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Polypyridyl ruthenium(II) complexes containing intramolecular hydrogen-bond ligand: syntheses, characterization, and DNA-binding properties
www.elsevier.nl/locate/jinorgbio Journal of Inorganic Biochemistry 76 (1999) 265–271
Polypyridyl ruthenium(II) complexes containing intramolecular hydrogen-bond ligand: syntheses, characterization, and DNA-binding properties Jin-Gang Liu a, Bao-Hui Ye a, Hong Li a, Qi-Xiong Zhen a, Liang-Nian Ji a,*, Yao-Hong Fu b b
a Department of Chemistry, Zhongshan University, Guangzhou, 510275 P.R. China Shanghai Institute of Organic Chemistry, Chinese Academy of Science, Shanghai, 200032 P.R. China
Received 6 May 1999; received in revised form 18 August 1999; accepted 31 August 1999
Abstract Two new ligands containing an intramolecular hydrogen bond, 2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline (HPIP) and 2-(2-hydroxy-1-naphthyl)imidazo[4,5-f][1,10]phenanthroline (HNAIP), and their complexes [Ru(bpy)2(HPIP)](PF6)2PH2O (1) and [Ru(bpy)2(HNAIP)](PF6)2P2H2O (2) (bpys2,29-bipyridine) have been synthesized and characterized by UV–Vis, IR, 1H NMR, and mass spectra. The electrochemical behaviors of the two complexes were studied by cyclic voltammetry. The binding of the two complexes with calf thymus DNA has been investigated by absorption, luminescence titrations, steady-state emission quenching, and viscosity measurements. The results suggest that complex 1 intercalates into DNA base pairs via the ligand HPIP, while complex 2 binds with DNA by partially intercalating the ligand HNAIP. Complex 1 shows higher affinity to DNA than complex 2. The intrinsic binding constants K for complexes 1 (6.5"0.3=105 My1) and 2 (8.3"0.4=104 My1) together with [Ru(bpy)2PIP]2q (4.7"0.2=105 My1, PIPs2-phenylimidazo[4,5f][1,10]phenanthroline) were determined by absorption titration. q1999 Elsevier Science Inc. All rights reserved. Keywords: Syntheses; Polypyridyl ligands; Ruthenium complexes; DNA binding
1. Introduction The potential application of metal complexes as photochemical and stereoselective probes of nucleic acid structure has been explored extensively over the past decade [1,2]. Despite a considerable amount of reported materials, however, the knowledge of the nature of binding of these complexes to DNA and their binding geometries has remained relatively modest. The binding mode of the prototype complex [Ru(phen)3]2q (phens1,10-phenanthroline) remains an issue of vigorous debate [3–6]. Transition metal complexes can interact non-covalently with nucleic acids by intercalation, groove binding, or external electrostatic binding. Metallointercalators have been particularly used as tools to probe DNA structure and observe the intercalation process, because the ligands or metal may be varied in an easily controlled way to facilitate the individual application. There is a consensus about classical intercalative binding of the recently developed complexes, such as [Ru(bpy)2(dppz)]2q and * Corresponding author: Fax: 86-20-840-36737; e-mail: cesjln@zsu. edu.cn
[Ru(phen)2(dppz)]2q (dppzsdipyrido[3,2-a:29,39-c]phenazine), in which the dppz ligand intercalates between the base pairs of double helical DNA [7–14]. Therefore, the binding of ruthenium(II) polypyridyl complex to DNA has initiated vigorous interest and many new structural analogues based on prototype [Ru(phen)3]2q have also been synthesized and investigated. As our ongoing focus on the interactions of polypyridyl ruthenium complexes with DNA, the cationic complexes have been found to bind with DNA in an intercalative [15– 19], electrostatic, or surface[20,21] interaction fashion. For the intercalating ligands of phenanthroline and its derivatives, inspection of models shows that, owing to the overhanging hydrogen atoms (2- and 3-positions) from the ancillary ligands, only the outer third phenyl ring of the phenanthroline ligand (5- and 6-positions) is available for stacking [22]. With this consideration in mind, we have designed and synthesized a series of novel ligands derived from 1,10-phenanthroline-5-6-dione to spread the planarity of phenanthroline. Based on our previous studies on 2-phenylimidazo[4,5-f][1,10]phenanthroline (PIP), in which the PIP molecule is almost coplanar [15], we now introduce an
0162-0134/99/$ - see front matter q1999 Elsevier Science Inc. All rights reserved. PII S 0 1 6 2 - 0 1 3 4 ( 9 9 ) 0 0 1 5 4 - 3
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Scheme 1.
ortho phenolic group that may form an intramolecular hydrogen bond with the nitrogen atom of the imidazole ring (Scheme 1) into the intercalated ligands to extend the planarity. Herein, we report the syntheses and characterization for the two novel ruthenium complexes [Ru(bpy)2 (HPIP)](PF6)2PH2O (1) (bpys2,29-bipyridine, HPIPs 2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline) and [Ru(bpy)2(HNAIP)](PF6)2P2H2O (2) (HNAIPs2(2-hydroxy-1-naphthyl)imidazo[4,5-f][1,10]phenanthroline). Their associations with calf thymus DNA were also investigated by electronic absorption, luminescence, steadystate emission quenching, and viscosity measurements.
Cyclic voltammetry was performed on an EG&G PAR 273 polarographic analyser. The electrochemical measurements were made in dried acetonitrile solutions with tetrabutylammonium hexafluorophosphate (0.1 M) as the supporting electrolyte. All samples were purged with nitrogen prior to measurements. A standard three-electrode system was used comprising a platinum microcylinder working electrode, platinum-wire auxiliary electrode, and a saturated calomel reference electrode (SCE). Viscosity measurements were carried out using a Ubbelodhe viscometer maintained at a constant temperature of 28.0("0.1)8C in a thermostatic bath. DNA samples of approximately 200 base pairs in average length were prepared by sonicating in order to minimize complexities arising from DNA flexibility [25]. Flow time was measured with a digital stopwatch and each sample was measured three times and an average flow time was calculated. Data were presented as (h/h0)1/3 versus binding ratio [26], where h is the viscosity of DNA in the presence of complex and h0 is the viscosity of DNA alone. 2.2. Syntheses
2. Experimental All reagents and solvents were purchased commercially (AR grade) and used without further purification unless otherwise noted. Acetonitrile was refluxed and distilled several times from P2O5 before electrochemical measurements.Solutions of calf thymus DNA (CT-DNA) in 50 mM NaCl–5 mM Tris–HCl (pHs7.2) gave a ratio of UV absorbance at 260 and 280 nm of 1.8–1.9:1, indicating that the DNA was sufficiently free of protein [23]. The concentration of DNA was determined spectrophotometrically using a molar absorptivity of 6600 My1 cmy1 (260 nm) [24]. Double-distilled water was used to prepare buffers. 2.1. Physical measurements Microanalyses (C, H, and N) were carried out with a Perkin–Elmer 240Q elemental analyser. Infrared spectra were obtained with a Nicolet 170SX-FTIR spectrophotometer with KBr discs and UV–Vis spectra on a Shimadzu MPS2000 spectrophotometer. 1H NMR spectra were recorded on a Bruker ARX-300 spectrometer with (CD3)2SO as solvent for the pro-ligands and CD3CN for the complexes at 300.13 MHz at room temperature. All chemical shifts were given relative to tetramethylsilane (TMS). Fast atom bombardment (FAB) mass spectra were measured on a VG ZAB-HS mass spectrometer with 3-nitrobenzyl alcohol as matrix and emission spectra on a Shimadzu RF-5000 spectrofluorophotometer at room temperature. Quenching experiments were conducted by adding small aliquots of a 25 mM ferrocyanide stock solution to the samples containing 4 mM metal and 160 mM nucleotides in a buffer solution consisting of 5 mM Tris– HCl and 50 mM NaCl (pHs7.2).
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The compounds cis-[Ru(bpy)2Cl2]P2H2O [27] and 1,10phenanthroline-5,6-dione [28] were prepared by the literature methods. 2.2.1. HPIP A mixture of salicylaldehyde (0.42 g, 3.5 mmol), 1,10phenanthroline-5,6-dione (0.525 g, 2.5 mmol), ammonium acetate (3.88 g, 50 mmol), and glacial acetic acid (10 ml) was refluxed for 2 h, then cooled to room temperature and diluted with water (ca. 40 ml). Dropwise addition of concentrated aqueous ammonia to neutralize gave a yellow precipitate, which was collected and washed with water. The crude product dissolved in ethanol was purified by filtration on silica gel (60–100 mesh, ethanol). The principal yellow band was collected. Then, evaporation of the solution gave yellow crystals. Yield 0.54 g, 70%. (Found: C, 72.9; H, 3.9; N, 17.7. Calc. for C19H12N4O: C, 73.1; H, 3.8; N, 17.9%.) IR data (KBr, cmy1): 3400–2600s (br), 1623m, 1588m, 1560m, 1510s, 1475s, 1398s, 1356m, 1293m, 1257s, 807s, 737s. lmax/nm (´/My1 cmy1) (CH3CN) 327 (39 200), 273 (46 600), 237 (36 300). 1H NMR (ppm, DMSO-d6): 13.94 (s, 1H), 12.75 (s, 1H), 9.06 (d, 2H, Js4.4 Hz), 8.94 (d, 2H, Js8.1), 8.20 (d, 1H, Js8.4), 7.87 (q, 2H), 7.42 (t, 1H), 7.13–7.08 (m, 2H). MS (FAB): 313, [Mq1]q. 2.2.2. HNAIP This compound was prepared by a similar procedure to that for HPIP; 2-hydroxy-1-naphthylaldehyde (0.60 g, 3.5 mmol) was used instead of salicylaldehyde. Yield 0.68 g, 75%. (Found: C, 72.7; H, 4.4; N, 14.9. Calc. for C23H14N4OPH2O: C, 72.6; H, 4.2; N, 14.7%.) IR data (KBr, cmy1): 3400–2600s (br), 1623s, 1567m, 1546m, 1503m, 1461m, 1398s, 1356s, 1243m, 807s, 737s. lmax/nm (´/My1
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cmy1) (CH3CN) 357 (24 600), 304 (31 800), 243 (53 600). 1H NMR (ppm, DMSO-d6): 13.82 (s, 1H), 10.64 (s, 1H), 9.05 (d, 2H, Js4.2 Hz), 8.91 (d, 2H, Js8.1), 8.13 (d, 1H, Js7.2), 7.99 (d, 1H, Js8.9), 7.92 (d, 1H, Js7.5), 7.83 (q, 2H), 7.48 (t, 1H), 7.38 (d, 1H, Js8.7), 7.37(t, 1H). MS (FAB): 363, [Mq1]q.
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of 1,10-phenanthroline-5,6-dione and salicylaldehyde or 2hydroxy-1-naphthylaldehyde in the presence of ammonium acetate and glacial acid. Their structures were confirmed by elemental analyses, mass and 1H NMR spectroscopies. The 1 H NMR spectra of the two ligands display a broad peak at 13.92 for HPIP and 13.82 ppm for HNAIP, respectively, which can be assigned to the peak of N–H from the imidazole ring [15,16]. The phenolic protons of HPIP and HNAIP appear at 12.73 and 10.64 ppm, respectively. These downshifts, compared with the other phenolic protons not involving an intramolecular hydrogen bond [30], can be attributed to the formation of an intramolecular hydrogen bond with the nitrogen atom of the imidazole ring [31,32]. This phenomenon is also identified by IR spectra in the solid state. The IR spectra display a broad band in the range 3400 to 2600 cmy1, which can be assigned to the stretching vibration of O–H involving the intramolecular hydrogen bond. This kind of intramolecular hydrogen bond has a great effect on the properties of the ligand. It has important consequences not only for the ligating ability of the ligand, but also for stabilizing the chelating conformation of the appropriate rotor to preorganize the phenyl ring planar to the central heterocyclic ring. These have been illustrated by crystal structure in a few analogous compounds containing pyridyl– or benzothiazole–phenol ligands [33–37]. In complexes 1 and 2, the proton on the nitrogen atom of the imidazole ring was not observed, probably because the proton exchanged quickly between the two nitrogens of the imidazole ring, characteristic of an active proton [15,16]. The electronic absorption spectra of the two complexes in acetonitrile, not different from those in water, are characterized by an intense ligand-centered transition in the UV and a metal-to-ligand charge transfer (MLCT) transition in the visible region (Table 1). The lowest-energy absorption bands at 458 and 456 nm for 1 and 2, respectively, are assigned to the MLCT transition. In the UV region, the intense, fairly sharp bands at 284 nm for 1 and 285 nm for 2 are attributed to the intraligand p–pU transitions by comparison with the spectrum of [Ru(bpy)3]2q [38]. The shoulder at 328 nm for 1 may be assigned to the HPIP ligand p–pU transition according to the absorption spectra of the ligand. Little variation in the energy of the MLCT bands with the annelation of a phenyl moiety to the HPIP ligand was observed. Similar phenomena have also been observed between [Ru(bpy)3]2q and [Ru(bpy)2dppz]2q [39].
2.2.3. [Ru(bpy)2(HPIP)](PF6)2PH2O (1) A mixture of cis-[Ru(bpy)2Cl2]P2H2O (0.261 g, 0.5 mmol), HPIP (0.156 g, 0.5 mmol), ethanol (20 ml), and water (10 ml) was refluxed under argon for 3 h to give a clear red solution. Upon cooling, the solution was then treated with saturated aqueous solution of NH4PF6 and gave a red precipitate. The crude product was purified by column chromatography on alumina with CH3CN–toluene (1:1, v:v) as an eluent. The mainly red band was collected. The solvent was removed under reduced pressure and red crystals were obtained. Yield 0.33 g, 66%. (Found: C, 45.5; H, 3.3; N, 10.6. Calc. for C39H28F12N8OP2RuPH2O: C, 45.3; H, 2.9; N, 10.8%.) 1H NMR (ppm, CD3CN): 12.30 (s, 1H), 9.07 (d, 2H, Js8.3 Hz), 8.60 (d, 2H, Js8.3), 8.56 (d, 2H, Js8.2), 8.19 (d, 1H, Js8.2), 8.16 (d, 2H), 8.07 (d, 2H), 8.03 (d, 2H), 7.92 (d, 2H, Js5.7), 7.81 (q, 2H), 7.66 (d, 2H, Js5.7), 7.51 (t, 2H), 7.44 (t, 1H), 7.27 (t, 2H), 7.14 (d, 1H, Js7.8), 7.12 (d, 1H). MS (FAB): m/z 871 ([MH2qqPF6y]q, 16%), 725 (M2q, 26). 2.2.4. [Ru(bpy)2(HNAIP)](PF6)2P2H2O (2) This complex was obtained by a similar procedure to that described for complex 1. Yield 0.38 g, 71%. (Found: C, 47.0; H, 3.4; N, 10.3. Calc. for C43H30F12N8OP2RuP2H2O: C, 46.9; H, 3.1; N, 10.2%.) 1H NMR (ppm, CD3CN): 10.46 (s, 1H), 9.12 (d, 2H, Js8.2 Hz), 8.58 (d, 2H, Js8.2), 8.53 (d, 2H, Js8.2), 8.13 (t, 2H), 8.01 (t, 2H), 7.94 (d, 2H), 7.92 (d, 2H), 7.90 (d, 1H), 7.85 (d, 2H, Js8.8), 7.71 (m, 4H), 7.49 (t, 2H), 7.43 (t, 1H), 7.34 (d, 2H, Js8.8), 7.25 (t, 2H). MS (FAB): m/z 920 ([M2qqPF6y]q, 22%), 775 (M2q, 100). 3. Results and discussion 3.1. Syntheses and characterization The ligands HPIP and HNAIP were prepared by a method similar to that described by Steck and Day [29], condensation Table 1 Electrochemical and UV–vis spectroscopic data Complex
Ru(bpy)2HPIP2q Ru(bpy)2HNAIP2q a b
Oxidation a
Reduction a E1/2(DEp, mV)
Absorption b
E1/2 (DEp, mV)
1
2
3
lmax/nm (10y4 ´/My1 cmy1)
1.29(60) 1.32(85)
y0.98(irr)
1.40(56) 1.43(58)
1.62(58) 1.62(56)
458(1.22), 3.28(sh), 284(6.25), 254(2.07) 456(1.26), 285(6.05), 253(3.63)
Redox potentials were quoted vs. SCE in 0.1 M TBAH–CH3CN. Scan rates200 m V sy1. Measured in CH3CN.
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Each complex displays well one oxidation and two reduction waves in the sweep range from y1.85 to q1.70 V. A small poorly shaped reduction wave appeared at ca. 0.97 V for complex 1. The electrochemical behavior of ruthenium(II) polypyridyl complex has been rationalized in terms of a metal-based oxidation and a series of reductions, which are ligand based and occur in a stepwise manner for each pU system [40]. As expected, the oxidation potential of complex 2 is 30 mV more positive than that of 1 (Table 1). The annelation of a phenyl ring to the HPIP moiety expands the p delocalization and thus decreases the s donor capacity of
HNAIP, which leads to a decrease of the electron density on the Ru ion and in turn stabilizes the metal dp orbital [41]. As a result, the oxidation potential shifts more positively. The first reduction, which was usually controlled by the ligand having the most stable lowest unoccupied molecular orbital (LUMO) [40], is proposed to be for the HPIP or HNAIP and appears irreversible, making a comparison between the two complexes difficult. The later two successive reversible reductions are characteristic of the two bpy ligands [38,40]. 3.2. DNA-binding studies 3.2.1. Electronic absorption titration The binding of intercalative molecular ligand to DNA has been well characterized classically through absorption titrations, following the hypochromism and red shift associated with the binding of the colored complex to the helix, due to the intercalative mode involving a strong stacking interaction between an aromatic chromophore and the DNA base pairs. The magnitudes of the hypochromism and red shift were commonly found to depend on the strength of the intercalative interaction [22]. The absorption spectra of complexes 1 and 2 in the absence and presence of CT-DNA (at a constant concentration of complexes) are given in Fig. 1. As the DNA concentration is increased, the MLCT transition bands of complexes 1 at 458 and 2 at 456 nm exhibit hypochromism of about 24.5 and 14.6%, and bathochromism of about 6 and 2 nm, respectively. These spectral characteristics may suggest a mode of binding that involves a stacking interaction between the aromatic chromophore and the DNA base pairs. In order to compare quantitatively the binding strength of the two complexes, the intrinsic binding constants K of the two complexes with CT-DNA were determined according to the following equation [42] through a plot of [DNA]/ (´ay´f) versus [DNA]. [DNA]/(´ay´ f)s[DNA]/(´ 0y´ f)q1/(K(´ 0y´ f))
Fig. 1. Absorption spectra of complexes 1 (a) and 2 (b) in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2) in the presence of increasing amounts of CT-DNA. ([DNA]s0–200.0 mM, [Ru]s20 mM).
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where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficients ´a, ´f, and ´0 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. In plots of [DNA]/(´ay´f) versus [DNA], K is given by the ratio of slope to intercept. Intrinsic binding constants K of complexes 1 and 2 of about (6.5"0.3)=105 My1 and (8.3"0.4)=104 My1, respectively, were obtained from the decay of the absorbance. For comparison, the intrinsic binding constant K of [Ru(bpy)2PIP]2q with DNA was also determined to be (4.7"0.2)=105 My1. These results indicate that complex 1 binds more strongly to CT-DNA than complex 2. The binding strength of complex 1 to CT-DNA is comparable to that of [Ru(NH3)4dppz]2q (1.24=105 My1) [43], but is not as strong as that of [Ru(bpy)2dppz]2q (4.9=106 My1) [44] or [Ru(IP)2dppz]2q (2.1=107 My1) [19].
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3.2.2. Luminescence titration The complexes 1 and 2 can emit luminescence in Tris buffer at ambient temperature under experimental conditions similar to those for [Ru(bpy)3]2q, with maxima at 610 and 607 nm, respectively. Despite the structural similarity of the two complexes, their interactions with double-strand CTDNA are obviously different, as revealed by the luminescence behavior in the presence of DNA. The results of the emission titrations for the two complexes with DNA are illustrated by the titration curves in Fig. 2. Solutions of complex 1 exhibit an obvious enhancement in emission intensity when DNA is added, and the enhancement is around 2.7 times larger than that of in the absence of DNA. However, in complex 2, the emission intensity, which approaches saturation with an overall gain of ca. 1.6, shows only a lesser enhancement in similar conditions. This implies that the HPIP ligand inserts more deeply and strongly than HNAIP.
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about the binding of the two complexes with DNA. As illustrated in Fig. 3, in the absence of DNA, complexes 1 and 2 were efficiently quenched by [Fe(CN)6]4y. However, on addition of DNA, the Stern–Volmer plots changed drastically. The efficiency of [Fe(CN)6]4y quenching of ruthenium complexes bound to DNA is decreased relative to that of the free ruthenium complexes. This may be because the bound [Ru(bpy)2L]2q (LsHPIP or HNAIP) cations are protected from the anionic water-bound quencher by the array of negative charges along the DNA phosphate backbone [3,45]. The curvature reflects different degrees of protection or relative accessibility of bound cations. From the results, complex 1 is more protected from the solvated anion of [Fe(CN)6]4y than 2, which is consistent with the results observed by electronic absorption and emission titrations.
Fig. 2. Plots of relative emission intensity vs. [DNA]/[Ru] ratio for complexes 1 (j) and 2 (d) in 5 mM Tris–HCl and 50 mM NaCl buffer at pH 7.2. ([Ru]s4 mM).
3.2.4. Viscosity measurements To further clarify the interactions between the two complexes and DNA, viscosity measurements were carried out. Photophysical probes generally provide necessary, but not sufficient, clues to support an intercalation binding model. Hydrodynamic measurements that are sensitive to length increases (i.e. viscosity, sedimentation, etc.) are regarded as the least ambiguous and the most critical tests of binding model in solution in the absence of crystallographic structural data [4,46]. A classical intercalation model results in lengthening of the DNA helix as base pairs are separated to accommodate the binding ligand, leading to an increase of DNA viscosity. However, a partial and/or non-classical intercalation of ligand may bend (or kink) the DNA helix, resulting in a decrease of its effective length and, concomitantly, its viscosity [4]. The effects of the complexes 1 and 2, together with [Ru(bpy)3]2q and [Ru(bpy)2dppz]2q, on the viscosity of rod-like DNA are depicted in Fig. 4. On increasing the amount of complex 1, the relative viscosity of DNA increases steadily, which is similar to the behavior of the complex [Ru(bpy)2dppz]2q. At low binding ratios, complex 2 exerts essentially no effect on DNA viscosity, but upon further bind-
Fig. 3. Emission quenching curves of the complexes 1 (h), 1qDNA (j), 2 (s) and 2qDNA (d) with increasing concentration of quencher [Fe(CN)6]4y. [Ru]s4 mM, [DNA]/[Ru]s40.
Fig. 4. Effects of increasing amounts of the complexes 1 (j), 2 (d), [Ru(bpy)2dppz]2q (h) and [Ru(bpy)3]2q (l) on the relative viscosities of calf thymus DNA at 28.0("0.1)8C, [DNA]s0.5 mM and rs[Ru]/ [DNA].
3.2.3. Steady-state emission quenching Steady-state emission quenching experiments using [Fe(CN)6]4y as quencher may give further information
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ing of the compound to DNA, the DNA viscosity decreases. Such behavior is consistent with bending or kinking of the helix upon binding racemic ruthenium(II) complex to DNA [4,46]. For complex [Ru(bpy)3]2q, which has been well known to bind with DNA only through the electrostatic mode, there is no effect on the relative viscosity of DNA solution. The results then suggest that complex 1 binds with CT-DNA intercalatively and complex 2 may bind with a partial or nonclassical intercalative mode. For the mixed-ligand complexes, the tendency of each of the ligands to intercalate may be compared. For the series [Ru(bpy)2PIP]2q, [Ru(bpy)2HPIP]2q and [Ru(bpy)2HNAIP]2q, where the ancillary bpy ligands are kept constant, the binding constants increase in the order HNAIP-PIP-HPIP. This variation likely reflects the different abilities of the ligands to stack and overlap well with the base pairs. Although the crystal structures of these complexes are unavailable at present, the structure of PIP ligand has been illustrated by single-crystal X-ray diffraction, in which the phenyl ring is almost coplanar with the imidazole ring [47]. We speculate that the ortho phenolic group of HPIP would be closely coplanar with the imidazole ring due to the formation of an intramolecular hydrogen bond with the nitrogen atom of imidazole ring, which have also been seen in the crystal structures of analogous compounds [33–37]. These results also suggest that the introduction of a phenolic group into the HPIP molecule may have some additional affinity to stabilize the binding of the complexes to DNA. Although the larger coplanar area of the HNAIP ligand is expected, complex 2 has the lowest affinity to CT-DNA. It is likely that the large naphthyl ring may lie out of the plane of the imidazole ring, resulting in the partial intercalation of the HNAIP ligand.
4. Conclusions Two new polypyridyl mixed-ligand ruthenium(II) complexes containing an intramolecular hydrogen-bond ligand and their DNA-binding properties have been demonstrated. In agreement with the spectra titrations and viscosity measurements, complex 1 binds to CT-DNA intercalatively via the HPIP ligand, while complex 2 binds with a partial intercalative mode. Complex 1 shows much high affinity to DNA than complex 2. In order to understand further the binding of these analogous complexes with DNA, efforts will be made to tune the properties of the complexes by introducing a substituent on the HPIP ligand or changing the nature of the ancillary ligands.
5. Abbreviations Bpy Dppz
2,29-bipyridine dipyrido[3,2-a:29,39-c]phenazine
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HNAIP HPIP IL LUMO MLCT Phen PIP SCE
2-(2-hydroxy-1-naphthyl) imidazo[4,5-f][1,10]phenanthroline 2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline intraligand lowest unoccupied molecular orbital metal-to-ligand charge transfer 1,10-phenanthroline 2-phenylimidazo[4,5-f][1,10]phenanthroline saturated calomel electrode
Acknowledgements We are grateful to the NNSF of China, the State Key Laboratory of Bio-organic and Natural Products Chemistry in Shanghai Institute of Organic Chemistry, the State Key Laboratory of Coordination Chemistry in Nanjing University and the NSF of Guangdong Province for their financial support.
References ´ P. Lincoln, B. Akerman, E. Tuite, in: A. Sigel, H. Sigel [1] B. Norden, (Eds.), Metal Ions in Biological Systems, vol. 33, Marcel Dekker, New York, 1996, p. 177. [2] A. Pyle, J.K. Barton, in: S.J. Lippard (Ed.), Progress in Inorganic Chemistry, vol. 38, New York, 1990, p. 413. [3] J.K. Barton, J.M. Goldberg, C.V. Kumar, N.J. Turro, J. Am. Chem. Soc. 108 (1986) 2081. [4] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry 32 (1993) 2573. ´ A. Rodger, J. Am. Chem. Soc. 112 (1990) 1971. [5] C. Hiort, B. Norden, ´ A. Graslund, Biochem[6] M. Eriksson, M. Leijon, C. Hiort, B. Norden, istry 33 (1994) 5031. [7] A.E. Friedman, J.-C. Chambron, J.-P. Sauvage, N.J. Turro, J.K. Barton, J. Am. Chem. Soc. 112 (1990) 4960. [8] C.M. Dupureur, J.K. Barton, Inorg. Chem. 36 (1997) 33. [9] Y. Jenkins, A.E. Freidman, N.J. Turro, J.K. Barton, Biochemistry 31 (1992) 10809. [10] C. Turro, S.H. Bossmann, Y. Jenkins, J.K. Barton, N.J. Turro, J. Am. Chem. Soc. 117 (1995) 9026. ´ B.Z. Chowdhry, J.B. Chaires, [11] I. Haq, P. Lincoln, D. Suh, B. Norden, J. Am. Chem. Soc. 117 (1995) 4788. ´ J. Am. Chem. Soc. 118 (1996) 2644. [12] P. Lincoln, A. Broo, B. Norden, ´ J. Am. Chem. Soc. 119 (1997) 239. [13] E. Tuite, P. Lincoln, B. Norden, [14] C.G. Coates, L. Jacquet, J.J. McGarvey, S.E.J. Bell, A.H.R. Al-Obaidi, J.M. Kelly, Chem. Commun. (Cambridge) (1996) 35. [15] J.-Z. Wu, B.-H. Ye, L. Wang, L.-N. Ji, R.-H. Li, J.-Y. Zhou, J. Chem. Soc., Dalton Trans. (1997) 1395. [16] J.-Z. Wu, L. Li, T.-X. Zeng, L.-N. Ji, J.-Y. Zhou, R.-H. Li, Polyhedron 16 (1997) 103. [17] X.-H. Zou, B.-H. Ye, H. Li, J.-G. Liu, Y. Xiong, L.-N. Ji, J. Chem. Soc., Dalton Trans. (1999) 1423. [18] Y. Xiong, X.-F. He, X.-H. Zou, J.-Z. Wu, X.-M. Chen, L.-N. Ji, R.H. Li, J.-Y. Zhou, K.-B. Yu, J. Chem. Soc., Dalton Trans. (1999) 19. [19] J.-G. Liu, B.-H. Ye, H. Li, L.-N. Ji, R.-H. Li, J.-Y. Zhou, J. Inorg. Biochem. 73 (1999) 117. [20] G. Yang, J.-Z. Wu, L. Wang, L.-N. Ji, X. Tian, J. Inorg. Biochem. 66 (1997) 141. [21] G. Yang, L. Wang, L.-N. Ji, J. Inorg. Biochem. 67 (1997) 289.
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J.-G. Liu et al. / Journal of Inorganic Biochemistry 76 (1999) 265–271 [22] A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar, N.J. Turro, J.K. Barton, J. Am. Chem. Soc. 111 (1989) 3053. [23] J. Marmur, J. Mol. Biol. 3 (1961) 208. [24] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954) 3047. [25] J.B. Chaires, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1982) 3933. [26] G. Cohen, H. Eisenberg, Biopolymers 8 (1969) 45. [27] B.P. Sullivan, D.J. Salmon, T.J. Meyer, Inorg. Chem. 17 (1978) 3334. [28] W. Paw, R. Eisenberg, Inorg. Chem. 36 (1997) 2287. [29] E.A. Steck, A.R. Day, J. Am. Chem. Soc. 65 (1943) 452. [30] T.E. Keyes, P.M. Jayaweera, J.J. McGarvey, J.G. Vos, J. Chem. Soc., Dalton Trans. (1997) 1627. [31] J. Catalan, F. Fabero, M.S. Guijarro, R.M. Claramunt, D.D.S. Maria, R. Sastre, J. Am. Chem. Soc. 112 (1990) 747. [32] P. Cornago, C. Escolastico, M.D.S. Maria, R.M. Claramount, D. Carmona, M. Esteban, L.A. Oro, C. Foces-Foces, A.L. Llamas-Siaz, J. Elguero, J. Organomet. Chem. 467 (1994) 293. [33] P. Stenson, ´ Acta Chem. Scand. 24 (1970) 3729. [34] R. Hage, J.G. Haasnoot, J. Reedijk, R. Wang, E.M. Ryan, J.G. Vos, A.L. Spek, A.J.M. Duisenberg, Inorg. Chim. Acta 174 (1990) 77.
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Polypyridyl ruthenium(II) complexes containing intramolecular hydrogen-bond ligand: syntheses, characterization, and DNA-binding properties Jin-Gang Liu a, Bao-Hui Ye a, Hong Li a, Qi-Xiong Zhen a, Liang-Nian Ji a,*, Yao-Hong Fu b b
a Department of Chemistry, Zhongshan University, Guangzhou, 510275 P.R. China Shanghai Institute of Organic Chemistry, Chinese Academy of Science, Shanghai, 200032 P.R. China
Received 6 May 1999; received in revised form 18 August 1999; accepted 31 August 1999
Abstract Two new ligands containing an intramolecular hydrogen bond, 2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline (HPIP) and 2-(2-hydroxy-1-naphthyl)imidazo[4,5-f][1,10]phenanthroline (HNAIP), and their complexes [Ru(bpy)2(HPIP)](PF6)2PH2O (1) and [Ru(bpy)2(HNAIP)](PF6)2P2H2O (2) (bpys2,29-bipyridine) have been synthesized and characterized by UV–Vis, IR, 1H NMR, and mass spectra. The electrochemical behaviors of the two complexes were studied by cyclic voltammetry. The binding of the two complexes with calf thymus DNA has been investigated by absorption, luminescence titrations, steady-state emission quenching, and viscosity measurements. The results suggest that complex 1 intercalates into DNA base pairs via the ligand HPIP, while complex 2 binds with DNA by partially intercalating the ligand HNAIP. Complex 1 shows higher affinity to DNA than complex 2. The intrinsic binding constants K for complexes 1 (6.5"0.3=105 My1) and 2 (8.3"0.4=104 My1) together with [Ru(bpy)2PIP]2q (4.7"0.2=105 My1, PIPs2-phenylimidazo[4,5f][1,10]phenanthroline) were determined by absorption titration. q1999 Elsevier Science Inc. All rights reserved. Keywords: Syntheses; Polypyridyl ligands; Ruthenium complexes; DNA binding
1. Introduction The potential application of metal complexes as photochemical and stereoselective probes of nucleic acid structure has been explored extensively over the past decade [1,2]. Despite a considerable amount of reported materials, however, the knowledge of the nature of binding of these complexes to DNA and their binding geometries has remained relatively modest. The binding mode of the prototype complex [Ru(phen)3]2q (phens1,10-phenanthroline) remains an issue of vigorous debate [3–6]. Transition metal complexes can interact non-covalently with nucleic acids by intercalation, groove binding, or external electrostatic binding. Metallointercalators have been particularly used as tools to probe DNA structure and observe the intercalation process, because the ligands or metal may be varied in an easily controlled way to facilitate the individual application. There is a consensus about classical intercalative binding of the recently developed complexes, such as [Ru(bpy)2(dppz)]2q and * Corresponding author: Fax: 86-20-840-36737; e-mail: cesjln@zsu. edu.cn
[Ru(phen)2(dppz)]2q (dppzsdipyrido[3,2-a:29,39-c]phenazine), in which the dppz ligand intercalates between the base pairs of double helical DNA [7–14]. Therefore, the binding of ruthenium(II) polypyridyl complex to DNA has initiated vigorous interest and many new structural analogues based on prototype [Ru(phen)3]2q have also been synthesized and investigated. As our ongoing focus on the interactions of polypyridyl ruthenium complexes with DNA, the cationic complexes have been found to bind with DNA in an intercalative [15– 19], electrostatic, or surface[20,21] interaction fashion. For the intercalating ligands of phenanthroline and its derivatives, inspection of models shows that, owing to the overhanging hydrogen atoms (2- and 3-positions) from the ancillary ligands, only the outer third phenyl ring of the phenanthroline ligand (5- and 6-positions) is available for stacking [22]. With this consideration in mind, we have designed and synthesized a series of novel ligands derived from 1,10-phenanthroline-5-6-dione to spread the planarity of phenanthroline. Based on our previous studies on 2-phenylimidazo[4,5-f][1,10]phenanthroline (PIP), in which the PIP molecule is almost coplanar [15], we now introduce an
0162-0134/99/$ - see front matter q1999 Elsevier Science Inc. All rights reserved. PII S 0 1 6 2 - 0 1 3 4 ( 9 9 ) 0 0 1 5 4 - 3
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Scheme 1.
ortho phenolic group that may form an intramolecular hydrogen bond with the nitrogen atom of the imidazole ring (Scheme 1) into the intercalated ligands to extend the planarity. Herein, we report the syntheses and characterization for the two novel ruthenium complexes [Ru(bpy)2 (HPIP)](PF6)2PH2O (1) (bpys2,29-bipyridine, HPIPs 2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline) and [Ru(bpy)2(HNAIP)](PF6)2P2H2O (2) (HNAIPs2(2-hydroxy-1-naphthyl)imidazo[4,5-f][1,10]phenanthroline). Their associations with calf thymus DNA were also investigated by electronic absorption, luminescence, steadystate emission quenching, and viscosity measurements.
Cyclic voltammetry was performed on an EG&G PAR 273 polarographic analyser. The electrochemical measurements were made in dried acetonitrile solutions with tetrabutylammonium hexafluorophosphate (0.1 M) as the supporting electrolyte. All samples were purged with nitrogen prior to measurements. A standard three-electrode system was used comprising a platinum microcylinder working electrode, platinum-wire auxiliary electrode, and a saturated calomel reference electrode (SCE). Viscosity measurements were carried out using a Ubbelodhe viscometer maintained at a constant temperature of 28.0("0.1)8C in a thermostatic bath. DNA samples of approximately 200 base pairs in average length were prepared by sonicating in order to minimize complexities arising from DNA flexibility [25]. Flow time was measured with a digital stopwatch and each sample was measured three times and an average flow time was calculated. Data were presented as (h/h0)1/3 versus binding ratio [26], where h is the viscosity of DNA in the presence of complex and h0 is the viscosity of DNA alone. 2.2. Syntheses
2. Experimental All reagents and solvents were purchased commercially (AR grade) and used without further purification unless otherwise noted. Acetonitrile was refluxed and distilled several times from P2O5 before electrochemical measurements.Solutions of calf thymus DNA (CT-DNA) in 50 mM NaCl–5 mM Tris–HCl (pHs7.2) gave a ratio of UV absorbance at 260 and 280 nm of 1.8–1.9:1, indicating that the DNA was sufficiently free of protein [23]. The concentration of DNA was determined spectrophotometrically using a molar absorptivity of 6600 My1 cmy1 (260 nm) [24]. Double-distilled water was used to prepare buffers. 2.1. Physical measurements Microanalyses (C, H, and N) were carried out with a Perkin–Elmer 240Q elemental analyser. Infrared spectra were obtained with a Nicolet 170SX-FTIR spectrophotometer with KBr discs and UV–Vis spectra on a Shimadzu MPS2000 spectrophotometer. 1H NMR spectra were recorded on a Bruker ARX-300 spectrometer with (CD3)2SO as solvent for the pro-ligands and CD3CN for the complexes at 300.13 MHz at room temperature. All chemical shifts were given relative to tetramethylsilane (TMS). Fast atom bombardment (FAB) mass spectra were measured on a VG ZAB-HS mass spectrometer with 3-nitrobenzyl alcohol as matrix and emission spectra on a Shimadzu RF-5000 spectrofluorophotometer at room temperature. Quenching experiments were conducted by adding small aliquots of a 25 mM ferrocyanide stock solution to the samples containing 4 mM metal and 160 mM nucleotides in a buffer solution consisting of 5 mM Tris– HCl and 50 mM NaCl (pHs7.2).
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The compounds cis-[Ru(bpy)2Cl2]P2H2O [27] and 1,10phenanthroline-5,6-dione [28] were prepared by the literature methods. 2.2.1. HPIP A mixture of salicylaldehyde (0.42 g, 3.5 mmol), 1,10phenanthroline-5,6-dione (0.525 g, 2.5 mmol), ammonium acetate (3.88 g, 50 mmol), and glacial acetic acid (10 ml) was refluxed for 2 h, then cooled to room temperature and diluted with water (ca. 40 ml). Dropwise addition of concentrated aqueous ammonia to neutralize gave a yellow precipitate, which was collected and washed with water. The crude product dissolved in ethanol was purified by filtration on silica gel (60–100 mesh, ethanol). The principal yellow band was collected. Then, evaporation of the solution gave yellow crystals. Yield 0.54 g, 70%. (Found: C, 72.9; H, 3.9; N, 17.7. Calc. for C19H12N4O: C, 73.1; H, 3.8; N, 17.9%.) IR data (KBr, cmy1): 3400–2600s (br), 1623m, 1588m, 1560m, 1510s, 1475s, 1398s, 1356m, 1293m, 1257s, 807s, 737s. lmax/nm (´/My1 cmy1) (CH3CN) 327 (39 200), 273 (46 600), 237 (36 300). 1H NMR (ppm, DMSO-d6): 13.94 (s, 1H), 12.75 (s, 1H), 9.06 (d, 2H, Js4.4 Hz), 8.94 (d, 2H, Js8.1), 8.20 (d, 1H, Js8.4), 7.87 (q, 2H), 7.42 (t, 1H), 7.13–7.08 (m, 2H). MS (FAB): 313, [Mq1]q. 2.2.2. HNAIP This compound was prepared by a similar procedure to that for HPIP; 2-hydroxy-1-naphthylaldehyde (0.60 g, 3.5 mmol) was used instead of salicylaldehyde. Yield 0.68 g, 75%. (Found: C, 72.7; H, 4.4; N, 14.9. Calc. for C23H14N4OPH2O: C, 72.6; H, 4.2; N, 14.7%.) IR data (KBr, cmy1): 3400–2600s (br), 1623s, 1567m, 1546m, 1503m, 1461m, 1398s, 1356s, 1243m, 807s, 737s. lmax/nm (´/My1
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cmy1) (CH3CN) 357 (24 600), 304 (31 800), 243 (53 600). 1H NMR (ppm, DMSO-d6): 13.82 (s, 1H), 10.64 (s, 1H), 9.05 (d, 2H, Js4.2 Hz), 8.91 (d, 2H, Js8.1), 8.13 (d, 1H, Js7.2), 7.99 (d, 1H, Js8.9), 7.92 (d, 1H, Js7.5), 7.83 (q, 2H), 7.48 (t, 1H), 7.38 (d, 1H, Js8.7), 7.37(t, 1H). MS (FAB): 363, [Mq1]q.
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of 1,10-phenanthroline-5,6-dione and salicylaldehyde or 2hydroxy-1-naphthylaldehyde in the presence of ammonium acetate and glacial acid. Their structures were confirmed by elemental analyses, mass and 1H NMR spectroscopies. The 1 H NMR spectra of the two ligands display a broad peak at 13.92 for HPIP and 13.82 ppm for HNAIP, respectively, which can be assigned to the peak of N–H from the imidazole ring [15,16]. The phenolic protons of HPIP and HNAIP appear at 12.73 and 10.64 ppm, respectively. These downshifts, compared with the other phenolic protons not involving an intramolecular hydrogen bond [30], can be attributed to the formation of an intramolecular hydrogen bond with the nitrogen atom of the imidazole ring [31,32]. This phenomenon is also identified by IR spectra in the solid state. The IR spectra display a broad band in the range 3400 to 2600 cmy1, which can be assigned to the stretching vibration of O–H involving the intramolecular hydrogen bond. This kind of intramolecular hydrogen bond has a great effect on the properties of the ligand. It has important consequences not only for the ligating ability of the ligand, but also for stabilizing the chelating conformation of the appropriate rotor to preorganize the phenyl ring planar to the central heterocyclic ring. These have been illustrated by crystal structure in a few analogous compounds containing pyridyl– or benzothiazole–phenol ligands [33–37]. In complexes 1 and 2, the proton on the nitrogen atom of the imidazole ring was not observed, probably because the proton exchanged quickly between the two nitrogens of the imidazole ring, characteristic of an active proton [15,16]. The electronic absorption spectra of the two complexes in acetonitrile, not different from those in water, are characterized by an intense ligand-centered transition in the UV and a metal-to-ligand charge transfer (MLCT) transition in the visible region (Table 1). The lowest-energy absorption bands at 458 and 456 nm for 1 and 2, respectively, are assigned to the MLCT transition. In the UV region, the intense, fairly sharp bands at 284 nm for 1 and 285 nm for 2 are attributed to the intraligand p–pU transitions by comparison with the spectrum of [Ru(bpy)3]2q [38]. The shoulder at 328 nm for 1 may be assigned to the HPIP ligand p–pU transition according to the absorption spectra of the ligand. Little variation in the energy of the MLCT bands with the annelation of a phenyl moiety to the HPIP ligand was observed. Similar phenomena have also been observed between [Ru(bpy)3]2q and [Ru(bpy)2dppz]2q [39].
2.2.3. [Ru(bpy)2(HPIP)](PF6)2PH2O (1) A mixture of cis-[Ru(bpy)2Cl2]P2H2O (0.261 g, 0.5 mmol), HPIP (0.156 g, 0.5 mmol), ethanol (20 ml), and water (10 ml) was refluxed under argon for 3 h to give a clear red solution. Upon cooling, the solution was then treated with saturated aqueous solution of NH4PF6 and gave a red precipitate. The crude product was purified by column chromatography on alumina with CH3CN–toluene (1:1, v:v) as an eluent. The mainly red band was collected. The solvent was removed under reduced pressure and red crystals were obtained. Yield 0.33 g, 66%. (Found: C, 45.5; H, 3.3; N, 10.6. Calc. for C39H28F12N8OP2RuPH2O: C, 45.3; H, 2.9; N, 10.8%.) 1H NMR (ppm, CD3CN): 12.30 (s, 1H), 9.07 (d, 2H, Js8.3 Hz), 8.60 (d, 2H, Js8.3), 8.56 (d, 2H, Js8.2), 8.19 (d, 1H, Js8.2), 8.16 (d, 2H), 8.07 (d, 2H), 8.03 (d, 2H), 7.92 (d, 2H, Js5.7), 7.81 (q, 2H), 7.66 (d, 2H, Js5.7), 7.51 (t, 2H), 7.44 (t, 1H), 7.27 (t, 2H), 7.14 (d, 1H, Js7.8), 7.12 (d, 1H). MS (FAB): m/z 871 ([MH2qqPF6y]q, 16%), 725 (M2q, 26). 2.2.4. [Ru(bpy)2(HNAIP)](PF6)2P2H2O (2) This complex was obtained by a similar procedure to that described for complex 1. Yield 0.38 g, 71%. (Found: C, 47.0; H, 3.4; N, 10.3. Calc. for C43H30F12N8OP2RuP2H2O: C, 46.9; H, 3.1; N, 10.2%.) 1H NMR (ppm, CD3CN): 10.46 (s, 1H), 9.12 (d, 2H, Js8.2 Hz), 8.58 (d, 2H, Js8.2), 8.53 (d, 2H, Js8.2), 8.13 (t, 2H), 8.01 (t, 2H), 7.94 (d, 2H), 7.92 (d, 2H), 7.90 (d, 1H), 7.85 (d, 2H, Js8.8), 7.71 (m, 4H), 7.49 (t, 2H), 7.43 (t, 1H), 7.34 (d, 2H, Js8.8), 7.25 (t, 2H). MS (FAB): m/z 920 ([M2qqPF6y]q, 22%), 775 (M2q, 100). 3. Results and discussion 3.1. Syntheses and characterization The ligands HPIP and HNAIP were prepared by a method similar to that described by Steck and Day [29], condensation Table 1 Electrochemical and UV–vis spectroscopic data Complex
Ru(bpy)2HPIP2q Ru(bpy)2HNAIP2q a b
Oxidation a
Reduction a E1/2(DEp, mV)
Absorption b
E1/2 (DEp, mV)
1
2
3
lmax/nm (10y4 ´/My1 cmy1)
1.29(60) 1.32(85)
y0.98(irr)
1.40(56) 1.43(58)
1.62(58) 1.62(56)
458(1.22), 3.28(sh), 284(6.25), 254(2.07) 456(1.26), 285(6.05), 253(3.63)
Redox potentials were quoted vs. SCE in 0.1 M TBAH–CH3CN. Scan rates200 m V sy1. Measured in CH3CN.
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Each complex displays well one oxidation and two reduction waves in the sweep range from y1.85 to q1.70 V. A small poorly shaped reduction wave appeared at ca. 0.97 V for complex 1. The electrochemical behavior of ruthenium(II) polypyridyl complex has been rationalized in terms of a metal-based oxidation and a series of reductions, which are ligand based and occur in a stepwise manner for each pU system [40]. As expected, the oxidation potential of complex 2 is 30 mV more positive than that of 1 (Table 1). The annelation of a phenyl ring to the HPIP moiety expands the p delocalization and thus decreases the s donor capacity of
HNAIP, which leads to a decrease of the electron density on the Ru ion and in turn stabilizes the metal dp orbital [41]. As a result, the oxidation potential shifts more positively. The first reduction, which was usually controlled by the ligand having the most stable lowest unoccupied molecular orbital (LUMO) [40], is proposed to be for the HPIP or HNAIP and appears irreversible, making a comparison between the two complexes difficult. The later two successive reversible reductions are characteristic of the two bpy ligands [38,40]. 3.2. DNA-binding studies 3.2.1. Electronic absorption titration The binding of intercalative molecular ligand to DNA has been well characterized classically through absorption titrations, following the hypochromism and red shift associated with the binding of the colored complex to the helix, due to the intercalative mode involving a strong stacking interaction between an aromatic chromophore and the DNA base pairs. The magnitudes of the hypochromism and red shift were commonly found to depend on the strength of the intercalative interaction [22]. The absorption spectra of complexes 1 and 2 in the absence and presence of CT-DNA (at a constant concentration of complexes) are given in Fig. 1. As the DNA concentration is increased, the MLCT transition bands of complexes 1 at 458 and 2 at 456 nm exhibit hypochromism of about 24.5 and 14.6%, and bathochromism of about 6 and 2 nm, respectively. These spectral characteristics may suggest a mode of binding that involves a stacking interaction between the aromatic chromophore and the DNA base pairs. In order to compare quantitatively the binding strength of the two complexes, the intrinsic binding constants K of the two complexes with CT-DNA were determined according to the following equation [42] through a plot of [DNA]/ (´ay´f) versus [DNA]. [DNA]/(´ay´ f)s[DNA]/(´ 0y´ f)q1/(K(´ 0y´ f))
Fig. 1. Absorption spectra of complexes 1 (a) and 2 (b) in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2) in the presence of increasing amounts of CT-DNA. ([DNA]s0–200.0 mM, [Ru]s20 mM).
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where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficients ´a, ´f, and ´0 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. In plots of [DNA]/(´ay´f) versus [DNA], K is given by the ratio of slope to intercept. Intrinsic binding constants K of complexes 1 and 2 of about (6.5"0.3)=105 My1 and (8.3"0.4)=104 My1, respectively, were obtained from the decay of the absorbance. For comparison, the intrinsic binding constant K of [Ru(bpy)2PIP]2q with DNA was also determined to be (4.7"0.2)=105 My1. These results indicate that complex 1 binds more strongly to CT-DNA than complex 2. The binding strength of complex 1 to CT-DNA is comparable to that of [Ru(NH3)4dppz]2q (1.24=105 My1) [43], but is not as strong as that of [Ru(bpy)2dppz]2q (4.9=106 My1) [44] or [Ru(IP)2dppz]2q (2.1=107 My1) [19].
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3.2.2. Luminescence titration The complexes 1 and 2 can emit luminescence in Tris buffer at ambient temperature under experimental conditions similar to those for [Ru(bpy)3]2q, with maxima at 610 and 607 nm, respectively. Despite the structural similarity of the two complexes, their interactions with double-strand CTDNA are obviously different, as revealed by the luminescence behavior in the presence of DNA. The results of the emission titrations for the two complexes with DNA are illustrated by the titration curves in Fig. 2. Solutions of complex 1 exhibit an obvious enhancement in emission intensity when DNA is added, and the enhancement is around 2.7 times larger than that of in the absence of DNA. However, in complex 2, the emission intensity, which approaches saturation with an overall gain of ca. 1.6, shows only a lesser enhancement in similar conditions. This implies that the HPIP ligand inserts more deeply and strongly than HNAIP.
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about the binding of the two complexes with DNA. As illustrated in Fig. 3, in the absence of DNA, complexes 1 and 2 were efficiently quenched by [Fe(CN)6]4y. However, on addition of DNA, the Stern–Volmer plots changed drastically. The efficiency of [Fe(CN)6]4y quenching of ruthenium complexes bound to DNA is decreased relative to that of the free ruthenium complexes. This may be because the bound [Ru(bpy)2L]2q (LsHPIP or HNAIP) cations are protected from the anionic water-bound quencher by the array of negative charges along the DNA phosphate backbone [3,45]. The curvature reflects different degrees of protection or relative accessibility of bound cations. From the results, complex 1 is more protected from the solvated anion of [Fe(CN)6]4y than 2, which is consistent with the results observed by electronic absorption and emission titrations.
Fig. 2. Plots of relative emission intensity vs. [DNA]/[Ru] ratio for complexes 1 (j) and 2 (d) in 5 mM Tris–HCl and 50 mM NaCl buffer at pH 7.2. ([Ru]s4 mM).
3.2.4. Viscosity measurements To further clarify the interactions between the two complexes and DNA, viscosity measurements were carried out. Photophysical probes generally provide necessary, but not sufficient, clues to support an intercalation binding model. Hydrodynamic measurements that are sensitive to length increases (i.e. viscosity, sedimentation, etc.) are regarded as the least ambiguous and the most critical tests of binding model in solution in the absence of crystallographic structural data [4,46]. A classical intercalation model results in lengthening of the DNA helix as base pairs are separated to accommodate the binding ligand, leading to an increase of DNA viscosity. However, a partial and/or non-classical intercalation of ligand may bend (or kink) the DNA helix, resulting in a decrease of its effective length and, concomitantly, its viscosity [4]. The effects of the complexes 1 and 2, together with [Ru(bpy)3]2q and [Ru(bpy)2dppz]2q, on the viscosity of rod-like DNA are depicted in Fig. 4. On increasing the amount of complex 1, the relative viscosity of DNA increases steadily, which is similar to the behavior of the complex [Ru(bpy)2dppz]2q. At low binding ratios, complex 2 exerts essentially no effect on DNA viscosity, but upon further bind-
Fig. 3. Emission quenching curves of the complexes 1 (h), 1qDNA (j), 2 (s) and 2qDNA (d) with increasing concentration of quencher [Fe(CN)6]4y. [Ru]s4 mM, [DNA]/[Ru]s40.
Fig. 4. Effects of increasing amounts of the complexes 1 (j), 2 (d), [Ru(bpy)2dppz]2q (h) and [Ru(bpy)3]2q (l) on the relative viscosities of calf thymus DNA at 28.0("0.1)8C, [DNA]s0.5 mM and rs[Ru]/ [DNA].
3.2.3. Steady-state emission quenching Steady-state emission quenching experiments using [Fe(CN)6]4y as quencher may give further information
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ing of the compound to DNA, the DNA viscosity decreases. Such behavior is consistent with bending or kinking of the helix upon binding racemic ruthenium(II) complex to DNA [4,46]. For complex [Ru(bpy)3]2q, which has been well known to bind with DNA only through the electrostatic mode, there is no effect on the relative viscosity of DNA solution. The results then suggest that complex 1 binds with CT-DNA intercalatively and complex 2 may bind with a partial or nonclassical intercalative mode. For the mixed-ligand complexes, the tendency of each of the ligands to intercalate may be compared. For the series [Ru(bpy)2PIP]2q, [Ru(bpy)2HPIP]2q and [Ru(bpy)2HNAIP]2q, where the ancillary bpy ligands are kept constant, the binding constants increase in the order HNAIP-PIP-HPIP. This variation likely reflects the different abilities of the ligands to stack and overlap well with the base pairs. Although the crystal structures of these complexes are unavailable at present, the structure of PIP ligand has been illustrated by single-crystal X-ray diffraction, in which the phenyl ring is almost coplanar with the imidazole ring [47]. We speculate that the ortho phenolic group of HPIP would be closely coplanar with the imidazole ring due to the formation of an intramolecular hydrogen bond with the nitrogen atom of imidazole ring, which have also been seen in the crystal structures of analogous compounds [33–37]. These results also suggest that the introduction of a phenolic group into the HPIP molecule may have some additional affinity to stabilize the binding of the complexes to DNA. Although the larger coplanar area of the HNAIP ligand is expected, complex 2 has the lowest affinity to CT-DNA. It is likely that the large naphthyl ring may lie out of the plane of the imidazole ring, resulting in the partial intercalation of the HNAIP ligand.
4. Conclusions Two new polypyridyl mixed-ligand ruthenium(II) complexes containing an intramolecular hydrogen-bond ligand and their DNA-binding properties have been demonstrated. In agreement with the spectra titrations and viscosity measurements, complex 1 binds to CT-DNA intercalatively via the HPIP ligand, while complex 2 binds with a partial intercalative mode. Complex 1 shows much high affinity to DNA than complex 2. In order to understand further the binding of these analogous complexes with DNA, efforts will be made to tune the properties of the complexes by introducing a substituent on the HPIP ligand or changing the nature of the ancillary ligands.
5. Abbreviations Bpy Dppz
2,29-bipyridine dipyrido[3,2-a:29,39-c]phenazine
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HNAIP HPIP IL LUMO MLCT Phen PIP SCE
2-(2-hydroxy-1-naphthyl) imidazo[4,5-f][1,10]phenanthroline 2-(2-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline intraligand lowest unoccupied molecular orbital metal-to-ligand charge transfer 1,10-phenanthroline 2-phenylimidazo[4,5-f][1,10]phenanthroline saturated calomel electrode
Acknowledgements We are grateful to the NNSF of China, the State Key Laboratory of Bio-organic and Natural Products Chemistry in Shanghai Institute of Organic Chemistry, the State Key Laboratory of Coordination Chemistry in Nanjing University and the NSF of Guangdong Province for their financial support.
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