The interesting DNA-binding properties of three novel dinuclear Ru(II) complexes with varied lengths of flexible bridges

Article abstract of DOI:10.1016/j.jinorgbio.2010.12.004

Three binuclear Ru(II) complexes with two [Ru(bpy)₂(pip)] ²⁺-based subunits {where bpy = 2,2′-bipyridine and pip = 2-phenylimidazo[4,5-f][1,10]phenanthroline} being linked by varied lengths of flexible bridges, were synthesized and characterized by ¹H NMR, elemental analysis, UV-visible (UV-vis) and photoluminescence spectroscopy. The structures of the three complexes were optimized by density functional theory calculations. The interaction of the complexes with calf thymus DNA was investigated by UV-vis and luminescence titrations, steady-state emission quenching by [Fe(CN)₆]^4-, DNA competitive binding with ethidium bromide, DNA melting experiments, and viscosity measurements. The experimental results indicated that the three complexes bound to the DNA most probably in a threading intercalation binding mode with high DNA binding constant values three orders of magnitude greater than the DNA binding constant value reported for proven DNA intercalator, mononuclear counterpart [Ru(bpy)₂(p-mopip)]²⁺ {p-mopip = 2-(4-methoxylphenyl) imidazo[4,5-f][1,10]phenanthroline}.

Full text of DOI:10.1016/j.jinorgbio.2010.12.004

Journal of Inorganic Biochemistry 105 (2011) 435–443
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
The interesting DNA-binding properties of three novel dinuclear Ru(II) complexes
with varied lengths of flexible bridges
Chuan-Chuan Ju, An-Guo Zhang, Chui-Li Yuan, Xiao-Long Zhao, Ke-Zhi Wang ⁎
College of Chemistry, Beijing Normal University, Beijing 100875, China
a r t i c l e i n f o
a b s t r a c t
Three binuclear Ru(II) complexes with two [Ru(bpy) (pip)]2+-based subunits {where bpy=2,2′-bipyridine and
Article history:
Received 10 January 2010
Received in revised form 11 December 2010
Accepted 14 December 2010
Available online 29 December 2010
2
pip=2-phenylimidazo[4,5-f][1,10]phenanthroline} being linked by varied lengths of flexible bridges, were
synthesized and characterized by 1H NMR, elemental analysis, UV–visible (UV–vis) and photoluminescence
spectroscopy. The structures of the three complexes were optimized by density functional theory calculations.
The interaction of the complexes with calf thymus DNA was investigated by UV–vis and luminescence titrations,
4−
steady-state emission quenching by [Fe(CN)
6
]
, DNA competitive binding with ethidium bromide, DNA melting
Keywords:
Ruthenium
Bipyridine
Phenanthroline
DNA
experiments, and viscosity measurements. The experimental results indicated that the three complexes bound to
the DNA most probably in a threading intercalation binding mode with high DNA binding constant values three
orders of magnitude greater than the DNA binding constant value reported for proven DNA intercalator,
2+
mononuclear counterpart [Ru(bpy) (p-mopip)]
2
{p-mopip=2-(4-methoxylphenyl)imidazo[4,5-f][1,10]
phenanthroline}.
©
2010 Elsevier Inc. All rights reserved.
1
. Introduction
However, a limited number of dinuclear Ru(II) complexes have been
reported for the DNA-binding studies [17,18,21–41].
Ruthenium(II) polypyridyl complexes have been investigated
Dinuclear Ru(II) complexes carrying a bridging ligand can be
classified into rigid and flexible ones according to the linking bridge
type. The binding to double-strand DNA by the two metal centers of
dinuclear complexes with rigid bridges are usually limited by their
rigidity. Because the two metal centers are rigidly linked and
consequently can not follow the curvature of the DNA, only one metal
center may bind deeply to the DNA with the second metal center
projecting out of the DNA [21–34]. The other kind of dinuclear Ru(II)
complexes carryinga flexible chain should overcomethelimitations and
potentially allow both the metal centers to bind optimally to the DNA
[35–41]. Since the chain length of the linker for dinuclear Ru(II)
complexes has been reported to be a crucial factor in determining the
binding efficiency [17], we have synthesized three novel dinuclear Ru
substantially because of their extensive applications in the field of
photochemistry, photophysics, and bioinorganic chemistry in the past
decades [1,2]. Due to their excellent chemical stability, facile electron
transfer, strong luminescent emission, and relatively long-lived
excited states, Ru(II) complexes have attracted much attention as
the DNA structural probes, DNA footprinting, and sequence specific
cleaving agents [3–6]. However, most of these Ru(II) complexes are
mononuclear [7–16]. There are some significant drawbacks for
mononuclear Ru(II) complexes as DNA binders and structural probes.
For example, if metal complexes are to approach the binding footprint
of a DNA-binding protein, the recognition of at least 8–10 base pairs is
essential. The complexes need to possess high DNA-binding affinity at
high ionic strength condition (such as 150 mM NaCl) [17,18].
However, the mononuclear Ru(II) complexes are relatively small
and can only span 1–2 DNA base pairs. Commonly, these mononuclear
(II) complexes with different lengths of flexible bridges as linkers of the
+
two [Ru(bpy)
2
(pip)]2 {bpy=2,2′-bipyridine; pip=2-phenylimidazo
[4,5-f][1,10]phenanthroline}-based subunit [42], and studied their DNA
binding properties.
4
6
−1
complexes have weak DNA-binding affinity (K
b
≈10 −10 M
,
depending on the intercalators) and are easily displaced from DNA at
high ionic strength [19,20]. Dinuclear Ru(II) complexes could
overcome above-mentioned drawbacks as they have increased size,
charge, varied molecular shapes, and greater DNA-binding affinity.
2. Experimental
2.1. Materials
2 2 2
cis-[Ru(bpy) Cl ]·2H O [43] and 1,10-phenanthroline-5,6-dione
[44] were prepared according to the literature routes. The three
dinuclear Ru(II) complexes 1–3 used in this study, were synthesized
according to a modified literature method for the synthesis of an
⁎
Corresponding author. Tel.: +86 10 58805476; fax: +86 10 58802075.
E-mail address: kzwang@bnu.edu.cn (K.-Z. Wang).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.12.004

436
C.-C. Ju et al. / Journal of Inorganic Biochemistry 105 (2011) 435–443
analogous Ru(II) complex [45]. The synthetic route is shown in
Scheme 1, and the synthetic details are described below. Other materials
were commercially available and used without further purification.
symbols are used in NMR spectral description: s (singlet), d (doublet),
dd (doublet of doublets), dt (doublet of triplets), and t (triplet). The
microanalyses (C, N, and H) were performed with a Vario EL elemental
analyzer.
2
.2. Physical measurements
All solutions involving DNA experiments, were prepared by thrice
distilled water. A solution of calf thymus DNA (ct-DNA) gave ratios
of UV absorbance at 260 and 280 nm of about 1.9:1, indicating that
the DNA is sufficiently free of protein [46]. The DNA concentrations
per nucleotide were determined spectrophotometrically by assuming
1H NMR spectra were collected with a Bruker DRX-400 NMR
spectrometer with (CD
chemical shifts were given relative to tetramethylsilane. The following
3 2
) SO as the solvent at room temperature. All
Scheme 1. Synthetic route to 1–3 and proton numbering scheme.

C.-C. Ju et al. / Journal of Inorganic Biochemistry 105 (2011) 435–443
437
260 nm =6600 M− cm−1 [47]. UV–visible (UV–vis) absorption spectra
1
2.3.4. 4,4′-[1,2-Ethanediylbis(oxy)]bis-benzaldehyde
ε
were recorded on a GBC Cintra10e UV–visspectrophotometer. Emission
spectra were obtained on a Varian Cary Eclipse fluorescence spectrom-
eter. The titrations were carried out by using 1-cm-path quartz cuvettes
at room temperature of 20 °C unless otherwise mentioned, in Tris–HCl
buffer (5 mM Tris–HCl, 50 mM NaCl, pH=7.10) by keeping the
concentrations of the complexes constant while varying the DNA
concentrations. After the solutions of the Ru(II) complexes were
allowed to incubate with the DNA for 10 min, the UV–vis spectra were
measured by using the buffer containing same amounts of the DNA as
To a mixture of 4-hydroxybenzaldehyde (2.07 g, 17 mmol) and
potassium carbonate (2.35 g, 17 mmol) in 100 ml N,N-dimethylformide,
was dropwise added ethane-1,2-diylbis(4-methylbenzenesulfonate)
(2.74 g, 8 mmol) in N,N-dimethylformide (150 ml) during a period of
20 min at constant stirring under the protection of N
temperature. The reaction mixture was heated at 80 °C under stirring
for 24 h under the protection of N and then was cooled to room
2
at room
2
temperature. A brown-yellow powder obtained after the removal of the
solvent under a reduced pressure, was directly used for following
synthesis without further purification.
the reference blanks. The values of intrinsic binding constant K
b
illustrating the binding strength of the complex with ct-DNA, and the
binding site size s were determined by fitting the titration data to
Eqs. (1) and (2) [48,49]
2.3.5. 4,4′-(2,2′-Oxybis(ethane-2,1-diyl)bis(oxy))dibenzaldehyde
This was synthesized as described above for 4,4′-[1,2-ethanediylbis
(
oxy)]bis-benzaldehyde except that 2,2′-oxybis(ethane-2,1-diyl) bis(4-
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
b
2
K
Ct½DNAꢀ
2
methylbenzenesulfonate) was used instead of ethane-1,2-diylbis(4-
methylbenzenesulfonate).
εa−ε
f
b− b −
s
=
ð1Þ
εb−εf
2K C
b t
2
.3.6. 4,4′-(2,2′-(Ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(oxy)
K ½DNAꢀ
b
b = 1 + K C +
ð2Þ
b
t
dibenzaldehyde
2
s
This was synthesized as described above for 4,4′-[1,2-ethanediylbis
(oxy)]bis-benzaldehyde except that 2,2′-(ethane-1,2-diylbis(oxy))bis
(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) was used instead of
ethane-1,2-diylbis(4-methylbenzenesulfonate).
where [DNA] is the concentration of DNA in base pairs, ε
a
, ε
f
, and ε
b
are
the apparent, free, and bound metal complex extinction coefficients,
respectively, s is the binding site size in base pairs, and C is the total Ru
II) complex concentration.
Steady-state emission quenching experiments were carried out
in the Tris–HCl buffer by using K [Fe(CN) ] as the quencher. The
t
(
2.3.7. 1,2-Bis(4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenoxy)
ethane (L )
1
4
6
experiments of DNA competitive binding with ethidium bromide (EB)
were carried out also in Tris–HCl buffer by keeping [DNA]/[EB]=5
and varying the concentrations of the Ru(II) complexes.
A mixture of 1,10-phenanthroline-5,6-dione (0.64 g, 3 mmol),
4,4′-[1,2-ethanediylbis(oxy)]bis-benzaldehyde (0.41 g, 1.5 mmol),
ammonium acetate (2.3 g, 30 mmol), and glacial acetic acid (25 ml)
was refluxed for 6 h. The cooled solution was neutralized to pH=7.0
with concentrated aqueous ammonia. The precipitate formed was
collected by filtration and was recrystallized from DMF-diethyl ether.
The resulting solid was directly used for following synthesis without
further purification due to solubility problem.
Thermal denaturation experiments were performed on a UV–vis
spectrophotometer in a phosphate buffer (1.5 mM Na
NaH PO , 0.25 mM Na EDTA, pH=7.0). With the use of the thermal
melting program, the temperature of the cell containing the cuvette was
2 4
HPO , 0.5 mM
2
4
2
−1
ramped from 50 to 90 °C at a rate of 1 °C min , and the absorbance at
2
60 nm was measured every 0.5 °C.
The viscosity measurements were carried out using an Ubbelodhe
viscometer immersed in a thermostated water bath maintained at
2.04± 0.02 °C. The DNA samples for viscosity measurements were
2.3.8. 2,2′-(4,4′-(2,2′-Oxybis(ethane-2,1-diyl)bis(oxy))
2
bis(4,1-phenylene))bis(1H-imidazo[4,5-f][1,10]phenanthroline) (L )
1
3
This was synthesized as described above for L except that 4,4′-
prepared by sonication in order to minimize complexities arising from
DNA flexibility [50]. The flow time was measured, and each sample
was measured at least five times, and an average flow time was
(2,2′-oxybis(ethane-2,1-diyl)bis(oxy))dibenzaldehyde was used in-
stead of 4,4′-[1,2-ethanediylbis(oxy)]bis-benzaldehyde.
1/3
calculated. Data were presented as (η/η
0
)
vs [Ru]/[DNA], where η is
2.3.9. 1,2-Bis(2-(4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)
3
the viscosity of DNA in the presence of the Ru(II) complex and η
0
is
phenoxy)ethoxy) ethane (L )
1
the viscosity of the DNA solution alone.
This was synthesized as described above for L except that 4,4′-
(
2,2′-(ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(oxy)diben-
2
2
1
.3. Synthesis
zaldehyde was used instead of 4,4′-[1,2-ethanediylbis(oxy)]bis-
benzaldehyde.
.3.1. Ethane-1,2-diyl bis(4-methylbenzenesulfonate)
A mixture of ethane-1,2-diol (1.25 g, 20 mmol), 4-methylbenzene-
-sulfonyl chloride (TsCl) (9.5 g, 50 mmol) and sodium hydroxide
1–3
2.3.10. [Ru
A suspension of [Ru(bpy)
(0.3 mmol) in ethanol (100 ml) was refluxed under the protection of
for 6 h. After most of the solvent was removed under a reduced
pressure, a red precipitate was obtained by the dropwise addition of a
4-fold excess of saturated aqueous NaClO solution. The purification
was carried out by column chromatography on silica gel with CH CN-
O-saturated aqueous KNO (40:4:1, v/v/v) as eluent followed by
reprecipitation with a saturated NaClO aqueous solution. Light-red
2 4 4 4
(bpy) (L )](ClO )
2
2 2
Cl ]·2H
O (0.291 g, 0.6 mmol) and L1–3
(
2.0 g, 50 mmol) was ground for about 10 min in mortar. Then a white
powder gained, was washed with water several times and then dried
in vacuo. The product was directly used for following synthesis
without further purification.
N
2
4
3
2
.3.2. 2,2′-Oxybis(ethane-2,1-diyl) bis(4-methylbenzenesulfonate)
H
2
3
This was synthesized as described above for ethane-1,2-diyl bis(4-
4
methylbenzenesulfonate) except that 2,2′-oxydiethanol was used
crystals were obtained in N60% yields. (Caution! All the perchlorate
instead of ethane-1,2-diol.
salts are potentially explosive and therefore should be handled in
small quantity with care.)
1
2
.3.3. 2,2′-(Ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)
[Ru
2
(bpy)
4
(L )](ClO
4
)
4
·3H
2
O 1∙3H
2
O: Yield: 60%. UV–vis in
cm ): 287 (2.02); 320 (0.81); 460
(0.37). H NMR (500 MHz, Me SO-d ) (see Scheme 1 for proton
numbering scheme): δ 14.22 (s, 2H ), 9.10 (d, 4H ), 8.87 (dd, 8Hd,e),
8.30 (d, 4H4,c), 8.23 (t, 4Hf,i), 8.12 (t, 4H ), 8.07 (d, 4H ), 7.95 (d, 4H ),
5
− 1
− 1
bis(4-methylbenzenesulfonate)
3
CH CN: λ/nm (ε/10 M
1
This was synthesized as described above for ethane-1,2-diyl bis(4-
methylbenzenesulfonate) except that 2,2′-(ethane-1,2-diylbis(oxy))
diethanol was used instead of ethane-1,2-diol.
2
6
l
k
b
h
a

438
C.-C. Ju et al. / Journal of Inorganic Biochemistry 105 (2011) 435–443
7
C
.86 (d, 4H
58Cl
b
), 7.60 (d, 8H
18Ru ·3H
g
), 7.34 (d, 8H
O: C, 49.79; H, 3.32; N, 11.62. Found: C, 49.81;
5
), 4.54 (s, 4H
8
). Anal. Calcd for
transition band at ~465 nm. These assignments are made on the basis
of the comparisons of the spectra of 1–3 with that for [Ru(bpy) ]
3
2+
80
H
4
N
16
O
2
2
H, 3.45; N, 11.49.
[56]. With increasing concentrations of ct-DNA, 1–3 exhibited smaller
hypochromicities of b8% for the MLCT bands than a hypochromicities of
2
[
Ru
2
(bpy)
4
(L )](ClO
4
)
4
·4H
2
O 2∙4H
2
O: Yield: 65%. UV–vis in
cm ): 287 (2.21); 320 (0.73); 460
0.38). H NMR (500 MHz, Me SO-d ) (see Scheme 1 for proton
numbering scheme): δ 14.23 (s, 2H ), 9.08 (d, 4H ), 8.88 (dd, 8Hd,e),
.28 (d, 4H4,c), 8.23 (t, 4Hf,i), 8.12 (t, 4H ), 8.06 (d, 4H
.85 (d, 4H ), 7.60 (t, 8H ), 7.35 (t, 4H ), 7.28 (d, 4H
.94 (s, 4H ). Anal. Calcd for C82 62Cl 19Ru ·4H
.52; N, 11.26. Found: C, 49.66; H, 3.54; N, 11.01.
5
−1
− 1
CH
(
3
CN: λ/nm (ε/10 M
11% reported for corresponding band of an analogous Ru(II) complex of
1
2+
2
6
[Ru(bpy)
2
(p-mopip)]
{p-mopip=2-(4-methoxylphenyl)imidazo
l
k
[4,5-f][1,10]phenanthrolin} [15], but more evident hypochromicities
of 47%, 39%, and 45% respectively for the π–π* transition bands at
8
7
3
3
b
h
), 7.94 (d, 4H
a
),
),
a
b
g
5
), 4.30 (s, 4H
8
2
285 nm than a hypochromicity of 34% reported for [Ru(bpy) (p-
+
9
H
4
N
16
O
2
2
O: C, 49.45; H,
mopip)]2 [15]. The little changes of the MLCT bands observed may
bebecause theMLCT bandis mainly Ru-to-bpy charge transfer in nature.
Similar behavior was also observed in other dinuclear Ru(II) complexes
[22,28,34]. The notable hypochromicities observed for the π–π*
transition bands of 1–3 at ~287 nm are much larger than those reported
3
[
Ru
λ/nm (ε/10 M cm ): 287 (1.99); 320 (0.61); 460 (0.33). H NMR
500 MHz, Me SO-d ) (see Scheme 1 for proton numbering scheme): δ
), 9.08 (d, 4H ), 8.86 (dd, 8Hd,e), 8.25 (t, 8H4,c), 8.22 (t, 4Hf,i),
), 8.06 (dt, 4H ), 7.85 (d, 4H ), 7.60 (t, 8H ), 7.34 (t, 4H ),
), 4.26 (s, 4H ), 3.85 (s, 4H ), 3.70 (s, 4H11). Anal. Calcd
20Ru ·5H O: C, 49.12; H, 3.70; N, 10.92. Found: C,
9.00; H, 3.84; N, 10.71.
2
(bpy)
4
(L )](ClO
4
)
4
·5H
2
O 3∙5H
2 3
O: Yield: 65%. UV–vis in CH CN:
5
−1
−1
1
(
2
6
1
8
7
4.19 (s, 2H
.11 (d, 4H
.27 (d, 4H
66Cl
l
k
for typical DNA intercalators [57,58] (see Table 1). Hiort et al. ever
(dppz)]2 {phen=1,10-phenan-
+
b
h
a
b
g
deduced that the dppz in [Ru(phen)
2
5
8
9
throline; dppz=dipyrido[3,2-a:2′,3′-c]phenazine} intercalates be-
tween the DNA base pairs because the hypochromism of the
intraligand transition of dppz is greater than that of the MLCT [58].
The evident spectral changes observed may suggest that 1–3 exhibit
high DNA binding affinity. After binding to the DNA, the π* orbital of the
binding ligand could couple with π orbit of base pairs in the DNA. The
coupling π* orbital was partially filled by electrons, thus decreasing
the transition probabilities, and concomitantly resulting in the
hypochromicity [59]. However, the ligand bpy was previously dem-
onstrated to be at best only minimally efficient at inducing intercalative
binding with the DNA [60]. The ligand pip-containing mononuclear
Ru(II) complex did not exhibit the notable hypochromicity as it
intercalated into the DNA [42]. These facts indicate that the flexible
bridges between the two Ru2 cores in 1–3 play an important role in
the DNA binding. In order to further illustrate the binding strength of
for C84
H
4
N
16
O
2
2
4
2
.4. Computational methods
Each of 1–3 is made of a Ru(II) ion, one of main ligands L1–3 and
two ancillary ligands bpy. The starting structures of 1–3 were
(pip)]2
2
+
constructed based on the optimized structure of [Ru(bpy)
51]. Geometry optimization computations were performed applying
[
the DFT-B3LYP method [52] and LanL2DZ basis set (ECP+DZ for the
Ru atom and D95 for C, N, O, H atoms) [53], and assuming the singlet
state for the ground state of the complex [54]. All the computations
were performed with the G03 quantum chemistry program-package
+
[55].
b
the complexes, the values of intrinsic binding constant K and the
3
. Results and discussion
binding site size s with ct-DNA were obtained by monitoring the
changes in absorbance at ~287 nm of the Ru(II) complexes, with
3
.1. UV–vis absorption spectra
increasing concentrations of DNA. The K
b
and s values of 1–3 were
and s=1.85± 0.02 for 1,
7
−1
derived to be K
b
=(5.7± 1.6)×10 M
7
− 1
The absorption spectra of 1–3 in the absence and the presence
K
b
=(7.5± 3.1)×10 M
and s=1.47± 0.02 for 2, and K
b
=(9.5±
7
− 1
of ct-DNA are shown in Figs. 1, S1, and S2. In the absence of the DNA,
UV–vis absorption spectra of the three complexes in water are quite
similar to each other, characterized by a bpy-centered π–π* transition
band at ~287 nm, and a metal-to-ligand charge transfer (MLCT)
2.5)×10 M
and s=0.92± 0.01 for 3, from data in the insets of
Figs. 1, S1, and S2. The values of hypochromicity and intrinsic DNA
binding constant of 1–3 in this study and analogous mononuclear and
dinuclear Ru(II) complexes are compared in Table 1. Obviously, the
values of the DNA binding constant derived for 1–3 are close to each
other within the experimental errors, while s values decease with
increasing linker lengths. The similar DNA binding affinities indicate
that 1–3 would have similar binding mode, and the strong
dependence of s values on the linker lengths of 1–3 suggests that
1
–3 interact with the DNA in a threading bis-intercalating binding
mode, namely both ends of the complex to bind to the same strand of
DNA rather than the interaction with two strands of the DNA through
interstrand binding. Kelley [17] reported DNA binding constant
Table 1
Comparisons of DNA binding parameters.
a
(M−1
Complex
H (%) (λmax) (nm)
K
b
)
Ref.
(pip)]2+
(p-hpip)]
22 (458)
4.7×10
6.9×10
2×10
5.0×10
1.3×10
5
[42]
[15]
[15]
[64]
[28]
[17]
[35]
[18]
[
[
[
[
[
[
[
[
Ru(bpy)
Ru(bpy)
Ru(bpy)
Ru(bpy)
2
2
2
2
2
+
4
34 (264);16 (458)
34% (264);11 (456)
14.5 (460)
36.6 (288)
16 (460)
2
+
4
(p-mopip)]
2
+
6
6
(dppz)]
4
)(bpy) ]
4
+
Ru
Ru
Ru
Ru
2
(ebipcH
2
(bmbh)(phen)
2
(cpdppz)(phen)
2
(phen-5-SOS-5-phen)
2
4
+
7
4
]
0.36×10
]4+
–
~10
9
4
7
–
(8.9± 4.3)×10
4
+
4
(dpq) ]
7
7
7
1
2
3
45 (287);5 (465)
38 (285);5 (465)
47 (286);5 (465)
(5.7± 1.6)×10
(7.5± 3.1)×10
(9.5± 2.5)×10
this work
this work
this work
Fig. 1. Changes in UV–vis spectra of 1 (3 μM) with increasing concentrations of ct-DNA
in 5 mM Tris–HCl buffer (pH=7.10, 50 mM NaCl). Arrows show spectral changes upon
increasing DNA concentrations.
a
b
See Abbreviations section. H%=100×(Afree −Abound)/Afree
.

C.-C. Ju et al. / Journal of Inorganic Biochemistry 105 (2011) 435–443
− 1
439
6
6
6
mopip)]2+, [Ru(bpy) (p-hpip)]2+ and [Ru(bpy) (p-npip)]2+ {p-
2 2
values of 2.4×10 , 3.6×10 and 3.0×10 M
for dinuclear Ru(II)
II
II
4+
complexes of [(phen)
2
Ru (Mebpy)-(CH
2
)
n
-(bpyMe)Ru (phen)
2
]
npip=2-(4-nitrophenyl)imidazo[4,5-f][1,10]phenanthroline} [15],
respectively, which agrees with the order of their DNA binding
constant values. Kelly [17] observed emission enhancement factors
(IDNA/Ifree) of 2.08, 2.36, and 1.68 for dinuclear Ru(II) complexes of
{
Mebpy=4-methyl-2,2′-bipyridyl-4′-} with n=5, 7, and 10, respec-
tively. In view of experimental uncertainties, Kelly's DNA binding
constant values seem insensitive to the linker lengths. Instead
variations in the linker lengths were reported to profoundly affect
values of binding site size s, s=6.4, 8.8, and 6.0 base pairs for
the complexes with linker lengths of n=5, 7, and 10, respectively,
which is in agreement with our observation. As shown in Table 1, the
4
+
[(phen)
2
Ru(Mebpy)-(CH
2
)
n
-(bpyMe)Ru(phen)
2
]
with n=5, 7,
and 10 in the presence of double-stranded DNA, respectively, which
are less than or comparable to a factor of 2.4 for mononuclear analog
bpy)]2 (Me
+
bpy=4, 4′-dimethyl-2,2′-bipyridyl).
of [Ru(phen)
2
(Me
2
2
4
−
K
4
b
values obtained for 1–3 are much larger than the K
b
values of
[Fe(CN)
6
]
was used as the quencher in steady-state emission
5
5
4
− 1
.7×10 , 1×10 and 2×10 M
clear complexes of [Ru(bpy)
reported for analogous mononu-
quenching experiments. As shown in Figs. 3, S5, and S6, the emissions
of 1–3 were efficiently quenched by [Fe(CN)
(pip)]2+ [42], [Ru(bpy)
2+
4−
in the absence of the
2
2
(p-hpip)] {p-
6
]
hpip=2-(p-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline} [61],
DNA, resulting in almost linear Stern–Volmer plots with slopes of
1213± 9, 736± 10, and 550± 10 mM for 1, 2 and 3, respectively. In
the presence of the DNA, the slopes of the Stern–Volmer plots for 1–3
2+
−1
and [Ru(bpy)
values of 5.0×10 and 1.3×10 M
2
(p-mopip)] [15], respectively, even larger than the K
b
6
6
−1
previously reported for a typical
+
DNA intercalator [Ru(bpy)
ing dinuclear complex [Ru (ebipcH
bis([1,10]-phenanthroline[5,6-f]imidazol-2-yl)carbazole}[28], respec-
tively. It is noteworthy that the K values for 1–3 are one order
of magnitude greater than a K value of 3.6×10 M
the dimeric complex of [Ru (phen)
methyl-2,2′-bipyridin-4-yl)heptane} [17], approach 8.9×10 M
reported for the threading bis-intercalator of [Ru (cpdppz)(phen)
]4+
cpdppz=N,N′-bis{12-cyano-12,13-dihydro-11H-cyclopenta[i]dipyrido
3,2-a:2′,3′-c]-phenazine-12-carboxmide-1,4-diaminobutane} [35]. The
2
(dppz)]2 [62], and a rigid bridge-contain-
were drastically decreased to 110± 10, 56.9± 0.4, and 11.20±
4+
−1
2
2 4
)(bpy) ]
{ebipcH
2
=N-ethyl-4,7-
0.2 mM , corresponding to R values (the ratio of the slop in the
absence of the DNA to that in the presence of DNA) of 11.0, 12.9, and
b
49.1, respectively. The R value has frequently been used to evaluate
6
−1
4−
b
reported for
(bmbh)]4 {bmbh=1,7-bis(4′-
accessibility of the complexes to [Fe(CN)
6
]
. The greater R value
+
2
4
corresponds to more protection from emission quenching of the
7
−1
Ru(II) complex by DNA, due to the repulsion of the highly anionic
4−
2
4
[Fe(CN)
6
]
by the DNA polyanion [60–63]. Although the R values
{
[
observed for 1–3 are qualitatively consistent with their DNA binding
affinities, these values are only at the same order of magnitude as a R
above mentioned facts indicate that 1–3 might bind to the DNA in a
threading bis-intercalating mode.
value of 20 reported for proven DNA intercalator, mononuclear
(pip)]2 [42]. Obviously, 1–3 were
+
analogous complex [Ru(bpy)
efficiently protected from the quenching by [Fe(CN)
2
4
−
6
]
as they bond
to the DNA, but less than anticipated from their DNA affinities,
probably due to both the threading binding effect and hydrogen
bonding formed between the oxygen atoms in the flexible linkers on
1–3 and the groove of the DNA [18] contributed to the DNA binding
affinities but little on the shielding of 1–3.
The competitive binding experiments with a well-established
quenching experiment based on the displacement of the intercalating
drug ethidium bromide (EB) from ct-DNA may give further informa-
tion about the DNA binding properties of 1–3. If a complex can
displace EB from DNA-bound EB, the fluorescence of EB will be sharply
decreased due to the fact that the free EB molecules are much less
fluorescent than the DNA bound EB molecules because the surround-
ing water molecules quench the fluorescence of free EB, and 1–3 as
3
.2. Luminescence spectra
–3 in Tris–HCl buffer at room temperature were luminescent
1
with maxima at 607, 608, and 612 nm, respectively, which were red-
shifted relative to that reported for analogous mononuclear complex
2
+
2
[Ru(bpy) (p-mopip)] (590 nm) [15]. Changes in emission spectra
of 1–3 with increasing DNA concentrations are shown in Figs. 2, S3,
and S4, respectively. As ct-DNA was added into the complex solutions,
the emission intensities of 1 were almost undisturbed, while those of
both 2 and 3 were increased by a factor of only b0.2. While Ji et al.
reported that addition of the DNA resulted in sharp enhancements in
2
the emission intensities by 0.9, 1.3, and 3.5 folds for [Ru(bpy) (p-
]4 in the
−
Fig. 2. Changes in emission spectra (λex =460 nm) of 1 (3 μM) with increasing
concentrations of ct-DNA in 5 mM Tris–HCl buffer (pH=7.10, 50 mM NaCl).
Fig. 3. Emission quenching of 1 with increasing concentrations of [Fe(CN)
6
absence (square) and presence (circle) of the DNA. [1]=3 μM, [DNA]/[1]=15.0.

440
C.-C. Ju et al. / Journal of Inorganic Biochemistry 105 (2011) 435–443
Fig. 4. Changes in emission spectra of EB bound to DNA upon successive additions of 1
0–27 μM). The arrows show the intensity changes upon increasing concentrations of 1.
Inset: a plot of percentage of free EB vs [1]/[EB]. [EB]=20 μM, [DNA]=100 μM,
ex =537 nm.
Fig. 5. Thermal denaturation curves of ct-DNA (100 μM) at varied concentration ratios
of [1]/[DNA].
(
λ
m
any particular type of noncovalent interaction, the ΔT values may
well as DNA bound 1–3 are also negligibly weakly emissive as excited
at the excitation wavelength of EB (λex =537 nm) [64]. However, not
only the DNA intercalators but also groove DNA binders could cause
the reduction in the emission intensities of DNA bound EB [65], but
only moderately for the latter case. As shown in Figs. 4, S7, and S8, a
remarkable reduction in emission intensities by 68%, 71%, and 53%
for 1–3 were achieved as 1–3 were added to EB–DNA system,
characteristic for the intercalative binding of 1–3 to the DNA [66–68].
To further illustrate the DNA binding affinities of 1–3, the values of the
apparent DNA binding constant Kapp were derived according to
Eq. (3),
give hints on the DNA binding mode. The DNA melting curves in the
absence and presence of different concentrations of the Ru(II)
complexes are presented in Figs. 5, S9, and S10. The T
DNA was found to be 67.1 °C in the absence of 1–3, and was enhanced
successively with increasing concentrations of 1–3. The ΔT values at
a concentration ratio of [DNA]/[Ru]=10:1 were found to be 1.5, N15,
and N15 °C for 1–3, respectively. In comparison with the ΔT values
reported for some DNA intercalative complexes shown in Table 2, the
ΔT value for 2 and 3 are typical for DNA intercalators, and are
reasonable as compared to a ΔT value of 20± 2 °C reported for the
duplex 5′-TCGGGATCCCGA-3′ in the presence of a threading bis-
m
value of ct-
m
m
m
m
4+
intercalator of [Ru
large Ru(II) complex concentration {[5′-TCGGGATCCCGA-3′]/[Ru]=
.5}[18]. Although the other spectral evidences except for the
thermal denaturation data seem in support of a threading inter-
calative DNA binding mode for 1, the small ΔT value observed for 1
2
(phen-5-SOS-5-phen)(dpq)
4
]
at a relatively
Kapp = KEB½EBꢀ50% = ½Ruꢀ50%
ð3Þ
0
where KEB is the DNA binding constant of EB, and [EB]50% and [Ru]50%
are the EB and Ru(II) complex concentrations at which 50% of EB were
replaced from EB–DNA complex. In the plots of percentage of free EB
vs [Ru]/[EB], we can see that 50% of the EB molecules were replaced
from DNA-bound EB at concentration ratios of [Ru]/[EB]=0.31, 0.21,
and 0.18 for 1, 2, and 3 respectively, as shown in the insets of Figs. 4,
m
may be because the linker length of 1 is too short to facilitate
the intercalation of the two Hpip moieties on L1 of 1 between the
base pairs of the DNA deeply. This lead us to draw a conclusion that
“the threading effect” would make a dominant contribution to the
DNA binding affinities observed for 1–3 compared to “the inter-
calative effect”, while “the intercalative effect” directly affect the
DNA denaturation temperature. It should also be emphasized that
7
−1
S7, and S8. By taking a DNA binding constant value of 1.0×10 M
for EB [11,69], the values of the apparent DNA binding constant for 1,
7
7
7
−1
2
, and 3 were derived to be 3.2×10 , 4.8×10 and 5.6×10 M
,
respectively, which are in agreement with those derived by UV–vis
absorption spectroscopy.
Table 2
The comparisons of the values of DNA binding constant
temperature difference ΔT .
m
K
b
and denaturation
3
.3. Thermal denaturation of ct-DNA
a
(M−1
DNA binder
K
b
)
[DNA]/[Ru] ΔT
m
(°C) Ref.
[65]
The DNA melting study is a further evidence for the intercalation of
the Ru(II) complexes into the DNA helix. The DNA melting experi-
ments were carried out to distinguish the different binding modes
7
6
7
EB
1.0×10
N1.0×10
8.9×10
10
25
13
6.0
[
Ru(phen)
Ru (phen-5-SOS-5-phen)
dpq)
Ru(phen)
2
(qdppz)]2+
[71]
[18]
b
[
2
0.5
20
(
binding by intercalation or binding externally). The intercalation of
4+
(
4
]
small molecules into the DNA double helix has been known to
increase the helix melting temperature at which the double helix
denatures into single stranded DNA. The melting of the helix can lead
to an increase in the absorption at 260 nm, because the extinction
coefficient of DNA bases at 260 nm in the double-helical form is much
less than in the single strand form [64,70–74]. Thus, the helix-to-coil
transition temperature can be determined by monitoring the
absorbance of the DNA bases at 260 nm as a function of temperature.
Although the increase in denaturation temperature is not specific of
2+
5
5
5
6
6
5
7
7
7
[
2
(hqdppz)]
1.0×10
4.3×10
9.7×10
1.3×10
5.1×10
1.8×10
25
1.0
1.0
10
10
10
10
10
10
6.0
[70]
[72]
[72]
[73]
[74]
[74]
This work
This work
This work
2
2
+
+
[Ru(NH
3
)
)
4
(pip)]
(pip)]
8.0
8.0
5.9
9.1
5.2
1.5
[
[
[
[
Ru(NH
3
4
2
+
Ru(bpy)(ppd)]
Ru(phen)
Ru(NH
2
(dppz)]2+
2+
)
3 4
(dppz)]
1
2
3
(5.7± 1.6)×10
(7.5± 3.1)×10
(9.5± 2.5)×10
N15
N15
a
b
See Abbreviations section. [5′-TCGGGATCCCGA-3′]/[Ru]=0.5

C.-C. Ju et al. / Journal of Inorganic Biochemistry 105 (2011) 435–443
441
the magnitude of ΔT
m
value does not invariably parallel the DNA
3.5. The optimized structures by density functional theory calculations
binding affinities even for structurally similar drugs with considerably
(qdppz)]2 {qdppz=
+
The optimized structures for 1–3 are shown in Fig. 7, and the
computational selected bond lengths, bond angles, and dihedral
angles of 1–3 using the DFT at the B3LYP/LanL2DZ level are shown in
different DNA binding affinity, e.g. [Ru(phen)
naphtho[2,3-a]dipyrido[3,2-h:2′,3′-f]phenazine-5,18-dione} and [Ru
2
2+
(phen)
2
(hqdppz)] {hqdppz=5,18-dihydroxynaphtho[2,3-a]dipyrido
[
3,2-h:2′,3′-f]phenazine} were reported to exhibit almost same ΔT
m
Table S1. The mean coordination bond lengths between the Ru and N
atoms of the main ligand L1 (Ru–N
–3
), and between Ru and N atoms
value of 6 °C at [DNA]/[Ru]=25:1 [70], but their DNA binding constant
values differed by one order of magnitude (see Table 2).
m
of the co-ligand bpy (Ru–Nco), and the mean coordination bond angles
between the central Ru and two N atoms of the main ligand (A ), and
between the central Ru and two N atoms of the co-ligand (Aco) in 1–3
are found to be Ru–N =0.2119–0.2124 nm, Ru–Nco =0.2108–
.2116 nm, A =78.38°–78.61° and Aco =77.69°–77.92°, which are
close to the corresponding data (Ru–N = 0.2108 nm, Ru–
co =0.2097 nm, A =79.26° and Aco =78.47°) previously reported
for [Ru(bpy) (pip)] [51], indicating that our computed results are
m
3
.4. Viscosity measurements
m
0
m
Optical photophysical probes provide necessary, but not sufficient,
m
clues to support a binding mode. Hydrodynamic measurements, such
as viscosity measurements which are sensitive to length change, are
regarded as the least ambiguous and the most critical tests of a
binding mode in solution in the absence of crystallographic struc-
tural data [75,76]. To further explore the interaction properties
between 1–3 and the DNA, the specific relative viscosities of the DNA
were measured by adding increasing concentrations of 1–3 and
known DNA intercalator ethidium bromide (EB) for comparison
purpose. A classical intercalation mode results in lengthening the DNA
helix, leading to an increase in the DNA viscosity. However, a partial or
nonclassical intercalation of the ligand could bend (or kink) the DNA
helix, reducing the effective length of the DNA, and therefore the DNA
viscosities. In contrast, an electrostatic or grooving binding mode has
little effects on the DNA viscosities [74,76]. As shown in Fig. 6, the
viscosities of the ct-DNA increased upon successive additions of 1–3,
N
m
2
reliable. Although 1–3 show some difference in linker orientations,
the two Ru(II) centers for each of 1–3 seem to have ample separation
for the orientation of the two pip moieties on 1–3 to thread the linkers
and intercalate two pip moieties between the base pairs of the DNA.
4. Conclusion
UV–vis absorption spectroscopy and EB competitive binding
experiments revealed that different lengths of flexible bridge-
containing 1–3 avidly bound to ct-DNA with DNA binding constant
7
−1
values on the order of magnitude of 10 M , three orders of
magnitude greater than that reported for analogous mononuclear
+
complex of [Ru(bpy)
2
(p-mopip)]2 . Emission spectrophotometric
similarly to proven DNA intercalators of EB and previously reported
DNA titrations, steady state emission quenching and DNA viscosity
measurements demonstrated that the intercalation depth or the
(p-mopip)]2 [15]. According to the
+
mononuclear complex [Ru(bpy)
2
values of DNA binding constant, 1–3 should result in more evident
extent of protection of 1–3 by the DNA are similar to those of [Ru
(bpy) (p-mopip)] . The much enhanced DNA binding affinities and
2
(p-mopip)]2 and EB,
+
2+
increases in DNA viscosities than [Ru(bpy)
since 1–3 has been evidenced to bind to the DNA more tightly than
2
comparable intercalation depth compared to their mononuclear
2
+
2+
[
Ru(bpy)
2
(p-mopip)] and EB. On the contrary, 1–3 were observed
analogous complex [Ru(bpy)
2
(p-mopip)]
led us to make a
to induce smaller increases in the DNA viscosities than EB. We
attribute these small viscosity increases in the presence of 1–3
with respect to EB, to that the threading effect of 1–3 fasten both ends
of 1–3 onto the same strand of the DNA, preventing evidently
lengthening the DNA helix and sharply increasing the DNA viscosities
accordingly.
conclusion that 1–3 bound to the DNA probably in a threading
intercalation binding mode, and the threading effect made a dominant
contribution to the DNA binding affinities, but little to the their
protection from emission quenching and the lengthening of the DNA
double strands.
Abbreviations
bpy
2,2′-bipyridine
bmbh
1,7-bis(4′-methyl-2,2′-bipyridin-4-yl)heptane
cpdppz N,N′-bis{12-cyano-12,13-dihydro-11H-cyclopenta[i] dipyr-
ido[3,2-a:2′,3′-c]phenazine-12-carboxamide}-1,4-
diaminobutane
DFT
density functional theory
ct-DNA calf thymus DNA
dppz
dpq
dipyrido[3,2-a:2′,3′-c]phenazine
pyrazino[2,3-f][1,10]phenanthroline
ebipcH N-ethyl-4,7-bis([1,10]-phenanthroline[5,6-f]imidazol-2-yl)
carbazole
EB
ethidium bromide
p-hpip 2-(p-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline
hqdpp
L1
5,18-dihydroxynaphtho[2,3-a]dipyrido[3,2-h:2′,3′-f]
phenazine
1,2-bis(4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)
phenoxy)ethane
L2
4,4′-[oxybis(2,1-ethanediyloxy)]bis-1H-Imidazo[4,5-f]
[
1,10]phenanthroline
L3
2,2′-[1,2-ethanediylbis(oxy-2,1-ethanediyloxy-4,1-phenyl-
ene)]bis-1H- imidazo[4,5-f][1,10]phenanthroline
mebpy 4-methyl-2,2′-bipyridyl-4′-
MLCT metal-to-ligand charge transfer
p-mopip 2-(4-methoxylphenyl)imidazo[4,5-f][1,10]phenanthroline
p-npip 2-(4-nitrophenyl)imidazo[4,5-f][1,10]phenanthroline
Fig. 6. Effects of increasing concentrations of 1–3 and EB on the relative viscosities of
ct-DNA in buffered 50 mM NaCl at 32.04± 0.02 °C, [DNA]=0.4 mM.

442
C.-C. Ju et al. / Journal of Inorganic Biochemistry 105 (2011) 435–443
Fig. 7. Optimized structures of 1 (a), 2 (b) and 3 (c).
phen
pip
1,10-phenanthroline
Appendix A. Supplementary materials
2-phenylimidazo[4,5-f][1,10] phenanthroline
pteridino[7,6-f][1,10]phenanthroline-1,13(10H,12H)-dione
naphtho[2,3-a]dipyrido[3,2-h:2′,3′-f]phenazine-5,18-dione
2-mercaptoethyl ether
Supplementary data to this article can be found online at
doi:10.1016/j.jinorgbio.2010.12.004.
ppd
qdppz
SOS
TsCl
p-toluenesulfonyl chloride
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