Synthesis, characterization, photophysical studies and interaction with DNA of a new family of Ru(II) furyl- and thienyl-imidazo-phenanthroline polypyridyl complexes

Article abstract of DOI:10.1016/j.ica.2011.07.020

A new family of Ru(II) polypyridyl complexes (C1 to C6) containing furyl- or thienyl-imidazo-phenanthroline ligands (4-6) were synthesized using microwave irradiation and characterized by elemental analysis, ¹H NMR, UV-Vis absorption and fluore

Full text of DOI:10.1016/j.ica.2011.07.020

Inorganica Chimica Acta 381 (2012) 95–103
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Inorganica Chimica Acta
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Synthesis, characterization, photophysical studies and interaction with DNA
of a new family of Ru(II) furyl- and thienyl-imidazo-phenanthroline polypyridyl
complexes
c
d
c
c,
Bruno Pedras a,b, , Rosa M.F. Batista , Laura Tormo , Susana P.G. Costa , M. Manuela M. Raposo
,
⇑
⇑
Guillermo Orellana d, , José Luís Capelo a,b, Carlos Lodeiro a
⇑
a Grupo BIOSCOPE, Departamento de Química-Física, Facultad de Ciencias de Ourense, Universidad de Vigo, Campus de Ourense, E-32004 Vigo, Spain
b REQUIMTE, Departamento de Química, FCT-UNL, 2829-516 Caparica, Portugal
c Centro de Química, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
d Departamento de Química Orgánica I, Facultad de Química, Universidad Complutense de Madrid, E-28040 Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 23 July 2011
A new family of Ru(II) polypyridyl complexes (C1 to C6) containing furyl- or thienyl-imidazo-phenan-
throline ligands (4–6) were synthesized using microwave irradiation and characterized by elemental
analysis, 1H NMR, UV–Vis absorption and fluorescence spectroscopy, FAB, ESI-MS and MALDI-TOF-MS
spectrometry. On the other hand, the novel furyl- or thienyl-imidazo-phenanthroline derivatives (5–6)
were synthesized through the Radziszewski reaction and completely characterized by the usual spectro-
scopic techniques. The interaction of the complexes with calf thymus DNA in the absence and in the
presence of different quenchers (ethidium bromide, potassium hexacyanoferrate(II) and methyl viologen)
has been studied by absorption spectroscopy, steady-state and single-photon timing luminescence
measurements. Their electronic spectra show visible absorption peaks at 457–463 nm, with red lumines-
cence at 603–613 nm. The emission quantum yields of these complexes are between 0.006 and 0.016 in
Fluorescence Spectroscopy: from Single
Chemosensors to Nanoparticles Science –
Special Issue
Keywords:
Ru(II) complexes
DNA
Phenanthroline
Thiophene
Furan
air-equilibrated DMSO solution. Luminescence lifetimes in water lie within the 0.4–1.0 ls range, with a
non-exponential behavior due to aggregation of the probe. Ru(II) complexes C3, C4, C5 and C6 show
intrinsic dsDNA-binding constants of 2.74 ꢀ 105, 3.02 ꢀ 105, 1.32 ꢀ 105 and 1.63 ꢀ 105 Mꢁ1, respectively.
The planar extended structure of the imidazo-phenanthroline ligands and the collected spectroscopic
data suggest a partial intercalative binding mode of the novel metal probes to double-stranded DNA.
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
the biopolymer tertiary structure, photocleavage agents and, in
recent times, as inhibitors of biological functions [4,1a]. One of
Among the multiple application fields of ruthenium(II) polypyr-
idyl complexes [1], its interaction with nucleic acids has been one
of the most thoroughly studied over more than two decades [2].
Featuring a unique set of chemical properties such as stability,
excited-state reactivity, redox potentials [3], luminescence and
excited state lifetimes, these complexes have attracted consider-
the most extensively studied metal complexes used as luminescent
probe is [Ru(bpy)2(dppz)]2+ (bpy = 2,20-bipyridine, dppz = dipyr-
ido[3,2-a:20,30-c]phenazine) [5]. This complex functions as a molec-
ular ‘‘light switch’’ for DNA because of the dramatic emission
enhancement experienced by this probe and related phenazine
complexes in the presence of double-stranded nucleic acids, being
otherwise weakly emissive in aqueous solution. The reason for this
behavior is the peculiar electronic nature of the phenazine ligand
and the lowest-lying excited state swap that occurs in protic sol-
vents [6], together with the intercalative binding mode to dou-
ble-stranded DNA of Ru(II)-dppz and related complexes [7]. For
all ruthenium(II) polypyridyl complexes, non-radiative vibrational
deactivation with the water molecules can be minimized by a close
interaction with a hydrophobic negatively charged surface [8], and
the intimate contact (e.g. DNA intercalation) with the biopolymer
protects the triplet excited state of the probe from the O2 quench-
ing [9], overall leading to a substantial increase in the 3MLCT
excited state lifetime.
able attention from
a great number of researchers, finding
applications in areas such as photophysics, photochemistry,
supramolecular chemistry, bioinorganic chemistry and catalysis.
Concerning their interaction with biological structures, Ru(II)
polyazaheteroaromatic compounds have been used as probes of
⇑
Corresponding authors at: REQUIMTE, Departamento de Química, FCT-UNL,
2829-516 Caparica, Portugal. Fax: +351 212 948 385 (B. Pedras), fax: +351 253
604382 (M.M.M. Raposo), fax: +34 91 394 4103 (G. Orellana).
E-mail addresses: b.pedras@dq.fct.unl.pt (B. Pedras), mfox@quimica.uminho.pt
(M.M.M. Raposo), orellana@quim.ucm.es (G. Orellana).
0020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2011.07.020

96
B. Pedras et al. / Inorganica Chimica Acta 381 (2012) 95–103
Very recently these complexes have been applied as multifunc-
100 MHz for 13C NMR, using the solvent residual peak as internal
reference. The solvents are indicated in parenthesis before the
chemical shift values (d in ppm relative to TMS). NMR spectra of
the complexes were recorded on a Bruker AVANCE II at 400 MHz
for 1H NMR, and processed with the TOPSPIN 2.0 software (Bruker).
Melting points were determined on a Gallenkamp apparatus and
are uncorrected. Infrared spectra were recorded on a BOMEM MB
104 spectrophotometer. UV–Vis absorption spectra of the ligands
(200–800 nm) were measured with a Shimadzu UV/2501PC appa-
tional biological agents for direct imaging of DNA in living cells
[10]. By varying the ligands that constitute the complexes, it is pos-
sible to modify the nature and strength of their binding to nucleic
acids. As mentioned above, all positively charged complexes are
expected to be attracted to the anionic DNA, and those containing
at least one extended heteroaromatic ligand in the coordination
sphere may insert such ligand between adjacent base-pairs of dou-
ble-stranded DNA (i.e., binding by intercalation) [2a,7,9a,11]. In-
deed, it has been observed that while [Ru(bpy)3]2+ binds in a
relatively weak manner to DNA (mostly through electrostatic
interaction in one of the grooves), the interaction of [Ru(phen)3]2+
is stronger [12].
Therefore, in order to study the factors that influence the DNA-
binding mode of Ru(II) complexes with extended and ancillary li-
gands, the structural diversity of the metal chelating structures
must be taken into account. Moreover, a further tuning of the
probe photophysical and DNA binding properties might be
achieved when new extended conjugation systems are composed
by different heterocyclic nuclei (molecular meccano concept).
Following our ongoing projects on fluorescent reporters and
their sensing applications [13], we have tackled the synthesis
and characterization of novel (oligo)thienyl- [14] and arylthienyl-
imidazo-phenanthrolines [15] due to their interesting emissive
properties (see below). Taking also into account that furans exhibit
high fluorescence quantum yields [16] and the recent studies con-
cerning the DNA binding and photocleavage of Ru(II) complexes
ratus and those of the complexes on
spectrophotometer.
a Varian Cary 3Bio
Mass spectrometry analyses of the ligands were performed at
the C.A.C.T.I. – Unidad de Espectrometria de Masas of the Univer-
sity of Vigo, Spain. MALDI-TOF-MS spectra have been performed
in a MALDI-TOF-MS model Voyager DE-PRO Biospectrometry
Workstation equipped with a nitrogen laser radiating at 337 nm
from Applied Biosystems (Foster City, United States) from the
MALDI-TOF-MS Service of the REQUIMTE, Chemistry Department,
Universidade Nova de Lisboa and in the MALDI-TOF-MS–MS model
4700 Applied Biosystems at the Faculty of Science of Ourense, Uni-
versity of Vigo. The acceleration voltage was 2.0 ꢀ 104 kV with a
delayed extraction (DE) time of 200 ns. The spectra represent accu-
mulations of 5 ꢀ 100 laser shots. The reflection mode was used.
The ion source and flight tube pressures were less than
1.80 ꢀ 10ꢁ7 and 5.60 ꢀ 10ꢁ8 Torr, respectively.
The MALDI mass spectra of the soluble compounds (1 or 2
lg/
lL) were recorded using the conventional sample preparation
of
a
2-(5-methyl-furan-2-yl)imidazo[4,5-f][1,10]phenanthroline
method for MALDI-MS without other MALDI matrix.
ligand [17], suggesting that these complexes bind to DNA through
intercalation and, when irradiated at 400 nm, promote the photoc-
leavage of the DNA, we set out to synthesize Ru(II) furyl-imidazo-
phenanthroline complexes that were hitherto unknown.
Elemental analyses were performed at the Analytical Services of
the Laboratory of REQUIMTE-Departamento de Química, Univer-
sidade Nova de Lisboa, on a Thermo Finnigan-CE Flash-EA 1112-
CHNS Instrument.
In this way, four new ligands and six Ru(II) polypyridyl com-
plexes, C1 to C6, are photophysically characterized in this work,
and the interaction of the latter with calf-thymus DNA has been
followed by steady-state and single-photon timing luminescence
measurements. The effect of three different quenchers on the emis-
sion properties of the DNA-bound complexes has also been stud-
ied, in an attempt to reveal the possible binding modes of the
complexes to the nucleic acid [18].
The steady-state luminescence measurements were carried out
with a Perkin-Elmer LS-5 spectrofluorometer. Luminescence life-
times were determined at 25 1 °C using ca. 10ꢁ5 M solutions of
the Ru(II) complexes by the single-photon timing (SPT) technique
with an Edinburgh Analytical Instruments LP900 kinetic spectrom-
eter. Excitation of the samples was carried out with a Horiba Nan-
oLED-07N 405-nm pulsed laser diode (<700 ps). A wide band-pass
405-nm interference filter (Edmund Scientific) was placed in front
of the laser source and cut-off filters (590 nm, Lambda Research
Optics) were used in the emission path to avoid distortions from
the laser light scattering. The luminescence decay profiles were
fit either to a single exponential function or to a sum of 2–3 expo-
nential functions with the original Marquardt algorithm-based EAI
decay analysis software. Satisfactory fits were obtained in all cases,
as judged from the weighted residuals, the goodness-of-fit v2
parameter and the autocorrelation function. No oxygen outgassing
was performed. The accuracy in the measured lifetimes for the
multi-exponential decay fits is estimated to be 3% (1% for the sin-
gle-exponential decays) and 10% for the relative weights. The pre-
2. Experimental
2.1. Materials
All reagents used in the present work were commercially avail-
able and used without further purification, unless otherwise sta-
ted. Progress of the reactions was monitored by thin layer
chromatography (0.25 mm thick pre-coated Merck Fertigplatten
Kieselgel 60F254 silica plates), while purification was carried out
by silica gel column chromatography (Merck Kieselgel 60; 230–
400 mesh). Calf thymus DNA was obtained from Pharmacia GE
Healthcare and purified by extensive dialysis against Tris buffer.
The concentration of stock solutions was determined spectropho-
tometrically using the molar absorption coefficient per base pair
(12 800 Mꢁ1 cmꢁ1) at 258 nm [9a] and found to be 2.82 mM (for
the stock solution used with C1 to C3) and 2.02 mM (for the stock
solution used with C4 to C6) in base pairs. All the experiments with
DNA were carried out in pH 7.0 3-mM Tris buffer.
exponential weighted emission lifetime (sm) is defined according
to Eq. (1), where si is the emission lifetime for each component
of the multi-exponential fit whose relative weight is (%)i.
X
ð%Þ
sm
¼
i si
ð1Þ
100
i
For the preliminary studies of complexes C1 to C3 in the pres-
ence of DNA, a solution of the metal complex in Tris buffer with
a concentration of ca. 10ꢁ5 M was obtained by dilution of a concen-
trated DMSO stock solution of each complex. Each solution was
mixed with DNA in different proportions, and their spectra
recorded both in the presence and in the absence of the biopoly-
mer. The mixtures were allowed to equilibrate for at least half an
hour between each addition and the respective measurement.
2.2. Instrumentation
NMR spectra of the ligands were obtained on a Varian Unity
Plus spectrometer at 300 MHz for 1H NMR and 75.4 MHz for 13C
NMR or a Bruker Avance III 400 at 400 MHz for 1H NMR and

B. Pedras et al. / Inorganica Chimica Acta 381 (2012) 95–103
97
For the subsequent studies with DNA the same procedure was
adopted, by measuring at different [DNA]/[Ru] ratios and choosing
the ratio after which no significant changes in the emission spec-
trum and lifetime were observed to be studied with the selected
quenchers (ethidium bromide, potassium hexacyanoferrate(II)
and methyl viologen).
(Liquid film,
1017, 896, 738, 704. Mp = 241.0–242.2 °C. UV–Vis (ethanol, nm):
kmax (log ) = 364 (4.20).
m
/cmꢁ1): 3436, 1567, 1498, 1444, 1421, 1265, 1072,
e
2.3.1.4. 2-(50-Phenylfuran-20-yl)-1H-imidazo[4,5-f][1,10]phenanthro-
line (6c). The compound was isolated as a yellow solid (0.061 g,
1
60%). H NMR (400 MHz, DMSO-d6): d 7.23 (d, 1H, J = 3.6 Hz, 30-
2.3. Synthesis
H), 7.34–7.38 (m, 2H, 400-H + 40-H), 7.49 (t, 2H, J = 8.0 and 7.6 Hz,
300-H + 500-H), 7.80–7.83 (m, 2H, 5-H + 10-H), 7.91 (d, 2H,
J = 7.6 Hz, 200-H + 600-H), 8.93 (dd, 2H, J = 8.4 and 1.6 Hz, 4-H + 11-
H), 9.02 (dd, 2H, J = 6.0 and 1.6 Hz, 6-H + 9-H) ppm. 13C NMR
(100.6 MHz, DMSO-d6): d 108.4, 111.9, 121.6, 123.3, 123.9, 128.1,
128.9, 131.3, 131.3, 143.2, 143.6, 145.3, 147.8, 153.9 ppm. MS
(FAB): m/z (%) 363 ([M+H]+, 100). HRMS (FAB): m/z Calc. for
The precursor complex, namely cis-[Ru(bpy)2Cl2], was synthe-
sized according to literature methods [19]. The syntheses of li-
gands 4a and 4b have been previously reported [14a].
2.3.1. General procedure for the synthesis of imidazo[4,5-f][1,10]-
phenanthrolines 5 and 6
C
23H15N4O: 363.12380. Found: 363.12404. IR (Nujol, m
/cmꢁ1):
A mixture of the corresponding aldehyde (1.2 mmol), NH4OAc
(20 mmol) and 1,10-phenanthroline-5,6-dione (1 mmol) in glacial
acetic acid (10 mL) was stirred and heated at reflux for 5 h. The
mixture was then cooled to room temperature and the product
precipitated during neutralization with NH4OH 5 m. The precipi-
tate was filtered out, washed with water and diethyl ether, recrys-
tallized from absolute ethanol and dried under vacuum to give the
expected product.
3371, 2911, 2853, 1619, 1565, 1490, 1397, 1299, 1277, 1190,
1150, 1124, 1073, 1027, 924, 803, 758, 688. Mp = 243.4–244.9.
UV–Vis (ethanol, nm): kmax (log e) = 353 (4.48).
2.3.2. Synthesis of the Ru(II) complexes
In 5 mL of ethylene glycol, the corresponding quantity of imi-
dazo-phenanthroline ligand and the Ru(bpy)2Cl2 complex were
dissolved. The mixture was heated in a Milestone microwave oven
(450 W) for 30 s, which led to a color change from violet to deep
orange, and then allowed to cool for a while. The mixture was
heated for three more periods of 30 s. The solvent was removed
by distillation at low pressure, the residue was dissolved in 2 mL
of water, and a saturated NH4PF6 aqueous solution was added.
The precipitate formed was then filtered through a frit, washed
with water (3ꢀ 10 mL) and diethyl ether (3ꢀ 10 mL), and dried un-
der vacuum.
2.3.1.1.
2-(40-(Thien-200-yl)phen-20-yl)-1H-imidazo[4,5-f][1,10]phe-
nanthroline (5). Yellow solid (0.139 g, 82%). 1H NMR (300 MHz,
DMSO-d6): d 7.18–7.21 (m, 1H, 400-H), 7.62 (d, 1H, J = 5.1 Hz, 300-
H), 7.66 (d, 1H, J = 3.6 Hz, 500-H), 7.81–7.85 (m, 2H, 5-H and 10-
H), 7.90 (d, 2H, J = 8.7 Hz, 20-H and 60-H), 8.31 (d, 2H, J = 8.4 Hz,
30-H and 50-H), 8.92 (dd, 2H, J = 8.1 and 1.5 Hz, 4-H and 11-H),
9.03 (dd, 2H, J = 4.5 and 1.8 Hz, 6-H and 9-H), 13.79 (s, 1H, NH)
ppm. 13C NMR (75.4 MHz, DMSO-d6): d 123.4, 124.5, 125.8,
126.5, 126.9, 128.7, 128.8, 129.8, 134.7, 135.9, 142.6, 146.4,
147.8, 150.1 ppm. MS (FAB): m/z (%) 379 ([M+H]+, 100). HRMS
(FAB): m/z Calc. for C23H15N4S: 379.1017. Found: 379.1015. IR
C1: About 30.2 mg (7.85 ꢀ 10ꢁ5 mol) of 4a and 42.7 mg
(8.2 ꢀ 10ꢁ5 mol) of Ru(bpy)2Cl2 were used. Color: deep orange.
Yield: 73.9 mg (87%). Anal. Calc. for C41H28F12N8P2RuS2: C, 45.30;
H, 2.60; N, 10.30; S, 5.90. Found: C, 45.40; H, 2.80; N, 10.05; S,
5.85%. 1H NMR (CD3CN): d = 8.69 (s, 1H); 8.56 (t, 4H); 8.36
(d, 2H); 8.11 (m, 4H); 7.85 (m, 7H); 7.47 (m, 7H); 7.13 (m, 3H)
ppm. FD-MS: m/z 943.06 ([MꢁPF6]+), 797.08 ([Mꢁ2PF6ꢁH]+),
399.04 ([Mꢁ2PF6]2+). ESI-MS: m/z 943.2 ([MꢁPF6]+), 797.2
([Mꢁ2PF6ꢁH]+), 399.1 ([Mꢁ2PF6]2+). MALDI-TOF MS: m/z 797.6
([Mꢁ2PF6ꢁH]+), 641.6 ([Mꢁ2PF6-bpyꢁH]+).
(KBr, m
/cmꢁ1): 3439, 2950, 1605, 1562, 1531, 1479, 1451, 1429,
1395, 1351, 1312, 1296, 1259, 1212, 1190, 1119, 1069, 1029,
957, 844, 803, 740. Decomposition at T > 320 °C. UV (ethanol,
nm): kmax (log e) = 345 (4.16).
2.3.1.2. 2-(Furan-20-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (6a).
The compound was isolated as an orange solid (0.114 g, 85%). 1H
NMR (300 MHz, DMSO-d6): d 6.76–6.79 (m, 1H, 40-H), 7.25 (d,
1H, J = 3.0 Hz, 30-H), 7.78–7.82 (m, 2H, 5-H + 10-H), 7.99 (d, 1H,
J = 1.2 Hz, 50-H), 8.90 (d, 2H, J = 7.8 Hz, 4-H + 11-H), 9.02 (d, 2H,
J = 1.8 Hz, 6-H + 9-H), 13.93 (br s, 1H, NH) ppm. 13C NMR
(75.4 MHz, DMSO-d6): d 109.9, 112.5, 123.3, 129.6, 143.0, 143.6,
144.4, 145.4, 147.9 ppm. MS (FAB): m/z (%) 287 ([M+H]+, 100),
226 (5). HRMS (FAB): m/z Calc. for C17H11N4O: 287.09389. Found:
C2: About 24.3 mg (5.85 ꢀ 10ꢁ5 mol) of 4b and 32.0 mg
(6.15 ꢀ 10ꢁ5 mol) of Ru(bpy)2Cl2 were used. A few drops of ethanol
were added in order to increase solubilization of the ligand. Color:
deep orange. Yield: 29.3 mg (45%). Anal. Calc. for C42H30F12N8O-
P2RuS2ꢂ1.5C2H5OH: C, 45.55; H, 3.30; N, 9.45; S, 5.40. Found: C,
45.88; H, 3.40; N, 9.70; S, 5.85%. 1H NMR (CD3CN): d = 8.80 (d,
2H); 8.53 (m, 4H); 8.10 (t, 2H); 7.99 (t, 2H); 7.87 (m, 4H); 7.66
(d, 3H); 7.56 (m, 2H); 7.44 (t, 2H); 7.25 (t, 2H); 7.02 (d, 1H); 6.91
(d, 1H); 6.24 (d, 1H); 3.92 (s, 3H) ppm. FD-MS: m/z 973.03
([MꢁPF6]+), 827.07 ([Mꢁ2PF6ꢁH]+). ESI-MS: m/z 973.2 ([MꢁPF6]+),
827.2 ([Mꢁ2PF6ꢁH]+), 414.1 ([MꢁRu(bpy)2(PF6)2]ꢀ. MALDI-TOF
MS: m/z 827.6 ([Mꢁ2PF6ꢁH]+).
287.09274. IR (Nujol,
m
/cmꢁ1): 3396, 2923, 2853, 1644, 1565,
1538, 1507, 1463, 1397, 1377, 1350, 1228, 1191, 1116, 1073,
1017, 976, 897, 885, 807, 739. Mp = 122.3–124.5 °C. UV–Vis (etha-
nol, nm): kmax (log e) = 318 (4.14).
C3: About 30.5 mg (8.06 ꢀ 10ꢁ5 mol) of
5 and 44.1 mg
2.3.1.3. 2-(50-(Thien-200-yl)furan-20-yl)-1H-imidazo[4,5-f][1,10]phe-
nanthroline (6b). The compound was isolated as a dark yellow solid
(0.110 g, 79%). 1H NMR (400 MHz, DMSO-d6): d 7.03 (d, 1H,
J = 3.6 Hz, 30-H), 7.20–7.22 (m, 1H, 400-H), 7.33 (d, 1H, J = 4.0 Hz,
40-H), 7.60 (dd, 1H, J = 3.6 and 1.2 Hz, 300-H), 7.66 (dd, 1H, J = 5.2
and 0.8 Hz, 500-H), 7.79–7.86 (m, 2H, 5-H + 10-H), 8.89–8.94 (m,
2H, 4-H + 11-H), 9.03 (dd, 2H, J = 4.0 and 2.0 Hz, 6-H + 9-H),
13.89 (s, 1H, NH) ppm. 13C NMR (100.6 MHz, DMSO-d6): d 108.0,
112.3, 119.2, 123.1, 123.4, 123.5, 124.4, 125.9, 126.4, 128.3,
129.7, 131.9, 135.8, 142.5, 143.6, 143.7, 144.3, 147.9, 147.9,
149.8 ppm. MS (FAB): m/z (%) 369 ([M+H]+, 100), 226 (4). HRMS
(FAB): m/z Calc. for C21H12N4OS: 369.08048. Found 369.08046. IR
(8.47 ꢀ 10ꢁ5 mol) of Ru(bpy)2Cl2 were used. Color: deep orange.
Yield: 26.3 mg (30%). Anal. Calc. for C43H30F12N8P2RuSꢂ6H2O: C,
43.40; H, 3.55; N, 9.40; S, 2.70. Found: C, 43.35; H, 3.25; N, 9.40;
S, 2.55%. 1H NMR ((CD3)2CO): d = 9.21 (d, 2H); 8.89 (dd, 4H); 8.50
(d, 2H); 8.31 (m, 4H); 8.20 (m, 4H); 7.99 (d, 2H); 7.91 (dd, 2H);
7.83 (d, 2H); 7.66 (m, 3H); 7.55 (d, 1H); 7.42 (t, 2H); 7.20 (m,
1H) ppm. FD-MS: m/z 790.82 ([Mꢁ2PF6ꢁH]+). ESI-MS: m/z 937.3
([MꢁPF6]+), 791.3 ([Mꢁ2PF6ꢁH]+), 396.1 ([MꢁRu(bpy)2(PF6)2]ꢀ).
MALDI-TOF-MS: m/z 791.28 ([Mꢁ2PF6ꢁH]+).
C4: About 27.2 mg (7.99 ꢀ 10ꢁ5 mol) of 6a and 44 mg
(8.39 ꢀ 10ꢁ5 mol) of Ru(bpy)2Cl2 were used. An orange solid was
obtained. Yield: 72.2 mg (91%). Anal. Calc. for C37H26F12N8OP2Ru:

98
B. Pedras et al. / Inorganica Chimica Acta 381 (2012) 95–103
C, 44.90; H, 2.65; N, 11.30. Found: C, 44.35; H, 2.85; N, 11.30%. 1H
NMR ((CD3)2CO): d = 9.07 (d, 2H); 8.86 (dd, 4H); 8.35 (m, 2H); 8.25
(t, 3H); 8.16 (m, 4H); 7.91 (m, 4H); 7.64 (m, 2H); 7.40 (m, 2H); 7.33
(s, 1H); 6.76 (s, 1H) ppm. MALDI-TOF-MS: m/z 699.73
([Mꢁ2PF6ꢁH]+), 543.71 ([Mꢁ2PF6-bpyꢁH]+).
3.2. Spectroscopic characterization
The absorption spectra of the Ru(II) complexes consist mainly of
three resolved bands in the 200–600 nm region. The bands around
280 nm are attributed to ligand-centered (LC)
bands around 350 nm can either be due to the n–p⁄ transitions or
inter-ligand
p⁄ transitions, and the lowest energy bands at
p–
p⁄ transitions; the
C5: About 25 mg (6.18 ꢀ 10ꢁ5 mol) of 6b and 33.8 mg
(6.49 ꢀ 10ꢁ5 mol) of Ru(bpy)2Cl2 were used. An orange solid was
obtained. Yield: 60.0 mg (91%). Anal. Calc. for C41H28F12N8OP2RuS:
C, 45.95; H, 2.65; N, 10.45; S, 3.00. Found: C, 45.90; H, 2.65; N,
10.60; S, 2.60%. 1H NMR ((CD3)2CO): d = 8.82 (m, 5H); 8.40 (m,
1H); 8.23–8.16 (m, 8H); 7.96 (m, 2H); 7.64–7.40 (m, 8H); 7.15 (s,
1H); 6.95 (s, 1H) ppm. MALDI-TOF-MS: m/z 781.79 ([Mꢁ2PF6ꢁH]+),
625.73 ([Mꢁ2PF6-bpyꢁH]+), 368.73 ([MꢁRu(bpy)2(PF6)2]ꢀ.
C6: About 23.5 mg (5.64Eꢁ05 mol) of 6c and 30.8 mg
(5.93Eꢁ05 mol) of Ru(bpy)2Cl2 were used. An orange solid was ob-
tained. Yield: 57.8 mg (96%). Anal. Calc. for C43H30F12N8OP2Ru: C,
48.45; H, 2.85; N, 10.50. Found: C, 48.35; H, 3.00; N, 9.90%. 1H
NMR ((CD3)2CO): d = 8.83 (m, 5H); 8.40 (m, 1H); 8.25–8.16 (m,
8H); 8.02 (m, 3H); 7.79 (s, 3H); 7.72 (s, 2H); 7.45–7.38 (m, 5H);
7.12 (s, 1H) ppm. MALDI-TOF-MS: m/z 775.65 ([Mꢁ2PF6ꢁH]+),
619.62 ([Mꢁ2PF6-bpyꢁH]+), 463.57 ([Mꢁ(bpy)2-2PF6ꢁH]+),
362.67 ([MꢁRu(bpy)2(PF6)2]ꢀ).
p–
around 460 nm are assigned to the metal-to-ligand charge transfer
(MLCT) transitions. The emission spectra are all centered above
600 nm according to what is expected for Ru(II) polypyridyl com-
plexes (see Figs. S1 and S2, Supplementary material). Table 2 sum-
marizes all the spectroscopic data.
3.3. Interaction of Ru(II) complexes with DNA
Electronic absorption spectroscopy is a very useful technique in
DNA-binding studies, since binding to double stranded (ds) DNA
through intercalation normally results in hypochromism and
bathochromism of the MLCT visible absorption band of the com-
plex [22]. The extent of the hypochromism usually parallels the
intercalative binding strength [22]. On the other hand, lumines-
cence studies usually show the enhancement of both the lumines-
cence intensity and lifetime of the Ru(II) complexes upon binding
to the DNA. This indicates that the complexes can interact with
DNA and are protected by this polynucleotide to some extent from
quenching by molecular oxygen and the solvent molecules. Emis-
sion quenching experiments may provide further information,
since the Stern–Volmer quenching constants can be used as a mea-
sure of the binding affinity.
3. Results and discussion
3.1. Synthesis
In order to compare the effect of the electronic nature of aryl
and heteroaryl moieties on the optical properties of linear
imidazo-phenanthrolines 4–6, formyl-derivatives containing bit-
hienyl 1a–b, arylthienyl 2, furyl 3a, thienylfuryl 3b and arylfuryl
In this way, preliminary studies were performed with com-
plexes C1, C2 and C3 to compare the interaction of these complexes
with DNA in a qualitative way. All the preliminary results obtained
with C1 to C3 in the presence of DNA are reported in the Supple-
mentary material.
3c
p-conjugated bridges were used as precursors of phenanthro-
lines 4–6. Compounds 1a, 2, 3a and 3c were commercially avail-
able. The synthesis of 50-formyl-2-methoxy-2,20-bithiophene 1b
has been reported [20].
The interaction of the stronger emissive C3 to C6 complexes
was studied quantitatively; Fig. 1 shows in more detail the spectro-
scopic study of complex C3 bearing a thienyl-aryl substituent in
the presence of increasing amounts of DNA. Fig. 1A indicates that
there is a hypochromicity in the MLCT band upon addition of
DNA, suggesting the intercalation of C3 into the double DNA helix.
At the same time the emission intensity (Fig. 1B) and lifetime
(Table T1 in Supplementary material) of the bound luminophore
are enhanced due to a combination of a more hydrophobic micro-
environment around the metal complex after binding (the O–H
oscillators help to deactivate the emissive 3MLCT state) [8] and
the protection from quenching by dissolved molecular oxygen
imparted by the polynucleotide strand. Inset in Fig. 1B shows the
increases observed both in the steady state spectra and in the lumi-
nescence lifetime in the presence of DNA.
Therefore, heterocyclic ligands 4–6 with either bithienyl, arylt-
hienyl, furyl, thienylfuryl and arylfuryl moieties (unsubstituted or
bearing a methoxy donor group) linked to the chelating imidazo-
phenanthroline system, were synthesized in good to excellent
yields (60–85%, Table 1) through the Radziszewski reaction [21],
using 5,6-phenanthroline-dione, formyl precursors 1–2 and
ammonium acetate in refluxing glacial acetic acid for 15 h
(Scheme 1).
In the 1H NMR spectra of most imidazo-phenanthroline deriva-
tives, a peak at about 13.8–13.9 ppm was detected as a broad sin-
glet that was attributed to the N–H in the imidazole moiety. The
NH was also identified by IR spectroscopy as a sharp band within
the spectral region of 3371–3439 cmꢁ1
.
The complexes were obtained by direct reaction of the ligands
with Ru(bpy)2Cl2 in ethylene glycol, under microwave irradiation
for 2 min (Scheme 2). Their purity was confirmed by elemental
analysis, 1H NMR and MALDI-TOF-MS (see Section 2).
A similar behavior was found for the other imidazo-phenan-
throline complexes, as can be seen for complex C5 in Fig. 2 and
Table T2 in Supplementary material. All the results for the
Table 1
Yields, IR absorption spectra and 1H NMR spectra of phenanthrolines 5–6.
a
Formyl derivative
Phenanthroline product
R
Yield (%)
IR
m
(cmꢁ1
)
dH (imidazole) (ppm)d
2
5
–
H
82
85
79
60
3439b
3396
3436c
3371
13.8
13.9
13.9
3a
3b
3c
6a
6b
6c
Thienyl
Phenyl
e
–
a
b
c
For the NH stretching band (recorded in Nujol).
For the NH stretching band (recorded in KBr).
For the NH stretching band (recorded in liquid film).
For the NH proton of the imidazole ring for compounds 5–6 (DMSO-d6).
Not observed.
d
e

B. Pedras et al. / Inorganica Chimica Acta 381 (2012) 95–103
99
N
HN
S
N
R
S
N
S
CHO
R
4a R = H
S
4b
R = MeO
1a R = H
1b R = MeO
O
O
N
+
NH4OAc /AcOH/ reflux/
H
N
N
N
N
S
N
CHO
S
5
2
N
HN
O
N
R
O
R
CHO
N
6a R = H
3a
R = H
6b
6c
R = thienyl
R = phenyl
3b R = thienyl
3c R = phenyl
Scheme 1. Synthesis of imidazo-furyl and imidazo-thienyl-phenanthroline derivatives.
would indicate a more intimate binding of the imidazo-phenan-
throline complex to the polynucleotide double strand than that
of the simple tris-phenanthroline complex although not as efficient
as those bearing a fused polycyclic (hetero)aromatic system that
can be inserted between adjacent base pairs of the DNA ladder.
It is interesting to note that the MLCT absorption band of com-
plexes C4 to C6 centered at ca. 455 nm undergoes a more pro-
nounced hypochromic effect upon binding to ds-DNA than the
MLCT band at ca. 430 nm. This observation would indicate that
the former corresponds to the electronic transition involving the
chelating ligand that interacts more closely with the polynucleo-
tide base-pair strand, presumably the intercalated 6a–c imidazo-
phenanthrolines (the small bpy ligands are unable to intercalate
into the double helix [26]). Surprisingly enough, the same differ-
ence is not observed for the Ru(II) complex C3 that contains the
imidazo-phenanthroline 5. In the absence of molecular modeling
studies, we can hypothesize that the crescent-shaped ligands 6a–
c intercalate more efficiently than the long, linear, twisted phe-
nyl-substituted imidazo-phenanthroline 5.
Except for C3, the emission profile of the luminophoric com-
plexes in buffer solution can only be fit to a double exponential
function (Tables T1–T4, Supplementary material). This fact may
be attributed to aggregation of the hydrophobic dyes; the observa-
tion of just two components is a consequence of the strong fitting
power of the bi-exponential function which does not require intro-
duction of additional components. However, a variety of self-
aggregates displaying different stoichiometries is to be expected,
the emission lifetime of which cannot be fully resolved. In the pres-
ence of just a fivefold (molar) concentration of DNA the lumines-
cence decay slows down for the same reasons outlined above for
the emission intensity and, although the individual lifetimes of
the observed components remain almost the same for higher
DNA concentrations, sm increases as a consequence of the rise of
the contribution of the long lifetime component to the overall de-
cay (except for C3 where the individual components remain un-
changed). These results indicate that the Ru(II) complexes in
buffer solution are already fully bound to the DNA under the used
experimental conditions even at a [DNA]bp/Ru ratio as low as 5.
Table 2
MLCT (d–p⁄) absorption and emission maxima, molar absorption coefficients (in Tris
buffer) and fluorescence quantum yields (in air-equilibrated DMSO) of complexes C1
to C6.
emax (Mꢁ1 cmꢁ1
)
UF
em
max
abs
max
Complex
k
(nm)
k
(nm)
C1
C2
C3
C4
C5
C6
463
459
460
459
458
457
12 700
17 800
11 900
15 900
14 300
12 000
608
607
603
613
613
611
0.006
0.015
0.016
0.009
0.008
0.008
remaining complexes are reported in Figs. S3–S6, and Tables T3
and T4, Supplementary material.
The intrinsic DNA binding constants (Kb), which provide a mea-
sure of the interaction strength, were obtained by monitoring the
changes in absorbance at 460 nm with increasing concentrations
of DNA. The experimental data were fit to the simple Scatchard
Eq. (2) [23], that is only valid for low binder-to-DNA ratios
(i.e., far from the DNA saturation) and assumes no binding
cooperativity:
½DNAꢃ=ðea
ꢁ
ef Þ ¼ ½DNAꢃ=ðeb
ꢁ
ef Þ þ 1=½Kbðeb
ꢁ
ef Þꢃ
ð2Þ
where [DNA] is the concentration of the nucleic acid in base pairs, ea
is the apparent absorption coefficient obtained by calculating Aobs
[Ru], and ef and eb are the absorption coefficients for the free and
the fully bound ruthenium complex, respectively.
/
In the [DNA]/(ea
ꢁ
ef) versus [DNA] plot, Kb is given by the ratio
of the slope to the intercept. The binding constants obtained there-
of for complexes C3, C4, C5 and C6 were, respectively, 2.7 ꢀ 105,
3.0 ꢀ 105, 1.3 ꢀ 105 and 1.6 ꢀ 105 Mꢁ1 (see Figs. S7–S10, Supple-
mentary material). These values are larger than those of the DNA
(minor) groove binding Ru(II) complexes such as tris(1,10-phenan-
throline)ruthenium(II) and related structures (1.1 ꢀ 104–4.8 ꢀ
104 Mꢁ1
) [23–25], but smaller than those observed for the
[Ru(bpy)2(dppz)]2+ DNA intercalator and other complexes contain-
ing an extended phenazine ligand (107–109 Mꢁ1) [4c]. This result

100
B. Pedras et al. / Inorganica Chimica Acta 381 (2012) 95–103
2+
N
H
N
N
N
N
N
H
N
Ru
N
N
Ru(bpy)2Cl2
S
N
S
N
S
N
ethylene glycol /M.W.
S
R
-
R
(PF6 )2
C1 R = H
C2 R = MeO
4a-b
2+
N
H
H
N
Ru(bpy)2Cl2
N
N
N
N
N
N
N
Ru
S
ethylene glycol /M.W.
S
N
N
N
-
5
(PF6 )2
C3
2+
N
H
N
N
N
H
N
N
N
Ru(bpy)2Cl2
N
N
Ru
O
N
R
ethylene glycol /M.W.
O
N
R
N
-
(PF6 )2
6a-c
C4 R = H
C5 R = thienyl
C6 R = phenyl
Scheme 2. Synthesis of ruthenium(II) complexes.
Further addition of the polynucleotide would just make the lumi-
nescent probes shift their binding mode, e.g. from aggregated on
the double helix to individually bound, as they become ‘‘diluted’’
with larger amounts of DNA. The longest emission lifetime would
correspond to the isolated Ru(II) complexes while the shortest
one is the signature of the aggregates. The lack of change in the rel-
ative contribution of the short and long lifetimes in the case of the
DNA-bound complex C3, and the preeminence of the fast compo-
nent of the decay even at large [DNA]bp/Ru ratios might be a con-
sequence of its different interaction mode with the polynucleotide
(see above) and a more difficult self-aggregation (unlike the other
imidazo-phenanthroline complexes, a single-exponential decay is
observed for the photoexcited C3 in the absence of DNA).
photoinduced electron transfer with rate constants in excess of
109 L molꢁ1 sꢁ1. Starting with C3, it was found out that the addition
of increasing amounts of ethidium bromide to a mixture of
C3 + DNA (ratio 1:30) promoted an enhancement of the absorption
values in the region corresponding to the MLCT absorption band
(Fig. S11, Supplementary material). However, since the intrinsic
absorption of ethidium takes place in the same wavelength range,
this absorption increase had to be discarded. A similar effect
occurred with the emission spectra (Fig. S11, Supplementary
material) and luminescence lifetime measurements (Table T5,
Supplementary material), since both compounds emit in the same
region, and the lifetime values for ethidium are significantly short-
er, thus masking the true sm values.
In the case of potassium hexacyanoferrate(II) quencher, no
changes in the luminescence lifetime were observed for the photo-
excited C3 bound to DNA (see Fig. S12 and Table T6, Supplemen-
tary material). These results suggest that the complex is fully
bound to DNA as the positively charged complex should be easily
quenched by this highly anionic quencher if both species were free
in solution [27], while the negative DNA phosphate backbone
hinders quenching of the bound complex emission. However, total
luminescence quenching is observed for the DNA-bound C3
complex, both in steady-state and time-resolved emission, in the
presence of methyl viologen (MV2+) (Fig. 3 and Table 3).
3.4. Emission quenching studies
After having studied the interaction of all the complexes with
DNA, it was observed that in all cases no significant changes oc-
curred, neither in the emission spectra nor in the luminescence
lifetimes, for DNA/Ru ratios in excess of 30. Therefore, this ratio
was chosen for subsequent studies with selected excited state
quenchers (ethidium bromide, potassium hexacyanoferrate(II)
and methyl viologen) [27]. It is well established that those quench-
ers deactivate the excited state of most Ru(II) polypyridyls by

B. Pedras et al. / Inorganica Chimica Acta 381 (2012) 95–103
101
0.2
0.1
0.075
0.05
0.025
0
A
A
0.15
A
0.1
0.05
0
A
400
450
500
550
320 360 400 440 480 520 560 600
Wavelength / nm
Wavelength / nm
4
3.5
3
1
0.8
0.6
0.4
0.2
0
2.4
2.2
2
B
1
0.8
0.6
0.4
0.2
0
B
2.5
2
1.8
1.6
1.4
1.2
1
1.5
1
0.5
0
20
40
60
80
100
0.8
DNA/Ru ratio
0
20
40
60
80
100
DNA/Ru ratio
500
550
600
650
700
750
800
500
550
600
650
700
750
800
Wavelength / nm
Wavelength / nm
Fig. 2. (A) Absorption spectra of C5, 10.5
increasing amounts of CT-DNA (DNA/Ru ratio of 0–80, in base pairs). (B) Emission
spectra of C5, 10.5 M in Tris buffer, in the presence of increasing amounts of CT-
DNA (DNA/Ru ratio of 0–80, in base pairs). kexc = 460 nm. Inset: changes in the
emission intensity, I/I0 (d), and emission lifetime, s/s0 (s), of C5 as a function of
DNA/Ru ratio.
lM in Tris buffer, in the presence of
Fig. 1. (A) Absorption spectra of C3, 4.9
the presence of CT-DNA in ratios of 5 (- - -) and 10 to 30 (—). (B) Emission spectra of
C3, 4.9 M in Tris buffer, in the presence of increasing amounts of CT-DNA (DNA/Ru
ratio of 0–80, in base pairs). kexc = 465 nm. Inset: changes in the emission intensity,
I/I0 (d), and emission lifetime, s0 (s), of C3 as a function of DNA/Ru ratio.
lM in Tris buffer, in the absence (ꢂꢂꢂ), and in
l
l
s/
Emission quenching experiments for complexes C4, C5 and C6
have also been performed using methyl viologen as a quencher,
and the results are comparable to those obtained for C3. Steady-
state emission spectra and luminescence lifetime decays for C5
are represented in Fig. 4 and Table 4, respectively (see absorption
spectra in Fig. S13, Supplementary material). All the results for
the remaining complexes are reported in Figs. S14 and S15 and
Tables T7 and T8, Supplementary material. The slight enhancement
of the lifetime values for the last methyl viologen additions
(Tables T7 and T8) suggest a partial displacement of the DNA
bound Ru(II) complexes to the bulk solution under these
conditions.
From the complexes C3 to C6 emission quenching studies with
methyl viologen the Stern–Volmer constants, KSV, and the corre-
sponding quenching rate constants, kq, were determined. When-
ever non-linear Stern–Volmer plots were obtained, the rate
constant data refer to the initial slope. The kq values obtained from
each lifetime component (fast and slow, respectively) of each com-
plex, were: 1.7 ꢀ 1011 and 1.0 ꢀ 1011 Mꢁ1 sꢁ1 (C3); 9.4 ꢀ 109 and
1.9 ꢀ 1010 Mꢁ1 sꢁ1 (C4); 3.9 ꢀ 109 and 7.6 ꢀ 109 Mꢁ1 sꢁ1 (C5);
2.0 ꢀ 1010 and 2.1 ꢀ 1010 Mꢁ1 sꢁ1 (C6). These values exceed by
one or two orders of magnitude those commonly obtained for
Ru(II) complexes with MV2+ (ca. 109 Mꢁ1 sꢁ1) [27]. Such accelera-
tion of the photoinduced electron transfer might be due to several
1
0.8
0.6
0.4
0.2
0
500
550
600
650
700
750
800
Wavelength / nm
Fig. 3. Emission spectra of CT-DNA + C3 (ratio 30/1) in the presence of increasing
amounts of methyl viologen (0–200 M). [C3] = 4.90 M. [DNA]stock = 2.82 mM.
[MV2+
stock = 5 mM. kexc = 465 nm.
l
l
]

102
B. Pedras et al. / Inorganica Chimica Acta 381 (2012) 95–103
Table 3
40
Luminescence lifetimes of 1:30 C3:DNA at different MV2+ concentrations.
[C3] = 4.90
l
M. kexc = 405 nm. kem = 610 nm. [DNA]stock = 2.82 mM. [MV2+
]
=
stock
35
30
25
20
15
10
5
5 mM.
[MV2+] (
l
M) s1 (ls) (%) s2 (ls) (%) s3 (ls) (%) sm (l
s) v2
0
5
15
25
50
–
0.74
0.43
0.22
0.21
0.13
0.10
0.09
0.08
0.07
0.07
0.07
67
60
50
37
41
35
30
32
29
25
22
1.34
0.84
0.54
0.55
0.40
0.38
0.44
0.40
0.40
0.41
0.47
33
19
13
10
5
0.94
0.45
0.21
0.17
0.10
0.07
0.05
0.05
0.04
0.04
0.04
1.10
1.04
1.03
1.10
1.04
1.05
0.91
1.04
0.95
0.98
0.98
0.14
0.08
0.08
0.04
0.03
0.03
0.02
0.02
0.02
0.02
21
37
56
54
62
69
66
70
73
77
Io/I
to/t fast
to/t slow
75
3
1
2
1
2
1
100
125
150
175
200
0
0
50
100
150
200
250
[MV2+] (μM)
Fig. 5. Stern–Volmer emission intensity (d) and lifetime quenching plot for
C3 + DNA (ratio 1:30) in the presence of increasing amounts of methyl viologen
(MV2+). Both fast (short) (N) and slow (long) (s) components of the emission decay
are represented. kexc = 405 nm. Emission collected at 610 nm.
1
0.8
0.6
0.4
0.2
0
4. Conclusions
Four new ligands and six Ru(II) polypyridyl complexes have
been synthesized, the latter through microwave-assisted reactions,
which considerably reduces the reaction times from hours to min-
utes, with satisfactory yields. Studies of their interaction with calf
thymus DNA have suggested a partial intercalation of the probes
into the nucleic acid double strand. According to the structural fea-
tures of the complexes and the obtained values for the binding
constants, it is possible that the crescent-shaped complexes C4,
C5 and C6 have a more intimate binding to the polynucleotide dou-
ble strand than the phenyl-substituted C3. Luminescence quench-
ing studies further support this hypothesis, since in the case of
potassium hexacyanoferrate(II) no quenching was observed
(which indicates that all the complex was bound to the DNA)
and with methyl viologen the quenching was accompanied by par-
tial displacement of the complexes to the bulk solution at high
quencher concentrations. All these results illustrate the feasibility
of fine tuning the DNA interaction mode of luminescent Ru(II)
complexes containing extended imidazo-phenanthroline ligands
by molecular engineering of their aryl substituents. Such feature
might be used in the future to design tailored molecular probes
of the polynucleotide features.
500
550
600
650
700
750
800
Wavelength / nm
Fig. 4. Emission spectra of CT-DNA + C5 (ratio 30/1) in the presence of increasing
amounts of methyl viologen (0–200 lM). kexc = 465 nm. [C5] = 10.5 lM. [DNA]s-
tock = 2.02 mM. [MV2+
]stock = 5 mM.
Table 4
Luminescence lifetimes of 1:30 C5:DNA at different MV2+ concentrations.
[C5] = 10.5
5 mM.
l
M. kexc = 405 nm. kem = 613 nm. [DNA]stock = 2.02 mM. [MV2+
]
=
stock
[MV2+] (
l
M) s1 (ls) (%) s2 (ls) (%) s3 (ls) (%) sm (l
s) v2
0
25
50
–
0.50
0.49
0.31
0.36
0.21
0.31
0.28
47
61
55
40
43
34
27
1.51
1.09
0.67
0.85
0.55
0.65
0.57
53
7
13
3
17
9
18
1.04
0.43
0.30
0.24
0.21
0.20
0.21
1.03
1.12
1.19
1.11
1.12
1.09
1.06
0.18
0.13
0.12
0.07
0.07
0.05
32
32
57
40
57
55
75
Acknowledgments
100
150
200
We are indebted to InOU Uvigo by project K914 122P 64702
(Spain), Xunta de Galicia (Spain) by project 09CSA043383PR and
FCT-Portugal by project PTDC/QUI/66250/2006 (FCOMP-01-0124-
FEDER-007428), for financial support. The NMR spectrometers
are part of the National NMR Network and were purchased in
the framework of the National Programme for Scientific Re-equip-
ment, contract REDE/1517/RMN/2005, with funds from POCI 2010
(FEDER) and FCT-Portugal.
J.L.C. and C.L. thank Xunta de Galicia, Spain, for the Isidro Parga
Pondal Research Program. B.P. and R.B. thank FC-MCTES (Portugal)
for their PhD Grants SFRH/BD/27786/2006 and SFRH/BD/36396/
2007, respectively. An UCM-Santander grant (GR 35/10-A) is also
acknowledged.
factors, one of them being the increase of the local concentration
(closer average distance) of donor and acceptor species on the
nucleic acid (since both the ruthenium(II) complex and the
quencher are bound to the DNA). Considering this effect, the calcu-
lated values for the quenching rate constants represent only appar-
ent values, since the quencher concentration values on the abscissa
correspond to different local quencher concentrations on the
polynucleotide.
The results for C3 + DNA in the presence of methyl viologen are
represented in Fig. 5. All the other results, including the data for C3
with ethidium bromide, are represented in Figs. S16–S19, Supple-
mentary material).
We are grateful to Dr. A. Jorge Parola from the REQUIMTE, Uni-
versidade NOVA de Lisboa, Portugal for his help with the synthesis
of the Ru(bpy)2Cl2 precursor.

B. Pedras et al. / Inorganica Chimica Acta 381 (2012) 95–103
103
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Appendix A. Supplementary material
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Absorption spectra of C1 to C6 complexes; luminescence
lifetimes for C3 to C6 at different ratios Ru:DNA; Absorption and
emission spectra of C4 and C6 with CT-DNA; Ethidium bromide,
methyl viologen and potassium ferrocyanide experiments and
Stern–Volmer experiments with DNA. Supplementary data associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.ica.2011.07.020.
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