Synthesis, structure, DNA interaction and nuclease activity of rhodium(III)-arylazoimidazole complexes

Article abstract of DOI:10.1016/j.ica.2012.08.005

The Rh(III) complexes of 1-alkyl-2-(arylazo)imidazoles (RaaiR′), [Rh(X)₂(RaaiR′)₂](ClO₄) (R = H (1, 3), Me (2, 4); R′ = Me (a), Et (b), CH₂Ph (c); X = Cl (1, 2), N ₃ (3, 4)), are synthesized and characterized by spectral (IR, UV-Vis, Mass and ¹H NMR) data. The structural confirmation has been done by single crystal X-ray diffraction study of [RhCl₂(MeaaiEt) ₂]ClO₄ (2b) (MeaaiEt, 1-ethyl-2-(p-tolylazo)imidazole). The DNA binding of the complexes (binding constant: 0.86 × 10⁴ to 1.05 × 10⁵) have been examined by absorption and fluorescence spectroscopic measurements. The nuclease activity of the complexes are examined by gel-electrophoresis and has revealed the highest activity of [Rh(N₃)₂(RaaiR′)₂]ClO₄. The DFT computation has been performed to the optimized geometry of the complexes to interpret the electronic structures and their spectral properties.

Full text of DOI:10.1016/j.ica.2012.08.005

Inorganica Chimica Acta 394 (2013) 98–106
Contents lists available at SciVerse ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis, structure, DNA interaction and nuclease activity
of rhodium(III)–arylazoimidazole complexes
Dibakar Sardar a,1, Papia Datta a,2, Sanju Das a, Biswarup Saha b, Saheli Samanta b, Debalina Bhattacharya b,
Parimal Karmakar b, Chung-De Chen c,d, Chun-Jung Chen c,d, Chittaranjan Sinha a,
⇑
a Department of Chemistry, Jadavpur University, Kolkata 700032, West Bengal, India
b Department of Life Science and Biotechnology, Jadavpur University, Kolkata 700 032, India
c Life Science Group, National Synchroton Radiation Research Centre, 101, Hsin Ann Road, Hsinchu 30076, Taiwan
d Department of Physics, National Tsing Hua University, Hsinchu 30043, Taiwan
a r t i c l e i n f o
a b s t r a c t
The Rh(III) complexes of 1-alkyl-2-(arylazo)imidazoles (RaaiR0), [Rh(X)2(RaaiR0)2](ClO4) (R = H (1, 3), Me
(2, 4); R0 = Me (a), Et (b), CH2Ph (c); X = Cl (1, 2), N3 (3, 4)), are synthesized and characterized by spectral
(IR, UV–Vis, Mass and 1H NMR) data. The structural confirmation has been done by single crystal X-ray
diffraction study of [RhCl2(MeaaiEt)2]ClO4 (2b) (MeaaiEt, 1-ethyl-2-(p-tolylazo)imidazole). The DNA
binding of the complexes (binding constant: 0.86 ꢀ 104 to 1.05 ꢀ 105) have been examined by absorption
and fluorescence spectroscopic measurements. The nuclease activity of the complexes are examined by
gel-electrophoresis and has revealed the highest activity of [Rh(N3)2(RaaiR0)2]ClO4. The DFT computation
has been performed to the optimized geometry of the complexes to interpret the electronic structures
and their spectral properties.
Article history:
Received 7 April 2012
Received in revised form 31 July 2012
Accepted 5 August 2012
Available online 29 August 2012
Keywords:
Rhodium(III)–arylazoimidazoles
Structure
DNA binding
Nuclease activity
DFT calculation
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
The DNA binding ability of the complexes is established by
spectroscopic studies and nuclease activity is considered by gel-
The interaction of transition metal complexes with DNA has re-
ceived vast attention in the last two decades of research both in
chemistry and biology [1–7]. The interaction of the metal com-
plexes with DNA is disturbing the replication and transcription
and ultimately leads to the cell death [8,9]. Over the past few years,
an intensive effort has been focused to develop metal-based drugs
with improved clinical effectiveness, reduced toxicity and broader
spectrum of activity [1–3,10–13]. The complexes of zinc [14],
molybdenum [15], palladium [16,17] and ruthenium [18,19] are
exhibiting anticancer activity. Many of them are very promising,
and show activity on tumors. Recently rhodium complexes have
been used in the development of metal-based drugs in cancer che-
motherapy [20–22]. They have been shown to bind nucleobases,
dinucleotides, and DNA dodecamer single strands [23–25]. This
has inspired us to study the interaction and nuclease activity of
rhodium complexes of N-donor ligands with DNA. In this article
the spectral and structural characterization of rhodium(III) com-
plexes of 1-alkyl-2-(arylazo)imidazoles (RaaiR0) are described.
electrophoresis. The electronic properties are correlated with DFT
calculation.
2. Experimental
2.1. Materials and methods
All reagents and solvents were of analytical grade except those
employed in DNA binding and cleavage experiments which were of
spectroscopic grade. RhCl3.3H2O was purchased from Arora-Mat-
they, India and used as it was received. NaN3, NaClO4 and the sol-
vents used were obtained from E. Merck, India. The ligands used in
this work were 1-alkyl-2-(arylazo)imidazole [RaaiR0, where R = H,
Me and R0 = CH3, CH2–CH3, CH2Ph] (Scheme 1) and were prepared
by reported procedure [26]. Chemicals used for syntheses were
analytical grade and solvents were dried before use [27]. The solu-
tion spectral studies were carried out by spectroscopic grade sol-
vents obtained from Lancester, UK.
Caution! Azide and perchorate salts are generally explosive.
Although no detonation tendencies have been observed, care is ad-
vised and handling of only small quantities recommended.
Microanalyses (C, H, N) were performed using Perkin-Elmer
2400 CHN elemental analyzer. Spectroscopic measurements were
⇑
Corresponding author. Fax: +91 033 2413 7121.
E-mail address: c_r_sinha@yahoo.com (C. Sinha).
Present address: Dinabandhu Andrews College, Garia, Kolkata 700084, India.
RCC Institute of Information Technology, Canal South Road, Kolkata 700015,
1
2
India.
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.08.005

D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106
99
Scheme 1. The ligands and the complexes.
carried out using the following instruments: UV–Vis spectra,
Lambda 25 Perkin Elmer; FT-IR spectra (KBr disk, 4000–
225 cmꢁ1), Perkin Elmer Spectrum BX1; 1H NMR spectra in CDCl3,
Bruker 300 MHz FT-NMR spectrometer in presence of TMS as inter-
nal standard. FAB-MS was collected from Jeol-AX 500 instrument.
Electrochemical measurements were carried out with a computer
controlled CH1 Electrochemical workstation using a Pt-disk work-
ing electrode and Pt-wire auxiliary electrode, [n-Bu4N][ClO4] sup-
porting electrolyte under inert (dry N2) environment at scan rate
50 mV Sꢁ1. The solution was IR compensated and the results were
collected at 298 K. The reported results were referenced to SCE in
MeCN and were uncorrected for junction potential.
4.11 (-N- CH3, s). The spectral data of other complexes are given
as Supplementary materials (Tables S1–S3).
2.2.2. Synthesis of [Rh(N3)2(RaaiR0)2]ClO4 (3, 4)
The complexes are synthesized following common procedure as
detailed below for 4a. The yield was varied 68–75%.
[Rh(N3)2(MeaaiMe)2]ClO4 (4a). To acetonitrile solution (10 ml)
of [RhCl2(MeaaiMe)2]ClO4 (2a) (0.2 g, 0.297 mmol), an aqueous
solution of AgNO3 (0.1 g, 0.594 mmol) was added and stirred for
few minutes. The orange brown mixture was then heated to reflux
for 30 min, then cooled and filtered through G-4 Gooch. To this fil-
trate NaN3 (0.038 g, 0.594 mmol) was added at room temperature
and stirred for 3 h. Or the addition of mixture of AgNO3 and NaN3
at once for metathetical reaction also resulted precipitate of AgCl.
It was filtered further and treated with aqueous NaClO4 (1 g in
3 ml water). A pale yellow precipitate immediately appeared. The
product was collected by filtration, washed with methanol and
ether and then dried under vacuum. Yield is 0.15 g (71%).
[Rh(N3)2(MeaaiMe)2]ClO4 (4a). Anal. Calc. for C22H24N14O4ClRh:
C, 38.57; H, 3.51; N, 28.63. Found: C, 38.63; H, 3.60; N, 28.73%. FT-
2.2. Synthesis of [RhCl2(RaaiR0)2]ClO4 (1, 2)
All the complexes were prepared following common procedure
as detailed below for 2a. Yield varied 65–75%.
2.2.1. [RhCl2(MeaaiMe)2]ClO4 (2a)
RhCl3.3H2O (0.10 g, 0.315 mmol) and 1-methyl-2-(ary-
lazo)imidazole (MeaaiMe) (0.125 g, 0.63 mmol) in 1:2 mol ratio
in methanol solution was refluxed for 6 h under stirring condition
and was cooled to room temperature. The volume of the solution
was reduced under pressure and chromatographed with silica gel
prepared in petroleum ether. A red solution was eluted with 1%
NaClO4 solution in MeOH and dried in vacuum and recrystallised
from acetonitrile–methanol (1:1, v/v) solution. The yield was
0.14 g (68%).
IR (KBr, cmꢁ1
)
m
(ClO4ꢁ), 624(w) and 1095(vs);
(N3), 2026(s). UV–Vis data (kmax(CH3CN)/nm; e,
m(N = N), 1390 (m);
m(C = N), 1593 (s);
m
103 Mꢁ1 cmꢁ1) 435 (1.62), 351 (1.10), 281 (1.18). MS(FAB+), m/z
587 (MꢁClO4ꢁ)+. 1H NMR (CDCl3) 8.12 (4-H, bs), 7.93 (5-H, bs),
8.02 (7,11-H, d, J = 8.0 Hz), 7.48 (8,10-H, d, J = 8.0 Hz), 2.50 (9-
CH3, s), 4.16 (–N–CH3, s). The spectral data of other complexes
are given as Supplementary materials (Tables S1–S3).
[RhCl2(MeaaiMe)2]ClO4 (2a): Anal. Calc. for C22H24N8O4Cl3Rh: C,
39.31; H, 3.57; N, 16.68. Found: C, 39.23; H, 3.50; N, 16.68%. FT-IR
2.3. X-ray crystallography
(KBr, cmꢁ1
)
m
(ClO4ꢁ), 622(w) and 1100 (vs);
m
((N = N), 1361 (m);
Dichloromethane solution of [RhCl2(MeaaiEt)2] ClO4 (2b) was
diffused into hexane and the crystals were grown within a week.
A suitable single crystal of was mounted on a CCD Diffractometer
equipped with fine-focus sealed tube graphite monochromated
m
(C = N), 1597 (s). UV–Vis data (kmax(CH3CN)/nm;
e
,103 Mꢁ1 cmꢁ1
)
500 (2.667), 405 (6.453), 214 (10.32)). MS(FAB+), m/z 490
(MꢁClO4ꢁ)+. 1H NMR (CDCl3) 8.14 (4-H, bs), 7.88 (5-H, bs), 7.90
(7,11-H, d, J = 8.0 Hz), 7.63 (8,10-H, d, J = 8.0 Hz), 2.49 (9-Me, s),
Mo-K (k = 0.71073 Å) radiation. Unit cell parameters were
a

100
D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106
determined from least-squares method. Summary of the crystallo-
graphic data and structure refinement parameters are given in
Table 1. Data were corrected for Lorentz and polarization effects.
Data reduction was carried out by using ‘Bruker SAINT’ programme.
The structure was solved by direct method using SHELXS-97 and suc-
cessive difference Fourier syntheses. All non-hydrogen atoms were
2.5. Interaction of complexes with DNA
To examine the interaction of the complexes with DNA we have
selected four complexes [RhCl2(HaaiMe)2]ClO4 (1a), [RhCl2(Mea-
aiMe)2]ClO4 (2a), [RhCl2(MeaaiEt)2]ClO4 (2b) and [Rh(N3)2(Meaai-
Et)2]ClO4 (4b) with reference to the variation of substituents in
chelated ligand (H/Me (1a/2a) and Me/Et (2a/2b)) and also substi-
tuting chloride by azide (2b/4b).
2
refined anisotropically. Full matrix least squares refinements on F0
were carried out using SHELXL-97 with anisotropic displacement
parameters for all non-hydrogen atoms. Hydrogen atoms were
constrained to ride on the respective carbon or nitrogen atoms
with anisotropic displacement parameters equal to 1.2 times the
equivalent isotropic displacement of their parent atom in all cases.
All calculations were carried out using SHELXS 97 [28], SHELXL 97 [29],
PLATON 99 [30] and ORTEP [31] programs.
2.5.1. Preparation of the complex solution for DNA binding and
nuclease studies
The title complexes were readily soluble in acetone free meth-
anol at a concentration of 2 mM each. The stock complex solutions
were diluted freshly in citrate–phosphate buffer (10 mM), pH 7.4
before each set of experiments.
2.4. Theory and computational methods
2.5.2. Preparation of calf thymus and pUC19 plasmid DNA
Purified calf thymus (CT) DNA (Bangalore Genei, India) and
pUC19 plasmid DNA (Bangalore Genei, India) were dissolved sepa-
rately at a concentration of 1 mg/ml stock in sterilized 10 mM cit-
rate–phosphate (CP) buffer, pH 7.4 at room temperature. Aliquots
were stored at 4 °C and diluted freshly before each set of
experiments.
In the framework of density functional theory (DFT) approach,
the B3LYP hybrid functional [32,33] one of the most preferred
methods since it proved its ability in reproducing various molecu-
lar properties, including structural parameters and vibrational
spectra is used. The combined use of B3LYP consisting of a hybrid
exchange functional, as defined by Becke’s three-parameter equa-
tion and the Lee–Yang–Par correlation and standard split valence
basis set 6-31G(d) is a common technique to provide an excellent
compromise between the accuracy and computational efficiency of
properties for large and medium-size molecules [34–36].
Ground-state electronic structure calculations of all compounds
had been performed using the Los Alamos effective core potential
plus double zeta (LanL2DZ) [37] basis set for rhodium and 6-
31G(d) for other elements. The ground-state geometries were ob-
tained in the gas phase by full geometry optimization and the opti-
mum structures, located as stationary points on the potential
energy surfaces, were verified by the absence of imaginary fre-
quencies. In all cases, vibrational frequencies were calculated to
ensure that optimized geometries represented local minima. Using
the respective optimized S0 geometries we employed time depen-
dent density functional theory (TD-DFT) at the B3LYP level to pre-
dict their absorptions characteristics [38]. The calculated data are
given as Supplementary material (Tables S4 and S5).
2.5.3. Ethidium bromide (EB) stock solution preparation
Ethidium bromide (EB) (Sigma–Aldrich, USA) was dissolved in
double distilled water at a concentration of 1 mM and stored at
4 °C in dark. The stock solution was diluted freshly before each
experiment.
2.5.4. Absorption spectroscopic studies of the complexes in presence of
CT DNA
Absorption spectroscopic studies were done on a spectropho-
tometer (Perkin Elmer, lambda-25), using either CT DNA (35
ml) with increasing concentrations of complex (0.5–20 M) or
each complex (40 M) with increasing concentrations of CT DNA
(0.13 g/ml to 11.4 g/ml). After each addition, the DNA and com-
lg/
l
l
l
l
plex mixtures were incubated at room temperature for 15 min and
scanned either from 210 nm to 310 nm for the DNA or from
290 nm to 600 nm for the complexes. The self-absorption of li-
gands in either case was eliminated in each set of experiments.
Each sample was scanned for a cycle number of 2, cycle time of
5 s at a scan speed of 100 nm/min. Modified Benesi–Hildebrand
[39] plot was used for the determination of ground state binding
constant between the complexes and CT DNA. The binding con-
stant ‘‘K’’ was determined by using the following relation:
Table 1
Crystallographic parameters of [RhCl2(MeaaiEt)2]ClO4 (2b).
Chemical_formula_sum
Crystal size (mm)
Chemical formula weight
C24H28N8O4Cl3Rh
0.1 ꢀ 0.1 ꢀ 0.1
701.80
A0=D
A ¼ A0=
D
Amax þ ðA0=
D
AmaxÞ ꢀ 1=K ꢀ 1=Lt
Crystal system
Space group
Monoclinc
C2/c
where
D
A = A0 ꢁ A,
D
Amax = maximum change in reduced absor-
a (Å)
b (Å)
c (Å)
12.9971(7)
17.6354(7)
37.9265(18)
95.5870(10)
8651.8(7)
12
bance, A0 = maximum absorbance of receptor molecules (without
any ligand), A = reduced absorbances of the receptor molecules (in
presence of ligand), Lt = ligand concentration.
b (°)
V (Å3)
Z
2.5.5. Fluorescence spectroscopic studies of the complexes with EB
bound DNA
T (K)
273(2)
1.616
0.840
Density (Mg/m3)
l
(mmꢁ1
)
The fluorescence spectroscopic studies of CT DNA (35 lg/ml)
F (000)
4056
0.71073
with varying concentrations of the complexes were done by using
a spectrofluorimeter (F3010, HITACHI). This study is based on the
competitive binding of the complex to the EB saturated DNA by
replacing EB. In this experiment at first, EB solution was gradually
added to the said concentration of DNA and at each time, the fluo-
rescence spectrum was scanned. The fluorescence intensity of EB
0
k (ÅA) (Mo-K )
a
hrange (°)
1.08 – 28.33
Index range
ꢁ17 6 h 6 17, ꢁ23 6 k 6 23, ꢁ50 6 l 6 47
Unique reflections
9856
R (Fo)a [I > 2
r
(I)]
(I)]
Goodness-of-fit (GOF)
0.0732
0.1456
1.417
wR (Fo)b [I > 2
r
bound DNA was saturated at 8.45
saturation level, the complex was added gradually (up to
34.7 M), incubated for 15 min after each addition and the fluores-
cence spectra were taken. The excitation wavelength was 546 nm
lM concentration of EB. At this
a
R =
wR = [
R
||Fo| ꢁ |Fc||/
R |Fo|.
b
2
R
w(Fo ꢁ Fc2)2/
R
w(Fo2)2]1/2
,
w = 1/[
r
2(Fo)2 + (0.0000P)2 + 99.7145P]
l
where P = (Fo2 + 2Fc2)/3.

D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106
101
Fig. 1. Molecular structure of two conformers of of cis-[RhCl2(MeaaiEt)2]ClO4 (2b).
Table 2
Selected bond distances (Å) and bond angles (°) for 2b (X-ray structure and theoretical).
Molecule-1
Molecule-2
Experimental
Calculateda
Experimental
Bond distances (Å)
Rh(1)–Cl(4)
Rh(1)–N(10)
Rh(1)–N(11)
N(9)–N(10)
2.3164(14)
2.051(4)
2.002(5)
1.286(6)
1.339(7)
2.4012
2.1496
2.0259
1.3088
1.3566
Rh(2)–N(7)
Rh(2)–N(5)
Rh(2)–N(1)
Rh(2)–N(2)
Rh(2)–Cl(2)
Rh(2)–Cl(1)
N(1)–N(4)
N(2)–N(3)
2.011(4)
2.013(4)
2.047(4)
2.057(4)
2.3224(13)
2.3242(13)
1.284(6)
1.289(6)
N(11)–C(26)
Bond angles(°)
N(10)–Rh(1)–N(10)b
N(11)–Rh(1)–N(10)b
N(11)–Rh(1)–N(10)
N(10)–Rh(1)–Cl(4)b
N(10)–Rh(1)–Cl(4)
N(11)–Rh(1)–N(11)b
N(11)–Rh(1)–Cl(4)b
N(11)–Rh(1)–Cl(4)
Cl(4)–Rh(1)–Cl(4)
84.0(2)
91.49
103.62
77.16
169.09
88.67
178.91
92.02
87.04
93.23
N(5)–Rh(2)–N(7)
N(5)–Rh(2)–N(1)
N(7)–Rh(2)–N(1)
N(5)–Rh(2)–N(2)
N(7)–Rh(2)–N(2)
N(1)–Rh(2)–N(2)
N(5)–Rh(2)–Cl(2)
N(7)–Rh(2)–Cl(2)
N(1)–Rh(2)–Cl(2)
N(2)–Rh(2)–Cl(2)
N(5)–Rh(2)–Cl(1)
N(7)–Rh(2)–Cl(1)
N(1)–Rh(2)–Cl(1)
N(2)–Rh(2)–Cl(1)
Cl(2)–Rh(2)–Cl(1)
169.85(18)
77.76(17)
94.28(16)
95.06(16)
77.74(16)
84.48(16)
91.49(13)
95.14(12)
92.22(12)
171.87(12)
94.82(13)
92.63(12)
171.56(12)
92.27(12)
91.85 (5)
96.53(18)
77.79(18)
170.14(13)
91.65(12)
172.4(3)
93.96(14)
91.20(14)
93.94(7)
a
As per atom numbering of molecule-1.
Symmetry: ꢁx + 1, y, ꢁz + 1/2.
b

102
D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106
and the emission spectra were scanned from 556 nm to 650 nm at
spectral response of 2s, along with a scanning speed of 60 nm/min
[40].
8000
7000
6000
5000
4000
3000
2000
1000
0
2.5.6. Nuclease activity of the complexes by agarose gel electrophoresis
study
Purified pUC19 plasmid DNA (500 mg) was incubated with
increasing concentrations of complex (17–100 lM) at 37 °C for
1 h in dark and then the DNA samples were loaded in 1% agarose
gel in 1ꢀ TASE buffer (pH 7.4) at a potential gradient of 6 volts/
cm for 2.5 h [40]. Each gel was then stained with EB solution
(500 lg/ml), photographs were taken in a gel documentation sys-
tem (Transilluminator, UVPRO) and analysed.
200
300
400
500
600
700
3. Results and discussions
Wavelength (nm)
3.1. The complexes and their formulation
Fig. 2. UV–Vis spectra of the complexes cis-[RhCl2(MeaaiEt)2]ClO4 (2b) (—) and cis-
[Rh(N3)2(MeaaiEt)2]ClO4 (4b) (---) in acetonitrile at room temperature.
The reaction of RhCl3 with 1-alkyl-2-(arylazo)imidazole (RaaiR0)
in 1:2 mol ratio in methanol followed by addition of saturated
aqueous solution of NaClO4 (Scheme 1, Eqs. (1) and (2) has sepa-
rated a red precipitate, [RhCl2(RaaiR0)2]ClO4. The azide derivative
[Rh(N3)2(RaaiR0)2 ]ClO4 (where R = H (3), Me (4) and R0 = Me (a),
Et (b). CH2Ph (c) is synthesised by metathesis with AgNO3 + NaN3
mixture.
The ligand, 1-alkyl-2-(arylazo)imidazole (RaaiR0), is N, N0 chelat-
ing system where N and N0 refer to N(imidazole) and N(azo) donor
centers, respectively (Scheme 1). The formulation of the com-
plexes, [RhCl2(RaaiR0)2]ClO4 (1, 2) and [Rh(N3)2(RaaiR0)2]ClO4 (3,
4) has been supported by microanalytical data (see Section 2)
and mass spectra. The structure of one of the complexes, [RhCl2(-
MeaaiEt)2]ClO4 (2b) has been established by single crystal X-ray
diffraction study. All the complexes are diamagnetic and 1:1 con-
ducting which indicates the presence of rhodium in +3 oxidation
state (d6). The complexes are soluble in common organic solvents.
3.2. Molecular structure of [RhCl2(MeaaiEt)2]ClO4 (2b)
cis-[RhCl2(MeaaiEt)2]ClO4 (2b) cis-[Rh(N3)2(MeaaiEt)2]ClO4 (4b)
The molecular structure of [RhCl2(MeaaiEt)2]ClO4 (2b) is given
in Fig. 1, and metric parameters are listed in Table 2. The asymmet-
ric unit of the lattice consists of one molecule in a general position
and half molecule which sits on a 2-fold axis i.e. the asymmetric
unit has 1.5 molecules. In terms of bond distances and angles these
two molecules have comparable values (see Supplementary mate-
rials). The complex comprises two chelating MeaaiEt ligands which
has two potential imidazolyl-N (abbreviated N) and azo-N (abbre-
viated N0) (Scheme 1) and two chloride, thus the central Rh atom
has a distorted octahedral coordination defined by a Cl2N2N02 do-
nor set. The atomic arrangement involves cis located chlorine,
and N0 donors, while Ns, are in trans position. The cis chlorine an-
gles are Cl(4)–Rh(1)–Cl(4) ( symmetry: ꢁx + 1, y, ꢁz + 1/2),
93.94(7)° and Cl(2)–Rh(2)–Cl(1), 91.98(5)°. The bond distances be-
tween rhodium and azo-N (Rh-N0) are Rh(1)–N(10), 2.051(4);
Rh(2)–N(1), 2.047(4); Rh(2)–N(2), 2.057(4) Å those are comparable
with reported results [35]. The rhodium–chlorine (Rh(1)–Cl(4),
2.3164(14); Rh(2)–Cl(1), 2.3242(13); Rh(2)–Cl(2), 2.3224(13) Å)
distances are also comparable with reported data [41,42]. The
Rh–N bond distances in the present example are Rh(1)–N(11),
2.002(5); Rh(2)–N(5), 2.013(4); Rh(2)–N(7), 2.011(4) Å) those are
lower than the reported Ru–N(imidazole) distances [43,44]. The
DFT calculation has been performed using optimized geometry of
the complex of C2 symmetry (molecule 1). The experimental struc-
ture data are comparable with the calculated data (Table 2). Gas-
phase calculation shows longer bond lengths than corresponding
experimental bond distances [45]. It is usual because in the gas
Fig. 3. Energy correlation diagram of cis-[RhCl2(MeaaiEt)2]ClO4 (2b) cis-[Rh(N3)2(-
MeaaiEt)2]ClO4 (4b).
phase molecules are isolated while in solid state molecules are
interacting themselves through some noncovalent interactions.
The theoretically calculated Rh–N0 and Rh–N distances of 2b are
longer than the crystallographic data by 0.02–0.08 Å. All other
bonds were found slightly longer than the corresponding solid
state bond lengths. One of the structures of asymmetric unit (mol-
ecule 1) agreed well with the theoretically determined structure
(Fig 1) of the complex. Bond angles are also in close agreement
with the crystallographic data and are varied by only ca. 0–7° from
crystal structure values.
3.3. Spectral studies and correlation with DFT data
In FT-IR spectra two moderately strong bands appear at 1360–
1390 and 1595–1598 cmꢁ1; these are assigned to
m(N@N) and m
(C@N), respectively. The azo and imine stretching frequency are
significantly shifted to lower frequency compared to free ligand va-
lue (
m m )
(N@N), 1400–1410 cmꢁ1 and (C@N), 1600–1610 cmꢁ1
[26] which supports coordination of Rh(III) with the ligand. The
complexes 1 and 2 show two m(Rh–Cl) stretches within the range
310–340 cmꢁ1 in the complexes which support their cis-RhCl2 type
configuration. The presence of ClO4 as a counter ion is confirmed
by a strong, broad peak at 1090–1100 cmꢁ1 and a weak stretch

D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106
103
Fig. 4. (a) Absorption spectroscopic study of 40
lM 2a with increasing concentrations of CT DNA (0, 1.14, 2.28, 3.42, 4.56, 5.7, 6.84, 7.98, 9.12, 10.26 and 11.4
lg/ml),
respectively (1 ? 11). (b) Absorption spectroscopic study of CT DNA (35
lg/ml) with increasing concentrations of 2a (0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 lM), respectively
(1 ? 11). (c) Modified Benesi–Hildebrand plot for the determination of ground state binding constant between CT DNA and 2a.
at 624 cmꢁ1. [Rh(N3)2(RaaiR0)2]ClO4 (3, 4) show characteristic
DFT calculation, it is observed (Fig. 3) that the electronic structures
of the complexes are sensitive to nature of the equatorially bonded
chloride or azide group. The energy of HOMO of 1a, 2a and 2b are
strong transmission at 2026–2030 cmꢁ1 with a weak band at
2045–2050 cmꢁ1 in infrared spectrum. These are corresponding
ꢁ
to
complexes.
The 1H NMR spectra of the complexes were recorded in CDCl3
m
asym(N3) [46–48]. This supports terminal N3 bonding in these
comparable (EHOMO = ꢁ9.03 eV) and more stable than 4b (EHOMO
=
ꢁ8.29 eV). The energy of LUMO of 4b (ELUMO = ꢁ6.24 eV) is compa-
rable with other complexes (ELUMO = ꢁ6.36 eV). In case of 1a, 2a, 2b
the major contribution (ꢂ60%) comes from chloride while azide
contribution is ꢂ80% to the HOMO of 4. The energy separation
between HOMO and LUMO is higher for 1a, 2a and 2b
and assigned on comparing with free ligand data [26]. Imidazole
protons, 4- and 5-H suffer downfield shifting by 0.7–0.8 ppm com-
pared to the free ligand position and appear as a broad singlet at
8.09–8.14 and 7.83–7.90 ppm, respectively. This supports the
strong preference of binding of imidazole-N to Rh(III). The singlet
nature of imidazole protons may be due to rapid proton exchange
at the NMR time scale. 1-Me appears as a singlet at 4.30 ppm; 1-
CH2–CH3 gives a quartet, 4.69–4.72 ppm (J = 8.0 Hz) and a triplet
1.61–1.70 ppm (J = 7.5 Hz) to –CH2– and –CH3 protons, respec-
tively. The 7- and 11-H in the complexes 3 and 4 appear at are
(
D
EHOMO–LUMO = ꢂ2.65 eV) compared to 4b (
DEHOMO–LUMO = 2.05
eV). The bonding scheme has unequivocally suggested the exis-
tence of two fragments in the complexes, namely electron donor
group Rh(X)2 (X = Cl, N3) and electron acceptor group MeaaiR
(R = Me, Et), respectively. Detail of composition of MOs are enlisted
in Table S4 (vide Supplementary material).
Time-dependent DFT (TDDFT) calculations are used to predict
observed transition energies and used to elucidate their origin.
The visible region of the experimental spectra of 1 and 2 are dom-
inated by intense asymmetric band with kmax ꢂ 405 nm along with
a shoulder at kmax ꢂ 500 nm. The longest wavelength calculated
signal at ꢂ565 nm in the visible region, is mainly attributed to
transitions from the HOMO-1 ? LUMO, HOMO-2 ? LUMO and
HOMO-3 ? LUMO. Detail of spectral assignment are given in
Table S5 (vide Supplementary material) Since LUMO of the ligand
is mainly composed of p⁄ of the azo bond, the type of transition
higher
d (8.04 ppm) compared to the complexes 1 and 2
(ꢂ7.92 ppm).
The absorption spectra of the complexes were investigated in
acetonitrile solution (Fig. 2). The red solution of the complexes ex-
hibit three bands in the wavelength range 200–500 nm. The visible
spectra of 1 and 2 are dominated by relatively intense asymmetric
band with kmax ꢂ 405 nm (
at kmax ꢂ 500 nm. These bands are assigned to the superposition of
(Cl) ? p ⁄ (azo) and d (Rh) ? p⁄ (azo). These bands experience
the blue shift (by 50–60 nm) upon replacement of Clꢁ by N3ꢁ. From
e
, 103 Mꢁ1 cmꢁ1) along with a shoulder
p
p
p
is attributed to the mixture of pp p
(Cl) ? p⁄ (azo) and d (Rh) ? p⁄

104
D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106
Fig. 5. Comparative fluorescence spectroscopic study of 2a with EB bound CT DNA. Excitation wavelength was 546 nm and the emission spectra were scanned from 556 to
650 nm in each case. (a) The mode of EB saturation in CT DNA (35 g/ml). The maximum fluorescence intensity of EB bound DNA complex was observed at 8.45 M of EB
concentration. At this point of saturation, 2a was added gradually up to 34.7 M till its saturation level, which is represented in figure b (see text for detail).
l
l
l
(azo). The experimentally obtained band at ꢂ405 nm region may
be assigned the admixture of
p⁄ (intraligand) and chloride-
to-ligand charge transfer transition i.e. p
(Cl) ? p⁄ (azo). In case
of 4 the lower energy transitions ca. 433 nm and 349 nm are blue
shifted compared to 1 or 2 and can be assigned as admixture of
intraligand, azide-to-ligand and metal-to-ligand charge transfer
transitions.
complex 4b (Supplementary material Fig. S2b). The hypochromic
effect of DNA by complex 4b may be due to the stringent configu-
ration of the aromatic bases in the negatively charged DNA mole-
cules in presence of heavy negative charge of the azide ligand in
the said complex. Moreover, the presence of more negative charges
in complex 4b renders the less binding affinity towards DNA. Thus
by changing the ligands in the rhodium complexes, we have ob-
served differential binding pattern of the same towards DNA.
p
?
p
3.4. Interaction of complexes with DNA
3.4.2. Fluorescence spectroscopic studies of the complexes with
ethidium bromide (EB) bound DNA
3.4.1. Absorption spectroscopic studies of the complexes in presence of
CT DNA
Fluorescence spectroscopic studies have been conducted to find
the interaction of DNA with rhodium complexes. Having clear evi-
dences that the rhodium complexes interact with DNA, we next
observed that whether the set of complexes can replace ethidium
bromide (EB) from DNA, which has an association constant of
3.4 ꢀ 103 Mꢁ1 towards any double-stranded DNA [48,49]. As a
matter of fact, either DNA or EB or even rhodium complexes do
not have fluorescence property alone. So we have used the fluores-
cence property of EB bound DNA to compare the DNA binding abil-
ity of the complexes with DNA.
When EB saturated DNA (Fig. 5a) is added to the solution of 1a
and 2a the complex partially replaces EB from DNA (Fig. 5b) which
is evidenced from the fluorescence intensity change. EB–DNA asso-
ciation constant is 3.4 ꢀ 103 Mꢁ1 [40]. Similarly, complexes 2b and
4b replace EB from DNA (Supplementary material Figs. S3 and S4,
respectively) partially. But comparing the results, it is obvious that
the degree of EB displacement from DNA by the complexes were
different, where the maximum EB displacement is achieved by
complex 4b and that of minimum by complex 2b. Though it is
not required to be an intercalator to reduce the fluorescence inten-
sity of the EB bound DNA, rather the complex might also interact
with DNA molecules in such a manner (possibly slight unwinding
The interaction of the complexes with chromosomal CT DNA are
investigated by spectrophotometric method. When CT DNA is
added with increasing concentrations to a fixed concentration of
complex 1a or 2a, the absorption is increased with a slight red shift
(Fig. 4a) while the addition of 1a or 2a to a fixed concentration of
DNA solution shows decrease in absorption of the DNA band
(Fig. 4b). Such differential absorption characteristics may be due
to the specific interaction of the complex with DNA molecules
resulting more relax structure of the complex but on the contrary,
a more rigid structure of the DNA bases. At the absorption maxi-
mum of DNA, we have calculated the binding constant between
the complexes and DNA by using modified Benesi–Hildebrand
(BH) plot (Fig. 4c) and the binding constant are 1.05 ꢀ 105 Mꢁ1
(1a) 1.02 ꢀ 105 Mꢁ1 (2a). Similar experiments were done for other
complexes and their binding constants with DNA are
3.479 ꢀ 104 Mꢁ1 (2b) and 1.36 ꢀ 104 Mꢁ1 (4b). In the complexes
2b and 4b, addition of DNA result hypochromic effects with slight
red shift (Supplementary material Figs. S1 and S2). The addition of
the complex to the DNA solution has resulted slight increase in the
absorption maxima of the DNA in the case of complex 2b (Supple-
mentary material Fig. S1b) and decrease the same in case of

D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106
105
Fig. 6. Agarose gel (1%) electrophoresis study of pUC19 plasmid DNA (500 ng) with increasing concentrations of 2a, 2b and 4b in figure a, b and c, respectively. Purified pUC19
plasmid DNA was treated with increasing concentrations of the said complex (0, 17, 33, 50, 67, 83 and 100 M in lanes 1 ? 7, respectively) for 1 h and was run at a potential
l
gradient of 6 volts/cm for 2.5 h. Lane 8 in figure a represents the control plasmid, treated with maximum amount of methanol used in lane 7. Bar diagram below each agarose
gel represents the normalized RF1 band intensity of the plasmid DNA in each lane with respect to that of mock treated DNA sample in lane 1.
of the DNA backbone) that induced a structural alteration in the
DNA molecules, resulting the release of EB from DNA, which might
not be associated with competitive EB and rhodium complex inter-
calation towards the DNA molecule. Such differential behavior of
the complexes may be due to the different atomic orientation
and associated charged property of the ligands in the complexes.
These rhodium complexes also have low-lying energy states
that might quench the emission of EB by means of energy transfer
when both molecules are bound to DNA in a close proximity. The
rhodium complexes in the solution absorb some of the excitation
light at 546 nm, where DNA–EB complex mostly absorbs. This
might results a reducing number of effective photons that can be
absorbed by EB absolutely. As a matter of fact, complex 4b reduces
the EB emission more than 2b, which can be understood by the
greater extinction coefficient of the former (ꢂ750 Mꢁ1 cmꢁ1) com-
pared to the later (ꢂ200 Mꢁ1 cmꢁ1) at this excitation wavelength.
The complexes may also absorb some of the emitted light of EB,
reducing the detected signal even further as their concentration
is increased.
constant, complex 4b was capable of inducing DNA double-strand
cleavage activity (RF3) directly from RF1 without forming any
intermediate RF2 form of DNA. As a result, it leads to a complete
degradation of the DNA molecules and therefore the RF1 form of
DNA were gradually vanished at the higher concentrations of com-
plex 4b (compare lanes 2–7 with respect to lane 1 in Fig. 6c). Thus
among all the four complexes, 1a and 2a show similar nuclease
activity and complex 4b has a unique nuclease activity.
4. Conclusion
We have prepared Rh(III) complexes of 1-alkyl-2-(arylazo)imi-
dazoles having N, N0 donor centers and chloride or azide ions as
other coordinating ligands. All the complexes are characterized
by elemental analyses and spectroscopic data and one of them is
structurally characterized. Although the complexes bind with
DNA at variable extent, complexes 1a and 2a bind most strongly
and least binding was observed in case of complex 4b. Among all
the complexes, complex 4b exhibits unique nuclease activity.
Changing of substituent within the heterocyclic ligand does not af-
fect much the nuclease activity but the replacement of chlorides
chloride with azide ions causes a significant improvement of the
nuclease activity.
3.4.3. Nuclease activity of the complexes by agarose gel electrophoresis
study
We have observed the nuclease property of rhodium complexes
on plasmid DNA by agarose gel electrophoresis study. The pUC19
plasmid DNA (500 ng) was incubated for an hour with increasing
concentrations of the complexes at 37 °C in dark and then sub-
jected to 1% agarose gel electrophoresis at a potential gradient of
6 volt/cm. After staining with ethidium bromide solution, it was
observed that complexes 1a and 2a (Fig. 6a), having highest bind-
ing affinity, induced slower migration of the super-coiled (RF1)
plasmid DNA and also induced DNA single-strand breakage (RF2)
in the plasmid back-bone, while complex 2b, having moderate
binding constant, mostly induced nicks (RF2) in the super-coiled
form of DNA (Fig. 6b). But interestingly, having least binding
Acknowledgements
Financial support from Council of Scientific and Industrial Re-
search (CSIR) and Department of Science and Technology (DST)
New Delhi are gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ica.2012.08.005.

106
D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106
[29] G.M. Sheldrick, SHELXL 97, Program for the Refinement of Crystal Structure,
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