Bi-nuclear Ru(ii) complexes of bis-chalcone and bis-flavonol: Synthesis, characterization, photo cleavage of DNA and Topoisomerase i inhibition

Article abstract of DOI:10.1039/c3ra41451e

Complexes of type [Ru₂(L¹/L²)(DMSO) ₆Cl₂] (L¹H₂ = 1-(2-Hydroxy-phenyl)- 3-{4-[3-(2-hydroxy-phenyl)-3-oxo-propenyl]-phenyl}-propenone 1, L ²H₂ = 1,4-bis-(3-hydroxy-4-oxo-4H-chromen-2-yl)-benzene) 2, are prepared and characterized using spectroscopic techniques. In complex 1, both ruthenium centres possess a similar coordination environment in which all S-dmso coordinate to ruthenium centres. However, in complex 2, one ruthenium centre is coordinated to three S-bonded dmso, and other ruthenium ion is coordinated to two S-bonded dmso and one O-bonded dmso. This difference in coordination around ruthenium centres in complex 2 is supported by two irreversible oxidation peaks observed at 0.84 V and 1.06 V and two MLCT transitions observed at λ_max 485 and 565 nm in its cyclic voltammetry and UV-visible spectrum respectively. The interaction of complexes with Calf Thymus DNA (CT-DNA) is monitored using UV-vis titrations (K _b = 2.1 × 10⁶ M^-1 for 1 and 1.3 × 10⁵ M^-1 for 2) and ethidium bromide displacement studies (K_sv = 1.37 and 0.98). The stronger binding of complex 1 with CT-DNA as compared to complex 2 could be attributed to its conjugated structural framework. In presence of complex 1 under photo-induced condition in visible region at λ 560 nm, supercoiled DNA is converted more in nicked circular form and supported its stronger binding with DNA as compared to complex 2. Complexes intercalate (1 stronger than 2) with DNA. They also prefer minor groove (1) and major groove (2) binding with DNA. Both complexes inhibit topoisomerase I relaxation activity at concentration > 50 μM. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra41451e

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Bi-nuclear Ru(II) complexes of bis-chalcone and bis-
flavonol: synthesis, characterization, photo cleavage of
DNA and Topoisomerase I inhibition3
Cite this: RSC Advances, 2013, 3,
12210
Ruchi Gaur and Lallan Mishra*
Complexes of type [Ru2(L1/L2)(DMSO)6Cl2] (L1H2 = 1-(2-Hydroxy-phenyl)-3-{4-[3-(2-hydroxy-phenyl)-3-oxo-
propenyl]-phenyl}-propenone 1, L2H2
= 1,4-bis-(3-hydroxy-4-oxo-4H-chromen-2-yl)-benzene) 2, are
prepared and characterized using spectroscopic techniques. In complex 1, both ruthenium centres
possess a similar coordination environment in which all S-dmso coordinate to ruthenium centres. However,
in complex 2, one ruthenium centre is coordinated to three S-bonded dmso, and other ruthenium ion is
coordinated to two S-bonded dmso and one O-bonded dmso. This difference in coordination around
ruthenium centres in complex 2 is supported by two irreversible oxidation peaks observed at 0.84 V and
1.06 V and two MLCT transitions observed at lmax 485 and 565 nm in its cyclic voltammetry and UV-visible
spectrum respectively. The interaction of complexes with Calf Thymus DNA (CT-DNA) is monitored using
UV–vis titrations (Kb = 2.1 6 106 M21 for 1 and 1.3 6 105 M21 for 2) and ethidium bromide displacement
studies (Ksv = 1.37 and 0.98). The stronger binding of complex 1 with CT-DNA as compared to complex 2
could be attributed to its conjugated structural framework. In presence of complex 1 under photo-induced
condition in visible region at l 560 nm, supercoiled DNA is converted more in nicked circular form and
supported its stronger binding with DNA as compared to complex 2. Complexes intercalate (1 stronger
than 2) with DNA. They also prefer minor groove (1) and major groove (2) binding with DNA. Both
complexes inhibit topoisomerase I relaxation activity at concentration . 50 mM.
Received 25th March 2013,
Accepted 24th April 2013
DOI: 10.1039/c3ra41451e
www.rsc.org/advances
molecular oxygen and light are typically utilized to destroy
cancer cells. There are two recognized mechanisms of action
Introduction
for photodynamic therapy.7 The first mechanism (type I)
involves light induced excitation of the photosensitizer
promoting an electron to a higher energy state. The excited
state photosensitizer can act as a reducing agent to create
reactive oxygen species (ROS) such as superoxide radical,
hydroxyl radicals that causes damage of biomolecules and
leads to cell death.8 The type II mechanism also involves
excitation of the photosensitizer with light but in this
mechanism energy is transferred to the triplet ground state
of molecular oxygen resulting in excited singlet state oxygen
which goes on to destroy cellular function.9 On the other hand,
DNA topoisomerases are important enzymes in the nucleus
that modify the topological state of DNA through introduction
of transient breaks in the phosphodiester backbone of the
DNA.10 Therefore, topoisomerase has been identified as an
important biochemical target in chemotherapy and microbial
infections.11 The wide variety of topoisomerase I inhibitors
including camptothecin, irinotecan, doxorubicin, novobiocin,
etoposide topotecan, bind both to enzyme and DNA with
hydrogen bonds and prevent DNA re-ligation. It, therefore
causes DNA damage which results in apoptosis.12
The binding and cleavage of DNA are critical events for
carcinogenesis and gene mutation in biological systems. It
enabled DNA as attractive target1 in the design of chemother-
apeutic and diagnostic agents. In this context, transition metal
complexes were identified as potential chemical nucleases and
ideal DNA-targeting molecules owing to their facilitated
transport inside the cells in addition to easy tuning in their
structural frameworks.2 It is well known that antitumor
efficiency of a drug relies on its binding character, binding
mode, selectivity toward specific DNA base pairs and kinetic
procedure followed during its reaction with DNA.3,4 Activation
of a molecule with low energy light provides a means to
localize the action of a nontoxic molecule to the affected area.
It is known as photodynamic therapy (PDT) and has gained
importance owing to its relevance to antitumor reagents used
in the treatment of cancer.5,6 In PDT, photosensitizers like
Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi
221005, India. E-mail: lmishrabhu@yahoo.co.in; Fax: +91 542 236 8174; Tel: +91
542 670 2449
Electronic supplementary information (ESI) available: CCDC 866469. For ESI
3
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c3ra41451e
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showed sharp bands of different intensities in 1620–400 cm21
region due to vibrations arising from the coordinated dmso,
chalcone and flavonol. The IR spectrum of L1H2 displayed
characteristic peaks at 3452, 1270 and 1639 cm21 assigned to
u(OH) and u(CLO) vibrations, respectively. However, L2H2
showed sharp and strong peaks at 3452, 1283 and 1636
cm21 assigned to u(OH) and u(CLO) vibrations respectively.
The u(CLO) vibration observed at 1614 and 1613 cm21 in the IR
spectra of complexes 1 and 2, respectively showed that CLO
group of the corresponding ligands had coordinated with
Ru(II) ion. However, no peak assignable to u(O–H) vibration
was observed in corresponding IR spectra of the complexes. It
is suggested that deprotonated OH has coordinated to metal
ion. Additional peaks observed at 1090, 1095 and 425, 423
cm21 are assigned to u(SLO) for S-bonded and u(Ru–S)
vibrations in complex 1 and 2 respectively. In the spectrum
of complex 2, u(SLO) for O-bonded DMSO appeared at 1017
cm21 and is found comparable with the peak reported earlier
for O-bonded ruthenium complex.20
In this context, chalcones and flavones were found ligands
of choice owing to their reported biological importance13,14
and high photoreactivity15,16 However, flavonols are well
known for their antiproliferative activities against cancer cell
lines and antioxidant activities comparable to vitamin C.17
Ruthenium complexes of chalcones and flavonols from our
laboratory have already been exploited in DNA cleavage and
cytotoxicity against several cell lines.18,19 Thus, in view of
excellent precedence, the present work stems from our interest
in the construction of dinuclear ruthenium(II) complexes of
chalcone and flavonol containing labile ligand like dmso
facilitating their easy substitution by nucleobases. Present
work embodies the synthesis and characterization of a bis-
chalcone and its bis-flavonol and their corresponding com-
plexes with cis,fac-[RuCl2(dmso-S)3(dmso-O)]. Binding of com-
plexes with DNA, their photo-nuclease and topoisomerase
activity have been studied.
The structures of ligands (L1H2, L2H2) as well as complexes
were further supported by their 1H NMR spectral data (S1). The
OH protons observed in the spectra of free bis chalcone and
bis flavonol disappeared in the spectra of corresponding
complexes. This again supports that deprotonated OH groups
had coordinated with the metal ion. Since there was no peak
observed at d y 2.72 ppm in complex 1 for O-bonded dmso as
reported earlier,21 so all dmso were assigned to S-coordinated
to the metal centre and displayed sharp singlet and multiplets
Result and discussion
Synthesis and description of molecular structure
Synthetic routes adopted for the preparation of ligands, their
Ru(II) complexes, proposed structures of complexes 1 and 2 are
depicted in Scheme 1. The complexes were found to be air
stable and soluble in organic solvents such as acetonitrile,
dimethylformamide (dmf), dimethyl sulfoxide (dmso) and
dichloromethane (dcm) and showed no electrical conductivity
in dmso (1023 M) solution. The IR spectra of these complexes
1
(36 H) in the region of (d) 3.09–3.59 ppm. However, H NMR
spectrum of complex 2 displayed a single peak at d 2.62 ppm
assigned to methyl protons of O-bonded one dmso molecule.
Other peaks observed at d 3.2–3.72 ppm were assigned to
methyl protons of three S-bonded dmso molecules. The sharp
and upfield shifted methyl protons of dmso at d 2.62 ppm
from the earlier reported O-bonded dmso (d y 2.72 ppm) were
attributed to two equivalent methyl groups of the dmso-O lying
in cis position to Cl atom.
The ESI-MS (S2) of complexes recorded in methanol showed
molecular ion peak at m/z 1133 and 1161 assigned to [M + Na]+
for complex 1 and 2 respectively.
In UV-vis spectra of complexes, the intense bands observed
at higher energy showed their dependence on the nature of
O,O-ligands as depicted in Fig. 1. In the electronic absorption
spectra, the peaks observed at lmax 377 and 407 nm for
complex 1 and 2 respectively were assigned to intra-ligand
transitions. These bands were red shifted as compared to the
band observed from respective free ligands (lmax 366 and 381
nm). The orbitals of low spin ruthenium(II) ion (d6 configura-
tion) provide filled metal orbitals of proper symmetry which
interact with relatively low lying p* orbitals of the ligand. In
this context, peak observed at l 511 nm in the spectrum of
complex 1 was assigned to MLCT transition. In the spectrum
of complex 2, peaks observed at l 485 and 565 nm were
assigned to two MLCT transitions arising from both ruthe-
nium centres. It occurred due to different electronic environ-
ments around ruthenium centres. Based on spectroscopic and
analytical data, the structures of both complexes were
tentatively proposed as shown in Scheme 1.
Scheme 1 Synthetic strategy for ligands, complexes and tentative structure of
complex 1 and 2.
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Fig. 1 UV–vis spectra of complexes L1H2, L2H2, 1 and 2 recorded in
dichloromethane (1 6 1024M).
Molecular structure of L1H2 obtained using single crystal
X-ray diffraction study is shown in S3. The crystallographic
data with bond length and bond angle are shown in S4 and S5.
The L1H2 was crystallized from dichloromethane: petroleum
ether (1 : 3) and was found monoclinic crystal system with
P21/c space group. In L1H2, the configuration of the keto
group was found syn with respect to olefinic double bond. It is
planar with the central CLC double bond symmetric trans
configuration to the phenyl ring and geometric parameters in
normal ranges. The bond length between O(1)–C(26) was
found as 1.246(3)Å while O(2)–C(9) = 1.359(3) Å. However,
bond between C(2)–C(4) = 1.393(4) Å was found closer to that
reported for aromatic ring system. It was found that C–H and
CLO groups are involved in hydrogen bonds formation. The
motifs C(7)–H(7) O(1) and O(2)–H(1) O(1) contacts with H–O
at a distance of 2.75 Å and 2.51 Å respectively. This resulted in
a ladder like supramolecular structure as depicted in S6.
Suitable crystals of L2H2 and complexes could not be obtained,
so their structures were supported by spectroscopic analysis
only.
Fig. 2 Cyclic voltammogram of complexes 1 (a) and 2 (b).
Electronic absorption measurements
CT-DNA interaction with complex using UV–vis spectro-
scopy. The binding of a complex with DNA through intercala-
tion usually results in hypochromic and bathochromic shifts
in its UV–vis absorption spectrum. It also involves strong
stacking interaction between an aromatic chromophore and a
base of DNA. The p* orbital of intercalated ligand coupled with
p orbital of DNA base, concomitantly lowers the p* transition
energy hence displays a bathochromic shift in the transition. It
may happen owing to strong stacking interaction between
aromatic rings of the ligand and DNA base pairs. If p* orbital
of intercalated ligand couples with p orbital of the DNA base
pair, p–p* transition energy decreases hence a red shift occurs
in the peak position. On the other hand, if p orbital of a ligand
is partially filled by electrons, it results hypochromic shift.24
Thus, such an observation prompted us to investigate the
binding of newly prepared complexes with DNA using spectro-
scopic titrations of a solution of the complex with varying
concentrations of CT DNA. Upon addition of calf-thymus DNA,
a hypochromic shifts in both chalcone centered and flavonol
centered bands and MLCT band were observed in complexes 1
and 2 without any shift in MLCT band positions as shown in
Fig. 3.
…
…
Cyclic voltammetry
The cyclic voltammetry of the complexes was carried out in
dichloromethane at 298 K using ferrocene/ferrocenium (Fc/
Fc+) as an internal standard (0.10 V (80 mv) vs. Ag/Ag+
electrode. The complex 1 showed irreversible oxidation peak at
0.80 V assigned to RuIII/RuII redox couplex. In complex 2, two
peaks observed at 0.84 V and 1.06 V. Two peaks arise as a
result of metal-based oxidation and are shown in Fig. 2. It
could be understood owing to two different electronic
environments around ruthenium centres as supported by
two MLCT bands. This irreversibility may be considered in
view of lower transient lifetime of the reduced state as reported
earlier.22 However, the lower value of oxidation potential for
Ru(III)/Ru(II) couple in complex 1 and 2 could be attributed to
the stronger s-donor and weaker p-acceptor ability of the
ligands, consequently stabilizing the higher oxidation state of
Ru(III).23 However, complex 1 displayed ligand centred reduc-
tion at 21.15 V whereas complex 2 showed this peak at 21.2 V.
The complexes can bind to double-stranded DNA in
different ways on the basis of their structure, charge and type
of ligands. As DNA double helix possesses many hydrogen
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Fig. 3 UV–vis absorption spectra of [Complex 1 (a) and Complex 2 (b)] = 10 mM
in the absence and presence of increasing amounts of DNA = 0–150 mM.
Fig. 4 Fluorescence quenching pattern of ethidium bromide bound to DNA in
the absence (……) and presence (—) of complexes. [EB] (——) = 10 mM, [DNA] =
10 mM, [Complexes 1 (a) and 2 (b)] = 0–0.325 mM. The arrow shows the intensity
changes upon increasing complex concentrations. (Inset: Plot of I0/I vs.
[complex]/[DNA]).
bonding motifs accessible in both the minor and major
grooves, it is likely that dmso and Cl group of complexes might
be involved in the formation of hydrogen bonds with DNA and
aromatic rings of the ligand in addition to stacking interac-
tions.25 Consequently, it may contribute to hypochromic shift
in its absorption spectrum. In order to compare the binding
strength of complexes with CT DNA, intrinsic binding
constants (Kb) were calculated from the ratio of slope to the
intercept from a plot of [DNA]/(ea 2 ef) vs. [DNA]. The
calculated Kb values of 2.1 6 106 M21 and 1.3 6 105 M21
for complexes 1 and 2 respectively suggested stronger binding
of complex 1 with DNA as compared to binding strength of
complex 2. The binding constant values for complexes 1 and 2
showed stronger binding with DNA as compared to the earlier
reported values for cisplatin26 as well as ruthenium–ammine
complexes.27
Competitive binding of complex with ethidium bromide
using fluorescence spectroscopy. Complexes did not fluores-
cence in aqueous solution or in organic solvents at room
temperature. Therefore, fluorescence titrations with DNA
could not be carried out. However, competitive binding studies
using an ethidium bromide (EB) bound to DNA was carried out
for these complexes irrespective of their binding modes.
Intense fluorescent light is emitted from EB in the presence of
DNA due to its intercalation between adjacent base pairs of
DNA. But a decrease in emission intensity results from the
displacement of EB from DNA helix by a quencher molecule.28
The extent of emission quenching could be used to determine
the extent of binding of a metal complex with DNA. The
emission spectra of EB–DNA system obtained in absence and
presence of complexes are displayed in Fig. 4. When
complexes 1 and 2 were added to DNA pre treated with EB,
the DNA-induced emission intensity of EB was decreased.
Addition of a second DNA binding molecule would quench the
EB emission by either replacing the DNA-bound EB (if it binds
to DNA more strongly than EB) or accepting an excited state
electron from EB. Because these complexes have planar
ligands, they efficiently compete with strong intercalators like
EB for intercalative binding sites on DNA by replacing EB,
which is reflected in quenching of emission intensity of DNA-
bound EB. The quenching plots of Io/I vs. [Complex]/[DNA]
(insets in Fig. 4(a) and (b)) are in good agreement with the
linear Stern–Volmer equation. Stern–Volmer quenching con-
stants (Ksv) were calculated as 1.37 and 0.98 for complex 1 and
complex 2 respectively.
In order to check, both the complexes displaces ethidium
bromide (EB) in the EB–DNA adduct, a UV-vis titration
experiment was performed. It is well known that free ethidium
bromide shows absorbance at 490 nm. After addition of CT-
DNA the intensity of the absorbance decreases.29 Addition of
increasing concentrations of complexes to the EB–DNA adduct
showed a linear increase in the absorption intensity, indicat-
ing increase in the free EB concentration ( S7). A control
3
experiment of reverse competitive binding of ethidium
bromide with complexes bound with DNA was carried out to
find out intercalation mode. Both complexes 1 and 2 did not
show emission, so the emission spectra of complexes bound
with DNA were recorded at excitation l 510 nm as shown in
S8. There is no effect on emission intensity after the addition
3
of increasing concentrations of EB to the complex-DNA
adduct, it indicated complex was fully bound with the DNA
and there is intercalating site remaining for EB.
Effect of ionic strength. The effect of ionic strength on
complex-DNA binding has been examined by studying the
binding in the presence of the strong electrolyte (NaCl) to
verify if there is significant electrostatic interaction between
the complex and DNA.30 An increase in ionic strength leads to
weakening of electrostatic attraction between the positively
charged molecule and DNA. In order to evaluate interaction
mode between DNA and complexes the effect of different
concentrations (0–70 mM) of salt such as NaCl was investi-
gated in sodium phosphate buffer. No significant changes
were observed in absorption spectra of the both the complexes
on increasing the concentration of salt ( S9). These results
3
showed that there are no electrostatic interactions between the
complexes and CT-DNA.31
CD spectral studies
CD spectroscopy is a useful technique in order to determine
the extent of change in conformation of the
B form
conformation of CT-DNA exhibits two conservative CD bands:
a positive band at lmax y 275 nm due to base stacking and a
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tion in migration of supercoiled DNA and a slight increase in
the mobility of open circular DNA. It is monitored by the
transition of the naturally occurring supercoiled form (Form I)
of DNA to open circular relaxed form (Form II) of DNA. This
occurs when one of the strands of the plasmid DNA is nicked;
it is determined by gel electrophoretic mobility assay of
plasmid DNA. If one strand is cleaved, the supercoils will relax
to produce a slower-moving nicked circular form (Form II).33
In order to assess the chemical nuclease activity of complexes
1 and 2 for DNA strand scission, cleavage reaction can be
monitored by gel electrophoresis. Control experiments have
shown that free ligands, complex 1 and 2 did not cleave DNA
in dark on 1 h incubation period at 100 mM concentration
(S10).
Photoinduced cleavage of plasmid DNA
Complexes capable of cleaving DNA on photo-activation are
considered most suitable for therapeutic applications, as they
can be photo-induced to give localized action and thereby
unwanted DNA damage can be avoided.34 Photonuclease
activity of complexes 1 and 2 was studied after exposure of
an aqueous solution of DNA-complexes to a visible light of l
560 nm for 10 minutes. The complexes caused a linear
increase in the intensity of NC DNA band accompanied by the
similar decline in SC DNA in a concentration dependent
manner in aqueous buffer. This suggested that complexes 1
and 2 upon photo irrradiation, were able to produce nicks in
the supercoiled plasmid DNA and hence showed a strong
nuclease activity in aqueous medium. Complex 1 converts
more than 50% of SC form into NC form at a concentration of
60 mM, whereas, for a similar level of conversion, 80 mM of
complex 2 was required as depicted in Fig. 6 and 7(a and b).
Fig. 5 CD spectra of CT-DNA (1 6 1024 M) in the absence and presence of
complexes 1 and 2 (a) DNA with complex 1; (b) DNA with complex 2.
DNA cleavage in presence of groove binders
negative band at lmax y 246 nm due to polynucleotide helicity
DNA upon binding of complexes. Simple groove binding and
electrostatic interaction of the complexes with DNA shows less
or no perturbation of the base stacking and helicity bands,
while an intercalator enhances the intensities of both bands
due to strong base stacking interactions and stable DNA
conformations (right-handed B conformation of CT-DNA).32
CD spectral variations of calf thymus DNA (100 mM, in 0.1
mM Na-phosphate buffer (pH = 7.4), were recorded in presence
of increasing amounts of complexes 1 and 2 until [complex]/
[DNA] molar ratios approached approximately 1. As shown in
Fig. 5, addition of complexes 1 and 2 to DNA, decrease in
intensity of positive CD band of DNA together with increase in
intensity of negative band was observed. However, more
decrease in intensity was observed in the presence of complex
1. Therefore, it was inferred that complex 1 binds DNA tightly
as compared to complex 2 and most likely through intercala-
tion mode. These data indicated that both complexes induced
the loss of characteristic B-DNA conformation, while the
changes did not simply reflect the formation of A-DNA or
Z-DNA.
In order to identify the potential interacting site of the
complexes with supercoiled plasmid pBR322 DNA, gel electro-
phoretic mobility assay was performed in presence of both
minor groove binding agent 49,6-diamidino-2-phenylindole
(DAPI) and major groove binding agent, methyl green (MG).
The supercoiled DNA was treated with DAPI and MG prior to
the addition of complexes 1 and 2 even in lower concentration
(20 mM). As depicted in Fig. 8, in presence of complex 1 and
DAPI, form I DNA is completely converted in form II and form
Fig. 6 (a) Agarose gel electrophoresis showing photocleavage patterns of
pBR322 plasmid DNA (300 ng) by the complex 1 in visible light 560 nm. Lane 1,
DNA control; Lane 2, 20 mM complex 1 + DNA; Lane 3, 40 mM complex 1 + DNA;
Lane 4, 60 mM complex 1 + DNA; Lane 5, 80 mM complex 1 + DNA; Lane 6, 100
mM complex 1 + DNA; (b) Cleavage of supercoiled pBR322 DNA showing the
decrease in SC DNA and the formation of NC DNA with increasing concentration
of complex.
DNA cleavage study
Changes in electrophoretic mobility of plasmid DNA on
agarose gel is commonly taken as evidence for direct DNA–
metal interactions. Alteration of DNA structure causes retarda-
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Fig. 9 Ethidium bromide stained agarose gel (1%) of pBR322 plasmid DNA (300
ng mL21) in the presence of complex 1 (20 mM) after 1 h of incubation: lane 1,
DNA control; lane 2, DNA + sodium formate (50 mM) + complex 1; lane 3, DNA +
NaN3 (50 mM) + complex 1; lane 4, DNA + dmso + complex 1; lane 5, DNA +
H2O2 + complex 1; lane 6, DNA + KI (50 mM) + complex 1; lane 7, DNA + sodium
ascorbate (50 mM) + complex 1; lane 8, DNA + D2O + complex 1.
Fig. 7 (a) Agarose gel electrophoresis showing photocleavage patterns of
pBR322 plasmid DNA (300 ng) by the complex 2 in visible light 560 nm. Lane 1,
DNA control; Lane 2, 20 mM complex 2 + DNA; Lane 3, 40 mM complex 2 + DNA;
Lane 4, 60 mM complex 2 + DNA; Lane 5, 80 mM complex 2 + DNA; Lane 6, 100
mM complex 2 + DNA; (b) Cleavage of supercoiled pBR322 DNA showing the
decrease in SC DNA and the formation of NC DNA with increasing concentration
of complex.
owing to the presence of a different ligand framework in this
complex.
III DNA. However, form I DNA is completely converted in form
II and form III DNA in presence of complex 2 together with
MG. This experiment supported that complex1 has a tendency
of binding with the minor groove of DNA where as complex 2
prefers major groove of DNA.
Topo I inhibition assay
DNA topoisomerases are proteins which take care of topolo-
gical problems as well as nuclear processes such as DNA
replication, transcription, repair and chromatin.37,38
Topoisomerase I (topo I) manipulates coiling by unwinding
duplex DNA, cleaves a phosphodiester bond of one strand and
results in a more relaxed structure. Supercoiled plasmid DNA
pBR322 is a suitable substrate for a study of topoisomerase I
inhibition. The supercoiled pBR322 (Form I) is very compact
and moves faster in gel during electrophoresis. When one
strand of supercoiled DNA is cut by topoisomerase, the
resultant unwound DNA is more relaxed.39,40 The reaction
between double-stranded DNA and topoisomerase I produces
Investigation of DNA cleavage in presence of activator and
radical scavengers
Visible light-induced DNA cleavage reactions of 1 and 2 have
been studied using different additives in visible light of l 560
nm. The nuclease efficiency of complexes is usually dependent
on activators and scavengers.35
As shown in Fig. 9, complex 1 did not cleave DNA in
?
presence of scavengers like sodium formate, dmso, KI (OH
a
covalent 39-phosphorotyrosyl adduct which is usually
radical scavenger) and sodium ascorbate (reducing agent). It
indicated that cleavage occurred due to formation of some
referred to as the cleavable complex. Under physiological
conditions, the DNA cleavage and ligation reactions catalysed
by the enzyme are tightly coordinated and the covalent
intermediate is rarely detected. The cleavage is coupled with
relegation to restore continuity in DNA duplex.41 A number of
drugs such as irinotecan, topotecan, camptothecin (CPT) can
convert topoisomerase I in a cell poison by blocking the
relegation step and enhancing the formation of persistent
DNA breaks responsible for cell death. The single-strand
breaks induced by topoisomerase I are considered non-toxic to
the cells because they can be efficiently and rapidly repaired
but their conversion in double strand breaks, generates
potentially lethal lesions.42,43
?
OH radical and follows type I mechanism. The cleavage
observed in presence of H2O2could also be understood due to
?
its photo decomposition in OH radical. The possibility of
singlet oxygen (1O2) pathway was discarded owing to cleavage
of DNA in the presence of activator NaN3 (singlet oxygen
trapper) and also in D2O.36
As depicted in Fig. 10, complex 2 also followed the similar
pathway as followed by complex 1 except that it could not
induce cleavage of DNA in presence of H2O2. This may occur as
photodecomposition of H2O2 that has not been catalyzed by 2
The results showed that complexes could inhibit Topo I and
inhibition was found to be concentration dependent. Visually,
Fig. 10 Ethidium bromide stained agarose gel (1%) of pBR322 plasmid DNA
(300 ng mL21) in the presence of complex 2 (20 mM) after 1 h of incubation: lane
1, DNA control; lane 2, DNA + sodium formate (50 mM) + complex 2; lane 3, DNA
+ NaN3 (50 mM) + complex 2; lane 4, DNA + dmso + complex 2; lane 5, DNA +
H2O2 + complex 2; lane 6, DNA + KI (50 mM) + complex 2; lane 7, DNA + sodium
ascorbate (50 mM) + complex 2; lane 8, DNA + D2O + complex 2.
Fig. 8 Agarose gel electrophoresis pattern for the photocleavage pattern of
pBR322 plasmid DNA (300 ng) by the complex (20 mM) in visible light 560 nm.
Lane 1, DNA control; Lane 2, DNA + complex 1 + DAPI; Lane 3, DNA + complex 1
+ methyl green; Lane 4, DNA + complex 2 + DAPI; Lane 5, DNA + complex 2 +
methyl green.
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and 570 nm at lexcitation 366 nm. Crystallographic data:
C24H18O4, fw = 370.38, monoclinic, P21/c, a = 4.967(5) Å, b =
27.181(14) Å, c = 7.764(7) Å, a = 90u, b = 117.55(6)u, c = 90u, V =
929.3(13) Å3, Z = 2, Dc = 1.324 Mg/m3, MoK\a radiation (l =
0.71073 Å), 3448 reflections, 2580 with full-matrix, least squares
on F2 used for refinement (R = 0.0532, wR2 = 0.0538, GoF = 0.735,
hydrogens calculated).
1,4-bis-(3-hydroxy-4-oxo-4H-chromen-2-yl)-benzene (bis-fla-
vonol L2H2).
Fig. 11 Effects of different concentrations of 1 and 2 on the activity of DNA
A solution of (L1H2) (0.370 g, 1 mmol) in CH3OH (15 mL)
containing aqueous solution of sodium hydroxide (10%, 15
mL) was stirred and then a warmed solution of H2O2 (30%, 10
mL) was added dropwise. After 15 min, the reaction mixture
was diluted with H2O then acidified with HCl (10%). The
precipitate obtained was extracted with CH2Cl2. The organic
layer was isolated and washed with NaHCO3 solution (5%) and
H2O. The CH2Cl2 layer was dried on anhydrous Na2SO4, and
the solvent was evaporated. The precipitate was recrystallized
from CH3OH. Yield: 40%, M.P. . 200 uC, (nmax/cm21):
3452(m), 1283 u(OH), 2987(m) u(CH, Ph), 1691, 1636 u(CO);
1H NMR (CDCl3, d ppm): 9.91 (s, 2H, –OH), 8.43(s, 4H, –Ar);
8.13(d, J = 7.8 Hz, 2H, Ar) 7.50 (m, 2H; Ar), 7.82(s, 4H, Ar); UV–
vis (DMF, 1024 M): lmax (nm) (emax 6 104 M21 cm21) 265
(5.974), 312(1.322), 381(1.896). lemission 438 nm and 570 nm at
lexcitation 381 nm.
topoisomerase I.
there seems to be no significant difference in the degree of
Topo I inhibition by both the complexes at lower concentra-
tion (10–50 mM) ( S11). As a preliminary investigation of the
3
mechanism of action of above Topo I inhibition, another panel
of different concentration of complexes varying (50–150 mM)
with DNA and Topo I was used (Fig. 11). Complex 1 completely
inhibited Topo I without promoting the formation of linear
DNA products, and similar results were also observed with
complex 2. The IC50 values for complexes 1 and 2 were found
to be approximately 87 and 112 mM respectively. Complexes 1
and 2 showed better topoisomerase inhibition as compared to
some of standard topoisomerases I inhibitors like camptothe-
cin, doxorubicin, novobiocin, etoposide.44,45
Synthesis of [Ru2(L1)(dmso)6Cl2] 1
A methanolic solution (30 mL) of partially dissolved cis,fac-
[RuCl2(dmso-S)3(dmso-O)] (0.484 g,
1 mmol) was added
Experimental
dropwise to a solution of L1H2 (0.185 g, 0.5 mmol) in methanol
(25 mL) containing equimolar amount of NEt3. After complete
addition, colour of the solution changed from orange to red
and stirring was continued for 24 h at room temperature till a
clear deep red solution was obtained. The volume of the
solution was then reduced and poured in water and finally
extracted with dichloromethane (DCM). Combined solution in
DCM was allowed to evaporate slowly and finally a red
coloured crystalline complex was obtained. Yield: 0.350 g
(59%). m.p.: . 200 uC. Anal. Calc. For C36H52O10S6Cl2Ru2
Found(calculated) (%): C 38.98(C 38.95); H, 5.22 (4.72%). ESI-
MS: m/z: 1133 [M + Na]+, 1055[M + Na-dmso]+, 977[M + Na–
2dmso]+. IR (KBr pellet, cm21): 2921(m) u(C–H), 1614(s)
u(CLO), 1090 u(SLO), 425(m) u(Ru–S). 1H NMR (CDCl3, d
ppm): 7.26–7.74(m, 8H, Ar, Ha and Ha), 7.1(d, 4H, Ar), 6.59(d,
4H, Ar), 6.54(t, 4H, Ar), 3.09–3.59 (s and m, 36H; dmso). UV–vis
(DCM, 1024 M): lmax (nm) (emax 6 104 M21 cm21) 377(2.02),
511(0.634). Emission excited at l 511 nm (dmso, 1024 M): non-
emissive.
Materials
Terephthalaldicarboxaldehyde,
2-hydroxy
acetophenone,
RuCl3?3H2O and agarose were purchased from Sigma Aldrich
Chemicals. Solvents were purchased from E. Merck and were
distilled prior to use. Calf thymus (CT) DNA and supercoiled
(SC) plasmid DNA pBR322 (as a solution in Tris buffer and
cesium chloride purified), with a length of 4361 base pairs
were purchased from Bangalore Genei, India. Infrared, UV-
visible and luminescence spectra were recorded on VARIAN
3100 FTIR, Jasco UV-630 spectrophotometer and Perkin Elmer
LS-45 spectrophotometer, respectively. Elemental analysis and
mass spectral measurements were carried out using a Carbo-
Erba elemental analyzer 1108 and JEOL SX-102 mass spectro-
meter respectively. However, 1H NMR spectra were recorded
on JEOL AL 300 MHz spectrometer using TMS as internal
reference. The starting material cis,fac-[RuCl2(dmso-S)3(dmso-
O)] was prepared from RuCl3?3H2O and dmso using an earlier
reported procedure.46
Synthesis of [Ru2(L2)(dmso)6Cl2] 2
Synthesis of ligands
1-(2-Hydroxy-phenyl)-3-{4-[3-(2-hydroxy-phenyl)-3-oxo-propenyl]-
phenyl}-propenone (L1H2) was synthesized by the condensation
of 29-hydroxy acetophenone and terephthalaldicarboxaldehyde
in presence of 50% NaOH solution using an earlier reported
method.47 Yield: 50%, M.P. 260 uC, IR (KBr pellet, nmax/cm21):
A methanolic solution (30 mL) of partially dissolved cis,fac-
[RuCl2(dmso-S)3(dmso-O)] (0.484 g,
1 mmol) was added
dropwise to a solution of L2H2 (0.199 g, 0.5 mmol) in methanol
(25 mL) containing an equimolar amount of NEt3. After
complete addition, colour of the solution changed from green
to brown. The stirring was continued for 24 h at room
temperature until a clear deep brown solution was obtained.
The volume of the reaction mixture was then reduced and
poured into water and extracted with chloroform. Chloroform
1
3452, 1270(m) u(OH), 2927(m) u(CH, Ph), 1639 (CO); H NMR
(CDCl3, d ppm): 12.74 (s, 2H, –OH), 6.94–7.95 (m, 12H; Ar), 7.25
(d, 1H, Ha), 6.97 (d, 1H, Ha), UV-vis (DCM, 1024 M): lmax(nm)
(emax 6 104 M21 cm21) 261 (3.614), 366 (1.754). lemission 452 nm
12216 | RSC Adv., 2013, 3, 12210–12219
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was then evaporated which gave a red coloured crystalline
compound. Yield: 0.388 g (60%). m.p.: . 200 uC. Anal. Calc.
For C36H48O12S6Cl2Ru2. Found (calculated) (%): C 38.31(C,
37.99); H, 4.30 (4.25%). ESI-MS: m/z: 1161 [M + Na]+. IR (KBr
pellet, cm21): 2922(m) u(C–H), 1613(s) u(CLO), 1095(s) u(SLO)
(S-bonded), 1017(s) u(SLO) (O-bonded), 423(m) u(Ru-S). 1H
NMR (CDCl3, d ppm): 8.71(s, 4H, –Ar), 8.19(d, J = 7.8 Hz, 4H,
Ar), 7.68(m, 4H, Ar), 3.21–3.71 (s and m, 30 H; dmso), 2.62 (s,
6H, dmso). UV–vis (DCM, 1024 M): lmax (nm) (emax 6 104 M21
cm21) 342(0.692), 407(0.490), 485(0.664), 565sh (0.347). Non-
emissive: at lexcitation 565 nm (dmso, 1024 M)
ruthenium(II) complex concentration. EB was found non-
emissive in phosphate buffer (pH 7.2) owing to fluorescence
quenching of free EB by the solvent molecules. In the presence
of DNA, EB showed enhanced emission intensity due to its
intercalative binding to DNA helix. A competitive binding of
ruthenium complexes to CT DNA resulted in the displacement
of bound EB decreasing its emission intensity. Stern-Volmer
quenching constants49 were calculated using the given
equation:
Io/I = 1 + Ksvr
(1)
Electrochemical studies
Where Io and I are the fluorescence intensities in absence
and presence of the complexes, Ksv is a linear Stern-Volmer
quenching constant and r is the ratio of the total concentra-
tion of complexes to that of DNA. The value of Ksv is given by
the ratio of slope to intercept in a plot of Io/I vs [complex]/
[DNA].
Cyclic voltammetry was performed on a CHI 620c electro-
chemical analyzer. A glassy carbon working electrode, plati-
num wire auxiliary electrode, and Ag/Ag+ reference electrode
were used in
a standard three-electrode configuration.
Tetrabutylammonium perchlorate (TBAP) was used as a
supporting electrolyte, and concentration of the solution was
kept as 1023 M.
CD spectral studies
CD spectra were recorded on a JASCO J-810 spectropolarimeter
equipped with Peltier cell temperature controller (¡0.2 uC) at
room temperature with increasing complex/DNA ratio. Each
sample solution was scanned in the range of 220–320 nm. The
CD spectrum thus generated represents an average of three
scans from which the buffer background had been subtracted.
The concentration of DNA solution was 1.0 6 1024 M.
Effect of ionic strength
Salt effect was monitored by subsequent addition of NaCl (0–
70 mM) to both complexes–CT DNA adducts (100 mM each) in
50 mM sodium phosphate buffer (pH 7.2).
Absorption titration
The concentration of CT-DNA was calculated from its known
extinction coefficient at l 260 nm (6600 M21 cm21). Solution of
calf thymus DNA in phosphate buffer gave a ratio of UV
absorbance at l 260 and 280 nm (A260/A280) 1.9 : 1 indicating
that DNA was sufficiently free from protein. Absorption
titrations were performed by maintaining a constant metal
complex concentration (10 mM) and varying DNA concentra-
tion (0–150 mM) in Na–phosphate buffer (pH 7.2). After
addition of DNA to the solution of complexes, the resulting
solution was allowed to equilibrate at 25 uC for 3 min then
corresponding absorbance was noted. From the absorption
data, the intrinsic binding constant Kb was calculated from a
plot of [DNA]/(ea 2 ef) vs. [DNA] using the equation:
DNA cleavage studies using agarose gel electrophoresis
In gel electrophoresis experiments, supercoiled pBR322 DNA
was treated with the solutions of complexes incubating at l560
nm for 15 min. After photoexposure, the samples were
separately incubated for 1 h at 37 uC, followed by their
addition to the loading buffer containing 25% bromophenol
blue, 0.25% xylene cyanol, and 30% glycerol (3 mL), and the
solution was finally loaded on 1% agarose gel containing 0.5
mg mL21 ethidium bromide. The electrophoresis was carried
out in a dark room for 2 h at 50 V in Tris-acetate-EDTA buffer
(pH 8.3). The bands were visualized by UV light and
photographed. The extent of cleavage of SC DNA was
determined by measuring intensities of the bands using a
Genosens 1510 Gel Documentation System. The experiments
were also carried out in presence of activators, viz., NaN3 (1O
singlet oxygen trapper), sodium formate (OH2 radical scaven-
ger), sodium ascobate (reducing agent), dmso, KI, H2O2 and
D2O, which were added to SC DNA prior to the addition of
complexes 1 and 2 (acetonitrile 0.01%) only.
[DNA]/(ea 2 ef) = [DNA]/(eb 2 ef) + [Kb(eb 2 ef)]21
Where, [DNA] is the concentration of DNA in base pairs. The
apparent absorption coefficients ea, ef and eb correspond to
Aobsd/[Ru], the extinction coefficient of free ruthenium (II)
complexes and extinction coefficient of ruthenium (II) com-
plexes in fully bound form, respectively.48 Kb is given by the
ratio of slope to the intercept.
Topoisomerase inhibition assay
DNA topoisomerase I (Topo I) from Escherichia coli was
purchased from New England Biolabs, and no further
purification was performed. One unit (U) of the enzyme was
defined as the amount that completely relaxed 0.5 mg of
negatively charged supercoiled pBR322 plasmid DNA in 30
min at 30 uC under standard assay conditions. The reaction
mixture (15 mL) consists of 50 mM potassium acetate, 20 mM
Tris-acetate, 10 mM magnesium acetate, 1 mM DTT (dithio-
threitol), 0.1 mg/mL BSA, and 1 U of Topo I together with
variable concentration of photoinduced Ru(II) complexes (0–
Competitive binding with ethidium bromide
The relative binding constants (Ksv) of complexes were
determined by fluorescence spectral technique using ethidium
bromide (EB)-bound CT DNA solution in a phosphate buffer
(pH, 7.2) solution. In a typical experiment, a 20 mL of CT-DNA
solution (A260 = 2.0) was added to a 2.0 mL of EB buffer
solution (pH 7.2) and changes in fluorescence intensities at l
600 nm (l 510 nm excitation) of EB bound to DNA were
recorded with the addition of increasing amount of
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RSC Advances
ˇ
´
Chemistry Edited by M. Melnık, P. Segla, M. Tatarko Slovak
University of Technology Press, Bratislava E 2011.
3 G. Marcon, S. Carotti, M. Coronnello, L. Messori, E. Mini,
P. Orioli, T. Mazzei, M. A. Cinellu and G. Minghetti, J. Med.
Chem., 2002, 45, 1672–1677.
150 mM) and 0.5 mg of pBR322 DNA. The corresponding
reaction mixtures were incubated at 37 uC for 1 h, and then
they were terminated by the addition of 3 mL of 5X stock
solution of dye consisting of 0.25% bromophenol blue, 4.5%
sodium dodecyl sulfate, and 45% glycerol. The electrophoresis
of the samples was carried out through 1% agarose in TAE
buffer at 50 V for 2 h. The gel was stained with 1 mg mL21 EB
and photographed under UV light. The concentration of the
inhibitor that prevented 50% of the supercoiled DNA being
converted into relaxed DNA (IC50 values) was calculated from
the midpoint concentration of the complex-induced DNA
unwinding. To check whether order of addition affected the
results, experiments were also carried out in ice, in which the
whole reaction mixture was assembled.
4 C. Metcalfe and J. A. Thomas, Chem. Soc. Rev., 2003, 32,
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complexes
1
and
2
of general composition [Ru2(L1/
L2)(dmso)6Cl2] have been obtained. The complex 1 is symme-
trical dinuclear complex with each Ru(II) ion in octahedral
coordination geometry with (g2) chelating binding mode of bis
chalcone, one chlorine, three S-bonded dmso. However
complex 2 possess one octahedral Ru(II) center with (g2)
chelating binding mode of bis-flavonol one chlorine, three
S-bonded dmso where as another octahedral Ru(II) center with
(g2) chelating binding mode of bis-flavonol, one chlorine, two
S-bonded dmso and one O-bonded dmso and makes different
electronic environment around both Ru(II) ions. Highly
conjugated structural framework of complex 1 enabled it to
bind strongly through intercalation and minor groove of DNA
as compared to complex 2 which prefers binding with major
groove of DNA as well as weakly intercalates with the DNA.
Complexes transform supercoiled DNA to nicked circular form
under photo-induced condition in visible region at l560 nm.
The order of photocleavage is 1 . 2. Results support that
?
complexes follow type I pathway by the formation of some OH
radical. Both complexes 1 and 2 inhibited Topoisomerase I.
The IC50 values for complexes 1 and 2 were found to be 87 and
112 mM respectively.
24 H. Chao, W. J. Mei, Q. W. Huang and L. N. Ji, J. Inorg.
Biochem., 2002, 92, 165–170.
Acknowledgements
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145–152.
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Photobiol. B, 2011, 103, 166–179.
Financial support received from CSIR New Delhi, India (Grant
No. 01 (2322)/09/EMR-II) is gratefully acknowledged. Authors
acknowledge A. L. Susmitha TIFR Mumbai for her help in
recording the CD spectra.
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