New [Ru(5,6-dmp/3,4,7,8-tmp)₂(diimine)]²⁺ complexes: non-covalent DNA and protein binding, anticancer activity and fluorescent probes for nuclear and protein components.

Journal of Inorganic Biochemistry 116 (2012) 151–162 Contents lists available at SciVerse ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio New [Ru(5,6-dmp/3,4,7,8-tmp)2(diimine)] 2+ complexes: Non-covalent DNA and protein binding, anticancer activity and ﬂuorescent probes for nuclear and protein components Venugopal Rajendiran a, 1, Mallayan Palaniandavar a,⁎, Vaiyapuri Subbarayan Periasamy b, Mohammad Abdulkader Akbarsha b a b School of Chemistry, Bharathidasan University, Tiruchirapalli 620 024, India Department of Animal Science, Bharathidasan University, Tiruchirapalli 620 024, India a r t i c l e i n f o Article history: Received 8 January 2012 Received in revised form 2 June 2012 Accepted 4 June 2012 Available online 18 June 2012 Keywords: Ru (II) polypyridyl complexes DNA binding Protein binding Anticancer activity Nuclear staining agent Protein staining agent a b s t r a c t A series of Ru(II) complexes of the type [Ru(5,6-dmp)2(diimine)] 2+ 1–3 and [Ru(tmp)2(diimine)] 2+ 4–6, where 5,6-dmp is 5,6-dimethyl-1,10-phenanthroline, tmp is 3,4,7,8-tetramethyl-1,10-phenanthroline and diimine is dipyrido-[3,2-d:2′,3′-f]-quinoxaline (dpq), dipyrido[3,2-a:2′,3′-c]phenazine (dppz) and 11,12dimethyl-dipyrido[3,2-a:2′,3′-c]phenazine (11,12-dmdppz), has been isolated and the DNA binding mode of the complexes studied by using emission and circular dichroic (CD) spectral techniques. All the complexes exhibit induced circular dichroism upon binding to calf thymus (CT) DNA and show preferential binding to AT and mixed (d(CGCGATCGCG)2) sequences rather than to GC sequences. The complex [Ru(tmp)2(dpq)]2+ 4 exhibits enhancement in luminescence higher than [Ru(5,6-dmp)2(dpq)]2+ 1 upon binding to DNA. In contrast, [Ru(5,6-dmp)2(dppz)]2+ 2 and [Ru(5,6-dmp)2(dmdppz)] 2+ 3 exhibit luminescence enhancement higher than [Ru(tmp)2(dppz)]2+ 5 and [Ru(tmp)2(dmdppz)] 2+ 6 respectively upon DNA binding, illustrating the importance of hydrophobic forces of interaction in determining the DNA binding afﬁnity. Among the complexes, 4 exhibits the highest enhancement in ﬂuorescence intensity upon binding to the protein bovine serum albumin (BSA). The cytotoxicity of the complexes has been studied by screening them against nonsmall lung carcinoma (NCI-H460) cell line. It is noteworthy that the complex showing the strongest DNA binding afﬁnity exhibits the highest cytotoxicity. The efﬁciency of the complexes as ﬂuorescent probes for detection of nuclear morphology and proteins has been evaluated by using ﬂuorescence microscopy. Remarkably, 4, which shows strong hydrophobic forces of interaction when bound to DNA and protein, acts as ﬂuorescent probes for detection of nuclear components in the head, and proteins in the tail, of sperms. © 2012 Elsevier Inc. All rights reserved. 1. Introduction Coordination complexes of Ru(II) with polyazaheterocyclic chelating ligands have found widespread application as protein markers in hydrodynamic and photoinduced electron-transfer studies [1–5], as molecular probes of DNA [6,7] and as stains for both isolated DNA and that in ﬁxed cell nuclei [8]. The most interesting complexes investigated so far are [Ru(bpy)2(dppz)] 2+ (bpy = 2,2′-bipyridine) [9] and [Ru(phen)2(dppz)] 2+ (phen = 1,10-phenanthroline) [10], both containing the intercalating ligand dipyrido[3,2-a:2′,3′-c]phenazine (dppz). They bind to DNA with the highest afﬁnity (Kb > 10 6 M − 1) ⁎ Corresponding author at: Department of Chemistry, School of Basic and Applied Sciences, Central University of Tamil Nadu, Thiruvarur 610 004, India. Fax: + 91 4312407043. E-mail addresses: palanim51@yahoo.com, palaniandavarm@gmail.com (M. Palaniandavar). 1 Present address: Department of Chemistry, School of Basic and Applied Sciences, Central University of Tamil Nadu, Thiruvarur 610 004, India. 0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2012.06.005 [9,10] and, interestingly, although lacking luminescence in aqueous solution, they show intense luminescence (molecular light switch effect) in the presence of double helical DNA, a property that makes them interesting as DNA stains. Also, a series of [Ru(phen)2(L)] 2+ complexes, where L is a substituted dppz ligand, have been investigated as luminescence reporters of DNA [11]. Very recently, Barton and her co-worker [12] have explored the cellular uptake of Ru(II) complexes and found that the complex cation [Ru(dip)2(dppz)]2+ (dip = 4,7-diphenyl-1,10-phenanthroline) is effectively transported into the cellular interior. These complexes have been exploited as probes to examine the local structural polymorphism of nucleic acids, as photo reactive reagents targeted to recognize mismatches or to repair thymine dimers and as probes of long range charge transport through the DNA helix [13–18]. Also, the DNA bisintercalator complex Δ–Δ[μC4(cpdppz)(phen)4Ru2] 4+, where C4(cpdppz)= N,N′ bis(cpdppz)-1,4diaminobutane; cpdppz = 12-cyano-12,13-dihydro-11 H-cyclopenta[b] dipyrido[3,2-h:2′3′-j]phenazine-12-carbonyl, acts as a DNA staining agent for V79 Chinese hamster cells [19,20]. 152 V. Rajendiran et al. / Journal of Inorganic Biochemistry 116 (2012) 151–162 Ru(II) complexes causes an increase in the excited state lifetime upon binding to CT DNA [34]. The DNA and protein binding of the present Ru(II) complexes has been studied by using a variety of physical methods like emission and circular dichroic spectroscopy, and attempts are made to exploit the new mixed ligand complexes [Ru(5,6-dmp/ tmp)2(diimine)] 2+ as ﬂuorescent DNA and protein probes. It is noteworthy that the complex [Ru(tmp)2(dpq)]2+ 4 acts as a ﬂuorescent probe for detection of cell morphologies in tissue section, DNA and proteins in the sperm head and tail respectively by using ﬂuorescent microscopy. Also, the cytotoxicity of the complexes has been studied by screening them against non-small lung carcinoma (NCI-H460) cell line, and it depends upon both the primary and secondary ligands. Since the metal-to-ligand charge transfer transition (MLCT) in [Ru(bpy)2(dppz)]2+ is located on the dppz ligand involved in intercalative DNA interaction leading to luminescence enhancement, substitution has been made in the diimine ligands in [Ru(diimine)2(dppz)]2+ to tune the redox, optical and DNA-binding properties of the complexes [21–23]. An increase in surface area of the primary ligand as in [Ru(ip)2(dppz)]2+, where ip= imidazo[4,5-f][1,10]-phenanthroline, enhances the DNA binding afﬁnity of the complex [24,25]. We have very recently shown that the 5,6-dmp ligand in rac-[Ru(5,6dmp)2(dppz)]2+ strongly favors the formation of a stable structural and electronic scaffold on the DNA surface for the unbound molecules to couple with the DNA-bound complexes facilitating spontaneous assembly of novel extended molecular aggregates [26]. Gyarfas and his co-workers have reported that [Ru(tmp)3] 2+, where tmp is 3,4,7,8tetramethyl-1,10-phenanthroline, exhibits tumor inhibiting properties [27]. Kruger and his co-workers [28] have reported that Ru(II) complexes of some of the methyl substituted derivatives of dpq exhibit unusual photophysical switching upon binding to DNA. Like DNA, protein is also considered to be one of the prime molecular targets for diagnostic and imaging agents, and so equal attention has been paid on designing novel probes for proteins [29–31]. Certain platinum containing compounds have been reported as molecular light-switches for proteins [32]. Very recently, we have demonstrated that the complex [Cu(tdp)(tmp)]+ (tmp= 3,4,7,8-tetramethyl-1,10phenanthroline), which binds to proteins more strongly and exhibits cytotoxicity higher than the analogous complexes [33]. In this study we have prepared a series of Ru(II) complexes of the type [Ru(5,6-dmp)2(diimine)] 2+ 1–3 and [Ru(tmp)2(diimine)]2+ 4–6, where diimine is an extended ligand (Scheme 1) such as dipyrido[3,2-d:2′,3′-f]-quinoxaline (dpq), dppz and 11,12-dimethyldipyrido [3,2-a:2′,3′-c]phenazine (11,12-dmdppz) on the optical, electrochemical and DNA and protein binding properties of the complexes. We have incorporated methyl substitution on positions 11 and 12 of the phenazine part of dppz (11,12-dmdppz) because Lincoln and his co-workers have found that incorporation of 11,12-dmdppz or 10mdppz (10-mdppz = 11-methyldipyrido[3,2-a:2′,3′-c]phenazine in 1 2 3 4 5 6 N' Ru N' N N H3 C N = The mixed ligand complexes [Ru(5,6-dmp)2(diimine)](PF6)2 1–3 and [Ru(tmp)2(diimine)](PF6)2 4–6 have been isolated by reacting the complexes [Ru(5,6-dmp)2Cl2]Cl and [Ru(3,4,7,8-tmp)2Cl2]Cl with the corresponding diimine ligands. The CHN analyses of the complexes are consistent with the formula [Ru(5,6-dmp/tmp)2(diimine)](PF6)2, which is supported by ESI-MS analysis. The NMR spectra show that the ligands are bound to Ru(II). The PF6 salts of the complexes are soluble in polar solvents like acetonitrile, DMF and DMSO etc but not in water and ether. The chloride salts of 1, 2, 4 and 5 are highly soluble in water but those of 3 and 6 are soluble in 5% DMF/5 mM Tris–HCl/50 mM NaCl buffer at pH 7.1. So the experimental solutions of all the complexes were prepared in 5% DMF/5 mM Tris–HCl/ 50 mM NaCl buffer at pH 7.1 and 5% DMF/NaH2PO4:NaHPO4 buffer at pH 7.1for DNA and protein binding studies respectively. For cytotoxic studies we have used 1% DMSO buffer solution as we found that 1% DMSO buffer is non-toxic to cells. The stability of the complexes in DMF and DMSO buffers has been checked by using the UV–visible (UV–VIS) spectral features of the solutions. Even after two weeks N' N' dpq dppz 11,12-dmdppz dpq dppz 11,12,-dmdppz H5 CH3 N CH3 N N N H9 / H9 ' H2 '/H2 H9 H2 CH3 H3 C H8 H3 H6 H3 C H7 H4 N 2.1. Synthesis and characterisation of Ruthenium(II) complexes N N 5,6-dmp " " 3,4,7,8-tmp " " N N 2. Results and discussion 5,6-dmp 3,4,7,8-tmp H8 H7 H6 N N' = N N N N H2 H2 dpq N H4' H4 H3 N H7' N H4 H3 CH 3 H7 N N H4 N' H3 C H3' H3 N H2' H2 dppz N 11,12-dmdppz Scheme 1. Structures of Ru(II) complexes and diimine ligands. V. Rajendiran et al. / Journal of Inorganic Biochemistry 116 (2012) 151–162 time we found that the spectral characteristics did not change, revealing that the compounds are stable enough in both the buffers. 2.2. Electronic absorption spectral and redox properties In 5% DMF/5 mM Tris–HCl/50 mM NaCl buffer at pH 7.1 all the complexes display a high energy intra-ligand π–π* band (234–386 nm, Table 1) [35] and the MLCT bands (420–475 nm, Table 1), which are typical of Ru(II) complexes with coordinated polyimine ligands [12]. The MLCT band energies (420–424 nm) of the tmp complexes 4–6 are higher than those for the 5,6-dmp complexes 1–3 (445–453 nm), possibly because the incorporation of four electron-releasing methyl groups to obtain 4–6 enhances the electron density on Ru(II), leading to the raise in energy of Ru(II) dπ orbitals and hence the observation of the MLCT band involving the co-ligands at lower energy. In acetonitrile solution all the complexes exhibit a quasi-reversible (ΔEp, 80–104 mV) metal-based oxidative response (Fig. 1, Table 2) with E1/2 values falling in the range of 0.869–1.029 V. The Ru(II)/ Ru(III) redox potentials of the 5,6-dmp complexes 1–3 are more positive than those of the corresponding tmp complexes 4–6. This trend is consistent with the lower MLCT band energies of the 5,6-dmp complexes 1–3 (cf. above). The number of electron-releasing methyl groups in 4–6 is higher than those in 1–3 leading to destabilize the Ru(II) oxidation state and hence the lower Ru(II)/Ru(III) redox potential of the former complexes. Also, the Ru(II)/Ru(III) redox potential becomes more positive as the dpq ligand in 1 and 4 is replaced by the dppz ligand with enhanced π-delocalization to obtain 2 and 5 respectively, revealing stabilization of Ru(II) oxidation state in the latter complexes. Further, the Ru(II)/Ru(III) redox potentials of 3 and 6 are less positive than those of 2 and 5 respectively, which is due to the increase in the electron density upon leading to destabilization of Ru(II) oxidation state (cf. above). All the complexes display two irreversible reduction waves in the potential range of − 1.191 to −1.852 V, which arise from the addition of electrons into the electrochemically accessible LUMO [36,37] of the more π-delocalized coligands. 2.3. DNA binding studies 2.3.1. Steady-state emission studies All the present Ru(II) complexes luminesce in aqueous solution (5% DMF/5 mM Tris–HCl/50 mM NaCl buffer at pH 7.1) upon irradiation in the MLCT band. No attempt was made to eliminate oxygen from solutions used in the emission experiments since the focus of the present study is on assessing the utility of the complexes as probes for DNA. The emission maxima vary, as do the intensities, but fall within the narrow range of 586–614 nm. The luminescence observed for the present complexes is relatively low in aqueous solution but is enhanced in the presence of calf thymus (CT) DNA (Fig. 2, Table 3). The observed enhancement is similar to that for the socalled [38] ‘molecular light-switches’ such as [Ru(bpy)2(dppz)] 2+ 153 and [Ru(phen)2(dppz)] 2+and the quinoxaline (1) and phenazine (2) nitrogen atoms of DNA-intercalated excited state complexes are well protected from access by water molecules leading to the observed emission enhancement. The relative emission intensity (I/I0) versus R (=[DNA]/[Ru(II) complex]) plot (Fig. 3) reveals that the luminescence enhancement for the 5,6-dmp complexes varies as 1 b 2 > 3 and that for the tmp complexes varies as 4 > 5 b 6 (cf. below). The steady-state luminescence titration data have been analyzed by using Mc Ghee von Hippel equation [39] for non-co-operative binding model by non-linear least-squares analysis. A known amount (10 μM) of 1–6 was titrated with DNA over a range of the DNA concentration (5.0 × 10 − 6–4.0 × 10 − 4 M, R = 0–40 = [DNA]/[Ru(II) Complex]). The concentration of the bound Ru(II) complex was calculated using Eq. (1): cb ¼ c½ðI−I0 Þ=ðImax −I0 Þ ð1Þ where c is the total Ru(II) complex concentration, I and I0 are the emission intensities in the presence and absence of DNA, and Imax is the ﬂuorescence of the totally bound complex. The concentration of the free complex, cf, is equal to (c − cb). A plot of r/cf vs r, where r is cb/[DNA], was constructed according to the Mc Ghee von Hippel equation: 2r=cf ¼ K b ð1−2nrÞ½ð1−2nrÞ=f1−2ðn−1Þrgðn−1 Þ ð2Þ where Kb represents the intrinsic binding constant of the complexes with DNA and n is the size of a binding site in base pairs. The binding data were ﬁtted (Figs. 4 and 5) to Eq. (2), and the observed intrinsic binding constants (Kb, 0.03–4.0 × 10 7 M − 1) along with the DNA binding site sizes (n, 6–10.5 base pairs), are collected in Table 3. The values of Kb are comparable to that of the known intercalator [Ru (bpy)2(phi)]Cl2 [24]. The binding site sizes are comparable to those of the well-known partially intercalating Ru(II) polypyridine complexes such as [Ru(bpy)2(phen)]2+, [Ru(phen)3]2+ and [Ru(phen)2(ﬂone)]2+ [40] (ﬂone= 4,5-diazaﬂuorene-9-one). Among the 5,6-dmp complexes the value of Kb varies as 1b 2 >3, which is in line with the observed enhancement in emission (cf. above). Thus 2 exhibits DNA binding afﬁnity higher than 1 because the coordinated dppz of 2 is inserted into the DNA base pairs much deeper than the dpq of 1, leading to higher stabilization of the excited state and hence higher enhancement in emission (cf. above). The methyl groups in dmdppz (3) prevents the partial intercalation of 3 into DNA and so DNA groove binding is preferred for 3, leading to weaker DNA binding of the complex. Also, the MLCT excited state localized on dmdppz ligand of 3 is less well preserved from access by water molecules and hence the ‘molecular light switch’ effect of 3 is less than that of its dppz analogue 2 (cf. above). In contrast to the 5,6dmp complexes, the DNA binding afﬁnity of the tmp complexes Table 1 Absorption spectral properties of Ru(II) complexesa. λ/nm (ε/M− 1 cm− 1) Complex 2+ [Ru (5,6-dmp)2(dpq)] 1 448 (12 250), 425 (sh) (11 650), 296 (sh) (28 136), 267 (34 540) [Ru (5,6-dmp)2(dppz)] 2 445 (17 820), 374 (19 090), 270 (32 660), 240 (59 840) [Ru (5,6-dmp)2(dmdppz)]2+ 3 453 (14,730), 383 (32,580), 275 (47,300) [Ru (3,4,7,8-tmp)2(dpq)]2+ 4 475 (sh) (8120), 421 (17,020), 302 (sh) (27,720), 266 (89,420) 2+ [Ru (3,4,7,8-tmp)2(dppz)] 5 424 (13,910), 376 (13,150), 360 (11,390), 272 (66,640), 234 (39,720) 2+ [Ru (3,4,7,8-tmp)2(dmdppz)] 6 420 (7190), 386 (8400), 268 (21,630) 2+ a Measured in 5% DMF/5 mM Tris–HCl/50 mM NaCl buffer at pH 7.1. Fig. 1. (a) Cyclic and (b) differential pulse voltammograms of 0.25 mM complex [Ru(tmp)2(dppz)]2+ 5 in acetonitrile solution at 25.0 ± 0.2 °C at 0.05 Vs- 1 scan rate. 154 V. Rajendiran et al. / Journal of Inorganic Biochemistry 116 (2012) 151–162 Table 2 Redox properties of Ru(II) complexesa,b,c at 25.0 ± 0.2 °C. Compound 1 Epa Epc ΔEp E1/2d E1/2e D × 106 V V mV V V cm2 s− 1 1.004 0.904 100 2 1.314 1.056 105 3 1.082 0.978 104 4 0.934 0.842 92 5 0.920 0.840 80 6 0.914 0.826 88 0.954 − 1.341 − 1.830 1.185 − 1.240 − 1.940 1.030 − 1.238 − 1.852 0.888 − 1.288 − 1.790 0.880 − 1.214 − 1.809 0.870 − 1.191 − 1.464 0.945 Table 3 Luminescence properties of the ruthenium complexes in the absence and presence of CT DNA (R = [DNA]/[Ru]). Complexes 10.7 1.189 1.8 1.029 9.5 0.875 7.2 [Ru (5,6-dmp)2(dpq)]2+ 1 [Ru (5,6-dmp)2(dppz)]2+ 2 [Ru (5,6-dmp)2(dmdppz)]2+ 3 [Ru (3,4,7,8-tmp)2(dpq)]2+ 4 [Ru (3,4,7,8-tmp)2(dppz)]2+ 5 [Ru (3,4,7,8-tmp)2(dmdppz)]2+ 6 [Ru (bpy)2(dpq)]2+ [Ru (phen)2(dpq)]2+ a λexcit b (nm) R = 0 R = 40 448 445 453 421 424 420 450 447 594 590 590 614 586 610 598 592 λemis (nm) 600 610 592 618 616 620 602 598 I/I0 Kb (M) n 4.6 6.2 1.8 10.1 2.2 6.3 3.3 3.1 1.6±0.1×106 1.8±0.1×107 3.3±0.3×105 3.0±0.2×105 1.0±0.09×106 6.0±0.3×106 1.2±0.09×106 1.0±0.05×106 6.8 2.0 6.9 7.9 6.0 7.9 8.9 6.7 a Excitation wavelength maximum of the complexes. Emission wavelength maximum of the complexes in the presence and absence of CT DNA. b 0.877 8.2 0.869 7.9 varies as 4 b 5 b 6. On replacing the 5,6-dmp ligands in 2 by tmp as in 5, a decrease in Kb value is observed as the bulky tmp co-ligands prevent the partial intercalation of dppz ligand. A similar decrease in DNA binding afﬁnity is observed on going from 1 to 4, supporting the importance of steric clash of tmp co-ligands with DNA. However, on going from 3 to 6 the Kb value increases because the latter with methyl groups on both the primary and co-ligands is involved in strong DNA groove binding (cf. above). This DNA binding mode also explains the inaccessibility of the MLCT excited state of 6 by water molecules, a Measured vs non-aqueous Ag/Ag+ reference electrode; add 544 mV [300 mV, Ag/ Ag+ to SCE + 244 mV, SCE to SHE] to convert to standard hydrogen electrode (SHE); scan rate, 50 mV s− 1; supporting electrolyte, [(C4H9)4 N](ClO4) (0.025 M). b Complexes 1–6 (0.25 mM) in acetonitrile. c Negative values are ligand reduction potential. d Redox potentials correspond to RuII/RuIII couple. e Differential pulse voltammetry, scan rate, 5 mV s− 1; pulse height, 50 mV. Intensity b b a a b Intensity b a a Intensity b a Fig. 2. (A) Emission spectra of the [Ru(5,6-dmp)2(dpq)]2+(a) in the absence and presence (b) of CT DNA. (B) Emission spectra of the [Ru(5,6-dmp)2(dmdppz)]2+(a) in the absence and presence (b) of CT DNA. (C) Emission spectra of the [Ru(3,4,7,8-tmp)2(dpq)]2+(a) in the absence and presence (b) of CT DNA. (D) Emission spectra of the [Ru(3,4,7,8-tmp)2(dppz)]2+ (a) in the absence and presence (b) of CT DNA. Emission spectra of the [Ru(3,4,7,8-tmp)2(dmdppz)]2+ (a) in the absence and presence (b) of CT DNA at R value of 40. V. Rajendiran et al. / Journal of Inorganic Biochemistry 116 (2012) 151–162 4 10 A 60 R = 40 50 8 2 6 6 Intensity I/I0 155 1 4 5 3 2 40 30 20 10 R=0 0 565 0 10 20 30 R = [DNA]/[Ru(II) Complex 40 665 Wavelength (nm) Fig. 3. Effect of addition of DNA on the emission intensity of the complexes 1, 600; 2, 590; 3, 592; 4, 618; 5, 616; 6, 620 nm in a 5% DMF/5 mM Tris–HCl/50 mM NaCl buffer at pH 7.1 and 25 °C, [Ru(II) complex] = 1 × 10− 5 M. leading to enhancement in emission for 6 higher than for 3 (cf. above). Thus the DNA binding afﬁnity of the mixed ligand 5,6-dmp and tmp complexes varies depending upon the extent of hydrophobic interaction of the diimine ligands, as determined by the number of methyl substituents, and the number of aromatic rings in the other ligand. B r/cf 0 2.4. Protein binding studies 2.4.1. Steady state emission All the present Ru(II) complexes luminesce in aqueous solution (5% DMF/NaH2PO4:NaHPO4 buffer at pH 7.1) upon irradiation in the MLCT band. Interestingly, on addition of BSA (5 μM), the emission intensity of the band (617–597 nm) observed for 2, 4 and 5 increases 1.4–2.1 folds (Fig. 6, Table 4). This enhancement in emission intensity is attributed to the strong binding of the complexes in the A 200 Intensity Fig. 5. (A) Emission spectra of [Ru (3,4,7,8-tmp)2(dmdppz)]2+ (1 × 10− 5 M) in 5% DMF/5 mM Tris–HCl/50 mM NaCl buffer at pH 7.1 (R = [DNA]/[complex] = 0) and Presence (R = 1 − 40) of increasing amounts of DNA. (B) Plot of r/cf vs r for [Ru (3,4,7,8-tmp)2(dpq)]2+. The best ﬁt line, superimposed on the data, according to McGhee and von Hippel Eqs. (2a), (2b) yields Kb = 6.0 ± 0.3 × 106 M− 1 and n = 7.9. R = 40 250 150 100 50 0 550 r R=0 600 650 700 Wavelength (nm) B hydrophobic pocket of serum albumin [32]. Interestingly, the increase in emission intensity observed for the well-known DNA molecular light-switch complex [Ru(phen)2(dppz)] 2+ under identical conditions is only 1.2. This reveals that methyl substitution on the phen ligand as in 2, 4 and 5 increases the hydrophobicity and hence the protein binding efﬁciency of the complexes. Among the present methyl substituted complexes, [Ru(tmp)2(dpq)]2+ 4 exhibits a higher protein binding efﬁciency as the dpq ligand is involved in hydrophobic interaction. As 3 and 6 are precipitated upon adding BSA even at lower concentrations of the complexes, their protein binding afﬁnity has not been determined. r/cf 2.5. Circular dichroic spectral measurements r Fig. 4. (A) Emission spectra of [Ru(3,4,7,8-tmp)2(dpq)]2+ (1 × 10− 5 M) in 5% DMF/ 5 mM Tris–HCl/50 mM NaCl buffer at pH 7.1 (R = [DNA]/[complex] = 0) and presence (R = 1 − 40) of increasing amounts of DNA. (B) Plot of r/cf vs r for [Ru(3,4,7,8tmp)2(dpq)]2+. The best ﬁt line, superimposed on the data, according to McGhee and von Hippel Eqs. (2a), (2b) yields Kb = 0.3 ± 0.02 × 106 M− 1 and n = 7.9. Circular dichroic spectra provide information about the chirality of spectroscopically active species in solution. They are particularly sensitive to the degenerate/non-degenerate exciton coupling expected to arise when the same or two different chromophores are closely located in space to interact strongly. Thus rac–metal complexes give a zero CD and show induced circular dichroism (ICD) signals due to enantiopreferential binding, if any, providing further and deﬁnitive conﬁrmation for their DNA binding [40]. Also, as CD signals are quite sensitive to the mode of DNA interactions of small molecules, the CD spectral technique is useful in diagnosing changes in DNA morphology during drug–DNA interaction [41,42]. Further, it is used to study the enantiopreferential DNA binding of metal complexes [17,40,43]. A solution of calf thymus DNA (2 × 10 − 5 M) exhibits a positive band (275 nm) due to base stacking and a negative band (248 nm) due to right-handed helicity of DNA. When the present rac-complexes are incubated with CT DNA at 1/R (=[Ru complex]/[DNA]) value of 156 V. Rajendiran et al. / Journal of Inorganic Biochemistry 116 (2012) 151–162 unity, the CD spectrum of DNA undergoes changes in both the positive and negative bands (Table 5). Upon addition of rac complexes 1–6 (Figs. 7 and 8) to DNA both these bands disappear and an inverted CD signal is observed with intensity much higher than that for free DNA. This is typical of exciton coupled ICD arising due to ligand-ligand interactions among DNA bound/unbound complexes [26,27]. The complexes 1, 2 [26] and 3 – 6 exhibit ICD upon interaction with different polynucleotides and the binding efﬁciency of DNA varies as CT DNA > poly(AT)12 ≅ d(CGCGATCGCG)2 > poly(GC)12 (Figs. 7 and 8). All these observations reveal the preferential binding of all the complexes to AT and mixed rather than GC sequences. 2.6. Cytotoxicity of Ruthenium(II) complexes Table 4 Luminescence properties of the ruthenium complexes in the absence and presence of BSA protein (R = [BSA]/[complex]). Complexes [Ru (5,6-dmp)2(dpq)]2+ 1 [Ru (5,6-dmp)2(dppz)]2+ 2 [Ru (5,6-dmp)2(dmdppz)]2+ 3 [Ru (3,4,7,8-tmp)2(dpq)]2+ 4 [Ru (3,4,7,8-tmp)2(dppz)]2+ 5 [Ru (3,4,7,8-tmp)2(dmdppz)]2+ 6 [Ru (bpy)2(dpq)]2+ [Ru (phen)2(dpq)]2+ a λexcit b (nm) R=0 R = 1.5 448 445 453 421 424 420 450 447 595 590 c – 616 617 c – 595 593 594 592 – 608 615 – 594 592 λemis (nm) I/I0 1.0 1.4 – 2.1 1.6 – 1.0 1.0 a Excitation wavelength maximum of the complexes. Emission wavelength maximum of the complexes in the presence and absence of BSA. c The complex was precipitated during the addition of BSA. b The cytotoxicity of 1–6 against non-small lung carcinoma cell line (NCI-H460) has been investigated in comparison with the widely used drug cisplatin under identical conditions by using MTT assay. As revealed by the IC50 values (Table 6), interestingly, the cytotoxicity of 5,6-dmp complexes varies as 1 b 2 > 3 while that of tmp complexes varies as 4 b 5 b 6, which is almost in line with the DNA binding afﬁnities of the complexes (cf. above). Thus the complex [Ru(5,6-dmp)2 (dppz)] 2+ 2, which exhibits the highest DNA binding afﬁnity, displays potency higher than the other complexes for 24, 48 and 76 h incubation. Similarly, the complex [Ru(tmp)2(dmdppz)] 2+ 6 exhibits cytotoxicity higher than all other complexes. Also, the complexes with hydrophobic ligands have the potential to bind with proteins strongly A b Intensity 10 8 a 4 2 500 550 600 650 700 Wavelength (nm) B 3.2 b 3.0 2.8 Intensity 2.7. Qualitative analysis of ﬂuorescence of Ru(II) complexes in live and ﬁxed cells The luminescence enhancement observed upon binding of the present low cytotoxic Ru(II) complexes to DNA has been studied further to explore their use as ﬂuorescent probes for DNA. The efﬁciency of the complexes as ﬂuorescent probes for the detection of nuclear morphology has been analyzed by using ﬂuorescent microscopy. 12 6 [44] and cause severe inhibition of fundamental enzyme function in cancer surveillance. Further, the cytotoxicity of the present complexes is time dependent and varies with the mode and extent of their interaction with DNA, and is lower than that of cisplatin, which is known to bind to DNA covalently, unlike the present complexes. 2.7.1. Cell morphology assessment Organic ﬂuorescent dyes such as acridine orange (AO), Hoechst 33258 (HO) and propidium iodide (PI) are among the most widely used ﬂuorescent dyes to analyze cell viability. All these dyes are principally applied to enumerate the proportion of live and dead cells in a given population. Fluorescence-based cell viability stains are generally less hazardous and less expensive than radioisotopic techniques, and are also more sensitive than brightﬁeld microscopy. These assays are reliable and easy to perform. Many popular ﬂuorescence-based cell viability stains are classiﬁed into two major categories: (i) dye exclusion stains, which do not enter eukaryotic cells due to impermeablitiy of the cell membrane unless the plasma membrane is damaged and (ii) dye uptake by viable cells in which the cell membrane allows the dye to permeate into cytoplasm. Despite the fact that ﬂuorescence is observed for all the six complexes, the complex 4 alone is a better reagent as it has less background emission. All the other complexes produce stronger emission making the morphology cloaked for visualization. Also, 4 possess low cytotoxicity supporting its use as stains in cell-labeling techniques without harming 2.6 Table 5 CD parameters for the interaction of calf thymus DNA with complexes 1–6. 2.4 Sample 2.2 2.0 a 1.8 520 540 560 580 600 620 640 660 680 700 720 Wavelength (nm) Fig. 6. (A) Emission spectra of the [Ru (3,4,7,8-tmp)2(dpq)]2+ (1 × 10− 5 M) (a) in the absence and presence (b) of BSA. (B) Emission spectra of the [Ru (3,4,7,8-tmp)2(dppz)]2+ (1× 10− 5 M) (a) in the absence and presence (b) of BSA at R value of 1.5. a CD spectral band wavelength (nm) 5 μM CT DNA DNA + 5 μM [Ru(5,6-dmp)2(dpq)]2+ 1 DNA + 5 μM [Ru(5,6-dmp)2(dppz)]2+ 2 DNA + 5 μM [Ru(5,6-dmp)2(dmdppz)]2+ 3 DNA + 5 μM [Ru(3,4,7,8-tmp)2(dpq)]2+ 4 DNA + 5 μM [Ru(3,4,7,8-tmp)2(dppz)]2+ 5 DNA + 5 μM [Ru(3,4,7,8-tmp)2(dmdppz)]2+ 6 245 242, 282 242, 282 245, 285 245, 295 245, 280 249, 276 276 260 262 267 262 265 265 a Measurement made at 1/R value of 1 for complexes 1–6. Where 1/R = [Ru]/[NP]; concentration of DNA solutions = 5 × 10− 5 M. Cell path length = 1 cm. V. Rajendiran et al. / Journal of Inorganic Biochemistry 116 (2012) 151–162 157 A b a B a b C b a D b a Fig. 7. (A) Circular dichroism spectra of CT DNA in the absence (a) and presence of [Ru (5,6-dmp)2(dpq)]2+ at 1/R = 1; [DNA] = 2 × 10− 5 M. (B) poly(GC)12, (C) poly(AT)12 and (D) d(GTCGAC)2 in the absence (a) and presence of [Ru (5,6-dmp)2(dpq)]2+ (b) at 1/R = 4; poly(GC)12, poly(AT)12 and d(GTCGAC)2 = 1 × 10− 5 M. the cell. When it is exposed to fresh cell suspension, it does not enter the cytoplasm because of dye exclusion phenomenon and only a few cells emit red color ﬂuorescence in fresh cell suspension (Figs. 9 (b), (c) Table 7). Therefore it is inferred that 4 can be used for cell viability assays. Comparative observation with phase contrast microscopy and AO/EB staining reveal the presence of monochromatic red ﬂuorescence only in dead cells (Figs. 9 (a)–(c) Table 7). Interestingly, these stains have slow permeability through live cell membrane and have a fast afﬁnity for dead cells and stain cytoplasm and nucleus, which help to visualize cell morphology of dead cells by ﬂuorescence microscopy with standard ﬁlters. This ﬁnding also might be used to distinguish live from dead cells in a population. However, it is to be noted that the staining efﬁciency [45] of [Ru(phen)2(dppz)]2+ is lower than those of the known dyes AO/EB and Hoechst 33258. Fig. 8. (A) Circular dichroism spectra of CT DNA in the absence (a) and presence of [Ru(3,4,7,8-tmp)2(dppz)]2+ at 1/R = 1; [DNA] = 2 × 10− 5 M. (B) poly(GC)12, (C) poly(AT)12 and (D) d(GTCGAC)2 in the absence (a) and presence of [Ru(3,4,7,8dmp)2(dppz)]2+ (b) at 1/R = 4; poly(GC)12, poly(AT)12 and d(GTCGAC)2 = 1 × 10− 5 M. 2.7.2. Detection of ﬁxed tissue sections A variety of nuclear binding DNA dyes have been evaluated for detecting cell and nuclear structure abnormalities in tissue sections [46]. In this study, the complex 4 has been employed for their ﬂuorescence speciﬁcity and sensitivity with cellular nucleus in parafﬁn-embedded tissue sections. Also, rat testis has been chosen as the tissue model as it has three different forms of nuclei such as germ cells (possessing tetraploid chromatin content, that is, after replication of DNA during M1 prophase), Leydig cells and Sertoli cells containing diploid nucleus, and sperm containing haploid nucleus, which helps us to interpret the speciﬁcities easily. Rehydrated parafﬁn sections were stained with 4, and the nuclear structures were then classiﬁed (Figs. 9 (k), (l), Table 7). The cell morphologies correlate with the appearance of tissue sections stained with AO/EB (Fig. 9 (o), Table 7). Since 4 stains the tissue sections neatly and the nuclear details are clearly distinguished more than AO/EB, they have the possibility of ﬁnding application as a counterstain for immunoﬂuorescence microscopy, permitting the identiﬁcation of various 158 V. Rajendiran et al. / Journal of Inorganic Biochemistry 116 (2012) 151–162 Table 6 In vitro cytotoxicity assays for complexes 1–6, cisplatin non-small lung carcinoma (NCI-H460) cell line (data are mean ± SD of four replicates each). a Complex IC50 values (μM) 2+ [Ru (5,6-dmp)2(dpq)] 1 [Ru (5,6-dmp)2(dppz)]2+ 2 [Ru (5,6-dmp)2(dmdppz)]2+ 3 [Ru (3,4,7,8-tmp)2(dpq)]2+ 4 [Ru (3,4,7,8-dmp)2(dppz)]2+ 5 [Ru (3,4,7,8-dmp)2(dmdppz)]2+ 6 [Ru (bpy)2(dppz)]2+ [Ru (phen)2(dppz)]2+ Cisplatin 24 h 48 h 76 h 30.0 ± 1.5 11.5 ± 1.0 13.0 ± 0.8 22.5 ± 2.0 12.5 ± 1.5 7.5 ± 0.5 42.0 ± 2.5 30.0 ± 2.0 5.0 ± 0.5 24.5 ± 1.2 6.5 ± 1.2 8.0 ± 0.7 10.0 ± 1.0 10.0 ± 0.9 7.0 ± 0.5 37 ± 1.7 20 ± 1.0 1.0 ± 0.1 10.0 ± 0.9 6.5 ± 1.2 8.0 ± 0.7 10 ± 1.0 7.5 ± 0.8 7.0 ± 0.5 30 ± 1.5 18 ± 0.5 1.0 ± 0.1 a IC50 = concentration of drug required to inhibit growth of 50% of the cancer cells (in μM). marker substances in speciﬁc cellular populations and the quantiﬁcation of particular cell types in tissue sections adopting ﬂuorescence microscopy. 2.7.3. Detection of ﬁxed cell suspension staining Recently, a variety of nucleic acid binding dyes have been developed [46] for ﬁxed cell staining methods. In this study the complex 4 was examined as a dye to determine whether it would be suitable for cellular pathology study by adopting ﬂuorescence microscopy. Three different cellular models were chosen for this study, in vitro cancer cells, human blood cells, and in vivo semen samples. They were ﬁxed in ethanol and smears were stained with 4 and the ﬁxed cell morphology detected is illustrated in Fig. 9 (b)–(j), Table 7. Speciﬁc emission was found in Human WBC cell types (e.g., neutrophils, eosinophils, basophils, lymphocyte and monocytes), cancer cells and also sperm heads with tails (Figs. 9 (e)–(h), Table 7). The nuclear morphology (Figs. 9 (m), (n), Table 7) reveals appreciable ﬂuorescence emission and good binding efﬁciency in the tail region in comparison with AO/EB and Hoechst when a standard ﬁlter (blue) is used. Therefore, the complex 4 stains adequately in tail region, thereby permitting rapid scanning of sperm abnormalities to ﬁnd out normal and defective sperms. When the DNA molecular light-switch complex [Ru(phen)2(dppz)] 2+ is used to stain sperm cells, it exhibits speciﬁc staining property, staining only the head region of the sperm. In contrast, the complex 4, which binds to DNA and protein through hydrophobic interaction, stains both the head and tail regions of the sperm. As the sperm head contains only nucleic acids, and the tail region only proteins [47], it is evident that 4 has the afﬁnity to bind to the protein in the tail region as well as DNA in the head region. Clapham and his co-workers have reported that if an agent blocks the CatSper protein, Fig. 9. Visualization of the morphology of In vitro, in vivo cells and tissues stained with [Ru(3,4,7,8-tmp)2(dpq)]2+ 4 using ﬂuorescent microscope (400× magniﬁcation). (a) In vitro cancer cells—phase contrast microscopy. (b) Unﬁxed fresh cells (dead cells stain red)—ﬂuorescence microscopy. (c) Simultaneous visualization in phase contrast and ﬂuorescence microscopy. (d) Ethanol ﬁxed cells—ﬂuorescent microscopy. (e–h) Human blood smear (nuclear lobules stain red). (i and j) Rat sperm (sperm heads (blue arrow) and tails (green arrow) stain red). (k and i) Rat testis tissue section. (m) Sperms stained by Hoechst. (n) In vitro cancer cells stained by Hoechst. (o) Testis section stained by acridine orange and ethidium bromide. (p) In vitro cancer cells stained by acridine orange and ethidium bromide. V. Rajendiran et al. / Journal of Inorganic Biochemistry 116 (2012) 151–162 Table 7 Optimum concentrations of ruthenium(II) complexes 1–6 used in various experiments in b1% EthOH/0.9% PBS Buffer (pH = 7.0). Complex [Ru (5,6-dmp)2(dpq)]2+ 1 [Ru (5,6-dmp)2(dppz)]2+ 2 [Ru (5,6-dmp)2(dmdppz)]2+ 3 [Ru (3,4,7,8-tmp)2(dpq)]2+ 4 [Ru (5,6-dmp)2(dpppz)]2+ 5 [Ru (5,6-dmp)2(dmdppz)]2+ 6 Optimum concentrations Morphology detection (μg/5 μl of cell suspension) Comet assay (μg/comet gel) 0.49 0.41 0.42 0.35 0.46 0.47 1.46 1.22 1.26 0.70 0.92 0.94 which is found exclusively in the tail of the sperm cell, then it would act as an effective non-hormonal contraceptive for both men and women [47]. So the complex 4 has the potential to act as a contraceptive under suitable conditions, and a deeper investigation on these protein binding complexes would pave the way for designing a promising non-hormonal metal-based contraceptive. 3. Conclusions A series of mixed ligand ruthenium(II) complexes have been isolated by incorporating methyl substituents on the phen and dppz ligands in [Ru(phen)2(dpq)] 2+ and [Ru(phen)2(dppz)] 2+ complexes. All the complexes produce a detectable level of luminescence in aqueous solution in the absence of DNA and display substantial enhancement in luminescence on DNA binding. Among the dpq complexes the tmp complex exhibits a larger enhancement in luminescence upon binding to DNA and among the dppz and methyl-substituted dppz complexes the 5,6-dmp complex exhibits a higher DNA binding afﬁnity suggesting the importance of ligand hydrophobic forces of interaction in DNA binding. All the complexes exhibit induced CD upon binding to CT DNA and a sequence speciﬁcity by binding to AT sequence more strongly than to GC sequence. It is noteworthy that upon increasing the number of methyl substituents on both the phen and dppz ligands the cytotoxicity of the complexes is increased. Interestingly, the [Ru(tmp)2(dpq)]2+ complex exhibits a higher enhancement in emission intensity when it binds with proteins and also stains proteins in the tail region of sperms. Our preliminary results reveal that the protein binding complex is expected to act as a contraceptive and this ﬁnding may pave the way for developing non-hormonal metal-based contraceptives. Thus the present study illustrates that increase in ligand hydrophobicity enhances on the DNA and protein binding abilities of Ru(II) complexes. 4. Experimental 4.1. Reagents and materials RuCl33H2O (Arora Matthey), 1,10-phenanthroline (Merck), 5,6-dimethyl-1,10-phenanthroline and 3,4,7,8-tetramethyl-1,10phenanthroline (Aldrich), CT DNA (highly polymerized stored at 4 °C), Hoechst 33258 (Sigma) and cisplatin (Bristol-Myers Squibb Co., Princeton), the self-complementary oligonucleotides d(GCGCGCGCGCGC) referred as d(GC)12, d(ATATATATATAT) referred as d(AT)12, d(CGCGATCGCG) referred as d(CGCGATCGCG)2 and protein BSA (bovine serum albumin, 66 kDa) were purchased from Sigma and stored at − 20 °C. The lyophilized oligonucleotides were digested in Tris buffer and annealed using standard procedures to make the double-stranded oligonucleotides and stored at 4 °C. The concentrations of the oligonucleotide solutions were determined using the procedures provided by the supplier. Ultra-pure Milli-Q water (18.2 mX) was used in all experiments. Reagent grade solvents were dried and distilled by usual methods and the solvents were stored over molecular sieves (4 Å). 159 The cancer cell line NCI-H460 was obtained from National Centre for Cell Science (NCCS), Pune, India. The cells were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA), as per the instruction by NCCS, supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and 100 U/mL penicillin and 100 μg/mL streptomycin as antibiotics (Himedia, Mumbai, India), in T25 cm2, T75 cm2, 6 well, 12 well, 24 well or 96 well culture plates (TPP, Switzerland), at 37 °C in a humidiﬁed atmosphere of 5% CO2 in a CO2 incubator (Heraeus, Hanau, Germany). All the experiments were performed by using cells from passage 15 or less. Institutional Animal Ethics Committee (IAEC), established under the auspices of Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, approved the experiment. Ninety-day-old Wistar strain male rats, raised from a stock obtained from the Indian Institute of Science, Bangalore, India, were used. Wistar rat was dissected under sodium pentobarbital anesthesia and the epididymis was removed. The cauda epididymal spermatozoa was taken and used for experimental purposes. Freshly prepared blood samples from healthy non-smoking donors at the University Biomedical center were used. The blood sampling has been approved by the Human Ethics Committee of the Bharathidasan University, India. 4.2. Methods and instrumentation Microanalysis (C, H and N) were carried out with Vario EL elemental analyzer. An LCQ DECA XP electrospray mass spectrometer was employed for ESI-MS analysis. UV–Vis spectroscopy was recorded on a Varian Cary 300 Bio UV–Vis spectrophotometer using cuvettes of 1 cm path length. The 1H NMR spectra were obtained at room temperature using a Bruker 400 MHz spectrometer. The chemical shift values in DMSO-d6 are reported with respect to tetramethylsilane as the internal standard and the values are reported as: d-values (multiplicity, assignment). Cyclic voltammetry and differential pulse voltammetry on a platinum sphere electrode were performed at 25.0 ± 0.2 °C. The temperature of the electrochemical cell was maintained by a cryocirculator (HAAKE D8-G). Voltammograms were generated with the use of an EG&G PAR Model 273 potentiostat. A Pentium IV computer along with EG&G M270 software was employed to control the experiments and acquire the data. A three-electrode system consisting of a platinum sphere (0.29 cm 2), a platinum auxiliary electrode and a reference electrode were used. The reference electrode for non-aqueous solution was Ag(s)/Ag +, which consists of a Ag wire immersed in a solution of AgNO3 (0.01 M) and tetra-n-butylammonium perchlorate (0.1 M) in acetonitrile placed in a tube ﬁtted with a vycor plug using a sleeve [48]. The E1/2 value observed under identical conditions for an Fc/Fc + couple in acetonitrile was 0.100 V with respect to the Ag/Ag + reference electrode. The cyclic voltammograms (CV) and differential pulse voltammograms (DPV) of 1–6 were obtained in MeCN solutions with 0.1 M [(C4H9)4N]ClO4 as the supporting electrolyte at ambient temperatures under N2. Redox potentials were measured relative to a Ag/Ag+ reference electrode. All the complexes are electroactive with respect to the metal as well as the ligand centers in the potential range of ±2.0 V. Emission intensity measurements were carried out using JASCO F 6500 spectroﬂuorometer. Circular dichroic spectra of DNA were obtained by using JASCO J-716 spectropolarimeter equipped with a peltier temperature control device. Visualization of the morphology of in vitro, in vivo cells and tissues stained by using ﬂuorescent microscope 450–490 nm ﬁlter (Carl Zeiss, Jena, Germany). Solutions of DNA in the buffer 50 mM NaCl/5 mM Tris HCl in water gave the ratio of UV absorbance at 260 nm and 280 nm, A260/A280, as 1.9 [49] indicating that the DNA was sufﬁciently free of protein. Concentrated stock solutions of DNA (13.5 mM) were prepared in buffer and sonicated for 25 cycles, where each cycle consisted of 30 s with 1 min intervals. The concentration of DNA in nucleotide phosphate (NP) was determined by UV absorbance at 260 nm after 1:100 dilutions. 160 V. Rajendiran et al. / Journal of Inorganic Biochemistry 116 (2012) 151–162 The extinction coefﬁcient, ε260, was taken as 6600 M− 1 cm− 1. Stock solutions were stored at 4 °C and used after more than 4 days or less. Concentrated stock solutions of metal complexes were prepared by dissolving calculated amounts of metal complexes in respective amount of solvent and diluted suitably with corresponding buffer to required concentrations for all the experiments. 4.3. Synthesis of ligands The ligands dpq [50], dppz [51] and 11,12-dmdppz [51] were synthesized according to literature methods. 4.4. Synthesis of complexes The complex [Ru(5,6-dmp)2(dppz)](PF6)2 (2) was prepared as reported already [26]. The precursor complex cis-[Ru(5,6-dmp)2Cl2] was synthesized according to literature methods [26] and cis-[Ru(3,4,7,8-tmp)2Cl2] was synthesized using a procedure reported for the synthesis of [Ru(bipy)2Cl2] [52]. 4.4.1. Synthesis of [Ru(5,6-dmp)2(dpq)](PF6)2 (1) [Ru(5,6-dmp)2(dpq)](PF6)2 was prepared by reﬂuxing the precursor cis-[Ru(5,6-dmp)2Cl2] (1 mmol) and dpq (1.1 mmol) in 50 mL of 75% ethanol for 8 h. The reaction volume was then reduced (20 mL), the solution was cooled, and excess KPF6 was added to it. The resultant orange precipitate was ﬁltered off and washed with water (100 mL) and then ether (50 mL). The solid was dissolved in acetonitrile (20 mL) and applied to the head of a column of activated aluminum oxide (neutral Brockmann 1). The orange band was eluted with acetonitrile. Yield, 0.31 g, 79%. Anal. Calc. for C28H20N6P2F12Ru: C, 40.45, H, 2.42, N, 10.11. Found: C, 40.25, H, 2.32, N, 10.06. ESI-MS: [Ru(5,6dmp)2(dpq)]2+ displays a peak at m/z = 375.40, calculated = 374.92. 1 H NMR (DMSO-d6, 400 MHz): δ (multiplicity, integration, assignment, J/Hz, coordination-induced shifts: c.i.s., δcomplex–δligand), ppm 5,6-dmp, 8.15 (d, 2 H, H2, 5.1, −0.93), 7.77 (t, 2 H, H3, 5.1, 0.21), 8.88 (d, 2 H, H4, 7.5, 0.58), 8.84 (d, 2 H, H7, 7.3, 0.53), 7.58 (t, 2 H, H8, 4.9, 0.02), 8.10 (d, 2 H, H9, 5.0, −0.98), 2.87 (s, 12 H, 5,6-CH3, 0.30); dpq, 8.33 (d, 2 H, H2, 7.5, −1.10), 7.921 (t, 2 H, H3, 4.8, 0.002), 9.53 (d, 2 H, H4, 7.5, 0.31), 9.39 (s, 2 H, H6, 0.25). The chloride salt was prepared from their PF6 salt by precipitating it from an acetone solution of the PF6 salt with tetra-n-butylammonium chloride. 4.4.2. Synthesis of [Ru(5,6-dmp)2(11,12-dmdppz)](PF6)2 (3) The complex [Ru(5,6-dmp)2(11,12-dmdppz)](PF6)2 was prepared by reﬂuxing the precursor cis-[Ru(5,6-dmp)2Cl2] (1 mmol) and dmdppz (1.1 mmol) in 75% ethanol (50 mL) for 8 h. The volume was then reduced to 20 mL, the solution was cooled, and excess KPF6 was added. The resultant orange precipitate was ﬁltered off, washed with water (100 mL) and then ether (50 mL). The product was dissolved in acetonitrile (20 mL) and applied to the head of a column of activated aluminum oxide (neutral Brockmann 1). The orange band was eluted with acetonitrile. Yield, 0.31 g, 79%. Anal. Calc. for C34H26N6P2F12Ru: C, 44.89, H, 2.88, N, 9.24. Found: C, 44.55, H, 2.82, N, 9.16. ESI-MS: [Ru(5,6-dmp) 2(dmdppz)] 2+ displays a peak at m/z = 413.50, calculated = 413.90. 1 H NMR (DMSO-d6, 400 MHz): δ (multiplicity, integration, assignment, J/Hz, coordination-induced shifts: c.i.s., δcomplex–δligand), ppm: 5,6-dmp, 8.200 (d, 2H, H2, 5.6, −0.891), 7.87 (t, 4H, H3 and H8, 4.1, 0.315), 8.53 (d, 2H, H4, 6.8, 0.220), 8.50 (d, 2H, H7, 6.4, 0.198), 8.18 (d, 2H, H9, 5.6, −0.913), 2.49 (s, 6H, 5-CH3, −0.084), 2.22 (s, 6H, 6-CH3, −0.351); 11,12-dmdppz, 8.09 (d, 2H, H2 and H2′, 5.4, −1.114), 7.72 (t, 2H, H3 and H3′, 4.0, −0.230), 9.56 (d, 2H, H4 and H4′, 7.2, 0.033), 8.48 (s, 2H, H7 and H7′, 0.309), 2.77 (s, 6H, 8,8′CH3, 0.179). The chloride salt was prepared from their PF6 salt by precipitating it from an acetone solution of the PF6 salt with tetra-nbutylammonium chloride. 4.4.3. Synthesis of [Ru(3,4,7,8-tmp)2(dpq)](PF6)2 (4) [Ru(3,4,7,8-tmp)2(dpq)](PF6)2 was prepared by reﬂuxing the precursor cis-[Ru (3,4,7,8-tmp)2Cl2] (1 mmol) and dpq (1.1 mmol) in 50 mL of 75% ethanol for 8 h. The volume was then reduced (20 mL), the solution was cooled, and excess KPF6 was added. The resultant orange precipitate was ﬁltered off washed with water (100 mL) and then ether (50 mL). The solid was dissolved in acetonitrile (20 mL) and applied to the head of a column of activated aluminum oxide (neutral Brockmann 1). The orange band was eluted with acetonitrile. Yield, 0.31 g, 79%. Anal. Calc. for C30H24N6P2F12Ru: C, 41.92, H, 2.81, N, 9.78. Found: C, 41.55, H, 2.72, N, 9.56. ESI-MS: [Ru(3,4,7,8-tmp)2(dpq)] 2+ displays a peak at m/z = 409.50, calculated = 409.90. 1H NMR (DMSO-d6, 400 MHz): δ (multiplicity, integration, assignment, J/Hz, coordinationinduced shifts: c.i.s., δcomplex–δligand), ppm 3,4,7,8-tmp, 8.475 (s, 4H, H2 and H9, −0.404), 7.80 (d, 2H, H5, 5.1, −0.057), 7.71 (d, 2H, H6, 4.8, −0.152), 2.22 (s, 6H, 3-CH3, −0.223), 2.20 (s, 6H, 8-CH3, −0.244), 2.77 (s, 12H, 4,7-CH3, 0.215); dpq, 8.10 (d, 2H, H2, 5.1, −1.329), 7.86 (t, 2H, H3, 4.2, −0.061), 9.47 (d, 2H, H4, 7.5, 0.246), 9.36 (s, 2H, H6, 0.229). The chloride salt was prepared from their PF6 salt by precipitating it from an acetone solution of the PF6 salt with tetra-n-butylammonium chloride. 4.4.4. Synthesis of [Ru(3,4,7,8-tmp)2(dppz)](PF6)2 (5) [Ru(3,4,7,8-tmp)2(dpq)](PF6)2 was prepared by reﬂuxing the precursor cis-[Ru(3,4,7,8-tmp)2Cl2] (1 mmol) and dpq (1.1 mmol) in 50 mL of 75% ethanol for 8 h. The volume was then reduced (20 mL), the solution was cooled, and excess KPF6 was added. The resultant orange precipitate was ﬁltered off and washed with water (100 mL) and then ether (50 mL). The solid was dissolved in acetonitrile (20 mL) and applied to the head of a column of activated aluminum oxide (neutral Brockmann 1). The orange band was eluted with acetonitrile. Yield, 0.31 g, 79%. Anal. Calc. for C34H26N6P2F12Ru: C, 44.89, H, 2.88, N, 9.24. Found: C, 44.55, H, 2.70, N, 9.16. ESI-MS: [Ru(3,4,7,8-tmp)2(dppz)]2+ displays a peak at m/z=428.47, calculated=428.01. 1H NMR (DMSO-d6, 400 MHz): δ (multiplicity, integration, assignment, J/Hz, coordinationinduced shifts: c.i.s., δcomplex–δligand), ppm 3,4,7,8-tmp, 8.48 (s, 4H, H2 and H9, − 0.398), 7.72 (s, 4H, H5 and H6, − 0.143), 2.23 (s, 12H, 3,8-CH3, −0.222), 2.77 (s, 12H, 4,7-CH3, 0.218); dppz, 8.06 (d, 2H, H2, 5.1, −1.397), 7.84 (t, 2H, H3, 4.6, −0.067), 9.47 (d, 2H, H4, 8.1, 0.286), 8.21 (s, 2H, H7, −0.119), 7.87 (s, 2H, H8, −0.144). The chloride salt was prepared from their PF6 salt by precipitating it from an acetone solution of the PF6 salt with tetra-n-butylammonium chloride. 4.4.5. Synthesis of [Ru(3,4,7,8-tmp)2(11,12-dmdppz)](PF6)2 (6) [Ru (3,4,7,8-tmp)2(11,12-dmdppz)](PF6)2 was prepared by reﬂuxing the precursor cis-[Ru (3,4,7,8-tmp)2Cl2] (1 mmol) and dmdppz (1.1 mmol) in 50 mL of 75% ethanol for 8 h. The volume was then reduced (20 mL), the solution was cooled, and excess KPF6 was added. The resultant orange precipitate was ﬁltered off and washed with water (100 mL) and then ether (50 mL). The solid was dissolved in acetonitrile (20 mL) and applied to the head of a column of activated aluminum oxide (neutral Brockmann 1). The orange band was eluted with acetonitrile. Yield, 0.31 g, 79%. Anal. Calc. for C36H30N6P2F12Ru: C, 46.11, H, 3.22, N, 8.96. Found: C, 46.00, H, 3.10, N, 8.66. ESI-MS: [Ru(3,4,7,8-tmp)2(dmdppz)]2+ displays a peak at m/z =442.47, calculated=442.03. 1H NMR (DMSO-d6, 400 MHz): δ (multiplicity, integration, assignment, J/Hz, coordination-induced shifts: c.i.s., δcomplex–δligand), ppm 3,4,7,8-tmp, 8.905 (s, 1H, H2, 0.026), 8.88 (s, 1H, H2′, 0.002), 8.09 (d, 2H, H5, 5.1, 0.232), 7.96 (d, 2H, H6, 4.8, 0.094), 8.86 (s, 1H, H9, −0.019), 8.83 (s, 1H, H9′, −0.047), 2.82 (s, 12H, 3,8-CH3, 0.369), 2.84 (s, 12H, 4,7-CH3, 0.289); 11,12-dmdppz, 8.34 (d, 1H, H2, 5.4, −0.865), 8.21 (d, 1H, H2′, 5.1, −0.998), 7.90 (t, 1H, H3, 5.0, −0.050), 7.75 (t, 1H, H3′, 4.6, −0.198), 9.57 (d, 1H, H4, 5.4, 0.040), 9.55 (d, 1H, H4′, 5.4, 0.016), 8.27 (s, 2H, H7 and H7′, 0.101), 2.64 (s, 6H, 8,8′-CH3, 0.041). The chloride salt was prepared from their PF6 salt by precipitating it V. Rajendiran et al. / Journal of Inorganic Biochemistry 116 (2012) 151–162 from an acetone solution of the PF6 salt with tetra-n-butylammonium chloride. 4.5. 1H NMR spectra To throw light on the nature of metal–ligand bonding and conformation of chelate rings of mononuclear Ru(II) complexes in dimethyl sulfoxide solutions, the chemical shifts of the free and coordindated ligands are summarized in the experimental section, along with coordinationinduced shifts (c.i.s.=δcomplex–δligand) and coupling constants [53–55]. Spectral assignments [56–58] were made on the basis of COSY spectra of the complexes and the J values are consistent with the assignment. The 1H NMR spectra of mononuclear complexes show seven (4,5) or ten (1,3) or 14 (6) aromatic signals and one (1) or two (5) or three (3,4,6) aliphatic signals due to the substitution of methyl groups indicating the presence of C2 symmetry in all the complexes. It is clear that the aromatic protons in the coordinated 5,6-dmp (1,3) and 3,4,7,8-tmp (4,5), dpq (1,4), 11,12-dmdppz (3) and dppz (5) are magnetically equivalent while 3,4,7,8-tmp (6) and 11,12-dmdppz (6) are magnetically nonequivalent due to the steric crowding imposed by both the ligands in the octahedral environment. When coordinated to Ru(II) the three ﬁvemembered chelate rings are expected to exist in envelope conformation. The aliphatic protons of methyl groups in the 5,6- (3) and 3,8- (4) positions are magnetically equivalent while 3,8- (5) position is magnetically non-equivalent show upﬁeld shifts indicating the importance of π-back donation, which would increase the electron density at these positions. On the other hand, the methyl groups in the 5,6- (1), 3,8- (6), 4,7- (4,5) and 8,8′- (3, 6) positions are magnetically equivalent and exhibit downﬁeld shift due to metal-to-ligand σ-donation. The aromatic H2 and H9 protons of 5,6-dmp (1,3) and 3,4,7,8-tmp (4,5), H2 proton of dpq (1,4), dppz (5) and H2 and H2′ proton of 11,12dmdppz (6) exhibit negative c.i.s. values, which are adjacent to the coordinated nitrogen resulting from through-space ring-current anisotropy effects since on coordination these protons lie directly over the shielding plane of another aromatic py ring. The H3 proton of 11,12-dmdppz (3,6), dpq (4) and dppz (5) also show negative c.i.s. value due to the magnetic anisotropy induced by proximate ring current; however the inﬂuence of the latter on H4 proton of 11,12-dmdppz (3,6), dpq (4) and dppz (5) is affected less. The H5 and H6 protons of 3,4,7,8-tmp (4,5) shifted upﬁeld due to Ru(II) to ligand π-back donation. The positive c.i.s. values observed for H3, H4, H7 and H8 protons of 5,6-dmp (1,3), H5 and H6 protons of 3,4,7,8-tmp (6), H4 and H6 protons of dpq (1,4), H4 and H7 protons of dppz (5) and 11,12-dmdppz (3,6) indicate the importance of σdonation of electrons to Ru(II) via the nitrogen lone pairs. Further, the 1 H NMR spectra of 1 and 3–6 show only single resonances for all the aromatic protons, substantiating the possibility of the presence of Λ and Δ enantiomers in equal proportions and thus a racemic mixture of 1 and 3–6 is obtained. 4.6. DNA binding experiments Concentrated stock solutions of metal complexes were prepared by dissolving them in 5% DMF/5 mM Tris–HCl/50 mM NaCl buffer at pH 7.1 of metal complexes and diluting suitably with corresponding buffer to required concentrations for all the experiments. For emission spectral experiments the DNA solutions were pretreated with the solutions of metal complexes to ensure no change in concentration of the metal complexes. Emission measurements were carried out by using a Hitachi F 4500 spectroﬂuorimeter. 5% DMF/5 mM Tris–HCl/50 mM NaCl buffer at pH 7.1 was used as a blank to make preliminary adjustments. The excitation wavelength was ﬁxed and the emission range was adjusted before measurements. All measurements were made at 25 °C in a thermostated cuvette holder with 5 nm entrance slit and 5 nm exit slit. For emission spectral titrations 1.0×10− 5 M concentration of ruthenium solutions were used and CT DNA was added in steps till R=40. The emission enhancement factors were measured by comparing the intensities at the 161 emission spectral maxima in the absence and presence of DNA, under similar conditions. The emission decay measurements of the DNAbound complexes (R=40) were carried out using the time correlated single photon counting technique (TCSPC) by exciting the sample at 445 nm (Model 5000U, LED, IBH, UK) with micro channel plate photomultiplier tube (MCP-PMT) as detector. Emission titration data were analyzed using McGhee von Hippel equation using non-co-operative binding model and non-linear least square analysis. Circular dichroic spectra of DNA were obtained by using JASCO J-716 spectropolarimeter equipped with a peltier temperature control device. All experiments were done using a quartz cell of 1 or 0.2 cm path length. Each CD spectrum was collected after averaging over at least 4 accumulations using a scan speed of 100 nm min−1 and a 1 s response time. Machine plus cuvette baselines were subtracted and the resultant spectrum zeroed 50 nm outside the absorption bands. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed in a single compartment cell with a three electrode conﬁguration on a EG&G PAR 273 potentiostat–galvanostat equipped with an PIV computer. The working electrode was a glassy carbon disk (0.384 cm2) and the reference electrode a saturated calomel electrode. A platinum plate was used as the counter electrode. The supporting electrolyte was 50 mM NaCl/5 mM Tris–HCl buffer at pH 7.1. Solutions were deoxygenated by purging with nitrogen gas for 15 min prior to measurements; during measurements a stream of N2 gas was passes over the solution. All the experiments were carried out at 25.0±0.2 °C maintained by a Haake D8-G circulating bath. The redox potential E1/2 was calculated from the anodic (Epa) and cathodic (Epc) peak potentials of CV traces as (Epa +Epc)/2 and also from the peak potential (Epa) of DPV response as Ep +ΔE/2 (ΔE is the pulse height). 4.7. Protein binding experiments Steady-state emission studies of the ruthenium(II) complexes were carried out by addition of the concentration of BSA (1.5 × 10 − 5 M) while keeping the concentration of metal complex constant. Concentrated stock solutions of metal complexes were prepared by dissolving them in 5% DMF/NaH2PO4:NaHPO4 buffer at pH 7.1 of metal complexes and diluting suitably with corresponding buffer to required concentrations for all the experiments. For emission spectral experiments the protein solutions were pretreated with the solutions of metal complexes to ensure no change in concentration of the metal complexes. Emission measurements were carried out by using a Hitachi F 4500 spectroﬂuorimeter. 5% DMF/NaH2PO4:NaHPO4 buffer at pH 7.1 was used as a blank to make preliminary adjustments. The excitation wavelength was ﬁxed and the emission range was adjusted before measurements. All measurements were made at 25 °C in a thermostated cuvette holder with 5 nm entrance slit and 5 nm exit slit. 4.8. Fixed cell staining The monolayer culture of NCI-H460 cells were harvested at exponential phase, human blood was collected from the vein [59] and 90 days old Wistar rat was dissected under sodium pentobarbital anaesthesia and the epididymis were removed. The cauda epididymal spermatozoa was taken. The cells were ﬁxed with 70% ethanol and cells washed with cold phosphate buffered saline, and incubated with Ru complex (Table 6) in buffer for 5–10 min at room temperature in the dark. Cells were then washed with buffer followed immediately by observation using a ﬂuorescent microscope using 450–490 nm ﬁlter (Carl Zeiss, Jena, Germany). 4.9. Cell viability assay MTT assay was carried out as described previously [60]. The complexes 1–5, in the concentration 0.05–100 μM, dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA), were added to the wells 24 h after 162 V. Rajendiran et al. / Journal of Inorganic Biochemistry 116 (2012) 151–162 seeding of 5×103 cells per well in 200 μL of fresh culture medium. DMSO was used as the vehicle control. After 24 and 48 h, 20 μL of MTT solution [5 mg/mL in phosphate-buffered saline (PBS)] was added to each well and the plates were wrapped with aluminum foil and incubated for 4 h at 37 °C. The purple formazan product was dissolved by addition of 100 μL of 100% DMSO to each well. The absorbance was monitored at 570 nm (measurement) and 630 nm (reference) using a 96 well plate reader (Bio-Rad, Hercules, CA, USA). The stock solutions of the metal complexes were prepared in DMSO and in all the experiments the percentage of DMSO was maintained in the range of 0.1–1%. DMSO by itself was found to be non-toxic to the cells till 1% concentration. Data were collected for four replicates each and used to calculate the mean. The percentage inhibition was calculated, from this data, using the formula: ¼ Mean OD of untreated cells ðcontrolÞ−Mean OD of treated cells 100 Mean OD of untreated cells ðcontrolÞ The IC50 values were calculated using Table Curve 2D version 5.01 Abbreviations AO acridine orange BSA bovine serum albumin CT DNA calf thymus cpdppz 12-cyano-12,13-dihydro-11H-cyclopenta[b]dipyrido[3,2-h:2′ 3′-j]phenazine-12-carbonyl CPCSEA Committee for Purpose of Control and Supervision of Experiments on Animals 5,6-dmp 5,6-dimethyl-1,10-phenanthroline 11,12-dmdppz 11,12-dimethyl-dipyrido[3,2-a:2′,3′-c]phenazine dip 4,7-diphenyl-1,10-phenanthroline 4,7-dmp 4,7-dimethyl-1,10-phenanthroline 2,9-dmp 2,9-dimethyl-1,10-phenanthroline 4,4′-dmb 4,4′-dimethyl-2,2′-bipyridine dpq dipyrido-[3,2-d:2′,3′-f]-quinoxaline dppz dipyrido[3,2-a:2′,3′-c]phenazine ﬂone 4,5-diazaﬂuorene-9-one DMF dimethylformamide MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide EB ethidium bromide ESI-MS electrospray ionization mass spectrometry \ HO Hoechst 33258 H(tdp) 2-[(2-(2-hydroxyethylamino)ethylimino)methyl] phenol ip imidazo[4,5-f] [1,10] phenanthroline IAEC Institutional Animal Ethics Committee ICD induced circular dichroism MCP-PMT micro channel plate photomultiplier tube MLCT metal-to-ligand charge transfer transition 10-mdppz 11-methyldipyrido[3,2-a:2′,3′-c]phenazine NCCS National Centre for Cell Science tmp 3,4,7,8-tetramethyl-1,10-phenanthroline TCSPC time correlated single photon counting technique PBS phosphate-buffered saline PI propidium iodide Acknowledgments Council of Scientiﬁc and Industrial Research, New Delhi, India (Grant No. 01(2101)/07/EMR-II) is gratefully acknowledged for ﬁnancial support. 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