TAE buffer (0.04 M Tris–Acetate, pH 8, 0.001 M
EDTA). 15 ␮L of reaction mixture contains 100 ␮g/mL plasmid DNA,
and complex. To study the effect of ionic strength, reaction mix-
ture contains plasmid DNA, complex and different concentration
of NaCl. Reaction mixture was incubated at 37 ◦C. All reactions
were quenched by addition of 3 ␮L loading buffer (0.25% bro-
mophenol blue, 40% sucrose, 0.25% xylene cyanole, and 200 mM
EDTA). The aliquots were loaded directly on to 1% agarose gel
and electrophoresed at 50 V in 1X TAE buffer. Gel was stained
with 0.5 ␮g/mL of EB, and was photographed on a UV illumina-
tor. After electrophoresis, the proportion of DNA in each fraction
was estimated quantitatively from the intensity of the bands using
AlphaDigiDocTM RT. Version V.4.0.0 PC-Image software.
2.5. Evaluation of binding constants
Influence of DNA on MLCT band of RuII complexes were mea-
sured via UV–Vis absorbance spectra [13–16]. The absorption
titration was carried out by keeping the concentration of complex
constant (20 ␮M) and varying the concentration of DNA. The change
in absorbance at MLCT band was recorded after each addition of
DNA. The intrinsic binding constant Kb was determined according
to the following equation [17]:
[DNA]
εa − εf
[DNA]
εb − εf
1
=
+
Kb(εb − εf)
where [DNA] is the concentration of DNA in base pairs, the appar-
ent absorption coefficient εa, εf and εb correspond to Aobs./[Ru],
the extinction coefficient for the free ruthenium complex for each
addition of DNA and the extinction coefficient for the ruthe-
nium complex in the fully bound form, respectively. In plots
[DNA]/(εa–εf) versus [DNA], Kb is given by the ratio of slope to the
y intercept.
3. Result and discussion
3.1. Thermogravimetric analysis and magnetic moment
measurement
TGA data of the complexes show no weight loss between the
temperature of 80 and 180 ◦C. So, there is an absence of coordi-
nated or lattice water molecules. Both the ligands decomposed in
single step between the temperature ranges 360–600 ◦C. Magnetic
moment value of all complexes is found to be zero, which suggests
low-spin configuration with d2sp3 hybridization for octahedral RuII
complexes.
2.6. Viscosity measurement
Cannon–Ubbelohde viscometer maintained at a constant tem-
perature of 27.0 ( 0.1) ◦C in a thermostatic jacket was used to
measure the relative viscosity of DNA solutions in the presence
of ethidium bromide or RuII complexes. The concentrations of the
RuII complex and Herring Sperm DNA were chosen to minimize the
volume of RuII complex added to a solution of DNA. DNA concen-
tration was chosen enough to make changes in the slope maximally
distinguishable. A 3.0 mM stock solution of each RuII complex was
prepared. A 0.40 mM solution of Herring Sperm DNA was titrated
with the RuII complex. The RuII complex to DNA concentration ratio
was maintained in the range of 0–0.16. Flow time was measured
with a digital stopwatch with an accuracy of 0.01 s. The flow time
3.2. Electronic absorption analysis
The electronic spectra of ligands show only one band at
∼280 nm. The electronic spectra of complexes consist of three well-
defined bands in the range 250–500 nm, similar to that observed for
[Ru(dpphen)(terpy)Cl]PF6 complex reported by Yoshikawa et al.
[19]. The band maxima and molar extinction coefficient of com-
plexes are listed in Table 1. The metal–ligand charge transfer

246
M.N. Patel et al. / Spectrochimica Acta Part A 84 (2011) 243–248
Table 1
Electronic spectral data for the ruthenium(II) complexes.
Complexes
ꢁmax/nm (ε/dm3 mol−1 cm−1
)
␲ → ␲*
MLCT
1
2
3
281.0 (70,150), 311.0 (67,100)
276.0 (61,900), 310.0 (42,100)
284.0 (43,100), 308.0 (44,850)
487.5 (27,100)
488.5 (17,000)
490.0 (17,600)
(MLCT) bands appear at 487.5, 488.5 and 490 nm for complexes
1, 2 and 3, respectively. Change in the substitution on terpyridine
from electron withdrawing group (–Cl) to electron donating group
(–CH3), red shift in the MLCT band is observed. The two higher
energy absorption bands were observed from 276 to 284 nm and
308 to 311 nm. These bands are attributed to the ligand centered
transitions dmphen(␲)→dmphen(␲*) and terpy(␲)→terpy(␲*),
respectively [19].
3.3. Infrared spectroscopy
Infrared spectroscopy of the ligands showed bands at
∼1580 cm−1 and ∼1420 cm−1 corresponding to C C and C N ring
stretching. The band appeared at ∼720 cm−1 is due to C–H out of
plane banding. An additional band appeared at 2934 cm−1 in 4-ttpy
is due to the C–H stretching of methyl. The presence of perchlorate
as a counter ion is confirmed by the very strong, broad band at
∼1085 cm−1 and the strong, sharp band at around 625 cm−1 [20].
A weak, broad band around 3070 cm−1 appeared due to aromatic
C–H stretching. A sharp band around 2922 cm−1 appeared due to
C–H stretching of methyl. A sharp band with medium intensity
appeared around 1600 and 1497 cm−1, characteristic of aromatic
ring stretching. An intense, sharp band appeared at ∼760 cm−1 is a
characteristic of ring deformations and C–H out-of-plane deforma-
tions. A weak, sharp band was around 487–496 cm−1, characteristic
of Ru–N stretching mode. A Ru–Cl stretching mode would be
expected in the region less than 400 cm−1 [21].
3.4. 1H NMR
The ligands and complexes display resolvable 1H NMR spectra
in CDCl3 and DMSO-d6. On coordination to ruthenium ion, chemical
ꢀ
ꢀ
ꢀꢀ
shift of the T3 ,5 and T6,6 show large downfield due to metal-to-
ligand ␲-back donation [10]. Tph2,6 and Tph3,5 of complex 1 shows
downfield shift, while that of complexes 2 and 3 show upfield shift.
One methyl group of dmphen appears in downfield than other due
to the diamagnetic anisotropic effect exerted by terpyridine.
Fig. 1. Electronic absorption spectra of (a) [RuII(4-cptpy)(dmphen)Cl]ClO4, (b)
[RuII(ptpy)(dmphen)Cl]ClO4 and (c) [RuII(4-ttpy)(dmphen)Cl]ClO4 with increasing
amount of DNA in phosphate buffer (Na2HPO4/NaH2PO4, pH 7.2). [Com-
plex] = 20 ␮M, [DNA] = 0–16.7 ␮M with incubation period of 15 min at 37 ◦C. Plots
of [DNA]/(εa − εf) versus [DNA] for the titration of DNA with RuII complexes.
3.5. Evaluation of binding constants
Electronic absorption titration is employed to study the bind-
ing of polypyridyl RuII complexes with DNA. Complex bind to DNA
through intercalation usually results in hypochromism (decrease
in absorbance) and bathochromism (red shift), because intercala-
tive mode involves a strong stacking interaction between aromatic
chromophore and the base pairs of DNA [13]. Electrostatic inter-
action of complex with DNA shows lower hypochromicity with
no bathochromic shift [10,22]. Complex binds to DNA through
partial intercalation, generally resulting in hypochromism and
small red shift in absorption titration [17,23–25]. The extent of
the hypochromism commonly parallels the intercalative binding
strength.
of 19.2% in the MLCT transition is observed for complex 1 with
red shift from 487.5 to 490.5 nm (ꢃꢁ = 3 nm). The hypochromism
of 4.7 and 17.6% with red shift of 0 and 1 nm is observed for
complexes 2 and 3, respectively. The hypochromism of complex 2
is much smaller than complexes 1 and 3. Also, complex 2 exhibit
Table 2
Electronic absorption data upon addition of Herring Sperm DNA.
Complex
ꢁmax (nm)
Hypochromism,
Binding constant,
Kb (M−1
Ha (%)
)
The absorption spectra of [RuII(4-cptpy)(dmphen)Cl]+,
[RuII(ptpy)(dmphen)Cl]+ and [RuII(4-ttpy)(dmphen)Cl]+ with
increasing concentration of Herring Sperm DNA are shown in
Fig. 1. As the concentration of DNA is increased, hypochromism in
the MLCT band of each complex is observed (Table 2). A decrease
Free
Bound
ꢃꢁ
1
2
3
487.5
488.5
490.0
490.5
488.5
491.0
3
0
1
19.2
4.7
17.6
9.21 × 104
3.71 × 103
7.88 × 104
a
H% = 100 × (Afree − Abound)/Afree
.

M.N. Patel et al. / Spectrochimica Acta Part A 84 (2011) 243–248
247
no red shift for MLCT band. These results suggest that the complex
2 interact with DNA through electrostatic interaction, while com-
plexes 1 and 3 may bind to DNA via classical intercalation or partial
intercalation. More confirmation regarding the binding mode of
the complexes will be obtained from viscosity measurement.
In order to compare quantitatively the DNA-binding affinities
of the three complexes, the intrinsic binding constants (Kb) of
the complexes with Herring Sperm DNA were determined. From
the decay of MLCT band absorbance, the Kb values obtained for
complexes 1–3 are 9.21 × 104, 3.71 × 103 and 7.88 × 104 M−1
,
respectively. These values are comparable to that observed
for [Ru(dmp)2(ipbp)]+2 (7.58 × 103 M−1), [Ru(dmb)2(ipbp)]+2
(1.18 × 104 M−1
)
[11] and [Ru(tpy)(pta)] (9.51 × 104 M−1),
[17] but smaller than those observed for [Ru(bpy)2(paip)]+2
(3.12 × 105 M−1) [1] and [Ru(phen)2PMIP]2+ (8.53 × 105 M−1) [26].
Non-planarity of terpyridines, and methyl substitution on
phenanthroline ligand, are the main reasons for the lower Kb values.
An absence of planarity of the phenyl ring with the basic terpyri-
dine unit affects the magnitude of binding [27]. Substitution on the
2- and 9-positions of the phenanthroline ligand may cause severe
steric constraints near the RuII core when the complex intercalates
into the DNA base pairs. The methyl groups may come into close
proximity of base pairs at the intercalation sites [11]. Among the
complexes, complex 2 shows the least binding strength to double
helical DNA, may be due to its electrostatic binding mode. The dif-
ference between the Kb values of complexes 1 and 3 is owing to
different substituents. The electron-withdrawing substituent (–Cl
in 4-cptpy) on the intercalative ligand increases the DNA bind-
ing affinity, whereas the electron-releasing substituent (–CH3 in
4-ttpy) decreases the DNA affinity [28].
Fig. 2. Effect on relative viscosity of DNA under the influence of increasing
amount of ethidium bromide (EB) and complexes at 27 0.1 ◦C in phosphate buffer
(Na2HPO4/NaH2PO4, pH 7.2).
Fig. 3. Agarose gel (1%) of pUC19 (100 ␮g/mL) at 37 ◦C in TE buffer (pH 8) with
200 ␮M [RuII(4-cptpy)(dmphen)Cl]ClO4 complex for increasing incubation time.
Lane 1, DNA control and lanes 2–8, 30, 60, 90, 150, 210, 270 and 360 min, respectively.
3.6. Viscosity measurement
Viscosity measurement study was carried out to confirm the
binding mode of complexes. In classical intercalation, the base pairs
are separated to accommodate the binding ligand results in length-
ening of the DNA helix and thereby increase the DNA viscosity. A
partial and/or non-classical intercalation of compound may bend
DNA helix, resulting in the decrease of its effective length and,
thereby its viscosity [29]. Electrostatic interaction of compound
with DNA does not affect relative viscosity of DNA [30]. The effect
of increasing amount of EB and complexes on the relative viscosity
of DNA is shown in Fig. 2. EB is a well-known classical intercalator,
which increases the relative viscosity of DNA solution. Complex
[Ru(bpy)3]2+ has been known to bind with DNA in electrostatic
mode, it exerts essentially no effect on DNA viscosity [28]. Com-
plexes 1 and 3 decrease the relative viscosity of DNA as shown by
the partial intercalators. No change in relative viscosity is observed
for complex 2, and leads to conclude that complex 2 interacts with
DNA via electrostatic interaction. Absorption titration also supports
the same binding mode for complex 2 (0 nm red shift). Consider-
ing the results of spectroscopic and viscosity measurements, we
may suggest that complex 2 shows lowest affinity towards DNA
and interacts electrostatically with DNA, while complexes 1 and 3
partially intercalate to DNA.
quantified by measuring the transformation of the SC form into OC
and linear forms.
The time dependence of the reaction was determined in the
presence and the absence of the complex. The pUC19 DNA
(100 ␮g/mL) was incubated with 200 ␮M complex 1 in TE buffer
at 37 ◦C for 30–360 min. The increase in the amount of OC and L
forms of DNA was observed to be associated with the increase of
reaction time (Fig. 3). The amount of linear DNA was 33% when the
reaction time was 360 min. Fig. 4 shows the extent of DNA cleavage
by the complex 1 with reaction time. The decrease in the amount
of SC form and the formation of OC form of DNA with time shows
3.7. Effects of time and ionic strength on DNA cleavage
DNA cleavage efficiency of complexes can be achieved by for-
mation of the naturally occurring supercoiled (SC) form to open
circular relaxed (OC) form. OC form produced when one of the
strands of the plasmid is nicked, and determined by gel elec-
trophoresis of the plasmid. If both strands of the plasmid are nicked,
linear (L) form will be generated [31]. SC form migrates faster, OC
form migrates slowly while L form migrates in between the SC form
and the OC form [32]. Hence, DNA cleavage ability of complexes was
Fig. 4. Decrease in the SC form and formation of the OC and L form of pUC19
DNA in the presence of the [RuII(4-cptpy)(dmphen)Cl]ClO4 complex (200 ␮M) with
incubation time. Inset: ln(% supercoiled form) versus time.

248
M.N. Patel et al. / Spectrochimica Acta Part A 84 (2011) 243–248
Conflicts of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.
Acknowledgments
Fig. 5. Agarose gel (1%) of pUC19 DNA (100 ␮g/mL) incubated for 270 min at 37 ◦C in
TE buffer (pH 8) with 200 ␮M [RuII(4-cptpy)(dmphen)Cl]ClO4 complex for increas-
ing NaCl concentrations. Lane 1, DNA control and lanes 2–8, NaCl concentration was
25, 50, 100, 200, 400, 600 and 800 mM, respectively.
Authors thank Head, Department of Chemistry, and Dr. Thakkar,
BRD School of Bioscience, Sardar Patel University, India for making
it convenient to work in laboratory and UGC for providing financial
support under “UGC Research Fellowship in Science for Meritorious
Students” scheme.
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