Ruthenium(II) Complexes of Bipyridine−Glycoluril and their Interactions with DNA

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Ruthenium(II) Complexes of Bipyridine#Glycoluriland their Interactions with DNA

Megha S. Deshpande, Anupa A. Kumbhar, Avinash S. Kumbhar, ManojKumbhakar, Haridas Pal, Uddhavesh B. Sonawane, and Rajendra R. Joshi

Bioconjugate Chem., 2009, 20 (3), 447-459• DOI: 10.1021/bc800298t • Publication Date (Web): 23 February 2009

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Ruthenium(II) Complexes of Bipyridine-Glycoluril and their Interactionswith 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 - 400085, 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 whereN-N is 2,2′-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq), dipyrido[3,2-a:2′,3′-c]phenazine (dppz), which incorporates bipyridine-glycoluril (BPG-4b,5,7,7a-tetrahydro-4b,7a-epimi-nomethanoimino-6H-imidazo[4,5-f][1,10]phenanthroline-6,13-dione) as the ancillary ligand, have been synthesizedand characterized. These complexes with the peripheral polypyridyl ligands have the ability to form conjugateswith 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 thesecomplexes has been studied to investigate the influence of the ancillary ligand. The binding ability of thesecomplexes to DNA is dependent on the planarity of the intercalative polypyridyl ligand, which is further affectedby the ancillary bipyridine-glycoluril ligand. The complexes 3, 4, 7, and 8 bind to CT-DNA with binding constantson the order of 104 M-1. Time-resolved emission measurements on the DNA-bound complexes 1, 3, 5-7, and 9show monoexponential decay of the excited states, whereas complexes 2, 4, and 8 show biexponential decay withshort- and long-lived components. Interaction of complexes 2-9 with plasmid pBR322 DNA studied by gelelectrophoresis experiments reveals that all complexes cleave DNA efficiently at micromolar concentrations underdark 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 dueto the high molecular weight of DNA-complex conjugates. However, the extent of the cleavage is augmented onirradiation in the case of complexes 3, 4, 7, and 8, which include the planar dpq and dppz ligands, suggesting acombination of hydrolytic and oxidative mechanism for the DNA scission. Molecular mechanics calculations ofthese systems corroborate the DNA binding and cleavage mechanisms.

INTRODUCTION

The interaction of transition metal complexes with DNA is avibrant area of research (1-4). An advantage of using thesecomplexes in such studies is that their ligands and metals canbe conveniently varied to suit individual applications. Ruthe-nium(II) complexes with intercalating polypyridyl ligands havebeen extensively studied in this context, as their luminescenceand photochemical reactivity are significantly altered on interac-tion with DNA (1-11). Ruthenium polypyridyl complexes areuseful probes of DNA structure and DNA oxidation chemistryand in addition are used as sensors (12-49). Among them,[Ru(bpy)2(dppz)]2+ and [Ru(phen)2(dppz)]2+ are found to behigh-affinity intercalators with interesting light switch behaviorwith potential applications in sensing and signaling, as well asin data storage and communication (14, 15). The versatility ofthese complexes is modulated by the ligand set, which controlswhether a complex is an intercalator, hemi-intercalator, orelectrostatic binder (12-49). In general, ruthenium polypyridylintercalators 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.

The affinity of dppz intercalators for DNA can be furtherenhanced by increasing the surface area of the ancillary ligand(45).

Adding groups to the edges of the ruthenium polypyridylcomplexes further expands the functionality of these complexes.For example, insertion of two polyamine tridentate arm-likesegments in a macrochelating ligand in the complex[Ru(DIP)2(macro)]n+ enables binding of certain divalent metalcations so as to deliver its coordinated nucleophile to thephosphate backbone for hydrolysis of the anionic diester (50).In a previous study, we have explored the possibility ofmodifying 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 extensiveH-bonding networks resulting in diverse frameworks encapsu-lating water/solvent molecules depending upon the number ofbipyridine-glycoluril ligands (51-53). We have also demon-strated that the urea groups of the BPG ligand are involved inDNA binding and facilitate hydrolytic cleavage of DNA by thecomplex [Ru(bpy)2(BPG)]2+ 1 (54). Thus, the molecules thatcan hydrolyze the DNA phosphodiester at specific positions arevaluable tools in biotechnology, thus facilitating DNA manipu-lation in a variety of applications.

To test whether the hydrolytic DNA cleavage mechanism isunique to a ruthenium polypyridyl complex containing a singleBPG ligand, we synthesized the series of compounds containingone, two, or three BPG ligands. In complexes with one or twoBPG ligands, the remaining ligands were varied to include

* 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).

Bioconjugate Chem. 2009, 20, 447–459 447

10.1021/bc800298t CCC: $40.75 2009 American Chemical SocietyPublished on Web 02/23/2009

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

EXPERIMENTAL SECTION

A. Materials. 1. General Details. All reagents and solventswere purchased commercially and were used as received.RuCl3 · xH2O and K4[Fe(CN)6] were obtained from S. D. FineChemicals Limited (India). Calf thymus DNA and plasmidpBR322 DNA were purchased from SRL (India). The enzymesuperoxide dismutase (SOD EC1.15.1.1) was purchased fromSigma Chemical Co. USA. Deionized water was used for thepreparation of the buffers. Supercoiled pBR322 DNA (CsCl)purified was obtained from Bangalore Genei (Bangalore, India)and used as received. The concentration of DNA in nucleotidephosphate (NP) was determined by UV absorbance at 260 nmusing the molar absorption coefficient as 6600 M-1 cm-1.Solutions of calf thymus DNA in phosphate buffer gave a ratioof 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 (phen-dione) (56), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq) (57), anddipyrido[3,2-a: 2′,3′-c]phenazine (dppz) (58) were synthesizedaccording to the literature protocols. BPG [4b,5,7,7a-tetrahydro-4b,7a-epiminomethanoimino-6H-imidazo[4,5-f] [1,10] phenan-throline-6,13-dione] was prepared by modifying the literaturemethod (59, 60, 51-54). The precursor complexes of the typecis-[Ru(N-N)2Cl2] (61, 62), [Ru(N-N)Cl4] (63) were preparedby 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 describedfor complex 1 (51, 54) using cis- [Ru(phen)2Cl2] ·2H2O in placeof cis-[Ru(bpy)2Cl2] ·2H2O. The precursor complex cis-[Ru(phen)2Cl2] ·2H2O (0.125 g, 0.21 mmol) and BPG (0.0608g, 0.21 mmol) in a 1:1 molar ratio were dissolved in 50%methanol/50% water (50 mL), and the mixture was heated toreflux for 8 h, whereupon the color of the solution changed fromdark purple to red. The red solution was filtered hot and wascooled to room temperature. After evaporation of the solvent,the red solid was collected and washed with small amounts ofmethanol and diethyl ether and then dried under vacuum. Theproduct was purified by column chromatography on activealumina using acetone and methanol as eluent. The red fractionwas collected and concentrated in vacuum, a small amount ofdiethyl ether was added to the concentrated solution, and a redsolid 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∼ ) 1708cm-1 (CdO), 3215 cm-1 (NsH), 3421 cm-1 (OsH), 1647 cm-1

(CdN), 1427 cm-1 (CdC). UV-visible (H2O), λmax, nm (logε): 441 (4.01), 263 (4.77), 223 (4.70). E1/2 (V vs Ag/AgCl inDMF 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 methoddescribed for complex 1, with cis-[Ru(dpq)2Cl2] ·2H2O and cis-[Ru(dppz)2Cl2] · 2H2O in place of cis-[Ru(bpy)2Cl2] · 2H2O.

However, in the case of 4 the precursor complex cis-[Ru(dppz)2Cl2] ·2H2O and BPG in a 1:1 molar ratio was refluxedin ethylene glycol (20 mL) for 12 h whereupon the color of thesolution changed to red. The purification was also carried outby column chromatography on an active alumina column.

[Ru(dpq)2(BPG)]Cl2 ·4H2O (3). Yield: (60%). Elementalanalysis 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 (300MHz, 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 (300MHz, 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∼ ) 1705cm-1 (CdO), 2923 cm-1 (NsH), 3336 cm-1 (OsH), 1602 cm-1

(CdN), 1407 cm-1 (CdC). UV-visible (H2O), λmax, nm (logε): 443 (4.05), 371 (4.03), 263 (4.83), 224 (4.68). E1/2 (V vsAg/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, 0.190mmol) and BPG (0.112 g, 0.381 mmol) in a 1:2 molar ratiowere refluxed in 50% methanol/50% water (50 mL) for 8 h,whereupon the color of the solution changed from dark greento red. The red solution was filtered hot and was cooled to roomtemperature. After evaporation of the solvent, the brownish redsolid was collected and washed with small amounts of methanoland diethyl ether and then dried under vacuum. The productwas purified by column chromatography on active alumina usingacetone and methanol as eluent. The red fraction was collectedand concentrated in vacuum, a small amount of diethyl etherwas added to the concentrated solution and red solid wasobtained.

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 describedfor 5, with the precursor complexes [Ru(phen)Cl4], [Ru(dpq)Cl4],and [Ru(dppz)Cl4] in place of [Ru(bpy)Cl4]. The purificationwas also carried out by column chromatography on an activealumina column. Single crystals were grown by slow evapora-tion of the methanol-water solution.

[Ru(phen)(BPG)2]Cl2 ·4.4H2O (6). Yield: (70%). Calcd forRuC40H36.8N14O8.4Cl2 (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 (logε): 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,

448 Bioconjugate Chem., Vol. 20, No. 3, 2009 Deshpande et al.

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 forRuC48N16O6H38Cl2: 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 inDMF 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 a1:3 molar ratio in 1:1 methanol/water at reflux for 12 hwhereupon the color of the solution changed to red. Theresulting red solution was cooled to room temperature andfiltered. After evaporation of the solvent, the complex wascollected and washed with water and diethyl ether. The productwas purified by column chromatography on active alumina usingmethanol and water as eluent. The red fraction was collected.

Yield: (75%). Elemental analysis calcd. for RuC42H38-N18O10Cl2 (1126.43): C 44.71, H 3.39, N 22.36%. Found: C43.80, 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∼ ) 1703cm-1 (CdO), 3213 cm-1 (NsH), 3417 cm-1 (OsH), 1643 cm-1

(CdN), 1454 cm-1 (CdC). UV-visible (H2O), λmax, nm (logε): 456 (4.10), 304 (4.48), 262 (4.29), 231 (4.52). E1/2 (V vsAg/AgCl in DMF 25 °C, 0.1 M [([But]4 NPF6)]): +1.14.

B. Methods. 1. Spectroscopy. The microanalyses (C, H, andN) were carried out with a Perkin-Elmer 240 Q elementalanalyzer at NCL, Pune. UV-vis spectra were recorded on aShimadzu UV-1601 spectrophotometer. 1H NMR spectra weremeasured on a Varian-Mercury 300 MHz spectrometer with (d6)DMSO as a solvent at room temperature, and all chemical shiftsare given relative to TMS. The infrared spectra of solid samplesdispersed in KBr were recorded on a Shimadzu FTIR-8400spectrophotometer. Electrochemical experiments with Ru(II)polypyridyl complexes in DMF solution were performed on aCH-660A (USA) electrochemical instrument in a conventionalthree-electrode cell assembly with a saturated Ag/AgCl referenceelectrode, platinum as working electrode for all measurements.Electrochemical measurements were performed using dimeth-ylformamide as solvent and 0.1 M tetrabutylammonium hexaflu-orophosphate ([But]4NPF6) as supporting electrolyte. Steady-state emission titrations were carried out on a Shimadzu RF-5301 spectrofluorometer at room temperature. The emissionlifetimes were measured with a time-correlated-single-photon-counting spectrometer from IBH, UK, using 455 nm nanosecondlight-emitting diode (NanoLed-01) for the excitation of thesamples.

2. DNA Binding and CleaVage Studies. a. Absorption Spec-tral Studies. For electronic absorption titration, a stock 10 µMsolution of the complex was made up in a phosphate buffer(pH 7.2); 3000 µL of the solution was loaded into an opticalglass cuvette with a path length of 1 cm, and 200 µL wasremoved with a micropipette and replaced with 200 µL of thecomplex solution. This cuvette was then loaded into thespectrometer sample block, controlled at 25 °C; 3000 µL ofthe buffer was loaded to an identical cuvette and placed in the

reference cell. Both the cuvettes were mixed 30 times with amicropipette, and all bubbles were removed. After the cuvetteshad been allowed to reach equilibrium over the course of 20min, a spectrum was recorded between 700 and 200 nm. Oneto 55 mL of CT-DNA was added to both cuvettes and mixed30 times. The spectrum was recorded after checking for bubblesand showed a drop in absorptivity showing interaction betweenthe DNA and the metal complex. The intrinsic binding constantof the complex with CT-DNA was determined from eq1 (64, 65) where [DNA] is the concentration of DNA in basepairs.

[DNA] ⁄ [εa - εf]) [DNA] ⁄ [εb - εf]+ 1 ⁄ Kb[εb - εf] (1)

The apparent absorption coefficients εa, εf, and εb correspondto Aobsd/[Ru], the extinction coefficient for the free complex andthe extinction coefficient for the complex in the fully boundform, respectively. The slope and the intercept of the linear fitof [DNA]/[εa - εf] versus [DNA] plot give 1/[εa - εf] and1/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 car-

ried out using a semimicro viscometer maintained at 28 °C ina thermostatic water bath. Flow time of solutions in phosphatebuffer (pH 7.2) was recorded in triplicate for each sample, andan average flow time was calculated. Data were presented as(η/η0)1/3 versus binding ratio, where η is the viscosity of DNAin the presence of complex and η0 is the viscosity of DNA alone(65).

c. Thermal Denaturation Study. DNA melting studies werecarried out with a JASCO V-630 spectrophotometer equippedwith a Peltier temperature-controlling programmer ETC-717((0.1 °C) in phosphate buffer. The DNA melting studies weredone by controlling the temperature of the sample cell with awater-circulating bath. UV melting profiles were obtained byscanning A260 absorbance was monitored at a heating rate of 1°C/min for solutions of CT-DNA (100 µM) in the absence andpresence of ruthenium(II) complexes (20 µM) from 30 to 90°C. The data were analyzed with the use of separate thermalmelting program; the temperature of the cell containing thecuvette was ramped from 50 to 90 °C. The melting temperatureTm which is defined as the temperature where half of the totalbase pairs are unbound was determined from the midpoint ofthe melting curves.

d. Steady-State and Time-ResolVed Luminescence SpectralStudies. For calculating emission quantum yields, the opticaldensities of the samples were adjusted to about 0.3 at theexcitation wavelength. Emission quantum yields (φ) werecalculated by integrating the area under the fluorescence curvesand by using the formula (66)

�Sample ) {ODStandard⁄ODSample} × {ASample ⁄ AStandard} × �Standard

(2)

where OD is optical density of the compound at the excitationwavelength (450 nm) and A is the area under the emissionspectral curve. The standard used for the fluorescence quantumyield measurements was [Ru(bpy)3]Cl2 (67). The rate constant,k0, includes both radiative kr and nonradiative knr contributionsto the rate of the 3MLCT excited-state decay. Radiative (kr) andnonradiative (knr) decay rate constants were determined usingthe values of τ and φr (radiative quantum yield) estimated atroom temperature (66).

The excitation and emission slit widths employed were 5 nmeach. Emission titration experiments were performed by usinga fixed metal complex concentration to which increments ofthe stock DNA solutions were added. Typical concentration ofmetal complex used was 0.025 mM and [DNA]/[Ru] ratio is inthe range 0-30. After the addition of DNA to metal complex,

Ru(II) Complexes of Bipyridine-Glycoluril Bioconjugate Chem., Vol. 20, No. 3, 2009 449

the resulting solution was allowed to equilibrate for 20-30 minat room temperature before being excited in their intense metalto ligand charge-transfer band between 400 and 500 nm.

Steady-state quenching experiments were conducted byadding 75-750 µL aliquots of a 4 mM ferrocyanide stocksolution to 3 mL sample solutions containing 0.8 mM nucleicacid concentration and 0.02 mM Ru(II) complex concentrationin phosphate buffer. All solutions were allowed to equilibratethermally for ∼15 min before measurements were made. TheStern-Volmer quenching constant is calculated according to theclassical Stern-Volmer equation (66, 68)

I0 ⁄ I) 1+KSV[Q] (3)

where I0 and I are the fluorescence intensities of the complexin the absence and presence of [Fe(CN)6]4-, and Ksv is the Stern-Volmer quenching constant, which is a measure of the efficiencyof fluorescence quenching by [Fe(CN)6]4-.

For time-resolved single photon counting measurements, thesamples were excited in their intense metal to ligand charge-transfer band between 400 and 500 nm. Emission was detectedin the wavelength range 590-630 nm depending on the sample,using a photomultiplier tube based detection module (modelTBX-04 from IBH). The instrument response function for thepresent setup is ∼1.2 ns (fwhm). The decay curves wereanalyzed by reconvolution procedure (69), using DAS-6 soft-ware, obtained from IBH, considering a suitable functional form(mono- or biexponential) of the decays. The quality of the fitswere judged by the reduced chi-square (�2) values and thedistribution of the weighted residuals among the data channels(69). For good fits, the �2 values were close to unity and theweighted residuals were distributed randomly among the datachannels (69).

e. DNA CleaVage. The DNA cleavage was carried out byagarose gel electrophoresis as described previously (54, 70, 71).A 10 µL total sample volume in 0.5 mL transparent Eppendorfmicrotubes containing pBR322 DNA (90 µM in base pairs). Forthe gel electrophoresis experiments, plasmid pBR322 DNA wastreated with the metal complex (20 µM) and the mixture wasincubated in the dark for 2 and 18 h at 37 °C for the darkexperiments. The samples were analyzed by 1% agarose gelelectrophoresis (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 ethidiumbromide and visualized by UV light and photographed foranalysis. The extent of cleavage of the plasmid pBR322 DNAwas determined by measuring the intensities of the bands usingthe Alpha Innotech Gel documentation system (AlphaImager2200). Irradiation experiments were carried out under il-luminated conditions using a UV lamp of 365 nm. In irradiationexperiments, pBR322 DNA (90 µM in base pairs) was treatedwith 20 µM samples of the metal complexes, and the mixtureswere incubated for 30 min in the dark followed by 20 minirradiation at 365 nm. For mechanistic investigations, experi-ments were carried out under irradiated conditions in thepresence of radical scavenging agents DMSO, mannitol, DAB-CO, NaN3, L-histidine, and SOD, which were added to theplasmid pBR322 DNA prior to the addition of the complexes.

f. Dialysis Experiments. A typical dialysis experiment wascarried out as follows. Three milliliters solution of the ruthe-nium(II) complexes 1-9 ([Ru] ) 20 µM) in the presence ofCT-DNA ([DNA] ) 200 µM) under dark conditions inphosphate buffer (pH 7.2) solution was transferred to the dialysistubing (molecular weight cutoff 12 000, 14 000) and dialysedwith gentle stirring against buffer solution in the dark, with threechanges of buffer over a 24 h period. Changes in the absorptionspectrum of complexes 1-9 in the presence of calf thymus DNAwith dialysis (24 h) compared with the dialyzed samples andundialyzed samples were monitored by UV-vis spectroscopy.

g. Molecular Modeling. Molecular modeling studies wereperformed on a Silicon Graphics Octane workstation using thesoftware Insight II 2000 (72) with the Discover 3 module. Initialmodels of right-handed B-DNA of sequence d(C:G)12 wereconstructed using the Biopolymer module of Insight II. Thecoordinates for the metal complex [Ru(bpy)2(BPG)]2+ andderivatives were taken from its crystal structure as a CIF fileand were converted to the PDB format using Mercury software(73). The all atom extensible systematic force field (ESFF) wasused for the entire modeling study. The [Ru(bpy)2(BPG)]2+ andderivative-bound DNA was then soaked in a water box ofdimensions 35.0 × 50.0 × 35.0, and periodic boundaryconditions were applied. A dielectric constant of 1.0 forelectrostatic energy and a cutoff of 9.5 A were used for boththe Coulomb as well as van der Waals energies. The nonbondedelectrostatic terms were calculated using the Ewald summationmethod (74). The accuracy of convergence for the EwaldCoulomb energy summation was kept to 0.0001 kcal/mol. Thesystem was minimized using the steepest descent gradientalgorithm for 1000 steps, followed by the conjugate gradientalgorithm for 1000 steps or until the maximum derivative wasbelow 0.1 kcal/mol/A. The Ewald summation method thatexpands the simple sum of the Coulomb’s law terms into severalsums including a direct, reciprocal term was used to calculatethe long-range electrostatics. As the Ewald sum method is validfor a neutral system, B-DNA-Ru(II) polypyridyl complex, theEwald summation method calculates charges by homogeneouscharge density distribution. The initial 1000 steps of steepestdescent minimization were performed to relax the initial strainon the molecule.

RESULTS AND DISCUSSION

A. Syntheses and Properties. The urea fused bipyridineligand bipyridine-glycoluril (BPG) (Scheme 1) and a series ofnine 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 racemicform by the reactions of precursor complexes cis-[Ru(N-N)2Cl2], [Ru(N-N)Cl4], and RuCl3 · xH2O by varying thenumber of BPG (Scheme 2). Crystal structures of BPG ligandand itscomplexes1,6, and9havebeenreportedelsewhere (51,52).

The electronic absorption spectra of complexes 1-9 aredominated by two sets of transitions (i) low-energy metal-to-ligand charge transfer (MLCT) transitions in the range 440-460nm and (ii) high-energy ligand-centered π-π* transitions (IL)in the range 200-380 nm for all complexes which are similarto the parent [Ru(bpy)3]2+ and other Ru(II) polypyridylcomplexes (75, 76). The peaks at 371 and 360 nm in compounds4 and 8 are characteristic of the π-π* transition of the dppz

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

450 Bioconjugate Chem., Vol. 20, No. 3, 2009 Deshpande et al.

ligand. The synthesized ruthenium(II) complexes 1-7 and 9emit in phosphate buffer at room temperature with maximumbetween 590 and 640 nm. The emission quantum yields for 1-7and 9 are calculated by using eq 2 and summarized in Table 1.The radiative rate constants for complexes 1-7 and 9 are onthe order of 104-105 and nonradiative rate constants are on theorder of 106 (Table 1) that are in the range expected for Ru(II)polypyridyl emitters (75, 76). No detectable luminescence isobserved for complex 8 in buffer solution. Single exponentialdecays are observed for 1-7 and 9 in buffer solutions, and theresults are tabulated in Table 1.

DNA Binding Studies. Absorption Spectroscopy Studies.Absorption spectral titrations are the most common method toinvestigate the interactions of metal complexes with DNA.Binding of the complexes to DNA through intercalation resultsin hypochromism and red shift of the bands due to noncovalentstacking of the planar aromatic chromophore between the basepairs of DNA (77-82). Changes in the electronic absorptionspectra of complexes 1-9 were measured as a function of DNAconcentration, and the titration curves for 3, 4, 7, and 8 areshown in Figure 1. There was insignificant change in theabsorption profile of complexes 1, 5, 6, and 9 on sequentialaddition of DNA, indicating an electrostatic binding similar tothat of [Ru(bpy)3]2+ (3, 78-82), as the nonplanar bpy and ureamoiety of BPG precludes intercalation. The binding constantsestimated 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 of8, whereas complex 4 shows 9.6% and 19.5% hypochromicityin both MLCT and IL bands, respectively, at the same [DNA]/[Ru] ratio. The extent of hypochromism and red shift is morein 4 than 3, indicating strong binding of this complex to CT-DNA. A comparison of the binding constants of 3 and 4 withstrong intercalators such as [Ru(phen)2dppz]2+ reveals a weakbinding due to hindrance to the intercalation by the secondplanar dppz and the nonplanar BPG ancillary ligand. For thecomplex 7, the hypochromism in the MLCT and IL bands are3.0% and 5.1% at the same [DNA]/[Ru] ratio. However, forcomplex 8, the absorption spectra on addition of DNA showspronounced hypochromism in both MLCT and IL bands ofabout 12.3% and 31.0%, respectively, at the ratio of [DNA]/[Ru] of 8. The extent of hypochromism and red shift isparticularly pronounced in the interligand absorption band (∼360nm) for 8 and is typical for stacking interaction of the dppzligand with the DNA base pairs. These spectral characteristicssuggest that complexes 7 and 8 interact with DNA through amode that involves a stacking interaction of the planar aromaticchromophore and the base pairs of DNA, but with a moderatebinding constant as compared to classical intercalators([Ru(phen)2(dppz)]2+, Kb ) 5.1 × 106 M-1) probably due tothe influence of ancillary BPG ligand. Similar results have beenobserved for the [Ru(NH3)4(dppz)]2+, Kb ) 1.8 × 105 M-1 (seeTable 2 for comparative data on dppz complexes) for whichthe NH3 ligand which has the potential for hydrogen bondingis found to be detrimental for DNA binding (46). In the presentseries, the ancillary ligand bipyridine-glycoluril has the po-tential for hydrogen bonding interactions with the phosphatesor bases of DNA, resulting in lowering the binding constantvalue as compared to classical intercalators.

Viscosity and Thermal Denaturation Studies. Viscositymeasurements, which are sensitive to length change, areregarded as most critical tests for binding mode and were studiedin order to assess the binding mode of these complexes withDNA. Changes in relative viscosity provide a reliable methodfor distinguishing between intercalators and electrostatic bindersof DNA. Intercalation of a ligand into DNA is known to causea significant increase in the viscosity of a DNA solution due toan increase in the separation of the base pairs at the intercalationsite and, hence, an increase in the overall DNA molecular length.In contrast, a ligand that binds in the DNA grooves causes aless pronounced change (positive or negative) or no change inthe viscosity of a DNA solution. The changes in the relativeviscosity of solutions containing DNA upon addition of increas-ing concentrations of 1-9 are shown in Supporting InformationFigures S1 and S2. The maximum increase in viscosity of DNAon increasing the [Ru]/[DNA] ratio for complexes 3, 4, 7, and8 suggest an intercalative binding mode, while a minute/negligible increase in viscosity for the complexes 1 and 2indicate an electrostatic binding. The incorporation of non-intercalating BPG ligands with bulky peripheral urea groupsare 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 ligandsresults in partial intercalation of the classical intercalator dpq(12). Addition of complexes 5, 6, and 9 has no effect on theDNA viscosity, suggesting an electrostatic binding similarlyobserved previously for [Ru(bpy)3]2+ (78, 87). The viscositydata taken together with the hypochromism observed in theabsorption data upon addition of DNA are in accord with thefact that the peripheral urea groups influence the ability of theintercalative ligand.

The thermal behavior of the DNA in the presence ofcomplexes would offer some information about the interaction

Scheme 2. Synthetic Route for Complexes 1-9

Table 1. Photophysical Data for Ruthenium(II) PolypyridylComplexes 1-7 and 9

emission (buffer)

complex λem (nm) τ (ns) φem Kr (s-1) Knr (s-1) K0 (s-1)

1 624 253 0.0096 3.8 × 104 3.9 × 106 3.9 × 106

2 622 369 0.0149 4.0 × 104 2.7 × 106 2.7 × 106

3 605 147 0.049 3.4 × 104 6.5 × 106 6.8 × 106

4 601 348 0.0119 3.4 × 104 2.8 × 106 2.8 × 106

5 612 346 0.027 7.7 × 104 2.8 × 106 2.9 × 106

6 609 384 0.032 8.4 × 104 2.5 × 106 2.6 × 106

7 598 332 0.033 1.0 × 105 2.9 × 106 3.0 × 106

9 596 342 0.0178 5.2 × 104 2.9 × 106 2.9 × 106

Ru(II) Complexes of Bipyridine-Glycoluril Bioconjugate Chem., Vol. 20, No. 3, 2009 451

affinities of these complexes with DNA and characterizes thetransition from double-stranded to single-stranded DNA. Theextinction coefficient of DNA bases at 260 nm in the doublehelical form is much less than in the single-stranded form; hence,melting of the helix leads to an increase in the absorption atthis wavelength. Thus, the melting temperature (Tm) of DNA,which can characterize the transition from double-stranded tosingle-stranded form of DNA, is usually determined (43, 49).Thermal denaturation Figures S3 and S4 in the SupportingInformation and the data for DNA in the presence and absenceof ruthenium(II) complexes 1-9 investigated in this study aresummarized in Table 3. Thermal denaturation experimentscarried out on CT-DNA in the absence of added complexrevealed that the Tm value for the duplex is 63° ( 1°. It is clearfrom the data given in the Table 3 that the complexes containingthe non-intercalative/partial intercalative ligands shift the Tm

values only by up to 2° compared to that of the pure DNAsample. However, the complexes containing the intercalativeligands dpq/dppz shift the Tm values by up to 4° compared tothat of the pure DNA sample. This increase in the helix melting

temperature indicates the increased stability of the double helixwhen these compounds 3, 4, 7, and 8 bind to DNA. However,the increase in the melting temperature is relatively lower thanthe classical intercalators, suggesting that the dpq/dppz com-plexes bind with DNA through a mode that involves a stackinginteraction of the planar aromatic chromophore and the basepairs of DNA, but with a moderate binding, probably due tothe 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 DNAare a diagnostic means to determine DNA binding (19, 76). Theemission spectra of complexes 1-9 have been measured in theabsence and in the presence of CT-DNA (Supporting Informa-tion Figures S5 and S6). The dependence of relative emissionintensities as a function of DNA concentration in bufferedsolution at pH 7.2 is shown in Figure 2 (in terms of [DNA]/[Ru]). The spectra profiles and emission maxima for thecomplexes 1-3, 5-7, and 9 exhibit weak luminescence

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 theconcentrations (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 bindingconstant.

Table 2. Spectroscopic Properties of the Complexes 3, 4, 7, and 8 inthe presence of CT-DNA

complexes Kb (M-1)

hypochromismb

H (%)MLCT LMCT ref

3 (3.14 ( 0.1) × 104 3.0 5.1 a this work4 (6.19 ( 0.1) × 104 9.6 19.5 a this work7 (2.87 ( 0.2) × 104 3.0 5.1 a this work8 (9.14 ( 0.2) × 104 12.3 31.0 a this work[Ru(bpy)2(dppz)]2+ 5.0 × 106 14.5 40.1 37, 20[Ru(phen)2(dppz)]2+ 5.1 × 106 - 35.0 78-82[Ru(dmp)2(dppz)]2+ 2.3 × 106 11.2 31.1 37, 43[Ru(dmb)2(dppz)]2+ 4.5 × 106 15.6 38.5 37[Ru(NH3)4(dppz)]2+ 1.8 × 105 13.6 - 46[Ru(tpm)(py)(dppz)]2+ 4.7 × 106 - - 41[Ru(terpy)(dppz)(OH2)]2+ 7.0 × 105 9.6 - 47[Ru(IP)2(dppz)]2+ 2.1 × 107 40.4 46.3 37, 42

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

Table 3. Luminescence and Thermal Properties of Complexes 1-9in the Absence and Presence of CT-DNA

Stern-Volmer quenchingconstantb (M-1)

complexes ∆Tm/°C I/I0a without DNA with DNA

1 2 1.13 2.12 × 103 1.96 × 103

2 2 1.23 1.02 × 103 1.00 × 103

3 2 1.69 2.59 × 103 2.69 × 102

4 3 3.70 2.35 × 103 1.49 × 102

5 2 1.09 3.32 × 103 2.68 × 103

6 2 1.13 3.09 × 103 2.31 × 103

7 3 1.65 2.64 × 103 4.11 × 102

8 4 23.31 - 2.83 × 102

9 1 1.05 2.45 × 103 2.40 × 103

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

452 Bioconjugate Chem., Vol. 20, No. 3, 2009 Deshpande et al.

enhancements in the range 1-1.7 after adding CT-DNA at aratio of [DNA]/[Ru] ) 30 indicating a weak binding of thesecomplexes with CT-DNA by partial intercalation or electrostaticassociation (Table 3). However, complex 4 exhibits lumines-cence enhancement of 3.7 indicating strong binding by inter-calation of this complex with CT-DNA. On the other hand,complex 8 is nonluminescent in aqueous solution but emitsintensely in the presence of CT-DNA. In this case, observedemission enhancement was ascribed to the protection of thephenazine nitrogen atoms of DNA-intercalated excited-state complex from accessibility by water molecules. Thefluorescence intensity is observed to vary with the [DNA]/[Ru]ratio. The remarkable sensitivity is observed for complex 8 inthe presence of CT-DNA (R ) 0-30) with the relative emissionintensity enhancement of a factor 23.31, much higher than thatfor the other complexes (Figure S6 in the Supporting Informa-tion) that supports the stacking interaction of the planar aromaticdppz ligand into the DNA base pairs, which is consistent withother spectroscopic studies.

Steady-State Emission Quenching Experiment UsingK4[Fe(CN)6]. The fluorescence intensities of the rutheniumpolypyridyl complexes, upon visible excitation, are enhancedon binding to DNA (83-85). Quenching of this luminescentexcited state with the use of an anionic quencher such as[Fe(CN)6]4- has been shown to be able to distinguish boundruthenium(II) species (86). A highly negatively charged quench-er is expected to be repelled by the negatively charged phosphatebackbone, and therefore, a DNA-bound cationic molecule shouldbe protected from quenching while free complexes should bereadily quenched. The results of steady-state emission quenchingexperiments using [Fe(CN)6]4- as the quencher are shown inFigures S7 and S8 in the Supporting Information. The resultsare interpreted in terms of two binding modes: electrostatic,which is easily quenched by ferrocyanide; and intercalative,which is protected from ferrocyanide quenching. Stern-Volmerquenching constants for complexes 1-7 and 9 in the absenceand presence of DNA are shown in Table 3. In the absence ofDNA, complexes 1-7 and 9 are efficiently quenched by[Fe(CN)6]4- with the Stern-Volmer quenching constants on theorder of 1.02-3.32 × 103. The nonlinear nature of the graphfor 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 obtainedwith the anionic quencher [Fe(CN)6]4- as having two bindingmodes, one intercalative and one groove-bound, with ∆ prefer-ring the former and Λ the latter mode in the case of[Ru(phen)3]2+ (87, 88). However, in the presence of DNA, themaximum decrease in the Stern-Volmer quenching constant isobtained for the complexes 3, 4, 7, and 8. For these complexes,the Stern-Volmer plot is a straight line, implying that theluminescent complex is homogeneous. The linear nature withnegligible difference in the Stern-Volmer quenching constants

for complexes 1, 5-6, and 9 indicates a single componentdonor-quencher system with electrostatic binding similar tothat for [Ru(bpy)3]2+.

Time-ResolVed Emission Measurements in the Presence ofDNA. The above-mentioned trends in DNA binding are furthercorroborated by time-correlated single photon counting fluo-rescence measurements. In the absence of DNA, all thecomplexes strictly exhibit monoexponential emission decay.Table S1 in the Supporting Information summarizes theluminescence lifetime of the complexes 1-9 in the presence ofCT-DNA for a ratio [DNA]/[Ru] of 2-30. Complexes 1, 5,and 9 show monoexponential decay with negligible change inτo value, indicating an electrostatic interaction of these com-plexes with DNA. Complex 3, 6, and 7 also exhibits monoex-ponential decay; however, the lifetime increases from 330 to432 ns for 3, 386 to 404 ns for 6, and 357 to 426 ns for 7exhibits monoexponential decay, indicating intercalative/partialintercalative binding mode with DNA. The luminescent char-acteristics of the complexes 2 and 4 bound to DNA showsbiexponential decay in emission indicating the presence of twodistinguishable DNA binding modes for the complexes. Twobinding modes were proposed for the complex 2; one may bepartial intercalation, while the other is groove-bound interactionor electrostatic interaction in which the excited-state lifetime iscomparable to that of the free form. When the binding ratio[DNA]/[Ru] is varied from 2:1 to 30:1 for the complex 4, theexcited-state lifetimes increased from 207 ns to 290 ns for short-lifetime component and from 677 to 1679 ns for long-lifetimecomponent and for the complex 2 from 183 to 334 ns for short-lifetime component and from 329 to 761 ns for long-lifetimecomponent. The steady-state quenching experiments and time-resolved emission measurements on complex 4 suggest that bothlifetime components could be assigned to partial intercalativebinding depending on the orientations of the two dppz ligandsin the complex. The results also are consistent with intercalationand electrostatic binding being the two binding modes. Thiscomplex contains the sterically demanding bipyridine glycolurilancillary ligand, and the other planar dppz ligand, whichincreases hydrophobicity (17). The presence of multiple bindingmodes for osmium and ruthenium polypyridyl complexes hasbeen suggested recently on the basis of DNA film voltammetry(21). The luminescent characteristics of the complex 8 boundto DNA show biexponential decay in emission indicating thepresence of two distinguishable DNA binding modes. Whenthe binding ratio [DNA]/[Ru] varied from 2:1 to 30:1 for thecomplex 8, the excited-state lifetimes increased up to 125 nsfor short-lifetime component and from 346 to 611 ns for long-lifetime component. The lifetimes obtained for long-lived speciesof complex 8 are higher than the others, confirming its strongerintercalative DNA binding, and does not differ from the excited-state lifetimes in the presence of DNA for [Ru(phen)2dppz]2+

(10, 11, 17-21) indicating that the intercalated dppz ligand iswell-protected from solvent.

Preliminary DNA CleaVage Studies under Dark and LightConditions. Majority of ruthenium-polypyridyl complexesnoncovalently bind to DNA by different modes, viz., electro-static, surface binding, or intercalation, and initiate DNAcleavage reactions on photoirradiation either by electron transferto base forming covalent photoadducts or by energy transfer tomolecular oxygen generating 1O2 or rarely by a hydrolyticmechanism (50, 54, 89-95). We have recently reported thehydrolytic cleavage of plasmid pBR322 DNA by [Ru(bpy)2-(BPG)]2+ 1 in an enzyme-like manner and detailed the kineticaspects of DNA cleavage by 1 under pseudo and trueMichaelis-Menten conditions (54). Therefore, in the presentstudy the potential of complexes 2-9 to cleave DNA under

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] ratiofrom 0-30.

Ru(II) Complexes of Bipyridine-Glycoluril Bioconjugate Chem., Vol. 20, No. 3, 2009 453

dark and light conditions was studied by gel electrophoresisusing plasmid pBR322 DNA.

When incubated for 2 h at 37 °C under dark conditions, it isobserved that micromolar concentration of complexes 2-4 and9 shows 22-40% (Figure 3A) and 5-8 shows 28-60% (Figure4A) DNA cleavage as evidenced by the disappearance of formI (supercoiled form) of the plasmid and the appearance of theform II (nicked circular form). A band migration of thesupercoiled form of DNA relative to the control lane is observedfor complexes 2-4 (Figure 3A; lanes 4-6) and for complexes7 and 8 (Figure 4A; lanes 6 and 7). An increase in DNAcleavage is observed for 2-9 after incubation for 2 h in argonatmosphere (Figures S9 and S10A in the Supporting Informa-tion) implying an oxygen-independent cleavage mechanismunder dark conditions. Extensive DNA cleavage is observedfor complexes 2-9 in dark conditions when incubated for 18 hat 37 °C (Figures S9B and S10B in the Supporting Information)indicating a hydrolytic mechanism, which suggests that thesecomplexes are oriented in such a way so as to facilitateintramolecular H-bonding with the phosphate group or with thenucleobases. Under similar conditions [Ru(bpy)3]2+ and[Ru(bpy)2(dppz)]2+, which binds to DNA by electrostaticbinding and intercalation, respectively, and are not capable ofH-bonding, cleave DNA to a lesser extent after 2 h incubationas compared to complexes 2-9. Here, we have observed DNAcleavage under dark conditions by ruthenium polypyridylcomplexes containing one, two, and three BPG ligands withoutany bound water molecule, but with the pheripheral urea moietyof the ancillary bipyridyl glycoluril ligand hydrolyzes thephosphodiester bond efficiently.

The rate of RNA hydrolysis is enhanced 3300-fold bymonometallic Zn(II) complexes of terpyridine-based ligandswith ammonium and guanidinium groups capable of H-bondingto the phosphodiester group compared to the parent complexwithout the functional H-bonding groups (96). A similar effectof enhancing the phosphodiester cleavage was observed by aZn(II) complex with three aminopyridyl hydrogen bond donors,which orient the complex and the substrate for efficientproximity interactions (97-99). A series of organometallic

ruthenium and cis-platin like ethylenediamine based complexesalso exploit these H-bonding interactions to direct DNA bindingby the NH2 groups of the metal-bound ethylenediamine ligandfavoring H-bonding interactions with nucleobases resulting insite-selectivity (100-102).

Rhodium(III) intercalators attached to peptide moieties[Rh(phi)2(bpy′-peptide)] (phi ) intercalative ligands), macro-cyclic complexes of lanthanides, polyamine derivatives such ascyclen, trpn, and tamen complexes of cobalt(III) can act ascatalysts for the hydrolysis of the phosphate esters of DNA andammonium-functionalized copper(II) complexes and copper(II)complexes of macrocycles, cis,cis-1,3,5-triaminocyclohexaneand neamine have been reported (103-105). Of those, however,the most highly efficient hydrolytic cleavage agents are mono-nuclear copper(II) complexes in which a copper-bound hydroxylgroup is the active species in the hydrolysis of the nucleic acidphosphate backbone. As far as we are aware, the only otherhydrolytic cleavage of DNA by the ruthenium-polypyridylcomplex was reported by Barton et al. for the [Ru(DIP)2-(macro)]2+, where Ru(DIP)2 binds to DNA via intercalation andmacro is a chelating ligand with two polyamine tridentate arm-like segments which bind certain divalent metal cations so asto deliver its coordinated nucleophile to the phosphate backbonefor the hydrolysis of the anionic diester (50).

Photocleavage of DNA by ruthenium(II) polypyridyl com-plexes on irradiation is well-documented in the literature (89-95).Therefore, the photocleavage of plasmid pBR322 DNA in thepresence of complexes 2-9 on irradiation at 365 nm was carriedout, and the results are shown in Figure 3B and Figure 4B. Thecomplexes 2 and 9 show only 3-12% increase in conversionof form I (SC) of DNA to form II (NC) of DNA, and thecomplexes 5 and 6 show only 6-7% increase in conversion ofform I (SC) of DNA to form II (NC) of DNA as compared toDNA cleavage under dark conditions. The fact that the observedphotocleavage for complexes 2, 5, 6, and 9 in air is significantlylower than what is observed for [Ru(bpy)3]2+ possibly indicateslower levels of 1O2 generation. The complexes 3, 4, 7, and 8and [Ru(bpy)2(dppz)]2+ shows extensive DNA cleavage in the

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) complexes2-4 and 9. (A) Dark experiments for 2-4 and 9 (incubation for 2 h in air atmosphere): Form I - supercoiled plasmid DNA, Form II - nickedcircular 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 ) 365nm): 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, Lane6 - 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) complexes5-8. (A) Dark experiments for 5-8 (incubation for 2 h in air atmosphere): Form I - supercoiled plasmid DNA, Form II - nicked circular plasmidDNA; 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, Lane6 - 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 - DNAcontrol, 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, Lane5 - DNA + 8.

454 Bioconjugate Chem., Vol. 20, No. 3, 2009 Deshpande et al.

presence of light with a shift in mobility of form I of DNAprobably due to the high molecular weight of DNA-complexconjugates.

In order to investigate the role of radicals in the DNA damageby these complexes 2-9, reactions were performed underaerobic conditions by incubating the complexes for half an hourand irradiation for 20 min with DNA in the presence of hydroxylradical scavengers (DMSO and mannitol), singlet oxygenscavengers (NaN3, histidine, and DABCO) and superoxidescavenger (superoxide dismutase, SOD) (Figures S11-S18,Tables S2-S9 in the Supporting Information). It is observedthat the photocleavage by complexes 2 and 9 was not inhibitedby reactive oxygen species scavengers indicating hydrolyticpathway for these complexes. However, complexes 5 and 6show small amounts of inhibition in the presence of reactiveoxygen species indicating hydrolytic mechanism. Complexes3, 4, 7, and 8 containing dpq and dppz intercalating ligandsshow extensive DNA cleavage in the presence of light, and thephotocleavage experiments in the presence of different radicalscavengers show inhibition indicating that hydroxyl radicals andsinglet oxygen are responsible for DNA cleavage by thesecomplexes upon irradiation. From the above results, it appearsthat the mechanism of DNA cleavage photoinduced by com-plexes 3, 4, 7, and 8 involve either singlet oxygen or hydroxylradicals. However, the photocleavage by these complexes ishigher than [Ru(bpy)2(dppz)]2+ complex implying that com-plexes 3, 4, 7, and 8 with an inherent intercalating ability cleaveDNA by a combination of oxidative and hydrolytic cleavage.The cleavage data for all complexes 2-9 under dark conditionsin the presence and absence of air and under light conditionsare tabulated in Table S10 in the Supporting Information anddepicted as a histogram in Figure 5.

DNA-Complex Conjugate InVestigation by UV-Vis Spec-troscopy: In order to further investigate the formation of theDNA-complex conjugates, dialysis experiments followed byUV-vis spectroscopy were performed on the ruthenium(II)bipyridine-glycoluril complexes in the presence of CT-DNAunder dark conditions in phosphate buffer (pH 7.2). If thecomplex is covalently bound to the DNA, then this can beverified by dialysis of the samples where the parent metalcomplex, but not the DNA, would pass through the dialysismembrane (106, 107). The formation of the product is monitoredby UV-vis spectroscopy, and the results are given in Figure 6and Figure S19 in the Supporting Information. Figure 6 showsthe spectrum of the complexes 1, 7, 8, and 9 kept in the darkfor 24 h in the presence of CT-DNA, both before and afterdialysis. With complexes 1 and 9, the large decrease inabsorption is observed showing that most of the product isdiffused through the dialysis membrane as would be expected

for the complexes which are not bound strongly to the CT-DNA. However, the absorption spectrum of the dialyzed samples2-8 in the presence of DNA reveals that the complex remainsbound to DNA inside the membrane (Figure 6 and Figure S19in the Supporting Information). The complexes 2-8 afterdialysis in the presence of CT-DNA show large increases inabsorption in the 345-500 nm region suggesting that thesecomplexes are strongly bound to the CT-DNA and the conjugateformed is retained after dialysis. The UV-vis spectroscopicobservations together with the electrophoresis results presentedearlier suggests that the DNA-complex conjugates are formedwith CT-DNA. A hyperchromic effect and mobility shift inelectrophoresis experiments were reported previously for thecomplexes 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 havebeen carried out for complexes 1-9 with the models of right-handed B-DNA of sequence d(C:G)12. Different modes ofbinding with different orientations of ruthenium complexes,including groove binding through major/minor groove, andintercalation through major and minor groove, were attemptedwith the model of right-handed B-DNA of sequence d(C:G)12.It was observed that the minimized structure maintains theoctahedral form of the complexes and shows the H-bonding tothe bases and phosphates of DNA without disrupting the helicalstructure of B-DNA. The H-bonding distances for 1-9 afterminimizations are summarized in Table S11 in the SupportingInformation. All complexes are stabilized by H-bonding interac-tions between the DNA and the complexes. It is found that 1and 2 orients in the minor and major groove, respectively, withhydrogen bonding interactions of the N-H and C-H groupspreferably to O1P and O10P phosphate oxygen atoms of DNAfor 1 and preferably to O2P phosphate oxygen atom in case of2 (Figure 7 and Figure S18 in the Supporting Information), whilefor 9 in the major groove with the H- bonding between the NsHand CdO groups of the bipyridine-glycoluril ligand and theN7 of the guanine. (Figure S24 in the Supporting Information).Such NsH · · ·O H-bonding interactions between one ethylene-diamine NH proton and the guanine O6 oxygen have beenshown by Sadler and co-workers in the case of organomatallicruthenium(II) arene complexes of the type [(η6-arene)RuII(en)-Cl](PF6) (en ) ethylenediamine, arene ) biphenyl, 5,8,9,10-tetrahydroanthracene, and 9,10-dihydroanthracene) specificallytarget guanine bases of DNA oligomers (102). It is found that

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

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

Ru(II) Complexes of Bipyridine-Glycoluril Bioconjugate Chem., Vol. 20, No. 3, 2009 455

the binding of 5 and 6 in the major groove with H-bondinginteractions of the N-H groups preferably to O2P phosphateoxygen atoms of DNA (Figure S21 and Figure S22 in theSupporting Information). In the complexes 1, 2, and 9, the ureagroups exclusively bind with phosphate oxygens of the nucleicacids. Recently, Farrell et al. have reported a phosphatebackbone binding mode for a polynuclear platinum(II) complexthat has planar arrays of hydrogen bond donors, leading toassociation with the DNA backbone (108). For complexes 3and 4, the molecular modeling reveals that the planar dpq anddppz ligands interact with the bases and the other planar ligandwith the phosphate oxygens. (Figure S19 and S20 in theSupporting Information). However, the complexes 7 and 8containing planar aromatic dpq and dppz ligands and with thetwo ancillary bipyridine-glycoluril ligands show the interca-lative binding mode in which the stacking interaction of theplanar dpq (Figure S23 in the Supporting Information) and dppzligand between the base pairs of DNA through major grooveside take place along with the phosphate interactions of the N-Hgroups of the two ancillary bipyridine-glycoluril ligands prefer-ably to O2P oxygen atoms (Figure 8), Such H-bonding suggeststhat the bipyridine-glycoluril ligand is located in a specificposition of the H-bond to the neighboring oxygen atoms of thebases or a phosphate group and thus influences the bindingability of these complexes with DNA.

CONCLUDING REMARKS

In summary, in the present study we have synthesized a seriesof new Ru(II) polypyridyl complexes containing urea fusedbipyridine-glycoluril ligand and characterized by variousphysical methods. By the incorporation of the simple modifica-tion on the ancillary ligand of bipyridine, different DNA binding

behaviors for complexes 1-9 were observed. The planararomatic ligands have the potential for intercalation, and thebipyridine-glycoluril ligand with inherent H-bond donor andacceptor groups are accessible for H-bonding with the phosphategroups or with the nucleobases of DNA. The DNA binding andphotocleavage measurements revealed that the complexes thatfavor electrostatic binding mode cleave DNA by hydrolyticmechanism while those that contain intercalating ligands formconjugates with and then cleave DNA by a combination ofoxidative and hydrolytic mechanism.

ACKNOWLEDGMENT

M.S.D. acknowledges Bhabha Atomic Research Centre(BARC) for providing research fellowship through collaborativeresearch scheme of Pune University - BARC, Mumbai, India.A.A.K. acknowledges the financial assistance from Departmentof Science and Technology (DST), New Delhi, for the awardof 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 spectrophotometerfacility in the Department was created by funding from theUniversity Grants Commission, New Delhi, under the Centrefor Advanced Studies funds (CAS). The authors thank Dr.Amitava Das CSMCRI, Bhavnagar, India, for cyclic voltam-metry data.

Supporting Information Available: Changes in the relativespecific viscosity of solutions 1-9 in the presence of CT-DNA(Figure S1 and S2); melting curves of CT-DNA in the absenceand presence of complexes 1-9 (Figure S3 and S4); emissionspectra of Ru(II) complexes 1-9 (25 µM) in phosphate bufferwith increasing [DNA]/[Ru] ratio from 0 to 30 (Figure S5 andS6), emission spectra of Ru(II) complexes 1-9 using anionicquencher K4[Fe(CN)6] in the presence and absence of DNA inphosphate buffer (Figure S7 and S8), luminescence decaylifetime of the complexes 1-9 in the presence of CT-DNA(Table S1), DNA cleavage experiment in the presence of 20µM Ru(II) complexes 2-9; dark experiments incubation for 2 hin argon atmosphere and dark experiments incubation for 18 hin air (Figure S9 and S10); DNA cleavage experiment in thepresence of different radical scavengers for Ru(II) complexes2-9 (Figure S11-S18 and Table S2-S9); DNA cleavageefficiency of ruthenium(II) complexes containing one, two, andthree BPG ligands 2-9 in the dark and light conditions (TableS10); changes in the absorption spectras of complexes 2-6 inthe presence of DNA before and after dialysis (Figure S19);the core view of the bound 2-7 and 9 (Figure S20-S26) toDNA, binding energies in kcal/mol and hydrogen bond informa-tion of Ru(II) polypyridyl complexes 1-9 with right-handedB-DNA of sequence d(C:G)12 (Table S11). This material isavailable free of charge via the Internet at http://pubs.acs.org.

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