Multispectroscopic DNA-binding studies of a tris-chelate nickel(II) complex containing 4,7-diphenyl 1,10-phenanthroline ligands

Article abstract of DOI:10.1016/j.molstruc.2010.02.048

A tris-chelate nickel(II) complex, [Ni(DIP)₃](NO₃)₂ · 3H₂O in which DIP = 4,7-diphenyl 1,10 phenanthroline, was synthesized and characterized by spectroscopic (¹H NMR and UV-vis) and elemental analysis techniques. Binding interaction of this complex with calf thymus-DNA (CT-DNA) was investigated using emission, absorption, circular dichroism (CD), viscosity and DNA thermal denaturation studies. In absorption spectrum of the complex, as the concentration of DNA increased, appearance of an isobestic point proved the new [Ni(DIP)₃]²⁺-DNA complex formation. The intrinsic binding constant (K_b = 4.34 × 10⁴ M^-1) is comparable to groove binding complexes. In fluorimetric studies, the dynamic enhancement constants (k_D) and bimolecular enhancement constant (k_B) were calculated and showed that the fluorescence enhancement was initiated by a static process in the ground state. Furthermore, the thermodynamic studies showed that the reaction is exothermic and enthalpy favored (ΔH = -58.41 kJ/mol). A strong CD spectrum in the UV and visible region develops upon addition of CT-DNA into the racemate solution of Ni(II) complex (Pfeiffer effect). This has revealed that a shift in diastereomeric inversion equilibrium takes place in the solution to yield an excess of one of the DNA complex diastereomers. Finally, all of the experimental results prove that the minor groove binding must be predominant.

Full text of DOI:10.1016/j.molstruc.2010.02.048

Journal of Molecular Structure 970 (2010) 90–95
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Multispectroscopic DNA-binding studies of a tris-chelate nickel(II) complex
containing 4,7-diphenyl 1,10-phenanthroline ligands
*
Nahid Shahabadi , Azadeh Fatahi
Department of Chemistry, Faculty of Science, Razi University, Kermanshah, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
A tris-chelate nickel(II) complex, [Ni(DIP)3](NO3)2 ꢀ 3H2O in which DIP = 4,7-diphenyl 1,10 phenanthro-
line, was synthesized and characterized by spectroscopic (1H NMR and UV–vis) and elemental analysis
techniques. Binding interaction of this complex with calf thymus-DNA (CT-DNA) was investigated using
emission, absorption, circular dichroism (CD), viscosity and DNA thermal denaturation studies. In absorp-
tion spectrum of the complex, as the concentration of DNA increased, appearance of an isobestic point
proved the new [Ni(DIP)3]2+–DNA complex formation. The intrinsic binding constant
(Kb = 4.34 ꢁ 104 Mꢂ1) is comparable to groove binding complexes. In fluorimetric studies, the dynamic
enhancement constants (kD) and bimolecular enhancement constant (kB) were calculated and showed
that the fluorescence enhancement was initiated by a static process in the ground state. Furthermore,
the thermodynamic studies showed that the reaction is exothermic and enthalpy favored
Received 28 November 2009
Received in revised form 17 February 2010
Accepted 18 February 2010
Available online 20 February 2010
Keywords:
Nickel
Enantioselective DNA binding
Circular dichroism
Pfeiffer effect
(
D
H = ꢂ58.41 kJ/mol). A strong CD spectrum in the UV and visible region develops upon addition of
Groove binding
CT-DNA into the racemate solution of Ni(II) complex (Pfeiffer effect). This has revealed that a shift in dia-
stereomeric inversion equilibrium takes place in the solution to yield an excess of one of the DNA com-
plex diastereomers. Finally, all of the experimental results prove that the minor groove binding must be
predominant.
Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
interaction [4–7]. Additionally, tris-phenanthroline metal com-
plexes and their analogs have attracted much attention for the chi-
Understanding the binding of small molecules to DNA is poten-
tially useful in developing principles to guide the synthesis of new
improved drugs which can recognize a specific site or conforma-
tion of DNA [1–3]. Generally, metal complexes upon binding to
DNA are stabilized through a series of weak interactions such as
ral recognition of DNA double helices with the enantiomeric
complexes and for the photochemical electron transfer reaction
initiated by the complex bound to DNA [8–10]. Since, DNA is an
optically active molecule, the specific binding sites may prefer
one of the enantiomeric forms of the metal complexes [11,12].
For that reason, metal complexes with phenanthroline and related
ligands, in particular ruthenium(II) complexes have been studied
extensively as structural probes [13–18]. A singular advantage in
using tris-chelate metal complexes is that the central metal or
the ligands in them may be varied in an easily controlled manner
to facilitate a certain application, which provides an easy access
for the detailed study of DNA-binding mechanism [19]. Moreover,
the types of different metal ions and their flexible valances, which
usually are responsible for the coordination geometry of com-
plexes, have significant influences on the intercalating ability of
transition metal complexes to DNA [20]. In addition, modification
of the ligand may lead to subtle or substantial changes in the bind-
ing modes, location and affinities of the complexes to DNA. It has
been found that complexes of phenanthroline lacking substitutions
can be mutagenic (by DNA intercalation) while substituted in some
positions are not [21].
the
p-stacking interactions of aromatic heterocyclic groups be-
tween the base pairs (intercalation), hydrogen bonding and van
der Waals interactions of functional groups bound along the
groove of the DNA helix [2]. It is believed that the non-covalent
interactions of these types take place in at least three distinct
modes, i.e. binding along the outside of the helix, binding along
the major and/or minor grooves, and intercalation of a planar mol-
ecule or a planar aromatic ring system between base pairs [3].
Identifying which of the above interaction modes is operative for
the molecule concerned, as well as which is predominant among
the series of the weak interactions, is essential in designing an
effective new drug and a DNA-probe molecule.
Metal complexes of the type [M(LL)3]+n, where LL is either 1,10-
phenanthroline (phen) or modified phen, are particularly attractive
because they can effectively bind to DNA in different modes of
For these reasons, we became interested in synthesis of a Ni(II)
tris-chelate complex with 4,7-diphenyl-1,10-phenanthroline (DIP)
* Corresponding author. Tel./fax: +98 831 8360795.
E-mail address: nahidshahabadi@yahoo.com (N. Shahabadi).
0022-2860/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2010.02.048

N. Shahabadi, A. Fatahi / Journal of Molecular Structure 970 (2010) 90–95
91
ligand (Fig. 1) as a means of better understand the effect of ligand
2.3. Instrumentation
substituents and the central metal on the behavior of this class of
compounds with DNA.
1H NMR spectra were recorded with using a Bruker Avance
DPX200 MHZ (4.7 T) spectrometer using d2-D2O as solvent.
The elemental analysis was performed using Heraeus CHN ele-
mental analyzer. Absorbance spectra were recorded using hp
spectrophotometer (agilent 8453) equipped with a thermo sta-
ted bath (Huber polysat cc1). Absorption titration experiments
were carried out by keeping the concentration of [Ni(DIP)3]2+
complex constant (5.0 ꢁ 10ꢂ5 M) while varying the DNA concen-
tration (R = [DNA]/[NiL3] = 0.0 ꢂ 1). Absorbance values were re-
corded after each successive addition of DNA solution and
equilibration (Ca. 40 min). To determine the intrinsic binding
constant (Kb) and the stoichiometry of the [Ni(DIP)3]2+–DNA
2. Experimental
2.1. Materials
Commercially pure chemicals such as 4,7-diphenyl-1,10-phe-
nanthroline (Merck), Tris–HCl (Sigma Co., Madrid, Spain), were
purchased and used without purification. All experiments were
carried out in Tris–HCl buffer solutions that prepared in double
distilled water (10 mM, pH 7.2). Highly polymerized calf thymus-
DNA (CT-DNA) was purchased from Sigma Co. All DNA samples
were dissolved in 50 mM NaCl/5 mM Tris–HCl buffer, pH 7.2. Solu-
tions of CT-DNA gave an UV absorbance ratio (260 over 280 nm) of
more than 1.8, indicating that the DNA was sufficiently free of pro-
tein [22]. The DNA concentration (monomer units) of the stock
solution (1 ꢁ 10ꢂ2 M per nucleotide) was determined by UV spec-
trophotometry in properly diluted samples using a molar absorp-
tion coefficient of 6600 Mꢂ1 cmꢂ1 at 258 nm [23]. The stock
solutions were stored at 4 °C and used within 4 days. Stock solu-
tions of [Ni(DIP)3]2+ complex were prepared by dissolving the com-
system, the quantity (a* ꢂ
ted as a function of the molar concentration of DNA, in mono-
meric units (DNAphosphate). eb ef and a* are, respectively, the
e
f)(eb
ꢂ
ef) at 318 nm has been plot-
,
molar extinction coefficients of free [Ni(DIP)3]2+ complex, of
[Ni(DIP)3]2+ complex bound to DNA and of the solution contain-
ing both free and bound [Ni(DIP)3]2+ complex. In particular, ef
was determined by calibration curve of the isolated metal com-
plex in aqueous solution, following the Beer–Lambert law. eb
was determined from the plateau of the DNA titration, where
addition of DNA did not result in further changes in the absorp-
tion spectrum. Finally, a* was determined as the ratio between
the measured absorbance and [Ni(DIP)3]2+ analytical molar con-
centration. Using Eq. (1) [25,26] was obtained the values of the
intrinsic binding constant (Kb) and of the binding size in base
pair(s) of the [Ni(DIP)3]2+–DNA complex.
plex in the 50 mM NaCl/5 mM Tris–HCl buffer to
a final
concentration of 0.5 mg mlꢂ1 and after mixing with DNA, incu-
bated at 37 °C for 24 h.
2.2. Synthesis of rac-[Ni(4,7-diphenyl-1,10-
phenanthroline)3](NO3)2ꢀ3H2O
2K2Ct½DNAꢅ
b ꢂ ðb2
Þ
1=2
b
aꢄ ꢂ ef
s
¼
eb
ꢂ
ef
2KbCt
The [Ni(4,7-diphenyl-1,10-phenanthroline)3](NO3)2 complex
([Ni(DIP)3]2+) (Fig. 1) was prepared by modification of the method
which used ethylene diamine as ligand [24]. Ni(NO3)2ꢀ6H2O
(0.356 g) and 4,7-diphenyl-1,10-phenanthroline (1.495 g) were
dissolved in 50 ml 1-butanol and refluxed for 5 h until the blue
precipitate was formed. The solution was filtered and the precipi-
tate was washed with 1-butanol and diethyl ether. The product ap-
peared as blue powders with a high yield (92–95%) and had high
solubility in water (0.1% DMSO). Anal. Calc. for C72H54N8O9Ni: C,
70.08; H, 4.41; N, 9.08. Found: C, 70.3; H, 5.0; N, 9.3. 1H NMR:
d = 8.75 (d, 2H atoms of phen ligand), d = 7.36 (d, 2H atoms of phen
ligand), d = 7.38 (d, 2H atoms of phen ligand), d = 7.12 (s, 10H
atoms of phenyl groups).
ð1Þ
Kb½DNAꢅ
2s
b ¼ 1 þ KbCt þ
where Ct is the total concentration of the metal complex. The Kb and
s values, obtained by nonlinear fitting of the experimental data by
Eq. (1), are Kb = (4.34 0.02) ꢁ 104 Mꢂ1 and s = 0.43 0.01.
Thermal denaturation experiments were carried out by moni-
toring the absorption of CT-DNA at k = 260 nm at various temper-
atures, both in the absence and presence of different amounts of
the complex (1/R = [Ni(DIP)3]2+/[DNA] = 0.0, 0.25, 0.5, 1.0)
([DNA] = 5.0 ꢁ 10ꢂ5 M). The melting temperature (Tm, the temper-
ature at which 50% of double-stranded DNA becomes single-
stranded) was calculated as reported [27,28].
In the 1H NMR spectrum of [Ni(DIP)3]2+ the signals due to the
,
Circular dichroism (CD) spectra of the racemic [Ni(DIP)3]2+
complex in the absence and presence of CT-DNA at various
[Ni(DIP)3]2+/[DNA] ratios (1/R = 0–0.2) were recorded on a JAS-
CO (J-810) spectropolarimeter operating at room temperature
(ca. 25 °C). The region of wavelength between 200 and
700 nm was scanned for each sample using 1 cm path quartz
cell.
various protons of 4,7-diphenyl-1,10-phenanthroline ligand
shifted to the high field (ꢃꢂ0.2 ppm) with corresponding free li-
gand and confirmed complexation.
Viscosity measurements were made using
a viscosimeter
(SCHOT AVS 450) which thermo stated at 25.0 0.5 °C by a con-
stant temperature bath. The DNA concentration was fixed at
5.0 ꢁ 10ꢂ5 M and flow time was measured with a digital stop
watch, the mean values of three replicated measurement were
N
N
N
N
N
N
Ni
Ni
N
N
N
N
used to evaluate viscosity
tive specific viscosity (
viscosity contributions of DNA in the absence (
g of the samples. The value of rela-
N
1/3
N
g/
go)
where go and
g
are the specific
o) and in the
(g), were plotted against 1/R
g
presence of the complex
(R = [DNA]/[Ni(DIP)3]2+ [29].
Fluorescence measurements were carried out with a JASCO
spectrofluorimeter (FP6200) using a fixed complex concentration
to which increments of the DNA stock solution were added.
Fig. 1. The chemical structure of Ni(II) enantiomers used for enantioselective DNA
binding study.

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N. Shahabadi, A. Fatahi / Journal of Molecular Structure 970 (2010) 90–95
[Ru(o-phen)3]2+ with binding constants of 4.9 ꢁ 104 Mꢂ1 and
3. Results and discussion
3.1. Absorption spectroscopy
2.8 ꢁ 104 Mꢂ1, respectively, which were assigned as non-intercala-
tors [32].
The electronic absorption spectroscopy is one of the most useful
techniques in DNA-binding studies [13]. The band at 260 nm of
3.2. Fluorescence studies
DNA arises due to the
p
–
p
* transitions of DNA bases. Stacking pat-
As the [Ni(DIP)3]2+ complex is luminescent in the absence of
DNA, it does show appreciable increase in emission upon addition
of CT-DNA. (Fig. 3). This figure shows that a regular increase in the
fluorescence intensity of the [Ni(DIP)3]2+ complex with a shift of
fluorescence emission maximum (414–416 nm) took place upon
increasing the concentration of DNA at 25.0 °C and at pH 7.2. These
fluorescence enhancements indicate that the [Ni(DIP)3]2+ complex
interacted with DNA and the quantum efficiency of [Ni(DIP)3]2+
was increased. Like to quenching process, the enhancement con-
stant can be obtained by Eq. (3) [33]:
tern changes, disruption of the hydrogen bonds between comple-
mentary strands, covalent binding of DNA bases and intercalative
mode involving a strong stacking interaction between aromatic
rings of molecules and the base pairs of DNA lead to the hypochro-
mism, red and/or blue shifts of this band [30,31]. The absorption
spectra of the [Ni (DIP)3]2+ complex in the absence and in the pres-
ence of increasing amounts of CT-DNA are shown in Fig. 2. The
electronic spectrum of this [Ni(DIP)3]2+ complex in water (0.1%
DMSO) is characterized by an intense ligand-centered transition
in the UV region at 284 nm and a metal-to-ligand charge transfer
(MLCT) in the visible region at 318 nm. The visible band around
F0
F
¼ 1 ꢂ KE½Eꢅ
ð3Þ
318 nm attributed to the overlap of Ni(d
ultraviolet band around 284 nm can be attributed (DIP)
p) ? DIP (p*) and the
If a dynamic process is part of the enhancing mechanism, the
above equation can be written as follows [33]:
p
? p
*.
The UV–vis absorption of the [Ni(DIP)3]2+ complex is significantly
perturbed by the addition of increasing amounts of CT-DNA. In de-
tails, the absorption band of the complex at k = 318 nm shows a red
shift about 3 nm and hypochromism (Fig. S1). Moreover, the inten-
sity of the absorption band at k = 284 nm is lowered by the addi-
tion of DNA in the range 0 6 [DNA]/[complex] 6 0.2 while it
increases at higher molar ratios. The DNA-binding constant of me-
tal complexes containing dinitrogen ligands are usually deter-
mined by the MLCT band because no light absorption for DNA is
found in the range of wavelength over 300 nm and thus no inter-
ference exists. The extent of hypochromism in the visible MLCT
band is commonly consistent with the strength of intercalative
interaction [13]. The extent of hypochromism of the spectra at
k = 318 nm was measured by Eq. (2) (H% = 15%).
F0
F
¼ 1 ꢂ kD½Eꢅ ¼ 1 ꢂ KBs0
ð4Þ
where kD is the dynamic enhancement constant (like to a dynamic
quenching constant), kB is the bimolecular enhancement constant
(like to a bimolecular quenching constant) and s0 is the lifetime
of the fluorophore in the absence of the enhancer. The dynamic
enhancement constants of [Ni(DIP)3]2+ complex at different tem-
peratures were calculated using Eq. (4) (Fig. 4, Table 1). Since fluo-
rescence lifetimes are typically near 10ꢂ8 s, the bimolecular
enhancement constant (kB) was calculated from kD = kBs0 (Table 1).
By considering the equivalency of the bimolecular quenching and
enhancement constants, it can be seen that the latter is greater than
the largest possible value (1.0 ꢁ 1010 Mꢂ1 sꢂ1) in aqueous medium.
Thus, the fluorescence enhancement is not initiated by a dynamic
process, it is suggested that a static process involves complex for-
mation in the ground state [33].
AFree ꢂ ABounded
H% ¼
ꢁ 100
ð2Þ
AFree
Similar spectral characteristics, i.e. bathochromic shift and hyp-
ochromism in the presence of DNA, observed for ruthenium(II)
complexes, have been attributed to a mode of binding which in-
volves a stacking interaction of aromatic chromophores with the
base pairs of DNA [22].
3.2.1. Equilibrium binding titration
The binding constant (Kf) and the binding stoichiometry (n) for
the complex formation between [Ni(DIP)3]2+ complex and DNA
were measured using the Eq. (5) [22,34].
The intrinsic binding constant, Kb, of the [Ni(DIP)3]2+ complex
was determined to be (4.34 0.02) ꢁ 104 Mꢂ1 using Eq. (1). This
value is comparable to those reported by Barton and coworkers
[12] for related ruthenium complex ([Ru(phen)2(DIP)]2+–DNA;
(1.15 ꢁ 104 Mꢂ1). Furthermore, the Kb value also is rather similar
to the binding constants of the delta and lambda isomers of
ðF ꢂ F0Þ
log
¼ log Kf þ n log½DNAꢅ
ð5Þ
F
here F0 and F are the fluorescence intensities of the fluorophore in
the absence and in the presence of different concentrations of
900
a=NiL3
b
ri=0.2
750
1
ri=0.6
0.9
ri=0.8
284
600
ri=1
0.8
0.7
0.6
0.5
b(ri=1.2)
450
a
300
150
0
0.4
318
0.3
327
390
400
410
420
430
440
450
0.2
0.1
200
wavelength
250
300
350
400
Fig. 3. Fluorescence spectra of the [Ni(DIP)3]2+ complex in the absence and
presence of the increasing amounts of DNA (solid lines) in aqueous solution at
25 °C. R = [DNA]/[Ni(DIP)3]2+ = 0.0, 0.2, 0.6, 0.8, 1, 1.2.
Fig. 2. Absorption spectra of [Ni(DIP)3]2+ complex in Tris–HCl buffer upon addition
of CT-DNA. [Ni] = 2 ꢁ 10ꢂ5 M, [DNA] = (0 ꢂ 1) ꢁ 10ꢂ5 M.

N. Shahabadi, A. Fatahi / Journal of Molecular Structure 970 (2010) 90–95
93
1
is also entropy disfavored. Formation of the complex therefore
results in a more ordered state, possibly due to the freezing (fixing)
of the motional freedom of both the NiL3 and DNA molecules.
These thermodynamic data are lower than those of previously re-
ported for an intercalator [36] and confirmed the non-intercalating
interaction mode.
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
The analogous [Ru(phen)3]2+ [35,37] which is suggested to bind
electrostatically to DNA, has been reported to exhibit a positive en-
thalpy change, but Mudasir et al. [38] found that the mode of bind-
310K
298 K
ing of CT-DNA with [Fe(phen)(DIP)2]2+
(D
H = ꢂ33.1 kJ molꢂ1) was
intercalation. Furthermore, Chaires studies of the thermodynamic
parameters of drug–DNA interactions revealed that for all the
intercalators except actinomycin, the binding enthalpy changes
are large and negative [1].
288K
277K
When cationic binding agent such as [Ni(DIP)3]2+ binds to DNA,
it is likely replace a countercation from the compact inner (stern)
layer or the defuse outer layer surrounding DNA. This counter
ion release process is believed to be nearly entirely entropic [39]
0
1
2
3
4
5
6
7
[DNA]×104 M
Fig. 4. Stern–Volmer plot for the observed fluorescence enhancement of
and the signs of
DH and DS obtained for DNA binding of complex
[Ni(DIP)3]2+ complex upon addition of CT-DNA at different temperatures.
should be positive. Since [Ni(DIP)3]2+ is also a cationic binding
agent and its structure is similar to other complexes which were
previously studied, [38] it is expected that counter ion release,
hydration and hydrophobic interaction, should occur in this com-
plex. Evidently, the overall negative enthalpy and entropy changes
are obtained for DNA binding of this complex. In addition to coun-
ter ion release there should be another type of molecular interac-
tion process in the DNA binding of [Ni(DIP)3]2+ complex from
which large negative enthalpy and entropy changes are produced.
Intercalation of the phenyl group and partial phenanthroline ring
Table 1
Dynamic enhancement and bimolecular enhancement constants of [Ni(DIP)3]2+
complex at different temperatures.
Temperature (K)
Linear equation
kD
kB
277
288
298
310
Y = 0.999 ꢂ 0.115X
Y = 0.99 ꢂ 0.094X
Y = 0.988 ꢂ 0.0866X
Y = 1 ꢂ 0.0798X
1150
940
866
798
1.15 ꢁ 1011
9.4 ꢁ 1010
8.6 ꢁ 1010
7.9 ꢁ 1010
of one DIP ligand in [Ni(DIP)3]2+ complex which involves
p-stack-
ing interaction between these planar substituents and DNA base
pairs seems to be consistent with the enthalpy and entropy
changes. Intercalation, in which two phenyl rings of the ligand
are inserted into the DNA base pairs, may be less favorable, be-
cause the sheer size of the DIP ligand would preclude simultaneous
intercalation of both phenyl rings as the diameter of the ligand is
much larger than the width of base pairs. In addition, the crystal
structure data of solid [Ru(DIP)3]2+ [17] and [Cu(bcp)2] BF4
(bcp = 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline) [40] com-
plexes show that all the phenyl groups are skewed about 40° out
of phenanthroline plane owing to steric effect between hydrogen
atoms in the phenyl and phenanthroline rings. Intercalation of
the entire DIP moiety thus would require that the phenyl groups
rotate into the plane of the phenanthroline moiety in order to min-
imize stacking. This may produce unfavorable intermolecular
interaction between the complex and DNA. In order to explain such
steric hindrance, a model of partial intercalation of the phenan-
throline ligand with only one phenyl substituent and partial phen
ring inserted between the base pairs in the major groove (an asym-
metric docking) has been proposed [41]. In this model, the other
phenyl group and non-intercalating DIP ligands are aligned along
the major groove and involved in hydrophobic non-stacking inter-
actions with the base pairs along the helical major groove. This
model does not require any rotation of the phenyl groups and takes
into account the participation of the non-intercalated phenyl group
and the other DIP ligands in supporting the binding. Furthermore,
even when the model is not operating, intercalative binding of this
complex could still be possible if the binding energy is large en-
ough to overcome the barrier against rotation of the phenyl group
into the plane.
DNA, respectively. The linear equations of log (F ꢂ F0)/F vs. log
[DNA] at different temperature are shown in Table 2 (Fig. S2). The
values of Kf clearly underscore the remarkably high affinity of
[Ni(DIP)3]2+ to DNA.
3.2.2. Thermodynamic parameters of DNA binding
To have a better understanding of thermodynamics of the
reaction between [Ni(DIP)3]2+ complex and DNA, it is useful to
determine the contributions of enthalpy and entropy of the reac-
tion. Therefore, the evaluation of formation constant for the
[Ni(DIP)3]2+–DNA complex at four different temperatures (277,
288, 298, and 310 K) allows determining the thermodynamic
parameters such as enthalpy
[Ni(DIP)3]2+–DNA formation by van’t Hoff equation and plotting
log Kf vs. 1/T. The positive slope of the plot (ꢂ H /R, R is the gas
constant) indicates that the reaction of DNA with [Ni(DIP)3]2+ com-
plex is exothermic and enthalpy favored. The H and S values of
(DH) and entropy (DS) of
D
D
D
the [Ni(DIP)3]2+–DNA complex were ꢂ58.41 0.6 kJ/mol and
ꢂ122.75 2 J/mol K, respectively. In general, electrostatic interac-
tions exhibit small enthalpy and positive entropy changes.
Hydrophobic interactions are generally indicated by positive en-
thalpy and entropy changes. Hydrogen bonding and van der Waals
interactions are usually characterized by negative standard enthal-
pies of interaction [34,35]. From the thermodynamic data, it is
quite clear that while complex formation is enthalpy favored, it
Table 2
Linear equations of log (F ꢂ F0)/F vs. log [DNA], and Kf of [Ni(DIP)3]2+ complex with
DNA at different temperatures.
Temperature (K)
Linear equation
Kf
3.3. Thermal denaturation experiments
277
288
298
310
Y = 0.71X + 4.61
Y = 0.69X + 4.24
Y = 0.68X + 3.87
Y = 0.67X + 3.43
4.1 ꢁ 104
1.74 ꢁ 104
7.4 ꢁ 103
2.7 ꢁ 103
The Tm experiments were carried out for DNA in the absence
and presence of different amounts of [Ni(DIP)3]2+ complex. The
melting plot of DNA (5.0 ꢁ 10ꢂ5 M) monitored by plotting the UV

94
N. Shahabadi, A. Fatahi / Journal of Molecular Structure 970 (2010) 90–95
maximum absorption of DNA at 260 nm vs. the temperature, in the
absence and presence of [Ni(DIP)3]2+ complex and at molar ratios
1/R = [NiL3]/[DNA] = 0.0, 0.25, 0.5, 1.0. The Tm for DNA in Tris–HCl
buffer and in the absence of any added of [Ni(DIP)3]2+ complex
was obtained 70 0.5 °C. An increase in the DNA melting temper-
ature by 2, 5 and 7 °C for the above mentioned molar ratios was ob-
served. In comparison with other studies [41,42], our finding
results suggest that the [Ni(DIP)3]2+ complex interacted with
DNA via groove binding.
equilibrium shift suggests that one of the enantiomers of the metal
complexes is preferentially bound to the optically active biopoly-
mers to afford their enantioselective binding. The shift in enantio-
meric equilibrium due to the enantioselective binding of metal
complexes to optically active biopolymers gives rise to the drastic
CD spectral changes in the UV–vis region because of the enrich-
ment of one enantiomeric conformation in the solution. This phe-
nomenon can be readily monitored in the laboratory by CD
spectroscopy. The enantioselective binding of optically active com-
pounds, e.g. optically active drugs to DNA, RNA or protein has also
been noticed as one of the reasons why one of the enantiomers of
optically active drugs exhibits a biological activity significantly dif-
ferent from the other, although both have chemically very similar
structures [48]. In order to understand such effects better at the le-
vel of molecular interactions, it is of vital importance to examine
enantiomeric effects on the DNA binding of metal complexes. Here,
we studied the CD spectroscopic behavior of racemic [Ni(DIP)3]2+
complex upon binding to CT-DNA to examine whether such selec-
tivity also happens to this complex. The UV and visible CD spectra
of racemic [Ni(DIP)3]2+ complex upon binding to CT-DNA and free
CT-DNA are shown in Fig. 6.
It is clearly seen that a few strong CD signals develop in the UV
and visible region of wavelength upon addition of CT-DNA to the
racemic solution of [Ni(DIP)3]2+ complex. The characteristic bands
of CT-DNA due to base stacking (275 nm) and that due to right
handed helicity (248 nm) disappear and a biphasic CD signal with
positive (261 nm) and negative (296 nm) with a cross over at
280 nm are observed. A positive signal (525 nm) is also observed
and the intensities of all these signals increase with increasing in
value of [Ni(DIP)3]2+ concentration. These observations suggest
that diastereomeric shift occurs in the solution due to the enantio-
selective binding of [Ni(DIP)3]2+ complex to CT-DNA. Indeed, no CD
signals are observed in the UV and visible region for the racemate
solution of [Ni(DIP)3]2+ complex. Furthermore, the development of
the CD spectra suggests that an enrichment of one of the complex-
DNA diastereomers occurs in the solution, probably owing to a
shift in the diastereomeric equilibria. Interestingly, the developed
CD spectrum has an exactly mirror-shape (opposite sign) with re-
3.4. Viscosity measurements
The hydrodynamic measurements are sensitive to length
change of DNA in absence and presence of foreign molecules. A
classical intercalation model demands that the DNA helix must
lengthen as base pairs are separated to accommodate the binding
ligand, leading to increase in DNA viscosity [1]. In contrast, a par-
tial and/or non-classical intercalation ligand could bend (or kink)
the DNA helix, reduce its effective length and concomitantly its vis-
cosity, while ligands that bind exclusively in the DNA grooves (e.g.,
netropsin, distamycin), under the same conditions, typically cause
less pronounced changes (positive or negative) or no changes in
DNA solution viscosity [39]. The values of relative specific viscosity
1/3
(g/g0)
– here g0 and g are the specific viscosity contributions of
DNA in the absence and in the presence of the present complex
were plotted against 1/R (R = [DNA]/[[Ni(DIP)3]2+]) (Fig. 5).
The results showed that the relative viscosity increases in rela-
tive low ratio, [Ni(DIP)3]2+/[DNA] = 0–0.2, while it decreases in high
ratios. The increase at the beginning is the same as intercalators
[43]. This equilibrium shift suggests [Ni(DIP)3]2+ complex bind to
CT-DNA by intercalation in very low ratios, whereas at higher con-
centration it binds partial and or in a non-classical intercalation
mode which can bend (or kink) the DNA helix reducing its effective
length and concomitantly its viscosity. This non-linearity is proba-
bly due to the phenyl groups so that the streric affect play a prom-
inent role in the DNA-binding process. It is also noteworthy that
the racemic mixture of [Ni(DIP)3]2+ complex is relatively labile to-
wards racemization and upon binding to DNA, diastereomeric shift
occurs in the solution, resulting in an enrichment of one enantio-
mer which is less or more favorable to the DNA binding [38,44].
spect to the
[38,49,50]. Therefore, it is immediately confirmed that the
tiomer of [Ni(DIP)3]2+ complex is preferentially bound to CT-DNA
rather than -enantiomer. Similar observation has been reported
K
-enantiomers which were studied previously
D
-enan-
K
3.5. Determination of enantioselectivity in DNA binding
previously for the DNA binding of racemic iron(II) complexes of
phen and bpy (bpy = 2,2’-bipyridine) [45,47,51], rac-[Fe(phen)
(DIP)2]2+ complex [38] [Co(phen)2(dppz)]2+, [46] [Fe(4,7-dmp)3]2+
The labile metal complexes toward racemization, e.g. iron(II),
nickel(II) and cobalt(II) complexes of phen and its derivatives have
been well known to undergo enantiomeric equilibrium shift in the
solution upon binding to optically active biopolymers such as DNA,
RNA or proteins, which is called Pffeifer effect [35,45–47]. This
[52] and
D-[Ru(bpy)2(pyip)](PF6)2 [53]. This result may be due to
different matching between the enantiomers and DNA or to the
different binding sites of the enantiomers, as DNA is a flexible
1.5
1.2
1
261
1
0.5
0
525
DNA
296
329
330
0.8
0.6
0.4
0.2
210
450
570
a
g
-0.5
-1
-1.5
Fig. 6. Circular dichroism (CD) spectra of free CT-DNA (blue line) of 24.5 mM in UV
and visible region at various 1/R ([Ni]/[DNA]) in Tris–HCl buffer (pH 7.2) and 50 ml
NaCl at 25 °C. Each spectrum was recorded at about 40 min after each incremental
addition of CT-DNA. The arrows for solid lines indicate the directions of CD spectral
changes as a function of ri. (For interpretation of color mentioned in this figure the
reader is referred to the web version of the article.)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1/ R
Fig. 5. Effects of increasing amounts of [Ni(DIP)3]2+ complex on the viscosity of CT-
DNA (5.0 ꢁ 10ꢂ5 M) in 10 mM Tris–HCl. (1/R = 0.0–1.5).

N. Shahabadi, A. Fatahi / Journal of Molecular Structure 970 (2010) 90–95
95
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DNA helix axis from any direction.
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5. Circular dichroism results suggest that diastereomeric shift
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Acknowledgments
Financial support from Razi University Research center is grate-
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