pH effects on optical and DNA binding properties of a thiophene-containing ruthenium(II) complex

Inorganica Chimica Acta 370 (2011) 132–140

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pH effects on optical and DNA binding properties of a thiophene-containing ruthenium(II) complex Zhen-Sheng Li a, Huai-Xia Yang a,b, An-Guo Zhang a, Hong Luo a, Ke-Zhi Wang a,⇑ a b

College of Chemistry, Beijing Normal University, Beijing 100875, China Pharmacy College, Henan University of Traditional Chinese Medicine, Zhengzhou 450008, China

a r t i c l e

i n f o

Article history: Received 6 August 2010 Received in revised form 9 January 2011 Accepted 15 January 2011 Available online 31 January 2011 Keywords: Ruthenium Bipyridine DNA pH Phenanthroline

a b s t r a c t A new ruthenium(II) complex, [Ru(bpy)2(Htip)]Cl2 {where bpy = 2,20 -bipyridine and Htip = 2-(thiophen2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline}, has been synthesized and characterized by 1H NMR spectroscopy, elemental analysis, and mass spectrometry. The pH effects on UV–Vis absorption and emission spectra of the complex have been studied, and the ground- and excited-state acidity ionization constant values have been derived. The calf thymus (ct) DNA binding properties of the complex have been investigated with UV–Vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)6]4, DNA competitive binding with ethidium bromide, DNA melting experiments, and viscosity measurements. The molecular structures and electronic properties of [Ru(bpy)2(Htip)]2+ and deprotonated form [Ru(bpy)2(tip)]+ have also been investigated by means of density functional theory calculations in an effort to understand the DNA binding properties. The results suggest that the complex undergo three-step successive protonation/deprotonation reactions with one of which occurring over physiological pH region, and act as a ct-DNA intercalator with an intrinsic DNA binding constant value on 105 M1 order of magnitude that is insensitive to pH. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Transition metal complex-based DNA binders have been focus of bioinorganic investigations for at least three decades due to their significant applications in DNA structural probes and molecular switches, disease defense, improvement in drug effectiveness, and new drug design and screening [1–4]. Among these DNA binders reported, ruthenium(II) complexes have attracted increasing attention due to their excellent thermodynamic and dynamic stability, facile electron transfer, strong luminescence emission and relatively long-lived excited states [5–9]. The electrostatic, groove, classic intercalative, and partially intercalative binding modes have been reported for non-covalent interactions [10]. The useful applications of such complexes generally require that the complex binds to DNA through the intercalative binding mode. The effects of the planarity and size of the intercalative ligand, and hydrophobicity, charge, and substituent of the complex on the DNA binding properties have attracted much attention [11,12]. On the other hand, some imidazole-containing Ru(II) complexes were shown to be capable of inducing a large energy perturbation by reversible acid–base interconversion, and be promising for making fundamental luminescence switching devices [13,14]. But studies on

the pH effects on biomolecule-binding properties and drug activities are particularly scarce [15,16]. We demonstrated that the delicately designed imidazole-containing bipyridyl ruthenium(II) complexes acted as pH-induced luminescence switches with emission intensity on–off ratios of as high as 100 [17,18]. Thiophene containing p systems have been extensively investigated due to their efficient electron transmission and inherent stability as well as their potential applications as electrode materials, organic semiconductors, nonlinear optical materials and so on [19–22]. Many of the theoretical studies have shown that a DNA molecule is an electron donor and an intercalated complex is an electron acceptor [23,24], so the introduction of thiophene to Ru(II) complex may mediate acid–base and DNA binding properties of the Ru(II) complex. Herein, we report a thiophene-containing bipyridyl Ru(II) complex of [Ru(bpy)2(Htip)]Cl2 {Htip = 2-(thiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline}, in which the thiophene grafted was shown to have significant effects on the acid–base and DNA binding properties of the complex.

2. Results and discussion 2.1. pH effects on UV–Vis and emission spectra

⇑ Corresponding author. Tel.: +86 10 58805476; fax: +86 10 58802075. E-mail address: [email protected] (K.-Z. Wang). 0020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ica.2011.01.039

The UV–Vis absorption spectrum of [Ru(bpy)2(Htip)]2+ in neutral aqueous solution mainly consists of three well-resolved bands

Z.-S. Li et al. / Inorganica Chimica Acta 370 (2011) 132–140

at 285, 323, and 461 nm. The two highest-energy bands are attributed to superposition of the p–p⁄ (bpy) and p–p⁄ (Htip) intraligand transitions (IL). The lowest-energy band at 461 nm is assigned to the singlet excited state of a metal-to-ligand charge-transfer (MLCT) transition based on comparisons with a MLCT band at 455 and 462 nm for analogous complexes of [Ru(bpy)2(Hip)]2+ {Hip = imidazo[4,5-f][1,10]phenanthroline} [25], and [Ru(bpy)2 (Hbtip)]2+ {Hbtip = 2-benzo[b]thien-2-yl-1H-imidazo[4,5-f][1,10] phenanthroline} [26], respectively. UV–Vis spectrophotometric pH titrations were carried out over the pH range from 0.10 to 13.20. The spectral changes (see Fig. 1) indicate that the complex underwent two protonation/deprotonation processes over the pH range studied. An increase in pH from 0.10 to 3.30 resulted in a red-shift of the absorption band from 282 to 285 nm, and a moderate enhancement in the intensities with appearance of an isosbestic point at 335 nm. The spectral changes observed above were due to the dissociation of one proton on the protonated imidazole ring. The second deprotonation step that took place upon raising pH from 3.30 to 10.00, was assigned to the proton dissociation of the neutral imidazole, resulting in decreased absorption intensities in all the bands. By sigmoidal fitting of the data in plots of the absorbance at 285 nm versus pH (see insets of Fig. 1), the negative logarithm values of ground-state acidity ionization constants, were derived to be pKa1 = 1.55 ± 0.04 and pKa2 = 7.3 ± 0.1, and are compared with those previously reported for analogous Ru(II) complexes in Table 1. Upon comparison of pKa values previously reported for [Ru(bpy)2(Hip)]2+ (pKa1 = 1.97, pKa2 = 10.48) [25], [Ru(bpy)2(Htip)]2+

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behaves as a weaker acid with its pKa1 and pKa2 values more than 0.4 and 3 pH units smaller than corresponding values of [Ru(bpy)2(Hip)]2+, respectively. The pKa2 value of [Ru(bpy)2(Htip)]2+ is close to a pKa2 = 7.84 reported for [(bpy)2Ru(bpibH2)Ru(bpy)2]4+ {bpibH2 = 1,4-bis([1,10]phenanthroline[5,6-d]imidazol-2-yl)benzene} [9]. These facts indicate that thiophene is capable of strongly stabilizing the deprotonated ip moiety on [Ru(bpy)2(tip)]+. The emission spectral changes in aqueous solution as a function of pH are shown in Fig. 2. We can see that the emission spectra are sensitive to pHs. Upon increasing pH from 0.1 to 4.0, the emission maxima had a blue shift from 625 to 615 nm, and the intensities almost remained constant. As the pH increased from 4.0 to 10.0, the emission maxima were almost unchanged, and the intensities at 615 nm decreased to original 52%. The insets of Fig. 2 clearly show that the profiles for the changes of relative emission intensities versus pH, consist of two sigmoidal curves of opposite gradient, indicative of two excited-state deprotonation processes, which are in accordance with the UV–Vis spectral changes. The values of excited-state acidity ionization constant, pK a , were roughly evaluated on the basis of the Föster cycle, which correlates pK a with pKa thermodynamically by Eq. (1):

pK a ¼ pK a þ ð0:625=TÞðmB  mHB Þ

ð1Þ

where mB and mHB are approximately equal to the emission maxima for the basic and acidic species, respectively [27]. The values of pK a1 and pK a2 were derived to be 2.08 and 7.24, respectively. The pK a1 value is 0.53 pH units greater than corresponding pKa1, while pK a1 value is close to pKa2, indicating that the electron density of [Ru(bpy)2(H2tip)]3+ in the excited state is higher than in the ground state, and the MLCT transition is localized on H2tip+ rather than bpy in [Ru(bpy)2(H2tip)]3+ [28]. 2.2. DNA binding studies 2.2.1. UV–Vis spectra The binding of a metal complex to DNA through intercalation usually results in hypochromism and bathochromism of the UV– Vis spectra due to a strong stacking interaction between an aromatic chromophore and DNA base pairs [26], so electronic absorption spectroscopy is one of the most useful techniques for DNAbinding studies of the metal complex. The absorption spectra of the complex in the absence and the presence of ct-DNA are shown in Fig. 3. As can be seen, the hypochromism for the bands at 246, 285, 323 and 460 nm in the presence of ct-DNA at a ratio of [DNA]/[Ru] 20 were found to be 37.5%, 34.3%, 29.2% and 24.3%, respectively, which are larger than corresponding hypochromism values of 11.5%, 3.8%, 0%, and 15.5% previously reported for [Ru(bpy)2(Hip)]2+, and of 28%, 20.0%, 0% and 21.9% for [Ru(bpy)2(Hpip)]2+ [13]. In order to compare quantitatively the DNA-binding strength, the value of intrinsic DNA-binding constant Kb is derived according to Eq. (2) [29]:

½DNA=ðea  ef Þ ¼ ½DNA=ðeb  ef Þ þ 1=K b ðeb  ef Þ

Fig. 1. pH effects on the UV–Vis spectra of [Ru(bpy)2(Htip)]Cl2 (5.4 lM), (A) pH 0.08–3.30 and (B) pH 3.30–12.20. Arrows show spectral changes upon increasing pH.

ð2Þ

where [DNA] is the concentration of DNA in base pairs (two times [DNA] in nucleotides), ea is the apparent absorption coefficient, which was obtained by calculating Aabs/[Ru], and ef and eb are the extinction coefficients for the free ruthenium complex and the ruthenium complex in the fully bound form, respectively. In a plot of [DNA]/(ea  ef) versus [DNA], Kb is given by the ratio of the slope to the y intercept. An intrinsic DNA binding constant of (2.2 ± 0.03)  105 M1 (285 nm), was obtained at 50 mM NaCl, which is larger than that of analogous complex [Ru(bpy)2(Hip)]2+ (Kb = 4.1  104 M1), demonstrating that the DNA-binding affinity is strengthened by introducing thiophene group due to the efficient charge transmission ability of thiophene group and the larger

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Table 1 Comparison of pKa and pK a values and luminescence enhancement factors (LEF) for [Ru(bpy)2(Htip)]2+ with those for analogous Ru(II) complexes. Complex

pK a

pKa 2+

[Ru(bpy)2(Hip)] [Ru(bpy)2(dmpip)]2+ [(bpy)2Ru(bpibH2)Ru(bpy)2]4+ [Ru(bpy)2(Htip)]2+

1.97; 1.97; 4.11; 1.55;

10.48 3.75; 10.56 7.84 7.3

2.88; 4.22; 4.34; 2.08;

Fig. 2. pH effects on the emission spectra (kex = 458 nm) of [Ru(bpy)2(Htip)]Cl2 (5.4 lM) over pH 0.08–4.0 (A) and pH 4.0–9.0 (B). Arrows show spectral changes upon increasing pH.

planarity of intercalative ligand Htip. However, the Kb value of the complex is slightly smaller than a Kb value of 4.7  105 M1 previously reported for [Ru(bpy)2(Hpip)]2+ [10]. It should be pointed out that the rule that larger size of the intercalative ligand would lead to tighter DNA binding, is not necessarily true, since larger planarity of Hbtip-containing [Ru(bpy)2(Hbtip)]2+ relative to [Ru(bpy)2 (Hpip)]2+ was reported to have a smaller Kb value of 2.8  104 M1 than [Ru(bpy)2(Hpip)]2+ [12]. Kb value was also determined by nonlinear regression analysis using Eqs. (3) and (4) [30]: 2

ðea  ef Þ=ðeb  ef Þ ¼ ðb  ðb  2K 2b C t ½DNA=sÞ1=2 Þ=ð2K b C t Þ

ð3Þ

b ¼ 1 þ K b C t þ K b ½DNA=2s

ð4Þ

where ea, ef and eb are extinction coefficients at a given DNA concentration, the complex free in solution and the complex fully bound to DNA, respectively. Ct is the total Ru(II) complex concentration, [DNA] is the DNA concentration in nucleotides, and s is the

10.66 10.71 7.46 7.34

LEF

Ref.

1.4(1.0–4.5); 2(4.5–13.0) 17(1.0–13.0) 2.5(4.0–5.9); 4(5.9–8.5) 1.8(4.0–9.0)

[25] [25] [9] This work

Fig. 3. (A) UV–Vis spectra of [Ru(bpy)2(Htip)]Cl2 (5.4 lM), in the presence of increasing amounts of DNA (0–110 lM) in buffer B 50 mM NaCl; inset shows a plot of [DNA]/(ea  ef) vs. [DNA] and the linear fit; (B) plot of (ea  ef)/(eb  ef) vs. [DNA] and the nonlinear fit.

binding site size. The values of Kb and s were derived to be s = 0.97 ± 0.17 and Kb = (4.0 ± 1.5)  105 M1, which is in good agreement with the Kb value derived according to Eq. (2). On the basis of the pKa1 (1.57) and pKa1 (7.24) values derived, the complex is expected to exist as 60% of [Ru(bpy)2(Htip)]2+ and 40% of [Ru(bpy)2(tip)]+ in aqueous pH 7.1 Tris–HCl buffer. We therefore carried out DNA binding studies also in the pH 4.0 and 9.0 buffer solutions (see Fig. S1 in supporting information), and the values of the DNA binding constant and binding bite size were obtained according to Eqs. (3) and (4) to be Kb = (8.1 ± 2.2)  105 M1 and n = 1.89 ± 0.22 at pH 4.0 at which the complex existed exclusively as [Ru(bpy)2(Htip)]2+, and Kb = (3.5 ± 2.3)  105 M1 and n = 0.71 ± 0.30 at pH 9.0 at which the complex existed exclusively as deprotonated form [Ru(bpy)2(tip)]+. Above facts indicate that the DNA binding constants obtained at pHs 4.0 and 9.0 are essentially comparable to each other, and comparable to Kb = (4.0 ± 1.5)  105 M1 we have mentioned at pH 7.1 in view of the experimental errors.

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2.2.2. Emission spectra As shown in Fig. 4, in the presence of saturated amount of ctDNA, emission peaks blue-shifted from 610 to 600 nm, and the fluorescence emission intensities at 610 nm increased by a factor of ca.1.9, which is close to emission enhancement factors of 1.7 and 2.6 previously reported for [Ru(bpy)2(Hip)]2+ and [Ru(bpy)2(Hpip)]2+, respectively [13]. A fitting of the fractional changes in emission intensities, (I  I0)/(If  I0), as a function of DNA concentrations according to the Bard–Torp–Murphy equation [31], gave a binding site size of s = 5.2 ± 0.3 base pairs and a binding constant of (1.1 ± 0.2)  106 M1. These facts indicate that [Ru(bpy)2(Htip)]2+ may intercalate between adjacent base pairs of the DNA, and be efficiently protected from the accessibility of solvent water, resulting in a decrease of non-irradiative vibration relaxation and accordingly enhanced emission [26]. Steady-state emission quenching experiments using [Fe(CN)6]4 as the quencher were also used to shed light on the DNA binding properties. [Fe(CN)6]4, an anionic quencher, very efficiently quenches the emission of ruthenium complex which is free in solution but weakly quenches the emission of the ruthenium complex which is tightly bound to DNA polyanion. Fig. 5 shows Stern– Volmer plots with the values of Stern–Volmer constant Ksv, of 6.5  104 and 936 M1 for the DNA-free and the DNA-bond Ru(II) complex solutions, respectively, according to Eq. (5):

I0 =I ¼ 1 þ K sv ½Q 

ð5Þ

where I0 and I are the emission intensities in the absence and the presence of [Fe(CN)6]4, respectively. [Q] is the concentration of [Fe(CN)6]4. A ratio of Ksv value derived in the absence of the DNA to that derived in the presence of the DNA, R, was found to be 70 for [Ru(bpy)2(Htip)]2+, which is even much larger than a R value of 20 previously reported for [Ru(bpy)2(Hpip)]2+ [32], indicating that the complex was protected from emission quenching, because the high repulsion between the highly anionic [Fe(CN)6]4 and the negatively charged DNA phosphate backbone hinders approaching of the quench to the bound complex.

135

Fig. 5. Emission quenching of [Ru(bpy)2(Htip)]2+ (5.4 lm) upon increasing concentrations of [Fe(CN)6]4 (0.00–165 lm) in the absence of (d) and the presence of (j) the DNA (0.5 mM).

nanthridinium ring between adjacent DNA base pairs on the double helix of the DNA. If a DNA intercalator is added successively to the DNA solution pretreated with EB, it would cause the displacement of EB molecules inserted between base pairs, resulting in the sharp decreases in emission intensities. The quenching extents of fluorescence of EB bound to DNA can be used to determine the relative DNA binding affinities of the drugs added. As shown in Fig. 6A, additions of the complex to the DNA-bound EB solution

2.2.3. EB competition assay The competitive ethidium bromide (EB) binding study was also performed by fluorescence spectroscopic titration in order to investigate whether drugs could replace proven DNA intercalator EB from strongly intercalatively binding EB–DNA complex. 3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide (EB), a typical indicator of intercalation [33,34], emits intense fluorescence in the presence of DNA due to strong intercalation of planar phe-

Fig. 4. Changes in fluorescent spectra of [Ru(bpy)2(Htip)]2+ (5.4 lM) in the presence of ct-DNA. Inset: plot of (Ia  If)/(Ib  If) vs. [DNA] and the nonlinear fitting (solid line).

Fig. 6. (A) Emission spectra of EB bound to DNA in the presence of [Ru(bpy)2(Htip)]2+(0–60 lM). Inset: florescence quenching curve of DNA-bound EB by the complex. (B) Plot of percentage of free EB vs. [Ru]/[EB]. [EB]/[DNA] = 1:5, kex = 537 nm.

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caused obvious reduction in emission intensities, indicating that the complex competitively bound to the DNA with EB. This is because the free EB molecules were much less fluorescent than the bound EB molecules, while [Ru(bpy)2(Htip)]2+ and DNA-bound [Ru(bpy)2(Htip)]2+ are also negligibly weakly emissive when excited at kex = 537 nm [35]. The quenching plot (the inset of Fig. 6A) of I0/I versus [Ru]/[DNA] is in good agreement with the linear Stern-Volmer equation of I0/I = 1 + KD[Ru]/[DNA], and a Stern– Volmer constant KD was derived to be 2.5. In addition, to further illustrate the DNA binding strength of [Ru(bpy)2(Htip)]2+, a competitive binding model can be used to calculate the apparent binding constants from the competition experiments using Eq. (6) [36]:

K app ¼ K EB ½EB50% =½Ru50%

ð6Þ

where Kapp is the apparent DNA binding constant of [Ru(bpy)2(Htip)]2+, KEB is the DNA binding constant of EB, and [EB]50% and [Ru]50% are the EB and [Ru(bpy)2(Htip)]2+ concentrations at which 50% of EB molecules were replaced from EB–DNA complex by the Ru(II) complex. In a plot of percentage of free EB, [EB]/([EB] + [EB  DNA]), versus [Ru]/[EB], we can see that 50% of EB molecules were replaced from DNA-bound EB at a concentration ratio of [Ru]/ [EB] = 1.55. Since the DNA binding constants of EB reported vary considerably, we employed the ct-DNA binding constant of KEB (4.94  105 M1) for EB that represents an average binding constant, yielding Kapp = 3.2  105 M1 for [Ru(bpy)2(Htip)]2+, which is in agreement with the Kb values of 2.2  105 M1 and 4.0  105 M1derived from UV–Vis and emission spectroscopy. 2.2.4. DNA melting experiments Other strong spectroscopic evidence for intercalation of the Ru(II) complex into the helix was obtained from the DNA melting studies. It is well accepted that as the double strands of the DNA gradually dissociates to single strands, the solution absorbance at 260 nm will increase since the absorption at 260 nm for the single strand is much stronger than that for the double strand [37]. The melting temperature (Tm) of DNA, which can characterize the transition from doubled-helical to single-stranded nucleic acids, was determined by monitoring the absorbance at 260 nm as a function of temperature. As shown in Fig. 7A, a Tm value of ct-DNA was obtained to be 67.0 °C by sigmoidal fitting of the data of percentage of single-strand DNA versus temperature in the absence of [Ru(bpy)2(Htip)]2+, and was successively increased upon increasing the concentrations of the complex (Fig. 7B). The increase in DNA melting point, DTm, was found to be 15.0 °C at a concentration ratio of [Ru]/[DNA] = 1:10. Compared to DTm values previously reported under the similar conditions for some DNA intercalators, e.g., 13 °C for EB [38], 6.4 °C for [Ru(bpy)2(ipbp)]2+ [39], the large increase in DTm suggests an intercalative binding mode of [Ru(bpy)2(Htip)]2+ to DNA. Furthermore, the values of the DNA binding constant K for the complex to ct-DNA at Tm were determined by McGee’s Eq. (7) [40]:

1=T 0m  1=T m ¼ ðR=DHm Þ lnð1 þ KLÞ1=n

ð7Þ

where T 0m is the melting point of ct-DNA alone, Tm is the melting temperature in the presence of the complex, DHm is the enthalpy of DNA melting (DHm = 6.9 kcal mol1) [41], R is the gas constant, L is the free Ru(II) complex concentration (approximated by the total complex concentration at Tm), and n is the binding site size. K was derived to be 5.4  104 M1 at 82.0 °C by taking n = 1.0 (approximated by the n values at 298 K), indicating that the complex still displays binding affinity at the melting point of the DNA, similarly to the behaviors of many DNA intercalators reported [27], but different from that of AMAC (AMAC = 9-anthrylmethylammonium chloride) which did not show the binding to the single-

Fig. 7. (A) Thermal denaturnation curves of ct-DNA (129 lM) at different [Ru(bpy)2(Htip)]2+ concentrations of [Ru]/[DNA] = 1/10, 1/20, 1/25, and DNA alone. (B) The plot of DNA helix melting temperature vs. [Ru]/[DNA].

strand DNA or to the phosphate backbone at the melting point of the DNA [42]. 2.2.5. Reverse salt effect The reverse salt effect experiment is an efficient method to distinguish DNA binding modes. The sensitivity of the DNA binding constants to ionic strength is expected to decrease in the order of the binding modes, electrostatic > groove > intercalative, accordingly providing information on the DNA binding modes in quantitative manners. The effects of the ionic strength on the emission yields were tested by addition of NaCl, and the changes in emission spectra of the Ru(II) complex in the presence of the DNA over the salt concentration range of 0.00–0.10 M are shown in Fig. 8. The values of DNA binding constant were found to be (3.14 ± 0.06)  105 M1 for 25 mM NaCl, (2.2 ± 0.03)  105 M1 for 50 mM NaCl, (1.13 ± 0.03)  105 M1 for 75 mM NaCl, and (0.82 ± 0.21)  105 M1 for 100 mM NaCl. Polyelectrolyte theory developed by Record et al. [43] was used to evaluate the electrostatic and nonelectrostatic contribution to the binding free energy changes. A plot of log Kobs against log [Na+] for the binding of the complex to ct-DNA is given in the inset of Fig. 8. Clearly, the DNA binding constants decreased with increasing salt concentrations. This is due to stoichiometry release of sodium ion following the binding of the Ru(II) complex to DNA, suggesting that the electrostatic interaction is involved in the DNA binding event. The slope of the linear fitting of the inset of Fig. 8 is equal to SK in the following Eq. (8):

SK ¼ Zw ¼ d log K obs =d log½Naþ 

ð8Þ

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leads to a significant increase in the viscosities of DNA solution. On contrary, a partial or nonclassical intercalation mode reduces the effective length of the DNA molecule by bending (or kinking) the strand and therefore would reduce DNA viscosities. In addition, electrostatic and grooving binding would not result in any change in viscosities. Here, the viscosities of DNA were found to steadily increase with the increment of [Ru(bpy)2(Htip)]2+, as shown in Fig. 9, suggesting that [Ru(bpy)2(Htip)]2+ intercalate between the base pairs of the DNA.

Fig. 8. Changes in emission spectra of DNA bound [Ru(bpy)2(Htip)]2+ upon successive additions of NaCl. [Ru]/[DNA] = 1:10; [NaCl] = 0–100 mM. Inset: salt dependence of DNA binding constant (K) of the complex.

where Z is the charge on the Ru(II) complex and w is the fraction of counterions associated with each DNA phosphate (w = 0.88 for double-stranded B-form DNA). The binding free energy change DGobs can be calculated based on the standard Gibbs Eq. (9).

DGobs ¼ RTInK obs

ð9Þ

DGpe ¼ ðSKÞRTIn½Naþ  DGt ¼ DGobs  DGpe

ð10Þ ð11Þ

Electrostatic (DGpe) and nonelectrostatic (DGt) portions of the free energy change were calculated base on Eqs. (10) and (11), respectively. A SK value of 1.1 yielded a charge Z of 1.25, which is less than two positive charges carried by the Ru(II) complex. At 50 mM NaCl, a nonelectrostatic free energy change DGt and electrostatic free energy change DGpe were derived to be 22.3 and 8.2 kJ mol1, respectively. It is obvious that the electrostatic contribution to the total binding energy change (DGpe/DGobs  100%), only accounts for 26.8%, which is much less than the nonelectrostatic portion, strongly supporting that [Ru(bpy)2(Htip)]2+ is a DNA intercalator as comparison to those (63–85%) (see Table 2) reported for such proven DNA intercalators as EB [44], [Ru(phen)2(dppz)]2+, and [Ru(bpy)2(Happip)]2+ {Happip = 2-(4-(b-D-allopyranoside) phenyl)imidazo[4,5-f][1,10]phenanthroline} [45,46]. 2.2.6. Viscosity measurements The optical experiments done above provide necessary but not sufficient evidence to support a DNA binding mode. Hydrodynamic measurements, sensitive to length change (i.e. viscosity and sedimentation) are regarded as the most critical tests of binding modes in solution in the absence of crystallographic structural data [11,48]. As for the behaviors of the known DNA binders, a classical intercalation mode demands that the DNA helix lengthens as base pairs are separated to accommodate the binding ligand, hence

2.2.7. Density functional theory calculations It was reported that at least there are three factors influencing DNA binding affinity of a drug molecule [10]. First, the planarity of intercalative moiety of the drug would make a dominant effect on the DNA binding affinity. Usually, the larger planarity area of the intercalative ligand would result in stronger DNA binding. The geometries of [Ru(bpy)2(Htip)]2+, [Ru(bpy)2(tip)]+ and [Ru(bpy)2(Hip)]2+ have been optimized by the density functional theory (DFT) calculations [49–52]. The selective bond lengths and angles listed in Table 3 are almost same as those previously reported by others (see Table 3) [10]. The dihedral angle data for [Ru(bpy)2(Htip)]2+ and [Ru(bpy)2(tip)]+ shown in Table 3 revealed that thiophenyl and ip moieties on both [Ru(bpy)2(Htip)]2+ and [Ru(bpy)2(tip)]+ are almost coplanar. [Ru(bpy)2(Htip)]2+ and [Ru(bpy)2(tip)]+ would have similar DNA binding affinity on view of their having similar planarity of the intercalative moieties. Second, the LUMO energy level was reported to influence DNA-binding affinity. The lower the lowest unoccupied molecular orbital (LUMO) energy of the complex is, the more favorable for the LUMO of the drug to accept the electron from the base pairs of the DNA thermodynamically, accordingly the greater the DNA binding affin-

Fig. 9. Effects of increasing amounts of [Ru(bpy)2(Htip)]2+ on the relative viscosities of ct-DNA in buffered 50 mM NaCl at 32 °C.

Table 2 Comparison of thermodynamic DNA binding parameters. DNA binder

Binding mode

Kobs (103M1)

DGobs (kJ/mol)

SK

Z

DGt/DGobs (%)

Ref.

Ethidium bromide Daynomycin

Intercalative Intercalative

494 4900

32.2 37.7

0.75 0.84

0.85 0.95

5.0 5.9

85 84

[(bpy)4Ru2(Mebpy) (CH2)7(Mebpy)]4+ [Ru(phen)2(dppz)]2+ [Ru(bpy)2(phen)]2+ [Ru(bpy)2(Htip)]2+

Electrostatic

780

23.5

2.2

2.50

16.0

32

[44] [44] [46] [47]

37.2 15.6 30.5

1.9 1.6 1.1

2.15 1.82 1.25

13.8 11.6 8.2

63 74 72

[45] [45] This work

Intercalative Semi-intercalative Intercalative

3200 0.55 220

DGpe (kJ/mol)

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Table 3 Computed selective bond lengths (nm) and angles Am and Aco (°)*. Complex

Ru–Nm

Ru–Nco

C–C(N)m

C–C(N)co

[Ru(bpy)2(Hip)]2+ [Ru(bpy)2(Hip)]2+ [Ru(bpy)2(Htip)]2+ [Ru(bpy)2(tip)]+

0.2108 0.2108 0.2124 0.2113

0.2098 0.2098 0.2115 0.2111

0.1401 0.1407 0.1397 0.1399

0.1400 0.1400 0.1389 0.1388

C6–C7

Am

Aco

0.1439 0.1449

79.2 79.17 78.46 79.20

78.5 78.44 77.70 77.71

Dihedral angles N5-C6-C7-S8

*

179.88 179.98

Ref. N5-C6-C7-C9

0.13 0.00

[10] This work This work This work

Subscript ‘‘m’’ stands for the main ligands of Hip, Htip and tip, and ‘‘co’’ represents for the co-ligand of bpy.

onstrated that the complex is of two successive protonation/ deprotonation processes with ground-state acidity ionization constant values of pKa1 = 1.55 and pKa2 = 7.3, and that the acid– base process for pKa2 occurred over physiological pH range. The spectroscopic titrations, steady-state emission quenching by [Fe(CN)6]4, DNA competitive binding with ethidium bromide, DNA melting experiments, and viscosity measurements showed that [Ru(bpy)2(Htip)]2+ bound to ct-DNA through intercalation with binding constant values on 105 M1 order of magnitude that are insensitive to pH. The grafting of thiophene moiety to Hip has evidenced to make the complex to have interesting acid–base properties and enhanced DNA binding affinity relative to parent complex [Ru(bpy)2(Hip)]2+. 4. Experimental 4.1. Materials Fig. 10. Schematic map of energies of some frontier molecular orbitals of [Ru(bpy)2(Hip)]2+, [Ru(bpy)2(Htip)]2+ and [Ru(bpy)2(tip)]+ (H: HOMO; H-1: next HOMO; L: LUMO; NL: next LUMO).

ity, if the sizes of intercalative plane moieties of drugs are similar. Third, the drugs carrying more positive charge would make more positive electrostatic contribution to the DNA binding affinity than less charged drugs. [Ru(bpy)2(tip)]+ has less positive charge, and higher LUMO level (see Fig. 10) than [Ru(bpy)2(Htip)]2+, would result in evidently reduced DNA binding affinity relative to [Ru(bpy)2(Htip)]2+, as opposed to the experimental observation. This lead us to make a conclusion that there are some unknown factors making a major contribution to the DNA binding of [Ru(bpy)2(Htip)]2+ and [Ru(bpy)2(tip)]+ by the interaction of base pairs of the DNA with Htip on [Ru(bpy)2(Htip)]2+ or tip on [Ru(bpy)2(tip)]+. 3. Conclusions A water-soluble thiophene-appended complex [Ru(bpy)2(Htip)]Cl2 has been synthesized and characterized. It has been dem-

cis-[Ru(bpy)2Cl2]2H2O [53], 2-(thiophen-2-yl)-1H-imidazo[4,5f][1,10]-phenanthroline (Htip) [22], and 1,10-phenanthroline-5,6dione [54] were prepared by the literature routes. Other materials were commercially available and used without further purification. Doubly distilled H2O was used to prepare all solutions. [Ru(bpy)2(Htip)]Cl2 was synthesized as detailed below according to a route described in Scheme 1. 4.2. Synthesis of [Ru(bpy)2(Htip)]Cl2 A solution of cis-[Ru(bpy)2Cl2]2H2O (102 mg, 0.20 mmol) and Htip (60 mg, 0.20 mmol) in ethanol (4 mL) was thoroughly deoxygenated. The solution was heated for 8 h at 80 °C under nitrogen atmosphere. The crude product obtained upon cooling the reaction solution to room temperature, was purified by column chromatography on alumina with CH2Cl2/CH3OH (v/v = 10:1) as eluent to afford [Ru(bpy)2(Htip)]Cl2 (91 mg, 59%). 1H NMR (500 MHz, Me2SOd6): d 8.97 (s, 2H); 8.90 (d, J = 5.0 Hz, 2H); 8.86 (d, J = 4.5 Hz, 2H); 8.21 (t, J = 7.5 Hz, 2H); 8.09 (t, J = 7.5 Hz, 2H); 7.87 (d, J = 5.0 Hz, 2H); 7.79 (s, 3H); 7.73 (d, J = 3.5 Hz, 2H); 7.59 (d, J = 5.0 Hz, 4H);

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139

Acknowledgements The authors thank Professor Kang-Cheng Zheng for helpful guidance for the theoretical calculations, and the National Natural Science Foundation (Nos. 20971016, 20771016, 90922004, 20871011), Beijing Natural Science Foundation (2072011), the Fundamental Research Funds for the Central Universities, Research Fund for the Doctoral Program of Higher Education (20060027002), and Measurements Fund of Beijing Normal University for financial supports.

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ica.2011.01.039. Scheme 1. Synthetic route to [Ru(bpy)2(Htip)]Cl2.

7.46 (s, 1H); 7.37 (t, J = 6.0 Hz, 2H); 7.13 (s, 1H) ppm. Anal. Calc. for [Ru(bpy)2(Htip)]Cl24.75H2O (F.W. = 872.18): C, 50.95; H, 4.10; N, 12.84; Found: C, 51.03; H, 4.12; N, 12.73. Anal. Calc. for MALDITOF MS m/z: 358.5 ([M–2Cl]2+); Found: m/z 357.7 ([M–2Cl]2+). 4.3. Physical measurements 1 H NMR spectra were obtained on a Bruker DRX-500 spectrometer with (CD3)2SO as solvent at room temperature. Microanalyses (C, N and H) were carried out with a Vario EL elemental analyzer. Matrix-assisted laser desorption ionization mass spectra (MALDI-TOF MS) were recorded on an API Q-star pulsar (Applied Biosystems) mass spectrometer. UV–Vis absorption spectra were recorded with a GBC Cintra 10e UV–Vis spectrometer. Emission spectra were obtained on a Cary Eclipse spectrofluorimeter. The UV–Vis absorption and emission spectrophotometric pH titrations of the complex were investigated in buffer A: 40 mM H3BO3, 40 mM H3PO4, and 40 mM CH3COOH. The pH values were read directly on a pHS-3B pH meter. The acidities were adjusted with concentrated HCl, saturated sodium hydroxide aqueous solution, and solid NaOH, respectively. The DNA binding experiments were performed in buffer B: 5 mM Tris-HCl, pH 7.1, 50 mM NaCl unless otherwise mentioned. A solution of calf thymus DNA (ctDNA) gave a ratio of UV absorbance at 260 and 280 nm of ca.1.9:1, indicating that the DNA was sufficiently free of protein [55]. The DNA concentration in nucleotides was determined spectrophotometrically by assuming e260nm = 6600 M1 cm1 [56]. Thermal denaturations of the DNA were performed on a UV–Vis spectrophotometer in buffer C: 1.5 mM Na2HPO4, 0.5 mM NaH2PO4, 0.25 mM Na2EDTA. DNA viscosities were measured by using an Ubbelodhe viscometer immersed in a thermostated water bath maintained at 32.10 ± 0.05 °C. DNA samples for viscosity measurements were prepared by sonicating in order to minimize complexities arising from DNA flexibility. Flow time was measured, and each sample was measured five times, and average flow time was calculated. Data were presented as (g/g0)1/3 versus [Ru]/ [DNA], where g is the viscosity of the DNA in the presence of the [Ru] and the g0 is the viscosity of the DNA alone.

4.4. Computational methods DFT calculations were performed using the B3LYP exchange correlation functional [49], as implemented in the Gaussian 03 program package [50]. The electronic structures of the complexes were determined using a general basis set with the Los Alamos effective core potential LanL2DZ basis set for ruthenium, and 6-31G for other atoms in vacuo [51,52].

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