In vitro tyrosinase, acetylcholinesterase, and HSA evaluation of dioxidovanadium (V) complexes: An experimental and theoretical approach

Article abstract of DOI:10.1016/j.jinorgbio.2019.110800

The present study reports the biological evaluation of vanadium(V) complexes (1–3) against three different proteins: tyrosinase, acetylcholinesterase (AChE), and human serum albumin (HSA), which were studied by spectroscopic techniques and molecular docki

Full text of DOI:10.1016/j.jinorgbio.2019.110800

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In vitro tyrosinase, acetylcholinesterase, and HSA evaluation of
dioxidovanadium (V) complexes: An experimental and theoretical
approach
Otávio Augusto Chaves, Márcia Cristina Campos de Oliveira,
Cristiane Martins Cardoso de Salles, Francisco Mainardi Martins,
Bernardo Almeida Iglesias, Davi Fernando Back
PII:
S0162-0134(19)30223-5
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.110800
JIB 110800
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
14 April 2019
7 August 2019
11 August 2019
Please cite this article as: O.A. Chaves, M.C.C. de Oliveira, C.M.C. de Salles, et al.,
In vitro tyrosinase, acetylcholinesterase, and HSA evaluation of dioxidovanadium (V)
complexes: An experimental and theoretical approach, Journal of Inorganic Biochemistry
(2019), https://doi.org/10.1016/j.jinorgbio.2019.110800
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In vitro Tyrosinase, Acetylcholinesterase, and HSA Evaluation of
Dioxidovanadium (V) Complexes: An Experimental and Theoretical
Approach
Otávio Augusto Chavesa,b, Márcia Cristina Campos de Oliveirab, Cristiane Martins Cardoso
de Sallesc, Francisco Mainardi Martinsd, Bernardo Almeida Iglesiase* and Davi Fernando
Backd*
aInstituto SENAI de Inovação em Química Verde. Rua Morais e Silva N° 53, Maracanã, 20271030,
Rio de Janeiro-RJ, Brazil.
bInstituto de Química, Departamento de Química Orgânica, Universidade Federal Rural do Rio de
Janeiro – UFRRJ, 23970-000 Seropédica, RJ, Brazil.
cInstituto de Química, Departamento de Bioquímica, Universidade Federal Rural do Rio de Janeiro –
UFRRJ, 23970-000 Seropédica, RJ, Brazil.
dLaboratório de Materiais Inorgânicos, Departamento de Química, Universidade Federal de Santa
Maria - UFSM, 97115-900 Santa Maria, RS, Brazil.
eLaboratório de Bioinorgânica e Materiais Porfirínicos, Departamento de Química, Universidade
Federal de Santa Maria – UFSM, 97105-900 Santa Maria, RS, Brazil.
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Abstract
The present study reports the biological evaluation of vanadium(V) complexes (1-3)
against three different proteins: tyrosinase, acetylcholinesterase (AChE), and human serum
albumin (HSA), which were studied by spectroscopic techniques and molecular docking.
Despite the synthesis and characterization of complexes 1 and 2 having already previously
described, complex 3 is a novel dioxidovanadium(V) derivative. Complex 1 can activate both
tyrosinase and AChE enzymes in about 11.5 and 47.0%, respectively. On the other hand,
complexes 2 and 3 inhibited the same enzymes (1.30 and 46.0% for tyrosinase and 20.0 and
21.9% for AChE, respectively). Molecular docking calculations suggested that the presence
of the hydroxyl group in complex 1 is essential to activate tyrosinase enzymes. According to
theoretical analysis, hydrogen bonding, van der Waals, and hydrophobic forces are the main
binding interactions for each V(V) complex and AChE. Moreover, the interaction between
HSA and vanadium(V) complexes occurs via ground-state association, being only
enthalpically driven for complexes 1 and 2 and entropically and enthalpically driven for
complex 3. The interaction is spontaneous for all samples and the binding modes do not
perturb significantly the secondary and surface structures of the albumin. As there are few
reported cases in the literature that explore vanadium complexes against these three proteins,
the present results may contribute to future studies by offering different scaffolds to design
new vanadium(V) complexes in the hyperpigmentation process and Alzheimer’s disease.
Keywords: cis-dioxidovanadium(V) complexes; Human serum albumin; Tyrosinase activity;
Acetylcholinesterase activity.
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Introduction
The field of metallopharmaceuticals has become an important field of medicinal
chemistry due to therapeutic uses of metal-based drugs. Thus, in addition to vanadium being
a relevant species in several biological processes, many of its complexes with organic
derivatives have been proposed for treating several types of diseases, including diabetes,
cancer, anti-parasitic, antiviral, anti-bacterial, phosphatase inhibitor, and cardioprotection
[1,2,3,4]. Additionally, the speciation of vanadium complexes in blood serum has also been
investigated mainly because of its direct impact on the pharmacokinetical profile of potential
drugs, although much remains to be done concerning in situ real-time investigations [5].
Despite vanadium complexes presenting positive biological activity, they have yet to be used
in the clinic phase and further research on vanadium-based drugs is still necessary not only in
pharmacodynamic profiles, but also in the pharmacokinetical point of view.
Melanogenesis is a complex process with different stages that involve melanin
synthesis and transport and melanosome release. Melanin is composed of pheomelanin and
eumelanin and is synthesized in the melanocyte melanosomes. Melanin synthesis is mainly
regulated by the enzyme tyrosinase, thus, an abnormal synthesis of melanin can be related to
disorders in the tyrosinase activity, leading to skin conditions such as vitiligo, melasma,
chloasma freckles, and inflammatory pigmentation [6,7]. Tyrosinase is a copper-containing
polyphenol oxidative enzyme that plays a vital role in melanogenesis by catalyzing the
hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), followed by L-
DOPA oxidation to L-Dopaquinone, although tyrosinase may lead to various dermatological
disorders including hyperpigmentation or hypopigmentation [8,9]. Recently, some hydrazide
and vanadium(V) hydrazide complexes were evaluated againt tyrosinase activity. The results
showed that vanadium complexes had better activity than hydrazides without the presence of
metal ions and variable degrees of inhibition potential [10]. Nevertheless, the development of
tyrosinase inhibitors or activators for use as skin agents is still under evaluation.
Acetylcholinesterase (AChE) is a very important enzyme used to control transmission
between neurons, which is when the process is either mediated or modulated by the
neurotransmitter acetylcholine (ACh). Acetylcholine is released by the axon terminal or
varicosities of the transmitter neuron into the extracellular space to interact with the receptors
of the other neuron [11]. Some reversible AChE inhibitors have medical applications and are
particularly important in treating Alzheimer’s disease. When people develop Alzheimer’s
disease, their neurons degenerate, which leads to low neurotransmitter production, a process
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that induces serious memory problems [12]. Mild AChE inhibition has shown to have
therapeutic relevance in treating Alzheimer’s disease. In contrast, strong AChE inhibition
may lead to cholinergic poisoning. In order to restore acetylcholine levels to normal
(removing the offending AChE inhibitor), novel series of organic/inorganic AChE
reactivators have been developed [13]. Recently, the possible effects of vanadium therapy on
the kinetic parameters of brain membrane-bound and soluble AChE forms in alloxan-induced
diabetic rats were reported and showed that sodium orthovanadate administration reversed
diabetic conditions by decreasing blood glucose levels and normalizing blood hemoglobin
A1C (HbA1C) levels. The same sodium orthovanadate may also normalize the levels of brain
AChE, glutathione-S-transferase, and thiobarbituric acid reactive substances compared to
diabetic state and control. Therefore, vanadate administration may protect against the direct
action of lipid peroxidation on brain AChE and, in this way, possibly help prevent cholinergic
neural dysfunction, which is one of the most common complications in diabetes [14].
Serum albumin is the most abundant plasma protein in the bloodstream. Human serum
albumin (HSA) is a highly soluble negatively charged protein that helps maintain oncotic
pressure of the human circulatory system and distribute different organic/inorganic
compounds by interacting with several endogenous (e.g., bilirubin and fatty acids) and
exogenous (e.g., drugs and xenobiotics) species [15]. Thus, HSA commonly plays a crucial
role in the pharmacokinetic process of commercial and potential drugs, as whatever the
therapeutic use envisaged for any compound, its pharmacokinetics must be addressed,
namely how it is transported in the bloodstream [16]. From a structural point of view, HSA is
a non-glycosylated polypeptide (585 amino acid residues and molecular weight 66500 Da)
that presents a heart-shaped structure containing three homologous domains (I, II, and III)
divided into a pair of subdomains termed subdomain-A and subdomain-B. There is just one
tryptophan residue (Trp-214) in the albumin structure and it is explored to investigate the
albumin binding ability through spectroscopic techniques [17,18].
To have a physiological role, the metal ion or vanadium complex must first be
transported and up-taken by cells. This process is most likely conducted by plasma proteins
such as human serum transferrin (hTF), HSA, and immunoglobulin G (IgG) [19]. The
interaction of V(IV) compounds, including bis(maltolato)oxovanadium(IV) (BMOV) and
[VO(dmpp)2] complexes, with apotransferrin (apo-Tf), human serum albumin (HSA), and
small bioligands present in blood serum has been studied by electron paramagnetic resonance
(EPR) and circular dichroism (CD) [20,21,22]. In the case of HSA, mixed species cis-
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[VO(carrier)2-(HSA)] complex involving hydrogen bonding and/or hydrophobic interactions
with the protein surface have been proposed [23].
Considering the biological importance of vanadium-based drugs in the
metallopharmaceutical field and the lack research on vanadium(V) derivatives against
hyperpigmentation/hypopigmentation processes and Alzheimer’s disease, the main goal of
the present study is the biological evaluation of three vanadium complexes (complexes 1-3)
against tyrosinase and AChE activity. Additionally, a preliminary evaluation of the
pharmacokinetic profile of complexes 1-3 was also performed using HSA as a model. All
experimental data (spectroscopic approach) were correlated with theoretical analysis by
molecular docking calculations. Complexes 1 and 2 have already been reported in the
literature [4], however, complex 3 is a novel dimeric derivative (Fig. 1).
Fig. 1. Chemical representation for each dioxydovanadium(V) ion complexes – complexes 1-3. It was chosen to
evidence the complex ions of dioxydovanadium(V) without the respective counterions. Both complexes 1 and 3
are triethylammonium molecules and in the case of complex 2, protonated DBU molecules are presented.
Material and Methods
General
Commercially available tyrosinase from mushroom lyophilized enzyme, (S)-2-amino-3-
(3,4-dihydroxyphenyl)-propanoic acid (L-DOPA), 2,2',2'',2'''-(ethane-1,2-diylbis(azanetriyl))-
tetraacetic acid (EDTA), dimethyl sulfoxide (DMSO - spectroscopic grade), HSA, AChE,
acetylthiocholine iodide, 5,5’-Dithiobis-(2-nitrobenzamide) (DTNB), disodium hydrogen
phosphate/sodium phosphate monobasic buffer (Na2HPO4/NaH2PO4, pH 7.5), phosphate
buffered saline (PBS, pH 7.4), o-vanillin, succinic dihydrazide, and oxyvanadium(IV)
acetylacetonate were purchased from Sigma-Aldrich Chemical Company. Acetonitrile
(spectroscopic grade) was obtained from Tedia Ltd. One tablet of commercial PBS dissolved
in 200 mL of Milli-Q water yields 1.00 × 10-2 M phosphate buffer, 2.70 × 10-3 M potassium
chloride and 1.37 × 10-1 M sodium chloride. The synthesis of the ligand L3 and complex 3
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are described in the supplementary material (Schemes S1-S2).
Elemental CHN% analyses were obtained using a Perkin-Elmer CHN% 2400
1
equipment. H and 13C NMR spectra were recorded on a Bruker DPX-400 spectrometer at
400 (1H) and 100 Hz (13C), respectively. DMSO-d6 was used as the solvent and as the internal
reference. The chemical shifts are expressed in δ (ppm). UV–vis spectra were recorded on a
Shimadzu UV-2600 spectrometer in DMSO solution. Cyclic voltammograms were recorded
with an AutoLab PGSTAT128N Eco Chemie system at room temperature under argon
atmosphere,
using
dry
DMSO
as
a
solvent.
Electrochemical
grade
tetrabutylammoniumhexafluorophosphate (TBAPF6) was used as supporting electrolyte.
Employing a standard three-component system we carried out the following experiments: a
glassy carbon working electrode; a platinum wire auxiliary electrode and a platinum wire
pseudo-reference electrode. Ferrocene was used as internal standard [24].
X-Ray crystallography
Data were collected on a Bruker D8 Venture Photon 100 diffractometer equipped with
an Incoatec IµS high brilliance Mo-Kα X-ray tube with two-dimensional Montel micro-
focusing optics. The structure was solved by direct methods using SHELXS [25]. Subsequent
Fourier-difference map analyses yielded the positions of the non-hydrogen atoms.
Refinements were carried out with the SHELXL package [25]. All refinements were made by
full-matrix least-squares on F2 with anisotropic displacement parameters for all non–
hydrogen atoms. Hydrogen atoms were included in the refinement in calculated positions but
the atoms (of hydrogens) that are commenting performing special bond were located in the
Fourier map. Drawings were done using DIAMOND for Windows [26]. Crystal data and
structure refinement for the ligand L3, complex 3 and ORTEP representation of L3 are
described in the supplementary material (Table S1-S4 and Fig. S2).
Tau parameter
A penta-coordinate complex can have its geometry and degree of distortion defined by
calculating the parameter tau (τ), which considers the largest angles α and β in Equation 1.
The τ values vary between 0 and 1.0. If τ is between 0 and 0.5, the geometry is distorted
quadratic pyramidal and, if it is between 0.5 and 1.0, the geometry is distorted trigonal
bipyramid. The integer values 0 and 1.0 indicate the corresponding ideal geometries without
distortion:
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( )
 
(1)
60
By considering the penta-coordination, the connection angles, and τ parameter equal to
0.22, it is concluded that the metallic center has a distorted quadratic pyramidal geometry
[4,27].
Spectroscopic analysis of the interaction HSA:complex 1-3
The interaction studies between HSA and vanadium(V) complexes (complexes 1-3)
were carried out through spectroscopic techniques. UV-vis and steady-state fluorescence
spectra were measured on a Jasco J-815 fluorimeter to which a thermostated cuvette holder
Jasco PFD-425S15F (0.1 °C accuracy) was coupled. All spectra were recorded with
appropriate background corrections. The UV-vis spectrum for each vanadium complex (1.32
× 10-5 M) was measured in the 200-500 nm range, in PBS solution at 310 K. For steady-state
fluorescence measurements (290-450 nm range and λexc = 280 nm, at 296 K, 303 K and 310
K), successive aliquots from a stock solution of each vanadium complex (1.00 × 10-3 M, in
acetonitrile) were added to 3.0 mL of HSA solution (1.00 × 10-5 M, in PBS), leading to a final
ligand concentration of 0.17; 0.33; 0.50; 0.66; 0.83; 0.99; 1.15 and 1.32 × 10-5 M. The steady-
state fluorescence data were analyzed at the maximum fluorescence emission wavelength
(λexc = 340 nm).
Since each vanadium complex showed significant absorption at the excitation and
emission wavelengths (280 and 340 nm, respectively – Fig. S1 in the supplementary
material), the inner filter correction was applied to the steady-state fluorescence data,
according to Equation 2 [28]:
F  F 10[( A A )/2]
ex
em
(2)
cor
obs
where Fcor and Fobs are the corrected and observed fluorescence intensity values. Aexc and Aem
are the absorbance values for the ligand at excitation (λ = 280 nm: ε = 33,859; 43,664; 50,329
M-1 cm-1 in PBS for complexes 1, 2, and 3, respectively) and emission wavelength (λ = 340: ε
= 24,960; 29,501 and 22,550 M-1 cm-1, in PBS for complexes 1, 2, and 3, respectively).
Time-resolved fluorescence measurements were performed on Edinburgh Instruments
fluorimeter model FL920 CD, equipped with an EPL laser (λexc = 280 ± 10 nm, λem = 340
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nm; pulse width = 850 ps and energy per pulse = 1.8 µW). Fluorescence decays of 1.00 × 10-5
M HSA (3.0 mL in PBS) were measured in the absence and presence of each vanadium
complex (1.32 × 10-5 M) at room temperature (ca 296 K). Time-resolved fluorescence decays
were analyzed using the deconvolution software presents in the Edinburgh Instrument.
Circular dichroism (CD) spectra were measured in a spectropolarimeter Jasco J-815
employing a thermostatic cuvette holder Jasco PFD-425S15F with 0.1 °C accuracy. All
spectra were recorded with appropriate background corrections. The CD spectra were
recorded in the 200-260 nm range for HSA solution (1.00 × 10-6 M) without and in the
presence of the maximum vanadium complex concentration used in the steady-state
fluorescence measurements (1.32 × 10-5 M) at 310 K. CD results were expressed in terms of
mean residue ellipticity (MRE) in deg cm2 dmol−1, according to Equation 3:

MRE 
(3)
(10.n.l.CP )
where θ, n, l, and Cp are the observed ellipticity (mdeg), number of amino acid residues (585
to HSA) [28], length of the optical cuvette (1.0 cm), and molar concentration of HSA (1.00 ×
10-6 M), respectively.
Synchronous fluorescence (SF) spectra were recorded in a spectrofluorimeter model
Xe900 from Edinburgh Instruments. The SF spectra were recorded for HSA (1.00 × 10-5 M)
without and in the presence of each vanadium complex, at the same concentration range used
in the steady-state fluorescence measurement. Spectra were recorded in the 240–320 nm
range by setting Δλ = 15 nm (for tyrosine) and Δλ = 60 nm (for tryptophan) at room
temperature (ca. 296 K).
Zeta potential analysis of the interaction HSA:complex 1-3
The surface charge of HSA in the absence and presence of each vanadium complex was
characterized in terms of zeta potential (ZP) using a Brookhaven Instruments NanoBrook
ZetaPALS. All measurements were performed with 10 runs at 296 K and results are reported
in terms of ZP ± SD. The ZP was measured for HSA solution (1.00 × 10-5 M in PBS solution,
pH 7.4) without and in the presence of complexes 1, 2 or 3 at the maximum concentration
used in the steady-state fluorescence measurements (1.32 × 10-5 M).
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Tyrosinase assays
The UV-vis absorption spectra for tyrosinase assays were recorded on Shimadzu UV-
vis spectrophotometer UV Mini 1240 (Kyoto, Japan), at room temperature (ca 296 K).
Tyrosinase inhibition activity was measured by the method of Amorim and co-workers [29].
Each vanadium complex was mixed with L-DOPA (0.17 mM), EDTA (0.022 mM) and
tyrosinase (50–100 units), in PBS solution (pH 7.4). Due to the fact that the vanadium(V)
complexes showed different solubility in water, the concentration of the complexes 1-3 used
to determine the inhibition porcentage was 100, 66 and 133 µM, respectively. When the
tyrosinase solution was added to the mixture, the absorbance was immediately measured at
475 nm and the formation of L-Dopaquinone was monitored for 30 min (Scheme 1).
Scheme 1. Oxidation of L-DOPA to L-Dopaquinone by tyrosinase enzyme.
The inhibition or activation percentage was calculated according to the Equation 4
[29]:
[(
-
-
B B0) - (A30 A0)]
30
(4)
Inhibition or Activation% 
x 100
-
(B30 B0)
where B0 and B30 are the absorbance of L-DOPA + tyrosinase at t = 0 min and t = 30 min,
respectively. A0 and A30 are the absorbance of L-DOPA + tyrosinase + inhibitor/activator at t
= 0 min and t = 30 min, respectively. In the Equation 4, the possible interference of the
absorbance of organic compounds was subtracted.
Acetylcholinesterase assays
Activity of AChE was determined using a Bio-Rad iMark microplate reader based on a
modification of the Ellman method [30]. Compounds were dissolved in DMSO (maximum
DMSO final concentration of 0.1%). The assay solution which contained 25 µL AChE, 10 µL
acetylthiocholine iodide (1.5 mM), 85 µL Na2HPO4/NaH2PO4 buffer (pH 7.5; 0.1 M), and 22
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µL tested compound solution (0.5 mM) were incubated at 298K for 10 min. Then, 100 µL of
DTNB (0.32 mM) were added and assay solution was incubated at 298 K for 10 min. The
absorbance of each assay mixture was measured at 412 nm by UV-vis spectroscopy. In
blanks solutions, tested compound solutions were not added.
Molecular docking analysis
The vanadium(V) derivatives structures 1-3 were obtained from X-ray crystallography
data, recently published by Siqueira and collaborators [4]. The crystallographic structure of
tyrosinase, AChE and HSA were obtained from the Protein Data Bank (PDB), with access
code 2Y9X [31], 1ACJ [32] and 1N5U [28], respectively. Molecular docking was performed
with GOLD 5.5 program (CCDC). Hydrogen atoms were added to the proteins according to
the data inferred by the program on the ionization and tautomeric states. For tyrosinase, a 10
Å radius spherical cavity around the dicopper center was defined as the binding site for the
molecular docking calculations. While, for AChE, a 10 Å radius spherical cavity around Phe-
330 residue was defined as the binding site for the molecular docking calculations. Finally,
for HSA a 10 Å radius spherical cavity around Trp-214 residue (in subdomain IIA) was
defined and the molecular docking calculations for each vanadium complex were carried out.
The scoring function used was “GoldScore”, “ChemPLP”, and “ChemPLP” for tyrosinase,
AChE, and HSA, respectively [33]. The figures of the docking pose for the largest docking
score value was generated with the PyMOL Delano Scientific LLC program.
Results and Discussion
Synthesis of ligand L3 and vanadium(V) complex 3
Siqueira and co-workers [4] previously described synthetical procedures and full
characterization of ligands/complexes 1 and 2. Complex 3 was obtained by the reaction
between L3 and dioxyvanadium(V)-bis(acetylacetonate) complex [VO2(acac)2] as starting
material using methanol as a solvent and triethylamine (Et3N) as a base. In this case, ligand
L3 and complex 3 were characterized by elemental analysis (CHN %), UV-vis, FTIR, nuclear
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magnetic resonance (1H and 13C for ligand L3), and cyclic voltammetry analysis.
Experimental procedures and selected spectra of ligand L3 and complex 3 are presented in
the supplementary material (Schemes S1-S2 and Figs. S1-S6).
Elemental analyses of compounds L3 and complex 3 are in accordance with the
assigned chemical composition of the compounds. The ligand L3 and complex 3 were
characterized and confirmed by single-crystal X-ray diffraction analysis and Fourier
Transformation Infrared spectroscopy (FTIR), showing characteristic stretches at (OH) 3450
cm-1, (C=O) 1667 cm-1, (C=N) 1576 cm-1, and (C-O)phenol in 1250 cm-1. In the case of
FTIR spectrum of the vanadium(V) complex 3, characteristic stretches attributed to (N+-H)
were observed at 3425 cm-1, (C=O) 1612 cm-1, (C=N) 1563 cm-1, (C-O)phenol in 1252 cm-
1, (VO2)asym in 958 cm-1, and (VO2)sym in 892 cm-1, respectively [34,35,36,37].
The nuclear magnetic resonance (1H and 13C NMR) spectra and chemical shift
attribution of ligand L3 are shown in supplementary material (Fig. S5-S6). Hydrogen signals
are assigned to iminic protons at the 11.68 - 11.28 ppm range, aromatic protons at the 8.35 -
6.83 ppm region, methoxyl groups at 3.80 ppm and aliphatic signals at the 2.93 - 2.55 ppm
13
range. The C NMR of ligand L3 showed carbon signals at the high frequency range of the
spectrum and chemical shifts at the 173.02 –118.81 ppm range, while C13 signals of the
electron donating group OCH3 appeared at 55.85 ppm and aliphatic signals were observed at
the 28.78 - 27.24 ppm region.
The absorption UV–vis spectrum of ligand L3 was recorded in DMSO solution and
revealed transition bands at 295 nm, which can be assigned to the intraligand π→π* transition
and 331 nm attributed to the n→π* transition band (Fig. 2 and Table 1). The absorption
electronic spectra of vanadium(V) complex 3 was conducted in DMSO and revealed
transitions at 323 nm and 396 nm, respectively (Fig. 2 and Table 1). The transition bands of
vanadium(V) compound is in accordance with dioxovanadium(V) derivatives in a distorted
trigonal bipyramidal environment, which had their structures confirmed by X-ray diffraction
analysis. No d-d type transitions were observed in the vanadium complex, revealing that the
vanadium center is in the vanadium(V) oxidation state.
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Fig. 2. Electronic UV-vis spectra for ligand L3 and complex 3 in DMSO solutions ([L3] = [complex 3] = 1.50
× 10-4 M).
The electrochemical behavior of ligand L3 and complex 3 were investigated by cyclic
voltammetry (CV) experiments in a dry DMSO solution in the 1.0 × 10-4 M range by using
0.1 M of TBAPF6 as a supporting electrode in an argon-saturated atmosphere at the -1.60 V
to +1.20 V range versus a normal hydrogen electrode (NHE). For all compounds, the Fc/Fc+
redox pair couple was used as the internal standard (see Materials and Methods). Cyclic
voltammograms of ligand L3 presented a broad irreversible oxidation peak close to +0.326 V
and irreversible reduction peak at -1.186 V, which may be attributed to ligand oxidation and
reduction processes (Fig. 3 and Table 1).
For cis-dioxovanadium(V) complex 3 at the cathodic region, one redox peak was
observed at -0.924 V, which is attributed to the reduction process V(V)/V(IV) center. At the
positive range, two irreversible redox processes were observed. The oxidation peak at +0.430
V may be attributed to the ligand oxidation coordinated to the vanadium (V) center,
following the next oxidation peak at +0.813 V, which can be assigned to the vanadium (V)
complexes and vanadium (IV) species (Fig. 3 and Table 1) [4,38]. The formal potentials were
calculated as the average of the cathodic (Epc) and anodic (Epa) peak potentials of this
process.
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Fig. 3. Cyclic voltammograms of ligand L3 and complex 3 solutions in dry DMSO, 0.1 M TBAPF6, using
glassy carbon electrode as work electrode, at scan rate 100 mV s-1.
Table 1. UV-vis transitions and redox potentials of ligand L3 and complex 3.
UV-vis
Redox Potential (vs NHE)
Compound
λ, nm (ε; M-1 cm-1)
Eox1
Eox2
-----
Ered1
295 (4,833) and 331 (1,720)
323 (5,113) and 396 (2,080)
+0.326 Va
+0.430Va
-1.186 Vb
-0.924Vb
L3
+0.813Va
Complex 3
aEpa = anodic peak potential; bEpc = cathodic peak potential;
Crystal structure of complex 3
Crystal data collection and refinement of complex 3 are presented in the supplementary
material (see Tables S3-S4). Complex 3 has two dioxovanadium centers coordinated at the
two extremities of a molecule of the ONO chelating ligand by three distinct coordination
sites: phenolate oxygen (O1), imine nitrogen (N1), and oxygen (O3) (Fig. 4) [39,40,41]. The
adjacent nitrogen of the hydrazide function presents deprotonation, which generates a
negative charge in the resonance between oxygen (O3) and nitrogen (N1). This causes the
complex to present itself as an anionic species and be neutralized by the presence of a
protonated triethylamine molecule (Et3NH+ - triethylammonium ion) [34,35,42,43]. The
anionic character of the complex is likely responsible for its solubility in water.
The carbon-oxygen bond length of this function (C(9)-O(3)) is 1.293(6) Å, which is
consistent with bonds for the enolate group [35,42,43]. The metal center is stabilized by
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double bonds with two oxygen atoms (O4 and O5), completing the coordination sphere and
providing a coordination geometry of NO4 type.
Fig. 4. Projection of the molecular structure of the complex 3 (complex ion). Thermal ellipsoids with 50%
probability level. For better visualization, the triethylammonium ions were omitted.
Regarding the bond lengths, the values found are 1.898 (3) Å for V-O(1) (vanadium ion
and phenolate group oxygen), 2.142 (3) Å for V-N(1) (vanadium ion and iminic nitrogen),
1.976(3) Å for VO(3) (vanadium ion and oxygen of enolate group), and 1.614(3)/1.647(3) Å
for VO(4)/VO(5). All values are in agreement with data of cis-dioxovanadium(V) complexes
already reported in the literature [4,35,42,43]. The presence of distortion in the coordination
spheres by the connection angle values found in the metal centers are 150.74°(14) for O(3)-
V-O (1), 99.86°(15) for O(3)-V-O(4), 137.27°(15) for N(1)-V-O(5), and 109.27°(18) for
O(4)-V-O (5). The selected values for connecting length and angle for vanadium complex 3
are listed in the supplementary material (Table S4).
Binding parameters for the interaction HSA:complexes 1-3
Human serum albumin (HSA) has strong intrinsic fluorescence at the 280-nm excitation
wavelength mainly due to the presence of aromatic amino acid residues: tryptophan (Trp),
tyrosine (Tyr), and phenylalanine (Phe). Tyrosine and phenylalanine residues have relatively
low fluorescence quantum yield when compared to Trp residues. Thus, the dominant
fluorescence at λexc = 280 nm is from Trp residue. This fluorescence emission is sensitive to
the ligand presence and often used as a probe to identify the interaction of small ligands
against serum albumin [44]. The steady-state fluorescence emission of HSA (1.00 × 10-5 M in
PBS solution) with and without the presence of the maximum complex 1 concentration
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addition (1.32 × 10-5 M) at 310 K is shown in Fig. 5. The steady-state results for complexes 2
and 3 are presented as Figure S7 in the supplementary material. Moreover, HSA exhibited
strong fluorescence emission band at 340 nm. The fluorescence intensities of HSA reduced in
the presence of each vanadium(V) complex and a weak red shift was also observed for
complexes 1 and 2 (from 340 to 347 nm and 340 to 345 nm, respectively). This suggests that
these ligands may be interacting with Trp-214 residue and the fluorescence chromophore of
serum albumin is placed in a more hydrophilic environment upon ligand binding [45].
Generally, the binding interaction may be subdivided into a ground state or excited
state complexation between fluorophore and quencher or structural change of protein around
the fluorophore [46]. The main mechanism of HSA fluorescence quenching due to binding of
quencher can be observed through steady-state and time-resolved fluorescence
measurements. In general, Stern-Volmer analysis (Equations 5A and 5B and inset in Fig. 4
for HSA:complex 1 and inset in the Figures S7-S8 in the supplementary material) are useful
to elucidate the mechanism involved in the quenching process [28]:
F0
F
KSV
=1+kq τ0

Q

=1+ KSV
Q
 
kq =
(A)
(B)
(5)
τ0
where F0 and F are the steady-state fluorescence intensities of HSA in the absence and
presence of each vanadium complex, respectively. Furthermore, Ksv and kq are the Stern-
Volmer quenching constant and bimolecular quenching rate constant, respectively, and [Q]
and τo are the vanadium complex concentration and fluorescence lifetime of HSA without the
quencher ((5.61±0.22) × 10-9 s).
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1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
3.0
296 K
303 K
310 K
HSA
2.7
2.4
2.1
1.8
1.5
1.2
3.0x10-6
6.0x10-6
9.0x10-6
1.2x10-5
[Complex 1] (M)
Complex 1
HSA:Complex 1
300
350
400
450
500
Wavelength (nm)
Fig. 5. Steady-state fluorescence emission spectra for HSA without and in the maximum addition of complex 1,
as well as complex 1 without the presence of albumin at pH 7.4 and 310 K. [HSA] = 1.00 × 10-5 M, [Complex 1]
= 1.32 × 10-5 M. Inset: Stern-Volmer plots for the interaction between HSA and complex 1 at 296 K, 303 K and
310 K.
Table 2 shows KSV and kq values for the interaction between HSA and each vanadium
derivative at 296 K, 303 K and 310 K (inset of the Fig. 4).Since KSV values decrease with the
increasing of temperature and kq values are larger than the diffusional collision quenching
constant (kdiff ≈ 7.40 × 109 M−1s−1, according to Smoluchowski-Stokes-Einstein theory at 298
K) [46] in all cases, indicates that the main fluorescence quenching mechanism is via static
process. Thus, there is a ground state association between HSA and each compound [44].
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Table 2. Binding parameters (KSV, kq, Ka, ΔH°, ΔS° and ΔG°) for the interaction HSA:complexes 1-3 at 296 K,
303 K and 310 K.
Complex
T
KSV (× 104)
(M-1)
kq (× 1012)
(M-1 s-1)
25.7
Ka (× 104)
(M-1)
ΔH°
(kJ mol-1)
ΔS°
ΔG°
(K)
296
303
310
296
303
310
296
303
310
(kJ mol-1 K-1) (kJ mol-1)
14.4±0.1
14.1±0.1
13.9±0.1
1.07±0.03
1.05±0.02
0.88±0.02
67.4±0.4
63.6±0.3
62.3±0.2
11.2±0.26
6.32±0.26
3.21±0.26
5.73±0.26
1.53±0.26
0.56±0.03
26.3±0.26
23.0±0.26
20.8±0.26
-28.7
25.2
-68.1±4.2
-127±8
-0.133±0.014
-0.339±0.026
0.0606±0.003
-27.8
-26.9
-27.5
-24.3
-22.7
-30.7
-31.2
-31.6
1
2
3
24.7
1.91
1.89
1.57
120
113
-12.8±1.0
111
Obs.: r2 for KSV, kq: 0.9802-0.9985; r2 for Ka: 0.9971-0.9999 and r2 for ΔH°, ΔS° and ΔG°: 0.9887-0.9981.
Time-resolved fluorescence spectroscopy is an extension of steady-state fluorescence,
which is a useful method to identify the main fluorescence quenching mechanism (static
and/or dynamic). Fluorescence lifetimes (τ) occur as emissive decays from the singlet-state
and can also be approximated as those decays occur in the time region from picoseconds to
nanoseconds. Nevertheless, HSA two fluorescence lifetimes (τ1 = 1.48±0.18ns and τ2 =
5.61±0.22 ns), being the second one the most relative (82.0%). This data is in good
accordance with the literature [47,48,49,50,51]. The presence of each vanadium derivative in
the HSA solution decreases both fluorescence lifetimes, however, τ1 and τ2 with and without
the presence of each complex are the same inside the standard deviation e.g. τ1 = 1.45±0.16
ns and τ1 = 5.57±0.20 ns, for HSA:complex 1. Thus, static process is the main fluorescence
quenching mechanism and in full agreement with the previously described KSV and kq values
[52]. All fluorescence lifetime plots and values are presented in the supplementary material
(Fig. S9 and Table S5).
Evaluating the binding constant between the serum albumin and a ligand is an
important parameter to understand drug bioavailability. If binding affinity is low, the initial
step of pharmacokinetics (drug absorption) is infeasible. In the case of moderate binding
affinity of bioactive substances to serum albumin, drug absorption and distribution to various
tissues are feasible. Finally, when binding affinity is high, drug absorption is feasible in spite
of distribution to the required tissues being limited as a result of complex stability, which, in
turn, adversely affects the pharmacokinetics of the drug [47,52]. The modified Stern-Volmer
equation (Equation 6) can be used to obtain information on the binding constant between
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HSA and ligands:
F
1
1
0
=
+
(6)
F - F fKa[Q]
f
0
where Fo and F are the steady-state fluorescence intensities of HSA in the absence and
presence of each vanadium complex, Ka, ƒ, and [Q] are the modified Stern-Volmer binding
constant, and the fraction of the initial fluorescence that is accessible to the quencher (f ≈
1.00) and vanadium complex concentration, respectively. Figure S9 in the supplementary
material depicts the modified Stern-Volmer plots for the interaction HSA:complexes 1-3 and
Table 2 shows Ka values at 296 K, 303 K, and 310 K. The Ka values for each vanadium
complex decrease as temperatures increase, which also confirms the static fluorecence
mechanism [53]. Additionally, the Ka values in the order of 105 M-1 for HSA:complex 3
indicate strong binding ability of this vanadium complex and albumin protein pocket. On the
other hand, Ka values in the range of 105– 104 M-1 and 104 – 103 M-1 for complexes 1 and 2,
respectively, indicate moderate to weak interactions [54].
The thermodynamics of the interaction process yield a close insight to the mechanistic
aspects involved. The interaction between drugs and biomacromolecules involve different
types of bindings, such as electrostatics, hydrogen bondings, van der Waals interactions,
hydrophobics, and steric contacts with the binding site [55]. The enthalpy (ΔH°) and entropy
changes (ΔS°) can be obtained using Vant’Hoff equation (Equation 7A) and from these
values Gibbs’ free energy change (ΔG°) can be obtained by employing Equation 7B [47,52]:
H0 S0
G0  H0 TS0
ln Ka  

(A)
(B)
(7)
RT
R
where ΔH°, ΔS°, and ΔG° are the enthalpy, entropy, and Gibbs’ free energy, respectively, R is
the gas constant (R = 8.314 × 10-3 kJ mol-1 K-1), T is the temperature (296 K, 303 K, and 310
K), and Ka the modified Stern-Volmer binding constant. Depending on the sign of
thermodynamic parameters, the mode of interactions are categorized as [50-56]: (i) ΔH° > 0,
ΔS° > 0 correspond to hydrophobic forces; (ii) ΔH° < 0, ΔS° < 0 correspond to van der
Waals interaction and hydrogen-bond formation; (iii) ΔH° < 0, ΔS° > 0 correspond to
electrostatic/ionic interaction.
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Figure S10 in the supplementary material depicts Vant’Hoff plot for the interaction
HSA:complexes 1-3 and Table 2 shows the thermodynamic parameters (ΔH°, ΔS°, and ΔG°)
for each sample. According to Ross and Subramanian’s theory [56], for the interaction
HSA:complex 1 and HSA:complex 2, negative values for ΔH° and ΔS° are related to van der
Waals interaction and/or hydrogen bonding. On the other hand, for the interaction
HSA:complex 3, the negative and positive value for ΔH° and ΔS°, respectively, suggest
electrostatic interactions as the main binding force. Additionally, ΔS° > 0 can also be related
to hydrophobic effects - desolvation of both protein cavity and complex 3 structure upon
binding [57]. These results are different from those obtained for cis-[VO(carrier)2-(HSA)]
complex in the literature [23].
The negative values for ΔG° support the assertion that the binding process is
spontaneous. According to Gibbs’ free energy equation, negative and positive values for ΔH°
and ΔS°, respectively, can contribute to the negative sign of ΔG°. Since complexes 1 and 2
showed ΔH° < 0 and ΔS° < 0, the binding process is only enthalpically driven. On the other
hand, complex 3 showed ΔH° < 0 and ΔS° > 0, indicating that the binding process is
entropically and enthalpically driven, which can contribute to higher Ka values [58].
Perturbation on the albumin structure upon compound binding
Circular dichroism (CD) spectroscopy is a useful technique to determine the possible
influence of the interaction process on the secondary structure of the proteins. As shown in
Fig. 6 for HSA:complex 1, two negative absorption peaks in the UV range (208 and 222 nm)
can be found in the CD spectra of HSA, which represent the α-helical structure through π →
π*and n → π*transition, respectively [59]. All CD spectra are presented in the supplementary
material – Fig. S11. The loss of α-helical content of the protein due to ligand binding was
calculated as significant molar residual ellipticities (MRE) to free and combined HSA from
MRE values at 208 and 222 nm using Equations 8A and 8B:
(
)
(
)
- MRE208 - 4000
- MRE222 - 2340
α - helix% =
x 100
α - helix% =
x 100
(8)
(A)
(B)
(
)
33000 - 4000
30300
where MRE208 and MRE222 are the significant molar residual ellipticities (deg.cm2.dmol-1) at
208 and 222 nm, respectively. The quantitative CD results for HSA:complexes 1-3 are
depicted in Table 3. The α-helix content of the secondary structure of HSA in the absence and
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presence of each vanadium complex barely changed, e.g. from 61.7 to 57.8% at 208 nm
(variation of 3.90%) and from 58.1 to 56.0% at 222 nm (variation of 2.10%) for
HSA:complex 1. These quantitative calculations further confirm that the binding
HSA:complexes 1-3 occurs with a relatively small change in the secondary structure of HSA
[60].
0
-20
-40
-60
-80
-100
HSA
-120
HSA:Complex 1
200
210
220
230
240
250
260
Wavelength (nm)
Fig. 6. CD spectra for HSA without and in the presence of complex 1 at pH 7.4 and 310 K. [HSA] = 1.00 × 10-6
M and [Complex 1] = 1.32 × 10-5 M.
Table 3. Quantitative α-helix % for the association HSA:complexes 1-3 at 208 and 222 nm (310 K). [HSA] =
1.00 × 10-6 M and [Complexes 1-3] = 1.32 × 10-5 M.
Free HSA α-helix %
HSA:complexes 1-3
Complex
208 nm
222 nm
58.1
208 nm
222 nm
56.0
61.7
61.9
62.5
57.8
59.7
60.7
1
2
3
58.4
56.9
58.9
55.9
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Zeta potential (ZP - ζ) is a useful and feasible parameter to characterize the surface
charge properties of proteins. Changes in the ZP of a protein may imply conformational
changes in its structure, surface modifications, aggregation, and/or unfolding/denaturation
processes. Therefore, the ZP of a protein can be used as an indicator of protein stability upon
ligand binding [61]. Table S6 in the supplementary material shows the ZP values for HSA
with and without the presence of each vanadium complex. The ZP values of HSA changed
after the addition of vanadium(V) complexes 1-3, although the changes are in accordance
with the standard error of the measurements, e.g. -9.06 ± 1.82mV and -8.87±1.50 mV for
HSA and HSA:complex 1, respectively. This suggests that there is no significant structural
change on the protein surface with the addition of each vanadium derivative [55].
Synchronous fluorescence (SF) spectroscopy is a convenient and fast method to study
changes in the microenvironments of tyrosine (Tyr) and tryptophan (Trp) residues of proteins
when the differences between the emission and excitation wavelengths are 15 nm and 60 nm,
respectively [58]. The effect of different concentrations of complexes 1-3 on Tyr and Trp
residues of HSA is depicted in Figures S12 - S14 of the supplementary material.
There is no significant evidence of blue or red shift in the maximum emission of HSA
upon successive ligand addition at Δλ = 15 nm in all samples, indicating that the
microenvironment around tyrosine (Tyr) has no obvious changes [62]. On the other hand,
there is a weak red shift upon successive addition of complex 1 (from 277 to 280 nm) and
complex 2 (from 277 to 281 nm) at Δλ = 60 nm, indicating that the amino acid residues
located near tryptophan Trp-214 are located in a more hydrophilic environment upon ligand
binding.
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Theoretical analysis of the albumin binding pocket
As previously described by Sudlow et al., there are two main binding sites in the HSA
structure: Sudlow’s site I and II, which are located in the subdomain IIA and IIIA,
respectively. The first one presents high affinity for warfarin, while the second presents high
affinity to ibuprofen [63]. According to the literature, Sudlow’s site I, which is where
tryptophan Trp-214 residue can be found, is the main binding site for some vanadium
complexes, such as bis(maltolato)oxovanadium(IV),
anionic
[VVO2(maltol)2]-,
[VVO2(dmpp)(OH)(H2O)]–, and [VVO2(dmpp)2]- complexes [23]. In order to analyze the main
intermolecular interactions between each studied compound and the amino acid residues
present in the interaction cavity located in the subdomain IIA (Sudlow’s site I), molecular
docking calculations were carried out.
The best docking pose for the interaction between HSA:complexes 1-3 inside Sudlow’s
site I (subdomain IIA) are depicted in Fig. 7. Table S7 in the supplementary material shows
the main amino acid residues and their corresponding intermolecular forces involved in the
binding process for HSA:complexes 1-3. Molecular docking results suggested hydrogen
bonding and van der Waals forces as the main intermolecular interactions for each inorganic
complex, however, hydrophobic interactions via t-Stacking was also detected for
HSA:complex 2 and HSA:complex 3. For example, the oxygen from vanadium group of
complex 1 interacts via hydrogen bonding with tryptophan Trp-214 residue within a distance
of 2.80 Å . The oxygen atom from the phenol group of the ligand structure is a potential
acceptor for hydrogen bonding with lysisne (Lys-198) residue within a distance of 3.50 Å .
The hydrogen atom from the hydroxyl group of the amino acid residue Ser-453 is also a
donor for hydrogen bonding with the nitrogen atom of the ligand structure within a distance
of 3.00 Å . On the other hand, valine (Val-343); leucine (Leu-480); arginine (Arg-483) and
proline (Pro-485) residues interact with complex 1 via van der Waals forces within a
distance of 3.60, 2.30, 2.40, and 3.90 Å , respectively. Overall, molecular docking results are
in good agreement with the intermolecular forces suggested by thermodynamic results.
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Fig. 7. Molecular docking results for the interaction between HSA:complex 1 (A), HSA:complex 2 (B) and
HSA:complex 3 (C) in Sudlow’s site I (ChemPLP function). Complexes 1-3 and selected amino acid residues
are in beige, brown, magenta and cyan, respectively. Elements’ colors: hydrogen, oxygen, nitrogen, and
vanadium are represented in white, red, dark blue, and orange, respectively.
Tyrosinase and AChE evaluation – experimental and theoretical analysis
Complex 1 activated tyrosinase activity at about 11.5%, while complexes 2 and 3
inhibited tyrosinase activity at about 1.30 and 46.0%, respectively. By comparing the results
of the complexes that can inhibit tyrosinase from others available in the literature (e.g.
hydrazides) [10], it is plausible that that hydrize-based vanadium complexes are more active
than vanadium complexes based on pyridoxals and resorcinols. Since complex 1 presented
the ability to activate tyrosinase activity and the other vanadium complexes inhibited less
than 50% of tyrosinase activity, the IC50 value was not determined. In order to offer an
atomic view of the activity of each cis-dioxovanadium(V) complex against tyrosinase,
molecular docking calculations were carried out (Fig. 8A-C). Molecular docking results
suggested that the hydroxyl group of complex 1 can interact with Cu(II) ions (cofactor
essential to the tyrosinase activity) within a distance of 2.20 Å , being essential to the
activation detected by experimental assays.
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On the other hand, complexes 2 and 3 do not interact with Cu(II) ions through the
hydroxyl group, but through the methyl group via van der Waals forces within a distance of
2.70 and 2.20 Å , respectively. Additionally, molecular docking also detected van der Waals
and hydrophobic interactions as the main intermolecular forces for the interaction between
each inorganic complex and amino acid residues in the tyrosinase active binding pocket,
however, hydrogen bonding was also suggested for complexes 1 and 2. The main amino-acid
residues that participate in the interaction tyrosinase:complexes 1-3 are shown in Table S8 of
the supplemenatry material. For example, the oxygen atom from the vanadium group of
complex 1 is a potential acceptor for hydrogen bonding with the amide group of the aspargine
(Asn-152) residue within a distance of 2.10 Å . The amino acid residues valine and
phenylalanine Val-140, Phe-156, Val-172, and Phe-181 interact via hydrophobic forces with
the aromatic rings of complex 1 within a distance of 2.70, 3.40, 3.70, and 2.40 Å ,
respectively. Finally, the interaction via van der Waals forces was also detected between
three histidines (His-47, His-136, and His-151 residues) and complex 1 within a distance of
2.00, 2.20, and 2.30 Å , respectively.
The biological assays of vanadium(V) derivatives against the AChE enzyme presented
similar ability when compared to tyrosinase (complex 1 can activate the enzyme, while
complexes 2 and 3 can inhibit it). According to the experimental data, complex 1 is a
potential activator in about 47.0% of AChE enzyme activity. On the other hand, complexes 2
and 3 inhibited enzyme activity in about 20.0 and 21.9%, respectively. The half maximal
inhibitory concentration (IC50) value can be determined only for compounds that present
inhibition ability, therefore, it cannot be determined in the case of AChE:complex 1.
Generally, compounds that present inhibition percentage 50% below the maximum
concentration of inhibitor that does not precipitate in the medium, the IC50 value is difficult to
be determined (complex 2 and 3 fits in this case).
In orde to offer an atomic view of the activity of each vanadium complex for AChE,
molecular docking calculations were also carried out (Fig. 8D-F). Molecular docking results
suggested hydrogen bonding, van der Waals, and hydrophobic interactions as the main
intermolecular forces. The main amino-acid residues that participate in the interaction
AChE:complexes 1-3 are shown in Table S8 of the supplemenatry material. For example, the
hydrogen from the phenol group of tyrosine Tyr-331 residue is a potential donor for hydrogen
bonding with oxygen from the vanadium group of complex 1 within a distance of 2.80 Å . On
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the other hand, the aromatic ring of complex 1 can interact via van der Waals forces with
serine Ser-119; tyrosine Tyr-127; tryphtophan Trp-429 and histidine His-437 residues within
a distance of 3.10, 2.80, 2.70, and 2.40 Å , respectively. Finally, amino acid residue
tryphtophan Trp-81 interacts via π-Stacking with the aromatic moiety of vanadium(V)
complex 1 within a distance of 3.70 Å and phenylalanine Phe-330 and isoleucine Ile-436
residues interact via hydrophobic forces with vanadium(V) complex 1 structure within a
distance of 2.60 and 2.10 Å , respectively.
Overall, the vanadium(V) complex 1 can activate both tyrosinase and AChE activity,
and vanadium(V) complexes 2 and 3 can inhibit both enzymes. In the case of tyrosinase
metalloenzyme, the interaction of each vanadium complex against Cu(II) ions is essential for
the biological activity, thus, theoretical calculations suggested that the presence of the
hydroxyl group in a vanadium complex can activate tyrosinase activity. In the case of AChE,
complex 1 presented high ability to activate this enzyme activity, being a good scaffold for
the design of potential AChE activators.
Fig. 8. Best docking poses for each inorganic complex toward tyrosinase (A, B and C), and AChE (D, E and F).
Complexes 1-3 and selected amino acid residues are in beige, brown, magenta and cyan, respectively. Elements’
colors: hydrogen, oxygen, nitrogen, and vanadium are represented in white, red, dark blue, and orange,
respectively. Cu(II) ions are represented as spheres in sandy brown.
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Conclusion
Ligand L3 and vanadium(V) complex 3 were synthetized and fully characterized by
spectroscopic and structural analysis. According to biological evaluation, vanadium(V)
complex 1 can activate both tyrosinase and AChE activity (11.5 and 47.0%, respectively),
and vanadium(V) complexes 2 and 3 can inhibit both enzymes (1.30 and 46.0% for tyrosinase
and 20.0 and 21.9% respectively for AChE). In the case of tyrosinase metalloenzyme, the
interaction of each vanadium complex against Cu(II) ions is essential for biological activity,
thus, theoretical calculations suggest that the presence of the hydroxyl group in a vanadium
complex can activate tyrosinase activity. In the case of AChE, complex 1 presented high
ability to activate this enzyme, being a good scaffold to design potential AChE activators.
Steady-state and time-resolved fluorescence analysis revealed that each vanadium complex is
able to interact with HSA via ground-state association (kq values higher than kdiff and
fluorescence lifetime values are the same with and without the presence of complexes 1-3).
The Ka values in the order of 105 M-1 for HSA:complex 3 indicate strong binding, while Ka
values in the range of 105– 104 M-1 and 104 – 103 M-1 for complexes 1 and 2, respectively,
indicate moderate to weak interaction. Zeta potential and CD results indicated that the
interaction HSA:complexes 1-3 does not perturb significantly the secondary and surface
structure of the albumin. Finally, molecular docking results suggested hydrogen bonding and
van der Waals forces as the main intermolecular interaction for each inorganic complex
inside subdomain IIA, however, hydrophobic interactions via t-Stacking was also detected for
HSA:complexes 2-3. Since in there are few cases reported in the literature that explore
vanadium complexes against tyrosinase, AChE, and HSA, the studied vanadium(V)
complexes presented good results to be used to design novel potential mettalodrugs, explored
in the hyperpigmentation process and Alzheimer’s disease.
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Abbreviations
HSA – Human serum albumin
AChE - acetylcholinesterase enzyme
L-DOPA - (S)-2-amino-3-(3,4-dihydroxyphenyl)-propanoic acid
HbA1C - Hemoglobin A1C
BMOV - bis(maltolato)oxovanadium(IV)
apo-Tf - apotransferrin
EDTA - 2,2',2'',2'''-(ethane-1,2-diylbis(azanetriyl))-tetraacetic acid
DMSO - Dimethyl sulfoxide
FTIR - Fourier Transformation Infrared Spectroscopy
1H NMR - Hydrogen nuclear magnetic resonance
13C NMR - Carbon nuclear magnetic resonance
CV - cyclic voltammetry
DTNB - acetylthiocholine iodide; 5, 5’-Dithiobis-(2-nitrobenzamide)
PBS – phosphate buffer saline
CD – Circular dichroism
ZP – Zeta potential
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Acknowledgements
This research was supported by the Brazilian funding agencies: Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do
Estado do Rio Grande do Sul (FAPERGS). D.F.B – acknowledges FAPERGS (Edital: Edital
02/2017 - Programa Pesquisador Gaúcho (PqG) – Process/Termo de Outorga N. 17/2551-
0000963-0), PROEEX/CAPES and CNPq (Universal-Process: 424515/2018-4 and PG-2016
(Process: 303011/2016-5). B.A.I. –acknowledges CNPq (Universal-Process: 409150/2018-5
and PG-2016 (Process: 304711/2018-7).
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