Zahra Kazemi

Amiri Hadi

Mehdi Rudbari

Valiollah Sahihi

Majid Mirkhani

Shahram Moghadam

Iraj Tangestaninejad

Sajjad Mohammadpoor-Baltork

Gharaghani

Hadi Amiri Rudbari

Mehdi Sahihi

Valiollah Mirkhani

Majid Moghadam

Shahram Tangestani

Iraj Mohammadpoor-Baltork

Sajjad Gharaghani

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

Keywords:

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

Introduction

The compounds containing azometine group (Schiff base) are of compounds that fascinate a large number of chemists and biochemists [START_REF] Ferlay | Cancer Incidence and MortalityWorldwide[END_REF], [START_REF] Chow | Discovery and investigation of anticancer ruthenium-arene Schiffbase complexes via water-promoted combinatorial threecomponent assembly[END_REF], [START_REF] Chen | Synthesis and biological evaluation of hydroxyl-substituted Schiff-bases containing ferrocenyl moieties[END_REF] due to their easy formation, high stability, their pharmacological properties and notably anticancer activity [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]. The researches have revealed that the bioactivity of many ligands significantly enhances by complexation to a metal ion.

Transition metal complexes can possess various molecular geometries by changing coordination number that is not achievable for purely organic molecule. Their tunable properties such as oxidation states are another advantage [START_REF] Chow | Discovery and investigation of anticancer ruthenium-arene Schiffbase complexes via water-promoted combinatorial threecomponent assembly[END_REF]. Furthermore, since discovery of Platinumbased anticancer drugs (cisplatin, carboplatin and oxaliplatin) as potent chemotherapeutic agents and then diagnose their drawbacks (drug resistance, normal tissue toxicity, nephrotoxicity and neurotoxicity) a growing interest in synthesis non-platinum metal complexes as a drug has been revealed [START_REF] Carreira | Cytotoxic hydrophilic iminophosphorane coordination compounds of d 8 metals. Studies of their interactions with DNA and HSA[END_REF], [START_REF] Yan | Cytotoxic palladium(II) complexes of 8-aminoquinoline derivatives and the interaction with human serum albumin[END_REF].

Some metal ions such as Copper [START_REF] Joseph | Syntheses, crystal structure and biological evaluation of Schiff bases and copper complexes derived from 4-formylpyrazolone[END_REF], Palladium [START_REF] Chu | Synthesis, cytotoxicity and DNA-binding properties of Pd (II), Cu (II) and Zn (II) complexes with 4′-(4-(2-(piperidin-1-yl) ethoxy) phenyl)-2, 2′: 6′, 2″-terpyridine[END_REF], Nickel [START_REF] Chellaian | Spectral characterization, electrochemical and anticancer studies on some metal (II) complexes containing tridentate quinoxaline Schiff base[END_REF] and Vanadium [START_REF] Correia | Hydroxyquinoline derived vanadium (IV and V) and copper (II) complexes as potential anti-tuberculosis and anti-tumor agents[END_REF] have significant biological activity and have been investigated by some workers over the years. Because of: a) remarkable analogy between Pd(II) and Pt(II) complexes [START_REF] Shoukry | Synthesis, DNA binding and complex formation reactions of 3-amino-5, 6-dimethyl-1, 2, 4-triazine with Pd (II) and some selected biorelevant ligands[END_REF]; Palladium complexes are regarded as potential anticancer substances that in some cases they have better anticancer activity than their Pt(II) analogue [START_REF] Kapdi | Anti-cancer palladium complexes: a focus on PdX2L2, palladacycles and related complexes[END_REF]. b) Less toxicity and higher activity of Cu(II) and Pd(II) complexes in compared to platinum ones. Therefore, Cu(II) complexes are known as good alternative to cisplatin. The significance of Copper in some biological processes and the reliance of catalytic activity of many enzymes on it causes Cu(II) complexes have attracted much attention [START_REF] Kapdi | Anti-cancer palladium complexes: a focus on PdX2L2, palladacycles and related complexes[END_REF], [START_REF] Ramakrishnan | Interaction of rac-[M (diimine)3]2+ (M=Co, Ni) complexes with CT DNA: role of 5, 6-dmp ligand on DNA binding and cleavage and cytotoxicity[END_REF]. c) Promising biological activities of these metal complexes [START_REF] Kyropoulou | Ni (II) complexes with non-steroidal anti-inflammatory drug diclofenac: structure and interaction with DNA and albumins[END_REF], [START_REF] Gao | Four relatedmixed-ligand nickel (ii) complexes: effect of steric encumbrance on the structure, DNA/BSA binding, DNA cleavage and cytotoxicity[END_REF], [START_REF] Ramachandran | Synthesis, X-ray crystal structure, DNA binding, antioxidant and cytotoxicity studies of Ni (II) and Pd (II) thiosemicarbazone complexes[END_REF] d) novel action mechanism of vanadium by the generation of reactive oxygen species (ROS); Vanadium is capable of increasing the ROS level, promoting apoptosis of cells and inhibiting tumour cell proliferation.

Therefore, the cell growth and spread of tumour is suppressed [START_REF] Dash | Syntheses and structural investigation of some alkali metal ion-mediated LVVO2 -(L2-= tridentate ONO ligands) species: DNA binding, photo-induced DNA cleavage and cytotoxic activities[END_REF]. e) Less investigation of interaction of Ni(II) and V(IV) compounds with biomacromolecules in compared to other metal ions such as Pd(II), Zn(II) and Cu(II), despite the fact that they play vital role in biological systems.

Hence, it is essential to synthesis and study the affinity of their complexes to some biomacromolecules.

The knowledge about interaction mechanisms of drug with plasma proteins as a biomacromolecule is required to understand pharmacodynamics and pharmacokinetics of drugs [START_REF] Cui | Interaction of anthracycline disaccharide with human serum albumin: investigation by fluorescence spectroscopic technique and modeling studies[END_REF]. As regards function of plasma proteins as drug carriers, there are a large number of literatures about binding of drugs to them [START_REF] Cui | Specific interaction of 4′-O-(aLCladinosyl) 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 s[END_REF]. Consequently, the interaction of plasma proteins with metal complexes as a drug candidate is worthy of evaluation.

Generally, proteins are known as the major targets for most of the drugs in organisms [START_REF] Lu | Interaction of human serum albuminwith N-(4-ethoxyphenyl)-N′-(4-antipyrinyl) thiourea using spectroscopies and molecular modeling method[END_REF]. Their interaction with drug affects on the drug absorption, distribution and elimination in the circulatory system [START_REF] Mccallum | A fluorescence-based high throughput assay for the determination of small molecule-human serum albumin protein binding[END_REF], and can prevent their rapid elimination from 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 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 drug can increase its solubility in plasma, decrease its toxicity, protect from oxidation, prolong its in vivo half-life as well as increase the pharmaceutical effect of drug [START_REF] Gou | Enhancing the copper (II) complexes cytotoxicity to cancer cells through bound to human serum albumin[END_REF]. Fig. 1 shows the way of HSA-binding of a metal complex: This interaction can be occurred through the donor atoms of ligands (the first coordination sphere), or with more remote located groups on the ligands (the second coordination sphere) [START_REF] Davies | Supramolecular interactions between functional metal complexes and proteins[END_REF]. In order to obtain information about HSA-binding of drugs ongoing development of accurate computational methods has been observed. Generally the researchers can receive reliable information such as geometries, energies and electronic properties using quantum mechanical (QM) methods. However, the use of purely QM method to calculate the structure of macromolecules is extremely time consuming. Many efforts of researchers led to initiation of hybrid methods and then QM/MM method. Despite the fact that HSA-binding study of drugs with both docking and ONIOM (our own N-layered Integrated molecular Orbital and molecular Mechanics) is feasible, the use of them are rarely seen in literatures [28].

In the current study, V(IV), Ni(II) Cu(II) and Pd(II) ions have been selected for synthesis of new metal complexes. Aware of the therapeutic activity of Schiff base metal complexes and in attempt to obtain more insight into their biological activity, the HSA-binding of the synthesized compounds have been investigated. The experimental methods including fluorescence quenching and UV-vis spectroscopies have been used to evaluate binding behaviour of the synthesized metal complexes to HSA, quantitatively. Secondary structural changes of HSA have been studied using circular dichroism (CD). In addition, to find out appropriate orientation of them into the binding site of HSA, two powerful computational methods, molecular docking and ONIOM have been used.

Experimental

Chemicals and Instrumentation

Human serum albumin (HSA) was purchased from Sigma-Aldrich. 2-Hydroxy-1-naphtaldehhyde and allylamine were obtained from Merck Co. and used without further purification. All salts used for buffer preparation were analytical grade and dissolved in double distilled water. 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 ) in KBr pellets. 1 H and 13 C NMR spectra were recorded on a Bruker Avance 400 spectrometer using DMSO as a solvent for the Palladium(II) complex. 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 circular dichroism (CD) spectra were recorded using Aviv spectropolarimeter model 215 (Proterion Corp., USA) at 25 °C. The scan speed was 20 nm min -1 . The CD spectra were recorded in the far-UV region (190-260 nm) using 0.1 cm path length cell.

Preparation of Complexes

General Synthesis

Methanolic solution (10 ml) of 2-hydroxy-1-naphtaldehyde (5 mmol) was added dropwise to a methanolic solution (10 ml) of allylamine (5 mmol). After continuously stirred for 2 h in ambient temperature, solution of triethylamine (7 mmol) in absolute methanol (5 ml) was added to the solution. The mixture was stirred for 10 min, and then a solution of appropriate metal salt (2.5 mmol NiCl2•6H2O, CuCl2•2H2O, PdCl2 and VO(acac)2 salts) in absolute methanol (20 cm 3 ) was added. The resulting solution was stirred for 2 h at ambient temperature for the preparation of VOL2, NiL2 and CuL2 complexes, while for Palladium(II) complex, the solution was refluxed overnight to proceed completely.

Copper(II) Complex CuL2

Dark green crystals were collected by filtration. The green flat crystals were obtained in chloroform by recrystallization. The typical yield was 87%. Anal. calc. for C28H24CuN2O2: C: 69.48, H: 5.0, N: 5.79. Found: C: 69.33, H: 4.67, N: 5.28. Selected IR data (KBr, cm -1 ): 1611 (νC N), 1459 (νC N), 1365 (νC O).

Palladium(II) Complex PdL2

The resulting green powder was isolated by filtration and washed by dry methanol several times.

Then an appropriate single crystal for X-ray crystallography was obtained by dissolving in DMSO and diethylether diffusion.

Nickel(II) Complex NiL2

The green powder was isolated from the solution and purified by washing with methanol. Then the precipitate was dried at room temperature.

Vanadium (II) Complex VOL2

The resultant clear yellow solution was allowed to stand overnight. After concentration at room temperature, dark yellow precipitate was collected by filtration. Appropriate single crystals for Xray crystallography were obtained directly from the reaction mixture.

Single Crystal Diffraction Studies

X-ray data for VOL2, NiL2, CuL2 and PdL2 complexes were collected on a STOE IPDS-II diffractometer with graphite monochromated Mo-Kα radiation. Suitable crystal was chosen using a polarizing microscope and they were mounted on a glass fiber which was used for data collection. Cell constants and an orientation matrix for data collection were obtained by leastsquares refinement of diffraction data. Data were collected at a temperature of 298(2) in a series of ω scans in 1° oscillations and integrated using the Stoe X-AREA [START_REF] Cie | Programfor the Acquisition and Analysis of Data XeAREA[END_REF] software package. A numerical absorption correction was applied using the X-RED [START_REF] Stoe&cie | Program for Data Reduction and Absorption Correction Xe[END_REF] and X-SHAPE [START_REF] Stoe&cie | Program for Crystal Optimization for Numerical Absorption Correction Xe[END_REF] software's.

The data were corrected for Lorentz and Polarizing effects. The structure was solved by direct methods using SIR2004. The non-hydrogen atoms were refined anisotropically by the full matrix least squares method on F2 using SHELXL [START_REF] Sheldrick | [END_REF]. All hydrogen atoms were added at ideal positions and constrained to ride on their parent atoms. Crystallographic data are listed in Table S1. Selected bond distances and angles are summarized in Table S2.

HSA Binding Experiments

Preparation of Compounds and Protein Solution

A stock solution of HSA was prepared by dissolving the desired amount of HSA in 50 mM phosphate buffer (pH = 7). The HSA stock solution was stored at 4 °C in the dark and used within 2 h. HSA concentration was determined by UV-vis spectrophotometry using the molar absorption coefficient 35,700 M -1 cm -1 at 278 nm [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]. The solution of the complexes was first prepared in DMSO, and then diluted suitably with phosphate buffer to required concentrations for all the experiments. The volume of DMSO never exceeded 1.5% (v/v), so the effect of DMSO is negligible. All the solutions were used freshly after preparation.

Fluorescence Spectroscopic Measurements

The interaction of HSA with the synthesized Schiff base complexes (V(II), Cu(II), Ni(II) and Pd(II) complexes) was investigated using fluorescence quenching experiment. In this experiment 2 ml of HSA solution (3 μM) was placed into the cell and various amount of the compounds (5 × 10 -5 M) were added to the cell. The fluorescence intensity was measured with excitation wavelength at 295 nm and emission wavelength rang of 300-450 nm. In each measurement the mixture was allowed to incubate for 2 min after addition of the complexes.

UV-vis Absorption Spectroscopy Measurements

Absorption titration experiment, an operational and very easy method, was also carried out to more investigation of HSA-binding of the compounds at room temperature. The UV-vis absorption spectra of the solution of HSA (5 μM) in the absence and presence of various amount of the compounds (5 × 10 -5 M) were recorded. Absorption curves of compounds-protein mixtures were corrected by subtracting the spectra of compounds solutions. All intensities were corrected for the dilution effect in fluorescence and UV absorption experiments.

Circular Dichroism Spectroscopy

The circular dichroism (CD) spectrum was recorded at room temperature in the range of 190-260 nm, using 0.1 cm quartz cell. The CD spectrum of HSA solution was recorded before and after addition of the synthesized compounds with molar ratio 0, 0.5, 1 and 2. The analysis CD spectra were carried out by CDNN software. The content of secondary conformation of HSA in each step was calculated from this technique. The spectra were obtained by subtracting the spectra of compounds as baseline.

Computational Details

Molecular Docking Simulation

In this work, docking study was carried out to indicate the HSA-binding site for the synthesized compounds. The 3D structures of the metal complexes were generated using the CIF files of their X-ray crystal structures. The CIF files were converted to the PDB format by using the Mercury software (http://www.ccdc.cam.ac.uk/). The 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, missing hydrogen atoms and Gasteiger charges were added.

Flexible-ligand docking was performed by AutoDock 4.2 molecular-docking program 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 charges were added to prepare the macromolecule input file for docking and the Auto Grid was used to calculate Grids. A blind docking with 126 lattice points along X, Y, and Z axes was performed to find the active site of ligands to the HSA. After determination of the active site, the dimensions of the grid map were selected 60 points 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.

Quantum Mechanical/Molecular Mechanics (QM/MM) Calculations

Present work investigates the interaction of the compounds with HSA accurately, using QM/MM calculations. In order 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, quantum mechanical method (QM) with a molecular mechanics (MM). Although this method was used as a two-layer QM/MM in this study, it is capable of combining any number of molecular orbital and molecular-mechanics methods and extending [START_REF] Dawson | Apoptosis induction in cancer cells by a novel analogue of 6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2naphthalenecarboxylic acid lacking retinoid receptor transcriptional activation activity[END_REF].

This method also is able to apply different ab initio or semi-empirical methods to different parts of a molecule/system and combine to produce reliable geometry and energy at reduced computational time [START_REF] Farrokhpour | A rare dihydroxo copper(II) complex with ciprofloxacin; a combined experimental and ONIOM computational study of the interaction of the complex with DNA and BSA[END_REF]. In the two layers ONIOM method, the total energy (EONIOM) of the whole system is a sum of the three independent energy calculations: [START_REF] Liao | Triaryl-substituted Schiffbases are high-affinity subtype-selective ligands for the estrogen receptor[END_REF] Real system contains full geometry of the molecule and is considered as MM layer while the model system contains the chemically most important (core) part of the system that is considered as QM layer. Furthermore, the stability of respective adducts was obtained by the evaluation the interaction energy (ΔE), which is calculated by the following equation:

where ΔEHSA/compound, ΔEHSA and ΔEcompound are the energy of the optimized adduct of compound-HSA, HSA receptor and compounds, respectively. A two layers QM/MM method was opted for all calculations. HSA is considered as low layer while the compounds are considered as high layer in the current work. Molecular mechanics method (UFF) and semi empirical method (PM6) were selected for the low layer and the high layer, respectively.

Result and Discussion

Synthesis and Characterization

The (Table 1).

The 1 H and 13 C NMR were run immediately after solution in DMSO and gave the expected simple spectra, indicating the integrity of the complexes. The spectra obtained after 12, 24 and 120 h were similar to the initial spectra indicating that it is stable in solution. The signal for the imine proton (CH N) in the Palladium(II) complex appears at 8.8 ppm. The aromatic protons were observed as multiplets in the aromatic region 7.1-8.2 ppm. No signal corresponded to hydroxyl protons suggested the hydroxyl group is fully deprotonated and the oxygen is most likely coordinated to the metal ion. The 13 C NMR spectrum shows 10 signals for PdL2 complex. The peak observed at 164.1 ppm is ascribed to the imine carbon atoms of PdL2 complex which supports the presence of the Schiff base in the complex [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].

Description of the Crystal Structures of Complexes

Structure Description of VOL2

An ORTEP view of VOL2 with the atom-numbering scheme is shown in Fig. 2 Fig. 2. Perspective view of VOL2 in the asymmetric unit showing the numbering scheme. Thermal ellipsoids are drawn at 30% probability level, while hydrogen size is arbitrary.

In this complex, the oxidovanadium(IV) unit is coordinated to two bidentate Schiff base ligands with the two nitrogen and two phenolate oxygen atoms in equatorial positions and one oxygen in the axial position to complete the distorted tetragonal pyramidal N2O3 coordination sphere.

The bond angles within the coordination sphere range from 84.53 [START_REF] Correia | Hydroxyquinoline derived vanadium (IV and V) and copper (II) complexes as potential anti-tuberculosis and anti-tumor agents[END_REF] [START_REF] Grivani | A new oxidovanadium (IV) complex containing an O, N-bidentate Schiff base ligand: synthesis, characterization, crystal structure determination, thermal study and catalytic activity for an oxidative bromination reaction[END_REF], [START_REF] Mahadevi | Cation-π interaction: its role and relevance in chemistry, biology, and material science[END_REF] and gives a strong absorption in the FT-IR spectrum at 977 cm -1 . The two Schiff base ligands coordinate to the vanadium centre in a trans geometry with respect to each other. The structural chemistry of small vanadium(IV) molecules is well-established, and, as with vanadium(V), the preponderance of five-coordinate vanadium(IV) complexes adopt a square pyramidal or distorted square pyramidal geometry. To quantitatively compare these structures in terms of their relative geometries about the vanadium atom, we have used the angular structural parameter, τ, which is defined in Scheme S1 [START_REF] Addison | Synthesis, structure, and spectroscopic properties of copper(II) compounds containing nitrogen-sulphur donor ligands; the crystal and molecular structure of aqua[1,7-bis(Nmethylbenzimidazol-2′-yl)-2,6-dithiaheptane]copper(II) perchlorate[END_REF]. For purely square pyramidal complexes τ is zero. Moving two trans ligands out of the equatorial square plane results in a distortion toward a "trigonal bipyramid" for which τ approaches 1 (τ = 1 for a perfect trigonal bipyramid). It should be noted that the symmetry change shown in Scheme S1 is C4ν to C2ν if all equatorial ligands are equivalent.

As reported in other papers [START_REF] Plass | Synthesis, reactivity, and structural characterization of dioxovanadium (V) complexes with tridentate Schiff base ligand: vanadium complexes in supramolecular networks[END_REF], [START_REF] Mahajan | Development of Pyrazolone and Isoxazol-5-one Cambinol Analogues as Sirtuin Inhibitors[END_REF], we can describe complexes with 0 ≤ τ ≤ 0.5 as distorted square pyramids and with 0. ] hydrogen bonds (Fig. S1). Additionally, the sum of the weak noncovalent interactions seems to play an important role in the crystal packing and the formation of the framework.

Structure Description of CuL2

An ORTEP view of CuL2 with the atom-numbering scheme is presented in Fig. 3 and the crystallographic data and selected bond lengths and angles are collected in Tables 2 andS1. The The Cu atom are located on the inversion centres and surrounded by the two N and the two O atoms of the symmetry related deprotonated 2-hydroxy-1-allyliminomethyl-naphthalen (L‾)

ligand. The four atoms in the equatorial planes around the Cu atom form slightly distorted squareplanar arrangements (Table S2).

The hydroxy-1-allyliminomethyl-naphthalen ligands are arranged with respect to each other in a trans orientation such that one allyl substituent lies above the [CuO2N2] plane and the other below. The Cu O distances of 1.880(2) Å and the Cu N bond lengths of 1.998(2) Å are similar to those seen in related complexes [START_REF] Poulter | Plasma deposited metal Schiff-base compounds as antimicrobials[END_REF]. For instance, distances of 1.902(2), 1.896(2) and 2.014(3), 2.003(3)Å for the Cu O and Cu N bonds, respectively, are found in a related copper(II) complex derived from allylamine and salicylaldehyde [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].

The C C distance in CuL2 (1.288 (4)) Å is well within the range expected for double bonds found in allyl derivatives. The C N bond distances is 1.291(4), which are consistent with a slight elongation of the C N double bond when coordinated to a late metal centre [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]. The allyl groups are centrosymmetric to one another but no significant interaction with a metal centre is observed, thereby allowing access for covalently bond on an insoluble support as heterogeneous catalysts [START_REF] Baleizão | Vanadyl salen complexes covalently anchored to single-wall carbon nanotubes as heterogeneous catalysts for the cyanosilylation of aldehydes[END_REF].

Although there are no classical hydrogen bonds in the structure, but four intramolecular C H ⋯ O contacts are observed in the solid state (Table S3). atoms (Fig. 3 and Table S3). In packing of CuL2, the supramolecular aggregations are stabilized by a combination of intermolecular π ⋯ π stacking interactions and M ⋯ π interactions. π-Stacking interactions between the π-electron clouds of aromatic systems have been extensively

observed in many fields such as chemistry [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] and biochemistry [START_REF] Baleizão | Vanadyl salen complexes covalently anchored to single-wall carbon nanotubes as heterogeneous catalysts for the cyanosilylation of aldehydes[END_REF]. They play an important role in the structures of biological macromolecules [START_REF] Mahadevi | Cation-π interaction: its role and relevance in chemistry, biology, and material science[END_REF] and are useful to study on the intercalation of drugs into DNA [46]. The nature of the π-electron is based on π ⋯ π stacking interactions by electron complementarity. The stability order of π ⋯ π stacking is π-electron-deficient-π-electrondeficient > π-electron-deficient-π-electron-rich > π-electron-rich-π-electron-rich [START_REF] Ni | Synthesis, structures and characterization of a series of Cu (i)-diimine complexes with labile N, N′-bis ((diphenylphosphino) methyl) naphthalene-1, 5-diamine: diverse structures directed by π-π stacking interactions[END_REF]. So, the enlargement of π-conjugated systems or polarized perturbation to π-system could change the intermolecular arrangement dramatically. In infinitely π-stacking complexes, solid-state properties such as electrical conductivity depend on the influence of infinite π ⋯ π forces [START_REF] Kar | A unique example of structural diversity tuned by apparently innocent o-, m-, and p-nitro substituents of benzoate in their complexes ofMn (II) with 4, 4′-Bipyridine: 1D ladder, 2D sheet, and 3D framework[END_REF]. 9) rings, respectively. As depicted in Fig. S2, the π-stacking between aromatic rings have paralleldisplaced configurations. The preference parallel-displaced geometry for a π ⋯ π interaction can be understood when keeping in mind the fact that a parallel face-to-face geometry for aromatic rings is not necessarily attractive [START_REF] Sinnokrot | Substituent effects in π-π interactions: sandwich and Tshaped configurations[END_REF]. Such a parallel or a parallel-displaced π-stacking with a ring separation between 3.3 and 3.8 Å is an important non-covalent organization possibility in supramolecular chemistry [START_REF] Janiak | A critical account on π-π stacking in metal complexes with aromatic nitrogen-containing ligands[END_REF]. Besides these π ⋯ π interactions, there also exists Cu ⋯ π interactions between the metal centre of one complex molecule and the phenyl ring of the deprotonated 2-hydroxy-1-allyliminomethyl-naphthalen (L -) ligand of the other two complex molecules (Fig. S3). This extended π ⋯ π and Cu ⋯ π interaction seems to be responsible for holding the crystal together. It may be relevant to note here that such non-covalent interactions are of significant importance in molecular recognition processes as well as in crystal engineering [START_REF] Burley | Aromatic-aromatic interactions in and around alpha helices[END_REF].

Structure Description of NiL2

The molecular structure of NiL2 is shown in Fig. 4. Selected bond distances and angles are listed in Table S2. S2). S1). As depicted in Fig. S4 (Scheme S2), in the crystal packing of NiL2, adjacent molecules are assembled through π-π stacking interaction. Centroid-centroid distances between two rings are 3.6992(15) and 3.9275 ( 16) Å, a typical distance for systems held by π ⋯ π stacking interactions [START_REF] Hunter | The nature of pi-pi interactions[END_REF]. Also, similar to CuL2 complex, there is M ⋯ π interaction in the crystal packing of NiL2 complex (Fig. S5). Despite the M ⋯ π interactions are probably weak; however, they are very import in stabilizing the polymeric structure of the complexes. As you can see in Figs. S4 and S5, crystal structure of NiL2 contains 1D arrays constructed by the intermolecular π ⋯ π and M ⋯ π interactions. These interactions are responsible for holding the crystal together.

Structure Description of PdL2

An ORTEP view of PdL2 with the atom-numbering scheme is presented in Fig. 5 and the crystallographic data and selected bond lengths and angles are collected in Tables 2 andS1. The (2)°] are close to 90°. Another interesting feature of the PdL2 complex is the presence of interaction between nitrogen atom and hydrogen of allyl group (C14 H14A ⋯ N1 [2.575( 5) Å]). Recently, we investigated this kind of interaction in Schiff base ligand derived from allylamine and 2,3-dihydroxybenzaldehyde and its Mo(VI) complex [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]. We suggest the use of pseudo-scorpionate term for this kind of ligand in compliance with the scorpionate ligand exploited for tridentate ligand which would bind to a metal in a fac manner [START_REF] Ponzano | Synthesis, biological evaluation, and 3D QSAR study of 2-methyl-4-oxo-3-oxetanylcarbamic acid esters as N-acylethanolamine acid amidase (NAAA) inhibitors[END_REF].

In order to understanding the nature of scorpion-like intramolecular interaction in the solid state of our reported Schiff base ligand, 3-((allylimino)methyl)benzene-1,2-diol, we have performed a series of ab-initio calculation [START_REF] Rudbari | Newasymmetric 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 aeruginosa PTTC 1570[END_REF]. The energy of the whole rotation around the N1-C8-C9-C10

(θ) torsion angle for one of the two molecules in the asymmetric unit was calculated. During the scan of 360° around θ we observed a series of maximum and minimum points. From the calculated result, we understood that the scorpion like interaction energy trend between hydrogen atom H10A and nitrogen atom N1 appears to be a subtle balance between the H10A ⋯ N1 and H9 ⋯ N1 [60], [START_REF] Blackburn | Nickel (II) and Palladium (II) complexes of azobenzene-containing ligands as dichroic dyes[END_REF], [START_REF] Hall | Structural investigations of palladium (II) and platinum (II) complexes of salicylhydroxamic acid[END_REF], [START_REF] Tsuno | Synthesis and structural characterization of isomeric palladium (II) complexes with chiral N, O-bidentate ligands[END_REF]), step configuration (with the distance between mean planes of the aromatic rings greater than d = 0.4 Å) (See for example

Refs. [START_REF] Gan | Effect of ortho-substituents on the stereochemistry of 2-(o-substituted phenyl)-1H-imidazoline-palladium complexes[END_REF]), bowl configuration (See for example Refs. [START_REF] Maverick | Flexible cofacial binuclear metal complexes derived from α, α-bis (salicylimino)-m-xylene[END_REF], [START_REF] Caruso | Facile synthesis of new Pd (II) and Cu (II) based metallomesogens from ligands containing thiophene rings[END_REF]). In the complexes Pd(N,O)2 with planar configuration the Pd atom lies in the plane of the chelate rings (See for example

Refs. [START_REF] Bahron | Bis {2methoxy-6-[(E)-(4-methylbenzyl) iminomethyl] phenolato} palladium (II) chloroform monosolvate[END_REF], [START_REF] Blackburn | Nickel (II) and Palladium (II) complexes of azobenzene-containing ligands as dichroic dyes[END_REF], [START_REF] Hall | Structural investigations of palladium (II) and platinum (II) complexes of salicylhydroxamic acid[END_REF], [START_REF] Tsuno | Synthesis and structural characterization of isomeric palladium (II) complexes with chiral N, O-bidentate ligands[END_REF]). The two six-membered chelate rings and their annulated phenyl rings are coplanar (Scheme S3a). The complexes have inversion symmetry. Frequently, however, a characteristic feature of Pd(N,O)2 complexes is an envelope configuration of the six-membered chelate rings with the Pd atom deviating from the plane of the other 5 atoms of the chelate rings.

The direct consequence is the alternative of a step or a bowl configuration (Scheme S3b and c). In the step configuration the planes between the aromatic rings are parallel to each other (See for example Refs. [START_REF] Gan | Effect of ortho-substituents on the stereochemistry of 2-(o-substituted phenyl)-1H-imidazoline-palladium complexes[END_REF]). The complexes still have Ci symmetry. Some Pd(II) complexes adopt a bowl configuration (See for example Refs. [START_REF] Maverick | Flexible cofacial binuclear metal complexes derived from α, α-bis (salicylimino)-m-xylene[END_REF], [START_REF] Caruso | Facile synthesis of new Pd (II) and Cu (II) based metallomesogens from ligands containing thiophene rings[END_REF]). At best they have C2 symmetry. According to the above description, the PdL2 complex has planner configuration. As we said in the above sentences, salicylidenimine palladium(II) complexes with planar configuration have inversion centre. But our palladium(II) complex, PdL2, is the first structure that have planner configuration without inversion centre. Orientation of the two allyl substituents in one side of the [MO2N2] plane is the reason of lack of inversion centre in structure PdL2 of complex.

Interaction With Human Serum Albumin (HSA)

UV-vis Absorption

The UV absorption spectrum of HSA shows a strong absorption peak at about 220 nm resulted of the n → π* transition for the peptide bond of α-helix and a weak peak at 278 nm assigned to π → π* transition of the phenyl rings in aromatic acid residues (Trp, Tyr and Phe) [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]. Through addition of various amount of the Schiff base metal complexes (5 × 10 -5 M), a hypochromic effect in spectrum of HSA is observed (Fig. 6). This observation indicates that the aromatic amino acid residues of HSA in a hydrophobic cavity were exposed to an aqueous upon binding to the compounds. Moreover, observation of hypochromic effect in UV-vis spectra of HSA with increase amount of the synthesized compounds reveals the π-π stacking interaction between the aromatic rings of the compounds and the phenyl rings of aromatic acid residues [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]. In order to assess the binding ability of the title compounds with HSA, the intrinsic binding constant (Kb) was determined by monitoring the changes of absorbance with increasing concentration of the compounds and using Benesi-Hildebrand plot and the following equation: [START_REF] Das | Binding studies of aristololactam-β-D-glucoside and daunomycin to human serum albumin[END_REF], [START_REF] Lown | Molecular mechanism of binding of Pyrrolo(1,4)benzodiazepine antitumour agents to deoxyribonucleic acid[END_REF] where A0 and A are the absorbance of HSA in the absence and presence of drug, respectively.

Fluorescence Spectroscopy

Fluorescence quenching experiment has been performed to obtain information including binding mode, binding constants, number of binding sites and intermolecular distances [START_REF] Mrkalić | Synthesis of novel palladium (II) complexes with oxalic acid diamide derivatives and their interaction with nucleosides and proteins. Structural, solution, and computational study[END_REF]. Although fluorescence of HSA protein 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 [START_REF] Ganeshpandian | New ruthenium (II) arene complexes of anthracenyl-appended diazacycloalkanes: effect of ligand intercalation and hydrophobicity on DNA and protein binding and cleavage and cytotoxicity[END_REF].

The fluorescence intensity of protein was quenched through the addition of the synthesized compounds. This implies that the compounds strongly interact with HSA, leading to changes of microenvironment around the Trp-214 residue in HSA [START_REF] Mrkalić | Synthesis of novel palladium (II) complexes with oxalic acid diamide derivatives and their interaction with nucleosides and proteins. Structural, solution, and computational study[END_REF], [START_REF] Wang | Interaction of the flavonoid hesperidin with bovine serum albumin: a fluorescence quenching study[END_REF]. Fig. 7 represents the fluorescence quenching of 3 × 10 -6 M HSA at the presence of various amount of the compounds (5 × 10 -5 M). The absorbance at excitation wavelengths was always < 0.1, in order to avoid inner filter effects.

In order to determine the binding ability between the compounds and HSA, the Stern-Volmer quenching plot was obtained by monitoring the fluorescence quenching of HSA with increasing the concentration of the compounds according to the Stern-Volmer equation: [START_REF] Mrkalić | Synthesis of novel palladium (II) complexes with oxalic acid diamide derivatives and their interaction with nucleosides and proteins. Structural, solution, and computational study[END_REF] where, F0 and F are the fluorescence intensity of HSA in absence and presence of the compounds. Ksv is the Stern-Volmer quenching constant, kq is the quenching rate constant of HSA and τ is the average lifetime of HSA without quencher which is typically equal to 10 -8 s for biomacromolecules [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]. Ksv is determined from the plot of F0/F vs. [Q] (Fig. 8). In the present study, the values of Ksv were obtained and presented in Table 2.

Fig. 8. Stern-Volmer plot for the synthesized compounds-HSA system and the number of binding sites on HAS at room temperature.

Fluorescence quenching is classified to two mechanisms: static quenching and dynamic quenching. In the static mechanism, the fluorophore and the quencher collide together in the ground state while fluorophore and quencher collide together in the excited state in dynamic mechanism. Linearity of the Stern-Volmer plot indicates that quenching fluorescence has only one mechanism, dynamic or static [START_REF] Frik | Cyclometalated iminophosphorane gold(III) and platinum(II) complexes. A highly permeable cationic platinum(II) compound with promising anticancer properties[END_REF].

In this study, the values of kq were obtained and represented in Table 2 that is greater than limiting diffusion rate constant of the diffusional quenching for biopolymers (2 × 10 10 M -1 S -1 ).

This observation supports that quenching fluorescence of HSA occurs by static mechanism [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].

In addition, the changes in the absorption spectrum of HSA shows the formation HSA-drug complex in the ground state, which is because of the change in the HSA structure. While dynamic quenching affects on the exited state and do not have impact on the absorption spectrum.

Moreover, the binding constant (Kb) have been determined using the following equation: [START_REF] Dighe | Synthesis of β-carboline-based N-heterocyclic carbenes and their antiproliferative and anti-metastatic activities against human breast cancer cells[END_REF] where, F0 and F are the fluorescence intensity of HSA in absence and presence of Schiff base complex, respectively. [Q] is the concentration of quencher that quencher is the synthesized compounds here. "Kb" is obtained from the plot of Ln((F0 -F)/F) versus Ln[Q] as a y-intercept.

Furthermore, "n" which is the number of binding site per protein is slope of the plot. The value of n is nearly 1, indicating that the synthesized compounds bind to HSA with molar ratio of 1:1.

The calculated results are shown in Table 2.

The Kb value reveals that the Pd-HSA complex is more stable than other compounds; in the other words this is more available for drug-cell interaction. Moreover, this result is in good agreement with the UV-vis spectroscopy result. In general, binding constant of a drug with a carrier protein such as HSA should be high enough to bind and transfer thought body. Moreover, in order to release a drug since arrival at its target, it should not be too high. With respect to above, HSAbinding constants of all the compounds are in a good range (2-10 × 10 4 ) [START_REF] Kyropoulou | Ni (II) complexes with non-steroidal anti-inflammatory drug diclofenac: structure and interaction with DNA and albumins[END_REF] and also are comparable to analogue compounds and some potent drugs (Table 3). In addition, our compounds are in the uncharged form which is an advantage for a drug. Because the researches show that the unionized drugs are soluble in lipid and capable of crossing through the membrane's lipid bilayer while the ionized analogue species fail to cross [START_REF] Abou-Zied | Revealing the ionization ability of binding site I of human serum albumin using 2-(2′-hydroxyphenyl) benzoxazole as a pH sensitive probe[END_REF]. The obtained results show that the title compounds with identical ligand and different metal centre bind to HSA differently, confirming the effect of metal ion on affinity of complexes to HSA.

Circular Dichroism (CD) Spectroscopy

CD spectral technique is very effective to diagnose alteration on the secondary structure of HSA causing from HSA-drug interaction and also to investigate the conformation of protein quantitatively. This spectroscopy was performed in the absence and presence of the synthesized metal complexes to prove the impact of them on the secondary structure of HSA. The CD spectra of HSA exhibits two negative peaks at 208 and 222 nm which are characteristic absorption peaks of α-helical structure of protein attributed to the n-π* transfer for the bonds of the α-helix [START_REF] Huang | Preparation of 2D hydroxylrich carbon nitride nanosheets for photocatalytic reduction of CO2[END_REF].

The band intensities of HSA decreased in negative ellipticity upon the binding of the synthesized complexes to HSA, which indicates the decrease of the α-helical content and increase of disorder structure and β-sheet contents of HSA. This is the result of the formation complex between HSA and the compounds. Moreover, after addition of these compounds the shape of the CD spectra was changed significantly. The results can be expressed in terms of the mean residue ellipticity (MRE) according to Eq. ( 6): [START_REF] Huang | Preparation of 2D hydroxylrich carbon nitride nanosheets for photocatalytic reduction of CO2[END_REF] where, Cp is the molar concentration of protein, l is the path length (0.1 cm), n is the number of amino acid residues that is 585 for HSA. The α-helical contents of free and combined HSA can be calculated from MRE values at 208 nm using the following equation:

where, MRE208 is the observed mean residue ellipticity value at 208 nm, 4000 is the MRE of the β-sheet and random coil conformation cross at 208, and 33,000 is the MRE value of the pure αhelix at 208 nm. The contents of different secondary structures in HSA are presented in Table 4.

As it can be seen, the percentage of α-helix of HSA decreased while those of random coil and βsheet increased. These results indicate that all the complexes bound to the amino acid residues of the main polypeptide chain of HSA that causes destroy the original hydrogen bonding networks [START_REF] Banerjee | Fluorescence resonance energy transfer: a promising tool for investigation of the interaction between 1anthracene sulphonate and serum albumins[END_REF]. According to the obtained results the maximum capacity of HSA is in molar ratio HSA/drug = 1 and the further addition of compounds caused only minor changes in the CD spectrum. The Fig. 9 represents the changes in the secondary structure of HSA upon addition of the complexes with molar ratio of HSA/complex = 1, indicating the HSA-PdL2 system has the maximum change that it is based on the magnitude of binding constant of HSA-complex. The obtained results from CD spectroscopy are in a good agreement with fluorescence and UV experiments. The existence of hydrogen bond between compounds with phenolic hydroxyl group and either C O or N H of the polypeptide chain leads to alter hydrogen bonding network and reduction of protein αhelix structure [START_REF] Xie | Interaction between hesperetin and human serum albumin revealed by spectroscopic methods[END_REF]. The utilized 2-hydroxy-1-naphtaldehyde in the synthesized metal complexes in this work raises the possibility of this type of hydrogen bond. This is in consistence with the docking results (see section docking study). The observation of considerably reduction in α-helix of HSA in the presence of the synthesized compounds can be as a result of hydrogen bond that can stabilize HSAcompound complex.

Energy Transfer From HSA to Compounds

Energy transfer between the compounds and HSA can provide valuable information about HSAcomplex binding. The fluorescence quenching of HSA upon its binding to metal complex can be indicative of energy transfer between HSA and metal complex. This energy transfer can be explained by fluorescence resonance energy transfer (FRET) theory. FRET also known as Förster's resonance energy transfer is an interaction between the excited molecule and its adjacent molecule. Upon this interaction, energy absorbed by donor molecule is transferred to an acceptor [START_REF] Fani | A combined spectroscopic, docking andmolecular dynamics simulation approach to probing binding of a Schiff base complex to human serum albumin[END_REF]. According to this theory, three conditions are required to energy transfer: (1) the donor should have fluorescence, (2) the fluorescence emission spectrum of the donor and the UV-vis spectrum of the acceptor should have sufficient overlap (3) the small distance between donor and acceptor (< 8 nm) [START_REF] Fani | A combined spectroscopic, docking andmolecular dynamics simulation approach to probing binding of a Schiff base complex to human serum albumin[END_REF]. The distance and efficiency of energy transfer (E) between tryptophan residue of protein (HSA) and drug (complex) has been calculated using this theory through the following equation:

where F0 and F are fluorescence intensities of HSA in the absence and presence of complex, respectively and R0 is the critical distance when the transfer efficiency is 50%; r is the distance between donor and acceptor. R0 can be calculated by Eq. ( 9) [START_REF] Huang | Preparation of 2D hydroxylrich carbon nitride nanosheets for photocatalytic reduction of CO2[END_REF].

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

where, F(λ) is the fluorescence intensity of the donor in the absence of the acceptor at wavelength λ and ε is the molar absorption coefficient of the acceptor at λ. In the present case, K 2 = 2/3, N = 1.336 and φ = 0.15 for HSA. Therefore, according to Eqs. ( 8), ( 9), [START_REF] Correia | Hydroxyquinoline derived vanadium (IV and V) and copper (II) complexes as potential anti-tuberculosis and anti-tumor agents[END_REF] the parameters for the synthesized compounds were calculated (Table 5). [HSA] = [PdL2] = 5 μM, λex = 295 nm.

Docking Study

It has been confirmed that the major factor affecting on the distribution, metabolism, the free concentration and the elimination rate of drugs is the binding affinity of drugs to HSA. Moreover, the half-life of drugs depends on their HSA-binding that affects on the dosage of drugs [START_REF] Ranjbar | Studies of the interaction between isoimperatorin and human serum albumin by multispectroscopic method: identification of possible binding site of the compound using esterase activity of the protein[END_REF].

Therefore the study of interaction between HSA and drug is essential to investigate the pharmacokinetics and availability of drugs in tissues. HSA as the most abundant carrier protein comprises three α-helical domains (I, II and III), each containing two subdomains (A and B) [START_REF] Zsila | Subdomain IB is the third major drug binding region of human serum albumin: toward the three-sites model[END_REF].

The previous crystal structure studies showed that many drugs bind to one of the two binding sites located in subdomains IIA and IIIA. However, these sites cannot be responsible for interaction with all drugs and a large number of endogenous and exogenous compounds bind to the D-shaped cavity in subdomain IB. Zsila reported the subdomain IB as the third major drug binding site on HSA [START_REF] Zsila | Subdomain IB is the third major drug binding region of human serum albumin: toward the three-sites model[END_REF]. The primary binding site for hemin, fusidic acid and sulphonamide derivative has been identified site IB. Moreover, researches demonstrated binding of some chemical compounds including dicoumarol, bile acids and biliverdin to this site. The researches revealed that anthracycline antitumor drugs such as doxorubicin and daunorubicin binds to subdomain IB [START_REF] Zsila | Subdomain IB is the third major drug binding region of human serum albumin: toward the three-sites model[END_REF], [START_REF] Zsila | Circular dichroism spectroscopic detection of ligand binding induced subdomain IB specific structural adjustment of human serum albumin[END_REF].

In this work, the synthesized compounds were docked to the crystal structure of HSA. The obtained results from molecular docking showed that the existence of metal in the compounds triggers the compounds bind to HSA in a suitable orientation and binding site. The appropriate insertion of Pd(II) complex in subdomain IB on HSA was observed with the highest value binding constant.

While the other compounds (VOL2, NiL2 and CuL2) binds to site IIA that is known as drug site 1. There are a large number of drugs that bind in this site such as warfarin, Amantadine, azapropazone and indomethacin [START_REF] Zsila | Subdomain IB is the third major drug binding region of human serum albumin: toward the three-sites model[END_REF]. The results indicated that GLN33, LYS106, TYR148 and ARG186 amino acid residues are involved in the hydrogen bond interaction in the HSA-compound systems. In all the VOL2-HSA, NiL2-HSA and CuL2-HSA systems TYR148 formed hydrogen bond. There is a bifurcated (or three centred) hydrogen bond interaction in the case of PdL2 complex between oxygen atom of Pd(II) complex and two hydrogen atom of ARG 186. It should be mentioned that proteins contain a large number of multifurcated hydrogen bonds [START_REF] Steiner | The hydrogen bond in the solid state[END_REF]. The binding energy for VOL2, NiL2, CuL2 and PdL2 are evaluated -9.57, -9.41, -9.24 and -7.52 kcal/mol, respectively. The larger negative value of binding energy for PdL2 means the higher affinity of it which can be as a result of the existence bifurcated hydrogen bond in PdL2-HSA system. This result is in a good agreement with experimental data (see related section). The results of docking are presented in Fig. 11, Fig. 12. Moreover, table S4 shows the adjacent amino acids with 3 Å distances to compounds.

QM/MM Calculation

In this 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 HSA (low layer) while semi-empirical quantum mechanics (QM) method (PM6) was opted for the compounds (high layer). The starting geometry of HSA-compounds for the two layer ONIOM study was obtained from the molecular docking simulation. The geometry was optimized using ONIOM calculation. All calculations were performed using Gaussian 09 quantum chemistry package. All calculations were performed while the structure of HSA and compounds were flexible. The partial atomic charges on the atoms of the compounds and HSA were considered to re-optimize the optimized geometry. In the structure optimization the van der Waals (vdw) and electrostatic interaction were considered. Moreover, the polarization of the wave function of the compounds was considered due to partial atomic charge of HSA in order to obtain more accurate structure [START_REF] Farrokhpour | A rare dihydroxo copper(II) complex with ciprofloxacin; a combined experimental and ONIOM computational study of the interaction of the complex with DNA and BSA[END_REF]. The results of ONIOM indicated that structure of the compounds deviate from the initial geometry due to the binding to HSA. Along with the interaction of the compounds with HSA some bond lengths and bond angles are decreased and/or increased. The existence of these changes can be indicative of the strength of the interaction between HSA and the compounds.

Fig. 1 .

 1 Fig. 1. Schematic illustration of non-covalent interactions of protein with a metal complex (Mn + = transition metal, D = donor atoms of exogenous ligands).

Scheme 1 .

 1 Scheme 1. Synthetic routes for the preparation of the complexes.

  5 ≤ τ ≤ 1 as distorted trigonal bipyramids. A search of the Cambridge Structural Database for monomeric five-coordinate vanadyl complexes with O/N donors yields > 100 structures. A few of them have distorted trigonal bipyramids structure, while most of them have distorted square pyramidal geometry. The paucity of trigonal bipyramids structures for vanadium(IV) suggests that there are electronic and/or steric requirements that preclude the formation of the trigonal bipyramids geometry. According to the above description, the geometry about the our synthesized oxovanadium(IV) Schiff base complex, VOL2, is best described as distorted square pyramidal with τ = 0.31. In the molecular packing of VOL2, three molecules are connected by intermolecular non-classical [C(17) H(17)⋯ O(3) [2.56(3) Å] C(12) H(12B)⋯ O(3) [2.51(3) Å]

XFig. 3 .

 3 Fig. 3. Perspective view of CuL2 constituted by the asymmetric unit (filled drawings) showing the numbering scheme and the centrosymmetric half part (empty drawings). Thermal ellipsoids are drawn at the 50% probability level, while the hydrogen size is arbitrary.

  But the design and synthesis of infinite π-stacking structures, especially supramolecular structures formed by different kinds of π-stacking interactions, are difficult. A selected view of the π ⋯ π interactions and bond parameters is shown in Fig. S2 (Scheme S2). The π ⋯ π interactions are exist between Cg(1)⋯ Cg(2), Cg(1)⋯ Cg(4) and Cg(2)⋯ Cg(2) with centroid-centroid distance of 3.8842(17), 3.6347(17) and 3.6684(17) Å, in which Cg(1), Cg(2) and Cg(4) are centres of Cu(1)/O(1)/C(1)/C(10)/C(11)/N(1);C(1)/C(2)/C(3)/C(4)/C(9)/C(10);C(4)/C(5)/C(6)/C(7)/C(8)/C(

Fig. 4 .

 4 Fig. 4. Perspective view of NiL2 constituted by the asymmetric unit (filled drawings) showing the numbering scheme and the centrosymmetric half part (empty drawings). Thermal ellipsoids are drawn at the 50% probability level, while the hydrogen size is arbitrary.

  Examination of the main metal-ligand distances shows that the Ni ⋯ N and Cu ⋯ N distances are longer than the Ni ⋯ O and Cu ⋯ O distance. The Ni ⋯ N and Ni ⋯ O distances are 1.993(2) and 1.8889(17) Å for NiL2 and the Cu ⋯ N and Cu ⋯ O distances are 1.998(2) and 1.880(2) Å for CuL2, respectively. Typical Ni ⋯ N and Cu ⋯ N distances range from 1.904 to 1.922 Å and from 1.939 to 2.019 Å, while the Ni ⋯ O and Cu ⋯ O distances range from 1.819 to 1.833 Å and from 1.879 to 1.893 Å, respectively [44], [52]. Similar to CuL2 complex, two non-classical intramolecular hydrogen bonds of the type C H ⋯ O are formed between the C H in the CH2 and CH groups as donors and O in the 2-hydroxy-1-naphthaldehyde moiety as acceptors (Fig. 4, Table

  crystallographic data reveal that the metal centre is four-coordinated by two phenolate oxygen and two imine nitrogen atoms of two Schiff base ligands. The geometry around the metal centre is slightly distorted square planer. The Schiff base ligands are arranged with respect to each other in a trans orientation such that two allyl substituents lies one side of the [MO2N2] plane. The Pd O distance [1.977(5) and 1.980(5) Å] and the Pd N bond length [1.988(6) and 2.012(5) Å] are similar to those seen in related complexes[START_REF] Coombs | Palladium (II) Schiff base complexes derived from sulfanilamides and aminobenzothiazoles[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]. For instance, distances of 1.987(3) Å for Pd O bond and 2.066(3) Å for Pd N bond are found in a related di-

Fig. 5 .

 5 Fig. 5. Perspective view of PdL2 constituted by the asymmetric unit (filled drawings) showing the numbering scheme and the centrosymmetric half part (empty drawings). Thermal ellipsoids are drawn at the 30% probability level, while the hydrogen size is arbitrary.

  interactions and the intramolecular hydrogen bonds O1 ⋯ H3 and O1 ⋯ H1. A search for salicylidenimine palladium(II) complexes with the Cambridge Structural Database System 2014 showed that except one case [59] the N,O-ligand atoms in bis(salicyli-denimine) palladium(II) complexes Pd(N,O)2 adopt a trans arrangement in the square planar Pd(II) configuration. As in salen-type complexes Pd(ONNO) the N,O-ligand atoms are cis to each other, they are excluded from the following analysis. The trans-Pd(N,O)2 complexes can subdivide into three classes (Scheme S3): planar configuration (with the distance between mean planes of the aromatic rings less than d = 0.4 Å) (See for example Refs.

Fig. 6 .

 6 Fig. 6. Electronic absorption spectra of HSA (5 μM) in the absence and presence of various concentrations of (a) VOL2, (b) NiL2, (c) CuL2 and (d) PdL2 ([compounds] = 0-25 μM).

Fig. 7 .

 7 Fig. 7. Changes in the fluorescence spectra of HSA in the presence of (a) VOL2, (b) NiL2, (c) CuL2(d) PdL2. [HSA] = 3 μM, [compound] = 0-25 μM, λex = 295 nm.

Fig. 9 .

 9 Fig. 9. The CD spectrum of HSA in the absence and presence of the complexes in the molar ratio [Complex]/[HSA] = 1.

  Fig. 10 represents the overlap of the fluorescence emission spectrum of HSA (5 μM) and the UV-vis spectrum of PdL2 (5 μM). The related Figs. to VOL2, NiL2 and CuL2 are shown as Figs. S6 to S8, respectively. The valuesof r for all the compounds are < 8 nm and 0.5 R0 < r < 1.5 R0, suggesting energy transfer from HSA to the Schiff base metal complexes occurs with high probability. Furthermore, the higher values of r than R0, indicating the presence of a static quenching mechanism in the interaction of HSA and compounds[START_REF] Huang | Preparation of 2D hydroxylrich carbon nitride nanosheets for photocatalytic reduction of CO2[END_REF].

Fig. 10 .

 10 Fig. 10. Spectral overlap of the HSA fluorescence with the PdL2 complex absorption spectrum.

Fig. 11 .

 11 Fig. 11. The docking pose of the synthesized compound-HSA.

Fig. 12 .

 12 Fig. 12. The docking pose of the synthesized compound-HSA.

Fig. 13 shows

 13 Fig.13shows the changes take place in some geometrical parameters including bond lengths and/or bond angles of the compounds along with binding to HSA. The calculated binding energies of the compounds and HSA (ΔE) are about -1.211 × 10 3 , -1.205 × 10 3 , -1.259 × 10 3 and

  13

Table 1 .

 1

	13 C NMR

1

H NMR and

13

C NMR data for PdL2.

1

H NMR

Table 2 .

 2 The HSA binding constant (Kb), the number of binding sites (n), the Stern-Volmer constant (Ksv) of the compounds and the quenching rate constant of HSA (kq).

	Compound	Kb/M -1	n	Ksv/M -1	kq/M -1 S -1
	VOL2	5.8 × 10 4	0.97	5.0 × 10 4	5.0 × 10 12

Table 3 .

 3 The value of Kb of some metal complexes and potent drugs.

	Compound

Table 4 .

 4 Changes in the secondary structure of HSA upon its interaction with the synthesized metal

	complexes in different concentration (R = [compound]/[HSA]).
	R	HSA HSA + VOL2	HSA + CuL2	HSA + NiL2		HSA + PdL2
		0	0.5 1	2	0.5 1	2	0.5 1	2	0.5 1	2
	α-Helix	42.9 21.4 14.0 12.4 18.5 13.9 13.4 15.2 16.1 17.0 21.4 13.5 12.4
	β-Sheet	30.4 39.6 44.1 44.0 37.4 40.7 42.1 40.3 42.8 44.6 39.6 44.1 44.0
	Random coil 26.7 37.8 40.3 43.0 43.0 45.2 43.8 44.2 39.7 38.7 37.8 41.3 43.0

Table 5 .

 5 The obtained results from FRET theory for the compounds.

	Compounds	J (Cm 3 L mol -1 )	R0 (nm)	r (nm)	E
	VOL2	6.12 × 10 -17	1.09	1.17	0.39
	CuL2	3.28 × 10 -15	2.1	2.14	0.47
	NiL2	9.0 × 10 -15	2.5	2.6	0.44
	PdL2	1.6 × 10 -14	2.7	2.8	0.45

Acknowledgements

The authors are grateful to the Research Council of the University of Isfahan for financial support of this work.

Discussion and Conclusions

In the current study, the synthesis of four novel Schiff base metal complexes including Cu(II), Ni(II), Pd(II) and V(IV) has been described. They were characterized by FT-IR, and elemental analysis (CHN). The Schiff base diamagnetic Pd(II) complex were also characterized by 1 H NMR and 13 C NMR. Furthermore, their structures were determined by single-crystal X-ray analysis. The crystallographic data show a square planar trans-[MN2O2] coordination geometry for the CuL2, NiL2 and PdL2 complexes while in the VOL2, the vanadium center has a distorted tetragonal pyramidal N2O3 coordination sphere. A unique structure for the PdL2 was observed in which there is planner configuration without inversion center. The lack of significant interaction between the allyl groups and the metal centres in the complexes allows them to bond on a drug carrier.

The HSA-binding of the synthesized compounds have been evaluated, using experimental (fluorescence quenching, UV-vis spectroscopy and circular dichroism (CD)) and computational methods (molecular docking and QM/MM). The obtained results indicate that the compounds bind to HSA as PdL2 > CuL2 > VOL2 > NiL2. The calculated binding constants between these compounds and HSA were about 3 to 9.5 × 10 4 M -1 . The results of fluorescence experiment as well as the changes in the absorption spectrum of HSA upon addition of the compounds show that the HSA-compound complexes formed in the ground state. In addition, the obtained results from CD spectroscopy indicates upon addition of the synthesized compounds to HSA the percentage of α-helix of HSA decreased while those of random coil and β-sheet increased. Molecular docking studies revealed that hydrogen bond interactions have major role in the interaction of these compounds to HSA. PdL2 complex with the highest binding constant interacts with IB site of HSA by formation of three centre hydrogen bond. The ONIOM method was carried out with the aim to investigate more accurately. Based on the ONIOM calculation, the structural parameters of compounds changed due to their interactions with HSA. Generally, the results of the present study exhibit the effect of the metal ion on the HSA-binding whether on the value of binding constant or the binding site.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jphotobiol.2016.07.003.


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

In-Silico Study on the Interaction of Saffron Ligands and beta-Lactoglobulin by Molecular Dynamics and Docking Approach

Keywords: lactoglobulin, Binding affinity, Molecular docking, Molecular dynamics simulation

Safranal, crocetin and dimethylcrocetin are secondary metabolites found in saffron and have a wide range of biological activities. An investigation of their interaction with a transport protein, such as --lg), at the atomic level could be a valuable factor in controlling their transport to biological sites. The i -lg, as a transport protein, was investigated using molecular docking and molecular dynamics simulation methods. The -lg.

However, dimethylcrocetin binds to the internal cavity of -lg. T -lg affinity for binding the saffron ligands decreases in the following order: crocetin > dimethylcrocetin > safranal. The analysis of MD simulation trajectories showed that the -lg and -lg ligand complexes became stable at approximately 3000 ps and that there was little conformational change in the -lg -lg dimethylcrocetin complexes over a 10 ns timescale. In addition, the profiles of atomic fluctuations showed the rigidity of the ligand binding site during the simulation time.

Introduction

Natural ligands have low toxicity and potent efficacy and are of particular interest as chemopreventive compounds [START_REF] Greenlee | Natural products for cancer prevention[END_REF]. Saffron (C. sativus L.) is a plant in the Iridaceae family, planted in Iran, China, Turkey, India, Spain and Algeria; it has a wide range of biological activities including oxytocic, anti-carcinogenic, exhilarant, anti-depressant and anti-asthma effects [START_REF] Javadi | A survey on saffron in major Islamic traditional medicine books[END_REF]. It has been widely used as a herbal medicine and spice from the distant past. Saffron enhances the bioavailability and absorption of other drugs [START_REF] Javadi | A survey on saffron in major Islamic traditional medicine books[END_REF]. Previous studies have shown that there are more than 150 components in saffron stigmas [START_REF] Bathaie | New applications and mechanisms of action of saffron and its important ingredients[END_REF]. The characterization of saffron showed the presence of vitamins, minerals, sugars, fats, and secondary metabolites. Among the secondary metabolites, carotenoids are the most important species because they control the color and taste of saffron and also have medicinal effects [START_REF] Gismondi | Biochemical, antioxidant and antineoplastic properties of Italian saffron (Crocus sativus L.)[END_REF]. Safranal, crocetin and dimethylcrocetin are the three main chemical constituents of the carotenoids (Figure 1). Safranal (2, 6, 6-trimethyl-1, 3-cyclohexadiene-1-carboxaldehyde, C 10 H 14 O) is an aromatic aldehyde which has anti-oxidant effects and reduces oxidative damages in various organs such as skeletal muscle, kidney and hippocampus [START_REF] Javadi | A survey on saffron in major Islamic traditional medicine books[END_REF][START_REF] Samarghandian | Anti-tumor activity of safranal against neuroblastoma cells[END_REF]. Some studies have shown that the genotoxic potency of methyl methanesulfonate (MMS)-induced DNA damage in the liver and lung of mice was inhibited by safranal [START_REF] Hosseinzadeh | Saffron and its constituents: new pharmacological findings[END_REF]. Furthermore, in-vitro studies showed the minor groove binding of safranal to DNA [START_REF] Bolhasani | Interaction of monoterpene aldehydes of Iranian saffronwith DNA[END_REF].

Crocetin is a natural carotenoid dicarboxylic acid whose chemical structure is the central core of crocins [START_REF] Bolhasani | Separation and purification of some components of Iranian saffron[END_REF]. Circular dichroism (CD) studies have showen that crocetin binds to the I-motif and G-quadruplex, unique secondary structures of telomeric DNA [START_REF] Hoshyar | Is there any interaction between telomeric DNA structures, g-quadruplex and I-motif, with saffron active metabolites? Nucleos[END_REF]. Recently, crocetin has been shown to inhibit pancreatic cancer cell proliferation and tumor progression in a xenograft mouse model [START_REF] Dhar | Crocetin inhibits pancreatic cancer cell proliferation and tumor progression in a xenograft mouse model[END_REF]. Furthermore, crocetin has a protective effect on the cyclophosphamide induced bladder toxicity [START_REF] Giaccio | Crocetin from saffron: an active component of an ancient spice[END_REF]. Crocetin scavenging free radicals can protect cells from oxidative stress [START_REF] Al-Qudsi | Effect of saffron on mouse embryo development[END_REF]. Crocetin reduces the growth of tumors by inhibiting nucleic acid synthesis, improving antioxidative systems, inducing apoptosis and hindering growth factor signaling pathways [START_REF] Gutheil | Crocetin: an agent derived from saffron for prevention and therapy for cancer[END_REF].

Dimethylcrocetin is synthesized from extracts of saffron [START_REF] Bolhasani | Separation and purification of some components of Iranian saffron[END_REF]. The hydroxyl unit in the carboxylic groups in crocetin makes it suitable for reaction with free radicals. Hence, crocetin is more effective than dimethylcrocetin. Furthermore, the antioxidant activity of crocetin increases as the concentration increases; but for dimethylcrocetin, there is a maximum point in its activity as a function of concentration [14 16]. However, safranal has even lower antioxidant activity than crocetin and dimethylcrocetin.

The delivery of safranal, crocetin and dimethylcrocetin are poorly understood. The interaction of these natural ligands and a transport protein, -lactoglobulin ( -lg), could be a valuable factor to control their transport to biological sites and could increase their bioavailability.

-lg is one of the natural lipocalin members that bind and transfer small hydrophobic ligands such as steroids, fatty acids, retinoids, vitamin D, and cholesterol [17-21 -lg is the major whey protein and has several genetic variants [START_REF] Sawyer | The core lipocalin, bovine b-lactoglobulin[END_REF][START_REF] Dong | Infrared and circular dichroism spectroscopic characterization of structural differences between blactoglobulin A and B[END_REF]. The crystal structure shows that the 162 amino acids along the single polypeptide chain of bovine -lg forms a calyx composed of an eight -sheet [START_REF] Brownlow | Bovine [beta]-lactoglobulin at 1.8 resolution-still an enigmatic lipocalin[END_REF]. Unfortunately, the clinical application of saffron ligands is limited due to their poor water solubility and low bioavailability caused by their hydrophobic structure. However, an important physicochemical property of -lg is its ability to bind physiologically relevant ligands. Hence,lg can serve as a potential transport protein, especially for poor water-soluble molecules such as the saffron ligands. Furthermore, finding novel bioactive ligand molecules that interact with -lg is of biotechnological and pharmacological importance. Such knowledge would contribute to a deeper understanding of molecular applications of this lipocalin. -lg shows significant resistance against both gastric and simulated duodenal digestions. Therefore, it seems an appropriate candidate for the safe delivery and protection of pH sensitive drugs to the stomach.

Moreover, the high stability of -lg under acidic condition guarantees the safe delivery of hydrophobic ligands through the acidic condition of the stomach.

We have previously studied the interaction of flavonoids and biguanides with -lg [START_REF] Sahihi | Binding of biguanides to -lactoglobulin: molecular-docking and molecular dynamics simulation studies[END_REF][START_REF] Sahihi | The Interaction of Polyphenol Flavonoids -lactoglobulin: Molecular Docking and Molecular Dynamics Simulation Studies[END_REF][START_REF] Khosravi | Computational Studies on the Interaction of Arctiin and Liquiritin With -lactoglobulin[END_REF][START_REF] Sahihi | An investigation of molecular dynamics simulation and molecular docking: Interaction of citrus flavonoids and -lactoglobulin in focus[END_REF]. The -lg and -lg flavonoid complexes are stabilized through hydrophobic interactions, hydrogen bond ng interactions. In the present study we examined the interaction and protein conformation of -lg with safranal, crocetin and dimethyl crocetin using molecular docking and molecular dynamics simulation studies. Finding new biologically active ligands to bind -lg is very important, and not only can contribute to a better understanding of the molecular properties of this protein, but also might pave the way for future studies about the transport of drugs to biological sites.

Experimental

Molecular docking

The geometry of the ligands was optimized using Gaussian 03 at the level of B3LYP/6-31G** [START_REF] Frisch | Revision C.02[END_REF]. The crystal structure of -lg (PDB ID: 3NPO) was taken from the Brookhaven Protein Data Bank (http://www.rcsb.org/pdb). The R-value of this file was 0.216, with the protein crystal prepared at pH=7.5. The water molecules of the protein .pdb file were removed, and missing hydrogen atoms and Gasteiger charges were added. Flexible-ligand docking was done using molecular docking software (AutoDock Version 4.2), the implemented empirical free energy function and the Lamarckian Genetic Algorithm (LGA) [START_REF] Morris | [END_REF]. A blind docking with 126 lattice points along each of the X, Y, and Z axes was performed to find the active site of the ligands related to the -lg. After determination of the active site, the dimensions of the grid map were selected as 60 points 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 for each molecule. Cluster analysis (RMS tolerance equal to 0.5 ) was then carried out on the docked results.

Molecular dynamics simulation

The lowest binding free energy conformation of each 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 GROMOS96 43a1 force field [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 saffron ligands were calculated using Gaussian 03 [START_REF] Frisch | Revision C.02[END_REF] at the level of B3LYP/6-31G**. The complex was located in the cubic box with the periodic boundary conditions. The box volume was 294.14 nm 3 (6.6504 × 6.6504 × 6.6504 nm 3 ) and the minimum distance between the protein surface and the box was 1.0 nm. The box, filled with extended simple point charges (SPC), water molecules [START_REF] Berendsen | Intermolecular Forces[END_REF], and the solvated systems, were neutralized by adding 8 sodium ions (Na + ). Energy minimization was done through using the steepest descent method for 8 ps. Then, the system was equilibrated for 40 ps at the temperature of 300 K. Finally, a 10 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 sumsin large systems[END_REF][START_REF] Essmann | A smooth particle mesh Ewald method[END_REF] for long range electrostatics, and a 7 Å cut off for van der Walls interactions and Coulomb interactions were used. The equation of motions was integrated by the leap-frog algorithm with the 2 fs time steps.

The atomic coordinates were recorded to the trajectory file every 0.5 ps for later analysis.

Finally, an all-bond constraint was used to keep the ligand from drifting in the MD. lg and dimethylcrocetin was placed within the internal cavity of -lg with the binding energies G bind ) of -4.9, -7.67 and -7.26 kcal.mol -1 for safranal, crocetin, and dimethylcrocetin, respectively. All of these compounds were near some of the hydrophobic residues of the -lg.

Results and discussion

Furthermore, the drugs were able to form hydrogen bond interactions with residues of the -lg.

Figure 3a shows that Gln(59) interacts with safranal by one hydrogen bond interaction. Also, as can be seen in Fig. 3b, Leu(1), Gly [START_REF] Hoshyar | Is there any interaction between telomeric DNA structures, g-quadruplex and I-motif, with saffron active metabolites? Nucleos[END_REF] and Lys(91) interact with crocetin by three hydrogen bond interactions. Finally, Fig. 3c shows that Asn(88), Asn(90) and Lys(69) also interact with dimethylcrocetin by three hydrogen bond interactions. Thus, it may be concluded that, in addition to the hydrophobic interactions which stabilize the protein-ligand complexes, the hydrogen bond interactions also play an important role in the stability of -lg ligand complexes.

The comparison of the G bind values with the results of the our previous study on the interaction -lg with citrus flavonoids (-10.91, -10.44, -9.15 and -7.93 kcal.mol -1 for naringenin, hesperetin, tangeretin and nobiletin, respectively [START_REF] Sahihi | An investigation of molecular dynamics simulation and molecular docking: Interaction of citrus flavonoids and -lactoglobulin in focus[END_REF]) indicates -lg has less affinity for saffron ligands than for the flavonoids.

Analysis of the MD simulation trajectories

The beginning structures for the MD analyses were selected from the conformations with lowest docking energies. The trajectories were analyzed in terms of root mean square deviation (RMSD), radius of gyration (R g ), total solvent accessible surface (SAS) and root mean square fluctuation (RMSF) using the GROMACS routines.

Root mean square deviation

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The stability of trajectories for unliganded -lg and -lg ligands was examined using the RMSD of the backbone of -lg (Figure 4). A glance at the analysis in Fig. 4 shows that the RMSD of all systems reached equilibrium and fluctuated around the mean value at about 3000 ps. In other words, the RMSD of the back bone atoms increased up to about 0.20 nm and then stabilized around 7 ns and remained stable till the end of the simulation, demonstrating that the molecular system was well behaved afterward.

Radius of gyration

The R g s for all systems were also quantified and plotted versus simulation time to examine the protein compactness (Figure 5). The results showed that all of the systems stabilized after about 2500 ps, indicating that the MD simulation equilibrated after 2500 ps. It can be clearly seen that the R g was larger upon binding of the safranal and dimethylcrocetin -lg and the bound crocetin, suggesting a less compact structure after the binding of these two ligands to -lg. Also, Fig. 5 shows that the R g -lg did not depend upon the complexation with crocetin. This -lg did not change during its interaction with crocetin.

Solvent accessible surface (RMSF)

The changes in the total solvent accessible surface (SAS) of -lg during the 10 ns simulation time are shown in Fig. 6. The variations of SAS were similar to the variations of R g . This similarity confirms the correctness of the MD simulation results. The mobility of the -lg residues was evaluated by analysis of the RMSF of the C atoms of -lg in the absence and presence of the saffron ligands (Figure 7). The results indicate that the fluctuations larger than 0.2 nm corresponded to the residues that are far from the binding sites of each ligand. Moreover, the residues that were in contact with the saffron ligands are the most stable and have lower RMSF values.

Root mean square fluctuation

In addition, the RMSF of the atomic positions of the saffron ligands was calculated to examine their conformational variations (Figure 8). The results indicate that the saffron ligands atoms showed limited fluctuations (<0.30 nm). Hence, it can be concluded that the interactions of -lg and the saffron ligands were stable during the simulation time.

Conclusions

In this work the interaction between saffron ligands and -lg has been investigated using molecular docking and molecular dynamics simulation techniques. On the basis of the molecular docking results, safranal and c -lg. However, dimethylcrocetin binds in the internal cavity of -lg. The -lg affinity for binding the saffron ligands decreases in the following order: crocetin > dimethylcrocetin > safranal. In addition, the docking results revealed that the hydrophobic and hydrogen bond interactions play a major role in the -lg ligands complex stability. MD simulation studies showed the stabilization of -lg and -lg ligand complexes at around 3 ns. It can be clearly seen that the R g and SAS increase upon binding of the safranal and dimethylcrocetin suggesting a less compact structure after the binding --lg did not change during its interaction with crocetin. Furthermore, the profiles of the atomic fluctuations showed the interactions of -lg and saffron ligands were stable during the simulation time. -lg using AutoDock.

Ligands, depicted as -lg, represented in cartoon ribbon.

3. 1 .

 1 Molecular docking studies-lg consists of 162 amino acids, with three potential binding sites: the internal cavity of thebarrel, the surface hydrophobic pocket in a groove between the -helix and the -barrel and the outer surface near Trp19-Arg124[START_REF] Roufik | Thermodynamics of Binding -Lactoglobulin A and t -Lg f142-148[END_REF]. The initial crystal structure analyses have shown that most ligands are bound to the internal cavity of the -barrel [41 43]. Besides the primary drug binding site in the internal cavity of the -barrel, some compounds, such as p-nitrophenyl phosphate, 5-fluorocytosine, ellipticine and protoporphyrin, bind to the outer surface site[START_REF] Sawyer | Lactoglobulin binds retinol and protoporphyrin IX at two different binding sites[END_REF] 45]. The best energy ranked results of the interaction between the ligands and -lg in the docking procedures are shown in Fig.2. As shown, safranal and crocetin were placed on the surface of -Downloaded by [Tulane University] at 05:19 05 December 2015

  Downloaded by [Tulane University] at 05:19 05 December 2015 10

Figure 1 .

 1 Figure 1. The chemical structures of a) safranal; b) crocetin and c) dimethylcrocetin.

Figure 2 .

 2 Figure 2.-lg using AutoDock.

Figure 4 Figure 5 .Figure 6 .Figure 8 .

 4568 Figure 4.--lg ligand

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Funding Support of this work by the University of Isfahan (Iran) (Grant No. 920916) is gratefully acknowledged. Downloaded by [Tulane University] at 05:19 05 December 2015

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