The effects of grafting of 2-pyridyl to [Ru(bpy)2(Hpip)]2+on acid-base and DNA-binding properties: Experimental and DFT studies

Article abstract of DOI:10.1080/07391102.2011.10508621

A new Ru(II) complex of [Ru(bpy)₂(Hppip)]²⁺ {bpy = 2,2′-bipyridine; Hppip = 2-(4-(pyridin-2-yl)phenyl)-1H-imidazo[4,5-f][1, 10]phenanthroline} has been synthesized by grafting of 2-pyridyl to parent complex [Ru(bpy)₂(Hpip)]²⁺ {Hppip = 2-(4-phenyl)-1H- imidazo[4,5-f] [1,10]phenanthroline}. The acid-base properties of [Ru(bpy) ₂(Hppip)]²⁺ studied by UV-visible and luminescence spectrophotometric pH titrations, revealed off-on-off luminescence switching of [Ru(bpy)₂(Hppip)]²⁺ that was driven by the protonation/deprotonation of the imidazolyl and the pyridyl moieties. The complex was demonstrated to be a DNA intercalator with an intrinsic DNA binding constant of (5.56 ± 0.2) × 10⁵ M^-1 in buffered 50 mM NaCl, as evidenced by UV-visible and luminescence titrations, reverse salt effect, DNA competitive binding with ethidium bromide, steady-state emission quenching by [Fe(CN)₆]^4-, DNA melting experiments and viscosity measurements. The density functional theory method was also used to calculate geometric/electronic structures of the complex in an effort to understand the DNA binding properties. All the studies indicated that the introduction of 2-pyridyl onto Hpip ligand is more favorable for extension of conjugate plane of the main ligand than that of phenyl, and for greatly enhanced ct-DNA binding affinity accordingly. Adenine Press (2011).

Full text of DOI:10.1080/07391102.2011.10508621

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The Effects of Grafting of 2-Pyridyl to
[Ru(bpy)2(Hpip)]2+ on Acid-Base and DNA-Binding
Properties: Experimental and DFT Studies
An-Guo Zhang a , Huai-Xia Yang a b & Ke-Zhi Wang a
a College of Chemistry, Beijing Normal University , Beijing , 100875 , China
b Pharmacy College, Henan University of Traditional Chinese Medicine , Zhengzhou ,
450008 , China
Published online: 21 May 2012.
To cite this article: An-Guo Zhang , Huai-Xia Yang & Ke-Zhi Wang (2011) The Effects of Grafting of 2-Pyridyl to
[Ru(bpy)2(Hpip)]2+ on Acid-Base and DNA-Binding Properties: Experimental and DFT Studies, Journal of Biomolecular Structure
and Dynamics, 28:6, 955-968, DOI: 10.1080/07391102.2011.10508621
To link to this article: http://dx.doi.org/10.1080/07391102.2011.10508621
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Journal of Biomolecular Structure &
Dynamics, ISSN 0739-1102
Volume 28, Issue Number 6, (2011)
©Adenine Press (2011)
An-Guo Zhang1
Huai-Xia Yang1,2
Ke-Zhi Wang1*
The Effects of Grafting of 2-Pyridyl to
[Ru(bpy)2(Hpip)]2+ on Acid-Base and DNA-Binding
Properties: Experimental and DFT Studies
http://www.jbsdonline.com
1College of Chemistry, Beijing Normal
University, Beijing 100875, China
2Pharmacy College, Henan University
of Traditional Chinese Medicine,
Zhengzhou 450008, China
Abstract
A new Ru(II) complex of [Ru(bpy)2(Hppip)]2+ {bpy = 2,2′-bipyridine; Hppip = 2-(4-(pyridin-
2-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} has been synthesized by grafting of
2-pyridyl to parent complex [Ru(bpy)2(Hpip)]2+ {Hppip = 2-(4-phenyl)-1H-imidazo[4,5-f]
[1,10]phenanthroline}. The acid-base properties of [Ru(bpy)2(Hppip)]2+ studied by UV-vis-
ible and luminescence spectrophotometric pH titrations, revealed off-on-off luminescence
switching of [Ru(bpy)2(Hppip)]2+ that was driven by the protonation/deprotonation of the
imidazolyl and the pyridyl moieties. The complex was demonstrated to be a DNA intercalator
with an intrinsic DNA binding constant of (5.56 0.2) × 105 M-1 in buffered 50 mM NaCl, as
evidenced by UV-visible and luminescence titrations, reverse salt effect, DNA competitive
binding with ethidium bromide, steady-state emission quenching by [Fe(CN)6]4-, DNA melt-
ing experiments and viscosity measurements. The density functional theory method was also
used to calculate geometric/electronic structures of the complex in an effort to understand
the DNA binding properties. All the studies indicated that the introduction of 2-pyridyl onto
Hpip ligand is more favorable for extension of conjugate plane of the main ligand than that
of phenyl, and for greatly enhanced ct-DNA binding affinity accordingly.
Key words: Ruthenium; DNA; Bipyridine; Phenanthroline; Density functional theory.
Introduction
The intriguing photophysical, photochemical, and electrochemical properties (1-3)
of Ru(II) polypyridyl complexes have attracted extensive attention in the past
decades with regard to their applications in several research areas, including bio-
inorganic and biomedicinal chemistry (4, 5), such as DNA cleavage reagents (6-8),
DNA molecular light switches (9-11), inhibiting of DNA transcription (12), DNA-
dependent electron transfer probes, chemical and stereoselective probes of nucleic
acid structures, as well as antitumor drugs (13-15). Ru(II) polypyridyl complexes
can interact with DNA noncovalently through electrostatic, groove, and intercalative
(including classic intercalation, semi-intercalction and quasi-intercalation) binding
modes (16). Many studies have revealed that the planarity of the main ligand plays
a key role in the binding mode and affinity (16-19). The ancillary ligand can also
indirectly affect the DNA binding properties through changing the planarity of the
main ligand and the hydrophobicity of the complex (20). Variations of substituents
or substitution position in the main ligand, even a subtle change of molecular struc-
ture, may result in some interesting differences in the space configuration and the
electron density distribution of Ru(II) polypyridyl complexes, and accordingly the
spectral properties and the DNA-binding behaviors of the complexes, providing a
chance to explore valuable conformation- or site- specific DNA probes (21, 22).
*Phone: +86-10-58805476
Fax: +86-10-58802075
E-mail: kzwang@bnu.edu.cn
955

The Ru(II) polypyridyl system with planarity and ancillary ligand presents simi-
larity and contrasts to many of the systems such as the actinomycins, porphyrins,
Hoechst and benzo[a]pyrene derivatives and others in forming complexes with the
nucleic acids (23-30).
956
Zhang et al.
Among Ru(II) complexes reported for DNA-biding studies, 2-phenylimidazo [4,5-f]
[1,10]-phenanthroline (Hpip)-based complexes have invoked considerable interest
(12, 31-42). Ji and co-workers performed systematic studies on the effects of the
electronic and geometric (the shape, size, planarity and the intramolecular hydrogen
bond) structures of the intercalative ligands on the DNA-binding affinity (31, 32)
and DNA cleavage ability (32-34) of the complexes. Our group have reported imi-
dazole-containing Ru(II) complexes with interesting pH-induced emission switch,
and/or DNA binding and DNA molecular light switching properties (35-42).
Studies on [Ru(bpy)2(Hbpip)]2+ {Hbpip = 2-(4-biphenyl)imidazo[4,5-f][1,10]
phenanthroline} carried out by Ji and co-workers (32) showed that it can interca-
late into DNA base pairs with a binding constant of 1.7 × 105 M-1, which is much
lower than that (4.7 × 105 M-1) of the parent complex [Ru(bpy)2(Hpip)]2+ (43).
It indicated that a grafting of benzene ring to Hpip ligand of the parent complex
[Ru(bpy)2(Hpip)]2+ leads to an unfavorable effect on the DNA-binding affinity
of the complex. This can be mainly attributed to the poor planarity of the inter-
calative ligand Hbpip of [Ru(bpy)2(Hbpip)]2+. However, in this work, we have
reported a Ru(II) complex of [Ru(bpy)2(Hppip)]2+ {Hppip = 2-(4-(pyridin-2-yl)
phenyl)imidazo[4,5-f][1,10]phenanthroline} with introduction of a 2-pyridyl ring
onto Hpip ligand of the parent complex [Ru(bpy)2(Hpip)]2+. The studies demon-
strated that the introduction of the pyridine ring onto Hpip ligand enhances the
DNA-binding affinity (Kb = 5.56 × 105 M-1) of [Ru(bpy)2(Hppip)]2+ moderately
compared to analogous complexes of [Ru(bpy)2(Hpip)]2+ and [Ru(bpy)2(Hbpip)]2+
(32, 43). Also, appending of 2-pyridyl to Hpip enriches the acid-base properties of
[Ru(bpy)2(Hppip)]2+ with pH-induced off-on-off emission switches.
Recently, theoretical studies have shown that density functional theory (DFT) can
predict some properties of Ru(II) complexes successfully (31, 43), because DFT
calculations can better consider electron correlation energies, obviously reducing
the computational expenses and suit such a kind of complexes with single state
(44, 45). These theoretical efforts are very significant in guiding the effects of
geometric/electronic structures on the DNA-interacting properties as well as the
functional molecular design of this kind of Ru(II) complex (46, 47). In this work,
DFT-B3LYP method was also used to calculate geometric/electronic structures of
the complex for revealing the factors affecting DNA-binding affinities of analo-
gous complexes of [Ru(bpy)2(Hppip)]2+.
Materials and Methods
Materials
Cis-[Ru(bpy)2Cl2]·2H2O (40) and 1,10-phenanthroline-5,6-dione (49) were syn-
thesized according to the literature methods. Solvents were purified and dried
according to the standard methods (50). The other materials were obtained from
commercial sources and were used without further purification. The synthetic route
to [Ru(bpy)2(Hppip)]2+ is shown in Scheme 1 (A), and the synthetic details are
given below.
Synthesis
2-(4-(Pyridin-2-yl)phenyl)imidazo[4,5-f][1,10]phenanthroline (Hppip): A mixture
of 4-(pyridin-2-yl)benzaldehyde (0.274 g, 1.5 mmol), 1,10-phenanthroline-5,6-
dione (0.315 g, 1.5 mmol) and ammonium acetate (2.47 g, 32 mmol) dissolved in

glacial acetic acid (20 mL), was refluxed for 6 h under N2 and was then cooled to
room temperature. The cooled solution was filtered, diluted with water (90 mL) and
neutralized with concentrated aqueous ammonia. A straw yellow precipitate was
filtered and washed with a small amount of water, diethyl ether, and then recrystal-
lized from EtOH. Yied: 0.326 g (58.2%). 1H NMR (400 MHz, Me2SO-d6) d/ppm:
9.06 (dd, J1 = 4.1, J2 = 1.7, 2H); 8.96 (dd, J1 = 7.9, J2 = 1.7, 2H); 8.74 (m, 1H); 8.40
(q, J1 = 6, J1 = 8.6, 4H); 8.12 (d, J = 7.9, 1H), 7.95 (td, J1 =7.9, J2 = 2.07, 1H); 7.86
(br, 2H); 7.42 (m, 1H). Anal. Calcd for C24H15N5·0.5H2O (F.W = 382.42) : C, 75.38;
N, 18.31; H, 4.22%. Found: C, 75.54; N, 18.31; H, 4.86%.
957
A New Ru(II) Complex
with DNA
[Ru(bpy)2(Hppip)](ClO4)2·2H2O: A solution of cis-[Ru(bpy)2Cl2]·2H2O (0.260 g,
0.5 mmol) and Hppip (0.187 g, 0.5 mmol) in EtOH/H2O (v/v = 2:1, 120 mL) was
heated at 80°C under N2 for 8 h. The solvent was removed by rotary evaporation,
and resulting crude product was purified by column chromatography on silica gel
1
Scheme 1: Synthetic routes to Hppip ligand and [Ru(bpy)2(Hppip)](ClO4)2 (A). H NMR spectra of Hppip (top) and [Ru(bpy)2(Hppip)](ClO4)2 (bottom) in
Me2SO-d6 (B).

with saturated aqueous KNO3-H2O-MeCN (1:4:40, v/v/v). After addition of satu-
rated NaClO4 aqueous solution and recrystallization from MeCN-diethyl ether, a
red product was obtained in a yield of 75.2% (0.322 g). Caution: Perchlorate salts
of metal complexes with organic ligands are potentially explosive. Although no
detonation tendencies have been observed, caution is advised and handling of only
958
Zhang et al.
1
small quantities is recommended. H NMR (400 MHz, Me2SO-d6) d/ppm: 9.15
(d, J = 7.6, 2H); 8.88 (dd, J1 = 15.2, J2 = 8.2, 4H); 8.75 (d, J = 3.9, 1H); 8.44 (q, J1
= 22.6, J2 = 8.7, 4H); 8.23 (td, J1 = 8.1, J2 = 1.4, 2H); 8.10 (m, 5H); 7.98 (m, 3H);
7.86 (d, J = 5.0, 2H); 7.06 (m, 4H); 7.45 (dd, J1 = 7.5, J2 = 4.8, 1H); 7.36 (t, J = 6.7,
2H). Anal. Calcd for C44H31Cl2N9O8Ru·2H2O (F.W = 1021.78): C, 51.75; N, 12.33;
H, 3.45%. Found: C, 51.80; N, 12.42; H, 3.32%. Anal. Calcd for MALDI-TOF
MS m/z: 886.12 ([M – ClO4-]+), 393.43 ([M – 2ClO4-]2+). Found: 886.15 ([M –
ClO4-]+), 393.56 ([M – 2ClO4-]2+).
The identity of the complex was characterized by 1H NMR, IR spectroscopy, lumi-
nescence, UV-vis spectroscopy, elemental analysis and MALDI-TOF mass spec-
trometry.The1HNMRspectraof[Ru(bpy)2(Hppip)](ClO4)2andHppiparecompared
in Scheme 1 (B). The proton resonance signals for the ligand and the complex were
assigned by comparisons with those of similar compounds [Ru(bpy)2(Hbpip)]2+
and [Ru(bpy)2(Happip)]2+ (32, 37).
Physical Measurements
Elemental analyses (C, N, and H) were performed on a Vario EL elemental ana-
1
lyzer. H NMR spectra were collected on a Bruker DRX-400(300) NMR spec-
trometer with Me2SO-d6 as solvent at room temperature. Matrix-assisted laser
desorption ionization mass spectra (MALDI-TOF MS) were run on API Q-star
pulsar (Applied Biosystems) mass spectrometer. Infrared spectra were recorded
on a Nicolet Avtar 360FT-IR spectrometer as KBr disks. UV-visible spectra were
obtained on a GBC Cintra 10e UV-visible spectrometer. Emission spectra were
obtained on a Shimadzu RF-5301PC spectrofluorimeter.
UV-visible and emission spectrophotometric pH titrations of the complex were
carried out in buffer A: 40 mM H3BO3, 40 mM H3PO4, 40 mM CH3COOH, with
additions of concentrated sulfuric acid, saturated sodium hydroxide aqueous solu-
tion, respectively, to cover the pH range studied. All the experiments involving the
interaction of the complex with ct-DNA were carried out in aerated buffer B: 5 mM
Tris-HCl, 50mM NaCl, pH = 7.10 0.02. The luminescence quantum yields were
calculated by comparison with [Ru(bpy)3]2+ (j = 0.033) (51) in aerated aqueous
solution at room temperature using eq 1, where j and jstd are the quantum yields,
A and Astd are the absorbances at the excitation wavelength, and I and Istd are the
integrated emission intensities for the unkown and standard samples, respectively.
j = jstd(Astd/A)(I/Istd)
[1]
A solution of DNA gave a ratio of UV absorbance at 260 and 280 nm of ca. 1.9:1,
indicating that the DNA was sufficiently free of protein. The concentration of DNA
in bp was determined spectrophotometrically by assuming e260 = 13200 M–1 cm–1
(52). The absorption titration with ct-DNA was carried out by keeping the concen-
tration of the Ru(II) complex constant while varying the DNA concentrations. The
competitive binding of ethidium bromide (EB) to the DNA with the Ru(II) complex
was carried out by measuring EB emission (lex = 537 nm) while keeping [DNA]/
[EB] = 5 constant but varying Ru(II) complex concentrations. Emission titrations
were carried out in buffer B but with varied concentrations of salts. Thermal dena-
turations of the DNA were performed on a UV-vis spectrophotometer in buffer C:
1.5 mM Na2HPO4, 0.5 mM NaH2PO4, 0.25 mM Na2EDTA. With the use of the ther-
mal melting program, the temperature of the cell containing the cuvette was ramped
from 50 to 90°C, and the absorbances at 260 nm were measured every 0.5°C.

Viscosity experiments used an Ubbelohde viscometer, immersed in a thermostated
water-bath maintained at 32.20 0.02°C. The DNA samples, approximately 200
base pairs in average length, were prepared by sonication in order to minimize
complexities arising from DNA flexibility (53). Data were presented as (h/h0)1/3
versus the ratio of the concentration of Ru(II) complex to that of DNA, where h and
h0 are the viscosities of DNA solutions in the presence and the absence of complex,
respectively. Viscosity values were calculated from the observed flowing time of
DNA containing solutions (t) corrected for that of buffer alone (t0), h = t – t0 (54).
959
A New Ru(II) Complex
with DNA
Computational Methods
Structural schematic diagrams of [Ru(bpy)2(Hppip)]2+ are shown in Scheme 2.
The complex forms from a Ru(II) ion, one main ligand Hppip and two ancillary
ligands (bpy). The complex has not any symmetry, whereas their parent complex
[Ru(bpy)3]2+ for comparison belongs to D3 symmetry. Geometry optimization
computations were performed applying the DFT-B3LYP method (55) and Lan-
L2DZ basis set (ECP+DZ for the Ru atom and D95 for C, N, O, H atoms) (56), and
assuming the singlet state for the ground state of the complex (57). All the compu-
tations were performed with the G03 quantum chemistry program-package (58).
Results and Discussion
Computed Geometrical Structures of the Complex
Computed data of the selected bond lengths and dihedral angles for
[Ru(bpy)2(Hppip)]2+ 1 were listed in Table I along with those for the
parent complex [Ru(bpy)2(Hpip)]2+ 2 (43) and the analogous com-
plex [Ru(bpy)2(Hbpip)]2+ 3 (32) for comparison. The computed geo-
metric structures using DFT-B3LYP and LanL2DZ level are rather
reliable as comparison with crystal structure of [Ru(bpy)3]2+ and
computed results for 2 and 3 previously reported (32, 43).
Some structural characteristics can be seen from Table I: (1)
Ru-N mean bond length (0.2107 nm) of the main ligand for
[Ru(bpy)2(Hppip)]2+ is slightly longer than that (0.2097 nm) of the
co-ligand; (2) the mean bond length (0.1400–0.1407 nm) of ligand
skeleton for the complex is very close to its standard bond length
(0.1400 nm) (60), and that of the main ligand skeleton is also slightly
larger than that of the co-ligand; (3) the calculated geometric data
of the complex indicate that the grafted pyridine ring is almost
co-planer with Hpip framework (dihedral angles are N5-C6-C7-C8
= -0.29° and C9-C10-C11-N12 = -0.03°) compared with those of the
parent complex [Ru(bpy)2(Hpip)]2+ (N5-C6-C7-C8 = 0.93°) and the
analogous complex [Ru(bpy)2(Hbpip)]2+ (N5-C6-C7-C8 = -0.13° and
C9-C10-C11-C12 = -31.45°). Energies of the frontier molecular orbitals
for 1 and 3 obtained based on the optimized geometrical structures
are shown in Scheme 3 along with those of complex 2 (43) for com-
parison. And the population and energy levels of the main frontier
molecular orbitals for 1 and 3 are compared in Figure S1 (supporting
information).
pH Effects on UV-visible and Emission Spectra
pH dependence of the ground state and excited state properties of
[Ru(bpy)2(Hppip)]2+ wasinvestigatedinbufferAoverthepHrangeof
0.17-12.86. All spectral changes with pH were reversible. As shown
in in Figure 1, changes of the UV-vis spectra of the complex as a func-
tion of pH, the complex underwent three successive deprotonation
Scheme 2: Structural schematic diagrams and atomic labels of
[Ru(bpy)2(Hppip)](ClO4)2 1, [Ru(bpy)2(Hpip)](PF6)2
[Ru(bpy)2(Hbpip)] (ClO4)2 3.
2
[43], and

processes upon increasing pH from 0.17 to 12.86. Upon increasing pH from 0.17
to 2.76, the absorbance of the bands at 280 and 458 nm increased visibly with a
bathochromic shift of 3 nm, the absorption intensities at 318 nm were decreased
sharply, and wave valley at 380 nm increased in the intensities with appearance
of one isosbestic point at 338 nm. The spectral changes observed above are due to
960
Zhang et al.
Table I
Computational selected bond lengths (nm), bond angles (°), and dihedral angels (°) using the DFT-B3LYP at the LanL2DZ level.
Dihedral anglesd
a
b
c
Com. (calc.)
Ru-Nm
Ru-Nco C-C(N)m
C-C(N)co
Am
Aco
C6-C7
C10-C(N)11 N5-C6-C7-C8 C9-C10-C(N)11-C(N)12
ref
1
2
3
0.2107
0.2108
0.2108
0.2097
0.2097
0.2097
0.1407
0.1406
0.1407
0.1400
0.1400
0.1400
79.31 78.47
79.26 78.47
79.29 78.48
0.1458
0.1463
0.1457
0.1491
—
0.1486
-0.29
0.93
-0.13
-0.03
—
-31.45
This work
43
This work
aRu-Nm expresses the mean coordination bond length between Ru and N atoms of the main ligand L, and Ru-Nco expresses that between Ru and N atoms of the
co-ligand (phen). bC-Cm expresses the mean bond length of the ring skeleton of the main ligand, and C-Cco expresses that of the co-ligand. cAm expresses the mean
coordination bond angle between central Ru and two N atoms of the main ligand, and Aco expresses that of the co-ligand. dThe dihedral angles of complexes 1, 2,
3 were computed by us.
one proton dissociation of the protonated imidazole ring of Hpip moiety. Further
increasing pH from 2.55 to 5.35 induced the second deprotonation step which is
assigned to the proton dissociation of the protonated pyridyl moiety, resulting in
the intensity increases in band at 283 nm with a bathochromic shift of 3 nm, the
wave valley at 310 nm increased sharply while the dexter wave shoulder of ppip
p-p∗-transition band became reduced with an isosbestic point appearing at about
333 nm. The third deprotonation, which was assigned to the deprotonation of the
proton on the neutral imidazole ring, took place over pH 7.27-10.86, accompany-
ing the following spectral features: the absorption at 286 nm decreased and the
MLCT broad band centered at 460 nm increased with a red shift of 2 nm. The three
successive protonation/deprotonation processes occurred for 1 are summarized in
Scheme 4. The negative logarithms of three ground-state ionization constant val-
ues, pKa1 = 1.77 0.01, pKa2 = 3.70 0.04, pKa3 = 9.03 0.04 were derived for
[Ru(bpy)2(Hppip)]2+ by nonlinear sigmoidal fit of the data in the insets of Figure 1.
The pKa1, pKa2 and pKa3 values fall within reasonably pH ranges as comparisons
with the pKa1 and pKa2 values (pKa1 = 0.31-2.48 and pKa2 = 8.21-9.65) we previ-
ously reported for the imidazole ring-containing Ru(II) complexes (35-39), and
pKa = 3.2-3.6 for uncoordinated pyridyl group on [Ru(bpy)2(qp)]2+
(qp = 4,4’:2’,2’’:4’’,4’’’-quaterpyridine) and [Ru(bpy)2(tpy)]2+ (tpy
= 2,2’:4’,4’’-terpyridine) reported by Ward (59).
Changes in luminescence spectra of the complex in water as a func-
tion of pH are shown in Figure 2, which shows that the emission
spectra are sensitive to pH, and consisting of two separate excited-
state proptonation/deprotonation processes. Upon increasing pH
from 0.17 to 4.56, the emission intensities increased sharply by a
factor of ~2.5, and the emission maxima are blue-shifted from 629
to 607 nm, which is different from the intensity enhancement by
only 11.6% exhibited by [Ru(bpy)2(Hpip)]2+. Obviously, the spectral
changes above are mainly contributed to the excited-state deproto-
nation of the pyridyl moiety on [Ru(bpy)2(Hppip)]2+. The emission
was quenched at the lower pH because the protonated pyridyl moi-
ety could act as an electron acceptor, similarly to the behaviors of
pyridyl-containing Ru(II) complexes previously reported (61). In
contrast, the further increases in pH from 5.35 to 10.27 reduced the
emission intensities by 39%, and elicited a big red-shift of the emis-
sion maxima from 607 to 618 nm. This behavior may involve rapid
Scheme 3: Schematic map of energies of some frontier molecular orbit-
als of [Ru(bpy)2L]2+ (L = Hpip 2 [43]; Hppip 1; Hbpip 3.

radiationless decay (57). It has been known that the energy of the metal-centered
excited states depends on the ligand field strength, which in turn depends on the
s-donor and p-acceptor properties of the ligands (57). The negative charge on the
deprotonated imidazol substituent may be delocalized over the whole p framework,
which decreases the s-donor and increases the p-acceptor capacity of the ppip
961
A New Ru(II) Complex
with DNA
Scheme 4: Protonation/deprotonation processes of [Ru(bpy)2(Hppip)]2+
.
ligand, resulting in weakening of the ligand-field strength around the metal center
and in turn lowering the metal s* orbitals (62-64). The grafting of 2-pyridyl group
on [Ru(bpy)2(Hpip)]2+ have made the complex [Ru(bpy)2(Hppip)]2+ to exhibit pH
induced off-on-off emission switches, although the emission intensity on-off ratio
need to be improved.
The ground- and excited-state ionization constants are related thermodynamically
by a Förster cycle (61). The Förster treatment results in eq 2, which describes the
relationship between the ground-state pKa and excited-state pKa* based on pure 0-0
transitions in wave numbers of nB and nHB for the basic and acidic species, respec-
tively (65).
pKa* = pKa + (0.625/T)(νB –νHB)
[2]
In reality, the νB and νHB values are often difficult or even impossible to obtain. A
good approximation is to use the emission maxima for νB and νHB since protonation
equilibrium is almost certainly established between the 3MLCT states. Therefore,
the energies of the emission maxima in wavenumbers were used in eq 2, and two
excited-stateionizationconstantsofpKa1* =2.98andpKa3 *=8.41werethusobtained.
*
The value of pKa3* is comparable to the corresponding pKa3 while the value of pKa1
is 1.21 pKa units greater than pKa1, indicating that the electron density was signifi-
cantly higher in the exited state than in the ground state for [Ru(bpy)2(H3ppip)]4+
and the excited electron was localized on [H3ppip]2+ rather than bpy ligands, while
the electron density in the excited state of [Ru(bpy)2(Hppip)]2+ was localized on
bpy rather than Hppip.

DNA Binding Properties
962
UV-Vis Absorption Spectra: UV-visible and luminescence spectroscopy are two of
the most useful techniques for DNA-binding studies of metal complexes. The UV-
vis absorption spectra of the complex in neutral aqueous solution mainly consist
of two well-resolved bands: one centered at ~286 nm are assigned to an intrali-
gand p-p*-transitions of bpy ligand, and the other one at ~460 nm are attributed to
the metal-to-ligand charge-transfer (MLCT) transition in comparisons with a p-p*
absorption band at 283, 284 and 285 nm and a MLCT band at 458, 458 and 459 nm
reported for analogous complexes of [Ru(bpy)2(Hpip)]2+, [Ru(bpy)2(Hcip)]2+ {Hcip
=2-(4-chloro-phenyl)imidazo[4,5-f][1,10]phenanthroline}, and[Ru(bpy)2(HOP)]2+
{HOP = 2-(4-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline}, respectively
(66–68). It can be concluded that there are no obvious substituent effects on the
bpy intraligand p-p* and MLCT transitions of the [Ru(bpy)2(Hpip)]2+ system.
Zhang et al.
The UV-vis absorption spectra of the complex in the absence and the presence of ct-
DNA are illustrated in Figure 3A. Upon increasing concentrations of the DNA from
0 to 13 μM (bp), the absorption bands at 286 nm, 320 nm and 460 nm displayed
clear hypochromicities with high hypochromism H% {H% = (Afree – Abound)/Afree
}
values of 32%, 35% and 26%, respectively. The presence of an isosbestic point at
472 nm indicated the coexistence of free and DNA-bound Ru(II) complex in the
solution. The intrinsic DNA binding constant Kb illustrating the binding strength of
the complex with ct-DNA was determined from the decay of the absorbance at 286
nm by employing eq 3 (69):
Figure 1: pH effects on the UV-vis spectra of
[Ru(bpy)2(Hppip)]2+ (5.0 μM). (A) pH = 0.17-2.76;
(B) pH = 2.55-5.35; (C) pH = 7.27-10.86. Arrows
show spectral changes upon increasing pH.
Figure 2: pH effects on the emission spectra (lex = 462 nm) of [Ru(bpy)2(Hppip)]2+ (5.0 μM) as a func-
tion of pH. (A) pH = 0.17-4.56; (B) pH = 5.35-10.27. Arrows show spectral changes upon increasing pH.

[DNA]/(ea –ef) = [DNA]/(eb –ef) + 1/{Kb(eb –ef)}
[3]
963
where [DNA] is the DNA concentration in base pairs, ea is the apparent absorp-
tion coefficient calculated as Aabs/[Ru], eb and ef are the extinction coefficients for
the free and bound forms of the ruthenium complex. In a plot of [DNA]/(ea –ef)
versus [DNA], Kb is given by the ratio of the slope to the y intercept. An intrinsic
DNA binding constants Kb for 1 was derived to be (5.56 0.2) × 105 M-1 in buff-
ered 50 mM NaCl, which is larger than Kb = 4.7 × 105 M-1 reported for analogous
intercalator of [Ru(bpy)2(Hpip)]2+ (43), 3.2-fold a Kb value of 1.7 × 105 M-1 for
[Ru(bpy)2(Hbpip)]2+ (32), 11.3-fold 4.9 × 104 M-1 for [Ru(bpy)2(Haip)]2+, indicating
that the introduction of pyridyl onto Hpip of the parent complex [Ru(bpy)2(Hpip)]2+
is very favorable for the complex to intercalate into the DNA, while the graft-
ing of phenyl to pip was reported to be unfavorable for the DNA-binding. How-
ever, the Kb value for [Ru(bpy)2(Hppip)]2+ is much lower than Kb > 106 M-1 for
[Ru(bpy)2(Hbopip)]2+ {Hbopip = 2-(4-benzoxazolyl)phenylimidazo[4,5-f][1,10]
phenanthroline}(39, 70) or even dipyrido- [3,2-a:2’,3’-c]phenazine (dppz)-based
DNA intercalators (71, 72). The DNA binding characteristics with respect to the
hypochromism, DNA binding constants, and induced red-shifts suggest that the
complex in this work most likely binds to the DNA in intercalative mode, involv-
ing a strong stacking interaction between the aromatic chromophore and the base
pairs of the DNA.
A New Ru(II) Complex
with DNA
In addition to the electrostatic contribution, the following two factors were reported
to influence DNA-binding affinity: (1) the planarity and plane area of an intercala-
tive ligand. Good planarity of an intercalative ligand is usually advantageous for the
complex binding to DNA in intercalative mode; (2) the energy (eLUMO) and popula-
tion of the lowest unoccupied molecular orbital (LUMO) of the complex. As is well-
established, an intercalative Ru(II) complex could act as an electron accepter, and
bind to electron-donating DNA by formation of p-p stacking interactions between
the intercalative ligand and DNA base pairs. Thus the lower the eLUMO of a complex
is, the stronger the p-p stacking interaction between the complex and DNA would be.
The related geometrical parameters of 1, 2 (43) and 3 are shown in Table I, and the
frontier molecular orbital energies of the complexes are illustrated in Scheme 3 (see
Figure S1). Since the LUMO energies of the three complexes are comparable to each
other, the planarity and plane area of the intercalative ligands of the complexes are
expected to play a dominant role in the DNA-binding affinities. The dihedral angles
in Table I show that the planarity of the main ligands of 1-3 follow the order of Hppip
> Hpip >> Hbpip. The poor planarity of Hbpip is due to the steric hindrance between
the hydrogen atoms on the two benzene rings of Hbpip ligand. DNA binding affini-
ties of Hppip , Hpip and Hbpip-containing complexes of 1-3 are anticipated to follow
the order of 1 > 2 >> 3, which agrees well with the experimental observations.
Figure 3: Changes in absorption spectra (A) and
emission spectra (lex
=
462 nm) (B) of
[Ru(bpy)2(Hppip)]2+ (5.0 μM) upon addition of ct-DNA
(0-13.8 μM) in buffered 50 mM NaCl (pH = 7.10).
Emission Spectroscopy: 1 in aerated aqueous solutions at room temperature emitted
strongly with luminescence peak at ~607 nm (j = 0.046), which is blue-shifted relative
to the peaks at 628 and 632 nm reported for [Ru(bpy)2(Hpip)]2+ and [Ru(bpy)2(Hip)]2+,
respectively (43). Changes in emission spectra of the complex with increasing DNA
concentrations ([DNA] = 0-13.8 μM) was shown in Figure 3B. As the DNA was
successively added into the complex solution, the emission intensities was increased
moderately with an intensity enhancement factor of 0.4 (j = 0.092) and a blue-shift in
the emission peak from 607 to 603 nm at [DNA]/[Ru] ≈ 3. The DNA-binding induced
emission enhancement most probably resulted from intercalative interaction since the
hydrophobic environment inside the DNA helix reduced the accessibility of water
molecules to the complex and the mobility of complex were restricted at the binding
site, leading to a decrease in radiationless vibrational relaxation.
[Fe(CN)6]4–, as an excellent anionic quencher, can efficiently quenches the emis-
sions of ruthenium complexes in the absence of the DNA due to ion pairing between
the complex cations and [Fe(CN)6]4– (73), but poorly quenches the emissions of

ruthenium complexes intercalating into the DNA polyanion due to that the electro-
static repulsion between highly anionic [Fe(CN)6]4 – and DNA polyanion hinders the
approaching of the quencher to the bound complex. Steady-state emission quench-
ing experiments using [Fe(CN)6]4 - as the quencher can therefore be an indispens-
able method to study DNA binding properties of a drug molecule. Figure 4 shows
Stern-Volmer plots for the emission quenching of 1 by [Fe(CN)6]4- in the absence
and the presence of ct-DNA. Clearly, in the absence of the DNA, the emission
of 1 was efficiently quenched by [Fe(CN)6]4–, resulting in an almost linear Stern-
Volmer plot with a slope of (8.8 0.2) ×103 M-1 (KQ1). However, in the presence of
DNA {[DNA]/[Ru] = 4}, the slope of the quenching plot decreased remarkably to
(7.6 0.3) ×102 M-1 (KQ2). A ratio (R) of the Stern-Volmer quenching constants, KQ1
to KQ2, is derived to be ~11.4, indicating that a better protection of 1 by the DNA
probably in an intercalative mode.
964
Zhang et al.
The competitive binding experiments with a well-established quenching assay
based on the displacement of the intercalating drug EB from ct-DNA was carried
out in order to get further information regarding the DNA binding properties of 1. If
1 could be a DNA intercalator, the successive additions of 1 to the DNA pretreated
with EB would cause the evident quenching in fluorescence of DNA-bound EB due
to the fact that the fluorescence of the free EB (lex = 537 nm) would be quenched
by the surrounding water molecules (74). On the contrary, if the evident reduction
in emission intensities of the EB-DNA system would not be observed, the complex
would not be a DNA intercalator. The groove DNA binders were reported to be also
capable of causing the reduction in EB emission intensities, but only moderately
(75). As shown in Figure 5, additions of 1 to the EB-DNA system resulted in obvi-
ous reduction in emission intensities of EB by following the linear Sterm-Volmer
equation {I0/I = 1 + KD[Ru]/[DNA]} with a Stern-Volmer constant KD of 2.82 (see
inset of Figure 5A), indicating that 1 is apparently a DNA intercalator. In addition,
a competitive binding model is used to calculate the apparent DNA binding con-
stant Kapp of 1 by using eq 4 (76)
Figure 4: Emission quenching of [Ru(bpy)2
(Hppip)]2+ (5.0 μM) with increasing concentrations
of [Fe(CN)6]4- (0.0-8.0 mM) in the absence of (solid
squares) and the presence of (hollow squares) the
DNA (20.0 μM).
Kapp = KEB([Ru]/[EB])50%
[4]
where KEB (4.94 × 105 M-1) (77) is the DNA binding constant of EB, and ([Ru]/
[EB])50% is the ratio of 1 and EB concentrations at 50% EB replacement. A Kapp
value was derived to be 8.00 × 105 M-1 for [Ru(bpy)2(Hppip)]2+, which is in agree-
ment with the Kb value derived from UV-vis spectral titration data.
Reverse Salt Effects on DNA Binding: As anticipated from polyelectrolyte theory,
the interaction of positively charged Ru(II) complex with nucleic acids would be
influenced by the presence of cations or the ionic strength of the solution (78).
The sensitivity to ionic strength was reported to decrease in the order of the DNA
binding modes: electrostatic > groove > intercalative, which has proved a quali-
tative and a quantitative manner to give information on the DNA binding mode.
In this study, the effects of the ionic strength on absorption and emission spectra
(lex = 462 nm) of 1 were tested by changing concentrations of NaCl in 5 mM Tirs-
HCl buffer solution at pH = 7.10 (see Figure 6), and the polyelectrolyte theory
was used to evaluate the relative electrostatic and nonelectrostatic contributions
to the binding free energy changes. The effects of the ionic strength on the emis-
sion spectra of 1 were test by the addition of NaCl from 0 to 100 mM because
the polyelectrolyte theory is strictly applicable to the system with salt concen-
trations lower than 0.100 M. The plot of log[Na+] against logKobs (Figure 6B)
clearly shows that the binding constant values of 1 decreased with increasing salt
concentrations. This is due to the stoichiometry release of sodium ion following
the binding of 1 to the DNA, suggesting that electrostatic interaction is involved in
the DNA-binding event. The slope of linear fitting in Figure 6(B) is equal to –Zy
in the following equation:
Figure 5: (A) Changes in emission spectra (lex
=
537 nm) of EB ([EB] = 20.0 μM) bound to DNA
([DNA] = 100.0 μM) upon successive additions of
[Ru(bpy)2(Hppip)]2+ (0.0-90.8 μM). The arrows
show the intensity changes upon increasing concen-
trations of the complex. Inset: florescence quenching
curve of DNA-bound EB by the complex. (B) Plot of
percentage of free EB vs. [Ru]/[EB].

–Zy = dlogKobs/dlog[Na+]
[5]
965
where Z is the charge on the Ru(II) complex, and y is the fraction of counterions
associated with each DNA phosphate (y = 0.88 for double-stranded B-form DNA).
The Z value for the DNA-bound complex was thus derived to be 2.The Gibbs free
energy change observed for DNA binding, DGobs, can be calculated on basis of the
standard Gibbs eq 6:
A New Ru(II) Complex
with DNA
DGobs = –RTlnKobs
DGpe = ZyRTln[Na+]
DGt = DGobs – DGpe
[6]
[7]
[8]
DGobs can be divided into two portions, electrostatic (DGpe) and nonelectrostatic
(DGt) components, which can be calculated from eqs 7 and 8, respectively. The
thermodynamic binding parameters of 1 along with those reported for representa-
tive DNA binders are summarized in Table II (37, 76, 79, 80). The percent non-
electrostatic contribution to DGobs was derived to be 53% for [Ru(bpy)2(Hppip)]2+
in 50 mM NaCl, which is comparable to those of the proven intercalators, such
as [Ru(bpy)2(dppz)]2+ (79) and [Ru(bpy)2(Happip)]2+ (37), and much more than
those of semi-intercalator [Ru(bpy)2(phen)]2+ (79) and electrostatically dominating
[(bpy)2Ru(tpphz)Ru(bpy)2]4+ (80), being consistent with the intercalative binding
mode of 1.
DNA Melting Experiments: The melting of the DNA double strands into the single
strand can lead to an intensity increase for the band at 260 nm, since the absorp-
tion in this wavelength for the bases of double-helical DNA is much less than that
in the single form (81). And the intercalation of small molecules into the helix is
known to increase the helix melting temperature (Tm), the temperature at which
the double helix denatures into single-stranded DNA (82). So the DNA melting
experiment offers another absolutely indispensable way to conclude how the com-
plex binds to the DNA, intercalatively or electrostatically. The values of melting
temperature, Tm, of the DNA double-strands were obtained by sigmoidal fitting of
the data of percentage of single-strand DNA versus temperature. From the data
shown in Figure 7, the Tm value of ct-DNA in the absence of 1 was determined to
be 67.5°C and was found to increase successively upon increasing the conconcen-
trations of 1. However, an accurate value of DNA melting temperature change,
DTm, at a concentration ratio of [Ru]/[DNA] = 1:10 could not be obtained because
the absorption band of DNA at 260 nm have not yet reached a platform at the
maximum temperature of the instrument. A DTm value of > 10°C were roughly
estimated. Such a large increase in DTm upon binding of 1 to the DNA, strongly
supports an intercalative DNA binding mode of 1, since the classical intercalators
were reported to generally give DTm values of > 5°C at the concentration ratio of
[DNA]/[Ru] = 10:1 (83, 84).
Figure 6: (A) Changes in emission spectra (lex
=
462 nm) of [Ru(bpy)2(Hppip)]2+ (5 μM) upon addi-
tion of NaCl (0-100 mM) in 5 mM Tirs-HCl buffer
(pH = 7.10). (B) Plot of log[Na+] vs. logKobs in 5 mM
Tris-HCl buffer solution. The slope of this plot
corresponds to the – Zy in eq 5.
Table II
Thermodynamic DNA binding parametersa of K in M–1 and DG in kJ mol–1.
DNA binder b
binding mode
Kobs/103
Z
Ref
DGobs
DGpe
DGt (DGt/DGobs
)
ethidium bromide
intercalative
intercalative
intercalative
intercalative
electrostatic
semi-intercalative
494
3200
295
556
51000
0.55
0.85
2.15
1.51
2.38
4.03
1.82
–32.2
–37.2
–31.2
–32.8
–43.5
-15.6
–5.0
–13.8
–9.9
–15.6
–26.4
-11.6
–27.2 (85%)
–23.4 (63%)
–21.3 (68%)
–17.2 (53%)
–17.2 (39%)
-4.0 (26%)
76
79
37
[Ru(phen)2(dppz)]2+
[Ru(bpy)2(Happip)]2+
[Ru(bpy)2(Hppip)]2+
[(bpy)2Ru(tpphz)Ru(bpy)2]4+
[Ru(bpy)2(phen)]2+
This work
80
79
aIn 50 mM NaCl, 5 mM Tris-HCl buffer at room temperature. bdppz = dipyrido[3,2-a:2′,3′-c]phenazine; phen=1,10-phenanthroline;Happip=2-(4-(β-D-
allopyranoside)phenyl)imidazo[4,5-f][1,10]phenanthroline; tpphz = tetrapyrido-[3,2-a:2′,3′-c:3″,2″-h:2″,3′″-j]phenazine.

DNA Viscosity Measurements: Hydrodynamic measurements, such as viscosity
and sedimentation, are critical evidences for a binding mode in solution in the
absence of crystallographic structural data because they are sensitive to the length
changes of nucleic acids. Therefore, to probe the nature of the interaction between
1 and the DNA, the effects of the successive addition of 1 on the viscosities of
aqueous ct-DNA solutions were investigated, since optical studies can not provide
sufficient evidence to support the binding mode. It is well-established that a classi-
cal intercalation mode results in lengthening the DNA helix as base pairs are sepa-
rated to accommodate the binding ligand, thus producing the increases in relative
specific viscosity of DNA. In contrast, a partial and/or nonclassical intercalation
of ligand could bend (or kink) the DNA helix, reducing its effective length and
concomitantly its viscosity. In addition, electrostatic and grooving binding were
reported to have little effects on DNA viscosities (17, 85). Effects of increasing
amounts of 1 and EB on the relative viscosities of ct-DNA (0.2 mM) in buffered
50 mM NaCl at 32.34 0.01°C are shown in Figure 8. As shown in Figure 8, the
viscosities of ct-DNA increased upon successive additions of 1, despite that the
increases in the relative viscosities of the DNA are even less evident than those of
the well-known DNA intercalator EB, and additions of higher concentrations of 1
were prohibited due to solubility problem, an intercalative DNA binding mode for
1 was clearly demonstrated.
966
Zhang et al.
Figure 7: Thermal denaturation curves of ct-DNA
(60 μM) at different concentrations of
[Ru(bpy)2(Hppip)]2+ as shown in the left corner.
Conclusions
In summery, a new Ru(II) complex of [Ru(bpy)2(Hppip)]2+ synthesized by graft-
ing 2-pyridyl to [Ru(bpy)2(Hpip)]2+ acts as a DNA intercalator in buffered 50 mM
NaCl, as evidenced by the following facts: (1) the high hypochromisms H% of
32%, 35% and 26% for absorption bands at 286, 320 and 460 nm respectively, and
the large intrinsic DNA binding constant Kb = (5.56 0.2) × 105 M-1 obtained from
UV-vis spectrophotometric DNA titrations; (2) 1 was effectively protected from
accessibility of the quencher [Fe(CN)6]4- in the presence of DNA, as demonstrated
by a large ratio of 11.4 for quenching constant derived in the absence of ct-DNA
to that in the presence of the DNA; (3) the sharp decreases in emission intensities
of EB-DNA system upon additions of 1, and the large apparent binding constants
Kapp = 8.0 × 105 M-1 validated by EB competition experiment; (4) the contribu-
tion from nonelectrostatic binding (53%) to the total binding free energy; (5) the
large difference of the DNA melting temperature, DTm > 10°C, in the absence and
the presence of 1 at a concentration ratio of [DNA]/[Ru] = 10; (6) the evidently
increasing trend for viscosities of ct-DNA with increasing concentrations of 1.
The DFT calculations indicated that the introduction of 2-pyridyl to Hpip extends
the conjugation plane of the intercalative ligand, and making 1 to bind to ct-DNA
more strongly than [Ru(bpy)2(Hpip)]2+ (43) and [Ru(bpy)2(Hbpip)]2+ (32). This
grafting of 2-pyridyl to Hpip also brought about pH-induced off-on-off lumines-
cent switching of 1.
Supplementary Material
Supplementray material dealing with some frontier MOs of [Ru(bpy)2(Hppip)]2+
and [Ru(bpy)2(Hbpip)]2+ calculated by DFT-B3LYP method is available at no
charge from the authors directly; the supplementary data can also be purchased
from Adenine Press for US $50.00.
Acknowledgement
TheauthorsthanktheNationalNaturalScienceFoundation(Nos.20971016,20771016,
90922004), Beijing Natural Science Foundation (2072011), the Fundamental Research
Funds for the Central Universities (2009SC-1), and Measurements Fund of Beijing
Normal University for financial supports.
Figure 8: Effects of increasing amounts of
[Ru(bpy)2(Hppip)]2+ (circles), and EB (squares) on
the relative viscosities of ct-DNA in buffered 50 mM
NaCl at 32.2°C.

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Date Received: October 6, 2010
Communicated by the Editor Ramaswamy H. Sarma