Spectral characteristics, DNA-binding and cytotoxicity of two functional Ru(ii) mixed-ligand complexes

Article abstract of DOI:10.1039/c2dt12402e

Two functional Ru(ii) mixed-ligand complexes, [Ru(phen)₂(ttbd)] ²⁺ (1) (ttbd = 4-(6-propenyl-pyrido[3,2-a]phenzain-10-yl-benzene-1,2- diamine, phen = 1,10-phenanthroline) and [Ru(bpy)₂(ttbd)] ²⁺ (2) (bpy = 2,2′-

Full text of DOI:10.1039/c2dt12402e

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PAPER
Spectral characteristics, DNA-binding and cytotoxicity of two functional
Ru(II) mixed-ligand complexes
Lifeng Tan,*a,b Jianliang Shen,a,c Jing Liu,a Leli Zeng,a Lianhe Jina and Chao Wenga
Received 12th December 2011, Accepted 13th January 2012
DOI: 10.1039/c2dt12402e
Two functional Ru(II) mixed-ligand complexes, [Ru(phen)2(ttbd)]2+ (1) (ttbd = 4-(6-propenyl-pyrido
[3,2-a]phenzain-10-yl-benzene-1,2-diamine, phen = 1,10-phenanthroline) and [Ru(bpy)2(ttbd)]2+ (2)
(bpy = 2,2′-bipyridine), have been synthesized and characterized. The spectral characteristics of
complexes 1 and 2 were investigated using fluorescence spectroscopy and revealed that both complexes
were very sensitive to solvent polarity and oxygen molecules in nonaqueous solvents. The binding
properties of the two complexes towards calf thymus DNA (CT-DNA) were investigated with different
spectrophotometric methods, viscosity measurements and quantum chemistry calculations, indicating that
both complexes could enantioselectively bind to CT-DNA by means of intercalation, but with different
binding strengths and discrimination. On the other hand, the cytotoxicity of both complexes have been
evaluated by MTT assays and Giemsa staining experiments. The main results reveal that the
hydrophobicity and surface area of the ancillary ligands have a significant effect on their DNA binding
behavior and both complexes are likely to be useful for optically probing nonaqueous and oxygen-free
environments.
understand the factors that determine the DNA binding mode are
necessary.
1. Introduction
During the past two decades, the binding of transition metal
complexes to DNA has been extensively studied.1–8 In particular,
ruthenium(II) complexes with polypyridine ligands have attracted
considerable attention9 due to a combination of their easily con-
structable rigid chiral structures, which span all three spatial
dimensions and their rich photophysical repertoire. Thus, a great
deal of attention has focused on the DNA binding mechanism of
Ru(II) polypyridyl complexes with DNA.1,9 Previous studies
have suggested that Ru(II) polypyridyl complexes can bind to
DNA by non-covalent interactions, such as electrostatic binding,
groove binding, intercalative binding and partial intercalative
binding.1 However, there is still no consensus regarding the
orientation and/or the location (major or minor groove) of the
enantiomers binding with DNA and the binding mode of the
prototype complex, [Ru(phen)3]2+ (phen = 1,10-phenanthroline),
remains an issue of vigorous debate.1,14,15 Therefore, further
studies using different structural ligands to evaluate and
On the other hand, ruthenium complexes are regarded as
promising alternatives to platinum complexes in cancer
therapies.10–12 Several ruthenium complexes have now been pro-
posed as potential anticancer substances.10 Some of the ruthe-
nium complexes demonstrate remarkable anticancer activity11,12
and the best example that describes the dependency of ruthenium
compounds on platinum drugs comes from the work on so called
RDCs (ruthenium derived complexes) by Prof. Pfeffer and co-
workers.12 In attempting to mimic cisplatin chemistry, they
obtained several compounds, which they ultimately patented as
potent cytotoxic agents for brain tumors.13 Our recent work also
showed that the Ru(II) complex, [Ru(bpy)2(hnip)]2+ {bpy = 2,2′-
bipyridine, hnip = 2-(2-hydroxy-1-naphthyl)imidazo[4,5-f][1,10]
phenanthroline}, possesses high anticancer activity against HeLa
cells.13
Herein, two new Ru(II) polypyridyl complexes, [Ru
(phen)2(ttbd)]2+ (1; phen = 1,10-phenanthroline, ttbd = 4-(6-pro-
penyl-pyrido[3,2-a]phenzain-10-yl-benzene-1,2-diamine)
and
[Ru(bpy)2(ttbd)]2+ (2; bpy = 2,2′-bipyridine), have been syn-
thesized and characterized. Their DNA binding behavior was
explored by spectroscopic titration, viscosity measurements and
thermal denaturation. Theoretical methods were used to explain
their different DNA binding affinities by applying density func-
tional theory (DFT). Additionally, their antitumor cell activities
were preliminarily evaluated by MTT {3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide} assays and Giemsa stain-
ing experiments.
aCollege of Chemistry, Xiangtan University, Xiangtan 411105,
P. R. China. E-mail: lfwyxh@yahoo.com.cn; Fax: +86 731 58292477;
Tel: +86 731 58293997
bKey Lab of Environmentally Friendly Chemistry and Application in
Ministry of Education, Xiangtan University, Xiangtan 411105,
P. R. China
cMOE Laboratory of Bioinorganic and Synthetic Chemistry, State Key
Laboratory of Optoelectronic Materials and Technologies, School of
Chemistry and Chemical Engineering, Sun Yat-sen University,
Guangzhou, 510275, P. R. China
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used instead of cis-[Ru(phen)2Cl2]·2H2O. Yield: 82%, 272 mg.
Elemental analysis (%) Calcd for C44H36N10F12O2P2Ru: C,
46.80; H, 3.22; N, 12.41; found: C, 46.69; H, 3.34; N, 12.31.
UV λmax/nm (ε/M−1 cm−1, MeCN): 414 (30 435), 293 (81 770),
2. Experimental
2.1. Materials
All solvents were of analytical reagent grade. 1,10-phenanthro-
1
261 (40 340). H NMR (400 MHz, d6-DMSO (dimethyl sulfox-
line-5,6-dione,14
cis-[Ru(bpy)2Cl2]·2H2O
and
cis-[Ru
ide); d, doublet; s, singlet; t, triplet; m, multiplet): 9.67 (m, 2H),
9.22 (s, 1H), 8.97 (d, 2H, J = 8.4), 8.90 (m, 4H), 8.75 (d, 1H, J
= 8.8), 8.64 (d, 1H, J = 9.2), 8.57 (m, 1H), 8.26 (m, 2H), 8.16
(m, 2H), 8.06 (m, 2H), 7.83 (d, 4H, J = 3.2), 7.71 (d, 2H, J =
8.8), 7.62 (t, 2H), 7.41 (t, 2H). ESI-MS (MeCN): m/z 947.5
([M–PF6]+), 401.4 ([M–2PF6]2+).
(phen)2Cl2]·2H2O were prepared according to the literature pro-
cedures.15 3,3′-Diaminobenzidine and K2PtCl4 were purchased
from Sigma Chemical Company (St. Louis, MO, USA). Double-
stranded calf thymus DNA (CT-DNA) was obtained from the
Sino-American Biotechnology Company. The Tris–HCl buffer
solution (5 mM Tris–HCl, 50 mM NaCl, pH 7.0, Tris = tris
(hydroxymethyl)aminomethane) was prepared using doubly dis-
tilled water. A solution of CT-DNA in Tris–HCl buffer gave a
ratio of UVabsorbance at 260 and 280 nm of 1.8–1.9 : 1, indicat-
ing that the DNA was sufficiently free of protein.16 The DNA
concentration per nucleotide was determined by absorption spec-
troscopy using the molar absorption coefficient (6600 M−1
cm−1) at 260 nm.17 Stock solutions were stored at 4 °C and used
within 4 days.
2.3. Physical measurements
2.3.1. General methods. Microanalyses (C, H and N) were
1
carried out on Perkin–Elmer 240Q elemental analyzer. H NMR
spectra were recorded on an Avance-400 spectrometer with d6-
DMSO (dimethyl sulfoxide) as the solvent at room temperature
and TMS (tetramethylsilane) as the internal standard. UV–vis
(UV–visible) spectra were recorded on a Perkin–Elmer Lambda-
25 spectrophotometer and the emission spectra were recorded on
a Perkin–Elmer LS-55 luminescence spectrometer at room temp-
erature. Fast atom bombardment mass spectroscopy (FAB-MS)
was performed on a VG ZAB-HS spectrometer in a 3-nitroben-
zyl alcohol matrix. Electrospray mass spectra (ES-MS) were
recorded on a LQC system (Finngan MAT, USA) using CH3CN
as the mobile phase. The spray voltage, tube lens offset, capillary
voltage and capillary temperature were set at 4.50 kV, 30.00 V,
23.00 V and 200 °C, respectively, and the quoted m/z values are
for the major peaks in the isotope distribution. Circular dichro-
2.2. Synthesis
2.2.1. Synthesis of 4-(6-propenyl-pyrido[3,2-a]phenzain-10-
yl-benzene-1,2-diamine. A mixture of 3,3′-diaminobenzidine
(0.43 g, 2.0 mmol), 1,10-phenanthroline-5,6-dione (0.42 g,
2.0 mmol) and glacial acetic acid (40 mL) was refluxed with stir-
ring for 2 h. The cooled solution was filtered and diluted with
water (10 mL) and then neutralized with concentrated aqueous
ammonia. The yellow precipitate was collected and purified by
column chromatography (CC, Alox; EtOH–toluene 3 : 1) to give
a pure yellow compound. Yield: 76%, 0.57 g. Elemental analysis
(%) Calcd for C24H20N6O2: C, 67.91; H, 4.75; N, 19.80; found:
C, 67.69; H, 4.82; N, 19.71. FAB-MS: m/z = 389.4 [M + 1].
ism (CD) spectra were measured on
spectropolarimeter.
a
JASCO-J810
2.3.2. DNA binding experiments. All DNA binding exper-
iments were performed in Tris–HCl buffer at 25 °C. The absorp-
tion titrations were performed at a fixed complex concentration,
to which the DNA stock solution was gradually added up to the
point of saturation. The mixture was allowed to equilibrate for
5 min before the spectra were recorded. The intrinsic binding
constants, Kb, of the Ru(II) complexes bound to DNAwere calcu-
lated from eqn (1):18
2.2.2. Synthesis of [Ru(phen)2(ttbd)](PF6)2·2H2O (1). A
mixture of cis-[Ru(phen)2Cl2]·2H2O (0.18 g, 0.30 mmol) and
ttbd (0.10 g, 0.30 mmol) in 20 mL of ethylene glycol was
thoroughly deoxygenated. The purple mixture was heated for 8 h
at 150 °C with stirring under an argon atmosphere, after which
time the the solution finally turned red. After it was cooled to
room temperature, an equal volume of saturated aqueous KPF6
solution was added under vigorous stirring. The red solid was
collected and washed with small amounts of water, ethanol and
diethyl ether, respectively, then dried under vacuum and purified
on a neutral alumina column with MeCN–toluene (2 : 1, v/v) as
an eluant. Yield: 74%, 0.26 g. Elemental analysis (%) Calcd for
C48H36N10F12O2P2Ru: C, 49.03; H, 3.09; N, 11.91; found: C,
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
b ꢀ ðb2 ꢀ 2Kb2Ct½DNAꢁ=sÞ
εa ꢀ εf
¼
ð1aÞ
ð1bÞ
εb ꢀ εf
2KbCt
b ¼ 1 þ KbCt þ Kb½DNAꢁ=ð2sÞ;
48.96; H, 3.21; N, 11.83. UV λmax/nm (ε/L mol−1 cm−1
,
where [DNA] is the concentration of DNA in the nucleotide
phosphate and εa, εf and εb are the apparent, free and bound
metal complex extinction coefficients, respectively. Kb is the
equilibrium binding constant in M−1, Ct is the total metal
complex concentration and s is the average binding size. When
plotting (εa − εb)/(εf − εb) vs. [DNA], Kb is given by the ratio of
the slope to the intercept.
Viscosity measurements were carried out using an Ubbelohde
viscometer maintained at a constant temperature of 25 0.1 °C
in a thermostatic bath. DNA samples of ca. 200 bp average
length were prepared by sonication.19 The flow time was
measured with a digital stopwatch and each sample was tested
MeCN): 405 (28 000), 311 (47 500), 262 (75 000). 1H NMR
(400 MHz, ppm, d6-DMSO (dimethyl sulfoxide); d, doublet; s,
singlet; m, multiplet.): 9.67 (t, 2H), 9.21 (s, 1H), 8.97 (d, 2H,
J = 8.4), 8.82 (t, 4H), 8.74 (d, 1H, J = 8.8), 8.43 (s, 4H), 8.31
(s, 2H), 8.24 (t, 2H), 8.08 (d, 2H, J = 5.6), 7.97 (m, 2H), 7.82
(m, 6H). ESI-MS (m/z, positive-ion mode, MeCN): 995.2
([M–PF6]+), 425.5 ([M–2PF6]2+).
2.2.3. Synthesis of [Ru(bpy)2(ttbd)](PF6)2·2H2O (2). The
procedure to prepare complex 2 was similar to that of complex 1,
except that cis-[Ru(bpy)2Cl2]·2H2O (130 mg, 0.25 mmol) was
4576 | Dalton Trans., 2012, 41, 4575–4587
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2.5. Cytotoxicity assays
three times to get an average calculated time. Data were pre-
sented as (η/η0)1/3 vs. the binding ratio,20 where η is the viscosity
of DNA in the presence of the complex and η0 is the viscosity of
free DNA.
Standard MTT assay procedures26 were performed using the
methodology described in detail previously.13 Two different
tumor cell lines (HeLa and HepG2) were the subjects of this
study. To study the apoptosis of the cancer cells induced by
either complex 1 or 2, HepG2 cells treated with the compounds
for 48 h were stained with Giemsa and then observed by
microscopy.
Thermal DNA denaturation experiments were carried out with
a Perkin–Elmer Lambda 850 spectrophotometer equipped with a
Peltier temperature-control programmer ( 0.1 °C). The tempera-
ture of the solution was increased from 50 to 95 °C at a rate of
1 °C min−1 and the absorbance at 260 nm was continuously
monitored for solutions of CT-DNA (42 μM) in the absence and
presence of the Ru(II) complex (20 μM). The data were presented
as (A − A0)/(Af − A0) vs. the temperature, where Af, A0 and A are
the final, initial and observed absorbances at 260 nm,
respectively.
3. Results and discussion
3.1. Synthesis and characterization
The synthetic routes to ttbd and its Ru(II) complexes, 1 and 2,
are presented in Scheme 1. The ligand, ttbd, is synthesized by
condensation of 1,10-phenanthroline-5,6-dione with the appro-
priate mole ratio of 3,3′-diaminobenzidine on the basis of a
method used for the preparation of pyrazine rings.27 Using ethyl-
ene glycol as a solvent, complexes 1 and 2 were then prepared
by direct reaction of ttbd with the appropriate mole ratios of the
precursor complexes, cis-[Ru(phen)2Cl2]·2H2O and cis-[Ru
(bpy)2Cl2]·2H2O, and obtained in yields of 76% and 74%,
respectively. The desired Ru(II) complexes were isolated as their
hexafluorophosphates and then purified by column chromato-
graphy. Both complexes were characterized by elemental analy-
sis, mass spectroscopy and NMR spectroscopy. In the ESI-MS
spectra of complexes 1 and 2 two signals of [M–PF6]+ and
[M–2PF6]2+ were observed and the determined molecular
weights were consistent with the expected values.
Equilibrium dialyses were conducted with 10 mL of CT-DNA
(1.0 mM) sealed in a dialysis bag and 10 mL of the complex
(50 μM) outside the bag with stirring of the solution for 36 h at
25 °C.
2.4. Theoretical calculations
The structural schemes of complexes 1 and 2 are shown in
Scheme 1. Each of them forms from the Ru(II) ion with one
main (intercalative) ligand (ttbd) and two ancillary ligands
(either phen or bpy). Full geometry optimization computations
were performed by applying the DFT-B3LYP method and
Land2DZ basis set21,22 and assuming a single state for the
ground state of the complexes.23 All computations were per-
formed with the G98 quantum chemistry program package.24 To
vividly depict the details of the frontier molecular orbital inter-
actions, the stereographs of some related frontier molecular orbi-
tals of the complexes were drawn with the Molden v3.7
program25 based on the obtained computational results.
Complexes 1 and 2 give well defined 1H NMR spectra
(Fig. 1), which further permit unambiguous identification and
assessment of their purity. In comparison to those of similar
Scheme 1 Synthesis of the ligand, ttbd, and its Ru(II) complexes, 1 and 2.
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compounds,28 the proton chemical shifts were assigned by allow-
ing for the influence of the steric, inductive and conjugative
effects. For each of the complexes, two sets of NMR signals
were observed. Because of the shielding influences of the adja-
cent ttbd and bpy (or phen) moieties, the two halves of each
phen are not chemically and magnetically equivalent, which
leads to eight signals that correspond to the bpy (or phen)
protons: one set of four is associated with the bpy (or phen) half
near the ttbd and the other set of four is associated with the bpy
(or phen) portion near the other bpy (or phen). Since the shield-
ing effect of ttbd is obviously greater than that of bpy (or phen),
the chemical shifts of the latter protons appear more downfield
than those of the former.
attributed to intraligand π → π* transitions.29 The lowest energy
band, at about 410 nm, is assigned to the metal–ligand charge
transfer (MLCT) transition, which is attributed to Ru(dπ) → ttbd
(π*) transitions. Obviously, this band is blue shifted in compari-
son to those of [Ru(phen)3]2+ (λmax = 448 nm) and [Ru(bpy)3]2+
(λmax = 452 nm), which may be due to the increased π delocali-
zation and, thus, the π-acceptor capacity of the ttbd ligand,
resulting in a decreased electron density on the central Ru(II)
and, in turn, stabilization of the metal dπ orbital.
3.2.2. Emission spectra. There has been increasing demand
for the design and development of transition metal complexes
that can act as luminescent probes for use in various environ-
ments.1 Complexes 1 and 2 showed no emission in aqueous sol-
utions at 25 °C in air. In contrast, distinct photoluminescence
behavior was observed for complexes 1 and 2 in nonaqueous sol-
vents, with emission maxima shifting over a range of ∼30 nm
around the generic ∼665 nm peak in air at 25 °C. In addition,
the emission spectra are somewhat solvatochromic (Fig. 2), indi-
cating that the more polar the solvent is, the smaller the relative
intensity is.30
3.2. Spectral characteristics
3.2.1. Absorption spectra. The absorption spectra of com-
plexes 1 and 2, upon dissolution in various nonaqueous solvents
(DMF, DMSO, MeCN and acetone), are very similar and consist
of three well resolved bands at about 410, 310 and 260 nm,
respectively. The bands at about 310 nm and 260 nm are
Fig. 1 The 1H NMR aromatic regions in the spectra of complexes 1 (top) and 2 (bottom) in d6-DMSO (400 MHz).
4578 | Dalton Trans., 2012, 41, 4575–4587 This journal is © The Royal Society of Chemistry 2012

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Notably, the luminescence of complexes 1 and 2 in organic
solvents is very sensitive to oxygen molecules (Table 1 and
Fig. 3). If air is removed from the solution of complex 1 (a nitro-
gen atmosphere), for example, a greater relative intensity is
observed; while if the oxygen-free system is replaced with air,
the greater relative luminescent intensity observed in the absence
of oxygen is brought back to the original level. This indicates
that oxygen-free radicals may be involved in the process of
complex light emission. Thus, both complexes can serve as
“oxygen molecule light switch” complexes. In particular, by the
successive introduction of nitrogen and air atmospheres a
cycling oxygen molecule light switch (with an “off and on”
effect) can be accomplished using both complexes . On the other
hand, the addition of low concentrations of H2O to both com-
plexes dissolved in nonaqueous solvents leads to their lumines-
cence being almost quenched. Fig. 4 shows the progressive
decrease in the emission intensity of complex 1 in MeCN upon
the addition of H2O. This result may indicate that the emission
quenching of the ruthenium complex proceeds via hydrogen
bonding to nearest-neighbor water molecules and may explain
the lack of emission for both complexes in aqueous solutions.31
Thus, the emission of both complexes is very sensitive to the
local environment and both complexes are likely to be useful for
the optical probing of nonaqueous and oxygen-free
environments.
Fig. 3 The fluorescence spectra of complex 1 (a) and 2 (b) (2 μM) in
MeCN at 25 °C in air-saturated solutions and oxygen-free solutions,
respectively.
Fig. 2 The fluorescence spectra of complex 1 (20 μM) in CH3CN,
DMF and DMSO at 25 °C.
Table 1 The emission characteristics of complexes 1 and 2 (2 μM) in nonaqueous solvents at 25 °C
Solvent
CH3CN
λmaxem, nm
Aira
DMF
DMSO
λmaxem, nm
Aira
I (a.u.)
λmaxem, nm
Aira
I (a.u.)
I (a.u.)
b
b
b
b
b
b
Complex
N2
Aira
N2
N2
Aira
N2
N2
Aira
N2
1
2
666
654
649
627
17
14
37
40
682
662
660
653
7
12
15
23
697
672
620
636
3
6
8
10
a, b The photoluminescence was determined for both complexes in air-saturated solutions and oxygen-free solutions.
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Fig. 4 The fluorescence spectra of complex 1 (2 μM) in MeCN at
25 °C in the absence (dotted line) and presence (solid lines) of water.
Increasing amounts of water up to 200 μL were added. The arrow shows
the intensity changes upon the addition of increasing amounts of water.
3.3. Electronic absorption titration of Ru(II) complexes and
DNA
The application of electronic absorption spectroscopy in DNA
binding studies is one of the most useful techniques.32 Complex
binding with DNA via intercalation usually results in hypochro-
mism and bathochromism, due to the intercalative mode invol-
ving
a strong stacking interaction between the aromatic
chromophore and the base pairs of DNA. The extent of the
hypochromism and red shift is commonly related to the intercala-
tive binding strength. The absorption spectra of complexes 1 and
2 in the absence and presence of CT-DNA are given in Fig. 5.
As the DNA concentrations were gradually increased, the
absorption spectra of complexes 1 and 2 showed significant per-
turbation. For complex 1, the hypochromism in the MLCT band
reaches about 33% at 415 nm with a red shift of 12 nm at a
[DNA]–[Ru] ratio of 1.84. With an increasing DNA concen-
tration, complex 2 showed a hypochromism of about 22% in the
MLCT band (414 nm) with a red shift of 7 nm at a [DNA]–[Ru]
ratio of 1.58. Comparing the hypochromism and red shifts of
both complexes with that of [Ru(phen)3]2+ (12% hypochromism
for the MLCT band at 445 nm and a 2 nm red shift),33 which
interacts with DNA through a semi-intercalation or quasi-inter-
calation,34 and [Ru(bpy)3]2+, which is a typical electrostatic
binding complex whose absorption was demonstrated to be
unchanged upon the addition of DNA,35 indicates that these
spectral characteristics suggest that complexes 1 and 2 interact
with DNA. This most likely proceeds through a mode that
involves a stacking interaction between the aromatic chromo-
phore and the base pairs of DNA.
Fig. 5 The absorption spectra of complexes 1 (a) and 2 (b) in Tris–
HCl buffer upon addition of CT-DNA at 25 °C. [Ru] = 20 μM, [DNA] =
(0–42) μM. The arrows show the absorbance change upon an increasing
DNA concentration. Insets: the plots of [DNA] − (εa − εf) vs. [DNA]
for the titration of DNAwith complexes 1 and 2.
(1.16 0.24) × 106 M−1 (s = 0.24 0.01), respectively. The Kb
values of both complexes are close to that of [Ru(phen)
(ppd)2]2+ with two intercalative ligands (1.55 × 106 M−1),36 but
smaller than those of [Ru(phen)2(dppz)]2+ and [Ru
(bpy)2(dppz)]2+ (dppz = dipyrido-[3,2-a-2′,3′-c]phenazine, >106
M−1), which are DNA intercalative Ru(II) complexes.37
However, the Kb values of complexes 1 and 2 are stronger than
those of other typical DNA intercalative Ru(II) complexes (1.1 ×
104 – 4.8 × 104 M−1 38
and are also stronger than the Kb values
)
of these Ru(II) complexes with two intercalative ligands, e.g. [Ru
(phen)(dicnq)2]2+ (dicnq = 6,7-dicyanodipyrido[2,2-d-2′,3′-f]
quinoxaline, 3.0 × 104 M−1),39 [Ru(phen)(pztp)2]2+ (4.8 × 104
M−1) and [Ru(bpy)(pztp)2]2+ (1.4 × 104 M−1).40 In particular,
the intrinsic binding constants of complexes 1 and 2 are much
To quantitatively compare the binding affinity of complexes 1
and 2 towards DNA, the intrinsic binding constants, Kb, were
determined to be (1.61 0.34) × 106 M−1 (s = 0.42 0.01) and
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stronger than those of their parent complexes, [Ru(bpy)3]2+
(4.7 × 103 M−1) and [Ru(phen)3]2+ (5.5 × 103 M−1),41 which
can be explained as follows: firstly, the planarity area (S) of the
intercalated ligand is Sttbd > Sphen/Sbpy; secondly, the intercalated
ligand, ttbd, contains two free amino groups, which may form
intermolecular hydrogen bonds with the base pairs. These two
factors are advantageous for the binding of complexes 1 and 2
with DNA. These results also indicate that the size and shape of
the intercalated ligand has a significant effect on the strength of
DNA binding and the most suitable intercalating ligand leads to
the highest affinity of the complexes for DNA. Additionally,
from these results, we could deduce that both 1 and 2 bind to
DNA by intercalation and their different DNA binding properties
may be due to the ancillary ligands. Comparing phen and bpy, it
is clear that the hydrophobicity and the surface area decrease in
bpy, resulting in a weaker DNA binding affinity for complex 2.
3.4. Competitive binding
Luminescence spectroscopy is one of the most common and sen-
sitive methods to analyze drug–DNA interactions. Support for
the aforementioned intercalative binding mode also comes from
the emission measurements of both complexes. Unfortunately, in
Tris–HCl buffer at 25 °C, both complexes showed no fluor-
escence emission, neither alone nor in the presence of CT-DNA.
The mechanism of the “light switch” effect for [Ru
(bpy)2(dppz)]2+ (dppz = dipyrido[3,2-a-2′,3′-c]phenazine) and
[Ru(phen)2(dppz)]2+ has been intensively studied and all of the
evidence points to hydrogen bonding and/or excited-state hydro-
gen atom transfer to the phenazine nitrogen atom as the mechan-
ism of deactivation of the complexes’ excited state.42 Upon
intercalation, DNA provides the metal-bound dppz ligand with a
hydrophobic environment, which in turn protects the Ru(II)
complex from the quenching effect of water.42 Comparing com-
plexes 1 and 2 with their parent complexes, [Ru(bpy)2(dppz)]2+
and [Ru(phen)2(dppz)]2+, may explain why they could not serve
as molecular “light switches” for DNA. The main reason may be
that when the ligand, ttbd, intercalates into the DNA helix, with
either the two nitrogen atoms on the pyrazine ring or the two
amino groups on the benzene ring, outside the DNA helix. In
this case, they would not be protected by DNA and could still
form hydrogen bonds with solvent water molecules, resulting in
the fluorescence emission of both complexes being fully
quenched by the solvent water molecules.
A steady-state competitive binding experiment using either
complex 1 or 2 as the quencher may afford further information
about the binding of the complex to DNA. Ethidium bromide
(EB) emits intense fluorescence light in the presence of DNA,
due to its strong intercalation between adjacent DNA base pairs.
It was previously reported that the enhanced fluorescence can be
quenched, at least partially, by the addition of a second mol-
ecule.43 The extent of fluorescence quenching an EB–DNA
system is used to determine the extent of binding of a second
molecule to DNA. The emission spectra of EB–DNA in the
absence and presence of both complexes are shown in Fig. 6.
Fig. 6 indicates that, upon addition of either complex 1 or 2 to
the EB–DNA system, an appreciable reduction in the emission
intensity of about 84% for complex 1 and 82% for complex 2 at
Fig. 6 The emission spectra of EB bound to DNA in the presence of
complex 1 (a, λex = 458 nm) and 2 (b, λex = 457 nm) at 25 °C. [EB] =
20 μM, [DNA] = 100 μM; [Ru]–[DNA] = 0.00, 0.04, 0.08, 0.12 and
0.16. The arrows show the intensity changes upon an increasing concen-
tration of the complexes. Inset: the plots of I0/I vs. [Ru]–[DNA].
a [Ru]–[DNA] ratio of 0.16 occurred. This indicates that both
complexes could compete with EB in binding to DNA and
complex 2 binds to DNA slightly stronger than complex 1.
Additionally, the quenching plots (inset in Fig. 5) illustrate that
the removal of EB bound to DNA by either complex 1 or 2 is
non-linear and not in agreement with the linear Stern–Volmer
equation, which implies that the competitive binding process is
both dynamic and static.44
3.5. Metal ion titration
The above discussion mentions that both complexes show no flu-
orescence emission even in the presence of DNA. Considering
the structure of the intercalative ligand, ttbd, it’s obvious that
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either the two nitrogen atoms on the pyrazine ring or the two
amino groups on the benzene ring (but not both) are inside the
DNA helix when the complex binds with DNA. Additionally, if
the two amino groups in ttbd are outside the DNA helix when
the complex binds, the two amino groups become coordination
sites and can bind with other metal cations, such Pt2+ and Cu2+.
Thus, in the absence and presence of DNA, a foreign metal ion
may disturb the absorption of the complexes to some extent,
affording further information to explain the lack of luminescence
for complexes 1 and 2 in the presence of DNA. Therefore, metal
ion titration was carried out with complex 1 as the representative
example.
[PtCl4]2− into the solution of DNA-free complex 1, a gradual
decrease in the absorption intensity is observed as a function of
[PtCl4]2−, which indicates that [PtCl4]2− coordinates to ttbd. The
decrease reaches a maximum at a [Ru]–[ PtCl4] ratio of 1 : 1
when essentially all of complex 1 is bound to [PtCl4]2−, reflect-
ing that the coordination ratio of [PtCl4]2− to complex 1 is
1. Note that the introduced [PtCl4]2− species may not coordinate
to the two nitrogen atoms on the pyrazine ring because of the
stereo-hindrance effect. In this case, the coordination ratio of
[PtCl4]2− to complex 1 is beyond 1. In other words, [PtCl4]2−
binding to the two nitrogen atoms on the pyrazine ring may not
occur. As Cu2+ has a relatively small size, it was used to further
confirm that the coordination site presented by the two amino
groups is the preferential site of metal ion coordination compared
to the two nitrogen atoms on the pyrazine ring. Similarly, the
Cu2+ titration into the solution of DNA-free complex 1 indicated
that the maximal decrease in the hypochromism also occurred at
a ratio of ca. 1 : 1 [Ru(phen)2(ttbd)2+]–[Cu2+], which further
suggests that the two nitrogen atoms on the pyrazine ring are not
the preferential coordination sites.
The absorption spectra for the titration of complex 1 with
[PtCl4]2− is presented in Fig. 7. Clearly, upon addition of
The absorption spectra in Fig. 8 show DNA-bound [Ru
(phen)2(ttbd)]2+ titrated with and without either [PtCl4]2− or
Cu2+. Clearly, no obvious decrease in absorption intensity of the
DNA-bound [Ru(phen)2(ttbd)]2+ system upon addition of either
[PtCl4]2− or Cu2+ is observed, which further affirms that the two
amino groups on the benzene ring are inside the DNA helix,
suggesting intercalation as a probable binding mode of both
complexes toward DNA. It is apparent that the two amino
groups, as the coordinated sites, become unavailable in the pres-
ence of DNA. Therefore, the reason why complexes 1 and 2
show no fluorescence emission in the presence of CT-DNA is
that when both complexes bind to DNA, the two nitrogen atoms
on the pyrazine ring are outside the DNA helix and can still
form hydrogen bonds with water molecules, resulting in the flu-
orescence emission of both complexes being fully quenched by
the water molecules.
3.6. Viscosity measurements
Further clarification of the interaction between the complexes
and DNA was carried out by viscosity measurements. Photophy-
sical probes provide necessary, but not sufficient, clues to
support a binding model. Hydrodynamic measurements that are
sensitive to the length change (i.e. viscosity and sedimentation)
are regarded as the least ambiguous and most critical tests of the
binding model in solution, in the absence of crystallographic
structural data.51 A classical intercalation model results in
lengthening of the DNA helix as the base pairs are separated to
accommodate the binding ligand, which leads to an increase in
the viscosity of DNA. However, a partial and/or non-classical
intercalation of the complex, such as with [Ru(phen)3]2+, may
bend (or link) the DNA helix and reduce its effective length and,
concomitantly, its viscosity.45 In addition, some complexes, such
as [Ru(bpy)3]2+, which interacts with DNA by an electrostatic
binding mode, have no obvious influence on DNAviscosity.46
The effects of complexes 1 and 2, together with [Ru(bpy)3]2+
and EB, on the viscosity of rod-like DNA are shown in Fig. 9.
EB, a well known DNA intercalator, results in a strong change in
Fig. 7 The absorption spectra of complex 1 (20.0 μM) in Tris–HCl
buffer with and without metal ions added at 25 °C. (a) With and without
20.0 μM PtCl4 added. (b) With and without 20.0 μM Cu2+− added.
2−
The spectra were collected after 5 min at 298 K. A reagent blank con-
taining 5 mM Tris and 20.0 μM PtCl42− or Cu2+ was used.
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Fig. 9 The effect of increasing amounts of EB (○), [Ru(bpy)3]2+ (■),
complex 1 (▼) and complex 2 (▲) on the relative viscosity of
CT-DNA at 25 0.1 °C. The total concentration of DNA is 0.5 mM.
leading to a greater DNA binding affinity for complex 1. Thus,
complex 1 is probably more deeply intercalated and more tightly
bound to adjacent DNA base pairs than complex 2, which is con-
sistent with the foregoing hypothesis.
3.7. Thermal denaturation study
DNA melting experiments are useful to establish the extent of
intercalation because the intercalation of the complex into DNA
base pairs causes stabilization of the base stacking and, therefore,
raises the melting temperature of double-stranded DNA.47 It is
well known that, when the temperature of the solution increases,
double-stranded DNA gradually dissociates into single strands,
which generates a hyperchromic effect on the absorption spectra
of the DNA bases (λmax = 260 nm). To identify this transition
process, the melting temperature, Tm, which is defined as the
temperature when half of the total base pairs are unbonded, is
usually introduced. According to a previous report,48 the interca-
lation of a complex into DNA generally results in a considerable
increase in the Tm. The melting curve of CT-DNA in the absence
and presence of either complex 1 or 2 is presented in Fig. 10.
Here the Tm of metal complex-free CT-DNA was determined to
be 75.11 0.16 °C. Upon increasing the concentration of either
complex 1 or 2, the Tm increases successively and reaches 81.20
0.11 °C and 83.49 0.20 °C, respectively, at a [Ru]–[DNA]
ratio of 10 : 21. The ΔTm values (6.09 °C and 8.28 °C) of
complex 1/2–DNA adducts are bigger than those of some Ru(II)
intercalators,49 but smaller than those of DNA-intercalating Ru
(II) polypyridyl complexes.6,7,49,50 However, the increases
(6.18 °C and 8.50 °C) in Tm are comparable to those observed
for classical intercalators,51 which reveals that the modes of both
complexes’ binding with DNA are intercalation.
Fig. 8 The absorption spectra of complex 1–DNA ([DNA]–[Ru] = 1.9,
[Ru] = 20 μM) in Tris–HCl buffer with and without metal ions added.
2−
(a) With and without 20.0 μM PtCl4 added. (b) With and without
20.0 μM Cu2+− added. The spectra were collected after 5 min at 25 °C.
A reagent blank containing 5 mM Tris–HCl buffer and 42 μM DNA was
used.
the DNA viscosity upon complexation and, upon increasing
amounts of either complex 1 or 2, the relative viscosity of DNA
increases steadily, similar as in the case of EB. The increase in
the relative viscosity, which is expected to correlate with the
compounds DNA intercalating potential, followed the order EB
> 1 > 2 > [Ru(bpy)3]2+. The result suggests that both complexes
bind to DNA through intercalation and the difference in the
binding strength is probably caused by the different ancillary
ligand. Comparing bpy to phen, it is clear that the surface area
and the hydrophobicity of the ancillary ligand increase in phen,
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Fig. 10 Melting temperature curves of DNA in the absence (○) and
presence of complex 1 (●) and complex 2 (▲). [Ru] = 20 μM,
[DNA] = 42 μM.
Fig. 11 Th CD spectra of complexes 1 (solid line) and 2 (dotted line)
after 32 h of dialysis against CT-DNA in stirred aqueous solutions at
25 °C. [Ru] = 20.0 μM, [DNA] = 1.0 mM.
3.8. Equilibrium-dialysis experiments
Equilibrium-dialysis experiments may offer the opportunity to
examine the enantioselectivity of complexes binding to DNA.52
The CD spectra in the UV region of complexes 1 and 2, after its
racemic solution has been dialysed against CT-DNA, are shown
in Fig. 11. The dialysate of complex 1 (solid line) shows a posi-
tive peak at 289 nm and a negative peak at 313 nm, while the
dialysate of complexes 2 (dotted line) shows a positive peak at
279 nm and a negative peak at 304 nm, indicating that both com-
plexes can interact enantioselectively with CT-DNA. On the
other hand, the stronger CD signal of complex 1 suggests a large
DNA binding discrimination between its two antipodes. Since
the intercalative ligands of complexes 1 and 2 are the same, the
difference should, again, be attributable to the ancillary ligands
and, more precisely, to the different hydrophobicity and surface
area of the phen and bpy moieties. These results indicate that the
hydrophobicity and surface area of the ancillary ligands have a
significant effect on the DNA binding discrimination.
titration indicate that the DNA binding affinity of complex 1 is
greater than that of complex 2, which further suggests that the
LUMO energy is not a decisive factor having an effect on the
affinities of complexes 1 and 2 and their binding with DNA.
Since the intercalative ligands of complexes 1 and 2 are the
same, the difference in the DNA binding affinity should be
attributed to the ancillary ligand effects. Clearly, one is the
hydrophobic effect of the ancillary ligands and the other is the
surface area of the ancillary ligands. The hydrophobicity and the
surface area of phen in complex 1 are greater than those of bpy
in complex 2. The results may reflect the two factors playing a
more important role than the LUMO and NLUMO energies of
the metal complex in the DNA binding affinity.54 In consider-
ation of these factors, the difference in the DNA binding affinity
of complexes 1 and 2 can be well understood.
3.10. In vitro cytotoxicity
3.9. A theoretical explanation for the difference in DNA
binding of complexes 1 and 2
MTT experiments were performed on two cancer cell lines,
HeLa and HepG2, using cis-Pt(NH3)2Cl2 as the control.6 The
IC50 values of complexes 1 and 2 are presented in Table 3,
which indicate that HepG2 cells are more sensitive to complexes
1 and 2 than the HeLa cancer cell line. In particular, the IC50
value of complex 1 against HepG2 cells is close to that of cis-Pt
(NH3)2Cl2.6 Notably, cis-Pt(NH3)2Cl2 exerts its cytotoxic effects
through covalently binding to DNA to form cis-DDP–DNA
adducts, which interferes with the DNA replication and tran-
scription and ultimately induces cell death.55 We speculate that
the anticancer activity of complexes 1 and 2 may not only be
related to intercalation, but also related to the specific molecular
shape of the complex, the chemical structure and the nature of
the intercalative ligand.
It has been documented that the LUMO energy of a metal
complex is an important factor (but not the only factor) in deter-
mining the DNA binding constant because a lower LUMO
energy of a metal complex is advantageous for an interaction
between the intercalative ligand and the double helical DNA.53
DFT calculations show that complex 2 is thermodynamically
more unstable than complex 1 since the computed total energy
of complex 1 is lower than that of complex 2 (see Table 2). In
contrast, the LUMO and NLUMO energies of complex 2 are
lower than those of complex 1 to some extent (see Table 2 and
Fig. 12–14), which is favorable for the electron flow from the
base pairs of DNA to the metal complex. Thus, the greater
LUMO energy of complex 2 is surely not advantageous to its
DNA binding affinity. In contrast, the above absorption spectra
To further study the apoptosis of the cancer cells induced by
complexes 1 and 2, Giemsa staining experiments were carried
4584 | Dalton Trans., 2012, 41, 4575–4587
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Table 2 Some frontier molecular orbital energies (εi/atomic unit) of complexes 1 and 2 (1 atomic unit = 27.21 eV)
c
d
Compound
H-2
NHOMOa
HOMO
LUMO
Virb
ΔεL-H
ΔεL-NH
ΔεL-(H-2)
ETotal
1
2
−0.3769
−0.3764
−0.3396
−0.3392
−0.3014
−0.3001
−0.2608
−0.2640
−0.2576
−0.2613
0.0406
0.0361
0.0788
0.0752
0.1161
0.1124
−2489.2155
−2308.4489
a Occ = occupied molecular orbital; HOMO (or H) = the highest Occ. NHOMO (or NH) = the next HOMO (or H-1). b Vir = virtual molecular orbital;
LUMO (or L) = the lowest Vir. c ΔεL–H = energy difference between the LUMO and HOMO, etc. d ETotal = the total energy of the complex.
out on HepG2 cells. Fig. 15 indicates that, similar to those cells
treated with cis-Pt(NH3)2Cl2, the majority of the HepG2 cells
treated with either complex 1 or 2 displayed the classic morpho-
logical features of apoptosis after exposure to 20 μM of the com-
plexes, including nuclear condensation, cell shrinkage,
cytoplasmic concentration, which is indicated by a dark red
color, and the formation of apoptotic bodies, which is indicated
by a purple color (Fig. 15c, d). In contrast, no obvious changes
were observed in the control cells (Fig. 15a). The results suggest
that both complexes can induce HepG2 cell death in viable cell
numbers, partially through induction of HepG2 cells apoptosis.
Further in detail studies are under-way to quantitatively deter-
mine the effect on cell apoptosis.
4. Conclusions
In conclusion, two novel Ru(II) complexes, [Ru(phen)2(ttbd)]2+
(1) and Ru(bpy)2(ttbd)]2+ (2), have been synthesized and charac-
terized. The photoluminescence of both complexes were very
sensitive to solvent polarity and oxygen molecules in nonaqu-
eous solvents and, by cycling the environment between oxygen-
1
Fig. 12 A schematic diagram of the energies and related MLCT tran-
sitions of complexes 1 and 2.
Fig. 13 Some related frontier molecular orbital stereographs of complexes 1 and 2.
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rich (air) and oxygen-deficient (nitrogen), an “off/on light
switch” could be accomplished for both complexes, revealing
that both complexes are likely to be useful in optically probing
nonaqueous and oxygen-free environments. Competitive binding
and viscosity studies yield convincing evidence for the true inter-
calative binding of both complexes to CT-DNA, while complex
1 possesses a greater binding affinity with DNA than complex 2,
as inferred by spectroscopic studies and DFT, which reveal that
the surface area and the hydrophobicity of the ancillary ligand
have a significant effect on the DNA binding. The IC50 value of
complex 1 against HepG2 cells is close to that of cis-Pt
(NH3)2Cl2, and both complexes can induce HepG2 cell apopto-
sis. These results further advance our knowledge of the inter-
action of metal complexes with DNA and may be useful in
developing DNA-targeted therapeutics, as well as optical probes
for nonaqueous and oxygen-free environments.
Fig. 14 The DFT-optimized structures and visualization of the orbitals
of complexes 1 (left) and 2 (right).
Acknowledgements
The authors are grateful for the support of the National Natural
Science Foundation of the People’s Republic of China
(21071120) and the Scientific Research Foundation of Hunan
Provincial Education Department (11A117). We also thank the
reviewers for their comments that enabled us to improve the
manuscript.
Table 3 IC50 values of cisplatin and complexes 1 and 2 towards
different cell lines determined by MTT assay following exposure of 72 h
IC50 (μM)
Complex
HeLa
HepG2
References
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1
2
5.85 0.60
11.10 0.98
13.85 0.68
11.18 2.48
12.67 1.15
14.55 1.33
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Dalton Trans., 2012, 41, 4575–4587 | 4587