A novel water-soluble tetranuclear copper (II) Schiff base cluster bridged by 2, 6-bis-[(2-hydroxyethylimino)methyl]-4-methylphenol in interaction with BSA: Synthesis, X-ray crystallography, docking and cytotoxicity studies

Article abstract of DOI:10.1016/j.jphotochem.2018.05.016

A water-soluble tetranuclear copper (II) Schiff base complex bridged by 2,6-bis-[(2-hydroxyethylimino)methyl]-4-methylphenol, namely [Cu₄(L¹)₂(O)(OAc)₄].2H₂O was synthesized. The complex was character

Full text of DOI:10.1016/j.jphotochem.2018.05.016

Accepted Manuscript
Title: A novel water-soluble tetranuclear copper (II) Schiff
base cluster bridged by 2,
6-bis-[(2-hydroxyethylimino)methyl]-4-methylphenol in
interaction with BSA: synthesis, X-ray crystallography,
docking and cytotoxicity studies
Authors: Zahra Asadi, Maryam Golchin, Vaclav Eigner,
Michal Dusek, Zahra Amirghofran
PII:
S1010-6030(18)30249-1
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2018.05.016
JPC 11286
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
21-2-2018
12-5-2018
12-5-2018
Please cite this article as: Asadi Z, Golchin M, Eigner V, Dusek M, Amirghofran Z,
A novel water-soluble tetranuclear copper (II) Schiff base cluster bridged by 2, 6-bis-
[(2-hydroxyethylimino)methyl]-4-methylphenol in interaction with BSA: synthesis, X-
ray crystallography, docking and cytotoxicity studies, Journal of Photochemistry and
Photobiology, A: Chemistry (2010), https://doi.org/10.1016/j.jphotochem.2018.05.016
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A novel water-soluble tetranuclear copper (II) Schiff base cluster bridged by 2, 6-bis-[(2-
hydroxyethylimino)methyl]-4-methylphenol in interaction with BSA: synthesis, X-ray
crystallography, docking and cytotoxicity studies
Zahra Asadi*a, Maryam Golchina, Vaclav Eignerb, Michal Dusekb, 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
Graphical Abstract
1

Highlights
 A water-soluble tetranuclear copper (II) Schiff base complex bridged by 2,6-bis-[(2-
hydroxyethylimino)methyl]-4-methylphenol, namely [Cu4(L1)2(O)(OAc)4].2H2O was
synthesized.
 The interaction with bovine serum albumin (BSA) was assessed.
 In vitro cytotoxicity against A549, Jurkat and Raji cell lines by MTT assay was done.
 Theoretical docking studies were investigated.
Abstract
A water-soluble tetranuclear copper (II) Schiff base complex bridged by 2,6-bis-[(2-
hydroxyethylimino)methyl]-4-methylphenol, namely was
[Cu4(L1)2(O)(OAc)4].2H2O
synthesized. The complex was characterized by 1HNMR, FT-IR, elemental analyses and single-
crystal X-ray diffraction. The crystal structure analyses revealed that four Cu atoms were
located around a central O atom in a distorted tetrahedral geometry. Beside the two ligands, four
acetates also bridged the four Cu atoms. The interaction of the complex with Bovine serum
albumin (BSA) was explored by absorption and emission titration methods the calculated
binding constants revealed high affinity of the complex to BSA. The antitumor activity of the
complex against Jurkat, Raji and A549 cell lines was assessed by the MTT colorimetric method.
A strong activity against the cultured tumor cells was observed which makes the complex a
possible candidate as an anticancer agent. Further investigation of the BSA-complex
interactions was done by docking studies.
Keywords: tetranuclear copper (II) Schiff base complex; bovine serum albumin; docking;
cytotoxicity.
Introduction
In recent years, research of the poly-nuclear complexes has a rapid development. Beside the
fascinating molecular architecture, their applications including catalysis, fluorescence, gas
storage, and biological activity are also attractive [1]. Schiff base ligands which contain alkoxo
and phenoxo groups can bridge metal atoms in different modes and produce mononuclear,
dinuclear or tetranuclear complexes [1]. A phenolato ion can bridge two Cu(II) centers and in
this case the coordination number of each Cu(II) center range from 4 to 6 which is typical for
2

the coordination chemistry of Cu(II) [2]. Special attention has been focused on the discrete
tetranuclear clusters, cubane-like oxygen-bridged Cu4O4 polynuclear complexes [3-6].
Copper, among the transition metals, is a biologically active one. The accessible redox
potentials of copper complexes and their combination with dioxygen or hydrogen peroxide
make them an effective DNA-cleavage agent through oxidative pathways. Multinuclear
complexes have been used in order to increase the selectivity and efficiency of them as metallo-
nuclease, due to cooperative effects between the metal centers [7].
Recently, the catecholase activity of dicopper(II) complexes with organic blocking ligands
have been reported. Some copper(II) mononuclear complexes also exhibit this property. In 1998
the crystal structure of the catechol oxidase was determined; demonstrating the active site of a
hydroxo-bridegd dicopper(II) center which coordinated to three histidine nitrogens [8].
Metal complexes and metal ions can transport through proteins in the blood stream. This
property is important because many drugs including anticoagulants, anti-inflammatory,
tranquilizers, and general anesthetics are delivered in the blood through combination with
albumin [9]. Thus metal complexes that can bind to protein or DNA under physiological
condition are interesting in designing the metal based drugs. Among them, water-soluble, inert
and stable complexes, which contain spectroscopically active metal centers, are used as probes
for biological systems. For example, copper complexes are good candidates for replacing
platinum drugs and reveal better cancer inhibiting activity [10-15]. One of the important effects
of the copper-based antineoplastic agents is changing the metabolism of cancer cells which
causes different response between normal and cancer cells [11, 15]. It is worthy to note that the
normal function of cells is critically dependent on the copper concentration in them. Copper
homeostasis and metabolism are also crucial to various human cancers [15-17]. In comparing
the cancerous tissues and normal tissues it is revealed that the concentration level of copper is
very high in cancerous tissues. Therefore, cancer cells may represent a suitable, selective target
for copper-based agents [15, 18]. Also, it has been established that copper compounds delay cell
cycle progression, increase cell death in different cell cultures, and cause apoptosis in cultured
mammalian cells which is considered to be crucial for drugs [19-21].
Herein, we synthesized an amino-alcohol ligand, namely; 2,6-bis-[(2-hydroxy
ethylimino)methyl]-4-methylphenol (L1) (Scheme 1) and investigated its self-assembly with
copper (II) centers. The phenoxo group of the Schiff base ligand bridged metal centers and
created a tetranuclear complex. This complex exhibited cytotoxicity effect against A549, Jurkat
3

and Raji cell lines. The interaction with BSA was also investigated by UV-Vis, fluorescence,
and synchronous fluorescence spectroscopic techniques.
CH3
CH3
H
N
+ OH
N
C
H
O
H
N
+
N
n
O
OH
O
OH
CH3
CH3
HO
+
NH2
N
OH
N
HO
O
OH
O
OH
CH3
CH3
OH
OH
N
O
O
N
Cu
Cu
O
O
CH3
CH3
O
+
Cu(OAc) .H O
O
O
H3C
O
2
2
N
N
O
O
O
O
Cu
Cu
H3C
HO
OH
OH
HO
N
N
CH3
Scheme 1: Synthetic route of the tetranuclear copper (II) Schiff base cluster.
4

Experimental
Materials
Paraformaldehyde, phenylbutazone, 2-aminoethanol,
bovine serum albumin (BSA),
ibuprofen, hexamethylenetetramine, p-cresol, copper(II) acetate, methanol, ethanol, sulfuric
acid, acetic acid, toluene, diethyl ether, n-hexane, chloroform, dimethylsulfoxide (DMSO), Tris-
HCl buffer solution, NaCl, CDCl3 and DMSO-d6 for NMR spectroscopy, potassium bromide
(KBr) for FT-IR spectroscopy were obtained from Sigma and Aldrich, Fluka and Merck.
Instruments
The NMR spectra were measured by Bruker Avance DPX 250 MHz spectrometer. Perkin-
Elmer (LAMBDA 25) UV-Vis. spectrophotometer was used for UV-Vis. measurements which
was equipped with LAUDA-ecoline-RE 104 thermostats. Shimadzu FT-IR 8300 infrared
spectrophotometer and Thermo Finnigan-Flash 1200 were employed for FT-IR spectra and
Elemental analysis (C.H.N.). Melting points of compounds were measured with BUCHI 535. X-
ray diffraction measurements were made at 120K on a Gemini diffractometer of Rigaku Oxford
Diffraction using the mirror-collimated CuKα radiation from a classical sealed X-ray tube, and
CCD detector Atlas S2. All biological experiments were performed in triple distilled water at
pH 7.4, 1 mM Tris buffer and 5 mM NaCl was used to create a biological medium. Variation in
fluorescence and synchronous fluorescence spectra of the interactions were measured by a
Perkin Elmer (LS 45) spectrofluorimeter.
Synthesis of 2,6-bis-[(2-hydroxyethylimino)methyl]-4-methylphenol (L1)
The di-aldehyde precursor (2,6-diformyl-4-methylphenol) was prepared according to the
procedure published by Verani et. al. [22]. To a methanolic solution of 2,6-diformyl-4-
methylphenol (0.164 g, 1 mmol) was added 2-aminoethanol (0.12 ml, 2 mmol). The reaction
mixture was refluxed for 3h after solvent evaporation; an orange product was collected by
filtration, washed with a minimum volume of methanol and diethyl ether. The product was
purified by recrystallization from methanol [23].(Fig. 1)
1
Yield: 0.18 g (72%); color: orange; m.p: 145 ºC; H NMR (DMSO-d6 , ppm): 14.36 (1H, s,
OHa), 8.54 (2H, s, HC=N), 7.50 (2H, s, Ar-H), 4.68 (2H, s, OHb), 3.62 (8H, s, CH2 ), 2.23 (3H,
s, CH3 ); FT-IR (KBr, cm-1): 3348 (υ O-H), 1635 (υ C=N), 1604 and 1450 (υ C=C), 1072 (υ C-O);
Elemental Analysis, Found (Calc.)%: C13H18N2O3 (MW=250 g.mol-1) C: 62.46 (62.38); H: 7.55
(7.25); N: 11.30 (11.19)
5

CH3
N
OH a
N
OH b
b OH
Fig. 1. The structure of 2,6-bis-[(2-hydroxyethylimino) methyl]-4-methylphenol (L1)
Synthesis of [Cu4 (L1)2 (O) (OAc)4]. 2H2O
To a methanolic solution (10 mL) of copper (II) acetate.H2O (0.39 g, 2 mmol), a solution of L1
(0.25 g, 1 mmol) in methanol was added slowly with continuous stirring at room temperature.
The solution turned green immediately and stirred for 3h. The solution was kept for solvent
evaporation. The green precipitate was filtered off and washed with diethyl ether. Green crystals
suitable for X-ray crystallography were obtained in a solution of mixed solvents; methanol and
n-hexane by the solvent penetration method; in which the complex completely dissolve in
methanol while it is insoluble in n-hexane. Slow penetration of n-hexane in the methanolic
solution of complex creates green crystals. (Fig. 2)
Yield: 0.27 g (26%); color: green; m.p: 175-186 ºC; FT-IR (KBr, cm-1): 3417 (υ OH), 1581 (υ
C=N), 1627, 1455 (υ C=O diatomic bridge), 1627, 1411 (υ C=O one O attached to Cu), 1110 (υ C-O); Elemental
Analysis, Found (Calc.)%: C34H43Cu4N4O15.2H2O (MW=1037.94 g.mol-1) C: 38.28 (39.34); H:
4.89 (4.56); N: 5.43 (5.40).
6

CH3
OH
N
O
O
N
Cu
Cu
Cu
O
O
CH3
CH3
O
O
O
H3C
O
O
O
O
O
Cu
H3C
HO
OH
N
N
CH3
Fig. 2. The structure of [Cu4 (L1)2 (O) (OAc)4]. 2H2O
X-ray crystallography
A saturated solution of the complex in a mixture of methanol 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 data were collected on Gemini diffractometer with Atlas CCD
detector using mirror-collimated Cu-Kα radiation (λ = 1.54184 Å) and corrected for absorption
using the CrysAlisPro software. The structure was solved by the charge-flipping method by
program Superflip [24] and refined by full matrix least squares on F2 with JANA 2006 program
[25].
Protein-binding experiments
Fluorescence spectroscopy
Proteins reveal intrinsic fluorescence which makes the fluorescence spectroscopy a valid
technique in the study of drug-protein interaction. BSA reveals an emission band at 260 nm thus
wavelength range between 300-500 nm was selected to study the fluorescence emission spectra
of BSA while the excitation wavelength was at 280 nm. The procedure was as follow: 2.5 mL
of serum albumin solutions (7.2×10-8 M) (pH=7.4) was added to the quartz cell of 1 cm optical
path and titrated by consecutive additions of complex (5×10-5 M). The equilibrium access
7

during 3 min after each injection, thus the change in fluorescence emission was measured at this
point of time. As the complex revealed band around 260 nm, the complex band was eliminated
by injecting equal amounts of complex solution to both reference cell and sample cell during
titration.
UV-Vis spectroscopy
For the absorption titrations, free protein solution (BSA) (2×10-5M) in Tris-HCl buffer (pH =
7.4) were poured into the cuvette, and the absorption in the absence and presence of the complex
(2×10-3M) was monitored in the wavelength range from 250 to 500 nm at 293, 298, 303 and 308
K.
Site marker competitive experiments
Bovine serum albumin contains several cavities, which can accommodate the whole or part
of a metal complex. The preference of metal complex for particular cavity (or site) is examined
by site marker competitive studies. Some specific compounds are used for this goal, for
example, phenylbutazone that occupies site I of BSA is used as a site marker of the site I, and
ibuprofen as a site marker of the site II of BSA. An effective procedure was supplied by mixing
2.5 mL BSA (7.2×10-8 M) and 10 μL phenylbutazone (or ibuprofen) (1.8×10-5 M in 50/50
V/V% water/acetone) and consequent titration with complex solution.
Synchronous fluorescence spectral measurements
This method consists in simultaneous scanning of the excitation and emission
monochromator (at a fixed wavelength differences (Δλ) between them) for tyrosine and
tryptophan amino acids. The addition of the complex causes some structural changes of BSA,
and these structural changes effect on the synchronous fluorescence spectra at the wavelength
interval Δλ =15 nm (tyrosine residues) and Δλ = 60 nm (tryptophan residue) of BSA [26].
The cytotoxicity experiments
The cytotoxic effects of the complex against two human leukemia\lymphoma cell lines
(Jurkat and Raji) and a lung cancer cell line (A549) were measured by 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described [27]. The cell lines
were grown in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf
serum (Gibco, Berlin, Germany). Cells were cultured at 37ºC with 5% CO2 and 95% humidity
until confluent and after trypsinization (for A549) placed into new culture flasks. Jurkat and
8

Raji cells (15×103/well/100µL) and A549 cells (7.5×103 /well/100μL) were cultured in a 96-
well plate in the presence of different concentrations of the complex (0.1–100 µg/mL). Cells
treated only with dimethyl sulfoxide (solvent) at the same concentration used in the test wells
was used as negative control and cells treated only with cisplatin considered as positive control.
After 48 h, 10 μL MTT solution (5mg mL-1 in RPMI 1640 medium) was added to the plate and
incubation was continued for 4 h. Finally, DMSO was added to dissolve the insoluble formazan
and then the optical density (OD) at 570 nm and 630 nm (as a reference wavelength) was
measured using a Bio-Tek microplate reader (ELx808, VT). The percentage of growth
inhibition was calculated according to the following formula: [1– (OD of test/OD of negative)]
× 100. The concentration of 50% cell inhibition (IC50) values was determined using Curve
Expert software.
Docking of the complex to BSA
In docking studies, the crystal structure of BSA (PDB ID: 3V03) was selected from the
Brook-haven Protein Data Bank (http://www.pdb.org). This structure model is complete, based
on data of good resolution, without any crystallized ligand.
Molecular docking of the complex in both two main sites of BSA was investigated using
Molegro Virtual Docker (Moldock) software [28]. In Moldock, the units are arbitrary, and an
ideal hydrogen bond contributes to the overall energy [28]. The energy based Moldock scoring
function includes terms accounting for short range interactions such as hydrophobic, dispersion,
or van der Waals, electrostatic interaction, loss of entropy upon ligand binding, hydrogen
bonding and solvation. In this work, we have selected score as Moldock score [GRID], with
GRID resolution of 0.30 Å, and for each docking calculation 10 different poses were requested.
Parameters setting, pose generation and simplex evolution were selected as default settings.
Results and Discussion
Synthesis and characterization of the compounds
1
The synthesized ligand and complex were identified by FT-IR, H NMR, electronic spectra
and elemental analysis. Single crystals of the complex were prepared and X-ray crystallography
was performed. The cluster complex was stable in atmosphere and soluble in water, DMSO,
methanol and slightly soluble in methanol and insoluble in non-polar solvents.
1
The H NMR spectrum of 2,6-diformyl-4-methylphenol was carried out in DMSO-d6 and
1
CDCl3 (Figs. S1,2). The H NMR spectrum of 2,6-bis-[(2-hydroxyethylimino)methyl]-4-
9

methylphenol in DMSO (Fig. S3) showed a singlet signal for OHa at 14.36 ppm, a singlet signal
for HC=N proton at 8.54 ppm, a singlet signal for aromatic protons at 7.50 ppm, a singlet signal
for OHb at 4.68 ppm, a singlet signal for CH2 protons at 3.62 ppm and a singlet signal for the
methyl protons at 2.23 ppm. For the distinction of OH signal one drop of D2O was added to the
DMSO-d6 solution of the 2,6-bis-[(2-hydroxyethylimino) methyl]-4-methylphenol (Fig. S4). OH
signals disappeared because of hydrogen binding with D2O.
The FT-IR spectrum of 2,6-diformyl-4-methylphenol is revealed in Fig. S5. The Schiff base
structure of 2,6-bis-[(2-hydroxyethylimino)methyl]-4-methylphenol was indicated by the
presence of strong imine (C=N) bands at 1581 (Fig. S6) [29, 30].
In the complex (Fig. S7), the frequency of imine bond (C=N) is shifted to lower values,
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[31]. Shift toward lower values is also observed for (C-O) stretching
vibration, again, as a result of coordination of the oxygen to the metal ion[32]. The vibration
around 3450 cm-1 is assigned to the presence of lattice water[33] and medium-weak band at
2923 cm-1 is due the modes of vibrations of (C-H) groups [34]. The ring skeletal vibrations in
the region of 1440–1465 were observed [35]. The M-O stretching vibration usually appears in
the region 400-500 while M-N stretching vibration appears at around 410-580 cm-1 [29]. Thus, a
close examination of the lower frequency infrared region helps to recognize M-O and M-N
coordination in complex.
Two intense bands in the IR spectrum of complex observed at 1627 and around 1455 cm-1
were assigned to υasym (OCO) and υsym (OCO), respectively. The acetate coordination mode is
assigned by the comparison of Δυ value of the acetate complex with the Δυ value of sodium
acetate [31, 36, 37]. The Δυ = 172 cm-1 value of the acetate complex is almost equal to the value
reported for sodium acetate (Δυ = 164 cm-1) and indicated a diatomic bridging mode of acetate
coordination that conforms to the structural data obtained by X-ray crystallography (vide infra).
Two intense bands at 1627 and 1411 cm-1 for υasym (OCO) and υsym (OCO), (the Δυ = 216 cm-1
is bigger than that reported for sodium acetate) are related to the acetate that attached by one
oxygen to the copper.
In the UV-Vis absorption spectrum of the complex (in Tris-HCl buffer, 2×10-5M) phenyl and
imine (π→π*) transitions, revealed two intense bands at 207 and 260 nm, respectively. Imine
(n→π*) transition was recognized as a wide weaker absorption band at 378 nm. (Fig. S8)
10

X-ray structure analysis
A molecule of the tetranuclear copper complex is shown in Fig. 3. Crystallographic data and
bond lengths and bond angles of the complex are listed in Tables 1, 2.
Fig. 3. A molecule of the tetranuclear copper complex. Labels of C, H atoms are omitted for
clarity
11

Table 1. Crystal data and structure refinement for the complex
Complex
Formula of refinement model
C34 H43 Cu4 N4 O15, 2(H2O), 0.497(O)
Temperature [K]
120
Crystal system
Space group
Triclinic
P -1
a [Å]
b [Å]
c [Å]
α [˚]
9.3168(5)
11.0701(7)
21.0231(8)
85.136(4)
86.012(4)
71.594(5)
2047.8(2)
2
β [˚]
γ [˚]
V[Å3]
Z
D[gcm-3]
1.696
3.025
µ [mm-1]
F(000)
1076.0
0.0599(5651)
0.1560(7162)
R(F2>3σ) (reflections)
wR( F2) (reflections)
Table 2. Selected bond lengths (Å) and angles () for the complex
Bond lengths (Å)
Bond angles ()
Cu1,O1cu,Cu2
Cu1,O1cu,Cu3
Cu1,O1cu,Cu4
Cu2,O1cu,Cu3
Cu2,O1cu,Cu4
121.6(2)
100.8(2)
111.3(2)
116.0(2)
101.8(2)
Cu1-O1cu
Cu1-O1b
Cu1-O2b
Cu1-N1b
Cu1-O1ca
Cu2-O1cu
Cu2-O2a
Cu2-N1a
Cu2-O2ca
Cu2-O1cb
Cu3-O1cu
Cu3-O2b
Cu3-N2b
Cu3-O2cb
Cu3-O1cc
Cu4-O1cu
Cu4-O2a
Cu4-N2a
Cu4-O2cc
Cu4-O1cd
1.902(4)
2.001(6)
1.969(3)
1.959(5)
2.237(3)
1.909(3)
1.990(3)
1.989(3)
1.931(5)
2.36(1)
1.943(4)
1.953(4)
1.979(4)
1.95(2)
2.484(4)
1.938(3)
1.960(4)
1.965(3)
1.922(7)
2.47(1)
Torsion angles ()
O2bCu1O1cuCu2
O2bCu1O1cuCu3
O2bCu3O1cuCu2
O2bCu3O1cuCu4
O2aCu2O1cuCu1
O2aCu2O1cuCu4
O2aCu4O1cuCu1
O2aCu4O1cuCu3
-128.1(2)
1.7(1)
131.6(2)
-117.3(2)
-133.8(2)
-9.5(2)
140.6(2)
-111.5(2)
12

The asymmetric unit of the copper(II) complex contains four Cu2+ cations with two 2,6-bis-[(2-
hydroxyethylimino)methyl]-4-methylphenol (L1) ligands and four acetate anions. L1 ligand, in
one side of the cluster, acts as N2O2 chelate while on the other side as N2O chelate. The μ2-O
phenolic oxygens in both sides of the cluster make a bridge between two Cu centers
(Cu(2),Cu(4) and Cu(1),Cu(3)); three acetates bridge two different Cu centers (Cu(3),Cu(4) and
Cu(1),Cu(2) and Cu(2),Cu(3)) bidentately; and one acetate is coordinated to Cu(4) center
monodentately. In the center of the structure, a μ4-O connects four metals. Each Cu(II) center is
five coordinated with one N atom and four O atoms (from acetates, central oxygen and ligand).
Four Cu centers around the central μ4-O display a distorted tetrahedral geometry (Fig. 4).
Fig. 4: Distorted tetrahedral geometry around central μ4-O.
The coordination geometry of each penta-coordinated copper ion is distorted square pyramidal.
To further investigate the distortion of the square pyramidal geometry a structural index
parameter (τ) can be calculated for each Cu center. In the ideal square pyramidal geometry the
metal center is displaced out of the square plane toward apical atom. Thus the angles between
two opposite atoms (α & β) are < 180°. τ values as a geometric parameters for five-coordinate
structures which define the degree of trigonality are calculated from τ = (β – α)/60 . For perfect
square pyramidal geometry τ is equal to zero and it became unity for perfect trigonal-
bipyramidal geometry [38]. By comparing the τ values (τ Cu1= 0.13, τ Cu2= 0.002, τ Cu3= 0.12, τ
Cu4= 0.12) it confirmed that the geometry around each Cu center is distorted square pyramid.
13

As usual for copper (II), the bond distances of the apical atoms are significantly longer than the
bond distanced of the basal atoms for all the metal centers [8]. For Cu(2,3,4) centers: N,
phenolic-O, central-O and one of the acetate-O create the basal plane (bond distances about 1.9
(Å)) and one of the acetate O creates the apical atom (bond distances about 2.36-2.48(Å)). For
Cu(1) center: N, ligand-O, phenolic-O, central-O create the basal plane (bond distances about
1.9-2.0(Å)) while the apical O (O1ca) bond distance is 2.23(Å). (Fig.5)
Fig. 5. Distorted square pyramidal geometry around Cu(1) center with the apical acetate oxygen and
four basal atoms. Other atoms were omitted for the sake of clarity.
Fluorescence spectral studies
Proteins are known to display intrinsic fluorescence caused by the presence of three aromatic
amino acids: tryptophan, tyrosine and phenylalanine. These three amino acids are relatively rare
in proteins; for example, the amount of the dominant intrinsic fluorophore tryptophan is about 1
mole% in proteins. However, measurements of fluorescence titration spectra are highly
sensitive and can be performed on dilute protein solutions. Because of the high sensitivity of
tryptophan to its local environment, intrinsic protein fluorescence allows us to study binding
interactions of proteins with different small molecules and monitor protein association
reactions. Emission maximum intensity or quantum yield of tryptophan residues often change
due to exposing to solvent or being in proximity of a quenching group [39].
Bovine serum albumin (BSA) is the most extensively studied serum albumin due to its
structural homology with human serum albumin (HSA) [40]. The fluorescence spectra of the
titration of BSA with various amount of complex are shown in Fig. 6. BSA has a strong
14

fluorescence emission peak around 345 nm after being excited at 280 nm.
The observed quenching of fluorescence emission intensity with a hypsochromic shift can be
regarded to the formation of a new complex between tetra-nuclear Cu complex and BSA[40].
The hypsochromic shift is a sign that upon the interaction of complex with BSA the
microenvironment around tryptophan residues becomes slightly hydrophobic [39].
500
450
400
350
300
250
200
150
100
50
0
300
350
400
450
500
Wavelength (nm)
Fig. 6. The effect of complex on the fluorescence spectra of BSA (λ EX=280),([complex] = 5×10-5 mol.L-
1
/ [BSA] = 7.2×10-8 mol.L-1): 1.4, 2.8, 4.2, 5.6, 6.9, 8.3, 9.7, 11.1, 12.5, 13.9, 15.3, 16.7, 18.1, 19.4,
20.8; (T = 298 K)
The fluorescence quenching data can be analyzed by the Stern–Volmer Equation (1) [41],
F0
[ ]
[ ]
( )
1
= 1 + KSV Q = 1 + 푘푞 τ0 Q
F
Here F and F0 are the fluorescence intensities in the presence and the absence of the
quencher, respectively. KSV is the Stern-Volmer quenching constant, [Q] is the concentration of
the quencher, kq is the bimolecular quenching constant, and τ0 is the lifetime of the fluorophore
in the absence of a quencher, which is 10–8 s for BSA[39].
The plot of F0/F vs. [Q] gives the KSV value. The Stern-Volmer quenching constant (KSV
=
3.8×105(M-1)) and quenching rate constant (kq = 3.8×1013 (M-1s-1)) were obtained. The linear Stern-
Volmer plot (Fig. 7) indicated that Equation (1) is applicable for the present system.
15

1.8
1.6
1.4
1.2
1
y = 385867x + 1.035
R² = 0.9989
0.8
0.6
0.4
0.2
0
0.00E+00 2.00E-07 4.00E-07 6.00E-07 8.00E-07 1.00E-06 1.20E-06 1.40E-06 1.60E-06
Q
Fig. 7. The Stern–Volmer plot of complex (5×10-5 mol.L-1) binding to BSA (7.2×10-8 mol.L-1); λEx = 280
nm, λEm = 345 nm.
Dynamic quenching and static quenching are two common fluorescence quenching
mechanisms. The dynamic quenching mechanism takes part due to the excited state when the
fluorophore and the quencher come into contact during the transient state. The static quenching
refers to the ground state when the fluorophore and the quencher form a new complex. UV-Vis
absorption spectroscopy is the easiest method used to determine the type of quenching[1, 42].
UV-Vis spectral studies
In order to distinguish between the quenching mechanisms of BSA by the complex, UV-Vis
absorption spectra were recorded. The quenching mechanism which affects the excited states of
the fluorophores is dynamic quenching. This mechanism induce no changes in the absorption
spectra but static quenching which affect by fluorophore-quencher complex formation in the
ground state induce changes in the absorption spectrum of fluorophore [40].
BSA has an absorption band at around 280 nm resulted from the aromatic amino acids (Trp,
Tyr and Phe). Upon the addition of the complexes to the BSA, the absorption is enhanced (Fig.
S9_and a slight blue shift occurs [44], manifesting there is an interaction between BSA and
complex [43]. This result also indicated that the microenvironment of the three aromatic acid
residues was altered and the interaction between the complex and BSA was mainly a static
quenching mechanism [44].
The intrinsic binding constant, Kb for all the complex was determined from the spectral
titration data using Equation (2) at four temperatures (293, 298, 303 and 308K) (Figs. 8, S10-
16

S12):
[푐표푚푝푙푒푥]
(ε – ε )
[푐표푚푝푙푒푥]
(ε – ε )
1
=
+
(2)
K (ε – ε )
a
f
b
f
b b
f
Here [complex] is the concentration of complex, εa corresponds to the extinction coefficient
observed (Aobsd/ [M]), εf corresponds to the extinction coefficient of the free compound, εb is the
extinction coefficient of the compound when fully bound to BSA, and Kb is the intrinsic binding
constant. The ratio of slope to intercept in the plot of [complex]/ (εa – εf) versus [complex] gave
the value of Kb (Fig. 9). The Kb values are presented in Table 3.
Dynamic or static quenching mechanisms can also be distinguished by their dependence on
temperature and viscosity, or by lifetime measurements. For dynamic interaction which mainly
depends on diffusion, increasing temperature causes faster diffusion and hence higher dynamic
quenching constants. On the other hand, in the static quenching mechanism, which refer to
formation of a complex between a bio-macromolecule and the quencher in the ground state,
increasing of temperature may cause the dissociation of weakly bound complexes and decrease
of the static quenching constants [45]. By comparing Kb values at different temperatures (Table
3) it was clear that the Kb decreased with increasing the temperature thus a static quenching
mechanism was confirmed.
1.4
1.2
1
0.8
0.6
0.4
0.2
0
250
270
290
310
330
350
Wavelength(nm)
Fig. 8. Titration of BSA (2×10-5M) (dot line) with various concentrations of complex (2×10-3M) (0-
45μL) at 20°C.
17

1.6E-09
1.5E-09
1.4E-09
1.3E-09
1.2E-09
1.1E-09
1E-09
y = 2E-05x + 8E-10
R² = 0.9924
y = 1E-05x + 9E-10
R² = 0.9914
y = 1E-05x + 1E-09
R² = 0.9902
y = 9E-06x + 9E-10
20
25
30
R² = 0.9906
9E-10
1.00E-06
1.10E-05
2.10E-05
3.10E-05 4.10E-05
35
[complex]
Fig. 9. The plot of [complex]/(ɛa - ɛf ) versus [complex] at 20, 25,
30 and 35°C.
Thermodynamic parameters and binding modes
Four types of non-covalent interactions, namely hydrophobic interactions, electrostatic
interactions, van der Waals forces and hydrogen bonds play an important role in the protein–
drug binding. In order to elucidate the binding modes the thermodynamic parameters such as
free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) are used, where ΔG
reveals the possibility of the reaction, ΔH and ΔS values are used to calculate the acting forces.
For small changes of the temperature, ΔH can be considered a constant. Then through the
binding constant Kb , thermodynamic parameters can be calculated from van’t Hoff Equation
(3) and Equation (4) :[39, 46]
∆H ∆S
ln K = −
+
(3)
b
RT
R
Here Kb is the binding constant at the corresponding temperature and R is the gas constant.
∆G = ∆H − T∆S
(4)
Resulting thermodynamic parameters are given in Table 3.
Table 3. Apparent binding constant and relative thermodynamic parameters for the interaction of
complex with BSA at different temperatures.
Compounds
T (K)
Kb (M-1)
R2
ΔH (kJ mol-1)
ΔG (kJ mol-1) ΔS (J mol-1 K-1)
293
298
303
1.8×104
1.5×104
1.2×104
0.9924
0.9902
0.9914
-29.80 ±0.92 -23.89
-23.78
-20.19 ±3.08
Complex
-23.68
308
1.0×104
0.9906
-23.58
18

From the data given in Table 3, negative value of free energy (ΔG) supported the assertion
that the binding process was spontaneous. Ross and Subramanian[47] have considered the signs
and magnitudes of the thermodynamic parameters to elucidate various kinds of interactions that
may take place in protein association processes. The negative ΔH and ΔS values indicated that
van der Waals’ forces and hydrogen bonds might play major roles in binding between complex
and BSA[46].
Binding stoichiometry
The average aggregation number of BSA, <J>, which potentially could be induced by complex,
can be determined by Equation (5).
[ ]
F
Q
1 − =< 퐽 >
F0
(5)
[
]
BSA
0
Where F and F0 are the fluorescence intensities in the presence and the absence of the
quencher, respectively and [Q] is the concentration of the quencher, the slope (<J> = 0.0159) of
the line presented in Fog. 10 is less than one, which indicated that the complex binding did not
induce any aggregation in BSA molecules and accordingly proved the 1:1 stoichiometry for
complex-BSA systems.
0.45
0.4
0.35
0.3
0.25
y = 0.0159x + 0.0676
0.2
R² = 0.99
0.15
0.1
0.05
0
0
5
10
15
20
25
[Complex]/[BSA]
Fig. 10. Determination of the average aggregation number of BSA (<J>) in presence of complex. λEx
=280 nm, λEm=345 nm.(T=298 K)
19

Binding constants and the number of binding sites
For further investigation on the proposed static quenching interaction, the fluorescence intensity
data were used to analyze the apparent binding constant (Kb) and the number of binding sites (n)
by using Equation (6) [42]. This Equation calculates the equilibrium between free and bound
small molecules when those bind independently to a set of equivalent sites on bio-
macromolecules.
퐹0 − 퐹
푙표푔 (
) = log 퐾푏 + 푛푙표푔[푄]
(6)
퐹
Here, F0 and F are the fluorescence intensities in the absence and presence of the quencher
(complex), respectively; Kb is the binding constant of the compound with BSA; n is the number
of binding sites per albumin molecules; and [Q] is the concentration of the quencher (complex).
From the plot of log [(F0 -F)/F] vs. log [Q] (Fig. 11), the binding constant (Kb =2.5×104 M-1) and
the number of binding sites (n = 0.796) were calculated. By considering the value of Kb, the
distribution of the complex in protein can be estimated. The large Kb illustrated a strong binding
between complex and BSA. The value of (n) was approximately equal to 1 which suggested that
there was only one binding site for the complex on BSA molecule [42].
0
-0.2
-0.4
-0.6
-0.8
y = 0.7961x + 4.3976
-1
-1.2
-1.4
R² = 0.9925
-7.2
-7
-6.8
-6.6
-6.4
-6.2
-6
-5.8
-5.6
log Q
Fig. 11. Plot of log [(F0-F)/F] vs. log[Q] for the complex
Site-selective binding of complex on BSA
In the molecular structure of Bovine serum albumin two main sites has been recognized for
drugs binding: site I, in which the tryptophan residue is located for drug binding, and site II, in
20

which the tyrosine residue is located for drug-BSA interaction. The former showed affinity for
warfarin, phenylbutazone, etc., whereas the binding pocket of the latter is well suited for
ibuprofen, diazepam, fluofenamic acid, etc. In order to identify the binding site of the complex
to BSA, displacement experiments were performed with phenylbutazone and ibuprofen as site
markers for site I and site II of BSA, respectively [48], by monitoring the changes in the
fluorescence of the ternary mixtures of complex (5×10-5 mol.L-1)(0-45μL), BSA(7.2×10-8 mol.L-1)
and site marker(1.8×10-5 mol.L-1) (Fig. 12). The binding constants in Table 4 revealed that after
addition of complex, Kb (the binding constant of complex-BSA) value of ibuprofen-BSA
mixture was remarkably small, while Kb in the phenylbutazone-BSA mixture remained almost
the same compared to a pure BSA solution [49]. These results revealed that the complex tended
to occupy site II which was occupied by ibuprofen molecules, and the complex couldn’t win in
this competitive binding reaction thus causing the small Kb .
a
b
c
500
450
400
350
300
250
200
150
100
50
500
450
400
350
300
250
200
150
100
50
500
450
400
350
300
250
200
150
100
50
0
0
0
300
400
500
300
400
500
300
400
500
Wavelength (nm)
Wavelenght (nm)
Wavelenght (nm)
Fig. 12. Quenching effect of complex (5×10-5 mol.L-1) (0-45μL) binding to BSA (7.2×10-8 mol.L-1) in the
a) absence of site marker, b) presence of phenylbutazone (1.8×10-5 mol.L-1) and c) presence
of ibuprofen (1.8×10-5 mol.L-1).
Table 4. Binding constants of competitive experiment of complex-BSA system.
Complex
Site marker
Without
Kb (M-1)
R2
2.498×104
0.211×104
2.191×104
0.9925
0.9933
0.9965
Complex
Ibuprofen
Phenylbutazone
21

Energy transfer and binding distance between complex and BSA
FRET (Förster resonance energy transfer) is a spectroscopic method used for estimating the
proximity and relative angular orientation of fluorophores. FRET has been recognized as a
"spectroscopic ruler" for measuring the distance between molecules in the biological and
macromolecular systems. Resonance energy transfer takes place when the fluorescence
emission band of donor overlaps with the excitation band of acceptor when they are in 2-8 nm
distance[50, 51]. For the occurrence of Förster’s resonance energy transfer (FRET) from donor
to acceptor the following conditions must be fulfilled: (1) donor must be a fluorophore; (2) the
fluorescence emission spectrum of the donor and UV–vis absorption spectrum of the acceptor
must be sufficiently overlapped; (3) the distance between the donor and the acceptor must be
within 2–8 nm[52]. Here the donor is BSA and the acceptor is the complex.
Using Förster theory (FRET) the distance between Complex and BSA could be calculated by
Equation (7):
R60
R60 + r6
F
E =
= 1 −
(7)
F0
Here E is the efficiency of transfer between the donor and the acceptor, F and F0 are the
fluorescence intensities of biomolecule (donor) in the presence and absence of acceptor
(complex), r is the donor–acceptor distance and R0 is the critical distance where the transfer
efficiency is 50% calculated by using Equation (8):
R60 = 8.8 × 10−25K2N−4ΦJ
(8)
In Equation (8) K2 is the spatial orientation factor related to the geometry of the donor and
acceptor of dipoles, taking the value of 2/3 for random orientation as in fluid solution, N is the
averaged refracted index of the medium in the wavelength range where spectral overlap is
significant, taking the value of 1.336 for the average refracted index of water and organics, Φ is
the fluorescence quantum yield of the donor taking the value of 0.118 for the fluorescence
quantum yield of tryptophan [52], J is the effect of the spectral overlap between the emission
spectrum of the donor and the absorption spectrum of the acceptor, which could be calculated
by using Equation (9):
∞
4
( )
F λ ε(λ)λ dλ
∫
0
J =
(9)
∞
( )
F λ dλ
∫
0
22

Here F(λ) is the corrected fluorescence intensity of the donor in the wavelength range of λ to
(l+Δλ) and ε(λ) is the molar extinction coefficient of the acceptor at λ.
The emission spectrum of BSA overlaps with the absorption spectrum of the complex in the
wavelength range of 300 to 450 nm (Fig. 13). This considerable overlap forms the basis of
FRET. Thus, the Förster’s non-radiative energy transfer theory can be used to determine the
distance between the amino acid residues on protein and the complex in the binding site[53]. By
using Equations 7-9 the values J = 2.62×10-15 (cm3 L mol-1), E = 0.38, R0 = 2.02nm and the
binding distance r = 2.19 nm were calculated. The value of r is less than 8 nm, and 0.5R0 < r <
1.5R0, indicating the energy transfer from BSA to the complex occurred with high probability
[54-56].
500
450
400
350
300
250
200
150
100
50
2
1.8
1.6
1.4
1.2
1
b
0.8
0.6
0.4
0.2
0
a
0
300
350
400
450
500
Wavelength (nm)
Fig. 13. Spectral overlap of (a) the absorption spectrum of complex (2×10-5 mol.L-1) with(b) the BSA
(7.2×10-8 mol.L-1) fluorescence spectrum.
Synchronous fluorescence spectroscopic studies
Synchronous fluorescence spectra provide detailed information about the molecular
microenvironment, particularly in the vicinity of the fluorophore in BSA [57]. In synchronous
fluorescence spectroscopy, the difference between excitation and emission wavelengths (Δλ=
λem - λex) reflects the spectra of a different nature of chromophores. The large Δλ value (60 nm)
is characteristic of tryptophan residue and a small Δλ value (15 nm) is characteristic of tyrosine
[58]. Shift in the position of the maximum emission wavelength (λmax) corresponds to changes
23

in the polarity around the chromophore molecule. The red shift of the maximum emission
wavelength indicates an increasing polarity of the surrounding environment, a lower
hydrophobicity and a looser structure of BSA, and vice versa[52].
The effect of the complex on the synchronous fluorescence spectra of BSA is shown in Fig.
14. In the synchronous fluorescence spectra at Δλ=15 nm for BSA, with increasing the
concentrations of complex a decrease in fluorescence intensity at 282 nm with slight red shift of
1 or 2 nm was observed, which suggested a more polar (or less hydrophobic) environment of
tyrosine residues. The synchronous fluorescence spectra of BSA at Δλ=60 nm exhibited a
decrease in fluorescence intensity at 280 nm without any appreciable shift in the position of the
band which indicated that there was no change of the microenvironment of tryptophan residues
[46]. It was in accordance with the results of the site selective studies mentioned above.
a
b
180
160
140
120
100
80
400
350
300
250
200
150
100
50
60
40
20
0
0
250
270
290
310
330
250
270
290
310
330
Wavelength (nm)
Wavelength (nm)
Figure 14. Synchronous fluorescence spectra of BSA (7.2×10-8 mol.L-1) with Δλ=15nm (a) and
Δλ=60nm (b) in the absence (dashed lines) and presence of complex (5×10-5 mol.L-1) (0-
40μL) (solid lines).
Inhibitory effects of the complex on various cancer cell lines
The antineoplastic activities of metal Schiff base complexes have been already established [59-
61]. In the present study, the synthetic Schiff base complex was tested for its inhibitory effects
on the growth of Jurkat human T cell leukemia, Raji Burkitt's lymphoma and A549 lung
carcinoma cell lines as the target. The cell lines were treated with various concentrations of the
complex to measure the cytotoxicity. Analysis of data showed a remarkable growth inhibitory
24

effect of the complex on all three cell lines. The Raji (IC50, 9.8 μg/mL) and Jurkat (IC50, 20.4
μg/mL) leukemia/lymphoma cells were more sensitive to the complex than the A549 solid
tumor cells (IC50, 48.4 μg/mL).
The IC50 values of cis-platin (as reference) for A549 cell line was 79.4 μg/mL, for Jurkat was 25
μg/mL and for Raji cells was 30 μg/mL.
Molecular docking study
The BSA molecule is made up of three homologous domains: domain I (residues 1–195), II
(196–383) and III (384–585) that are divided into nine loops (L1–L9) by 17 disulfide bridges.
Each domain is composed of two sub-domains (A and B) (Fig. 15).[62]
In the present study, the MVD program [63] was chosen to obtain the binding mode of the
complex at the active site of BSA. The ranked results are reported in Table 5, showing that the
best ranked result was obtained for site II of BSA. As shown in Fig. 16(a) the complex is
inserted into the hydrophobic residues of site II (subdomain IIIA). The binding energy for the
complex to BSA was found to be −198.276. These results are in agreement with those obtained
by the experimental method. According to the Fig. 16(b) the complex is surrounded by a
number of amino acid residues, namely: Ala489, Arg409, Arg484, Asn385, Asn390, Gln389,
Gln393, Glu382, Ile387, Leu386, Leu490, Lys413, Phe487, Pro383, Pro485, Ser488, and
Thr491. Non-coordinated OH groups of the complex could create H-bonding between the
complex and some amino-acid residues of BSA. The main H-bonding interactions are collected
in Table 6.
25

Figure 15. The structure of BSA
Table 5: Ranked results of the docking of the complex in site I and site II of BSA.
Mol Dock Score
Rank
Site I
Site II
1
2
3
4
5
6
7
8
9
10
-183.896
-198.276
-176.502 -191.27
-173.262 -175.719
-171.421 -174.68
-171.314 -170.997
-169.474 -169.085
-156.631 -165.172
-155.65
-156.596
-152.507 -152.96
-149.138 -142.378
26

a
b
The complex
Figure 16: (a) The docking pose of the complex with BSA (b) the amino acid residues which
surrounding the complex
Table 6: The main H-bonding between the amino-acids of BSA and complex
Hydrogen bond interaction
Arg409 With N atom of ligand
Asn390 With N atom of ligand
Ser488 With O atom of ligand
Gln389
Glu382
Leu386
Arg484
With OH group
With OH group
With OH group
With OH group
27

Conclusion
A new tetranuclear copper (II) Schiff base complex was synthesized from 2,6-diformyl-4-
methylphenol and 2-aminoethanol and copper(II) acetate as a metal source. The output of the
synthesis was a tetranuclear water-soluble copper (II) cluster. The ligand and acetate ions
bridged Cu(II) centers and arranged them in a distorted tetrahedral geometry around one central
1
oxygen atom. The complex was characterized by elemental analysis, FT-IR, H NMR and UV-
Vis spectroscopy, and X-ray structure analysis. Some spectroscopic methods such as
absorption, fluorescence and synchronous fluorescence spectroscopy were used to investigate
the binding interaction of the complex with BSA. By the fluorescent titration the quenching rate
constant were calculated by Stern-Volmer equation (KSV = 3.8×105(M-1)). Competitive binding
experiments also revealed that the complex was located in site II of serum albumin. From the
Förster energy transfer theory, the probability of the transfer efficiency of energy and the
distance between the complex and protein were r = 2.19 nm.
Using the synchronous fluorescence spectroscopy, it was concluded that the interaction of
complex with albumins did not change the conformation of tryptophan microenvironment,
while the hydrophobicity near the tyrosine residues was changed.
The binding constants (Kb) were calculated from the UV-Vis spectroscopy in four different
temperatures. The negative ΔH and ΔS values indicated that van der Waals’ forces and
hydrogen bonds were important in binding between complex and BSA and ΔG value indicated
that the binding process is spontaneous.
Finally, the complex was examined for its anticancer activity against three human Jurkat,
Raji and A549 tumor cell lines as the target. According to IC50 values, the results suggested that
this complex might be a potential anticancer agent.
Acknowledgements
We are grateful to the Shiraz university research council for its financial support. The
crystallographic part was supported by the project 18-10504S of the Czech Science Foundation
using instruments of the ASTRA lab established within the Operation program Prague
Competitiveness - project CZ.2.16/3.1.00/24510.
Supplementary material
CCDC 1544390 contains the supplementary crystallographic data for this paper. These data
can be obtained free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
28

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