Synthesis, characterization, crystal structure and DNA, HSA-binding studies of four Schiff base complexes derived from salicylaldehyde and isopropylamine

Article abstract of DOI:10.1016/j.ica.2017.05.035

Four new Schiff base complexes (NiL₂, CoL₂, CuL₂ and ZnL₂) (HL: ((E)-2-((isopropylamino) methyl) phenol) were synthesized and characterized by CHN elemental analysis, FT-IR and single crystal X-ray diffraction t

Full text of DOI:10.1016/j.ica.2017.05.035

Accepted Manuscript
Research paper
Synthesis, characterization, crystal structure and DNA, HSA-binding studies of
four Schiff base complexes derived from salicylaldehyde and isopropylamine
Monireh Dehkhodaei, Mahsa Khorshidifard, Hadi Amiri Rudbari, Mehdi
Sahihi, Gholamhassan Azimi, Neda Habibi, Salman Taheri, Giuseppe Bruno,
Reza Azadbakht
PII:
S0020-1693(16)30826-X
http://dx.doi.org/10.1016/j.ica.2017.05.035
ICA 17604
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
7 November 2016
8 May 2017
15 May 2017
Please cite this article as: M. Dehkhodaei, M. Khorshidifard, H.A. Rudbari, M. Sahihi, G. Azimi, N. Habibi, S.
Taheri, G. Bruno, R. Azadbakht, Synthesis, characterization, crystal structure and DNA, HSA-binding studies of
four Schiff base complexes derived from salicylaldehyde and isopropylamine, Inorganica Chimica Acta (2017),
doi: http://dx.doi.org/10.1016/j.ica.2017.05.035
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Synthesis, characterization, crystal structure and DNA, HSA-binding studies
of four Schiff base complexes derived from salicylaldehyde and
isopropylamine
a
a
a,*,1
a,*,1
Monireh Dehkhodaei , Mahsa Khorshidifard , Hadi Amiri Rudbari , Mehdi Sahihi
,
a
b
c
d
e
Gholamhassan Azimi , Neda Habibi , Salman Taheri , Giuseppe Bruno , Reza Azadbakht
a
Department of Chemistry, University of Isfahan, Isfahan 81746-73441, Iran.
b
Department of Biomedical Engineering, College of Engineering, The University of Texas at San
Antonio, One UTSA Circle, San Antonio, TX 78249-0619, USA.
c
Chemistry and Chemical Engineering Research Center of Iran, Tehran, Iran.
d
Department of Chemical Sciences, University of Messina, Via F. Stagno d'Alcontres 31, 98166
Messina, Italy.
e
Department of Chemistry, Payame Noor University, P.O. Box 19395-3697, Iran.
1
These authors contributed equally to this work
*Corresponding authors
1
) Dr. Hadi Amiri Rudbari
2) Dr. Mehdi Sahihi
Assistant Professor of Biophysical Chemistry
Assistant Professor of Inorganic Chemistry
University of Isfahan
University of Isfahan
Isfahan 81746-73441
Isfahan 81746-73441
Iran
Iran
Tel.: +98-313-7934918
Fax: +98-313-36689732
E-mail: h.a.rudbari@sci.ui.ac.ir
hamiri1358@gmail.com
Tel.: +98-313-7934918
Fax: +98-313-36689732
E-mail: m.sahihi@chem.ui.ac.ir
1

Abstract
Four new Schiff base complexes (NiL
2
, CoL , CuL
2
2
and ZnL ) (HL: ((E)-2-((isopropylamino)
2
methyl) phenol) were synthesized and characterized by CHN elemental analysis, FT-IR and
single crystal X-ray diffraction technique. The crystallographic data reveal that in all complexes
the metal centers are four-coordinated by two phenolate oxygen and two imine nitrogen atoms of
two moles of Schiff base ligand HL and geometry around the metal center in all of them is
1
13
distorted tetrahedral. In addition, H and C NMR techniques were employed for
characterization of diamagnetic ZnL complex. The binding affinity of complexes with DNA
fish sperm DNA, FS-DNA) and Human Serum Albumin (HSA) were investigated using
2
(
fluorescence quenching, chemometrics, UV-Vis spectroscopy, viscosity measurements and
molecular docking methods. The obtained results revealed that the DNA and HSA affinity for
binding to complexes are in the following order: CuL
CuL > CoL . The distance between complexes and HSA was obtained based on the Förster’s
theory of non-radiative energy transfer. The computational molecular docking results showed
that H-bond interactions, hydrophobic interactions, π-π stacking and π-cation interactions have
dominant role in the stability of HSA-ML (M: Cu, Co, Ni and Zn). The computational docking
2
> ZnL
2
> CoL
2
> NiL
2
and NiL
2
> ZnL
2
>
2
2
2
and viscosity results suggest that all metal complexes interact with DNA presumably by the
groove binding mechanism.
Keywords: DNA interaction, HSA binding, Schiff base complex, Crystal structure.
2

1
. Introduction
The medicinal properties of transition metal complexes depend on both the nature of the ligands
and metal ions [1]. Schiff base ligands have attracted great interest because of their broad range
of biological as well as pharmaceutical properties such as antibacterial, antioxidant, anti-malarial
and antitumor activities [2-13]. The azomethine linkage in Schiff base is responsible
2
antimicrobial activity. Presence of lone pair electrons in sp -hybridized orbital of nitrogen atom
in the azomethine group plays a remarkable chemical and biological role [14-15]. Among the
Schiff base ligands, bidentate and tetradentate Schiff base ligands involving N,O donor sites
possess many advantages such as facile synthesis, readily adjusted ancillary ligands, and tunable
steric and electronic coordination environments on the metal center. These complexes represent
interesting models for metallo-enzymes which efficiently catalyze the reduction of dinitrogen
and dioxygen [16-19].
The metal ion of the complexes accelerates the drug activity and increases the effectiveness and
efficiency of the ligands [20]. In recent decades, considerable effort has been devoted to the
design and development of complexes by using different metal ions, which possess excellent
antioxidant, antibacterial and antitumor activities. Investigations showed that metal ions such as
cobalt, nickel, copper and zinc could have remarkable biological activity [21-29].
Previously, we have reported the bi-dentate ON Schiff base ligand derived from condensation of
isopropylamine with salicylaldehyde (HL) and its oxovanadium(IV) complexes [30]. Continuing
of our research in design of new Schiff base ligands, complexes [31-45] and their interaction
with bio macromolecules, herein we present the results of a detailed investigation on the HSA
and DNA interaction of new distorted tetrahedral complexes, NiL
2
, CoL , CuL
2
2
and ZnL ,
2
derived from HL ligand. The new complexes have been characterized by IR, elemental analysis
3

1
13
and single-crystal X-ray diffraction. The H and C NMR techniques were employed for
characterization of the ZnL complex. The HSA and DNA binding affinity of complexes were
2
investigated by using fluorescence quenching, chemometrics, UV–Vis spectroscopy, viscosity
measurements as well as molecular docking methods.
2
. Experimental section
2
.1. Chemicals and instrumentation
Fish sperm DNA (FS-DNA), human serum albumin (HSA) and ethidium bromide (3,8-diamino-
-ethyl-6-phenylphenanthridinium bromide, EtBr) were purchased from Sigma-Aldrich. Other
chemicals were purchased from Merck Co. and used without further purification. The FT-IR
5
-1
spectra were recorded on a JASCO, FT-IR 6300 spectrometer (4000–400 cm ) in KBr pellets.
1
13
H and C NMR spectra of Zn(II) complexes were recorded on a Bruker Avance 400
spectrometer using deuterated chloroform (CDCl ) as solvents. Elemental analysis was
3
performed on Leco, CHNS-932 and Perkin-Elmer 7300 DV elemental analyzers. UV-Vis spectra
were recorded on a JASCO V-670 spectrophotometer. Fluorescence and Viscosity measurements
were carried out at room temperature using Shimadzu RF-5000 spectrofluorometer and a
Brookfield rotational viscometer, respectively.
2
.2. Preparation of complexes
General procedures
A MeOH solution (30 mL) of isopropylamine (5 mmol) was added dropwise to a MeOH solution
(
30 mL) of the salicylaldehyde (5 mmol), stirred for 2 h at ambient temperature (yellow solution
A). Triethylamine (7 mmol) in MeOH (10 mL) was added to yellow solution A and stirred for 10
4

min with formation of a dark yellow solution (solution B). A solution of NiCl
Co(NO .6H O, Cu(OAc) or Zn(NO .6H O, (2.5 mmol) in MeOH (30 mL) was added drop
wise to solution B.
2
.6H
2
O,
3
)
2
2
2
3
)
2
2
2
.2.1. Cobalt(II) complex (CoL
2
)
An immediate precipitation of CoL
2
complex occurred on addition of Co(NO
3
)
2
.6H O to
2
solution B. The reaction mixture was stirred for 8 h in ambient temperature and orange
precipitates were collected by filtration, washed with cold methanol, and dried in air with a
typical yield of 82%. Orange crystals suitable for single crystal X-ray diffraction analysis were
obtained from the filtrate standing for 2 days at room temperature. Anal. calc. for
C
20
H24CoN
2
O
2
: C: 62.66, H: 6.31, N: 7.31. Found: C: 62.69, H: 6.36, N: 7.28. Selected IR data
-1
(
KBr, cm ): 3058-2867 (νC=N), 1605 (νC=N), 1536 (νC=C), 1143 (νC-O).
2
.2.2. Nickel(II) complex (NiL
2
)
An immediate precipitation of NiL
2
complex occurred on addition of NiCl
2
.6H O to solution B.
2
The reaction mixture was stirred for 8 h in ambient temperature and green precipitates were
collected by filtration, washed with cold methanol, and dried in air with a typical yield of 82%.
Green crystals suitable for single crystal X-ray diffraction analysis were collected from the
filtrate standing for 2 days at room temperature. Anal. calc. for C20
N: 7.31. Found: C: 62.75, H: 6.27, N: 7.30. Selected IR data (KBr, cm ): 3051-2864 (νC=N),
609 (νC=N), 1536 (νC=C), 1142 (νC-O).
H24NiN
2
2
O : C: 62.70, H: 6.31,
-1
1
5

2
.2.3. Copper(II) complex (CuL
2
)
A red solution was obtained on addition of Cu(OAc)
2
to solution B. The reaction mixture stirred
for 8 h at ambient temperature and allowed to stand overnight. Big red crystals were collected by
filtration with a typical yield of 91%. Appropriate single crystals for X-ray crystallography were
obtained directly from the reaction mixture. Anal. calc. for C20
H24CuN
2
2
O : C: 61.92, H: 6.24, N:
-1
7
.22. Found: C: 61.87, H: 6.30, N: 7.23. Selected IR data (KBr, cm ): 3045-2857 (νC=N), 1614
(
νC=N), 1536 (νC=C), 1143 (νC-O).
2
2
.2.4. Zinc(II) complex (ZnL )
A yellow solution was obtained on addition of Zn(NO
3
)
2
.6H O to solution B. The reaction
2
mixture refluxed for 8 h and slowly evaporated to dryness at room temperature. The yellow
solid was purified by washing with diethyl ether (50 mL) and dried in air with a typical yield of
8
5%. Appropriate single crystals for X-ray crystallography were obtained directly from the
reaction mixture. Anal. calc. for C20 Zn: C: 61.62, H: 6.21, N: 7.19. Found: C: 61.59, H:
.25, N: 7.23. Selected IR data (KBr, cm ): 3051-2896 (νC=N), 1617 (νC=N), 1539 (νC=C), 1144
H
24
N
2
O
2
-
1
6
1
13
(
ν
C-O). HNMR and CNMR (DMSO-d , 400 MHz, 298 K): Table 3.
6
2
.3. Single crystal diffraction studies
The X-ray data of complexes (NiL
2
, CoL
2
, CuL
2
and ZnL ) were collected at room temperature
2
with a Bruker APEX II CCD area detector diffractometer using Mo Kα radiation (λ = 0.71073
Å). Data collections, cell refinements, data reductions and absorption corrections were
performed using multiscan methods with Bruker software [46]. The structures were solved by
direct methods using SIR2004 [47]. The non-hydrogen atoms were refined anisotropically by the
6

2
full matrix least squares method on F using SHELXL [48]. All hydrogen atoms were added at
ideal positions and constrained to ride on their parent atoms. Molecular graphics
were prepared with the Olex2 program [49]. Crystallographic data for complexes are listed in
Table 1. Selected bond distances and angles are summarized in Table 2.
2
2
.4. DNA binding studies
.4.1. Preparation of compounds and DNA stock solutions
The stock solution of FS-DNA was prepared by 50 mM Tris buffer at pH 7.5 using double-
o
distilled deionized water and stored at 4 C. The FS-DNA concentration per nucleotide was
determined using absorption intensity at 260 nm after adequate dilution with the buffer and using
-1
-1
the reported molar absorptivity of 6600 M .cm [50]. Purity of FS-DNA solution was confirmed
by ratio of UV absorbance at 260 and 280 nm (A260/A280=1.9), indicating that FS-DNA is free
from protein impurity [51]. The complex solutions in methanol as co-solvent were diluted with
corresponding buffer to the required concentration for all experiments. The volume of co-solvent
never exceeded 0.5% (v/v), so the effect of methanol is negligible. To confirm the stability of the
metal complexes in the buffer solution at room temperature, a UV–Vis study was performed. The
spectral features of the complexes exhibited no change in the position of absorption bands over a
period of 24 h and no precipitation or turbidity was observed even after long storage at room
temperature (at least 1 month after preparation). This indicates the stability of the compounds in
different media. However, all the solutions were used freshly after preparation.
2
.4.2. UV-Vis spectroscopy measurements
7

Absorption titration experiment as an operational and very easy method was carried out to
investigate of DNA-binding of the compounds at room temperature. Absorption spectral titration
-4
experiments were performed by addition of various amounts of DNA (0-5×10 M) to the
-4
compounds (1×10 M). All compounds–DNA solutions were allowed to incubate for 2 min
before recording the related spectra. Absorption curves of compounds–DNA mixtures were
corrected by subtracting the spectra of DNA and all intensities were corrected for the dilution
effect.
2
.4.3. Fluorescence spectroscopic measurements
Fluorescence quenching experiments was carried out using quartz cuvette with 1 cm optical path
length and the excitation and emission slits were set at 5 and 10 nm, respectively. In our primary
experiments, ethidium bromide (EtBr) emission was checked in the presence of various amounts
of DNA. The results showed that the emission of EtBr was increased up to DNA:EtBr=10:1
mole ratio and there was no significant increasing in emission after the mentioned mole ratio.
The FS-DNA solution was stirred with EtBr with molar ratio of DNA:EtBr 10:1 and incubated
for 1 h at 4 °C to completion of interaction between DNA and EtBr. Various amounts of metal
complexes (0–250 µM) were added to the mixture of DNA:EtBr. The fluorescence spectra were
measured in the range of 500–700 nm with exciting wavelength at 520 nm. In each measurement
after addition of metal complexes, the mixture was allowed to stand for 2 min. Moreover, the
measured fluorescence intensities were corrected for the dilution and the inner-filter effect. To
eliminate the inner filter effects, absorption measurements were carried out at the fluorescence
excitation and emission wavelengths. The extent of this effect can be roughly evaluated with the
following relationship [52]:
8

ꢀ
= ꢀꢂꢄꢅ × ꢆ(ꢇꢈꢉꢊꢇꢈꢋ)/ꢌ
ꢂꢃꢃ
(1)
Where Fcorr and Fobs are the corrected and observed fluorescence intensities, respectively, while
ex and Aem are the absorption of compounds at excitation and emission wavelengths,
respectively. It was assumed that fluorescence spectra of solutions with low concentration
ꢁ
A
(
A
FLꢀ=ꢀ0.1) are not disturbed by reabsorption effect [53].
2
.4.4. Viscosity measurements
Viscosity experiments were carried out using a rotational viscometer and the measurements were
performed at 200 rpm at room temperature. The viscosity of DNA solution was measured in the
presence of increasing amounts of the metal complexes. The obtained data are presented as
1
/3
(
η/η
0
) versus [complex]/[DNA], where η and η are the viscosity of DNA in the absence and
0
presence of the metal complexes, respectively.
2
2
.5. HSA binding experiments
.5.1. Preparation of protein stock solution
A stock solution of HSA was prepared by dissolving the desired amount of HSA in 50 mM
phosphate buffer (pH=7). The HSA stock solution was stored at 4 °C in the dark and used within
2
h. HSA concentration was determined by UV-Vis spectrophotometry using the molar
-1
-1
absorption coefficient 35700 M .cm at 278 nm [54].
9

2
.5.2. Fluorescence spectroscopic measurements
The interaction of HSA with the synthesized Schiff base complexes (ZnL
CoL ) was investigated using fluorescence quenching experiment. In this experiment 2 mL of
2
, CuL
2
, NiL and
2
2
HSA solution (5 µM) was placed into the cell and various amounts of the compounds (0-50 µM)
were added to the cell. The fluorescence intensity was measured with investigation of excitation
wavelength at 295 nm and emission wavelength range of 300-450 nm. In each measurement, the
mixture was allowed to incubate for 2 min after addition of the complexes. All intensities were
corrected for the dilution and inner filter effect in fluorescence experiments.
2
.5.3. UV-Vis absorption measurements
Absorption titration experiment as an operational and very easy method was also carried out to
investigate the HSA binding of complexes at room temperature. The UV-Vis absorption spectra
of the complexes solution (10 µM) in the absence and presence of various amounts of the HSA
(
0-50 µM) were recorded. Absorption curves of compounds–protein mixtures were corrected by
subtracting of HSA solution spectra.
2
.6. Molecular docking simulation
In this work, docking study was carried out to indicate DNA and HSA binding site for the
synthesized compounds. The 3D structures of the metal complexes were generated using the CIF
files of their X-ray crystal structures. The CIF files were converted to the PDB format by using
the Mercury software (http://www.ccdc.cam.ac.uk/). The known crystal structures of DNA (PDB
ID: 423D) with sequence d(ACCGACGTCGGT)
2
and HSA (PDB ID: 1AO6) were taken from
the Brookhaven Protein Data Bank (http://www.rcsb.org/pdb). The resolution of these files was
10

0
.160 and 0.25 Å for DNA and HSA, respectively. Water molecules of the PDB files were
removed and missing hydrogen atoms were added. Flexible-ligand docking was performed by
AutoDock 4.2.5.1 molecular-docking program using the implemented empirical free energy
function and the Lamarckian Genetic Algorithm [55]. The Gasteiger charges were added to
prepare the macromolecule input file for docking and the Auto Grid was used to calculate Grids.
A blind docking with 126 lattice points along X, Y, and Z axes was performed for docking of
metal complexes with DNA to find the binding site of complexes on DNA with a grid point
spacing of 0.375 Å and to allow the complex to rotate freely. The centre of the grid box was
located at the binding site and the second docking was performed using a cubic box with
6
0×60×60 dimensions. For the docking of synthesized metal complexes to HSA the grid box was
centred on C of Trp-214 residue and a docking with 90 lattice points along X, Y, and Z axes
α
was performed to find the binding site of complexes on HSA. 250 docking runs with 25,000,000
energy evaluations for each run were performed.
3
. Results and discussion
3
.1. Synthesis and spectroscopic characterization of the complexes
The reaction of Schiff base ligand with metal ions is shown in Scheme 1. All of complexes were
obtained in good yield (more than 80%). The elemental analyses of the complexes were
consistent with their proposed compositions. These results are also confirmed by X-ray
crystallography. Stability of all complexes in the most common polar and non-polar solvents
including H
2
O, MeOH, EtOH, CH
3
CN, CHCl , DMSO and DMF was tested and the results
3
showed the stability of complexes in solvents.
11

The most characteristic feature in the IR spectra of the metal complexes comes from the C=N
-1
stretching vibrations. This band appeared at 1605, 1609, 1614 and 1617 cm for the cobalt(II),
nickel(II), copper(II), zinc(II) complexes, respectively [31,32,34]. Compared to the free Schiff
-1
base ligand (HL) (1630 cm ) [30], this band was shifted to lower wavenumber upon
coordination. Formation of M-N bonds leads to weakening of C=N band and this can be
explained by the donation of electrons from the nitrogen atom to the empty orbitals of the metal
-1
atom. The presence of several medium intensity bands between 3051-2857 cm suggests the
existence of C-H stretching vibrations of aliphatic and aromatic protons. The C-O stretching
-1
-1
-1
vibration, 1143 cm (CoL
2
and CuL
2
), 1142 cm (NiL
2
) and 1144 cm for ZnL , confirms the
2
presence of phenolate group in the complexes. Additional support for the formation of the
-1
complexes were provided by the existence of weak intensity bands at ~ 480 cm attributed to the
formation of M-N and M-O bonds.
1
13
The diamagnetic zinc(II) complex, ZnL
2
was studied by H and C NMR experiments (Table
1
13
3
). The H and C NMR spectra of ZnL
2
complex showed the expected simple spectra,
indicating the integrity of the complex. The spectra of ZnL
2
complex obtained after 12, 24 and
1
20 h were similar to the initial spectrum, indicating that the complex is stable in deuterated
chloroform. The signal for the imine proton in the zinc(II) complex appears at 8.24 ppm. This
signal shifted downfield with respect to the corresponding signal in the free ligand (8.36 ppm),
which is consistent with the coordination of the azomethine nitrogen atom to the metal ion and
1
also indicates that the metal-nitrogen bond is retained in solution. Also, H NMR spectrum
shows the hydrogens of methyl group as two doublets at 1.22 and 1.32 ppm and methine protons
1
as one septet at 3.64 ppm. The protons of each ‒CH
3
group give different signal in H NMR
spectrum (Table 3) and confirms that protons of each ‒CH
3
group are in different chemical
12

environments in ZnL
2
complex. The splitting reason of ‒CH and ‒CH
3
peaks is the coupling of
hydrogens of ‒CH and ‒CH
3
with together.
No signal corresponded to hydroxyl proton at 13.7 ppm and this suggested that the hydroxyl
group was fully deprotonated and the oxygen was most likely coordinated to the metal ion. The
1
3
13
C NMR spectrum of ZnL
spectrum of ZnL assignable to the imine carbon atoms, confirms the presence of the Schiff base
ligand in the complex [34,45]. The results shown in Table 3 confirmed that carbons of two ‒CH
groups are in different chemical environments (24.3 and 23.9 ppm) and that they appeared as two
2
complex show 10 signals. The peak at 169.1 ppm in C NMR
2
3
1
3
signals in C NMR spectrum.
3
.2. Description of the crystal structures
View of the molecular structures of NiL
2
, CoL , CuL
2
2
and ZnL complexes with common atom
2
numbering scheme are shown in Figs. 1‒4, respectively. The crystallographic data and selected
bond lengths and angles are collected in Tables 1 and 2, respectively. The crystallographic data
reveal that in all complexes the metal center is four coordinated by two phenolate oxygen and
two imine nitrogen atoms of two moles of Schiff base ligand HL. The geometry around the
metal center in all complexes is distorted tetrahedral [32]. The C=N bond distances are 1.288(2)
(
N1=C7) and 1.284(3) Å (N2=C18) for CoL
NiL , 1.298(4) (N1=C7) and 1.284(5) Å (N2=C18) for CuL
.282(3) Å (N2=C18) for ZnL consistent with the C=N bond when coordinated to a metal
center [32].
2
, 1.282(3) (N1=C7) and 1.288(2) Å (N2=C18) for
2
2
and 1.287(3) (N1=C7) and
1
2
Examination of the main metal-ligand distances shows that the M—N distances are longer than
the M—O distances due to stronger ability of the oxygen atom to bond to the metal than the
13

nitrogen atom. The M—N distances are 2.0004(17) and 1.9896(18) Å for CoL
2
, 1.9768(17) and
and 2.0110(16) and 2.0028(18) Å for
, 1.8943(15) and
and 1.9098(16) and 1.9205(16) Å for
1
.9840(16) Å for NiL
ZnL
.8879(14) Å for NiL
ZnL
CoL
in CuL
and lower respectively than other complexes.
Structures of Cobalt(II), Copper(II), Zinc(II) and Palladium(II) Schiff base complexes, CoL'
CuL' , ZnL' and PdL' derived from salicylaldehyde and tert-butylamine have been previously
reported by our group [32]. The crystal structure of theses complexes also show a distorted
2
, 1.993(3) and 1.998(3) Å for CuL
2
2
. While, the M—O distances are 1.9050(15) and 1.9117(15) Å for CoL
2
1
2
, 1.902(2) and 1.900(2) Å for CuL
2
2
. The N(1)–M–N(2) angle in CuL
; 120.61(7)° for NiL ; 137.70(13)° for CuL
, the distance and steric hindrance between the two isopropyl groups in CuL
2
is larger than the other complexes (122.51(7)° for
and 122.62(7)° for ZnL ). With such an angle
are greater
2
2
2
2
2
2
2
,
2
2
2
tetrahedral geometry around the metal center. There was no structural change in the complexes
by changing the tert-butylamine to isopropylamine, except some change in distances and angles
(
Fig. 5). The most important change is the M-N bond lengths in two types of complexes (Fig. 5).
The M-N bond lengths in complexes derived from salicylaldehyde and isopropylamine (CoL
2
,
CuL
2
and ZnL
2
) are shorter than complexes derived from salicylaldehyde and tert-butylamine
(
CoL'
2
, CuL'
2
and ZnL' ) (Fig. 5). The steric hindrance of ancillary groups on nitrogen atom of
2
azomethine group (tert-butyl or isopropyl) are responsible in increase or decrease of M-N bond
lengths. For example see comparison between CuL and CuL' complexes in Fig. 6. The X-ray
results showed that the hydrogen atoms of two tert-butyl group coordinated to metal center in
CoL' , CuL' and ZnL' complexes have higher steric crowding than hydrogen atoms of two
isopropyl group in CoL , CuL and ZnL complexes (Fig. 6). This steric hindrance reduced the
2
2
2
2
2
2
2
2
14

M-N bond lengths in CoL'
CuL and ZnL
2
, CuL'
2
and ZnL'
2
and increased the M-N bond lengths in CoL ,
2
2
2
.
3
3
.3. Interaction with DNA
.3.1. UV-Vis absorption
UV-Vis spectroscopy is an effective method to obtain valuable information about binding mode
and binding affinity of various types of compounds to biomacromolecules [56]. Generally,
hyperchromic or hypochromic effect and red or blue shift are observed in the UV–vis spectrum
of a drug upon its DNA-binding. Both hypochromic and red shift are indicative of intercalation
mode involving an interaction between π*-orbital of drug with π-orbital of DNA base pairs.
Therefore, energy level of π*-orbital of drug decreases which causes red shift in its UV–vis
spectrum. Furthermore, the coupled π*-orbital is filled and so the probability of electron
transition decreased and hypochromic is observed. While a groove binding or electrostatic
interaction leads to hyperchromic effect along with blue shift. A hyperchromic effect can be due
to breakage of the secondary structure of DNA [57,58]. The absorption spectra of the synthesized
metal complexes both in the absence and presence of different concentrations of FS-DNA were
given in Figs. 7 and S1. The intensity of these spectra decreased with the addition of FS-DNA.
To quantitatively evaluate the affinity of compounds with FS-DNA, the intrinsic binding
constant K
b
was determined by monitoring the changes in absorbance by using the following
equation [59]:
1
1
1
=
+
ꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕ(1)
ꢍꢎ − ꢍꢄ ꢍꢄ − ꢍꢏ ꢐ ꢑꢍ − ꢍ ꢒ[ꢓꢔ]
ꢄ
ꢄ
ꢏ
15

Where [BM] is the concentration of biomacromolecule while the [BM] is concentration of FS-
DNA; ε , ε and ε are the apparent extinction coefficient, the extinction coefficient for free
compounds and the extinction coefficient for the compounds in a fully bound form, respectively.
was determined by calibration curve and ε is the ratio of Aobs to [complex]. A plot of 1/(ε -ε
versus 1/[DNA] gives K as ratio of y-intercept to slope. The binding constants of FS-DNA with
a
f
b
ε
f
a
a
f
)
b
4
4
4
3
-
Cu(II), Zn(II), Co(II) and Ni(II) complexes were 2.46×10 , 2.09×10 , 2.03×10 , and 9.85×10 M
1
,
respectively.
3
.3.2. Fluorescence spectroscopy
The fluorescence measurements were performed in order to investigate the interaction
mode between the synthesized metal complexes and FS-DNA. Ethidium bromide (EtBr)
is a standard intercalating agent of DNA that is used to study DNA-binding of
compounds. It was known that EtBr has only a weak fluorescence emission in solution,
while a significant increase of its fluorescence intensity can be observed when it binds to
DNA in view of its strong intercalation between the adjacent base pairs of DNA [60-63].
The fluorescence quenching of DNA-EtBr with addition of metal complexes are shown in
Figs. 8 and S2. Synthesized metal complexes are not able to have efficiently competition
with EtBr as strong intercalators due to their non-planer structure. However, these
complexes can displace EtBr in a non-competitive manner by changing the DNA
conformation. Consequently, the DNA-bound EtBr molecules are converted to their free
form in solution and cause fluorescence quenching. In order to determine the binding
ability between the compounds and FS-DNA, the Stern–Volmer quenching plot was
16

obtained by monitoring the fluorescence quenching of EtBr-DNA with increasing the
concentration of the compounds according to the Stern–Volmer equation [64]:
ꢖꢗ
=
1 + ꢐ [ꢚ] = 1 + ꢛ ꢝ[ꢚ]
(2)
ꢘꢙ
ꢜ
ꢖ
Where,
F
0
and
F
are the fluorescence intensity of EtBr-DNA in absence and presence of
SV is the Stern-Volmer quenching constant, is the quenching rate
is the average lifetime without quencher which is typically equal to 10
sv for synthesized complexes are
ꢕꢕvs. [Q] (Figs. 8 and S2) and were presented in Table 4.
the compounds.
constant of and
K
k
q
-
8
τ
s for biomacromolecules [65]. The values of
determined from the plot of
K
0
F /F
Fluorescence quenching is classified to two mechanisms including static quenching and
dynamic quenching. In the static mechanism, the fluorophore and the quencher collide are
both in the ground state while fluorophore and quencher collide together in the excited
state in dynamic mechanism [65]. Linearity of the Stern–Volmer plot indicates that
quenching fluorescence has only one mechanism, either dynamic or static [66]. In this
1
1
-1 -1
study, the values of
q
k were about 10 M .s which are greater than the limiting
1
0
-1 -1
diffusion rate constant of the diffusional quenching for biopolymers (2×10 M .s ). This
observation supports that quenching fluorescence occurs through static mechanism [65].
Moreover, the binding constant (
equation [66]:
b
K ) have been determined by using the following
ꢀꢡ − ꢀ
ꢞꢟ ꢠ
ꢢ = ꢞꢟ(ꢐ ) + ꢟꢞꢟ[ꢚ]ꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕ(3)
ꢄ
ꢀ
17

Where, [Q] is the concentration of quencher that is the synthesized compounds here. “
b
K ”
is obtained from the plot of ꢞꢟ((ꢀ − ꢀ)/ꢀ)ꢕ versusꢕꢕꢞꢟ[ꢚ]ꢕ as a y-intercept.
ꢡ
Furthermore, “
n
” which is the number of binding site per DNA is slope of the plot (Fig.
is nearly 1, indicating that the synthesized complexes bind to DNA
S5). The value of
n
with molar ratio of 1:1. The calculated results are shown in Table 4 and are in good
agreement with the UV-Vis spectroscopy results.
3.3.3. Viscosity measurements
To further verify of interaction mode between the metal complexes and DNA, viscosity
measurements of DNA upon addition of the complexes were carried out. A classical
intercalative mode causes an increase in the DNA viscosity. Because of base pairs are
separated in order to accommodate the binding ligand and then the overall length of DNA
increases [51]. Non-classical mode of interactions such as groove binding and
electrostatic could bend the DNA helix, reduces its length and causes the reduction or no
change in the DNA viscosity [51]. The effect of the metal complexes on the viscosity of
DNA is illustrated in Fig. 9. As can be seen, the viscosity of DNA decreased slightly or
remained constant with an increasing amounts of complexes, indicating the binding mode
of all complexes may be groove binding. This result is consistent with molecular docking
results (section 3.3.4.).
3.3.4. Molecular docking
To obtain a deep insight about DNA binding of complexes, molecular docking studies
were carried out. The docked model results suggest that all complexes bind to the minor
18

groove of DNA. Table 5 represents the binding mode and the nucleotides around each of
metal complexes. The CuL ZnL CoL and NiL complexes formed two, three, one
2
,
2
,
2
2
and one hydrogen bond interactions with DNA, respectively. Also, there are two π-π
stacking interactions between Cu(II) complex and DG4 nucleotide of DNA. The docking
poses of metal complexes-DNA and participant nucleotides in H-bond and π-π stacking
interactions are presented in Fig. 10. The standard binding free energies (∆
G°),
describing the affinity of the complexes for binding to DNA with the best scores, are -
−
1
7
.14, -6.82, -6.17 and -6.11 kcal.mol for CuL
2
,
ZnL
2
,
CoL
2
and NiL complexes,
2
respectively. The docking results are in good agreement with spectroscopic results (see
Table 4). Both experimental and computational results collectively suggest that Cu(II)
complex has more DNA binding affinity than the other synthesized complexes and all of
the synthesized complexes are DNA groove binder. The binding of a ligand to the minor
groove of DNA simplifies the interaction between ligand and minor groove binding
proteins which are needed for gene expression [67].
3
.4. Interaction with Human Serum Albumin (HSA)
Drug-protein interaction affects the drug absorption, distribution and elimination in the
cardiovascular system [68]. Moreover, this binding can prevent rapid elimination of drugs from
bloodstream [69], and also increase drug solubility in plasma, decrease its toxicity, protect from
oxidation and prolong its in vivo half-life [70]. The requirement of HSA-binding study in design
and synthesis new drugs interested us to investigate HSA-binding behavior of the synthesized
metal complexes. Consequently, fluorescence quenching experiment together with chemometrics
method have been carried out to obtain information including binding mode, binding constants,
19

number of binding sites and intermolecular distances [71]. The UV–Vis absorption spectroscopy
was performed in order to calculate binding constant. More details about binding mode of
synthesized metal complexes to HSA were detected by means of molecular docking calculations.
3
.4.1. UV-Vis absorption
UV-Vis absorption spectroscopy is an effective and simple technique to study the HSA-drug
binding. The absorption spectra of the complexes in the absence and presence of different
concentrations of HSA were given in Figs. 11 and S3. The intensity of these spectra decreased
with the addition of HSA. To quantitatively evaluate of complexes affinity with HSA, the
intrinsic binding constant K
b
was determined by monitoring the changes in absorbance using
equation (1). The binding constants of HSA with NiL
2
, ZnL
2
, CuL
2
and CoL complexes are
2
4
4
3
3
-1
determined as 3.72×10 , 3.08×10 , 9.80×10 , and 3.38×10 M , respectively.
3
.4.2. Fluorescence spectroscopy
Fluorescence quenching experiment has been performed to investigate the mechanism of
interaction between HSA and the compounds. Although the fluorescence of HSA arise from
tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues, its intrinsic fluorescence is
mainly due to tryptophan [72]. The fluorescence intensity of protein was quenched through the
addition of complexes. This implies that the compounds strongly interact with HSA, leading to
changes of microenvironment around the Trp-214 residue in HSA [70, 73]. Figs. 12 and S4 show
-6
the fluorescence quenching of 5×10 M HSA at the presence of various amount of the
20

-5
compounds (0-5×10 M). It has been found that the fluorescence emission of synthesized metal
complexes has considerable overlap with fluorescence emission of HSA. Hence, using F in
equations (2) and (3) is an approximation; thus, these equations cannot predict the actual binding
constant. In order to overcome to this shortcoming, the chemometrics method was used. This
method allows concentration information of an individual component to be extracted in the
presence of other constituents. Thus, it is highly useful for solving analytical problems involving
a complex matrix. Separation of the experimental results obtained of fluorescence emission into
its components was performed by regression analysis using a written program in our laboratory
running in the MATLAB 7.2 (The MathWork Co.) environment. Calculations were performed
by an iterative procedure by using the Newton–Gauss–Levenberg/Marquardt (NGL/M)
algorithm of nonlinear least squares fitting [74].
The data were processed by using the proposed chemical equilibria (model; eg. P+A↔AP) and
an initial estimate of the binding constant (K) values. The program then minimizes the following
equation based on the variation of K in each titration and considers some restrictions such non-
negativity in concentration and the fluorescence intensities [75].
ꢩꢅ
ꢣ(ꢀꢤꢥꢦ − ꢀ )ꢌ ≅ 0ꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕ(4)
ꢁꢎꢧꢁ ꢨ
ꢨꢪꢡ
Where ns is the number of solution (total titration steps) and “exp” and “calc” are referred to
recorded and calculated signals, respectively. The output of program comprises the refined
parameters (net fluorescence of HSA, metal complex and HSA-metal complex systems), the
sum-of-squares and the standard deviation of the binding constant.
The computer fit of the fluorescence-mole ratio data for compounds is shown in Fig. 13.
21

In order to determine the binding ability between the compounds and HSA, the Stern–Volmer
quenching plot was obtained by monitoring the net fluorescence quenching of HSA (extracted
from chemometrics results) with increasing the concentration of the compounds according to the
Stern–Volmer equation (equation (2)). The values of Ksv for the synthesized complexes are
determined from the plot of F
0
/Fꢕꢕvs. [Q] (Figs. 12 and S4) and were presented in Table 6.
1
2
13
-1 -1
Moreover, the obtained values for k
q
are about 10 -10 M .S that are greater than maximum
1
0
-1 -1
collision quenching constant (2 × 10 M .S ) of various quenchers with the
biomacromolecules. This result represents the existence of static quenching mechanism. The
binding constants (K ) of the metal complexes and HSA are calculated using Eq. (3) (Fig. S6)
b
and are presented in Table 6. Comparison between obtained results from fluorescence quenching
with UV–Vis absorption showed a good agreement between them and represents high affinity of
HSA for all synthesized metal complexes. In general, binding constant of a drug with a carrier
protein such as HSA should be high enough to bind and transfer throughout the body. Moreover,
in order to release a drug on target, it should not be too high. HSA Binding constants of all the
4
-1
synthesized compounds are in a good range (2-7×10 M ) [76] and also are comparable to
analogue compounds and some potent drugs (Table 7).
Furthermore, these synthesized complexes are uncharged which and this is an advantage for a
drug. Several researches show that the unionized drugs are soluble in lipid and capable of
crossing through the membrane’s lipid bilayer while the ionized analogue species fail to cross
[
82].
3
.4.3. Energy transfer from HSA to complexes
22

Energy transfer between the compounds and HSA can provides valuable information about
HSA-complex binding. The fluorescence quenching of HSA upon its binding to metal complex
can be indicative of energy transfer between HSA and metal complex. This energy transfer can
be explained by fluorescence resonance energy transfer (FRET) theory. FRET also known as
,
Förster s resonance energy transfer which is an interaction between the excited molecule and its
adjacent molecule. Upon this interaction, energy absorbed by donor molecule is transferred to an
acceptor [83]. According to this theory, three conditions are required to energy transfer: (1) the
donor should have fluorescence, (2) the fluorescence emission spectrum of the donor and the
UV-Vis spectrum of the acceptor should have sufficient overlap (3) a small distance between
donor and acceptor (< 8 nm) [83]. The distance and efficiency of energy transfer (E) between
tryptophan residue of protein (HSA) and drug (complex) has been calculated by using this theory
through the following equation:
ꢭ
ꢀ
ꢬ
ꢡ
ꢫ = 1 −
=
ꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕ(5)
ꢭ
ꢭ
ꢀꢡ ꢬ + ꢮ
ꢡ
Where R
0
is the critical distance when the transfer efficiency is 50% ; r is the distance between
can be calculated by Eq. (6) [84].
donor and acceptor. R
0
ꢭ
ꢯꢌꢰ
ꢌ
ꢯꢲ
ꢬ = 8 ∙ 79 × 10 ꢐ ꢱ ꢳꢴꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕ(6)
ꢡ
2
In the above equation, the term K is the orientation factor of the dipoles; N is the refracted index
of medium, J is the overlap integral of the fluorescence spectrum of the donor with absorption
spectrum of the acceptor and φ is the fluorescence quantum yield of the donor. The value of J
can be calculated by the following expression:
23

ꢲ
ꢵ
ꢀ(ꢶ)ꢍ(ꢶ)ꢶ ꢷꢶ
ꢳ =
ꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕꢕ(7)
ꢵ
ꢀ(ꢶ)ꢷꢶ
Where, F(λ) is the fluorescence intensity of the donor in the absence of the acceptor at
2
wavelength λ and ɛ is the molar absorption coefficient of the acceptor at λ. In the present case, K
=
2/3, N = 1.336 and φ=0.15 for HSA. The parameters for the synthesized compounds were
calculated according to Eqs. (5)-(7) (Table 8). Fig. 14 represents the overlap of the fluorescence
emission spectrum and the UV-Vis spectrum of HSA-ML complexes with 1:1 molar ratio of
HSA to ML . The values of r for all the compounds are less than 8 nm and 0.5 R < r < 1.5 R
suggesting that energy transfer from HSA to metal complexes occurs with high probability.
2
2
0
0
,
3
.4.4. Molecular docking of the metal complexes with HSA
In this work, the CuL
2
, CoL
2
, NiL
2
and ZnL complexes were docked to the crystal structure of
2
HSA. The molecular docking results showed that all synthesized metal complexes are bound to
the boundary of IIA-IIB subdomains which is the main binding site for some drugs such as
diflunisal, ibuprofen and halothane (Table 9). The results indicated that H-bond interactions,
hydrophobic interactions, π-π stacking and π-cation interactions have a dominant role in the
stability of CuL
stacking or π-cation interactions in the CoL
complexes-HSA are shown in Fig. 15. The binding energy for NiL
2
, ZnL
2
and NiL
2
complexes-HSA. While, there is no H-bond interactions, π-π
-HSA complex. The docking poses of all metal
, ZnL , CuL and CoL
2
2
2
2
2
-1
complexes are evaluated about -6.75, -6.72, -6.61 and -6.04 kcal.mol , respectively. The larger
negative value of binding energy for Ni(II) complex means the higher affinity for HSA binding
which is in good agreement with UV-Vis and fluorescence experimental data. Additionally, the
24

distances between Trp-214 and metal complexes were 0.39, 0.32, 0.39 and 0.44 nm for NiL
ZnL , CuL and CoL complexes, respectively. These values are in consistent with obtained r
values from FRET calculations.
2
,
2
2
2
4
. Conclusion
In conclusion, we have synthesized four new Schiff base complexes, NiL
ZnL , derived from a bidentate Schiff-base ligand (HL: ((E)-2-
(isopropylamino)methyl)phenol). The structures of all complexes have been established by
2
, CoL
2
, CuL and
2
2
(
single crystal X-ray diffraction. The crystallographic data reveal that in all the complexes the
metal centers are four-coordinated by two phenolic-O atoms and two azomethine-N atoms to
form a distorted tetrahedral geometry. The DNA- and HSA-binding of the synthesized metal
complexes were investigated by using both experimental (fluorescence quenching, viscosity
measurements and UV-Vis spectroscopy) and computational methods (chemometrics and
molecular docking). Chemometrics method was carried out in order to study HSA-binding of
complexes, quantitively. The obtained HSA-binding constant values from experimental and
computational methods stated that the Ni(II) complex-HSA is more stable than the others.
Molecular docking studies showed that H-bond interactions, hydrophobic interactions, π-π
stacking and π-cation interactions have a dominant role in the stability of complex-HSA. The
computational docking and viscosity data suggest that all metal complexes interact with DNA by
the groove binding mechanism. The results showed that Cu(II) complex exhibited stronger DNA
binding affinity than other complexes.
25

Acknowledgement
The authors are grateful to the Research Council of the University of Isfahan for financial
support of this work.
Appendix A. Supplementary data
CCDC 1487336, 1487337, 1487338 and 1487339 contain the supplementary crystallographic
data for NiL
2
, CoL
2
, CuL
2
, and ZnL , respectively. These data can be obtained free of charge
2
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
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