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Accepted Manuscript

Synthesis, structural characterization and DNA interaction of new copper-ter-


pyridine complexes
Natalia Alvarez, Nicols Veiga, Sebastin Iglesias, Mara H. Torre, Gianella
Facchin
PII: S0277-5387(13)00747-X
DOI: http://dx.doi.org/10.1016/j.poly.2013.11.002
Reference: POLY 10410
To appear in: Polyhedron
Received Date: 30 September 2013
Accepted Date: 4 November 2013
Please cite this article as: N. Alvarez, N. Veiga, S. Iglesias, M.H. Torre, G. Facchin, Synthesis, structural
characterization and DNA interaction of new copper-terpyridine complexes, Polyhedron (2013), doi: http://
dx.doi.org/10.1016/j.poly.2013.11.002
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Synthesis, structural characterization and DNA interaction of new copper-terpyridine
complexes
REVISED VERSION
Natalia Alvarez
1
, Nicols Veiga
1
, Sebastin Iglesias
1
, Mara H. Torre
1
, Gianella Facchin
1

1
Facultad de Qumica, General Flores 2124, UdelaR, Montevideo, Uruguay, gfacchin@fq.edu.uy

Abstract
As a part of our studies of copper(II) complexes with possible antitumor activity, the
synthesis and structural characterization of two new copper terpyridine (terpy) complexes:
[Cu(terpy)(SO
4
)(H
2
O)]2H
2
O and [Cu
2
(terpy)
2
(SO
4
)
2
]4H
2
O are presented. The crystal
structures show that the copper ion in both complexes is in a square pyramidal environment,
coordinated equatorially to the terpy N atoms and completing the coordination with O atoms
from the sulfate or water molecules. The second compound is a sulfate bridged dinuclear
complex. In aqueous solution the coordination scheme is modified. Taking the crystal
structure as a starting point the geometries of the complexes were optimized and vibrational
spectroscopy calculations were performed allowing an assignation of the experimental IR
spectra. Finally, the interaction of the complexes with DNA was studied, by UV titration and
docking studies of the complexes. The results suggest that the interaction occur by minor
groove interaction and partial insertion of the terpy into the DNA bases and that terpy
interaction with DNA is strongly influenced by the Cu-coordination process.
Key words: Cu-terpy, X-ray diffraction, DNA interaction, docking studies

1. Introduction
There has been widespread interest in investigating the potential antitumor activity of
transition-metal complexes following the discovery of Cisplatin as an anticancer drug. An
extensive search of new anticancer metallodrugs has been pursued with the aid of structure
activity relationship studies. These studies led to the discovery of novel coordination
compounds containing ruthenium [1, 2], rhodium [3], gold [4, 5] and copper [6], which are
reported to have promising chemotherapeutic potential, and different mechanisms of action
than those of the platinum based drugs.

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To date, several copper(II) coordination complexes bearing cytotoxic activity have been
identified. In some cases families of related complexes have been obtained allowing the
development of structure-activity relationships [7, 8]. As an example, a series of mixed
chelate copper complexes named Casiopeinas, present antitumor activity and two of
them: [(Cu(II))(4,7-dimethyl-phen)(glycinate)(NO
3
)(H
2
O)] and [(Cu(II))(4,4-dimethyl-2,2-
bipyridine)(acetylacetonate)(NO
3
)(H
2
O)] are approved to enter phase I clinical studies [9,
10].
Another series that has been extensively studied to different ends is the Cu-terpy system.
[11-16]. There are several crystalline structures reported in the Cambridge Structure
Database with hexaflourophosphate, bromide, cyanide, among other anions.
To the best of our knowledge, these reports did not include either theoretical calculation on
the optimized structure or complete spectroscopic studies useful in the structural
characterization of related terpy complexes.
Besides, as a part of our research, Cu-terpy complexes were synthesized and evaluated as
antiproliferative compounds showing interesting results [17]. For this reason, to continue with
the structural characterization both in solid state and in solution, as well as its interaction
with DNA, has been considered a priority.
Following these studies, in this work the synthesis and structural characterization of two new
Cu-terpy complexes is presented. Taking the crystal structure as a starting point, the
geometry of the complexes was optimized and vibrational spectroscopy calculations were
performed in order to assign the IR spectra, providing a tool to characterize other Cu
complexes containing terpy whose crystalline structure are not obtainable. In addition efforts
were made to model the complex in solution. Finally, we present studies of the interaction of
the complexes with DNA, by means of UV titrations (determination of K of binding) and
docking studies.
2. Experimental
Reagents: 2,2':6'2''-terpyridine (terpy, Sigma, 98%), cupric sulfate pentahydrate (Fluka,
99%), KBr (Merck IR grade), Calf Thymus DNA (CT-DNA, SIGMA), as well as the organic
solvents and distilled water were used without further purification.

2.1 Synthesis of [Cu(terpy)(SO
4
)(H
2
O)]2H
2
O (1)
To an aqueous solution of CuSO
4
5H
2
O (25 mM) an ethanolic solution of 2,2':6'2''-terpyridine
(terpy) (50 mM) was added while constant stirring (Cu/terpy ratio 1:2). A change of color in

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the solution from light blue to dark green was observed instantly. Dark green crystals were
obtained by gas-phase diffusion of acetone at room temperature.

2.2 Synthesis of [Cu
2
(terpy)
2
(SO
4
)
2
]4H
2
O (2)
The complex was synthesized following the procedure in 2.1, using CuSO
4
5H
2
O (25 mM)
and terpy (25 mM) (Cu/terpy ratio 1:1). The change of color from light blue to dark green in
the solution was observed instantly. Dark green crystals were obtained by gas-phase
diffusion of acetone at room temperature.

2.3 Physical measurements
Electronic absorption spectra were recorded on aqueous solutions of the complexes with a
Shimadzu UV1603 spectrophotometer. FT-IR spectra were measured as KBr pellets on a
Bomen MB 102 instrument. Aqueous solution FT-IR spectrum was measured using CaF
2

windows on a Shimadzu IR Prestige-21 instrument. Conductivity measurements of 1mM
complex and copper sulfate solutions at 22C were determined in a Jenway Conductivity
Meter 4310.

2.4 Crystal structure determination
Suitable single-crystal samples were mounted on a micromount tip and crystallographic data
were collected at 200(2)K on a Bruker SMART X2S diffractometer using graphite
monochromated MoK radiation (k=0.71073 ). The structures were solved by direct
methods and all non-hydrogen atoms were refined on F
2
by full-matrix least-squares
procedure using anisotropic displacement parameters within Brukers SHELXTL-PC
software package [18]. Hydrogen atoms of the organic ligands were inserted in calculated
positions and assigned fixed isotropic thermal parameters at 1.2 times the equivalent
isotropic U of the atoms to which they are attached. Water hydrogen atoms were found in
the difference Fourier map, positionally fixed and their isotropic displacement parameters
fixed to 1.5 times the U
iso
of the oxygen atom they are bonded to. Molecular structure
graphics were prepared using MERCURY program [19]. A summary of crystallographic data,
experimental details and refinement results is listed in Table 1.

Table 1. Crystal data and structure refinement data for complexes 1 and 2.
[Cu(terpy)(SO
4
)(H
2
O)]2H
2
O (1) [Cu
2
(terpy)
2
(SO
4
)
2
]4H
2
O (2)

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Empirical formula CuC
15
H
17
N
3
O
7
S Cu
2
C
30
H
30
N
6
O
12
S
2

F
w
446.92 857.84
Temperature 296 (2) 296 (2)
Crystal system Monoclinic Triclinic
Space group P2
1
/c P-1
Z 4 1
Unit cell parameters
a () 13.061 (3) 9.6150 (19)
b () 18.371 (4) 9.7490 (19)
c () 7.0175 (14) 10.600 (2)
() 90.00 96.27 (3)
() 90.007 (6) 106.51 (3)
() 90.00 118.18 (3)
V () 1683.8 (6) 804.1 (3)

calc
(mg.m
-3
) 1.763 1.771
F(000) 916 438
Absorption coefficient () 1.468 1.529
Gaussian transmition
coefficient
Tmin: 0.710
Tmax: 0.791
Tmin: 0.700
Tmax:0.807
Rad. wavelength (MoK)
() graphite
monochromator
0.71073 0.71073
range () for data
collection
2.71-25.38 2.47-25.11
Limiting indices -15h15 -11h11
-22k22 -11k11
-8l8 -12l12
Reflections
collected/Unique
15260/3055 [R
int
:0.0245] 5299/2819 [R
int
:0.0245]
Data completeness (%) 98.8 97.9
Residuals: R
1
a
, wR
2

(I>2(I))
R
1
: 0.0526 wR
2
: 0.1406 R
1
: 0.0535 wR
2
: 0.1480
Residuals: R
1
, wR
2
b,c
(all
data)
R
1
: 0.0471 wR
2
: 0.1363 R
1
: 0.0566 wR
2
: 0.1518
Goodness of fit on F
2
1.087 (0 restraints) 1.1010(0 restraints)
a
R
1
=F
o
-F
c
/F
c

b
wR
2
={|w(F
o
2
-F
c
2
)
2
]/ |w(F
o
2
)
2
]}


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c
w=1/[o
2
(F
o
2
)+(s1*P)
2
+(s2*P)]; P=(F
o
2
+2F
c
2
)/3
2.5 DFT calculations
Geometry optimizations were performed in gas phase by means of the methods described
hereinafter as implemented in Gaussian 03, Revision B.01 package [20]. The final structures
obtained were all minima in the potential energy surface, being the nature of the stationary
points verified through vibrational analysis.
For the molecular modelling approach to terpy and its complexes, DFT geometry
optimization runs were performed. The initial geometries for complexes 1 and 2 were taken
from the reported crystal structures presented in this paper. Taking into account the possible
loss of the sulfate ligand in aqueous solution, the following complexes were considered:
[Cu(terpy)(H
2
O)
3
]
2+
and [Cu(terpy)(H
2
O)
2
]
2+
. The initial geometries for the latter were built
deleting the sulfate anion and linking two or three water molecules to the copper cation. All
the systems were finally optimized by means of the B3LYP/6-31G(d) method.
The calculated infrared spectra of terpy and complex 1 were computed, and the obtained
wavenumbers were scaled to avoid systematic errors by using the recommended factor for
B3LYP/6-31G(d) method (0.9613) [21].
2.6 DNA binding: UV absorption titration experiments
Absorption titration experiments were carried out varying the concentration of CT-DNA,
SIGMA (0 to 15 M), while keeping the metal complex concentration constant (26 M) in
buffer solution of pH=7.4. The intrinsic binding constant (Kb) for the interaction of the
complexes with CT-DNA was determined from the A
0
/(A-A
0
) versus 1/[DNA] plot using
absorption spectral titration data and the BenesiHildebra equation: A
0
/(A-A
0
) = c
G
/(c
G-H
- c
G
)
+ c
G
/(c
G-H
- c
G
) *1/K |DNA| (Eq. 1) where K is the association/binding constant, A
0
and A are
the absorbances of the copper complex and its complex with DNA, respectively, and c
G
and
c
HG
are the absorption coefficients of the free copper complex and the copper(II) complex
DNA complex, respectively. The Kb value is calculated as the ratio of the slope to the
intercept [22]. The concentration of DNA was determined from the intensity of the 260 nm
band with a known extinction coefficient value (
260
= 6600 M
-1
cm
-1
).

2.7 DNA binding: docking studies
The crystal structure of calf thymus DNA (ct-DNA) was extracted from the Protein Data Bank
(identifier 453D) [23] having all the water molecules removed. The molecular geometries of

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terpy and its complexes tested as substrates were taken from those optimized according to
section 2.5. Molecular-docking experiments were carried out by using the GOLD docking
program (v 4.1.2) [24]. GOLD genetic algorithm was applied to dock the flexible model of
substrates into ct-DNA. The binding site was defined taking into account all the nucleotides
in ct-DNA, except from those located in the extremes of the biomolecule. Each substrate
was docked to ct-DNA a total of 40 times and the docking modes that achieved the highest
Goldscore fitness were retained and compared. Goldscore fitness is determined as the
negative of the sum of five energy terms: receptor-substrate hydrogen bond energy,
receptor-substrate van der Waals energy (vdw), substrate internal vdw energy, substrate
torsional strain energy, and substrate intramolecular hydrogen bond energy. For the most
stable poses obtained, the total free energy change that occurs on substrate binding was
estimated through the ChemScore scoring function [25, 26]. Other miscellaneous
parameters were assigned the default values given by the GOLD program. The output from
GOLD was rendered with Discovery Studio Visualizer software [27].

3. Results and discussion
3.1 Description of the crystalline structure of [Cu(terpy)(SO
4
)(H
2
O)]2H
2
O (1)
The system crystallizes in the monoclinic P2
1
/c space group, consistent with what is
observed in literature for similar complexes [28, 29]. The X-ray diffraction data shows a
Cu(II) center with the terpy acting as a tridentate equatorial ligand through its nitrogen
atoms, the sulfate anion acting as a monodentate ligand with one oxygen atom completing
the fourth position of the basal plane and an oxygen atom from a water molecule filling the
apical position of a geometry that is best described as square pyramidal with a distortion
towards trigonal bipyramidal. factor [30] was calculated in order to describe geometrys
distortion in the coordination sphere and the value obtained was 0.10 ( = 0 for perfect
square pyramidal and = 1 for perfect trigonal-bipyramidal geometry). An ORTEP
representation of the structure including atom numbering scheme is shown in Fig. 1.

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Fig. 1. ORTEP of [Cu(terpy)(SO
4
)(H
2
O)]2H
2
O (1).

Cu1-O5 bond distance is slightly larger than Cu1-O1, due to the axial elongation
experienced by copper(II) complexes. Although the copper atom shows a square-based
pyramid environment, the sulfate oxygen atom O4 is oriented towards the electron deficient
copper center approaching the sixth coordination position. The Cu1O4 distance (3.032 )
does not strictly correspond with a bond but represents a weak electrostatic interaction.
Selected bond lengths and angles are listed in Table 2.
All potential hydrogen bond donors (sulfate oxygen, water oxygen) and acceptors (water
hydrogen) from the complex and hydration water molecules in the lattice participate in
intermolecular hydrogen interactions, creating a network with a one dimensional channel
filled with water molecules along the c axis (Fig. 2). Hydrogen bond distances range from
2.70 to 2.85 . The formation of molecular channels is also assisted by the presence of -
stacking along the c axis between the central pyridines in the terpy of contiguous complex
molecules (centroid-to-centroid distance: 3.519 , interplanar distance: 3.295 , dislocation
angle: 20.52).

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Fig. 2. A) Space lling model of the crystal lattice showing channels and molecule assembly
leading to the channel formation along the c axis, compound 1. B) Water molecules
arrangement in the channel, view along the a axis, compound 1.
Table 2. Selected bond lengths and angles in the crystalline structures.
[Cu(terpy)(SO
4
)(H
2
O)]2H
2
O (1) [Cu
2
(terpy)
2
(SO
4
)
2
]4H
2
O (2)
Cu1-N1 2.0270 (27) Cu1-N1 2.0394 (29)
Cu1-N2 1.9496 (26) Cu1-N2 1.9462 (28)
Cu1-N3 2.0189 (27) Cu1-N3 2.0136 (32)
Cu1-O1 1.9240 (22) Cu1-O1 2.2025 (27)
Cu1-O5 2.2233 (23) Cu1-O3 1.9168 (25)
N1-Cu1-N2 80.10 (10) N1-Cu1-N2 78.64 (12)
N1-Cu1-N3 158.98 (11) N1-Cu1-N3 158.93 (13)
N1-Cu1-O1 101.25 (10) N1-Cu1-O1 90.44 (11)
N1-Cu1-O5 94.19 (0.10) N1-Cu1-O3 91.54 (11)
N2-Cu1-O1 164.96 (10) N2-Cu1-O1 98.79 (12)
N2-Cu1-N3 79.05 (11) N2-Cu1-N3 80.87 (12)
N2-Cu1-O5 101.82 (9) N2-Cu1-O3 161.42 (12)
N3-Cu1-O1 98.18 (11) N3-Cu1-O1 97.52 (11)
N3-Cu1-O5 92.69 (10) N3-Cu1-O3 106.70 (11)
O1-Cu1-O5 93.05 (9) O1-Cu1-O3 97.00 (12)

3.2 Description of the crystalline structure of [Cu
2
(terpy)
2
(SO
4
)
2
]4H
2
O (2)

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The structure consists of a sulfate bridged dinuclear complex. The system crystallizes in the
triclinic P-1 space group, as observed for complexes of the same family [31, 32]. The
pentacoordinated Cu atoms in the dimeric unit are centrosymmetric and present a
predominantly square pyramidal geometry ( =0.04).
The Cu(II) coordination environment consists of three pyridine nitrogen atoms from the terpy
and two oxygen atoms from the different coordinated sulfate anions, which also act as
bridges between the [Cu(terpy)]
2+
cations forming the dimeric complex. An ORTEP
representation of the structure including atom numbering scheme is shown in Fig. 3.

Fig. 3. ORTEP plot of [Cu
2
(terpy)
2
(SO
4
)
2
]4H
2
O (2).
Nitrogen atoms N1, N2, N3 and oxygen atom O3 occupy the corners of the equatorial plane,
whereas the oxygen atom O1 completes the apical position at a significantly longer distance
than the equatorial one as a consequence of the Jahn-Teller effect. Selected bond lengths
and angles are listed in Table 2.
In the crystal, hydration water molecules act as hydrogen acceptor and donor, participating
in hydrogen bonding among themselves and with the oxygen atoms of the sulfate anion,
constructing 1-D molecular chains along the c axis (Fig. 4 A). Hydrogen bond distances
range from 2.85 to 2.91 . Meanwhile, two of the pyridine groups in the terpy of adjacent
chains stack via - interactions (Fig. 4 B) maintaining the stability of the supramolecular
framework. Intercentroid and interplanar distances are 3.697 and 3.310 , respectively, with
a dislocation angle of 26.5.

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Fig. 4. A) 1-D molecular chain formed through O-HO interactions along the c-axis,
compound 2. B) - interactions, view along the c axis, compound 2.
3.3 Computational studies
3.3.1 terpyridine ligand
In order to calculate the IR spectrum of terpy, its structure was first optimized. The three
nitrogen atoms are directed to the same side of the molecule, although the whole molecule
is not planar. The theoretical IR frequencies obtained reproduce quite well the absorption
bands observed experimentally, further validating the calculation. Additionally, this allows us
to assign the different bond vibrations associated to the different normal modes, as
presented in table 3.
3.3.2 [Cu(terpy)(SO
4
)(H
2
O)]2H
2
O (1) and [Cu
2
(terpy)
2
(SO
4
)
2
(H
2
O)
2
] (2)

The predicted B3LYP/6-31G(d) geometry for [Cu(terpy)(SO
4
)(H
2
O)]2H
2
O is similar to that
found in the solid state. The most representative difference between the theoretical model
and the crystalline structure is observed in the Cu1O4 distance (2.26 vs. 3.03 ), being
the distance in the model closer to a covalent bond. This effect is due to the fact that the
theoretical model does not take into account the intermolecular interactions in the lattice. In
fact, O4 participates in hydrogen bonding with a contiguous terpy, forming a 1D
macromolecular channel along the c-axis that stabilizes the crystalline structure, as
discussed in section 3.1.
Since the predicted and crystalline structure for complex 1 are similar, the theoretical
infrared spectrum was used to assist in the assignation of the most important experimental
IR bands. Results are shown in Table 3.

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47
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49
50
51
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65
Table 3. Assignment of the most important infrared bands of terpy and
Cu(terpy)(SO
4
)(H
2
O)]2H
2
O.
terpy v
exp
(cm
-1
) terpy v
theo
(cm
-1
) C1 v
exp
(cm
-1
) C1 v
theo
(cm
-1
) Assignment
- - 3448 3648-3018 v (O-H) water
3046, 3009, 2924,
2855
3094-3045
3061, 3059, 3053 3115-3077 v (C-H) terpy
- - 1595, 1576, 1561 1701-1652 (H-O-H) water
1581, 1560, 1467,
1454, 1423, 1262,
1151
1580-1240
1497, 1473, 1440,
1405
1597-1561
v (C-N) + v (C-C)
terpy
1103, 1076, 1039,
989
1156-1058
1330, 1302 1477-1394 (C-H) terpy

1256 1319-1253
v (C-N) + v (C-C)
+ (C-H) terpy

1107 1193
v (O3-S-O2)
sulfate

1033 1108
v(O4-S-O2/3)
sulfate
830, 763, 732 965-731 983-963 1057-1037 (C-H) terpy

982, 964, 910 1011-991
(C-C-C) terpy
(C-C-N) terpy
v (Cu-N)
830, 763, 732 965-731 980-959 (C-H) terpy

829 922
v(O4-S-O2/3)
sulfate
778, 730 882-775 (C-H) terpy

1
2
3
4
5
6
7
8
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23
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31
32
33
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62
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64
65

667, 652 761, 755
(C-H) terpy
v (S-O1) sulfate
894 969-967
616 654-630
Ring breathing
terpy
(O-H) water
v (Cu-N)


613
602
(O-H) water
v (Cu-O1/O4)

509
528
522
(N-Cu-O1)
(N-Cu-N)

437 480-466
(O-H) water
(Cu-O1/4)

408 435-401
(Cu-N) out of
plane

The experimental IR spectrum for 1 is in agreement with the theoretical results and all the
band assignments are in accordance with the complex structure found in solid state.
In the case of complex [Cu
2
(terpy)
2
(SO
4
)
2
(H
2
O)
2
] (2) no significant differences are observed
among the theoretical model and the experimental X-ray data for complex 2. The
experimental and theoretical IR spectra, as well as the normal modes analysis, for the
dimeric species (not presented) are similar to the one presented for complex 1.
3.4 Aqueous solution studies of [Cu(terpy)(SO
4
)(H
2
O)]2H
2
O (1) and
[Cu
2
(terpy)
2
(SO
4
)
2
(H
2
O)
2
] (2)

As an approach to the structure of the complex in solution, we performed molecular
modeling studies of the complexes. To this end, and taking into account that the structures
observed in solid state are likely to change in the dissolution process, we firstly checked if
the ligands remained coordinated in aqueous solution.

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The visible spectra present one pick with
max
= 675 nm (c= 80 M-1 cm-1). These spectra are
characteristic of Cu(II) in an octahedral environment and the main absorption can be
assigned to the d
xy
d
x2-y2
transition. The infrared spectra in aqueous solution show the loss
of the asymmetry of some of the sulfate anions bands (partial disappearance of the bands
around 1033 cm-1). The bands corresponding to (C-C-C) and (C-C-N) observed in solid
state IR are maintained, confirming that the terpy ligand is still coordinated in solution. This
phenomenon is observed for both the monomeric and dimeric species. Conductivity
measurements for both complexes were carried out and confirm this data, showing nearly
complete formation of ions. The results are consistent with a model in solution in which the
[Cu(terpy)(H
2
O)
3
]
2+
and SO
4
2-
ions are the most abundant species, as expected.
Considering these results, we carried out the molecular modeling of [Cu(terpy)(H
2
O)
3
]
2+
, as it
is one of the most abundant species in solution. After deleting the sulfate anion the results,
shown in Fig. 5 A, indicate that the terpy remains bounded to the Cu
2+
ion. However, only
two water molecules remain linked to the metal centre, giving rise to a pentacoordinated
environment. During the optimization steps, the third H
2
O molecule is transferred to the
second coordination sphere, and stays attached to the complex by means of a strong
hydrogen bond. This information suggests that, in solution, it is possible that one axial
position of the copper coordination sphere would be occupied partially and dynamically by
the solvent molecules in the surroundings.

Fig. 5. B3LYP/6-31G(d) geometries of [Cu(terpy)(H
2
O)
3
]
2+
(A) and [Cu(terpy)(H
2
O)
2
]
2+
(B).
The intramolecular hydrogen bonds are shown as dashed lines and the distances for some
of the interactions are given in .

Therefore, the [Cu(terpy)(H
2
O)
3
]
2+
complex in solution would be best represented by the
formula [Cu(terpy)(H
2
O)
2
]
2+
, considering only the first coordination sphere. To obtain the
most stable geometry for the latter species, we deleted the non-coordinated water molecule

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3
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13
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59
60
61
62
63
64
65
in [Cu(terpy)(H
2
O)
3
]
2+
, and let the system to optimize under the same conditions. The
obtained structure (Fig. 5 B) is analogous to that shown in Fig. 5 A, in which the copper
cation is coordinated by three nitrogen atoms from terpy and two oxygen atoms from the
water molecules, in a distorted square pyramidal environment. Given the fact that terpy
chelates the copper ion in the same way in [Cu(terpy)(H
2
O)
2
]
2+
and in [Cu(terpy)(SO
4
)(H
2
O)],
it is not surprising that the (C-C-C) and (C-C-N) bands fall in the same IR frequency region
both in solution and in the solid state.

3.6 DNA interaction: UV titration

The determined binding constant was 5,9 E 3. This is a moderate value, minor to that of that
of the classical intercalator ethidium bromide (1.4x10
6
) |35|.

3.7 Docking studies

To gain knowledge about the mechanism whereby the terpy and its complexes,
[Cu(terpy)(SO
4
)(H
2
O)] and [Cu(terpy)(H
2
O)
2
]
2+
, bind to the DNA, molecular docking
calculations were performed. Given that GOLD software is not parameterized for metal ions,
we focused our efforts in analyzing the interactions between the terpy, sulfate anion and
coordinated water molecules with the DNA, as they are expected to establish first in the
formation of the complex-DNA adduct.
In order to help in the following analysis, Table 4 shows an estimation (ChemScore) of the
change in the free energy related to the 1:1 docking of all the substrates tested to the ct-
DNA (G
binding
). Additionally, the breakdown of the most important terms that contribute to
the G
binding
is also listed. These correspond to three factors: one due to electrostatic
interactions and hydrogen bonds formed (G
H-bond
), one given by lipophilic interactions
(G
lipo
) and the latter is associated with the loss of ligand rotational states due to the binding
to the active site (G
rot
). Of these, the first two are usually negative, thus adding to the
protein-substrate affinity. G
rot
is generally positive, and corresponds to the loss of entropy
of the system by immobilization of the substrate in the active site.
Table 4. ChemScore estimation of the change in the free energy related to the 1:1 docking of
the different substrates to the ct-DNA. The most important terms that contribute to the
G
binding
are also listed.
Substrate G
binding
(kJ/mol) G
H-bond
(kJ/mol) G
lipo
(kJ/mol) G
rot
(kJ/mol)

1
2
3
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5
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22
23
24
25
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27
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29
30
31
32
33
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49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
Terpy -24.6 0 -19.1 0
[Cu(terpy)(SO
4
)(H
2
O)] -26.2 -5.6 -15.1 0
[Cu(terpy)(H
2
O)
2
]
2+
-21.5 0 -16.0 0

The most stable docked structures of terpy, [Cu(terpy)(SO
4
)(H
2
O)] and [Cu(terpy)(H
2
O)
2
]
2+
to
the calf thymus DNA are shown in Fig. 6 A, B and C, respectively. All the complexes fit
within the minor groove of the DNA, where the pyridine moieties of terpy are positioned in
order to allow an effective interaction between the aromatic rings and the DNA bases. This is
in agreement with the fact that minor groove binders generally have aromatic rings
connected by single bonds that allow for torsional rotation, in order to fit into the narrower
helical curvature of the groove with displacement of water molecules [33].
For terpy, the docking results (Fig. 6 A and D) show that the N1 and N3 pyridine rings rotate
nearly 180 from the most stable conformation (in relation to the calculated one for terpy
alone) before the ligand is bonded to the DNA. This preorganization of the terpy molecule is
necessary for maximizing the lipophilic interaction with the DNA (Table 4), because not only
does the ligand succeed in following the helical curvature of the minor groove, but also
exposes two of the nitrogen atoms to the polar skeleton of the DNA.
When terpy chelates the copper ion the electronic density changes substantially, as
previously discussed. Consequently, the spatial distribution of [Cu(terpy)(SO
4
)(H
2
O)] and
[Cu(terpy)(H
2
O)
2
]
2+
into the DNA are different from the one obtained for terpy (Fig. 6 B and
C). In both cases, the copper ion and its co-ligands point towards the phosphate-sugar
skeleton, while the aromatic moiety of terpy faces the nitrogenated bases. The conformation
of terpy is frozen upon metal coordination, therefore is no longer capable of preorganizing its
geometry to effectively couple to the curvature of the minor groove. This explains the higher
value of G
lipo
for the two copper complexes with respect to the one calculated for terpy
(Table 4). Only for [Cu(terpy)(H
2
O)
2
]
2+
this phenomenon implies a decrease in the DNA
affinity with respect to terpy, because both substrates establish only lipophilic interactions
with the DNA. In the case of [Cu(terpy)(SO
4
)(H
2
O)], three hydrogen bonds are formed
between the complex and the DNA skeleton (Fig. 6 E), adding to the stability of the DNA-
complex adduct and decreasing the estimated G
binding
(Table 4).
G
rot
is zero for all the substrates tested, indicating that there is not a significant loss of
rotational states due to the binding to the DNA. While for the complexes this is mainly owing
to the rigidity brought about by copper complexation, this is not the case for terpy. This data
suggests that the binding of terpy to the DNA is relatively weak, in agreement with the

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65
experimentally determined constant of binding, permitting slightly dynamic rearrangements
of the molecule docked into the groove. This phenomenon is likely to occur to some extent
also for the complexes, and mostly for [Cu(terpy)(H
2
O)
2
]
2+
, for which only weaker lipophilic
interactions are established with the DNA molecule. In this context, it is probable that the
DNA structure would not be drastically affected by the union of terpy or its complexes, a fact
that is in agreement with the results of Circular Dicroism studies, were the addition of the
complex causes only small changes on the DNA spectra.
All the results point towards one fact: the complexation of terpy modulates the substrate-
DNA affinity. The docked conformation allows the copper ion and its co-ligands to face the
DNA skeleton, giving rise to a moderately stable complex-DNA adduct, permitting the
interaction of the aromatic moieties with the nitrogenated bases.
This studies constitute further evidence that, as suggested by Galindo-Murillo et al [34], the
single fact of having a planar ligand is not indicative of complex-DNA intercalation as
previously assumed.


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64
65
Fig. 6. Model of terpy (A), [Cu(terpy)(SO
4
)(H
2
O)] (B) and [Cu(terpy)(H
2
O)
2
]
2+
(C) docked into
the minor groove of ct-DNA. For the ct-DNA, the solvent-accessible surface is depicted, with
the interpolated atomic charges mapped on it. In (D), (E) and (F), a zoom of the docked pose
of (A), (B) and (C) is shown, along with the most relevant hydrogen bonds between the
substrate and the ct-DNA.
4. Conclusions
Two new Cu-terpy complexes have been obtained and its crystalline structures resolved.
Taking as a starting point these data and with the aid of theoretical calculations we were
able to perform a detailed interpretation of the IR spectra and gain some insight of the
structure of the complex in solution, which is relevant for biochemical and biological
processes.
Finally, docking studies allowed us to understand the initial interaction of the complex with
DNA, which is suggested to occur by minor groove interaction and partial insertion of the
terpy into the DNA bases. This study also remarks the modification of the interaction of the
terpy with the DNA as a consequence of coordination.
Appendix A. Supplementary data

CCDC 960828 and CCDC 960732 contain the supplementary crystallographic data for
compounds 1 and 2 respectively. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:
deposit@ccdc.cam.ac.uk.

Fig. S1: RB3LYP/6-31G(d) geometry for terpyridine (A) and experimental (above) and
calculated (B3LYP/6-31G(d)) (below) infrared spectra (B) of terpy.
Fig. S2: Structure of [Cu(terpy)(SO
4
)(H
2
O)]2H
2
O (1) (A and B) and [Cu
2
(terpy)
2
(SO
4
)
2
(H
2
O)
2
]
(2) (C and D). A and C present the predicted B3LYP/6-31G(d) geometries, while B and D
show the crystalline structure for both complexes. The strongest hydrogen bonds are shown
as dashed lines, and the through-space interactions are depicted as dotted lines. Selected
distances are given in .
Fig. S3: Experimental (above) and calculated (B3LYP/6-31G(d)) (below) infrared spectra of
[Cu(terpy)(SO
4
)(H
2
O)]2H
2
O (1).

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65
Fig. S4: First 10 docking poses having the highest Goldscore fitness values superimposed
for terpy (A), [Cu(terpy)(SO
4
)(H
2
O)] (B) and [Cu(terpy)(H
2
O)
2
]
2+
(C).
Fig. S5: CD spectra of Calf thymus DNA alone and in presence of [Cu(terpy)(SO
4
)(H
2
O)] (r
pbDNA/Cu=6.25).

Acknowledgements
The authors thank the American Crystallographic Association for a scholarship to N. A. to
attend to ACA Summer School on Small Molecule Crystallography, where the structures
presented in this paper were solved, with the guidance of Prof. Peter Muller; and to Prof.
Antonio Costa Filho for the use of the CD equipment.

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Two new Cu-terpy complexes were characterized by X-ray diffraction. By means of theoretical
calculations the IR spectra is interpreted in detail. Docking studies of the complexes with DNA
suggest a minor groove interaction and partial insertion of the terpy into the DNA, which is strongly
influenced by the coordination process.

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