Synthesis, structure, DNA/protein binding, and cytotoxic activity of a rhodium(III) complex with 2,6-bis(2-benzimidazolyl)pyridine.

Accepted Manuscript Synthesis, structure, DNA/protein binding, and cytotoxic activity of a rhodium(III) complex with 2,6-bis(2-benzimidazolyl)pyridine Roya Esteghamat-Panah, Hassan Hadadzadeh, Hossein Farrokhpour, Jim Simpson, Amir Abdolmaleki, Fatemeh Abyar PII: S0223-5234(16)30943-6 DOI: 10.1016/j.ejmech.2016.11.005 Reference: EJMECH 9038 To appear in: European Journal of Medicinal Chemistry Received Date: 17 September 2016 Revised Date: 1 November 2016 Accepted Date: 3 November 2016 Please cite this article as: R. Esteghamat-Panah, H. Hadadzadeh, H. Farrokhpour, J. Simpson, A. Abdolmaleki, F. Abyar, Synthesis, structure, DNA/protein binding, and cytotoxic activity of a rhodium(III) complex with 2,6-bis(2-benzimidazolyl)pyridine, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT European Journal of Medicinal Chemistry Synthesis, Structure, DNA/Protein Binding, and Cytotoxic Activity of a Rhodium(III) Complex with 2,6-Bis(2-benzimidazolyl)pyridine T Roya Esteghamat-Panah,a Hassan Hadadzadeh,*a Hossein Farrokhpour,a Jim Simp P son,b Amir Abdolmaleki,a and Fatemeh Abyar c I R a Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran b Department of Chemistry, University of Otago, P.O. Box 56, Dunedin C 9054, New Zealand c Department of Engineering, Ardakan University, Ardakan S89518-95491, Iran U N Number of PAages: 48 Number oMf Schemes: 1 Number of Tables: 7 D Number of Figures: 17 E *Corresponding author, T P Hassan Hadadzadeh E Professor of Inorganic and Bioinorganic Chemistry Department of CChemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran E-mail address: hadad@cc.iut.ac.ir C A Crystallographic data for [Rh(bzimpy)Cl ]·3DMSO have been deposited with the 3 Cambridge Crystallographic Data Centre, CCDC 1500565. 1 ACCEPTED MANUSCRIPT European Journal of Medicinal Chemistry Synthesis, Structure, DNA/Protein Binding, and Cytotoxic Activity of a Rhodium(III) Complex with 2,6-Bis(2-benzimidazolyl)pyridine T P Roya Esteghamat-Panah,a Hassan Hadadzadeh,*a Hossein Farrokhpour,a Jim Simpson,b Amir Abdolmaleki,a and Fatemeh Abyar c I R a Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Iran C b Department of Chemistry, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand c Department of Engineering, Ardakan University, Ardakan S89518-95491, Iran U Abstract N A new mononuclear rhodium(III) complex, [Rh(bzimpy)Cl ] (bzimpy = 2,6-bis(2- 3 A benzimidazolyl)pyridine), was synthesized and characterized by elemental analysis and M spectroscopic methods. The molecular structure of the complex was confirmed by single- crystal X-ray crystallography. The interac tion of the complex with fish sperm DNA (FS- D DNA) was investigated by UV spectroscopy, emission titration, and viscosity measurement E in order to evaluate the possible DNA-binding mode and to calculate the corresponding T DNA-binding constant. The results reveal that the Rh(III) complex interacts with DNA P through groove binding mode with a binding affinity on the order of 104. In addition, the E binding of the Rh(III) complex to bovine serum albumin (BSA) was monitored by UV–Vis C and fluorescence emission spectroscopy at different temperatures. The mechanism of the C complex interaction was found to be static quenching. The thermodynamic parameters (∆H, A ∆S, and ∆G) obtained from the fluorescence spectroscopy data show that van der Waals interactions and hydrogen bonds play a major role in the binding of the Rh(III) complex to BSA. For the comparison of the DNA- and BSA-binding affinities of the free bzimpy ligand with its Rh(III) complex, the absorbance titration and fluorescence quenching experiments of the free bzimpy ligand with DNA and BSA were carried out. Competitive experiments using 2 ACCEPTED MANUSCRIPT eosin Y and ibuprofen as site markers indicated that the complex was mainly located in the hydrophobic cavity of site I of the protein. These experimental results were confirmed by the results of molecular docking. Finally, the in vitro cytotoxicity properties of the Rh(III) T complex against the MCF-7, K562, and HT-29 cell lines were evaluated and compared with P those of the free ligand (bzimpy). It was found that the complexation process improved the I anticancer activity significantly. R Keywords: Rhodium(III) complex, 2,6-bis(2-benzimidazolyl)pyridine based ligand, C DNA/protein binding, Cytotoxicity, Cancer cells, Molecular docking. S * Corresponding author. Tel.: +98 3133913240; fax: +98 3U133912350. E-mail address: hadad@cc.iut.ac.ir (H. Hadadzadeh). N A 1. Introduction M In recent years, the interaction of the transition metal complexes with DNA and protein D molecules has been an active research area in bioinorganic chemistry and has been shown to E play a very important role in medicine, pharmacy and diagnostics [1]. T It is well known that DNA is the primary pharmacological target of many anticancer P compounds [2]. The interaction of metal complexes with specific DNA sequences has been E studied in the hope of understanding the mechanism of their tumor inhibition for the design C of new drugs and recognizing specific sites or conformations targeted on the DNA [3,4]. It is C commonly known that small molecules can interact with double-stranded DNA through the A following three non-covalent modes: intercalation, groove binding, and electrostatic interactions [5]. Intercalative binding occurs when planar aromatic molecules are sandwiched between the DNA base pairs thereby distorting the backbone conformation of the DNA, while groove binding occurs due to hydrogen bonding or van der Waals interaction of the small molecules with bases of the DNA in the major or minor groove without causing any 3 ACCEPTED MANUSCRIPT significant distortion to the DNA backbone. An electrostatic interaction can occur between the negatively charged phosphate backbone of the DNA helix and the positively charged small molecules [6]. T In addition, binding of the metal complexes (drugs) with proteins is an important factor in P the pharmacokinetics and pharmacodynamics of the drugs [7]. Proteins are the most I important chemical substance in living organisms. The interactions of the proteins with R surfactants have been studied for several decades due to their various applications in C biological and pharmaceutical systems [8]. Serum albumin such as bovine serum albumin S (BSA) is the most abundant soluble protein in the plasma and plays an important role in the U transport and deposition of many drugs to the target sites [2,9]. The principal binding mode in N the interactions of proteins and drugs is non-covalent [7]. The investigation of drug–protein A interaction is essential for an understanding of the drug action mechanism and in designing M new medicines [3,10]. Designing effective anti-cancer drugs with high selectivity and low D toxicity is an active research area in the field of pharmaceutical chemistry [4,5]. The efficacy of the currently used anticancer Edrug, cisplatin, is reduced by several side-effects such as T neurotoxicity, nephrotoxicity, and drug resistance [11,12]. Therefore, considerable attempts P are being made to replace this drug with suitable anticancer agents [13]. As it is known that E rhodium complexes show interesting antitumor and antibacterial activities, they can be C introduced as promising candidates for the development of compounds related structurally to C the better known platinum analogues [14]. In the literature, there are many rhodium A complexes that interact with DNA and show high anticancer activity including [RhCl (N– 3 N)(DMSO)] (N–N = phen, bpy, dpphen, 1,10-phenanthroline-5,6-dione, and dmbpy) [15], Rh (µ-O CR) (R = Me, Et, Pr, and Bu) [16] and [(η5-C Me )RhCl(L)] (L = benzylidene(4- 2 2 4 5 5 tert-butylphenyl) amine 4-methyl ester) [17]. 4 ACCEPTED MANUSCRIPT The synthesis of transition metal complexes with 2,6-bis(2-benzimidazolyl)pyridine (bzimpy) (Scheme 1) has received considerable attention due to the biological properties of both the ligand and its complexes [18,19]. The tridentate ligand 2,6-bis(2- T benzimidazolyl)pyridine, constructing from both pyridyl and benzimidazolyl units [20,21], P exhibits a variety of biological activities, which include antiallergic, antibacterial, I antiproliferative [22], antitumor [23], and antiviral [24] properties. The benzimidazole moiety R is part of the chemical structure of vitamin B and contains an imidazole ring [18]. Despite 12 C the fact that the imidazole ring is a critical metal binding site in metalloproteins [18], ligands S containing imidazoles have been relatively less studied, although interest in their chemistry is U increasing [25]. Until now, the DNA-binding properties of some transition metal complexes N of bzimpy have been reported, e.g., three bzimpy complexes including A [Mn(bzimpy) ](pic) ·2DMF [18], [Zn(bzimpy)NO ]NO [19], and [Ni(bzimpy) ](pic) ·2DMF 2 2 M3 3 2 2 [26] bind to CT-DNA via an intercalative mode. Also, [Cr(bzimpy) ]Cl [27] and 2 D [Ru(bzimpy) ]Cl [28] complexes can bind to DNA with moderate affinity. 2 2 In this paper, we report the synEthesis, structural characterization, and biological properties T of the neutral mononuclear rhodium(III) complex ([Rh(bzimpy)Cl ]) and 2,6-bis(2- 3 benzimidazolyl)pyridine Pligand. The study of the biological properties of the complex has E been focused on (i) the binding properties with fish sperm DNA (FS-DNA) investigated by C electronic absorption titration, fluorescence spectroscopy, and viscosimetric measurement, C and (ii) the affinity for bovine serum albumin (BSA) investigated by UV–Vis and A fluorescence spectroscopy. The binding site of the Rh(III) complex on BSA was also assigned using fluorescence competition experiments and the nature of its interaction was analyzed based on thermodynamic parameters. In addition, the cytotoxicity of the Rh(III) complex against three human cancer cell lines, viz., breast carcinoma (MCF-7), erythroleukemia carcinoma (K562), and colorectal adenocarcinoma (HT-29) was evaluated in 5 ACCEPTED MANUSCRIPT vitro. Finally, the molecular docking studies were carried out to obtain detailed binding information of the complex and bzimpy with FS-DNA and BSA. T P I R C Scheme 1. The molecular structure of bzimpy. S 2. Experimental section U 2.1. Materials N The bzimpy ligand was synthesized according to the reported procedure [29]. All chemicals A and solvents were of high purity materials from Merck and were used without any further M purification. RhCl ·3H O and tris(hydroxymethyl)-aminomethane (Tris) buffer were of 3 2 analytical reagent grade also purchased from Merck. Double-stranded fish sperm D deoxyribonucleic acid (ds-FS-DNA), bovine serum albumin (BSA), RPMI 1640 culture E medium, site markers (eosin Y and ibuprofen), and 3-(4,5-dimethylthiazole-2-yl)-2,5- T diphenyl tetrazolium bromide (MTT) were obtained from Sigma-Aldrich. The solution of FS- P DNA gave a ratio ofE UV absorbance at 260 and 280 nm, A 260 /A 280 , of 1.8 – 1.9, indicating that the DNA wCas sufficiently free of protein [30]. The DNA concentration was determined by measurinCg the UV absorption at 260 nm, taking the molar absorption coefficient (ε 260 ) of FS-DN A A as 6600 M−1 cm−1 [31]. 2.2. Methods Elemental analysis (C, H, and N) was performed on a Heraeus CHN–O-Rapid elemental analyzer. IR spectra were recorded as KBr pellets on an FT-IR JASCO 680-PLUS spectrophotometer in the region of 4000–400 cm–1. UV–Vis spectra were measured on a 6 ACCEPTED MANUSCRIPT JASCO-570 spectrophotometer. Fluorescence measurements were performed on a SHIMADZU RF-5301PC spectrofluorimeter. 1H NMR data were obtained by a Bruker DRX- 400 MHz Avance spectrometer at ambient temperature in DMSO-d with tetramethylsilane 6 T (TMS; δ = 0 ppm) as the internal standard. Viscometric titrations were carried out using an P Ubbelohde viscometer maintained at a constant temperature of 298.0 (± 0.1) K in a I thermostatic water bath. R 2.3. Synthesis of the bzimpy ligand C The bzimpy ligand was prepared according to the Addison’s method [29]. Yield: 51%. Anal. S Calcd for C H N : C, 73.30; H, 4.21; N, 22.49%. Found: C, 73.26; H, 4.23; N, 22.52%. IR 19 13 5 U (KBr, cm–1): 3170 [ν(N–H)]; 1600 [ν(C=C)]; 1572 [ν(C=N)]. UV–Vis (DMSO): λ /nm (ε/L N max mol–1 cm–1): 307 (22600), 327 (25400). 1H NMR (DMSO-d , 400 MHz): δ/ppm; 7.29 (t, 2H), A6 7.36 (t, 2H), 7.75 (d, 2H), 7.79 (d, 2H), 8.19 (t, 1H), 8.36 (d, 2H), 12.99 (s, 2H). M 2.4. Synthesis of [Rh(bzimpy)Cl ]·3DMSO 3 D To a stirred solution of 2,6-bis(2-benzimidazolyl)pyridine (0.156 g, 0.50 mmol) in hot EtOH (10 mL) was added RhCl ·3EH O (0.132 g, 0.50 mmol) solution in 5 mL of ethanol. The 3 2 T mixture was refluxed for 6 h. The resulting precipitate was filtered off, washed with ethanol, and then air-dried. The Pdried precipitate was dissolved in DMSO and the yellow crystals E suitable for X-ray diffraction studies were obtained by slow diffusion of diethylether into this C solution of the complex over one week at room temperature. Yield: 89%. Anal. Calcd for C H Cl N C O RhS Anal. Calcd for C H Cl N O RhS : C, 39.77; H, 4.14; N, 9.28%. 25 31 3 5 3 3 25 31 3 5 3 3 A Found: C, 39.68; H, 4.09; N, 9.35%. IR (KBr, cm–1): 3163 [ν(N–H)]; 1610 [ν(C=C)]; 1590 [ν(C=N)]. UV–Vis (DMSO): λ /nm (ε/L mol–1 cm–1): 314 (30000), 338 (33150), 420 max (5650). 1H NMR (DMSO-d , 400 MHz): δ/ppm; 7.55 (t, 2H), 7.59 (t, 2H), 7.87 (d, 2H), 8.54 6 (d, 2H), 8.62 (t, 1H), 8.71 (d, 2H), 15.17 (s, 2H). 2.5. DNA binding studies 7 ACCEPTED MANUSCRIPT All the experiments involving FS-DNA were performed in buffer solution (5 mM Tris– HCl/10 mM NaCl, pH = 7.2). Tris-HCl buffer was prepared using doubly-distilled deionized water. A stock solution of FS-DNA was obtained by dispersing the desired amount of FS- T DNA in the buffer solution, stored at 4°C and used within 4 days. A concentrated stock P solution of the Rh(III) complex, because of its moderate solubility in water, was prepared by I dissolving it in an aqueous solution of DMSO as the co-solvent and diluting suitably with the R corresponding buffer to the required concentrations. The final DMSO concentration never C exceeded 0.1% v/v. The UV–Vis spectral feature of [Rh(bzimpy)Cl ] did not change on 3 S keeping its buffered solution for 48 h and no precipitation or turbidity was observed even U after long storage at room temperature (at least 3 weeks after the preparation of its solution), N which indicates the stability of the Rh(III) complex in the buffer media. In all the A experiments, the Rh(III) complex–DNA solutions were incubated for 7 min before the spectra M were recorded. D The interaction of the Rh(III) complex with FS-DNA was studied by UV spectroscopy in order to investigate the possible bEinding mode to DNA and to calculate the binding constant T to DNA (K ). The absorption titration experiment was carried out at a constant concentration b of the Rh(III) complex, Pviz. 20 µM, and various concentrations of DNA (0.0–49.0 µM) in E Tris-buffer. C The binding specificity (Stern–Volmer quenching constant) of the Rh(III) complex to FS- C DNA was also studied by the fluorescence titration method. Since the Rh(III) complex has a A fluorescence emission in the buffer solution, the binding of the complex to DNA can be directly predicted from its emission spectra. The fluorescence spectra were recorded at room temperature with excitation at 330 nm and emission at about 420 nm. The experiment was carried out by titrating DNA into the Rh(III) complex solution (5 × 10–6 M). 8 ACCEPTED MANUSCRIPT The binding mode of the Rh(III) complex was further investigated by viscosity measurements. In this experiment, the FS-DNA sample solution was prepared by sonication in order to minimize complexities arising from DNA flexibility [32]. The viscosity of the T DNA solution was measured in the absence and presence of increasing amounts of the P complex. Flow time of the DNA solution was measured using a digital stopwatch, taking the I average of three measurements. The data obtained were presented as (η/η )1/3 versus R0 [complex]/[DNA], where η and η are the viscosity measured for a DNA solution in the 0 C absence and presence of the complex, respectively. The relative viscosity values were S calculated according to the relation η = (t − t )/t , where t and t represent the flow times of 0 0 U0 the blank buffer and the DNA-containing solutions, respectively [33]. N 2.6. BSA binding studies A In order to study the BSA-complex interaction, solutions of BSA and the Rh(III) complex M were prepared by dissolving them in Tris–HCl/NaCl buffer solution (pH = 7.2) to the D required concentrations. The absorption titration expeEriment was done by keeping the concentration of BSA constant (6 × 10–5 M) while a T dding various concentrations the Rh(III) complex (0.0–1.2 µM). During the measurement Pof the absorption spectra, to eliminate the absorbance due to Rh(III) E complex, an equal amount of the Rh(III) complex solution was added to both the BSA and C the reference solutions. All of the data were obtained after each successive addition and C equilibration (ca. 7 min). A The emission quenching of the tryptophan residues of BSA was performed using the Rh(III) complex as a quencher. The fluorescence spectra were recorded after equilibration (ca. 5 min) at room temperature with an excitation wavelength of BSA at 290 nm and the emission at 342 nm, keeping the concentration of BSA constant (2 × 10–6 M) while varying 9 ACCEPTED MANUSCRIPT the complex concentration from 0.0 to 2.4 µM. The inner–filter effect was also taken into account but, it was not found to be significant for these measurements. 2.7. Site marker competitive experiments T The site competitive replacement experiments were performed in the presence of two site P markers (Eosin Y and Ibuprofen) using the fluorescence titration methods to identify the I binding location of the Rh(III) complex on BSA. Equimolar concentrations (2 × 10–6 M) of R BSA and eosin Y/ibuprofen were used and the Rh(III) complex was then added gradually. C The fluorescence spectra were recorded at 298 K with an excitation wavelength of 290 nm S with emission range of 300-500 nm. U 2.8. In vitro growth inhibition assay N 2.8.1. Cell culture A Human breast (MCF-7), leukemia (K562), and colorectal (HT-29) cancer cell lines were M purchased from the cell bank of the Pasteur Institute of Iran. The cell lines were cultured in D an RPMI 1640 medium. The medium was supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 2 mM glEutamine, and 1% penicillin and streptomycin (Invitrogen). T The cells were grown at 37°C under a 5% CO atmosphere. 2 2.8.2. Assessment of cytoPtoxicity E The cytotoxicity of the Rh(III) complex and the bzimpy ligand were studied using an MTT C assay [17]. To investigate the cytotoxic effect of the Rh(III) complex and the free bzimpy ligand, the c C ells were placed in 96-well plates at a density of 5 × 103 cells per well and were A grown at 37°C in a humidified 5% CO incubator. Then, the cells were treated with different 2 concentrations of the Rh(III) complex in an aqueous DMSO solution for 24 h. The control cultures were supplemented with the same amount of solvent containing DMSO. After the drug treatment, 20 µL of the MTT solution were added to each plate and incubation was performed at 37°C for 4 h. Then, 200 µL of DMSO were added to dissolve the formazan 10 ACCEPTED MANUSCRIPT crystals that formed and the absorbance was read at 570 nm using an ELISA reader (Bio-Tek, Elx 808, Germany). The cytotoxicity effect was recorded as the percentage of treated cells relative to untreated cells at A . The IC values were used to express the sensitivity of the 570 50 T cancer cells to the drug treatment and were obtained using Curve Expert software v1.3. P 2.9. Docking studies I The molecular docking calculations of the Rh(III) complex and the free bzimpy ligand with R DNA and BSA were performed using Autodock 4.2 software [34]. The Autodock 4.2 C software can be used to identify active sites at which the ligand binds to the receptor [34]. S The structure of DNA (PDB ID: 423D) with sequence d (ACCGACGTCGGT) and BSA U 2 (PDB ID: 4F5S) were taken from the protein data bank (PDB) [35] (RCSB) at a resolution of N 1.60 and 2.47 Å, respectively. Before starting the docking calculations, the structure of the A free bzimpy ligand was optimized using density functional theory (DFT) employing M062X M functional [36] and the 6-311+G(d,p) basis set for the C, H, and N atoms was selected The D structure of the complex was taken from the X-ray crystallography data. Firstly, a blind docking (BD) process was used tEo examine the interactions of the Rh(III) complex and the T bzimpy ligand with DNA and BSA [37-39]. The blind docking can identify the possible binding sites by scanninPg the entire surface of the target (DNA and BSA) and select the E location with the highest binding affinity. Then, the structures of DNA–complex, BSA– C complex, DNA–ligand, and BSA–ligand systems, which obtained from the blind docking, C were used as input structures for subsequent focus docking calculations. For the docking A studies of the Rh(III) complex and the free ligand with DNA, a grid map with dimensions of 126 × 126 × 126 Å3 along the x, y, and z directions, respectively, with grid-point spacing of 0.15 Å was generated and for the docking calculations of the Rh(III) complex and the free ligand with BSA, the grid map was set to 126 × 126 × 126 Å3 with a grid-point spacing of 0.375 Å in the x, y, and z directions, respectively. 11 ACCEPTED MANUSCRIPT 3. Results and discussion 3.1. Synthesis and crystal structure [Rh(bzimpy)Cl ] was synthesized in high yield by reaction of rhodium(III) chloride with 3 T bzimpy in ethanol at reflux temperature. The free bzimpy ligand is soluble in polar aprotic P solvents but insoluble in water. The synthesized Rh(III) complex is soluble in DMF and I DMSO, and is moderately soluble in water at room temperature. Air-stabRle yellow crystals of [Rh(bzimpy)Cl ]·3DMSO were grown by ether diffusion into a saturated solution of the 3 C complex in DMSO. The crystal structure was characterized successfully by X-ray diffraction S analysis. The crystal structure determination and the detaiUls of the X-ray measurements and crystal data for the Rh(III) complex (Table S1) have been reported in the Supporting N Information. The ORTEP drawing of the complexA is shown in Fig. 1. M D E T P E C C A Fig. 1. An ORTEP drawing of [Rh(bzimpy)Cl ] showing the atom numbering. The solvent 3 molecules (DMSO) have been omitted for clarity. 12 ACCEPTED MANUSCRIPT The structure comprises a [Rh(bzimpy)Cl ] complex molecule and three solvent DMSO 3 molecules. During refinement one of these solvent molecules was found to be seriously T disordered and a suitable disorder model could not be found. The SQUEEZE procedure [40] P was therefore used to remove the disordered molecule from the final refinement cycles, vide I supra. The Rh(III) cation is 6-coordinate with an N Cl coordination environment. The 3 3 R bzimpy ligand is predictably planar coordinating to the Rh center through the pyridyl N1 and C the two non-protonated imidazole N23 and N33 atoms. The chloride (Cl1) anion lies trans to S N1 and completes the equatorial plane of the distorted octahedral coordination environment U with two remaining chloride anions, Cl2 and Cl3, mutually trans in axial positions. The N degree of distortion in the octahedron is indicated by the trans angles that vary from A 178.89(8) to 160.04(11)o, Table 1, while the cis angles range from 80.00(11) to 100.27(7)o. M The bzimpy ligand is close to planar with an rms deviation of 0.0190 Å from the best fit D plane through all 24 non-hydrogen atoms. The Rh1 and Cl1 atoms lie close to this plane with deviations of -0.0254 (7) and -0E.0860 (15) Å, respectively. The observed planarity of the T bzimpy ligand fits well with the results of additional investigations showing the Rh(bzimpy)Cl complex iPnteract with DNA in a groove binding mode. 3 E Structures of bzimpy complexes of the transition metals are reasonably common with 91 C examples in the Cambridge crystallographic database [41]. However, to our knowledge this is C the first report of a structure of a rhodium complex of this ligand. Structures of transition A metal bzimpy complexes with an N Cl coordination environment are also rare with only 3 3 Cr(III) [42] and Fe(III) [43] derivatives reported previously. In the crystal structure, an extensive series of classical and non-classical hydrogen bonds are found (Table S2) and these are augmented by C—H···π contacts linking the complex to the DMSO solvent molecules. An offset π···π stacking interaction between the five and six 13 ACCEPTED MANUSCRIPT membered rings of the N31 benzimidazolyl ring system is also present with a centroid to centroid distance of 3.6505(19) Å well below the value of 4.0 Å considered to constitute a contact of reasonable strength [44,45]. These contacts combine to stack complex molecules T and DMSO solvent molecules along the b axis direction, Fig. S1. P I Table 1 Selected bond lengths and bond angles for [Rh(bzimpy)Cl ]·3DMSO. 3 R Bond lengths (Å) C Rh(1)-N(1) 1.962(3) S Rh(1)-N(23) 2.031(3) U Rh(1)-N(33) 2.039(3) Rh(1)-Cl(1) 2.3519(7) N Rh(1)-Cl(2) 2.3366(7) A Rh(1)-Cl(3) 2.3422(7) M Bond angles (°) N(1)-Rh(1)-N(23) 80.00(11) D N(1)-Rh(1)-N(33) 80.06(11) N(23)-Rh(1)-Cl(1) E100.27(7) N(33)-Rh(1)-Cl(1) T99.69(8) N(33)-Rh(1)-Cl(2) 90.69(7) P N(1)-Rh(1)-Cl(2) 88.62(8) E N(23)-Rh(1)-Cl(2) 89.59(7) C Cl(1)-Rh(1)-Cl(2) 90.31(3) Cl(1)-Rh(1)-CCl(3) 92.84(3) N(23)-RAh(1)-Cl(3) 88.83(8) N(33)-Rh(1)-Cl(3) 89.80(7) N(1)-Rh(1)-Cl(3) 88.24(8) N(23)-Rh(1)-N(33) 160.04(11) N(1)-Rh(1)-Cl(1) 178.89(8) Cl(2)-Rh(1)-Cl(3) 176.69(3) 14 ACCEPTED MANUSCRIPT 3.2. Spectroscopic studies The 1H NMR spectral data for the free bzimpy ligand (Fig. S2) and its Rh(III) complex (Fig. S3) were measured in DMSO-d using TMS as an internal standard. The 1H NMR 6 T spectrum of the complex indicates a diamagnetic behavior at room temperature. As shown in P Figs. S2 and S3, the spectral pattern of the bzimpy ligand changes in its complex due to the I coordination to the Rh(III) cation. R The IR spectral data for the free bzimpy ligand and the Rh(III) complex have been studied C to characterize the structure of the complex. The N–H stretching and bending vibrations of S the free ligand at 3170 and 1460 cm–1, remain either unperturbed or undergo only a slight U shift on complexation, suggesting that the amine (N–H) protons remain bound to their N N atoms [46]. In the free ligand, the C=N stretching frequency appears at 1572 cm–1. In the A Rh(III) complex, the C=N stretching vibration shifts to a higher frequency 1590 cm–1 and is M greatly reduced in intensity, which indicates binding of the imine nitrogen atom to the metal ion [47]. Also, the IR spectrum of th D e free ligand shows a sharp band at 1600 cm–1 due to ν(C=C) (ring) vibration, which caEn be found at about 1610 cm–1 in the complex [46]. T The electronic spectra of free bzimpy and its Rh(III) complex were recorded at 298 K in DMSO (Fig. 2). The bzimPpy ligand shows absorbance bands at 307 and 327 nm, which are E attributed to the π → π* transitions [18], whereas for the Rh(III) complex, the same C transitions occur at 314 and 338 nm. A metal-to-ligand charge-transfer transition [48-50] (MLCT, dπ C (Rh(III)) → π*(bzimpy)) is also observed as a shoulder at 420 nm that is not A present in the spectrum of the free ligand. 15 ACCEPTED MANUSCRIPT T P I R C Fig. 2. Electronic spectrum of (A) the free bzimpy ligand, andS (B) the Rh(III) complex (2 × U 10–5 M in DMSO) at room temperature. N A 3.3. DNA-binding properties M The interaction of the Rh(III) complex with FS-DNA was studied by UV–Vis spectroscopy, emission titration, and viscosity measurem ents. D 3.3.1. Electronic absorption titration E Upon the incremental addition of FS-DNA to the Rh(III) complex, the intensity of the T ligand centered π → π* absorption band (338 nm) decreases (Fig. 3A). The observed P hypochromic change without any shift in the UV spectrum of the complex suggests that the E complex binds to DNA through the groove binding mode [51]. The extent of the C hypochromism is normally found to correlate with the groove binding strength. The binding C constant of the complex with FS-DNA (K ) was determined by monitoring the changes of the A b absorbance at 338 nm using the equation (1) [52]: [DNA]/(ε – ε) = [DNA]/(ε – ε) + 1/K (ε – ε) (1) a f b f b b f where, [DNA] is the concentration of DNA in its base pairs, ε is the apparent extinction a coefficient calculated as A /[complex], ε is the extinction coefficient of the complex, and ε obs f b is the extinction coefficient of the complex when fully bound to DNA. A plot of [DNA]/(ε _ a 16 ACCEPTED MANUSCRIPT ε) versus [DNA] gives a slope and an intercept equal to 1/(ε – ε) and 1/K (ε – ε), f b f b b f respectively (Fig. 3B). The intrinsic binding constant, K b , is the ratio of the slope to the intercept and the value of K obtained for the Rh(III) complex has been found to be 2.50 × b T 104 M–1. P I R C S U N A M Fig. 3. (A) Electronic absorption spectra of the Rh(III) complex (2 × 10–5 M) in buffer solution (5 mM Tris–HCl/10 mM NaDCl at pH 7.2) in the presence of increasing amounts of FS-DNA (0.0–49.0 µM). The arrEow shows the intensity changes upon increasing the DNA concentration. (B) Plot of [DNTA]/(ε – ε) vs. [DNA]. a f P To calculate theE binding constant (K ) of the free bzimpy ligand with DNA, the b absorbance titratCion experiment of bzimpy was carried out in the same way as described for its Rh(III) Ccomplex (Fig. 4A). The value of K obtained for the free bzimpy ligand by b monitor A ing the changes of the absorbance at 327 nm has been found to be 1.20 × 104 M–1 (Fig. 4B). 17 ACCEPTED MANUSCRIPT T P I R C S Fig. 4. (A) Electronic absorption spectra of the bzimpy ligand (2 × 10–5 M) in buffer solution U (5 mM Tris–HCl/10 mM NaCl at pH 7.2) in the presence of increasing amounts of FS-DNA N (0.0–43.6 µM). The arrow shows the intensity changes upon increasing the DNA A concentration. (B) Plot of [DNA]/(ε – ε) vs. [DNA]. a f M D 3.3.2. Fluorescence quenching The absorption titration results Eindicate that the Rh(III) complex effectively binds to DNA. T To further investigate the binding mode between the complex and DNA, a fluorescence experiment was employePd. Fig. 5A shows the fluorescence emission spectra of the Rh(III) E complex and its fluorescence titration with FS-DNA. Upon each successive addition of DNA, C a gradual quenching of the fluorescence intensity was observed without any significant C change to the emission maxima. This provides a direct evidence for the interaction between A the Rh(III) complex and DNA through the groove binding mode [6]. To calculate the fluorescence quenching constant (K ), the observed fluorescence sv intensities of the Rh(III) complex were plotted against the DNA concentration (Fig. 5B) according to the Stern-Volmer equation (2) [53]: I /I = 1 + K [Q] (2) 0 sv 18 ACCEPTED MANUSCRIPT where, I and I are the fluorescence intensities of the Rh(III) complex in the absence and 0 presence of DNA (quencher) , respectively. [Q] is the concentration of the quencher and K sv is the Stern-Volmer quenching constant, which is a measure of the efficiency of quenching by T DNA. The K value of 2.1 × 104 M–1 for the Rh(III) complex was obtained which is sv consistent with values observed in other groove binding systems [54]. P I The Stern-Volmer plot is linear, indicating that only one type quenching process occurs, R either static or dynamic quenching which can be differentiated using equation (3) [55]: C k = K /τ (3) q sv 0 S where, τ is the average lifetime of the biomolecule without any quencher and taken as 0 U 10–8 s [56] and k is the apparent bimolecular quenching rate constant. Apparently, the value q N of k (2.1 × 1012 M–1 s–1) was greater than the value of the maximum scatter collision q A quenching constant (2.0 × 1010 M–1 s–1) [57]. Thus, the interaction between the Rh(III) M complex and DNA is a static quenching process [58]. D E T P E C C A Fig. 5. (A) Emission spectra of the Rh(III) complex at room temperature in Tris–HCl/NaCl buffer solution (pH = 7.2) in the absence and presence of FS-DNA, λ = 330 nm. The arrow ex shows the intensity changes upon increasing the concentration of FS-DNA (0.0–38.1 µM). (B) Plot of I /I vs. [DNA]. 0 19 ACCEPTED MANUSCRIPT To calculate the fluorescence quenching constant (K ) and quenching rate constant (k ) for sv q the free bzimpy ligand with DNA, the fluorescence quenching experiment was performed T with the similar method that was carried out in the case of its Rh(III) complex (Fig. 6A). The K and k values obtained for the free bzimpy ligand are 1.03 × 104 M–1 and 1 P .03 × 1012 M–1 sv q I s–1 , respectively (Fig. 6B). R C S U N A M D E T Fig. 6. (A) Emission spectra of the free bzimpy ligand (5 × 10–6 M) at room temperature in P Tris–HCl/NaCl buffer solution (pH = 7.2) in the absence and presence of FS-DNA, λ = 320 ex E nm. The arrow shows the intensity changes upon increasing the concentration of FS-DNA C (0.0–54.5 µM). (B) Plot of I /I vs. [DNA]. 0 C A 3.3.3. Viscosity measurements Viscosity measurements were carried out by keeping the DNA concentration constant (200 µM) and varying the concentration of the Rh(III) complex. A slight increase in the viscosity value is observed which is not as pronounced as the increase observed for the classical intercalator EtBr (Fig. 7). In a classic intercalation binding mode, the DNA base pairs are 20 ACCEPTED MANUSCRIPT separated in order to host the bound compound leading to an overall increase in the length of the DNA double helix with a subsequent increase in the DNA viscosity [59,60] whereas a small molecule, that binds exclusively in the DNA grooves under the same conditions, causes T lesser or no change to the viscosity of the DNA solution [6,61,62]. These results again P suggest that the Rh(III) complex interacts with the groove of DNA, in agreement with the I above experimental results. R C S U N A M Fig. 7. The effect of adding the Rh(III) complex and EtBr to FS-DNA; the relative viscosity D (η/η )1/3 vs. [complex]/[DNA]; [DNA] = (200 µM). 0 E T 3.4. BSA-binding properties P The interaction between BSA and [Rh(bzimpy)Cl ] was investigated by UV–Vis 3 E spectroscopy and a tryptophan emission-quenching experiment. C 3.4.1. Absorption spectral studies C The UV-Visible absorption titration of BSA with the Rh(III) complex was done to A investigate the nature of the quenching process. The quenching mechanism may follow a static or dynamic process. Static quenching typically results from a complex formation reaction between the fluorophore and a quencher in the ground state, while dynamic quenching arises from an interaction between the fluorophore and a quencher in a short-lived 21 ACCEPTED MANUSCRIPT excited state [63]. As shown in Fig. 8A, the BSA displays an absorption band at 278 nm due to the presence of three aromatic amino acids (Trp, Tyr, and Phe) [64,65]. Addition of the Rh(III) complex to BSA leads to an enhancement of the intensity of T absorption without affecting the position of the absorption band. This result confirms that the P Rh(III) complex interacts with BSA molecule, resulting in an alteration of the I microenvironment of the three aromatic amino acid residues [65]. Also, this suggests the R presence of a static interaction between the complex and BSA due to the formation of a C ground state complex-BSA system [10]. S In order to investigate the interaction of the free bzimpy ligand with BSA, the absorbance U titration experiment of BSA with bzimpy was carried out in the same way as described for its N Rh(III) complexes (Fig. 9A). The results indicate that the free bzimpy ligand interacts with A BSA and the microenvironment of the three aromatic amino acid residues is altered. M 3.4.2. Fluorescence spectroscopic studies D In order to examine the binding characteristics of the Rh(III) complex with BSA and to confirm the static complex formEation between BSA and the Rh(III) complex that was T indicated by the absorption spectroscopy results, a fluorescence quenching experiment was carried out. The emissionP spectrum of BSA in the buffer medium (excited at 290 nm) shows a E characteristic peak at 342 nm that is mainly due to the presence of tryptophan residues [66]. C Addition of successive amounts of the Rh(III) complex to BSA results in a progressive C decrease in the fluorescence intensity as shown in Fig. 8B. A 22 ACCEPTED MANUSCRIPT T P I R Fig. 8. (A) UV–Vis absorption spectra of BSA (6 × 10–5 M) in the absence and presence of C the various concentrations of the Rh(III) complex (0.0–1.2 µM) in Tris–HCl/NaCl buffer. S The arrow shows the intensity changes upon increasing the concentration of the Rh(III) U complex. (B) The fluorescence quenching curve of BSA (2 × 10–6 M) in the absence and N presence of the Rh(III) complex (0.0–2.4 µM). The arrow indicates changes in emission A intensity of BSA upon increasing the concentration of the Rh(III) complex. M After correction for the inner-filter effDect [3] using equation (4) [67]: I = I exp(½A + ½A ) (4) corr obs ex em E (where, I is the correctedT fluorescence in the absence of inner-filter effect, I is the corr obs measured fluorescence bPefore correction for the inner-filter effect, and A ex and A em are the absorption values aEt excitation and emission wavelength, respectively,) the corrected fluorescence intCensities at three different temperatures (298, 303, and 308 K) were used to investigate Cthe interaction between the Rh(III) complex and BSA. The fluorescence quenchAing data were further analyzed by the Stern−Volmer relation which again can be expressed in terms of a bimolecular quenching rate constant and average life time of the fluorophore as shown in equation (5) [68]: I /I = 1 + K [Q] = 1 + k τ [Q] (5) 0 sv q 0 23 ACCEPTED MANUSCRIPT I and Iare the fluorescence intensities in the absence and presence of the Rh(III) complex (as 0 a quencher) after correction for the inner-filter effect, respectively, K is the Stern–Volmer sv quenching constant, [Q] is the quencher concentration, k the quenching rate constant, and τ q 0 T the average lifetime of the molecule without quencher. Its value is considered here to be 10−8 s [69]. The K and k values obtained from the plot of I /I versus [Q], a P t temperatures sv q 0 I ranged from 298 to 308 K (Fig. 10), are given in Table 2. The calculated value of k is 5.58 × Rq 1013 M–1 s–1 at 298 K, that is about three orders of magnitude higher than that expected for a C purely dynamic quenching mechanism (2.0 × 1010 M–1 s–1). This confirms that the S fluorescence quenching of BSA by the Rh(III) complex is a static quenching process [70]. In U addition, the type of the quenching mechanism can be distinguished by its differing N dependence on the temperature. In the case of static quenching, the bound complex becomes A less stable with increase in the temperature of the solution, and hence, the Stern–Volmer M quenching constant decreases, whereas for a dynamic quenching process, higher temperatures D result in faster diffusion, and consequently, K values increase [71]. With increase in the sv temperature, the K values shoEw a decreasing trend, indicative of a static quenching sv T mechanism (Fig. 10). To calculate the fluoresPcence quenching constant (K ) and quenching rate constant (k ) for sv q E the free bzimpy ligand with BSA, the tryptophan quenching experiment was carried out with C the similar method that was performed in the case of its Rh(III) complex (Fig. 9B). The K sv C and k values obtained for the free bzimpy ligand after correction for the inner-filter effect are q A 4.51 × 105 M–1 and 4.51 × 1013 M–1s–1 at 298 K, respectively.. 24 ACCEPTED MANUSCRIPT T P I R C Fig. 9. (A) UV–Vis absorption spectra of BSA (6 × 10–5 M) in the absence and presence of S the various concentrations of the free bzimpy ligand (0.0–1.0 µM) in Tris–HCl/NaCl buffer. U The arrow shows the intensity changes upon increasing the concentration of bzimpy. (B) The N fluorescence quenching curve of BSA (2 × 10–6 M) in the absence and presence of the free A bzimpy ligand (0.0–2.4 µM), λ = 290 nm. The arrow indicates changes in emission intensity ex M of BSA upon increasing the concentration of bzimpy. Inset: Stern–Volmer plot of I /I vs. the 0 concentration of bzimpy after correctiDon for the inner-filter effect. E T P E C C A Fig. 10. Stern–Volmer plots of I /I vs. the concentration of Rh(III) complex at different 0 temperatures after correction for the inner-filter effect. 25 ACCEPTED MANUSCRIPT Table 2 Quenching parameters for the Rh(III) complex–BSA system at different temperaturesa. T (K) K (L mol–1) k (L mol–1 s–1) R sv q CC 298 5.58 × 105 5.58 × 1013 0.9988 T 303 2.45 × 105 2.45 × 1013 0.9996 P I 308 1.27 × 105 1.27 × 1013 0.9853 R a Note: R is the correlation coefficient for the Stern–Volmer plot. CC C S 3.5. Binding parameters U When a static quenching mechanism is involved in a reaction, the relationship between the N fluorescence quenching intensity and the concentration of the quencher can be described A using the Scatchard equation (6) [72]: M log (I – I/I) = log K + n log [Q] (6) 0 b where, K and n are the binding constant and the number of the binding sites, respectively. b D The number of the binding sites and the binding constant have been obtained from the slope E and intercept of the linear plot of log[(I – I)/I] versus log [Q]. At 298 K, the number of the T0 binding sites for the Rh(III) complex on DNA (Fig. 11A) and BSA (Fig. 11B) are 0.70 and P 1.06, respectively. The calculated values of the number of the binding sites (n) on both DNA E and BSA are therefore around 1 indicating the existence of a single available binding site for C the Rh(III) complex in both systems [73]. The values of K determined from the fluorescence C b quenchAing experiments show a good conformity with the values of the binding constants obtained from the UV–Vis titrations. 26 ACCEPTED MANUSCRIPT T P I R Fig. 11. Determination of the binding constant and number of the binding sites for the Rh(III) C complex on (A) DNA, and (B) BSA at room temperature. S U The K and n values for the reaction of BSA with the Rh(III) complex at different b N temperatures are summarized in Table 3. The values of the binding constant decrease with A increasing temperature, because of the reduction in the stability of the complexes between the M BSA and the Rh(III) complex [74]. The number of the binding sites, n, for the Rh(III) complex on BSA varies only slightly with increase in temperature. D E Table 3 Regression equation, binding constant (K ), number of the binding sites (n), and T b correlation coefficient (R), for the interaction of the Rh(III) complex with BSA at different P temperatures. E T (K) RegrCession equation K b (L mol–1) n R 298 log[(I – I)/I] = 5.956 + 1.067 log[Q] 9.04 × 105 1.067 0.9994 C0 303 log[(I – I)/I] = 5.467 + 1.014 log[Q] 2.93 × 105 1.014 0.9996 A0 308 log[(I – I)/I] = 5.167 + 1.013 log[Q] 1.47 × 105 1.013 0.9877 0 3.6. Thermodynamic parameters and the nature of the binding interaction The binding forces between small molecules and biological macromolecules include van der Waals interactions, hydrophobic forces, hydrogen bonds, and electrostatic interactions 27 ACCEPTED MANUSCRIPT [75]. In order to identify the type of the interaction forces that act between the Rh(III) complex and BSA, various thermodynamic parameters such as the enthalpy change (∆H) and entropy change (∆S) of the interaction were calculated using the Van’t Hoff equation (7) T [76]: P lnK = –∆H/RT + ∆S/R (7) b I where, R is the gas constant, T is the experimental temperature, and K is the binding constant b R at T. Also, the free energy change (∆G) was determined using equation (8): C ∆G = ∆H – T∆S (8) S The values of ∆H, ∆S, and ∆G are summarized in Table 4. U Table 4 Thermodynamic parameters of the binding of the Rh(III) complex with BSA at the N different temperatures a. A T (K) ∆H (kJ mol–1) ∆S (J mol–1 K–1) ∆G (kJ mol–1) R M CC 298 –138.77 –352.23 –33.80 0.9836 303 D –32.04 308 –30.28 E a Note: R is the correlation coefficient for the Van't Hoff plot. CC T P The negative values of the free energy, ∆G, at different temperatures support the assertion E that the binding process between the Rh(III) complex and BSA is spontaneous. The negative C values of ∆H and ∆S indicate that van der Waals interactions and hydrogen bonds play major C roles in the binding process [77] and contribute to the stability of the resulting complex. A 3.7. Site-selective binding of the Rh(III) complex on BSA The BSA protein consists of three domains (I–III) with two subdomains (A and B) on each domain [73]. The principal complex-binding sites on the albumin are located in subdomains IIA (Sudlow’s site I) and IIIA (Sudlow’s Site II) [78]. To locate whether binding of the Rh(III) complex to BSA occurs at site I and/or II, competitive site marker competitive 28 ACCEPTED MANUSCRIPT experiments were performed using eosin Y and ibuprofen as site markers for Sudlow's site I and II, respectively [79,80]. Information about the complex-binding site on the BSA can be obtained by monitoring the changes in the emission of the Rh(III) complex bound to BSA in T the absence and presence of both of the markers separately. As shown in Fig. 12A, the Trp P emission intensity at 342 nm decreases after the addition of ibuprofen. The fluorescence I intensity is clearly lower than that without ibuprofen indicating that the ibuprofen molecule R has already bound to BSA. When the Rh(III) complex is added to the ibuprofen–BSA system, C the complex must compete with ibuprofen in order to bind to BSA, that is if they bind on the S same site. A similar phenomenon can be observed with eosin Y in Fig. 12B. U N A M D E T P E C C A Fig. 12. Fluorescence spectra of BSA in the absence and presence of the site marker (A) Ibuprofen, and (B) Eosin Y in the presence of the various concentrations of the Rh(III) complex. Both the concentration of BSA and the site markers are 2 × 10−6 mol L−1. The blue arrow shows the emission intensity changes upon increasing the concentration of the Rh(III) complex. 29