Experimental and theoretical studies on Cu(II) complex of N,N'-disalicylidene-2,3-diaminopyridine ligand reveal indirect evidence for DNA intercalation

Article abstract of DOI:10.1016/j.poly.2015.11.037

A potential DNA intercalating Cu(II) complex ([CuL]) of the N,N'-disalicylidene-2,3-diaminopyridine ligand (H₂L; dianion = L^2-) has been synthesized and characterized. The photophysical and the DNA binding behaviors of the

Full text of DOI:10.1016/j.poly.2015.11.037

Accepted Manuscript
Experimental and theoretical studies on Cu(II) complex of N,N’-disalicyli-
dene-2,3-diaminopyridine ligand reveals indirect evidence for DNA intercala-
tion
Abdulkadir Kocak, Ozlem Faiz, Hakan Yilmaz, Omer Andac
PII:
S0277-5387(15)00723-8
DOI:
http://dx.doi.org/10.1016/j.poly.2015.11.037
POLY 11682
Reference:
Polyhedron
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Accepted Date:
21 September 2015
19 November 2015
Please cite this article as: A. Kocak, O. Faiz, H. Yilmaz, O. Andac, Experimental and theoretical studies on Cu(II)
complex of N,N’-disalicylidene-2,3-diaminopyridine ligand reveals indirect evidence for DNA intercalation,
Polyhedron (2015), doi: http://dx.doi.org/10.1016/j.poly.2015.11.037
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Experimental and theoretical studies on Cu(II) complex of N,N’-
disalicylidene-2,3-diaminopyridine ligand reveals indirect evidence for
DNA intercalation
Abdulkadir Kocaka,*, Ozlem Faizb, Hakan Yilmazc, and Omer Andacc
a Department of Chemistry, Gebze Technical University, 41400 Kocaeli,
Turkey
b Department of Chemistry, Recep Tayyip Erdogan University, 53100 Rize,
Turkey.
c Department of Chemistry, Ondokuz Mayis University, 55200 Samsun,
Turkey.
Abstract
A potential DNA intercalating Cu(II) complex ([CuL]) of the N,N'-
disalicylidene-2,3-diaminopyridine ligand (H2L; dianion=L2-) has been
synthesized and characterized. The photophysical and the DNA binding
behaviors of the neutral [CuL] have been investigated both experimentally and
theoretically. Experimental studies reveal that the [CuL] has a quite strong
interaction with the Calf Thymus DNA (ctDNA) with Kb = (1.53 0.48) x 106.
Experimental bathochromism of 4 nm and hypochromism of ~50% on the
absorption band of the [CuL] at 408 nm by intercalation were reproduced by
calculations. The competitive displacement experiments were carried out
using methylene blue (MB) and ethidium bromide (EB). Viscosity
measurements totally supported the intercalative interaction. Quantum
mechanical calculations using time dependent density functional theory
*
Corresponding author: Phone: +90 262 605 3084; Fax: +90 262 605 3005; E-Mail:
kocak@gtu.edu.tr
1

(TDDFT) coupled with polarizable continuum model were carried out in the
proposed dimer of deoxyguanosine-monophosphate-deoxycytidine, d(GpC)2,
intercalation pocket. The calculations qualitatively confirmed the intercalative
binding. In addition, vertical excitation calculations showed that electronic
excitations of H2L are affected by the environment and Cu2+ ion. The
electronic transitions of the [CuL] are involved in mostly π-π* transitions but
includes significant contribution from the charge transfer. According to the
calculations, the electronic spectrum of the [CuL] is sensitive to the DNA
intercalation because of the π-π stacking interaction between the DNA base
pairs and aromatic rings of the [CuL].
Keywords
DNA intercalation, Schiff base, salphen, absorption, density functional
theory, π-π stacking.
2

1. Introduction
Attaching small molecules to DNA with high affinity has been a goal of
drug design in the recent years due to the possibility of developing anticancer
and antibiotics drugs since DNA is ultimately responsible for all the cell
functions [1]. Comparing to proteins, studies on DNA as a drug target are still
very limited as opposed to well-known structure of DNA [1]. The attachment
(and thus disfunctioning of the DNA) can be done by a) irreversible covalent
binding or metal coordination to the bases b) reversible non-covalent as
minor/major groove bindings c) intercalation and d) sugar-phosphate
(phosphodiester) backbone binding [2-4].
Salen and salphen type ligands and their corresponding metal complexes,
apart from their outstanding applications such as efficient molecular sensors
related to fluorescence detections of biologically common ions [5-13] and as
on-off light switch behaviors by multi-fold fluorescence enhancement or
quenching upon saturation [14-25], are excellent DNA interacting systems.
The findings of the literature studies on metal complexes of salen and salphen
type ligands such as Al [26], V [27], Mn [28-31], Fe [32, 33], Co [30, 34], Ni
[2,5,6,11] Cu [27, 30, 35, 36] and Zn [27] show that the interaction depends on
the nature of the ligand and coordination around the metal ion [37].
The interaction between the DNA and small metal complexes can be
measured by direct methods such as X-ray crystallography and NMR
techniques or by indirect methods such as electronic absorption, fluorescence
spectroscopy, viscosity measurements and circular dichroism [3, 38]. Since
computational treatment of the interaction phenomenon can provide useful
3

information at the atomistic level, there have been numerous computational
efforts on both large or small scale [39, 40]. These include molecular
dynamics (MD) simulations of small molecule – DNA oligomer interaction,
performed for imaging particularly groove binding modes [41-44]; hybrid
quantum mechanics (QM) and molecular mechanics (MM) calculations for
modeling both intercalative and groove binding modes of the ligand - DNA
oligomers [38, 45, 46]; or pure QM calculation including only the DNA
dimers at the intercalation pocket [37, 45, 47, 48].
A new salphen type Schiff base, N,N’-disalicylidene-2,3-diaminopyridine
(H2L), was first prepared and characterized by Bosner and co-workers [49].
Later, Atakol [50] et al. successfully crystalized neutral Cu(II) complex
([CuL]) of dianion of H2L, but it was never characterized in detail until
recently [51-53]. Asadi et al. [53] characterized the [CuL] complex (in
methanol) using UV-Vis, FT-IR and H-NMR techniques. The potential DNA
binding features and photophysical behaviors have never been investigated
(Figure 1).
Here, we report experimental studies of the [CuL] to explore DNA
intercalation properties along with theoretical calculations on both [CuL] and
its insertion to double stranded DNA dimer via intercalation.
2. Experimental and Computational Methods
2.1 Materials
The 2-3-diaminopyridine, salicylaldehyde, and solvents (DMF and EtOH)
were purchased from Sigma-Aldrich and used without further purification. For
4

the intercalation experiments, Calf Thymus DNA (ctDNA), Ethidium Bromide
(EB), Methylene Blue (MB) and all the solvents and reagents used to prepare
buffer were purchased from the same commercial supplier (Sigma-Aldrich).
Single crystal x-ray data were collected using STOE X-AREA
diffractometer at 293 2 K using Mo Kα radiation (λ=0,71073Å). The
structures were solved by direct methods using SHELXS-97 and refined by a
full-matrix least-squares procedure using the program SHELXL-97[54, 55].
ESI-MS spectrum was taken by LC/MS (HPLC unit: Agilent 1260 Infinity
Series, TOF-MS unit: Agilent 6230), at Central Research Laboratory, Giresun
University.
The ctDNA binding studies of [CuL] along with its competitive
displacement experiments with MB and EB were carried out by electronic
spectroscopy on Shimadzu UV 1601. Viscosity measurements were performed
at 25 °C on Ostwald-type viscometer.
The DFT calculations were carried out using Gaussian 09 (G09) Rev. D01
software package [56].
2.2 Synthesis and Characterization
2.2.1 H2L Ligand
The procedure for the synthesis of the H2L was described elsewhere [52].
Briefly, 2,3-diaminopyridine (in EtOH) was treated with hot solution of
salicylaldehyde (in EtOH), with the ratio of 1:2 for 3-4 h at 50 °C. Upon
cooling, the solid orange product was immediately filtered, washed with
ethanol, ether, and dried in air.
5

2.2.2 [CuL]
For the synthesis of the [CuL], the H2L ligand was simply mixed with
Cu(CH3COO)2 (1:1 ratio) in an abundant solvent (EtOH) environment and
stirred ~1 hour in hot solvent bath. Hot solution was filtered and cooled for
crystallization.
Elemental Analysis: [CuL] with empirical formula of C19H13CuN3O2
Observed: 59.83% C, 3.77% H, and 10.93% N. Calculated ([CuL]): 60.23% C,
3.46% H and 11.09% N.
ESI-MS of [CuL]: The characteristic molecular ion peak of [CuL] was
observed at m/z=379 (100%), confirming the formation of the [CuL] complex.
65
The relative abundance of the Cu isotopomer peak at m/z=381 also supports
the structure. (Figure S1)
XRD of H2L and [CuL]: The structure of the [CuL] complex confirms the
square planar geometry while the H2L ligand is non-planar. Selected bond
distances and dihedral angles for the H2L ligand and [CuL] complex are given
in Table 1. The crystallographic data are summarized in Table S1. Both H2L
ligand and [CuL] complex adopt monoclinic P21/c space group. The structures
will be discussed in detail in the results section.
6

Figure 1. Molecular structures of H2L (left) and [CuL] (right) from the X-ray
crystallographic data. The bond distances and dihedral angles of the labeling
are discussed in section 3.1.
2.3 DNA Interaction Experiments
The ratio of observed absorption of ctDNA at 260 and 280 nm was
A260/A280 = 1.9, indicating sufficient purity of the DNA (devoid of proteins).
For the binding studies, 500 µM stock solution of ctDNA in 5mM the
tris(hydroxymethyl)aminomethane (Tris)/HCl buffer with pH=7.2 and I=0.1
M (NaCl) was kept in the dark at 4 °C and used within 2 days of preparation.
The concentration of the DNA in monomer units was determined using the
molar extinction coefficient (ꢀꢁꢂꢃ = 6600ꢄꢅ. ꢆꢇꢈꢉꢊꢋꢆꢉꢊ ) at 260 nm [57].
Due to the low solubility in water, the stock solutions of [CuL] (1 mM) were
first prepared in DMF (Sigma Aldrich, purity >99%) and then diluted to 15
µM with the Tris buffer, in which the DMF content was negligible (5% DMF
in the Tris buffer). The isolated [CuL] in the solvent mixture was verified for
no shift on the spectra for 10 minutes, which has been used as standard
incubation time for the DNA binding experiments.
7

For the binding studies, the [CuL] concentration was kept constant at 15
µM and ctDNA concentration was increased step by step (0-32 µM). For each
step, the same amounts of the ctDNA were added to both the [CuL] and the
reference solutions [58].
For the competitive displacement experiments, 20 µM solutions of the MB
and EB intercalators in the same buffer were prepared and 20 µM of ctDNA
solution was added to each solution. Keeping the intercalator and DNA
concentrations constant, the [CuL] with varying (0-10 µM) concentrations
were mixed and absorption spectra were measured. For the relative viscosities,
ctDNA concentration was kept constant at 20 µM and the [CuL] concentration
was varied (0-10 µM).
2.4 Theoretical Calculations
2.4.1 The Systems of Interest
In calculations, a double stranded DNA base pairs of 5’-Deoxyguanosine –
phosphate- Deoxycytidine-3’, d(GpC)2, was used as the intercalation region.
All the calculations were carried out in gas phase and water solvent [56].
During the calculations, no restrictions have been applied. All the
geometries for the [CuL] ligand, the DNA pairs, and the [CuL]-DNA
intercalated complexes were relaxed to their minimum energies without
freezing any atom or applying any restrictions. In addition, symmetry
constraints were also removed from all the calculations. All the optimizations
were confirmed for their minima by frequency calculations.
8

For the calculations of the free [CuL], the initial structure was taken from
our XRD data. For the optimization of the DNA base pairs (without the
ligand), we have used two different starting molecules. First, d(GpC)2 slice of
the dodecamer B-DNA (PDB id=1BNA) was directly used. Second, we
removed the ligand of a known intercalated DNA pairs, in which the base
pairs were wide open, and let the DNA pairs relax to the normal distances.
Both starting geometries were converged to the same structure.
Similarly, for the calculations the [CuL] intercalated DNA pairs, initial
structures of, two starting geometry were considered. First, the X-ray structure
of a known structure (with PDB id= 454D), in which a different ligand is
intercalated to the d(GpC)2 pairs, was taken and cut to include only the
intercalated region. Then, [CuL] was replaced with the ligand, and
optimization was carried out. In the second starting geometry, a dummy
insertion of the [CuL] into the d(GpC)2, which was extracted from the
dodecamer (PDB id=1BNA) without any modification. Both structures were
converged to the same intercalated complex.
2.4.2 The Method & Basis Set
Here, we report the interaction of the [CuL] with only the intercalation
pocket d(GpC)2 in the QM level ignoring the mechanical part in order to show
the possibility of intercalation alone.
Due to the large size of the systems, the calculations can easily be too
costly. Thus, the systems should be as small as single base pairs for post
Hartree-Fock methods such as additional second order correlation functions
(MP2) [59] or computationally more efficient DFT methods. Most DFT
9

methods failed [59-62] for the inclusion of long distance dispersion forces,
hydrogen bonds between the base pairs, and π-π stacking interaction that is
responsible for the intercalation [63, 64]. Recent DFT studies such as B3LYP,
CAM-B3LYP, M05-2X and M06-2X have been successfully performed to
describe the covalency, π-π stacking interaction and hydrogen bonding [3, 38,
45, 47, 65, 66]. Among these, M06-2X [3, 38, 45, 47, 66] and BP86 [65] are
widely used for their capabilities of describing hydrogen bonding in Watson-
Crick base pairs as well as π-π stacking interactions [67].
Thus, in our calculations we have used M06-2X with Stuttgart/Dresden
(SDD) basis set with (quasi relativistic) effective core (10 electrons) potential
(ECP) on Cu2+ and 6-31G(d) for the rest of the atoms. We have evaluated that
increasing the basis set for the non-copper atoms to 6-311++G(d,p) is affecting
only in ~1 kJ/mol for the [CuL] complex. And the basis set superposition error
(BSSE) was negligible. Thus, we believe this basis set is sufficient for
describing the C, H, O, N, and P atoms.
Integral Equation Formalism of a Polarizable Continuum Model (IEF-
PCM) was used to include the solvent effects. And default water parameters
were used as the PCM water solvent. Routinely, after each optimization
process, a frequency calculation was carried out in order to make sure the
minimum reached.
10

3. Results & Discussions
3.1 Experimental and Theoretical Study of [CuL]
In order to understand the nature of the intercalation, we first analyzed the
photophysical behavior of the [CuL] complex. The bimorphous crystal
structure of H2L ligand shows that one of the aromatic salicyl rings is twisted
by 46°, distorting the Cs symmetrical structure (Table 1). The twist on the
other aromatic salicyl ring is very little (2°) due to the intra-ligand hydrogen
bonding to the pyridine N atom. Upon addition of the Cu(II) ion, the salicyl
rings are restricted to stay aligned to the pyridine ring. The highly twisted ring
on H2L is rotated by ~50° to become 4° in [CuL]. The untwisted ring is not
affected by Cu2+. The calculations in PCM water solvent are in excellent
agreement in estimating structural change upon complexation of the H2L
ligand. According to the calculations, the H2L ligand holds the twisted ring by
42° and [CuL] structure adopts a Cs symmetrical structure with both salicyl
rings 0°. We believe the larger size of the planar group in the [CuL] structure
can activate it for the intercalation since the size of the planar groups is critical
for intercalation.
Table 1. Selected bond distances and dihedral angles for the H2L ligand and
[CuL] complex. Only the heavy atoms around the Cu(II) are given. The
dihedral angles belong to the ones between pyridine ring and salicyl rings (See
Figure 1). All the calculations are in water PCM environment
Cu-O1 Cu-O2 Cu-N2 Cu-N3
Molecule
φ (°) φ' (°)
(Å)
(Å)
(Å)
(Å)
Experimental H2L
Calculated H2L
-46
-42
4
-2
-3
-2
0
Experimental [CuL]
Calculated [CuL]
1.89
1.92
1.88
1.91
1.93
1.99
1.94
2.00
0
11

In the [CuL] complex, experimental bond lengths of Cu—O and Cu—N
are 1.89-1.88 Å and 1.93-1.94 Å, respectively (Table 1). The calculations
predict similar bond distances. These strong bonds are typical for the metal
ions with empty 4s orbitals and partially occupied 3d orbitals. The effect of
size and electronic configuration of metal ions such as Mn+(3d54s1) [68],
Fe+(3d64s1 or 3d74s0) [69], Co+ (3d84s0) [70, 71], Ni+(3d94s0) [70, 72],
Cu+(3d104s0) [73], and Ag+(4d105s0) [73] on the bond strength and
coordination is well documented in literature. Since the size of 4s orbital is
larger than that of 3d orbitals, the repulsion between the metal and ligand is
smaller in the systems in which 4s orbitals are empty, so the ligand can come
closer to the metal ion. In addition, the 3d orbitals are directional while 4s
orbitals are spherical. This means the electrons on 3d orbitals can be localized
in order to minimize the repulsion. Thus, the ligand can come closer to the
metal ion, resulting with stronger M-L bond [69-72].
Demir et al. reported the experimental UV-VIS spectra of the H2L ligand
and [CuL] complex in methanol solutions [51]. Our experimental findings for
the [CuL] in the Tris buffer (with 5% DMF) are very similar. The Figure 2
shows the absorption spectrum of the [CuL] in the buffered aqueous solution.
There is an intense absorption band at 262 nm (ε=15930 L.mol-1.cm-1), a broad
and intense band at 322 nm (ε=21530 L.mol-1.cm-1) with two shoulders at 293
nm and 342 nm, and another broad band starting at 364 nm and peaking at 408
nm (ε=21600 L.mol-1.cm-1). The overall pattern of the spectrum is very similar
to those of Fe(III), Zn(II), Ni(II) and Cu(II) –salen and salphen complexes, all
of which have been reported to adopt a square planar coordination around the
metal center [3, 67, 74]. For such systems, it was reported that the low lying
12

band is due to π−π* transition while the higher wavelength bands are due to
metal and solvent perturbed intra-ligand electronic transition [3, 74, 75].
Figure 2. The electronic absorption spectra of [CuL] (a) experimental (top)
and theoretical UV-Vis spectra of [CuL] (b) in vacuo (middle), and (c) in
PCM water environment (bottom). Vertical lines and numbering show the
transition from the ground state to the regarding electronic excited state.
.
13

The ground and excited state calculations (Figure S2) show that [CuL]
complex has an additional band in the high wavelength region suggesting
ligand-to-metal charge transfer (LMCT) at 375 nm besides the intra-ligand
electronic transitions at 350 nm, which is also observed for the H2L ligand at
331 nm. The Cu2+ ion causes the planarization of the system increasing the
electron fluency and reducing the band gap between the charge transfer
orbitals, thus, resulting with red shift on the transition.
In order to understand the solvent effect, we compared calculations in
solution environment. Figure 2 shows comparison of the experimental UV-
VIS spectrum of [CuL] to the calculated ones in gas phase and water. As it can
be seen, the bands of calculated spectra are consistently blue-shifted from the
experiment, and the shift is smaller in the low wavelength region.
Surprisingly, this blue-shift gets even larger upon inclusion of the water
solvent effect. However, in terms of relative transition intensities, the solvent
effect makes the spectra better match to the experiment.
To investigate the insights for the transitions with high oscillator strengths,
we also performed Natural Transition Orbital (NTO) analysis [76]. This
method is a compact orbital representation for electronic transition density
matrix and gives a maximal correspondence between the excited and empty
orbital pairs (particle/hole NTO pairs) [76]. Figure 3 shows the most
dominant NTO pairs and their contributions to related transitions. It shows that
the bands at 375 nm and 350 nm are mostly due to intra-ligand transitions, and
metal involvement in the transitions is very small. However, it carries some
charge transfer and planarization plays an important role on that. The
14

transitions that are just below 300 nm are also involved some charge transfer
character but these are barely perturbed by the metal ion (Figure S2). On the
other hand, the lowest wavelength transition is strongly affected by the metal.
For the allowed transitions, vertical excitation energies in the PCM water
solvent, the molecular orbital contributions and corresponding NTOs are listed
in Table 2. Highly mixed molecular orbitals, especially at higher energy
region, is much more explained by the NTO analysis. However, we should
note that the Cu2+ is open-shell (3d94s0) and the percent contributions are not
normalized. Thus only alpha molecular orbitals are listed here (for the beta
orbitals, see Table S2).
The transitions due to DNA sandwiched π-stacking can be observed at
200-300 nm region, depending on the buffer environment, which overlaps
with the low lying transitions of the [CuL] complex. However, the DNA
shows almost no absorption above 300 nm.
.
15

Hole
Particle
Hole
Particle
Excited State 8
73%
Excited State 15
56%
26%
44%
Excited State 10
58%
Excited State 23
64%
40%
36%
Excited State 12
77%
Excited State 25
69%
33%
34%
Figure 3. Most dominant NTO pairs belonging the transitions with oscillator strength, f>0.1,
for [CuL] in PCM water solvent. The contributions of alpha spin NTOs for each transition are
also shown. Corresponding oscillator strengths and amplitudes of SCF orbitals are presented
in Table 2 (For beta spin NTOs, see Table S2).

Table 2. Transition energies, intensities and orbitals from vertical excitations.
Calculations are in water (PCM) environment. For the orbital descriptions
only alpha orbitals are given without normalization. For the corresponding
beta contributions, see Table S2
Excited
State
SCF Orbitals (SCFO)
Description
∆E
(eV)
f
NTO Description
- 0.33997(ψΗ−1ꢀψ'L+1
+ 0.59649(ψΗꢀψ'L)
0.53284(ψΗ−1ꢀψ'L)
)
0.73(φΗꢀφ'L)
8
3.31 0.2049
3.54 0.5462
+ 0.26(φΗ−1ꢀφ'L+1
0.58(φΗꢀφ'L)
)
)
10
- 0.43925(ψΗꢀψ'L+1
)
+ 0.40(φΗ−1ꢀφ'L+1
0.11533(ψΗ−5ꢀψ'L+1
)
+ 0.27160(ψΗ−2ꢀψ'L+1
+ 0.40256(ψΗ−1ꢀψ'L)
)
)
0.77(φΗꢀφ'L)
12
15
23
4.01 0.1124
4.16 0.3349
4.54 0.3031
+ 0.33(φΗ−1ꢀφ'L+1
)
)
)
+ 0.53439(ψΗꢀψ'L+1
0.34583(ψΗ−2ꢀψ'L)
)
0.56(φΗꢀφ'L)
+ 0.51581(ψΗ−1ꢀψ'L+1
+ 0.32441(ψΗꢀψ'L)
+ 0.44(φΗ−1ꢀφ'L+1
- 0.26706(ψΗ−3ꢀψ'L+1
+ 0.51958(ψΗ−2ꢀψ'L)
- 0.30336(ψΗ−1ꢀψ'L+1
- 0.22812(ψΗꢀψ'L)
)
0.64(φΗꢀφ'L)
)
+ 0.36(φΗ−1ꢀφ'L+1
- 0.33435(ψΗ−3ꢀψ'L)
+ 0.55642(ψΗ−2ꢀψ'L+1
- 0.21928(ψΗ−1ꢀψ'L)
)
0.69(φΗꢀφ'L)
25
4.70 0.2256
+ 0.34(φΗ−1ꢀφ'L+1
)
- 0.19027(ψΗꢀψ'L+1
)
+ 0.11084(ψΗꢀψ'L+3
)
H:HOMO; L:LUMO; ψ: Occupied SCFO; ψ': Virtual SCFO;
φ: Occupied NTO; φ':Virtual NTO
17

3.2 DNA Intercalation
The [CuL] complex synthesized, characterized and analyzed in the first
part is a good candidate for being a DNA intercalator. Therefore, we
investigated its intercalative behavior both experimentally and theoretically.
3.2.1 DNA Titration by [CuL]
Previous studies have shown that when the ligand-DNA interaction is
intercalative, the absorption bands of the ligand shift to higher wavelength
(red-shift) and the relative intensities drop drastically (hypochromism) [45,
75]. The absorption spectrum of the [CuL] is perturbed by the addition of
ctDNA at increasing concentrations (Figure 4). The spectra show that the
absorption band at 323 nm is red shifted by 1 nm and shows 60% of
hypochromism. Similarly, the band at 408 nm is red shifted by 4 nm and
shows a 50% of drop in the intensity. This clearly shows the intercalative
interaction between DNA and [CuL] complex. However, the band at 262 nm is
red shifted and the intensity drops upon addition of the DNA up to reaching
the equivalent DNA concentration ([DNA]/[CuL]=1). After that it behaves
differently and 3 nm blue-shifted band at 259 nm raises with further additions
of DNA. This behavior is explainable and actually confirms the intercalative
interaction. The idea behind that is as follows. In the 250-260 nm region both
the free DNA and free [CuL] absorbs in the πꢀπ* transition. However, when
the intercalation occurs, the π−π stacking interaction between the aromatic
rings of the [CuL] and DNA base pairs, which results with less πꢀπ*
transitions in the [CuL]-DNA complex. Initially, the absorption is due to
completely free [CuL] since no DNA is available. With the addition of DNA
18

up to the stoichiometric ratio, the [CuL] concentration is diminished by the
formation of the intercalated complex which has very little absorption due to
the stacking interaction. Thus, for these concentrations the absorption
decreased. On the other hand, when there is excess amount of DNA, the
absorption is mostly due to DNA base pairs and it increases depending on the
free DNA in the media. This behavior is also observed for Cu(II) complexes of
similar salphen type ligands [74].
19

Figure 4. a) Absorption spectra (top) for the titration of [CuL] by
increasing concentrations of ctDNA in the Tris/HCl buffer pH=7.2 and I=0.1
M (NaCl). The vertical arrows show the trend in the absorption intensity with
the increasing DNA concentration. b) The nonlinear-fit (bottom) using the eq (
4 ) of [DNA] at 408 nm.
20

The typical red shift value for strong interaction is ~15-25 nm [45, 74].
Our experimental red-shifts (1-4 nm) are suggesting somewhat weak
interaction.
From the absorption titrations, the interaction strength can be quantified by
finding the binding constant of the [CuL] to DNA. The quadratic formula to
find the binding constant derived by Bard and co-workers [77] can be utilized
for the titration. Considering the binding of the ligand, L, to a DNA binding
site of, D, which is composed of s base pairs of total number of nucleotides, N,
forming an intercalated species, D-L, a non-cooperative interaction, in which
there is only one type of binding site and the ligand does not bind sequence
selectively, can be represented as [77]:
ꢅ + ꢌ = ꢌ − ꢅ
( 1 )
The binding constant is given with;
ꢏꢎ
ꢍꢎ =
ꢏ ꢏꢑ
ꢐ
( 2 )
Where, ꢏꢎ, ꢏ , and ꢏꢑ are equilibrium concentrations of bound ligand, free
ꢐ
ligand, and free binding site, respectively.
ꢏꢒ = ꢏꢎ + ꢏ
The total ligand concentration, ꢏꢒ is:
ꢐ
[ꢌꢓꢔꢕꢖꢗꢖ
2. ꢘ
]
The total DNA concentration in monomer unit:
= ꢏꢎ + ꢏꢑ
Where, s is the number of the base pairs that are interacting with ligand.
Including these terms in the equation (2),
21

ꢍꢎ[ꢌꢓꢔꢕꢖꢗꢖ
2ꢘ
]
ꢍꢎꢏꢒ[ꢌꢓꢔꢕꢖꢗꢖ
2ꢘ
]
ꢍꢎꢏꢎꢁ − (1 + ꢍꢎꢏꢒ +
)ꢏꢎ +
= 0
( 3 )
The eq. ( 3 ) can be readily solved for ꢏꢎ using the quadratic formula.
Assuming the apparent Absorption (ꢔꢙ) to be sum of free (ꢔꢐ) and bound
absorptions (ꢔꢎ),
ꢔꢙ = ꢔꢐ + ꢔꢎ
ꢀꢙꢏꢒ = ꢀꢐꢏꢐ + ꢀꢎꢏꢎ = ꢀꢐ(ꢏꢒ−ꢏꢎ) + ꢀꢎꢏꢎ
Thus ꢏꢎ can be represented as [78];
ꢀꢙ − ꢀꢐ
ꢏꢎ = ꢏ
ꢒ ꢀꢎ − ꢀꢐ
Thus, solution to the equation becomes;
ꢁ
[
]
2ꢍꢎ ꢏꢒ ꢌꢓꢔꢕꢖꢗꢖ
ꢁ
ꢛ
ꢚ − ꢚ −
ꢀꢙ − ꢀꢐ
ꢀꢎ − ꢀꢐ
( 4 )
ꢘ
=
2ꢍꢎꢏꢒ
ꢍꢎ[ꢌꢓꢔꢕꢖꢗꢖ
2ꢘ
]
Where,
ꢚ = 1 + ꢍꢎꢏꢒ +
In the eq. ( 4 ) [77], the ꢀꢐ, ꢀꢎ, and ꢀꢙ are the molar extinction coefficients of
a) free [CuL] ligand in the absence of DNA b) bound [CuL]-DNA in
stoichiometric ratio c) apparent mixture of [CuL], DNA and [CuL]/DNA when
both [CuL]-DNA complex and either one of the [CuL] or DNA is freely,
respectively, available. The ꢀꢐis a constant value and can be determined from
the calibration curve of the free [CuL] complex, using the Beer-Lambert law.
The ꢀꢎ constant value can be determined from the plateau of the titration curve
where no more change in the absorption is observed. The ꢀꢙis a varying value
and is determined from the ratio of the measured absorbance to the constant
22

ꢜ ꢉꢜ
ꢝ
ꢞ
[CuL] initial concentration value. The experimental titration curve of
ꢜ ꢉꢜ
ꢟ
ꢞ
vs. ꢄ[ꢌꢓꢔꢕꢖꢗꢖ] is shown in Figure 4. The Kb and s coefficients can be
obtained by nonlinear least-squares fitting of the experimental data using the
eq. ( 3 ).
The band at 408 nm in Figure 4 includes no contribution from the DNA
absorption. For this reason, the experimental binding constant Kb and binding
site size s were determined from this band as (1.53 0.48) x 106 M-1 and (0.82
0.04). This Kb value confirms that the interaction is strong. The proximity of
the s value to 1 confirms that the binding is intercalative in which 1 ligand per
base pair interacts. This binding constant is similar to those of EB and MB
(For EB-ctDNA, Kb=1.7 x 105 M-1 [79] (1.4 x 106 M-1 on ref. [80]) and for
MB-ctDNA, Kb=2.32 x 105 M-1 [81] (1.13 x 106 M-1 on ref. [82]). Both the EB
and MB have been studied numerously as their excellent fluorometric
behavior upon binding to the DNA. Thus, these two compounds are widely
used as competitive probes.
3.2.2 MB and EB Competitive Displacement Assays
We also carried out further experiments in order to ensure that the [CuL]
interacts with the ctDNA more strongly than the EB and MB. The idea is that
the absorption spectra of EB and MB are red shifted by DNA intercalation,
and if the ligand exchange reactions occur between the ligands of [CuL] and
of EB and MB, the free EB and MB concentrations will increase recovering
the shift in the spectra. The Figure 5 (bottom) shows change in the absorption
spectra of the intercalated EB-DNA (20 µM, 1:1 solution) by addition of
[CuL]. As can be seen from the spectra, the free EB (solid blue line) maximum
23

absorption band at 480 nm shifts to 493 nm and the intensity is reduced by
35% due to the DNA intercalation (solid black line). However, with the
addition of increasing concentrations of the [CuL], the intensity (dashed red
lines) re-increases and it blue-shifts, almost fully recovering to the free EB
absorption spectrum. This confirms that the [CuL] strongly binds to the DNA
in the same region by kicking the EB. The Figure 5 (top) shows the same
procedure applied to the MB. The free MB ligand (solid blue line) has an
absorption maximum at 662 nm, which shifts to 668 nm when intercalated to
the ctDNA (solid black line). When MB-DNA intercalated complex is treated
with the increasing concentrations of [CuL] ligand, the intensity and red-shift
are recovered by ~35% (dashed red lines).
24

Figure 5. Absorption spectra for competitive displacement assays of [CuL]
with (a) MB (top) and (b) EB (bottom). The solid black lines represent
absorption of MB or EB fully intercalated in ctDNA with the absent of [CuL].
By addition of the [CuL] (dashed lines, increasing thickness according to the
concentration), the absorption approaches the free MB/EB spectra.
25

3.2.3 Viscosity
Lerman [83] showed that viscosity of rod-like calf thymus (double
stranded) DNA increases with the increase of the intercalating ligand amounts.
The increase in viscosity happens due to an increase of the DNA duplex length
(L) upon intercalation [84, 85]. The viscosity of DNA can be expressed as
flow times of the buffer (ꢠꢃ) and the DNA (ꢠ) solutions.
ꢡ = (ꢠ − ꢠꢃ)/ꢠꢃ
Since the viscosity is proportional to L3, relative viscosity values of the
DNA the presence (ꢡ) and in the absence (ꢡ°) of ligand is given as (ꢡ/ꢡ°)ꢊ/ꢢ
[ꢥꢦꢧꢙꢗꢑ]
versus the binding ratio[86], ꢣ
=
.
[ꢨꢩꢪ]
ꢒꢖꢒꢙꢤ
The Figure 6 shows the relative viscosity increase by addition of [CuL]
ligand to DNA containing buffer, which confirms the intercalation of the DNA
by the [CuL].
Figure 6. Plot of relative viscosity (η/η°)1/3 of ctDNA versus rtotal at 25 °C in
the Tris buffer with pH=7.2 and I=0.1 M (NaCl). Each measurement was
made with average flow rates of 3 repetitive flow times.
26

3.3 Comparison of calculations to experiments
The [CuL] complex can interact with the DNA in an intercalative way or
minor/major groove electrostatic interaction. As the experimental studies
suggested, here we report only intercalative interaction on d(GpC)2 pocket.
Since the effects of sugar and charged phosphate groups are important [45, 47]
we have optimized the structures including these moieties. The Figure 7 shows
the optimized structures of both intercalated [CuL]-DNA complex and free
d(GpC)2 in water (PCM).The changes on the geometry of the DNA upon
interacting with the [CuL] are summarized in Table 3.
Figure 7. Optimized molecular structure of [CuL] intercalated in d(GpC)2.
Calculations were carried out using M06-2X level with SDD-ECPs for the
Cu2+ and 6-31G(d) for the rest of the atoms. Two different starting geometries,
inserting at the intercalation pocket of the free B-DNA (pdb id: 1BNA) and
replacing the [CuL] with a known intercalated ligand (pdb id:454D), were
converged to the same structure.
27

Table 3. Some selected bond lengths and angles of X-ray crystallography and
geometry optimization calculations. Labels of the first column are referring
the centroids and planes (described in the text).
Experimental
Calculations
Base
Base
Intercalated
[CuL]-DNA
(454D)
5.6
Base pairs
only (1BNA)
pairs only pairs only
(1BNA)
(454D)
C1-G1
C2-G2
5.4
5.6
5.6
5.5
5.7
5.5
5.6
G1-C2
3.5
3.4
3.8
6.6
C1-G2
4.3
4.4
4.1
6.2
<PC1-PG2
<PG1-PC2
<PC1-PG1
<PC2-PG2
26.3
4.7
22.3
12.9
26.3
4.8
22.4
12.6
6.1
3.7
12.2
7.8
20.6
3.7
18.3
5.1
We describe the distances and angles by creating a centroid on each six
membered ring of DNA bases and a plane for each six membered ring,
covering all the atoms on the ring. Upon binding, distance of the inter-base
pairs (base pair step) rises. The G1-C2 goes from 3.4 Å (in 454D, same value
is 3.8 Å in 1BNA) to 6.6 Å and C1-G2 distance of 4.4 Å (in 454D, same value
is 4.1 Å in 1BNA) increases to 6.2 Å (Table 3). Such a nearly doubling of the
distances is typical for an intercalation. The intra-base pair hydrogen bonds are
maintained in the intercalated complexes, thus the distances of C1-G1 and C2-
G2 in [CuL]-DNA are almost the same as free DNA, 5.6 0.1 Å. The angles
of the planes, on the other hand, show quite differences upon the intercalation.
The PG2 plane appears to be the most flexible and rotates, which affects
mostly <PC1-PG2, changing from 26.3° (on 454D) to 20.6°, and <PC2-PG2,
from 12.6° (on 454D) to 5.1°. Although, this change can be attributed to
opening of the double strand, we know that the angles don’t have great impact
on the energy of the molecule. Therefore, having great change on the angles
may not mean great change in the stability of the molecule. In addition, the
28

correct plane angles in free DNA molecules are already quite different
between the 454D and 1BNA structures.
Calculated binding energies are given in Table 4 for both gas and water
solution phases. The values are in qualitatively agreement with the
experimental value. The binding energy in the gas phase is larger. This shows
that water solvent destabilizes the DNA-[CuL] complex.
Table 4. The calculated binding energies of [CuL] in the DNA base pairs. The
values are bottom of the well values and do not include zero point energies
and thermal energies.
Gas Phase
Water (PCM) Solvent
Binding
Binding
SCF Energy
Energy
SCF Energy
(Hartree)
Energy
(kJ/mol)
-
(Hartree)
(kJ/mol)
[CuL] Ligand
DNA Base Pair,
d(GpC)2
-1243.5827
-4539.1101
-1243.60039
-4539.34549
-
-
-
[CuL]-DNA
Complex
-5782.7218
-76.2
-5782.953215
-19.3
Figure 8 compares the experimental spectra of isolated [CuL] and [CuL]-
ctDNA complex (top) along with calculated spectra of [CuL] and [CuL]-
d(GpC)2 intercalated systems. The change in the experimental spectrum upon
binding to the ctDNA is totally reproduced by the calculations. The overall
intensity is reduced by intercalation in both experimental and calculated
spectra. All of the three bands in the spectra are affected by intercalation. In
addition, perfectly agreeing with the experiment, the intercalation is calculated
to have a 9 nm of red shift in the lowest energy band, which is observed as 4
nm.
29