Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 150 (2015) 290–300

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Synthesis, structure, protein binding of Cu(II) complexes with a tridentate NNO Schiff-base ligand Mei Li ⇑, ShuJuan Huang, Cheng Ye, YongRong Xie ⇑ Key Laboratory of Jiangxi University for Functional Material Chemistry, College of Chemistry & Chemical Engineering, Gannan Normal University, Ganzhou, Jiangxi, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Four new Cu(II) Schiff-base

Four new Cu(II) complexes with a tridentate NNO Schiff-base ligand have been synthesized and well characterized. The interactions of the complexes with HSA have been investigated by spectroscopic methods and a molecular docking technique. The results provide valuable information for designing HSA targeting carriers to deliver Cu(II) complex drugs and to study pharmacological behavior of drugs.

complexes 1–4 were synthesized and characterized.  The interactions of 1–4 with HSA have been investigated.  1–4 quench the fluorescence of HSA through a static quenching mechanism.  The active binding sites for 1, 2 and 4 are site III in IB and for 3 is site II in III A.  1–4 bind to HSA through hydrophobic forces as well as hydrogen bonds.

a r t i c l e

i n f o

Article history: Received 1 December 2014 Received in revised form 11 May 2015 Accepted 23 May 2015 Available online 30 May 2015 Keywords: Cu complexes HSA Spectroscopy Docking

a b s t r a c t Four new Cu(II) complexes (1, 2, 3 and 4) in the presence of different anions (Cl, Br, I and ClO 4 ) have been prepared by tridentate NNN Schiff-base ligand (N,N-dimethyl-N0 -[phenyl(2-pyridyl)methylene]et hane-1,2-diamine) and well characterized by single-crystal X-ray diffraction, elemental analysis, IR and UV–Vis spectroscopy. The interactions of complexes 1–4 with human serum albumin (HSA) have been investigated in Tris–HCl buffer solution at pH 7.4 by spectroscopic methods and a molecular docking technique. Experimental results proved that the four complexes quench the fluorescence of HSA through a static quenching mechanism. Thermodynamic parameters were calculated from Van’t Hoff equation. The distance r between the donor (HSA) and acceptor (complexes 1–4) has been obtained by means of Förester resonance energy transfer (FRET). Molecular docking results indicated that the main active binding sites for complexes 1, 2 and 4 are site III in subdomain IB and for complex 3 is site II in subdomain III A. The combination of molecular docking results and fluorescence experimental results indicate that the interaction between 1–4 and HSA are dominated by hydrophobic forces as well as hydrogen bonds. Ó 2015 Published by Elsevier B.V.

Introduction The metal-based cancer drug discovery is one of the most rapidly changing areas of pharmaceutical research. Study of the ⇑ Corresponding authors at: Gold Development Zone, Ganzhou, Jiangxi 341000, China. Tel.:/fax: +86 797 8268653. E-mail addresses: [email protected] (M. Li), [email protected] (Y. Xie). http://dx.doi.org/10.1016/j.saa.2015.05.064 1386-1425/Ó 2015 Published by Elsevier B.V.

interaction between metal-based drugs and plasma proteins becomes an important research field in chemical biology and pharmacology [1]. Human serum albumin (HSA) is the most abundant protein in plasma. HSA displays an extraordinary ligand binding capacity, which makes it the most important drug carrier protein. HSA plays an important role in the transport and disposition of endogenous and exogenous ligands such as fatty acids, hormones,

M. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 150 (2015) 290–300

and foreign molecule including drugs. Binding of a metal-based drug to HSA, results in an increased drug solubility in plasma, decreased toxicity, and protection against oxidation of the bound drug. It has been exposed that the distribution, free concentration and the metabolism of various metal-based drugs can be significantly altered as a result of their binding to HSA [2,3]. Structurally, HSA has a molecular mass of 66,500 Da composed of 585 amino acids and has 67% of a-helix. HSA consists of three homologous domains (I–III), each domain being divided into subdomains A and B, and the overall structure is stabilized by 17 disulfide bridges [4]. Since HSA serves as a drug transport carrier, the investigation of interaction between drugs and HSA is of major importance to understand the drug pharmacokinetics and pharmacodynamics [5]. Copper is an essential trace element in the body, which plays a vital role in biological processes like electron transfer, endogenous oxidative DNA damage associated with aging and cancer. Current interest in copper(II) complexes is stemming from their potential use as antimicrobial, antiviral, anti-inflammatory, antipyretic, enzyme inhibitors, or chemical nucleases [6–11]. For example, Ma et al. have synthesized Cu(II) complexes which could inhibit different enzymatic activities and induce cell apoptosis [12]. Majouga et al. have reported new binuclear mixed valence Cu(I,II) complexes containing substituted 2-alkylthio-5-arylmethylene-4H-im idazolin-4-ones, and these complexes are shown to be cytotoxic for various cell lines [13]. Loganathan et al. have prepared mixed ligand l-phenoxo-bridged dinuclear copper(II) complexes with diimine co-ligands, and all the complexes exhibit efficient chemical nuclease and protease activities and cytotoxicity against human breast cancer cell lines (MCF-7) [14]. Previous studies have demonstrated that Schiff base copper(II) complexes, which containing the ligand L1 (N,N-dimethyl-N0 -[ph enyl(2-pyridyl)methylene]ethane-1,2-diamine) and its derivates, have magnetic and spectroscopic properties [15–17], but very little research focused on their biological properties and applications in cancer treatment. What’s more, the modification of complexes with different anion could cause the structural transformation and result in the variation of optical and electrical parameters [18,19]. In this context, we have employed the ligand L1 and different anion to synthesize and seek metal-based drugs with higher biological activity. In the interaction of complexes with HSA, the anion would affect binding to HSA through altering association constants, thermodynamic parameters, number of binding sites, binding forces, and energy transfer distance, and thus lead to changes in complexes’ biological activity. In this study, we describe synthesis and characterization of four novel copper(II) complexes 1–4 derive from 2-benzoylpyridine and N,N-dimethylethylenedi amine. A comparative study of interactions of these complexes with major carrier protein HSA was investigated by spectroscopic and molecular docking methods.

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Vis) absorption spectra were performed on a Varian Cary100 UV– Visible spectrophotometer. Fluorescence measurements were obtained by using a Shimadzu RF-5301/PC spectrofluorop hotometer. Synthesis of N,N-dimethyl-N0 -[phenyl(2-pyridyl)methylene]ethane1,2-diamine, L1 The ligand L1 was synthesised by refluxing a methanolic solution (50 ml) of 2-benzoylpyridine (10 mmol) and 2-dimethylaminoethylamine (10 mmol) for 30 min. The resulting mixture obtained without further separation was a yellow solution containing the liquid ligand L1. Synthesis of 1, 2, 3 and 4

Experimental

1: To a solution of CuCl22H2O (170.48 mg, 1 mmol) in 10 ml methanol, the liquid Schiff base ligand L1 (1 mmol) was added, and refluxed for 2 h. The mixture obtained was filtered and left to stand at room temperature for slow evaporation, and the dark blue crystals of 1 were collected after several days. Yield: 262 mg, 64.2%. Selected IR (KBr, cm1): 2923 m (C–H), 1637s (C@N), 1597s, 1495w, 1445s. UV–Vis in DMSO, k nm; (e M1 cm1): 282 (17,190), 223 (54,322). Elemental analysis (%): calc. for C16H19Cl2CuN3O: C, 47.55; H, 4.71; N, 10.40. Found: C, 47.43; H, 4.66; N, 10.52. 2: The synthesis was carried out using the same procedure as in 1, by using CuBr2 (1 mmol, 223.35 mg) instead of CuCl22H2O for 2. The dark blue crystals of 2 suitable for X-ray analysis were collected after several days. Yield: 246 mg, 49.9%. Selected IR (KBr, cm1): 2964 m (C–H), 1636s (C@N), 1594s, 1492w, 1444s. UV– Vis in DMSO, k nm; (e M1 cm1): 282 (30,995), 229 (66,516). Elemental analysis (%): calc. for C32H38Br4Cu2N6O2: C, 38.97; H, 3.86; N, 8.52. Found: C, 38.92; H, 3.68; N, 8.59. 3: To a solution of Cu(OAc)2H2O (199.65 mg, 1 mmol) in 10 ml methanol, the liquid Schiff base ligand L1 (1 mmol) was added with constant stirring for 1 h. Then KI (166.00 mg, 2 mmol) was added, and refluxed for 0.5 h. The mixture obtained was filtered and left to stand at room temperature, and the dark blue crystals of 3 formed after several days. Yield: 330 mg, 49.56%. Selected IR (KBr, cm1): 2912 m (C–H), 1651s (C@N), 1593s, 1492w, 1442s. UV–Vis in DMSO, k nm; (e M1 cm1): 281 (38,258), 225 (73,684). Elemental analysis (%): calc. for C32H38Cu3I5N6: C, 28.83; H, 2.85; N, 6.31. Found: C, 28.75; H, 2.79; N, 6.42. 4: The procedure of synthesis is same as that of 1, only additional NaClO4 (122.44 mg, 1 mmol) were added. The dark blue crystals of 4 suitable for X-ray diffraction were collected after several days. Yield: 217 mg, 48.0%. Selected IR (KBr, cm1): 2914 m (C–H), 1651s (C@N), 1598s, 1571w, 1495w, 1445s, 1090s (ClO 4 ). UV–Vis in DMSO, k nm; (e M1 cm1): 282 (36,555), 222 (52,485). Elemental analysis (%): calc. for C16H19Cl2CuN3O4: C, 42.50; H, 4.21; N, 9.30. Found: C, 42.47; H, 4.15; N, 9.40.

Materials and physical measurements

X-ray crystallography

All chemicals and reagents were purchased commercially and were all used as received without further purification. Cupric chloride dihydrate, N,N-dimethylethylenediamine, 2-benzoylpyridine, copper(II) bromide, sodium perchlorate, potassium iodide and cupric acetate were purchased from Alfa Aesar Chemicals Co. (USA). HSA was purchased from Sigma Chemicals Co. (USA). The Tris– HCl buffer solution was prepared with triple-distilled water. Elemental analyses (C, H, N) were carried out on a Perkin Elmer Series II CHNS/O 2400 elemental analyzer. Infrared spectra were obtained on a Perkin-Elmer FT-IR Spectrometer. UV–Visible (UV–

The X-ray diffraction data for the complexes were collected on a Bruker Smart Apex II CCD diffractometer equipped with graphite monochromated Mo Ka radiation (k = 0.71073) at room temperature. The structures were solved with direct methods and refined using SHELX-97 programs [20,21]. The non-hydrogen atoms were located in successive difference Fourier synthesis. The final refinement was performed by full-matrix least-squares methods with anisotropic thermal parameters for non-hydrogen atoms on F2. The hydrogen atoms were added theoretically and riding on the concerned atoms.

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Interaction with HSA

Molecular docking

The binding interactions of complexes 1, 2, 3 and 4 with HSA were carried out using standard Trp fluorescence with excitation at 280 nm and the corresponding emission at 343 nm by fluorescence spectra. Various concentrations of complexes (0–13 lM) were added to a solution containing 1 lM HSA and 20 mM NaCl/Tris–HCl buffer (pH = 7.4). Fluorescence spectra were obtained by recording the emission spectra (300–450 nm).

The crystal structure of HSA was downloaded from the Protein Data Bank (PDB ID: 1BJ5). The complex.cif of complexes 1–4 structure has been obtained from single-crystal X-ray diffraction. The structure of complex 4 is in disorder because of thermal vibration of the perchlorate, hence we optimize the structure of 4 before the molecular docking experiment. The complex.cif and sdf file types were converted to pdb file type using Molegro Virtual Docker

Scheme 1. Synthetic route of Cu(II) complexes 1–4.

Table 1 Crysallographic data and structure refinement parameters for 1–4. Complexes

1

2

3

4

CCDC number Empirical formula Formula weight Temperature (K) Crystal system Space group a (Å) b (Å) c (Å) a (°) b (°) c (°) Volume (Å3) Z qcalc (mg mm3) l (mm1) F(0 0 0) Crystal size (mm3) 2H range for data collection Index ranges

976,009 C16H19Cl2CuN3O 403.80 293(2) Monoclinic P21/c 13.677(3) 11.0524(15) 13.448(3) 90.00 119.34(3) 90.00 1772.1(6) 4 1.513 1.540 2163 0.20  0.08  0.05 3.03–26.37° 16 6 h 6 17 13 6 k 6 12 16 6 l 6 15 11,832 3610[R(int) = 0.1474] 3610/0/210 1.006 R1 = 0.0762 wR2 = 0.1931 R1 = 0.1047 wR2 = 0.2429 0.874/1.005

976,011 C16H19Br2CuN3O 492.70 296.15 Monoclinic P21/c 13.849(6) 11.184(5) 13.512(6) 90.00 117.754(6) 90.00 1852.1(14) 4 1.767 5.500 972 0.35  0.25  0.11 1.66–26.37° 16 6 h 6 17 13 6 k 6 13 16 6 l 6 16 19,146 3759[R(int) = 0.0621] 3759/0/210 1.132 R1 = 0.0520 wR2 = 0.1261 R1 = 0.0655 wR2 = 0.1317 0.886/0.941

976,010 C32H38Cu3I5N6 1331.80 293(2) Monoclinic C2/c 13.7951(3) 11.93576(19) 24.3645(6) 90.00 90.580(3) 90.00 4011.54(16) 4 2.205 5.454 2496 0.20  0.08  0.05 5.9–52.74° 17 6 h 6 17 14 6 k 6 13 30 6 l 6 30 16,174 4096[R(int) = 0.0342] 4096/0/211 1.067 R1 = 0.0277 wR2 = 0.0504 R1 = 0.0366 wR2 = 0.0541 0.531/0.756

976,012 C16H19Cl2CuN3O4 451.78 294(2) Monoclinic P21/c 11.735(2) 13.078(2) 12.993(2) 90.00 107.822(3) 90.00 1898.5(6) 4 1.581 1.458 924 0.40  0.30  0.24 2.27–26.48° 14 6 h 6 13 16 6 k 6 13 15 6 l 6 16 10,364 3902[R(int) = 0.0376] 3902/112/298 1.014 R1 = 0.0441 wR2 = 0.1101 R1 = 0.0786 wR2 = 0.1341 0.712/0.595

Reflections collected Independent reflections Data/restraints/parameters Goodness-of-fit on F2 Final R indexes [I > 2r(I)] Final R indexes [all data] Largest diff. peak/hole (e Å3)

M. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 150 (2015) 290–300 Table 2 Selected bond lengths (Å) and angles (°) for 1–4. 1 Cu1–Cl1 Cu1–N1 Cu1–N3 Cl1–Cu1–Cl2 N1–Cu1–Cl2 N2–Cu1–Cl1

2.2773(16) 2.021(5) 2.053(5) 108.80(6) 90.06(13) 146.81(13)

Cu1–Cl2 Cu1–N2 N1–C1 N1–Cu1–Cl1 N1–Cu1–N3 N2–Cu1–Cl2

2.4803(18) 1.985(4) 1.339(7) 97.57(13) 161.89(19) 104.25(13)

2 Br1–Cu1 Cu1–N1 Cu1–N3 Br2–Cu1–Br1 N1–Cu1–Br2 N2–Cu1–Br1

2.6305(13) 2.034(5) 2.076(5) 106.81(5) 97.96(14) 104.26(15)

Br2–Cu1 Cu1–N2 N1–C1 N1–Cu1–Br1 N1–Cu1–N3 N2–Cu1–Br2

2.4285(12) 1.978(5) 1.336(8) 89.47(15) 162.0(2) 148.78(15)

3 I1–Cu1 I2–Cu2 Cu1–N1 I1–Cu1–I2 N1–Cu1–I1 N1–Cu1–N3

2.6200(5) 2.5489(5) 2.028(3) 101.454(16) 96.89(8) 161.79(11)

I2–Cu1 I3–Cu2 Cu1–N2 Cu2–I2–Cu1 N1–Cu1–I2 N2–Cu1–I1

2.8241(5) 2.4752(8) 1.973(3) 112.375(17) 89.51(8) 149.87(9)

4 Cu1–Cl1 Cu1–N2 N1–C1 N1–Cu1–Cl1 N2–Cu1–Cl1 N2–Cu1–N3

2.2158(9) 1.960(2) 1.336(4) 97.62(7) 176.14(8) 84.12(9)

Cu1–N1 Cu1–N3 N1–C5 N1–Cu1–N3 N2–Cu1–N1 N3–Cu1–Cl1

2.023(2) 2.045(2) 1.350(3) 164.29(9) 80.20(9) 98.09(7)

(MVD). Water molecules were removed, and hydrogen atoms were added. AutoDock Vina [22] and MGLTools of The Scripps Research Institute are mainly used to perform the docking calculations. The results are also verified with AutoDock 4 [23] and on PatchDock [24] server. The later uses a different algorithm from that of AutoDock. The PyMOL [25] molecular viewer and the MGLTools are used to render the output and to calculate the distance between nearest atoms. Results and discussion Spectroscopic characterization of complexes 1–4 In the IR spectra of the Schiff base ligands [26], the presence of an intense band at around 1620 cm1 has been assigned to the stretching vibrations of the azomethine groups. The bands at

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1630–1660 cm1 are present in the IR spectra of complexes 1–4 and have shifted to higher wave numbers upon coordination. This band is shifted in the complexes toward higher frequencies because of the coordination of the nitrogen to the metal ion [27]. Several weak bands in the range 2970–2910 cm1 assignable to aliphatic C–H stretching vibration are routinely observed. In the UV– Vis electronic spectrum, the intense band at about 225 nm is attributed to bpy (p–p⁄) transition, while the less intense band at about 280 nm is typical of charge transfer between bpy and metal (ligand-to-metal charge transfer) [28]. These spectroscopic data of complexes 1–4 are in accord with the following determination of the crystal structures. Structures of complexes 1–4 We have synthesized four complexes in the presence of four different anions (Cl, Br, I and ClO 4 ) in Cu(II) system. The synthesis pathway of complexes 1–4 are summarized in Scheme 1. Crystal data as well as data collection and refinement for complexes 1–4 are summarized in Table 1. Selected bond distances and bond angles are given in Table 2. Various anions are capable of playing an important role in the construction of molecular architectures and the properties of complexes through the subtle tuning effect of their bonding abilities and/or structural geometries as bonds to metal ions [29–32]. In complexes 1–4, the similar Cl, Br, I have a stronger bonding ability relative to ClO 4 . Two similar basically five-coordinate geometry were formed in 1 and 2, while two different coordination modes was obtained in 3 with the Cu1 acting as five-coordinate sphere and Cu2 adopting three-coordinate geometry. For 4, the Cu atom is basically four-coordinate, but with the O atom, O30 of the ClO 4 group occupying a five-coordinate position at a distance greater than 2.4 Å, namely 2.565 Å, to give (4 + 1⁄)-type coordination [33]. Apparently, the various anions (Cl, Br, I and ClO 4 ) are driving forces for the selection of different structures. 1 crystallizes in the monoclinic system, space group P21/c. As shown in Fig. 1A, the asymmetric unit of 1 consists of one copper(II) ion, a Schiff base unit, two Cl and one unbounded water molecule. The Cu(II) ion is coordinated by three nitrogen atoms from the Schiff base ligand and two Cl to form a CuN3Cl2 distorted square pyramidal coordination geometry [Cu1Cl1 = 2.2773(16), Cu1Cl2 = 2.4803(18), Cu1N1 = 2.0 21(5), Cu1N2 = 1.985(4) and Cu1N3 = 2.053(5) Å], and the

Fig. 1. (A) ORTEP view of the molecular structure and atom-labeling scheme of 1. H atoms and H2O are omitted for clarity. (B) Schematic representation of the 4-connected sql topological net of 1.

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Fig. 2. ORTEP view of the molecular structure and atom-labeling scheme of 2. H atoms and H2O are omitted for clarity.

angles around it fall in the range of 82.81(17)161.89(19)°. The distortion of the square pyramidal can be indicated by the calculated value of the s parameter introduced by Addison [34] to describe the geometry of a five-coordinate metal system, which is 0.25 for Cu1 (for perfect square pyramidal geometry, s = 0). In addition, the benzene ring of the Schiff base ligand and the pyridine ring of the Schiff base ligand were twisted, at an angle of 55.25° with respect to each other. Intermolecular C–H  Cl interaction also has been reported in several recent literature sources [35,36]. Whilst exploring the acting forces in the crystal lattice, we discovered that there exist C–H  Cl weak interactions: C11– H11  Cl1i = 2.876 Å; C2–H2  Cl2ii = 2.876 Å. When above C– H  Cl interaction weak interactions are taken into account, the resulting structure displays a 2D 4-connected net with sql (44.62) topology (Fig. 1B). 2 crystallizes in the monoclinic P21/c space group. There is one copper(II) ion, one Schiff base unit, two Br and one H2O in the asymmetric unit. As shown in Fig. 2, Cu1 atom is five-coordinated and exhibits distorted square pyramidal geometry. Cu1 atom is surrounded by three nitrogen atoms from the Schiff base ligand and two Br ligands (Cu1Br1 = 2.628(15), Cu1Br2 = 2.431(14), Cu1N1 = 2.028(6), Cu1N2 = 1.978(6) and Cu1N3 = 2.071(7) Å). The angles subtended at the Cu in the square pyramidal geometry (CuN3Br2) are in the range from

Fig. 3. ORTEP view of the molecular structure and atom-labeling scheme of 3. H atoms are omitted for clarity.

79.1(2)° to 162.0(3)°. The s values is 0.219, which is close to that of a regular square pyramidal CuN3Br2 chromophore, s = 0. Br1– Cu1–Br2 angle of 106.83(5)° is slightly narrower than the corresponding Cl1–Cu1–Cl2 angle (108.80(6)°) found in 1. The C– H  Br weak interactions [C2–H2  Br1i = 2.909 Å, C11– H11  Br2ii = 2.972 Å] arrange the motifs into a 2D network. Interestingly, when these C–H  Br interactions are taken into account, 2 displays topology structure the same as 1. In addition, the benzene ring of the Schiff base ligand and the pyridine ring of the Schiff base ligand were twisted, at the angles of 55.96° with respect to each other, which is relatively close to 1. 3 crystallizes in the monoclinic C2/c space group. The Cu(II) ions exhibit a slightly distorted square pyramidal geometry (Cu1) and a planar triangular I3-coordination geometry (Cu2) in the asymmetric unit. As shown in Fig. 3, the Cu1 atom is surrounded by three nitrogen atoms and two I atoms (Cu1I1 = 2.6200(5), Cu1I2 = 2.8241(5), Cu1N1 = 2.028(3), Cu1N2 = 1.973(3) and Cu1N3 = 2.069(3) Å), while the Cu2 bonds to three I atoms (Cu1I2 = 2.5489(5), Cu1I2i = 2.5489(5) and Cu1I3 = 2.47 52(8)), and the angles around it fall in the range of 79.44(11)16 1.79(11)°. The Cu2 atom in the CuI3 unit is coplanar by symmetry requirement In 3. The Cu1 atom is five coordinated. The coordination polyhedron around the Cu1 center could be described as distorted square pyramidal, and the index of the degree of square pyramidal, s, has a value 0.19, indicating a much less distorted structure compared to 1 and 2. The longer Cu1I2 apical bond length as compared to the Cu1I1 distance is associated to the pseudo-Jahn–Teller effect on the copper(II) ion. The benzene ring of the Schiff base ligand and the pyridine ring the Schiff base ligand were twisted, at an angle of 58.28° with respect to each other, which is larger than 1 and 2. 4 crystallizes in the monoclinic system, space group P21/c and consists of Schiff base ligand and ClO 4 coordinated to copper(II) in a 1:1:1 ratio. As shown in Fig. 4, the Cu1 atom is surrounded by three nitrogen atoms from the Schiff base ligand and one Cl ligand (Cu1Cl1 = 2.2158(9), Cu1N1 = 2.023(2), Cu1N2 = 1.9 60(2) and Cu1N3 = 2.045(2) Å), and the angles around it fall in the range of 80.20(9)176.14(8)°. The stereochemistry of the CuN3O30 Cl chromophore in 4 is basically four-coordinate with a second oxygen of the ClO 4 anion occupying a five position at a distance greater than 2.4 Å, to give a (4 + 1⁄)-type coordination sphere. The benzene ring of the Schiff base ligand and the pyridine ring of the Schiff base ligand were twisted, at an angle of 63.89° with respect to each other, which is larger than 1, 2 and 3.

Fig. 4. ORTEP view of the molecular structure and atom-labeling scheme of 4. H atoms are omitted for clarity.

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Fig. 5. The emission spectra of HSA (1 lM) in the absence (dashed line) and presence (solid lines) of complexes 1 (A) (0–12 lM), 2 (B) (0–1.2 lM), 3 (C) (0–1.2 lM) and 4 (D) (0–13 lM). The arrow shows the fluorescence quenching upon increasing the concentrations of 1–4.

Fig. 6. Plots of F0/F vs. [Q] for the fluorescence titration of 1–4 to HSA. 1 (A), 2 (B), 3 (C), 4 (D).

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Table 3 Binding and thermodynamic parameters for the interaction between 1–4 and HSA at different temperatures, pH 7.4.

1

2

3

4

T (K)

KSV(105) (L mol1)

kq(1013) (L mol1 s1)

K(104) (M1)

n

DG (kJmol1)

DH (kJmol1)

DS (Jmol1K1)

293 298 310 293 298 310 293 298 310 293 298 310

0.46 ± 0.02 0.41 ± 0.03 0.37 ± 0.03 2.27 ± 0.02 1.82 ± 0.01 1.59 ± 0.03 2.70 ± 0.01 2.29 ± 0.02 2.00 ± 0.01 0.33 ± 0.02 0.29 ± 0.03 0.26 ± 0.01

0.46 ± 0.02 0.41 ± 0.03 0.37 ± 0.03 2.27 ± 0.02 1.82 ± 0.01 1.59 ± 0.03 2.70 ± 0.01 2.29 ± 0.02 2.00 ± 0.01 0.33 ± 0.02 0.29 ± 0.03 0.26 ± 0.01

0.75 ± 0.01 0.26 ± 0.03 0.11 ± 0.02 0.94 ± 0.01 0.46 ± 0.02 0.18 ± 0.03 1.34 ± 0.04 1.11 ± 0.01 0.54 ± 0.02 0.35 ± 0.01 0.13 ± 0.02 0.06 ± 0.01

0.9 0.8 0.7 0.8 0.7 0.7 0.8 0.8 0.7 0.8 0.7 0.7

21.74 19.48 18.05 22.29 20.90 19.32 23.15 23.08 22.15 19.88 17.76 16.49

55.06

119.40

65.18

145.20

37.94

50.84

63.91

154.86

Fig. 7. Plots of log [(F0  F)/F] vs. log [Q] for the fluorescence titration of 1–4 to HSA. 1 (A), 2 (B), 3 (C), 4 (D).

Complexes 1–4 binding to HSA Fluorescence spectroscopy is an effective technique used for qualitative analysis of the binding of compounds to HSA. HSA contains three aromatic amino acid (tryptophan, tyrosine and phenylalanine). In fact, the intrinsic fluorescence of HSA is essentially contributed by tryptophan residue alone, because of the very low quantum yield of phenylalanine and tyrosine [37,38]. Fig 5 shows the effects of 1–4 on the fluorescence intensity of HSA at 298 K. The fluorescence intensities of HSA decrease with the increasing concentrations of complexes for the four studied complexes. This trend was also observed at the other studied temperatures (293 and 310 K). Upon addition of complexes 1, 2, 3 and 4, the fluorescence intensities of HSA decreases by 33.9% (ratio 12:1/1:HSA), 19.0% (ratio 1.2:1/2:HSA), 21.1% (ratio 1.2:1/3:HSA) and 28.8% (ratio 13:1/4:HSA), respectively. The results suggest a definite interaction of all of the complexes with HSA protein.

The fluorescence quenching data at different temperatures (293, 298 and 310 K) have been analyzed by the Stern–Volmer equation [39]: F0/F = 1 + KSV[Q], where F0 and F are the fluorescence intensities of the fluorophore in the absence and presence of quencher, respectively, KSV is the Stern–Volmer quenching constant, and [Q] is the quencher concentration. As shown in Fig. 6 and Table 3, the quenching constants KSV for 1–4 are all inversely correlated with temperatures. The KSV values are 4 < 1 < 2 < 3 obviously, because the complexes are coordinated by different anion ions. The lifetime of HSA protein is on the order of 108 s [40], the calculated bimolecular quenching rate constants (kq) using KSV = kqs0 were found to be higher than the maximum collisional quenching (kq) of various kinds of quenchers to biopolymers (2.0  1010 M1 s1) [39]. Hence, fluorescence quenching results from the formation of complexes between 1–4 and HSA. Fluorescence quenching may occur by different mechanisms, typically classified as static or dynamic quenching. Dynamic quenching

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Fig. 8. Spectral overlap between absorption of 1–4 (1.5 lM) and normalized emission of HSA (1.5 lM). HSA (the solid line), 1–4 (the dashed line). 1 (A), 2 (B), 3 (C), 4 (D).

Table 4 Parameters of energy transfer between 1–4 and HSA. Complexes

J (1015cm3 L mol1)

R0 (nm)

r (nm)

E

1 2 3 4

7.79 9.81 15.62 6.57

2.29 2.38 2.44 2.23

3.73 3.18 2.96 3.75

0.051 0.152 0.241 0.042

depends on the diffusion, since higher temperatures result in greater diffusion coefficients, the bimolecular quenching constants are expected to increase with increasing temperature. On the other hand, an increase in temperature results in a decrease of the complexes stability and thus lower values of static quenching constants [39]. The decrease of quenching constants in temperature results indicate that the quenching mechanisms are all static quenching. The binding parameters between complexes 1–4 and HSA can be determined using the equation [41]: log [(F0  F)/F] = l og K + nlog [Q], where K and n are the binding constant and the number of binding sites, respectively. F0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively. [Q] is the quencher concentration. Fig. 7 shows the binding equilibrium plots for the fluorescence quenching of HSA by complexes 1–4 at 293, 298 and 310 K. The values of K, n are listed on Table 3. These results show that the association constants K decreased with the temperature, which may indicate the formation of stable complexes between 1–4 and HSA. The values of n were approximately equal to 1, this indicating the existence of a single binding site in HSA for these four copper complexes. So, it can be concluded that complexes 1–4 bind to HSA forming 1:1 adducts. The binding constants K are 4 < 1 < 2 < 3 at the same temperature obviously, which are consistent with the above results. The minimum K of 4 may be attributed to its largest steric effects in the four complexes in the aqueous solution, which might decrease the

accessibility of the copper complex center to the active site in HSA. While the maximum K of 3 may be that the trinuclear 3 show stronger HSA-binding interaction than mononuclear 1, 2 and 4. The interaction forces between small molecules and biomolecules include van der Waals forces, hydrogen bonds, electrostatic forces, and hydrophobic interaction forces. The thermodynamic parameters, enthalpy (DH), and entropy (DS) of reaction are important for confirming binding mode. if DH < 0, DS > 0, the main force is electrostatic; if DH < 0, DS < 0, van der Waals or hydrogen bond interactions play major roles in the reaction; If DH > 0, DS > 0, hydrophobic interaction is implied as the main force [42]. The temperatures were chosen at 293, 298, and 310 K, and thus HSA will not undergo any structural degradation. Besides, when there is no significant change in temperature, the enthalpy of the reaction can be considered as a constant, so the thermodynamic parameters can be calculated from Van’t Hoff equation [43]: ln K = DH/RT + DS/R and DG = DH  TDS = RTln K. Where K is the binding constant at the corresponding temperature, R is the gas constant, and T is the temperature. DH, DG, and DS are enthalpy change, free enthalpy change and entropy change, respectively. The thermodynamic parameters calculated are presented in Table 3. The negative value of DG reveals that the interaction process is spontaneous. The negative DH and negative DS reveal that the interaction forces between complexes 1–4 and HSA are owing to van der Waals force or hydrogen bonds. Energy transfer between complexes 1–4 and HSA Fluorescence resonance energy transfer (FRET) between small molecules and biomolecules has been widely used to study protein–ligand interaction. The fluorescence quenching of HSA after binding with complexes 1–4 indicated the occurrence of energy transfer between complexes and HSA. FRET is a distance-dependent interaction in which the excitation energy is

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Scheme 2. Structure of human serum albumin showing the subdomains and binding sites of HSA.

Fig. 9. The interaction modes between 1–4 (showing stick representation) and HSA (cartoon form). 1 (A), 2 (B), 3 (C). 4 (D). The black dashed line showing hydrogen bond interaction between 1 and residue Tyr 161.

transferred non-radiatively from the donor molecule (protein) in the excited state to the acceptor molecule (ligand) in the ground state. The overlap of the emission spectrum of the donor and the absorption spectrum of the acceptor is the essential prerequisite for the possibility of an energy transfer mechanism [39,44]. The absorption spectra of complexes 1–4 have been performed by

maintaining complexes concentration fixed to 1.5 lM. Fig. 8 shows the overlap between the absorption spectra of complexes 1–4 with the fluorescence spectrum of HSA. The overlap is large, which indicate high energy transfer efficiency between HSA and complexes 1–4.

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Fig. 10. Molecular docked model of complexes 1–4 (stick representation) located within the hydrophobic pocket in subdomain IB of HSA (1, 2 and 4) and IIIA of HSA (3).

According to Förester resonance energy transfer theory, energy transfer is controlled by the following three aspects: (1) the donor should have strong fluorescence quantum yield, (2) more spectral overlap between the donor emission and the acceptor absorption, and (3) the distance r between the acceptor and the donor should be within 7 nm. The efficiency of energy transfer, E, is calculated by [45]: E = 1  F/F0 = R60/(R60 + r6). Where r is the distance between donor and accepter, and R0 is the distance at 50% transfer efficiency [46]. R0 is calculated by the equation [45]: R60 = 8.8  1025K2n4UJ. Where K2 is the orientation factor related to the geometry of the donor–accepter of dipole and K2 = 2/3 for random orientation as in fluid solution, n is the refractive index of medium, U is the fluorescence quantum yield of the donor, J is the spectra overlap of the donor emission and the acceptor absorption [47]. J is given by the equation [45]: J = RF(k)e(k)k44k/RF(k)4k. Where F(k) is the fluorescence intensity of the fluorescence donor in wavelength k, e(k) is the molar absorbance coefficient of the acceptor in wavelength k. From these relationships, J, E, R0 and r can be calculated. The obtained results are stated in Table 4. All binding distances r are much lower than 7 nm and 0.5R0 < r < 2.0R0, which indicates that non-radiation energy transfer from HSA to complexes 1–4 with high probability [48]. However, the value of r is higher than the respective critical distance R0, so, the static quenching is more likely responsible for fluorescence quenching other than the dynamic quenching [48]. The short distance values between bound ligand and tryptophan residue by this method suggested the significant interaction between complexes 1–4 and HSA. Docking results Crystalline albumin have revealed that human serum albumin molecular docking technique was further employed to understand the interaction between ligands and HSA. Descriptions of the 3D structure of crystalline albumin have revealed that HSA comprises three homologous domains (denoted I, II, and III): I (residues 1– 195), II (196–383) and III (384–585); each of which contains two subdomains, denominated subdomain IA, IB, IIA, IIB, IIIA and IIIB, which assemble to form heart shaped molecule [4] (Scheme 2). In general, the main regions of ligand binding to HSA are located in hydrophobic cavities in subdomains of IIA and IIIA, which are consistent with site I and site II, respectively, and one tryptophan residue (Trp-214) of HSA is in sub-domain IIA [49–52]. The hydrophobic, D-shaped cavity in subdomain IB termed as site III is also the primary binding site of some compounds [53–55]. Several studies have shown that HSA is able to bind many ligands in several binding sites, which play an important role in absorption, metabolism, and transportation of HSA. In the context of

the present study, the docking results showed that 1, 2 and 4 bind within the binding pocket of subdomain IB (Fig. 9A, B and D) whereas 3 bind within the binding pocket of subdomain IIIA (Fig. 9C). Complexes 1 and 4 are stabilized by hydrogen bonds between N atoms of complexes and Tyr 161 amino acid residues of the protein of length 3.55 and 3.31, respectively. The computationally calculated binding energy of lowest energy conformers of 1, 2, 3 and 4 are 8.4, 8.3, 6.9 and 7.7 kcal/mol, respectively. The overall structure of 1–4 binding with HSA is shown in Fig. 10. The moiety of 1–4 was located within the hydrophobic binding pocket and several groups of 1–4 interact with the several residues of subdomain IB and IIIA. Almost all the interactions between the active site and 1–4 are present within the 4 Å moiety [56] which consists of several hydrophobic residues such as PHE 157, TRY 161, ALA 158, LEU 139, HIS 146, PHE 156, LEU 154, IIE 142, ARG 186 for HSA-1 complex; PHE 157, PHE 156, TYR 161, LEU 154, PHE 134, LEU 182, ARG 186, MET 123, LEU 135, LEU 115, LEU 139 for HSA-2 complex; PRO 180, ARG 428, LYS 432, ASP 187, LYS 436, ASP 183, GAN 522, GLU 184, TYR 401 SER 435 for HSA-3 complex; LEU 182, LEU185, LEU154, PHE157, IIE 142 for HSA-4 complex. The results suggest the existence of hydrophobic interaction between them. The distance between Trp-214 and the binding sites found by docking was 3.53, 3.07, 2.56 and 3.62 nm, respectively. The result is very close to that obtained by FRET calculation (3.73, 3.18, 2.96 and 3.75 nm). Hence, this finding provides a good structural basis to explain the efficient fluorescence quenching of HSA emission in the presence of the complexes 1–4. The combination of molecular docking results and fluorescence experimental results indicate that the interaction between 1–4 and HSA are dominated by hydrophobic forces as well as hydrogen bonds. Conclusions In this work, we have synthesized and characterized four novel copper(II) complexes 1–4 derived from 2-benzoylpyridine and 2-dimethylaminoethylamine. 1–3 exhibit distorted square pyramidal geometry around Cu(II) metal ion and the Cu atom of 4 is basically four-coordinate with the O30 atom of the ClO 4 group occupying a five-coordinate position to give (4 + 1⁄)-type coordination. The interaction between complexes 1–4 and HSA was investigated employing different spectroscopic and molecular docking techniques. The experimental results indicated that 1–4 bind to HSA by static quenching mechanisms and are owing to hydrogen bonds and hydrophobic forces. The distance r between HSA and 1–4 has been calculated by means of FRET. Our results provide

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valuable information to rationally design HSA targeting carriers to deliver copper complex drugs and to study pharmacological behavior of drugs. Acknowledgments This work was supported by the Natural Science Foundation of China (21241005, 21461002, 21363001 and 21401026), and Major Project for Science and Technology of Jiangxi Province (20152ACB21016), and Education Department of Jiangxi Province (15YB106). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2015.05.064. References [1] M.Y. Tian, X.F. Zhang, L. Xie, J.F. Xiang, Y.L. Tang, C.Q. Zhao, J. Mol. Struct. 892 (2008) 20–24. [2] F. Faridbod, M.R. Ganjali, B. Larijani, S. Riahi, A.A. Saboury, M. Hosseini, P. Norouzi, C. Pillip, Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 78 (2011) 96–101. [3] B. Sudhamalla, M. Gokara, N. Ahalawat, D.G. Amooru, R. Subramanyam, J. Phys. Chem. B 114 (2010) 9054–9062. [4] J. Ghuman, P.A. Zunszain, I. Petitpa, A.A. Bhattacharya, M. Otagiri, S. Curry, J. Mol. Biol. 353 (2005) 38–52. [5] F. Samari, M. Shamsipur, B. Hemmateenejad, T. Khayamian, S. Gharaghani, Eur. J. Med. Chem. 54 (2012) 255–263. [6] M.C. Bof Oliveira, D. Mazera, M. Scarpellini, P.C. Severino, A. Neves, H. Terenzi, Inorg. Chem. 48 (2009) 2711–2713. [7] M. Barcelo-Oliver, A.A. Garcıa-Raso, A.A. Terron, E. Molins, M.J. Prieto, V. Moreno, J. Martınez, V. Llado, I. Lopez, A. Gutierrez, P.V. Escriba, J. Inorg. Biochem. 101 (2007) 649–659. [8] B. Selvakumar, V. Rajendiran, P.U. Maheswari, H.S. Evans, M. Palaniandavar, J. Inorg. Biochem. 100 (2006) 316–330. [9] D. Montagner, V. Gandin, C. Marzano, A. Erxleben, J. Inorg. Biochem. 145 (2015) 101–107. [10] M.T. Klepka, A. Drzewiecka-Antonik, A. Wolska, P. Rejmak, K. Ostrowska, E. Hejchman, H. Kruszewska, A. Czajkowska, I. Młynarczuk-Biały, W. Ferenc, J. Inorg. Biochem. 145 (2015) 94–100. [11] R.A. Steiner, D. Foreman, H.X. Lin, B.K. Carney, K.M. Fox, L. Cassimeris, J.M. Tanski, L.A. Tyler, J. Inorg. Biochem. 137 (2014) 1–11. [12] X. Qiao, Z.Y. Ma, J. Shao, W.G. Bao, J.Y. Xu, Z.Y. Qiang, J.S. Lou, Biometals 27 (1) (2014) 155–172. [13] A.G. Majouga, M.I. Zvereva, M.P. Rubtsova, D.A. Skvortsov, A.V. Mironov, D.M. Azhibek, O.O. Krasnovskaya, V.M. Gerasimov, A.V. Udina, N.I. Vorozhtsov, E.K. Beloglazkina, L. Agron, L.V. Mikhina, A.V. Tretyakova, N.V. Zyk, N.S. Zefirov, A.V. Kabanov, O.A. Dontsova, J. Med. Chem. 57 (14) (2014) 6252–6258. [14] R. Loganathan, S. Ramakrishnan, E. Suresh, M. Palaniandavar, A. Riyasdeen, M.A. Akbarsha, Dalton Trans. 43 (16) (2014) 6177–6194. [15] H. Mohammad, D. Babulal, K.C. Swapan, J. Indian Chem. Soc. 88 (4) (2011) 491–496. [16] S. Smita, D. Sumitra, B. Kishalay, K. Ramachandran Krishna, M. Tapas Kumar, G. Barindra Kumar, Polyhedron 30 (2) (2011) 387–396. [17] R. Hafijur, C. Habibar, B. Doyel, G. Rajarshi, H. Chen-Hsing, G. Barindra Kumar, Polyhedron 24 (13) (2005) 1755–1763. [18] R.H. Wang, D.Q. Yuan, F.L. Jiang, L. Han, Y.Q. Gong, M.C. Hong, Cryst. Growth Des. 6 (6) (2006) 1351–1360.

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