Tris-heteroleptic ruthenium(II) polypyridyl complexes: Synthesis, structural characterization, photophysical, electrochemistry and biological properties

Article abstract of DOI:10.1016/j.jinorgbio.2019.110903

Three water-soluble tris-heteroleptic ruthenium(II) polypyridyl complexes [Ru(bpy)(phen)(bpg)]²⁺ (1), [Ru(bpy)(dppz)(bpg)]²⁺ (2), and [Ru(phen)(dppz)(bpg)]²⁺ (3) (where bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2′,3′-c] phenazine, bpg = 4b,5,7,7a-tetrahydro-4b,7a-epiminomethanoimino-6H-imidazo[4,5-f] [1,10] phenanthroline-6,13-dione) have been synthesized and characterized. Molecular structures of complexes 1 and 3 are confirmed by single crystal X-ray structure determination. Interaction of complexes 1–3 with DNA is explored by various spectroscopic techniques. The complexes 1–3 show solvent dependent photophysical properties. Complexes 2 and 3 show extensive “molecular light switch” effect for DNA. The complexes 1–3 are low toxic towards HeLa (human cervical cancer) and HL-60 (human promyelocytic leukemia) cell lines. Further, the cellular uptake of complexes 2 and 3 by cells shows that complexes mainly localised on the nucleus of the cells.

Full text of DOI:10.1016/j.jinorgbio.2019.110903

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Tris-heteroleptic ruthenium(II) polypyridyl complexes: Synthesis,
structural characterization, photophysical, electrochemistry and
biological properties
Satish S. Bhat, Avinash S. Kumbhar, Neeraja Purandare, Ayesha
Khan, Günter Grampp, Peter Lönnecke, Evamarie Hey-Hawkins,
Ruchi Dixit, Kumar Vanka
PII:
S0162-0134(19)30366-6
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.110903
JIB 110903
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
4 June 2019
14 October 2019
18 October 2019
Please cite this article as: S.S. Bhat, A.S. Kumbhar, N. Purandare, et al., Tris-heteroleptic
ruthenium(II) polypyridyl complexes: Synthesis, structural characterization,
photophysical, electrochemistry and biological properties, Journal of Inorganic
Biochemistry (2019), https://doi.org/10.1016/j.jinorgbio.2019.110903
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Tris-Heteroleptic Ruthenium(II) Polypyridyl Complexes: Synthesis,
Structural Characterization, Photophysical, Electrochemistry and
Biological Properties
Satish S. Bhat,a Avinash S. Kumbhar,*a Neeraja Purandare,b Ayesha Khan,b Günter Grampp,c
Peter Lönnecke,d Evamarie Hey-Hawkinsd Ruchi Dixite and Kumar Vankae
a Department of Chemistry, Savitribai Phule University of Pune, Pune-411007, India
b Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University of Pune,
Pune- 411007, India.
C Institute of Physical and Theoretical Chemistry, Technical University Graz, A-8010,
Austria.
d Faculty of Chemistry and Mineralogy, Institute of Inorganic Chemistry, Johannisallee-29,
Universität Leipzig, 04103 Leipzig, Germany.
e Physical and Materials Chemistry division, CSIR - National Chemical Laboratory, pune,
411008, India.
* Corresponding authors
E-mail: askum@chem.unipune.ac.in
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Abstract
Three water-soluble tris-heteroleptic ruthenium(II) polypyridyl complexes
[Ru(bpy)(phen)(bpg)]2+ (1), [Ru(bpy)(dppz)(bpg)]2+ (2), and [Ru(phen)(dppz)(bpg)]2+ (3)
(where bpy = 2,2-bipyridine, phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]
phenazine, bpg = 4b,5,7,7a-tetrahydro-4b,7a-epiminomethanoimino-6H-imidazo[4,5-f] [1,10]
phenanthroline-6,13-dione) have been synthesized and characterized. Molecular structures of
complexes 1 and 3 are confirmed by single crystal X-ray structure determination. Interaction
of complexes 1-3 with DNA is explored by various spectroscopic techniques. The complexes
1-3 show solvent dependent photophysical properties. Complexes 2 and 3 show extensive
“molecular light switch” effect for DNA. The complexes 1-3 are low toxic towards HeLa
(human cervical cancer) and HL-60 (human promyelocytic leukaemia) cell lines. Further, the
cellular uptake of complexes 2 and 3 by cells shows that complexes mainly localised on the
nucleus of the cells.
Keywords: ruthenium• polypyridyl• fluorescence• circular dichroism • cytotoxicity
1. Introduction
Luminescent molecules having low toxicity and ability to undergo cellular uptake are of
great importance as molecular probes for cellular imaging, across cell biology, molecular
biology, and flow cytometry applications[1-3]. The understanding of DNA structure and
organization within the cell is of great importance as DNA is a carrier of genetic material[3].
For this purpose, biologists are using organic dyes which have major disadvantages such as
low water solubility, high toxicity, photo bleaching effect, small Stokes shift and require UV
light irradiation leading to cleavage of DNA[4-6]. Another main task in cellular imaging is to
differentiate the endogenous fluorescence of biological species (auto-fluorescence) from that
of the organic dyes which are applied. If the fluorophore dye has the large stoke shift then the
auto-fluorescence can be eliminated from the required signal because the typical Stokes shifts
for species involved in auto-fluorescence is small[4-6]. Additionally, the auto-fluorescence
can be eliminated if the emission lifetime of the fluorophore is much longer than the short-
lived auto-fluorescence by using time-resolved microscopy. However, it is still difficult to
differentiate the area occupied by the fluorophore autofluorescence[6-8].
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In this context, the exceptional photophysical properties such as intense polarized
luminescence, large Stokes shift, high water solubility, red emission wavelength, and good
photostability of ruthenium(II) polypyridyl complexes make them potentially valuable probes
for cellular imaging and probe for DNA structure determination [3, 9-22]. The Ruthenium(II)
dipyridophenazine complexes {[Ru(NN)2(dppz)]2+, NN = 2,2-bipyridine (bpy), 1,10-
phenanthroline (phen), dipyrido[3,2-a:2,3-c]phenazine (dppz) has low background emission
and long emission life time, when bound to DNA, make them attractive candidates as
molecular probes for cellular imaging using fluorescence microscopy[10, 23]. J. A. Thomas
et.al. have reported the dinuclear ruthenium(II) polypyridyl complex as an in cellulo nuclear
stain for eukaryotic and prokaryotic cells[3, 24]. Research on ruthenium polypyridyl
complexes has mainly focused on the design of in vitro probes, but the use of these
complexes in cellular imaging of nucleic acids in cells is limited due to poor cell membrane
permeability and structure-specific binding[3, 10, 12, 21, 25-29]. Recently Liang-Nian Ji and
coworker have reported the effect of modification of ancillary ligand on DNA binding modes
and cytotoxicity of ruthenium complexes[13, 14, 30].
As a part of our project aimed to investigate the effect of modification of ancillary
ligands of ruthenium complexes on DNA interaction properties, we previously demonstrated
the DNA binding and DNA condensation properties of the complexes[24, 31-36]. Herein, we
report on structural, photophysical, electrochemical, DNA binding and cellular imaging
properties of three new tris-heteroleptic ruthenium(II) polypyridyl systems (Scheme 1).
2.0 Results and Discussion
2.1 Synthesis and Characterization
Tris-heteroleptic ruthenium(II) polypyridyl complexes were synthesized as shown in
Scheme 1 by reacting appropriate ligands followed by chromatographic purification using
neutral alumina column and obtained as racemic mixtures. The compounds were
characterized by NMR, IR, elemental analysis, UVVisible spectroscopy and electrospray
mass spectrometry (see experimental section). In ESI-MS (Electrospray Ionization Mass
Spectrum) spectra peaks due to [M – Cl] + and [M – 2Cl]2+ were observed. Complexes 1 and
3 were characterized by single-crystal X-ray structure analysis. Further physicochemical and
biological studies were carried out using the racemic mixtures.
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2.2 Single Crystal Structures
Single crystals of complexes 1 and 3 suitable for single-crystal X-ray data collection
were grown by slow evaporation of complexes dissolved in dimethylsulfoxide/water and
methanol/water, respectively. Relevant crystallographic information for complexes 1 and 3 is
given in Table S1 (Supporting Information), and a list of selected bond lengths and angles
can be found in Tables 1 and 2. ORTEP (Oak Ridge Thermal Ellipsoisal Plot) representations
of ruthenium complexes 1 and 3 are shown in Figure 1. Both complexes crystallize in the
monoclinic space group P21/c. The ruthenium(II) cation is chelated by three different
polypyridyl ligands namely bpy, phen and bpg for complex 1, and phen, dppz and bpg in case
of complex 3. The N-Ru-N “bite” angles for the bidentate ligands are 83.00(3), 75.80(3),
79.1(1) for complex 1, and 80.0(2), 79.5(2), 79.2(2) for complex 3, which are typical of 5-
membered chelate rings formed by such diimine ligands[11, 22, 37-40]. The mean Ru-N
(phen) bond length is 2.063 Å, the Ru-N (bpy) bond length of 2.074 Å and the Ru-N (bpg)
(bpg= bpg = 4b,5,7,7a-tetrahydro-4b,7a-epiminomethanoimino-6H-imidazo[4,5-f] [1,10]
phenanthroline-6,13-dione) bond length of 2.058 Å for complex 1 and is similar to that found
in other analogous mixed ligand polypyridyl ruthenium(II) complexes[11, 22, 39, 40]
including tris-heteroleptic ruthenium(II)-diimine complexes[37, 38]. The molecular structure
of 3 shows “dimers" formed by intermolecular hydrogen donor and acceptor bonds between
bpg ligands (Figure S4, Supporting Information).
2.3 Photo-physical Studies
The UV/Visible and emission data of complexes 1-3 in different solvents are
summarized in Table 3 and the absorption and luminescence spectra of the complexes 1-3 are
shown in Figure S5 and Figure S6 (Supporting Information). The low energy broad band
around 450 nm for the complexes 1-3 is assigned to the MLCT (Metal to Ligand Charge
Transfer) Ru (d)  ligand (*) transitions[13, 32, 41] typical of polypyridyl ruthenium(II)
complexes. The intense bands between 240 to 300 nm corresponding to   * transitions of
the aromatic polypyridyl ligands. In case of complexes 2 and 3 a band around 360 nm
corresponds to the characteristic of a   * (dppz) transition[32, 33, 35, 42].
Complexes 1-3 show strong luminescence in organic solvents with an emission peak
centered between 600 and 750 nm but these complexes have weak emission in aqueous
solutions. The emission maxima and quantum yield with respect to [Ru(bpy)3]2+ as standard
are compiled in Table 3.
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2.4 Electrochemistry
The oxidation and reduction potentials of the complexes in acetonitrile are compiled
in Table 4. Complexes 1-3 exhibit one oxidation and three reduction waves in the sweep
range from -2.0 to +2.0 V (Figure 2) and display ruthenium(II/III)-based half-wave oxidation
potential at 0.96, 0.99, 0.99 versus Fc0/+, respectively. The anodic shift relative to
[Ru(bpy)3]2+ (0.88 V) is attributed to the more electronegative character or stronger π-
accepting feature of the ligands that stabilize the ruthenium-based HOMO (Highest Occupied
Molecular Orbital), rendering the oxidation of the metal more difficult, similar to other
ruthenium(II) mixed polypyridyl complexes[13, 43-45]. All complexes exhibit three
reversible or quasi-reversible redox couples. The ruthenium(II/III)-based half-wave oxidation
potential of complex 2 and 3 are almost same due to the almost same HOMO to LUMO
(Lowest Occupied Molecular Orbital) gap in both the complexes, which is evidenced by
density functional theoretical calculation (discussed in electronic structure calculation
section). Due to the similarity between the ligands used here, our data do not allow the order
in which the ligands are reduced for either complex to be determined.
2.5 Spectroelectrochemistry
The bulk electrolysis experiments were conducted to further probe the metal-based
(RuII/RuIII) redox process. For each experiment, the oxidation of RuII to RuIII in situ was
followed by absorption spectroscopy by applying a constant potential of 200 mV higher than
that of the oxidation potential (vs Fc/Fc+) of the complex at a platinum gauze electrode to a
30 µM complex solution in acetonitrile. For complex 1, a potential of 1120 mV (vs Fc/Fc+)
was applied to a platinum gauze electrode, and the oxidation of RuII to RuIII in situ was
monitored (Figure 3). In the absorption spectrum, the bands at 262 and 286 nm decreased in
intensity accompanied by the growth of a band at 325 nm. In the visible region, the MLCT
band at 454 nm decreased in intensity and the oxidized species display a weak band at 430
nm. Isosbestic points were observed at 356, 296, 269 and 256 nm indicating a clean oxidation
reaction. After the potential was removed reconversion to [RuII(bpy)(phen)(bpg)]2+ was not
observed, even though low concentration of the complex was used. However, by changing
the applied potential to 800 mV (vs Fc/Fc+) almost complete reversion to
[RuII(bpy)(phen)(bpg)]2+ was achieved[37, 46]. The three bands observed in the high energy
region for the electrogenerated RuIII complex at 262 nm and 325 nm have been assigned to
intra-ligand (*) transitions of the diimine ligands[47, 48]. The weak band observed in
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the visible region at 430 nm is attributed to an LMCT transition (→ t2) characteristic of
RuIII[36].
2.6 Electronic structure calculations
To probe the excitation in complexes 1-3 in comparison with reported [Ru(bpy)3]2+,
[Ru(bpy)2(dppz)]2+ complexes a series of TD-DFT (Time Dependent Density Functional
Theory) computations were carried out. The HOMO-LUMO energy level diagram is given in
Figure 4. The UV-Vis absorption originates from HOMO to LUMO electronic transition,
where HOMO is located on the metal center which has a significant amount of dz2 character
and LUMO is delocalized over the carbon ring with significant π- character. From the
theoretically calculated first excitation energy (S0S1) it has been found that the excitations
occur almost at the same wavelength.
2.7 DNA BINDING STUDIES
2.7.1 Steady-state Emission Studies
The investigation of the interaction of the molecules with the DNA by monitoring the
changes in the luminescence spectra are diagnostic means to determine the DNA binding[32,
36, 49]. In the aqueous buffer solution complexes, 1-3 show an increase in luminescence
intensity with the successive addition of CT-DNA (Calf Thymus-DNA) (Figure 5).
Complexes 2 and 3 shows negligible luminescence in aqueous solution, but in the presence of
DNA are strongly luminescent displaying a “molecular light switch effect” (Figure 5) with
luminescence enhancement of around 95-fold, indicating strong binding of these complexes
with CT-DNA. However, complex 1 shows only a small increase in emission intensity
around 1.2 times upon addition of DNA indicating weak binding. This implies that
complexes 2 and 3 interact strongly with DNA by intercalation[23]. The dipyridophenazine
(dppz) containing complexes 2 and 3 acts as “molecular light switch” for DNA due to
intercalation of dppz ligand between the base pairs of CT-DNA this behaviour is similar to
that observed for the complex {[Ru(NN)2(dppz)]2+, NN = 2,2-bipyridine (bpy), 1,10-
phenanthroline (phen), dipyrido[3,2-a:2,3-c]phenazine (dppz) in the presence of DNA[23].
The molecular light switch behavior of complexes 2 and 3 for DNA may be mainly due to
two reasons: first, these complexes have dppz ligand with stack between base-pairs of DNA
by intercalation mode of binding and the hydrophobic environment inside the DNA helix
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reduces the accessibility of water molecules with the N-atom of the phenazine moieties of the
complex, interaction of water molecules quenches the emission and second, the complex
mobility is restricted at the binding intercalation binding site and so the vibrational mode of
relaxation decreases[15-18].
DNA binding of these complexes with DNA was further confirmed by time-correlated
single-photon counting luminescence measurements. For complex 1 no increase in lifetime
was observed at [CT-DNA]: [Ru] = 20; complexes 2 and 3 do not have any fluorescence but
in the presence of CT-DNA showed strong luminescence with emission lifetime of 1056 ns
and 1078 ns, respectively, at [CT-DNA]: [Ru] = 20 confirming the strong binding of these
complexes. A similar effect was found in other ruthenium polypyridyl complexes which
exhibit a molecular light switch effect[23, 36].
2.7.2 Absorption Spectroscopy Studies
The interaction of molecules with DNA can be followed by absorption spectroscopy,
which is one of the wide techniques to understand the mode of interaction of molecules with
DNA.[32, 50] The interaction of molecules with the DNA through intercalation results in
hypochromism and a redshift, due to the interaction of the complex and between the base
pairs of the DNA[19, 32, 34, 35, 50-55]. The electronic spectra of complexes 2 and 3 in the
presence and absence of DNA are shown in Figure S7 (supporting information), a redshift of
the MLCT band by 10 nm and 6 nm, respectively, was observed for complexes 2 and 3 in the
presence of DNA. The percentage of hypochromicity for the MLCT band of complex 2 (450
nm) and 3 (438 nm) upon binding to DNA was observed to be 15% and 12%, respectively
(Table 5). The intrinsic binding constants of complexes 2 and 3 with CT-DNA were
estimated from equation 2 (Supporting Information). The CT-DNA binding constants for
thecomplexes 2 and 3 Figure S7 (supporting information) were found to be 2.3×104 and
6.8×104 M-1respectively. These values are comparable to that of some known DNA
intercalators such as 1.2×105 M-1 for [Ru(NH3)4(dppz)]2+,[56] 3.9×105 M-1 for
[Ru(bpy)2(dcdpq)]2+ (dcdpq = dicyano-dipyrido[3,2-d:2,3-f]quinoxaline),[57] 4.7×104 M-1
for [Ru(bpy)2(dpq)]2+,[55] 6.3×104 M-1 for [Ru(bpy)2(dpt)]2+ (dpt = 3-(pyrasinyl)- as-
triazino[5,6-f]phenanthrene),[58] 4.9 × 104 M-1 for [Ru(bpy)2(aip)]2+ (aip = 2-(9-anthryl)-
1H-imidazo[4,5-f][1,10] phenanthroline),[20] but smaller than that of the parent complex
[Ru(bpy)2(dppz)]2+ (Kb = 5.0×106 M-1)[13] probably due to hindrance caused by the non-
planar bpg ancillary ligand for intercalative binding. The spectral characteristic of
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hypochromism and redshifts, as well as large Kb values, suggest that the two complexes bind
to DNA by partial intercalation mode. The complex 2 and 3 have higher DNA binding
constants due to the presence of planar dppz ligand which intercalates between the base-pairs
of DNA, but complex 1 planarity of the ligands bpy, bpg, and phen ligands is much less
compared to dppz so this complex weakly interacts with DNA.
2.7.3 DNA Melting Experiments
The DNA helix melting temperature increases with the intercalation of small
molecules into the double helix[32, 59-62]. The DNA melting temperature is the point where
the double helix denatures into single-stranded DNA[32]. The double-stranded DNA has very
small extinction coefficient at 260nm compared to the single-stranded form of DNA.
Therefore, the melting of the helix leads to an increase in the absorption at this wavelength.
In the absence of complex, CT-DNA melts at 65±1 °C (phosphate buffer). A large increase in
melting temperature, 6 °C for 2 and 7 °C for 3, was observed and is comparable to that
observed for classical intercalators[59-62]. These results are consistent with the other
spectroscopic studies indicating the intercalation of complexes 2 and 3 into the DNA helix.
2.7.4 Viscosity Studies
Intercalation of molecules with DNA is known to cause a substantial increase in
viscosity of a DNA solution due to an increase in separation of base pairs at the intercalation
site, and hence, an increase in overall DNA molecular length[32, 63, 64]. In contrast, a
complex that binds to the DNA grooves causes less-pronounced changes or no change in
viscosity of a DNA solution[63, 64]. Typical data of such experiments on complexes 1-3 in
comparison with DNA intercalator ethidium bromide (EtBr) is shown in Figure 6. The
complexes 1, 2 and 3 induces viscosity changes that are indicative of stronger DNA binding
and are similar to that of previously reported complexes[32, 64]. The viscosity changes for
complex 2 and 3 are similar to that of well-known DNA intercalator ethidium bromide
suggesting intercalation mode of DNA binding by complexes 2 and 3. These results are
inconsistent with absorption and emission titration, and thermal melting experiments showing
stronger binding of complexes 2 and 3 to the DNA by intercalation.
2.7.5 Circular Dichroism (CD) Spectral Analysis
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The conformational changes in the DNA by the interaction of molecules with the
DNA can be investigated by CD spectral analysis[65-67]. To confirm the interaction of
complexes 1-3 with DNA, CD spectra of DNA were recorded in the presence and absence of
complexes 1-3. A very small change in the CD spectrum of DNA was observed when
complex 1 was added to DNA due to an electrostatic mode of interaction (Figure 7). Addition
of complexes 2 or 3 to DNA increased the intensity of the positive band which may be due to
strong interactions (Figure 7). These results show that complexes 2 and 3 interact with DNA
and change their conformation.
2.7.6 Cytotoxicity Studies
Low toxicity is a prerequisite for cellular imaging probes for long-term monitoring.
Hence toxicity profiles for complexes 1-3 were evaluated by MTT {3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide} assay against HeLa (human cervical cancer) and HL-
60 (human promyelocytic leukemia) cell lines (Figure 8 and Figure 9). Complexes 1-3 did
not display noticeable cytotoxicity towards both cell lines; cell viability is more than 80 %
even after 72 h incubation of cells with complexes 1-3, and after 24 h of incubation rapid
growth of cells is observed, hence there is an increase in cell viability. The low toxicity of the
complexes may be due to the electrostatic binding by complex 1 and low order of DNA
binding constant of the complexes of the order 104 for complexes 2 and 3.
2.7.7 Cellular Imaging Studies
The cellular uptake and localization properties of complexes 1-3 were evaluated using
fluorescence microscopy. For fluorescence microscopy experiments the cells were treated
with complexes 2 and 3 and co-stained with DAPI (DAPI = 4,6-diamidino-2-phenylindole)
for 24 and 72 h. Both complexes undergo rapid cellular internalization and clearly visible
within the cell after 24 h incubation (Figure 10 and 11). To a co-staining experiment with the
DNA-specific dye DAPI was performed and strong co-localization of complex signal with
that of DAPI was observed (Figure 10 and 11, overlay image, and Figure S8 and S9,
Supporting Information). From these studies, it is proposed that complexes 2 and 3 can be
used as potential DNA-staining probes.
3.0 Conclusions
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In summary, three novel non-toxic water-soluble tris-heteroleptic ruthenium(II) polypyridyl
complexes 1-3 have been designed, and the photophysical and electrochemical properties
have been investigated. Electrochemical studies of 1-3 showed a reversible RuII/RuIII
oxidation process at 0.96, 0.99, 0.99 V (vs Fc/Fc+), respectively. The complexes 2 and 3
show an extensive “molecular light switch effect” for DNA. The DNA binding significantly
stronger for the complexes 2 and 3 than the complex 1. The complexes 1-3 are low toxic
towards HeLa and HL-60 cell lines. Further, the complexes 2 and 3 specifically stains the
nucleus of the cells.
4.0 Experimental Section
4.1 Chemicals
All chemicals and solvents were purchased commercially and were used as received.
RuCl3·3H2O was purchased from S. D. Fine Chemicals, Mumbai and calf thymus DNA was
purchased from SRL, Kolkata and used as received.
4.2 Synthesis
The ligands 1,10-phenanthroline-5,6–dione (phendione),[68] and dipyrido[3,2-a:2,3-c]
phenazine
(dppz),[69,
70]
4b,5,7,7a-tetrahydro-4b,7a-epiminomethanoimino-6H-
and
imidazo[4,5-f][1,10]phenanthroline-6,13-dione (bpg),[71, 72] [RuCl2(dmso)4][73]
[Ru(NN)(NN)′Cl2)] (where, NN, NN′ are different polypyridine ligands) were synthesized
according to the literature[74].
4.2.1 [Ru(bpy)(phen)(bpg)]Cl2 (1)
The precursor complex cis-[Ru(bpy)(phen)Cl2] ·2H2O (100 mg, 0.196 mmol) and bpg
(57 mg, 0.196 mmol) were dissolved in methanol-water (1:1, 50 mL) and the mixture was
heated to reflux for 8 h, whereupon the color of the solution changed from dark purple to red.
The red solution was filtered hot and was cooled to room temperature. The solvent was
removed under vacuum to obtain a red solid. The product was purified by column
chromatography on neutral alumina using acetone and methanol as solvents. The orange-red
fraction was collected and concentrated under vacuum to get the orange-red pure product.
Yield: 130 mg (82.3%). Crystals were grown by slow evaporation of a solution of the
perchlorate salt of the complex which was prepared by adding aqueous sodium perchlorate
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solution to the purified product in water. The bright red precipitate formed was collected by
1
filtration and washed with diethyl ether. H NMR (DMSO-d6, 300 MHz, 25 °C);  = 8.84
(m, 5H), 8.72 (t, 2H), 8.46 (t, 2H), 8.38 (s, 2H), 8.22 (m, 3H), 8.11 (m, 2H), 7.89 (m, 2H),
7.79 (d, 1H), 7.69-7.43 (m, 6H), 7.35 (t, 1H); IR (KBr pellet, cm-1) 
̃ = 3448 (H2O), 3176
(NH), 3101, 3084 (ArH), 1707 (C=O), 1635, 1608, 1454, 1423 (C=C, C=N); ESI-MS (m/z,
positive mode) : ([M–2Cl]2+) 366 (100), ([M–2Cl–H]+) 731 (5%); Anal. Calcd. for
C36H26N10O2Cl2Ru 4H2O: C, 49.41; H, 3.92; N, 16.02; found: C, 49.19 ; H, 4.01; N, 16.23.
Caution!! Perchlorate salts of metal complexes with organic ligands are potentially
explosive, and only small amounts of material should be prepared and these should be
handled with great care.
4.2.2 [Ru(bpy)(dppz)(bpg)]Cl2 (2)
The synthesis and purification of compound 2 was similar to that of 1 using cis-
[Ru(bpy)(dppz)Cl2]·2H2O (100 mg, 0.164 mmol) and bpg (48 mg, 0.164 mmol). Yield: 95
mg (64.2%). 1H NMR (DMSO-d6, 300 MHz, 25 °C);  = 9.64 (dd, 2H), 8.86 (t, 2H), 8.74 (d,
2H), 8.52 (m, 4H), 8.34 (m, 2H), 8.25-8.12 (m, 6H), 8.08 - 8.01 (m, 2H), 7.89 (d, 2H), 7.78 -
7.62 (m, 4H), 7.51 (m, 1H), 7.39 (t, 1H); IR (KBr pellet, cm-1); 
̃ = 3416 (H2O), 3217 (NH),
3090, 2924 (ArH), 1703 (C=O), 1649, 1456, 1384 (C=C, C=N).; ESI-MS: (m/z, (% positive
mode): ([M–2Cl–H]+) 833 (7%), ([M–2Cl]2+) 417 (100%); Anal. Calcd. for
C42H28N12O2Cl2Ru 2.5H2O; C, 53.12; H, 3.54; N, 17.69; found: C, 53.27; H, 3.49; N, 17.41.
4.2.3 [Ru(phen)(dppz)(bpg)]Cl2 (3)
The synthesis and purification of compound 3 was similar to that of 1 using cis-
[Ru(phen)(dppz)Cl2]·2H2O (100 mg, 0.157 mmol) and bpg (46 mg, 0.157 mmol). Yield: 88
mg (60.2%). Crystals were grown by slow evaporation of solution of complex in
1
water:methanol mixture. H NMR (DMSO-d6, 300 MHz, 25 °C)  = 9.68 (d, 1H), 9.57 (d,
1H), 8.86 (d, 1H), 8.74 (m, 3H), 8.52 (m, 3H), 8.44 (d, 2H), 8.40 (d, 2H), 8.29 (d, 2H), 8.19-
8.07 (m, 5H), 8.05-7.97 (m, 2H), 7.85 (m, 2H), 7.76 (m, 1H), 7.64-7.50 (m, 3H); IR (KBr
pellet, cm-1); 
̃ = 3456 (H2O), 3209 (NH), 3091 2924 (ArH), 1701 (C=O), 1651, 1433, 1373
(C=C, C=N).; ESI-MS: (m/z, (%) positive mode): ([M–Cl]+) 894 (7%), ([M–2Cl]2+) 429
(100%); Anal. Calcd. for C44H28N12O2Cl2Ru 3H2O: C, 53.77; H, 3.49; N, 17.11; found: C,
53.62; H, 3.61; N, 16.93.
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4.3 Methods and Instrumentation
Details of instruments used and the methods/bioassays are given in supplementary material.
4.4 Supporting Information. Relevant crystallographic information for complexes 1 and 3 is
given in Table S1. ESI-MS spectra of complexes 1-3 (Figures S1-S3), Packing diagram of 3
(Figure S4), absorption spectra of complexes 1-3 (Figure S5), emission spectra of complexes
1-3 (Figure S6), Absorption titration with DNA (Figure S7), fluorescence microscopy images
after 72 h incubation (Figures S8 and S9).
5.0 Table of Abbreviations:
bpy = 2,2-bipyridine
phen = 1,10-phenanthroline
dppz = dipyrido[3,2-a:2',3'-c] phenazine
bpg
=
4b,5,7,7a-tetrahydro-4b,7a-epiminomethanoimino-6H-imidazo[4,5-f]
[1,10]
phenanthroline-6,13-dione
ESI-MS = Electrospray Ionization Mass Spectrum
CT-DNA = Calf Thymus – DNA
ORTEP = Oak Ridge Thermal Ellipsoisal Plot
MLCT =Metal to Ligand Charge Transfer
HOMO = Highest Occupied Molecular Orbital
LUMO = Lowest Occupied Molecular Orbital
TD-DFT = Time Dependant Density Functional Theory
MTT ={3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide}
EtBr = ethidium bromide
CD = Circular Dichroism
HeLa = human cervical cancer
HL-60 = human promyelocytic leukemia
DAPI = 4,6-diamidino-2-phenylindole
6.0 Acknowledgements
SSB acknowledges the Department of Science and Technology (DST), New Delhi, India, for
providing a fellowship under the scheme SR/S5/BC-25/2006. ASK acknowledges the
Department of Science and Technology (DST), New Delhi, India, for financial support under
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“New Initiatives in Bioinorganic Chemistry” (SR/S5/BC-25/2006). A.S.K. and E. H-H.
acknowledges financial support from the Indo-German (DST-DAAD) joint Research Project
(INT/FRG/DAAD/P-225/2013). SSB acknowledges the OEAD fellowship for short term
research visit to the Graz University of Technology. We thank SAIF, CDRI-Lucknow, India
for providing ESI-MS data.
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Scheme/Figure, Tables Captions
Scheme 1. Synthetic route for complexes 13.
Figure 1. ORTEP diagram of the cations of complexes (a) 1 and (b) 3; anions and solvent
molecules are omitted for clarity. All ellipsoids are drawn at 50% probability level.
Figure 2. Cyclic voltammograms obtained at platinum working electrode for oxidation of
complex 1 (1 mM) oxidation (top) and reduction (bottom) in acetonitrile (0.1 M
tetrabutylammonium hexafluorophosphate (TBA)PF6) for different scan rates.
Figure 3. UV-Visible spectroelectrochemical responses of complex 1 (30 µM) in acetonitrile
(0.1 M tetrabutylammonium hexafluorophosphate (TBA)PF6), during oxidation.
Figure 4. HOMO-LUMO energy level diagram for [Ru(bpy)3]2+, [Ru(bpy)2(dppz)]2+ and
complexes 1-3.
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Figure 5. Emission spectra of complexes (a) 1, (b) 2, (c) 3, 20 µM in phosphate buffer, pH
7.2, at 298 K with increasing [CT-DNA]/[Ru] ratio 0-30, (d) plot of relative integrated
emission intensity versus [CT-DNA]/[Ru] for the complexes ( ) 1, ( ) 2, ( ) 3.
Figure 6. Effect of increasing amounts of complexes 1-3 and ethidium bromide (EtBr) on the
relative viscosity of calf-thymus DNA at 28.0 °C, [DNA] = 300 µM.
Figure 7. CD spectra of CT-DNA in the presence and absence of complexes 1-3 in phosphate
buffer (pH 7.2) [DNA]/[Complex] = 4, [DNA] = 200µM.
Figure 8. Cytotoxicity of complexes 1-3 against HL-60 cell line evaluated by MTT assay. (a)
Time-dependent, [complex] =5µM (b) Concentration-dependent, [complex] =2-10µM.
Results are mean values of three identical experiments.
Figure 9. Cytotoxicity of complexes 1-3 against HeLa cell line evaluated by MTT assay, (a)
time-dependent, [complex] =5µM (b) concentration dependent, [complex] =2-10µM. Results
are mean values of three identical experiments.
Figure 10. Fluorescence microscopy images of HL-60 (103) cells incubated (24 h) with 5 µM
of complexes 2, 3 and DNA-specific stain DAPI, (a) with complex 2 (left), DAPI (centre),
overlay image (right); (b) with complex 3 (left), DAPI (centre), overlay image (right).
Figure 11. Fluorescence microscopy images of HeLa cells incubated (24 h) with 5 µM of
complexes 2 or 3 and DNA-specific stain DAPI; (a) cells incubated with complex 2 (left),
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DAPI (centre), overlay image (right); (b) cells incubated with complex 3 (left), DAPI
(centre), overlay image (right). Images were taken after 24 h of incubation.
Table 1 Selected bond lengths [Å] and angles [°] for complex 1
Table 2 Selected bond lengths [Å] and angles [°] for complex 3
Table 3 UV/Vis and emission data for complexes 13
Table 4 Electrochemical data for complexes in Acetonitrile
Table 5 Electronic absorption data of complexes 2 and 3 upon addition of CT-DNA
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Scheme 1.
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(a)
(b)
Figure 1.
(a)
(b)
Figure 2.
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Figure 3.
Figure 4.
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Figure 5.
Figure 6.
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Figure 7.
(a)
(b)
Figure 8.
(a)
(b)
Figure 9.
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(a)
(b)
Figure 10.
(a)
(b)
Figure 11.
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Table 1 Selected bond lengths [Å] and angles [°] for complex 1.
Bond lengths
Ru(1)-N(1)
Ru(1)-N(2)
Ru(1)-N(3)
2.060(3)
2.066(3)
2.077(3)
Ru(1)-N(4)
Ru(1)-N(5)
Ru(1)-N(6)
2.070(3)
2.055(3)
2.060(3)
Bond angles
N(2)-Ru(1)-N(1)
N(3)-Ru(1)-N(1)
N(4)-Ru(1)-N(1)
N(5)-Ru(1)-N(1)
N(6)-Ru(1)-N(1)
N(4)-Ru(1)-N(2)
N(5)-Ru(1)-N(2)
N(6)-Ru(1)-N(2)
83.00(3)
96.50(2)
N(2)-Ru(1)-N(3)
N(4)-Ru(1)-N(3)
84.4(2)
75.80(3)
99.2(1)
172.8(2)
91.1(2)
97.10(2)
79.1(1)
170.70(3) N(5)-Ru(1)-N(3)
95.20(3)
90.7(1)
91.0(2)
176.2(1)
97.5(1)
N(6)-Ru(1)-N(3)
N(5)-Ru(1)-N(4)
N(6)-Ru(1)-N(4)
N(5)-Ru(1)-N(6)
Table 2 Selected bond lengths [Å] and angles [°] for complex 3.
Bond Lengths
Ru(1)-N(1)
Ru(1)-N(2)
Ru(1)-N(3)
Ru(2)-N(13)
Ru(2)-N(14)
Ru(2)-N(15)
2.067(4)
2.068(4)
2.059(4)
2.094(9)
2.069(8)
2.066(4)
Ru(1)-N(4)
Ru(1)-N(7)
Ru(1)-N(8)
Ru(2)-N(16)
Ru(2)-N(19)
Ru(2)-N(20)
2.071(4)
2.069(4)
2.025(5)
2.077(4)
2.047(5)
2.053(4)
Bond Angles
N(2)-Ru(1)-N(1)
N(3)-Ru(1)-N(1)
N(4)-Ru(1)-N(1)
N(7)-Ru(1)-N(1)
N(8)-Ru(1)-N(1)
N(3)-Ru(1)-N(2)
N(4)-Ru(1)-N(2)
N(8)-Ru(1)-N(2)
N(16)-Ru(2)-N(13)
79.95(2)
93.38(2)
89.52(2)
96.02(2)
N(8)-Ru(1)-N(3)
N(8)-Ru(1)-N(7)
N(3)-Ru(1)-N(7)
N(2)-Ru(1)-N(7)
91.65(2)
79.18(2)
95.90(2)
93.88(2)
95.63(2)
79.45(2)
172.99(2)
173.39(2) N(8)-Ru(1)-N(4)
160.70(2) N(3)-Ru(1)-N(4)
91.30(2)
95.75(2)
92.0(3)
N(7)-Ru(1)-N(4)
N(20)-Ru(2)-N(16) 174.62(2)
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N(14)-Ru(2)-N(13)
N(15)-Ru(2)-N(13)
N(20)-Ru(2)-N(13)
N(19)-Ru(2)-N(13)
N(14)-Ru(2)-N(16)
N(15)-Ru(2)-N(16)
N(19)-Ru(2)-N(20)
77.8(4)
87.4(3)
92.7(3)
172.1(3)
93.2(3)
79.93(2)
79.58(2)
N(19)-Ru(2)-N(16) 95.79(2)
N(15)-Ru(2)-N(14) 163.5(3)
N(20)-Ru(2)-N(14) 90.4(3)
N(19)-Ru(2)-N(14) 100.2(3)
N(20)-Ru(2)-N(15) 97.67(2)
N(19)-Ru(2)-N(15) 95.42(2)
Table 3 UV/Vis and emission data for complexes 13.
Complex[a]
Absorbance
max/ (M-1cm-1)
(Acetonitrile)
Emission
[c]
[d]
[c]
[d]
[c]
[d]

em
em
em em
em
Water
635
--
--
606 0.063 601 0.062 603

em
Ligand
transitions
264/49945
MLCT
Acetonitrile
DMF
616 0.075 619 0.065
0.022
--
1
286/35840 449/14015
278/76095
351/17690 454/16360
611
0.081 614 0.051
2
265/85370
610 0.095 616 0.047
--
3
366/17565
438/1607
--
[b][Ru(bpy)3]2+
0.042
a
b
c
[Ru] = 10 (±0.2) µM, data taken from ref.[75], error limit: max = ± 2 nm,  = ±2%,
emission maxima, d  = emission quantum yield excited at 450 nm.
Table 4 Electrochemical data for complexes in Acetonitrile.
[a]Complex
Metal-based E0 in Ligand-based (V)
f
V
[Ru(bpy)3]2+
0.88
0.96
0.99
0.99
-
-
-
0.92 1.12 1.36
1
2
3
-
-
-
0.79 1.03 1.30
-
-
-
0.53 1.07 1.43
-
-
-
0.48 0.96 1.18
a Data reported for 1 mM solutions in CH3CN with (TBA)PF6 as the supporting electrolyte.
Scan rate was 100 mV/s, vs Fc/Fc+, error limit: E0 = ± 0.01 V.
f
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