A novel Cu(II) distorted cubane complex containing Cu4O4 core as the first tetranuclear catalyst for temperature dependent oxidation of 3,5-di-tert-butyl catechol and in interaction with DNA & protein (BSA)

Article abstract of DOI:10.1016/j.saa.2019.117593

The tri-dentate Schiff base ligand 3-(2-hydroxyethylimino)-1-phenylbut-1-en-1-ol (L) produced the tetra-nuclear Cu(II) distorted cubane complex which contain Cu₄O₄ core, upon reaction with Cu(II)acetate.H₂O. The complex was structurally characterized by X-ray crystallography and found that, in this tetrameric and tetra-nuclear distorted cubane structure, each two-fold deprotonated Schiff base ligand coordinated to a Cu(II) center with their alcoholic oxygens and imine nitrogens and formed six and five-membered chelate rings. At the same time, each ligand bridged to a neighboring Cu(II) atom by its alcoholic oxygen, thus the metal centers became penta-coordinated. The copper(II) complex with μ-?²-hydroxo bridges and Cu….Cu distance about 3 ? was structurally similar to the active site of natural catechol oxidase enzyme and exhibited excellent catecholase activity in aerobic oxidation of 3,5-di-tert-butyl catechol to its o-quinone. The kinetics and mechanism of the oxidation of 3, 5-DTBCH₂ catalyzed by [CuL]₄ complex, were studied at four different temperatures from 283 to 313K by UV-Vis spectroscopy. Interaction of [CuL]₄ complex with FS-DNA was investigated by UV-Vis and fluorescence spectroscopy, viscosity measurements, cyclic voltammetry (CV), circular dichroism (CD) and agarose gel electrophoresis. The main mode of binding of the complexes with DNA was intercalation. The interaction between [CuL]₄ complex and bovine serum albumin (BSA) was studied by UV-Vis, fluorescence and synchronous fluorescence spectroscopic techniques. The results indicated a high binding affinity of the complex to BSA. In vitro anticancer activity of the complex was evaluated against A549, Jurkat and Ragi cell lines by MTT assay. The complex was remarkably active against the cell lines and can be a good candidate for an anticancer drug. Theoretical docking studies were performed to further investigate the DNA and BSA binding interactions.

Full text of DOI:10.1016/j.saa.2019.117593

Journal Pre-proof
A novel Cu(II) distorted cubane complex containing Cu O core as the first
4 4
tetranuclear catalyst for temperature dependent oxidation of 3,5-di-tert-butyl catechol
and in interaction with DNA & protein (BSA)
Zahra Asadi, Leila Zarei, Maryam Golchin, Eliska Skorepova, Vaclav Eigner, Zahra
Amirghofran
PII:
S1386-1425(19)30983-7
https://doi.org/10.1016/j.saa.2019.117593
SAA 117593
DOI:
Reference:
To appear in: Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
Received Date: 8 August 2019
Revised Date: 1 October 2019
Accepted Date: 1 October 2019
Please cite this article as: Z. Asadi, L. Zarei, M. Golchin, E. Skorepova, V. Eigner, Z. Amirghofran,
A novel Cu(II) distorted cubane complex containing Cu O core as the first tetranuclear catalyst for
4 4
temperature dependent oxidation of 3,5-di-tert-butyl catechol and in interaction with DNA & protein
(BSA), Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2019), doi: https://
doi.org/10.1016/j.saa.2019.117593.
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For Table of Contents Only
A new synthesized tetra-nuclear Cu(II) distorted cubane complex containing Cu4O4 core;
nominated ([CuL]4); exhibited excellent catecholase activity in aerobic oxidation of 3,5-di-tert-
buthyl catechol to its o-quinone. The kinetics and mechanism of the oxidation of 3,5-DTBCH2
catalyzed by the complex, were studied at four different temperatures. Interaction of complex
with FS-DNA and bovine serum albumin (BSA) was investigated. In vitro anticancer activity
of the complex and DNA cleavage activity was evaluated.

A novel Cu(II) distorted cubane complex containing Cu4O4 core as the first
tetranuclear catalyst for temperature dependent oxidation of 3,5-di-tert-butyl
catechol and in interaction with DNA & protein (BSA)
a
Zahra Asadi , Leila Zareia, Maryam Golchina, Eliska Skorepovab, Vaclav Eignerb, Zahra
Amirghofranc
a) Department of Chemistry, College of Sciences, Shiraz University, Shiraz 71454, Iran
b) Institute of Physics ASCR, v.v.i, Na Slovance 2, 182 21 Prague, Czech Republic
c) Department of Immunology and Autoimmune Diseases Research Center, Shiraz University of
Medical Sciences, Shiraz 71454, Iran
Abstract
The tri-dentate Schiff base ligand 3-(2-hydroxyethylimino)-1-phenylbut-1-en-1-ol (L)
produced the tetra-nuclear Cu(II) distorted cubane complex which contain Cu4O4 core, upon
reaction with Cu(II)acetate.H2O. The complex was structurally characterized by X-ray
crystallography and found that, in this tetrameric and tetra-nuclear distorted cubane structure,
each two-fold deprotonated Schiff base ligand coordinated to a Cu(II) center with their alcoholic
oxygens and imine nitrogens and formed six and five-membered chelate rings. At the same time,
*
Corresponding author. E-mail address: zasadi@shirazu.ac.ir
Tel: +98 713 6137118; Fax: +98 713 646 0788
1

each ligand bridged to a neighboring Cu(II) atom by its alcoholic oxygen, thus the metal centers
became penta-coordinated. The copper(II) complex with µ-ɳ2-hydroxo bridges and Cu….Cu
distance about 3Å was structurally similar to the active site of natural catechol oxidase enzyme
and exhibited excellent catecholase activity in aerobic oxidation of 3,5-di-tert-butyl catechol to
its o-quinone. The kinetics and mechanism of the oxidation of 3, 5-DTBCH2 catalyzed by [CuL]4
complex, were studied at four different temperatures from 283 to 313K by UV-Vis spectroscopy.
Interaction of [CuL]4 complex with FS-DNA was investigated by UV-Vis and fluorescence
spectroscopy, viscosity measurements, cyclic voltammetry (CV), circular dichroism (CD) and
agarose gel electrophoresis. The main mode of binding of the complexes with DNA was
intercalation. The interaction between [CuL]4 complex and bovine serum albumin (BSA) was
studied by UV-Vis, fluorescence and synchronous fluorescence spectroscopic techniques. The
results indicated a high binding affinity of the complex to BSA. In vitro anticancer activity of the
complex was evaluated against A549, Jurkat and Ragi cell lines by MTT assay. The complex
was remarkably active against the cell lines and can be a good candidate for an anticancer drug.
Theoretical docking studies were performed to further investigate the DNA and BSA binding
interactions.
Keywords: Cu(II) cubane complex; catecholase activity; FS-DNA; BSA; docking study;
anticancer activity.
Introduction
Currently, metal-organic molecules are developed by using organic/inorganic bridging
ligands, which create di- or poly-nuclear clusters, by one-, two- or three-dimensional self-
assembly [1, 2]. The organic ligands and the reaction conditions effect on the composition and
2

topology of the molecules. Among the poly-nuclear clusters, tetranuclear copper (II) complexes
with a Cu4O4 cubane core are particularly interesting. These complexes have medium size; they
are not as small as simple binuclear complexes, nor as large as the bulky poly-nuclear
complexes, which effects the magnetic properties of these complexes. Cubane-like complexes
are classified according to two different methods based on the Cu-O distances [3] and the
Cu….Cu distances [4].
Cu complexes have been used in various areas of research, such as crystal engineering [5, 6]
supramolecular chemistry, molecular magnetism [7, 8], to mimic the active sites of metallo-
enzymes and as an anti-cancer treatment [9-13]. Since the discovery of cis-platin as an efficient
anticancer drug, the pharmaceutical research has focused on other metal-based drugs for cancer
therapy [14, 15]. Because of the serious side effects of cis-platin as a covalent DNA binder [16],
researchers now focus on the less toxic, more-efficient and target-specific anticancer drugs with
non-covalent mode of binding. The most important limitations of the drugs under development
are their side-effects and cell membrane resistance [17]. Copper (II) complexes are promising
anticancer drugs of this type. Up to now, DNA-binding, cytotoxicity, the ability of DNA-
cleavage and the possible utility of the Cu(II) complexes in photodynamic therapy have received
considerable attention.[18-23] Recently many researches focused on the interaction of copper(II)
complexes with serum albumins, e.g. BSA & HSA, because, same as for DNA, proteins are one
of the targets for anticancer drugs [9, 18, 24-26].
Beside the potential for the cancer treatment, we have decided to study also the catecholase
oxidation activity of our Cu complex. The oxidation of reagents with the atmospheric oxygen in
mild conditions is of great importance especially in synthetic and industrial processes. In
biological systems, this is done by oxidation catalysts such as metallo-proteins, which contain
3

copper or iron metals [27]. In type (III) copper proteins, e.g. hemocyanin, tyrosinase, catechol
oxidase, a coupled binuclear copper center is the active site. Certain copper(II) complexes with
oxide bridges can oxidize different kinds of o-diphenols (catechols) to o-quinones with
simultaneous 2e- reduction of O2 to H2O [28]. This process is called the catecholase activity. The
active site of the catecholase protein contains two copper (II) centers (Cu….Cu distance is about
2.9Å), which are connected through di-µ-hydroxo bridges. Each Cu(II) center has a trigonal
pyramidal structure with three histidine nitrogens. To mimic the catecholase activity of the
bicopper(II) complexes, the structural parameters must be considered. The geometry of
bicopper(II) complexes affects the catalytic activity [29]. For example, the mononuclear square-
planar copper(II) complexes are less active than mononuclear non-planar copper(II) complexes.
In the binuclear copper(II) complexes, the Cu….Cu distance must be less than 5Å for the activity
to be retained. One explanation is that, for the electron transfer to be possible, the two metal
centers must be in an appropriate distance, in which it is possible for the two hydroxyl oxygen
atoms of catechol to bind to the metals and act as a bridging ligand [10].
We have chosen 3,5-di-tert-butylcatechol as a suitable model substrate to identify new
efficient oxidation catalysts. Considering the above-explained geometric requirements, we aimed
to synthesize model complexes containing at least two copper centers in close proximity.
Overally, according to the importance of synthesizing the artificial enzymes, and because of
the significance of mimicking the anti-cancer metal-based drugs, designing a complex with all
these properties is a success. Thus, in this paper we synthesized and characterized a tetra-nuclear
copper(II) complex with µ-ɳ2-hydroxo bridge as a potential structural and functional model for
the active site of the catechol oxidase enzyme. We also provide the results related to the
4

copper(II) complexes ability to function as anti-cancer agents, particularly the affinity of the
complex to bind to HS-DNA & BSA and the ability of the complex for DNA cleavage.
Experimental Section
Material and methods
All used materials, instruments and reaction conditions are mentioned in the supporting
information.
Synthesis and characterization
The Synthesis route of 3-(2-hydroxyethylimino)-1-phenylbut-1-en-1-ol (L) (Fig. S1) and
1
[CuL]4 complex with HNMR, FT-IR, Elemental Analysis, UV–Vis. spectral data of the
compounds are mentioned in the supporting information.
X-ray structure analysis
A saturated solution of the complex in a mixture of chloroform and n-hexan solvents was
allowed to evaporate at room temperature for two weeks and then green crystals of tetranuclear
copper compound were obtained. The X-ray diffraction measurements were carried out at 120K
on a Xcalibur, Gemini ultra diffractometer using the MoKα radiation ( λ = 0.71073 Å) from a
fine-focus sealed X-ray tube with a graphite monochromator and CCD detector Atlas S2. The
data were corrected for absorption using the CrysAlisPro software. The structure was solved by
the charge-flipping method by program Superflip [30] and refined by full matrix least squares on
F2 with JANA2006 program [31].
Catecholase activity
5

The catalytic activity of [CuL]4 complex for oxidation of 3,5-di-tert-butylcatechol (3,5-
DTBCH2) to 3,5-ditert-butylquinone (3,5-DTBQ) under aerobic condition (using only
atmospheric oxygen) were monitored spectrophotometrically by using a Lambda 25 Perkin–
Elmer spectrophotometers. The reaction conditions and formulas are mentioned in the supporting
information.
DNA binding studies, Molecular docking of DNA, DNA cleavage experiment and ethidium
bromide (EB) competitive study, Protein-binding studies, Molecular docking to proteins
The reaction conditions and methods of the experimental and theoretical studies are
mentioned in the supporting information.
In vitro anticancer studies
The cytotoxic effects of the complex against various cell lines comparing with cis-platin
were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT)
colorimetric assay as previously described [32]. Cell lines including Jurkat (human T cell
leukemia), Raji (Burkitt's lymphoma) and A549 (lung carcinoma) were cultured in RPMI 1640
medium (Sigma, St. Louis, MO) containing 10% fetal calf serum (Gibco, Berlin, Germany) at
37ºC in a CO2 incubator until confluent. Jurkat and Ragi cells at 15×103 cells/100µl and A549
cells at 7.5×103 cells/100µl were seeded into 96-well culture plates in triplicate. Cells were
treated with various concentrations of the complex (0.1–100 µg/mL) for 48 h. Negative control
was cells treated only with DMSO solvent at a concentration equal to the test wells and positive
control was cells treated only with cis-platin. After incubation time, 10µL MTT solution (5mg
mL-1 in RPMI medium) was added for 4 h and then cells treated with 100 µl DMSO to dissolve
formazan crystals while shaking. The optical density (OD) of the wells were measured at 570nm
6

with a 630nm reference wavelength using a microplate reader (Bio-Tek’s, VT). The percent
inhibition of cell growth was calculated using the following formula; [1– (test OD/negative
control OD)] × 100. Fifty percent cell growth inhibition (IC50) values were determined from
percent inhibition graph for different concentration the complex.
Results and discussion
Synthesis and characterization of compounds
Synthesis route of [CuL]4 complex is shown in Scheme 1. Schiff base ligand (L) was
synthesized by the condensation of benzoylacetone and 2-aminoethanol in 1:1 mole ratio. L
seems to behave as a tridentate ligand. After addition of Cu(II) acetate to the ethanolic solution
of L, a cubane like complex was synthesized. The affinity of Cu(II) center for a penta-
coordinated geometry with John-teller distortion produced a distorted cubane complex
containing Cu4O4 core.
7

a)
OH
CH3
OH
CH3
NH2
Toluene
N
O
+
H
OH
Reflux
5h
H
L
OH
CH3
b)
N
CH3
N
O
Cu
R.T
O
O
O
L
+
Cu(II)acetate.H2O
O
Cu
O
3 h
Cu
N
Cu
H3C
O
N
O
CH3
Scheme 1. Synthesis route of [CuL]4 complex.
1
The synthesized ligand and complex were identified by FT-IR, H NMR and elemental
analysis. Redox properties of the Cu(II) complex were studied by cyclic voltammetry. The
single-crystals of complex were prepared for X-ray crystallography. The complex was stable in
atmosphere and soluble in DMSO, methanol and ethanol and insoluble in non-polar solvents.
1H NMR spectrum of 3-(2-hydroxyethylimino)-1-phenylbut-1-en-1-ol (L) carried out in
DMSO-d6 (Fig. S2) and CDCl3 (Fig. S3). In DMSO-d6, a singlet signal for OHa at 11.34 ppm
and a multiple signal for aromatic protons at 7.81-7.40 ppm were observed additionally, a singlet
a
signal at 5.72 ppm for C=CH, a multiple signal at 4.93 ppm for OHb and a triplet signal for CH2
b
protons at 3.55 ppm (CH2 was hidden under DMSO signal) and a singlet signal for the methyl
protons at 2.05 ppm were assigned. In order to distinct between the signals of OHb and Ha one
8

drop of D2O was added to the DMSO-d6 solution of L. Hydrogen bonding between OHa, OHb
and D2O removed the related signals of hydroxyl groups. On the other hand, hydrogen bonding
b
caused a slight shift for DMSO signals and so the signal of CH2 could be seen at 3.52 ppm and
a
CH2 at 3.35 ppm in this spectrum (Fig. S2). To confirm the existence and the signal positions of
a
b 1
CH2 and CH2 , H NMR analysis in CDCl3 was repeated. In this spectrum, a singlet signal for
OHa at 11.51 ppm, a multiple signal for aromatic protons at 7.85-7.37 ppm, a singlet signal for
a
b
C=CH proton at 5.66 ppm, a triplet signal for CH2 protons at 3.83 ppm, a quartet signal for CH2
at 3.48 ppm, a singlet signal for OHb at 2.62 ppm and a singlet signal for the methyl protons at
2.05 ppm were observed (Fig. S3).
FT-IR spectrum of the 3-(2-hydroxyethylimino)-1-phenylbut-1-en-1-ol (L) (Fig. S4) was
indicated by the presence of strong imine (C=N) bands at 1604 cm-1 and OH vibrations at 3328
cm-1 [33, 34]. In the complex (Fig. S5), υC=N was generally shifted to lower frequencies relative
to the free ligand, indicating a decrease in the C=N bond order due to the coordination of the
imine nitrogen to the metal and back bonding from the Cu(II) to the π* orbital of azomethine
group [35]. Also, C-O stretching vibrations shift toward lower values as a result of coordination
of the oxygen to the metal ion [34]. Vibrations around 3448 cm-1 was attributed to the presence
of lattice and coordinated water [36]. Medium-weak bands at 3062 cm-1 is related to (C-H)
modes of vibrations [37]. The ring skeletal vibrations (C=C) were consistent in the region of
1440–1465 [38]. In the lower frequency region medium-weak bands observed at 551–563 and
435–458 cm-1 which have been assigned to (M–N) and (M–O) vibrations, respectively [39, 40].
The UV-Vis spectra (200-700nm) of the ligand in Tris-HCl buffer solution (pH 7.2) (10 µM)
and complex in methanol/Tris-HCl buffer solution (30 µM) were recorded. The electronic
absorption spectrum of the complex, displayed three specific absorption bands. Two absorption
9

bands at 209 and 256 nm were assigned to the π-π* transition in aromatic rings and azomethine
groups. The first band at higher energies were attributed to π→π* transitions associated with the
phenyl ring and the second band at lower energy arise from π→π* transitions associated with the
carbonyl and azomethine chromophores. A wide intense absorption band at 346 nm was
attributed to the n-π* transition in carbonyl group and oxygen of Schiff base to phenyl ring (Fig.
S6). For the free ligand, the band in the region 346 nm was attributed to the n→π* transition of
the imine chromophore, and the band in the higher energy region at 247 nm was assigned to the
π→π* transition of the phenyl ring [41-44] (Fig. S7).
Crystal structure of [CuL]4
The molecular structure of tetra-nuclear copper (II) complex along with atom labeling is
shown in Fig. 1. Crystallographic data and bond lengths and bond angles of the complex are
listed in Tables 1 and S1.
10

Fig. 1. The [CuL]4 complex with thermal ellipsoids at 50% probability level (except the cubane
cube, which is highlighted and displayed as balls) and the atom-labeling scheme. The labels of H
and C atoms are omitted for clarity.
Table 1. Crystal data and structure refinement for [CuL]4 complex
Formula of refinement model
Temperature [K]
Crystal system
C48 H52 Cu4 N4 O8, 3.195(O)
120
Trigonal
R 3
Space group
11

a [Å]
29.951(1)
29.951(1)
14.7438(5)
90
b [Å]
c [Å]
α [˚]
β [˚]
90
γ [˚]
120
V[Å3]
11454.1(7)
9
Z
D[gcm-3]
1.459
µ [mm-1]
1.709
F(000)
5162.0
R(F2>3σ) (reflections)
wR( F2) (reflections)
0.0203 (12347)
0.0579 (12936)
The synthesized complex created a tetrameric and tetra-nuclear cubane complex, in which
each two-fold deprotonated Schiff base ligand coordinated to a Cu(II) center with their hydroxyl
oxygen and imine nitrogen atoms and formed six and five-membered chelate rings. At the same
time, each ligand bridged to a neighboring Cu(II) atom by its hydroxyl oxygen (CH2CH2OH)
thus the metal centers were penta-coordinated (three donor atoms from one Schiff base ligand,
12

two oxygens from two other neighboring Schiff base ligands). The torsion angles between six
and five-membered chelate rings were in the range ±178.7-179.8° confirming that these two
rings were almost coplanar (Fig.2) (Table S1).
Fig. 2. The torsion angle between six and five-membered chelate rings.
The tetramer formed as a distorted cube, in which four Cu(II) atoms and four oxygen atoms
alternatively occupied the vertices. In this distorted cube, four of the twelve edges (Cu-O bonds)
were longer. The eight Cu-O shorter bonds were about 1.9 Å and out of the longer ones, three
were about 2.3 Å and one was 2.592 Å. Because the six Cu….Cu distances are all similar and
about 3 Å in the distorted cube (Cu1….Cu2 = 3.246 Å, Cu1….Cu3 = 3.253 Å, Cu1….Cu4 =
13

3.097 Å, Cu2….Cu3 = 3.130 Å, Cu2….Cu4 = 3.388 Å, Cu3….Cu4 = 3.138 Å) the structure is
classified as [6+0] cubane complex [4] (Fig. S8).
The geometry around each Cu(II) center was a distorted square pyramid (Fig. S9) with τ
values of 0.17, 0.06, 0.19, 0.16 for Cu1, Cu2, Cu3, Cu4, respectively. A. W. Adison et.al [45]
determined τ=1 for a regular trigonal bipyramide and τ=0 for a regular square pyramide by using
the formula: ꢀ = (ꢁꢂꢃ) where α, β are the angles between each trans donor atoms forming the
ꢄꢅ
plane in a square pyramidal geometry (Table 1). τ value is applicable for five coordinate
structures.[45] By comparing the τ values, we see that distortion from square pyramidal structure
around Cu2 was less than around the other Cu centers. Moreover, the degree of distortion from
square pyramidal structure for Cu1, Cu3, Cu4 was almost the same. The four basal positions of
each square pyramide around a Cu center contained three oxygen and a single nitrogen atom. An
oxygen occupied the apical position. Bond length of basal Cu-O and Cu-N were 1.906-1.967 Å.
The bond lengths for the apical Cu-O were in the range 2.375-2.592 Å due to Jahn-Teller
distortion.
The packing diagram of complex is shown in Fig. 3. Nine independent distorted cubane are
packed in a unit cell (Z=9). There are no regular H-bond donors present in the complex, so there
is no H-bonding present in the structure. Neighboring [CuL]4 units are connected through edge-
to-face π-π interactions between the benzene rings of the ligand. Beside the complex itself, the
structure contains disordered low-occupancy water molecules as a solvent, making the structure
a non-stoichiometric hydrate. On average, there are 3.195 water molecules per one [CuL]4
complex.
14

Fig. 3. Crystal packing of the complex. Disordered water molecules shown as red balls.
Kinetic studies of catecholase activity
Mechanistic studies on catechol oxidation by the [CuL]4 complex. The substrate 3,5-di-
tert-butylcatechole was used to study the catecholase activity of the model complex [CuL4]
because of its low redox potential which accelerate its oxidation to the corresponding quinone
and the presence of bulky tert-butyl substituents which prevent further reactions such as ring-
opening and polymerization and also the stability and presence of a characteristic band around
400nm of the resulted quinone that help the monitoring of the reaction. (Scheme S1)
Different approaches have been proposed for the mechanism of catecholase activity of Cu(II)
complexes base on the crystallographic, spectroscopic, structural and chemical evidence. For the
initial step, deprotonated catechol or its derivatives should bind to Cu(II) center(s). Different
15

binding modes of the catechol (or deprotonated catechol) to copper(II) centers has been proposed
as: i) ɳ2 chelating catechol, ii) ɳ2:ɳ1 bidentate bridging, iii) syn-syn bidentate bridging, iv) anti-
anti bidentate bridging, etc [10]. According to structural properties (metal-metal distances, nature
of bridging ligands between Cu centers, rigidity of the structure) and electrochemical properties
of the complexes, each mode of binding mentioned above is possible. However, it was reported
that the highest catecholase activity was observed for the complexes, which bound catechol in a
bidentate bridging manner. It means that the Cu…Cu separation in these complexes must be less
than 4 Å, otherwise simultaneous binding of catechol to both Cu centers is prevented. It is worth
noting that small bridging ligands bring Cu centers to the close proximity of each other and thus
promote the catalytic activity. Examples of such bridging ligands are: hydroxides, alkoxides, and
phenoxides [10].
In the [CuL]4 complex, hydroxide moiety of the Schiff base ligands bridge between three
Cu(II) centers and bring them close to each other. All the Cu...Cu distances are in the range
3.097-3.388 Å, which allow 3, 5-ditertbutylcatecholate dianion to bind as a bridging ligand in a
syn-syn fashion (Fig. S10).
Casella and co-workers [46] proposed two consecutive steps for the catalytic reaction
including step one: a fast stoichiometric reaction between a molecule of 3,5-DTBCH2 and
dicopper(II) complex and step two: a slower catalytic reaction. They also proposed that, in the
first stoichiometric step, electron transfer between the bound catechol and dicopper(II) centers
occurred, thus, the first step, to some extent, is dependent to CuII/CuI redox potential. In the
catalytic process, two copper (II) centers reduce to copper (I) simultaneously and one molecule
of 3, 5-DTBCH2 is then oxidized to the related quinone molecule. Dicopper (I) centers reoxidize
16

to copper (II) centers in a relatively slow step in the presence of O2. A proposed cycle of
mechanism is shown in Scheme 2.
2H+
OH
OH
II
O
Cu
O
Cu
Cu
O
II
O
Cu
O
O
II
Cu
O
O
O
Cu
O
II
O
Cu
O
Cu
H2O
I
O
Cu
I
O2
Cu
O
O
I
Cu
O
O
I
Cu
O
Scheme 2. Catalytic cycle for the oxidation of 3, 5-DTBCH2 catalyzed by [CuL]4 complex.
To confirm this hypothesis, we have performed the following: In a typical procedure; after
mixing 3, 5-DTBCH2 with the complex, the spectral changes at intervals of 4 min were
monitored (Fig. 4). The quinone band at 410 nm grew; therefore, the oxidation of catechol has
occurred.
17

1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
after 56 min
after 4 min
385
405
425
445
465
485
Wavelength (nm)
Fig.4. Spectral profile showing the increase of quinone band at about 410 nm after the addition
of 600 fold of 3,5-DTBCH2 to a solution of complex (50µM) in DMF at 283K. Spectra were
recorded after each 4 min.
The kinetics of the oxidation of 3,5-DTBCH2 catalyzed by [CuL]4 complex were studied
under pseudo first order conditions. The ratio of [3, 5-DTBCH2]/[complex] varied from 10 to
800 fold, in which 3,5-DTBCH2 concentrations were varied in the range 500 (µM) to 40000
(µM) and the complex concentration was constant at 50 µM. A first-order dependence was
observed at these concentrations of the substrate. The experiments were done at four different
temperatures from 283-313K, under aerobic conditions. The reaction process of the mixture of 3,
5-DTBCH2 and complex at the maximum of the 3, 5-DTBQ band (410 nm) were followed for a
period of 90 min. For the blank experiment containing 3,5-DTBCH2 without catalyst, no
formation of the 3,5-DTBQ was observed up to 2h in DMF. Pseudo-first-order rate constants
18

(kobs) in different temperatures are available in Table 2. kobs values during the temperature change
from 283 to 303K were obtained in catechol concentrations from 0.01 to 0.04M (100-800 folds)
and at 313K from 0.0005 to 0.008M (10-140 folds). At 313K, the reaction at catechol
concentrations from 0.01 to 0.04M was too fast to obtain proper spectral changes thus lower
concentrations of catechol were used. These clearly revealed that the catecholase activity of the
complex [CuL]4 is temperature dependent.
Table 2. Pseudo-first-order rate constants, 104kobsa(s-1), for the oxidation of 3,5-DTBCH2
(0.0005-0.04 M) catalyzed by [CuL]4 complex (50µM) in DMF at different temperatures (283-
313K).
[catechol]
(M)
0.0005
0.002
0.0035
0.005
0.0065
0.008
0.01
0.015
0.02
0.025
0.03
0.035
0.04
T(K)
283
0.55
0.63
0.68
0.72
0.78
0.82
(0.01) (0.01) (0.01) (0.01) (0.01) (0.01)
0.87 0.95 1.14 1.22 1.33
(0.02) (0.02) (0.02) (0.01) (0.02) (0.01)
1.01 1.24 1.55 1.70 1.95
(0.01) (0.01) (0.01) (0.04) (0.07)
293
303
0.69
0.97
(0.01)
313
1.13
1.23
1.33
1.46
1.54
1.65
(0.01)
(0.02)
(0.01)
(0.02)
(0.03)
(0.09)
a) The numbers in parentheses are the standard deviations of kobs.
k2 values are obtained from the slope of the linear plots of kobs versus [catechol] (Fig. S11).
By increasing the temperature, k2 values increased (103k2 = 1.06 (283K), 2.55 (293K), 4.17
(303K), 6.95 (313K)) indicating an endothermic oxidation reaction which might be very
important in designing more efficient catecholase model complexes.
The activation parameters were obtained from Eyring plots of ln(k2/T) vs. 1/T at four
different temperatures (Fig. S12). The positive value of free energy at 293 K (∆G# =∆H#-T∆S#
=28.705 KJ mol-1) indicated that the oxidation process was nonspontaneous. The positive ∆H#
values (42.788 KJ mol-1) confirmed endothermic process and the positive value of ∆S# (48.064 J
mol-1 K-1) revealed that the disorder of the system was raised during the process due to the
19

production of H2O molecules. In each catalytic cycle, one mole of 3, 5-DTBCH2 produced one
mole of 3, 5-DTBQ, successively, atmospheric O2 diffuse in the solution and one mole of H2O
was produced, thus the disorder of the medium was increased.
To investigate the enzymatic kinetics of catecholase activity of various copper(II) complexes
Michaelis–Menten equation was used repeatedly. Many reports in this field revealed that the
reaction rate might be dependent to the concentration of the substrate (catechol), the kind of
catalyst (metal complex), the concentration of dioxygen in the reaction medium and the kind and
concentration of some additives such as dyhydrogenperoxide and Kojic acid[10]. It is worthy to
mention that few studies repot the dependence of the reaction rate to the catalyst concentration
[47].
Similar to other enzymatic mimicking complexes, [CuL]4 revealed saturation kinetics and the
treatment was investigated bases on Michaelis–Menten model. Michaelis−Menten constant (KM),
maximum initial rate (Vmax) were calculated by Michaelis–Menten equation and Lineweaver–
Burk plot [48] (Fig. S13) . The turnover number (kcat) value can be calculated by dividing the
Vmax value by the concentration of the complex. The determined kinetic data, Vmax = 4.7461×10-5
Mmin-1, KM = 0.0178 M, and kcat = 56.95 h-1.
Because the solvent is one of the main factors in the enzymatic activity of the catalysts, some
kcat of various copper (II) complexes in DMF was reported for comparison: [{Cu2L(µ-
OH)(H2O)}(µ-ClO4)]n(ClO4)n , kcat = 39.0h-1 [1]; [{Cu2L(µ1,1-N3)(ClO4)}2(µ1,3-N3)2], kcat
=
39.9h-1 [1] thus, [CuL]4 complex is located in the upper range of functional models for
catecholase active complexes.
DNA-binding studies
20

DNA is the primary pharmacological targets for anticancer drugs thus studying the
interaction of metal complexes with DNA is of great importance. Different techniques are used
commonly to study the binding strength and binding modes. Some of these techniques are purely
experimental and some of them are theoretical.
Electronic absorption spectroscopic studies. In many DNA interaction studies, UV-Vis
absorption spectroscopy has been employed as a simple instrumental technique, which is used
for either studying the DNA stability or drug-DNA interactions. In this method the changes in
UV-Vis absorption spectra of the drug (complex) or DNA is monitored. An easy way to
investigate the interaction between drug and DNA, is comparing the shift in the maximum of the
absorption band of drug when it is free in solution or is bound to DNA [49]. The extent of this
shifting could be related to the strength of the interaction between DNA and drug. Fig. 5
describes the interaction of our complex with DNA. The absorption band at 346nm revealed
hypochromism. Also two isosbestic points near 367 nm and 302 nm were recognized. These
spectral changes suggest covalent binding of complex to DNA or intercalation, in which a strong
stacking interaction between the aromatic chromophore of the complex and base pairs of DNA
has occurred [19]. The absorption band at 346 nm revealed a blue shift about of 6 nm along with
hyperchromism (21%). These spectral changes correspon to groove binding of complex to DNA
[50]. Hyperchromism along with hypsochromism suggest an electrostatic attraction or groove
binding between Cu(II) complex and DNA which can cause changes in DNA conformation
including partial uncoiling of the helix structure of DNA [51, 52].
Besides the qualitative investigation of the interaction, by calculating Kb, an insight into the
quantitative behaviour was can be gained by using these equations (1a ,1b) [53, 54]
21

2Kꢈ²Cꢊ [DNA]
ꢋ/ꢉ
b − (bꢉ −
)
εꢆ − εꢇ
εꢈ − εꢇ
s
=
(1a)
(1b)
2KꢈCꢊ
Kꢈ[DNA]
b = 1 + KꢈCꢊ +
2s
In these equations, the absorption coefficients are denoted by εa, εb and εf for the absorption
band (at a given DNA concentration) for the complex in its free form and for the complex in the
fully bound form, respectively. Kb and s values were obtained by nonlinear least squares fitting
of the experimental data to Eqs. (1a, 1b). The observed binding constant (104 M–1) corresponds
to the intercalation binding mode of interaction suggested above.
22

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
0.8
0.6
0.4
0.2
0
0
2
4
6
8
[DNA] (×10-4 M)
280
330
380
430
wavelength (nm)
Fig. 5. Absorption spectra of the complex (12 µM) in DMSO, in the absence and presence of
increasing amounts of DNA (0-110-220-330-430-540-650-750 and 860 µM). Inset: plot of (Ԑa-
Ԑf)/( Ԑb-Ԑf) vs.[DNA].
The intrinsic binding constant was calculated as Kb=1×104 M–1 and s value is 1.5. The Kb
value for ethidium bromide as a classical intercalator is 1×107 M-1, which is much larger than the
Kb of [CuL]4 complex. It is worthy to note that the Kb value for [CuL]4 complex is comparable to
some transitional metal complexes either mono- or multi-centers, which were bound to DNA
through intercalation or groove binding mode. For example: [Cu2(nap)4(H2O)2] Kb=2.27×104M-1,
[Cu2(dicl)4(H2O)2] Kb=1.74×104M-1, [Cu(nap)2(bipy)] Kb=3.86×104M-1 [Cu(dicl)2(phen)]
Kb=1.81×104M-1 with intercalation mode of binding and the Kb values of all compounds were
lower than that of the classical intercalator [55]. [Cu (imda) (5,6-dmp)] Kb=3.9×103 bound to
23

DNA through groove binding and [Cu (imda) (dpq)] Kb=1.7×104 through partial intercalation
[56]. [Cu (tdp) (bpy)]+ Kb=7.1×103M-1, [Cu (tdp) (phen)]+ Kb=9.0×104M-1, [Cu (tdp) (tmp)]+
Kb=7.0×105M-1, [Cu (tdp) (dpq)]+ Kb=9.0×105M-1 are examples of complexes with intercalation
mode of binding.[18]
Viscosity measurements. A critical test in proposing the mode of binding of the small
molecules to DNA is the viscosity measurement, which is a hydrodynamic measurement that is
very sensitive to the DNA length change. Classical intercalators locate between DNA base pairs
and cause an increase of the length of DNA and concomitantly its viscosity. However, non-
classical intercalators cause bending (or kinking) of the DNA helix and reduce the length of
DNA and concomitantly its viscosity. Groove binders, which bind in the grooves of DNA, cause
less changes (positive or negative) or no changes in DNA viscosity [57, 58]. The values of
relative specific viscosity (η/η0)1/3, where η is viscosity of pure DNA and η0 is the viscosity of
DNA in the presence of the different concentration of complex, were plotted against r (r =
[complex]/[DNA]). From (Fig. S14), it is concluded that non classical intercalation (i.e. van der
Waals interactions, groove binding or external binding) was the main mode of binding [59, 60].
Fluorescence studies (competitive binding affinity of the complex with ethidium
bromide). A common fluorescence probe, which can bind to DNA, is ethidium bromide (EB), a
planar cationic dye. In the presence of DNA, the fluorescence of EB increases according to its
intercalation between adjacent DNA base pairs. If the enhanced fluorescence quenches by
addition a second molecule, it competes with EB in binding to DNA. The base of displacement
technique is the quenching of fluorescence due to the displacement of EB from the DNA
sequence by another molecule. Quenching owns to the decreasing of the number of binding sites
on the DNA, which had been occupied by EB. The extent of quenching of EB/DNA can be
24

correlated straightforward to the extent of binding of the second molecule to DNA via
intercalation.[9, 19, 61, 62]
The fluorescence quenching spectra of the DNA-bound EB ([DNA]/[EB]=10) by addition of
[CuL]4 complex ([DNA]/[complex]=10, is shown in Fig. S15.
Quenching data were analyzed according to Stern−Volmer Equation 2 [63]:
ꢌꢅ
[ ]
= 1 + ꢍꢎ ꢏ
(2)
ꢌ
I0 denotes the emission intensity in the absence of quencher and I the emission intensity in the
presence of a quencher, Kq is the quenching constant, and [Q] is the quencher concentration. The
Kq value is 1.77×105 that were obtained from the slope of the plot of I0 /I versus [Q] (Inset Fig.
S15). The value of apparent DNA binding constant (Kapp) was calculated using Equation 3.
[
]
[
]
ꢍꢐꢑ ꢒꢓ = ꢍꢔꢕꢕ ꢖꢗꢘꢙꢚꢛꢜ
(3)
where KEB = 1.0 × 107 M−1 and [EB] = 1.25 µM [complex] is the concentration of complex
causing a 50% reduction in the fluorescence intensity of EB. Kapp calculated as 7.89 ×106 M−1.
The higher value of Kapp revealed that [CuL]4 bind to FS-DNA by intercalation.
Circular dichroism spectral analysis. It is obvious that during drug-DNA interactions the
morphology of DNA changes to some extent. A useful technique for diagnosing these changes
and proposing the mode of binding is circular dichroism (CD). It means that drug-DNA
interactions can affect the structure of DNA and thus the intrinsic CD spectrum of DNA must be
changed. The intrinsic CD spectrum of FS-DNA is characterized by a positive band at 275 nm
assigned to the base stacking and a negative band at 245nm assign to the right handed helicity of
B-form DNA [64, 65], which the latter is very sensitive to the mode of drug-DNA binding.
25

Electrostatic interaction or groove binding of complexes with DNA reveals less or no alteration
on the helicity band or base stacking band but intercalators perturb both the bands [66]. As
revealed in Fig. 6, by addition of the Cu(II) complex to the DNA solution, no significant changes
in the form and no distinguished shift in the CD spectrum of B-DNA was recognized, which
indicated that the FS-DNA kept the double helix B-form structure in the presence of Cu(II)
complex. The only indicated diffrence is that by adding the complex to the DNA solution, the
intensity of the both bands at 275 nm 245nm increased. The increasing of the negative peak
intensity (245 nm) showed that the interaction of the complex with DNA tightened the double
helix structure of DNA [67]. Thus, the possible mode of binding concluded as groove binding or
electrostatic interaction.
26

DNA
15
10
5
[DNA]/[Complex]=40
[DNA]/[Complex]=20
0
-5
230
250
270
290
310
330
Wavelength/nm
Fig. 6. The CD spectra of DNA (200µM) in the presence of complex in Tris–HCl buffer
solution/0.5% DMSO. The concentrations of complex were 0, 5µM and 10µM.
Electrochemical behavior of complex in the presence of DNA. Cyclic voltammograms
were run in the potential range from -1.00 to +1.00 V in DMSO (starting value: -1.00 V). The
cyclic voltammograms of [CuL]4 complex revealed two anodic peaks at 0.873 V, 0.373 V and
two cathodic peaks at -0.024 V and -0.436 V (Fig. S16). Upon addition of DNA solution to the
metal complex solution (10 mL, 103 µM), the anodic peaks shifted to a more positive value of
0.904 V and negative value of 0.357 V and a new anodic peak at 0.016 V appears. The cathodic
peaks shifted to value that is more negative. Therefore, there is a mixed mode of binding
(intercalation and groove binding) between metal complex and DNA. The observed decrease in
27

the anodic current and an increase in peak currents of the cathodic waves in the CV responses,
suggesting the binding of the complex to the large slowly diffusing DNA molecule [68].
DNA cleavage and ethidium bromide competitive studies. The interaction between DNA
and complex is important due to its effect on cell cycle arrest, gene expression modification and
DNA cleavage activity. These incidences caused special programmed cell death means
apoptosis. The cleavage of DNA including single or double strand cleavage caused by metal
complex alone or with the cooperation of oxidant like H2O2 and transition metal ions like Cu2+.
In comparison, double strand breakage induces the most harmful damage to the cell because
cellular repair systems such as NHEJ (Non-Homologous End Joining) repair the strand less
accurate and with strand insertion/deletion, which cause genome instability and finally apoptosis.
As an applicable method, gel electrophoresis was used to assess the structural changes induced in
DNA by the Cu(II) complex. In this manner, supercoiled (SC) plasmid pBluescript KS (+) DNA
was incubated with various concentrations of the complex to study their DNA cleavage ability.
Agarose gel electrophoresis was used to monitor the conversion of supercoiled plasmid
pBluescript KS (+) DNA (SC DNA) to the nicked circular (NC DNA) and linear open circular
DNA (LC DNA). The control experiments suggested that untreated DNA (line 7 left) and
untreated DNA+50 µM H2O2 (line 7 right) did not show any notable DNA cleavage. As shown
in Fig. 7 the Cu(II) complex could not cleave DNA in physiological (PBS buffer) condition but
in the presence of H2O2 the complex converted supercoiled (SC) DNA to nicked circular (NC)
form in concentrations higher than 25 µM (Fig. 7). It may be interesting that Cu(II) complex
didn’t convert SC DNA to LC DNA despite the presence of aromatic moiety in the complex
structure[69]. These results suggested the strong DNA cleavage activity of [CuL]4 complex in
the presence of H2O2.
28