Interaction of calf thymus DNA with N,N'-bis(3,4-dihydroxybenzylidene)-1,2- diaminobenzene ligands

Article abstract of DOI:10.1134/S0036024410130133

In the present study, at first, N,N'-bis(3,4-dihydroxyhenzylidene)-1,2- diaminobenzene (BDBDAB), has been synthesized by combination of 1,2-diaminobenzene and 3,4-dihydroxybenzaldehyde in a solvent system. These ligand containing ortho-quinone functional

Full text of DOI:10.1134/S0036024410130133

ISSN 0036ꢀ0244, Russian Journal of Physical Chemistry A, 2010, Vol. 84, No. 13, pp. 2284–2289. © Pleiades Publishing, Ltd., 2010.
PHYSICAL CHEMISTRY
OF SOLUTIONS
Interaction of Calf Thymus DNA
with N,N'ꢀBis(3,4ꢀdihydroxybenzylidene)ꢀ1,2ꢀdiaminobenzene Ligands1
,2
M.ꢀR. Azania , A. Hassanpoura, A.ꢀK. Bordbarb, and M. A. Mehrgardib
a Departamento de Química Inorgánica, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain
b Department of Chemistry, Faculty of Science, University of Isfahan, Isfahan, 81746ꢀ73441, I.R. Iran
eꢀmail: mr.azani@yahoo.com
Received October 18, 2009
Abstract—In the present study, at first, N,N'ꢀbis(3,4ꢀdihydroxyhenzylidene)ꢀ1,2ꢀdiaminobenzene
(BDBDAB), has been synthesized by combination of 1,2ꢀdiaminobenzene and 3,4ꢀdihydroxybenzaldehyde
in a solvent system. These ligand containing orthoꢀquinone functional groups were characterized using
UVꢀVIS and IR spectroscopies. Subsequently, the interaction between native calf thymus deoxyribonuꢀ
cleic acid (ctꢀDNA) and BDBDAB was investigated in 10 mM TrisꢀHCl buffer solution, pH 7.2, using
UVꢀvisible absorption and fluorescence spectroscopies, thermal denaturation technique, viscosity meaꢀ
surement, and cyclic voltametry. From spectrophotometric titration experiments, the binding constants
of BDBDAB with double stranded DNA were found to be (0.9 0.1)
interaction between the ligands and DNA can be quantified by using the Stern–Volmer equation. From
the plot of I0 versus [DNA]/[BDBDAB] the quenching constants ( SV) were obtained and found to be
×
104 M–1. The magnitude of the
/
I
K
5.6 0.2. Thermal denaturation experiments represent the increasing of melting temperature of DNA
(about 3.2°C) due to binding of BDBDAB. There is no increasing of viscosity was observed in addition of
BDBDAB to DNA. The electroactivity of the quinone moiety in N,N'ꢀbis(3,4ꢀdihydroxybenzylidene)ꢀ
1,2ꢀdiaminobenzene bound to DNA could be employed as cyclic voltametry to obtain binding constant.
These results suggest that interaction with the grooves could be the principal mode of binding of
BDBDAB to double stranded ctꢀDNA.
DOI: 10.1134/S0036024410130133
INTRODUCTION
side binding with selfꢀstacking in which the Schiff
bases are stacked along the DNA helix. The interacꢀ
tion of H2 Salen transition metal complexes, including
Cu [5, 6], Ni [7–9], Mn [10], Co [11], and Ru [12],
with DNA was studied and great changes of spectroꢀ
scopic properties were generally noticed, indicating
binding interactions between DNA and such comꢀ
pounds. An intercalating interaction mode was proꢀ
posed for Co (Salen) [5, 11], an external binding with
the surface of the double helix was suggested for a
functionalized Cu (Salen) complex [5, 6], while Ni
(Salen) presented a high affinity toward the N7 atom
of guanosine residues [7, 9].
The study of the interaction of Schiff bases with
DNA has been the focus of some recent research works
[1]. This is partly because that, chemical carcinogens,
radiation and many chemical antitumor agents share a
common property in that they exert their biological
effects through mechanisms involving DNA damage.
On the one hand, certain DNAꢀbound forms of cheꢀ
motherapeutic agents are responsible for arresting
tumor cell growth. On the other hand, some of the
damages induced by carcinogenic chemicals are the
mediators of mutational change, which, in turn, are
likely to be necessary prerequisites for carcinogenic
transformation. The studies of the molecular details of
these binding interactions have been crucial in rational
design of new antitumor agents as well as developing
sensitive probes of local nucleic acid structure [2]. The
interaction of multidentate aromatic ligands with
DNA has recently gained much attention following
the important biological and medical roles played by
these intercalators [3, 4]. Three major binding modes
have been proposed for the binding of Schiff bases to
DNA: intercalation, outside groove binding and outꢀ
In this work, we present a comprehensive study on
the interaction between DNA and this new bifuncꢀ
tional Schiff base molecule containing both aromatic
domains and electroactive functional groups. This new
molecule can be easily synthesized in high yield using
inexpensive starting materials. The mode of interacꢀ
tion with DNA has been elucidated by UVꢀvisible
absorption, fluorescence spectroscopies, thermal
denaturation technique, viscosity measurement and
cyclic voltametry. Experimental results suggest that
this molecule is capable of binding specifically to
ctꢀDNA sequences.
1
The article is published in the original.
The corresponding author.
2
2284

INTERACTION OF CALF THYMUS DNA
2285
EXPERIMENTAL
Spectroscopy
The IR spectra were recorded on KBr pressed pelꢀ
lets using a Philips spectrometer. The absorbance
measurements were carried out using UVꢀVIS, Carryꢀ
500 double beam spectrophotometer, operating from
200 to 800 nm in 1.0 cm quartz cells. The absorbance
titrations were performed at a fixed concentration of
the specific BDBDAB ligand and varying the concenꢀ
tration of double stranded ctꢀDNA. Fluorescence
spectra were recorded with a RFꢀ5000 Shimadzu
spectrofluorimeter equipped with a Peltier system to
control the temperature inside the cuvettes.
Chemicals and Solutions
1,2ꢀDiaminobenzene and the 3,4ꢀdihydroxybenzꢀ
aldehyde, were obtained from Aldrich Chemical Co.
and were used as received. Anhydrous dimethylformaꢀ
mide (DMF) was purchased from Sigma. All other
chemicals used in this work, were reagent quality,
obtained from Sigma–Aldrich Co. and used as
received without further purification. BDBDAB has
been synthesized by combination of 1,2ꢀdiaminobenꢀ
zene and the 3,4ꢀdihydroxybenzaldehyde. This ligand
containing ortoꢀquinone functional groups was charꢀ
acterized using UVꢀVIS and IR spectroscopies, in solꢀ
vents, such as dimethylformamide (DMF). Stock
solutions of N,N'ꢀbis(3,4ꢀdihydroxybenzylidene)ꢀ
1,2ꢀdiaminobenzene (typically 10 mM) were prepared
just prior to use by dissolving the solids in DMF. Water
was purified with a Millipore MilliꢀQ system and all
the experiments were carried out at room temperature.
Double stranded calf thymus DNA (ctꢀDNA, actiꢀ
vated and lyophilized) was purchased from Sigma. ctꢀ
DNA stock solutions (2 mg/ml) were prepared in
10 mM TrisꢀHCl, pH 7.2 buffer. The DNA solutions
Melting Experiments
Melting curves were performed using an UVꢀVIS
Carryꢀ500 double beam spectrophotometer in conꢀ
junction with a thermostated cell compartment. The
measurements were carried out in 10 mM TrisꢀHCl,
pH 7.2. The temperature inside the cuvette was deterꢀ
mined with a platinum probe and was increased over
the range 20–100°C at a heating rate of 1 K/min. The
melting temperature, Tm, was obtained from the midꢀ
point of the hyperchromic transition.
gave a UV absorbance ratio (A260/A280) of about 1.9,
indicating that the DNA was sufficiently free from
protein [13]. The concentration in base pairs of DNA
was determined using an extinction coefficient of
6600 cm–1 M–1 at 260 nm [14].
Viscosity Measurements
The viscosity of ctꢀDNA solutions was measured at
25 0.1°C using an Ubbelohde viscometer. Typically,
10 mM TrisꢀHCl buffer solution, pH 7.2 was transꢀ
ferred to the viscometer to obtain the reading of flow
time. For determination of solution viscosity, 10.0 ml
Synthesis of N,N'ꢀbis(3,4ꢀdihydroxybenzylidene)ꢀ
1,2ꢀdiaminobenzene Ligand
of buffered solution of 160 M ctꢀDNA was taken to
μ
the viscometer and a flow time reading was obtained.
An appropriate amount of Schiff base was then added
According to the traditional procedure of synthesis
of tetradentate Schiff base ligands [15], the reaction of
salicylaldehydes with 0.5 mol equivalent of diamines,
in refluxing methanol for a few hours, gives rise to the
final products (75–85% yield) which were analytically
pure solids after recrystalization. In a 25 ml rounded
bottom flask 1,2ꢀdiaminobenzene (0.1080 g, 1.00 mmol)
was mixed with 3,4ꢀdihydroxybenzaldehyde (0.2762 g,
1.00 mmol) then added 25 ml ethanol 95% and
refluxed. Afterwards the mixture was refluxed for 12 h
a fine light brown solid mixture was obtained. The
obtained precipitate was filtered and washed several
times with ethanol and ether. The product was
recrystalized by methanol to obtain brown needles
of pure N,N'ꢀbis(3,4ꢀdihydroxybenzylidene)ꢀ1,2ꢀ
diaminobenzene (0.2819 g, 76%). The chemical
structure of BDBDAB was shown below.
to the viscometer to give a certain
r (r = [Schiff
base]/[DNA base pair]) value while keeping the
ctꢀDNA concentration constant, and the flow time
was read. The flow times of samples were measured after
the achievement of thermal equilibrium (30 min). Each
point measured was the average of at least five readꢀ
ings. The obtained data were presented as relative visꢀ
cosity,
η
/
η0, versus
r
, where
η
is the reduced specific
viscosity of DNA in the presence of Schiff base and
η0 is the reduced specific viscosity of ctꢀDNA alone
[16, 17].
Electrochemical Studies
Electrochemical measurements were carried out at
room temperature using an Autolab PGSTAT 30
potentiostat from EcoꢀChemie. Experiments were perꢀ
formed in a home made three compartment electroꢀ
chemical cell. Goldꢀdisk electrodes obtained from Bioꢀ
analytical Systems (BAS, geometric area 0.018 cm2)
were used as working electrodes. A coiled platinum
wire served as auxiliary electrode and the potentials are
reported against a Ag+/AgCl electrode without regards
for the liquid junction. Prior to electrochemical meaꢀ
C
N
N
C
OH OH
OH OH
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
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2286
AZANI et al.
UVꢀVIS Spectral Studies
Abs
0.6
The binding of certain complex to DNA can proꢀ
duce hypochromism, a broadening of the envelope,
and a red shift of the complex absorption band. These
effects are particularly pronounced for intercalators,
with groove binders, a large wavelength shift usually
correlates with a complex conformational change on
binding or complex–complex interactions. A spectral
change of BDBDAB due to addition of DNA was
shown in Fig. 1. For obtaining these spectra, the fixed
amount of Schiff base in TricꢀHCl buffer pH 7.2 was
titrated with a stock solution of ctꢀDNA. The changes
in absorbance of the Soret band upon addition of ctꢀ
DNA were monitored at the maximum of the Soret
band.
0.4
0.2
0
280
300
320
340
, nm
It exhibited the low hypochromism and negligible
red shift due to the incremental addition of ctꢀDNA
indicating groove binding mode. An isosbestic point is
evident at 325 nm and low redꢀshift associated with
the decrease in the absorbance is observed.
λ
Fig. 1. The spectrum of N,N'ꢀbis(3,4ꢀdihydroxybenꢀ
zylidene)ꢀ1,2ꢀdiaminobenzene in the presence of various
concentration of ctꢀDNA. Measurements were done in
10 mM TrisꢀHCl buffer, pH 7.2 and at 25°C.
The apparent binding constant, Kapp, for the interꢀ
action between the BDBDAB ligand and ctꢀDNA can
be determined by analysis of absorption spectrophotoꢀ
metric titration data at room temperature [19]:
surements the solutions were bubbled with preꢀpuriꢀ
fied N2 for at least 20 min and kept under a nitrogen
atmosphere throughout the entire procedure of cyclic
voltammetry (CV). In all of the experiments, for the
pH measurement, we used a potentiometer (Metrohm
model, 744).
[DNA]T
[DNA]T
1
ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ = ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ + ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ,
(1)
( εapp – εf )
K
app( εb – εf ) ( εb – εf )
where [DNA]T, εapp, εf, and εb correspond to total conꢀ
centration of ctꢀDNA, Aobsd/[Schiff base], the extincꢀ
tion coefficient for the free Schiff base, and the
extinction coefficient for the Schiff base in the fully
bound form, respectively. This equation is followed
RESULTS AND DISCUSSION
by overall linear equation
[DNA]T/(|εapp εf|), = [DNA]T,
and = 1/(|εb εf|). In the plot of [DNA]T/(|εapp
versus [DNA]T
to the intercept. The apparent binding constants of
BDBDAB was calculated to be (0.9 0.1)
104 M–1.
The value of b described in the literature for intercaꢀ
lating molecules is at least two orders of magnitude
higher than that obtained for BDBDAB ligand. These
results suggest that intercalation between the base
pairs is not the main mode of interaction of these molꢀ
ecules with DNA.
y = ax + b in which y =
Structural Characterization of N,N'ꢀbis(3,4ꢀ
dihydroxybenzylidene)ꢀ1,2ꢀdiaminobenzene
–
–
K
x
a
= 1/
Kapp(|εb
–
–
εf|)
εf|)
b
IR spectra of tetradentate BDBDAB ligand, synꢀ
thesized according with the procedure described
before, had this IR information (KBr): νC=N
app is given by the ratio of the slope
=
×
1607 cm–1; νAr–O–H = 1280 cm–1, νC=C = 1430 cm–1,
νAr–H = 740 cm–1. The significant frequencies were
selected by comparing the IR spectra of the purified
ligands with those obtained for the commercially
available Schiff base. These Schiff base ligands present
a characteristic band in the range of 1605–1630 cm–1
attributable to the stretching vibration of the azomeꢀ
thine (C=N) group. The presence of this band is conꢀ
comitant with the absence of absorption bands in the
range of 1680–1690 cm–1 corresponding to the startꢀ
ing carboxaldehyde groups.
K
Fluorescence Spectroscopic Studies
To corroborate the above results, fluorescence
studies were also carried out. In fluorescence titraꢀ
tions, the concentration of the corresponding
BDBDAB ligand was fixed, while the concentration of
In addition, the presence of phenolic hydroxylic
groups can be ascertained by the presence of strong
absorption bands in the range of 1270–1290 cm–1. The
absorbance spectrum of BDBDAB also shows the
maximum wave length at 331 nm. These results have a
good harmony with pervious references [18].
ctꢀDNA was varied from 50 to 2000
shows the excitation spectrum of 20
μ
M. Figure 2
μ
M in 10 mM
TrisꢀHCl buffer, pH 7.2. Upon the addition of ctꢀ
DNA, the excitation intensities are efficiently miniꢀ
mized with no detectable shifts in the excitation waveꢀ
length, even in the presence of a large excess of DNA.
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INTERACTION OF CALF THYMUS DNA
2287
Using as excitation wavelength 313 nm, the emission
Abs
spectrum of free BDBDAB shows a broad maximum
intensity at 370 nm. As in the case of excitation experꢀ
iments, the addition of increasing amounts of ctꢀDNA
gives rise to a gradual decay in the emission intensities
with a negligible red shift.
The magnitude of the interaction between the
ligands and DNA can be quantified by using the
Stern–Volmer equation [20]:
300
200
100
0
I0/I = 1 + Kr,
(2)
where I0 and
I are the fluorescence area peak in the
absence and the presence of ligand, respectively, is a
K
340
360
380
400
420
440
, nm
linear Stern–Volmer quenching constant, is the ratio
of total concentration of ctꢀDNA to that of ligand.
This equation is directly proportional to linear equaꢀ
r
λ
Fig. 2. The emission spectrum of N,N'ꢀbis(3,4ꢀdihydroxꢀ
ybenzylidene)ꢀ1,2ꢀdiaminobenzene (20 M) bound to in
tion y = ax + b so y = I0/I, x = r, a = K, b = 1.
µ
the presence of various amounts of DNA. Measurements
were done in 10 mM TrisꢀHCl buffer, pH 7.2 and at 25°C.
The peak area is decreased with increasing of DNA conꢀ
centration in the direction of arrow. The excitation waveꢀ
length was 313 nm.
The quenching plots illustrate that the quenching
of BDBDAB bound to ctꢀDNA is in good agreement
with the linear Stern–Volmer equation, which proves
that the Schiff base bind to DNA. In the plot of I0
versus [Schiff base]/[DNA], is given by the ratio of
the slope to intercept. The value for N,N'ꢀbis(3,4ꢀ
dihydroxybenzylidene)ꢀ1,2ꢀdiaminobenzene was 5.14.
The relative value of obtained respect to other
/I
K
K
critical tests of the binding models in solution. The
DNA helix lengthens as the base pairs are separated to
accommodate the bound complex for the groove bindꢀ
ing of the molecule, leading to low increasing in DNA
viscosity. Hydrodynamic methods are thus suitable to
detect such changes and, in the absence of crystalloꢀ
graphic structural data, provide essential evidence to
support the intercalation model. In contrast, partial
and/or no classical intercalation of complex could
bend (or kink) the DNA helix, reduce its effective
length and in turn, its viscosity. The relative viscosity
of DNA shows small increasing with increase in the
concentration of the BDBDAB, which is not similar to
that of the classical intercalation (i.e. ethidium broꢀ
mide) [25, 26]. The viscosity results unambiguously
show that BDBDAB bind with DNA by groove bindꢀ
ing mode, this results are in agreement with optical
absorption experiments.
K
known intercalators represents the nonꢀintercalation
mode for BDBDAB [21].
Thermal Denaturation Experiments
When a molecule intercalates to ctꢀDNA, the staꢀ
bility of the helix increases and, as a result, the temperꢀ
ature at which the helix denatures (Tm) goes up around
5–12°C in unit molar ratio [22, 23]. Thus, this paramꢀ
eter is most useful in analyzing the mode of interacꢀ
tion. The melting plot of ctꢀDNA was monitored by
plotting the variation of maximum absorption of ctꢀ
DNA solution (100 M) at 258 nm vs. temperature.
μ
The denaturation curves were measured at various
[BDBDAB]/[DNA] molar ratios. The melting temꢀ
perature of ctꢀDNA (Tm) was estimated from the midꢀ
point of transition curve. The melting temperature of
ctꢀDNA has been increased about 3.2°C in molar
ratio 1 in the presence of BDBDAB. This observed
Cyclic Voltammetry of N,N'ꢀbis(3,4ꢀ
dihydroxybenzylidene)ꢀ1,2ꢀdiaminobenzene
in Presence of ctꢀDNA
small change in the Tm of ctꢀDNA in the presence of
BDBDAB suggests that the interaction of these comꢀ
pounds with ctꢀDNA does not involve intercalation
between the base pairs and could be ascribed to interꢀ
actions with the DNA grooves [24].
Figure 3 shows the cyclic voltammetric profile of
BDBDAB (50 M) at a bare gold electrode in 10 mM
μ
TrisꢀHCl buffer solution, pH 7.2. An electrochemical
process corresponding to the oxidation and subseꢀ
quent reduction of the hydroquinone functional group
is observed. As can be seen, this process has a large
Viscosity Measurements
Optical or photo physical probes generally provide peak potential separation (Ep = 250 mV) suggesting
necessary, but not sufficient, clues to support kind of severe limitations in the charge transfer kinetics. In
binding model. Hydrodynamic measurements that are addition, continuous potential cycling results in an
sensitive to length increases (i.e., viscosity, sedimentaꢀ increase in the peak potential separation and a gradual
tion, rotational diffusion as measured by transient decrease in the anodic and cathodic peak currents
electric diffusion) are the least ambiguous and most (data not shown). This behavior suggests that
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
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2288
AZANI et al.
106, A
These results prove previous results and suggest that
intercalation between the bases pairs is not the main
mode of interaction of these molecules with DNA.
I
×
2
1
0
1
CONCLUSIONS
The results obtained from fluorescence, UVꢀVIS
and thermal denaturation measurements exclude
DNA outside groove binding. From spectrophotometꢀ
ric and cyclic voltametry titration experiments, the
binding constants of N,N'ꢀbis(3,4ꢀdihydroxybenꢀ
zylidene)ꢀ1,2ꢀdiaminobenzene with ctꢀDNA were
−
104 M–1. The results
−
0.4
0
0.4
0.8
, V
found to be about (1.0 0.2)
×
E
show that BDBDAB bind to DNA by groove binding
mode. Our research should be valuable for seeking and
designing new antitumor drugs, as well as for underꢀ
standing the mode of the Schiff base binding to DNA
and helical conformations of nucleic acids.
These results are consistent with a binding mode
dominated by interactions with the groove of ctꢀDNA,
analogously to what reported for a number of porꢀ
phyrazines and metal–porphyrazine complexes interꢀ
acting with DNA [21].
Fig. 3. The cyclic voltametry spectrum of N,N'ꢀbis(3,4ꢀ
dihydroxybenzylidene)ꢀ1,2ꢀdiaminobenzene (50 M)
bound to in the presence of various amounts of DNA.
Measurements were done in 10 mM TrisꢀHCl buffer,
pH 7.2 and at 25°C. The nips of peaks are decreased with
increasing of DNA concentration in the direction of arrow.
Sweep rater 100 mV s
µ
–1
.
BDBDAB is deposited onto the gold surface after each
cycle giving rise to a nonꢀelectroactive film and hence
a gradual passivation of the electrode surface. In order
to minimize these adsorption effects, solutions conꢀ
taining a mixture of BDBDAB and ctꢀDNA in 10 mM
TrisꢀHCl buffer solution, pH 7.2 were incubated durꢀ
ing 1 h at room temperature, to reach binding equilibꢀ
rium. Then, bare gold electrodes were placed in the
solution just prior to carrying out the electrochemical
measurements and only the first voltammogram was
recorded. Under these conditions, the cyclic voltamꢀ
mograms of BDBDAB obtained in the presence of
increasing amounts of DNA exhibited a significant
shift in the anodic and cathodic peak potentials to
more negative values followed by a decrease in both
peak currents.
ACKNOWLEDGMENTS
We wish to acknowledge the financial support from
the Research Council of Isfahan University.
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