Synthesis, DNA-binding and photocleavage of "light switch" complexes [Ru(bpy)2(pyip)]2+ and [Ru(phen) 2(pyip)]2+

Article abstract of DOI:10.1016/j.saa.2010.06.030

Two novel Ru(II) complexes [Ru(bpy)₂(pyip)]²⁺ 1 and [Ru(phen)₂(pyip)]²⁺ 2 (bpy = 2,2′-bipyridine; phen = 1,10-phenanthroline; pyip = 2-(pyridine-2-yl)imidazo-[4,5-f][1,10]- phenanthroline), have been synthesized and characterized by elemental analysis, ES-MS, ¹H NMR, UV-Vis. The DNA-binding behaviors of both complexes were studied by spectroscopic methods and viscosity measurements. The results indicate that the two complexes can bind to CT-DNA in an intercalative mode, and also show that these two Ru(II) complexes can promote the photocleavage of pBR322 DNA. In addition, In the presence of Co²⁺, the emission of DNA-[Ru(L)₂pyip]²⁺ can be quenched, which exhibited the DNA "light switch" properties.

Full text of DOI:10.1016/j.saa.2010.06.030

Spectrochimica Acta Part A 77 (2010) 522–527
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, DNA-binding and photocleavage of “light switch” complexes
[Ru(bpy)2(pyip)]2+ and [Ru(phen)2(pyip)]2+
Xue-Wen Liua,b,∗, You-Ming Shena, Ji-Lin Lua,∗, Yuan-Dao Chena,b, Lin Lia, Da-Shun Zhanga
a College of Chemistry and Chemical Engineering, Hunan University of Arts and Science, ChangDe 415000, PR China
b Key Lab of Environment-Friendly Chemistry and Application in Ministry of Education, Xiangtan University, Xiangtan 411105, PR China
a r t i c l e i n f o
a b s t r a c t
Two novel Ru(II) complexes [Ru(bpy)2(pyip)]2+ 1 and [Ru(phen)2(pyip)]2+ 2 (bpy = 2,2ꢀ-bipyridine;
phen = 1,10-phenanthroline; pyip = 2-(pyridine-2-yl)imidazo-[4,5-f][1,10]-phenanthroline), have been
synthesized and characterized by elemental analysis, ES-MS, 1H NMR, UV–Vis. The DNA-binding behav-
iors of both complexes were studied by spectroscopic methods and viscosity measurements. The results
indicate that the two complexes can bind to CT-DNA in an intercalative mode, and also show that these
Article history:
Received 30 April 2010
Received in revised form 4 June 2010
Accepted 17 June 2010
Keywords:
two Ru(II) complexes can promote the photocleavage of pBR322 DNA. In addition, In the presence of Co2+
,
Ru(II) complex
Polypyridyl ligand
Light switch
DNA-binding
Photocleavage
the emission of DNA-[Ru(L)2pyip]2+ can be quenched, which exhibited the DNA “light switch” properties.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
aqueous solution at ambient temperature but displays strong pho-
toluminescence in DNA solution [13]. Furthermore, it is interesting
During the past decades, the DNA-binding properties of
Ru(II) polypyridyl complexes have captured an extensive inter-
est. Ru(II) polypyridyl complexes can bind to DNA through a
noncovalent modes such as electrostatic binding, groove bind-
ing, or intercalation, in which the intercalation mode is the most
interesting one because there is strong ␲–␲ stacking interac-
tion between the intercalative ligand of the complex and base
pairs of DNA. Many important applications of these complexes
require that the complexes could bind to DNA in an intercala-
tive mode. Recently, Ru(II) polypyridyl complexes have been
widely studied due to their potential applications in stereoselec-
tive probes of nucleic acid structures, molecular “light switches”,
DNA-photocleavage, optical reagents and solar energy utiliza-
tions [1–12]. Of these, some Ru(II) polypyridyl complexes function
as “molecular light switch” for DNA in aqueous solution, turn-
ing the switch on or off in the presence of DNA. As is well
known, [Ru(L)2(dppz)]2+ (L = bpy (2,2ꢀ-bipyridine) or phen = (1,10-
phenanthroline); dppz = dipyrido[3,2-a:2ꢀ,3ꢀ-c]phenazine) is the
most extensively investigated complex as molecular “light switch”
for DNA, because the complex shows negligible luminescence in
that [Ru(bpy)2(tpphz)]2+ [14,15] exhibits the DNA light switch
properties through the introduction of Co2+ or Cu2+. In addition,
pH- and DNA-induced dual molecular light switches have also been
reported [16–18]. Such excellent performances of these complexes
come from their binding to DNA in a typical intercalative mode as
well as effective spectral properties.
Although much work has been done, the other metal complexes
that possess DNA light switch properties are rare. In order to further
develop ruthenium(II) complexes as DNA “light switches”, in this
paper, we report the synthesis and characterization of two novel
ruthenium complexes [Ru(L)2pyip]2+ (L = bpy (2,2ꢀ-bipyridine) or
phen (1,10-phenanthroline); pyip = 2-(pyridine-2-yl)imidazo-[4,5-
f][1,10]phenanthroline). The ligand pyip can coordinate metal ion
via two different sites: one is the nitrogen atoms of phen ring,
and the other is composed of one of the pyridyl ring and one
of the imidazole ring. The results show that [Ru(L)2pyip]2+ pos-
sesses “molecular light switch” properties similar to those of
[Ru(bpy)2(tpphz)]2+ [14,15] and [Co(bpy)2(odhip)]3+ [19]. Herein,
we find that the emission of [Ru(L)2pyip]2+ binding to DNA can
be quenched by metal ions (Co2+). Furthermore, the DNA-binding,
DNA-photocleavage and spectral properties of these novel Ru(II)
complexes were carefully studied.
∗
Corresponding authors at: College of Chemistry and Chemical Engineering,
2. Experimental
Hunan University of Arts and Science, 170 dongting Road, ChangDe 415000, PR
China. Tel.: +86 736 7186115; fax: +86 736 7186133.
E-mail addresses: liuxuewen050@sina.com (X.-W. Liu), lu jilin10@163.com
(J.-L. Lu).
The complexes methyl pyridine-2-carboximidate [20,21],
cis-[Ru(bpy)2Cl2]·2H2O, cis-[Ru(phen)2Cl2]·2H2O [22], 5-nitro-
1386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2010.06.030

X.-W. Liu et al. / Spectrochimica Acta Part A 77 (2010) 522–527
523
1,10-phenanthroline [23,24], 6-amino-5-nitro-1,10-phenanthro-
line [25], [Ru(bpy)2(5,6-(NH2)2-phen)]2+ and [Ru(phen)2(5,6-
(NH2)2-phen)]2+ [26] were prepared by the literature methods.
Other reagents and solvents were purchased commercially and
used without further purification unless otherwise noted.
Viscosity measurements were carried out using an Ubbelohde
viscometer maintained at a constant temperature of 30.0 0.1 ◦C
(in a thermostatic bath). Flow time was measured with a digital
stopwatch and every sample was measured three times and an
average flow time was calculated. Data were presented as (Á/Á0)1/3
vs. binding ratio [29], where Á is the viscosity of DNA in the presence
of complex and Á0 is the viscosity of DNA alone.
2.1. Physical measurement
Equilibrium dialysis measurements were conducted at room
temperature with 5 cm3 of CT-DNA (0.5 mM) sealed in a dialysis
bag (cellulose membrane) and 10 cm3 of the complexes (20 ␮M)
outside the bag with the solution stirred for 48 h. Before use, the
dialysis membranes were boiled for ca. 1 h in a 1% EDTA and 3%
NaHCO3 solution, and then rinsed in doubly deionized water.
For the gel electrophoresis experiments, supercoiled pBR322
DNA (0.1 ␮g) was treated with Ru(II) complexes in buffer B (50 mM
Tris, 18 mM NaCl, pH = 7.8), and the solutions were incubated for
1 h in the dark, then irradiated at room temperature with an UV
lamp (365 nm, 10 W). The samples were analyzed by electrophore-
sis for 1.5 h at 75 V in TBE buffer C (89 mM Tris, 89 mM boric acid,
2 mM EDTA) containing 1% agarose gel. The gel was stained with
0.5 ␮g/ml ethidium bromide and then photographed under UV
light.
Microanalyses (C, H, and N) were carried out with a Perkin-
Elmer 240Q elemental analyzer. Electrospray mass spectra were
recorded on a LQC system (Finngan MAT, USA) using CH3CN as
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 m/z values were quoted for the major
peaks in the isotope distribution. 1H NMR spectra were recorded on
a Bruker ARX-500 spectrometer with (CD3)2SO for the complexes
at 500 MHz at room temperature. All chemical shifts relative to TMS
(tetramethylsilane) were given. Absorption spectra were recorded
with a Perkin-Elmer Lambda850 spectrophotometer and emission
spectra on a Perkin-Elmer Ls55 spectrofluorophotometer at room
temperature. CD spectra were recorded on a JASCO J-810 spec-
trometer in a quartz cuvette with a 10 mm path-length at 25 ◦C.
10 acquisitions per spectrum were run from 200 to 400 nm and
scan speed was 50 nm/min.
2.3. Synthesis
2.2. DNA-binding and photocleavage experiments
2.3.1. [Ru(bpy)2pyip](ClO4)2·H2O (1)
A solution of methyl pyridine-2-carboximidate (ca. 0.4 mmol)
in CH3OH was added to a solution of [Ru(bpy)2(5,6-(NH2)2-
phen)](ClO4)2 (0.261 g, 0.3 mmol) in 10 ml glacial acetic acid, and
the mixture was refluxed under argon for 4 h. Upon cooling, the
resulting clear red solution was diluted with water (ca. 40 ml),
neutralized with concentrated aqueous ammonia and treated with
a saturated aqueous solution of NaClO4. The orange precipitate
was collected and purified by column chromatography on a neu-
tral alumina with acetonitrile-toluene (5:1, v/v) as an eluent.
Yield: 0.179 g, 64.5%. Anal (%):(Found: C, 49.22; H, 3.20; N, 13.57%.
Calcd for C38H29N9O9RuCl2: C, 49.19; H, 3.15; N, 13.59%). ES-
MS (CH3CN): m/z = 810.1 ([M−ClO4−]+), 710.3 ([M−2ClO4−−H]+),
All DNA-binding experiments were carried out in buffer A
(50 mM NaCl, 5 mM Tris–HCl, pH = 7.2). Solutions of CT-DNA in
buffer A gave a ratio of UV–Vis absorbance of 1.8–1.9:1 at 260 and
280 nm, indicating that the DNA was sufficiently free of protein
[27]. The concentration of DNA was determined spectrophotomet-
rically (ε260 = 6600 M−1 cm−1) [28].
The absorption titrations of Ru(II) complexes in buffer A were
performed by using a fixed ruthenium concentration (20 ␮M),
to which increments of the DNA stock solution were added.
Ruthenium-DNA solutions were allowed to incubate for 10 min
before the absorption spectra were recorded.
Scheme 1. The synthetic routes of the Ru(II) complexes [Ru(bpy)2(pyip)]2+ and Ru(phen)2(pyip)]2+
.

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X.-W. Liu et al. / Spectrochimica Acta Part A 77 (2010) 522–527
2+). 1H NMR (500 MHz, ppm, DMSO-d6): 9.23
−
]
complex 2 according to Eqs. (1a) and (1b) [34].
355.2 ([M−2ClO4
ꢀ
ꢁ
(s, 2H), 8.87 (d, 2H, J = 8.5 Hz), 8.83 (d, 3H, J = 7.5 Hz), 8.47 (d, 1H,
J = 7.5 Hz), 8.22 (t, 2H), 8.11 (t, 3H), 8.04 (d, 2H, J = 5.0 Hz), 7.92 (d,
1H, J = 5.0 Hz), 7.90 (d, 1H, J = 5.5 Hz), 7.85 (d, 2H, J = 5.5 Hz), 7.62 (d,
2H, J = 5.5 Hz), 7.59 (t, 3H), 7.34 (t, 2H).
εa − εf
εb − εf
b − (b2 − 2K2Ct[DNA]/s)1/2
2KCt
=
(1a)
(1b)
K[DNA]
b = 1 + KCt +
2s
2.3.2. [Ru(phen)2(pyip)](ClO4)2·H2O (2)
where εa is the extinction coefficient observed for the 1MLCT
absorption band at a given DNA concentration, εf is the extinc-
tion coefficient of the complex in the absence of DNA, εb is the
extinction coefficient of the complex fully bound to DNA. K is the
equilibrium binding constant in M−1, Ct is the total metal complex
concentration, [DNA] is the concentration of DNA in M (nucleotide),
and s is the binding site size. The experimental absorption titration
data were fitted to obtain the binding constants by a non-linear
least-square method. The intrinsic binding constants K of com-
plexes 1 and 2 were 5.3 0.2 × 105 M−1 (s = 2.22, Fig. 1 (a)) and
7.0 0.6 × 105 M−1 (s = 1.76, Fig. 1 (b)), respectively. The values
are smaller than that of those Ru(II) complex reported in the lit-
erature, such as [Ru(bpy)2(dppz)]2+ (Table 1) (Kb = 1.1 × 106 M−1
[35]) or [Ru(bpy)2L-pterin]2+ (Kb = 1.1 × 106 M−1 [35]) (L-pterin = 2-
diamino-4(3H)-oxopteridino[6,7-f]phenanthroline). This may be
attributed to the different planarity of the intercalative lig-
and. From the results, we could deduce that the two com-
plexes bind to DNA by intercalation. Here, complex 2 exhibits
This red complex was obtained by
a similar procedure
as described for complex 1, and the only difference is that
[Ru(phen)2(5,6-(NH2)2-phen)](ClO4)2 (0.261 mg, 0.3 mmol) was
used instead of [Ru(bpy)2(5,6-(NH2)2-phen)](ClO4)2. Yield: 0.180 g,
61.4%. Anal (%):(Found: C, 51.71; H, 3.03; N, 12.88%, Calc
for
C42H29N9O9RuCl2: C, 51.69; H, 3.00; N, 12.93%). ES-MS
(CH3CN): m/z = 858.0 ([M−ClO4−]+), 759.0 ([M−2ClO4−−H]+),
−
379.2 ([M−2ClO4
]
2+). 1H NMR (500 MHz, ppm, DMSO-d6): 9.22
(s, 2H), 8.85 (d, 1H, J = 4.5 Hz), 8.78 (d, 2H, J = 1.5 Hz), 8.76 (d, 2H,
J = 1.5 Hz), 8.47 (d, 1H, J = 8.0 Hz), 8.40 (s, 4H), 8.15 (d, 2H, J = 5.5 Hz),
8.08 (m, 3H), 8.02 (d, 2H, J = 5.5 Hz), 7.79 (m, 6H), 7.62 (t, 1H).
3. Results and discussion
3.1. Synthesis and characterization
An outline of the synthesis of the two complexes is presented
in Scheme 1. The ligand pyip was reported by Wang et al. [30].
Obviously, the ligand pyip can coordinate metal ion via two dif-
ferent sites: one is the nitrogen atoms of phen ring, and the other
is composed of one of the pyridyl ring and one of the imidazole
ring. Therefore, to prepare the mononuclear complexes, we have
carried out the condensation of methyl pyridine-2-carboximidate
with the precursor complexes [Ru(bpy)2(5,6-(NH2)2-phen)]2+ or
[Ru(phen)2(5,6-(NH2)2-phen)]2+ on the basis of the method estab-
lished by Schaefer and Peters [21,22] to avoid the formation of
the dinuclear comlpex. The desired Ru(II) complexes were iso-
lated as the corresponding perchlorates, and purified by column
chromatography. In the ES-MS spectra for the complexes, only the
a
stronger DNA-binding affinity than complex 1 due to
signals of [M−ClO4−]+, [M−2ClO4−−H]+ and [M−2ClO4
]
were
−
2+
observed. The measured molecular weights were consistent with
expected values.
These novel Ru(II) complexes gave well-defined 1H NMR spectra
(Fig. S1). The proton chemical shifts were assigned via comparison
with those of similar compounds [8,27,31–33]. The chemical shifts
of all the protons in aromatic region were presented in Section 2.
3.2. Electronic absorption titration
A complex bound to DNA through intercalation can result in
hypochromism and red shift (bathochromism), due to the interca-
lation mode involving a strong ␲–␲ stacking interaction between
aromatic chromophore and the base pairs of DNA. The extent of the
hypochromism in the visible 1MLCT band is commonly consistent
with the strength of intercalative interaction [8].
The absorption spectra of complexes 1 and 2 in the absence
and presence of CT-DNA (at a constant concentration of complexes,
[Ru] = 20 ␮M) are shown in Fig. 1. When the amount of DNA was
increased to a certain extent, for complex 1, the decreases in the
1MLCT transitions reach as high as 23.2% at 458 nm with red shift
of 2 nm at a ratio of [DNA]/[Ru] = 15.2. For complex 2, upon addi-
tion of DNA, the MLCT band at 454 nm exhibits hypochromism
of about 26.9% with 1 nm red shift at a ratio of [DNA]/[Ru] = 13.1,
respectively.
In order to compare quantitatively the DNA-binding affinities
of the complexes, the intrinsic binding constants K of these two
complexes to DNA were obtained by monitoring the changes of
the 1MLCT absorbance at 458 nm for complex 1 and at 454 nm for
Fig. 1. Absorption spectra of complexes 1 (a) and 2 (b) in Tris–HCl buffer upon the
addition of CT-DNA, [Ru] = 20 ␮M, [DNA] = 0–300 ␮M. Arrow shows the absorbance
changing upon the increase in DNA concentration. Inset: plots of (εa − εf)/(εb − εf)
vs. [DNA] for the titration of DNA to Ru(II) complexes.

X.-W. Liu et al. / Spectrochimica Acta Part A 77 (2010) 522–527
525
Table 1
DNA-binding constants Kb of Ru(II) complexes.
Complex
Absorption titration data
Kb (M−1
)
S
H %a
1
2
3
4
[Ru(bpy)2pyip]2+
[Ru(phen)2pyip]2+
[Ru(bpy)2L-pterin]2+
[Ru(bpy)2dppz]2+
5.3 × 105
7.0 × 105
1.1 × 106
2.9 × 106
2.22
1.76
1.4
23.2 (458 nm)
26.9 (454 nm)
21.0 (420 nm)
38.0 (369 nm)
0.84
a
Hypochromism (H %).
the different plane area and hydrophobicity of the ancillary
ligands.
3.3. Steady-state emission studies
In the absence of DNA, complexes 1 and 2 can emit strong
luminescence in buffer A at ambient temperature, with maximum
appearing at 597 and 595 nm, respectively. Upon the addition of CT-
DNA, the emission intensity increases steadily to ca. 2.17 times of
the original for complex 1 and 2.29 times for complex 2 (Fig. 2).
This implies that two complexes can interact with CT-DNA and
be protected by DNA efficiently, since the hydrophobic environ-
ment inside the DNA helix reduces the accessibility of solvent water
molecules to the complex and the complex mobility is restricted
at the binding site, leading to the decrease of vibration modes of
relaxation.
Fig. 3. Emission quenching curves of the complexes 1 (ꢀ), 1 + DNA (ꢁ), 2 (ꢁ), 2 + DNA
(ꢂ) with increasing concentration of [Fe(CN)6]4−. [Ru] = 5 ␮M, [DNA]/[Ru] = 40.
Steady-state
emission
quenching
experiments
using
[Fe(CN)6]4− as quencher may give more information about
the binding of the two complexes with DNA. As shown in Fig. 3,
in the absence of DNA, the emission of complexes 1 and 2 were
efficiently quenched by [Fe(CN)6]4−. However, in the presence of
DNA, the Stern–Volmer plots changed drastically, the emission of
the two complexes were hardly quenched by [Fe(CN)6]4−. This may
be explained by repulsion of the highly negative [Fe(CN)6]4− from
the DNA polyanion backbone which hinders access of [Fe(CN)6]4−
to the DNA-bound complexes [36]. The curvature reflects different
extent of protection, a larger slope for the Stern–Volmer curve
parallels poorer protection and lower binding. From Fig. 3, we can
see that complex 2 binds to DNA more strongly than complex
1, which is consistent with the results observed by electronic
absorption titration.
Fig. 4 gives the emission spectra of DNA-[Ru(L)2pyip]2+ in
the absence and presence of Co2+. From Fig. 4, it could be seen
that after binding to DNA (switch on), the emission of DNA-
Fig. 4. Emission spectra of DNA-[Ru(bpy)2pyip]2+ (a), and DNA-[Ru(phen)2pyip]2+
(b) in Tris–HCl buffer at 298 K in the absence (dash line) and the presence (solid line)
of Co2+. [Ru] = 5 ␮M, [DNA] = 200 ␮M, [Co2+] = 5 ␮M.
[Ru(phen)2pyip]2+ can be quenched by Cobalt(II) ion, thus turning
the light switch off. The addition of 5 ␮M Co2+ to 5 ␮M com-
plex bound to 200 ␮M DNA results in the loss of almost 94% of
the luminescence due to the formation of Co2+-[Ru(phen)2pyip]2+
heterometallic complex. In order to further provide additional
evidence for the quenching originated the formation of het-
erometallic complex, the emission spectra of [Ru(phen)2pyip]2+
without DNA in the absence and presence of ca. an equimolar
concentration Co2+ are measured (Fig. S2). Similar quenching of
luminescence is observed. This observation is consistent with for-
mation of a nonluminescent species, most likely the heterometallic
complex Co2+-[Ru(phen)2pyip]2+. Similar quenching behavior of
[Ru(bpy)2pyip]2+ is also observed upon addition of Co2+
.
3.4. Viscosity properties
Fig. 2. Emission spectra of Ru(II) complexes (2 ␮M) 1 (a) and 2 (b) in Tris–HCl buffer
at 298 K in the absence and the presence of CT-DNA. Arrow shows the intensity
change upon the increase in DNA concentration.
In order to further elucidate the binding mode of the present
complex, the viscosity measurements were carried out on CT-DNA

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X.-W. Liu et al. / Spectrochimica Acta Part A 77 (2010) 522–527
Fig. 6. CD spectra of complex 1 (dash line) and 2 (solid line) in the presence of
CT-DNA after 48 h dialysis with the stirred solution, [Ru] = 20 ␮M, [DNA] = 0.5 mM.
difference between the interactions of its two isomers with DNA.
This difference may be attributed to the different ancillary ligand.
Fig. 5. Effects of the increase in amounts of complexes 1 (ꢁ), 2 (ꢂ) and [Ru(bpy)3]2+
(ꢁ) on the relative viscosity of CT-DNA at 30 ( 0.1) ◦C, respectively. The total con-
centration of DNA is 0.25 mM.
3.6. Photocleavage of pBR322 DNA by Ru(II) complexes
by varying the concentration of the added complex. Hydrodynamic
measurements being sensitive to length change of DNA (i.e. vis-
cosity and sedimentation) are regarded as the least ambiguous and
most critical tests for a classical intercalation model in solution in
the absence of crystallographic structural data [38,39]. A classical
intercalation model demands that the DNA helix lengthens as base
pairs are separated to accommodate the bound ligand, leading to
the increase of DNA viscosity. In contrast, a partial, non-classical
intercalation of ligand could bend (or kink) the DNA helix, reducing
its length and, concomitantly, its viscosity [37,38].
Ru (II) complexes with polypyridyl ligands, as well-known pho-
tocleavers [40–43], have attracted considerable attention. Upon
irradiation, most of them can generate singlet oxygen, thus induce
single-strand or double-strand cleavage of DNA [44]. The effec-
tive cleavage activity is due to the well-behaved redox-active and
photochemical properties.
The potential of the present complexes to cleavage DNA was
studied by gel electrophoresis using supercoiled pBR322 DNA in
TBE buffer (pH = 7.8). When circular plasmid DNA is subjected to
gel electrophoresis, relatively fast migration will be observed for
the intact supercoil form (Form I). If scission occurs on one strand
(nicked circulars), the supercoil will relax to generate a slower-
moving nicked circular form (Form II). If both strands are cleaved,
a linear form (Form III) that migrates between Form I and II will be
generated [45].
Fig. 7 shows gel electrophoresis separation of pBR322 DNA after
incubation with each of Ru(II) complexes and irradiation at 365 nm.
No obvious DNA cleavage was observed for controls in the absence
of the complex (Fig. 7a and b: lane 0). With the increase in con-
centration of complex 1 (Fig. 7a) and 2 (Fig. 7b), the amount of
Form I of pBR322 DNA is decreased, whereas that of Form II is
increased. Complex 1 induces the single-strand scissions in super-
coiled DNA; however, at the concentration of 80 ␮M, complex 2 can
almost promote the complete conversion of DNA from Form I to
Form II. These results show that two complexes 1 and 2 can cleave
DNA upon irradiation and complex 2 exhibits a higher efficiency
in DNA-photocleavage than complex 1. However, here the nature
of reactive intermediates involved in the DNA-photocleavage by
complexes 1 and 2 has not been clear yet, and further studies on
the mechanism are currently under way.
The effects of complexes 1, 2 and [Ru(bpy)3]2+ on the vis-
cosity of CT-DNA are shown in Fig. 5. For complex [Ru(bpy)3]2+
,
which has been known to bind to DNA in an electrostatic mode,
it exerts essentially no effect on DNA viscosity. With the increase
in the amount of complex 1 or 2, the relative viscosity of DNA
increases steadily. The extent of increase in viscosity, which may
depend on the DNA-binding mode and affinity, follows the order
of 2 > 1 > [Ru(bpy)3]2+. The experimental results suggest that these
two complexes could bind DNA in a classical intercalation mode.
Due to the more hydrophobic ability of co-ligand phen, complex 2
can intercalate into DNA base pairs deeper and thus show stronger
DNA-binding affinity than complex 1. This is also consistent with
the above spectroscopic results.
3.5. Circular dichroism spectra
Equilibrium dialysis experiments provide the means of examin-
ing the enantioselectivity of the complex binding to DNA. According
to the DNA-binding model, the ꢀ enantiomer binds preferentially
to the right-handed helix [31,39]. The circular dichroism spectra
in UV region of complexes 1 and 2 after their racemic solutions
have been dialyzed against CT-DNA for 48 h are shown in Fig. 6.
The strong CD signals appear for complex 1 with a negative peak
at 280 nm and a positive peak at 291 nm, and for complex 2 with
a negative peak at 257 nm and a positive peak at 267 nm, respec-
tively. Although neither of the complexes has been resolved into
their pure enantiomers, and we cannot determine which enan-
tiomer binds to DNA enantioselectively for both complexes, it is
sure that complexes 1 and 2 interact with CT-DNA enantioselec-
tively. The stronger CD signals of complex 2 suggest the large
Fig. 7. Photoactivated cleavage of pBR322 DNA in the presence of Ru(II) complexes
after 60 min irradiation at 365 nm. Lane 0: DNA alone; Lanes 1–4: (a) complex 1 at
30, 60, 90 and 120 ␮M and (b) 2 at 20, 40, 60 and 80 ␮M.

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4. Conclusions
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We are grateful to the supports of the Natural Science Foun-
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