Arezoo Jamshidvand

Mehdi Sahihi

Valiollah Mirkhani

Majid Moghadam

Iraj Mohammadpoor-Baltork

Shahram Tangestaninejad

Amiri Hadi

Hadi Rudbari

Reza Kargar

Sajjad Keshavarzi

Gharaghani

Iraj Mohamma Dpoor-Baltork

Rudbari

Hadi Kargar

Reza Keshavarzi

Sajjad Gharaghani

Studies on DNA binding properties of new Schiff base ligands using spectroscopic, electrochemical and computational methods: Influence of

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Cancer is a large family of diseases that involves abnormal cell growth with potential to invade or spread to the other parts of the body. Nowadays, these sorts of diseases are known as the main cause of worldwide death. However, it can be treated by disordering the DNA of cancer cells. Schiff base ligands are significant compounds which biochemists are focusing on their design and synthesis as anticancer and play an impressive role in human welfare. These compounds are the urgent ligands in modern coordination and medicinal chemistry [ [START_REF] Habibi | Synthesis, Structural Characterization, Thermal, and Electrochemical Investigations of a Square Pyramid Manganese (III) Complex with a Schiff Base Ligand Acting as N2O2 Tetradentate in Equatorial and as O Monodendate in Axial Positions: Application as a Precursor for Preparation of Mn-doped ZnO Nanoparticle, Synthesis and Reactivity in Inorganic, Metal-organic[END_REF], [START_REF] Hathaway | Copper (II) ammonia complexes[END_REF], [START_REF] Jandera | Description of adsorption equilibria in liquid chromatography systems with binary mobile phases[END_REF], [START_REF] Kadry | Pharmacokinetics of benzophenone-3 after oral exposure in male rats[END_REF], [START_REF] Kashanian | In vitro study of calf thymus DNA interaction with butylated hydroxyanisole[END_REF], [START_REF] Kashanian | Spectroscopic studies on the interaction of isatin with calf thymus DNA[END_REF], [START_REF] Kumar | Applications of Metal Complexes of Schiff Bases -A Review[END_REF]]. Schiff bases are derived by substitution aldehyde/ketone with a primary amine. The azomethine ( HC N ) linkage present in Schiff base ligands demonstrate a wide range of medicinal activities such as antibacterial [START_REF] Raman | A novel bioactive tyramine derived Schiff base and its transition metal complexes as selective DNA binding agents[END_REF][START_REF] Subbaraj | Novel mixed ligand complexes of bioactive Schiff base (E)-4-(phenyl (phenylimino) methyl) benzene-1, 3-diol and 2-aminophenol/2-aminobenzoic acid: synthesis, spectral characterization, antimicrobial and nuclease studies[END_REF], antifungal [START_REF] Raman | DNA cleavage and antimicrobial activity studies on transition metal (II) complexes of 4aminoantipyrine derivative[END_REF], herbicidal, anti-inflammatory [START_REF] Kumar | Applications of Metal Complexes of Schiff Bases -A Review[END_REF], anticancer, anti-diabetic [START_REF] Raman | DNA cleavage and antimicrobial activity studies on transition metal (II) complexes of 4aminoantipyrine derivative[END_REF] and antitumor [START_REF] Osowole | Synthesis, spectral, thermal, in-vitro antibacterial and anticancer activities of some metal (II) complexes of 3-(-1-(4-methoxy-6methyl)-2-pyrimidinylimino) methyl-2-napthol[END_REF] activities. The interaction between Schiff base ligands and DNA is one the most essential step to design and synthesis of new pharmaceutical molecules, due to the importance of DNA as the main target for anticancer drugs [START_REF] Shahabadi | DNA binding and DNA cleavage studies of a water soluble cobalt (II) complex containing dinitrogen Schiff base ligand: the effect of metal on the mode of binding[END_REF][START_REF] Jayamani | Synthesis, structural, electrochemical, DNA interaction, antimicrobial and molecular docking studies on dimeric copper (II) complexes involving some potential bidentate ligands[END_REF]. There are three possible ways for interaction of the drugs and DNA: (i) electrostatic binding between cation species and negative charged DNA phosphate residues (ii) interaction with grooves of DNA by hydrogen bond or van der Waals interactions, and (iii) intercalative binding [ [START_REF] Radi | Electrochemical and spectral studies on the interaction of the antiparasitic drug nitazoxanide with DNA[END_REF], [START_REF] Behnamfar | Experimental and molecular modeling studies of the interaction of the polypyridyl Fe (II) and Fe (III) complexes with DNA and BSA[END_REF], [START_REF] Ozluer | In vitro DNA binding studies of anticancer drug idarubicin using spectroscopic techniques[END_REF]]. Recently, we have focused on designing and synthesis of novel Schiff base ligands, and investigation of their assembly with metals as well as their biological activities [ [START_REF] Kazemi | Synthesis, characterization and separation of chiral and achiral diastereomers of Schiff base Pd (II) complex: a comparative study of their DNA-and HSA-binding[END_REF], [START_REF] Kazemi | Synthesis, characterization and biological application of four novel metal-Schiff base complexes derived from allylamine and their interactions with human serum albumin: experimental, molecular docking and ONIOM computational study[END_REF], [START_REF] Khosravi | Synthesis, characterization, crystal structure and HSA binding of two new N, O, O-donor Schiff-base ligands derived from dihydroxybenzaldehyde and tert-butylamine[END_REF]]. Herein, five new Schiff base ligands were synthesized and characterized by various spectroscopic techniques such as FT-IR, 1 H NMR, 13 C NMR and elemental analysis (CHNS). Also, the molecular structures of two ligands were determined by single crystal X-ray diffraction technique. Furthermore, DNA binding of the synthesized Schiff base ligands was investigated using fluorescence quenching, UV-Vis spectroscopy, circular dichroism (CD), cyclic voltammetry, molecular docking and molecular dynamics simulation methods. The effect of substituents on the Schiff base ligands on their DNA-binding ability was also investigated.

Experimental

Chemicals and instrumentation

Fish sperm DNA (FS-DNA), tris(hydroxymethyl)aminomethane (Tris), ethidium bromide (3,8diamino-5-ethyl-6-phenylphenanthridinium bromide, EB), 4-aminobenzoic acid, 3-aminobenzoic acid, 3-amino-4-methylbenzoic acid and 3-ethoxy-2-hydroxybenzaldehyde were purchased from Sigma-Aldrich. 2-Hydroxy-5-bromobensaldehyde, 3-methoxy-2-hydroxybenzaldehyde, Sodium bicarbonate, sulfuric acid, methanol, ethanol and DMF were obtained from Merck Co. and used without further purification. All of the solutions were used freshly after preparation. The FT-IR spectra were recorded on a JASCO, FT/IR-6300 spectrometer (4000-400 cm -1 ) as KBr pellets. The 1 H and 13 C NMR spectra were recorded on a Bruker Avance III 400 spectrometer using CDCl3 and DMSO-d6 as solvents for Schiff base ligands. The elemental analysis was performed on Leco, CHNS-932 and Perkin-Elmer 7300 DV elemental analyzers. The UV-Vis spectra were recorded on a JASCO V-670 spectrophotometer. Fluorescence measurements were carried out on Shimadzu RF-5000 spectrofluorometer at room temperature. The cyclic voltammetry measurements (CV) were performed using an Autolab (PGSTAT 30), with a three-electrode system: a 0.10-cm-diameter Glassy carbon (GC) disc as working electrode, a calomel electrode as reference electrode, and a Pt wire as counter electrode. The circular dichroism (CD) spectra were recorded using Aviv spectropolarimeter model 215 (ProterionCorp., USA) at 25 °C. The scan speed was 20 nm min -1 . The circular dichroism (CD) spectra were recorded using 0.1 cm quartz cell in farand near-UV region (215-310) for DNA to clarify the its structural changes during the interaction with the ligand. The CD spectra of DNA solutions were recorded before and after addition of the ligand with the most binding affinity with molar ratio of 1:1. 1 2-Hydroxy-5-bromobenzaldehyde (4.02 g, 20 mmol) was added to an ethanolic solution (50 mL) of 4-aminobenzoic acid (2.75 g, 20 mmol) and the mixture was refluxed for 1 h. The yellow precipitates of L 1 were collected by filtration, washed with ethanol and then dried under vacuum at room temperature. Yield 5.31 g (83% 2 2-Hydroxy-5-bromobenzaldehyde (4.02 g, 20 mmol) was added to an ethanolic solution (50 mL) of 3-aminobenzoic acid (2.75 g, 20 mmol) and the mixture was refluxed for 1 h. The yellow precipitates of L 2 were collected by filtration, washed with ethanol and then dried in vacuum at room temperature. Yield 5.12 g (80% 3 2-Hydroxy-5-bromobenzaldehyde (4.02 g, 20 mmol) was added to an ethanolic solution (50 mL) of 3-amino-4-methylbenzoic acid (3.02 g, 20 mmol) and the mixture was refluxed for 1 h. The yellow precipitates of L 3 were collected by filtration, washed with ethanol and then dried in vacuum at room temperature. Yield 5.68 g (85% For synthesis of L 4 and L 5 ligands, 4-aminobenzoic acid was converted to 4-aminobenzoate according to the following procedure: 4-Aminobenzoic acid (2.75 g, 20 mmol) was dissolved in MeOH (100 mL) and refluxed in the presence of 3 mL concentrated sulfuric acid for 4 h. After the reaction was completed, the solution was concentrated to approximately 10 mL and neutralized by adding NaHCO3 aqueous solution (10%). The crude crystal was filtered and recrystallized from MeOH/H2O (1/1) to obtain the methyl 4-aminobenzoate (yield 63%) [START_REF] Yamada | Development of a fluorescent chelating ligand for scandium ion having a Schiff base moiety[END_REF].

Preparation of ligands

Synthesis of L

Synthesis of L

Synthesis of L

Synthesis of L

Single crystal diffraction studies

X-ray data for L 4 and L 5 were collected on a STOE IPDS-II diffractometer with graphite monochromated Mo Ka radiation. The suitable crystals were chosen using a polarizing microscope and were mounted on a glass fiber which were used for data collection. Data were collected at 298(2) K in a series ω scans in 1° oscillations and integrated using the StöeX-AREA [START_REF] Cie | Program for the Acquisition and Analysis of Data XeAREA[END_REF] software package. A numerical absorption correction was applied using the X-RED [START_REF]Program for Data Reduction and Absorption Correction[END_REF] and X-SHAPE [START_REF] Stoe | X-SHAPE, Version 2.05, Program for Crystal Optimization for Numerical Absorption Correction[END_REF] software for the ligands. The data were corrected for Lorentz and Polarizing effects. The structures were solved by direct methods using SIR2004. The non-hydrogen atoms were refined anisotropically by the full-matrix least-squares method on F 2 using SHELXL [START_REF] Sheldrick | A short history of SHELX[END_REF]. All hydrogen atoms were added at ideal positions and constrained to ride on their parent atoms. Crystallographic data are listed in Table 1. Selected bond distances and angles are summarized in Table S1. Table 1. Crystal data and refinement parameters for L 4 , L 5 .

Empirical formula C17H17NO4(L

DNA binding studies

Preparation of the ligands and DNA stock solutions

The stock solution of FS-DNA was prepared in 50 mM Tris buffer at pH = 7.5 and was 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 the reported molar absorptivity of 6600 M -1 .cm -1 [START_REF] Reichmann | A further examination of the molecular weight and size of desoxypentose nucleic acid[END_REF]. 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 [START_REF] Zheng | Synthesis, structure and molecular docking studies of dicopper (II) complexes bridged by N-phenolato-N′-[2-(dimethylamino) ethyl] oxamide: the influence of terminal ligands on cytotoxicity and reactivity towards DNA and protein BSA[END_REF]. Also, the stock solutions of the ligands were prepared in dimethylformamide (DMF) and then was diluted to the desired concentrations with corresponding buffer. The volume of DMF in all final solutions were <0.5% (v/v), so the effect of DMF was negligible.

Fluorescence spectroscopy measurements

Fluorescence spectroscopy is a sensitive and effective method to study binding of drugs to biomacromolecules. Fluorescence quenching experiment can helps us to obtain the binding mode, binding constants, number of binding sites and intermolecular distances [START_REF] Fu | Water-soluble DNA minor groove binders as potential chemotherapeutic agents: synthesis, characterization, DNA binding and cleavage, antioxidation, cytotoxicity and HSA interactions[END_REF]. The interactions of DNA with the synthesized Schiff base ligands were investigated using fluorescence quenching experiment. Quartz cuvette with 1 cm optical path length was used and the excitation and emission slits were set at 5 and 10 nm, respectively. To investigate the binding of the ligands with DNA, the FS-DNA solution was stirred with ethidium bromide (EB) with molar ratio of DNA:EB 10:1 for 1 h at 4 °C. Significant increase of fluorescence intensity of EB is observed at the presence of FS-DNA due to intercalation of the EB molecules into the double helix of DNA [ [START_REF] Arjmand | Synthesis, characterization and in vitro DNA binding and cleavage studies of Cu (II)/Zn (II) dipeptide complexes[END_REF], [START_REF] Fromherz | Photoinduced electron transfer in DNA matrix from intercalated ethidium to condensed methylviologen[END_REF], [START_REF] Fu | Synthesis, characterization, DNA binding and cleavage, HSA interaction and cytotoxicity of a new copper (II) complex derived from 2-(2′-pyridyl) benzothiazole and glycylglycine[END_REF], [START_REF] Waring | Complex formation between ethidium bromide and nucleic acids[END_REF]]. Then, various amounts of the ligands (0-250 μM) were added to this mixture. The fluorescence spectra were recorded in the range of 500-700 nm with excitation wavelength of 520 nm. The mixture was allowed to incubate for 2 min after addition of the ligands. Moreover, in all of the DNA binding experiments, the measured fluorescence intensities were corrected for the dilution and the inner filter effect.

UV-Vis absorption spectroscopy measurements

Electronic absorption spectroscopy is one of the used methods for studying the binding affinity.

To confirm the binding of the ligands to DNA, absorption titration experiments were carried out at room temperature. The UV-Vis absorption spectra of the ligands solutions (10 μM) in the absence and presence of various amounts of FS-DNA (0-50 μM) were recorded. In all of the measurements, the mixture was allowed to incubate for 2 min before recording the related spectra. Absorption curves of ligands-DNA mixtures were corrected for both absorption of DNA solutions and the dilution effect.

Molecular docking procedure

Studying the interaction between drug molecules and biomacromolecules, is one of the interesting topics in biochemistry [START_REF] Tian | The effect of Cu 2+ on the interaction between an antitumor drug-mitoxantrone and human serum albumin[END_REF]. Molecular docking is one of the known theoretical techniques for prediction of interaction between drugs and biomacromolecules. To dock our ligands to DNA, The 3D structures of the L 4 and L 5 ligands was generated using the .cif file of their X-ray crystal structures. The .cif files were converted to the .pdb format using the Mercury software (http://www.ccdc.cam.ac.uk/). The geometry of the L 1 , L 2 , L 3 ligands were optimized using Gaussian 03 [START_REF] Khorshidifard | Synthesis, characterization and X-ray crystal structures of Vanadium (IV), Cobalt (III), Copper (II) and Zinc (II) complexes derived from an asymmetric bidentate Schiffbase ligand at ambient temperature[END_REF] at the level of B3LYP/6-31G** [START_REF] Pace | How to measure and predict the molar absorption coefficient of a protein[END_REF][START_REF] Reichmann | A further examination of the molecular weight and size of desoxypentose nucleic acid[END_REF]. The crystal structure of DNA (PDB ID: 423D) with sequence d(ACCGACGTCGGT)2 were taken from the Brookhaven Protein Data Bank (http://www.rcsb.org/pdb). The resolution of this file was 1.6 Å. Water molecules were deleted from the .pdb files and missing hydrogen atoms were added. Flexibleligand docking was carried out by AutoDock 4.2.5.1 molecular docking program using the implemented empirical free energy function and the Lamarckian Genetic Algorithm (LGA) [START_REF] Morris | Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function[END_REF].

The Gasteiger charges were added to the macromolecule input file and the AutoGrid was used to calculate grids. For docking of the synthesized ligands to DNA, in first step a blind docking with 126 lattice points along X, Y, and Z axes was performed to find the binding site of complexes on DNA with a grid point spacing of 0.375 Å. In the next step, the centre of the grid box was located at the binding site and the second docking was performed using a cubic box with 60 × 60 × 60 dimensions. 250 docking runs with 25,000,000 energy evaluations for each run were performed.

Molecular dynamics (MD) simulation

MD simulation method was used to compare the structural changes of DNA in the absence and presence of the ligands. The Schiff base-DNA complexes with the most negative free binding energy were considered as the initial conformations for the MD studies. GROMACS 5.1.2 package [START_REF] Berendsen | A message-passing parallel molecular dynamics implementation[END_REF] and Amber99 force field [START_REF] Pérez | Refinement of the AMBER force field for nucleic acids: improving the description of α/γ conformers[END_REF][START_REF] Wang | Development and testing of a general amber force field[END_REF] were used to carry out all MD studies. The partial atomic charges of the ligands were calculated using Gaussian 03 [START_REF] Frisch | Gaussian 03[END_REF] at the level of B3LYP [START_REF] Lee | Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density[END_REF], while the other intramolecular force-field parameters were generated with ACPYPE [START_REF] Da Silva | ACPYPE-Antechamber python parser interface[END_REF]. Free DNA and Schiff base-DNA complexes were located in the cubic box with the periodic boundary conditions in the three directions. The solutes were placed in the centre of box and the minimum distance between solute surface and the box was 1.0 nm. The box was filled with TIP3P water molecules [START_REF] Jorgensen | Comparison of simple potential functions for simulating liquid water[END_REF][START_REF] Mark | Structure and dynamics of the TIP3P, SPC, and SPC/E water models at 298 K[END_REF], and the solvated systems were neutralized by adding appropriate amounts of sodium ions (Na + ) and chloride ions (Cl -). After energy minimization using the steepest descent method, the systems were equilibrated for 200 ps at the temperature of 300 K. Finally, a 10 ns MD simulation was carried out at 1 bar and 300 K. Parrinello-Rahman barostate [START_REF] Parrinello | Polymorphic transitions in single crystals: a new molecular dynamics method[END_REF][START_REF] Rahman | Molecular dynamics study of liquid water[END_REF] at 1 bar, Berendsen thermostat [START_REF] Lemak | On the Berendsen thermostat[END_REF] at 300 K. 9 Å cut off for Van der Walls (VdW) and Coulomb interactions and the particle mesh Ewald (PME) method [START_REF] Darden | Particlemesh Ewald: an N•log (N)method for Ewald sums in large systems[END_REF][START_REF] Essmann | A smooth particle mesh Ewald method[END_REF] for long range electrostatics were used. The leap-frog algorithm with the 2 fs time step was used to integrate the equation of motions. Finally, an all-bond constrain was used to keep the ligands from drifting and the atomic coordinates were recorded to the trajectory file every 0.5 ps for later analysis.

Result and discussion

o-Hydroxy Schiff-bases ligands are an important class of compounds from crystallography and biological activity points of view mostly because of their intramolecular hydrogen bonds. The o-Hydroxy ligands synthesized in this work are stable in air for an extended period of time and are soluble in the most common solvents such as methanol, ethanol, DMF, chloroform and DMSO. Scheme 1, Scheme 2 present the synthetic procedure of these ligands. The ligands were characterized by FT-IR, 1 H and 13 C NMR spectroscopies. Moreover, the structures of L 4 and L 5 were determined by single crystal X-ray analysis. Analytical and physical data of Schiff base ligands are listed in Table 3.

Scheme 1. Synthetic routes for the preparation of the ligands L 1 , L 2 and L 3 .

Scheme 2. Synthetic routes for the preparation of the ligands L 4 and L 5 .

IR spectra

The most characteristic feature in the IR spectra of the Schiff base ligands comes from the C N stretching vibrations. In the FT-IR spectra of these ligands, the stretching vibration of imine group (C N) appeared at 1595, 1619, 1611, 1591 and 1591 cm -1 for L 1 , L 2 , L 3 , L 4 and L 5 ligands, respectively that confirmed the synthesis of Schiff bases. The strong band related to the C O stretching were observed at the region of 1279, 1274, 1279, 1278 and 1261 cm -1 for L 1 , L 2 , L 3 , L 4 and L 5 ligands, respectively. In the same way, the bands at 1680, 1677, 1683, 1706, 1721 cm -1 , attributed to COO -(asymmetric) and the bands at 1428, 1421, 1422, 1455 and 1467 cm -1 assigned to the COO -(symmetric) stretching vibrations for L 1 , L 2 , L 3 , L 4 and L 5 ligands, respectively confirmed the successful synthesis of these ligands. The presence of several medium intensity bands at 2800-3100 cm -1 in FT-IR spectra of ligands suggests the existence of C H stretching vibrations of aliphatic and aromatic protons. In Table 4 characteristic IR stretching bands of Schiff base ligands are listed.

NMR spectra

The 1 H and 13 C NMR spectra were run in DMSO-d6 (for L 1 , L 2 , L 3 ) and CDCl3 (for L 4 and L 5 ) and gave the expected simple spectra, indicating the integrity of the Schiff base ligands. The spectra obtained after 12, 24 and 120 h were similar to the initial spectra indicating that the Schiff base ligands are stable in solution. The 1 H NMR spectrum of the L 1 in DMSO-d6 showed the aromatic protons as a multiplet in the range of 6.97 - 8.03 ppm. The OH protons of the phenolic and carboxylic acid groups are appeared at 12.63 ppm and 12.97 ppm, respectively. The imine proton was appeared as a sharp singlet at 8.96 ppm (Fig. S1 (a)). The 1 H NMR spectrum of L 2 in DMSO-d6 showed the singlet peak of the imine proton at 9.01 ppm. The signal observed at 12.80 ppm is assigned to OH proton of the phenolic group and OH proton of the COOH group was appeared at 13.19 ppm. The aromatic protons were observed as a multiplet at region 6.96-7.93 ppm (Fig. S2 (a)). In the 1 H NMR spectrum (Fig. S3 (a)) of the L 3 , the aliphatic protons of the ligand are in 2.50-2.51 ppm. The aromatic protons were appeared as a multiplet in the range of 6.96-7.97 ppm and the OH protons of the phenolic and COOH groups are in the range of 13.06 ppm. The imine proton was appeared as a sharp singlet at 8.96 ppm. The 1 H NMR spectra (Figs. S4 and S5 (a)) of L 4 and L 5 in CDCl3 showed signal of the imine protons (CH N) were appeared at 8.67 and8.65 ppm, respectively. In these ligands, the signals observed at 13.39 and 13.31 ppm are assigned to OH proton of the phenolic groups of L 4 and L 5 , respectively. The aromatic protons were observed as multiplets at 6.88-8.13 and 6.90-8.12 ppm while the aliphatic protons of the L 4 and L 5 ligands are in the range of 1.51-4.20 and 3.94-3.95 ppm, respectively. The 13 C NMR spectra of the ligands are consistent with the number of carbons in their structures (Figs. S1-S5. (b)). The peaks observed at 166.79, 166.84, 166.94, 166.57 and 166.54 ppm are ascribed to the imine carbon atoms of L 1 , L 2 , L 3 , L 4 and L 5 , respectively. The existence of these peaks in the spectra of ligands supports the presence of the Schiff base in them.

Description of the crystal structure of L 4 , L 5

The formation of L 4 and L 5 ligands was confirmed by single crystal X-ray diffraction analysis. Crystallographic data are collected in Table 1. Also, the selected bond lengths and angles are listed in Table S1. The L 4 and L 5 ligands crystallize in space groups P21/c and P212121, respectively and their representative ORTEP view with the atom-numbering scheme are presented in (Fig. 1) As seen in these figures, L 4 and L 5 have adopted E-configuration about the C( 9)

N( 1) double bond with a C6-N1-C9-C10 torsion angle of 178.88° and 178.91°, respectively. In both ligands N(1)⋯H( 3) O( 3) intramolecular hydrogen bond between N atom and the OH group generates a S [START_REF] Kashanian | Spectroscopic studies on the interaction of isatin with calf thymus DNA[END_REF] motif that helps to stabilize the planarity of the molecule [START_REF] Khosravi | Synthesis, characterization, crystal structure and HSA binding of two new N, O, O-donor Schiff-base ligands derived from dihydroxybenzaldehyde and tert-butylamine[END_REF][START_REF] Özek | Three (E)-2-[(bromophenyl) iminomethyl]-4-methoxyphenols[END_REF] C1in L 4 and O3⋯H5 C5 and O2⋯H1A C1in L 5 ) (Table 2). According to the hydrogenbonding classification [START_REF] Steiner | The hydrogen bond in the solid state[END_REF] these intramolecular and intermolecular hydrogen bonding are moderate and weak interactions, respectively. Table 2. Intra-and intermolecular hydrogen bond geometries (Å,°) for L 4 and L 5 .

D-H⋯A

Absorption spectroscopic studies

The UV-Vis absorption spectroscopy is also a useful technique which has been frequently used to examine binding process. The photometric titration was carried out by adding various amount of FS-DNA to the ligands solutions in mole ratio range of [DNA]/[ligand] = 0-4.5 (Fig. S6). Generally, hyperchromic or hypochromic effect and red or blue shift are observed in the UV-Vis spectrum of a drug upon its DNA-binding [START_REF] Kazemi | Synthesis, characterization and separation of chiral and achiral diastereomers of Schiff base Pd (II) complex: a comparative study of their DNA-and HSA-binding[END_REF]. Through addition of various amount of the FS-DNA, a hypochromic effect was observed in the ligands absorption spectra. These spectral changes suggest that the ligands can interact with FS-DNA through groove binding mode [START_REF] Kazemi | Mohammadpoor-Baltork, Synthesis, characterization, crystal structure, DNA-and HSA-binding studies of a dinuclear Schiff base Zn (II) complex derived from 2hydroxynaphtaldehyde and 2-picolylamine[END_REF].

Fluorescence quenching experiments

EB is a standard intercalating agent of DNA that is used to study DNA-binding of compounds. A significant increase in fluorescence intensity of EB is observed upon addition of DNA, while EB shows a weak fluorescence emission in solution due to hydrogen transfer from one of its amino group into solution which causes non-radiative decay [ [START_REF] Arjmand | Synthesis, characterization and in vitro DNA binding and cleavage studies of Cu (II)/Zn (II) dipeptide complexes[END_REF], [START_REF] Fu | Characterization, DNA binding and cleavage, HSA interaction and cytotoxicity of a new copper (II) complex derived from 2-(2pyridyl) benzothiazole and glycylglycine[END_REF], [START_REF] Waring | Complex formation between ethidium bromide and nucleic acids[END_REF], [START_REF] Fromherz | Photoinduced electron transfer in DNA matrix from intercalated ethidium to condensed methylviologen[END_REF]]. In order to determine the binding ability of the ligands, the Stern-Volmer quenching plot (Eq. ( 1)) was obtained by monitoring the fluorescence quenching of DNA-EB with increasing the concentration of the ligands [START_REF] Steiner | The hydrogen bond in the solid state[END_REF].

where, F0 and F are the fluorescence intensity of DNA-EB in the absence and presence of the ligands. Ksv is the Stern-Volmer quenching constant, kq is the quenching rate constant of biomolecule, [Q] is concentration of the quencher (complexes) and τ is the average lifetime of biomolecule without quencher (typically equal to 10 -8 s for biomacromolecules) [START_REF] Kazemi | Synthesis, characterization and separation of chiral and achiral diastereomers of Schiff base Pd (II) complex: a comparative study of their DNA-and HSA-binding[END_REF]. Ksv is determined from the plot of F0/F vs. [Q] (Fig. 2). The values of Ksv were presented in Table 5.

There are two mechanisms for fluorescence quenching: static quenching and dynamic quenching.

In the static mechanism, the fluorophore and the quencher collide together in the ground state, but the fluorophore and quencher collide together in the excited state in dynamic mechanism [START_REF] Kazemi | Synthesis, characterization and separation of chiral and achiral diastereomers of Schiff base Pd (II) complex: a comparative study of their DNA-and HSA-binding[END_REF]. The Stern-Volmer plots indicate that fluorescence quenching may have only one of the above mechanisms or combination of them [START_REF] Kazemi | Mohammadpoor-Baltork, Synthesis, characterization, crystal structure, DNA-and HSA-binding studies of a dinuclear Schiff base Zn (II) complex derived from 2hydroxynaphtaldehyde and 2-picolylamine[END_REF]. Our results showed that the plots are linear and therefore, the mechanism should be dynamic or static. The values of kq were obtained for DNA (Table 5) and the values were about 10 11 M -1 •S -1 . This confirms that fluorescence quenching of biomolecule occurs by static mechanism since kq values are greater than limiting diffusion rate constant of the diffusional quenching for biopolymers (2 × 10 10 M -1 •S -1 ).

The binding constants (Kb) were determined using the following equation [START_REF] Kazemi | Mohammadpoor-Baltork, Synthesis, characterization, crystal structure, DNA-and HSA-binding studies of a dinuclear Schiff base Zn (II) complex derived from 2hydroxynaphtaldehyde and 2-picolylamine[END_REF]:

"Kb" is obtained from the plot of Ln((F0 - F)/F) versus Ln[Q] as y-intercept. These plots are present in Fig. 3. The Kb values (Table 5) reveal that the L 4 -DNA complex is more stable than the other compounds. Moreover, this result is in good agreement with the molecular docking results. Moreover, L 4 has higher binding affinity to FS-DNA than L 5 . This indicates that the FS-DNA binding ability in this group of the synthesized ligands rises with increasing the electron donating ability of the substituent on their phenyl ring. This result is in good agreement with previous results for binding of DNA, protein to palladium(II) complexes [START_REF] Kalaivani | DNA, protein binding, cytotoxicity, cellular uptake and antibacterial activities of new palladium (II) complexes of thiosemicarbazone ligands: effects of substitution on biological activity[END_REF][START_REF] Kalaivani | Influence of terminal substitution on structural, DNA, protein binding, anticancer and antibacterial activities of palladium (II) complexes containing 3-methoxy salicylaldehyde-4 (N) substituted thiosemicarbazones[END_REF].

Cyclic voltammetry (CV) measurements

Cyclic voltammetry is one of the known methods to investigate the interaction of DNA with drugs. CV measurements were performed using an Autolab (PGSTAT 30), with a three-electrode system: a 0.10-cm-diameter Glassy carbon (GC) disc, a calomel electrode and a Pt wire as working, reference and counter electrodes, respectively. L 4 ligand which has the most binding constant among the five synthesized Schiff base ligands, was chosen for cyclic voltammetry measurements. The supporting electrolyte was 50 mM of Tris-HCl buffer solution (pH = 7.4). The ligand and DNA concentrations were constant (5.0 × 10 -5 M) and interaction time was 2 min. Fig. 4 shows the cyclic voltammograms of L 4 ligand in the absence and presence of DNA. The cathodic peak around -0.55 V in the curve can be attributed to the reduction peak of guanine oxidation intermediate [START_REF] Psciuk | Computational prediction of one-electron reduction potentials and acid dissociation constants for guanine oxidation intermediates and products[END_REF]. This observation confirms that L 4 has appropriate interaction with DNA.

Circular dichroism (CD) spectroscopy

The CD spectra of DNA in free and bonded form to the L 4 ligand are shown in Fig. 5. The CD spectrum of free double stranded DNA exhibits three marker peaks at about 222 (positive), 245 (negative) and 280 nm (positive). The positive peak at about 280 nm due to base stacking and negative peak at 245 nm due to polynucleotide helicity are main peaks of DNA for its B-type, which are highly sensitive towards the DNA interaction with small molecules [START_REF] Dehkhodaei | Studies of DNA-and HSA-binding properties of new nano-scale green synthesized Ni (II) complex as anticancer agent using spectroscopic methods, viscosity measurement, molecular docking, MD simulation and QM/MM[END_REF]. The L 4 ligand which showed the most binding constant with FS-DNA was selected for CD spectroscopy studies. Fig. 5 shows that the intensity of DNA peaks has been altered due to its interaction with the L 4 ligand. The results indicate that binding of the L 4 ligand alters the DNA conformation. This observation is in consistent with the molecular dynamics (MD) simulation results (Section 3.4.6).

Molecular docking studies

Molecular docking is a valuable technique to predict the stable structure of receptor-ligand complex for better recognition of the interaction details in drug discovery process. This method is frequently employed as virtual searching tools in primary steps of drug design and development. In order to find out the preferred location of the ligands on DNA, molecular docking studies were carried out. Table 6 represents the obtained binding modes and docking energies of the ligands during their interaction with DNA. The docked models suggest that the ligands bind with the minor groove of DNA with standard binding free energy (ΔG°) of -6.98, -6.94, -7.13, -7.34 and -7.25 kcal.mol -1 for L 1 , L 2 , L 3 , L 4 and L 5 , respectively. These scores are related to the docking cluster with maximum population and minimum binding energy. These results are in good agreement with the obtained fluorescence results (see Section 3.4.2). Also, docking studies showed that H-bond interactions have the main role in the stability of ligand-DNA complexes.

Molecular dynamics (MD) simulation

The beginning structures for the MD analyses were selected from the conformations with the lowest docking energies. The stability of the systems (DNA and the ligands) properties was examined by means of RMS deviations (RMSD) of unbound and ligand conjugated DNA with respect to the initial structure and RMS fluctuations (RMSF) of atoms of the ligands. Preliminary simulations over 8 ns time were performed on DNA with the ligands. Table 7 provides the average values of RMSD for free and conjugated DNA. Fig. 6a indicates that the trajectories of both systems are stable and their RMSD reached equilibrium and fluctuated around its mean value after about 1 ns simulation time. In addition, the RMSF values of the atomic positions of the ligands were calculated to examine their conformational variations (Fig. 6b). The results indicate that the ligands atoms showed limited fluctuations (<0.20 nm). Hence, it can be concluded that the interactions of DNA and the ligands were stable during the simulation time.

Conclusion

In the current study, five novel Schiff base ligands containing hydroxyl groups were synthesized and characterized using IR, 1 H and 13 C NMR techniques. In addition, the structures of L 4 and L 5 were determined using single crystal X-ray method. DNA-binding of the ligands was investigated using fluorescence quenching, UV-Vis spectroscopy and cyclic voltammetry methods. Based on similarity of the ligands structures and their Kb values, their affinity for binding to FS-DNA follow as: L 3 > L 1 > L 2 and L 4 > L 5 . Furthermore, the atomic details of ligands-DNA interactions were investigated using molecular docking and MD simulation methods. Molecular docking results show that all of the synthesized ligands are minor groove binders and H-bond interactions have dominant role in the stability of ligand-DNA complexes. Also, the MD results indicate that the interactions of DNA and the ligands were stable during the simulation time. All of the results indicate that the FS-DNA binding ability of the synthesized ligands rises with increasing the electron donating ability of the substituent on their phenyl ring. Also, both of the CD spectroscopy and MD simulation results (RMSD) show that Schiff base binding destabilizes DNA double helix.

Final

  Largest diff. peak and hole0.390 and -0.631 e.Å -3 0.121 and -0.164 e.Å -3

Fig. 1 .

 1 Fig. 1. a) ORTEP representation of L 4 . Displacement ellipsoids are drawn at the 30% probability level and H atoms are shown as small spheres of arbitrary radii. Intramolecular H-bond is illustrated by dashed line. b) ORTEP representation of L 5 . Displacement ellipsoids are drawn at the 30% probability level and H atoms are shown as small spheres of arbitrary radii. Intramolecular H-bond is illustrated by dashed line.

Table 6 .Table 7 .

 67 Binding energies and binding modes of the ligands during their interaction with DNA. (H-bond interactions are shown by green spheres). Mean RMSD values for free and conjugated DNA during 8 ns of MD simulation.

Fig. 2 .

 2 Fig. 2. Stern-Volmer plot for the synthesized ligands-DNA systems.

Fig. 3 .

 3 Fig. 3. Plot of Ln(F0-F)/F vs. Ln[ligand] for DNA-binding.Based on the structural similarity of the ligands, they can be arranged in two groups: (i) L 1 , L 2 , L 3 and (ii) L 4 and L5 . The comparison of L 1 , L 2 and L 3 shows that L 3 has the most affinity for FS-DNA. This difference can be due to existence of the electron donating group (methyl) on the phenyl ring of L3 . Furthermore, all substituents in L 1 and L 2 are the same and they differ only in

Fig. 4 .

 4 Fig. 4. Cyclic voltammetry of L 4 + DNA.

Fig. 5 .

 5 Fig. 5. The CD spectra of DNA in the absence and presence of L 4 ligand in the molar ratio [L 4 ]/[DNA] = 1.

Fig. 6 .

 6 Fig. 6. a) Time dependence of RMS deviation of distance (RMSD) between atoms from the crystal structure as a function of simulation time for free DNA and conjugated DNA. b) Atomic fluctuations of the DNA conjugated Schiff base ligands during 8 ns of MD simulation.

  ). Anal. Calc. for C14H10BrNO3: C, 52.53; H, 3.15; N, 4.38. Found: C, 52.51; H, 3.17; N, 4.40%. IR (KBr, cm -1 ): νasym (COO -) 1680, νsym (COO -) Hz, 4 J = 2.4 Hz, 1H), 7.48 (d, 3 J = 8.8 Hz, 2H), 6.98 (d, 3 J = 8.8 Hz, 1H)• 13 CNMR (DMSO-d6, 100 MHz, 298 K) δ/ppm: 166.79,162.95, 159.23, 138.44, 135.96, 133.77, 131.17, 130.71, 130.42, 128.96, 121.52, 119.12, 112.49, 110.08.

	1428, ν (C	N) 1595, ν (C	N) 1279.

1 HNMR (DMSO-d6, 400 MHz, 298 K) δ/ppm: 12.97 (br, 1H, OH); 12.63 (s, 1H, OH), 8.96 (s, 1H, N CH); 8.02 (d, 3 J = 8.8 Hz, 2H), 7.92 (d, 4 J = 2.4 Hz, 1H), 7.59 (dd, 3 J = 8.8

4

56.20, 52.23. 2.2.5. Synthesis of L 5 3

	3-Ethoxy-2-hydroxybenzaldehyde (3.32 g, 20 mmol) was added to an ethanolic solution (50 mL)
	of 4-aminobenzoate (3.02 g, 20 mmol) and the mixture was refluxed for 1 h. The resultant red
	solution was filtered. Red single crystals of the title compound were re-crystallized from
	methanol by slow evaporation of the solvents at room temperature over several days. Yield
	4.96 g (83%). Anal. Calc. for C17H17NO4: C, 68.22; H, 5.72; N, 4.68. Found: C, 68.19.51; H,
	5.74; N, 4.66%. IR (KBr, cm -1 ): νasym (COO -) 1706, νsym (COO -) 1455, ν (C	N) 1591, ν (C
	N) 1278.
	1 HNMR (CDCl3, 400 MHz, 298 K) δ/ppm: 13.39 (s, 1H, OH), 8.67 (s, 1H, N	CH); 8.12
	(d, 3 J = 8.4 Hz, 2H), 7.33 (d, 3 J = 8.4 Hz, 2H), 7.06 (dd, 3 J = 7.6 Hz, 4 J = 1.2Hz, 2 H), 6.91
	(d, 3 J = 8.0 Hz, 1H), 4.17 (q, 3 J = 7.2Hz, 2 H, -CH2-CH3); 3.95 (s, 3H, -OCH3); 1.53
	(t, 3 J = 7.2 Hz, 3H, CH3-CH2-). 13 CNMR (DMSO-d6, 100 MHz, 298 K) δ/ppm: 166.57, 164.22,
	152.21, 151.46, 148.49, 131.60, 131.06, 128.40, 124.05, 121.200, 118.86, 118.84, 115.20,
	113.78, -Methoxy-2-hydroxybenzaldehyde (3.04 g, 20 mmol) was added to an ethanolic solution
	(50 mL) of 4-aminobenzoate (3.02 g, 20 mmol) and the mixture was refluxed for 1 h. The
	resultant red solution was filtered. Red single crystals of the title compound were re-crystallized
	from methanol by slow evaporation of the solvents at room temperature over several days. Yield
	4.62 g (81%). Anal. Calc. for C16H15NO4: C, 67.36; H, 5.30; N, 4.91. Found: C, 67.39; H, 5.35;
	N, 4.90%. IR (KBr, cm -1 ): νasym (COO -) 1721, νsym (COO -) 1467, ν (C	N) 1591, ν (C	N)
	1261.
	1 HNMR (CDCl3, 400 MHz, 298 K) δ/ppm: 13.31 (s, 1H, OH), 8.65 (s, 1H, N	CH), 8.11
	(d, 3 J = 8.4 Hz, 2H), 7.32 (d, 3 J = 8.4 Hz, 2H), 7.05 (dd, 3 J = 8.0 Hz, 4 J = 1.2Hz, 2 H), 6.92
	(d, 3 J = 8.0 Hz, 1H), 3.95 (s, 3H,	OCH3), 3.94 (s, 3H,	OCH3)•

13

CNMR (DMSO-d6, 100 MHz, 298 K) δ/ppm: 166.54, 164.15, 152.09, 151.71, 147.74, 131.05,128.36, 124.09, 121.15, 118.95, 118.79, 116.65, 64.61, 52.21, 14.89.

Table 3 .

 3 Analytical and physical data of Schiff base ligands.

	D-H	H⋯A	D⋯A	D-H⋯A	Empty Cell

Table 4 .

 4 Characteristic IR stretching bands of Schiff base ligands in cm -1 .

	Compound υ C-N
	υ C	N (

azomethine) υ as(COO-(carboxylate) υ s(COO-(carboxylate)

	L 1	1279 1595	1680	1428
	L 2	1274 1619	1677	1421
	L 3	1279 1611	1683	1422
	L 4	1278 1591	1706	1455
	L 5	1261 1591	1721	1467

Table 5 .

 5 The values of Kb, n, Ksv and kq for binding of the ligands to FS-DNA.

	Type of ligand	Kb (M -1 )	N	Ksv (M -1 )	kq (M -1 •S -1 )
	L 1	8.9 × 10 4	0.45	4.1 × 10 3	4.1 × 10 11
	L 2	6.8 × 10 4	0.42	3.2 × 10 3	3.2 × 10 11
	L 3	1.2 × 10 5	0.28	5.6 × 10 3	5.6 × 10 11
	L 4	2.3 × 10 5	0.24	3.1 × 10 3	3.1 × 10 11

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 1549535 and 1549534 contain the supplementary crystallographic data for L4 and L5, respectively. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or fromthe Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK. Fax: +44 1223 336 033; or e-mail: deposit@ccdc. cam.ac.uk. Supplementary data associated with this article can be found in the online version, at https://doi.org/10.1016/j.molliq.2018. 01.029.


Monireh Dehkhodaei

Mehdi Sahihi
email: m.sahihi@chem.ui.ac.ir

Amiri Hadi

Rudbari
email: h.a.rudbari@sci.ui.ac.ir

Fariborz

Hadi Amiri

Fariborz Momenbeik

Vanadium (IV), Copper (II), and Zinc (II) complexes derived from an asymmetric bidentate Schiff-base ligand: multi spectroscopic, viscosity measurements, molecular docking, and ONIOM studies

Keywords: DNA interaction, HSA binding, Schif base, Molecular docking, ONIOM

    DNA and HSA interaction of Vanadium (IV), Copper (II), and Zinc (II) complexes derived from an asymmetric bidentate Schiff-base ligand: multi spectroscopic, viscosity measurements, molecular docking, and ONIOM studies

Introduction

Schif-base ligands and their metal complexes have been studied increasingly in past decades. These compounds show wide variety of chemical structures with diferent physicochemical properties [1][2][3][4]. On the other hand, there are many reports in the literatures based on their potential applications in diferent sciences, e.g., solar cells, molecular recognition, catalysis, and nano-materials, due to mild reaction conditions and high synthesis rates of Schif-base complexes [5][6][7][8]. In addition, in the recent years, remarkable attention has been paid to the biological applications of Schif-base compounds due to their stability, biocompatibility, and biological activities [9]. Biochemists believe that the imine group existence in the chemical structure of Schif bases is the main cause of their biological activity of Schif-base compounds. Furthermore, complexation of Schif-base ligands with transition metal ions enhances their biological activities [10][11][12]. In addition, metal ion compounds are attractive candidates in biological felds and medicinal applications. The role of Copper(II) as antitumor and antibacterial agent [13,14], Vanadium (IV) as antitumor, antimicrobial, an insulin-mimetic, and antidiabetic agent [15][16][17] and Zinc (II) in peptidase enzymes, its antimicrobial activity, its function as enzyme activator or inhibitor, and its partnership in intracellular and intercellular signal transduction [18][19][20] are the best examples for biological importance of these metal ions. Therefore, studying the interaction of Schif-base metal complexes with biomacromolecules such as DNA and proteins is the frst step for the intellectual design and fabrication of new and more efcient pharmaceutical molecules. Proteins and DNA are vital members of our life and the major target in medicine and pharmacy felds. In general, proteins are known as the major targets for most of the drugs in organisms [21]. The interaction mechanism of a drug with plasma proteins is key point to understand its pharmacodynamics and pharmacokinetics [22]. Their interaction with drugs has great infuence on drug absorption/distribution in the circulatory system [23] and can prevent rapid elimination of drug from blood stream [24]. Human serum albumin (HSA) is the most abundant plasma protein. In addition, it is known as the dominant transporter plasma protein for endogenous and exogenous ligands (e.g. fatty acids and hormones) [25] and is one of the most important targets in binding of various drugs (e.g. warfarin, diazepam, and ibuprofen) [26] and metal ions [27]. The HSA binding of a drug increases its solubility in plasma, decreases its toxicity, protects it from oxidation, prolongs its in vivo half-life, and increases its pharmaceutical efect [28][29][30][31]. Understanding of these interaction mechanisms helps us to know the pharmacodynamics and pharmacokinetics efects of drugs. In addition, regarding DNA as one of the frst targets of intracellular anticancer drugs, investigation of metal complexes interactions with this double helix is key stage in the design of new drugs with anticancer activities [32,33]. Herein, the Fish Sperm DNA (FS-DNA)-and HSA-binding of the Vanadium (IV), Copper (II), and Zinc (II) complexes derived from an asymmetric bidentate Schif-base ligand, which have been synthesized in our research group earlier [34], have been evaluated using both experimental (fuorescence quenching, UV-Vis spectroscopy and viscosity measurements) and computational methods (molecular docking and ONIOM).

Experimental section

Chemicals and instrumentation

HSA and DNA were purchased from Sigma-Aldrich. Other chemicals were purchased from Merck and were used without further purification. Buffer solutions were prepared using analytical grade salts and double distilled water. All the solutions were used freshly after preparation. The UV-Vis spectra were recorded by Shimadzu UV-160 spectrophotometer. Fluorescence and Viscosity measurements were carried out at room temperature using Shimadzu RF-5000 spectrofluorometer and a Brookfield rotational viscometer, respectively.

HSA-and DNA-binding experiments

Preparation of the complexes, and HSA and DNA stock solutions

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 dark and was used within 2 h. HSA concentration was determined using UV-Vis spectrophotometry and the molar absorption coefficient 35,700 M -1 cm -1 at 278 nm [35]. The stock solution of FS-DNA was prepared in 50 mM Tris buffer at pH = 7.5 and was 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 the reported molar absorptivity of 6600 M -1 cm -1 [36]. Purity of FS-DNA solution was confirmed by ratio of UV absorbance at 260 and 280 nm (A 260/A 280 = 1.9), indicating that FS-DNA is free from protein impurity [37]. Also, the stock solutions of the complexes were prepared in dimethylformamide (DMF) and then were diluted to the desired concentrations with corresponding buffer. The volume of DMF in all final solutions was less than 0.5% (v/v), so the effect of DMF was negligible.

Fluorescence spectroscopy measurements

Fluorescence spectroscopy is a sensitive and effective method to study binding of drugs to biomacromolecules. Fluorescence quenching experiment can help us to obtain the binding mode, binding constants, number of binding sites, and intermolecular distances [38]. The interactions of HSA and DNA with the synthesized Schiff-base complexes [Zn(II), Cu(II) and V(IV) complexes] were investigated using fluorescence quenching experiment. Quartz cuvette with 1 cm optical path length was used, and the excitation and emission slits were set at 5 and 10 nm, respectively. In HSA-binding experiments, 2 ml of HSA solution (5 µM) was placed into the cell and various amounts of the complexes solutions (0-50 µM) were added to it. The fluorescence emission spectra were recorded using 295 nm as excitation wavelength and 300-450 nm as emission wavelength range. Although fluorescence of proteins is due to the presence of three amino acids, i.e., tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) residues, the intrinsic fluorescence of HSA comes from tryptophan [39]. In addition, to investigate the binding of the complexes with DNA, the FS-DNA solution was stirred with ethidium bromide (EtBr) with molar ratio of DNA:EtBr 10:1 for 1 h at 4 °C. Significant increase of fluorescence intensity of EtBr is observed at the presence of FS-DNA due to intercalation of the EtBr molecules into the double helix of DNA [40,41,42,43]. Then, various amounts of the metal complexes (0-250 μM) were added to this mixture. The fluorescence spectra were recorded in the range of 500-700 nm with excitation wavelength of 520 nm. The mixture was allowed to incubate for 2 min after addition of the complexes (Scheme 1).

Synthetic routes for the preparation of the complexes

Moreover, in all the HSA-and DNA-binding experiments, the measured fluorescence intensities were corrected for the dilution and the inner filter effect.

UV-Vis absorption spectroscopy measurements

Electronic absorption spectroscopy is one of the used methods for studying the binding affinity. To confirm the binding of the complexes to HSA and DNA, absorption titration experiments were carried out at room temperature. The UV-Vis absorption spectra of the metal complexes' solutions (10 µM) in the absence and presence of various amounts of HSA and FS-DNA (0-50 µM) were recorded. In all the measurements, the mixture was allowed to incubate for 2 min before recording the related spectra. Absorption curves of complexes-biomacromolecule mixtures were corrected for both absorptions of biomacromolecule solutions and the dilution effect.

Viscosity measurements

Viscosity experiments were carried out by a rotational viscometer and the measurements were performed at 200 rpm at room temperature. The viscosity of FS-DNA solution was measured in the presence of increasing amounts of the metal complexes. The obtained data are presented as (η/η 0) 1/3 versus [complex]/[DNA], where η0 and η are the viscosities of FS-DNA in the absence and presence of the metal complexes, respectively.

Molecular docking procedure

Studying the interaction between drug molecules and biomacromolecules is one of the interesting topics in biochemistry [44]. Molecular docking is one of the known theoretical techniques for the prediction of interaction between drugs and biomacromolecules. To dock our complexes to HSA and DNA, the 3D structures of the metal complexes were obtained using the .cif files of their Xray crystal structures [34]. The .cif files were converted to .pdb format using the Mercury software (http://www.ccdc.cam.ac.uk/). The crystal structure of HSA (PDB ID: 1AO6) and DNA (PDB ID: 423D) with sequence d(ACCGACGTCGGT)2 were taken from the Brookhaven Protein Data Bank (http://www.rcsb.org/pdb). The resolution of these files was 2.6 and 1.6 Å for HSA and DNA, respectively. Water molecules were deleted from the .pdb files and missing hydrogen atoms were added. Flexible-ligand docking was carried out by AutoDock 4.2.5.1 molecular docking program using the implemented empirical free energy function and the Lamarckian Genetic Algorithm (LGA) [45]. The Gasteiger charges were added to the macromolecule input file and the AutoGrid was used to calculate grids. For docking of the synthesized metal complexes to HSA, the grid box was centred on C α of the Trp-214 residue of protein. Ninety lattice points along X, Y, and Z axes were selected to find the active site of complexes on HSA with a grid point spacing of 0.375 Å. For the docking of metal complexes with DNA, in the first step, a blind docking with 126 lattice points along X, Y, and Z axes was performed to find the binding site of complexes on DNA with a grid point spacing of 0.375 Å. In the next step, the centre of the grid box was located at the binding site and the second docking was performed using a cubic box with 60 × 60 × 60 dimensions. Two hundred and fifty docking runs with 25,000,000 energy evaluations for each run were performed.

Quantum mechanical/molecular-mechanics (QM/MM) calculations

QM/MM calculations were used to investigate the conformational changes on interaction of the complexes with HSA and DNA. To carry out QM/MM calculations, Our own N-layered Integrated molecular Orbital and molecular Mechanics (ONIOM) methodology was employed. The ONIOM can be considered as a hybrid method including quantum mechanical method (QM) and a molecular mechanics (MM). Although this method was used as a two-layer QM/MM in this study, it is capable for combining any number of molecular orbital and molecular-mechanics methods [46]. Using this, method one is also able to apply different ab initio or semi-empirical methods to different parts of a molecule/system. This resulted in producing reliable geometry and energy data at reduced computational time [47]. Real system contains full geometry of the molecule which is considered as MM layer, while the model system contains the chemically most important (core) part of the system is considered as QM layer. In current work, a two-layer QM/MM method was opted for all calculations. Molecularmechanics method (UFF) was applied to HSA or DNA as low layer. In similar way, semiempirical method (PM6) was selected for the complexes as high layer.

Results and discussion

Fluorescence spectroscopy

Figures 1,2 show the fluorescence quenching of HSA and DNA (5 × 10 -6 M) at the presence of various amounts of the complexes, respectively. The fluorescence intensity of protein was quenched through the addition of the complexes. This implies that the complexes strongly interact with HSA, leading to microenvironment changes around the Trp-214 residue. In addition, these complexes can displace EtBr by changing the DNA conformation. Consequently, the DNA-bound EtBr molecules are converted to their free form in solution and cause fluorescence quenching [48,49].

Fluorescence emission spectra of HSA upon its titration with various amounts of metal complex: a Zn(II), b V(IV), c Cu(II). To determine the binding ability of the complexes, the Stern-Volmer quenching plot (Eq. 1) was obtained by monitoring the fluorescence quenching of HSA and DNA-EtBr with increasing the concentration of the complexes [50]:

F0F=1+Ksv[Q]=1+kqτ[Q],�0�=1+�sv[�]=1+�q�[�], (1)
where F 0 and F are the fluorescence intensity of HSA or DNA-EtBr in the absence and presence of the compounds. K sv is the Stern-Volmer quenching constant, k q is the quenching rate constant of biomolecule, [Q] is concentration of the quencher (complexes), and τ is the average lifetime of biomolecule without quencher (typically equal to 10 -8 s for biomacromolecules) [51]. K sv is determined from the plot of F0/F�0/� versus.

[Q]. The values of K sv were presented in Tables 1 and2, for HSA and DNA, respectively. Table 1 HSA-binding constant (K b), binding energy, the number of binding site (n), the Stern-Volmer constant (K SV), and the quenching rate constant (k q) of the metal complexes

of the metal complexes

There are two mechanisms for fluorescence quenching: static quenching and dynamic quenching.

In the static mechanism, the fluorophore and the quencher collide together in the ground state, but the fluorophore and quencher collide together in the excited state in dynamic mechanism [51]. The Stern-Volmer plots indicate that fluorescence quenching may have only one of the above mechanisms or combination of them [52]. Our results showed that the plots are linear, and therefore, the mechanism should be dynamic or static. The values of k q were obtained for HSA and DNA (Tables 1 and2, respectively), and the values were about 10 11 M -1 S -1 . This confirms that fluorescence quenching of biomolecules occurs by static mechanism, since k q values are greater than limiting diffusion rate constant of the diffusional quenching for biopolymers (2 × 10 10 M -1 S -1 ). The binding constants (K b) were determined using the following equation [52]:

Ln(F0-FF)=Ln(Kb)+nLn[Q].Ln(�0-��)=Ln(�b)+�Ln[�]. (2)
"K b" is obtained from the plot of Ln((F0-F)/F)Ln((�0-�)/�) versus Ln[Q] as y-intercept. These plots are present in Figs. 1 and2 for HSA and DNA, respectively. The K b values (Table 1) reveal that the Zn(II)-HSA complex is more stable than the other compounds; in the other words, Zn(II)-HSA complex is more available for drug-cell interaction. Moreover, this result is in good agreement with the UV-Vis spectroscopy and molecular docking results (see Sects. "UV-Vis absorption" and "Docking study"). Furthermore, "n" which is the number of binding site per protein (slope of the plot) is near to 1 (Table 1), indicating that the complexes bind to HSA with molar ratio of 1:1. In general, binding constant of a drug with a carrier protein such as HSA should be high enough to bind and transfer it throughout the body. On the other hand, to release a drug in its target, K b should not be too high. The obtained HSA-binding constants of all the complexes are in a good range (1-6 × 10 4 ) [53]. In addition, our complexes are in uncharged form and, therefore, capable to cross through the membrane's lipid bilayer [54]. The obtained results show that our compounds bind to HSA differently which arises from their different central metal ions and their corresponding affinities to HSA. In addition, the binding constants (K b) for the interaction of metal complexes with FS-DNA have been determined using Eq. ( 4) and are presented in Table 2. The K b values reveal that V(IV) forms more stable complex with FS-DNA than the other complexes.

UV-Vis absorption

The UV-Vis absorption spectroscopy is also a useful technique which has been frequently used to examine binding process. The photometric titration was carried out by adding various amounts of HSA or DNA to the complexes solutions in mole ratio range of [HSA]/[complex] = 0-4.5 (Figs. S1 andS2). Through addition of various amounts of the HSA or DNA, a hypochromic effect was observed in the complexes absorption spectra. To assess the binding ability of the complexes with HSA and DNA, the intrinsic-binding constant (K b) was estimated by monitoring the titration curves and using the following equation [55]:

1(εa-εf)=1(εb-εf)+1Kb(εb-εf)×1[BM].1(εa-εf)=1(εb-εf)+1�b(εb-εf)×1[BM]. (3)
Here, [BM] is the concentration of biomacromolecule (HSA or DNA); ε a, ε f, and ε b are the apparent molar absorptivity, the molar absorptivity for free compounds, and the molar absorptivity for the compounds in fully bound form, respectively. ε f was estimated from calibration curve and ε a is the ratio of Aobs to [complex]. A plot of 1/(ε a -

ε f) versus 1/[BM]
gives K b as ratio of y-intercept to slope. The binding constants for Zn(II), V(IV), and Cu(II) complexes with HSA are about 4.58 × 10 4 , 2.38 × 10 4 , and 5.14 × 10 3 M -1 , respectively. In the same way, binding constants for DNA-complex adducts are about 1.83 × 10 3 , 1.27 × 10 3 , and 1.19 × 10 3 M -1 for V(IV), Zn(II), and Cu(II), respectively.

Energy transfer from HSA to the complexes

Energy transfer between the complexes and HSA can provide valuable information about HSAcomplex binding. The fluorescence quenching of HSA upon its binding to metal complexes can be deduced from energy transfer between HSA and metal complexes. This energy transfer can be explained by fluorescence resonance energy transfer (FRET) theory. FRET "known as Förster ' s resonance energy transfer" is an interaction between the excited molecule and its adjacent molecule, upon it; energy absorbed by donor molecule is transferred to an acceptor [56].

According to this theory, energy transfer will observe if: (1) the donor has fluorescence, (2) the fluorescence emission spectrum of the donor and the UV-Vis spectrum of the acceptor have sufficient overlap, and (3) the distance between donor and acceptor is less than 8 nm [56]. The distance (r) and efficiency of energy transfer (E) between tryptophan residue of protein (HSA) and drug (complex) were calculated using this theory through the following equation:

E=1-FF0=R60R60+r6,�=1-��0=�06�06+�6, (4)
where F 0 and F are fluorescence intensities of HSA in the absence and presence of complex, respectively. R 0 is the critical distance when the transfer efficiency is 50% and r is the distance between donor and acceptor. R 0 can be calculated by the following equation [57]: R60=8⋅79×10-25K2N-4Jφ.�06=8⋅79×10-25K2N-4Jφ.

(

In the above equation, the term K 2 is the orientation factor of the dipoles; N is the refractive index of medium, J is the overlap integral of the fluorescence spectrum of the donor with absorption spectrum of the acceptor, and φφ is the fluorescence quantum yield of the donor. The value of J can be calculated as follows:

J=∑F(λ)ε(λ)λ4Δλ∑F(λ)Δλ.�=∑�(�)ε(�)�4Δ�∑�(�)Δ�. (6)
Here, F(λ) is the fluorescence intensity of the donor in the absence of the acceptor at wavelength λ and εε is the molar absorption coefficient of the acceptor at λ. In general, K 2 = 2/3, N = 1.336 and φφ = 0.15 for HSA. Therefore, according to Eqs. (4-6), the parameters for the complexes were calculated and the related results are shown in Table 3 and Fig S3. The values of r for all the complexes are less than 8 nm and 0.5 R 0 < r < 1.5 R 0, suggesting that energy transfer from HSA to the complexes occurs with high probability. Table 3 Obtained results from FRET theory for the metal complexes

Viscosity measurements

To further verify the interaction mode of the metal complexes with FS-DNA, viscosity measurements of DNA solution upon addition of the complexes were carried out. A classical intercalation mode causes an increase in DNA solution viscosity. This resulted from separation of base pairs by accommodated compound and subsequent increase of the DNA overall length [58]. Non-classical mode of interactions such as groove-binding and electrostatic interactions could bend the DNA helix, reduce its length, and may cause the reduction of the DNA solution viscosity [58]. The effect of the metal complexes on the viscosity of FS-DNA solution is illustrated in Fig. 3. As can be seen, the viscosity of DNA solution decreased slightly or remained constant with increasing amounts of the complexes, indicating that the binding mode of all complexes may be groove-binding. This result is in consistent with molecular docking results (Sect. "Docking study").

Effect of increasing amounts of metal complexes on the viscosity of FS-DNA.

[Complex]/[DNA] = 0-0.4.

[DNA] = 10 μM

Docking study

The complexes were docked to the crystal structure of HSA and DNA. The docking results for HSA are collected in Table 4, and revealed that V(IV), Cu(II), and Zn(II) are bound to the IIA subdomain of HSA, which is the main binding site for some drugs such as thyroxin, ibuprofen, and warfarin [26]. The Cu(II) complex has hydrogen-bond interaction with Arg-222 and π-cation interactions with Arg-222 and Lys-199 residues. In addition, hydrophobic interactions with hydrophobic residues of HSA can also stabilize the Cu(II) complex in its binding site. Furthermore, there is one hydrogen bond with Lys-199 and two π-cation with Lys-195 residue which can stabilize the V(IV) complex in its binding site. Finally, Zn(II) complex-HSA system is stabilized by one hydrogen-bond interaction with Arg-222 and four π-cation interactions with Lys-199, Arg-222, and Arg-257 residues. In addition, hydrophobic interactions have dominant role in stability of Zn(II) complex-HSA system. The obtained binding energy for all metal complex-HSA adducts are represented in Table 1. The larger negative value of binding energy for Zn(II) complex means the higher affinity for HSA binding which is in good agreement with UV-Vis and fluorescence experimental data. Moreover, the distances between Trp-214 and metal complexes were 3.151, 3.605, and 3.202 nm for Cu(II), V(IV), and Zn(II) complexes, respectively.

Table 4 Molecular docking results for the interaction of metal complexes with HSA

The docking results revealed that V(IV) and Cu(II) are bound to the minor groove of DNA. On the other hand, Zn(II) is bound to the major groove of DNA. Table 6 represents the binding mode and the nucleotides around each of metal complexes. Molecular docking results show that there are no particular interactions (such as hydrogen bond, π-π stacking, or π-cation interactions) between the Cu(II) or V(IV) complexes and nucleotides in minor groove of DNA. However, one hydrogen-bond interaction and one π-cation interaction with DG4 nucleotide stabilize the Zn(II) complex in the major groove of DNA. The standard binding-free energies (ΔG°), describing the affinity of the complexes for binding to DNA with the best scores, are -5.66, -5.58, and -5.50 kcal mol -1 for V(IV), Zn(II), and Cu(II) complexes, respectively. The docking results are in good agreement with spectroscopic results (see Table 5). Both the experimental results and computational docking data collectively suggest that V(IV) complex has more DNA-binding affinity than the other metal complexes which it may be due to its larger structural volume than the two other ones (990.73, 967.38, and 966.75 Å 3 for V(IV), Zn(II), and Cu(II) complexes, respectively). It seems that as the structural volume of the complex increases, the orientation of the complex in the groove of DNA gets appropriate, and this can affect the fitting of the complex in the binding site. The appropriate orientation of the complex leads to the increasing of hydrophobic interactions and the stability of DNA-complex adduct.

Table 5 Molecular docking results for the interaction of metal complexes with DNA

QM/MM calculation

In the present work, a two-layer ONIOM calculation including PM6:UFF was employed to perform QM/MM calculation. The molecular mechanics (MM) was described using the UFF force field for biomolecule (low layer), while semi-empirical quantum mechanics (QM) method (PM6) was opted for the complexes (high layer). The starting geometry of HSA-complex or DNA-complex adducts for the two-layer ONIOM study was obtained from the molecular docking simulation. The geometry was optimized using ONIOM calculation. All calculations were carried out using Gaussian 09 quantum chemistry package. The partial atomic charges on the atoms of the complexes, HSA, and DNA were used to re-optimize the optimized geometries. The results of ONIOM indicated that structures of the complexes deviate from the initial geometry due to the binding to HSA or DNA. Along with the interaction of the complexes with HSA or DNA, some bond lengths and bond angles are changed. These changes can be resulted from the strength of the interaction between the complexes with biomacromolecules. Tables 6 and7 show the changes in bond angles of the complexes along with binding to HSA or DNA. Table 6 Geometry changes of the compounds during binding to HSA using ONIOM Table 7 Geometry changes of the compounds during binding to DNA using ONIOM

Conclusion

In the current study, the binding ability of three Schiff-base metal complexes including Cu(II), V(IV), and Zn(II) to DNA or HSA was described. These metal complexes have been synthesized in our research group, previously [34]. Herein, they were resynthesized by the same procedure and in water as a green solvent. The experimental DNA-binding results (spectroscopic and viscosity measurements) as well as computational docking and ONIOM data collectively suggest that all metal complexes interact with DNA, presumably by the groove-binding mechanism.

According to this result, V(IV) complex showed stronger DNA-binding affinity than the other metal complexes which it may be due to its larger structural volume than the two other ones. Moreover, the HSA binding of the complexes was evaluated using experimental (fluorescence quenching and UV-Vis spectroscopy) and computational (molecular docking, ONIOM) methods.

The obtained results indicated that the compounds bind to the IIA subdomain of HSA. The calculated binding constants between these compounds and HSA were about 1.07 to 5.26 × 10 4 M -1 . The results of fluorescence experiment as well as the changes in the absorption spectrum of HSA upon addition of the complexes show that the HSA-complex adducts formed in the ground state. In addition, molecular docking studies revealed that hydrogen-bond, hydrophobic, and π-cation interactions have dominant role in the binding of these complexes to HSA. In addition, the ONIOM calculation was employed to investigate the effects of the HSA interaction on geometry of the compounds. Based on the ONIOM calculations, the structural parameters of the complexes changed due to their appropriate interactions with HSA or DNA. In general, the results of the present study exhibit the effect of the metal ion on the binding of the complexes to DNA or HSA.

  [HSA] = 5 μM, [Comp] = 0-50 μM, λ ex = 295 nm Fluorescence emission spectra of EtBr-DNA system in the presence of various amounts of metal complexes: a Cu(II), b V(IV), and c Zn(II). [DNA] = 25 μM, [Comp] = 0-250 μM, λ ex = 520 nm. Kb values were obtained from slope of the insets

Table 2 DNA

 2

-binding constant (K b), binding energy, the Stern-Volmer constant (K SV), and the quenching rate constant (k q)


est destinée au dépôt

 jcis.2008.03.001

  Structural stability of β-lactoglobulin in the presence of cetylpyridinum bromide: spectroscopic and molecular docking studies Zahra Chavoshpour-Natanzi, Mehdi Sahihi, Sajjad Gharaghani To cite this version: Zahra Chavoshpour-Natanzi, Mehdi Sahihi, Sajjad Gharaghani. Structural stability of β-lactoglobulin in the presence of cetylpyridinum bromide: spectroscopic and molecular docking studies. Journal of Biomolecular Structure and Dynamics, 2018, 36 (3), pp.753-760. 10.1080/07391102.2017.1297254. hal-04086087 12 Chamani, J., & Heshmati, M. (2008). Mechanism for stabilization of the molten globule state of papain by sodium n-alkyl sulfates: spectroscopic and calorimetric approaches. Journal of colloid and interface science, 322(1), 119-127. doi: 10.1016/j.


Hanieh Abolhassani

Abdol-Khalegh Bordbar
email: bordbarbordbar@chem.ui.ac.ir

Najmeh Fani

Zahra Adibipour

Mehdi Sahihi

Elham Sattarinezhad

Samira Gholami

Maryam Atrian Abdol-Khalegh

Spectroscopic and molecular modeling probing of biophysical influence of β-casein nanoprotein on adrenaline and arachidonoyl adrenaline •

The interaction mechanism of adrenaline and arachidonoyl adrenalin with β-casein milk protein as a nano-carrier was investigated by fluorescence spectroscopy, molecular docking, and molecular dynamics simulation approaches. The results of fluorescence studies and thermodynamic parameters revealed the negative values of enthalpy and entropy changes for both compounds which illustrate the vital role of hydrogen bond and van der Waals interaction in stabilizing protein-ligand complexes. The docking studies showed that adrenaline binds to several polar and non-polar residues in the inner hydrophobic core of β-casein with binding energy of -27.25 kJ mol -1 but arachidonoyl adrenalin binds in a core near the surface of this protein with the binding energy of -28.67 kJ mol -1 . Finally, the molecular dynamics simulation results suggested that the interactions between protein and both compounds are very stable and during the whole simulation time no significant protein structure changes were observed, consequently βcasein can be considered as a suitable carrier for adrenaline and arachidonoyl adrenalin.

Introduction

When a person perceives a situation as threatening or exciting, the hypothalamus in the brain signals to the adrenal glands to produce adrenalin (AD) by transforming the amino acid tyrosine into dopamine. In fact, the oxygenation of dopamine produces noradrenaline (norAD), which is then converted into AD. AD as a catecholamine is structurally similar to norAD and is the Nmethylated product of this compound. Both the adrenal hormones have the same chemical precursors and they both are vasoconstrictors [1] but AD is 20-100 times more active than norAD. It has been shown that various forms of stress, thermal burn, muscular exercise, centrifugation, and trauma activate the sympatho-adrenal system and this activation is manifested by an increase in the urinary output of AD and norAD [2]. These effects can reverse severe low blood pressure, wheezing, severe skin itching, hives, and other symptoms of an allergic reaction. It was shown that AD does this by its effects on alpha and beta receptors [START_REF] Rhoades | Medical physiology: principles for clinical medicine[END_REF]. AD binds to these receptors on the heart and arteries and increases heart rate and respiration. Moreover, this hormone inhibits the production of insulin and stimulates the synthesis of sugar and fat by binding to the receptors on the pancreas, liver, muscles and fatty tissue. Arachidonoyl adrenalin (AA-AD) is strictly related to endocannabinoid/endovanilloid N-arachidonoyl dopamine (NADA) as an endogenous capsaicin-like compound. NADA displays high potency for transient receptor potential vanilloid type 1 (TRPV1) channels and cannabinoid type 1 receptors (CB1) [START_REF] Bezuglov | [END_REF]5]. Endocannabinoids modulate a multitude of physiological functions which is only partially based on their binding to the classical cannabinoid receptors CB1 and CB2. These signaling lipids have been implicated in the regulation of the immune system [6], the gastrointestinal tract [7] and the cardiovascular system [8]. Moreover, endocannabinoids have psychoactive effects on the central nervous system (CNS) of human body [9]. Experimental studies showed that extreme amounts of circulating catecholamines are oxidized to aminochromes that could affect their physiological benefits and cause some disorders in body. Moreover, formation of oxyradicals during the oxidation process may result in sudden cardiac death [10]. The binding of catecholamines to nano-carrier proteins can protect them from oxidation, increase drug efficacy, solubility, and stability and can decrease drug toxicity [11]. Hence, it would be of interest to obtain insight into the binding mechanism of these compounds with some nano-carrier proteins. Caseins are a family of phosphoproteins which commonly make up 80% of the proteins in cow's milk and between 20 and 45% of the proteins in human milk [12]. Bovine beta-casein (βCN) that constitutes up to 45% of the casein of bovine milk is a single-chain protein containing 209 residues with molecular weight of 23.983 kDa. βCN is a highly amphiphilic calcium-sensitive phosphoprotein which consists of a hydrophilic N-terminal part with a cluster of five phosphoryl residues and a hydrophobic C-terminal part [13]. This protein has been known as a dynamic protein with non-compact and flexible conformation [14]. Many studies have demonstrated that βCN micelles may be utilized as natural nano-delivery vehicles for lipid-soluble drugs. Moreover, βCN evolved to be easily digestible and this basic nano-carrier is expected to readily release its chemotherapeutic compound in the stomach [15]. Some studies have investigated the binding properties of some drug candidates with bovine βCN nanoparticles using various experimental and computational techniques [16]. In this paper, fluorescence spectroscopy, molecular docking and molecular dynamics (MD) simulation methods have been used for investigation of the interaction mechanism between AD and AA-AD (Fig. 1) with bovine βCN nano-carrier. Structural information regarding binding modes, binding energies, and the effect of AD and AA-AD on the protein stability and protein structure are stated here.

Results and discussion

Fluorescence spectroscopy

Measuring the intrinsic fluorescence of protein in the presence of different concentrations of ligands is a way in which we can compute binding parameters. βCN has one Trp residue that the intrinsic fluorescence of its indole chromospheres is chiefly sensitive to their polarity of environments. Essentially, this Trp residue is placed in a primarily hydrophobic domain of βCN, and can provide important information about the interaction and formation of AD-βCN and AA-AD-βCN complexes. As shown in Figs. 2a and3a, when a fixed concentration of βCN was titrated with various amounts of AA and AA-AD, a remarkable increase of the fluorescence intensity was observed. The enhancement of fluorescence is based on the photophysical phenomenon whereby the intensity of a fluorophore increases upon proximal binding of a ligand. There are two different mechanisms, static and dynamic, for enhancing of fluorescence intensity. In the static mechanism, the ligand binds to protein and a complex with higher molar absorptivity formed while in the dynamic mechanism, the ligand interacts with excited state of protein and reduced the rate constant of irradiative mechanisms. By calculating the surfaces under the fluorescence plots and using Eq. ( 1) the increasing constants can be estimated [START_REF] Lakowicz | Principles of fluorescence spectroscopy[END_REF]18]. where S 0 and S are the total area under the emission spectrum in the absence and presence of ligand, [E] is the ligand concentration, and K E is the increasing constant. Plots of S 0/S versus [AD] and [AA-AD] at 293 K are shown in Figs. 2b and3b, respectively. The calculated K E at four temperatures from 293 to 308 K for AD and AA-AD are presented in Table 1. The K E values decrease with the increase of temperature which demonstrates static mechanism for interaction between these ligands with βCN. Table 1 Increasing constants for interaction of AD and AA-AD with βCN in PBS, pH 7

If there are n substantive binding sites on the surface of protein to accommodate the ligand molecules, the equilibrium between free and bound molecule is given by the following equation [START_REF] Lakowicz | Principles of fluorescence spectroscopy[END_REF] where K a is the associative binding constant:

The corresponding plots of this equation are shown in Figs. 2c and3c for AD and AA-AD, respectively. Binding constants and number of binding sites for interaction of AD and AA-AD with βCN are listed in Table 2.

Table 2 Binding constants and number of binding sites for interaction of AD and AA-AD with βCN in PBS, pH 7

The calculated binding constant for interaction of AD and AA-AD with βCN is lower than the binding constants of curcumin, diacetyl curcumin, and quercetin with this protein [16,19,20]. Thermodynamic parameters such as the standard Gibbs free energy, ΔG 0 , the standard molar enthalpy, ΔH 0 , and the standard molar entropy, ΔS 0 , can be estimated by analyzing K a values at different temperatures using the following equations:

where R and T are gas constant and the absolute temperature, respectively. If the temperature does not vary considerably, the standard enthalpy change (ΔH 0 ) can be regarded as a constant. Plots of ΔG 0 /T vs. 1/T for AD and AA-AD are shown in Fig. 4 and calculated thermodynamic parameters are reported in Table 3.

The variation of ∆G 0 /T vs. 1/T for binding of AD (a) and AA-AD (b) to βCN at pH 7 in PBS buffer Table 3 Thermodynamic parameters for the association of AD and AA-AD with βCN

The driving forces for interaction of AD and AA-AD with βCN can be concluded from the sign and magnitude of the thermodynamic parameters which were characterized by Ross and Subramanian. They express that hydrophobic force might increase ΔH 0 and ΔS 0 of a system, hydrogen bond and van der Waals interactions decreased them and electrostatic force usually made ΔH 0 ≈ 0 and ΔS 0 > 0 [21,22]. Just as Table 3 shows, negative values of standard free energy explain the spontaneity of the binding reaction and relative high affinity of these ligands to βCN and the negative values of ΔH 0 and ΔS 0 represent an enthalpy-driven process. In advance, the negative value obtained for ΔH 0 points to the importance of hydrogen binding in the interaction process and the negative values of ΔS 0 shows the van der Waals interactions in solution. Therefore, it can be concluded that both hydrogen bonding and van der Waals interactions play an essential role in the binding of AD and AA-AD to βCN.

Molecular docking study

Docking studies showed that AD and AA-AD bind to several polar and non-polar residues in the inner hydrophobic core of β-casein and in the core near the surface of this protein. Docking results for binding of AD and AA-AD to βCN are shown in Fig. 5. AD is close to some hydrophobic residues in the core of βCN such as Leu31, Ile45, Phe102, Val92, and Phe67. Moreover, this compound is in contact with some polar residues such as Glu106, Gln71, Lys43, and Lys44. AA-AD interacts with hydrophobic residues: Ile202, Tyr195, Val224, Phe205, Val212, Ile222, Phe102, and Ile223 and polar residues: Lys191 and Asp199. Moreover, hydroxyl groups of AD were found to interact with Ile45, Glu106, and Lys43 through hydrogen bonds and nitrogen atom of this compound formed one hydrogen bond with Phe67. The representative build structure derived from the best pose for interactions between AD and βCN has the minimal binding energy of -27.25 kJ mol -1 and for AA-AD-βCN complex has the minimal binding energy of -28.67 kJ mol -1 . The interaction of the ligands with these residues could change the microenvironment of Trp158 and consequently enhances its quantum yield. The calculated binding energy for interaction of AD and AA-AD with βCN is higher than the binding energies of curcumin, diacetylcurcumin and quercetin with this protein [16,19,20].

The structure of AD (a) and AA-AD (b) in the binding site of βCN as a mesh surface image and the schematic description of the environment of ligands bound to protein generated by Ligplot+. Only the more important residues for binding are shown

Molecular dynamics simulation study

The structure with the lowest binding energy from docking calculation was selected to perform another MD simulation (40 ns) on the AD-βCN and AA-AD-βCN complexes. To compare the structural stability of free and bound βCN and ligands and determination of involving interactions, some dynamic structural properties such as root mean square deviations (RMSD), radius of gyration, root mean square fluctuations (RMSF), surface area of protein, and hydrogen bonds have been analyzed. In the following, the details of investigated parameters are explained.

Root mean square deviations (RMSD)

The RMSD of backbone atoms of protein from the initial structure as a function of time have been deliberated to analyze the trajectory stability of free βCN and βCN in complex with AD and AA-AD. As Fig. 6a shows the RMSD of βCN, AD-βCN, and AA-AD-βCN achieve stability after 19, 25, and 17 ns, respectively. Moreover, the mean RMSD values of protein backbone atoms from last 20 ns trajectory were 0.429 ± 0.073, 0.431 ± 0.078, and 0.407 ± 0.070 nm for βCN, βCN-AD, and βCN-AA-AD complexes, respectively. a RMSD (nm) of backbone atoms of βCN in the absence and presence of AD and AA-AD. b Time dependence of the radius of gyration (R g) for the backbone atoms of βCN during the simulation in the absence and presence of AD and AA-AD

Radius of gyration (R g)

The compactness of protein structure during the MD simulation time was assessed by the radius of gyration (R g) using g_gyrate command. The R g for the free and bound form of protein with AD and AA-AD was determined and plotted as a function of time (Fig. 6b). For all cases the R g values were stabilized at about 20 ns, indicating that the system achieved equilibrium after this time. The values of R g for bound form of βCN is less than free protein implying the compacting of βCN structure after binding with AD and AA-AD.

Root mean square fluctuations (RMSF)

The time-averaged RMSF values for free and bound βCN were measured to investigate local protein mobility. The results were plotted against residue number at the simulation trajectory and shown in Figs. 7 and8. RMSF values demonstrate that protein structure in the presence of both ligands remained stable during the simulation. a RMSF of residues of protein from their time-averaged positions for free and bound βCN with AA. b RMSF of AD atoms during the simulation time a RMSF of residues of protein from their time-averaged positions for free and bound βCN with AA-AD. b RMSF of AA-AD atoms during the simulation time

The particular regions directly in contact with ligands including Leu31, Ile45, Phe102, Val92, Phe67, Glu106, Gln71, Lys43, Pro105, Lys44 residues for βCN-AD, and Ile202, Tyr195, Val224, Phe205, Val212, Ile222, Phe102, Ile223, Pro201, Lys191, Asp199 residues for βCN-AA-AD (pointed in Figs. 7a and8a, respectively) showed more significant reduction of RMSF that is related to intermolecular interactions of these residues with these ligands. The same residues are in contact with ligands with respect to docking results representing insignificant displacement of ligands inside the binding site during the simulation time.

Surface area of the protein

Another parameter for evaluating a MD simulation is the variation of accessible surface area (ASA) of free and bound forms of protein as function of time to compute hydrophobic, hydrophilic, and total solvent accessible surface area and to judge the stability of the system. Figure 9a shows the area of the protein in the presence and absence of the ligands and indicates that the surface of βCN due to its complexation reduces which is in good agreement with investigation of the radius of gyration.

Hydrogen bonds

g_hbond is a command to measure the number of hydrogen bonds, hbond distances and angles between molecules or groups through the course of the simulation and these are shown in Fig. 9b,c. As seen in these figures, maximum of seven hydrogen bonds can be exist between ligands and βCN but at most of the time only three and two hydrogen bonds exist in βCN-AD and βCN-AA-AD complexes, respectively, which is in good agreement with the docking results.

Conclusions

In this study, the interactions of AD and AA-AD with βCN were investigated using fluorescence spectroscopy, molecular docking and molecular dynamics simulation studies. The enhancement of fluorescence emission of βCN in the presence of these ligands was observed which represents the changing of microenvironment of Tyr due to the binding of AD and AA-AD. Analysis of fluorescence binding data at various temperatures represents the formation of 1:1 complexes, the higher affinity of AD, enthalpy-driven nature of process and indicates both hydrogen bond and van der Waals interaction as the main driving forces in the binding process. The results of molecular docking calculation represent different binding sites for these ligands located in the hydrophobic domain of the βCN. The obtained RMSD and R g profiles from MD simulations proved the stability of βCN-ligand complexes and validity of docking results. In the selected conformations of βCN-AD, five hydrogen bonds were observed and the RMSF outlines reveal negligible movement of ligands from their poses during the MD simulation. The results of computational studies are in agreement with the experimental results as both approaches assign the same kind of binding interactions and higher binding affinity of AD.

Materials and methods

Phosphate buffer solution (10 mM, pH 7.0, containing 80 mM NaCl, with an ionic strength of 0.1) (PBS) was prepared. The stock solution of bovine βCN (> 99%; Sigma-Aldrich) was made up by dissolving it in the PBS. To avoid large protein aggregation the protein solution was filtered through a porous membrane. The protein concentration was determined from the absorbance at 280 nm by a UV-Vis spectrophotometer using an extinction coefficient of 4.6 (1%) for βCN [this is the extinction coefficient on the basis of mass concentration (the absorbance of protein solution with weight concentration of 1% equals to 4.6)]. AD was purchased from Merck Company and AA-AD (purity not less than 97%, HPLC check) was synthesized according to the literature [23] and stored only as ethanol stocks under argon atmosphere at -52 °C. UV spectra: AA-AD λ max = 282 nm (ε = 2770, ethanol). Due to the low water solubility of ligands; their solutions were made up by dissolving them in ethanol. The exact concentration of these two solutions was measured by determining light absorption at λ max = 279 nm and extinction coefficient (ε) of 3981.1 M -1 cm -1 for AD and λ max = 282 nm (ε = 2770 M -1 cm -1 , ethanol) for AA-AD. All of the solutions were prepared using double-distilled water and were used freshly after preparation.

Fluorescence spectroscopy

We performed fluorescence measurements on a RF-5000 spectrofluorimeter (Shimadzu, Japan) equipped with a xenon lamp source, a 1.0-cm quartz cell and a thermostat bath that keeps temperature constant within ± 0.1 ○ C. In our experiments, 2 cm 3 of βCN solution (16.30 μM) was put in quartz cell and titrated with specified volumes of AD stock solution. Emission spectra were recorded from 300 to 450 nm at an excitation wavelength of 295 nm. The spectrofluorometric titration experiments were run at four different temperatures from 293 to 308 K. Both excitation and emission slit widths were set at 5 nm and control experiment was done on βCN micelle without AD. Ethanol concentration did not exceed 1% v/v during the fluorescence measurements. A similar procedure has been used for running the spectrofluorometric titration experiments for AA-AD with βCN. In these measurements, the emission spectra were recorded from 290 to 430 nm, the concentration of βCN solution and excitation wavelengths were 9.60 μM and 280 nm, respectively. The obtained data were used for binding and thermodynamic analysis.

Preparation of βCN, AA and AA-AD structures

There is no crystal structure of βCN in protein data bank; hence, homology modeling techniques and I-Threading ASSEmbly Refinement (I-TASSER) server were used to build its structure. Structural templates are first identified from PDB by multiple threading approach LOMETS; fulllength atomic models are then constructed by iterative template fragment assembly simulations. Finally, function insights of the target are derived by threading the 3D models through protein function database BioLiP. The structure of βCN which was made with I-TASSER server consists of 1 helix and 5 strands and other positions were made up of random coils. Better and more stable structure can be achieved by running a molecular dynamic simulation on the proposed model of I-TASSER. For this purpose, a 50-ns molecular dynamic simulation was performed on the βCN structure using GROMACS 4.5.4 software package and the final constructed structure was used as the beginning point of molecular docking calculations. Finally, to get the optimized and stable geometry of ligands, AD and AA-AD, the structureoptimizing calculations were carried out at the 6-31G** level by employing the Becke threeparameter Lee-Yang-Parr (B3LYP) hybrid density functional theory using the quantum chemistry software Gaussian 03 [24].

Molecular dynamics simulation

GROMACS 4.5.4 package with the GROMOS96 43a1 force field [25] and the SPC water model [26] was employed to carry out the MD simulations. First, protein was solvated in a cubic box with periodic boundary conditions, and then an appropriate number of Na + counterions were added instead of solvent molecules to neutralize the system. Steepest descent method [27] was employed to relax the system, energy minimization and ease from unfavorable interactions. To equilibrate the systems at 300 K, a 100-ps MD simulation in the NVT ensemble with the Berendsen thermostat coupling method was run. After stabilization of temperature an isothermal-isobaric ensemble (NPT) with the constant pressure of 1.0 bar and time duration of 100 ps was performed by employing Berendsen barostat. The Particle Mesh Ewald (PME) method [28] with 1.0-nm cutoff was used for long-range electrostatics. For van der Waals interactions, Lennard-Jones potential with 1.0-nm cutoff was employed. Finally, a 40-ns MD simulation was done to record trajectories using the leap-frog algorithm [29] to solve the equations of motion with a time step of 2 fs.

Molecular docking calculation

To find the best pose of ligands on protein, molecular docking calculation was done using Autodock 4.2 software. Hydrogens were added to the protein and during docking the macromolecule was kept rigid, while all the torsional bonds of ligands were set free to rotate. First, a blind docking was done with grid size of 126 × 126 × 126 along X-, Y-, and Z-axes; then the dimensions of grid map around the binding site which was found from blind docking were reduced and a grid size of 60 × 60 × 60 was constructed with a grid point spacing of 0.375 Å. Lamarckian genetic algorithm (LGA) method for 200 docking runs with 25,000,000 energy evaluations for each run and AutoDock empirical free energy function were used. The docking results were clustered with a tolerance criterion of 2.0 Å and the structure with lowest free energy of binding in the highest populated cluster was chosen as the optimal docking pose. The binding pose and important residues of protein that involved in the binding can be determined from docking results but dynamics of binding process and conformational changes during the binding cannot be understood from docking; hence, MD simulations of protein in the presence and absence of ligands were performed in which the topology parameters of protein were created from the GROMACS program and the force field parameters of ligands were taken from PRODRG web server [30].

a

  Fluorescence increase of βCN solution by AD in PBS, pH 7, T = 293 K, λ ex = 295 nm, and [βCN] = 16.30 μM. The concentration of AD is increased in the direction of arrow. b The variation of S 0/S versus total concentration of AD. c The variation of log [(S 0 -S)/S] vs. log [E] a Fluorescence increase of βCN solution by AA-AD in PBS, pH 7, T = 293 K, λ ex = 280 nm, and [βCN] = 16.30 μM. The concentration of AA-AD is increased in the direction of arrow. b The variation of S 0/S vs. total concentration of AA-AD. c The variation of log [(S 0 -S)/S] vs. log [E]

a

  Total surface area of the protein in the presence and absence of AD and AA-AD. b Number of hydrogen bonds that exist in the 40-ns simulation between βCN and AD. c Number of hydrogen bonds that exist in the 40-ns simulation between βCN and AA-AD Accessible surface area values for βCN, βCN-AD, and βCN-AA-AD were obtained as 125.135 ± 1.564, 118.684 ± 3.076, and 122.617 ± 3.567 nm 2 , respectively.


Christian Mougin

René Scalla

Ferdinand Cabanne

Monooxygenases responsible for the metabolism of chlorotoluron in wheat microsomes

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Mougin C., Scalla R., and Cabanne F.

Laboratoire des herbicides, INRA, BV 1540, 21034 Dijon, France

MONOOXYGENASES RESPONSIBLES FOR THE METABOLISM OF CHLOROTOLURON IN WHEAT MICROSOMES

Microsomes isolated from achlorophyllous cells of wheat (Triticum aestivum L. var. Koga II) metabolized the herbicide chlorotoluron by two enzymatic reactions, namely a ring-methyl hydroxylation and a N-demethylation. These activities, and also lauric acid in-chain hydroxylation and concentrations of cytochrome P-450, were increased following cell treatment with cyometrinil or 2,4-D. The last compound was also an inducer of cinnamic acid 4-hydroxylase.

Cofactor requirements and susceptibility to inhibitors of the reactions lead to the assumption that chlorotoluron ring-methyl hydroxylase and N-demethylase probably belong to the family of cytochrome P-450 monooxygenases. However, the properties of the N-demethylase appeared to be somewhat different from those of ring-methyl hydroxylase. N-demethylation was not sensitive to CO and could be supported by cumene hydroperoxide acting as oxygen donor.

Other extraction conditions and buffer compositions, as well as incubations of microsomes with chlorotoluron in the presence of inhibitors of peroxydases, peroxygenases, flavin-containing monooxygenases and cytochromes P-450 confirmed the involvement of cytochrome P-450(s) in the oxidative metabolism of chlorotoluron. However, no inhibition of ring-methyl hydroxylation and Ndemethylation could be obtained with polyclonal antibodies prepared against NADPH cytochrome P-450 reductase purified from Jerusalem artichoke. Substrate specificity of enzymes oxidizing chlorotoluron was investigated. Wheat cell microsomes were not able to metabolize diclofop, which has been shown to be hydroxylated in wheat plants by a P-450 monooxygenase, nor to transform diuron, a structural analog of chlorotoluron. Until now, cell treatments with classical inducers of P-450(s) systems such as phenobarbital and aminopyrine did not lead to any specific induction of chlorotoluron metabolism. Microsomes from wheat seedlings exhibited only basal rates of ringmethyl hydroxylation and N-demethylation without any detectable effects of inducers.

Cellular localisation of activities showed that they belong to the endoplasmic reticulum. Improvement of specific activities in in progress through enrichment of the endoplasmic reticulum in the microsomal fraction.