Synthesis, characterization, and DNA binding of complexes [Pt(bpy)(pip)]2+ and [Pt(bpy)(hpip)]2+

Article abstract of DOI:10.1007/s00775-013-0991-7

Two new platinum(II) complexes, [Pt(bpy)(pip)](NO₃)₂ (1) and [Pt(bpy)(hpip)](NO₃)₂·2H₂O (2) (bpy is 2,2′-bypyridine; pip is 2-phenylimidazo[4,5-f][1,10] phenanthroline; hpip is 2-(2-hydroxyphenyl) imidazo[4,5-f][1,10]phenanthroline), have been synthesized and fully characterized by CHN analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, ¹H-NMR spectroscopy, IR spectroscopy (attenuated total reflection), and UV-vis spectroscopy. The DNA-binding behaviors of both complexes have been studied by spectroscopic methods and viscosity measurements, and their ability to inhibit DNA transcription was measured. The results indicate that both complexes show some degree of binding to DNA in an intercalative mode, resulting in intrinsic binding constants of (2.88 ± 0.4) × 10⁴ and (5.38 ± 0.8) × 10⁴ for 1 and 2, respectively. The comparatively observed difference in the DNA-binding affinities of the two complexes can be reasonably explained by the presence of intramolecular hydrogen bonding between the ortho phenolic group and the nitrogen atom of the imidazole ring. The extended coplanarity of the hpip ligand due to intramolecular hydrogen bonding may lead to an enhancement of the DNA-binding affinity of the hpip complex. In addition, the complexes can promote photocleavage of pUC19 DNA on irradiation as revealed by the spectroscopic and viscometric measurements, with 2 promoting cleavage of pUC19 DNA at lower concentration. Moreover, increasing concentrations of both complexes inhibited DNA transcription, and as expected 2 was shown to be a better antitumor agent than 1.

Full text of DOI:10.1007/s00775-013-0991-7

J Biol Inorg Chem
DOI 10.1007/s00775-013-0991-7
ORIGINAL PAPER
Synthesis, characterization, and DNA binding of complexes
[Pt(bpy)(pip)]2+ and [Pt(bpy)(hpip)]2+
•
Burak Coban Ufuk Yildiz Abdurrahman Sengul
•
Received: 6 November 2012 / Accepted: 22 February 2013
Ó SBIC 2013
Abstract Two new platinum(II) complexes, [Pt(bpy)(pip)]
(NO3)2 (1) and [Pt(bpy)(hpip)](NO3)2ꢀ2H2O (2) (bpy is 2,20-
bypyridine; pip is 2-phenylimidazo[4,5-f][1,10]phenanthro-
line; hpip is 2-(2-hydroxyphenyl) imidazo[4,5-f][1,10]
phenanthroline), have been synthesized and fully charac-
terized by CHN analysis, matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry, 1H-NMR
spectroscopy, IR spectroscopy (attenuated total reflection),
and UV–vis spectroscopy. The DNA-binding behaviors of
both complexes have been studied by spectroscopic methods
and viscosity measurements, and their ability to inhibit DNA
transcription was measured. The results indicate that both
complexes show some degree of binding to DNA in an in-
tercalative mode, resulting in intrinsic binding constants of
complexes inhibited DNA transcription, and as expected 2
was shown to be a better antitumor agent than 1.
Keywords Pt(II) complex ꢀ Polypyridyl ligand ꢀ
DNA binding ꢀ Photocleavage
Introduction
In recent decades, much attention has been paid to the
binding of small molecules with DNA, as a result of
decisive advantages of these molecules as potential drugs.
Many natural or synthetic drugs serve as analogs in
research on protein–nucleic acid recognition and provide
site-specific reagents for molecular biology. Therefore, the
investigation of drug–DNA interaction is important to
understand the molecular mechanisms of drug action and
for the design of specific DNA-targeted drugs. Since the
concept of intercalation into DNA was first formulated by
Lerman in 1961, it has become widely recognized that the
biological and therapeutic activities of many compounds of
pharmacological interest, including anticancer drugs and
antibiotics, are correlated with the ability of intercalative
interaction with DNA. This noncovalent binding has an
important function in life phenomena at the molecular
level, indicating the interaction specificity of a drug with
DNA [1].
(2.88
0.4) 9 104 and (5.38
0.8) 9 104 for 1 and 2,
respectively. The comparatively observed difference in the
DNA-binding affinities of the two complexes can be rea-
sonably explained by the presence of intramolecular
hydrogen bonding between the ortho phenolic group and the
nitrogen atom of the imidazole ring. The extended coplan-
arity of the hpip ligand due to intramolecular hydrogen
bonding may lead to an enhancement of the DNA-binding
affinity of the hpip complex. In addition, the complexes can
promote photocleavage of pUC19 DNA on irradiation as
revealed by the spectroscopic and viscometric measure-
ments, with 2 promoting cleavage of pUC19 DNA at lower
concentration. Moreover, increasing concentrations of both
In particular, cationic metal complexes possessing pla-
nar aromatic ligands may bind to DNA by intercalation,
which involves stacking of the planar ligand between
adjacent base pairs of the DNA duplex [2–4]. Such me-
tallointercalators tend to be strongly mutagenic, and some
have shown promising chemotherapeutic activity, which
correlates well with DNA-binding affinity [5, 6]. Addi-
tionally, they have also been particularly useful in probing
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-013-0991-7) contains supplementary
material, which is available to authorized users.
B. Coban (&) ꢀ U. Yildiz ꢀ A. Sengul
Department of Chemistry, Faculty of Arts and Sciences,
Bulent Ecevit University, Zonguldak 67100, Turkey
e-mail: burakcoban@yahoo.com
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J Biol Inorg Chem
DNA structure and function and the intercalation process
itself, because the ligands or metal may be varied in an
easily controlled manner to facilitate the individual appli-
cation [7]. Certain metallointercalators which cleave DNA
owing to the redox activity of the metal center have further
been successfully employed in footprinting studies of drug
binding and in the examination of charge transport through
DNA [8, 9].
were recorded with a Bruker Ultra Shield Plus ultra-long-
hold-time spectrometer with (CD3)2SO or CDCl3 as the
solvent for the ligands, (CD3)2SO as the solvent for the
complexes, and tetramethylsilane as an internal standard at
400 MHz at room temperature. All chemical shifts are
given relative to tetramethylsilane. UV–vis spectra were
recorded with a Varian Cary 100 spectrophotometer and
emission spectra were recorded with a PerkinElmer LS 55
spectrofluorophotometer at room temperature.
Although interaction of cobalt and ruthenium complexes
of 2-phenylimidazo[4,5-f][1,10]phenanthroline (pip) and
2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline
(hpip) with DNA has been studied, interaction of platinum
complexes of these ligands with DNA has not been shown
yet [10–12]. Platinum complexes have a large planar aro-
matic surface because of their square planar geometry. This
planarity allows ligands to bind to DNA base pairs by
intercalation. We report here the synthesis and character-
ization of two new Pt(II) complexes, [Pt(bpy)(pip)](NO3)2
(1) and [Pt(bpy)(hpip)](NO3)2ꢀ2H2O (2) (bpy is 2,20-
bypyridine) (Fig. 1). The DNA-binding properties of the
Pt(II) complexes were explored by spectroscopic methods
and viscosity measurements. Their DNA-cleavage behav-
iors toward pUC19 and their inhibition effects on in vitro
transcription were also investigated.
Absorption and emission titrations
For the absorption and emission titrations, Pt(II) complexes
were dissolved in a minimum amount of DMSO, and were
then diluted in buffer (5 mM ammonium acetate, pH 7.5)
to a final concentration of 20 lM. Titrations were per-
formed in a 10-mm stoppered quartz cell by using a fixed
concentration of the complex (20 lM), to which the
CT-DNA stock solution was added in increments to a
DNA-to-platinum concentration ratio of 6:1. Analysis was
performed by means of a UV–vis or fluorescence spec-
trophotometer by recording the spectrum after each addi-
tion of DNA. Platinum(II) complex–DNA solutions were
incubated for 10 min each time before the spectra were
recorded. A control solution of 20 lM platinum complex in
the same buffer was also treated in the same manner. Cell
compartments were thermostated at 25 0.1 °C.
Materials and methods
For emission intensity measurements, the excitation
wavelength was fixed and the emission range was adjusted
before the measurements. Ammonium acetate (5 mM), pH
7.5 buffer was used as a blank to make preliminary
adjustments. All measurements were performed with a
5-nm entrance slit and a 5-nm exit slit.
All reagents and solvents were of commercial origin and
used without further purification unless otherwise noted.
Solutions of calf thymus DNA (CT-DNA; purchased from
Sigma) in 50 mM ammonium acetate (pH 7.5) had a
UV–vis absorbance ratio of 1.8–1.9:1 at 260 and 280 nm
(A260/A280 = 1.9), indicating that the DNA was sufficiently
free of protein [13]. The concentration of DNA was
determined spectrophotometrically using a molar absorp-
tivity of 6,600 M-1 cm-1 (260 nm) [13]. Double-distilled
water was used to prepare buffers. Stock solutions of
CT-DNA were stored at 4 °C and used within 4 days. The
compounds Pt(bpy)(H2O)2 [14] and 1,10-phenanthroline-
5,6-dione (phendione) [15] were prepared by literature
methods. Solutions of Pt(II) complexes were prepared by
dissolving a weighed amount in 0.5 mL dimethyl sulfoxide
(DMSO) for solubility reasons and were then diluted (up to
150 times without precipitation) with 50 mM ammonium
acetate (pH 7.5) to the concentration needed.
Competitive studies
The competitive behavior of each Pt(II) complex with
ethidium bromide (EB) was investigated by fluorescence
spectroscopy in order to examine whether the Pt(II) com-
plex is able to displace EB from the DNA–EB complex.
DNA was pretreated with EB at a DNA-to-EB concentra-
tion ratio of 50:1 for 30 min at 27 °C to prepare the initial
DNA–EB complex. The intercalating effect of the Pt(II)
complexes with the DNA–EB complex was studied by
adding a certain amount of a solution of the Pt(II) complex
in increments to the solution of the DNA–EB complex. The
influence of each addition of Pt(II) complex to the solution
of the DNA–EB complex was obtained by recording the
change in the fluorescence spectrum. To study the com-
petitive binding of the Pt(II) complexes with EB, EB was
excited at 545 nm in the presence of DNA alone as well as
in the presence of the Pt(II) complexes.
Physical measurements
Microanalyses (C, H, and N) were performed with a Leco
932 CHNS analyzer. IR spectra were recorded with a
1
PerkinElmer Spectrum 100 instrument. H-NMR spectra
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J Biol Inorg Chem
Fig. 1 Complexes 1 and 2 and
the other ligands mentioned in
the text
Viscosity measurements
borate–EDTA running buffer solution. Reaction mixtures
(10 mL) containing 0.1 lg pUC9 together with different
amounts of Pt(II) complexes (0, 15, 75, and 150 lM for
experiments in the dark; 0, 10, 20, and 40 lM for experi-
ments with irradiation) in 50 mM ammonium acetate buffer,
pH 7.5 were prepared at 0 °C, were then incubated at 36 °C
for 1 h in the dark, and were then irradiated at room tem-
perature with a UV lamp (365 nm, 10 W). Prior to the
samples being loaded onto the gel, 2.5 mL of 0.25 % bro-
mophenol blue loading buffer and sucrose in water (40 %
w/v) was added to the reaction mixtures. Gels were obtained
at room temperature by using a Thermo midi horizontal
agarose gel electrophoresis system and applying a potential
of 35 V for 4 h. The resulting gels were stained with EB
solution (0.5 mg mL-1) for 45 min, after which they were
soaked in water for a further 20 min. Gels were visualized
under UV light and photographed.
Viscosity measurements were performed using a common
Ubbelohde viscometer maintained at a constant tempera-
ture of 30.0 0.1 °C (in a thermostatic bath). The DNA
concentration was 160 lM. The flow time was measured
with a digital stopwatch; every sample was measured three
times and an average flow time was calculated. Data are
presented as (g/g0)1/3 versus binding ratio [16], where g is
the viscosity of DNA in the presence of the complex and g0
is the viscosity of DNA alone.
Gel electrophoresis
Gel electrophoresis experiments were performed using
pUC9 negatively supercoiled plasmid DNA and 1 % agarose
gels together with a tris(hydroxymethyl)aminomethane–
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J Biol Inorg Chem
In vitro transcription experiments
Preparation of [Pt(bpy)(pip)](NO3)2 (1)
In vitro transcription experiments were conducted using a
pGEM express positive control DNA template and a Ri-
boMAX large-scale RNA production system with T7 RNA
polymerase purchased from Promega. Reaction mixtures
containing the DNA template, 59 T7 transcription buffer
(specified by the provider), 25 lM nucleoside 50-triphos-
phate (ATP, UTP, CTP, and GTP) and differing amounts of
the complexes were mixed together prior to addition of the
T7 enzyme mix (specified by the provider). Transcription
inhibition experiments were conducted by incubating the
DNA template with increasing concentrations of the com-
plexes prior to the addition to the reaction mixture. Tran-
scription reactions were allowed to proceed for 1 h at
37 °C. The resulting mixtures were analyzed by gel elec-
trophoresis using 1 % agarose gels and a tris(hydroxy-
methyl)aminomethane–acetate–EDTA running buffer
solution. Gels were run for 2 h at 30 V, and then imme-
diately stained with EB, visualized under UV light, and
photographed.
A concentrated solution of [Pt(bpy)(H2O)2]2? in water and a
solution of pip (0.030 g, 0.1 mmol) in ethyl alcohol were
mixed and set to reflux for 2 h. On cooling, the homogenous
clear-orange solution was diluted with water (60 mL). After
filtration, the solution was added to a saturated aqueous
solution of NaNO3 at room temperature to yield a dark-
yellow precipitate, which was obtained by filtration, and was
washed with acetone and ether. Yield: 0.048 g, for 63 %
C29H20N6Pt. Matrix-assisted laser desorption/ionization
(MALDI) MS: m/z = 648 [M ? 1] (Fig. S3). Anal. Calcd
for C29H20N8O6Pt (%): C, 45.13; H, 2.61; N, 14.53. Found
(%): C, 45.14; H, 2.58; N, 14.51. 1H-NMR (400 MHz,
DMSO-d6, d, ppm): 13.75 (broad singlet, 1H, NH), 8.98 (m,
2H), 8.50 (d, 2H, J = 7.96 Hz), 8.41 (m, 6H), 8.18 (d, 2H,
J = 7.09 Hz), 7.94 (dd, 2H, J = 7.65 Hz, J = 4.50 Hz),
7.85 (m, 2H), 7.53 (m, 3H) (Fig. S4). UV–vis (H2O) [kmax
,
nm (e, M-1 cm-1)]: 292 (95,000), 350 (60,000). FT-IR
(attenuated total reflection) (m, cm-1): 3,335vw (NH),
3,200–2,400 (H-bonded OH), 3,050 (CHar), 1,620vw (d NH),
1,586 (C=N ? C=C), 1,564 (C=N ? C=C), 1,508
(C=N ? C=C), 1,478, 1,406, 1,355, 1,299, 1,254, 1,153,
1,122, 1,070, 1,031, 955, 798, 763, 715 (x NH), 694, 661.
Preparation of pip
The ligand was synthesized according to a previously
published procedure [17], as follows. A solution of phe-
nanthrolinedione (0.105 g, 0.5 mmol) and benzaldehyde
(0.51 mL, 0.5 mmol) in glacial acetic acid (20 mL) was
heated at reflux for 3 h. The yellow color at the beginning
turned brown. The mixture was treated with ice and then
neutralized with concentrated ammonia solution. The
yellow precipitate was filtered and washed with cold
ethanol, water, and finally ether and vacuum-dried. Yield:
0.100 g, 66 % for C19H12N4. Electrospray ionization
mass spectrometry (MS): m/z = 297 [M ? 1]. 1H-NMR
(400 MHz, CDCl3, d, ppm): 13.48 (br, 1H, NH), 8.97 (dd,
2H), 8.89 (dd, 1H), 8.76 (dd, 1H), 8.15 (dd, 2H), 7.58
(m, 2H), 7.41 (dd, 2H), 7.3 (t, 1H) (Fig. S1). Fourier
transform (FT) IR (m, cm-1): 2,965 (aromatic C–H), 1,603
(C=N).
Preparation of [Pt(bpy)(hpip)](NO3)2 (2)
This complex was obtained by a procedure similar to that
described for complex 1, but hpip (0.031 mg, 0.1 mmol)
was used instead of pip. Yield: 0.048 g, 61 %. Found: C,
49.30; H, 2.89; N, 12.27 % for C29H20N6OPt. MALDI-MS:
m/z = 810 ([M2? ? 2NO3 ? Na]?) (Fig. S5). Anal. Calcd
for C29H24N8O9Pt (%): C, 42.28; H, 2.94; N, 13.61. Found
(%): C, 42.27; H, 2.96; N, 13.60. 1H-NMR (400 MHz,
DMSO-d6, d, ppm): 13.88 (broad singlet, 1H, NH), 12.41
(broad singlet, 1H, OH), 9.10 (m, 2H), 8.84 (m, 2H), 8.72
(m, 4H), 8.64 (m, 2H), 8.41 (m, 2H), 8.02 (m, 4H), 7.52 (m,
2H) (Fig. S4). UV–vis (H2O) [kmax, nm (e, M-1 cm-1)]:
277 (46,000), 352 (33,000). FT-IR (attenuated total
reflection) (m, cm-1): 3,330vw (NH), 3,200–2,400 (H-bon-
ded OH ? H2O), 3,054 (CHar), 1,630–1,603vw (d H-O-
H), 1,564–1,517vw (d NH), 1,504vw (C=N ? C=C), 1,478,
1,459, 1,395, 1,350, 1,277, 1,191, 1,124, 1,088 (C-O),
1,068, 1,044, 1,031, 951, 923 (s OH), 875, 853, 801
(s NH), 777, 737, 715 (x NH), 666.
Preparation of hpip
This compound was prepared by a procedure similar to that
described for pip, but 2-hydroxybenzaldehyde (0.53 mL,
0.5 mmol) was used instead of benzaldehyde. Yield:
0.080 g, 51 % for C19H12N4O. Electrospray ionization MS:
m/z = 313 [M ? 1]. 1H-NMR (400 MHz, CDCl3, d, ppm):
13.31 (br, 1H, NH), 11.19 (s, 1H, OH), 9.19 (dd, 2H), 8.83
(dd, 1H), 8.67 (dd, 1H), 8.15 (dd, 1H), 7.75 (dd, 2H), 7.64
(dd, 1H), 7.44 (dd, 2H) (Fig. S2). FT-IR (m, cm-1): 2,706
(aromatic C–H), 1,627 (C=N).
Results and discussion
Synthesis of ligands and complexes
The ligands pip and hpip were prepared following a
well-established imidazole synthetic procedure. The
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J Biol Inorg Chem
spectroscopic and physical data are identical with the data
reported in [17]. An equimolar mixture of phenanthrolin-
edione and benzaldehyde for pip (2-hydroxybenzaldehyde
for hpip) was heated under reflux in glacial acetic acid
containing an excess of ammonium acetate. The compound
precipitated as a yellow powder on cooling. Metathesis of
the chloride ions from the Pt(bpy)Cl2 source to hydrate
groups was realized prior to metallation of the phenan-
throline end of the ligand. A methanolic solution of
Pt(bpy)(H2O)2 was heated to reflux in the presence of the
ligand. [Pt(bpy)(pip)](NO3)2 (1) and [Pt(bpy)(hpip)](-
NO3)2ꢀ2H2O (2) were isolated on addition of an excess of
NaNO3 in a concentrated solution of the reacting mixture.
The structures of the ligands were confirmed by CHN
analysis, MALDI time-of-flight (TOF) MS, IR (attenuated
and was attributed to the singly charged species
[Pt(bpy)(hpip)](NO3)2 ? Na] (C29H20N8O7PtNa). The
isotopic distribution pattern of this peak matches perfectly
with the simulated one. The CHN analysis is consistent
with a structure consisting of two molecules of water of
crystallization in the lattice, that is, consistent with the IR
spectrum of 2 showing strongly hydrogen bonded water
molecules at 3,200–2,400 cm-1, along with the additional
bending vibration bands at around 1,630–1,603 cm-1 (d
H-O-H).
Electronic absorption titration
Electronic spectra are the commonest way to investigate
the interactions of complexes with DNA. A complex bound
to DNA through intercalation usually results in hypochro-
mism and redshift (bathochromism) owing to the interca-
lation mode involving a strong stacking interaction
between an aromatic chromophore and the base pairs of
DNA. The extent of the hypochromism in the visible
metal-to-ligand charge transfer band is commonly consis-
tent with the strength of the intercalative interaction [20].
The absorption spectra of 1 and 2 in the absence and
presence of CT-DNA (at a constant concentration of
complexes, 20 lM Pt) are shown in Fig. 2. With increasing
DNA concentration, the hypochromism increases, although
no obvious redshift in the metal-to-ligand charge transfer
band of the complexes was observed. When the amount of
DNA was increased, the decreases in the intensities of the
p-p* transitions were 25.8 % for 1 at a DNA-to-Pt con-
centration ratio of 4.8 and 23.4 % for 2 at a DNA-to-Pt
concentration ratio of 2.4.
1
total reflection) spectroscopy, and H-NMR spectroscopy
and are consistent with the literature data [18, 19]. The 1H-
NMR spectrum of pip displays a broad peak at 13.48 due to
N–H of the imidazole ring. The phenolic proton of hpip
appears at 11.19 ppm but the proton on the nitrogen atom
of the imidazole ring was not observed presumably owing
to the quick proton exchange between the two nitrogens of
the imidazole ring, which is characteristic of an active
proton [18]. The IR spectrum of hpip shows a broad band at
2,600–3,400 cm-1 due to a stretching vibration of phenolic
O-H involving the intramolecular hydrogen bond. The
1
purity of the complexes was examined by H-NMR spec-
troscopy, MALDI-TOF MS, and elemental analysis. In the
1H-NMR spectra of 1 and 2 (Figs. S4, S6, respectively), the
imidazolic NH proton was observed at 13.75 and
13.88 ppm, respectively. In the spectrum of 2, the qua-
druple singlet attributed to the OH was deshielded to
12.41 ppm, whereas the corresponding peak for the hpip
ligand was observed at 11.76 ppm. Both spectra show a
downfield shift for almost all protons, but the shifts are
more pronounced for those para and ortho to coordinating
nitrogen atoms compared with the corresponding spectra of
the ligands (vide supra). This clearly shows the effect of
decreasing electron density on the molecule on coordina-
tion to the metal center. Owing to low solubility of both
complexes, most of the peaks appear as a multiplet and the
signals are very weak especially for 2. Therefore, the
proper assignments of the aromatic protons remained
To quantitatively compare the DNA-binding affinities of
the two complexes, the intrinsic binding constants Kb of the
two complexes with regard to DNA were obtained by
monitoring the changes of the p-p* absorbance at 292 and
277 nm for 1 and 2, respectively, according to the fol-
lowing equation:
½DNAꢁ=ðea ꢂ efÞ ¼ ½DNAꢁ=ðeb ꢂ efÞ þ 1=Kbðeb ꢂ efÞ
ð1Þ
where [DNA] is the concentration of the nucleic acid in
base pairs, ea is the apparent absorption coefficient obtained
by calculating Aobs/[Pt], and ef and eb are the absorption
coefficients of the free and the fully bound platinum
complex, respectively. In the [DNA]/(ea - ef) versus
[DNA] plot, Kb is given by the ratio of the slope to the
intercept. The values of the intrinsic binding constants Kb
for 1 and 2 were derived to be (2.88 0.4) 9 104 and
(5.38 0.8) 9 104, respectively. These values are com-
parable to those of Pt(II) complexes such as [Pt(py)
(terpy)]2? (py is pyridine) (Kb = 3.5 9 104 M-1),
[Pt(py)2(dpq)]2? (Kb = 2.2 9 105), and [Pt(py)2(dppz)]2?
1
unclear. However, the integration of the H-NMR spectra
in DMSO-d6 unambiguously confirmed the stoichiometry
of 1 and 2 and also bpy to planar pip or hpip ratios. In the
MALDI-TOF mass spectrum of 1 (Fig. S3), a major peak
at m/z 648 was observed, and was attributed to the doubly
positively charged species [Pt(bpy)(pip)]2? (C29H20N6Pt).
The isotopic distribution pattern of this peak matches
perfectly with the simulated one. The CHN analysis further
confirmed the structure. In the MALDI-TOF mass spec-
trum of 2 (Fig. S5), a major peak at m/z 810 was observed,
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J Biol Inorg Chem
Fig. 2 Absorption spectra of 1 (left) and 2 (right) in 50 mM
ammonium acetate buffer on the addition of calf thymus DNA:
20 lM Pt(II) complex, 0–64 lM DNA. Arrows show the absorbance
changing with increase of DNA concentration. The insets show plots
of DNA concentration divided by the difference between the apparent
absorption coefficient (ea) and the absorption coefficient of the free
platinum complex (ef) versus DNA concentration for the titration of
DNA to Pt(II) complexes
(Kb = 1.3 9 103 M-1) [3, 21]. The values are smaller than
those of the Ru(II) complexes [Ru(NH4)4(pip)]2? (Kb =
4.3 9 105 M-1) and [Ru(NH4)4(hpip)]2? (9.7 9 105 M-1
)
[11], but are comparable to those observed for
[Ru(phen)2(p-mopip)]2? (2.0 9 104 M-1), [Ru(phen)2
(p-hpip)]2? (6.9 9 104 M-1), [Ru(phen)2(p-nmip)]2?
(1.56 9 105 M-1) [22], [Ru(bpy)2nmip]2? [(1.15 0.3) 9
104 M-1] [23], [Ru(bpy)2ppip]2? (4.3 9 104 M-1) [24],
and [Ru(bpy)2hnaip]2? (8.3 9 104 M-1) [19]. The struc-
tures of the ligands are shown in Fig. 1.
Fig. 3 [Pt(bpy)(hpip)]?2 complex with intramolecular hydrogen
bonding
As reported before, DNA-binding affinity of complexes
can be influenced by electronic effects of intercalative
ligands. The electron-withdrawing substituent (–NO2) on
the intercalative ligand can increase the DNA-binding
affinity of the complex, whereas the electron-donating
substituent (–OH) has the opposite effect [21]. However,
here the findings show an opposite trend. The binding
constant of the hpip complex is higher than that of the pip
complex, revealing the involvement of the phenolic
hydroxyl group in DNA binding. A similar enhancement in
DNA-binding affinity was observed earlier for [Co(phen)2
(hpip)]2? (Kb = 4.1 9 105 M-1) relative to [Co(phen)2
(pip)]2? (Kb = 2.2 9 105 M-1) [25, 26] and [Ru(NH4)4
(pip)]2? (Kb = 4.3 9 105 M-1) and [Ru(NH4)4(hpip)]2?
(Kb = 9.7 9 105 M-1), respectively [11]. As for the pip
ligand, the phenyl ring in hpip is expected to be almost
coplanar with the imidazole ring, possibly supported by the
formation of an intramolecular hydrogen bond between the
ortho phenolic group and the nitrogen atom of the imid-
azole ring. The extended coplanarity of the hpip ligand
leads to an enhanced DNA-binding affinity of the hpip
complex compared with the pip complex (Fig. 3). Simi-
larly, the hqdppz (Fig. 1) complex exhibits a binding
constant (Kb, 7.7 9 106 M-1) higher than that of the oxi-
dized qdppz complex (Kb, 5.4 9 106 M-1) [11]. The
intramolecular hydrogen bonding in the hqdppz complex
(Fig. 3) enhances the surface area of the planar hqdppz
ligand involved in the intercalative mode of interaction and
hence leads to the higher DNA-binding affinity [11]. Also
the flipping of the quinone part of the qdppz ligand and its
possible involvement in hydrogen bonding with suitable
donors on the DNA base pairs would decrease the extent of
p–p stacking interactions with the DNA base pairs.
At neutral pH, [Pt(bpy)(pip)]2? and [Pt(bpy)(hpip)]2?
are dicationic complexes, and thus one should bear in mind
that the interaction between the Pt(II) complex and nucleic
acids may be as a result of electrostatic interaction and
influenced by the presence of other cations or the ionic
strength of the solution [27]. The effects of the ionic
strength on the binding equilibrium constant of the com-
plexes were tested by the addition of KCl solution in
increments. Salt concentrations of 0.01–0.100 M were
selected in this study because the polyelectrolyte theories
[27] which will be used for subsequent analysis are based
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J Biol Inorg Chem
on limiting laws that are strictly applicable to salt con-
centration lower than 0.100 M. The salt concentration
dependence of the DNA binding of the complexes was
evaluated and the binding equilibrium constant (Kb) of
each Pt(II) complex with regard to CT-DNA was deter-
mined by spectrophotometric titration with KCl concen-
trations of 25, 50, 75, and 100 mM. The new Kb values for
a KCl concentration of 100 mM were 2.58 9 10-4 and
4.13 9 10-4 for complexes 1 and 2, respectively. Variation
of the Kb value with and without KCl in the medium was
very small: the initially calculated Kb values were
(2.88 0.4) 9 104 and (5.38 0.8) 9 104 for complexes
1 and 2, respectively. This clearly indicates that the elec-
trostatic contribution to the overall binding constant is
significantly small. In addition, from these titrations the
binding constant can be used to calculate the Gibbs free
energy (DG°), using the standard equation
Fig. 4 Salt concentration dependence of DNA binding constants for
1 (diamonds) and 2 (squares). The slopes of these plots give
SK = 0.06 and SK = 0.14 for 1 and 2, respectively
DGꢃ ¼ ꢂRT ln K
ð2Þ
Emission spectra
where R is the gas constant and T is the temperature in
kelvins. The salt concentration dependence of the binding
constant defines the slope
In the absence of DNA, complexes 1 and 2 can emit weak
luminescence in ammonium acetate buffer at ambient
temperature, with the maximum at 383 and 455 nm,
respectively. On addition of CT-DNA, the emission
intensity increases slightly (Fig. 5). This implies that the
complexes can interact with CT-DNA and are protected by
DNA efficiently, since the hydrophobic environment inside
the DNA helix reduces the accessibility of solvent water
molecules to the complex and the mobility of the complex
is restricted at the binding site, leading to a decrease of the
vibrational modes of relaxation. But the complexes fail to
show steady-state emission even in the presence of CT-
DNA. So the extent of binding of these complexes was
evaluated using competitive binding studies involving EB,
which is known to emit strongly owing to its intercalative
interaction with DNA.
SK ¼ d log K=d log ½Kþꢁ
ð3Þ
and SK may be used to calculate the polyelectrolyte
contribution (DGpe) at a given salt concentration by the
relation
Gpe ¼ ðSKÞ RT ln ½Kþꢁ
ð4Þ
The difference between the Gibbs free energy and DGpe
gives the nonelectrostatic free energy contribution DGt
[27]:
DGt ¼ DGꢃ ꢂ DGpe
ð5Þ
The salt concentration dependence of the DNA binding
constants for 1 and 2 was studied, and Eq. 3 is resolved
in Fig. 4. The slopes of these plots give SK = 0.06 and
SK = 0.14 for 1 and 2, respectively. The ratios of the non-
electrostatic free energy contributions to the Gibbs free
energy expressed as percentages were 94 and 86 % for 1
and 2, respectively, using Eqs. 4 and 5. This suggests that
the DNA binding constants of the Pt(II) complexes largely
result from non-electrostatic interactions. Thus, the above-
mentioned variation in Kb values demonstrates that the
binding affinity of the complexes is dictated (more than
85 %) by the surface area of the extended planar p system
of the intercalating ligand. Further, the binding constants of
the complexes show a DNA-binding affinity less than that
for proven classic intercalators such as the Ru(II) complex
of dppz (Kb: 1.1 9 106 M-1) [28]. So it is clear that in all
the present complexes the modified phenanthroline ligands
are involved in less strong insertion into the DNA base
pairs, i.e., a nonclassic mode of intercalation.
Competitive binding experiments
Competitive binding experiments with a well-established
quenching assay based on the displacement of the inter-
calating drug EB from CT-DNA may give further infor-
mation regarding the DNA-binding properties of the
complex. A complex which is involved in strong DNA
intercalation would compete with the intercalatively bound
EB for DNA binding and quench the EB emission. When a
complex displaces EB from DNA-bound EB, the emission
of the solution will be greatly quenched because the free
EB molecules are readily quenched by the surrounding
water molecules [29]. DNA intercalators were reported to
cause evident reduction in EB emission intensities [30], but
only moderate decays in emission intensities were reported
for groove DNA binders. The results can be interpreted by
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J Biol Inorg Chem
Fig. 5 Emission spectra of 1 (left) and 2 (right) (10 lM) in
ammonium acetate buffer at 25 °C in the presence of 0–20 lM calf
thymus DNA. Arrows show the intensity change with increasing DNA
concentration. The complexes were excited at 383 and 400 nm,
respectively; the emission spectra were monitored between 440 and
600 nm
the Stern–Volmer equation [31], which is used to calculate
the degree of binding of the complexes:
also quenches the EB emission completely and more effi-
ciently than the hqdppz complex in spite of its higher
DNA-binding affinity [11 (the structures of qdppz and
hqdppz are shown in Fig. 1).
I0=I ¼ 1 þ Kr
where I0 and I represent the fluorescence intensities in the
absence and presence of the complex, respectively, K is a
linear Stern–Volmer quenching constant dependent on the
ratio of the concentration of bound EB to the concentration
of DNA, and r is the ratio of the total concentration of the
complex to that of DNA.
Viscosity properties
Hydrodynamic measurements (i.e., viscosity and sedi-
mentation), being sensitive to length changes, are regarded
as the least ambiguous and most critical tests for a classic
intercalation model in solution in the absence of crystal-
lographic structural data [33, 34]. A classic intercalation
model demands that the DNA helix lengthens as base pairs
are separated to accommodate the bound ligand, leading to
an increase of DNA viscosity. In contrast, a partial, non-
classic intercalation of ligand could bend (or kink) the
DNA helix, reducing its length and, concomitantly, its
viscosity [33, 34]. The effects of 1, 2, and EB on the vis-
cosity of rodlike DNA are shown in Fig. 7. For EB, the
proven classic intercalator will exert the maximum effect
on the viscosity of DNA. On increasing the amount of 1 or
2, the relative viscosity of DNA increases steadily. The
extent of the increase in viscosity, which may depend on
the DNA-binding affinity, follows the order EB [ 2 [ 1.
This is also consistent with the spectroscopic results
described earlier, and Ru(II) complexes of the same ligands
also change the viscosity of CT-DNA in the same order
[11].
As shown in Fig. 6, the emission of EB is quenched by
both complexes. Appreciable reduction in emission inten-
sity was achieved on addition of both complexes to the
EB–DNA system. The quenching plots shown in the insets
in Fig. 6 are in good agreement with the linear Stern–
Volmer equation with slopes of 2.78 and 1.89 for 1 and 2,
respectively.
With use of a DNA binding constant of 4.54 9 105 M-1
for EB [32], apparent DNA binding constants of
7.09 9 104 M-1 and 9.45 9 104 M-1 were derived for 1
and 2, respectively, by using the Stern–Volmer equation..
These values are comparable with those derived by direct
spectroscopic measurements, where 2 was found to bind
more strongly than 1, and a similar order is expected for the
quenching of the EB emission by displacement of the
DNA-bound EB. However, complex 2 was found to quench
the emission less efficiently than 1. Exactly the same
conclusion was drawn for the quenching of the EB emis-
sion by the addition of Ru(II) complexes of pip and hpip in
the literature. It was suggested that the DNA helix simul-
taneously accommodates both the complex and EB in the
grooves and the enhanced hydrophobicity of the phenyl
ring in the DNA-bound pip complex perturbs the DNA
helix more than the enhanced planarity of the hpip ligand
does; thus, the bound EB is displaced more efficiently [11].
Similarly, it is interesting to note that the qdppz complex
Photocleavage of pUC19 DNA by Pt(II) complexes
The potential of the present complexes to cleave DNA was
studied by gel electrophoresis using supercoiled pUC19
DNA in ammonium acetate buffer (pH 7.5). When circular
plasmid DNA is subjected to gel electrophoresis, relatively
fast migration will be observed for the intact supercoiled
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J Biol Inorg Chem
Fig. 6 Emission spectra of ethidium bromide bound to DNA in the
presence of 1 (left) and 2 (right) (0.0–92.0 M). The arrows show
the intensity changes on increasing the concentration of the complex.
The insets show the emission quenching curve of DNA-bound
ethidium bromide
Fig. 7 Effects of increasing amounts of complex 1 (squares),
complex 2 (triangles), and ethidium bromide (EtBr; diamonds) on
the relative viscosity of calf thymus DNA at (25 0.1) °C. The total
concentration of DNA is 0.5 mM
Fig. 8 Photoactivated cleavage of pUC19 DNA in the presence of
Pt(II) complexes on irradiation at 365 nm. Lanes 1 and 5, DNA alone;
lanes 2–4 no irradiation and 1 h incubation, in different concentra-
tions of 1 for a and 2 for b (1 0 lM; 2 15 lM; 3 75 lM; 4 150 lM);
lanes 5–8 irradiation and 1 h incubation, in different concentrations of
1 for a and 2 for b (5 0 lM; 6 10 lM; 7 20 lM; 8 40 lM)
form (form I). If scission occurs on one strand (nicked
circular), the supercoiled form 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 [35]. Figure 8 shows
separation of pUC19 DNA by gel electrophoresis after
incubation with each of the Pt(II) complexes and irradia-
tion at 365 nm. With increasing concentration of both
complexes (Fig. 8, lanes 1–4), the amount of form I pUC19
DNA diminished gradually. However, DNA nicking was
observed for pUC19 DNA treated with both complexes and
irradiated with 365-nm light, and the amount of form I
diminished gradually, whereas that of form II increased.
These results show that both 1 and 2 can cleave DNA on
irradiation. When the complexes are compared, it can be
clearly seen that complex 2 cleaves DNA at lower
concentrations. Similar results were obtained in other
studies [36–38]. However, the nature of reactive interme-
diates involved in the DNA cleavage by 1 and 2 is not clear
yet. Further studies on the mechanism are currently under
way.
Inhibition of transcription
A variety of small organic molecules and metal complexes
are known to function as anticancer agents as a result of their
ability to interfere with DNA transcription [39–43]. Inhibi-
tion of transcription can occur because the compounds of
123

J Biol Inorg Chem
complexes have been investigated. The results show that
both complexes can bind to DNA in an intercalative mode
but with different affinities, and that the intramolecular
hydrogen bonding involving the intercalative ligand has a
significant effect on the DNA-binding affinity, resulting in
intrinsic binding constants Kb of (2.88 0.4) 9 104 and
(5.38 0.8) 9 104 M-1 for 1 and 2, respectively. Such a
difference in the DNA-binding affinities between 1 and 2
can be reasonably explained by the intramolecular hydro-
gen bond between the ortho phenolic group and the
nitrogen atom of the imidazole ring. The extended co-
planarity of the hpip ligand leads to enhanced DNA-bind-
ing affinity of the hpip complex. In addition, 1 and 2 are
efficient photocleavers on irradiation at 365 nm, and
complex 2 can cleave pUC19 DNA at lower concentra-
tions. Moreover, increasing concentrations of both com-
plexes inhibited DNA transcription, and as expected 2 was
shown to be a better antitumor agent than 1.
Fig. 9 Ethidium bromide stained agarose gel (1 %) of transcribed
messenger RNA in the presence of increasing concentrations (100,
150, 200, 300 lM) of complexes 1 and 2: lane 1 positive control;
lanes 2–5 100, 150, 200, and 300 lM 2; lanes 6–9 100, 150, 200, and
300 lM 1
interest bind either to RNA polymerase or to the double-
stranded DNA (dsDNA) template. In the latter case, the
binding stabilizes the duplex structure and inhibits DNA
strand separation, which is a key event in transcription. This
was shown where ruthenium and rhodium complexes con-
taining phenanthroline and quinine diimine ligands are able
to inhibit transcription to differing degrees as a result of their
ability to bind to dsDNA [44, 45].
Acknowledgments We are grateful for the support of Bulent Ecevit
University with grant 2011-03-13-06. We also thank Zehra Safi Oz
for the use of her laboratory to make it possible to visualize and
photograph the gel electrophoresis results.
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