Synthesis, characterization and DNA-binding and DNA-photocleavage studies of two Ru(II) complexes containing two main ligands and one ancillary ligand

Article abstract of DOI:10.1016/j.poly.2011.04.047

DNA-binding and DNA-photocleavage properties of two Ru(II) complexes, [Ru(L¹)(dppz)²](PF⁶)⁴ (1) and [Ru(L²)(dppz)²](PF⁶)⁴ (2) (L ¹ = 5,50′-di(1-(triethylammonio)methyl)-2,2′-dipyridyl cation; L² = 5,5′-di(1- (tributylammonio)methyl)-2,2′- dipyridyl cation; dppz = dipyrido[3,2-a:2′,3′-c]phenazine, have been investigated. Experimental results show that the DNA-binding affinity of complex 1 is greater than that of 2, both complexes emit luminescence in aqueous solution, either alone or in the presence of DNA, complex 1 can bind to DNA in an intercalative mode while 2 most likely interacts with DNA in a partial intercalation fashion, and complex 2 serves as a better candidate for enantioselective binding to CT-DNA compared with 1. Moreover, complex 1 reveals higher efficient DNA cleavage activity than 2, during which supercoiled DNA is converted to nicked DNA with both complexes. Theoretical calculations for the two complexes have been carried out applying the density functional theory (DFT) method at the level of the B3LYP/LanL2DZ basis set. The calculated results can reasonably explain the obtained experimental trends in the DNA-binding affinities and binding constants (K_b) of these complexes.

Full text of DOI:10.1016/j.poly.2011.04.047

Polyhedron 30 (2011) 1953–1959
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Synthesis, characterization and DNA-binding and DNA-photocleavage studies
of two Ru(II) complexes containing two main ligands and one ancillary ligand
a,b
c
b
b
c,
⇑
b
b,
⇑
Jing Sun , Shuo Wu , Huo-Yan Chen , Feng Gao , Jie Liu , Liang-Nian Ji , Zong-Wan Mao
a
School of Pharmacy, Guangdong Medical College, Dongguan 523808, China
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China
Department of Chemistry, Jinan University, Guangzhou 510632, China
b
c
a r t i c l e i n f o
a b s t r a c t
1
Article history:
2 6 4
DNA-binding and DNA-photocleavage properties of two Ru(II) complexes, [Ru(L )(dppz) ](PF ) (1) and
Received 20 December 2010
Accepted 26 April 2011
Available online 18 May 2011
2
1
0
0
2
0
[Ru(L )(dppz)
2 6 4
](PF ) (2) (L = 5,5 -di(1-(triethylammonio)methyl)-2,2 -dipyridyl cation; L = 5,5 -di(1-
tributylammonio)methyl)-2,2 -dipyridyl cation; dppz = dipyrido[3,2-a:2 ,3 -c]phenazine, have been
0
0
0
(
investigated. Experimental results show that the DNA-binding affinity of complex 1 is greater than that
of 2, both complexes emit luminescence in aqueous solution, either alone or in the presence of DNA, com-
plex 1 can bind to DNA in an intercalative mode while 2 most likely interacts with DNA in a partial inter-
calation fashion, and complex 2 serves as a better candidate for enantioselective binding to CT-DNA
compared with 1. Moreover, complex 1 reveals higher efficient DNA cleavage activity than 2, during
which supercoiled DNA is converted to nicked DNA with both complexes. Theoretical calculations for
the two complexes have been carried out applying the density functional theory (DFT) method at the
level of the B3LYP/LanL2DZ basis set. The calculated results can reasonably explain the obtained exper-
Keywords:
Synthesis
Ru(II) complexes
DNA-binding
Photocleavage
Density functional theory (DFT)
b
imental trends in the DNA-binding affinities and binding constants (K ) of these complexes.
Ó 2011 Elsevier Ltd. All rights reserved.
1
. Introduction
excited state of [Ru(L)
2
(dppz)]2+ in water was strongly enhanced
due to the intercalation of the planar dppz ligand between the base
pairs of DNA [7–9]. An important characteristic of these complexes
is that their main ligands possess extended and planar aromatic
structures, which could insert and stack between the base pairs
of double helical DNA [10–11]. Consequently, Ru(II) complexes
with one ancillary ligand and two main ligands have attracted
much attention [12–15]. We previously synthesized [Ru(b-
The interaction between Ru(II) polypyridine complexes and
DNA has attracted much attention [1–3]. It is well established that
the geometry of the DNA-binding complex plays a very important
role in the interactions with nucleic acids. Thus, modification to the
polypyridyl ligands allows fine tuning of the space configurations
and electronic structures of Ru(II)-polypyridyl complexes. As a re-
sult, the DNA-binding behaviors of these complexes are changed.
2
+
2+
py)(pztp)
2
]
and [Ru(phen)(pztp)
2
]
(pztp = 3-(pyrazin-a-yl)-as-
0
In this regard, ligands derived from 2 2-bipyridine (bpy) and
triazino[5,6-f]-1,10-phenanthroline) [16], and found that both
exhibited enhanced luminescence in the presence of CT-DNA and
functioned as good ‘‘light switches’’ for DNA, though they had no
extended aromatic structures. Varying the number of main ligands,
on the other hand, might yield a clearer understanding of the DNA
binding mechanism.
1
,10-phenanthroline (phen) with various modifications have been
employed to address different issues [4]. Recently, a great deal of
effort has been directed toward complexes containing one main li-
gand and two ancillary ligands because of their stability, strong
DNA affinity, photochemical properties and proven applications
in several areas [5–6]. Barton and co-workers showed that the
This paper mainly concerns the DNA-binding affinities of two
(dppz)]2 complexes (L = bpy or phen,
+
complexes, [Ru(L )(dppz)
1
](PF
)
[17] and a new complex
strong binding of [Ru(L)
dppz = dipyrido[3,2-a:2 ,3 -c]phenazine) to DNA gave rise to a
2
0
2
6
4
0
0
2
1
[Ru(L )(dppz)
2
](PF
6
)
4
(L = 5,5 -di(1-(triethylammonio)methyl)
0
2
0
‘
‘molecular light-switch’’ effect, where the nearly undetectable
-2,2 -dipyridyl cation; L = 5,5 -di(1-(tributylammonio)methyl)
0
emission from the triplet metal-to-ligand charge transfer (MLCT)
-2,2 -dipyridyl cation), and their differences in several related
properties as well as their DFT calculations resulting from slight
ligand differences. The ligand system design in the two complexes
may provide more understanding on how steric hindrance affects
these properties. These results will hopefully be of value in further
⇑
Corresponding authors. Tel.: +86 20 84113788, fax: +86 20 84112245.
E-mail addresses: tliuliu@jnu.edu.cn (J. Liu), cesmzw@mail.sysu.edu.cn (Z.-W.
Mao).
0
277-5387/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2011.04.047

1
954
J. Sun et al. / Polyhedron 30 (2011) 1953–1959
DNA binding studies, improve efficiency in DNA recognition and
cleavage by Ru(II) complexes, and lay foundations for the rational
design of new photoprobes and photonucleases of DNA.
2.4. DNA-binding experiments
The absorption and emission titrations of the Ru(II) complexes
in buffer A were performed using a fixed complex concentration,
to which increments of the DNA stock solution were added. The
concentration of the [Ru(L)(dppz)
volume of the complex was 3000
2
. Materials and methods
4
+
2
]
l
solution was 10
lM and the
L. Complex-DNA solutions were
2.1. Materials
allowed to incubate for 5 min before the spectra were recorded.
The titration processes were repeated several times until no
change was observed in the spectra, indicating that binding satura-
tion was achieved. Changes in the Ru(II) complex concentration
due to dilution at the end of each titration were negligible.
Equilibrium dialysis was carried out in the dark and held at
room temperature for 36 h with CT-DNA (1.0 mM, 5.0 mL) sealed
inside a dialysis bag and a Ru(II) complex (50 lM, 10 mL) outside,
the solution stirring varying with the dialysis time. For each sam-
ple, the spectrum was scanned at least three times and accumu-
CT-DNA was purchased from Sino-American Biotechnology
Company and pBR322 DNA from Sangon Biotechnology Company,
Canada. Buffer A (5.0 mM tris(hydroxymethyl)aminomethane tris-
hydrochloride, 50 mM NaCl, pH 7.2) was used for absorption titra-
tion, luminescence titration, dialysis and viscosity experiments,
and buffer B (50 mM Tris–HCl, 18 mM NaCl, pH 7.2) was used for
DNA photocleavage experiments. The solution of CT-DNA in buffer
A gave a ratio of UV absorbance of 1.8–1.9:1 at 260 and 280 nm,
suggesting that the DNA was sufficiently free of protein [18]. The
concentration of DNA was determined spectrophotometrically,
lated over
a wavelength range of 200–400 nm. During the
ꢀ1
ꢀ1
equilibrium dialysis and CD studies, a blank experiment was also
carried out on the complex to ensure no obvious CD signal.
Viscosity measurements were carried out using an Ubbelohde
viscometer maintained at a constant temperature of 30.0 ± 0.1 °C
in a thermostatic bath. DNA samples with an approximate average
length of 200 base pairs were prepared by sonication in order to
minimize complexities arising from DNA flexibility [25]. The flow
time was measured with a digital stopwatch. Each sample was
measured three times and an average flow time was calculated
assuming the molar absorption was 6600 M cm
(260 nm)
[
19]. The dialysis membrane was purchased from Union Carbide
Co. and treated by general procedures prior to use [20]. All reagents
and solvents were purchased commercially and used without fur-
ther purification unless specially noted. Double distilled water was
used to prepare the buffer solutions.
2
.2. Physical measurements
0
1/3
and used. Data were presented as (
g/g
)
versus binding ratio
Elemental analyses (C, H and N) were carried out with a Perkin–
(
[Ru]/[DNA]) [26], where
g
was the viscosity of DNA in the pres-
1
Elmer 240C elemental analyzer. H NMR spectra were recorded on
0
ence of the complex and
g was the viscosity of DNA alone.
a Varian Mercury-plus 300 NMR spectrometer with DMSO-d
the solvent and SiMe as an internal standard at 300 MHz at room
temperature. Electrospray ionization mass spectrometry (ESI-MS)
was recorded on an LQC system (Finngan MAT, USA) using CH CN
6
as
4
2.5. DNA photocleavage experiments
3
During the gel electrophoresis experiments, supercoiled
pBR322 DNA (0.10 lg) was treated with a Ru(II) complex in buffer
as the mobile phase. UV–Vis and emission spectra were measured
on a Perkin–Elmer Lambda-850 spectrophotometer and an Ls55
spectrofluorophotometer, and circular dichroism (CD) spectra were
measured on a Jasco J-810 spectropolarimeter.
B, and the solution was subsequently irradiated at room tempera-
ture with a UV lamp (365 nm, 10 W) for 60 min. The samples were
analyzed by electrophoresis for 3 h at 60 V on 1.0% agarose gel in
TBE buffer (89 mM tris–borate acid, 2.0 mM EDTA, pH 8.3). The
gel was stained with EB (3,8-diamino-5-ethyl-6-phenylphenanth-
2
.3. Preparation of the ligands and complexes
ꢀ1
ridinium bromide, 1.0 lg mL ) and photographed with an Alpha
0
0
5
,5 -Dimethyl-2,2 -dipyridyl was purchased from Aldrich
Innotech IS-5500 fluorescence chemiluminescence and visible
imaging system.
0
0
1
Chemical Co. 5,5 -Dibromomethyl-2,2 -dipyridyl, L Br
2
ꢁ4H
were synthesized and characterized according to our previ-
ous procedures [21]. 1,10-Phenanthroline-5,6-dione [22], dppz
23], cis-[Ru(dppz) Cl O [24], and complex 1 [17] were pre-
2
O and
2
L Br
2
2.6. Theoretical section
[
2
2
]ꢁ2H
2
pared and characterized according to methods reported in the
literature.
Each complex is formed from a Ru(II) ion, two main ligands
1
2
(
dppz) and one ancillary ligand (L or L ). Full geometry optimiza-
2
tion computations were performed applying the DFT-B3LYP meth-
od [27–30] and LanL2DZ basis set [31,32], and assuming the singlet
state for the complexes [33]. All computations were performed
with the GAUSSIAN03 quantum chemistry program-package [34]. In
order to vividly depict the details of the frontier molecular orbital
interactions, stereographs of some related frontier molecular orbi-
tals of the complexes were drawn with the MOLDEN v4.4 program
2
.3.1. [Ru(L )(dppz)
2 6
](PF )4. (2)
A solution of cis-[Ru(dppz)
2
Cl
2
]ꢁ2H
2
O (0.26 g, 0.34 mmol) and
L2Br
3
2
(0.24 g, 0.34 mmol) in ethylene glycol (30.0 cm ) was heated
at 130 °C under the protection of argon for 6 h. In the process, the
solution turned dark red. The solution was allowed to cool down to
room temperature. After filtration, dropwise addition of saturated
4 6
NH PF resulted in a deep red precipitate, which was further fil-
[
35] based on the DFT calculational results.
tered. The solid was washed with small amounts of water and
diethyl ether, dried under vacuum, and then purified by column
chromatography on alumina using acetonitrile as the eluent. Yield:
3. Results and discussion
0
.24 g (39%). Anal. Calc. for C72
84 24 12 4
H F N P Ru (1798.44): C, 48.08;
H, 4.71; N, 9.35. Found: C, 47.83; H, 4.74; N, 9.38%. ESI-MS: m/
3.1. Synthesis
ꢀ
+
ꢀ 2+
z = 1653.3 [MꢀPF
6
]
(45), 754.3 [Mꢀ2PF
(26). H NMR (DMSO-d ) d: 9.77 (dd, 2H), 9.63 (dd,
H), 9.10 (d, 2H), 8.53 (m, 6H), 8.30 (d, 2H), 8.20 (m, 4H), 8.10
6
]
(100), 454.5
ꢀ
3+
1
[
Mꢀ3PF
6
]
6
Similar to that of complex 1, the synthetic route to complex 2 is
summarized in Scheme 1. Each synthetic step involved here was
quite straightforward and provided a moderate yield of the desired
product in a pure form. The products were characterized by ele-
2
(
2
dd, 2H), 8.04 (d, 2H), 7.91 (s, 2H), 7.85 (dd, 2H), 4.43 (s, 4H),
.98 (t, 12H), 1.48 (m, 12H), 1.05(m, 12H), 0.76 (t, 18H).

J. Sun et al. / Polyhedron 30 (2011) 1953–1959
1955
N
N
N
N
N
NH
2
2
3 2
RuCl ·3H O
N
L Br
2
N
N
O
O
N
N
Cl
NH
2
LiCl
N
N
NH
4
PF
6
N
N
N
Ru
CH
3
OH
DMF
ClOHCH
2
CH OH
N
2
Ru
N
N
N
N
N
N
N
N
N
N
(
6 4
PF )
2
Scheme 1. Synthetic route to complex 2.
1
mental analysis, H NMR and electrospray ionisation mass spectra
1.2
1
0
0
.0
.8
.6
(
ESI-MS).
1
.0
.8
3
.2. Absorption spectra
1
0.4
0
Electronic absorption spectroscopy serves as the most common
0
.2
.0
means to study the interactions between metal complexes and
DNA [18]. A complex binding to DNA through intercalation usually
results in hypochromism and bathchromism, due primarily to the
intercalation mode involving a strong stacking interaction between
an aromatic chromophore and the base pairs of DNA. It is generally
accepted that the extent of the hypochromism in a UV–Vis band is
consistent with the strength of interaction [36–38]. The absorption
spectra of complexes 1 and 2 in the absence and presence of CT-
DNA are compared in Fig. 1, and other related data are listed in Ta-
ble 1.
0
0.6
0
2
4
6
8
10
[DNA]*100000
0.4
0.2
0.0
300
400
500
600
The absorption spectra of complexes 1 and 2 exhibit similar
characters, i.e., there are three bands with comparable intensity
ranging between 250 and 550 nm. Two intra-ligand (IL) transitions
in the UV region, at 282 and 360 nm, are observed for complexes 1
Wavelength(nm)
1
1
0
0
.2
.0
.8
.6
1.0
0
.8
0.6
.4
.2
0.0
and 2, respectively. There is also a metal-to-ligand charge transfer
1
(
MLCT) in the visible region around 450 nm, likely arising from
) to dppz [39].
Ru(II) (d
p
2
0
With an increase in concentration of DNA, all the absorption
bands of the complexes display clear hypochromism, and a red
shift is observed in the MLCT band of the complexes. The hypo-
chromism (H%) of the MLCT bands in complexes 1 and 2, defined
as H% = 100% (Afree ꢀ Abound)/Afree, is determined to be about
0
0
2
4
6
8
10
[
DNA]*100000
(
11.18 ± 0.05)% and (11.04 ± 0.05)%, respectively. In order to com-
0.4
pare the DNA-binding affinities of the two complexes quantita-
tively, their intrinsic binding constants K to DNA are obtained
from monitoring the changes in the MLCT absorbance for both
complexes according to Eq. (1) [39–43], where [DNA] is the DNA
b
0
.2
.0
0
concentration in nucleotides,
M]) observed for the MLCT absorption band at a given DNA con-
centration, and and are, respectively, the extinction coeffi-
cients for the free Ru(II) complex and Ru(II) complex in the fully
a
e is the extinction coefficient (Aabs/
300
400
500
600
[
Wavelength(nm)
e
f
e
b
Fig. 1. Absorption spectra of complexes 1 and 2 in buffer A in the presence of
increasing amounts of CT-DNA. [Ru] = 10 M, [DNA] = 0–100 from top to
bottom. Arrows indicate the change in absorbance upon increasing the DNA
concentration. Inset: plot of ( )/( ) versus [DNA] and the non-linear fit for
the titration of DNA to Ru(II) complexes.
ꢀ
1
b t
bound form. K is the equilibrium binding constant in M , C is
l
lM
the total Ru(II) complex concentration and s is the binding site size.
Eq. (1) is applied to absorption titration data of non-cooperative
metallointercalators binding to CT-DNA.
e
a
ꢀ
e
f
b f
e ꢀ e
2
2
b
1=2
From the decay of the absorbance, the intrinsic binding con-
stants of complexes and are measured to be
1.9 ± 0.6) ꢃ 10 M and (0.58 ± 0.2) ꢃ 10 M , respectively. The
ð
e
a
ꢀ
e
f
Þ=ð
e
b
ꢀ
e
þ K
f
Þ ¼ ðb ꢀ ðb ꢀ 2K C
t
½DNAꢂ=sÞ =2K
b
C
t
ð1aÞ
ð1bÞ
K
b
1
2
b ¼ 1 þ K
b
C
t
b
½DNAꢂ=2s
6
ꢀ1
6
ꢀ1
(

1
956
J. Sun et al. / Polyhedron 30 (2011) 1953–1959
Table 1
6
5
4
3
2
1
6
ꢀ1
Absorption spectra (kmax/nm) and DNA-binding constants K
plexes 1 and 2.
b
(ꢃ10
M
) of com-
25
ꢀ1
Complex
k
max(free)
k
max(bound)
D
k/nm H/(%)
K
b
/106
M
s
20
15
10
1
444
3
2
453
3
2
459
364
294
455
365
293
15
4
12
2
5
11
11.18 1.9 ± 0.6
26.56
1.5 ± 0.1
60
82
1
29.30
40
80
120
160
2
11.04 0.58 ± 0.2
23.75
1.5 ± 0.2
[
DNA](μM)
60
82
28.27
b
binding constant K of complex 1 is greater than that of 2, revealing
a stronger DNA-binding affinity of 1 compared to 2. Since the two
complexes have the same main ligand, the difference comes mostly
from the ancillary ligands, where the methyl group in complex 1
gives less steric hindrance than the ethyl group in 2. The two
5
0
550
600
650
700
750
b
complexes in the current study are found to have K values larger
2
+
4
ꢀ1
Wavelength(nm)
than [Ru(phen)(dicnq)
2
]
(K
b
= 3.0 ꢃ 10 M , dicnq = 6,7-dicyan-
0
0
2+
odipyrido[2,2-d:2 3 -f]quinoxaline) [12], [Ru(bpy)(pztp)
2
]
and
2
+
4
ꢀ1
4
ꢀ1
[
[
Ru(phen)(pztp)
2
]
(K
b
= 1.4 ꢃ 10 M
and K
b
= 4.8 ꢃ 10 M
)
150
120
7
6
5
4
3
2
1
16], but smaller K
b
values than complexes having extended-aro-
2+
matic structures, such as [Ru(bpy)(pp[2,3]p)
2
]
and [Ru(phen)-
2
+
6
ꢀ1
6
ꢀ1
(
[
pp[2,3]p)
2
]
(K
b
= 3.08 ꢃ 10 M
and K
b
= 6.53 ꢃ 10 M , pp
0
0
2,3]p = pyrido[2 3 :5,6]pyrazino[2,3-f][1,10]phenanthroline) [13].
2
This is mainly because the extended-aromatic structures of the
dppz ligand increase the action between the complexes and DNA,
but the steric hindrance of quaternary ammonium cation weakens
the effect. Although a charge increase in the ligands gives no affin-
ity enhancement between the complexes and DNA, the above re-
sults indicate that the ancillary ligands (L1 or L ) directly affect
the DNA-binding affinity.
9
6
3
0
40
80
120
160
[DNA](μM)
0
0
2
3.3. Luminescence studies
0
5
50
600
650
700
750
The results of the luminescence titration for the two complexes
Wavelength(nm)
with DNA are shown in Fig. 2. Upon excitation using wavelengths
of 447 and 453 nm for complexes 1 and 2, respectively, both com-
plexes exhibit luminescence in buffer A with a maximum wave-
length of about 630 nm in the presence of CT-DNA. The
luminescence intensity of the complexes increases with the in-
crease in CT-DNA concentration and reaches a maximum at a ratio
of [DNA]/[Ru] of about 18:1. We can also derive the binding con-
stants of the two complexes interacting with DNA from the emis-
sion spectra using the luminescence titration method [44]. The
binding data obtained from the emission spectra were fitted using
the McGhee and Von Hippel equation [45] to acquire the binding
Fig. 2. Emission spectra of complexes 1 and 2 in buffer A in the presence of
increasing amounts of CT-DNA, [Ru] = 10 M, [DNA] = 0–175 M. Arrow shows the
emission intensity changes upon increasing the DNA concentration. is the
fluorescence intensity in the absence of DNA, I is the observed fluorescence
emission intensity at the given DNA concentration.
l
l
I
0
The CD spectra in the UV region 250–50 nm are shown in Fig. 3
for complexes 1 and 2 after their racemic solutions are dialyzed
against CT-DNA. As shown in the spectra, complex 2 exhibits a
stronger CD signal than 1. Although neither of the complexes is re-
solved into pure enantiomers, and it cannot be determined which
enantiomer preferentially binds to DNA for each complex, it is
rather certain that both of the complexes interact with CT-DNA
enantioselectively, while complex 2 is a better candidate for
enantioselectively binding to CT-DNA.
parameters. The intrinsic binding constants K
b
of 6.4(±0.4) ꢃ
6
ꢀ1
6
ꢀ1
1
0 M for complex 1 and 2.0(±0.6) ꢃ 10 M for complex 2 were
determined. Comparing these values with those obtained from
absorption spectra, although the binding constants obtained from
the fluorescence with the McGhee–von Hippel method are differ-
ent from those obtained from absorption, both sets of binding con-
stants show that complex 1 binds to DNA more avidly than
complex 2.
3
.5. Viscosity studies
Optical photophysical probes provide necessary, albeit not suf-
3
.4. Enantioselective binding studies
ficient, insight to support a binding model. Hydrodynamic mea-
surements, i.e. viscosity and sedimentation, that are sensitive to
length changes are regarded as the least ambiguous and most crit-
ical tests to a binding model in solution in the absence of crystal-
lographic structural data [44,47]. A classical intercalation model
usually results in lengthening of the DNA helix as base pairs are
separated to accommodate the bound ligand, leading to an in-
crease in the DNA viscosity. In contrast, semi-intercalation of a
ligand could bend or kink the DNA helix, and thus reduce its effec-
tive length and, concomitantly, its viscosity. Moreover, certain
According to the proposed binding mode [18,46], the
D-enan-
tiomer of the complex, a right-handed propeller-like structure, will
display a greater affinity for the right-handed CT-DNA helix than
the
The enantiospecific binding of complexes to DNA can be examined
clearly from circular dichroism spectra, with the presence of CD
signals indicative of enrichment in the enantiomer less favorably
binding to DNA in dialysate.
K-enantiomer, owing to the more appropriate steric matching.

J. Sun et al. / Polyhedron 30 (2011) 1953–1959
1957
1
0
1
2
3
4
1
2
-
-
-
-
Fig. 5. Photocleavage of supercoiled pBR322 DNA in the presence of complexes 1
(
lane 1–4) and 2 (lane 5–8) in 2, 4, 6, and 8
lM, respectively. All lanes are under
irradiation at 365 nm for 60 min.
E/a.u
-0.36
L+2
L+1
-
0.38
L+2
L+1
2
75
300
325
350
Wavelength(nm)
-0.40
0.42
-0.44
L
-
Fig. 3. CD spectra of complexes 1 and 2 in buffer A in the presence of CT-DNA,
Ru] = 50
L
[
l
M, [DNA] = 1.0 mM.
-
-
-
-
0.46
0.48
0.50
0.52
1
1
1
1
1
.20
.15
.10
.05
.00
H
H
H-3
H-3
1
2
EB
Fig. 6. Schematic diagram of the energies and related energy transitions of
complexes 1 and 2 (L = LUMO, H = HOMO).
2+
[Ru(bpy)3]
the other dppz ligand being left outside the helix [16]. In contrast,
complex 2 can decrease the viscosity of DNA at low binding ratios
r < 0.04) and gradually increase it upon further molar ratio (r) in-
crease. The results suggest that the binding modes of complexes 1
and 2 differ markedly, and while 1 can intercalate between DNA
base-pairs, complex 2 most likely interacts with DNA in a partially
intercalating mode [49].
(
0.00
0.02
0.04
0.06
0.08
[
EB]/[DNA] or [Ru]/[DNA]
3
.6. Photocleavage of pBR 322 DNA by Ru(II) complexes
Fig. 4. Effects of increasing amounts of complexes 1 (Ç), 2 (.), EB (j) and
2+
Ru(bpy)
3
(d) on the relative viscosities of CT-DNA at 30.0 ± 0.1 °C,
There is substantial and continuing interest in DNA endonucleo-
[
DNA] = 0.5 mM, r = [Ru]/[DNA].
lytic cleavage reactions that are activated with metal ions [50,51].
The cleavage reaction on plasmid DNA can be conveniently moni-
tored by agarose gel electrophoresis. When circular plasmid DNA is
subjected to electrophoresis, relatively fast migration will be ob-
served for the intact supercoil form (form I). If scission occurs on
one strand (nicking), the supercoil will relax to generate a
slower-moving open circular form (form II). If both strands are
cleaved, a linear form (form III) that migrates between forms I
and II will be generated. Fig. 5 shows the gel electrophoresis sepa-
ration of pBR322 DNA after incubation with complex 1 or 2 and
irradiation at 365 nm. Only a little DNA cleavage is observed for
a control study, where there is no metal complex (lane 0). For com-
complexes, such as [Ru(bpy)
electrostatic binding mode, have no influence on the DNA viscosity
48].
The changes in the relative viscosity of rod-like CT-DNA in the
]2+, which interact with DNA in an
3
[
4+
1
2
2+
presence of [Ru(L)(dppz)
2
]
(L or L ), EB and [Ru(bpy)
3
]
are
shown in Fig. 4. EB, a well-known DNA intercalator, can strongly
raise the relative viscosity by lengthening the DNA double helix
2+
3
through intercalation. On the contrary, [Ru(bpy) ] , which binds
to DNA in an electrostatic mode, exerts essentially no effect on
the DNA viscosity. As revealed in Fig. 4, upon increasing the
amounts of complex 1, the relative viscosity of DNA increases
similarly to the behavior of EB. This observation suggests that
the principal mode of DNA binding by 1 involves base-pair interca-
lation, with one dppz ligand intercalating into the base pairs and
plex 1, at a concentration of 2
the supercoiled plasmid has been converted to the nicked form;
at a concentration of 6 M (line 3), it can promote the complete
lM (line 1), approximately half of
l
conversion of DNA from Form I to Form II. However, complex 2
only promotes the complete conversion of DNA from Form I to
Table 2
I
Some frontier molecular orbital energies (e /atomic unit) of the complexes (1 atomic unit = 27.21 eV).
Complex HOMO ꢀ 8 HOMO ꢀ 7 HOMO ꢀ 6 HOMO ꢀ 5 HOMO ꢀ 4 HOMO ꢀ 3 HOMO ꢀ 2 HOMO ꢀ 1 HOMO
LUMO
LUMO + 1 LUMO + 2
1
2
ꢀ0.53475
ꢀ0.52915
ꢀ0.53463
ꢀ0.52829
ꢀ0.52606
ꢀ0.51958
ꢀ0.52315
ꢀ0.51925
ꢀ0.51989
ꢀ0.51557
ꢀ0.51833
ꢀ0.51466
ꢀ0.51820
ꢀ0.51439
ꢀ0.49747
ꢀ0.49411
ꢀ0.49734 ꢀ0.42509 ꢀ0.38944 ꢀ0.38582
ꢀ0.49398 ꢀ0.41622 ꢀ0.38138 ꢀ0.38058

1
958
J. Sun et al. / Polyhedron 30 (2011) 1953–1959
Fig. 7. Contour plots of some related frontier molecular orbital stereographs of complexes 1 and 2 using the DFT method at the B3LYP/LanL2DZ level.
Form II at a concentration of 8
ability may originate from the different DNA-binding affinity.
l
M. The difference in DNA-cleavage
molecular orbital theory [52–54]. Some frontier MO energies are
listed in Table 2. A schematic diagram of the energies and related
MLCT transitions are shown in Fig. 6, and the related orbital ster-
1
3.7. Theoretical explanation of DNA binding behavior
eographs of the two complexes are presented in Fig. 7.
As is well-established, there are p–p stacking interactions in the
The different DNA-binding behavior of the two title complexes
DNA-binding of these complexes upon intercalation (or partial
can be reasonably explained by DFT calculations and frontier
intercalation) [16], and many theoretical studies [55,56] have also

J. Sun et al. / Polyhedron 30 (2011) 1953–1959
[6] M.J. Hannon, Chem. Soc. Rev. 36 (2007) 280.
1959
shown that the DNA molecule is an electron-donor and the inter-
[
[
[
7] A.E. Friedman, J.C. Chambron, J.P. Sauvage, N.J. Turro, J.K. Barton, J. Am. Chem.
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affecting the DNA-binding affinities of the complex can usually
be considered from the planarity and plane area of the main ligand,
and the energy and population of the lowest unoccupied molecular
orbital (LUMO, and even LUMO + x, x = 1, 2, 3) of the complex mol-
ecule. A lower LUMO (and LUMO + x) energy of the complex easily
accepts electrons from the HOMO (and HOMO ꢀ x) of the DNA
base-pairs and the more population of the LUMO (and LUMO + x)
on the intercalative ligand is advantageous to the orbital interac-
tion between the LUMO (and LUMO + x) of the complex and the
HOMO (and HOMO ꢀ x) of DNA according to frontier molecular
orbital theory [53,54].
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[
1
Since complexes 1 and 2 have the same main ligand, dppz, the
planarity area of the main ligand in each case is equal. The energy
and population of the lowest unoccupied molecular orbital (LUMO,
even and LUMO + x) of the molecule can be considered as the main
factor affecting the DNA-binding affinities of the complexes. In fact,
we can see that the related frontier MO contour plots of the two
title complexes are very alike from Fig. 7, the LUMO (and LU-
MO + x) of complexes 1 and 2 are mostly distributed on the ancil-
lary ligands, and thus their LUMO (and LUMO + x) should play an
important role in accepting electrons from base pairs of DNA. From
Table 2 and Fig. 6, we can clearly see that the order of the energies
[
[
[
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of the LUMO (and LUMO + x) of the two complexes are
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MO + x must be advantageous to accepting the electrons of the
HOMO of DNA in the interaction based on the frontier MO the-
ory, the experimental trend in the DNA-binding constants (K ), i.e.
(1) > K (2), can be reasonably explained.
L L
e (2) > e (1)
e
e
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K
b
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4
. Conclusion
In this paper, we studied the DNA-binding and photocleavage
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to CT-DNA through intercalation, while 2 binds to DNA in a partial
intercalative mode. When irradiated at 365 nm, the two Ru(II)
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DFT/TDDFT calculations and frontier molecular orbital theory, the
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Appendix A. Supplementary data
[
1H NMR spectra of dppz, complex 2 and their assignments (Figs.
S1, S2, S3) are included as supplementary materials. Supplemen-
tary data associated with this article can be found, in the online
version, at doi:10.1016/j.poly.2011.04.047.
[
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