Synthesis, characterization, redox behavior, DNA and protein binding and antibacterial activity studies of ruthenium(II) complexes of bidentate schiff bases

Article abstract of DOI:10.1080/15257770.2017.1332763

Two new ruthenium(II) complexes of Schiff base ligands (L) derived from cinnamaldehyde and ethylenediamine formulated as [Ru(L)(bpy)₂](ClO₄)₂, where L¹ = N,N’-bis(4-nitrocinnamald-ehyde)ethylenediamine and L² = N,N’-bis(2-nitrocinnamaldehyde)-ethylenediamine for complex 1 and 2, respectively, were isolated in pure form. The complexes were characterized by physicochemical and spectroscopic methods. The electrochemical behavior of the complexes showed the Ru(III)/Ru(II) couple at different potentials with quasi-reversible voltammograms. The interaction of the complexes with calf thymus DNA (CT-DNA) using absorption, emission spectral studies and electrochemical techniques have been used to determine the binding constant, K_b and the linear Stern–Volmer quenching constant, K_SV. The results indicate that the ruthenium(II) complexes interact with CT-DNA strongly in a groove binding mode. The interactions of bovine serum albumin (BSA) with the complexes were also investigated with the help of absorption and fluorescence spectroscopy tools. Absorption spectroscopy proved the formation of a ground state BSA-[Ru(L)(bpy)₂](ClO₄)₂ complex. The antibacterial study showed that the Ru(II) complexes (1 and 2) have better activity than the standard antibiotics but weak activity than the ligands.

Full text of DOI:10.1080/15257770.2017.1332763

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: http://www.tandfonline.com/loi/lncn20
Synthesis, characterization, redox behavior, DNA
and protein binding and antibacterial activity
studies of ruthenium(II) complexes of bidentate
schiff bases
Hena Paul, Buddhadeb Sen, Tapan Kumar Mondal & Pabitra Chattopadhyay
To cite this article: Hena Paul, Buddhadeb Sen, Tapan Kumar Mondal & Pabitra Chattopadhyay
(2017): Synthesis, characterization, redox behavior, DNA and protein binding and antibacterial
activity studies of ruthenium(II) complexes of bidentate schiff bases, Nucleosides, Nucleotides and
Nucleic Acids, DOI: 10.1080/15257770.2017.1332763
To link to this article: http://dx.doi.org/10.1080/15257770.2017.1332763
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Date: 17 July 2017, At: 20:25

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
, VOL. , NO. , –
https://doi.org/./..
Synthesis, characterization, redox behavior, DNA and
protein binding and antibacterial activity studies of
ruthenium(II) complexes of bidentate schiff bases
Hena Paula, Buddhadeb Sena, Tapan Kumar Mondalb, and Pabitra Chattopadhyaya
aDepartment of Chemistry, Burdwan University, Golapbag, Burdwan, India; bDepartment of Chemistry,
Jadavpur University, Jadavpur, Kolkata, India
ABSTRACT
ARTICLE HISTORY
Received  June 
Accepted  May 
Two new ruthenium(II) complexes of Schiff base ligands (L)
derived from cinnamaldehyde and ethylenediamine formulated
as [Ru(L)(bpy)2](ClO4)2, where L1 = N,N’-bis(4-nitrocinnamald-
ehyde)ethylenediamine and L2 = N,N’-bis(2-nitrocinnamaldehyde)-
ethylenediamine for complex 1 and 2, respectively, were isolated in
pure form. The complexes were characterized by physicochem-
ical and spectroscopic methods. The electrochemical behavior
of the complexes showed the Ru(III)/Ru(II) couple at different
potentials with quasi-reversible voltammograms. The interaction
of the complexes with calf thymus DNA (CT-DNA) using absorp-
tion, emission spectral studies and electrochemical techniques
have been used to determine the binding constant, Kb and the
linear Stern–Volmer quenching constant, KSV. The results indicate
that the ruthenium(II) complexes interact with CT-DNA strongly
in a groove binding mode. The interactions of bovine serum albu-
min (BSA) with the complexes were also investigated with the
help of absorption and fluorescence spectroscopy tools. Absorp-
tion spectroscopy proved the formation of a ground state BSA-
[Ru(L)(bpy)2](ClO4)2 complex. The antibacterial study showed that
the Ru(II) complexes (1 and 2) have better activity than the stan-
dard antibiotics but weak activity than the ligands.
KEYWORDS
Ruthenium(II) complex;
Schiff base; DNA and BSA
interaction; antibacterial
activity
Introduction
The chemistry of metal complexes with multidentate ligands and delocalized
π-orbitals such as Schiff bases[1,2] has attracted much attention because they act as a
versatile model of metallic biosites.[3] Schiff bases are reagents which are becoming
increasingly important in the pharmaceutical, dye and plastic industries as well as
for liquid crystal technology.[4–7] Furthermore, Schiff bases enhance the solubility
and stability of either homogeneous or heterogeneous catalysts.[8] Schiff base com-
pounds have been regarded as excellent fluorescent materials because of their ability
CONTACT Pabitra Chattopadhyay
Golapbag, Burdwan-, India.
pabitracc@yahoo.com
Department of Chemistry, Burdwan University,
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lncn.
©  Taylor & Francis Group, LLC

2
H. PAUL ET AL.
to achieve high thermal stability as well as high photoluminescent efficiently. Tran-
sition metal complexes of these Schiff bases are also important not only due to the
interesting structural and bonding modes they possess, but also because of their var-
ious industrial applications.[9] Particularly, the use of ruthenium complexes to cat-
alyze oxidation of alcohols by oxygen atom donors has been well documented.[10,11]
The use of cinnamaldehyde to synthesis the Schiff base has been well estab-
lished[12] because cinnamaldehyde is effective in inhibiting growth of bacteria, yeast
and filamentous molds. Antimicrobial activity of cinnamaldehyde is also attributed
to its membrane action.[13] Antifungal MICs of cinnamaldehyde are high and its
use is further limited by its irritant effect on skin which is attributed to aldehyde
function.[14] Substituted cinnamaldehydes are known to be poorer skin sensitizer
because they react slowly with amines.[15]
Among the transition metals, the chemistry of ruthenium is currently receiv-
ing a lot of attention, primarily because of the fascinating electron transfer, pho-
tochemical and catalytic properties displayed by the complexes of this metal.[16]
Ruthenium(II) complexes with heterocyclic N-donor ligands have also been exten-
sively investigated because of their interesting photochemical, catalytic, biological
and electrochemical[17–19] properties. Ruthenium-Schiff base complexes, contain-
ing oxygen and nitrogen as donor atoms were found to be very efficient catalysts in
the oxidation of alcohols using N-methylmorpholine-N-oxide as co-oxidant.[20–23]
As a part of our continuing efforts to synthesis and characterize ruthenium
chelates using various types of N-donor ligands, in this paper, we describe the
synthesis, characterization and DNA and protein binding studies of ruthenium(II)
complexes.
Materials and methods
Reagents and measurements
All chemicals and reagents were obtained from commercial sources and used as
received. Solvents were distilled from an appropriate drying agent. RuCl3.3H2O
(Aldrich) was used without further purification. [RuII(bpy)2Cl2].2H2O was syn-
thesized according to the literature method.[24] Tetra-n-butylammonium per-
chlorate (TBAP) was prepared by addition of sodium perchlorate (taking the
usual precaution of handling perchlorate salts!) to a hot solution of tetra-n-
butylammoniumbromide (Aldrich).
The C, H, N elemental analyses were performed on a Perkin Elmer model
2400 elemental analyzer and ruthenium analyses were carried out by means of
a Varian atomic absorption spectrophotometer (AAS) model-AA55B, GTA using
graphite furnace. Electronic absorption spectra were recorded on a JASCO UV-
Vis/NIR spectrophotometer model V-570 in the range of 1100–200 nm. IR spec-
tra were recorded using a Perkin-Elmer FTIR model RX1 spectrometer (using KBr
discs, 4000–300 cm−1). 1H NMR spectra were recorded on a Bruker AC300 spec-
trometer using TMS as internal standard in CDCl3. Room temperature magnetic

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
susceptibility measurements were performed with a vibrating sample magnetome-
ter PAR 155 model. Molar conductances (ꢀ ) were measured in a Systronics
M
conductivity meter (model 304) in acetonitrile with ∼10−3 mol L−1 complex con-
centration. The measurement of pH of the reaction mixture was done with a systron-
ics digital pH meter (Model 335). Electrochemical measurements were recorded on
a CH-Instrument electrochemical system (Model 620D) using Pt-wire and Ag/AgCl
as working and reference electrodes, respectively, with TBAP as supporting elec-
trolyte. All the measurements were made at 298 K by using complex concentration
of ca. 10−3–10−4 M in acetonitrile purged with dry nitrogen for 3–4 min in order to
remove dissolved oxygen.
Preparation of the ligands
The syntheses of the ligands were carried out following the common procedure. The
procedure for the preparation of N,N’-bis(4-nitrocinnamaldehyde)ethylenediamine
(L1) is here described in detail, being the other ligands obtained as solid products in
the same way.
N,N’-Bis(4-nitrocinnamaldehyde)ethylenediamine (L1):
A
solution of
4-nitrocinnamaldehyde (2 mmol) was added to an ice cooled methanolic solu-
tion of ethylenediamine (1 mmol). The mixture was then stirred for ࣈ3.0 h. A
yellowish white coloured precipitate was obtained. The precipitate was filtered off
and washed with 10 ml cold absolute methanol. The crude product was recrystal-
lized from ethanol. The yield was 87%. The yield of the other ligand (L2) was almost
90% following a similar method (Scheme 1). Yield: 87%. m.p. 215 1°C.
C20H18N4O4: Anal. Found: C, 63.48; H, 4.75; N, 14.81; Calc.: C, 63.34; H, 4.63;
1
N, 14.61. IR (KBr, cm−1): νC=N, 1633. H NMR (δ, ppm in CDCl3): 8.11 (d, 4H,
j = 4.5); 7.51 (m, 6H); 6.64 (d, 2H, j = 8.53); 5.326(m, 2H); 1.073 (d, 4H, j = 3.24).
N,N’-Bis(2-nitrocinnamaldehyde)ethylenediamine (L2): The ligand (L2) was pre-
pared taking 2-nitrocinnamaldehyde (2.0 mmol) instead of 4-nitrocinnamaldehyde
following similar method as described above. Yield: 90%. m.p. 163 1°C.
C20H18N4O4: Anal. Found C, 63.22; H, 4.66; N, 14.78; Calc.: C, 63.34; H, 4.63; N,
14.61. IR (KBr, cm−1): νC=N, 1637. 1H NMR (δ, ppm in CDCl3): 8.043 (d, 2H, j =
4.35); 7.452 (m, 10H); 5.723 (d, 2H, j = 4.3); 1.102 (d, 4H, j = 7.63).
Preparation of [Ru(L1)(bpy)2](ClO4)2 (1) and [Ru(L2)(bpy)2](ClO4)2 (2)
Syntheses of the complexes were performed following a common procedure. To
the ethanolic solution of L1 (0.378 g, 1.0 mmol) in case complex 1 or L2 (0.378 g,
1.0 mmol) complex 2, a solution of [RuCl2(bpy)2] (bpy = bipyridyl) (0.484 g,
1 mmol) in dry EtOH (20 mL) previously purged with N2 was added dropwise and
the resulting solution was refluxed for ∼6 h under N2 atmosphere. The solvent of the
resulting solution was reduced to one-third of the total volume, and water solution
of NaClO4 (0.140 g 1mmol) was added in stirring condition. The resulting mixture
was stirred for ∼2 h and then by filtering a red solid mass was obtained. The product

4
H. PAUL ET AL.
Scheme . Synthetic procedure of organic moieties L and ruthenium(II) complexes.
was dissolved in the minimum amount of dichloromethane and chromatographed
over a silica gel column prepared in dichloromethane. Only one band was obtained
(Scheme 1).
[Ru(L1)(bpy)2](ClO4)2 (1): C40H34N8RuCl2O12: Anal. Found: C, 48.42; H, 3.48;
N, 11.11, Ru, 10.28 Calc.: C, 48.48; H, 3.43; N, 11.31, Ru, 10.20. IR (KBr cm−1):
ν
C=N, 1597; ν(ClO4-), 1107 and 623. Conductance ꢀo (ohm−1 cm2 mol−1) in
1
methanol: 52. H NMR (δ, ppm in CDCl3): 8.748 (d, 4H, j = 4.7); 8.741 (d, 4H,
j = 5.1); 7.725 (m, 4H); 7.306 (t, 2H); 7.234 (m, 6H); 7.114 (m, 6H); 6.828 (m, 2H);
6.415 (d, 2H, j = 6.8; 1.153 (t, 4H). Yield: 70%.
[Ru(L2)(bpy)2](ClO4)2 (2): C40H34N8RuCl2O12: Anal. Found: C, 48.34; H, 3.40;
N, 11.03, Ru, 10.18 Calc.: C, 48.48; H, 3.43; N, 11.31, Ru, 10.20. IR (KBr cm−1): ν
,
C=N
1599; ν(ClO4-), 1098 and 624. Conductance ꢀ (ohm−1 cm2 mol−1) in methanol: 51.
o
1H NMR (δ, ppm in CDCl3): 8.001 (d, 4H, j = 5.2); 7.702 (d, 4H, j = 4.6); 7.633
(t, 4H); 7.455 (m, 8H); 7.360–7.263 (m, 6H); 6.868 (m, 2H); 6.305 (d, 2H, j = 9.8);
0.857(t, 4H). Yield: 63%.
DFT calculation
Full geometry optimization was carried out using the density functional theory
method at the B3LYP level for 1 and 2.[25] All elements except ruthenium were
assigned the 6–31G(d) basis set. The SDD basis set with effective core potential

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
was employed for the ruthenium atom.[26] The vibrational frequency calculations
were performed to ensure that the optimized geometries represent the local min-
ima and there are only positive eigen values. All calculations were performed with
Gaussian03 program package. Vertical electronic excitations based on B3LYP opti-
mized geometry was computed using the time-dependent density functional theory
(TD-DFT) formalism[27] in acetonitrile using conductor-like polarizable continuum
model (CPCM). Gauss Sum[28] was used to calculate the fractional contributions of
various groups to each molecular orbital.
DNA binding experiments
The tris-HCl buffer solution (pH 7.4), used in all the experiments involving CT-
DNA, was prepared by using deionized and sonicated HPLC grade water (Merck).
The used CT-DNA was sufficiently free from protein, being the ratio of UV
absorbance of the DNA in tris-HCl solution at 260 and 280 nm (A260/A280) of ca.
1.9.[29] The concentration of DNA was determined with the help of its extinction
coefficient ε of 6600 L mol−1 cm−1 at 260 nm.[30] The stock solution of DNA was
always stored at 4°C and used within 4 days. Concentrated stock solution of com-
plex was prepared by dissolving the ruthenium (II) complex in DMSO and suitably
diluting with tris-HCl buffer to the concentration required for all the experiments.
Absorption spectral titration experiment was performed by keeping constant the
concentration of the ruthenium (II) complex while varying the CT-DNA concen-
tration. To eliminate the absorbance of DNA itself, an equal solution of CT-DNA
was added both to the ruthenium (II) complex solution and to the reference one.
In the fluorescence displacement experiment with ethidium bromide (EB), 5 µL
of EB solution (1.0 mmol L−1) in tris-HCl were added to 1.0 mL of DNA solution
at saturated binding levels[31] and stored in the dark for 2.0 h. The ruthenium (II)
complex solution was titrated into the DNA/EB mixture and then diluted in tris-HCl
buffer to 5.0 mL, making the solutions with the varied mole ratio of the metal com-
plex to CT-DNA. Prior to measurements, the mixture was shaken up and incubated
at room temperature for 30 min. The fluorescence spectra of EB bound to DNA were
obtained at an emission wavelength of 584 nm. The viscosity values of the solutions
were calculated from the observed flow time of CT-DNA-containing solution cor-
rected from the flow time of buffer alone (t0), η = t – t0.[32] The calculated data were
used to plot the (η/η )1/3 versus the ratio of the concentration of 1 and CT-DNA,
0
where η is the viscosity of CT-DNA in the presence of the compound and η is the
0
viscosity of CT-DNA alone.
Protien (bovine serum albumin) binding experiments
Samples for spectroscopic measurements were prepared by dissolving bovine serum
albumin in water and administering the appropriate concentration of the Ru(II)
complexes. The samples were carefully degassed using pure nitrogen gas for 15 min.
Quartz cells with high vacuum Teflon stopcocks were used for degassing.

6
H. PAUL ET AL.
Cyclic voltammetry (CV) experiments
Voltammetric measurements were carried out in a one-compartment cell using a
glassy carbon (area, 0.088 cm2) or a Pt disk (area, 0.023 cm2) working electrode,
a Pt flag counter electrode, and a Ag/AgCl reference electrode (SCE). Typical CV
curves for 0.1 mM complexes in tris-buffer, in the absence and in the presence of
CT DNA were carried out.
In vitro antibacterial assay
The biological activities of synthesized ligands (L1 and L2) and the Ru (II) com-
plexes have been studied for their antibacterial activities by agar well diffusion
method.[33–35] The antibacterial activities were done at 100 µg/mL concentrations
of different compounds in DMF solvent by using four pathogenic gram negative
bacteria (Escherichia coli, Vibrio cholerae, Streptococcus pneumoniae, Shigella spp)
and one gram positive pathogenic bacteria (Cereus bacillus). Stock cultures of the
test bacterial species were maintained on Nutrient Agar media by sub culturing in
slants. The media were prepared by adding beef extract 3 g, peptone 5 g, agar 15 g,
distilled water 1000 ml and the pH was adjusted at 7.2. Media was sterilized in the
autoclave at 15 lbs pressure for 20 min. 20 mL of media was poured in each Petri
dish and allowed to solidify. After solidification, nutrient agar plates were swabbed
by sterile cotton swabs with 12 h old 0.1 ml broth culture of respective bacteria. The
wells were bored with corkborer and the agar plugs were removed. Then solution
of ligands and the Ru(II) complexes were added to the agar wells. DMF was used as
a negative control. The Petri dishes were incubated at 37°C for 24 h. After incuba-
tion plates were observed for the growth inhibition zones. The diameter of the zone
of inhibition was measured in millimeters. The well diameter was deducted from
the zone diameter to get the actual zone of the inhibition and the values have been
tabulated in Table 2.
Result and discussion
Synthesis and characterization
The organic moiety was synthesized by stirring p/o-nitrocinnamaldehyde and
ethylenediamine in 2:1 ratio in methanol in an ice bath for ∼3 h (Scheme 1). The
yellowish white compound (L) N,Nꢀ-bis(p/o-nitrocinnamaldehyde)ethylenediamine
Table . UV-vis spectral data, electrochemical dataa .
Compd.
λ nm (ε) (ε, dm.mol−.cm−
)
Electrochemical data E/
,
E (V) for Ru(II)/Ru(III)
1
2
 (),  (),  ()
 (),  (),  ()
. (.)
. (.)
aIn acetonitrile.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
Table . Antibacterial data of the ligands and Ru(II) complexes ( µg/ml).
Inhibition zone in mm
Treatment
E. coli
V. cholerae
S. pneumoniae
Cereus bacillus
Shigella sp.
DMF(control)

























L1
1
L2


2






Bore diameter
Ampicillin


was obtained as the end product. The structural analysis by spectroscopic tools con-
firmed the product. These organic moieties act as bidentate N,N chelators.
The complexes 1 and 2 were obtained in good yield from the reaction of the
[RuCl2(bpy)2] (bpy = bipyridyl) with equimolar amount of the organic moiety (L1,
L2) in dry EtOH medium in refluxing condition under dinitrogen (N2) atmosphere
followed by the addition of water solution of sodium perchlorate to the reaction
mixture at cold condition (Scheme 1).
Neutral complexes 1 and 2 are soluble in DCM, acetonitrile, methanol, and DMF.
Microanalytical data confirms the formulation of complexes 1 and 2. The conduc-
tivity measurements indicate that both the ruthenium (II) complex species are non-
electrolytes in methanolic solution and the magnetic moment studies demonstrate
that the complexes are diamagnetic in nature.
DFT computations: Explanation of spectral and redox properties
DFT calculations have been performed for both 1 and 2. The optimized
structures of these molecules are developed using GAUSSIAN 03 (Figure 1). The
calculated bond angles and lengths for 1 and 2 are given in Table 3. The orbital
Figure . Optimized structure of 1 and 2 at DFT-BLYP/–G(d) level of theory.

8
H. PAUL ET AL.
˚
Table . Some selected optimized bond distances (A) and angles (°) of 1 and 2.
Bonds
Ru-N
Ru-N
Ru-N
Ru-N
Ru-N
Ru-N
.
.
.
.
.
.
.
.
.
.
.
.
Angles
N-Ru-N
N-Ru-N
N-Ru-N
N-Ru-N
N-Ru-N
N-Ru-N
N-Ru-N
N-Ru-N
N-Ru-N
N-Ru-N
N-Ru-N
N-Ru-N
N-Ru-N
N-Ru-N
N-Ru-N
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
energies along with contributions from the ligands and metal are given in Table 4
and Figure 2, which depict selected occupied and unoccupied frontier orbitals for 1.
The HOMO of 1 is constituted by 39% contribution from L, whereas to HOMO-
2 bpy contributes 17% and the metal ion 78%. The LUMO-2 is composed of L
by 90%. Thus, HOMO → LUMO is considered as major transition corresponding
to dπ(Ru) / π(L1) → π∗(bpy). The other transitions are HOMO-1 → LUMO+1
Table . Energy and composition of some selected MOs of 1.
% of Composition
MO
Energy (eV)
Ru
L
Bpy
LUMO+
LUMO+
LUMO+
LUMO+
LUMO+
LUMO+
LUMO+
LUMO+
LUMO+
LUMO+
LUMO
− .
− .
− .
− .
− .
− .
− .
− .






















































− .
− .
− .
− .
− .
− .
− .
− .
− .
− .
− .
− .
− .
− .
HOMO
HOMO -
HOMO -
HOMO -
HOMO -
HOMO -
HOMO -
HOMO -
HOMO -
HOMO -
HOMO -













NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
Figure . Contour plots of some selected MOs of 1.
[dπ(Ru) / π(L1) → π∗(bpy)], HOMO-1→LUMO [dπ(Ru) / π(L1) → π∗(bpy)],
and HOMO-2 → LUMO+2 [dπ (Ru) → π∗(L1)] that are intra-ligand and ligand-
to-ligand charge transfer transitions. All these transitions are observed for 1, and
the calculated transitions are reported in Table 5. Solvent polarity stabilizes occu-
pied MO’s more efficiently than unoccupied MO’s. Thus, the energy separation (DE)
between HOMO and LUMO increase on going from gas phase to MeCN solution.
Figure 3 depicts selected occupied and unoccupied frontier orbitals of 2. The
metal significantly contributes to the occupied MO’s (76%, comparable to 78% of 1).
Bipyridine contributes 18% to HOMO-2 and 94% to LUMO in 2. The orbital ener-
gies of 2 along with contributions from the ligands and metal are given in Table 6
Table . Calculated vertical electronic transitions of 1.
Eex (eV) λex (nm) Osc. Strength (f)
Key transitions
Character
λexpt. (M− cm−
)
.
.
.
.
.
.
.
.
.
(%) HOMO→LUMO
dπ(Ru)/ π(L) → π∗(bpy)
(%) HOMO-→LUMO+ dπ(Ru)/ π(L) → π∗(bpy)
(%) HOMO-→LUMO
dπ(Ru)/ π(L) → π∗(bpy)
()
(%) HOMO→LUMO+
.
.
.
.
.
.
.
.
.
.
.
.
.
.
(%) HOMO-→LUMO+ dπ(Ru) → π∗(L)
(%) HOMO-→LUMO+ dπ(Ru)/ π(L) → π∗(bpy)
(%) HOMO-→LUMO
π(L) → π∗(bpy)
dπ(Ru)/ π(L) → π∗(bpy)
()
(%) HOMO-→LUMO+ dπ(Ru) → π∗(bpy)
.
(%) HOMO-→LUMO
(%) HOMO-→LUMO
.
.
.
(%) HOMO-→LUMO+ π(L) → π∗(bpy)

10
H. PAUL ET AL.
Figure . Contour plots of some selected MOs of 2.
Table . Energy and composition of some selected MOs of 2.
% of Composition
L
MO
Energy (eV)
Ru
bpy
LUMO+
LUMO+
LUMO+
LUMO+
LUMO+
LUMO+
LUMO+
LUMO+
LUMO+
LUMO+
LUMO
− .
− .





























































− .
− .
− .
− .
− .
− .
− .
− .
− .
− .
− .
− .
− .
− .
HOMO
HOMO −
HOMO −
HOMO −
HOMO −
HOMO −
HOMO −
HOMO −
HOMO −
HOMO −
HOMO −
− .
− .
− .





− .
− .
− .

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
Table . Calculated vertical electronic transitions of 2.
Eex (eV) λex (nm) Osc. Strength (f)
Key transitions
Character
λexpt. (M− cm−
)
.
.
.
.
.
.
(%)HOMO→LUMO
dπ(Ru) → π∗(bpy)
dπ(Ru) → π∗(L)
dπ(Ru) → π∗(bpy)
dπ(Ru) → π∗(bpy)
dπ(Ru) → π∗(L)
dπ(Ru) → π∗(L)
dπ(Ru) → π∗(bpy)
(%)HOMO→LUMO+
(%)HOMO-→LUMO+
(%)HOMO-→LUMO+
(%)HOMO→LUMO+
(%)HOMO-→LUMO+
(%)HOMO-→LUMO+
 ()
.
.
.
.
.
.
.
.
.
.
.
.
(%)HOMO-→LUMO+ dπ(Ru) → π∗(bpy)
(%)HOMO-→LUMO+
dπ(Ru) → π∗(L)
(%)HOMO-→LUMO+ dπ(Ru) → π∗(L)
 ()
(%)HOMO-→LUMO
π(L) → π∗(bpy)
and the calculated transitions are in Table 7. The intensity of these transitions has
been assessed from oscillator strength (f). In MeCN, the longest wavelength band is
calculated at 546.1 nm (f, 0.1160) for 1 followed by transitions at 506.6 (f, 0.2505),
486.2 (f, 0.2899), and 435.8 (f, 0.1543) nm along with a large number of transitions
in the UV region (<400 nm). In 2, the calculated transitions are 506.8 (f, 0.0985),
480.68 (f, 0.1365), 477.65 (f, 0.2064), and 437.61 (f, 0.1522) nm. The observed tran-
sitions are in agreement with the calculated ones for both complexes.
Spectral properties
Infrared spectral studies
The IR spectra of the complexes are compared with the free ligands in order to con-
firm the ligand coordination to the metal, and for this there are some reference peaks
that are of good help for achieving this goal. The νC=N band in the free ligand, L1 is
1633 cm−1 and it is shifted to lower wave numbers in the complex1, suggesting coor-
dination to the metal ion. The similar result obtained for 2. The characteristic bands
1
2
at 1595–1600 cm−1 is assigned to ν(C N) of the coordinated ligands (L / L ) in the
=
2
2
2
2
∗1
=
complexes. As we know, in going from –C N–(σ π ) to (σ π π ), the bond order
decreases from 2 to 1.5; therefore, it was observed that the vibrational frequency of
ν
C=N decreases significantly from the free ligand value (1630–1640 cm−1). The com-
plexes [Ru(L)(bpy)2](ClO4)2 show sharp absorption bands around 1107 cm−1 and
623 cm−1 assignable to ν
and ν
), which are absent in the spectra of
−
−
s(ClO4
)
as(ClO4
the free organic moieties (L1 and L2).
1HNMR spectra
1
The HNMR spectra of the organic moieties and the [Ru(L)(bpy)2](ClO4)2 com-
plexes were recorded in CDCl3. The characteristic signals for the protons of L1 and
L2 appeared in the spectra as usual fashion in support of the proposed structural
formulas. In the 1H NMR spectrum of the complexes the protons appeared at char-
acteristic δ values and these values are in accordance with the proposed structure. It

12
H. PAUL ET AL.
Figure . Cyclic voltammogram of complex 1 in MeCN (. M n-BuNClO) at  K, Ag/AgCl reference
electrode, and scan rate  mV/s.
was observed that characteristic signals for the protons of the complexes appeared
at higher δ values with respect to the free organic moieties.
Electronic absorption spectra
The electronic absorption spectra of the complexes were recorded at room tempera-
ture using acetonitrile as the solvent and data are reported in Table 1. The spectra of
the complexes exhibit the characteristic transitions in the range of 240–245 nm and
335–340 nm corresponding to intramolecular π → πࢩ and n → πࢩ transitions,
respectively. A band at 473 and 456 nm in 1 and 2, respectively, were observed due
to the d(Ru) → π∗ (ligand) MLCT transitions.
Electrochemical behavior
The redox properties of the complexes were examined by cyclic voltammetry using
a Pt-disk working electrode, a Pt-wire auxiliary electrode and Ag/AgCl electrode as
a reference electrode in dry MeCN using [n-Bu4N]ClO4 (0.1 M) as the supporting
electrolyte. Voltammetric data are given in Table 1. Each complex exhibits a quasi-
reversible RuII/RuIII redox couple arround a redox wave at ca. E1/2 of 0.797 V (versus
Ag/AgCl) for 1 (Figure 4) and at ca. E1/2 of 0.935 V (versus Ag/AgCl) for 2 in MeCN
solution at 25°C. No cathodic response for RuII → RuI reduction was obtained for
the complexes up to −1.50 V. The voltammetric parameters were studied in the scan
rate interval 50–400 mV s−1. The ratio between the cathodic peak current and the
square root of the scan rate (Ipc / ν1/2) is approximately constant. The peak potential
shows a small dependence on the scan rate. The ratio Ipc to Ipa is close to unity. From
these data, it can be concluded that the redox couple is related to a quasi-reversible
one-electron transfer process controlled by diffusion.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
DNA-binding studies
The binding interaction of the Ru (II) complex 1 (complex 2 give same groove bind-
ing interaction) with calf thymus DNA (CT-DNA) has been investigated by using
absorption and emission spectra.
Spectrophotometric study
Electronic absorption spectroscopy is an effective method to examine the binding
modes of metal complexes with DNA. In general, binding of the ruthenium (II)
complex to the CT-DNA helix is examined by an increase of the absorption band
(ca. 304 nm) of ruthenium (II) complex. This increasing absorbance indicates that
there is the involvement of strong interactions between complex and the base pairs
of DNA. The absorption spectra of the Ru (II) complex 1 in the absence and presence
of CT-DNA are given in (Figure 5). The spectral change might be interpreted as due
to the groove binding nature of the adducts[36] since ruthenium (II) complexes con-
taining Schiff base ligands, which likely facilitates the formation of Vander-Waals
contacts or hydrogen bonds during interaction with DNA grooves. In order to fur-
ther illustrate the binding strength of the Ru(II) complex with CT-DNA, the intrin-
sic binding constant Kb was determined from the spectral titration data using the
following equation[37]
DNA /(ε −ε ) = DNA /(ε −ε ) + 1/[K (ε −ε )]
[
]
[
]
a
f
b
f
b
b
f
where [DNA] represents the DNA concentration, ε and ε are the extinction coef-
f
b
ficients for the free and fully bound ruthenium(II) complex, respectively, and ε the
a
metal complex extinction coefficient during each addition of DNA. The [DNA] / (ε
a
– ε ) plot against [DNA] gave a linear relationship (Figure 6). The intrinsic binding
f
Figure . Electronic spectra of complex 1 through titration with CT-DNA in tris-HCl. The increase of
DNA concentration is indicated by an arrow.

14
H. PAUL ET AL.
Figure . Plost of [DNA]/(εa-εf) vs [DNA] for the titration of CT-DNA with complex 1 in tris-HCl buffer;
binding constant Kb = . ×  M− (R = . for five points).
constants (Kb) for the complex 1 were calculated from the slope to intercept ratio
(Kb = 9.1866 × 104 M−1, R = 0.99798 for five points). The value is in close agree-
ment with those of the well-established groove binding rather than classical inter-
calation agent.[38]
Spectroflurometric study
Fluorescence intensity of EB bound to CT-DNA at excitation wavelength of 522 nm
shows a decreasing trend with the increasing concentration of the complex 1
(Figure 7). The quenching of EB bound to DNA by the complex 1 is in agreement
with the linear Stern–Volmer equation[39]
I0/I = 1 + K Q
sv [ ]
Figure . Emission spectra of the CT-DNA-EB system in tris–HCl buffer based on the titration of com-
plex. λex =  nm.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
Figure . Plot of I/I vs [complex] for the titration of CT-DNA–EB system with complex 1 using spec-
trofluorimeter; linear Stern–Volmer quenching constant (Ksv) = . × ; (R = . for five
points).
where I0 and I represent the fluorescence intensities in the absence and presence
of quencher respectively. Ksv is a linear Stern–Volmer quenching constant, Q is the
concentration of quencher. The Ksv value calculated from the plot (Figure 8) of I0/I
versus [complex] for the complex 1 is 2.5 × 104 (R = 0.99506 for five points), sug-
gesting a strong affinity of the complex 1 to CT-DNA. The value is in close agreement
with those of the well-established groove binding rather than classical intercalation
agent.[40]
Number of binding sites can be calculated from fluorescence titration data using
the following equation[41]
log (I −I) /I = logK + nlog Q
[
]
[ ]
0
K and n is the binding constant and binding site of complex 1 to CT-DNA, respec-
tively. The number of binding sites (n) determined from the slope of log[(I0 – I)/I]
versus log[Q] is 0.84 which indicates less association of the complex 1 to the number
of DNA bases, also suggesting strong affinity of the complex 1 through surface or
groove binding.
Viscosity technique
From spectroscopic technique it was observed that the complex 1 has a strong inter-
action with DNA, so to further clarify the interactions study between the complex
and DNA, viscosity measurements were carried out. From this experiment it was
observed that there is almost no effect on the relative viscosity of the DNA solu-
tion. This experiment suggests that the binding mode is groove binding which is in
support of the above results obtained in spectroscopic study, otherwise if it be inter-
calative candidate, then the change of the relative viscosity of the DNA solution was

16
H. PAUL ET AL.
Figure . Cyclic voltammograms of the ruthenium (II) complex in Tris-HCl buffer in (a) absence (black
colored curve) and (b) presence of CT-DNA (red colored curve).
observed because intercalation leads to an increase in the DNA viscosity by length-
ening the DNA helix, or the non-classical intercalation could bend (or kink) the
DNA helix and reduce its effective length and, concomitantly, its viscosity.[42,43]
Electrochemical study
Electrochemical investigations are useful technique to analyze metal-DNA interac-
tions over spectroscopic methods.[44] The binding nature of the ruthenium(II) com-
plex 1 with DNA, has been shown in (Figure 9). Cyclic voltammograms of complex
1, in the absence and presence of CT-DNA, exhibited significant shifts in the anodic
peak potentials followed by decrease in peak currents, indicating the interaction
occurring between the metal complex and CT-DNA. The shift in the value of the
formal potential ( E0) is used to calculate the ratio of equilibrium binding constants
K2+ /K3+ according to the following equation described by Bard and Carter.[45]
E◦ = Eb −Ef = 0.059 log (K2+/K3+
)
◦
◦
where Eb° and Ef° are the formal potentials of the bound and free complex forms,
respectively, and K2+ and K3+ are the corresponding binding constants for the bind-
ing of reduction and oxidation species to DNA, respectively. Ratio of equilibrium
binding constants, K2+ / K3+ is calculated to be 2.55 that indicates a strong binding
of DNA with reduced form of ruthenium complex, i.e., Ru(II) species.
Protien (bovine serum albumin) binding experiments
Absorption characteristics of BSA-Ru(II) complex 1
The absorption spectra of BSA in the absence and presence of Ru(II) complex 1
(complex 2 give same type of binding interaction) were studied at different concen-
trations (Figure 10). From this study we observed that absorption of BSA increases

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
Figure . Absorption spectrum of BSA in the presence of complex in the concentration range –.
× − M.
regularly upon increasing the concentration of the complex. It is may be due to the
adsorption of BSA on the surface of the complex. From these data the apparent asso-
ciation constant (Kapp) determined of the complexes with BSA has been determined
using the following equation.[37]
ꢀ
ꢁ
1/ (Aobs − A0) = 1/ (Ac − A0) + 1/Kapp (Ac − A0) comp
Where, Aobs is the observed absorbance of the solution containing different con-
centrations of the complex at 280 nm, A0 and Ac are the absorbances of BSA and
the complex at 280 nm, respectively, with a concentration of complex and Kapp
represents the apparent association constant. The enhancement of absorbance at
280 nm was due to absorption of the surface complex, based on the linear relation-
ship between 1/(Aobs – A0) vs reciprocal concentration of the complex with a slope
equal to 1/Kapp(Ac – A0) and an intercept equal to 1/(Ac – A0) (Figure 11). The value
of the apparent association constant (Kapp) for complex 1 determined from this plot
is 6.12 × 103M−1(R = 0.99896 for four points).
Fluorescence quenching of BSA by the complex 1
The effect of increasing the concentration of the complex on the fluorescence
emission spectrum of BSA were studied and represented in (Figure 12). With the
addition of complex BSA fluorescence emission is quenched. The fluorescence
quenching is described by the Stern–Volmer relation[39]
:
ꢀ
ꢁ
I0/I = 1 + KSV complex
where I0 and I represent the fluorescence intensities of BSA in the absence and pres-
ence of quencher, respectively. KSV is the linear Stern–Volmer quenching constant
and [complex] the molar concentration of the quencher. A linear plot (Figure 13)
between I0/I against [complex] was obtained and from the slope we calculated the

18
H. PAUL ET AL.
Figure . Plot of /(A - A) vs /[Complex] for the titration of BSA with complex 1 in tris HCl buffer;
apparent association constant (Kapp) = . × M−(R = . for four points).
Figure . Fluorescence Quenching of BSA in the presence of complex 1in the concentration range
–. × − M.
KSV as 8.4 × 104 (R = 0.99321 for five points), suggesting a strong affinity of the
complex 1 to BSA.
Antibacterial activity
The in vitro antimicrobial screening effects of the ligands(L1 and L2) and their ruthe-
nium complexes(1 and 2) were performed against five pathogenic bacteria viz.,
(Escherichia coli, Vibrio cholerae, Streptococcus pneumoniae, Shigella sp. and Cereus
bacillus) by the agar disc diffusion method and the results are shown in Table 2. The
activities of the compounds were compared with standards Antibiotic such as ampi-
cillin. The results indicate that the ruthenium (II) complexes are more active when
compared with parent ligands, this would suggest that the chelation could facilitate

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
19
Figure . The Stern–Volmer plot, the linear Stern–Volmer quenching constant (Ksv) = . ×  (R =
. for four points).
the ability of a complex to cross a cell membrane and can be explained by Tweedy’s
chelation theory.[46] Such a chelation could enhance the lipophilic character of the
central metal atom, which subsequently favors its permeation through the lipid layer
of the cell membranes and blocks the metal binding sites in enzymes of microorgan-
isms. However, the Ru(II) complexes (1 and 2) have better activity than the standard
antibiotics.
Conclusions
In this article, we have presented the synthesis of two Schiff base lig-
ands, L1 = N,N’-bis(4-nitrocinnamaldehyde)ethylenediamine and L2 = N,N’-bis
(2-nitrocinnamaldehyde) ethylenediamine and their corresponding ruthenium(II)
complexes. The ligands L1 and L2 and the complexes (1 and 2) have been charac-
terized by IR, UV-Vis, NMR, electrochemical techniques and spectroscopic anal-
ysis. The aim of this research was devoted to study the interaction of the com-
plexes with calf thymus DNA and BSA and the antibacterial activity of the com-
plexes and the organic moieties. The interaction of the Ru (II) complexes with CT-
DNA at physiological pH shows very good agreement between spectrophotometric
and fluorimetric methods of measurement, the data lend support to the validity of
the methods used in the experiments and all results indicate that the Ru (II) com-
plexes bind to CT-DNA through surface or groove binding mode and it is also in
accordance with the unchanged values of the viscosity of the DNA solution upon
addition of complex 1. This study clearly indicates that 1 binds to calf thymus DNA
in a groove binding interaction[47–49] but not in a intercalative mode like reported
octahedral ruthenium (II) complexes.[50–53] However, the Kb of 1 is comparable to
that of the reported ruthenium (II) complexes.[47–49] The electrochemical studies

20
H. PAUL ET AL.
indicated that the reduced form of the complex Ru(II) has a stronger association to
DNA than the oxidized Ru(III) form. The absorption and emission spectroscopy, as
well as fluorescence spectroscopy tools are also used to study the interaction of the
complex 1 with bovine serum albumin that proves the formation of a ground state
BSA-[Ru(L1)(bpy)2](ClO4)2 complex. From the antibacterial study it was observed
that Ru(II) complexes are highly active than their parent ligands but lesser activ-
ity than standard antibiotic against pathogenic bacteria viz.(Escherichia coli, Vibrio
cholerae, Streptococcus pneumoniae, Shigella sp. and Cereus bacillus).
Acknowledgment
Financial support from Council of Scientific and Industrial Research (CSIR), New Delhi, India is
gratefully acknowledged.
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