Ruthenium(II) Complex of Hbopip: Synthesis, Characterization, pH

Su-Hua Fan , An-Guo Zhang , Chuan-Chuan Ju , Li-Hua Gao and Ke-Zhi ..... Jing Sun , Shuo Wu , Yan An , Jie Liu , Feng Gao , Liang-Nian Ji , Zong-Wan M...
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2364

J. Phys. Chem. B 2006, 110, 2364-2371

Ruthenium(II) Complex of Hbopip: Synthesis, Characterization, pH-Induced Luminescence “Off-On-Off” Switch, and Avid Binding to DNA Mei-Jiao Han,† Li-Hua Gao,†,‡ Yuan-Yuan Lu1 ,† and Ke-Zhi Wang*,† Department of Chemistry and Key Laboratory of Radiopharmaceuticals, Ministry of Education, Beijing Normal UniVersity, Beijing 100875, China, and School of Chemical and EnVironment Engineering, Beijing Technology and Business UniVersity, Beijing 100037, China ReceiVed: August 27, 2005; In Final Form: December 17, 2005

A novel ruthenium(II) complex of [Ru(bpy)2(Hbopip)](ClO4)2 (in which bpy ) 2,2′-bipyridine, Hbopip ) 2-(4-benzoxazolyl)phenylimidazo[4,5-f][1,10]phenanthroline) was synthesized and characterized. The spectrophotometric pH titrations of the complex showed that it acted as a pH-induced luminescence “off-onoff” switch: a luminescence off-on switch with a luminescence enhancement factor of IpH)3.0/IpH)1.0 ) 20 occurring over a narrow pH range of 1.00-3.00 plus a luminescence on-off switch with a luminescence enhancement factor of 3 over a pH range of 3.20-9.40. The excited-state ionization constant of the complex derived, pKa1* ) 3.06, is 1.36 pKa units greater than the ground-state pKa1 ) 1.70, and pKa2* ) 5.01 and pKa3* ) 8.22 are comparable to the ground-state pKa2 ) 5.23 and pKa3 ) 8.22, respectively. The complex avidly bound to calf thymus DNA with a large binding constant of (1.2 ( 0.3) × 107 M-1 in buffered 50 mM NaCl, as evidenced by UV-vis and luminescence titrations, steady-state emission quenching by [Fe(CN)6]4-, DNA competitive binding with ethidium bromide, viscosity measurements, and DNA melting experiments.

1. Introduction 2+

The interesting chemistry of Ru(bpy)3 has stimulated the preparation and characterization of many new octahedral ruthenium(II) polypyridyl complexes in order to elucidate the effects of ligand structures on the ground- and excited-state redox, photochemical, and photophysical properties,1-8 and to develop novel DNA structural probes and new chemotherapeutic agents.9-16 Polypyridyl Ru(II) complexes can bind to DNA in noncovalent binding fashions of electrostatic, groove, and intercalative binding including classical intercalation, semiintercalation, and quasi-intercalation.17 Many important applications of these complexes require that the complex binds to DNA through an intercalative mode. The factors influencing the DNA binding affinity and selectivity also arose intensive efforts. The planarity of the main ligand is thought to play a key role in the binding mode and affinity;17,18 also the ancillary ligand can indirectly affect the DNA binding properties through changing the planarity of the main ligand and the hydrophobicity of the complex.19,20 All the studies indicated that a subtle change in the molecular structure may exert significant effects on binding modes, locations, and affinities and provide a chance to explore various valuable information regarding conformation- or sitespecific DNA probes. On the other hand, the pH responsive transition-metal complexes containing N-heterocyclic ligands are one family of fundamental molecular devices with adjustable ground- and excited-state properties21-27 and biological activities28 by changing the pH of molecular environment. The Ru(II) complex-based pH-induced on-off luminescence switches have been well documented.21 However, the on-off-on or offon-off type luminescence switches as well as the studies on both acid-base and DNA binding properties of Ru(II) com* To whom correspondence should be addressed. Fax: +86-1058802075. E-mail: [email protected]. † Beijing Normal University. ‡ Beijing Technology and Business University.

plexes have been relatively few. We have recently investigated acid-base and DNA binding properties of several polypyridyl Ru(II) complexes,29-31 and a large luminescence on-off ratio of 100 over a narrow pH range of 8.00-10.0 on a dinuclear imidazole-containing Ru(II) complex has recently been achieved by us.30 However, the large luminescence on-off ratio over a narrow acidic region on imidazole-containing Ru(II) complexes has not yet been reported. In this paper we report on the acid-base and DNA binding properties of a Ru(II) mixed ligand complex with 2,2′-bipyridine and Hbopip integrating oxazole and pip (2-phenylimidazo[4,5f][1,10]phenanthroline) moieties. The imidazole group is wellknown for its reversible acid-base interconversion which often induces a large energy perturbation.26,27,29-31 The oxazolecontaining compounds were found to be interesting luminescent materials,32-35 whitening agents, laser dyes, and optical fiber sensors.36-38 Here, we demonstrate that a bipyridyl Ru(II) complex containing Hbopip acted as a pH-induced off-onoff luminescence switch with a large luminescence on-off ratio of 20 over a narrow acidic region, and a strong DNA intercalator with a large binding constant of (1.2 ( 0.3) × 107 M-1 in buffered 50 mM NaCl. 2. Experimental Section 2.1. Materials. cis-[Ru(bpy)2Cl2]‚2H2O39 and 1,10-phenanthroline-5,6-dione40 were prepared by the literature routes. Other materials were commercially available and used without further purification. 2.2. Syntheses. 2.2.1. Synthesis of 2-(4-benzoxazolyl)phenylimidazo[4,5-f][1,10]phenanthroline (Hbopip). The compound was synthesized by following the procedure reported before.41 A mixture of 1,10-phenanthroline-5,6-dione (0.210 g, 1 mmol), 4-benzoxazolylbenzaldehyde (0.210 g, 1 mmol), and ammonium acetate (1.54 g, 20 mmol) dissolved in glacial acetic acid (15.0

10.1021/jp0548570 CCC: $33.50 © 2006 American Chemical Society Published on Web 01/17/2006

pH-Induced Luminescence Switch and DNA Binding

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cm3), was refluxed for 6 h under the protection of N2 and then was cooled to room temperature. A light-orange precipitate was vacuum filtered and washed with a small amount of water, diethyl ether, and then recrystallized from EtOH. Yield: 0.200 g (50.0%). IR νmax (KBr, cm-1): 3428 (vs, N-H), 3065 (w, C-H), 1616 (s, CdN), 1565 (m, ring), 1478 (m, ring), 1242 (s, C-O), 808 (m, ring), 739 (vs). 1H NMR (500 MHz, Me2SO-d6): δ 14 (br 1H), 9.05 (dd, 2H), 8.94 (dd, 2H), 8.51 (d, 2H), 8.43 (d, 2H), 7.85 (m, 4H), 7.46 (m, 2H). 2.2.2. Synthesis of [Ru(bpy)2(Hbopip)](ClO4)2. A solution of cis-[Ru(bpy)2Cl2]‚2H2O (0.13 g, 0.25 mmol) and Hbopip (0.10 g, 0.25 mmol) in ethylene glycol (7.0 cm3) was heated at 126 °C under the protection of N2 for 12 h, during which the solution turned red. The solution was cooled to room temperature. After filtration dropwise addition of saturated NaClO4 resulted in a red-orange precipitate which was filtered and recrystallized with CH3CN/1,4-dioxane. Caution! All the perchlorate salts are potentially explosive and therefore should be handled in small quantity with care. Yield: 0.169 g (66%). IR νmax (KBr, cm-1): 3425 (vs, N-H), 3072 (w, C-H), 1617 (m, CdN), 1602 (w, ring), 1570 (w, ring), 1445 (m, ring), 1242 (m, C-O), 1091 (vs, ClO4-), 763 (s, ring), 622 (s, ClO4-). 1H NMR (500 MHz, Me2SO-d6): δ 14.6 (s, 1H), 9.13 (t, 2H), 8.90 (d, 2H), 8.86 (d, 2H), 8.56 (d, 2H), 8.52 (d, 2H), 8.23 (dt, 2H), 8.12 (m, 4H), 7.99 (t, 1H), 7.95 (dd, 1H), 7.87 (d, 4H), 7.62 (m, 4H), 7.47 (m, 2H), 7.37 (t, 2H). Anal. Calcd For C46H31N9Cl2O9Ru: C, 53.86; H, 3.05; N, 12.29. Found: C, 54.07; H, 3.62; N, 12.78. 2.3. Physical Measurements. Infrared spectra were recorded on a Nicolet Avtar 360 FT-IR spectrometer as KBr disks. 1H NMR spectra were collected on a Bruker DRX-500 NMR spectrometer with (CD3)2SO as solvent at room temperature. All shifts were given relative to TMS. Microanalyses (C, N, and H) were performed on a Vario EL elemental analyzer. UVvis absorption spectra were recorded on a GBC Cintra 10e UVvis spectrophotometer. Emission spectra were obtained on a Shimadzu RF-5301PC spectrofluorimeter. The luminescence quantum yields were calculated by comparison with [Ru(bpy)3]2+ (φ ) 0.033)42 in aerated aqueous solution at room temperature using eq 1, where φ and φstd are the quantum yields of the unknown and the standard sample; A and Astd are the absorbances at the excitation wavelength; I and Istd are the integrated emission intensities.

φ ) φstd(Astd/A)(I/Istd)

(1)

The buffers used were as follows. Buffer A: 40 mM H3BO3, 40 mM H3PO4, 40 mM CH3COOH. Buffer B: 5 mM TrisHCl, 50 mM NaCl, pH ) 7.1. Buffer C: 1.5 mM Na2HPO4, 0.5 mM NaH2PO4, 0.25 mM Na2EDTA. The UV-vis and emission spectrophotometric pH titrations of the complex were investigated in buffer A with 0.2 M NaCl to keep constant ionic strength in order to avoid any changes arising from a change in the environment of the medium. The absorption and emission titrations with calf thymus (ct) DNA were performed in buffer B by keeping the concentrations of the Ru(II) complex constant while varying the DNA concentrations. A solution of ct-DNA gave ratios of UV absorbance at 260 and 280 nm of about 1.9:1, indicating that the DNA was sufficiently free of protein. The DNA per nucleotide was determined spectrophotometrically by assuming 260nm ) 6600 M-1 cm-1.43 Thermal denaturation experiments were performed on a UV-vis spectrophotometer in buffer C. With the use of the thermal melting program, the temperature of the cell containing the cuvette was ramped from 50 to 90 °C, and the absorbance at 260 nm was measured every 0.5 °C. Steady-state emission quenching experiments were

Figure 1. pH effects on the UV-vis spectra of [Ru(bpy)2(Hbopip)]2+ (3.50 µM). (a) pH ) 0.00-3.00; (b) pH ) 3.20-7.60; (c) pH ) 6.609.60. Arrows show spectral changes upon increasing pH.

carried out in buffer B by using Fe(CN)64- as the quencher. Viscosity measurements were carried out using an Ubbelodhe viscometer immersed in a thermostated water bath maintained at 32.10 ( 0.02 °C. The DNA samples for viscosity measurements were prepared by sonication in order to minimize complexities arising from DNA flexibility.18 Flow time was measured, and each sample was measured at least three times, and an average flow time was calculated. Data were presented as (η/η0)1/3 versus [Ru]/[DNA], where η is the viscosity of DNA in the presence of the Ru(II) complex and η0 is the viscosity of the DNA solution alone. Due to the solubility limitation, the viscosity measurements were made below a concentration ratio of [Ru]/[DNA] ) 0.05. The experiments of DNA competitive binding with ethidium bromide (EB) were carried out in buffer B by keeping [DNA]/[EB] ) 5 and varying the concentrations of the Ru(II) complex. 3. Results and Discussion 3.1. pH Effects on UV-Vis and Emission Spectra. 3.1.1. UV-Vis Spectra and the Ground-State pKa. UV-vis spectrophotometric pH titrations were carried out over the pH range from 0.02 to 10.00, and the spectral changes were reversible. The changes in the UV-vis absorption spectra for the complex in buffer A as a function of pH are shown in Figure 1. The spectra of the complex in aqueous solution mainly consist of three well-resolved bands at 286, 340, and 460 nm. The bands centered at 286 and 340 nm are attributed to the π-π* (bpy) and π-π* (Hbopip) intraligand transitions (IL) by analogy to the parent Ru(bpy)32+ and analogous polypyridyl Ru(II) complexes.44 The lowest-energy band at 460 nm is assigned to

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SCHEME 1: Molecular Structure and Protonation/Deprotonation Processes of [Ru(bpy)2(Hbopip)]2+

MLCT and consists of overlapping Ru(dπ) f Hbopip(π*) and Ru(dπ) f bpy(π*) transitions. As the pHs of aqueous [Ru(bpy)2(Hbopip)](ClO4)2 were raised from 0.02 to 3.00, the absorption bands at 282 and 332 nm were red-shifted by 4 and 6 nm, respectively, with two isobestic points occurring at 294 and 377 nm, and were moderately decreased in intensities. The spectral changes observed here are due to the dissociation of one proton on the protonated imidazole ring of the pip moiety. The second deprotonation step, which took place over pH 3.00 to 7.50, is assigned to the proton dissociation of the protonated oxazole moiety, resulting in decreased absorption intensities in all the bands. Upon further increasing the pH from 7.50 to 10.0, the third deprotonation process, which was assigned to the deprotonation of the proton on the neutral imidazole ring, occurred accompanying the following spectral features: the absorbance of the IL bands at 286 and 340 nm decreased and a new absorption band at 357 nm appeared. In Scheme 1 are summarized the three successive deprotonation processes that occurred for the complex upon raising the pH from 0.02 to 10.0. The changes of absorbance at 337 nm as a function of pH are shown in the insets of Figure 1. Three ground-state ionization constants were derived to be pKa1 ) 1.70 ( 0.03, pKa2 ) 5.23 ( 0.06, and pKa3 ) 8.22 ( 0.05 by nonlinear sigmoidal fit of the data in the insets of Figure 1 and were compared with those for the analogous Ru(II) complexes in Table 1. The first ionization constant of the protonated imidazole ring on the complex, pKa1 1.70, is comparable to a corresponding pKa1 value of 1.97 for [Ru(bpy)2(pidbH2)]2+ (pidbH2 ) (1-[1,10]phenanthroline[5,6-d]imidazo-2-yl)-4-N,N-dimethylaminobenzene)46 and is much smaller than a value of 4.16 for [(bpy)2Ru(ebpibcH2)Ru(bpy)2]4+ (ebipcH2 ) N-ethyl-4,7-bis([1,10]phenanthroline[5,6-f]imidazo-2-yl)carbazole)30 and 4.11 for [(bpy)2Ru(bpibH2)Ru(bpy)2]4+ (bpibH2 ) 1,4-bis([1,10]phenanthroline[5,6-d]imidazo-2-yl)benzene)46 due to the strong electron-withdrawing effect of the protonated oxazole group on [Ru(bpy)2(H3bopip)]4+. The ionization constant of the neutral imidazole moiety on the complex, pKa3 ) 8.22, is reasonably smaller than

Figure 2. pH effects on the emission spectra (λex ) 467 nm) of [Ru(bpy)2(Hbopip)]2+ (3.50 µM) as a function of pH. (a) pH ) 0.003.60; (b) pH ) 3.40-9.60; (c) pH ) 9.00-10.6. Arrows show spectral changes upon increasing pH.

the corresponding ionization constant for the electron-donorcontaining complex of [Ru(bpy)2(pidbH2)]2+ (pKa3 ) 10.6)45 while being 0.38 pKa units greater than pKa2 ) 7.84 for [Ru(bpy)2(bpibH2)]2+ containing the electron-withdrawing fragment of bis(2,2′-bipyridine)(imidazo[4,5-f][1,10]phenanthroline)ruthenium(II).46 3.1.2. Luminescence Spectra and the Excited-State pKa*. The complex in aerated aqueous solutions at room temperature emitted strongly with an emission peak at 609 nm (λex ) 467 nm), which is characteristic of the 3MLCT (dπ(Ru) f dπ* (ligand)) emission state. The emission quantum yields were determined to be φ ) 0.063 for an air-equilibrated aqueous solution of the Ru(II) complex and φ ) 0.096 for an aqueous solution degassed with nitrogen for 15 min. The emission spectra of the complex were strongly pH dependent as shown in Figure 2. The emission intensities versus pH profiles (the insets of Figure 2) of the complex were composed of three sigmoidal curves of opposite gradients which were due to three separate protonation/deprotonation processes over the pH region from

TABLE 1: Comparison of pKa and pKa* Values and Luminescence On/Off Ratios for[Ru(bpy)2(Hbopip)]2+ with Those for Analogous Ru(II) Complexes complexa

pKa

pKa*

on/off ratio (pH region)

ref

[Ru(bpy)2(pidbH2)]2+ [(bpy)2Ru(bpibH2)Ru(bpy)2]4+ [(bpy)2Ru(ebipcH2)Ru(bpy)2]4+ [Ru(bpy)2(ppi)]2+ [Ru(bpy)2(Hbopip)]2+

1.97, 3.57, 10.6 4.11, 7.84 4.16, 5.07, 9.65, 12.1 8.80 1.70, 5.23, 8.22

4.22, 10.7 4.34, 7.46 4.54, 5.07, 9.76, 12.1 9.10 3.06, 5.01, 8.22

16.0 (0.6-12.0) 2.50 (0-6.0); 4.00 (6.0-10.0) 100 (8.0-10.0) 3.00 (7.5-11.5) 20 (1.0-3.0); 3.0 (3.6-9.0)

45 46 30 31 this work

a pidbH2 ) (1-[1,10]phenanthroline[5,6-d]imidazo-2-yl)-4-N,N-dimethylaminobenzene; bpibH2 ) 1,4-bis([1,10]phenanthroline[5,6-d]imidazo2-yl)benzene; ebipcH2 ) N-ethyl-4,7-bis([1,10]phenanthroline[5,6-f]imidazo-2-yl)carbazole); ppi ) 2-pyridin-2-yl-1H-phenanthro[9,10-d]imidazole.

pH-Induced Luminescence Switch and DNA Binding

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0.02 to 10.0. In the strongly acidic solutions of pH below 1.00, the luminescence of the complex was quenched as the imidazole and oxazole rings were protonated because the protonated species are better electron acceptors than [Ru(bpy)3]2+ itself.47,48 The emission intensities were found to increase sharply from pH 1.00 to 3.00 with the emission maximum blue-shifted from 633 to 609 nm. Over this narrow pH region the complex acted as an “off-on” emission switch with an emission enhancement factor of ∼20. This factor is the largest value among those for imidazole-containing Ru(II) complexes operated over the acidic pH range (see Table 1).31,45-49 However, the emission of the complex did not completely vanish at the low pHs. In the second gradient (from pH 3.20 to 9.40), the emission intensities decreased by about 67%, accompanying a slight red-shift of the emission maxima from 611 to 620 nm. In the pH region aforementioned the complex acted as an “on-off” emission switch with a small on-off ratio of ∼3. As increasing pH from 9.40 to 10.2, the emission intensities increased slightly. Clearly, the emission spectral changes discussed above were associated with three excited-state deprotonation processes, and each of them is considered to deal with the same protons as those involved in the UV-vis spectral titrations. The ground- and excited-state ionization constants are related thermodynamically by a Fo¨rster cycle.48 The Fo¨rster treatment results in eq 2, which describes the relationship between the ground-state pKa and excited-state pKa* based on pure 0-0 transitions in wavenumbers of νB and νHB for the basic and acidic species, respectively.21,25

pKa* ) pKa + (0.625/T)(νB - νHB)

(2)

In reality, the νB and νHB values are often difficult or even impossible to obtain. A good approximation is to use the emission maxima for νB and νHB since protonation equilibrium is almost certainly established between the 3MLCT states. Therefore, the energies of the emission maxima in wavenumbers were used in eq 2, and three excited-state ionization constants of pKa1* ) 3.06, pKa2* ) 5.01, and pKa3* ) 8.22 were thus obtained. The values of pKa2* and pKa3* are comparable to the corresponding pKa2 and pKa3 values, while the value of pKa1* is 1.36 pKa units greater than pKa1, indicating that the electron density was significantly higher in the excited state than in the ground state for [Ru(bpy)2(H3bopip)]4+ and the excited electron was directed on [H3bopip]2+ rather than bpy on [Ru(bpy)2(H3bopip)]4+. The increase in electron density on the [H3bopip]2+ ligand increased its basicity and, therefore, increased the excitedstate pKa* value, while the excited electrons on [Ru(bpy)2(H2bopip)]3+ and [Ru(bpy)2(Hbopip)]2+ were localized on bpy rather than H2bopip+ or Hbopip. 3.2. DNA Binding Properties. 3.2.1. UV-Vis Spectra. The absorption spectra of the complex in the absence and the presence of ct-DNA are illustrated in Figure 3. In the absence of the DNA, the complex in aqueous solution showed the lowest-energy metal-ligand charge transfer (MLCT) transition absorption at 459 nm. The energy in the MLCT band increased in the order [Ru(bpy)2(Hbopip)]2+ (459 nm) < [Ru(bpy)2(pip)]2+ (458 nm) < [Ru(bpy)2(cip)]2+ (457 nm) < [Ru(bpy)2(ip)]2+ (455 nm). The increases in energies can also be observed in the IL transition: [Ru(bpy)2(Hbopip)]2+ (286 nm) < [Ru(bpy)2(pip)]2+ (283 nm) < [Ru(bpy)2(ip)]2+ (280 nm) (Table 2), suggesting enhanced conjugation on going from ip to Hbopip.51,52 Upon increasing concentrations of DNA, all the absorption bands of the complex displayed clear hypochromicities and red-shifts, and related spectral data are listed in Table 2. The hypochromisms H%, as defined by H% ) 100(Afree - Abound)/Afree,

Figure 3. Changes in absorption spectra of [Ru(bpy)2(Hbopip)]2+ (3.40 µM) upon addition of ct-DNA (0.00-11.6 µM). Inset: plots of (a f)/(b - f) vs [DNA] and the nonlinear fit for the titration of the complex with the DNA.

for IL bands at 286 and 340 nm reached as high as 44% and 39% with the red-shifts by 4 and 2 nm, respectively, at a ratio of [DNA]/[Ru] ≈ 2.3. The MLCT band at 459 nm showed hypochromism by about 22% and a red-shift of 5 nm under the same experimental condition. As shown in Figure 3 and Table 2, a hypochromism of 44% was found for the IL band at 286 nm of the complex which is much larger than those observed for analogous Ru(II) complexes, 12% (457 nm) for [Ru(bpy)2(cip)]2+, 15.5% (458 nm) for [Ru(bpy)2(ip)]2+, 21.9% (458 nm) for [Ru(bpy)2(pip)]2+, and 26% (457 nm) for [Ru(bpy)2(hpip)]2+,53 and even larger than those observed for typical DNA intercalators, 32.1% (372 nm) for ∆-[Ru(phen)2(dppz)]2+ and 29.8% (372 nm) for Λ-[Ru(phen)2(dppz)]2+,54 indicating a high binding affinity of the complex to DNA. The intrinsic binding constant K illustrating the binding strength of the complex with ct-DNA was determined from the eq 3 through a plot of (a f)/(b - f) vs [DNA], where [DNA] is the concentration of DNA in base pairs.

(a - f)/(b - f) ) [b - (b2 - 2Kb2Ct[DNA]/n)1/2]/(2KbCt) (3) b ) 1 + KbCt + Kb[DNA]/2n The apparent absorption coefficients a, f, and b correspond to Aobsd/[Ru], the extinction coefficient for the free ruthenium complex, and the extinction coefficient for the ruthenium complex in the fully bound form, respectively, Ct is the total Ru(II) complex concentration, [DNA] is the DNA concentration in nucleotides, and n is the binding site size.12 The intrinsic binding constant Kb and binding site size of the complex with DNA were derived to be Kb ) (1.2 ( 0.3) × 107 M-1, n ) 0.65 ( 0.03, as shown from the inset in Figure 3 by monitoring the decay of the absorbance at 286 nm. The hypochromism and intrinsic binding constant of [Ru(bpy)2(Hbopip)]2+ are compared in Table 2 with those reported for representative Ru(II) complexes. The intrinsic binding constant derived for [Ru(bpy)2(Hbopip)]2+ is much larger than 1.7 × 106 M-1 for Λ-[Ru(phen)2(dppz)]2+, 3.2 × 106 M-1 for ∆-[Ru(phen)2(dppz)]2+, and 20 times greater than 4.7 × 105 M-1 for the parent complex [Ru(bpy)2(pip)]2+ under the same experimental condition.52 The spectral characteristics of the large hypochromism and clear redshifts as well as the large Kb value observed may suggest that the complex most likely intercalatively binds to DNA, involving a strong stacking interaction between the aromatic chromophore and the base pairs of the DNA. 3.2.2. Luminescence Studies. The complex in aerated aqueous solutions at room temperature emitted strongly with a lumines-

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TABLE 2: Interaction Parameters of the Ru(II) Complexes with ct-DNA complexa

hypochromism H/% (λmax/nm)b

[Ru(phen)3]2+ [Ru(bpy)2(ip)]2+ [Ru(bpy)2(pip)]2+ [Ru(bpy)2(cip)]2+ [Ru(bpy)2(hpip)]2+ ∆-[Ru(phen)2(dppz)]2+ Λ-[Ru(phen)2(dppz)]2+ [Ru(bpy)2(Hbopip)]2+

12 (445) 15.5 (455) 21.9 (458) 12 (457) 26 (458) 32.1 (372); 29.8 (372) 44 (286)

Kb/M-1

claimed binding mode

5.5 × 103

semi-intercalation classic intercalation classic intercalation classic intercalation classic intercalation classic intercalation

50 51, 52 51, 52 52 53 54

classic intercalation

this work

4.7 × 105 6.5 × 105 3.2 × 106 1.7 × 106 1.2 × 107

ref

ip ) imidazo[4,5-f][1,10]phenanthroline; pip ) 2-phenylimidazo[4,5-f][1,10]phenanthroline; cip ) 2-(2-chlorophenyl)imidazo[4,5f][1,10]phenanthroline; hpip ) 2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline; dppz ) dipyrido[3,2-a:2′,3′-c]-phenazine. b The maximum hypochromism selected among the UV-vis absorption peaks. a

Figure 4. Changes in the emission spectra (λex ) 467 nm) of [Ru(bpy)2(Hbopip)]2+ (3.40 µM) with increasing concentrations of ct-DNA (0.00-34.3 µM).

cence peak at 609 nm which is blue-shifted relative to those observed for [Ru(bpy)2(pip)]2+ (615 nm) and [Ru(bpy)2(ip)]2+ (625 nm).56 The changes in emission spectra of the complex with increasing DNA concentrations are shown in Figure 4. As the DNA was successively added into the complex solution, the emission intensities of the complex were first reduced and reached a saturated value at [DNA]/[Ru] ) 0.4 and thereafter gradually increased upon further increasing [DNA]/[Ru] until a stable emission intensity at [DNA]/[Ru] ) 5.3 was observed. The reduction in emission intensities at initial titrations was also accompanied by a slight blue-shift from 609 to 603 nm. These emission spectral characteristics imply the presence of at least two possible binding conformations: one was less emissive and was formed at the low DNA concentrations, the other one formed at the high DNA concentrations and was highly emissive which most probably resulted from intercalative interaction since the hydrophobic environment inside the DNA helix reduced the accessibility of water molecules to the complex and the complex mobility was restricted at the binding site, leading to a decrease in radiationless vibrational relaxation. This effect prevailed over the other emission quenching processes, e.g., the potential photoinduced electron transfer from the guanine base of DNA to the 3MLCT of the complex.30,57 The results of steady-state emission quenching experiments using [Fe(CN)6]4- as the quencher are shown in Figure 5. In the absence of DNA, the emission of the complex was efficiently quenched by [Fe(CN)6]4-, resulting in an almost linear SternVolmer plot with a slope of 42.1 mM-1, which is much larger than a slope of 2.2 mM-1 for [Ru(bpy)2(ip)]2+ and 2.0 mM-1 for [Ru(bpy)2(pip)]2+.52 However, addition of DNA made the slope drastically decrease to near zero, which is much smaller than a slope of 0.6 mM-1 for [Ru(bpy)2(cip)]2+ and 0.1 mM-1 for [Ru(bpy)2(pip)]2+.51 This behavior may be explained by the repulsion of the highly anionic [Fe(CN)6]4- by the DNA

Figure 5. Emission quenching of [Ru(bpy)2(Hbopip)]2+ (2.50 µM) with increasing concentrations of [Fe(CN)6]4- (0.00-1.00 mM) in the absence of (solid squares) and the presence of (hollow squares) the DNA (44.0 µM).

Figure 6. Emission spectra of EB bound to DNA in the presence of [Ru(bpy)2(Hbopip)]2+ (0.00-33.0 µM). The arrows show the intensity changes upon increasing concentrations of the complex. Inset: florescence quenching curve of DNA-bound EB by the complex. [DNA] ) 100 µM, [EB] ) 20.0 µM, λex ) 537 nm.

polyanion which hinders the bound complex from quenching of the emission. The slope reflects different degrees of protection or relative accessibility of bound cations and can be taken as a measure of binding affinity.58,59 Since a larger slope corresponds to poorer protection and weaker binding affinity, [Ru(bpy)2(Hbopip)]2+ bound to the DNA more strongly than did [Ru(bpy)2(ip)]2+ and [Ru(bpy)2(pip)]2+. The competitive binding experiments with a well-established quenching assay based on the displacement of the intercalating drug EB from ct-DNA may give further information regarding the DNA binding properties of the complex to DNA (Figure 6). If a complex could replace EB from DNA-bound EB, the fluorescence of the solution would be greatly quenched due to the fact that the free EB molecules were ready to be quenched by the surrounding water molecules.57 However, not only the DNA intercalators but also groove DNA binders can cause the

pH-Induced Luminescence Switch and DNA Binding

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Figure 7. Thermal denaturation curves of ct-DNA (60.0 µM) at the concentration ratios of [Ru]/[DNA] ) 0([), 1/10(1), 1/20(2), 1/40(b), 1/60(9).

(Hbopip)]2+ to ct-DNA at Tm were determined by McGee’s equation.65-67

TABLE 3: The Comparison of ∆Tm of the DNA with Different Binders complexa

Kb

2+

Ru(bpy)3 EB Ru(NH3)4(ip)2+ Ru(NH3)4(pip)2+ Ru(NH3)4(hpip)2+ ∆-[Ru(phen)2(dppz)]2+ Λ-[Ru(phen)2(dppz)]2+ [Ru(bpy)2(Hbopip)]2+ a

1.0 × 107 3.0 × 105 4.3 × 105 9.7 × 105 3.2 × 106 1.7 × 106 1.2 × 107

[DNA]/[Ru] ∆Tm/°C 10 10 1.0 1.0 1.0 1.0 1.0 15 10