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Nickel(II), copper(II) and zinc(II) metallo-

Cite this: Dalton Trans., 2014, 43,
intercalators: structural details of the DNA-binding
6108 by a combined experimental and computational
investigation†
Antonino Lauria,a Riccardo Bonsignore,a Alessio Terenzi,a Angelo Spinello,a
Francesco Giannici,b Alessandro Longo,c,d Anna Maria Almericoa and
Giampaolo Barone*a,e

Received 30th October 2013,
Accepted 20th January 2014
DOI: 10.1039/c3dt53066c
www.rsc.org/dalton
We present a thorough characterization of the interaction of novel nickel(II) (1), copper(II) (2) and zinc(II) (3)
Schiff base complexes with native calf thymus DNA (ct-DNA), in buffered aqueous solution at pH 7.5.
UV-vis absorption, circular dichroism (CD) and viscometry titrations provided clear evidence of the inter-
calative mechanism of the three square-planar metal complexes, allowing us to determine the intrinsic
DNA-binding constants (Kb), equal to 1.3 × 107, 2.9 × 106, and 6.2 × 105 M−1 for 1, 2 and 3, respectively.
Preferential affinity, of one order of magnitude, toward AT compared to GC base pair sequences was
detected by UV-vis absorption titrations of 1 with [ poly(dG-dC)]2 and [ poly(dA-dT)]2. Structural details of
the intercalation site of the three metal complexes within [dodeca(dA-dT)]2 were obtained by molecular
dynamics (MD) simulations followed by density functional theory/molecular mechanics (DFT/MM) calcu-
lations. The calculations revealed that three major intermolecular interactions contribute to the strong
affinity between DNA and the three metal complexes: (1) the electrostatic attraction between the two
positively charged triethylammoniummethyl groups of the metal complexes and the negatively charged
phosphate groups of the DNA backbone; (2) the intercalation of the naphthalene moiety within the four
nitrogen bases of the intercalation site; (3) the metal coordination by exocyclic donor atoms of the bases,
specifically the carbonyl oxygen and amine nitrogen atoms. Remarkably, the Gibbs formation free energy
calculated for the intercalation complexes of 1, 2 and 3 with [dodeca(dA-dT)]2 in the implicit water solu-
tion is in agreement with the experimental Gibbs free energy values obtained from the DNA-binding con-
stants as ΔG° = −RT ln(Kb). In particular, the DNA-binding affinity trend, 1 > 2 > 3, is reproduced. Finally,
the first shell coordination distances calculated for the intercalation complex 3/[dodeca(dA-dT)]2 are in
excellent agreement with the experimental distances extracted from the extended X-ray absorption fine
structure (EXAFS) spectrum of the corresponding 3/ct-DNA solutions. The latter results provided the first
evidence of metal ion coordination by native DNA in aqueous solution.

a
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche,
Viale delle Scienze, Edificio 17, I-90128 Palermo, Italy.
E-mail: giampaolo.barone@unipa.it; Fax: +39 091 596825
b
Introduction
Dipartimento di Fisica e Chimica, Università di Palermo, Viale delle Scienze,
Edificio 17, I-90128 Palermo, Italy The interaction of metal complexes with DNA is associated
c
Istituto per lo Studio su Materiali Nanostrutturati del Consiglio Nazionale delle
with interesting chemical and biological properties of the
Ricerche (ISMN-CNR), Via Ugo La Malfa n. 153, I-90146 Palermo, Italy
d
Dubble CRG at ESRF, Netherlands Organization for Scientific Research (NWO),
resulting supramolecular system. In fact, the cytotoxic activity
c/o ESRF, BP 220, F-38043 Grenoble Cedex, France of metallodrugs has often been correlated to their DNA-
e
Istituto EuroMediterraneo di Scienza e Tecnologia, Via Emerico Amari 123, binding properties.1–3 For this reason, we have been recently
90139 Palermo, Italy involved in the synthesis of novel metal complexes and organic
† Electronic supplementary information (ESI) available: Additional figures
compounds as potential DNA binders.4–10
(Fig. S1–S5) and tables (Tables S1–S3), reporting NMR spectra and DFT energies,
in vacuo and in solution, thermal corrections, Cartesian coordinates. See DOI: The interaction modes of a small molecule with the DNA
10.1039/c3dt53066c macromolecule have been broadly categorized into the following

6108 | Dalton Trans., 2014, 43, 6108–6119 This journal is © The Royal Society of Chemistry 2014
Dalton Transactions
three types:4,11,12 (1) covalent binding DNA-alkylators,13,14 or
with Lewis acid metal ions, such as platinum in many clinically
used anticancer drugs;15–17 (2) major or minor grooves-
binding;18,19 and (3) intercalation.20,21 The latter two categories
are classified as non-covalent interactions. Grooves binding
involves the contributions of electrostatic interaction of polar or
cationic molecules with the negatively charged phosphate
groups, and/or the formation of hydrogen bonds with the
oxygen and/or nitrogen atoms of the DNA bases or of the sugar
fragment, as both donor and acceptor atoms. Intercalation
involves specifically the π–π interaction of the planar aromatic
section of the small molecules with the stacked aromatic planes
of the nitrogen bases.
Mixed modes of interactions have been also advised. For
example, many intercalators, such as anthracyclines, also
interact by electrostatic attraction or by hydrogen bonding with
the atoms exposed in the grooves.22–27 Moreover, it has been
recently shown that transition metal complexes of planar aro-
matic ligands may combine coordination with intercalation in
their DNA-binding.20,28 Such a feature confers them enhanced
anticancer properties.
Of course, fine structural details of the molecule–DNA
binding mode in solution cannot be completely obtained by
techniques such as UV-vis absorption, CD and viscometry.
Nonetheless, the above-mentioned techniques are interesting
because they can be routinely applied to solution samples
under physiological conditions.
Computational chemistry may support the interpretation
of experimental data with atomistic models. In this
context, quantum mechanics/molecular mechanics (QM/MM)
calculations provide reliable structural information on a
local region of biomolecular systems, such as the active site
of metalloproteins.29–31 We have recently shown that
molecular dynamics (MD) simulations, followed by QM/MM
calculations, with the QM part described by density
functional theory (DFT), provided a detailed structure of the
intercalation complexes of dodecanucleotide double-helices
with a copper(II) metallointercalator, and formation energy
data in good agreement with the experimental DNA-binding
constant.32
On the other hand, detailed local structural information
can be experimentally obtained by X-ray crystallography or by
NMR spectroscopy only of drug–DNA supramolecular com-
plexes involving small synthetic DNA oligonucleotides, typi-
cally in the range 2–12-mer oligonucleotides.33–41 Luckily, the
analysis of the X-ray absorption spectrum allows detecting
changes in the coordination structure of metal complexes, in
particular the formation of new covalent bonds, when a metal
complex interacts with biomolecules in aqueous solution.42
For such reasons, in the present paper we have exploited also
this technique. Even though the extended X-ray absorption
fine structure (EXAFS) has been widely used to characterize the
active site of metalloproteins,43–46 to the best of our knowl-
edge, this is the first time this spectroscopic technique is used
to detect the bonds between native DNA and a metal complex
in water solution.
This journal is © The Royal Society of Chemistry 2014
Paper
Experimental section
Materials and methods
Solvents and reagents (reagent grade) were all commercially
available and used without further purification. 1H NMR and
13
C NMR spectra were recorded in DMSO-d6 solution, with TMS
as an internal reference, on a Bruker Avance 200 MHz NMR
spectrometer at 200 and 50 MHz, respectively. IR spectra were
recorded on a Perkin-Elmer Spectrum 100 FT-IR spectrometer.
Lyophilized calf thymus DNA (Fluka, BioChemika) was
resuspended in 1.0 mM tris-hydroxymethyl-aminomethane
(Tris-HCl) at pH = 7.5 and dialyzed as described in the litera-
ture.47 DNA concentration, expressed in monomer units
([DNAphosphate]), was determined by UV spectrophotometry
using 7000 M−1 cm−1 as the molar absorption coefficient at
258 nm.48 Lyophilized synthetic polynucleotides [ poly(dG-
dC)]2 and [poly(dA-dT)]2 (Sigma) were dissolved in 1.0 mM
Tris-HCl, at pH = 7.5, and their molar concentration, in
monomer units, was determined using 8400 M−1 cm−1 and
6650 M−1 cm−1 as the molar absorption coefficient at 254 nm
and 262 nm, respectively.49,50
UV-vis spectra were collected on a Varian UV-vis Cary 1E
double beam spectrophotometer equipped with a Peltier temp-
erature controller. Circular dichroism spectra were recorded on
a Jasco J-715 spectropolarimeter. 1 cm path-length quartz cuv-
ettes were used. Viscosity measurements were performed on
an Ubbelodhe viscometer maintained at 25.0 ± 0.1 °C. The
flow time was measured with a digital stopwatch. UV-visible
absorption titrations were carried out adding increasing
amounts of ct-DNA to a metal-complex solution with constant
concentration. DNA melting experiments were carried out by
recording the absorbance of ct-DNA solutions at 258 nm while
the temperature was elevated gradually from 20 to 95 °C at a
rate of 0.5 °C min−1. CD, melting temperature and viscometry
titrations were carried out adding increasing amounts of metal
complex stock solution to a DNA solution with constant con-
centration. All experiments were carried out in Tris-HCl 1 mM
aqueous buffer at pH = 7.5.
X-Ray absorption spectroscopy (XAS) experiments were
carried out at the BM26A beamline of European Synchrotron
Radiation Facility (ESRF), Grenoble. The spectra were recorded
in fluorescence mode with a 9-element Ge detector on Ni (8333
eV), Cu (8979 eV) and Zn (9659 eV) respectively, on buffered
aqueous solutions of [1] = 503 μM, [2] = 95 μM and [3] = 457 μM,
both with and without DNA at a molar concentration of 4 : 1
DNA–metal complex. The sample solutions were quenched in
liquid nitrogen and the spectra recorded at 80 K using a cryo-
stat, averaging 10 spectra per sample for about 6 hours. XAS
spectra were averaged and normalized with Athena.51 EXAFS
spectra were fitted with Viper.52 FEFF8.4 was used to calculate
theoretical amplitudes and phases for the EXAFS analysis, and
full multiple scattering simulations on the XANES.53
Synthesis and characterization
Complexes 1–3 (Scheme 1) were synthesized by the reaction
of 5-(triethylammoniummethyl)salicylaldehyde chloride, 2,3-
Dalton Trans., 2014, 43, 6108–6119 | 6109
Paper
Scheme 1 Reaction pathway leading to the nickel(II), copper(II) and zinc(II)
complexes, 1–3, of N,N’-bis-5-(triethyl ammonium methyl)-salicy-
lidene-2,3-naphthalendiiminato).
diaminonaphthalene and metal(II) perchlorate in a
2 : 1.1 : 1 molar ratio. Salicylaldehyde in EtOH–H2O basic solu-
tion was added dropwise to the diamine and the metal
perchlorate in ethanol. The 5-(triethylammoniummethyl)sali-
cylaldehyde chloride ligand was prepared from 5-chloromethyl
salicylaldehyde and triethylamine in tetrahydrofuran, as
reported.54 The products were characterized by NMR, IR,
elemental analysis, and UV-vis spectroscopy.
1: 5-(triethylammoniummethyl)salicylaldehyde chloride
(135.5 mg, 0.50 mmol) was dissolved in H2O–EtOH with two
equivalents of NaOH (20.0 mg, 0.50 mmol). This solution was
added to an ethanol solution of 2,3 naphthalendiamine
(39.5 mg, 0.25 mmol) and Ni(ClO4)2·6H2O (92.3 mg,
0.25 mmol). The resulting mixture was stirred for 4 h at room
temperature. The brilliant orange precipitate was washed with
cold ethanol and diethyl ether, and recrystallized from 50%
methanol–water solutions to afford the compound as an
orange solid (yield 106.0 mg, 48%).
1
H NMR δ ( ppm): 1.32–1.38 (m, 18H, CH3); 3.17 (d, 12H, J =
9.6 Hz, CH2); 4.37 (s, 4H, CH2); 6.94 (d, 2H, J = 13.2 Hz, Ar);
7.43 (d, 2H, J = 12.9 Hz, Ar); 7.54–7.58 (m, 2H, Ar); 7.71 (s, 2H,
Ar); 7.88–7.93 (m, 2H, Ar); 8.61 (s, 2H, Ar); 10.16–10.68 (s, 2H,
CH).
13
C NMR δ ( ppm): 7.4 (CH3); 51.4 (CH2); 58.8 (CH2); 99.5
(Ar); 114.1 (Ar); 114.7 (Ar); 121.0 (Ar); 127.3 (Ar); 128.0 (Ar);
132.1 (Ar); 138.7 (Ar); 138.9 (Ar); 139.8 (Ar); 157.5 (Ar); 166.2
(CH).
Elemental analysis for C38Cl2H52N4O12Ni (1, 2ClO4−, 2H2O).
Found: C, 50.05%, H, 5.61%, N 5.90%; calc.: C, 51.49%, H,
5.91%, N, 6.32%.
IR: ν (CvN) 1619 cm−1; ν (O–Ni) 506 cm−1; ν (N–Ni)
475 cm−1. UV [nm (ε, M−1 cm−1)]: 256 (5.93 × 104), 346 (2.55 ×
104), 467 (7.67 × 103).
2: 5-(triethylammoniummethyl)salicylaldehyde chloride
(140.0 mg, 0.52 mmol) was dissolved in H2O–EtOH with two
equivalents of NaOH (20.0 mg, 0.50 mmol). This solution was
added to an ethanol solution of 2,3 naphthalendiamine
(39.8 mg, 0.25 mmol) and Cu(ClO4)2·6H2O (92.6 mg,
0.25 mmol). The resulting mixture was stirred for 4 h at room
temperature. The brownish-green precipitate was washed with
cold ethanol and diethyl ether, and recrystallized from 50%
methanol–water solutions to afford the compound as a green
solid (yield 165.3 mg, 74%).
6110 | Dalton Trans., 2014, 43, 6108–6119
Dalton Transactions
Elemental analysis for C38Cl2H52N4O12Cu (2, 2ClO4−,
2H2O). Found: C, 51.72%, H, 5.46%, N, 6.55%; calc.: C,
51.21%, H, 5.88%, N, 6.29%). IR: ν (CvN) 1625 cm−1; ν (N–Cu)
503 cm−1; ν (O–Cu) 469 cm−1. UV [nm (ε, M−1 cm−1)]: 247 (5.14
× 104), 316 (2.26 × 104), 406 (1.91 × 103).
3: 5-(triethylammoniummethyl)salicylaldehyde chloride
(135.4 mg, 0.50 mmol) was dissolved in H2O–EtOH with
two equivalents of NaOH (20.1 mg, 0.50 mmol). This solution
was added to an ethanol solution of 2,3-naphthalendiamine
(39.2 mg, 0.25 mmol) and Zn(ClO4)2·6H2O (94.0 mg,
0.25 mmol). The resulting mixture was stirred for 4 h at room
temperature. The brilliant yellow precipitate was washed with
cold ethanol and diethyl ether, and recrystallized from 50%
methanol–water solutions to afford the compound as a yellow
solid (yield 169.0 mg, 79%).
1
H NMR δ ( ppm): 1.18–1.44 (m, 18H, CH3); 3.13–3.15
(m, 12H, CH2); 4.32 (s, 4H, CH2); 6.70 (d, 2H, J = 12.6 Hz, Ar);
7.27 (d, 2H, J = 12.9 Hz, Ar); 7.45–7.60 (m, 4H, Ar); 7.89–8.02
(m, 2H, Ar); 8.22–8.34 (m, 2H, Ar); 9.07 (s, 2H, CH).
13
C NMR δ ( ppm): 7.5 (CH3); 51.1 (CH2); 59.5 (CH2); 110.8
(Ar); 114.1 (Ar); 119.5 (Ar); 123.8 (Ar); 126.4 (Ar); 127.7 (Ar);
132.1 (Ar); 137.4 (Ar); 139.2 (Ar); 140.9 (Ar); 163.4 (Ar); 173.2
(CH).
Elemental analysis for C38Cl2H48N4O10Zn (3, 2ClO4−):
Found: C, 53.01%, H, 5.76%, N, 5.90%; calc.: C, 53.25%, H,
5.64%, N, 6.54%. IR: ν (CvN) 1626 cm−1; ν (N–Zn) 552 cm−1;
ν (O–Zn) 468 cm−1. UV [nm (ε, M−1 cm−1)]: 244 (6.27 × 104),
304 (2.80 × 104), 384 (2.81 × 103).
Computational details
Molecular dynamics simulations. An alternating adenine–
thymine sequence of 12 base pairs, [dodeca(dA-dT)]2, was con-
structed in the double-helix B-DNA conformation by the
NUCLEIC routine of the TINKER program package,55
as recently described.32 To create the intercalation pocket in
the starting DNA structure, the torsion angles α–ζ and χ of the
sugar phosphate backbone were opportunely modified.56 The
naphthalene moiety of the zinc(II) complex 3 was intercalated
from the major groove side between the 6th and 7th base pairs
using a home-made routine. MD simulations on the 3/[dodeca-
(dA-dT)]2 complex were performed by the GROMACS 4.5.3
software package57,58 using the Amber99 force field59 with
Parmbsc0 nucleic acid torsions.60,61 The starting geometry and
the partial atomic charges of the metal complex were obtained
by DFT calculations (see below), while other intramolecular
force-field parameters were generated using the ACPYPE
software.62–64
A triclinic box of TIP3P water molecules was added around
the oligonucleotides to a depth of 1.5 nm on each side of the
solutes, for a total of 20 187 atoms, to obtain a solution
density of about 1.02 g ml−1. 20 Na+ counterions were added
to neutralize the negative charges of the phosphate groups,
while other 18 Na+ and Cl− ions were added to set the solution
ionic strength to about 0.15 M (see ESI, Fig. S1†). van der
Waals parameters for zinc (σ = 0.195998 nm, ε = 0.05230 kJ
mol−1), sodium (σ = 0.332840 nm, ε = 0.0115897 kJ mol−1) and
This journal is © The Royal Society of Chemistry 2014
Dalton Transactions
chlorine (σ = 0.440104 nm, ε = 0.418400 kJ mol−1) were taken
from the Amber99 force field implemented in GROMACS.
Explicit solvent simulations were performed in the isother-
mal–isobaric NPT ensemble, at a temperature of 300 K, under
the control of a velocity rescaling thermostat.65,66 The particle
mesh Ewald method67 was used to describe long-range electro-
static interactions. The timestep for integration was 2 fs and
all covalent bonds, including the four bonds between the
metal ion and the tetracoordinate Schiff base ligand, con-
strained with the LINCS algorithm. There were two tempera-
ture coupling groups in these simulations, the first for the
dodecanucleotide and, if present, for the metal complex, and
the second for water and ions. Preliminary MD simulations
showed that the structure of the isolated metal complex is
maintained in solution. Preliminary energy minimizations
were run for 5000 steps with the steepest descent algorithm.
During the equilibration, the oligonucleotide and the metal
complex/oligonucleotide system were harmonically restrained
with a force constant of 1000 kJ mol−1 nm−2, gradually relaxed
into five consecutive steps of 100 ps each, to 500, 200, 100
and 50 kJ mol−1 nm−2. The MD simulations were carried out
for 50 ns.
DFT/MM calculations. The relaxed geometries of the com-
plexes between [dodeca(dA-dT)]2 and the metal complexes 1–3
were optimized by two-layer quantum mechanics/molecular
mechanics (QM/MM) hybrid calculations, as implemented in
the ONIOM method,68,69 with the aim to perform a high-level
calculation on the intercalation pocket and to take into
account the constraining effects of the double-helical structure
at lower levels of theory. The M06-2X70 DFT functional and the
dzvp basis set71 were used in the higher QM layer, to suitably
model the hydrogen bonding and π–π stacking interactions
between the sixth and seventh Watson–Crick base pairs. As
recently described,32 the Amber99 force field was used in the
lower MM layer of the DFT/MM calculations. The highest
layer of the model includes the sixth and seventh base
pairs and the cationic complexes 1–3, with charge set to +2
and spin multiplicity 1 or 2, the latter in the presence of the
copper ion. Default atomic partial charges were used for the
dodecanucleotide atoms, implicitly included in the force field
parameters.
Vibration frequency calculations, within the harmonic
approximation, were performed on the optimized geometries
by the same DFT/MM method. Solvent effects were evaluated
by performing M06-2X/dzvp single point calculations on the
high layer model extracted by the DFT/MM optimized geome-
try, with the implicit water solvent reproduced by the polariz-
able continuum model (PCM),72,73 using default settings for
PCM cavities. Standard enthalpy and Gibbs free energy values,
at 298.15 K, of each energy minimum structure, both in vacuo
and in solution, were calculated by adding the thermal correc-
tion obtained by vibration frequency analysis of the DFT/MM
systems to the DFT energy calculated for the high layers both
in vacuo and in water solution (see ESI, Table S1†). The PCM
energy data contain also non-electrostatic effects. Cartesian
coordinates of the optimized structures are reported in the ESI
This journal is © The Royal Society of Chemistry 2014
Paper
(Tables S2–S4†). All calculations were performed using the
Gaussian 09 program package.74
Results and discussion
Synthesis and characterization
1–3 were synthesized as reported in Scheme 1 and character-
ized by NMR, IR and elemental analysis.
While the NMR spectra of the paramagnetic copper
complex 2 were broadened by the magnetic interaction with
the copper ion, the square planar N2O2 coordination around
the metal atom is confirmed by the NMR spectra obtained for
1 on the basis of the diamagnetic nature of the compound,
typical of planar nickel compounds with a d8 electronic con-
figuration. According to the literature, the diamagnetism of
NiII-Salen complexes is also maintained in solutions of coordi-
nating and non-coordinating solvents.75 It should be noted
that elemental analysis suggests that the nickel(II) and
copper(II) complexes, 1 and 2, present two crystallization water
molecules.
Experiments in aqueous solution
The metal complexes under investigation share an intense
absorption band at about 250 nm. A characteristic absorption
band, related to metal perturbed infra-ligand electronic tran-
sition, is noticeable in 1 (346 nm), 2 (316 nm) and 3 (304 nm).
Finally, a weak transition band can be observed at 467 nm for
1, which is remarkably stronger at 406 nm for 2 and at 384 nm
for 3 (black lines in Fig. 1a–c). Such spectra are significantly
modified by the addition of increasing amounts of calf thymus
DNA. In detail, the absorption bands of 1 at 346 nm, of 2 at
316 nm and of 3 at 304 nm are red shifted by 5, 8 and 11 nm
and show hypochromism of about 42%, 35% and 26%,
respectively (Fig. 1a–c). These results, attributable to stacking
interaction between the aromatic naphthalene rings of the
complexes and the base pairs of DNA, collectively suggest that
the three metal complex cations act as DNA intercalators.4,76,77
To determine the intrinsic binding constant, Kb, and the
stoichiometry, s, of the metal complex–DNA systems, the quan-
tity (εa − εf )/(εb − εf ) at 346 nm for 1, at 316 nm for 2 and at
304 nm for 3, has been plotted as a function of the molar con-
centration of DNA (inset in Fig. 1a–c), and analyzed by eqn
(1a) and (1b):78
1=2
2K b 2 Ct DNAphosphate


b b2
εa εf s
¼ ð1aÞ
εb εf 2K b Ct
K b DNAphosphate
 
b ¼ 1 þ K b Ct þ ð1bÞ
2s
where Ct is the total concentration of the metal complex, εf,
determined by a calibration curve of the isolated metal com-
plexes in aqueous solution. εb was determined from the
plateau of the plot, where further addition of DNA did not
cause any changes in the absorption spectrum. Finally, εa was
Dalton Trans., 2014, 43, 6108–6119 | 6111
Paper
Fig. 1 Absorption spectra of 1 (a), 2 (b) and 3 (c) in the presence of
increasing amounts of ct-DNA in Tris-HCl 1 mM buffer. (a) [1] = 8 μM,
[DNAphosphate] = 0–100 μM. (b) [2] = 13 μM, [DNAphosphate] = 0–60 μM. (c)
[3] = 35 μM, [DNAphosphate] = 0–193 μM. The arrows indicate the trend of
the spectral change upon DNA addition.
determined as the ratio between the measured absorbance
and the analytical molar concentration of 1–3.
The values of Kb and s are reported in Table 1. These
results confirm that each metal complex tightly binds DNA
and that the binding strength decreases in the order 1 > 2 > 3,
previously observed for similar Salphen-like complexes of the
same three metals, presenting the same triethylammonium-
methyl groups.54,79 Finally, the complexes show an interaction
stoichiometry that also increases going from about 0.7 with 1
to 1 with 3.
6112 | Dalton Trans., 2014, 43, 6108–6119
Dalton Transactions
Table 1 DNA-binding constant (Kb) and stoichiometry (s) of the three
metal complexes
1 2 3
Kb (1.03 ± 0.05) × 107 (2.9 ± 0.2) × 106 (6.2 ± 0.3) × 105
s 0.74 ± 0.06 0.87 ± 0.07 1.01 ± 0.07
Fig. 2 Absorption spectra of 1 in the presence of increasing amounts of
[ poly(dA-dT)]2 (a) and of [ poly(dG-dC)]2 (b) in Tris-HCl 1 mM buffer. [1] =
9 μM, [DNAphosphate] = 0–80 μM. The arrows indicate the trend of the
spectral change upon polynucleotide addition.
Analogous spectrophotometric titrations of the nickel(II)
complex 1 were carried out with synthetic polynucleotides of
alternating AT and GC sequences, as shown in Fig. 2, with the
aim of determining its preferential affinity toward specific
base pair sequences.
The values of Kb and s obtained, reported in Table 2, show
that the binding affinity toward AT sequences is the same as
that toward ct-DNA, within the associated error and is one
order of magnitude stronger than that toward GC sequences.
These results suggest that the strong affinity of the title metal
complexes toward ct-DNA is essentially attributable to their
binding with AT sequences. Interestingly, the base-pair compo-
sition of native ct-DNA is roughly 60% AT and 40% GC.80
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Dalton Transactions
Table 2 Binding constant (Kb) and stoichiometry (s) of compound 1
with [ poly(dA-dT)]2 and [ poly(dG-dC)]2
[Poly(dA-dT)]2 [Poly(dG-dC)]2
Kb (1.3 ± 0.3) × 107 (2.0 ± 0.2) × 106
s 0.70 ± 0.03 0.73 ± 0.01
Fig. 3 Circular dichroism spectra of ct-DNA (black) in the presence
of increasing amounts of 1 (a), 2 (b) and 3 (c) in 1.0 mM Tris-HCl. (a)
[DNAphosphate] = 50 μM, [1] = 0–30 μM. (b) [DNAphosphate] = 50 μM, [2] =
0–26 μM. (c) [DNAphosphate] = 50 μM [3] = 0–20 μM. The arrows indicate
the trend of the spectral change upon DNA addition.
Circular dichroism (CD) is a useful technique to monitor
changes in the DNA morphology due to drug–DNA inter-
actions, since CD signals are sensitive to small variations in
the chiral conformation of DNA.81–83 For this reason, CD
spectra of ct-DNA 50 µM, in 1.0 mM Tris-HCl (Fig. 3), were
recorded in the presence of increasing amounts of 1, 2, and 3,
This journal is © The Royal Society of Chemistry 2014
Paper
up to [ML2+]/[DNAphosphate] molar ratios of approximately 0.6,
0.6 and 0.4 respectively. The CD of native DNA is drastically
modified by the addition of increasing amounts of the three
metal complexes. In particular, a decrease and a blue shift of
the positive CD band of DNA is observed in both titrations of 1
(Fig. 3a) and 2 (Fig. 3b).
The positive CD band of DNA becomes negative at metal
complex concentrations of 1 and 2 higher than 5 μM and
10 μM, respectively (Fig. 3a–b). Induced CD bands are also
evident for both complexes, at 356 and 479 nm for 1 (Fig. 3a)
and at 325 and 426 nm for 2 (Fig. 3b). Moreover, the DNA
dichroic band at 275 nm (black line in Fig. 3c) is mono-
tonously increased and split into two bands at 266 and 287 nm,
by the addition of increasing amounts of 3. Induced CD bands
appear at approximately 405, 447 and 513 nm. These results
indicate that deep conformational changes occur in the
double-helical structure of DNA, due to the interaction with
the metal complexes. The appearance of induced CD bands
shows that each of the three metal complexes supplies a
further chromophore tightly appended to the chiral backbone
of the DNA double helix.84 Interestingly, the CD spectrum
recorded in the presence of 1 and 2 at concentrations higher
than 15 μM is similar to that observed for the condensed ψ-
DNA forms.85 This would indicate that the nickel(II) and
copper(II) complexes at higher concentrations induce the for-
mation of supramolecular DNA aggregates. On the other hand,
the zinc complex 3 does not induce such supramolecular DNA
organization, possibly following its relatively lower DNA-
binding affinity. Similar results were obtained with analogue
intercalator Salphen-like nickel(II), copper(II) and zinc(II)
complexes.54,77,79
Thermal denaturation profiles of calf thymus DNA solu-
tions, in the presence of increasing amounts of 1–3, are shown
in Fig. 4 and were obtained by plotting the absorbance
at 258 nm as a function of temperature. As a matter of fact,
with the increasing of temperature the double stranded DNA
gradually dissociates into single strands: the DNA melting
Fig. 4 Examples of thermal denaturation profiles of ct-DNA 50 μM in
the presence of the three complexes 1 (diamonds), 2 (triangles) and 3
(circles), in 1.0 mM Tris-HCl, at the metal complex/DNAphosphate molar
ratio, R1, 0.10. The thermal denaturation profile of ct-DNA is represented
by squares.
Dalton Trans., 2014, 43, 6108–6119 | 6113
Paper
Table 3 Increase of the DNA melting temperature (ΔTm) in the pres-
ence of the three metal complexes at the indicated metal complex/
DNAphosphate molar ratio (R1)
R1 1 2 3 confirm that the three metal complexes are DNA-intercalators.
0.05 10 °C 9 °C 6 °C
0.10 19 °C 14 °C 13 °C
temperature (Tm) is defined as the temperature where half of
the total base pairs is unpaired,86 and corresponds to the
inflection point in the sigmoidal plots of Fig. 4. The DNA
melting temperature is therefore strictly related to the stability
of the helix and any interaction of chemicals with DNA alters
its Tm, stabilizing or destabilizing the final complex. Intercala-
tion of any compound between DNA base pairs results in a
quite strong stabilization of the helix that produces an increase
of melting temperature of DNA.87 Moreover, the presence of
positive charges on the DNA-intercalator, precisely on the two
triethylammoniummethyl groups, besides providing a remark-
able water solubility to the three complexes, further improves
the interaction by means of attractive interactions with the
negatively charged sugar-phosphate backbone of DNA. In
detail, the melting temperature of ct-DNA 50 μM in Tris-HCl
1 mM (63.5 ± 1 °C, black line in Fig. 4) increases to 10, 9 and
6 °C, at the metal complex/DNAphosphate molar ratio, R1 = 0.05,
and of 19, 14 and 13 °C at R1 equal to 0.10, for 1, 2 and 3,
respectively (Table 3).
These results are indicative of a strong metal complex–DNA
interaction, which leads to a stabilization of the native DNA
conformation. They also confirm the order of DNA-binding
strength obtained for the three metal complexes by spectro-
photometric titrations: 1 > 2 > 3.
The relative viscosity of a solution of DNA 5 × 10−5 M line-
arly increases with the addition of 1, 2 and 3, at metal
complex/DNAphosphate molar ratios equal to 0, 0.05, 0.10, 0.15
and 0.20, where η0 is the viscosity of the ct-DNA solution
(Fig. 5). It is known that an intercalative interaction causes an
Fig. 5 Relative viscosity of ct-DNA solutions 50 μM in the presence of
the three metal complexes 1 (green diamonds), 2 (blue triangles) and 3
(red circles), in 1.0 mM Tris-HCl, at the indicated metal complex/
DNAphosphate molar ratio, R1.
6114 | Dalton Trans., 2014, 43, 6108–6119
Dalton Transactions
increase in viscosity of DNA since it increases separation of
base pairs at intercalation sites, hence an increase in overall
double-helical axial length occurs.88 Therefore these results
Moreover, the slope of the three linear trends decreases in the
order 1 > 2 > 3. More in detail, the three correlation coeffi-
cients of the linear fit of the data are higher than 0.99 and the
slopes of the three linear trends are 0.074, 0.042 and 0.023, in
the presence of 1, 2 and 3, respectively. These results indicate
that the relative viscosity raise in the presence of the nickel(II)
and copper(II) complexes, 1 and 2, is more than three times
and roughly two times, respectively, than that observed in the
presence of the zinc(II) complex 3. In conclusion, the interca-
lating affinity of the three complexes perfectly follows the same
decreasing binding affinity order found with both spectro-
photometric and thermal denaturation experiments.
MD simulations and DFT/MM calculations
MD simulations have been performed on the intercalation
complex 3/[dodeca(dA-dT)]2. The root mean square deviation
(RMSD) along the simulation for all non-hydrogen atoms is
shown in the ESI, Fig. S1.† The metal complex remains inside
the binding pocket for the whole simulation time.
The equilibrium geometry, after about 50 ns, has been used
as a starting point for further geometry optimizations, by
hybrid two-layer QM/MM calculations, using DFT as the QM
method and the Amber99 force field as the MM method, as
recently reported,32 of the intercalation complexes of the three
metal complexes 1–3 with [dodeca(dA-dT)]2 (Fig. 6).
The Cartesian coordinates of the three intercalation com-
plexes are reported in the ESI.† The higher layer of the DFT/
MM structures shown in Fig. 6 involves the four nitrogen bases
of the intercalation pocket and the metal complex. The
selected DFT functional, M06-2X, allows to reliably describe
weak interactions in implicit water solution, notably H-bond
and π–π stacking89 that are fundamental in the binding
between the metal complex and the DNA biomolecule.
Significant structural details can be obtained by the ana-
lysis of the optimized structures reported in Fig. 6, which
provide atomic level models explaining the strong DNA-
binding experimentally detected. In particular, the following
three are the complementary contributions ruling the DNA–
metal complex interaction: (1) the electrostatic attraction
between the two positively charged residues of the metal com-
plexes, the triethylammoniummethyl groups and the two nega-
tively charged phosphate groups; (2) the intercalation of the
naphthalene moiety between the stacked and H-bonded nitro-
gen bases of the intercalation pocket; (3) the metal coordi-
nation by exocyclic donor atoms of the nitrogen bases, as
summarized in Table 4.
Concerning the first contribution, electrostatic stabilizing
attraction, it is interesting to see that the triethylammonium-
methyl groups are at a short distance from the nearest-
neighbor phosphate groups of the DNA backbone, with
minimum P–N distances (of the phosphate and of the
This journal is © The Royal Society of Chemistry 2014
Dalton Transactions
Fig. 6 Two different views of the intercalation site of the supramolecu-
lar complexes between the three metal complexes 1–3 with [dodeca-
(dA-dT)]2, highlighting the interatomic distances of the metal ion with
the O4 keto atom of thymine {in red, 3.02 Å (1), 2.57 Å (2) and 2.94 Å (3)}
and with the N6 amine atom of adenine {in blue, 3.46 Å (1), 3.05 Å (2)
and 2.41 Å (3)}. DFT and MM regions are represented by “ball and
stick” and “sticks” styles, respectively.
Table 4 Calculated coordination distances (Å) of the metal ion of 1, 2
and 3 (M = Ni, Cu, Zn) with the oxygen keto atom of thymine O4 and
with the amine nitrogen atom of adenine N6, in the intercalation pocket
of [dodeca(dA-dT)]2
1 2 3 provide an explanation of the observed trends. The role of the
O4 3.02 2.57 2.94
N6 3.46 3.05 2.41
triethylammoniummethyl groups, respectively) in the range
5–11 Å for the three metal complex–dodecanucleotide systems.
With regard to the intercalation of the naphthalene residue
between the stacked and H-bonded DNA bases, the pictures in
Fig. 6 show that such a process is more pronounced for the
nickel complex 1 and, in decreasing order, for the copper and
the zinc ones 2 and 3. This trend is evidenced by the distortion
from linearity of the N–M–O angle between the metal ion and
the axial exocyclic atoms N and O of the amine and keto
groups interatomic distances, highlighted in blue and red,
respectively. In detail, this angle assumes the following values:
172°, 175° and 168° for 3, 2 and 1, respectively.
Finally, concerning metal coordination, it has been
reported that N,O coordination distances within 2.5–3.0 Å,
revealed by X-ray crystallography for copper and zinc ions in
active sites of biomolecules, are considered weak coordination
bonds.90 Following this rule, our DFT/MM calculations support
This journal is © The Royal Society of Chemistry 2014
Paper
Table 5 Formation energy,a in kJ mol−1, in terms of standard enthalpy
(ΔH°) and Gibbs free energy (ΔG°), calculated at 298.15 K for the com-
plexes of 1, 2 and 3 with [dodeca(dA-dT)]2
ΔH° ΔG° ΔH° ΔG°
Model system (vacuo) (vacuo) (water) (water)
1/[dodeca(dA-dT)]2 −141.1 −96.5 −102.8 −58.2
2/[dodeca(dA-dT)]2 −153.3 −89.5 −112.8 −49.0
3/[dodeca(dA-dT)]2 −192.8 −171.2 −54.3 −32.7
a
The formation energy was evaluated by the following equation, where E
can be either H or G: ΔE° = E°(ML2+/[dodeca(dA-dT)]2) − E°([dodeca(dA-
dT)]2) − E°(ML2+).
the hypothesis that, in the intercalation complexes with DNA,
while the copper and zinc ions are axially coordinated in an
asymmetrical octahedral environment, the nickel ion is not and
preserves its square planar coordination geometry (Table 4).
Interestingly, the analysis of the optimized geometries
allows to nicely show that the decrease in the DNA binding
strength monotonously follows the distortion from the co-
planarity of the MO2N2 atoms in the three metal complexes, as
it often occurs in nickel(II), copper(II) and zinc(II) complexes of
the same chelating ligand.4
Standard enthalpy and Gibbs free energy values, calculated
at 298.15 K, were used to evaluate, in vacuo and in solution,
the formation energy of the supramolecular complexes of 1, 2
and 3 with [dodeca(dA-dT)]2 (Table 5).
The analysis of the data in Table 5 allows us to make inter-
esting considerations on the energetic contributions involved
in the DNA binding of the title metal complexes. First, the for-
mation of the intercalation complexes is always exothermic,
in vacuo and in solution. However, both entropy and solvation
seem to destabilize the DNA-binding energy. The structure and
electronic properties of the three intercalation complexes may
polar solvent can be rationalized taking into account that there
is a considerable electrostatic character in the interaction
energy that is screened going from the gas phase to water solu-
tion. However, the solvent destabilization of the zinc(II)
complex 3 is much larger than those of the nickel(II) and
copper(II) complexes 1 and 2. This result finds nice support in
the calculated APT charge on the zinc atom, which is about 1.4,
also in the intercalation complexes, while those on the nickel and
copper atoms are about 1.0 and 1.2, respectively. The formation
free energy is always smaller than the formation enthalpy, both
in vacuo and in solution, indicating that the entropic contri-
bution, in the equation ΔG° = ΔH° − TΔS°, is always negative.
However, such entropic destabilization is slightly larger for the
intercalation complexes of [dodeca(dA-dT)]2 with 1, 2 and smaller
for that with the zinc complex 3. This result also follows the DNA-
binding affinity, which is greater for 1 and 2 and smaller for 3.
Remarkably, excellent agreement exists between the calcu-
lated formation free energy values of the intercalation com-
plexes with [dodeca(dA-dT)]2 and the analogous formation free
energy values experimentally obtained from the DNA-binding
constants reported in Table 1, as ΔG° = −RT ln(Kb). In
Dalton Trans., 2014, 43, 6108–6119 | 6115
Paper Dalton Transactions

Fig. 7 Linear correlation plot between the experimental and calculated

formation free energies for 1, 2 and 3 with native ct-DNA and with
[dodeca(dA-dT)]2, respectively.

particular, the latter are −40.0, −36.9 and −33.1 kJ mol−1, for
1, 2 and 3, respectively. Moreover, the experimental DNA-
affinity trend 1 > 2 > 3 is reproduced by the calculation. In fact,
a good linear correlation (R = 0.995) was found between the
experimental and calculated formation Gibbs free energies, as
shown in Fig. 7. In conclusion, although it is known that there
are ten possible ways in which the four nitrogen bases can
produce intercalation sites,80 the DNA model selected for our
calculations correctly describes the DNA-binding interaction
mechanism of the title metal complexes.
Fig. 8 Zn K-edge XANES (a) and FT-EXAFS (b) of 3 457 μM (blue) and in
X-ray absorption spectroscopy the presence of ct-DNA 1.692 mM (green) in Tris-HCl 1 mM aqueous
solutions.
The XANES spectra of the zinc(II) complex 3 in aqueous solu-
tion, isolated (blue line) and in the presence of ct-DNA (green
line) are reported in Fig. 8a. The corresponding Fourier trans-
formed EXAFS (FT-EXAFS) spectra for the same samples are
shown in Fig. 8b. By comparison with the literature,91 it can be
concluded that the near-edge features in the XANES spectra of
both 3 and of 3/ct-DNA solutions are typical of Zn2+ ions in an
octahedral coordination environment.
The FT-EXAFS spectrum of 3 (Fig. 8b, blue line) shows a
broad first coordination peak, between 1.4 and 2.0 Å (un-
corrected for phase shift), and several other contributions
between 2 and 3 Å, arising from the multiple scattering paths
of the C, O and N atoms of the chelating ligand. Such features
are nicely reproduced by using as guess structure the opti-
mized geometry of the diaquo complex reported in the inset of
Fig. 9. There is in fact good agreement between the refined
structures obtained from EXAFS and DFT simulations: the Fig. 9 FT EXAFS data (blue), model (red) and residual (brown) of 3
457 μM in Tris-HCl 1 mM aqueous solutions. The DFT optimized geome-
experimental equatorial distances, Zn–N and Zn–O, are 2.1
try of the distorted octahedral complex 3, with two apically coordinated
and 1.9 Å, respectively, while those calculated are 2.1 and water molecules, is also shown.
2.0 Å, respectively. The DFT calculations predict the two water
molecules to be placed at 2.1 and 2.2 Å, tilted away from the che-
lating atoms, while the model used to fit the EXAFS shows the [DNAphosphate] molar ratio of about 0.27. Under these con-
best agreement with the apical O of the water molecules at 2.1 Å. ditions, the solution experiments reported above have shown
The EXAFS spectrum of the isolated complex is significantly the metal complex to be fully intercalated. Analogous aqueous
modified by the addition of an excess of DNA, at a [3]/ solutions of the nickel(II) and copper(II) complexes 1 and 2

6116 | Dalton Trans., 2014, 43, 6108–6119 This journal is © The Royal Society of Chemistry 2014
Dalton Transactions
Fig. 10 FT EXAFS data (green), model (magenta) and residual (brown)
of 3 457 μM in the presence of ct-DNA 1.692 mM in Tris-HCl 1 mM
aqueous solutions.
undergo photoreduction and/or photodegradation to some
extent, both as isolated species and in the presence of DNA,
when exposed to the synchrotron X-ray beam. The presence of
metal–metal distances (either from the formation of metal
nanoparticles or from polymerization of the complexes) was
detected in the spectra, which were not analyzed further.
In the presence of DNA, an overall change in the local struc-
ture of Zn2+ is evident. Firstly, the first coordination shell loses
one or two apical H2O molecules, as witnessed by the decrease
in the broad contribution around 1.5 Å (Fig. 8b). Moreover, a
sharp increase of the feature around 2.4 Å is visible. The high-
layer structure optimized by DFT/MM calculations (Fig. 6) has
been used to reproduce the simulated FT-EXAFS of 3/ct-DNA
solutions (Fig. 10).
Interestingly, the peak at 2.4 Å can be modelled by two N
and two O atoms at 2.1 Å and 2.0 Å, respectively, corres-
ponding to the four equatorial atoms of the Schiff base, plus
one further N atom at about 2.5 Å and one O atom around
2.9 Å (Fig. 10). The six coordination distances are in excellent
agreement with the calculated distances for Zn–N and Zn–O,
in 3/[dodeca(dA-dT)]2 (Fig. 6), in particular, with the Zn–N6
and Zn–O4 apical distances, 2.41 and 2.94 Å, respectively,
involving the amine group of adenine and the keto group of
thymine (Table 4). The latter prove the occurrence of a further
stable coordination of the metal ion with respect to the metal
complex alone.
Conclusions
Detailed information on the DNA-binding of three square
planar nickel(II) (1), copper(II) (2) and zinc(II) (3) Schiff-base
complexes was obtained through the complementary appli-
cation of experimental investigations in water solution
and quantum mechanics/molecular mechanics (DFT/MM)
calculations.
Spectroscopic and viscometry measurements confirmed
that 1, 2 and 3 are DNA-intercalators with DNA-affinity
This journal is © The Royal Society of Chemistry 2014
Paper
decreasing in the order Ni > Cu > Zn and with selective affinity
of the three metal complexes toward AT with respect to GC
sequences. DFT/MM calculations provided detailed local infor-
mation on the DNA-binding site, which consists of (1) electro-
static attraction between the lateral cationic groups of the
metal complexes and the anionic phosphate groups of the
DNA backbone, (2) the DNA-intercalation of the planar aro-
matic fragment of the chelating Shiff-base ligand, (3) the metal
ion coordination by the exocyclic keto-oxygen and amine-nitro-
gen of the nitrogen bases. The values of the DNA-binding con-
stants and their decreasing trend in the order 1 > 2 > 3 are
correctly reproduced by the calculated formation Gibbs free
energy values of the supramolecular complexes of 1, 2 and 3
with [dodeca(dA-dT)]2 in solution.
Remarkably, for the first time, the occurrence of DNA–
metal ion coordination in aqueous solution was confirmed for
the zinc(II) complex 3 by the modifications of the experimental
EXAFS spectra of the metal complex in the presence of DNA
and by the excellent agreement between the first shell coordi-
nation distances, found by analysis of the EXAFS, with those
obtained in the structure of 3/[dodeca(dA-dT)]2, optimized by
DFT/MM calculations.
Acknowledgements
We gratefully acknowledge the CINECA award N. IsB05, year
2012, under the ISCRA initiative, for the availability of high
performance computing resources and support, and the Euro-
pean Synchrotron Radiation Facility (ESRF) for the provision of
synchrotron beam time (experiment SC-3581) and for financial
support. We thank Dr S. Nikitenko, Dr D. Banerjee and the
staff of the BM26 beamline of ESRF for assistance during the
XAS measurements.
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