Synthesis, structure, DNA interaction and nuclease activity of rhodium(III)–arylazoimidazole complexes

Inorganica Chimica Acta 394 (2013) 98–106

Contents lists available at SciVerse ScienceDirect

Inorganica Chimica Acta journal homepage: www.elsevier.com/locate/ica

Synthesis, structure, DNA interaction and nuclease activity of rhodium(III)–arylazoimidazole complexes Dibakar Sardar a,1, Papia Datta a,2, Sanju Das a, Biswarup Saha b, Saheli Samanta b, Debalina Bhattacharya b, Parimal Karmakar b, Chung-De Chen c,d, Chun-Jung Chen c,d, Chittaranjan Sinha a,⇑ a

Department of Chemistry, Jadavpur University, Kolkata 700032, West Bengal, India Department of Life Science and Biotechnology, Jadavpur University, Kolkata 700 032, India Life Science Group, National Synchroton Radiation Research Centre, 101, Hsin Ann Road, Hsinchu 30076, Taiwan d Department of Physics, National Tsing Hua University, Hsinchu 30043, Taiwan b c

a r t i c l e

i n f o

Article history: Received 7 April 2012 Received in revised form 31 July 2012 Accepted 5 August 2012 Available online 29 August 2012 Keywords: Rhodium(III)–arylazoimidazoles Structure DNA binding Nuclease activity DFT calculation

a b s t r a c t The Rh(III) complexes of 1-alkyl-2-(arylazo)imidazoles (RaaiR0 ), [Rh(X)2(RaaiR0 )2](ClO4) (R = H (1, 3), Me (2, 4); R0 = Me (a), Et (b), CH2Ph (c); X = Cl (1, 2), N3 (3, 4)), are synthesized and characterized by spectral (IR, UV–Vis, Mass and 1H NMR) data. The structural confirmation has been done by single crystal X-ray diffraction study of [RhCl2(MeaaiEt)2]ClO4 (2b) (MeaaiEt, 1-ethyl-2-(p-tolylazo)imidazole). The DNA binding of the complexes (binding constant: 0.86  104 to 1.05  105) have been examined by absorption and fluorescence spectroscopic measurements. The nuclease activity of the complexes are examined by gel-electrophoresis and has revealed the highest activity of [Rh(N3)2(RaaiR0 )2]ClO4. The DFT computation has been performed to the optimized geometry of the complexes to interpret the electronic structures and their spectral properties. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction The interaction of transition metal complexes with DNA has received vast attention in the last two decades of research both in chemistry and biology [1–7]. The interaction of the metal complexes with DNA is disturbing the replication and transcription and ultimately leads to the cell death [8,9]. Over the past few years, an intensive effort has been focused to develop metal-based drugs with improved clinical effectiveness, reduced toxicity and broader spectrum of activity [1–3,10–13]. The complexes of zinc [14], molybdenum [15], palladium [16,17] and ruthenium [18,19] are exhibiting anticancer activity. Many of them are very promising, and show activity on tumors. Recently rhodium complexes have been used in the development of metal-based drugs in cancer chemotherapy [20–22]. They have been shown to bind nucleobases, dinucleotides, and DNA dodecamer single strands [23–25]. This has inspired us to study the interaction and nuclease activity of rhodium complexes of N-donor ligands with DNA. In this article the spectral and structural characterization of rhodium(III) complexes of 1-alkyl-2-(arylazo)imidazoles (RaaiR0 ) are described. ⇑ Corresponding author. Fax: +91 033 2413 7121. E-mail address: [email protected] (C. Sinha). Present address: Dinabandhu Andrews College, Garia, Kolkata 700084, India. RCC Institute of Information Technology, Canal South Road, Kolkata 700015, India. 1 2

0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ica.2012.08.005

The DNA binding ability of the complexes is established by spectroscopic studies and nuclease activity is considered by gelelectrophoresis. The electronic properties are correlated with DFT calculation. 2. Experimental 2.1. Materials and methods All reagents and solvents were of analytical grade except those employed in DNA binding and cleavage experiments which were of spectroscopic grade. RhCl3.3H2O was purchased from Arora-Matthey, India and used as it was received. NaN3, NaClO4 and the solvents used were obtained from E. Merck, India. The ligands used in this work were 1-alkyl-2-(arylazo)imidazole [RaaiR0 , where R = H, Me and R0 = CH3, CH2–CH3, CH2Ph] (Scheme 1) and were prepared by reported procedure [26]. Chemicals used for syntheses were analytical grade and solvents were dried before use [27]. The solution spectral studies were carried out by spectroscopic grade solvents obtained from Lancester, UK. Caution! Azide and perchorate salts are generally explosive. Although no detonation tendencies have been observed, care is advised and handling of only small quantities recommended. Microanalyses (C, H, N) were performed using Perkin-Elmer 2400 CHN elemental analyzer. Spectroscopic measurements were

D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106

99

Scheme 1. The ligands and the complexes.

carried out using the following instruments: UV–Vis spectra, Lambda 25 Perkin Elmer; FT-IR spectra (KBr disk, 4000– 225 cm1), Perkin Elmer Spectrum BX1; 1H NMR spectra in CDCl3, Bruker 300 MHz FT-NMR spectrometer in presence of TMS as internal standard. FAB-MS was collected from Jeol-AX 500 instrument. Electrochemical measurements were carried out with a computer controlled CH1 Electrochemical workstation using a Pt-disk working electrode and Pt-wire auxiliary electrode, [n-Bu4N][ClO4] supporting electrolyte under inert (dry N2) environment at scan rate 50 mV S1. The solution was IR compensated and the results were collected at 298 K. The reported results were referenced to SCE in MeCN and were uncorrected for junction potential. 2.2. Synthesis of [RhCl2(RaaiR0 )2]ClO4 (1, 2) All the complexes were prepared following common procedure as detailed below for 2a. Yield varied 65–75%. 2.2.1. [RhCl2(MeaaiMe)2]ClO4 (2a) RhCl3.3H2O (0.10 g, 0.315 mmol) and 1-methyl-2-(arylazo)imidazole (MeaaiMe) (0.125 g, 0.63 mmol) in 1:2 mol ratio in methanol solution was refluxed for 6 h under stirring condition and was cooled to room temperature. The volume of the solution was reduced under pressure and chromatographed with silica gel prepared in petroleum ether. A red solution was eluted with 1% NaClO4 solution in MeOH and dried in vacuum and recrystallised from acetonitrile–methanol (1:1, v/v) solution. The yield was 0.14 g (68%). [RhCl2(MeaaiMe)2]ClO4 (2a): Anal. Calc. for C22H24N8O4Cl3Rh: C, 39.31; H, 3.57; N, 16.68. Found: C, 39.23; H, 3.50; N, 16.68%. FT-IR (KBr, cm1) m(ClO4), 622(w) and 1100 (vs); m((N = N), 1361 (m); m(C = N), 1597 (s). UV–Vis data (kmax(CH3CN)/nm; e,103 M1 cm1) 500 (2.667), 405 (6.453), 214 (10.32)). MS(FAB+), m/z 490 (MClO4)+. 1H NMR (CDCl3) 8.14 (4-H, bs), 7.88 (5-H, bs), 7.90 (7,11-H, d, J = 8.0 Hz), 7.63 (8,10-H, d, J = 8.0 Hz), 2.49 (9-Me, s),

4.11 (-N- CH3, s). The spectral data of other complexes are given as Supplementary materials (Tables S1–S3). 2.2.2. Synthesis of [Rh(N3)2(RaaiR0 )2]ClO4 (3, 4) The complexes are synthesized following common procedure as detailed below for 4a. The yield was varied 68–75%. [Rh(N3)2(MeaaiMe)2]ClO4 (4a). To acetonitrile solution (10 ml) of [RhCl2(MeaaiMe)2]ClO4 (2a) (0.2 g, 0.297 mmol), an aqueous solution of AgNO3 (0.1 g, 0.594 mmol) was added and stirred for few minutes. The orange brown mixture was then heated to reflux for 30 min, then cooled and filtered through G-4 Gooch. To this filtrate NaN3 (0.038 g, 0.594 mmol) was added at room temperature and stirred for 3 h. Or the addition of mixture of AgNO3 and NaN3 at once for metathetical reaction also resulted precipitate of AgCl. It was filtered further and treated with aqueous NaClO4 (1 g in 3 ml water). A pale yellow precipitate immediately appeared. The product was collected by filtration, washed with methanol and ether and then dried under vacuum. Yield is 0.15 g (71%). [Rh(N3)2(MeaaiMe)2]ClO4 (4a). Anal. Calc. for C22H24N14O4ClRh: C, 38.57; H, 3.51; N, 28.63. Found: C, 38.63; H, 3.60; N, 28.73%. FTIR (KBr, cm1) m(ClO4), 624(w) and 1095(vs); m(N = N), 1390 (m); m(C = N), 1593 (s); m(N3), 2026(s). UV–Vis data (kmax(CH3CN)/nm; e, 103 M1 cm1) 435 (1.62), 351 (1.10), 281 (1.18). MS(FAB+), m/z 587 (MClO4)+. 1H NMR (CDCl3) 8.12 (4-H, bs), 7.93 (5-H, bs), 8.02 (7,11-H, d, J = 8.0 Hz), 7.48 (8,10-H, d, J = 8.0 Hz), 2.50 (9CH3, s), 4.16 (–N–CH3, s). The spectral data of other complexes are given as Supplementary materials (Tables S1–S3). 2.3. X-ray crystallography Dichloromethane solution of [RhCl2(MeaaiEt)2] ClO4 (2b) was diffused into hexane and the crystals were grown within a week. A suitable single crystal of was mounted on a CCD Diffractometer equipped with fine-focus sealed tube graphite monochromated Mo-Ka (k = 0.71073 Å) radiation. Unit cell parameters were

100

D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106

determined from least-squares method. Summary of the crystallographic data and structure refinement parameters are given in Table 1. Data were corrected for Lorentz and polarization effects. Data reduction was carried out by using ‘Bruker SAINT’ programme. The structure was solved by direct method using SHELXS-97 and successive difference Fourier syntheses. All non-hydrogen atoms were refined anisotropically. Full matrix least squares refinements on F02 were carried out using SHELXL-97 with anisotropic displacement parameters for all non-hydrogen atoms. Hydrogen atoms were constrained to ride on the respective carbon or nitrogen atoms with anisotropic displacement parameters equal to 1.2 times the equivalent isotropic displacement of their parent atom in all cases. All calculations were carried out using SHELXS 97 [28], SHELXL 97 [29], PLATON 99 [30] and ORTEP [31] programs. 2.4. Theory and computational methods In the framework of density functional theory (DFT) approach, the B3LYP hybrid functional [32,33] one of the most preferred methods since it proved its ability in reproducing various molecular properties, including structural parameters and vibrational spectra is used. The combined use of B3LYP consisting of a hybrid exchange functional, as defined by Becke’s three-parameter equation and the Lee–Yang–Par correlation and standard split valence basis set 6-31G(d) is a common technique to provide an excellent compromise between the accuracy and computational efficiency of properties for large and medium-size molecules [34–36]. Ground-state electronic structure calculations of all compounds had been performed using the Los Alamos effective core potential plus double zeta (LanL2DZ) [37] basis set for rhodium and 631G(d) for other elements. The ground-state geometries were obtained in the gas phase by full geometry optimization and the optimum structures, located as stationary points on the potential energy surfaces, were verified by the absence of imaginary frequencies. In all cases, vibrational frequencies were calculated to ensure that optimized geometries represented local minima. Using the respective optimized S0 geometries we employed time dependent density functional theory (TD-DFT) at the B3LYP level to predict their absorptions characteristics [38]. The calculated data are given as Supplementary material (Tables S4 and S5).

Table 1 Crystallographic parameters of [RhCl2(MeaaiEt)2]ClO4 (2b). Chemical_formula_sum Crystal size (mm) Chemical formula weight Crystal system Space group a (Å) b (Å) c (Å) b (°) V (Å3) Z T (K) Density (Mg/m3) l (mm1) F (0 0 0) 0

k (Å A) (Mo-Ka) hrange (°) Index range Unique reflections R (Fo)a [I > 2r (I)] wR (Fo)b [I > 2r (I)] Goodness-of-fit (GOF)

C24H28N8O4Cl3Rh 0.1  0.1  0.1 701.80 Monoclinc C2/c 12.9971(7) 17.6354(7) 37.9265(18) 95.5870(10) 8651.8(7) 12 273(2) 1.616 0.840 4056 0.71073 1.08 – 28.33 17 6 h 6 17, 23 6 k 6 23, 50 6 l 6 47 9856 0.0732 0.1456 1.417

a

R = R||Fo|  |Fc||/R |Fo|. wR = [Rw(Fo2  Fc2)2/Rw(Fo2)2]1/2, where P = (Fo2 + 2Fc2)/3. b

w = 1/[r

2

(Fo)2 + (0.0000P)2 + 99.7145P]

2.5. Interaction of complexes with DNA To examine the interaction of the complexes with DNA we have selected four complexes [RhCl2(HaaiMe)2]ClO4 (1a), [RhCl2(MeaaiMe)2]ClO4 (2a), [RhCl2(MeaaiEt)2]ClO4 (2b) and [Rh(N3)2(MeaaiEt)2]ClO4 (4b) with reference to the variation of substituents in chelated ligand (H/Me (1a/2a) and Me/Et (2a/2b)) and also substituting chloride by azide (2b/4b). 2.5.1. Preparation of the complex solution for DNA binding and nuclease studies The title complexes were readily soluble in acetone free methanol at a concentration of 2 mM each. The stock complex solutions were diluted freshly in citrate–phosphate buffer (10 mM), pH 7.4 before each set of experiments. 2.5.2. Preparation of calf thymus and pUC19 plasmid DNA Purified calf thymus (CT) DNA (Bangalore Genei, India) and pUC19 plasmid DNA (Bangalore Genei, India) were dissolved separately at a concentration of 1 mg/ml stock in sterilized 10 mM citrate–phosphate (CP) buffer, pH 7.4 at room temperature. Aliquots were stored at 4 °C and diluted freshly before each set of experiments. 2.5.3. Ethidium bromide (EB) stock solution preparation Ethidium bromide (EB) (Sigma–Aldrich, USA) was dissolved in double distilled water at a concentration of 1 mM and stored at 4 °C in dark. The stock solution was diluted freshly before each experiment. 2.5.4. Absorption spectroscopic studies of the complexes in presence of CT DNA Absorption spectroscopic studies were done on a spectrophotometer (Perkin Elmer, lambda-25), using either CT DNA (35 lg/ ml) with increasing concentrations of complex (0.5–20 lM) or each complex (40 lM) with increasing concentrations of CT DNA (0.13 lg/ml to 11.4 lg/ml). After each addition, the DNA and complex mixtures were incubated at room temperature for 15 min and scanned either from 210 nm to 310 nm for the DNA or from 290 nm to 600 nm for the complexes. The self-absorption of ligands in either case was eliminated in each set of experiments. Each sample was scanned for a cycle number of 2, cycle time of 5 s at a scan speed of 100 nm/min. Modified Benesi–Hildebrand [39] plot was used for the determination of ground state binding constant between the complexes and CT DNA. The binding constant ‘‘K’’ was determined by using the following relation:

A0 =DA ¼ A0 =DAmax þ ðA0 =DAmax Þ  1=K  1=Lt where DA = A0  A, DAmax = maximum change in reduced absorbance, A0 = maximum absorbance of receptor molecules (without any ligand), A = reduced absorbances of the receptor molecules (in presence of ligand), Lt = ligand concentration. 2.5.5. Fluorescence spectroscopic studies of the complexes with EB bound DNA The fluorescence spectroscopic studies of CT DNA (35 lg/ml) with varying concentrations of the complexes were done by using a spectrofluorimeter (F3010, HITACHI). This study is based on the competitive binding of the complex to the EB saturated DNA by replacing EB. In this experiment at first, EB solution was gradually added to the said concentration of DNA and at each time, the fluorescence spectrum was scanned. The fluorescence intensity of EB bound DNA was saturated at 8.45 lM concentration of EB. At this saturation level, the complex was added gradually (up to 34.7 lM), incubated for 15 min after each addition and the fluorescence spectra were taken. The excitation wavelength was 546 nm

101

D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106

Fig. 1. Molecular structure of two conformers of of cis-[RhCl2(MeaaiEt)2]ClO4 (2b).

Table 2 Selected bond distances (Å) and bond angles (°) for 2b (X-ray structure and theoretical). Molecule-1

a b

Molecule-2 Experimental

Calculateda

Bond distances (Å) Rh(1)–Cl(4) Rh(1)–N(10) Rh(1)–N(11) N(9)–N(10) N(11)–C(26)

2.3164(14) 2.051(4) 2.002(5) 1.286(6) 1.339(7)

2.4012 2.1496 2.0259 1.3088 1.3566

Rh(2)–N(7) Rh(2)–N(5) Rh(2)–N(1) Rh(2)–N(2) Rh(2)–Cl(2) Rh(2)–Cl(1) N(1)–N(4) N(2)–N(3)

2.011(4) 2.013(4) 2.047(4) 2.057(4) 2.3224(13) 2.3242(13) 1.284(6) 1.289(6)

Bond angles(°) N(10)–Rh(1)–N(10)b N(11)–Rh(1)–N(10)b N(11)–Rh(1)–N(10) N(10)–Rh(1)–Cl(4)b N(10)–Rh(1)–Cl(4) N(11)–Rh(1)–N(11)b N(11)–Rh(1)–Cl(4)b N(11)–Rh(1)–Cl(4) Cl(4)–Rh(1)–Cl(4) 

84.0(2) 96.53(18) 77.79(18) 170.14(13) 91.65(12) 172.4(3) 93.96(14) 91.20(14) 93.94(7)

91.49 103.62 77.16 169.09 88.67 178.91 92.02 87.04 93.23

N(5)–Rh(2)–N(7) N(5)–Rh(2)–N(1) N(7)–Rh(2)–N(1) N(5)–Rh(2)–N(2) N(7)–Rh(2)–N(2) N(1)–Rh(2)–N(2) N(5)–Rh(2)–Cl(2) N(7)–Rh(2)–Cl(2) N(1)–Rh(2)–Cl(2) N(2)–Rh(2)–Cl(2) N(5)–Rh(2)–Cl(1) N(7)–Rh(2)–Cl(1) N(1)–Rh(2)–Cl(1) N(2)–Rh(2)–Cl(1) Cl(2)–Rh(2)–Cl(1)

169.85(18) 77.76(17) 94.28(16) 95.06(16) 77.74(16) 84.48(16) 91.49(13) 95.14(12) 92.22(12) 171.87(12) 94.82(13) 92.63(12) 171.56(12) 92.27(12) 91.85 (5)

As per atom numbering of molecule-1. Symmetry: x + 1, y, z + 1/2.

Experimental

102

D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106

and the emission spectra were scanned from 556 nm to 650 nm at spectral response of 2s, along with a scanning speed of 60 nm/min [40].

8000 7000

-1

4000

ε / M cm

5000

-1

6000

2.5.6. Nuclease activity of the complexes by agarose gel electrophoresis study Purified pUC19 plasmid DNA (500 mg) was incubated with increasing concentrations of complex (17–100 lM) at 37 °C for 1 h in dark and then the DNA samples were loaded in 1% agarose gel in 1 TASE buffer (pH 7.4) at a potential gradient of 6 volts/ cm for 2.5 h [40]. Each gel was then stained with EB solution (500 lg/ml), photographs were taken in a gel documentation system (Transilluminator, UVPRO) and analysed.

3000 2000 1000 0 200

3. Results and discussions

300

400

500

600

700

Wavelength (nm) 3.1. The complexes and their formulation 0

The reaction of RhCl3 with 1-alkyl-2-(arylazo)imidazole (RaaiR ) in 1:2 mol ratio in methanol followed by addition of saturated aqueous solution of NaClO4 (Scheme 1, Eqs. (1) and (2) has separated a red precipitate, [RhCl2(RaaiR0 )2]ClO4. The azide derivative [Rh(N3)2(RaaiR0 )2 ]ClO4 (where R = H (3), Me (4) and R0 = Me (a), Et (b). CH2Ph (c) is synthesised by metathesis with AgNO3 + NaN3 mixture. The ligand, 1-alkyl-2-(arylazo)imidazole (RaaiR0 ), is N, N0 chelating system where N and N0 refer to N(imidazole) and N(azo) donor centers, respectively (Scheme 1). The formulation of the complexes, [RhCl2(RaaiR0 )2]ClO4 (1, 2) and [Rh(N3)2(RaaiR0 )2]ClO4 (3, 4) has been supported by microanalytical data (see Section 2) and mass spectra. The structure of one of the complexes, [RhCl2(MeaaiEt)2]ClO4 (2b) has been established by single crystal X-ray diffraction study. All the complexes are diamagnetic and 1:1 conducting which indicates the presence of rhodium in +3 oxidation state (d6). The complexes are soluble in common organic solvents. 3.2. Molecular structure of [RhCl2(MeaaiEt)2]ClO4 (2b) The molecular structure of [RhCl2(MeaaiEt)2]ClO4 (2b) is given in Fig. 1, and metric parameters are listed in Table 2. The asymmetric unit of the lattice consists of one molecule in a general position and half molecule which sits on a 2-fold axis i.e. the asymmetric unit has 1.5 molecules. In terms of bond distances and angles these two molecules have comparable values (see Supplementary materials). The complex comprises two chelating MeaaiEt ligands which has two potential imidazolyl-N (abbreviated N) and azo-N (abbreviated N0 ) (Scheme 1) and two chloride, thus the central Rh atom has a distorted octahedral coordination defined by a Cl2N2N0 2 donor set. The atomic arrangement involves cis located chlorine, and N0 donors, while Ns, are in trans position. The cis chlorine angles are Cl(4)–Rh(1)–Cl(4)  ( symmetry: x + 1, y, z + 1/2), 93.94(7)° and Cl(2)–Rh(2)–Cl(1), 91.98(5)°. The bond distances between rhodium and azo-N (Rh-N0 ) are Rh(1)–N(10), 2.051(4); Rh(2)–N(1), 2.047(4); Rh(2)–N(2), 2.057(4) Å those are comparable with reported results [35]. The rhodium–chlorine (Rh(1)–Cl(4), 2.3164(14); Rh(2)–Cl(1), 2.3242(13); Rh(2)–Cl(2), 2.3224(13) Å) distances are also comparable with reported data [41,42]. The Rh–N bond distances in the present example are Rh(1)–N(11), 2.002(5); Rh(2)–N(5), 2.013(4); Rh(2)–N(7), 2.011(4) Å) those are lower than the reported Ru–N(imidazole) distances [43,44]. The DFT calculation has been performed using optimized geometry of the complex of C2 symmetry (molecule 1). The experimental structure data are comparable with the calculated data (Table 2). Gasphase calculation shows longer bond lengths than corresponding experimental bond distances [45]. It is usual because in the gas

Fig. 2. UV–Vis spectra of the complexes cis-[RhCl2(MeaaiEt)2]ClO4 (2b) (—) and cis[Rh(N3)2(MeaaiEt)2]ClO4 (4b) (- - -) in acetonitrile at room temperature.

cis- [RhCl2(MeaaiEt)2]ClO4 (2b) cis-[Rh(N3)2(MeaaiEt)2]ClO4 (4b) Fig. 3. Energy correlation diagram of cis-[RhCl2(MeaaiEt)2]ClO4 (2b) cis-[Rh(N3)2(MeaaiEt)2]ClO4 (4b).

phase molecules are isolated while in solid state molecules are interacting themselves through some noncovalent interactions. The theoretically calculated Rh–N0 and Rh–N distances of 2b are longer than the crystallographic data by 0.02–0.08 Å. All other bonds were found slightly longer than the corresponding solid state bond lengths. One of the structures of asymmetric unit (molecule 1) agreed well with the theoretically determined structure (Fig 1) of the complex. Bond angles are also in close agreement with the crystallographic data and are varied by only ca. 0–7° from crystal structure values. 3.3. Spectral studies and correlation with DFT data In FT-IR spectra two moderately strong bands appear at 1360– 1390 and 1595–1598 cm1; these are assigned to m(N@N) and m (C@N), respectively. The azo and imine stretching frequency are significantly shifted to lower frequency compared to free ligand value (m (N@N), 1400–1410 cm1 and m (C@N), 1600–1610 cm1) [26] which supports coordination of Rh(III) with the ligand. The complexes 1 and 2 show two m(Rh–Cl) stretches within the range 310–340 cm1 in the complexes which support their cis-RhCl2 type configuration. The presence of ClO4 as a counter ion is confirmed by a strong, broad peak at 1090–1100 cm1 and a weak stretch

D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106

103

Fig. 4. (a) Absorption spectroscopic study of 40 lM 2a with increasing concentrations of CT DNA (0, 1.14, 2.28, 3.42, 4.56, 5.7, 6.84, 7.98, 9.12, 10.26 and 11.4 lg/ml), respectively (1 ? 11). (b) Absorption spectroscopic study of CT DNA (35 lg/ml) with increasing concentrations of 2a (0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 lM), respectively (1 ? 11). (c) Modified Benesi–Hildebrand plot for the determination of ground state binding constant between CT DNA and 2a.

at 624 cm1. [Rh(N3)2(RaaiR0 )2]ClO4 (3, 4) show characteristic strong transmission at 2026–2030 cm1 with a weak band at 2045–2050 cm1 in infrared spectrum. These are corresponding to masym(N3) [46–48]. This supports terminal N3 bonding in these complexes. The 1H NMR spectra of the complexes were recorded in CDCl3 and assigned on comparing with free ligand data [26]. Imidazole protons, 4- and 5-H suffer downfield shifting by 0.7–0.8 ppm compared to the free ligand position and appear as a broad singlet at 8.09–8.14 and 7.83–7.90 ppm, respectively. This supports the strong preference of binding of imidazole-N to Rh(III). The singlet nature of imidazole protons may be due to rapid proton exchange at the NMR time scale. 1-Me appears as a singlet at 4.30 ppm; 1CH2–CH3 gives a quartet, 4.69–4.72 ppm (J = 8.0 Hz) and a triplet 1.61–1.70 ppm (J = 7.5 Hz) to –CH2– and –CH3 protons, respectively. The 7- and 11-H in the complexes 3 and 4 appear at are higher d (8.04 ppm) compared to the complexes 1 and 2 (7.92 ppm). The absorption spectra of the complexes were investigated in acetonitrile solution (Fig. 2). The red solution of the complexes exhibit three bands in the wavelength range 200–500 nm. The visible spectra of 1 and 2 are dominated by relatively intense asymmetric band with kmax  405 nm (e, 103 M1 cm1) along with a shoulder at kmax  500 nm. These bands are assigned to the superposition of pp (Cl) ? p ⁄ (azo) and dp (Rh) ? p⁄ (azo). These bands experience the blue shift (by 50–60 nm) upon replacement of Cl by N3. From

DFT calculation, it is observed (Fig. 3) that the electronic structures of the complexes are sensitive to nature of the equatorially bonded chloride or azide group. The energy of HOMO of 1a, 2a and 2b are comparable (EHOMO = 9.03 eV) and more stable than 4b (EHOMO = 8.29 eV). The energy of LUMO of 4b (ELUMO = 6.24 eV) is comparable with other complexes (ELUMO = 6.36 eV). In case of 1a, 2a, 2b the major contribution (60%) comes from chloride while azide contribution is 80% to the HOMO of 4. The energy separation between HOMO and LUMO is higher for 1a, 2a and 2b (DEHOMO–LUMO = 2.65 eV) compared to 4b (DEHOMO–LUMO = 2.05 eV). The bonding scheme has unequivocally suggested the existence of two fragments in the complexes, namely electron donor group Rh(X)2 (X = Cl, N3) and electron acceptor group MeaaiR (R = Me, Et), respectively. Detail of composition of MOs are enlisted in Table S4 (vide Supplementary material). Time-dependent DFT (TDDFT) calculations are used to predict observed transition energies and used to elucidate their origin. The visible region of the experimental spectra of 1 and 2 are dominated by intense asymmetric band with kmax  405 nm along with a shoulder at kmax  500 nm. The longest wavelength calculated signal at 565 nm in the visible region, is mainly attributed to transitions from the HOMO-1 ? LUMO, HOMO-2 ? LUMO and HOMO-3 ? LUMO. Detail of spectral assignment are given in Table S5 (vide Supplementary material) Since LUMO of the ligand is mainly composed of p⁄ of the azo bond, the type of transition is attributed to the mixture of pp (Cl) ? p⁄ (azo) and dp (Rh) ? p⁄

104

D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106

Fig. 5. Comparative fluorescence spectroscopic study of 2a with EB bound CT DNA. Excitation wavelength was 546 nm and the emission spectra were scanned from 556 to 650 nm in each case. (a) The mode of EB saturation in CT DNA (35 lg/ml). The maximum fluorescence intensity of EB bound DNA complex was observed at 8.45 lM of EB concentration. At this point of saturation, 2a was added gradually up to 34.7 lM till its saturation level, which is represented in figure b (see text for detail).

(azo). The experimentally obtained band at 405 nm region may be assigned the admixture of p ? p⁄ (intraligand) and chlorideto-ligand charge transfer transition i.e. pp (Cl) ? p⁄ (azo). In case of 4 the lower energy transitions ca. 433 nm and 349 nm are blue shifted compared to 1 or 2 and can be assigned as admixture of intraligand, azide-to-ligand and metal-to-ligand charge transfer transitions.

complex 4b (Supplementary material Fig. S2b). The hypochromic effect of DNA by complex 4b may be due to the stringent configuration of the aromatic bases in the negatively charged DNA molecules in presence of heavy negative charge of the azide ligand in the said complex. Moreover, the presence of more negative charges in complex 4b renders the less binding affinity towards DNA. Thus by changing the ligands in the rhodium complexes, we have observed differential binding pattern of the same towards DNA.

3.4. Interaction of complexes with DNA 3.4.1. Absorption spectroscopic studies of the complexes in presence of CT DNA The interaction of the complexes with chromosomal CT DNA are investigated by spectrophotometric method. When CT DNA is added with increasing concentrations to a fixed concentration of complex 1a or 2a, the absorption is increased with a slight red shift (Fig. 4a) while the addition of 1a or 2a to a fixed concentration of DNA solution shows decrease in absorption of the DNA band (Fig. 4b). Such differential absorption characteristics may be due to the specific interaction of the complex with DNA molecules resulting more relax structure of the complex but on the contrary, a more rigid structure of the DNA bases. At the absorption maximum of DNA, we have calculated the binding constant between the complexes and DNA by using modified Benesi–Hildebrand (BH) plot (Fig. 4c) and the binding constant are 1.05  105 M1 (1a) 1.02  105 M1 (2a). Similar experiments were done for other complexes and their binding constants with DNA are 3.479  104 M1 (2b) and 1.36  104 M1 (4b). In the complexes 2b and 4b, addition of DNA result hypochromic effects with slight red shift (Supplementary material Figs. S1 and S2). The addition of the complex to the DNA solution has resulted slight increase in the absorption maxima of the DNA in the case of complex 2b (Supplementary material Fig. S1b) and decrease the same in case of

3.4.2. Fluorescence spectroscopic studies of the complexes with ethidium bromide (EB) bound DNA Fluorescence spectroscopic studies have been conducted to find the interaction of DNA with rhodium complexes. Having clear evidences that the rhodium complexes interact with DNA, we next observed that whether the set of complexes can replace ethidium bromide (EB) from DNA, which has an association constant of 3.4  103 M1 towards any double-stranded DNA [48,49]. As a matter of fact, either DNA or EB or even rhodium complexes do not have fluorescence property alone. So we have used the fluorescence property of EB bound DNA to compare the DNA binding ability of the complexes with DNA. When EB saturated DNA (Fig. 5a) is added to the solution of 1a and 2a the complex partially replaces EB from DNA (Fig. 5b) which is evidenced from the fluorescence intensity change. EB–DNA association constant is 3.4  103 M1 [40]. Similarly, complexes 2b and 4b replace EB from DNA (Supplementary material Figs. S3 and S4, respectively) partially. But comparing the results, it is obvious that the degree of EB displacement from DNA by the complexes were different, where the maximum EB displacement is achieved by complex 4b and that of minimum by complex 2b. Though it is not required to be an intercalator to reduce the fluorescence intensity of the EB bound DNA, rather the complex might also interact with DNA molecules in such a manner (possibly slight unwinding

D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106

105

Fig. 6. Agarose gel (1%) electrophoresis study of pUC19 plasmid DNA (500 ng) with increasing concentrations of 2a, 2b and 4b in figure a, b and c, respectively. Purified pUC19 plasmid DNA was treated with increasing concentrations of the said complex (0, 17, 33, 50, 67, 83 and 100 lM in lanes 1 ? 7, respectively) for 1 h and was run at a potential gradient of 6 volts/cm for 2.5 h. Lane 8 in figure a represents the control plasmid, treated with maximum amount of methanol used in lane 7. Bar diagram below each agarose gel represents the normalized RF1 band intensity of the plasmid DNA in each lane with respect to that of mock treated DNA sample in lane 1.

of the DNA backbone) that induced a structural alteration in the DNA molecules, resulting the release of EB from DNA, which might not be associated with competitive EB and rhodium complex intercalation towards the DNA molecule. Such differential behavior of the complexes may be due to the different atomic orientation and associated charged property of the ligands in the complexes. These rhodium complexes also have low-lying energy states that might quench the emission of EB by means of energy transfer when both molecules are bound to DNA in a close proximity. The rhodium complexes in the solution absorb some of the excitation light at 546 nm, where DNA–EB complex mostly absorbs. This might results a reducing number of effective photons that can be absorbed by EB absolutely. As a matter of fact, complex 4b reduces the EB emission more than 2b, which can be understood by the greater extinction coefficient of the former (750 M1 cm1) compared to the later (200 M1 cm1) at this excitation wavelength. The complexes may also absorb some of the emitted light of EB, reducing the detected signal even further as their concentration is increased.

3.4.3. Nuclease activity of the complexes by agarose gel electrophoresis study We have observed the nuclease property of rhodium complexes on plasmid DNA by agarose gel electrophoresis study. The pUC19 plasmid DNA (500 ng) was incubated for an hour with increasing concentrations of the complexes at 37 °C in dark and then subjected to 1% agarose gel electrophoresis at a potential gradient of 6 volt/cm. After staining with ethidium bromide solution, it was observed that complexes 1a and 2a (Fig. 6a), having highest binding affinity, induced slower migration of the super-coiled (RF1) plasmid DNA and also induced DNA single-strand breakage (RF2) in the plasmid back-bone, while complex 2b, having moderate binding constant, mostly induced nicks (RF2) in the super-coiled form of DNA (Fig. 6b). But interestingly, having least binding

constant, complex 4b was capable of inducing DNA double-strand cleavage activity (RF3) directly from RF1 without forming any intermediate RF2 form of DNA. As a result, it leads to a complete degradation of the DNA molecules and therefore the RF1 form of DNA were gradually vanished at the higher concentrations of complex 4b (compare lanes 2–7 with respect to lane 1 in Fig. 6c). Thus among all the four complexes, 1a and 2a show similar nuclease activity and complex 4b has a unique nuclease activity. 4. Conclusion We have prepared Rh(III) complexes of 1-alkyl-2-(arylazo)imidazoles having N, N0 donor centers and chloride or azide ions as other coordinating ligands. All the complexes are characterized by elemental analyses and spectroscopic data and one of them is structurally characterized. Although the complexes bind with DNA at variable extent, complexes 1a and 2a bind most strongly and least binding was observed in case of complex 4b. Among all the complexes, complex 4b exhibits unique nuclease activity. Changing of substituent within the heterocyclic ligand does not affect much the nuclease activity but the replacement of chlorides chloride with azide ions causes a significant improvement of the nuclease activity. Acknowledgements Financial support from Council of Scientific and Industrial Research (CSIR) and Department of Science and Technology (DST) New Delhi are gratefully acknowledged. 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.ica.2012.08.005.

106

D. Sardar et al. / Inorganica Chimica Acta 394 (2013) 98–106

References [1] L.D. Dale, J.H. Tocher, T.M. Dyson, D.I. Edwards, D.A. Tocher, Anti-cancer Drug Des. 7 (1992) 3. [2] E.R. Jamieson, S.J. Lippard, Chem. Rev. 99 (1999) 2467. [3] H.M. Pineto, J.H. Schornagel, Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy, Plenum, New York, 1996. [4] J. Suh, Acc. Chem. Res. 33 (2003) 562. [5] W.H. Ang, S. Pilet, R. Scopelliti, F. Bussy, L. Juillerat-Jeanneret, P.J. Dyson, J. Med. Chem. 48 (2005) 8060. [6] B. Rosenberg, L. VanCamp, T. Krigas, Nature 205 (1965) 698. [7] M.A. Fuertes, C. Alonso, J.M. Perez, Chem. Rev. 103 (2003) 645. [8] B.K. Keppler (Ed.), Metal Complexes in Cancer Chemotherapy, VCH, Weinheim, 1993. [9] P.M. Takahara, A.C. Rosenzweig, C.A. Frederick, S.J. Lippard, Nature 377 (1995) 649. [10] C. Metcalfe, J.A. Thomas, Chem. Soc. Rev. 32 (2003) 215. [11] K.E. Erkkila, D.T. Odom, J.K. Barton, Chem. Rev. 99 (1999) 2777. [12] C.B. Spillane, J.L. Morgan, N.C. Fletcher, J.G. Collins, F.R. Keene, Dalton Trans. (2006) 3122. [13] P.U. Maheswari, V. Rajendiran, H. Stoeckli-Evans, M. Palaniandavar, Inorg. Chem. 45 (2006) 37. [14] A. Banerjee, S. Ganguly, T. Chattopadhyay, K.S. Banu, A. Patra, S. Bhattacharya, E. Zangrando, D. Das, Inorg. Chem. 48 (2009) 8695. [15] J.B. Waern, M.M. Harding, Inorg. Chem. 43 (2004) 206. [16] E. Budzisz, M. Małecka, I.P. Lorenz, P. Mayer, R.A. Kwiecien, R.A. Paneth, U. Krajewska, Inorg. Chem. 45 (2006) 9688. [17] J. Reedijk, Eur. J. Inorg. Chem. (2009) 1303. [18] E. Corral, A.C.G. Hotze, H. den Dulk, A. Leczkowska, A. Rodger, M.J. Hannon, J. Reedijk, J. Biol. Inorg. Chem. 14 (2009) 439. [19] G. Sava, A. Bergamo, S. Zorzet, B. Gava, C. Casrsa, M. Cocchietto, A. Furlani, V. Scarcia, B. Serli, E. Iengo, E. Alessio, G. Mestroni, Eur. J. Cancer 38 (2002) 427. [20] J.D. Aguirre, D.A. Lutterman, A.M. Angeles-Boza, K.R. Dunbar, C. Turro, Inorg. Chem. 46 (2007) 7494–7502. [21] R. Bieda, I. Ott, R. Gust, W.S. Sheldrick, Eur. J. Inorg. Chem. (2009) 3821. [22] K. Sorasaenee, Inorg. Chem. 41 (2002) 433. [23] M. Dobroschke, Y. Geldmacher, I. Ott, M. Harlos, L. Kater, L. Wagner, R. Gust, S. William Sheldrick, P. Aram, ChemMedChem 4 (2009) 177. [24] C.A. Crawford, E.F. Day, V.P. Saharan, K. Folting, J.C. Huffman, K.R. Dunbar, G. Christou, Chem. Commun. (1996) 1113. [25] H.T. Chifotides, K.M. Koshlap, J. Am. Chem. Soc. 125 (2003) 10703. [26] T.K. Misra, D. Das, C. Sinha, Polyhedron 16 (1997) 4163. [27] A.I. Vogel, A.R. Tatchell, B.S. Furnis, A.J. Hannaford, P.W.G. Smith, in: Text Book of Practical Organic Chemistry, fifth ed., Prentice Hall, 1996. [28] G.M. Sheldrick, SHELXS 97, ‘‘Program for the Solution of Crystal Structure’’, University of Gottingen, Germany, 1997.

[29] G.M. Sheldrick, SHELXL 97, Program for the Refinement of Crystal Structure, University of Gottingen, Germany, 1997. [30] A.L. Spek, PLATON, ‘‘Molecular Geometry Program’’, University of Utrecht, The Netherlands, 1999. [31] L.J. Farrugia, J. Appl. Cryst. 30 (1997) 565. [32] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H.P. Hratchian, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, J.A. Pople, Gaussian 03, Revision C.02, Gaussian, Inc., Wallingford, CT, 2004. [33] C. Lee, W. Yang, R.G. Parr, Phys. Rev. B 37 (1988) 785. [34] H.G. Korth, M.I. De Heer, P. Mulder, J. Phys. Chem. A 106 (2002) 8779. [35] B.G. Johnson, P.M.W. Gill, J.A. Pople, J. Chem. Phys. 98 (1993) 5612. [36] V. Chis, Chem. Phys. 300 (300) (2004) 1. [37] P.J. Hay, W.R. Wadt, J. Chem. Phys. 82 (1985) 270. [38] EMSL, Basis Set Library. Available at: . [39] H.A. Benesi, J.H. Hildebrand, J. Am. Chem. Soc. 71 (1949) 2703. [40] B. Saha, A. Mukherjee, C.R. Santra, A. Chattopadhyay, A.N. Ghosh, U. Choudhuri, P. Karmakar, J. Biomol. Struct. Dyn. 26 (2009) 421. [41] S.P. Dutta, Shie-Ming, S. Bhattacharya, J. Chem. Soc., Dalton Trans. (2000) 4623–4627. [42] K. Pramanik, U. Das, B. Adhikari, D. Chopra, H. Stoeckli-Evans, Inorg. Chem. 47 (2008) 429. [43] S. Pal, T.K. Misra, C. Sinha, A.M.Z. Slawin, J.D. Woollins, Polyhedron 19 (2000) 1925. [44] P. Byabartta, S.K. Jasimuddin, G. Mostafa, Polyhedron 22 (2003) 849. [45] W.J. Hehre, A Guide to Molecular Mechanisms and Quantum Chemical Calculations, Wavefunction Inc., Irvine, CA, 2003. [46] P. Bhuina, U.S. Ray, G. Mostafa, J. Ribas, C. Sinha, Inorg. Chim. Acta. 359 (2006) 4660. [47] U.S. Ray, B.K. Ghosh, M. Monfort, J. Ribas, G. Mostafa, T.-H. Lu, C. Sinha, Eur. J. Inorg. Chem. (2004) 250–259. [48] R.F. Ziolo, M. Allen, O.D. Titus, H.B. Gray, Z. Dori, Inorg. Chem. 11 (1972) 3044. [49] P.V. Scaria, R.H. Shafer, J. Biol. Chem. 266 (1991) 5417.