Iman Khosravi
email: iman.khos2012@gmail.com

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

Hadi Amiri-Rudbari

Ghazal Borhan

Zahra Chavoshpour-Natanzi

The interaction of a new Schiff base ligand with human serum albumin: molecular docking and molecular dynamics simulation studies

Keywords: Human serum albumin, molecular docking, molecular dynamics simulation, Schiff base ligand

The interaction of a new heterocyclic Schiff base bearing pyridine and pyrimidine cycles, with human serum albumin (HSA) using molecular docking and molecular dynamics simulation methods was examined. Molecular docking studies showed that the ligand was bonded to the IB domain of the protein. It was found that there was one hydrogen bond interaction between HSA and the ligand. The standard Gibbs free energy for binding of the ligand to HSA was calculated as -9.63 kcal.mol -1 . The results of the molecular dynamics simulation showed that the root mean square deviation (RMSD) of the non-liganded HSA and the HSA ligand complex reached equilibration after 1000 ps. The study of the radius of gyration revealed that there was a conformational change when the HSA ligand complex was formed. Finally, analyzing the RMS (RMSF) suggested that the structure of the ligand binding site remained approximately rigid during the simulation.

Introduction

Schiff bases fascinate a large number of chemists and biochemists due to their easy formation, high stability, their pharmacological properties and, notably, their anticancer activity [START_REF] Ferlay | Cancer incidence and mortality patterns in Europe: Estimates for 40 countries in 2012[END_REF][START_REF] Chow | Discovery Base Complexes via Water-Promoted Combinatorial Three Component Assembly[END_REF][START_REF] Chen | Synthesis and biological evaluation of hydroxyl-substituted Schiff-bases containing ferrocenyl moieties[END_REF][START_REF] Ghosh | Synthesis, crystal structure and antibacterial activities of mixed ligand copper (II) and cobalt (II) complexes of a NNS Schiff base[END_REF]. Most Schiff base ligands show biological activities, such as antibacterial [START_REF] Popp | Synthesis of potential anticancer agents. V. Schiff bases and related Compounds1-2[END_REF], anticancer [START_REF] Chohan | Antifungal cobalt (II), copper (II), nickel (II) and zinc (II) complexes of furanyl-, thiophenyl-, pyrrolyl-, salicylyl-and pyridyl-derived cephalexins[END_REF][START_REF] Siddiqi | Characterization and toxicity of lanthanide complexes with nitrogen-and Sulphur-containing Schiff bases[END_REF], anti- [START_REF] Demirbas | Synthesis and Antitumor Activities of Some New 4-(1-Naphthylidenamino)-and 4-(1-Naphthylmethylamino)-1,2,4-Triazol-5-one Derivatives[END_REF], antiparasitic [START_REF] Rathelot | 1,3-Diphenylpyrazoles: synthesis and anti-parasitic activities of azomethine derivatives[END_REF], antiviral [START_REF] Chavan | Total synthesis of nothapodytine B and (±)-mappicine[END_REF] and anti-HIV [START_REF] Pandeya | Synthesis, antibacterial, antifungal and anti-HIV activities of Schiff and Mannich bases derived from isatin derivatives and N-[4-chlorophenyl)thiazol-2-yl] thiosemicarbazide[END_REF].

The knowledge about interaction mechanisms of a drug with plasma proteins as a biomacromolecule is required to understand the drug`s pharmacodynamics and pharmacokinetics [START_REF] Cui | Interaction of anthracycline disaccharide with human serum albumin: investigation by fluorescence spectroscopic technique and modeling studies[END_REF]. Due to the function of plasma proteins as drug carriers, there are a large number of literatures about their binding to drugs [START_REF] Cui | Yao -O-(aL-Cladinosyl) daunorubicin with human serum albumin: the binding site II on HSA molecular using spectroscopy and modeling[END_REF][START_REF] Khan | Interaction of mitoxantrone with human serum albumin: spectroscopic and molecular modeling studies[END_REF]. The interaction of plasma proteins with a drug has effects on the drug absorption, distribution and elimination in the circulatory system [START_REF] Mccallum | A fluorescence-based high throughput assay for the determination of[END_REF], and can prevent their rapid elimination from the blood stream [START_REF] Li | Protein binding by dinuclear polypyridyl ruthenium (II) complexes and the effect of cucurbit [10] uril encapsulation[END_REF]. Human serum albumin (HSA) is the most abundant plasma protein; it also is known as the dominant transporter plasma protein for endogenous and exogenous ligands (fatty acids, hormones and some drugs) [START_REF] Domonkos | Synthesis and serum protein binding of novel ring-substituted harmine derivatives[END_REF]. The accommodation of a wide range of lipophilic molecules in HSA is possible as a result of its flexible structure [START_REF] Ghuman | Structural Basis of the Drug-binding Specificity of Human Serum Albumin[END_REF]. The HSA-binding of a drug can increase its solubility in plasma, decrease its toxicity, protect it from oxidation, prolong its in vivo half-life as well as increase the pharmaceutical effect of the drug [START_REF] Gou | Enhancing the copper (II) complexes cytotoxicity to cancer cells through bound to human serum albumin[END_REF].

Recently, Ozedmir et. al. synthesized a new, heterocyclic Schiff base bearing pyridine and pyrimidine cycles (Figure 1) [START_REF] Ozdemir | Synthesis of a new -5-benzoyl-4phenyl-1-((pyridin-2-ylmethylene)amino) pyrimidin-2(1H)-An experimental and computational modeling study[END_REF]. Aware of the therapeutic activity of Schiff bases and in an attempt to obtain more insight into their biological activity, the HSAbinding of the mentioned compound has been investigated. The computational molecular docking method was used to evaluate the binding behaviour of the Schiff base ligand to HSA, quantitatively. In addition, to find out the behaviour of the Schiff base-HSA complex, a powerful computational method, molecular dynamics (MD) simulation has been used.

Methods

Molecular docking procedure

In this work, a docking study was carried out to indicate the HSA-binding site for the Schiff base compound. The 3D structures of the compound were generated using GaussView 5.0 software [23] and its geometry was optimized using Gaussian 03 [START_REF] Frisch | Gaussian Development Version, Revision B.01[END_REF] software by the B3LYP (Becke, three-parameter, Lee-Yang-Parr) method [START_REF] Becke | Density-functional thermochemistry. III. The role of exact exchange[END_REF] at the level of 6-31g** [START_REF] Francl | Self-consistent molecular orbital methods. XXIII. A polarization type basis set for second-row elements[END_REF]. The crystal structure of HSA (PDB ID: 1AO6) was taken from the Brookhaven Protein Data Bank (http://www.rcsb.org/pdb). The R-value and resolution of this file were 0.218 and 0.25 Å, respectively. It was prepared in pH=7.5.

Water molecules of the protein pdb file were removed and missing hydrogen atoms and Gasteiger charges were added. Flexible-ligand docking was performed by AutoDock 4.2 molecular-docking program (http://autodock.scripps.edu) using the implemented empirical free energy function and the Lamarckian Genetic Algorithm [START_REF] Darden | Particle mesh Ewald: an N log(N) method for Ewald sums in large systems[END_REF]. The Gasteiger charges were added to prepare the macromolecule input file for docking and the Auto Grid was used to calculate the grids. A blind docking with 126 lattice points along the X, Y, and Z axes was performed to find the active site of the Downloaded by [Australian Catholic University] at 00:49 20 August 2017 ligands with the HSA. After determination of the active site, the dimensions of the grid map were selected as 60 points on a side with a grid point spacing of 0.375 Å, to allow the ligand to rotate freely. 200 docking runs with 25,000,000 energy evaluations for each run were performed.

Molecular Dynamics Simulation

The lowest binding free energy conformation of the complex was considered as the initial conformation for the MD studies. All MD studies were carried out using the GROMACS 4.5.6 (University of Groningen, Netherlands) package [START_REF] Berendsen | GROMACS: a message-passing parallel molecular dynamics implementation[END_REF][START_REF] Lindah | GROMACS 3.0: a package for molecular simulation and trajectory analysis[END_REF] and the [START_REF] Van Gunsteren | Biomolecular Simulation: the GROMOS96 Manual and User Guide[END_REF][START_REF] Van Gunsteren | Encyclopedia of Computational Chemistry[END_REF]. The Dundee PRODRG2.5 server was used to generate the topology parameters of the ligands [START_REF] Schuttelkopf | PRODRG: a tool for high-throughput crystallography of protein ligand complexes[END_REF]. The partial atomic charges of the ligand were calculated using Gaussian 03 [START_REF] Frisch | Gaussian Development Version, Revision B.01[END_REF] at the level of B3LYP/6-31G** [START_REF] Becke | Density-functional thermochemistry. III. The role of exact exchange[END_REF][START_REF] Francl | Self-consistent molecular orbital methods. XXIII. A polarization type basis set for second-row elements[END_REF]. The complex was located in a cubic box with periodic boundary conditions.

The box volume was 11.36381 × 11.36381 × 11.36381 nm 3 and the minimum distance between the protein surface and the box was 1.0 nm. The box contents extended simple point charges (SPC), water molecules [START_REF] Berendsen | Intermolecular Forces[END_REF], and the solvated systems, were neutralized by adding 15 sodium ions (Na + ). Energy minimization was done through using the steepest descent method. Then, the system was equilibrated for 100 ps at the temperature of 300 K. Finally, a 20 ns MD simulation was carried out at 1 bar and 300 K. A Berendsen thermostat [START_REF] Berendsen | Molecular dynamics with coupling to an external bath[END_REF] at 300 K, the particle mesh Ewald (PME) method [START_REF] Darden | Particle mesh 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, and a 9 Å cut off for van der Walls . In this work, the selected Schiff base ligand was docked to the crystal structure of HSA. The obtained results from molecular docking showed that the ligand binds to the IB subdomain of HSA. The standard Gibbs free energy for binding of the ligand to HSA was -9.63 kcal.mol -1 . The results indicated that the Arg (117) amino acid residue is involved in the hydrogen bond interaction in the HSAligand system. The results of the docking are presented in Fig. 2. The other amino acid residues shown in Fig. 2 (lower right) are involved in hydrophobic interactions with the Schiff base ligand. Thus it can be concluded that the interaction of ligand with HSA is not mainly hydrophobic and that the carbonyl groups in the structure of the

Analysis of MD trajectories

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The beginning structure for the MD analyses was selected from the conformation with lowest docking energies. The trajectories were analyzed in terms of root mean square (RMSF) using the GROMACS routines.

Root mean square deviation of the trajectory of the HSA backbone

The stabilities of the trajectories for HSA and the HSA-ligand were examined using the RMSD of the backbone of HSA (Figure 3). A glance at the analysis in Fig. 3 shows that the RMSD of the two systems reached 0.027 and 0.017 nm for HSA and HSA-ligand, respectively at about 300 and 1000 ps, respectively. This means that both systems were equilibrated well and the RMSDs had only low s around their mean values.

Radius of gyration

The Rg for both systems were also determined and plotted against simulation time to study the protein compactness (Figure 4) showed that both systems stabilized after about 500 and 4000 ps for HSA and HSA-ligand, respectively. It can be clearly seen that the Rg was larger upon binding of the ligand, suggesting a more compact structure before the binding of Schiff base ligand to HSA. This indicates that the environment of the HSA changed during its interaction with the Schiff base ligand.

3.2.3.

The mobility of the HSA residues was evaluated by analysis of the RMSF of the C atoms of HSA in the presence of the Schiff base ligand (figure is not shown). The es of the the Downloaded by [Australian Catholic University] at 00:49 20 August 2017 HSA ligand complex. The obtained results clearly indicate that the residues at the binding site had only small in their interactions with the ligand. Also, the structure of the ligand binding site remained approximately rigid during simulation. In addition, the RMSFs of the atomic positions of the Schiff base ligand were calculated to examine its conformational variations (Figure 5). The results indicated that the ligand atoms showed limited 30 nm). Hence, it can be concluded that the interactions of HSA and the Schiff base ligand were stable during the simulation time.

Conclusions

Interaction of a new heterocyclic Schiff base bearing pyridine and pyrimidine cycles with HSA was examined since HSA plays a major role in transporting ligands target places. The molecular docking studies indicated that the Schiff base ligand binds to the IB domain of HSA with one hydrogen bonding interaction. Therefore, it can be concluded that the interaction of this Schiff base ligand with HSA is not mainly hydrophobic. The standard Gibbs free energy for binding of ligand to the HSA was calculated to be -9.63 kcal.mol -1 . The MD study, in particular, made an important contribution toward understanding the effect of the binding of this ligand on the conformational changes of HSA and the stability of HSA ligand complex system in aqueous solution. The MD simulation studies revealed that the RMSD of the HSA and HSA-ligand systems reached equilibrium and oscillated around the average value after

  interactions and Coulomb interactions were used. The equation of motions was integrated by the leap-frog algorithm with 2 fs time steps. The atomic coordinates Downloaded by [Australian Catholic University] at 00:49 20 August 2017 6 -bond constraint was used to keep the ligand from drifting in the MD.

3 .

 3 Results and Discussion 3.1. Molecular docking study HSA is the most abundant protein in human blood plasma and consists of 585 amino acids; it has three -helical domains (I, II and III), each containing two subdomains (A and B) [36]. HSA transports drugs, hormones, fatty acids, and other compounds, and maintains osmotic pressure, among other functions [37,38]. Previous crystal structure studies have shown that most ligands are bound to binding site in subdomains IIA or IIIA [39 43]. Also, the D-shaped cavity in subdomain IB is the binding site of some compounds [44 46]

  300 and 1000 ps simulation time, respectively. Analysis of Rg indicated that the ligand changed the conformation of the HSA during MD simulation. The similarity of Downloaded by [Australian Catholic University] at 00:49 20 August 2017 the HSA and the HSA ligand complex suggested that the structure of the ligand-binding site remained approximately rigid during the simulation.

Figure captions:Figure 1 .

 1 Figure captions:

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2. The docking pose of the HSA-ligand complex.

Figure 2 .Figure 3 .

 23 Figure 2.

Figure 4 .

 4 Figure 4. Time dependence of Rg. Rg values for non-liganded HSA and HSA ligand complex

Figure 4 .Figure 5 .

 45 Figure 4.

Figure 5 .

 5 Figure 5.

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Acknowledgment

Support for this research by the Islamic Azad University (Qeshm branch), Iran, is acknowledged.

Downloaded by [Australian Catholic University] at 00:49 20 August 2017


Dr Iman Khosravi
email: iman.khos2012@gmail.com

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

Maryam Dashtbani
email: maryam.dashtbani70@yahoo.com

Amiri Hadi

Ghazal Rudbari

Borhan

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

Ghazal Borhan

Deoxyribonucleic Acid and Bovine Serum Albumin Interaction with the Asymmetric Schiff Base Ligand and Its Molybdenum (VI) Complex: Multi Spectroscopic and Molecular Docking Studies

Keywords: DNA interaction, BSA binding, Fluorescence quenching, Molecular docking, Schiff base

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

Introduction

Schiff bases are a class of compounds that have attracted a large number of researchers due to their easy formation, high stability, their pharmacological properties and, notably, anti-cancer activity [START_REF] Ferlay | Cancer Incidence and Mortality Worldwide[END_REF][START_REF] Chow | Discovery and Investigation of Anticancer Ruthenium--Arene Schiff-Base Complexes via Water-Promoted Combinatorial Three-Component Assembly[END_REF][START_REF] Chen | Synthesis and biological evaluation of hydroxyl-substituted Schiff-bases containing ferrocenyl moieties[END_REF][START_REF] Ghosh | Synthesis, crystal structure and antibacterial activities of mixed ligand copper(II) and cobalt(II) complexes of a NNS Schiff base[END_REF]. Also, in recent years, remarkable attention has been paid to the biological applications of the metal complexes of Schiff bases due to their stability, biocompatibility and biological activity [START_REF] Pravin | Investigation of in vitro anticancer and DNA strap interactions in live cells using carboplatin type Cu(II) and Zn(II) metalloinsertors[END_REF][START_REF] Arjmand | Synthesis of new chiral heterocyclic Schiff base modulated Cu(II)/Zn(II) complexes: Their comparative binding studies with CT-DNA, mononucleotides and cleavage activity[END_REF]. Biochemists believe that the cause of the biological activity of these compounds is the imine group existence in the chemical structure of Schiff bases [START_REF] Raman | Metallation of ethylenediamine based Schiff base with biologically active Cu(II), Ni(II) and Zn(II) ions: Synthesis, spectroscopic characterization, electrochemical behaviour, DNA binding, photonuclease activity and in vitro antimicrobial efficacy[END_REF][START_REF] Antony | Synthesis, spectroscopic and catalytic studies of Cu(II), Co(II) and Ni(II) complexes immobilized on Schiff base modified chitosan[END_REF][START_REF] Guo | Antifungal properties of Schiff bases of chitosan, N-substituted chitosan and quaternized chitosan[END_REF]. Furthermore, results of recent investigations indicated that after complexation of Schiff base ligands with transition metal ions, their biological activity was enhanced [START_REF] Anacona | Synthesis, characterization and antibacterial activity of a Schiff base derived from cephalexin and sulphathiazole and its transition metal complexes[END_REF][START_REF] Sundararajana | Synthesis of metal complexes involving Schiff base ligand with methylenedioxy moiety: Spectral, thermal, XRD and antimicrobial studies[END_REF][START_REF] Tarushi | Interaction of Zn(II) with quinolone drugs: Structure and biological evaluation[END_REF]. Therefore, investigation of their interaction with biomacromolecules, such as DNA and proteins, is the first step for the intellectual design and fabrication of new and more efficient pharmaceutical molecules. Proteins are vital and impartible members of our life and a major target in medicine and pharmacy fields, similar to DNA. The interaction mechanism of a drug with plasma proteins is a key point to understanding the drugs pharmacodynamics and pharmacokinetics [START_REF] Cui | Interaction of anthracycline disaccharide with human serum albumin: Investigation by fluorescence spectroscopic technique and modeling studies[END_REF].

Generally, proteins are known as the major targets for most of the drugs used in organisms [START_REF] Vuignier | Drug--protein binding: a critical review of analytical tools[END_REF].

Their interaction with a drug affects the drug absorption, distribution and elimination in the circulatory system [START_REF] Mccallum | A fluorescence-based high throughput assay for the determination of Anal[END_REF], and can prevent its rapid elimination from the blood stream [START_REF] Li | Protein binding by dinuclear polypyridyl ruthenium(II) complexes and the effect of cucurbit [10] uril encapsulation[END_REF]. Bovine serum albumin (BSA) is the most abundant plasma protein; it also is known as the dominant transporter plasma protein for endogenous and exogenous ligands (fatty acids, hormones and drugs) [START_REF] Domonkos | Synthesis and serum protein binding of novel ring-substituted harmine derivatives[END_REF]. The BSA-binding of a drug can increase its solubility in plasma, decrease its toxicity, protect it from oxidation, prolong its in-vivo half-life as well as increase the pharmaceutical effect of the drug [START_REF] Gou | Enhancing the copper(II) complexes cytotoxicity to cancer cells through bound to human serum albumin[END_REF].

As a part of our ongoing investigation into designing new Schiff base ligands and complexes [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] Khorshidifard | Cobalt(II), copper(II), zinc(II) and palladium(II) Schiff base complexes: Synthesis, characterization and catalytic performance in selective oxidation of sulfides using hydrogen peroxide under solvent-free conditions[END_REF][START_REF] Amiri Rudbari | New asymmetric Schiff base ligand derived from allylamine and 2,3-dihydroxybenzaldehyde and its molybdenum(VI) complex: Synthesis, characterization, crystal structures, computational studies and antibacterial activity together with synergistic effect against Pseudomonas aeroginosa PTTC 1570[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][23], and studying their interaction with biomacromolecules, herein the DNA-and BSA-binding of the asymmetric Schiff base ligand derived from allylamine and 2, 3-dihydroxybenzaldehyde (L) and its molybdenum(VI) complex (MoL 2 ) (Fig. 1), which had been synthesized in our research group earlier [START_REF] Amiri Rudbari | New asymmetric Schiff base ligand derived from allylamine and 2,3-dihydroxybenzaldehyde and its molybdenum(VI) complex: Synthesis, characterization, crystal structures, computational studies and antibacterial activity together with synergistic effect against Pseudomonas aeroginosa PTTC 1570[END_REF], was evaluated using both experimental (fluorescence quenching and UV--Vis spectroscopy) and computational methods (molecular docking).

Experimental

Chemicals and instrumentation

All the chemicals for synthesis of the compounds were purchased from the Merck Co.

(Germany) and used without further purification. BSA and DNA were purchased from the Sigma-Aldrich Corp. (France). All used salts for buffer preparation were analytical grade and were dissolved in double distilled water. All of the solutions were used freshly after preparation.

The UV-Vis spectra were recorded on a V-670 spectrophotometer (Jasco Inc., Japan).

Fluorescence quenching experiments were carried out at room temperature using RF-5000 spectrofluorometer (Shimadzu Corp., Japan).

BSA binding experiments

Preparation of L and ML 2 compounds and protein stock solutions

A stock solution of BSA was prepared by dissolving the desired amount of BSA in 50 mM phosphate buffer (pH = 7). The BSA stock solution was stored at 4°C in the dark and was used within 2 h. The BSA concentration was determined by UV-Vis spectrophotometry using the molar absorption coefficient 44300 M 1 cm 1 at 280 nm [START_REF] Anjomshoa | Synthesis, crystal structure and Hirshfeld surface analysis of copper(II) complexes: DNA-and BSA-binding, molecular modeling, cell imaging and cytotoxicity[END_REF].

The solutions of the L and ML 2 compounds were first prepared in DMF and then were diluted suitably, with phosphate buffer to the required concentrations for all the experiments. The volume of DMF never exceeded 0.5% (v/v), so the effect of DMF was negligible. All the solutions were used freshly after preparation.

Fluorescence spectroscopy measurements

The interactions of BSA with the L and MoL 2 were investigated using fluorescence quenching experiments. In this experiment 2 mL of BSA solution (5 µM) was placed into the cell and various amounts of the L and MoL 2 compounds (0-50 µM) were added to it. The fluorescence intensity was measured with an excitation wavelength at 295 nm and emission wavelength range of 300-450 nm. In each measurement the mixture was allowed to incubate for 2 min after addition of the compounds to the cell. Moreover, the measured fluorescence intensities were corrected for the dilution and the inner filter effects.

UV-Vis absorption spectroscopy measurements

Electronic absorption spectroscopy is one of the methods used for studying the binding modes of drugs to biomacromolecules. Absorption titration experiment was carried out to further investigate the BSA-binding of the compounds at room temperature. The UV-Vis absorption spectra of the solution of BSA (10 M) in the absence and presence of 1-100 of L and MoL 2 were recorded at room temperature and the changes in the BSA absorption were recorded after each addition of the ligand or complex. Absorption curves of the compounds--protein mixtures were corrected by subtracting the spectra of the L and The stock solution of FS-DNA was prepared in 50 mM Tris buffer at pH 7.5 using double--DNA concentration per nucleotide was determined using the absorption intensity at 260 nm after adequate dilution with the buffer and using the reported molar absorptivity of 6600 M 1 .cm 1 . The purity of the FS-DNA solution was confirmed by the ratio of UV absorbance at 260 and 280 nm with a value of A 260 /A 280 = 1.9 indicating that the FS-DNA was free from protein impurity [START_REF] Zheng | Synthesis, structure and molecular docking studies of dicopper(II) complexes bridged by N-phenolato--[2-(dimethylamino)ethyl]oxamide: the influence of terminal ligands on cytotoxicity and reactivity towards DNA and protein BSA[END_REF]. The solutions of the compounds were first prepared in DMF as co-solvent, and then diluted with the corresponding Tris buffer to the required concentration for all experiments. The volume of the co-solvent never exceeded 0.5% (v/v), so the effect of the DMF was negligible.

Fluorescence spectroscopic measurements

Fluorescence quenching experiments were carried out using a quartz cuvette with 1 cm optical path length; the excitation and emission slits were set at 5 and 10 nm, respectively. For this purpose the FS-DNA solution was stirred with ethidium bromide (EtBr), with molar ratio of DNA:EtBr 10:1, for 1 h at 4°C. Then various amounts of the Mo complex (0--ere added to this mixture. The fluorescence spectra were measured in the range of 500--700 nm with exciting wavelength at 520 nm. In each measurement, after addition of FS-DNA, the mixture was allowed to stand for 2 min prior to the measurements. Moreover, the measured fluorescence intensities were corrected for the dilution and inner-filter effects.

UV-Vis spectroscopy measurements

Absorption titration experiments were carried out to further investigate the DNA-binding of the compounds at room temperature. Absorption spectral titration experiments were performed by addition of various amounts of FS-DNA-Tris buffer solution (0-5 × 10 5 M) to the L and ML 2 compounds-Tris buffer solutions (1 × 10 5 M). All compounds--DNA solutions were allowed to incubate for 2 min before recording the related spectra. Absorption curves of the compounds--DNA mixtures were corrected by subtracting the spectrum of DNA and all intensities were corrected for the dilution effect.

Molecular docking procedure

Study of the interaction between drug molecules and biomacromolecules is one of the interesting topics in biochemistry [START_REF] Tian | The effect of Cu2+ on the interaction between an antitumor drug--mitoxantrone and human serum albumin[END_REF]. Molecular docking helps investigations of these interactions. Owing to this, we examined the docking of L and MoL 2 with BSA and DNA, as important biomacromolecules. The 3D structures of the L and MoL 2 were obtained using the. cif (Crystallographic Information File) files of their X-ray crystal structures [START_REF] Amiri Rudbari | New asymmetric Schiff base ligand derived from allylamine and 2,3-dihydroxybenzaldehyde and its molybdenum(VI) complex: Synthesis, characterization, crystal structures, computational studies and antibacterial activity together with synergistic effect against Pseudomonas aeroginosa PTTC 1570[END_REF]. The .cif files were converted to the .pdb (Protein Data Bank) format by using the Mercury software (http://www.ccdc.cam.ac.uk/). The crystal structure of BSA (PDB ID: 4F5S) 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 were 2.47 and 1.6 Å for BSA and DNA, respectively. Water molecules in the structures of the .pdb files were deleted and missing hydrogen atoms were added.

Flexible-ligand docking was carried out by the AutoDock 4.2.5.1 molecular-docking program (http://autodock.scripps.edu/) using the implemented empirical free energy function and the Lamarckian Genetic Algorithm [START_REF] Morris | Automated Docking Using a Lamarckian Genetic Algorithm and an Empirical Binding Free Energy Function[END_REF]. The Gasteiger (Gasteiger-Marsili) charges were added to prepare the macromolecule input file for docking and the Auto Grid was used to calculate Grids. For the docking of L and MoL 2 to BSA and DNA, a blind docking with 126 lattice points along each of the X, Y, and Z axes was performed to find the binding sites of the compounds on BSA and DNA with a grid point spacing of 0.375 Å, to allow the compounds to rotate freely. In the next step, the center of the grid box was located at the binding site and a 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.

Results and Discussion

Interactions with Bovine Serum Albumin (BSA)

Serum albumin (SA) is one of the most important targets in binding of various molecules such as drugs (warfarin, diazepam, Ibuprofen) [START_REF] Ghuman | Structural Basis of the Drug-binding Specificity of Human Serum Albumin[END_REF], metal ions [START_REF] Bal | Binding of transition metal ions to albumin: Sites, affinities and rates[END_REF], fatty acids [30], etc.

SA is recognized as a transporter protein of blood plasma. Hence, in recent years much attention has been focused on drug-SA interactions. Understanding of these interaction mechanisms help us to know the pharmacodynamics and pharmacokinetics effects of drugs. Several experimental and computational methods have been used to study these binding processes. In the current study the BSA-binding of the prepared compounds was investigated using both experimental and theoretical methods.

Fluorescence spectroscopy.

Fluorescence spectroscopy is a sensitive and effective method to study the binding of drugs to biomacromolecules. A fluorescence quenching experiment can characterize the availability of quenchers to protein fluorophore, which 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 fluorescence intensity of the protein was quenched through the addition of the synthesized compounds. This implies that the compounds strongly interacted with BSA, leading to changes of the microenvironment around the Trp residues in BSA [32,[START_REF] Wang | Interaction of the flavonoid hesperidin with bovine serum albumin: A fluorescence quenching study[END_REF]. Fig. 2 indicates the fluorescence quenching of 5 × 10 6 M BSA in the presence of 0.5-50 µM of the compounds.

In order to determine the binding ability between the compounds and BSA, the Stern--Volmer quenching plot was obtained by monitoring the fluorescence quenching of BSA with increasing concentration of the compounds, according to the Stern--Volmer equation [START_REF] Dighe | -Carboline-Based N-Heterocyclic Carbenes and Their Antiproliferative and Antimetastatic Activities against Human Breast Cancer Cells[END_REF]:

0 s v q 1 K Q 1 k F F (1)
where F 0 and F are the fluorescence intensity of BSA in the absence and presence of the compounds. K sv is the Stern-Volmer quenching constant, k q is the quenching rate constant of BSA and is the average lifetime of BSA without quencher which is typically equal to 10 8 s for biomacromolecules [START_REF] Darabi | The piroxicam complex of cobalt(II): Synthesis in two different ionic liquids, structure, DNA-and BSA interaction and molecular modeling[END_REF]. K sv is determined from the plot of 0 / F F vs.

[Q]

(insets in Fig. 2). In the present study the values of K sv were obtained and are listed in Table 1.

Fluorescence quenching is proposed to occur according to one of two mechanisms: static quenching and dynamic quenching. In the static mechanism, while the fluorophore and the quencher collide together in the ground state while the fluorophore and quencher collide together in the excited state in the dynamic mechanism [START_REF] Darabi | The piroxicam complex of cobalt(II): Synthesis in two different ionic liquids, structure, DNA-and BSA interaction and molecular modeling[END_REF]. Linearity of the Stern-Volmer plot indicates that the quenching fluorescence has only one mechanism, dynamic or static [START_REF] Lakowicz | Plasmonics in Biology and Plasmon-Controlled Fluorescence[END_REF]. The value of k q was obtained and is also listed in Table 1. In this study, the values of k q were greater than the limiting diffusion rate constant for the diffusional quenching for biopolymers (2 × 10 10 M 1 .S 1 ); this observation supports that the quenching fluorescence of BSA occurred by the static mechanism.

The affinity of the biomacromolecules for binding the drugs can be stated by the equilibrium constant (binding constant) for biomacromolecule complex equlilibrium. The binding constants (K b ) were determined using the following equation [START_REF] Lakowicz | Plasmonics in Biology and Plasmon-Controlled Fluorescence[END_REF]:

0 b F F L n L n K n L n Q F (2)
where F 0 and F are the fluorescence intensity of BSA in the absence and presence of the compounds, respectively. [Q] is the concentration of quencher; i.e., the synthesized compounds here.

K b is obtained from the plot of 0 L n / F F F
versus Ln[Q] as the yintercept (figures not shown). The K b values revealed that the MoL 2 -BSA complex was more stable than the L-BSA; in other words, the MoL 2 -BSA complex was more available for drug-cell interaction. Moreover, this result was in good agreement with the UV-Vis spectroscopy and molecular docking results. Furthermore, n , which is the number of binding sites per protein molecules, is the slope of the plot. The value of n was nearly 1, indicating that the synthesized compound binds to BSA with a molar ratio of 1:1. The calculated results are shown in Table 1.

In general, the binding constant of a drug with a carrier protein, such as BSA, should be high enough to bind sufficiently to permit transfer through the body. Moreover, in order to release a drug following arrival at its target, it should not be too high. With respect to the above, the BSA-binding constants of both compounds were in a good range (1-6 × 10 4 M 1 ) [START_REF] Kyropoulou | Ni(II) complexes with nonsteroidal anti-inflammatory drug diclofenac: Structure and interaction with DNA and albumins[END_REF]. In addition, our compounds were in an uncharged form which is an advantage for a drug; research has shown that a unionized drug is soluble in lipid and capable of cross [START_REF] Abou-Zied | Revealing the ionization ability of binding site I of human serum albumin using 2--hydroxyphenyl)benzoxazole as a pH sensitive probe[END_REF]. The obtained results show that L and MoL 2 bind to BSA differently, confirming the effect of the metal ion on the affinity of the two compounds to BSA.

UV-Vis absorption.

UV-Vis absorption spectroscopy is a useful technique that has been frequently used to examine binding processes. The absorption titration was carried out by adding various amounts of L or MoL 2 compound to the BSA ([Compound]/[BSA] = 0-4.5); their absorption spectra are shown in Fig. 3. Through addition of various amounts of the compounds, a hypochromic effect in the spectrum of BSA was observed. In order to assess the binding ability of the two compounds with BSA, the intrinsic binding constant (K b ) was estimated by monitoring the changes of absorbance with increasing concentration of compounds and using the following equation [START_REF] Das | Binding studies of aristololactam--D-glucoside and daunomycin to human serum albumin[END_REF]:

12 0 b 1 1 1 1 K M m a x m a x A A A A A A (3)
where, A 0 and A are the absorbance of BSA in the absence and presence of the compound, respectively. A max is the obtained absorbance at saturation and [M] is the concentration of the compound. The plot of 1/(A-A 0 ) versus 1/[M] gives K b as the ratio of the y-intercept to the slope (Table 1). The binding constants for L and MoL 2 are equal to 1.4 × 10 3 and 2.9 × 10 3 M 1 , respectively. and drug (L and MoL 2 ) was calculated using this theory through the following equation:

13 6 0 6 6 0 0 R 1 R r F E F (4)
where F 0 and F are fluorescence intensities of BSA in the absence and presence of the 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 Eq. ( 5) [START_REF] Huang | Preparation of 2D hydroxylrich carbon nitride nanosheets for photocatalytic reduction of CO2[END_REF].

6 -2 5 2 -4 0 R = 8 .7 9 × 1 0 K N J (5)
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 the absorption spectrum of the acceptor and is the fluorescence quantum yield of the donor. The value of J can be calculated by the following expression: the parameters for the synthesized compounds were calculated and the related results are listed in Table 2 and Figure 4. The values of r for both compounds were less than 8 nm and (0.5 R 0 ) < r < (1.5 R 0 ), suggesting energy transfer from BSA to the compounds occurred with high probability.

3.1.4. Docking study.

One of the known theoretical techniques in biochemistry that can give us information about the atomic details of the interaction of various molecules with biomacromolecules is molecular docking. Therefore, the synthesized compounds were docked to the conformation of BSA in its crystal. The docking results are shown in Fig. 5; it shows that MoL 2 and L were bound to the IIIB subdomain of BSA. The interactions of the compounds within the binding sites of BSA are also shown in this figure. The binding site is considerably hydrophobic, so the L and MoL 2 were in hydrophobic interaction with hydrophobic residues in their binding site. Also, Fig. 5a shows that there were two hydrogen bond interactions of MoL 2 , with Tyr 419 and one with Lys 533 residues.

Furthermore, t -cation interactions with Lys 499 stabilized the MoL 2 --BSA complex.

Figure 5b shows that L is in hydrogen bond interaction with Ala 527 residue andinteractions with Phe 550 residue. The obtained binding energies for the compound-BSA systems are listed in Table 1. The larger negative value of binding energy for MoL 2 means a higher affinity for BSA binding, which is in good agreement with the UV-Vis and fluorescence experimental data.

Interaction with DNA

Fluorescence spectroscopy

The fluorescence measurements were performed to further investigate the interaction mode between the synthesized compounds and FS-DNA (Fig. 6). Hydrogen transfer from one of the amino groups of ethidium bromide (EtBr) into the solution caused nonradiative decay and a weak fluorescence emission. However, a significant increase of fluorescence intensity of EtBr was observed in the presence of FS-DNA due to intercalation of the EtBr molecules into the double helix of the 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] Fu | 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][START_REF] Fromherz | Photoinduced electron transfer in DNA matrix from intercalated ethidium to condensed methylviologen[END_REF]. Figure 6 represents the fluorescence enhancement of the DNA-EtBr by addition of the compounds. In order to determine the binding ability between the compounds and FS-DNA, the enhancement plot was obtained by monitoring the fluorescence enhancement of EtBr-DNA with increasing concentration of the compounds according to the equation ( 6) [START_REF] Shahabadi | In Vitro Study of DNA Interaction with a Water-Soluble Dinitrogen Schiff Base[END_REF].

E 0 1 K E F F (6)
where F 0 and F are the fluorescence intensity of the EtBr-DNA complex in the absence and presence of the compounds. K E is the enhancement constant and [E] is the concentration of the enhancer that causes the enhancement of fluorescence intensity (L and MoL 2 ). The values of K E for the synthesized compounds were determined from the plot of F/F 0 vs. [Q] (Fig. 6) and are presented in Table 3. In this study, the binding constants (K b ) for the interaction of L and MoL 2 with FS-DNA were determined using equation [START_REF] Raman | Metallation of ethylenediamine based Schiff base with biologically active Cu(II), Ni(II) and Zn(II) ions: Synthesis, spectroscopic characterization, electrochemical behaviour, DNA binding, photonuclease activity and in vitro antimicrobial efficacy[END_REF] and are presented in Table 3 [START_REF] Shahabadi | In Vitro Study of DNA Interaction with a Water-Soluble Dinitrogen Schiff Base[END_REF].

0 b F F L n L n K n L n E F (7)
where F 0 and F are the fluorescence intensity of EtBr-DNA in the absence and presence of the compounds, respectively. [E] is the concentration of enhancer; i.e., the synthesized compounds here. K b is obtained from the plot of

UV-Vis absorption

The absorption spectra of L and MoL 2 in the absence and presence of various concentrations of FS-DNA are given in Fig. 7. The intensity of these spectra decreased with the addition of FS-DNA. To quantitatively evaluate the affinity of the compounds with FS-DNA, the intrinsic binding constant K b was determined by monitoring the changes in absorbance using equation ( 8). (insets in Fig. 7). The binding constants for L and MoL 2 were about 1.7 × 10 3 and 1.9 × 10 3 M 1 , respectively.

Docking study

Conclusions

The DNA-and BSA-binding of an asymmetric Schiff base ligand derived from allylamine and 2, 3-dihydroxybenzaldehyde and its molybdenum (VI) complex are described. These compounds had been previously synthesized in our research group [START_REF] Amiri Rudbari | New asymmetric Schiff base ligand derived from allylamine and 2,3-dihydroxybenzaldehyde and its molybdenum(VI) complex: Synthesis, characterization, crystal structures, computational studies and antibacterial activity together with synergistic effect against Pseudomonas aeroginosa PTTC 1570[END_REF] and were resynthesized using the same procedure. The DNA binding results indicate that the both compounds interact with DNA, presumably by the groove binding mechanism; the Mo(VI) complex exhibited stronger DNA binding affinity compared to the Schiff base ligand. The BSA-binding of the synthesized compounds were also evaluated, using experimental (fluorescence quenching and UV-Vis spectroscopy) as well as computational methods (molecular docking). The obtained results indicated that the Mo(VI) complex also bound BSA stronger than the Schiff base ligand. The calculated binding constants between these compounds and BSA were about 10 4 M 1 . The results of the fluorescence experiment as well as the changes in the absorption spectra of the compounds upon addition of the BSA show that the BSA-compound complexes formed in the ground state.

In addition, molecular docking studies revealed that hydrogen bond interactions, hydrophobic interactions --cation interactions have the dominant roles in the interaction of these compounds with BSA. Generally, the results of the present study exhibited the effect of the metal ion on the DNA-and BSA-binding of the drugs and their values of binding constants.

MoL 2

 2 solutions. All intensities were corrected for the dilution effect in the UV absorption experiments. 2.3. DNA binding studies 2.3.1. Preparation of compounds and Fish Sperm DNA (FS-DNA) stock solutions

3. 1 . 3 .

 13 Energy transfer from BSA to compounds.Knowledge of the energy transfer between the compounds and BSA can provide valuable information about the BSA-complex binding. The change in the fluorescence quenching of BSA upon its binding to L and MoL 2 can be indicative of energy transfer between BSA and the compounds. This energy transfer can be explained by the fluorescence resonance energy transfer (FRET) theory. FRET, also known as Förster s resonance energy transfer; it is the result of an interaction between the excited molecule and its adjacent molecule. Upon this interaction, the absorbed energy by the donor molecule is transferred to an acceptor[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]. According to this theory, three conditions are required for energy transfer: (1) the donor should have fluorescence, (2) the fluorescence emission spectrum of the donor and the UV-Vis spectrum of the acceptor should have sufficient overlap (3) a small distance between donor and acceptor (< 8 nm)[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]. The distance (r) and efficiency of energy transfer (E) between the tryptophan residue of the protein (BSA)

  intensity of the donor in the absence of the acceptor at wavelength K 2 = 2/3, N = 1.336 and = 0.15 for BSA. Fig.4revealed that there is sufficient overlap between emission spectrum of BSA and absorption spectrum of the compounds. Therefore, according to Eqs.[START_REF] Ghosh | Synthesis, crystal structure and antibacterial activities of mixed ligand copper(II) and cobalt(II) complexes of a NNS Schiff base[END_REF][START_REF] Pravin | Investigation of in vitro anticancer and DNA strap interactions in live cells using carboplatin type Cu(II) and Zn(II) metalloinsertors[END_REF][START_REF] Arjmand | Synthesis of new chiral heterocyclic Schiff base modulated Cu(II)/Zn(II) complexes: Their comparative binding studies with CT-DNA, mononucleotides and cleavage activity[END_REF]

  E] as the yintercept (Figures are not shown).The K b values reveal that the MoL 2 formed a more stable complex with FS-DNA than L.

  apparent extinction coefficient, the extinction coefficient for free compounds and the extinction coefficient for the compound in the fully bound form, f was determined based on the a is the ratio of A obs to [compound]. af ) versus 1/[DNA] gives K b as the ratio of the y-intercept to slope

  Molecular docking studies were carried out to obtain a deeper insight into the DNA binding of L and MoL 2 . The docking results revealed that L and MoL 2 were bound to the minor groove of DNA. Figures8a and 8bpresent the binding modes and the nucleotides around MoL 2 and L, respectively. The molecular docking results show hydrogen bond interactions for L and MoL 2 in the minor groove of DNA. The standard binding free G°), describing the affinity of the L and MoL 2 for binding to DNA with the best scores, were -5.50 and -8.77 kcal.mol 1 , respectively. The docking results were in good agreement with the spectroscopic results. Both the experimental results and computational docking data, collectively, suggested that the MoL 2 complex had more DNA binding affinity than the L Schiff base ligand.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Chemical structure of (a) MOL 2 , (b) L.

Fig. 3 .

 3 Fig. 3. Electronic absorption spectra of BSA (10 mM) in the absence and presence of various concentrations of (a) MoL 2 and (b) L.

Fig. 4 .

 4 Fig. 4. Spectral overlap of the BSA fluorescence with the (a) MoL 2 and (b) L absorption spectrum.

Fig. 5 .

 5 Fig. 5. The docking diagrams of the BSA-compound complexes. a) MoL 2 and b) L. Mo atom, Hydrogen bond --interactions are shown as blue circle, green spheres, yellow cones and yellow cylinders, respectively.

Fig. 6 .

 6 Fig. 6. Fluorescence enhancement of EtBr-DNA complex in the presence of various amounts of (a) MoL 2 , (b) L.

Fig. 7 .

 7 Fig. 7. Electronic absorption spectra of (a) MoL 2 and (b) L in the absence and presence of various concentrations of DNA.

Fig. 8 .

 8 Fig. 8. The docking diagrams of the DNA-compound complexes. a) MoL 2 and b) L. Mo atom and Hydrogen bond interactions are shown as blue circle and green spheres, respectively.

Table 1 .

 1 BSA-binding constants, binding energy, number of binding site (n), and Stern--Volmer constant

	Type of Complex	K binding /M -1 (Fluorescence)	K binding /M -1 (UV-Vis)	Binding Energy/kcal.mol -1 (Docking)	n	K sv /M -1	k q /M -1 S -1
	L	1.01 × 10 4	1.4 × 10 3	-5.89	0.8671	6.2 × 10 3	6.2 × 10 11
	MoL 2	3.4 × 10 4	2.9 × 10 3	-7.05	1.02	4.1 × 10 4	4.1 × 10 12
	(K SV ) of the compounds.

Table 2 .

 2 FRET theory results for the compounds.

	Type of Complex	R 0 (nm)	r (nm)	E
	L	0.4682	0.4495	0.5608
	MoL 2	0.5050	0.4004	0.8011

Table 3 .

 3 DNA-binding constants, binding energy and the enhancement constant (K E ) of the compounds.

	Type of Complex	K binding /M 1 (Fluorescence)	K binding /M 1 (UV-Vis)	Binding (Docking) Energy/kcal.mol 1	K E /µM 1
	L	9.6 × 10 3	1.7 × 10 3	-5.50	0.0705
	MoL 2	1.5 × 10 5	1.9 × 10 3	-8.77	0.023

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Acknowledgement

The authors are grateful to the Research Council of the Islamic Azad University (Qeshm branch) for financial support of this work.