Synthesis and the effect of a peripheral N-arylcarbazole moiety on the acid-base and DNA binding properties of a novel RuII complex

Article abstract of DOI:10.1002/ejic.200500748

A novel Ru^II complex of [Ru(bpy)₂(cpipH)](ClO ₄)₂ (where bpy = 2,2′-bipyridine, cpipH = 2-[4-(9H-carbazol-9-yl)phenyl]-1H-imidazol[4,5-f][1,10]phenanthroline) was synthesized. The binding of the complex to calf thymus DNA was investigated with UV/Vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)₆]^4-, DNA competitive binding with ethidium bromide, and thermal denaturation. The pH effects on the UV/Vis absorption and emission spectra of the complex were also studied, and the ground- and excited-state ionization constants were derived. The results indicated that the complex intercalatively bound to the DNA, with an intrinsic binding constant of (8.2 ± 0.8) × 10⁵ M^-1 in buffered 50 mM NaCl, is stronger than the parent complex, [Ru(bpy)₂(pip)]²⁺ (pip = 2-phenylimidazo[4,5-f][1,10]phenanthroline), and its excited states are 0.7-0.8 pK_a units more basic than those of the ground states. Wiley-VCH Verlag GmbH & Co. KGaA, 2006.

Full text of DOI:10.1002/ejic.200500748

FULL PAPER
DOI: 10.1002/ejic.200500748
Synthesis and the Effect of a Peripheral N-Arylcarbazole Moiety on the
II
Acid-Base and DNA Binding Properties of a Novel Ru Complex
[a]
[a,b]
[a]
[a]
Yuan-Yuan Lü, Li-Hua Gao,
Mei-Jiao Han, and Ke-Zhi Wang*
Keywords: Ruthenium / N ligands / DNA / Sensors / Luminescence / UV/Vis spectroscopy
II
A novel Ru complex of [Ru(bpy)
=
2
(cpipH)](ClO
4
)
2
(where bpy
ground- and excited-state ionization constants were derived.
The results indicated that the complex intercalatively bound
2,2Ј-bipyridine, cpipH = 2-[4-(9H-carbazol-9-yl)phenyl]-
1
H-imidazol[4,5-f][1,10]phenanthroline) was synthesized.
to the DNA, with an intrinsic binding constant of
(8.2±0.8)×105
M
–1
in buffered 50 m
(pip)]2+ (pip = 2-phenylimid-
azo[4,5-f][1,10]phenanthroline), and its excited states are
0.7–0.8 pK units more basic than those of the ground states.
M NaCl, is stronger than
The binding of the complex to calf thymus DNA was investi-
gated with UV/Vis absorption and luminescence titrations,
steady-state emission quenching by [Fe(CN) ] , DNA com-
6
petitive binding with ethidium bromide, and thermal dena-
turation. The pH effects on the UV/Vis absorption and emis-
sion spectra of the complex were also studied, and the
the parent complex, [Ru(bpy)
2
4
–
a
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
sion.[6a,9–12] However, studies of the RuII complexes with
both the DNA-binding and acid-base interconversion prop-
Introduction
Over the past two decades ruthenium(ii) polypyridyl erties remain relatively scarce. We have been interested in
complexes have attracted much attention with regard to the syntheses and studies of the acid-base and DNA bind-
DNA interaction studies because of their potential applica- ing properties of both mononuclear and dinuclear imid-
II
[6,13]
tions as nonradioactive structural probes of nucleic acids, azole-containing Ru complexes.
[
1,2]
II
in DNA cleaving, and as chemotherapeutic agents.
The
In this paper we wish to report on a novel Ru complex,
results have shown that the subtle changes in the molecular [Ru(bpy) (cpipH)](ClO ) , in which cpipH was synthesized
2
4 2
II
structures of Ru complexes might bring about substantial by grafting an N-substituted carbazole group to 2-phenyl-
effects on binding modes, sites, and affinities, and provide imidazo[4,5-f][1,10]phenanthroline (pip). Attention was
a chance to explore valuable information on conformation- paid to the effects of the peripheral carbazole group on the
or site-specific DNA probes. The various factors of ligand acid-base and DNA binding properties of this complex by
2+
planarity, the charge carried on the complex, enantio- comparison with the parent complex, [Ru(bpy) (pip)] .
2
selectivity, the formation of intramolecular hydrogen bond- 2,2Ј-Bipyridine (bpy) was chosen as the ancillary ligand for
ing, and the hydrophobicity of ancillary ligands, etc., have the complex since the “parent” complex [Ru(bpy)3]2 was
+
II
been effectively explored, but the new Ru complexes are reported to bind extremely weakly to double-stranded
[
14]
still needed to establish the DNA binding property-struc- DNA. Hence, it is reasonable for us to examine the “in-
[
1,2]
II
ture relationships.
On the other hand, the Ru com- spected” ligand on the DNA binding properties of the com-
plexes that append protonatable/deprotonatable groups, e.g. plex. We demonstrate here the interesting findings regard-
II
2+
hydroxy, carboxyl and amino groups may respond sensi- ing this novel Ru complex, [Ru(bpy) (cpipH)] , interca-
2
tively to the changes in environmental pH, providing a lated to the DNA with
a
binding constant of
5
–1
chance to make pH sensing or switching molecular de- (8.2± 0.8)×10 m in buffered 50 mm NaCl, which is
[
3]
2+
vices, and to greatly modulate their biological functionali- greater than that for [Ru(bpy) (pip)] . The excited states
2
ties.[ The ruthenium(ii) complexes containing imidazole of this new complex are 0.7–0.8 pK units more basic than
4]
a
groups have been well studied with respect to their interac- those of the ground states.
[
5–8]
tion with DNA
or reversible acid-base interconver-
Results and Discussion
[
[
a] Department of Chemistry and Key Laboratory of Radiophar-
maceuticals, Ministry of Education, Beijing Normal University,
Beijing 100875, China
Synthesis and Common Spectral Characteristics
Fax: +86-10-58802075
E-mail: kzwang@bnu.edu.cn
The synthetic route to the complex is summarized in
Figure 1. CpipH was synthesized by coupling 1,10-phenan-
throline-5,6-dione with 4-(carbazol-9-yl)benzaldehyde ac-
b] School of Chemical and Environment Engineering, Beijing
Technology and Business University,
Beijing 100037, China
430
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2006, 430–436

DNA Binding Properties of a Novel RuII Complex
FULL PAPER
cording to protocols reported by Steck and Day.[15] The were found to be 4, 2 and 6 nm, respectively. The hypochro-
complex [Ru(bpy) (cpipH)](ClO ) was synthesized by di- mism of 23.7% for the MLCT band for the complex is
2
4 2
2+
rect reaction of Ru(bpy) Cl ·2H O with cpipH in EtOH/ larger than 15.5% for [Ru(bpy) (ip)]
H O (4:1, v/v), and purified by column chromatography on [Ru(bpy) (pip)] .
silica gel with CH CN/H O/saturated aqueous KNO
and 21.9% for
The hypochromism observed for the
IL transition of 54.2% (285 nm) for our complex appreci-
2
2
2
2
2+ [5a]
2
2
3
2
3
(
400:7:1, v/v/v) as eluent. The complex was obtained in sat- ably prevailed over the 3.8% (280 nm) observed for
2+
2+ [5a]
isfactory purity, which was verified by elemental analysis [Ru(bpy) (ip)] and 20% (283 nm) for [Ru(bpy) (pip)] .
and H NMR spectroscopy.
2
2
1
The spectroscopic changes suggest that the complex has a
stronger interaction with DNA than [Ru(bpy) (ip)] and
2+
2
2+
[
Ru(bpy) (pip)] .
2
Figure 1. The synthetic route to the RuII complex.
The complex, in aqueous solution, showed the lowest-
energy metal-ligand charge transfer (MLCT) transition ab-
sorption band at 462 nm. The energy in the MLCT band
2
+
increased in the order: [Ru(bpy) (cpipH)] (462 nm) Ͻ
2
Figure 2. UV/Vis absorption spectra of the RuII complex (4.6 μm),
in the absence and presence of increasing concentrations of ct-
DNA (0–25 μm).
2
+
2+
[5a]
[
Ru(bpy) (pip)] (458 nm) Ͻ [Ru(bpy) (ip)] (455 nm).
2
2
The high-energy absorption bands at 285 and 338 nm are
attributed to the intraligand (IL) π-π* transition by com-
2
+ [16]
parison with those of [Ru(bpy) ] . The order of energies
To further illustrate the binding strength of the complex
3
in the IL bands parallels that of the MLCT bands: [Ru- quantitatively, the intrinsic DNA binding constant of the
2
+
2+
(
[
bpy) (cpipH)] (285 nm) Ͻ [Ru(bpy) (pip)] (283 nm) Ͻ complex was determined by monitoring the changes in ab-
2
2
2
+
Ru(bpy) (ip)] (280 nm), indicating enhanced π delocaliza- sorbance at 285 nm with increasing concentrations of
2
2+
[20]
tion upon grafting carbazole to [Ru(bpy) (pip)] . The DNA, according to Equation (1),
where [DNA] is the
complex, in TRIS buffer at room temperature, was strongly concentration of DNA in base pairs; the apparent absorp-
emitting under visible light excitation at 465 nm with emis- tion coefficients ε , ε and ε correspond to the extinction
sion maxima occurring at 608 nm, which is characteristic coefficients at a given DNA concentration, the free complex
2
a
f
b
of MLCT luminescence,[ and is assigned to the MLCT in solution and the complex that is fully bound to DNA,
dπ(Ru) Ǟ π* (ligand)] state. An emission quantum yield respectively; and K is given by the ratio of the slope to the
for the complex in aerated H O was determined to be intercept.
17]
3
[
b
2
2
+
0
.0497 by comparison with [Ru(bpy) ] (φstd = 0.033) in
3
[
DNA]/(ε
a
– ε
f
) = [DNA]/(ε
b
– ε
f
) + 1/K
b
(ε
b
– ε
f
)
(1)
[18,19]
aerated aqueous solution.
The MLCT emission max-
ima were bathochromically shifted on going from [Ru-
An intrinsic binding constant was derived to be
2
+
2+
2+
5
–1
2+
(
(
bpy) (cpipH)] to [Ru(bpy) (ip)] : [Ru(bpy) (cpipH)]
(8.2± 0.8)×10 m for [Ru(bpy) (cpipH)] , and ca. twice
2
2
2
2
2
+
2+
5
–1 [20]
λem = 608 nm) Ͻ [Ru(bpy) (pip)] (λem = 615 nm) Ͻ that for [Ru(bpy)
(pip)] (4.7×10 m ).
The hypochro-
2
2
2
+
[5a]
[
Ru(bpy) (ip)] (λ = 625 nm).
mism (H%) and the intrinsic DNA binding constant of
2 em
2+
[
Ru(bpy) (cpipH)] are compared with those reported for
2
representative DNA intercalators in Table 1. The K value
of the complex is larger than those for most of the DNA
intercalators collected in Table 1, while smaller than those
b
DNA-Binding Studies
UV/Vis Spectra
2+
for the strong DNA intercalators of Δ-[Ru(phen)(dppz)]
6
–1
2+
6
–1 [21]
The electronic absorption spectra traces of the complex (3.2×10 m ), Λ-[Ru(phen)(dppz)] (1.7×10 m )
and
The H%
2+
6
–1 [6a]
that was titrated with DNA are given in Figure 2. With in- [Ru(bpy) (ebipcH )(bpy) Ru] (1.3×10 m ).
2
2
2
2+
creasing DNA concentrations, the hypochromisms for the value listed in Table 1 for [Ru(bpy) (cpipH)] is even larger
2
bands at 285, 338 and 462 nm were found to be 54.2%, than those for the typical DNA intercalators mentioned
37.3% and 23.7%, respectively, and the bathochromisms above. The large hypochromism and clear red shifts, as well
Eur. J. Inorg. Chem. 2006, 430–436
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
431

Y.-Y. Lü, L.-H. Gao, M.-J. Han, K.-Z. Wang
(cpipH)]2+ with those for analogous Ru(ii) complexes.
2
FULL PAPER
Table 1. A comparison of DNA binding data for [Ru(bpy)
Complex[a]
Hypochromism
[b]
K
[m ]
×105
R[c]
Ref.
b
b
–1
H /% (λmax/nm)
(ip)]2+
[5a]
[
[
[
[
[
[
Ru(bpy)
Ru(bpy)
Ru(bpy)
Ru(bpy)
Ru(bpy)
Ru(bpy)
2
2
2
2
2
2
15.5 (455)
21.9 (458)
34.8 (248)
17.4 (458)
54.2 (285)
36.6 (288)
32.1 (372)
29.8 (372)
–
0.25
0.051
Ϸ0
3.43
0.0093
Ϸ0
–
–
(pip)]2+
[5a,20a]
[20a]
4.7
1.7
0.42
8.2
13.1
32
2
+
(taptp)2]
+
[20b]
(ipbp)]
(cpipH)]2+
this work
(ebipcH
2
)(bpy)
2
Ru]2+
[15]
2
+
[21]
[21]
Δ-[Ru(phen)
Λ-[Ru(phen)
2
(dppz)]
2
(dppz)]2
+
17
[
a] ip
=
imidazo[4,5-f][1,10]phenanthroline; pip
=
2-phenylimidazo[4,5-f][1,10]phenanthroline; taptp
=
4,5,9,18-tetraazaphen-
anthreno[9,10-b]triphenylene; ipbp = 3-(1H-imidazo[4,5-f][1,10] phenanthrolin-2-yl)-4H-1-benzopyran-4-one; ebipcH
2
= N-ethyl-4,7-
bis([1,10]phenanthroline[5,6-f]imidazo-2-yl)carbazole; dppz = dipyrido[3,2-a:2Ј,3Ј-c]-phenazine. [b] The maximum hypochromism selected
4
–
from the UV/Vis absorption peaks. [c] R is the ratio of the Stern–Volmer quenching constant by using [Fe(CN)
6
]
as the quencher, and
obtained from the ratio of the presence of DNA to that obtained in the absence of DNA.
as a relatively large K value observed for the interaction
b
II
of the Ru complex with the DNA, indicate that complex
Ru(bpy) (cpipH)] might bind to the DNA by intercal-
2
+
[
2
ation.
Luminescence Studies
Steady-state emission quenching experiments for the
4
–
complex, using [Fe(CN)6] as the quencher, further support
the intercalation interaction. As illustrated in Figure 3, in
2
+
the absence of DNA, the emission of [Ru(bpy) (cpipH)]
2
4
–
is efficiently quenched by [Fe(CN) ] , resulting in a slope
6
of 92.6 for a linear Stern–Volmer plot, but in the presence
of DNA the slope of the quenching plot decreases remarka-
bly to 0.49. The positively charged free complex ions should
4
–
be readily quenched by [Fe(CN) ] , but when bound to
6
DNA, the complex can be protected from the quencher be-
4
–
cause the highly negatively charged [Fe(CN)6] would be
repelled by the negative DNA phosphate backbone, hinder- Figure 3. Emission quenching of the Ru complex with increasing
II
4
–
concentrations of [Fe(CN)
6
]
in the absence (᭿) and presence (᭹)
ing quenching of the emission of the bound complex. A
of ct-DNA:[Ru] = 4.6 μm, [DNA]/[Ru] = 9.
ratio (R) of Stern–Volmer quenching constants, using
4
–
[
Fe(CN)6] and obtained from the ratio of the presence of
cause the evident reduction in emission intensities of the
EB–DNA system. The changes in the emission spectra of
the EB–DNA system in the absence and presence of the
complex are shown in Figure 4. The emission of the EB–
DNA system was efficiently quenched by the complex, re-
sulting in a linear Stern–Volmer plot according to Equa-
DNA to that of the absence of DNA, can be taken as a
measure of the binding affinity. A large R value corre-
sponds to a poorer protection and a weaker binding to
DNA. As anticipitated, an R value of 0.0093 found for the
complex we studied in this paper, is smaller than a value
2
+
0
.051 reported for [Ru(bpy) (pip)] , and 0.25 for [Ru(bpy) -
2
2
2
+ [5a]
tion (2), where I and I represent the fluorescence intensities
(ip)] ,
providing further evidence that the binding af-
0
2+
in the absence and presence of complex, respectively; K is a
linear Stern–Volmer quenching constant dependent on the
ratio of the bound concentration of EB to the concentration
of DNA; and r is the ratio of the total concentration of the
complex to that of DNA ([Ru]/[DNA]).
finity to the DNA is in the order of [Ru(bpy) (cpipH)]
Ͼ
2
2
+
2+ [5a]
[
Ru(bpy) (pip)] Ͼ [Ru(bpy) (ip)] , and the interaction
2 2
mode between the complex and the DNA is intercalative.
The fluorescence of ethidium bromide (EB) itself in
aqueous solution is very weak, but when it intercalates be-
tween adjacent DNA base pairs of double-stranded DNA
I
0
/I = 1 + Kr
(2)
6
–1 [22]
(
K = 1.4×10 m ), the fluorescence is greatly enhanced.
b
If a second DNA intercalator is added to the EB–DNA
As shown in the inset of Figure 4, the Stern–Volmer plot
system, it will compete with EB for the DNA binding site, has of a slope of K = 6.17, which is larger than 2.72 for
2
+
and the fluorescence of the EB–DNA system will be [Ru(dmp) (obpip)]
quenched.
(dmp = 2,9-dimethyl-1,10-phenan-
2
[
23,24]
So, the quenching extent of fluorescence of throline; obpip = 2-(2-bromophenyl)imidazo[4,5-f]-1,10-
2+
the EB–DNA system can reflect the DNA binding strength phenanthroline), and 5.98 for [Ru(dmp) (pbip)] (pbip =2-
2
of the second molecule. Otherwise, if the second added (4-bromophenyl)imidazo[4,5-f]-1,10-phenanthroline),[
25]
molecule is not a DNA intercalator, its addition will not but smaller than a K value of 19.3 for [Ru(bpy)-
432
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© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Inorg. Chem. 2006, 430–436

DNA Binding Properties of a Novel RuII Complex
FULL PAPER
Figure 5. Melting curves of ct-DNA in the absence (᭿) and pres-
II
ence (᭹) of the Ru complex. [Ru] = 5.8 μm, [DNA] = 58 μm.
Figure 4. Emission quenching of the EB–DNA system with in-
creasing concentration of the Ru complex. [EB] = 20 μm, [DNA]
II
=
100 μm, [Ru]:[DNA] = 0–0.25.
And on the basis of the neighbor exclusion principle the n
value for the complex was assumed to be 2.0 base pairs. As
(
pp[2,3]p)2]2+ (bpy = 2, 2Ј-bipyridine, pp[2,3]p = pyr-
ido[2Ј,3Ј:5,6]pyrazino[2,3-f][1,10]phenanthroline), and 31.2
4
–1
a result K was calculated to be 9.14×10 m at 73.5 °C. As
the complex binds to ct-DNA, the changes in standard en-
thalpy, standard entropy and standard free energy can be
determined according to Equations (4), (5), and (6), where
K and K are the DNA intrinsic binding constants of the
2
+
for [Ru(phen)(pp[2,3]p) ]
(phen
From the data in Figure 4, we also know
that 50% of EB was replaced by [Ru(bpy)2-
cpipH)]2+ from the DNA bound EB at a concentration
ratio of [Ru]/[EB] Ϸ 0.8. Hence, an apparent DNA binding
=
1,10-phenan-
2
[
13a]
throline).
1
2
(
complex at temperature T and T , respectively; ΔG°, ΔH°,
1
2
and ΔS° are the corresponding standard free energy change,
standard enthalpy change and standard entropy change,
respectively.
6
–1
constant of 1.8×10 m was derived [K (EB)/0.8].
b
Thermal Denaturation Studies
The thermal melting study of the DNA is a technique ln(K /K ) = (ΔH°/R)(T – T )/T T
1 2
(4)
(5)
(6)
1
2
1
2
used to evaluate the stability of a DNA double helix. When
a complex intercalates into DNA base pairs, the base stack-
ing will be more stable and hence the melting temperature
of the double-stranded DNA will rise. So the DNA melting
ΔG°
T
= –RTlnK
ΔG°
= ΔH° – TΔS°
T
temperature (T ) is useful in establishing the extent of inter-
m
[
26]
5
–1
calation.
The melting curves of ct-DNA in the absence
By substituting K = 8.21×10 m (T = 298 K) and K2
1 1
4
–1
and presence of the complex are presented in Figure 5. Tm = 9.14×10 m (T = 346.5 K) into Equations (4)–(6), the
of ct-DNA was found to be 66.3 °C. In the presence of the values of ΔH°, ΔG
2
°
and ΔS° were found to be –
2
98
–1
–1
–1 –1
complex at a concentration ratio [Ru]/[DNA] = 1:10, Tm of 38.9 kJmol , –33.8 kJmol and –17.1 Jmol K at 25 °C,
ct-DNA was raised to 73.5 °C. The large increase of 7.2 °C respectively. The negative ΔG° value suggests that the en-
in Tm is comparable to that observed for a classical DNA ergy of the adduct is lower than the sum of the energies of
[
22–29]
intercalator.
the free complex and DNA. The negative ΔH° suggests that
The DNA intrinsic binding constant at 73.5 °C can be the binding of the complex to DNA at 25 °C is exothermic
obtained from the McGhee equation [Equation (3)], where and enthalpically driven.
0
Tm is the melting temperature of ct-DNA alone, T is the
m
II
melting temperature in the presence of the Ru complex,
Spectrophotometric pH Titrations
ΔH is the enthalpy of DNA (per base pair), R is the gas
constant, K is the DNA binding constant at T , L is the
m
UV/Vis Absorption Spectra
free complex concentration (approximated at the Tm by the
total complex concentration), and n is the size of the bind-
ing site.
As shown in Figure 6, [Ru(bpy) (cpipH)]2 underwent
two successive deprotonation processes upon increasing the
pH from –0.08 to 11.23. When the pH was increased from
+
2
1
/T 0
m
– 1/T
m
= (R/ΔH)ln(1 + KL)1/n
(3)
0.10 to 3.73, the bands at 286 and 342 nm increased in in-
For the ct-DNA used in these studies, under identical tensity and an isosbestic point appeared at 360 nm. These
–
1
solution conditions, a melting enthalpy of 6.9 kcalmol
was determined by differential scanning calorimetry.
spectral changes resulted from the dissociation of the pro-
ton on the protonated imidazole ring. The second depro-
[
30]
Eur. J. Inorg. Chem. 2006, 430–436
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433

Y.-Y. Lü, L.-H. Gao, M.-J. Han, K.-Z. Wang
FULL PAPER
tonation step, which takes place over the pH range of 7.00– Luminescence Spectroscopic Studies
1
0.45, is assigned to the deprotonation of the proton on the
As shown in Figure 7, the emission spectra of the com-
plex are sensitive to pH. When the pH is increased from
neutral imidazole ring, accompanying the following spectral
features: the band at 342 nm and the wave valley at 490 nm
increase, and the band at 286 nm is red-shifted from 286 nm
to 289 nm without any changes in the intensities. The
changes in absorbance at the fixed wavelengths as a func-
tion of pH are shown in the insets of Figure 6. Two ground-
state ionization constants of pKa1 = 0.31± 0.06, pKa2
8
data, shown in the insets of Figure 6(a) and Figure 6(b).
As compared with the two ionization constants of the
protonated imidazole ring on some representative Ru
0.10 to 3.31, the emission maxima are blue-shifted from 628
to 613 nm and the intensities decrease by about 13.9%. As
the pH increases from 6.21 to 10.45, the emission maxima
become red-shifted from 610 to 622 nm, and the intensities
decrease by about 38.1%. The insets of Figure 7, which are
the changes of relative emission intensities vs. pH, clearly
show that the profiles consist of two sigmoidal curves of
opposite gradients, indicative of two deprotonation pro-
cesses. This is the same result as that described in the sec-
tion on UV/Vis absorption spectral titrations. The proton-
ation/deprotonation processes are summarized in Figure 8.
=
.24± 0.03 were obtained by nonlinear sigmoidal fits of the
II
analog complexes, the complex we studied in this paper
is more acidic than [Ru(bpy) (ip)]
2
+
(pKai
=
1.97,
2
2
+
10.46) and [Ru(bpy) (ebipcH )(bpy) Ru]
{ebipcH2
=
2
2
2
N-ethyl-4,7-bis([1,10]phenanthroline[5,6-f]imidazo-2-yl)-
carbazole} (pKai = 4.16, 9.65).[
6a,10]
Figure 7. The changes in the emission spectra of the RuII complex
upon raising the pH: (a) pH = 0.10–3.31; (b) pH = 6.21–10.45.
Figure 6. The changes of electronic absorption spectra of the RuII
complex upon raising the pH: (a) pH = –0.08 to 3.73; (b) pH =
Excited-state ionization constants, pK *, could be
a
roughly evaluated on the basis of the Förster cycle, which
[
3]
6.21 to 10.45.
thermodynamically correlates pK * with pK according to
a
a
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Eur. J. Inorg. Chem. 2006, 430–436

DNA Binding Properties of a Novel RuII Complex
FULL PAPER
Its interesting DNA-binding and pH responsive spectro-
scopic properties make it attractive for applications involv-
ing molecular optical devices.
Experimental Section
Physical Measurements: Elemental analyses were performed with a
Vario EL elemental analyzer. Infrared spectra were measured with
1
a Nicolet–Avatar 360 FT-IR spectrometer using KBr disks.
H
NMR spectra were obtained with a Bruker DRX-500 spectrometer.
UV/Vis absorption spectra were determined with a GBC Cintra 10e
UV/Vis spectrophotometer. Emission spectra were recorded with a
Shimadzu RF-5301PC spectrofluorimeter. The interaction of the
complex with DNA was conducted in buffer A (5 mm TRIS, 50 mm
NaCl, pH = 7.1). A solution of ct-DNA gave ratios of UV ab-
sorbance at 260 and 280 nm of about 1.8–1.9:1 indicating that the
DNA was sufficiently free of protein. The DNA per nucleotide was
determined spectrophotometrically by assuming
ε
260
=
–
1
–1
6
600 m cm . Thermal denaturation experiments of the DNA were
performed on a GBC Cintra 10e UV/Vis spectrophotometer in
Buffer B (1.5 mm Na HPO , 0.5 mm NaH PO , 1 mm Na EDTA).
2
4
2
4
2
Using the thermal melting program, the temperature of the cell
containing the cuvette was increased from 50 °C to 90 °C and the
absorbance at 260 nm was measured every 0.5 °C. The data were
smoothed at 0.5 °C intervals with a filter of 5, and the derivative
of the resulting curve was taken at a data interval of 0.5 °C. The
pH effects on the UV/Vis and emission spectra of the complex were
carried out in DMF/Britton–Roberson buffer (4 mm H
3 3
BO , 4 mm
Figure 8. The acid–base equilibria of the RuII complex.
H PO , 4 mm CH COOH) (1:1, v/v).
3
4
3
[31]
Materials: 4-(Carbazol-9-yl)benzaldehyde,
5,6-dione and cis-Ru(bpy) Cl ·2H O
2 2 2
1,10-phenanthroline-
were prepared according
Equation (7), where ν and ν are pure 0–0 transitions in
[32]
[33]
B
HB
–
1
cm for the basic and acidic species, respectively.
to literature methods.
2
-[4-(9H-Carbazol-9-yl)phenyl]-1H-imidazol[4,5-f][1,10]phenanthro-
pK
a
* = pK
a
+ (0.625/T)(ν
B
– νHB
)
(7)
line (cpipH):[
34]
A
solution of 1,10-phenanthroline-5,6-dione
In practice, ν and ν are often difficult or even imposs- (0.21 g, 1 mmol), 4-(carbazol-9-yl)benzaldehyde (0.27 g, 1 mmol),
B
HB
ible to obtain. A good approximation is obtained by using and ammonium acetate (1.62 g, 21 mmol) dissolved in acetic acid
the emission maxima for νB and νHB, since protonation (30 mL) was refluxed at 110 °C for 5 h under nitrogen. The solution
2
was cooled to room temperature, and H O (60 mL) was added.
equilibrium is almost certainly established between the
3
MLCT states.[18]
The suspension was then neutralized with concentrated aqueous
ammonia. The yellow precipitate that formed was washed with
water, dichloromethane and ethanol, and then dried at 100 °C in
vacuo. Yield 0.185 g (40%). IR (KBr): ν˜ max = 3435 (s), 1610 (m),
By using the emission band maxima from both the pro-
tonated and deprotonated forms of the ruthenium complex
for ν and ν in Equation (7), two pK * values of pK *
B
HB
a
a1
–1
1
1
525 (m), 1482 (m), 1452 (s), 1352 (m), 739 (s), 720 (m) cm . H
=
1.1± 0.1 and pK * = 8.9± 0.1, were obtained. The ex-
a2
NMR ([D ]DMSO): δ = 13.95 (s, 1 H), 9.07 (s, 2 H), 8.98 (d, J =
6
cited state pK * values are 0.7–0.8 pK units more basic
a
a
7.7 Hz, 2 H), 8.59 (d, J = 8.4 Hz, 2 H), 8.30 (d, J = 7.8 Hz, 2 H),
than the respective ground-state ones, indicating that the 7.94 (d, J = 8.4 Hz, 2 H), 7.88 (m, 2 H), 7.56 (d, J = 8.2 Hz, 2 H),
excited electron was delocalized on the cpipH moiety rather 7.50 (t, J = 7.3 Hz, 2 H), 7.35 (t, J = 7.2 Hz, 2 H) ppm.
than on that of the bpy moiety.
31 19 5 2 5
C H N ·0.2C H OH (470.7): calcd. C 80.13, H 4.29, N 14.87;
found C 81.90, H 4.61, N 14.63.
Caution! All the perchlorate salts are potentially explosive and there-
fore should be handled in small quantities with care.
Conclusions
In summary, a newly synthesized RuII complex of
[Ru(bpy) (cpipH)](ClO ) : A mixture of cis-[Ru(bpy) Cl ]·2H O
2
4 2
2
2
2
(
2 5
0.052 g, 0.1 mmol), cpipH·0.2C H OH (0.046 g, 0.98 mmol), etha-
[
Ru(bpy) (cpipH)](ClO ) was demonstrated to be a DNA
2 4 2
nol (8 mL), and H O (2 mL) was refluxed at 100 °C for 9 h under
2
intercalator by evident hypochromism and clear batho-
chromic shifts of the band at 286 nm, and protection from
emission quenching by [Fe(CN)6]4 upon binding to the
DNA, as well as comparable competitive binding to the
DNA with the proven DNA intercalator EB and the large
nitrogen. The solution was cooled to room temperature and was
filtered to remove the insoluble substance. An orange precipitate
was obtained by dropwise addition of a fourfold excessive of aque-
–
ous NaClO
chromatography on silica gel with CH
4
solution. The product was purified by column
CN/H O/saturated aqueous
3
2
increase of 7.2 °C in the melting temperature of the double- KNO3 (400:7:1, v/v/v) as eluent followed by reprecipitation with
stranded DNA at a concentration ratio [Ru]/[DNA] = 1:10. aqueous NaClO solution. Red crystals were obtained. Yield
4
Eur. J. Inorg. Chem. 2006, 430–436
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
435

Y.-Y. Lü, L.-H. Gao, M.-J. Han, K.-Z. Wang
FULL PAPER
0
.064 g (60%). C51
H
35Cl
2
N
9
O
8
Ru (1073.9): calcd. C 57.04, H 3.28,
zuya, T. Yamada, M. D. Hossain, M. Inoue, Dalton Trans.
2003, 2069–2079.
N 11.92; found C 56.60, H 3.55, N 11.86. IR (KBr): ν˜ max = 3431
[
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Received: August 25, 2005
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Published Online: December 6, 2005
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