Synthesis and characterisation of RuII polypyridyl complexes: DNA-binding, photocleavage, and topoisomerase i and II inhibitory activity

Article abstract of DOI:10.1071/CH13329

Two mixed-ligand ruthenium(ii) complexes [Ru(phen)2(cptcp)]2+ (Ru1; phen≤1,10-phenanthroline, cptcp≤2-(4-carbazol-9-yl-phenyl)-1H-1,3,7,8- tetraaza-cyclopenta-[l]-phenanthrene) and [Ru(phen)2(btcpc)]2+ (Ru2; btcpc≤9-butyl-6-(1H-1,3,7,8-tetraaza-cyclo-cyclopenta-[l]-phenanthren-2-yl) -9H-carbazole-3-carbaldehyde) have been synthesised and characterised. The DNA-binding behaviours of the two complexes have been investigated by using spectroscopic and viscosity measurements. Results suggest that the two complexes bind to DNA by intercalation. The photocleavage of plasmid pBR322 DNA indicates that Ru1 exhibits more effective DNA cleavage activity in comparison to that exhibited by Ru2 under the same conditions, and different cleavage mechanisms are determined. Topoisomerase inhibition and DNA strand passage assay confirm that Ru1 may act as an efficient dual inhibitor of topoisomerases I and II, whereas Ru2 may only act as a single inhibitor of topoisomerases II. CSIRO 2013.

Full text of DOI:10.1071/CH13329

CSIRO PUBLISHING
Aust. J. Chem. 2013, 66, 1406–1414
Full Paper
http://dx.doi.org/10.1071/CH13329
Synthesis and Characterisation of RuII Polypyridyl
Complexes: DNA-Binding, Photocleavage,
and Topoisomerase I and II Inhibitory Activity
A C
,
A C
,
A
Xiaojun He,
Guang Yang,
Xiaonan Sun,
A
A B D
, ,
Lingjun Xie, and Lifeng Tan
A
College of Chemistry, Xiangtan University, Xiangtan 411105, China.
Key Lab of Environment-friendly Chemistry and Application in Ministry of Education,
Xiangtan University, Xiangtan 411105, China.
B
C
These two authors are both first authors.
D
Corresponding author. Email: lfwyxh@yahoo.com.cn
Twomixed-ligandruthenium(II) complexes [Ru(phen)2(cptcp)]2þ (Ru1; phen ¼ 1,10-phenanthroline, cptcp ¼ 2-(4-carbazol-
9-yl-phenyl)-1H-1,3,7,8-tetraaza-cyclopenta-[l]-phenanthrene) and [Ru(phen)2(btcpc)]2þ (Ru2; btcpc¼ 9-butyl-6-
(1H-1,3,7,8-tetraaza-cyclo-cyclopenta-[l]-phenanthren-2-yl)-9H-carbazole-3-carbaldehyde) have been synthesised and
characterised. The DNA-binding behaviours of the two complexes have been investigated by using spectroscopic
and viscosity measurements. Results suggest that the two complexes bind to DNA by intercalation. The photocleavage of
plasmid pBR322 DNA indicates that Ru1 exhibits more effective DNA cleavage activity in comparison to that exhibited by
Ru2 under the same conditions, and different cleavage mechanisms are determined. Topoisomerase inhibition and DNA
strand passage assay confirm that Ru1 may act as an efficient dual inhibitor of topoisomerases I and II, whereas Ru2 may only
act as a single inhibitor of topoisomerases II.
Manuscript received: 27 June 2013.
Manuscript accepted: 23 July 2013.
Published online: 20 August 2013.
During the past decade, the interest in the field of RuII
polypyridyl complexes–nucleic acid interactions has burgeoned
due to RuII polypyridyl complexes with rich photochemical
properties and varied coordination forms.[8] However, surpris-
ingly, and in contrast to studies on RuII polypyridyl complexes–
nucleic acid interactions, investigations of the inhibition of
topoisomerases activity by RuII polypyridyl complexes and
the relationship between the structures of RuII complexes and
the enzymatic inhibition activities are very scarce.[6f]
In addition, the emergence of resistance phenomena to
Topo I inhibitors is often accompanied by a concomitant rise
of the level of Topo II expression and vice-versa, resulting in the
failure of clinical therapies.[4] In this regard, a single compound
able to inhibit both Topo I and II may present the advantage of
improving antitopoisomerase activity, with reduced toxic side
effects, with respect to the combination of two inhibitors.[9]
Recently, although some DNA-intercalating RuII polypyridyl
complexes exhibited inhibition activities on Topo II,[7e,8a,10]
studies involving the dual inhibition of Topo I and II by RuII
polypyridyl complexes are very scarce.[8c,11] Therefore, studies
on the interaction between polypyridyl-based RuII complexes and
topoisomerases are very important for developing novel antitu-
mour drugs and elucidating the underlying molecular mechanism.
Considering all the above, two new RuII polypyridyl
complexes (Fig. 1), [Ru(phen)2(cptcp)]2þ (Ru1; phen ¼ 1,10-
phenanthroline, cptcp ¼ 2-(4-carbazol-9-yl-phenyl)-1H-1,3,7,8-
tetraaza-cyclopenta-[l]-phenanthrene) and [Ru(phen)2(btcpc)]2þ
Introduction
DNA topoisomerases are crucial nuclear enzymes that regulate
the topological state of DNA during replication, transcription,
recombination, and chromosome segregation at mitosis.[1] Two
types of DNA topoisomerases have been isolated from prokar-
yotes and eukaryotes. In general, topoisomerase I (Topo I)
catalyses the relaxation of superhelical DNA generating a
transient single strand nick in the DNA duplex,[2] whereas
topoisomerase II (Topo II) mediates the ATP-dependent
induction of coordinated nicks in both strands of the DNA
duplex.[3] Under normal conditions, the step of DNA relegation
is much faster than that of DNA cleavage, which may be tol-
erated by the cell. However, conditions that significantly change
either the physiological concentration or the lifetime of the
breaks are responsible for DNA alterations, playing a crucial
role in inhibiting cell cycle progression.[4] Thus, inhibition of
topoisomerases has been considered as an effective strategy for
the design of many anticancer agents.[5]
In recent years, numerous natural and synthetic compounds
have been chosen to detect the inhibition of topoisomerase
activity.[2a,6] Most of such studies at present, however, have
mainly revolved around organic compounds and, to a far lesser
extent, around metal complexes.[6f,7] Metal complexes with a
coordination centre have many advantages over organic mole-
cules, such as a larger variety of structures, higher environ-
mental stability, and a much greater diversity of tunable
electronic properties.
Journal compilation Ó CSIRO 2013
www.publish.csiro.au/journals/ajc

Studies on the Properties of RuII Complexes
1407
2+
2ꢀ
N
N
N
N
H
N
N
N
N
H
N
C4H9
N
Ru
N
N
Ru
N
N
N
N
N
N
OHC
Ru1
Ru2
Fig. 1. Chemical structures of Ru1 and Ru2.
O
N
O
N
H
F
CHO
N
N
N
N
NH
N
CHO
N
DMF/Potassium
tert-butoxide
HAc/NH4AC
cptcp
2ꢀ
Ru(phen)2Cl2
Ethylene glycol,
Ar, reflux, 8 h
N
N
H
N
N
Ru
N
N
N
N
N
Ru1
OHC
OHC
KOH
POCl3
DMF
CH3(CH2)CH2Br
N
C4H9
NH
ꢀ
N
C4H9
N
O
O
2ꢀ
HAc/NH4AC
N
N
N
H
N
N
N
C4H9
H
N
Ru
N
N
N
Ru(phen)2Cl2
C4H9
N
N
N
Ethylene glycol, Ar,
reflux, 8 h
N
N
OHC
OHC
Ru2
btcpc
Scheme 1. Synthesis of the ligands cptcp (2-(4-carbazol-9-yl-phenyl)-1H-1,3,7,8-tetraaza-cyclopenta-[l]-phenanthrene) and btcpc (9-butyl-6-(1H-
1,3,7,8-tetraaza-cyclo-cyclopenta-[l]-phenanthren-2-yl)-9H-carbazole-3-carbaldehyde) and their complexes Ru1 and Ru2.
(Ru2;
btcpc ¼ 9-butyl-6-(1H-1,3,7,8-tetraaza-cyclopenta-[l]-
Results and Discussion
phenanthren-2-yl)-9H-carbazole-3-carbaldehyde) have been
synthesised and characterised. The DNA binding, DNA photo-
cleavage, and topoisomerase inhibitory activity of the two com-
plexes have been analysed.
Synthesis and Characterisation
The synthetic routes to cptcp, btcpc, and their RuII complexes
are shown in Scheme 1. According to protocols reported by

1408
X. He et al.
Steck and Day,[12] cptcp and btcpc were synthesised by cou-
pling 1,10-phenanthroline-5,6-dione with 4-carbazol-9-yl-
benzaldehyde and 9-butyl-9H-carbazole-3,6-dicarbaldehyde,
respectively. Using a mixture of water and ethanol as solvent,
Ru1 and Ru2 were prepared by direct reaction of the precursor
complex cis-[Ru(phen)2Cl2]ꢀ2H2O with the appropriate mole
ratios of cptcp and btcpc, and were obtained in yields of 67 and
62 %, respectively. The desired RuII complexes were isolated as
their perchlorates and then purified by column chromatography
to afford satisfactory purity, which was verified by elemental
analysis, matrix-assisted laser desorption ionisation time-of-
flight (MALDI-ToF) mass spectrometry (Fig. S1 in the Sup-
plementary Material), and NMR spectroscopy (Fig. S2 in the
Supplementary Material).
The absorption spectra of Ru1 and Ru2 (Fig. S3 in the
Supplementary Material) in acetonitrile are very similar and
consist of three well resolved bands at ,460, 350, and 264 nm.
The bands at ,350 and 264 nm are attributed to intraligand (IL)
p - p* transitions.[13] The lowest energy band, at ,460 nm, is
assigned to a metal–ligand charge transfer (MLCT) transition,
which is attributed to Ru(dp) - cptcp or btcpc (p*) transitions.
In comparison to the spectra of other polypyridyl RuII
complexes, such as [Ru(phen)3]2þ (lmax 448 nm),[13] [Ru
(bpy)2(ip)]2þ (lmax 455 nm), [Ru(bpy)2(pip)]2þ (lmax 458 nm)
(where bpy ¼ 2,20-bipyridine, ip ¼ imidazo[4,5-f] [1,10]-
1.0
0.8
0.6
0.4
0.2
0
0.75
0.60
0.45
0.30
0.15
0
0
0.6 1.2 1.8 2.4 3.0 3.6 4.2
[DNA] ꢁ 105
250
300
350
400
450
500
550
600
Wavelength [nm]
1.0
0.8
0.6
0.4
0.2
0
1.50
1.25
1.00
0.75
0.50
0.25
0
phenanthroline,
and
pip ¼ 2-phenylimidazo[4,5-f]1,10-
0
0.5 1.0 1.5 2.0 2.5 3.0 3.5
phenanthroline),[14] the MLCT bands of Ru1 and Ru2 are
obviously red shifted, which may be due to the increased p
delocalisation and, thus, the p-acceptor capacity of the ligands
btcpc and cptcp, resulting in a decreased electron density on the
central RuII and, in turn, stabilisation of the metal dp orbital.
Complexes Ru1 and Ru2, upon dissolution in various
solvents, such as acetonitrile and water, could emit lumines-
cence in the absence of DNA at 258C. As shown in Fig. S4 in
the Supplementary Material, Ru1 and Ru2 showed emission
in acetonitrile with a maximum appearing at 597 and 585 nm,
respectively. Note that distinct red shifts around 12 nm were
observed for Ru1 and Ru2 in aqueous solution. In addition,
the emission spectra of Ru1 and Ru2 are somewhat solvato-
chromic, reflecting that the more polar the solvent, the smaller
the relative intensity.[15,16]
[DNA] ꢁ 105
250
300
350
400
450
500
550
600
Wavelength [nm]
Fig. 2. Absorption spectra of Ru1 (top) and Ru2 (bottom) in Tris–HCl
buffer upon addition of calf thymus (CT)-DNA. [Ru1] ¼ 10 mM, [Ru2] ¼
20 mM, [DNA] ¼ 0–34 mM. Arrow shows the absorbance changing upon the
increase of DNA concentration. Inserts: plots of (ea ꢃ ef)/(eb ꢃ ef) v. [DNA]
for the titration of DNA to complexes Ru1 and Ru2. ea, ef and eb are the
apparent, free, and bound metal complex extinction coefficients,
respectively.
Electronic Absorption Titration
The application of electronic absorption spectroscopy in DNA-
binding studies is one of the most useful techniques.[17] Upon
gradual addition of DNA, the electronic absorption spectros-
copy of the complex is perturbed due to the stacking interactions
between the aromatic chromophore of the intercalative ligand in
the complex and the base pairs of DNA. Therefore, binding of
the complex to DNA by intercalation usually results in hypo-
chromism and bathochromism. In general, the extent of the
hypochromism commonly parallels the intercalative binding
strength. The absorption spectra of Ru1 and Ru2 in the absence
and presence of calf thymus (CT)-DNA are given in Fig. 2. As
the concentrations of DNA are increased to saturation, for Ru1,
the hypochromism at 351 nm reaches as high as 14.1 % with
a red shift of 4 nm at a [DNA]/[Ru] ratio of 2.31; for Ru2, the
hypochromism at 367 nm reaches ,9.8 % with a red shift of
2 nm at a [DNA]/[Ru] ratio of 3.55. These spectroscopic char-
acteristics obviously suggest that both complexes can interact
with DNA, which most likely proceeds through a mode that
involves a stacking interaction between the aromatic chromo-
phore and the base pairs of DNA.
From the decrease of the absorbance of both complexes
(at 351 nm for Ru1 and 367 nm for Ru2), the intrinsic binding
constants Kb of Ru1 and Ru2 to DNA were determined as
(6.8 ꢁ 0.45) ꢂ 106 Mꢃ1 (s ¼ 0.67 ꢁ 0.15), and (4.61 ꢁ 0.22) ꢂ
106 Mꢃ1 (s ¼ 0.53 ꢁ 0.12), respectively. The Kb values of
the two complexes are comparable to that of [Ru
(bpy)2(pip)]2þ [14]
,
[Ru(bpy)2(ppd)]2þ (where ppd ¼ pteridino
[7,6-f][1,10]phenanthroline-1,13(10H,12H)-dione),[16] and the
known DNA intercalator ethidium bromide (EB).[18] However,
the Kb values of Ru1 and Ru2 are stronger than their parent
complexes, [Ru(bpy)3]2þ (4.7 ꢂ 103 Mꢃ1) and [Ru(phen)3]2þ
(5.4 ꢂ 103 Mꢃ1).[19] These data indicate that the size and the
shape of the intercalated ligand of the RuII complexes have a
significant effect on the strength of DNA binding, and the most
suitable intercalating ligand leads to the highest affinity of
complexes with DNA. Although the hydrophobicity of the
intercalative ligand in Ru2 is greater than that in Ru1, the steric
hindrance caused by a normal-butyl group is not advantageous
to the DNA-binding of Ru2, resulting in a lower DNA-binding
affinity. Therefore, synthetically considering these factors, the

Studies on the Properties of RuII Complexes
1409
1.7
1.30
1.25
1.20
1.15
1.10
1.05
1.00
70
60
50
40
30
20
10
0
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0
3.5 7.0 10.5 14.0 17.5 21.0
[DNA]/[Ru]
500
550
600
650
700
750
800
Wavelength [nm]
1.3
1.2
1.1
1.0
40
30
20
10
0
0
0.02
0.04
0.06
0.08
0
3.5 7.0 10.5 14.0 17.5 21.0
[DNA]/[Ru]
[Ru]/[DNA]
Fig. 4. Effect of increasing amounts of ethidium bromide (EB) (£),
[Ru(bpy)3]2þ (bpy ¼ 2,20–bipyridine) (’), Ru1 (¢), and Ru2 (ꢄ) on the
relative viscosity of claf-thymus (CT)-DNA. Total DNA concentration:
0.5 mM, T ¼ 28 ꢁ 0.18C.
bound molecules with the base pairs of DNA. Regarding Ru2,
the intercalative ligand btcpc containing a normal-butyl group is
not advantageous to the interaction between btcpc and DNA,
resulting in a lower DNA-binding affinity relative to Ru1.
500
550
600
650
700
750
800
Wavelength [nm]
Fig. 3. Emission spectra of Ru1 (top) and Ru2 (bottom) in Tris–HCl buffer
in the absence and presence of calf-thymus (CT)-DNA. Arrow shows the
intensity change upon increasing DNA concentrations.
Determination of the Binding Mode by Viscosity Studies
To further clarify the interactions between complexes and
DNA, viscosity measurements were carried out on CT-DNA by
varying the concentration of the added RuII complex. Hydro-
dynamic measurements that are sensitive to length change
(i.e. viscosity and sedimentation) are regarded as the least
ambiguous and the most critical tests of a binding model in
solution in the absence of crystallographic structural data.[21]
Fig. 4 indicates that upon increasing the concentrations of either
Ru1 or Ru2, the relative viscosity of DNA increases steadily,
similar to the behaviour of EB. The increased degree of vis-
cosity, which may depend on its affinity to DNA, follows the
order of EB . Ru1 . Ru2 . [Ru(bpy)3]2þ, suggesting that Ru1
and Ru2 bind to DNA by intercalation with different affinities.
The difference of Ru1 and Ru2 binding with DNA could be
caused by their different intercalative ligands. Obviously, the
normal-butyl group in complex 2 is expected to give rise to steric
hindrance, which is not advantageous to the DNA-binding
of complex 2. Thus, Ru1 containing the intercalative ligand
cptcp can intercalate more deeply into adjacent DNA base
pairs than Ru2 does, causing the helix to extend and the DNA
viscosity to increase.
difference of the DNA-binding affinity of Ru1 and Ru2 can be
well understood.
Luminescence Spectroscopic Studies
Luminescence spectroscopy is one of the most common and
sensitive methods to analyse drug–DNA interactions. Support
for the aforementioned intercalative binding mode also comes
from the emission measurements of both complexes. In Tris–
HCl buffer at 258C, Ru1 and Ru2 showed fluorescence emission
with a maximum appearing at ,600 nm in the absence of DNA
(Fig. 3, dotted line). The emission spectroscopic changes of the
two complexes upon increasing DNA concentrations are also
given in Fig. 3 (solid lines). Fig. 3 indicates that the intrinsic
fluorescence of either Ru1 or Ru2 grows steadily upon addition
of DNA and eventually reaches 1.6 times or 1.3 times that of
either Ru1 or Ru2 without DNA, which implies that the location
of the bound Ru1 and Ru2 is in a hydrophobic environment
similar to an intercalated state. The reason for this is that the
hydrophobic environment inside DNA may reduce the acces-
sibility of water molecules to the complex and the complex
mobility is restricted at the binding site, leading to a decrease of
the vibrational modes of relaxation in the excited state.[20]
Compared with Ru2, the greater fluorescence changes in the
presence of DNA are indicative of stronger association of Ru1 to
DNA. Therefore, Ru1 is protected more efficiently than Ru2 by
DNA, resulting presumably from a more effective overlap of the
Thermal Denaturation Study
DNA melting experiments are useful to determine the extent of
intercalation, because the intercalation of the complex into
DNA base pairs causes stabilisation of the base stacking and,
therefore, raises the melting temperature of the double-stranded
DNA.[22] It is well accepted that when the temperature of the

1410
X. He et al.
Form II
Form I
1.0
0.8
0.6
0.4
0.2
0
(a)
(b)
0
1
2
3
4
5
Form II
Form I
0
1
2
3
4
5
Fig. 6. Photoactivated cleavage of pBR322 DNA in the presence of
(a) Ru1 and (b) Ru2, after irradiation at 365 nm for 45 min. Lanes 0–5 are
the different concentrations of either Ru1 or Ru2: (0) 0; (1) 0.5; (2) 2.0;
(3) 5.0; (4) 10; (5) 15 mM, respectively.
50
60
70
80
90
Form II
Temperature [ꢃC]
(a)
Form I
J
Fig. 5. Melting temperature curves of DNA in the absence ( ) and
presence of Ru1 (ꢄ) and Ru2 (’), respectively. [Ru] ¼ 10 mM,
[DNA] ¼ 100 mM.
0
1
2
3
4
5
solution increases, the double-stranded DNA gradually dis-
sociates into single strands, which generates a hyperchromic
effect on the absorption spectra of the DNA bases (lmax 260 nm).
To identify this transition process, the melting temperature Tm,
which is defined as the temperature where half of the total base
pairs are unbonded, is usually introduced. According to previous
reports,[23] the intercalation of a complex into DNA generally
results in a considerable increase of Tm. The melting curve of
CT-DNA in the absence and presence of either Ru1 or Ru2 is
presented in Fig. 5. Here the Tm of metal complex-free CT-DNA
was determined to be 73.17 ꢁ 0.128C. In the presence of either
Ru1 or Ru2, the Tm increases successively and reaches
82.81 ꢁ 0.26 and 80.07 ꢁ 0.338C, respectively, at a [Ru]/[DNA]
ratio of 1 : 10. The DTm values (9.64 and 6.908C) of Ru1/2–DNA
adducts are larger than those of some RuII intercalators,[23]
which reveals that the modes of both complexes binding to DNA
are intercalation.
Form II
Form I
(b)
0
1
2
3
4
5
Fig. 7. Photoactivated cleavage of pBR322 in the presence of either
(a) Ru1 (10 mM) or (b) Ru2 (10 mM) and different inhibitors after irradiation
at 365 nm for 60 min. Lane 0, no complex; lane 1, no inhibitor; lanes 2–5:
(2) in the presence of mannitol (100 mM); (3) in the presence of DMSO
(10.0 mM); (4) in the presence of superoxide dismutase (1000 U mLꢃ1); and
(5) in the presence of histidine (1.2 mM).
converted into nicked form; at a concentration of 15 mM
((a), lane 5), it can promote the complete conversion of DNA
from Form I into Form II. However, the photoactivated cleavage
of DNA is not sensitive to the concentrations of Ru2. Fig. 6 also
indicates that even at a concentration of 15 mM, Ru2 cannot
promote the complete conversion of DNA from Form I into
Form II. The results indicate that Ru1 exhibits more effective
DNA cleavage activity than Ru2.
Photocleavage of pBR322 DNA by RuII Complexes
The cleavage reaction on plasmid DNA can be monitored by
agarose gel electrophoresis. When circular plasmid DNA is
subject to electrophoresis, relatively fast migration will be
observed for the intact supercoil form (Form I). If scission
occurs on one strand (nicking), the supercoil will relax to gen-
erate a slower moving open circular form (Form II). If both
strands are cleaved, a linear form (Form III) that migrates
between Form I and Form II will be generated.[24]
Fig. 6 shows the photoactivated cleavage of pBR322 DNA in
the presence of different concentrations of either Ru1 or Ru2
after irradiation at 365 nm for 45 min. As seen in Fig. 6, control
photoreactions with DNA alone ((a) and (b), lane 0) resulted in
little or no DNA cleavage. In contrast, photoreactions using
365 nm ((a) and (b), lanes 1–5) resulted in different production
of nicked DNA depending on the concentrations of both com-
plexes used. For Ru1, at a concentration of 10 mM ((a), lane 5),
approximately half of the supercoiled plasmid has been
In order to identify the nature of the reactive species that are
responsible for the photoactivated cleavage of the plasmid DNA
induced by either Ru1 or Ru2, further investigations were
carried out to evaluate the influence of different potentially
inhibiting agents. In the case of Ru1 (Fig. 7a), studies with the
facile hydroxyl radical (ꢄOH) scavengers mannitol and DMSO
were carried out, and no obvious inhibition was observed (lanes
2 and 3), which indicated that the hydroxyl radical (ꢄOH) was
not the reactive species.[25] Similarly, in the presence of super-
oxide dismutase (SOD), a facile superoxide anion radical (O2ꢄꢃ
)
quencher, no inhibition was observed (lane 4), this indicated that
the superoxide anion radical (Oꢄ2ꢃ) was not the reactive species
either. However, in the presence of the singlet oxygen (1O2)
quencher, histidine, obvious inhibition was observed (lane 5),
which suggested that singlet oxygen (1O2) was involved in the

Studies on the Properties of RuII Complexes
1411
Ru1 (I)
Ru1 (IIα)
2
5
10
15
2
5
10
15
Fig. 10. Effects of different concentrations of Ru1 on the activity of DNA
Topo IIa.
Fig. 8. Effects of different concentrations of Ru1 on the activity of DNA
Topo I.
drug to the rate of supercoiling of the relaxed plasmid with EB.
Fig. 9 indicated that the rate of supercoiling of relaxed plasmid
in the presence of Ru1 was much lower than that of relaxed
plasmid in the presence of EB. EB is identical to the rate of Topo
I-catalysed DNA relaxation in the absence of drug. The result
reflected that Ru1 was a Topo I poison, similar to that observed
for other RuII complexes.[28]
EB [min]
10
Ru1 [min]
10
2
5
15
2
5
15
The results of a concentration-dependent Topo II inhibition
assay of Ru1 and Ru2 are given in Fig. 10 and Fig. S6
(Supplementary Material), respectively. As can be seen from
Fig. 10 and Fig. S6, the amount of negatively supercoiled
plasmid suffered different degrees of reduction upon increasing
the concentrations of either Ru1 or Ru2, which suggested that
both complexes could inhibit the activity of Topo II. The value
of IC50 for Topo II inhibition by Ru1 is ,6.6 mM. However, for
Ru2, its value of IC50 is smaller than 2 mM, suggesting that Ru2
exhibits more effective Topo II inhibition activity than Ru1
under the same conditions. Similar to that described above for
Topo I, a DNA strand passage assay was also used to distinguish
the effects of the complexes on Topo IIa function from their
effects on DNA topology. As depicted in Fig. 11 and Fig. S7
(Supplementary Material), compared with the re-ligation rate of
the relaxed plasmid in the presence of EB, the re-ligation rate
of the relaxed plasmid in the presence of either Ru1 or Ru2
didn’t slow down with the passage of time. The results indicate
that Ru1 and Ru2 are suppressors of human topoisomerase IIa.
Fig. 9. The time dependence of Topo I DNA strand passage assays in the
presence of ethidium bromide (EB) and Ru1.
cleavage.[26] On the contrary, for Ru2 (Fig. 7b), the obvious
inhibitions were observed in the presence of the facile hydroxyl
radical (ꢄOH) scavengers mannitol (lane 2) and DMSO (lane 3).
This indicated that for Ru2 the hydroxyl radical (ꢄOH) plays a
significant role in the photocleavage mechanism and the photo-
reduction of RuII complexes with concomitant hydroxide oxi-
dation is an important step in the DNA cleavage reaction.[25]
Topoisomerases I and II Inhibition by Ru1 and Ru2
Topoisomerase inhibitory reactions were performed using Ru1
and Ru2. Fig. 8 indicates that significant amounts of the
supercoiled plasmid gradually increase upon increasing
the concentrations of Ru1, which reflects that Ru1 can inhibit
the ability of Topo I to relax negatively supercoiled plasmid
DNA by blocking the DNA strand passage event of the enzyme.
For Ru1, the value of IC50 (half maximal inhibitory concentra-
tion) is ,15.4 mM. However, in the case of Ru2 (Fig. S5 in the
Supplementary Material), no obvious topoisomerase inhibitions
are observed by increasing its concentrations from 0 to 20 mM,
which indicates that Ru2 can hardly inhibit the ability of Topo I
to relax negatively supercoiled plasmid DNA at concentrations
below 20 mM. The results suggest that Ru1 may serve as a cat-
alytic inhibitor of Topo I. Note that the complexes as DNA
intercalators can induce constrained negative and unconstrained
positive superhelical twists in plasmid DNA, resulting in
directly altering the topological state of the negatively super-
coiled DNA substrate. The reason for this is that Topo I could
only remove the unconstrained positive supercoils. Therefore,
the negatively supercoiled DNA product would be identical to
the topological state of the original plasmid substrate. Thus, the
inhibition of enzyme catalysis may also arise in the presence of
the complexes as DNA intercalators.
Conclusions
Two mixed-ligand RuII complexes [Ru(phen)2(cptcp)]2þ (Ru1)
and [Ru(phen)2(btcpc)]2þ (Ru2) have been synthesised and
characterised. The binding properties of the two complexes
towards CT-DNA indicated that both complexes bind to
CT-DNA by means of intercalation, while the binding affinity of
Ru1 with DNA is greater than that of Ru2 with DNA. Ru1 also
exhibits more effective DNA cleavage activity than Ru2 under
the same condition. For Ru1, the singlet oxygen (1O2) is likely to
be the reactive species responsible for the DNA cleavage,
whereas the hydroxyl radical (ꢄOH) may play a significant role
in the DNA cleavage by Ru2. In addition, topoisomerases
inhibition by the two complexes indicate that Ru1 may serve
as an efficient dual inhibitor of topoisomerases I and II, whereas
Ru2 may only act as a single inhibitor of topoisomerases II.
Furthermore, these results reveal that there are no direct rela-
tions between the DNA binding affinities of the two RuII com-
plexes and their abilities to inhibit the activity of topoisomerases
I and II. We hope that this work will aid in advancing our
knowledge of the interaction between polypyridyl-based RuII
complexes and DNA, as well as laying the foundation for the
rational design of dual topoisomerases I and II inhibitors as
novel anti-cancer agents.
To determine whether Ru1 interferes with the DNA relaxa-
tion reaction by inhibiting Topo I catalysis or by altering the
apparent topological state of DNA, the DNA strand passage
assay was performed.[27] The effects of the complex on enzyme-
catalysed DNA strand passage were assessed by comparing the
rate of relaxation of negatively supercoiled plasmid without

1412
X. He et al.
EB [min]
10
Ru1 [min]
2
5
15
2
5
10
15
Fig. 11. ThetimedependenceofTopo IIDNAstrandpassageassays inthepresenceofethidiumbromide(EB)andRu1.
Experimental
tubes containing supercoiled pBR322 DNA (0.1 mg) and a var-
iable concentration of RuII complexes (0–15 mM) in buffer B
(50 mM TRIS-HCl, 18 mM NaCl, pH 7.2). Irradiation of the
solutions was performed at room temperature with a UV lamp
(365 nm, 10 W). Following irradiation, all the samples were
analysed by 1 % agarose gel electrophoresis for 80 min at 75 V
in buffer C (89 mM Tris, 89 mM boronhydroxide, 2 mM
ethylenediaminetetraacetic acid). Gels were stained with EB
(1 mg mLꢃ1) and then photographed and analysed on the
FluorChem FC2.
Reagents and Physical Measurements
All chemicals used were obtained from commercial sources
and directly used without additional purification. 1,10-
Phenanthroline-5,6-dione,[29]
cis-[Ru(phen)2Cl2]ꢀ2H2O,[30]
4-carbazol-9-yl-benzaldehyde, and 9-butyl-9H-carbazole-3,6-
dicarbaldehyde[31] were synthesised according to the literature
methods. Doubly distilled water was used to prepare buffers.
CT-DNA was obtained from the Sino-American Biotechnology
Co. A solution of CT-DNA in buffer gave a ratio of UV
absorbance at 260 and 280 nm of ,1.8–1.9 : 1, indicating that
the DNA was sufficiently free of protein.[32] The DNA con-
centration per nucleotide was determined by absorption spec-
troscopy using the molar absorption coefficient (6600 Mꢃ1 cmꢃ1
at 260 nm.[33] DNA Topo I from calf thymus together with
human Topo IIa were purchased from TopoGen Inc.
Microanalyses (C, H and N) were carried out on a Perkin–
Elmer 240Q elemental analyser. UV-Vis spectra were recorded
with a PerkinElmer Lambda 25 spectrophotometer, emission
spectra were recorded with a PerkinElmer LS 55 luminescence
spectrometer at room temperature. 1H NMR spectra were
recorded on an Avance-400 spectrometer with d6-DMSO as
solvent at room temperature and tetramethylsilane as the inter-
nal standard. Mass spectrometry was measured on an Autoflex
III Maldi-Tof mass spectrometer (Bruker) using DMSO as the
mobile phase. The data of agarose gel electrophoresis were
recorded on the FluorChem FC2.
Topoisomerase Inhibition Assay
Supercoiled pBR322 DNA (0.1 mg) was incubated with 1 unit of
Topo I (one unit of the enzyme was defined as the amount that
completely relaxes 1 mg of negatively supercoiled pBR322
DNA at 378C in 30 min under the standard assay conditions) and
variable concentrations of RuII complexes (0–15 mM) at 378C
for 30 min in buffer D (35 mM TRIS-HCl, pH 8.0, 2.5 mM
dithiothreitol (DTT), 5 mM MgCl2, 2 mM spermidine, 72 mM
KCl, 0.1 mg mLꢃ1 bovine serum albumin (BSA)), and the
total reaction volume was 20 mL. Reactions were terminated
by adding 4 mL of 5 ꢂ stop solution consisting of 0.25 % bro-
mophenol blue, 45 % glycerol, and 4.5 % sodium dodecyl sul-
phate (SDS). DNA samples were then carried out through 1 %
agarose in buffer C at 75 V for 2 h at room temperature. Gels
were stained with EB (1 mg mLꢃ1), and photographed under the
FluorChem FC2. The concentrations of the inhibitor that pre-
vented 50 % of the supercoiled DNA from being converted into
relaxed DNA (IC50 values) were calculated by the midpoint
concentration for drug-induced DNA unwinding.
)
DNA-Binding Experiments
The methods for spectroscopic titrations and viscosity mea-
surements of Ru1 and Ru2 binding with CT-DNA were as
published.[15] Using the McGheeeVon Hippel (MVH) mod-
el,[34] the intrinsic binding constants (Kb) of either Ru1 or Ru2 to
CT-DNA were determined by monitoring the changes of
absorbance at 351 nm for Ru1 and 367 nm for Ru2 upon addition
of DNA.
Topo II functional activity was assayed in buffer E (10 mM
TRIS-HCl, 50 mM NaCl, 50 mM KCl, 5.0 mM MgCl2, 0.1 mM
Na2H2edta, 15 mg mLꢃ1 BSA, 1.0 mM ATP, pH 7.9) containing
0.1 mg of pBR322 DNA and 2 units of Topo II in the absence and
presence either Ru1 or Ru2. The reaction mixture was incubated
at 308C for 15 min. Except for these, other subsequent operations
were similar to the previous paragraph.
Thermal DNA denaturation experiments were carried out
with a Perkin–Elmer Lambda-25 spectrophotometer equipped
with a Peltier temperature-control programmer (ꢁ0.18C). The
temperature of the solution was increased from 50 to 958C at a
rate of 18C minꢃ1, and the absorbance at 260 nm was continu-
ously monitored for solutions of CT-DNA (100 mM) in the
absence and presence of the RuII complex (10 mM). The data
were presented as (A ꢃ A0)/(Af ꢃ A0) versus temperature, where
Af, A0, and A are the final, initial, and observed absorbance at
260 nm, respectively.
DNA Strand Passage Assay
The DNA strand passage activity of Topo I was determined by
investigating the ability of Topo I to relax negatively super-
coiled plasmid DNA or to supercoil plasmid DNA.[35,36] The
reaction was carried out without drug or with 10 mM RuII
complexes or EB in buffer D. Topo I was added after a 5 min
incubation of DNA with drug or water, and reactions were
incubated up to 15 min at 378C. Reactions were stopped, pro-
cessed, and subjected to gel electrophoresis as above.
Regarding the DNA strand passage activity of Topo II,
reactions contained relaxed or supercoiled pBR322 plasmid
DNA (0.3 mg) and 10 units of Topo II in buffer E. Following a
5 min incubation, Topo II was added and then reactions were
DNA Photocleavage Experiments
The DNA photocleavage experiments were carried out with a
10 mL total sample volume in 0.5 mL transparent Eppendorf

Studies on the Properties of RuII Complexes
1413
incubated up to 15 min at 308C. The rest of the operations were
the same as described above.
Supplementary Material
MALDI-TOF mass spectrometry, 1H NMR spectra, absorption
spectrum and fluorescence spectra of Ru1 and Ru2, effects
of different concentrations of Ru2 on the activity of DNA Topo
I/II, and the time dependence of Topo II DNA strand passage
assays in the presence of Ru2 are available on the Journal’s
website.
Synthesis of Cptcp
The procedure for the synthesis of ligand 2-(4-carbazol-9-yl-
phenyl)-1H-1,3,7,8-tetraaza-cyclopenta-[l]-phenanthrene was
carried out as below. A mixture of 1,10-phenanthroline-5,6-
dione (0.42 g, 2.0 mM), 4-carbazol-9-yl-benzaldehyde (0.54 g,
2.0 mM), ammonium acetate (3.2 g, 42 mM), and glacial acetic
acid (40 cm3) was refluxed with stirring for 5 h, and then cooled
to room temperature and diluted with water (60 mL). Dropwise
addition of concentrated aqueous ammonia gave a yellow pre-
cipitate, which was collected and washed with distilled
water, dichloromethane, and ethanol. The crude product was
completely dissolved in ethanol and then purified by recrys-
tallisation. The pure yellow crystalline solid was filtered from
the solution and dried at 1008C under vacuum. Yield: 0.66 g,
72 %. Anal. Calc. for C31H19N5: C 80.68, H 4.15, N 15.17.
Found: C 80.79, H 4.26, N 15.01 %. m/z (MALDI-ToF,
CH3COOH) 461.96 ([M þ H]þ).
Acknowledgements
The authors are grateful for the support of the National Natural Science
Foundation of the People’s Republic of China (21071120), the Scientific
Research Foundation of Hunan Provincial Education Department (11A117),
Hunan Provincial Natural Science Foundation of China (12JJ2011) and the
Key Project of Chinese Ministry of Education (212127). They also thank
the reviewers for the comments that enabled them to improve upon the
manuscript.
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