DNA interaction with Al-N,N′-bis(salicylidene)2,2′-phenylendiamine complex

Article abstract of DOI:10.1016/j.saa.2006.08.017

The Al(III) complex, [Al(salophen)2H₂O]NO₃, was synthesized and characterized by spectroscopic (NMR and FT-IR) techniques. Then the binding of Schiff base complex of [Al(salophen)]⁺ type, where salophen denotes N,N′-bis(sa

Full text of DOI:10.1016/j.saa.2006.08.017

Spectrochimica Acta Part A 67 (2007) 472–478
ꢀ
DNA interaction with Al–N,N -bis(salicylidene)
ꢀ
2
,2 -phenylendiamine complex
a,∗
a
a
a
b
S. Kashanian , M.B. Gholivand , F. Ahmadi , A. Taravati , A. Hosseinzadeh Colagar
a
Department of Chemistry, Faculty of Science, Razi University, Kermanshah, Iran
Department of Biology, Faculty of Science, Mazandaran University, Babolsar, Iran
b
Received 1 March 2006; received in revised form 2 August 2006; accepted 2 August 2006
Abstract
The Al(III) complex, [Al(salophen)2H
binding of Schiff base complex of [Al(salophen)] type, where salophen denotes N,N -bis(salicylidene) 2,2-phenylendiamine to calf thymus DNA,
has been investigated by spectrophotometric, circular dichroism, spectrofluorometric, melting temperature and viscosimetric techniques. This
Al(III) complex showed absorption hyperchromism in the range of 310–390 nm, increase in melting temperature, some structural changes in
specific viscosity, when bound to calf thymus DNA. The binding constant has been determined using absorption measurement and found to be
2
O]NO
3
+
, was synthesized and characterized by spectroscopic (NMR and FT-IR) techniques. Then the
ꢀ
3
−1
3
−1
1
.82 × 10 M and 1.31 × 10 M in HEPES and Tris–HCl buffers, respectively. Also the fluorescence spectral characteristics and interaction
of Al–salophen complex with DNA have been studied. Al–salophen bound to DNA showed a marked increase in the fluorescence intensity along
with a bathochromic shift (5 nm). The intersection point of the binding isotherm indicated a binding site size of 12 bp per bound complex molecule
in both HEPES and Tris–HCl buffers. The experimental results showed that the Al–salophen complex bound to DNA by non-intercalative mode
and major groove binding was the preferred mode of interaction
©
2006 Published by Elsevier B.V.
Keywords: CT-DNA; Salophen; Al–salophen complex; Schiff base; Circular dichroism
1
. Introduction
subject of many investigations and indicating the binding inter-
actions between DNA and these compounds with significant
changes of spectroscopic properties. For example, an intercalat-
ing interaction mode was proposed for Co (Salen) [15] while an
external binding with the surface of double helix was suggested
for a functionalized Cu (Salen) [4].
The interaction of metalocomplexes containing multidentate
aromatic ligands with DNA has recently gained a growing inter-
est [1,2]. In addition, the interaction of cationic metal complexes
of Schiff bases, as a new agent in order to study anticancer
characteristics and analytical application with DNA has been
developed [3–7]. The cationic metalocomplexes bind to DNA
through a series of following interaction: (i) electrostatic forces,
Aluminum is the third most abundant element in the crust of
the earth and it was not considered to be a toxic element until the
mid 1970s [17]. It was found that the abnormal accumulation of
aluminum in certain tissues of the human body was associated
with various diseases such as Alzimer’s disease [18], Osteo-
malacia [19], and Parkinsonism [20]. Pervious studies revealed
that the Al3 has great affinity for phosphate ligating sites [21].
(ii) hydrophobic interactions with minor and major grooves, (iii)
hydrogen bonding, and (iv) ␲-stacking interactions associated
with the interactions of aromatic heterocyclic groups between
the base pairs. It is valuable to understand the type of interactions
involved between the complex and DNA sites. The interactions
and binding modes of several transition metals such as Cr (IV)
+
3+
Although some researchers have supposed that Al should bind
to DNA in the cell nucleus, Martin predicted in his paper [22]
that in the nuclear chromatin, Al3 do not bind to DNA, but to
ATP or other comparable ligating sites in the presence of ATP.
In this work, we studied the interaction of native calf
thymus-DNA with [Al(salophen)2H2O]NO3, where salophen
+
[
(
3], tin (IV) [7,8], iron (II) [9] iron (III) [10], Ni (II) [11,12], Mn
II) [13,14], Co (II) [15] and Ru [16] with DNA have been the
ꢀ
∗
is the anion of the Schiff base (N,N -bis(salicylidene)-1,2-
Corresponding author. Tel.: +98 918 3311450; fax: +98 831 4274559.
E-mail address: kashanian s@yahoo.com (S. Kashanian).
phenylenedamine) by spectrophotometric, circular dichroism,
1
386-1425/$ – see front matter © 2006 Published by Elsevier B.V.
doi:10.1016/j.saa.2006.08.017

S. Kashanian et al. / Spectrochimica Acta Part A 67 (2007) 472–478
473
fluorimetric, melting temperature and viscosimetric techniques.
It has been shows that, some Schiff base complexes may cause
apoptosis in lymphocyte cell cultures [3]. Hence, the knowledge
of binding mode of these compounds to DNA and the changes
brought about in DNA by these Schiff base complexes are of pri-
mary importance. Finally, we believed that, such investigations
could be beneficial to understand the possible anti carcinogenic
effect of Schiff bases and if Al3 can be reduced [41].
+
2
. Experimental
2
.1. Materials
ꢀ
Fig. 1. The structure of N,N -bis(salicylidene)-1,2-phenylenediaminoalu-
minum(III) nitrate.
All chemicals were purchased from Merck and highly poly-
merizedcalfthymus-DNA(CT-DNA)fromSigmaCo. Thestock
solution of DNA was prepared by dissolving of DNA in 10 mM
of the HEPES and Tris–HCl buffers at pH 7.0 and dialyzing
exhaustively against the same buffers for 24 h. A solution of CT-
DNA gave a ratio of UV absorbance at 260 and 280 nm more
than 1.8, indicating that DNA was sufficiently free from protein.
The DNA concentration (monomer units) of the stock solu-
tons, which appears at δ 13.0 in the free salophenH2 spectrum,
was absent in the spectra of the complex.
2.4. Instrumentation
The FT-IR measurement were carried out using Shimadzu
(IR Prestige-21). The NMR characteristics of salophen and
−
2
tion (1 × 10 M per nucleotide) was determined by UV spec-
Al–salophen complex were determined by using Bruker Avance
DPX 200 MHZ (4.7 T).
Absorbance spectra were recorded using a hp spectropho-
tometer (Agilent 8453) equipped with a thermostated bath
trophotometery, in properly diluted samples, using the molar
−
1
−1
absorptioncoefficient7000 M cm at258 nm[23]. Thestock
◦
solutions were stored at −20 C.
(
Huber polystat cc1). For complexation studies the temperature
2
.2. Synthesize of salophen
◦
of the cell holder was maintained at 25 ± 0.1 C.
Circular dichroism (CD) measurements were recorded on
a JASCO (J-810). Solutions of DNA and Al–salophen were
scanned in 0.5 cm (1 ml) quartz cuvette. The spectrums were
recorded by progressive addition of Al–salophen to the pure
DNA solution.
The ligand was prepared according to our pervious work [24]
as follows: 1.08 g of 1,2-phenylenediamine (10.0 mmol), was
dissolved in 25 ml of ethanol, then transferred into a 250-ml
three necked flask. Under reflux, 2.44 g (20.0 mmol) of salicy-
laldehyde in 25 ml of ethanol was added drop wise to the flask.
The stirred mixture was kept reacting for 45 min under reflux,
then cooled to room temperature. The solid mass was filtered,
For viscosity measurements a viscosimeter (SCHOT AVS
◦
4
50), which thermostated at 25 C by a constant temperature
ꢀ
bath was used. Flow time was measured with a digital stopwatch;
the mean values of three replicated measurements were used to
evaluate the viscosity η´ of the samples. The data were reported
and the product, N,N -bis(salicylidene)-1,2-phenylenediamine
(
salophenH2),wasrecrystallizedfromethanolandvacuum-dried
1
for 2 h. H NMR δ: 6.8–7.6 (m, 12H, phenyl), 8.8 (s, 2H,
◦
−1
as ( η´ / η´ ) versus the [Al(Salophen)2H2O]NO3/[DNA] ratio [3],
–
1
8
CH N–), 13.0 (s, 2H, OH); IR (ν, cm ): 1620 s, 1590 s,
550 s, 1475 s, 1450 s, 1290 s, 1210 m, 1150 m, 1020 m, 930 m,
30 m, 750 s.
◦
where η´ is the viscosity of the DNA solution alone.
All fluorescence measurements were carried out with a
JASCO spectrofluorometer (FP6200). Excitation and emission
wavelengths of 255 and 425 nm were used respectively.
2
.3. Synthesize of Al–salophen complex
ꢀ
The complex of Al–salophen, [N,N -bis(salicylidene)-1,2-
3. Results and discussion
phenylenediaminoaluminum(III) nitrate] (Fig. 1), was synthe-
sized by modification of the reported procedures [25–27] as
follows: to a 1.58 g (5.0 mmol) sample of salophenH2 in 50 ml
of ethanol, 1.88 g (5.0 mmol)of Al(NO3)3·9H2O in 30 ml of
ethanol was added drop by drop. After being stirred for 1 h at
room temperature, the mixture was refluxed for 2 h and cooled to
room temperature; the synthesized complex was recrystallized
In order to determine the stoichiometry and stability of the
resulting salophen complex with Al3 ion in a methanol solu-
tion, the spectra of a series of solutions containing a constant
+
−
5
concentration of ligand (5.0 × 10 M) at a fixed ionic strength
−
3
of 1.0 × 10 M of tetraethylammoniumperchlorate (TEAP) at
◦
25 C and varying amounts of the metal ion were obtained
from ethanol by diethyl ether, filtered and dried under vacuum at
and the result is shown in Fig. 2a. As can be seen, the com-
plexation was accompanied by increasing and decreasing in
absorption bands of the salophen at wavelengths of 225 and
270 nm, respectively, and producing a new absorption band at
390 nm in methanol solution. The obtained mole ratio plots
◦
5
0 C. There was no obvious frequency corresponding to ν(OH)
in the IR spectra of the complex as in the case of the free ligand
spectrum in which hydrogen bonding occurred, also the appear-
−
1
ance of a wide peak in frequency of 3600 cm corresponding to
the presence of water in the complex. The resonance of OH pro-
3+
(absorbance versus [Al ]/[salophen]) at wavelengths of 270,

4
74
S. Kashanian et al. / Spectrochimica Acta Part A 67 (2007) 472–478
Table 1
3
+
Formation constants of 1:1 complex of Al with salophen in methanol
Wavelength
log K
2
3
3
70
50
90
4.98 ± 0.051
5.00 ± 0.048
4.99 ± 0.035
by following the changes in absorbance (hypochromism) and
shift to longer wavelengths (red shift) [30]. In the present
investigation, it has been used to monitor the interaction of
+
[
Al(salophen)2H2O] in aqueous solution with CT-DNA. The
Al–salophen complex shows an absorbance, which is due to
metal-to-ligand charge transfer interactions. The Al–salophen
complex has two replaceable H2O molecules and hence can
coordinate with nucleotide bases. The intensity of Al–salophen
metal-to-ligand charge transfer (MLCT) band of the Al complex,
observed at the range of 310–390 nm (Fig. 3a), was monitored
Fig. 2. (a) Electronic spectra of a 5.0 × 10−5 M solution of salophen in MeOH
3
+
in the presence of increasing concentration of Al and (b) absorbance vs.
3
+
[
Al ]/[salophen] molar ratio plots.
3
50, and 390 nm for salophen are shown in Fig. 2b. As can be
seen from Fig. 2b, in the case of all absorbance–mole ratio plots
3+
revealed one distinct inflection point at [Al ]/[salophen] molar
ratio of 1, emphasizing the formation of 1:1 (metal to ligand)
complex in solution.
The formation constants of the resulting complex between
Al3+ and salophen was evaluated by computer fitting of the cor-
responding mole ratio data to a ML complex derived equation
[
28] using a non-linear curve fitting program, KENFIT [29]. The
results are summarized in Table 1.
3
.1. Electronic spectra
+
−5
Fig. 3. (a) Charge transfer spectra of [Al(salophen)2H2O (5 × 10 M) in the
absence (- - -) and presence (—) of increasing amounts of CT DNA in HEPES
and (b) typical plots of [DNA]/( ε´ A − ε´ F) vs. [DNA] for absorption titration of
The binding of intercalative drugs to DNA helix has been
+
characterized classically through absorption spectral titrations,
CT DNA with [Al(salophen)2H2O] in HEPES and Tris–HCl buffers.

S. Kashanian et al. / Spectrochimica Acta Part A 67 (2007) 472–478
475
as a function of added DNA. The complex shows an increase
in molar absorptivity (hyperchromism) as well as a slight red
shift (3 nm). To understand whether the hyperchromism is due
to higher formation of the complex (which may dissociate in
dilute solutions) upon the addition of DNA, spectral titrations
were carried out by varying the concentration of DNA.
The results show that the origin of hyperchromism might lie
in the mechanism of interaction of the complex with DNA. A
similar hyperchromism has been observed for the sort bands
of certain porphyrins and phenantrolines when interacted with
DNA [31,32].
The absorbance measurements were performed by keeping
−
4
the concentration of the complex constant (1 × 10 M) while
−
4
−3
varying the DNA concentrations (1 × 10 to 2 × 10 M). The
spectrum of Al–salophen complex was recorded after each addi-
tion of DNA. The intrinsic binding constant K was determined
b
for Al–salophen–DNA according to Eq. (1) [33]:
[
DNA]
[DNA]
1
−
+
(1)
(
ε´ A − ε´ F) ( ε´ B − ε´ F) K ( ε´ A − ε´ F)
b
where ε´ A, ε´ F, and ε´ B correspond to Aobsd./[Al], the extinction
coefficient for the free aluminum complex and the extinction
coefficient for the aluminum complex in the fully bound form,
respectively. A plot of [DNA]/( ε´ A − ε´ F) versus [DNA], gives Kb
as the ratio of the slope to intercept (Fig. 3b). The binding con-
Fig. 4. Three-dimension fluorescence spectra of excitation and emission of
Al–salophen (5 × 10 M) in absence of DNA, excitation λmax is 220 nm and
emission λmax is 425 nm.
−
5
stants, K in HEPES and Tris–HCl buffers have been calculated
b
3
−1
3
−1
ing agents [3,36]. Molecules showing large binding site sizes
are indicative of non-intercalation as a probable mode of bind-
ing and they require correspondingly lower concentrations to
saturate the sites. The binding isotherm of the complex is again
indicative of non-intercalative binding of the Al(III)–salophen
complex to DNA.
to be (1.82 × 10 M ) and (1.31 × 10 M ), respectively. The
K values are lower than those observed for typical classical
b
7
−1
intercalators, ethidium–DNA, 7 × 10 M in 40 mM Tris–HCl
6
−1
buffer, pH 7.9 [34] and 1.4 × 10 M in 40 mM NaCl–25 mM
Tris–HCl [35].
This indicating that DNA complex bonding has an affinity
less than the classical intercalators.
3.3. Circular dichroism
3
.2. Fluorescence studies of Al–salophen–DNA
The CD spectrum of the CT DNA exhibits a positive band at
The excitation and emission wavelengths of Al–salophen
complex were 255 and 425 nm, respectively (Fig. 4).
270 nm due to base stacking and a negative band at 240 nm due
to helixity of B DNA [37]. The interaction of the Al–salophen
with DNA induces a change in the CD spectrum of B DNA
(Fig. 6a and b). The intensities of both the negative and posi-
tive bands decrease significantly (shifting to zero levels). This
suggests that the DNA binding of the complexes induces certain
conformational changes, such as the conversion from a more
B-like to a more C-like structure within the DNA molecule
[32]. Interaction with cations effectively screens the negative
charge on N(7) base sites as well as phosphate oxygens simul-
taneously, both along the deoxyribophosphate backbone and in
the groove of the helix to promote such a trans conformational
change [38].
The CD spectral changes induced by [Al–salophen] in
HEPES buffer are more significant than those by Al–salophen
in the Tris–HCl buffer. This suggesting the electrostatic inter-
action in HEPES has more influence on trans-conformational
changes of DNA than Tris–HCl buffer. Also the one isodichroic
point at 258 and 255 nm for HEPES and Tris–HCl buffers,
respectively, indicates the existence of a single binding mode
[4].
Type of buffer has no effect on the excitation and emission
wavelengths of Al–salophen complex. Fluorescence titration
experiments were performed by keeping the Al–salophen com-
plex concentration constant and stoichiometrically varying the
DNA concentration. Fig. 5a shows the fluorescence emission
spectra in both the absence and presence of different concentra-
tions of DNA in HEPES buffer.
Upon the addition of DNA the fluorescence spectra of
complex shows an increase in the intensity of excitation and
emission along with a red shift (5 nm), indicating the inter-
action of Al–salophen with DNA. The binding site size was
determined from the binding stoichiometry of the complex of
Al–salophen–DNA isotherm as shown in Fig. 5b.
Knowledge about the binding stoichiometery of the
Al–salophen–DNA complex would be helpful in characteriz-
ing the geometry of the drug binding to DNA. The intersection
point of the binding isotherm, gave a binding site size of 12 bp
per bound complex molecule. The binding site size allows one
to distinguish between intercalating and non-intercalating bind-

4
76
S. Kashanian et al. / Spectrochimica Acta Part A 67 (2007) 472–478
−
4
Fig. 6. Circular dichroism spectra of CT DNA (2.0 × 10 M) in: (a)
HEPES (10 mM), (b) Tris–HCl (10 mM), in the presence of increas-
ing amounts of Al–salophen at the following stoichiometric ratios:
R = [Al–salophen]/[DNA] = 0.0, 0.25, 0.5, 0.75, 1.0.
Fig. 5. (a) Effect of DNA on the fluorescence spectra of Al–salophen
−
5
−5
plotting the UV maximum absorption of DNA at 260 nm versus
the temperature, in the absence and presence of Al–salophen
at molar ratios R = [Al–salophen]/[DNA] = 0.0, 0.25, 0.5, 1.0
(
(
3
3.5 × 10 M) in HEPES, DNA concentrations: (1) none, (2) 3.5 × 10 M,
−
5
−4
−4
−4
3) 7.0 × 10 M, (4) 1.75 × 10 M, (5) 2.45 × 10 M, (6) 3.15 × 10 M, (7)
−
4
−4
−4
.5 × 10 M, (8) 4.2 × 10 M, (9) 5.25 × 10 M, and (b) fluorescence bind-
ing isotherms for the association of Al–salophen with DNA (M per nucleotide)
(Fig. 7).
in 10 mM HEPES (ꢀ) and Tris–HCl (ꢁ) buffers.
The Tm for CT DNA in the absence of any added complex
◦
was obtained 67 ± 0.5 and 59 ± 0.5 C for HEPES and Tris–HCl
Also, molar ellipticity values were calculated according to
the formula [37]:
buffers, respectively. An increase in the DNA melting temper-
◦
◦
ature by 5, 11 and 15 C in HEPES buffer, 3, 7 and 10 C
in Tris–HCl buffer for the above mentioned molar ratio was
1
00 θ(λ)
[
θ] =
◦
λ
observed. This increase (3–15 C) in the melting temperature of
lC
DNA, in different buffers, indicates that an electrostatic binding
interaction between the stacked Al–salophen cation and DNA
where [θ]λ is the molar ellipticity value at a particular wave-
2
−1
length expressed in deg cm dmol , C the concentration in
moles of nucleotide phosphate per liter, l the length of the cell
◦
Table 2
in dm, and θλ is the observed rotation in degrees ( ). The results
Effect of Al–salophen on CD spectra of CT DNA
are summarized in Table 2.
2
45 nm
275 nm
HEPES
245 nm
Tris–HCL
275 nm
Tris–HCL
θ
θ
θ
θ
HEPES
3
.4. Thermal denaturation experiments
−9440
9600
8880
8130
7580
6730
−9790
−9720
−8960
−8810
−7710
1060
9990
9180
8340
7050
−
−
−
−
8840
8340
7970
7070
The Tm experiments were carried out for CT DNA in the
absence and presence of different amounts of Al–salophen com-
plex. The melting plot of DNA 2.0 × 10 M, monitored by
−
4

S. Kashanian et al. / Spectrochimica Acta Part A 67 (2007) 472–478
477
Fig. 7. Melting curves of CT DNA (2.0 × 10− M per nucleotide) in the absence
and in the presence of different amounts of Al–salophen complex in HEPES
buffer.
4
were performed, also the low change in the Tm indicative of
non-intercalative binding [39].
3
.5. Viscosity measurements
As a means for further exploring the binding of the present
Al–salophen complex, viscosity measurements were carried out
on CT DNA by varying the concentration of the added complex.
◦
The values of relative specific viscosity ( η´ / η´ ) versus 1/R
(R = [DNA]/[Al(salophen)2H2O]NO3) in the absence and in the
presence of Al–salophen complex in different buffers, were plot-
ted (Fig. 8a). As it is observed from Fig. 8a in the presence of
the Al–salophen complex, viscosity of DNA exhibits a depen-
dence on the concentration of Al–salophen complex. The results
show that the specific viscosity of DNA in HEPES buffer first
increased then decreased and at 1/R = 0.5 reached to its low-
est level, then increased again. The similar behavior has been
observed for Tris–HCl buffer. Under appropriate conditions,
intercalation of classical compounds such as ethidium bromide
Fig. 8. (a) Effect of [Al–salophen]+ on the viscosity of CT DNA in 10 mM
of HEPES (ꢀ) and 10 mM of Tris–HCl (ꢁ) buffers and (b) charge transfer
+
−5
spectra of [Al(salophen)2H2O (5 × 10 M) and NaCl 50 mM in the presence
of increasing amounts of CT DNA in Tris–HCl.
by spectrophotmetrically titration and no change in spectrum
was observed (Fig. 8b). This is due to uncharging of oxygen
(
EthBr) causes a significant increase in viscosity of DNA solu-
+
atoms of phosphate groups by Na ions, to prevent electrostati-
tions due to increase in separation of base pairs at intercalation
sites and hence an increase in overall DNA contours length.
Since the increase of viscosity is less than that observed for
an intercalator like EthBr, interaction involving classical inter-
calation is ruled out for the present complex, and exclusively
DNA grooves binding is suggested. McMillin and co-workers
cally interaction of aluminum-salophen complex with CT-DNA.
[
40] have hypothesized that such viscosimetery results could
be explained in terms of bridged structure to promote exten-
sion of duplexes. Also our result indicating that there are at
4. Conclusion
+
least two phases of binding for [Al–salophen] . The hydropho-
+
bic interaction of [Al–salophen] with DNA occurs first, which
The small-observed changes in the CD spectra, Tm and vis-
cosity of DNA due to its interacting with Al–salophen complex
are indicative of a non-intercalative mode of binding. The sec-
ond point is that in the presence of Tris–HCl buffer, changes are
less pronounced, so higher concentration of complex is required
in the presence of Tris–HCl, compared to HEPES, to produce
the same conformational changes on the DNA structure.
is followed by the bridging of duplexes involving the ligand.
We suggest that the complex exhibits the electrostatic binding,
involve inner sphere complex formation via phosphate oxygen
negative charge with the positive charge of complex [32].
For this purpose, the interaction of Al–salophen complex
with CT-DNA in the presence of 50 mM of NaCl was carried out

4
78
S. Kashanian et al. / Spectrochimica Acta Part A 67 (2007) 472–478
The lower values of K in comparison with those of Al–DNA
[17] E.J. Underwood, Trace Elements in Human and Animal Nutrition,
Academic Press, New York, 1977, pp. 430.
b
complex [41] and no evidence of DNA precipitation in the pres-
ence of Al–salophen complex in comparable concentrations,
indicating that the toxicity of aluminum ion is decreased remark-
ably when Al–salophen is formed, so proper complexation of
[
[
18] J.F. Foncin, Nature 326 (1987) 136.
19] B.F. Boyce, H.Y. Elder, H.L. Ellio, I. Fogelman, G.S. Fell, B.J. Juners,
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[
20] D.P. Perl, D.C. Gajdusek, R.M. Garruto, R.T. Yanagihara, C.J. Gibbs,
Sciences 27 (1982) 1053.
3
+
Al would diminish its dramatic toxicity.
[
[
[
[
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