Ruthenium(II) Complexes of BipyridineâGlycoluril and their

Bioconjugate Chem. 2009, 20, 447–459

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Ruthenium(II) Complexes of Bipyridine-Glycoluril and their Interactions with DNA Megha S. Deshpande,† Anupa A. Kumbhar,† Avinash S. Kumbhar,*,† Manoj Kumbhakar,‡ Haridas Pal,‡ Uddhavesh B. Sonawane,§ and Rajendra R. Joshi§ Department of Chemistry, University of Pune, Pune - 411 007, Radiation and Photochemistry Division, BARC, Mumbai - 400 085, and Bioinformatics Team, Scientific and Engineering Computing Group, Centre for Development of Advanced Computing (C-DAC), Pune University Campus, Pune - 411 007, India. Received July 15, 2008; Revised Manuscript Received January 12, 2009

Nine complexes of the type [Ru(N-N)2(BPG)]Cl2 1-4, [Ru(N-N)(BPG)2]Cl2 5-8, and [Ru(BPG)3]Cl2 9 where N-N is 2,2′-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq), dipyrido[3,2a:2′,3′-c]phenazine (dppz), which incorporates bipyridine-glycoluril (BPG-4b,5,7,7a-tetrahydro-4b,7a-epiminomethanoimino-6H-imidazo[4,5-f][1,10]phenanthroline-6,13-dione) as the ancillary ligand, have been synthesized and characterized. These complexes with the peripheral polypyridyl ligands have the ability to form conjugates with DNA. The DNA binding (absorption spectroscopy, steady-state and time-resolved emission measurements, steady-state emission quenching measurements) and cleavage (under dark and irradiated conditions) by these complexes has been studied to investigate the influence of the ancillary ligand. The binding ability of these complexes to DNA is dependent on the planarity of the intercalative polypyridyl ligand, which is further affected by the ancillary bipyridine-glycoluril ligand. The complexes 3, 4, 7, and 8 bind to CT-DNA with binding constants on the order of 104 M-1. Time-resolved emission measurements on the DNA-bound complexes 1, 3, 5-7, and 9 show monoexponential decay of the excited states, whereas complexes 2, 4, and 8 show biexponential decay with short- and long-lived components. Interaction of complexes 2-9 with plasmid pBR322 DNA studied by gel electrophoresis experiments reveals that all complexes cleave DNA efficiently at micromolar concentrations under dark and anaerobic conditions probably by a hydrolytic mechanism. Complexes 3, 4, 7, 8, and [Ru(bpy)2(dppz)]2+ show extensive DNA cleavage in the presence of light with a shift in mobility of form I of DNA probably due to the high molecular weight of DNA-complex conjugates. However, the extent of the cleavage is augmented on irradiation in the case of complexes 3, 4, 7, and 8, which include the planar dpq and dppz ligands, suggesting a combination of hydrolytic and oxidative mechanism for the DNA scission. Molecular mechanics calculations of these systems corroborate the DNA binding and cleavage mechanisms.

INTRODUCTION The interaction of transition metal complexes with DNA is a vibrant area of research (1-4). An advantage of using these complexes in such studies is that their ligands and metals can be conveniently varied to suit individual applications. Ruthenium(II) complexes with intercalating polypyridyl ligands have been extensively studied in this context, as their luminescence and photochemical reactivity are significantly altered on interaction with DNA (1-11). Ruthenium polypyridyl complexes are useful probes of DNA structure and DNA oxidation chemistry and in addition are used as sensors (12-49). Among them, [Ru(bpy)2(dppz)]2+ and [Ru(phen)2(dppz)]2+ are found to be high-affinity intercalators with interesting light switch behavior with potential applications in sensing and signaling, as well as in data storage and communication (14, 15). The versatility of these complexes is modulated by the ligand set, which controls whether a complex is an intercalator, hemi-intercalator, or electrostatic binder (12-49). In general, ruthenium polypyridyl intercalators like [Ru(bpy)2(dppz)]2+ and [Ru(phen)2(dppz)]2+ have K ) 106-107 M-1, whereas electrostatic binders like [Ru(bpy)3]2+ have binding affinities on the order of 103 M-1. * E-mail: [email protected]. Tel: (+91)-020-25601225 (534); Fax:(+91)-020-25691728. † University of Pune. ‡ Radiation and Photochemistry Division, BARC. § Centre for Development of Advanced Computing (C-DAC).

The affinity of dppz intercalators for DNA can be further enhanced by increasing the surface area of the ancillary ligand (45). Adding groups to the edges of the ruthenium polypyridyl complexes further expands the functionality of these complexes. For example, insertion of two polyamine tridentate arm-like segments in a macrochelating ligand in the complex [Ru(DIP)2(macro)]n+ enables binding of certain divalent metal cations so as to deliver its coordinated nucleophile to the phosphate backbone for hydrolysis of the anionic diester (50). In a previous study, we have explored the possibility of modifying reactivity by using the urea-fused bipyridine ligand (BPG) that contains hydrogen bond donor (NsH) and acceptor (CdO) groups. This ligand is capable of forming extensive H-bonding networks resulting in diverse frameworks encapsulating water/solvent molecules depending upon the number of bipyridine-glycoluril ligands (51-53). We have also demonstrated that the urea groups of the BPG ligand are involved in DNA binding and facilitate hydrolytic cleavage of DNA by the complex [Ru(bpy)2(BPG)]2+ 1 (54). Thus, the molecules that can hydrolyze the DNA phosphodiester at specific positions are valuable tools in biotechnology, thus facilitating DNA manipulation in a variety of applications. To test whether the hydrolytic DNA cleavage mechanism is unique to a ruthenium polypyridyl complex containing a single BPG ligand, we synthesized the series of compounds containing one, two, or three BPG ligands. In complexes with one or two BPG ligands, the remaining ligands were varied to include

10.1021/bc800298t CCC: $40.75  2009 American Chemical Society Published on Web 02/23/2009

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ligands that favor a particular binding mode (i.e., intercalation, electrostatic binding, etc.) to assess the influence of the BPG ligands on the binding mode and reactivity. If the reactivity and binding mode of these complexes is dominated by the BPG-DNA interactions, these complexes should cleave DNA via a hydrolytic mechanism, consistent with our previous work on [Ru(bpy)2(BPG)]2+ 1. Here, we present data on the DNA binding and cleavage ability of these complexes probed by spectroscopic methods, gel electrophoresis, and molecular mechanics calculations.

EXPERIMENTAL SECTION A. Materials. 1. General Details. All reagents and solvents were purchased commercially and were used as received. RuCl3 · xH2O and K4[Fe(CN)6] were obtained from S. D. Fine Chemicals Limited (India). Calf thymus DNA and plasmid pBR322 DNA were purchased from SRL (India). The enzyme superoxide dismutase (SOD EC1.15.1.1) was purchased from Sigma Chemical Co. USA. Deionized water was used for the preparation of the buffers. Supercoiled pBR322 DNA (CsCl) purified was obtained from Bangalore Genei (Bangalore, India) and used as received. The concentration of DNA in nucleotide phosphate (NP) was determined by UV absorbance at 260 nm using the molar absorption coefficient as 6600 M-1 cm-1. Solutions of calf thymus DNA in phosphate buffer gave a ratio of UV absorbance at 260 and 280 nm, A260/A280, of 1.8-1.9:1, indicating that the DNA was sufficiently free of protein (55). 2. Syntheses. The ligands 1,10-phenenthroline-5,6-dione (phendione) (56), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq) (57), and dipyrido[3,2-a: 2′,3′-c]phenazine (dppz) (58) were synthesized according to the literature protocols. BPG [4b,5,7,7a-tetrahydro4b,7a-epiminomethanoimino-6H-imidazo[4,5-f] [1,10] phenanthroline-6,13-dione] was prepared by modifying the literature method (59, 60, 51-54). The precursor complexes of the type cis-[Ru(N-N)2Cl2] (61, 62), [Ru(N-N)Cl4] (63) were prepared by using the literature methods, and the synthesis of the complex [Ru(bpy)2(BPG)]Cl2 1 (51, 54) was reported previously by us. 3. Series I - Ru/BPG Ratio (1:1). [Ru(phen)2(BPG)]Cl2 · 4H2O (2). This complex was prepared by the method described for complex 1 (51, 54) using cis- [Ru(phen)2Cl2] · 2H2O in place of cis-[Ru(bpy)2Cl2] · 2H2O. The precursor complex cis[Ru(phen)2Cl2] · 2H2O (0.125 g, 0.21 mmol) and BPG (0.0608 g, 0.21 mmol) in a 1:1 molar ratio were dissolved in 50% methanol/50% water (50 mL), and the mixture was heated to reflux for 8 h, whereupon the color of the solution changed from dark purple to red. The red solution was filtered hot and was cooled to room temperature. After evaporation of the solvent, the red solid was collected and washed with small amounts of methanol and diethyl ether and then dried under vacuum. The product was purified by column chromatography on active alumina using acetone and methanol as eluent. The red fraction was collected and concentrated in vacuum, a small amount of diethyl ether was added to the concentrated solution, and a red solid was obtained. Yield: (67%). Elemental analysis calcd for RuC38H34N10O6Cl2 (898.35): C 50.76, H 3.81, N 15.59%. Found: C 49.80, H 3.71, N 14.90%. 1H NMR (300 MHz, DMSO-d6, 25 °C): δ ) 8.92 (2H), 8.80 (2H), 8.51 (2H), 8.45 (4H), 8.41 (2H), 8.24 (2H), 8.03 (4H), 7.74 (4H), 7.57 (2H) ppm. IR (KBr): ν∼ ) 1708 cm-1 (CdO), 3215 cm-1 (NsH), 3421 cm-1 (OsH), 1647 cm-1 (CdN), 1427 cm-1 (CdC). UV-visible (H2O), λmax, nm (log ε): 441 (4.01), 263 (4.77), 223 (4.70). E1/2 (V vs Ag/AgCl in DMF 25 °C, 0.1 M [([But]4 NPF6)]): +1.18. Complexes [Ru(dpq)2(BPG)]Cl2 · 4H2O (3) and [Ru(dppz)2(BPG)]Cl2 · 4H2O (4) were prepared similarly to the method described for complex 1, with cis-[Ru(dpq)2Cl2] · 2H2O and cis[Ru(dppz)2Cl2] · 2H2O in place of cis-[Ru(bpy)2Cl2] · 2H2O.

Deshpande et al.

However, in the case of 4 the precursor complex cis[Ru(dppz)2Cl2] · 2H2O and BPG in a 1:1 molar ratio was refluxed in ethylene glycol (20 mL) for 12 h whereupon the color of the solution changed to red. The purification was also carried out by column chromatography on an active alumina column. [Ru(dpq)2(BPG)]Cl2 · 4H2O (3). Yield: (60%). Elemental analysis calcd for RuC42H34N14O6Cl2 (1002.38): C 50.29, H 3.41, N 19.56%. Found: C 50.78, H 3.60, N 19.10%. 1H NMR (300 MHz, DMSO-d6, 25 °C): δ ) 9.57 (d, 2H), 9.47 (d, 2H), 9.33 (s, 4H), 8.69 (s, 2H), 8.47 (s, 2H), 8.40 (d, 2H), 8.15 (m, 6H), 7.83 (dd, 2H), 7.75 (d, 2H), 7.49 (dd, 2H) ppm. IR (KBr): ν∼ ) 1703 cm-1 (CdO), 3217 cm-1 (NsH), 3413 cm-1 (OsH), 1645 cm-1 (CdN), 1448 cm-1 (CdC). UV-visible (H2O), λmax, nm (log ε): 452 (4.13), 296 (4.60), 256 (4.85), 205 (4.77). E1/2 (V vs Ag/AgCl in DMF 25 °C, 0.1 M [([But]4 NPF6)]): +1.16. [Ru(dppz)2(BPG)]Cl2 · 4H2O (4). Yield: (25%). Elemental analysis calcd for RuC50H38N14O6Cl2 (1102.42): C 56.06, H 3.57, N 18.32%. Found: C 55.99, H 3.87, N 17.98%. 1H NMR (300 MHz, DMSO-d6, 25 °C): δ ) 8.85 (m, 8H), 8.45 (s, 4H), 8.12 (m, 5H), 8.02 (d, 4H), 7.82 (m, 9H) ppm. IR (KBr): ν∼ ) 1705 cm-1 (CdO), 2923 cm-1 (NsH), 3336 cm-1 (OsH), 1602 cm-1 (CdN), 1407 cm-1 (CdC). UV-visible (H2O), λmax, nm (log ε): 443 (4.05), 371 (4.03), 263 (4.83), 224 (4.68). E1/2 (V vs Ag/AgCl in DMF 25 °C, 0.1 M [([But]4 NPF6)]): +1.24. 4. Series II - Ru/BPG Ratio (1:2). [Ru(bpy)(BPG)2]Cl2 · 4H2O (5). The precursor complex [Ru(bpy)Cl4] (0.100 g, 0.190 mmol) and BPG (0.112 g, 0.381 mmol) in a 1:2 molar ratio were refluxed in 50% methanol/50% water (50 mL) for 8 h, whereupon the color of the solution changed from dark green to red. The red solution was filtered hot and was cooled to room temperature. After evaporation of the solvent, the brownish red solid was collected and washed with small amounts of methanol and diethyl ether and then dried under vacuum. The product was purified by column chromatography on active alumina using acetone and methanol as eluent. The red fraction was collected and concentrated in vacuum, a small amount of diethyl ether was added to the concentrated solution and red solid was obtained. Yield: (65%). Elemental analysis calcd for RuC38H36N14O8Cl2 (988.38): C 46.13, H 3.67, N 19.84%. Found: C 46.70, H 3.70, N 19.20%. 1H NMR (300 MHz, DMSO-d6, 25 °C): δ ) 8.82 (1H), 8.63 (2H), 8.51(3H), 8.38 (2H), 7.79 (12H), 7.60 (8H). IR (KBr, cm-1): ν∼ ) CdO (1709), NsH (3207, 3411), CdN (1616), CdC (1454). UV-visible (H2O), λmax, nm (log ε): 460 (4.07), 301.5 (4.48), 287.5 (4.53), 255 (4.29), 232.5 (4.47), 204.5 (4.74). E1/2 (V vs Ag/AgCl in DMF 25 °C, 0.1 M [([But]4 NPF6)]): + 1.18. Complexes [Ru(phen)(BPG)2]Cl2 · 4.4H2O (6), [Ru(dpq)(BPG)2]Cl2 · 3H2O (7), and [Ru(dppz)(BPG)2]Cl2 · 2CH3OH (8) were prepared similarly to the method described for 5, with the precursor complexes [Ru(phen)Cl4], [Ru(dpq)Cl4], and [Ru(dppz)Cl4] in place of [Ru(bpy)Cl4]. The purification was also carried out by column chromatography on an active alumina column. Single crystals were grown by slow evaporation of the methanol-water solution. [Ru(phen)(BPG)2]Cl2 · 4.4H2O (6). Yield: (70%). Calcd for RuC40H36.8N14O8.4Cl2 (1019.59): C 47.08, H 3.64, N 19.23%. Found: C 47.63, H 3.83, N 19.63%. 1H NMR (300 MHz, DMSO-d6, 25 °C): δ ) 8.57(m, 6H), 8.33(s, 8H), 8.17(s, 2H), 8.05 (d, 2H), 7.97 (d, 3H), 7.70 (m, 3H), 7.54 (d, 2H), 7.27 (d, 2H). IR (KBr, cm-1): ν∼ ) CdO (1703), NsH (3207, 3408), CdN (1616), CdC (1452). UV-visible (H2O), λmax, nm (log ε): 457 (4.11), 375.5 (3.87), 301 (4.43), 262.5 (4.72), 224 (4.70), 205 (4.84). E1/2 (V vs Ag/AgCl in DMF 25 °C, 0.1 M [([But]4 NPF6)]): +1.17. [Ru(dpq)(BPG)2]Cl2 · 3H2O (7). Yield: (63%). Calcd for RuC42H36N16O8Cl2 (1046.39): C 48.16, H 3.27, N 21.41%. Found: C 47.86, H 3.90, N 21.70%. 1H NMR (300 MHz,

Ru(II) Complexes of Bipyridine-Glycoluril

DMSO-d6, 25 °C): δ ) 9.43 (dd, 2H), 9.14 (dd, 2H), 8.85 (s, 6H), 8.34 (m, 3H), 8.22 (m, 6H), 8.19 (dd, 2H), 7.91 (dd, 2H), 7.88 (m, 4H), 7.41 (dd, 1H). IR(KBr, cm-1) ν∼ ) CdO (1699), NsH (3091, 3178), CdN (1614), CdC (1450). UV-visible (H2O), λmax, nm (log ε): 454 (4.20), 297.5 (4.67), 256.5 (4.84), 206.5 (4.85). E1/2 (V vs Ag/AgCl in DMF 25 °C, 0.1 M [([But]4 NPF6)]): +1.17. [Ru(dppz)(BPG)2]Cl2 · 2CH3OH (8). Yield: (67%). Calcd for RuC48N16O6H38Cl2: C 52.01, H 3.48, N 20.23%. Found: C 52.06, H 3.43, N 19.46%. 1H NMR (300 MHz, DMSO-d6, 25 °C): δ ) 9.60 (dd, 2H), 9.18 (d, 1H), 8.78 (d, 4H), 8.46 (s, 8H), 8.39 (d, 4H), 8.25 (m, 5H), 8.09 (m, 3H), 7.75 (m, 3H). IR (KBr, cm-1): ν∼ ) CdO (1699), NsH (3095, 3186), CdN (1616), CdC (1454). UV-visible (H2O), λmax, nm (log ε): 457 (3.90), 359.5 (4.10), 277.5 (4.64), 205.5 (4.73). E1/2 (V vs Ag/AgCl in DMF 25 °C, 0.1 M [([But]4 NPF6)]): +1.18. 5. Series III - Ru/BPG Ratio (1:3). [Ru(BPG)3]Cl2 · 4H2O (9). This complex was prepared by the reaction of RuCl3 · xH2O (0.100 g, 0.444 mmol) and BPG (0.392 g, 1.331 mmol) in a 1:3 molar ratio in 1:1 methanol/water at reflux for 12 h whereupon the color of the solution changed to red. The resulting red solution was cooled to room temperature and filtered. After evaporation of the solvent, the complex was collected and washed with water and diethyl ether. The product was purified by column chromatography on active alumina using methanol and water as eluent. The red fraction was collected. Yield: (75%). Elemental analysis calcd. for RuC42H38N18O10Cl2 (1126.43): C 44.71, H 3.39, N 22.36%. Found: C 43.80, H 3.09, N 22.63%. 1H NMR (300 MHz, DMSO-d6, 25 °C): δ ) 8.79 (s, 6H), 8.50 (s, 6H), 8.28 (d, 6H), 7.79 (d, 6H), 7.75 (d, 6H) ppm. 1H NMR (300 MHz, D2O, 25 °C): δ ) 8.15 (d, 6H), 7.90 (d, 6H), 7.57 (d, 6H) ppm. IR (KBr) ν∼ ) 1703 cm-1 (CdO), 3213 cm-1 (NsH), 3417 cm-1 (OsH), 1643 cm-1 (CdN), 1454 cm-1 (CdC). UV-visible (H2O), λmax, nm (log ε): 456 (4.10), 304 (4.48), 262 (4.29), 231 (4.52). E1/2 (V vs Ag/AgCl in DMF 25 °C, 0.1 M [([But]4 NPF6)]): +1.14. B. Methods. 1. Spectroscopy. The microanalyses (C, H, and N) were carried out with a Perkin-Elmer 240 Q elemental analyzer at NCL, Pune. UV-vis spectra were recorded on a Shimadzu UV-1601 spectrophotometer. 1H NMR spectra were measured on a Varian-Mercury 300 MHz spectrometer with (d6) DMSO as a solvent at room temperature, and all chemical shifts are given relative to TMS. The infrared spectra of solid samples dispersed in KBr were recorded on a Shimadzu FTIR-8400 spectrophotometer. Electrochemical experiments with Ru(II) polypyridyl complexes in DMF solution were performed on a CH-660A (USA) electrochemical instrument in a conventional three-electrode cell assembly with a saturated Ag/AgCl reference electrode, platinum as working electrode for all measurements. Electrochemical measurements were performed using dimethylformamide as solvent and 0.1 M tetrabutylammonium hexafluorophosphate ([But]4NPF6) as supporting electrolyte. Steadystate emission titrations were carried out on a Shimadzu RF5301 spectrofluorometer at room temperature. The emission lifetimes were measured with a time-correlated-single-photoncounting spectrometer from IBH, UK, using 455 nm nanosecond light-emitting diode (NanoLed-01) for the excitation of the samples. 2. DNA Binding and CleaVage Studies. a. Absorption Spectral Studies. For electronic absorption titration, a stock 10 µM solution of the complex was made up in a phosphate buffer (pH 7.2); 3000 µL of the solution was loaded into an optical glass cuvette with a path length of 1 cm, and 200 µL was removed with a micropipette and replaced with 200 µL of the complex solution. This cuvette was then loaded into the spectrometer sample block, controlled at 25 °C; 3000 µL of the buffer was loaded to an identical cuvette and placed in the

Bioconjugate Chem., Vol. 20, No. 3, 2009 449

reference cell. Both the cuvettes were mixed 30 times with a micropipette, and all bubbles were removed. After the cuvettes had been allowed to reach equilibrium over the course of 20 min, a spectrum was recorded between 700 and 200 nm. One to 55 mL of CT-DNA was added to both cuvettes and mixed 30 times. The spectrum was recorded after checking for bubbles and showed a drop in absorptivity showing interaction between the DNA and the metal complex. The intrinsic binding constant of the complex with CT-DNA was determined from eq 1 (64, 65) where [DNA] is the concentration of DNA in base pairs. [DNA] ⁄ [εa - εf] ) [DNA] ⁄ [εb - εf] + 1 ⁄ Kb[εb - εf]

(1)

The apparent absorption coefficients εa, εf, and εb correspond to Aobsd/[Ru], the extinction coefficient for the free complex and the extinction coefficient for the complex in the fully bound form, respectively. The slope and the intercept of the linear fit of [DNA]/[εa - εf] versus [DNA] plot give 1/[εa - εf] and 1/Kb[εb - εf], respectively. The intrinsic binding constant Kb can be obtained from the ratio of the slope to the intercept (64). b. Viscosity Measurements. Viscosity experiments were carried out using a semimicro viscometer maintained at 28 °C in a thermostatic water bath. Flow time of solutions in phosphate buffer (pH 7.2) was recorded in triplicate for each sample, and an average flow time was calculated. Data were presented as (η/η0)1/3 versus binding ratio, where η is the viscosity of DNA in the presence of complex and η0 is the viscosity of DNA alone (65). c. Thermal Denaturation Study. DNA melting studies were carried out with a JASCO V-630 spectrophotometer equipped with a Peltier temperature-controlling programmer ETC-717 ((0.1 °C) in phosphate buffer. The DNA melting studies were done by controlling the temperature of the sample cell with a water-circulating bath. UV melting profiles were obtained by scanning A260 absorbance was monitored at a heating rate of 1 °C/min for solutions of CT-DNA (100 µM) in the absence and presence of ruthenium(II) complexes (20 µM) from 30 to 90 °C. The data were analyzed with the use of separate thermal melting program; the temperature of the cell containing the cuvette was ramped from 50 to 90 °C. The melting temperature Tm which is defined as the temperature where half of the total base pairs are unbound was determined from the midpoint of the melting curves. d. Steady-State and Time-ResolVed Luminescence Spectral Studies. For calculating emission quantum yields, the optical densities of the samples were adjusted to about 0.3 at the excitation wavelength. Emission quantum yields (φ) were calculated by integrating the area under the fluorescence curves and by using the formula (66) φSample ) {ODStandard⁄ODSample} × {ASample ⁄ AStandard} × φStandard (2) where OD is optical density of the compound at the excitation wavelength (450 nm) and A is the area under the emission spectral curve. The standard used for the fluorescence quantum yield measurements was [Ru(bpy)3]Cl2 (67). The rate constant, k0, includes both radiative kr and nonradiative knr contributions to the rate of the 3MLCT excited-state decay. Radiative (kr) and nonradiative (knr) decay rate constants were determined using the values of τ and φr (radiative quantum yield) estimated at room temperature (66). The excitation and emission slit widths employed were 5 nm each. Emission titration experiments were performed by using a fixed metal complex concentration to which increments of the stock DNA solutions were added. Typical concentration of metal complex used was 0.025 mM and [DNA]/[Ru] ratio is in the range 0-30. After the addition of DNA to metal complex,


Deshpande et al.

the resulting solution was allowed to equilibrate for 20-30 min at room temperature before being excited in their intense metal to ligand charge-transfer band between 400 and 500 nm. Steady-state quenching experiments were conducted by adding 75-750 µL aliquots of a 4 mM ferrocyanide stock solution to 3 mL sample solutions containing 0.8 mM nucleic acid concentration and 0.02 mM Ru(II) complex concentration in phosphate buffer. All solutions were allowed to equilibrate thermally for ∼15 min before measurements were made. The Stern-Volmer quenching constant is calculated according to the classical Stern-Volmer equation (66, 68) I0 ⁄ I ) 1 + KSV[Q]

Scheme 1. Structure of BPG and Various Polypyridyl Ligands Used in the Present Study

(3)

where I0 and I are the fluorescence intensities of the complex in the absence and presence of [Fe(CN)6]4-, and Ksv is the SternVolmer quenching constant, which is a measure of the efficiency of fluorescence quenching by [Fe(CN)6]4-. For time-resolved single photon counting measurements, the samples were excited in their intense metal to ligand chargetransfer band between 400 and 500 nm. Emission was detected in the wavelength range 590-630 nm depending on the sample, using a photomultiplier tube based detection module (model TBX-04 from IBH). The instrument response function for the present setup is ∼1.2 ns (fwhm). The decay curves were analyzed by reconvolution procedure (69), using DAS-6 software, obtained from IBH, considering a suitable functional form (mono- or biexponential) of the decays. The quality of the fits were judged by the reduced chi-square (χ2) values and the distribution of the weighted residuals among the data channels (69). For good fits, the χ2 values were close to unity and the weighted residuals were distributed randomly among the data channels (69). e. DNA CleaVage. The DNA cleavage was carried out by agarose gel electrophoresis as described previously (54, 70, 71). A 10 µL total sample volume in 0.5 mL transparent Eppendorf microtubes containing pBR322 DNA (90 µM in base pairs). For the gel electrophoresis experiments, plasmid pBR322 DNA was treated with the metal complex (20 µM) and the mixture was incubated in the dark for 2 and 18 h at 37 °C for the dark experiments. The samples were analyzed by 1% agarose gel electrophoresis (Tris-Boric acid-EDTA (TBE) buffer, (pH 8.2) for 2 h at 60 mV. The gel was stained with a 0.5 µg/mL ethidium bromide and visualized by UV light and photographed for analysis. The extent of cleavage of the plasmid pBR322 DNA was determined by measuring the intensities of the bands using the Alpha Innotech Gel documentation system (AlphaImager 2200). Irradiation experiments were carried out under illuminated conditions using a UV lamp of 365 nm. In irradiation experiments, pBR322 DNA (90 µM in base pairs) was treated with 20 µM samples of the metal complexes, and the mixtures were incubated for 30 min in the dark followed by 20 min irradiation at 365 nm. For mechanistic investigations, experiments were carried out under irradiated conditions in the presence of radical scavenging agents DMSO, mannitol, DABCO, NaN3, L-histidine, and SOD, which were added to the plasmid pBR322 DNA prior to the addition of the complexes. f. Dialysis Experiments. A typical dialysis experiment was carried out as follows. Three milliliters solution of the ruthenium(II) complexes 1-9 ([Ru] ) 20 µM) in the presence of CT-DNA ([DNA] ) 200 µM) under dark conditions in phosphate buffer (pH 7.2) solution was transferred to the dialysis tubing (molecular weight cutoff 12 000, 14 000) and dialysed with gentle stirring against buffer solution in the dark, with three changes of buffer over a 24 h period. Changes in the absorption spectrum of complexes 1-9 in the presence of calf thymus DNA with dialysis (24 h) compared with the dialyzed samples and undialyzed samples were monitored by UV-vis spectroscopy.

g. Molecular Modeling. Molecular modeling studies were performed on a Silicon Graphics Octane workstation using the software Insight II 2000 (72) with the Discover 3 module. Initial models of right-handed B-DNA of sequence d(C:G)12 were constructed using the Biopolymer module of Insight II. The coordinates for the metal complex [Ru(bpy)2(BPG)]2+ and derivatives were taken from its crystal structure as a CIF file and were converted to the PDB format using Mercury software (73). The all atom extensible systematic force field (ESFF) was used for the entire modeling study. The [Ru(bpy)2(BPG)]2+ and derivative-bound DNA was then soaked in a water box of dimensions 35.0 × 50.0 × 35.0, and periodic boundary conditions were applied. A dielectric constant of 1.0 for electrostatic energy and a cutoff of 9.5 A were used for both the Coulomb as well as van der Waals energies. The nonbonded electrostatic terms were calculated using the Ewald summation method (74). The accuracy of convergence for the Ewald Coulomb energy summation was kept to 0.0001 kcal/mol. The system was minimized using the steepest descent gradient algorithm for 1000 steps, followed by the conjugate gradient algorithm for 1000 steps or until the maximum derivative was below 0.1 kcal/mol/A. The Ewald summation method that expands the simple sum of the Coulomb’s law terms into several sums including a direct, reciprocal term was used to calculate the long-range electrostatics. As the Ewald sum method is valid for a neutral system, B-DNA-Ru(II) polypyridyl complex, the Ewald summation method calculates charges by homogeneous charge density distribution. The initial 1000 steps of steepest descent minimization were performed to relax the initial strain on the molecule.

RESULTS AND DISCUSSION A. Syntheses and Properties. The urea fused bipyridine ligand bipyridine-glycoluril (BPG) (Scheme 1) and a series of nine new ruthenium(II) polypyridyl complexes of the type [Ru(N-N)2(BPG)]Cl2 1-4, [Ru(N-N)(BPG)2]Cl2 5-8, and [Ru(BPG)3]Cl2 9 were synthesized and obtained in a racemic form by the reactions of precursor complexes cis-[Ru(NN)2Cl2], [Ru(N-N)Cl4], and RuCl3 · xH2O by varying the number of BPG (Scheme 2). Crystal structures of BPG ligand and its complexes 1, 6, and 9 have been reported elsewhere (51, 52). The electronic absorption spectra of complexes 1-9 are dominated by two sets of transitions (i) low-energy metal-toligand charge transfer (MLCT) transitions in the range 440-460 nm and (ii) high-energy ligand-centered π-π* transitions (IL) in the range 200-380 nm for all complexes which are similar to the parent [Ru(bpy)3]2+ and other Ru(II) polypyridyl complexes (75, 76). The peaks at 371 and 360 nm in compounds 4 and 8 are characteristic of the π-π* transition of the dppz



Scheme 2. Synthetic Route for Complexes 1-9

Table 1. Photophysical Data for Ruthenium(II) Polypyridyl Complexes 1-7 and 9 emission (buffer) complex λem (nm) τ (ns) 1 2 3 4 5 6 7 9

624 622 605 601 612 609 598 596

253 369 147 348 346 384 332 342

φem

Kr (s-1)

0.0096 0.0149 0.049 0.0119 0.027 0.032 0.033 0.0178

× × × × × × × ×

3.8 4.0 3.4 3.4 7.7 8.4 1.0 5.2

Knr (s-1) 4

10 104 104 104 104 104 105 104

3.9 2.7 6.5 2.8 2.8 2.5 2.9 2.9

× × × × × × × ×

6

10 106 106 106 106 106 106 106

K0 (s-1) 3.9 2.7 6.8 2.8 2.9 2.6 3.0 2.9

× × × × × × × ×

106 106 106 106 106 106 106 106

ligand. The synthesized ruthenium(II) complexes 1-7 and 9 emit in phosphate buffer at room temperature with maximum between 590 and 640 nm. The emission quantum yields for 1-7 and 9 are calculated by using eq 2 and summarized in Table 1. The radiative rate constants for complexes 1-7 and 9 are on the order of 104-105 and nonradiative rate constants are on the order of 106 (Table 1) that are in the range expected for Ru(II) polypyridyl emitters (75, 76). No detectable luminescence is observed for complex 8 in buffer solution. Single exponential decays are observed for 1-7 and 9 in buffer solutions, and the results are tabulated in Table 1. DNA Binding Studies. Absorption Spectroscopy Studies. Absorption spectral titrations are the most common method to investigate the interactions of metal complexes with DNA. Binding of the complexes to DNA through intercalation results in hypochromism and red shift of the bands due to noncovalent stacking of the planar aromatic chromophore between the base pairs of DNA (77-82). Changes in the electronic absorption spectra of complexes 1-9 were measured as a function of DNA concentration, and the titration curves for 3, 4, 7, and 8 are shown in Figure 1. There was insignificant change in the absorption profile of complexes 1, 5, 6, and 9 on sequential addition of DNA, indicating an electrostatic binding similar to that of [Ru(bpy)3]2+ (3, 78-82), as the nonplanar bpy and urea moiety of BPG precludes intercalation. The binding constants estimated using eq 1 for complexes 2, 3, 4, 7, and 8 are (3.86 ( 0.2) × 103, (3.14 ( 0.1) × 104, (6.19 ( 0.1) × 104, (2.87 ( 0.2) × 104, and (9.14 ( 0.2) × 104, respectively (Table 2). The

absorption spectra of 3 on addition of DNA shows 5.1% hypochromism in the MLCT band at the [DNA]/[Ru] ratio of 8, whereas complex 4 shows 9.6% and 19.5% hypochromicity in both MLCT and IL bands, respectively, at the same [DNA]/ [Ru] ratio. The extent of hypochromism and red shift is more in 4 than 3, indicating strong binding of this complex to CTDNA. A comparison of the binding constants of 3 and 4 with strong intercalators such as [Ru(phen)2dppz]2+ reveals a weak binding due to hindrance to the intercalation by the second planar dppz and the nonplanar BPG ancillary ligand. For the complex 7, the hypochromism in the MLCT and IL bands are 3.0% and 5.1% at the same [DNA]/[Ru] ratio. However, for complex 8, the absorption spectra on addition of DNA shows pronounced hypochromism in both MLCT and IL bands of about 12.3% and 31.0%, respectively, at the ratio of [DNA]/ [Ru] of 8. The extent of hypochromism and red shift is particularly pronounced in the interligand absorption band (∼360 nm) for 8 and is typical for stacking interaction of the dppz ligand with the DNA base pairs. These spectral characteristics suggest that complexes 7 and 8 interact with DNA through a mode that involves a stacking interaction of the planar aromatic chromophore and the base pairs of DNA, but with a moderate binding constant as compared to classical intercalators ([Ru(phen)2(dppz)]2+, Kb ) 5.1 × 106 M-1) probably due to the influence of ancillary BPG ligand. Similar results have been observed for the [Ru(NH3)4(dppz)]2+, Kb ) 1.8 × 105 M-1 (see Table 2 for comparative data on dppz complexes) for which the NH3 ligand which has the potential for hydrogen bonding is found to be detrimental for DNA binding (46). In the present series, the ancillary ligand bipyridine-glycoluril has the potential for hydrogen bonding interactions with the phosphates or bases of DNA, resulting in lowering the binding constant value as compared to classical intercalators. Viscosity and Thermal Denaturation Studies. Viscosity measurements, which are sensitive to length change, are regarded as most critical tests for binding mode and were studied in order to assess the binding mode of these complexes with DNA. Changes in relative viscosity provide a reliable method for distinguishing between intercalators and electrostatic binders of DNA. Intercalation of a ligand into DNA is known to cause a significant increase in the viscosity of a DNA solution due to an increase in the separation of the base pairs at the intercalation site and, hence, an increase in the overall DNA molecular length. In contrast, a ligand that binds in the DNA grooves causes a less pronounced change (positive or negative) or no change in the viscosity of a DNA solution. The changes in the relative viscosity of solutions containing DNA upon addition of increasing concentrations of 1-9 are shown in Supporting Information Figures S1 and S2. The maximum increase in viscosity of DNA on increasing the [Ru]/[DNA] ratio for complexes 3, 4, 7, and 8 suggest an intercalative binding mode, while a minute/ negligible increase in viscosity for the complexes 1 and 2 indicate an electrostatic binding. The incorporation of nonintercalating BPG ligands with bulky peripheral urea groups are expected to influence the efficacy of the intercalative ligand. A similar effect was observed in the case of [Ru(bpyMe2)2(dpq)]2+ where the orientation of the peripheral bpyMe2 ligands results in partial intercalation of the classical intercalator dpq (12). Addition of complexes 5, 6, and 9 has no effect on the DNA viscosity, suggesting an electrostatic binding similarly observed previously for [Ru(bpy)3]2+ (78, 87). The viscosity data taken together with the hypochromism observed in the absorption data upon addition of DNA are in accord with the fact that the peripheral urea groups influence the ability of the intercalative ligand. The thermal behavior of the DNA in the presence of complexes would offer some information about the interaction


Deshpande et al.

Figure 1. Changes in the electronic absorption spectra of (A) 3 (10 µM), (B) 4 (10 µM), (C)7 (10 µM), and (D) 8 (10 µM) with increasing the concentrations (0-100 µM) of CT-DNA (phosphate buffer pH 7.2); the inset graph shows a fit of the absorbance data used to obtain the binding constant. Table 2. Spectroscopic Properties of the Complexes 3, 4, 7, and 8 in the presence of CT-DNA

complexes

Kb (M-1)

3 4 7 8 [Ru(bpy)2(dppz)]2+ [Ru(phen)2(dppz)]2+ [Ru(dmp)2(dppz)]2+ [Ru(dmb)2(dppz)]2+ [Ru(NH3)4(dppz)]2+ [Ru(tpm)(py)(dppz)]2+ [Ru(terpy)(dppz)(OH2)]2+ [Ru(IP)2(dppz)]2+

(3.14 ( 0.1) × 104 (6.19 ( 0.1) × 104 (2.87 ( 0.2) × 104 (9.14 ( 0.2) × 104 5.0 × 106 5.1 × 106 2.3 × 106 4.5 × 106 1.8 × 105 4.7 × 106 7.0 × 105 2.1 × 107

a

hypochromismb H (%) MLCT LMCT 3.0 9.6 3.0 12.3 14.5 11.2 15.6 13.6 9.6 40.4

5.1 19.5 5.1 31.0 40.1 35.0 31.1 38.5 46.3

Table 3. Luminescence and Thermal Properties of Complexes 1-9 in the Absence and Presence of CT-DNA Stern-Volmer quenching constantb (M-1)

ref a

this work this work a this work a this work 37, 20 78-82 37, 43 37 46 41 47 37, 42 a

[DNA]/[Ru] ) 8:1 H % ) 100(Afree - Abound)/Afree. b

affinities of these complexes with DNA and characterizes the transition from double-stranded to single-stranded DNA. The extinction coefficient of DNA bases at 260 nm in the double helical form is much less than in the single-stranded form; hence, melting of the helix leads to an increase in the absorption at this wavelength. Thus, the melting temperature (Tm) of DNA, which can characterize the transition from double-stranded to single-stranded form of DNA, is usually determined (43, 49). Thermal denaturation Figures S3 and S4 in the Supporting Information and the data for DNA in the presence and absence of ruthenium(II) complexes 1-9 investigated in this study are summarized in Table 3. Thermal denaturation experiments carried out on CT-DNA in the absence of added complex revealed that the Tm value for the duplex is 63° ( 1°. It is clear from the data given in the Table 3 that the complexes containing the non-intercalative/partial intercalative ligands shift the Tm values only by up to 2° compared to that of the pure DNA sample. However, the complexes containing the intercalative ligands dpq/dppz shift the Tm values by up to 4° compared to that of the pure DNA sample. This increase in the helix melting

complexes 1 2 3 4 5 6 7 8 9

∆Tm/°C 2 2 2 3 2 2 3 4 1

I/I0a

without DNA

with DNA

1.13 1.23 1.69 3.70 1.09 1.13 1.65 23.31 1.05

2.12 × 10 1.02 × 103 2.59 × 103 2.35 × 103 3.32 × 103 3.09 × 103 2.64 × 103 2.45 × 103

1.96 × 103 1.00 × 103 2.69 × 102 1.49 × 102 2.68 × 103 2.31 × 103 4.11 × 102 2.83 × 102 2.40 × 103

3

a Relative emission intensity enhancement in the presence of CTDNA at R ) 30. b Stern-Volmer constants for the quenching of the complexes by K4[Fe(CN)6] in the absence and presence of DNA.

temperature indicates the increased stability of the double helix when these compounds 3, 4, 7, and 8 bind to DNA. However, the increase in the melting temperature is relatively lower than the classical intercalators, suggesting that the dpq/dppz complexes bind with DNA through a mode that involves a stacking interaction of the planar aromatic chromophore and the base pairs of DNA, but with a moderate binding, probably due to the influence of ancillary BPG ligand, thus lowering the Tm values for these complexes. Steady-State Emission Studies. The changes in the emission spectra of Ru(II) polypyridyl complexes in the presence of DNA are a diagnostic means to determine DNA binding (19, 76). The emission spectra of complexes 1-9 have been measured in the absence and in the presence of CT-DNA (Supporting Information Figures S5 and S6). The dependence of relative emission intensities as a function of DNA concentration in buffered solution at pH 7.2 is shown in Figure 2 (in terms of [DNA]/ [Ru]). The spectra profiles and emission maxima for the complexes 1-3, 5-7, and 9 exhibit weak luminescence


Figure 2. Plots of relative integrated emission intensity versus [DNA]/ [Ru] for the complexes: (A) Complexes (9) 1, (b) 2, ([) 3, (2) 4, and (1) 9 (24 µM). (B) Complexes (b) 5, (9) 6, (2) 7, (1) 8 (24 µM) in phosphate buffer, pH 7.2 at 298 K with increasing [DNA]/[Ru] ratio from 0-30.

enhancements in the range 1-1.7 after adding CT-DNA at a ratio of [DNA]/[Ru] ) 30 indicating a weak binding of these complexes with CT-DNA by partial intercalation or electrostatic association (Table 3). However, complex 4 exhibits luminescence enhancement of 3.7 indicating strong binding by intercalation of this complex with CT-DNA. On the other hand, complex 8 is nonluminescent in aqueous solution but emits intensely in the presence of CT-DNA. In this case, observed emission enhancement was ascribed to the protection of the phenazine nitrogen atoms of DNA-intercalated excitedstate complex from accessibility by water molecules. The fluorescence intensity is observed to vary with the [DNA]/[Ru] ratio. The remarkable sensitivity is observed for complex 8 in the presence of CT-DNA (R ) 0-30) with the relative emission intensity enhancement of a factor 23.31, much higher than that for the other complexes (Figure S6 in the Supporting Information) that supports the stacking interaction of the planar aromatic dppz ligand into the DNA base pairs, which is consistent with other spectroscopic studies. Steady-State Emission Quenching Experiment Using K4[Fe(CN)6]. The fluorescence intensities of the ruthenium polypyridyl complexes, upon visible excitation, are enhanced on binding to DNA (83-85). Quenching of this luminescent excited state with the use of an anionic quencher such as [Fe(CN)6]4- has been shown to be able to distinguish bound ruthenium(II) species (86). A highly negatively charged quencher is expected to be repelled by the negatively charged phosphate backbone, and therefore, a DNA-bound cationic molecule should be protected from quenching while free complexes should be readily quenched. The results of steady-state emission quenching experiments using [Fe(CN)6]4- as the quencher are shown in Figures S7 and S8 in the Supporting Information. The results are interpreted in terms of two binding modes: electrostatic, which is easily quenched by ferrocyanide; and intercalative, which is protected from ferrocyanide quenching. Stern-Volmer quenching constants for complexes 1-7 and 9 in the absence and presence of DNA are shown in Table 3. In the absence of DNA, complexes 1-7 and 9 are efficiently quenched by [Fe(CN)6]4- with the Stern-Volmer quenching constants on the order of 1.02-3.32 × 103. The nonlinear nature of the graph for complex 2 indicates that two differential binding modes, viz., partial intercalation and surface binding, may be possible. Barton et al. interpreted the curved Stern-Volmer plots obtained with the anionic quencher [Fe(CN)6]4- as having two binding modes, one intercalative and one groove-bound, with ∆ preferring the former and Λ the latter mode in the case of [Ru(phen)3]2+ (87, 88). However, in the presence of DNA, the maximum decrease in the Stern-Volmer quenching constant is obtained for the complexes 3, 4, 7, and 8. For these complexes, the Stern-Volmer plot is a straight line, implying that the luminescent complex is homogeneous. The linear nature with negligible difference in the Stern-Volmer quenching constants


for complexes 1, 5-6, and 9 indicates a single component donor-quencher system with electrostatic binding similar to that for [Ru(bpy)3]2+. Time-ResolVed Emission Measurements in the Presence of DNA. The above-mentioned trends in DNA binding are further corroborated by time-correlated single photon counting fluorescence measurements. In the absence of DNA, all the complexes strictly exhibit monoexponential emission decay. Table S1 in the Supporting Information summarizes the luminescence lifetime of the complexes 1-9 in the presence of CT-DNA for a ratio [DNA]/[Ru] of 2-30. Complexes 1, 5, and 9 show monoexponential decay with negligible change in τo value, indicating an electrostatic interaction of these complexes with DNA. Complex 3, 6, and 7 also exhibits monoexponential decay; however, the lifetime increases from 330 to 432 ns for 3, 386 to 404 ns for 6, and 357 to 426 ns for 7 exhibits monoexponential decay, indicating intercalative/partial intercalative binding mode with DNA. The luminescent characteristics of the complexes 2 and 4 bound to DNA shows biexponential decay in emission indicating the presence of two distinguishable DNA binding modes for the complexes. Two binding modes were proposed for the complex 2; one may be partial intercalation, while the other is groove-bound interaction or electrostatic interaction in which the excited-state lifetime is comparable to that of the free form. When the binding ratio [DNA]/[Ru] is varied from 2:1 to 30:1 for the complex 4, the excited-state lifetimes increased from 207 ns to 290 ns for shortlifetime component and from 677 to 1679 ns for long-lifetime component and for the complex 2 from 183 to 334 ns for shortlifetime component and from 329 to 761 ns for long-lifetime component. The steady-state quenching experiments and timeresolved emission measurements on complex 4 suggest that both lifetime components could be assigned to partial intercalative binding depending on the orientations of the two dppz ligands in the complex. The results also are consistent with intercalation and electrostatic binding being the two binding modes. This complex contains the sterically demanding bipyridine glycoluril ancillary ligand, and the other planar dppz ligand, which increases hydrophobicity (17). The presence of multiple binding modes for osmium and ruthenium polypyridyl complexes has been suggested recently on the basis of DNA film voltammetry (21). The luminescent characteristics of the complex 8 bound to DNA show biexponential decay in emission indicating the presence of two distinguishable DNA binding modes. When the binding ratio [DNA]/[Ru] varied from 2:1 to 30:1 for the complex 8, the excited-state lifetimes increased up to 125 ns for short-lifetime component and from 346 to 611 ns for longlifetime component. The lifetimes obtained for long-lived species of complex 8 are higher than the others, confirming its stronger intercalative DNA binding, and does not differ from the excitedstate lifetimes in the presence of DNA for [Ru(phen)2dppz]2+ (10, 11, 17-21) indicating that the intercalated dppz ligand is well-protected from solvent. Preliminary DNA CleaVage Studies under Dark and Light Conditions. Majority of ruthenium-polypyridyl complexes noncovalently bind to DNA by different modes, viz., electrostatic, surface binding, or intercalation, and initiate DNA cleavage reactions on photoirradiation either by electron transfer to base forming covalent photoadducts or by energy transfer to molecular oxygen generating 1O2 or rarely by a hydrolytic mechanism (50, 54, 89-95). We have recently reported the hydrolytic cleavage of plasmid pBR322 DNA by [Ru(bpy)2(BPG)]2+ 1 in an enzyme-like manner and detailed the kinetic aspects of DNA cleavage by 1 under pseudo and true Michaelis-Menten conditions (54). Therefore, in the present study the potential of complexes 2-9 to cleave DNA under


Deshpande et al.

Figure 3. Ethidium bromide-stained agarose gel (1%) of plasmid pBR322 DNA (90 µM in base pairs) in the presence of 20 µM Ru(II) complexes 2-4 and 9. (A) Dark experiments for 2-4 and 9 (incubation for 2 h in air atmosphere): Form I - supercoiled plasmid DNA, Form II - nicked circular plasmid DNA; Lane 1 - DNA control, Lane 2 - DNA + [Ru(bpy)3]2+, Lane 3 - DNA + [Ru(bpy)2(dppz)]2+, Lane 4 - DNA + 2, Lane 5 - DNA + 3, Lane 6 - DNA + 4, Lane 7 - DNA + 9. (B) Light experiments for 2-4 and 9 in air (incubation 30 min, irradiation 20 min; λirr ) 365 nm): Lane 1 - DNA control, Lane 2 - DNA + [Ru(bpy)3]2+, Lane 3 - DNA + [Ru(bpy)2(dppz)]2+, Lane 4 - DNA + 2, Lane 5 - DNA + 3, Lane 6 - DNA + 4, Lane 5 - DNA + 9.

Figure 4. Ethidium bromide-stained agarose gel (1%) of plasmid pBR322 DNA (90 µM in base pairs) in the presence of 20 µM Ru(II) complexes 5-8. (A) Dark experiments for 5-8 (incubation for 2 h in air atmosphere): Form I - supercoiled plasmid DNA, Form II - nicked circular plasmid DNA; Lane 1 - DNA control, Lane 2 - DNA + [Ru(bpy)3]2+, Lane 3 - DNA + [Ru(bpy)2(dppz)]2+, Lane 4 - DNA + 5, Lane 5 - DNA + 6, Lane 6 - DNA + 7, Lane 7 - DNA + 8. (B) Light experiments for 5-8 in air (incubation 30 min, irradiation 20 min; λirr ) 365 nm): Lane 1 - DNA control, Lane 2 - DNA + [Ru(bpy)3]2+, Lane 3 - DNA + [Ru(bpy)2(dppz)]2+, Lane 4 - DNA + 5, Lane 5 - DNA + 6, Lane 6 - DNA + 7, Lane 5 - DNA + 8.

dark and light conditions was studied by gel electrophoresis using plasmid pBR322 DNA. When incubated for 2 h at 37 °C under dark conditions, it is observed that micromolar concentration of complexes 2-4 and 9 shows 22-40% (Figure 3A) and 5-8 shows 28-60% (Figure 4A) DNA cleavage as evidenced by the disappearance of form I (supercoiled form) of the plasmid and the appearance of the form II (nicked circular form). A band migration of the supercoiled form of DNA relative to the control lane is observed for complexes 2-4 (Figure 3A; lanes 4-6) and for complexes 7 and 8 (Figure 4A; lanes 6 and 7). An increase in DNA cleavage is observed for 2-9 after incubation for 2 h in argon atmosphere (Figures S9 and S10A in the Supporting Information) implying an oxygen-independent cleavage mechanism under dark conditions. Extensive DNA cleavage is observed for complexes 2-9 in dark conditions when incubated for 18 h at 37 °C (Figures S9B and S10B in the Supporting Information) indicating a hydrolytic mechanism, which suggests that these complexes are oriented in such a way so as to facilitate intramolecular H-bonding with the phosphate group or with the nucleobases. Under similar conditions [Ru(bpy)3]2+ and [Ru(bpy)2(dppz)]2+, which binds to DNA by electrostatic binding and intercalation, respectively, and are not capable of H-bonding, cleave DNA to a lesser extent after 2 h incubation as compared to complexes 2-9. Here, we have observed DNA cleavage under dark conditions by ruthenium polypyridyl complexes containing one, two, and three BPG ligands without any bound water molecule, but with the pheripheral urea moiety of the ancillary bipyridyl glycoluril ligand hydrolyzes the phosphodiester bond efficiently. The rate of RNA hydrolysis is enhanced 3300-fold by monometallic Zn(II) complexes of terpyridine-based ligands with ammonium and guanidinium groups capable of H-bonding to the phosphodiester group compared to the parent complex without the functional H-bonding groups (96). A similar effect of enhancing the phosphodiester cleavage was observed by a Zn(II) complex with three aminopyridyl hydrogen bond donors, which orient the complex and the substrate for efficient proximity interactions (97-99). A series of organometallic

ruthenium and cis-platin like ethylenediamine based complexes also exploit these H-bonding interactions to direct DNA binding by the NH2 groups of the metal-bound ethylenediamine ligand favoring H-bonding interactions with nucleobases resulting in site-selectivity (100-102). Rhodium(III) intercalators attached to peptide moieties [Rh(phi)2(bpy′-peptide)] (phi ) intercalative ligands), macrocyclic complexes of lanthanides, polyamine derivatives such as cyclen, trpn, and tamen complexes of cobalt(III) can act as catalysts for the hydrolysis of the phosphate esters of DNA and ammonium-functionalized copper(II) complexes and copper(II) complexes of macrocycles, cis,cis-1,3,5-triaminocyclohexane and neamine have been reported (103-105). Of those, however, the most highly efficient hydrolytic cleavage agents are mononuclear copper(II) complexes in which a copper-bound hydroxyl group is the active species in the hydrolysis of the nucleic acid phosphate backbone. As far as we are aware, the only other hydrolytic cleavage of DNA by the ruthenium-polypyridyl complex was reported by Barton et al. for the [Ru(DIP)2(macro)]2+, where Ru(DIP)2 binds to DNA via intercalation and macro is a chelating ligand with two polyamine tridentate armlike segments which bind certain divalent metal cations so as to deliver its coordinated nucleophile to the phosphate backbone for the hydrolysis of the anionic diester (50). Photocleavage of DNA by ruthenium(II) polypyridyl complexes on irradiation is well-documented in the literature (89-95). Therefore, the photocleavage of plasmid pBR322 DNA in the presence of complexes 2-9 on irradiation at 365 nm was carried out, and the results are shown in Figure 3B and Figure 4B. The complexes 2 and 9 show only 3-12% increase in conversion of form I (SC) of DNA to form II (NC) of DNA, and the complexes 5 and 6 show only 6-7% increase in conversion of form I (SC) of DNA to form II (NC) of DNA as compared to DNA cleavage under dark conditions. The fact that the observed photocleavage for complexes 2, 5, 6, and 9 in air is significantly lower than what is observed for [Ru(bpy)3]2+ possibly indicates lower levels of 1O2 generation. The complexes 3, 4, 7, and 8 and [Ru(bpy)2(dppz)]2+ shows extensive DNA cleavage in the



Figure 5. DNA cleavage efficiency of ruthenium(II) complexes containing one, two, and three BPG ligands 2-9 in the dark and light conditions.

presence of light with a shift in mobility of form I of DNA probably due to the high molecular weight of DNA-complex conjugates. In order to investigate the role of radicals in the DNA damage by these complexes 2-9, reactions were performed under aerobic conditions by incubating the complexes for half an hour and irradiation for 20 min with DNA in the presence of hydroxyl radical scavengers (DMSO and mannitol), singlet oxygen scavengers (NaN3, histidine, and DABCO) and superoxide scavenger (superoxide dismutase, SOD) (Figures S11-S18, Tables S2-S9 in the Supporting Information). It is observed that the photocleavage by complexes 2 and 9 was not inhibited by reactive oxygen species scavengers indicating hydrolytic pathway for these complexes. However, complexes 5 and 6 show small amounts of inhibition in the presence of reactive oxygen species indicating hydrolytic mechanism. Complexes 3, 4, 7, and 8 containing dpq and dppz intercalating ligands show extensive DNA cleavage in the presence of light, and the photocleavage experiments in the presence of different radical scavengers show inhibition indicating that hydroxyl radicals and singlet oxygen are responsible for DNA cleavage by these complexes upon irradiation. From the above results, it appears that the mechanism of DNA cleavage photoinduced by complexes 3, 4, 7, and 8 involve either singlet oxygen or hydroxyl radicals. However, the photocleavage by these complexes is higher than [Ru(bpy)2(dppz)]2+ complex implying that complexes 3, 4, 7, and 8 with an inherent intercalating ability cleave DNA by a combination of oxidative and hydrolytic cleavage. The cleavage data for all complexes 2-9 under dark conditions in the presence and absence of air and under light conditions are tabulated in Table S10 in the Supporting Information and depicted as a histogram in Figure 5. DNA-Complex Conjugate InVestigation by UV-Vis Spectroscopy: In order to further investigate the formation of the DNA-complex conjugates, dialysis experiments followed by UV-vis spectroscopy were performed on the ruthenium(II) bipyridine-glycoluril complexes in the presence of CT-DNA under dark conditions in phosphate buffer (pH 7.2). If the complex is covalently bound to the DNA, then this can be verified by dialysis of the samples where the parent metal complex, but not the DNA, would pass through the dialysis membrane (106, 107). The formation of the product is monitored by UV-vis spectroscopy, and the results are given in Figure 6 and Figure S19 in the Supporting Information. Figure 6 shows the spectrum of the complexes 1, 7, 8, and 9 kept in the dark for 24 h in the presence of CT-DNA, both before and after dialysis. With complexes 1 and 9, the large decrease in absorption is observed showing that most of the product is diffused through the dialysis membrane as would be expected

Figure 6. Changes in the absorption spectras of complexes 1 and 7-9 in the presence of calf thymus DNA under dark conditions with dialysis (24 h) compared with the dialyzed samples and undialyzed samples in phosphate buffer pH 7.2 solutions. ([Ru] ) 20 µM, [DNA] ) 200 µM).

for the complexes which are not bound strongly to the CTDNA. However, the absorption spectrum of the dialyzed samples 2-8 in the presence of DNA reveals that the complex remains bound to DNA inside the membrane (Figure 6 and Figure S19 in the Supporting Information). The complexes 2-8 after dialysis in the presence of CT-DNA show large increases in absorption in the 345-500 nm region suggesting that these complexes are strongly bound to the CT-DNA and the conjugate formed is retained after dialysis. The UV-vis spectroscopic observations together with the electrophoresis results presented earlier suggests that the DNA-complex conjugates are formed with CT-DNA. A hyperchromic effect and mobility shift in electrophoresis experiments were reported previously for the complexes of the type [Ru(bpy)3-n(TAP/HAT)n]2+ (n ) 2, 3), which result in the formation of adducts on irradiation (106, 107). Molecular Modeling. Molecular mechanics calculations have been carried out for complexes 1-9 with the models of righthanded B-DNA of sequence d(C:G)12. Different modes of binding with different orientations of ruthenium complexes, including groove binding through major/minor groove, and intercalation through major and minor groove, were attempted with the model of right-handed B-DNA of sequence d(C:G)12. It was observed that the minimized structure maintains the octahedral form of the complexes and shows the H-bonding to the bases and phosphates of DNA without disrupting the helical structure of B-DNA. The H-bonding distances for 1-9 after minimizations are summarized in Table S11 in the Supporting Information. All complexes are stabilized by H-bonding interactions between the DNA and the complexes. It is found that 1 and 2 orients in the minor and major groove, respectively, with hydrogen bonding interactions of the N-H and C-H groups preferably to O1P and O10P phosphate oxygen atoms of DNA for 1 and preferably to O2P phosphate oxygen atom in case of 2 (Figure 7 and Figure S18 in the Supporting Information), while for 9 in the major groove with the H- bonding between the NsH and CdO groups of the bipyridine-glycoluril ligand and the N7 of the guanine. (Figure S24 in the Supporting Information). Such NsH · · · O H-bonding interactions between one ethylenediamine NH proton and the guanine O6 oxygen have been shown by Sadler and co-workers in the case of organomatallic ruthenium(II) arene complexes of the type [(η6-arene)RuII(en)Cl](PF6) (en ) ethylenediamine, arene ) biphenyl, 5,8,9,10tetrahydroanthracene, and 9,10-dihydroanthracene) specifically target guanine bases of DNA oligomers (102). It is found that


Deshpande et al.

behaviors for complexes 1-9 were observed. The planar aromatic ligands have the potential for intercalation, and the bipyridine-glycoluril ligand with inherent H-bond donor and acceptor groups are accessible for H-bonding with the phosphate groups or with the nucleobases of DNA. The DNA binding and photocleavage measurements revealed that the complexes that favor electrostatic binding mode cleave DNA by hydrolytic mechanism while those that contain intercalating ligands form conjugates with and then cleave DNA by a combination of oxidative and hydrolytic mechanism.

ACKNOWLEDGMENT

Figure 7. The core view of the bound 1 to DNA with the H-bond patterns observed showing phosphate interactions.

Figure 8. The core view of the bound 8 to DNA showing the intercalation of the dppz ligand between the base pairs of DNA along with the H-bonding interactions of the two BPG ligands with the phosphate groups of DNA.

the binding of 5 and 6 in the major groove with H-bonding interactions of the N-H groups preferably to O2P phosphate oxygen atoms of DNA (Figure S21 and Figure S22 in the Supporting Information). In the complexes 1, 2, and 9, the urea groups exclusively bind with phosphate oxygens of the nucleic acids. Recently, Farrell et al. have reported a phosphate backbone binding mode for a polynuclear platinum(II) complex that has planar arrays of hydrogen bond donors, leading to association with the DNA backbone (108). For complexes 3 and 4, the molecular modeling reveals that the planar dpq and dppz ligands interact with the bases and the other planar ligand with the phosphate oxygens. (Figure S19 and S20 in the Supporting Information). However, the complexes 7 and 8 containing planar aromatic dpq and dppz ligands and with the two ancillary bipyridine-glycoluril ligands show the intercalative binding mode in which the stacking interaction of the planar dpq (Figure S23 in the Supporting Information) and dppz ligand between the base pairs of DNA through major groove side take place along with the phosphate interactions of the N-H groups of the two ancillary bipyridine-glycoluril ligands preferably to O2P oxygen atoms (Figure 8), Such H-bonding suggests that the bipyridine-glycoluril ligand is located in a specific position of the H-bond to the neighboring oxygen atoms of the bases or a phosphate group and thus influences the binding ability of these complexes with DNA.

CONCLUDING REMARKS In summary, in the present study we have synthesized a series of new Ru(II) polypyridyl complexes containing urea fused bipyridine-glycoluril ligand and characterized by various physical methods. By the incorporation of the simple modification on the ancillary ligand of bipyridine, different DNA binding

M.S.D. acknowledges Bhabha Atomic Research Centre (BARC) for providing research fellowship through collaborative research scheme of Pune University - BARC, Mumbai, India. A.A.K. acknowledges the financial assistance from Department of Science and Technology (DST), New Delhi, for the award of Fast Track Project for Young Scientist (SR/FTP/CSA-15/ 2003). A.S.K. thanks University of Pune for partial funding. The time correlated single photon-counting spectrophotometer facility in the Department was created by funding from the University Grants Commission, New Delhi, under the Centre for Advanced Studies funds (CAS). The authors thank Dr. Amitava Das CSMCRI, Bhavnagar, India, for cyclic voltammetry data. Supporting Information Available: Changes in the relative specific viscosity of solutions 1-9 in the presence of CT-DNA (Figure S1 and S2); melting curves of CT-DNA in the absence and presence of complexes 1-9 (Figure S3 and S4); emission spectra of Ru(II) complexes 1-9 (25 µM) in phosphate buffer with increasing [DNA]/[Ru] ratio from 0 to 30 (Figure S5 and S6), emission spectra of Ru(II) complexes 1-9 using anionic quencher K4[Fe(CN)6] in the presence and absence of DNA in phosphate buffer (Figure S7 and S8), luminescence decay lifetime of the complexes 1-9 in the presence of CT-DNA (Table S1), DNA cleavage experiment in the presence of 20 µM Ru(II) complexes 2-9; dark experiments incubation for 2 h in argon atmosphere and dark experiments incubation for 18 h in air (Figure S9 and S10); DNA cleavage experiment in the presence of different radical scavengers for Ru(II) complexes 2-9 (Figure S11-S18 and Table S2-S9); DNA cleavage efficiency of ruthenium(II) complexes containing one, two, and three BPG ligands 2-9 in the dark and light conditions (Table S10); changes in the absorption spectras of complexes 2-6 in the presence of DNA before and after dialysis (Figure S19); the core view of the bound 2-7 and 9 (Figure S20-S26) to DNA, binding energies in kcal/mol and hydrogen bond information of Ru(II) polypyridyl complexes 1-9 with right-handed B-DNA of sequence d(C:G)12 (Table S11). This material is available free of charge via the Internet at http://pubs.acs.org.

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