Synthesis and Characterization of Cobalt(II) and Manganese(II) Schiff Base Complexes: Metal Effect on the Binding Affinity with β-Casein

Article abstract of DOI:10.1007/s10895-020-02612-0

In this study, two metal Schiff base complexes (cobalt(II) and manganese(II), were synthesized and characterized by ¹H-NMR and FT-IR analyses. The in vitro binding behavior of this complexes with β-Casein (β-CN) was investigated by using biophy

Full text of DOI:10.1007/s10895-020-02612-0

Journal of Fluorescence
https://doi.org/10.1007/s10895-020-02612-0
ORIGINAL ARTICLE
Synthesis and Characterization of Cobalt(II) and Manganese(II) Schiff
Base Complexes: Metal Effect on the Binding Affinity with β-Casein
Nafiseh Jahandari1 & Fatemeh S. Mohseni-Shahri1 & Farid Moeinpour1
Received: 23 July 2020 /Accepted: 24 August 2020
#
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
In this study, two metal Schiff base complexes (cobalt(II) and manganese(II), were synthesized and characterized by 1H-NMR
and FT-IR analyses. The in vitro binding behavior of this complexes with β-Casein (β-CN) was investigated by using biophys-
ical techniques. For evaluation the thermodynamics parameters of interaction between Schiff base complexes and β-CN,the
fluorescence data at different temperatures were done. The results showed that the intrinsic fluorescence of the β-CN was
quenched by increasing the complexes through the dynamic quenching mechanism. Also, these complexes demonstrated a
considrable binding affinity to β-CN and the process is mainly entropy driven. Fluorescence resonance energy transfer was used
in order to estimating the distance between donor (β-CN) and acceptor (complex). Results demonstrated that the sequence of
tendency of the complexes to β-CN was as follows: Mn-Schiff base complex > Co-Schiff base complex. All these experimental
results propose that β-CN might act as carrier protein for Co(II) and Mn (II) complexes to deliver it to the target molecules.
.
.
.
Keywords β-Casein binding Schiff base complex Fluorescence quenching Thermodynamic parameters
Abbreviations
soluble, but increasing them to the formulation is very diffi-
β-CN
β-Casein
cult. Nomerous hydrophobic compounds, like essential fatty
acids and lipid-soluble vitamins, are sensitized to oxidation,
and additionally the requirement to stabilize in the aqueous
conditions, as well as they need conservation from destructive
agents. The organization of nano-carriers to deliver these nu-
tritious has conduced to increased protection and solubility.
Milk proteins have significant functional properties, contaning
the capability to bond to hydrophilic molecules, the capability
to organize gels, interact with biopolymers, and stabilize
emulsions. Due to these properties, casein is known as a per-
fect tool for the delivery and enticement of bioactive mole-
cules [1].
β-Casein (β-CN), classified among four major caseins
in bovine milk, contains 209 residues in a individual chain
with the molecular weight of 23.983 kDa. Bovine β-CN is
an extremely amphiphilic protein consist of a hydrophilic
N-terminal segment with a hydrophobic C-terminal seg-
ment and a group consist of five phosphoryl residues [2].
Radius of gyration (Rg) of single β -CN molecules are
4.6 nm and its isoelectric pH (pI) is 5.33. The micelles
including about 16–60 molecules of β-CN have a Rg value
of 7.2 to 13.4 nm [3]. It must be attended that bovine β-CN
is recognized as a dynamical protein that consist of flexible
and non-condensed conformation [2].
DMSO Dimethyl sulfoxide
TMS
TYR
TRP
Phe
Tetramethylsilane
Tyrosine
Tryptophan
Phenylalanine
Introduction
Due to the growing knowledge of the significance of food in
preventing disease, modern technologies for enriching differ-
ent types of foods and drinks have been recommended using
several types of bioactive compounds to produce supplemen-
tary foods. As well as, because of the growing utilization of
low-fat and non-fat foods, the requirement to add lipid-soluble
nutritious to other foods is more than the types of water
* Fatemeh S. Mohseni-Shahri
fmohsenishahri@gmail.com; fmohsenishahri@iauba.ac.ir
1
Department of Chemistry, Islamic Azad University, Bandar Abbas
Branch, Bandar Abbas, Iran

J Fluoresc
Previous studies have shown that the average accumulation
number of β -CN micella is within 45.75 [4]. Therefore, β-
CN micelles probably used in the role of naturally nano-
delivery transporters for lipid-soluble drugs (for example: su-
crose esters, vitamin A, K, E and D) [5]. Also β-CN nanopar-
ticles can capture and carry hydrophobic chemotherapeutics in
order that binding of lipidsoluble molecules on β-CN hydro-
phobic interactions take part in a significant role [6–8]. The
simple digestion of β-CN guaranties the easy liberation of
encapsulated chemotherapeutics drugs in the stomach [9].
Compounds with a functional group that contains -C=N-
(azomethine functional group) are well-known in the role of
Schiff bases, which are generally prepared by the condensa-
tion reaction of carbonyl groups and primary amines [10].
Schiff bases are mainly studied because of their sensitivity
to the metallic ions, synthetic flexibility and selectivity [11].
Schiff bases possess a diversity of biological applications in
medicinal and pharmaceutical region. They possess antimi-
crobial, antibacterial, antifungal, antioxidant, antimalarial
and anticancer properties [12–14]. As Schiff bases are poten-
tially anticancer drugs, it was of concern to investigation their
manners of binding to the plasma carrier proteins.
were of analytic quality and were dissolved in doubled dis-
tilled water.
For prepare of β-CN micelle solutions, the β-CN solution
was filtrated via a 0.45 μm aporous membrane. Next, they
were dialyzed versus the phosphate buffer for 24 h at 4 °C,
to prevent formation of Ca2+ bridge using 1 mM EDTA solu-
tion [17]. After dialysis the β-CN concentration was specified
spectrophotometrically employing ε = 0.44 ml mg−1 cm−1 at
280 nm [18].
Synthesis and Characterization
Schiff base 2,2′-((1E,1′E)-(1,2-phenylenebis(azaneylylidene))
bis(methaneylylidene)) diphenol was prepared by condensa-
tion of Salicylaldehyde (2 mmol) with 1,2-Phenylenediamine
(1 mmol). The reaction solvent was absolute ethanol. The
solution was refluxed for 5 h and next was made cold at
ambient temperature. Yellow production was precipitated,
then it was filtered and washed [19] (Scheme 1).
A solution of metal(II) acetate tetrahydrate (2 mmol) was
adding to the stirring solution of the ligand (2 mmol). The
melange was strongly stirred for 6 h at 40 °C, and then ethanol
(5 mL) was adding and stirring was persisted to precipitate the
production. The resulting powder was gathered by filtration,
washed with ethanol then recrystallized and dried at 55 °C in
vacuum.
Schiff bases and their first-row transition metal complexes
(such as Co(II), Mn(II), Ni(II), etc.) were accounted to display,
antiviral, antibacterial, antifungal and antitubercular activity
[15]. Manganese Schiff base complexes can be beneficial in
the field of pharmaceutical manufacturing because of their
biological responsibilities as excellent antibacterial in addition
to antifungal agents [16].
Characterization
In continuing our research on the interaction of transition
metal complexes with β-CN, Co(II) and Mn(II) Schiff base
complexes have been synthesized to study the effect of metal
on the binding constants of the complexes with β-CN. These
studies are beneficial for the design and synthesis of new base
metal drugs. At first the Schiff base complexes were synthe-
Reaction was detected using thin layer chromatography
(TLC). Melting point was controled on a apparatus
Electrothermal IA9100. A THERMO IR100 FT-IR spectrom-
eter was used to recording the IR spectrum for investigate the
functional groups of Schiff base ligand. 1H-NMR spectrum of
Schiff base ligand was registered in DMSO using TMS as
1
sised and specified by H-NMR and FT-IR spectroscopies.
After that, the β-CN interaction of these complexes in aque-
ous solutions were explored using fluorescence and UV-Vis
spectroscopy methods.
Experimental
Materials and Methods
Bovine milk β-CN (98%, Sigma Company) was dissolved in
pH 7.0 phosphate buffer comprising 80.0 mM NaCl, 5.60 mM
Na2HPO4, and 3.10 mM NaH2PO4 with an ionic strength of
0.10. 1,2-Phenylenediamine, Salicylaldehyde, Cobalt(II) ace-
tate tetrahydrate and Manganese(II) acetate tetrahydrate were
purchased from Merck company and employed without addi-
tional purifiying. Each of salts employed for buffer preparing
Scheme 1 The chemical structure of 2,2′-((1E,1′E) -(1,2-
phenylenebis(azaneylylidene)) bis(methaneylylidene)) diphenol

J Fluoresc
standard on a Bruker Vector 33 NMR spectrometer
(400 MHz, SF:300) to recognize its molecular structure.
Fluorescence Quenching
To decrease the inner filter effect, absorption spectra of β-CN
at 280 nm (excitation wavelength) was registered and the
fluorescence intensities used in this research were all corrected
using the relationship equation [24]:
Results & Discussion
Schiff Base Characterization
ꢀ
ꢁ
Aex þ Aem
Fc ¼ F*antilog
ð1Þ
2
The synthesized Schiff base is colored solid, water insoluble,
but soluble in ethanol. The compound is persistent and has
melting point (169 °C). Its structure was proved through spec-
troscopic data (1H-NMR and FTIR).
where F and Fc are initial and corrected intensity of fluores-
cence, respectively, Aex is absorption of complex in the exci-
tation wavelength and Aem is absorption of complex in the
emission wavelength.
The intrinsic fluorescence of β-CN is related to Trp-143
(tryptophan). This tryptophan residue is mainly placed in the
hydrophobic region of this protein. When other molecules
interact with β-CN dependent upon the effect of interaction
on the protein configuration, the fluorescence of tryptophan
may change [25]. The effect of Cobalt(II) and Manganese(II)
Schiff Base Complexes on β-CN fluorescence intensity is
shown in Fig. 2. It can be noted that schiff base complexes
quenched fluorescence of β-CN and maximum emission
wavelength has only a weak blue shift (Fig. 2a and b). The
slight blue shift in the fluorescence spectra correlated with a
decreased polarity of the microenvironment after binding [26].
For analysis of β-CN fluorescence quenching, the fluores-
cence intensity data of β-CN at 298, 305 and 310 K was
analysed in accordance with the Stern–Volmer equation [26]:
1H-NMR Analysis
The 1H-NMR spectrum affirms the identity of the synthesized
Schiff base ligand, untill identify its structure. The 1H-NMR
spectrum of synthetic Schiff base ligand (Scheme 1) exhibited
the azomethine protons at 8.94 ppm as singlet [20]. The com-
pound has aromatic rings, the hydrogens of the aromatic rings
were confirmed in the range of 7.00–7.66 ppm. The phenolic
OH peak was observed at 12.91 ppm.
FT-IR Analysis
The FT-IR spectrum of synthesized Schiff base are represent-
ed in Fig. 1. The FT-IR spectrum displays a powerful band
allocated to azomethine stretching vibrations revealed at
1608 cm−1 affirming the Schiff base structure [21–23]. A
weakly absorption bands in the around 3120–3040 cm−1 allo-
cated to the CH aromatic stretching vibrations. The broad
band at 3325 cm−1 wich attributed to phenolic OH stretching
vibrations. The different absorption bands in the area 1500–
1580 cm−1 allocated to C=C stretching vibrations of the aro-
matic rings.
F0
F
¼ 1 þ Kqτ0½QŠ ¼ 1 þ Ksv½QŠ
ð2Þ
where F and F0 are the intensities of fluorescence in the pres-
ence and absence of quencher, respectively; Kq is the
quenching rate constant for the biomolecule, KSV is the
Stern–Volmer dynamic quenching constant, τ0 is the mean
lifetime of the fluorophore whitout quencher (τ0 = 3.3 ns for
Fig. 1 FT-IR spectrum of 2,2′-
((1E,1′E)-(1,2-
phenylenebis(azaneylylidene))
bis(methaneylylidene)) diphenol
Wavelength (cm-1)
4.00E+03 3.50E+03 3.00E+03 2.50E+03 2.00E+03 1.50E+03 1.00E+03 5.00E+02
0.00E+00
5.00E-02
1.00E-01
1.50E-01
2.00E-01
2.50E-01
3.00E-01
3.50E-01

J Fluoresc
number, <J>, possibly induced by the complexes can be spec-
ified by using the following equation (Table 1):
F
F0
½complexŠ
½proteinŠ
1−
¼< J >
ð3Þ
The slope, <J > <1, demonstrated that the complex binding
did not bring about any ggregation on β-CN molecules and
consequently evidenced the 1:1 stoichiometry for
complex:protein systems [19].
Identification of Binding Parameters
The Scatchard equation is applied to evaluate the binding
constant of ligand to protein (Ka) and the binding sites number
(n) concerned with the binding interaction; it is known as [27]:
ðF0−FÞ
log
¼ logKa þ nlog½QŠ
ð4Þ
F
Figure 5 shows the plots of log (F0 − F)/F against log[Q] for
the interaction between β-CN and Co-Schiff base complex at
different temperatures. n and Ka obtained from the plots are
listed in Table 2. It may be considered from Table 2 with
increasing the temperatures, Ka increased slightly. The order
of the Ka values was as follows: Mn-Shiff base complex > Co-
Shiff base complex. Therefore, it is deduced that Mn(II) com-
plex had more tendency to interact with β-CN than Co(II).
Fig. 2 Emission fluorescence spectra of β-CN at varying concentrations
of (a) Co complex (0–9.68 μM), (b) Mn complex (0–9.68 μM), at λex
=
280 nm, T = 298 K, pH 7.0
β-CN), and [Q] is the quencher concentration. The plots of F0/
F against schiff base complex concentration at three different
temperatures were presented in Fig. 3. The quantities of KSV
at different temperatures are given in Table 2. The values of
KSV demonstrated the respective affinity of complex for β-
CN. The data indicated that with increasing temperature, the
values of KSV increased so the eventual quenching mecha-
Thermodynamic Parameters
The interaction forces between proteins and ligands contain
four types: H-bonds; hydrophobic and electrostatic interac-
tions; and van der Waals forces. In accordance with the
Van’t Hoff equation, when the temperature does not change
extremely,the enthalpy (ΔH) and entropy (ΔS) changes of the
nism is dynamic quenching.20 The plots of 1− vs. [com-
F
F0
plex]/[protein] affirmed the 1:1 stoichiometry between β-CN
1.4
1.3
1.2
1.1
1
and quencher (Fig. 4). The average β-CN aggregation
Table 1 Stern−Volmer quenching constants and average aggregation
number of β-CN molecules (<J>) for the interaction of Schiff base com-
plexes with β-CN at different temperatures (pH = 7.0)
Complex
T(K) Ksv (M−1) × 10−4 R2
<J>
Co-Schiff base complex
298
305
310
4.55
5.81
7.02
6.59
7.94
8.864
0.983 0.118
0.995 0.137
0.995 0.185
0.984 0.221
0.996 0.247
0.995 0.273
0.9
0.8
0
1
2
3
4
5
Mn-Schiff base complex 298
[complex] µM
305
310
Fig. 3 The Stern–Volmer plots of the fluorescence titration of β-CN with
Co-Schiff base complex at different temperatures ((Δ) 298 K, (■) 303 K,
(•) 310 K), pH = 7.0

J Fluoresc
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
[complex]/[protein]
Fig. 4 Determination of the average aggregation number of β-CN (<J>)
in the presence of Co-Shiff base complex at different temperatures (■)
298 K, (♦) 305 K, (•) 310 K, pH = 7.0
Fig. 5 Plot of log (F0 − F)/F versus log[complex] at different
temperatures (■) 298 K, (♦) 305 K, (•) 310 K, pH = 7.0
Fluorescence Resonance Energy Transfer (FRET) be-
tween β-CN and Schiff Base Complexes
binding reaction can give proof for clarification interaction
types. If ΔH does not alter remarkably through the tempera-
ture range considered, the values of ΔH, ΔS, and ΔG are
calculated by the following equation:
The energy transfer phenomena, is classified into radiative and
nonradiative energy transfer modes in fluorescence. In accor-
dance with Forster’s theory [29], the energy transfer is influ-
enced by the distance amongst the donor and acceptor, (r) and
the critical energy transfer distance (R0).
ΔH ΔS
ln Ka ¼ −
þ
ð5Þ
RT
R
Somewhere R is the universal gas constant, T is the expe-
riential temperature (K), Ka is the binding constant. The en-
tropy (ΔS) and the enthalpy (ΔH) changes can be achieved
from the intercept and slope of the fitted curve of ln Ka against
1/T, respectively (Fig. 6). Later the Gibbs free energy, ΔG,
was obtained employing the subsequent equation [28]:
The relationship between FRET efficiency and distance is
as follows:
F
F0
R06
R60 þ r6
E ¼ 1−
¼
ð7Þ
somewhere E is energy transfer effciency; F and F0 are inten-
sities of fluorescence of β-CN with and whithout of ligand. R0
is calculated using the subsequent equation:
ΔG ¼ ΔH−TΔS
ð6Þ
Ross and Subramanian described that the sign and value of
the thermodynamic variables envolved in the protein associa-
tion processes. The calculated thermodynamic variables are
summarised in Table 2.
It can be concluded from results that the binding reactions
have spontaneously occurred (ΔG < 0). Additionally, the neg-
ative enthalpy (ΔH>0) and entropy (ΔS>0) demonstrate the
important roles of hydrophobic interactions in the both of
metal Schiff base complexes binding to β-CN.
R0 ¼ 8:79 Â 10−25 K2 N−4 Ф J
ð8Þ
6
somewhere K2 implies the spatial orientation factor; medium
refractive index is “N” that N = 1.53. Ф is the donor’s fluores-
cence quantum yield without acceptor which is equal to Ф =
1.49; and J is the result of spectra overlap between absorption
spectrum of the acceptor and the fluorescence emission spec-
trum of the donor and is presented by:
Table 2 Binding constant (Ka), binding site (n) and thermodynamic parameters for the interaction of Schiff base complexes with β-CN at different
temperatures
Complex
T(K)
Ka(M−1) × 10−4
n
R2
ΔH (kJ mol−1
)
ΔS (J mol−1 K−1
)
ΔG (kJ mol−1
)
Co-Schiff base complex
298
305
310
298
305
310
1.21
2.66
7.59
6.43
9.65
14.11
1.39
1.42
1.59
1.48
1.63
1.73
0.991
9.996
0.985
0.987
0.991
0.994
114.86
462.97
−23.105
−26.346
−28.660
−27.390
−29.204
−30.500
Mn-Schiff base complex
49.830
259.13

J Fluoresc
12
11
10
9
that the synchronous fluorescence of β-CN was characteristic
of TRP residue and Δλ = 15 nm was characteristic of TYR.
The synchronous fluorescence spectra of β-CN at various
concentrations of Co-Schiff base complex at Δλ = 60 and
Δλ = 15 nm are shown in Fig. 8a and b, respectively.
It is obvious from Fig. 8 that the emission peak of the TRP
residue (Δλ = 60 nm) showed a slow quenching and a slight
blue shift, denoting that the metal complex was placed in near
vicinity to the TRP residues and the polarity near the TRP
residues was decreased and so the hydrophobicity was in-
creased slightly. When the hydrophobic part of the complex
was close sufficient to the phenyl moiety of TRP, hydropho-
bicity near the TRP residues increased unwillingly. However
the emission peak of the TYR residues (Δλ = 15 nm)
displayed quenching without considrable shift, which sug-
gested that referred complex was near to TYR residues but
had no effect on the conformation of TYR microenvironment.
As well as, the quenching of the fluorescence intensity of
TYR residues was stronger than that of the TRP residues,
proposing that TYR residues contributed significantly to the
quenching of the intrinsic fluorescence [24].
8
0.0032
0.00324
0.00328
0.00332
0.00336
0.0034
1/T
Fig. 6 The van’t Hoff plot of Co-Schiff base complex at different
temperatures
J ¼ Σ F ðλÞ ε ðλÞ λ4 Δλ=Σ F ðλÞ Δλ
ð9Þ
soemwhere F(λ) is the emission spectrum fluorescent of do-
nor; ε(λ) implies molar absorption coefficient of the acceptor.
Using the above equations the r values were estimated 2.87
and 4.02 nm for Mn-Schiff base and Co-Schiff base com-
plexes, respectively. Figure 7 displays an overlap among the
absorption spectra of Co-Schiff base complex and the emis-
sion spectra of β-CN.
Since the distance amongst acceptor and donor is found to
be lower than 7 nm, indicates a high opportunity of
nonradiative energy transfer between β-CN and the both met-
al Schiff base complexes [30].
Similar spectral specifications were found for the interac-
tion of the Mn-Schiff base complex with β-CN.
Protein Conformational Changes Study
The fluorescence of proteins originates from the tryptophan
(TRP), tyrosine (TYR) and phenylalanine (Phe) residues. The
spectrum of proteins was susceptible to the microenvironment
of such chromophores. To investigate molecular environment
surrounding of β-CN, synchronous fluorescence spectra can
be used. Discrimination of the disagreement between excita-
tion wavelength and emission wavelength (Δλ) demonstrates
the spectra of diverse chromophores. Δλ = 60 nm mentioned
Fig. 7 Spectral overlap of β-CN fluorescence (a) with Co-Schiff base
complex absorption (b)
Fig. 8 Synchronous fluorescence spectra of β-CN at (a) Δλ = 60 nm and
(b) Δλ = 15 nm in the presence of Co-Schiff base complex

J Fluoresc
Conclusion
In this study, two metal Schiff base complexes were synthe-
sised and specified by diverse spectroscopic techniques.
Binding of Schiff base complexes with β-CN were studied
by multi-spectroscopic techniques. The outcoms of fluores-
cence demonstrated that Schiff base complexes be able to bind
to β-CN and quench the fluorescence of protein with dynamic
mechanism. As well as, it was deduced that complex binding
did not cause any aggregation on β-CN molecules and so
evidenced the 1:1 stoichiometry for metal Schiff base com-
plex: β-CN systems. Binding parameters between β-CN and
Schiff base complexes such as the number of binding site and
binding constant were determined. The thermodynamic re-
sults displayed that hydrophobic interactions playing a princi-
pal role in the binding of these complexes to β-CN. ΔG
values indicated a high affinity of the β-CN for these com-
plexes which made clear the role of the β-CN as endogenic
carrier for these complexes in the body.
Fig. 9 The absorption spectra of β-CN in the presence of increasing
amounts of Co-Schiff base complex at T = 298 K
Absorption Study
The UV–Vis absorption spectra of β-CN in the presence of
different concentrations of Co-Shiff base complex show in
Fig. 9. With increasing the Co-Shiff base complex concentra-
tion, β-CN represented increasing of the absorption band.
Changes in maximum absorbance are powerful evidences
for the formation of complex between protein and ligand [31].
To assessment the binding constant Ka between protein and
ligand, the absorption information.
The sequence of the Ka values was as follows: Mn-Shiff
base complex> Co-Schiff base complex. Therefore, it is con-
cluded that Mn(II) Schiff base complex had more tendency to
interact with β-CN than Co(II).
All results revealed that both of metal Schiff base com-
plexes bind to β-CN efficiently,which might be a benificial
guide for Schiff-base drug design and β-CN seems to be the
promising Schiff base complex carrier.
were analyzed employing the subsequent equation [32,
33]:
Acknowledgements The authors are grateful for the financial support of
the Islamic Azad University Bandar Abbas Branch for this study.
A0
εP
εP
1
Compliance with Ethical Standards
¼
þ
Â
ð10Þ
A−A0 εB εB  Ka ½QŠ
Competing Interests The authors declare no competing financial
interest.
Where A0 and A are the absorbance of β-CN with and
without of metal Schiff base complex and εPand εB are the
extinction coefficients of protein and its complex with
Schiff base complex. Ka is the binding constant at the re-
lated temperature and [Q] is the concentration of metal
Schiff base complex. So, the double reciprocating chart
of A0/(A-A0) against 1/[Q] is straight line and the Ka could
be evaluated from the proportion of the intercept to the
slope. Ka values are listed in Table 3. The Ka values are
of the similar order of magnitude achieved by fluorescence
spectroscopy.
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