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Electronic effects on the interactions of complexes [Ru(phen)2(p-L)]2+ (L=MOPIP, HPIP, and NPIP) with DNA
Inorganica Chimica Acta 357 (2004) 285–293 www.elsevier.com/locate/ica
Electronic effects on the interactions of complexes [Ru(phen)2(p-L)]2þ (L ¼ MOPIP, HPIP, and NPIP) with DNA Jie Liu, Wen J. Mei, Li J. Lin, Kang C. Zheng *, Hui Chao, Feng C. Yun, Liang N. Ji
*
School of Chemistry and Chemical Engineering, Zhongshan University, Guangzhou 510275, PR China Received 4 July 2003; accepted 12 July 2003
Abstract In order to explore the electronic effects of Ru(II) complexes binding to DNA, a series of Ru(II) complexes [Ru(phen)2 (pMOPIP)]2þ (1), [Ru(phen)2 (p-HPIP)]2þ (2), and [Ru(phen)2 (p-NPIP)]2þ (3) were synthesized and characterized by elementary, 1 H NMR, and ES-MS analysis. The binding properties of these complexes to CT-DNA were investigated with spectroscopic methods and viscosity experiments. Furthermore, the computations for these complexes applying the density functional theory (DFT) method have also been performed. The results show that all of these complexes can well bind to DNA in intercalation mode and DNA-binding affinity of these complexes is greatly influenced by electronic effects of intercalating ligands. The intrinsic binding constants for 1, 2, and 3 are 0.20, 0.69, and 1.56 105 M1 , respectively. This order is in accordance with that of the electronwithdrawing ability of substituent [–OR < –OH < –NO2 ]. Such a trend in electronic effects of Ru(II) complexes binding to DNA can be reasonably explained by the DFT calculations. Ó 2003 Elsevier B.V. All rights reserved. Keywords: Ru(II) complex; DNA; Intercalation mode; Density functional theory (DFT); Electronic effect
1. Introduction The interactions of transition metal complexes with DNA have attracted many attentions for many years, because of their potential utilities for DNA structure probes, DNA-dependent electron transfer (ET) probes, DNA footprinting and sequence-specific cleaving agents, etc. [1–12]. In general, Ru(II) complexes can bind to DNA with intercalating, groove binding, and/or electrostatic binding modes. However, the binding af-
Abbreviations: phen ¼ 1,10-phenanthroline; p-MOPIP ¼ 2-(4-methoxylphenyl)imidazo[4,5-f][1,10]phenanthroline; p-HPIP ¼ 2-(4-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline; p-NPIP ¼ 2-(4-nitrophenyl) imidazo[4,5-f][1,10]phenanthroline; phi ¼ 9,10-phenanthrenequinone diimine); dppz ¼ dipyridophenazine; ip ¼ imidazole[4,5-f][1,10] phenanthroline. * Corresponding authors. Tel.: +862084110696; fax: +862084035497. E-mail addresses: [email protected] (K.C. Zheng), cesjln@zsu. edu.cn (L.N. Ji). 0020-1693/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0020-1693(03)00478-X
finity and binding sequence depend on not only the conformations of DNA, but also the structures of Ru(II) complexes and salts [13–16]. In particular, the structures of intercalating ligands play a very important role in the DNA-binding of complexes. It has been found that 2-phenylimidazo[4,5-f ][1,10]-phenanthroline (pip) and its substituted derivates are excellent intercalating ligands in DNA-binding [17–20]. Factors affecting the interactions of Ru(II) complexes with DNA have been experimentally studied and effects of planarity [20] and intramolecular hydrogen bond of intercalating ligands have also been reported [19]. However, studies on the electronic effects of Ru(II) complexes have been still untapped, and it is very important and significant to understand the electronic effects and modify substituents in order to design new complexes applied to the field of DNA-probes. In addition to the experimental studies, transition metal complexes, especially Ru(II) polypyridine complexes have attracted more and more attentions from theoretical chemists. Recently, many computations
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applying the density functional theory (DFT) method [21–24] on transition metal complexes have been reported [25–34]. Rillema and co-workers [25] suggested that the HOMO and LUMO distributions for Ru(II) two ring diimine complex cations from the DFT calculations support the idea that the lowest energy transition is a metal-to-ligand charge transfer and that the LUMO for the mixed ligand complexes is mainly located on the ligand with the lowest LUMO energy in the corresponding complex [RuL3 ]2þ . Kurita and Kobayashi [26] further reported density functional MO calculations for stacked DNA base-pairs with backbones. We have also reported the studies on the disubstitution effects, electron structures and related properties in some Ru(II) polypyridyl complexes with the DFT method [30–34]. These direct theoretical efforts on the level of molecular electronic structures of the complexes are very significant in guiding experimental works, and quantum chemical computations can offer important information on the electronic structures of complexes and DNA base-pairs with backbones, and help us to understand the electronic effects on the interaction of Ru(II) complexes with DNA. In order to explore the electronic effects of Ru(II) complexes binding to DNA experimentally and theoretically, a series of Ru(II) complexes [Ru(phen)2 (pMOPIP)]2þ (1), [Ru(phen)2 (p-HPIP)]2þ (2), and [Ru(phen)2 (p-NPIP)]2þ (3) (see Scheme 1) have been synthesized and characterized by elementary, 1 H NMR, and ES-MS analysis. The binding properties of these complexes to CT-DNA were investigated with spectroscopic methods and viscosity experiments. Furthermore, the theoretical computations for these complexes applying the DFT method have also been performed.
2. Experimental 2.1. Chemicals CT-DNA was purchased from the Sino-American Biotechnology Company. All reagents and solvents were purchased commercially and used without further purification unless otherwise noted, and doubly distilled water was used to prepare buffers. Solutions of DNA in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2) gave a ratio of UV absorbance at 260 and 280 nm of ca. 1.8–1.9:1, indicating that the DNA was sufficiently free of protein [35]. The concentration of calf thymus DNA was determined spectrophotometrically using the molar absorptivity 6600 mol1 cm1 (260 nm) [36]. Stock solutions were stored at 4 °C and used in no more than four days. 2.2. Synthesis and characteristics [Ru(phen)2 Cl2 ] 2H2 O was prepared following the literature and determined to be the identical compound [37]. 2.2.1. p-MOPIP (1a) The ligand 2-(4-methoxylphenyl)imidazo[4,5-f ] [1,10]phenanthroline (p-MOPIP) was prepared using the similar method to that as the literatures [38,39] with some modifications. A solution of phenanthraquinone (0.26 g, 1.2 mmol), p-anisaldehyde (0.24 g, 1.8 mmol), and ammonium acetate (1.9 g, 25 mmol) in 10 cm3 glacial acetic acid was refluxed for 2 h. The cooled deep red solution was diluted with 25 cm3 water and neutralized with ammonium hydroxide. Then the mixture was filtered and the
N
N N
N
N
NH
Ru N
N
N
N
NH
N
N
Ru
OMe N
OH
N
N
[Ru(phen) 2 (p-HPIP)]2+ (2)
[Ru(phen)2 (p-MOPIP)]2+ (1)
N N
N
NH
N
N
Ru N
NO2
N
[Ru(phen)2 (p-NPIP)]2+ (3)
Scheme 1. Molecular structural schemes and computational models of Ru(II) complexes.
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precipitates were washed with water and acetone, then dried, and purified by chromatography over 60–80 mesh SiO2 using methanol as an eluent, yields: 0.35 g (85%, calculated from phenanthraquinone). Calculated for C20 H14 N4 O H2 O (%): C, 69.8; H, 4.68; N, 16.3; Found (%): C, 69.3; H, 4.66; N, 16.2. ES-MS (in MeOH): m=z 327.0. 2.2.2. p-HPIP (2a) p-HPIP was synthesized by the same method as above, but with phenanthraquinone (0.26 g, 1.2 mmol) and 4-hydroxybenzaldehyde (0.22 g, 1.8 mmol), yield: 0.32 g (81%). Calculated for C19 H12 N4 O H2 O (%): C, 69.1; H, 4.27; N, 17.0; Found (%): C, 68.8; H, 4.22; N, 16.4. ES-MS (in MeOH)): m=z 312.3. 2.2.3. p-NPIP (3a) p-NPIP was synthesized by the same method as above, but with phenanthraquinone (0.26 g, 1.2 mmol) and 4-nitrobenzaldehyde (0.27 g, 1.8 mmol), yield: 0.37 g (86%). Calculated for C19 H11 N5 O2 H2 O (%): C, 63.5; H, 3.65; N, 19.5; Found (%): C, 63.6; H, 3.71; N, 19.4. ES-MS: (in MeOH): m=z 341.8. 2.2.4. [Ru(phen)2 (p-MOPIP)](PF6 )2 2H2 O (1) [Ru(phen)2 (p-MOPIP)]2þ was synthesized as the literatures [39,40] with some modifications. [Ru(phen)2 Cl2 ] 2H2 O (0.090 g, 0.17 mmol) and 1a (0.058 g, 0.17 mmol) were added to 10 cm3 ethylene glycol. The mixture was refluxed for 2 h under an argon atmosphere. The cooled reaction mixture was diluted with water (20 cm3 ) and filtered to remove solid impurities. The complex was then separated from soluble impurities by precipitation with NH4 PF6 . The precipitated complex was dried, dissolved in a small amount of acetonitrile, and purified by chromatography over alumina oxide using MeCN–toluene (2:1, v/v) as an eluent, yield: 0.16 g (84%, calculated from [Ru(phen)2 Cl2 ] 2H2 O). 1 H NMR (DMSO-d6 , d ppm): 9.04 (2H, d); 8.75 (4H, d); 8.38 (4H, s); 8.27 (2H, d); 8.11 (2H, s); 8.07 (2H, d), 8.00 (2H, d); 7.79 (6H, m); 7.23 (2H, d); 3.89 (3H, s); ES-MS of the PF6 salt in MeCN: m=z 933.0 (calc: 932.8); 394.3 (calc: 393.9). Resolution of the peak 394.3 shows that the species is double charged and the isotopic distribution corresponds to the calculated one. For absorption UV–Vis in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2), kmax (e=104 M1 cm1 ): 263 nm (8.6), 455 nm (1.7). Uncorrected emission maximum in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2):590 nm. 2.2.5. [Ru(phen)2 (p-HPIP)](PF6 )2 2H2 O (2) [Ru(phen)2 (p-HPIP)]2þ was prepared following the same method as above but with 2a (0.056 g, 0.17 mmol); yield: 0.15 g (81%). 1 H NMR (DMSO-d6 , d ppm): 10.00 (1H, s); 9.06 (2H, d); 8.78 (4H, d); 8.39 (4H, s); 8.16 (2H, d); 8.15 (2H, d); 8.13 (2H, d); 8.00 (2H, d), 7.78 (6H, m);
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7.04 (2H, d); ES-MS of the PF6 salt in MeCN: m=z 919.0 (calc: 918.8); 773.3 (calc: 772.8); 387.3 (calc: 386.9). For absorption UV–Vis in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2), kmax (e=104 M1 cm1 ): 264(7.9), 456(1.7). Uncorrected emission maximum in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2): 588 nm. 2.2.6. [Ru(phen)2 (p-NPIP)](PF6 )2 2H2 O (3) [Ru(phen)2 (p-NPIP)]2þ was prepared following the same method as above but with 3a (0.061 g, 0.17 mmol); yield: 0.14 g (73%). 1 H NMR (DMSO-d6 , d ppm): 8.96 (2H, d); 8.77 (4H, d); 8.60 (2H, d); 8.38 (4H, s); 8.34 (2H, d); 8.12 (4H, 2d); 7.79 (6H, m), 7.65 (2H, m); ESMS of the PF6 salt in MeCN: m=z 947.9 (calc: 947.8); 802.3 (calc: 801.8); 401.8 (calc: 401.4). Resolution of the peak 401.8 shows that the species is double charged and the isotopic distribution corresponds to the calculated one. For absorption UV–Vis in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2), kmax (e=104 M1 cm1 ): 265 nm (6.1), 392 nm (1.8), 455 nm (1.8). Uncorrected emission maximum in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2): 589 nm. 2.3. Physical measurements Microanalysis was carried out on an Elementar Vario EL elemental analyser. Electrospray mass spectra (ESMS) were recorded on a LCQ system (Finnigan MAT, USA). The spray voltage, tube lens offset, capillary voltage, and capillary temperature were set at 4.50 kV, 30.00 V, 23.00 V, and 200 °C, respectively, and the quoted m=z values are for the major peaks in the isotope distribution. UV–Vis absorption spectra were recorded on a Shimadzu UVPC-3000 spectrophotometer and emission spectra on a Shimadzu RF-5000 spectrofluorophotometer. The intrinsic binding constants of Ru(II) complexes to DNA were obtained via electronic titration experiments. Equal small aliquots of DNA stock solution were added to both Ru(II) complex and reference solution to eliminate the effect of absorbance of complex. The binding constants Kb were calculated, according to the decay at MLCT absorption bands following Eq. (1) [41]: ½DNA ½DNA 1 ¼ þ ea ef eb ef Kb ðeb ef Þ
ð1Þ
where [DNA] is the concentration of DNA in base pairs. ea , ef , and eb are the apparent extinction coefficient (Aobsd =½M), the extinction coefficient for free ruthenium complex, and the extinction coefficient for the metal(M) complex in the fully bound form respectively. In plot of ½DNA=ðea ef Þ versus [DNA], Kb is given by the ratio of slope to intercept.
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Viscosity experiments were performed on an Ulbbelodhe viscometer, immersed in a thermostatted water-bath maintained at 30.0 0.1 °C. Data were presented as (g=g0 Þ1=3 versus the concentration of presence of [complex]/[DNA] [42]. Viscosity values were calculated from the observed flow time of DNAcontaining solutions (t > 100 s) corrected for the flow time of buffer alone (t0 ), g ¼ t t0 .
3. Theoretical section Each of the octahedral complexes [Ru(phen)2 (p-L)]2þ (L ¼ MOPIP, HPIP, and NPIP) forms from Ru(II) and one main ligand (L) or intercalated ligand and two called co-ligands(phen). There is not any symmetry in these complexes. The full geometry optimization computations were performed for these complexes applying the DFT-B3LYP method [21–24] and LanL2DZ basis set [43]. The structural models of the studied compounds were shown in Scheme 1 and the ground states of these complexes were regarded as a singlet state [44]. All computations were performed with the G98 quantum chemistry program-package [45]. In order to vividly depict the detail of the frontier molecular orbital interactions, the contour plots of some related frontier MOs of the complexes were drawn with the Molden v3.6 program [46] based on the obtained computational results.
4. Results and discussion 4.1. Electronic effects of [Ru(phen)2 L]2þ binding to DNA from experiments 4.1.1. Electronic absorption spectra and binding constants The electronic spectra are the most common ways to investigate the interactions of complexes with DNA. In the presence of DNA, the electronic absorption spectra of Ru(II) complexes are usually broadened and undergone apparent hypochromism and red shift [47,48]. The electronic spectra of Ru(II) complexes 1, 2, and 3 in the absence and in the presence of CT-DNA in 5
mM Tris–HCl and 50 mM NaCl buffer were listed in Table 1. The electronic absorption spectra of Ru(II) complex 1 were characterized by a metal to ligand charge-transfer (MLCT) transition band and intraligand (IL) absorption band at about 456 and 264 nm, respectively. For complex 2, the MLCT and IL absorption bands also exhibit at about 456 and 264 nm, respectively. For complex 3, the MLCT absorption band appears at about 441 nm, accompany with a shoulder at about 365 nm, and the IL absorption appears at 264 nm. Upon the addition of CTDNA, both the MLCT and IL absorptions of these Ru(II) complexes exhibit obviously hypochromisms (H) and red shifts (Dk), as shown in Table 1. The intrinsic binding constants for complexes 1, 2, and 3 were calculated according to the Eq. (1) at the MLCT absorption bands, and the data were listed in Table 1. These values are comparable to those observed for [Ru(phen)2 phi]2þ (4.7 104 M1 ) [49], but smaller than those observed for [Ru(bpy)2 dppz]2þ (>106 M1 ) [50] and [Ru(ip)2 dppz]2þ (2.1 107 M1 ) [51]. These results show that the DNA binding affinity of these complexes closely correlates to the electronic effects of their intercalating ligands. The electron-withdrawing substituent (–NO2 ) on intercalating ligand can improve the DNA-binding affinity of the complex, whereas the electron-pushing substituent (–OCH3 or –OH) is reverse. This trend suggests us that binding strength of the complex to DNA can be effectively controlled by the substituents. 4.1.2. Emission spectra To further clarify the interaction of these Ru(II) complexes with DNA, the emission spectra of 1, 2, and 3 have been measured in the absence and in the presence of CT-DNA, the results are shown in Fig. 1. When excited at the MLCT absorption, all of 1, 2, and 3 emit weak fluorescence in the range of 500–700 nm at room temperature with the maximum (I0 ) at ca. 590, 588, and 589 nm, respectively. However, when calf thymus DNA was added into the solution, the enhanced emission (I) for all of these complexes was observed (Fig. 1). It can be explained that the hydrophobic environment inside the DNA helix reduces the accessibility of water
Table 1 Electronic spectra, hypochromism (H%), and DNA-binding constants of Ru(II) complexes 1, 2, and 3 Compound
kmax (free) (nm)
k0max (bound) (nm)
Dk (nm)
H (%)
Kb (105 M1 )
1 (–OCH3 ) 2 (–OH) 3 (–NO2 )
456 264 456 264 456 264
464 266 466 266 465 266
8 2 10 2 9 2
)11 )34 )16 )34 )15 )20
0.20
[Ru] ¼ 20 lM, [DNA] ¼ 2.5 mM.
0.69 1.56
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16
289
10
14 8
Intensity (a.u.)
Intensity (a.u.)
12 10 8 6 4
6 4 2
2 0 500
0 500
550
600
650
700
750
550
600
650
700
Wavelength / nm
Wavelength / nm 1
2 1.0
Intensity (a.u.)
0.8 0.6 0.4 0.2 0.0 500
550
600
650
700
750
Wavelength / nm 3 Fig. 1. Emission spectra of 1, 2 and 3 in the absence and in the presence of increasing amount of CT-DNA in 5 mM Tris–HCl and 50 mM NaCl buffer (pH ¼ 7.2). [Ru] ¼ 10 lM, [DNA] ¼ 2.5 mM.
4.1.3. Viscosity properties The viscosity experiments were performed to investigate the binding mode of these complexes to DNA. Optical photophysical probes provide necessary but not sufficient clues to support a binding mode. Viscosity experiment is considered as one of the least ambiguous
5
4
3
I/I 0
molecules to the complex and the complex mobility is restricted at the binding site, leading to decrease of the vibrational modes of relaxation [52,53]. The emission intensity for complex 1 increases steadily to around 1.9 times than the original and saturates at a [DNA]/[Ru] ratio of ca. 13:1 (Fig. 2). However, for complexes 2 and 3, the emission intensities increase by about 2.3 and 4.5 times under the same conditions, respectively. These data are in agreement with that of previously reported within error [39]. These results also imply that the DNA binding affinities (A) of these complexes increase in the sequence: Að1Þ < Að2Þ < Að3Þ, indicating that the electronwithdrawing substituent on intercalating ligand can stabilize the DNA-complex system more strongly than the electron-pushing substituent.
2
1 0
4
8
12
[DNA]/[Ru] Fig. 2. Plots of relative emission intensity vs. [DNA]/[Ru] ratio for complexes 1 (j), 2 (d), and 3 (N). [Ru] ¼ 10 lM.
and the most critical tests of a binding mode in solution in the absence of crystallographic structure data [54]. In general, a classical intercalation mode may increase the relative viscosity of DNA, because the binding ligand
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Fig. 3. Effect of increasing amount of [Ru(bpy)3 ]2þ (.), 1 (j), 2 (d), and 3 (N) on the relative viscosity of CT-DNA at 30.0 0.1 °C. [DNA] ¼ 0.5 mM.
will separate base pairs of double helix DNA, and thus lengthen the DNA helix. On the contrary, a partial and/ or non-classical intercalation of complex will reduce the relative viscosity of DNA, since the binding ligand may bend (or kink) the DNA helix and reduce its effective length [54]. The relative viscosities of rod like CT-DNA in the presence of complex 1, 2, and 3, as well as [Ru(bpy)3 ]2þ , respectively, are shown in Fig. 3. The relative viscosity of CT-DNA is almost kept unchanged in the presence of [Ru(bpy)3 ]2þ , which is in agreement with an electrostatic binding mode. However, the relative viscosity of CTDNA increases apparently in the presence of complex 1, and more in the presence of complex 2 and the most in the presence of complex 3. These results indicate unambiguously that complexes 1, 2, and 3 behave as intercalators of DNA base pairs. 4.2. Theoretical explanation on the electronic effects of complexes binding to DNA The above-mentioned trend in the electronic effects of complexes binding to DNA can be well explained by our theoretical computations with DFT method. Some frontier molecular orbital energies, and the schematic diagram of energies and related 1 MLCT transitions of [Ru(phen)2 (p-L)]2þ , as well as molecular orbital contour
of [Ru(phen)2 (p-L)]2þ based on the computed results were given in Table 2, Fig. 4 and Fig. 5, respectively. As well-known, there are p–p interactions in the DNA-binding of these complexes in intercalation mode. According to the frontier molecular orbital theory [55– 57], for a reaction controlled by orbital interactions between reactant molecules, a higher HOMO energy of one reactant molecule and a lower LUMO energy of other one are more advantageous to the reaction between the two molecules, because electrons more easily transfer from the HOMO of one reactant to the LUMO of another one in the orbital interaction. A simple calculation model and computed results by the DFT method for stacked DNA base-pairs with backbones have been reported by Kruita and Kobayashi [26]. It should be a perfect simplified approximation model for DNA, and thus should be useful and feasible for discussion on some trends in interaction between the complexes and DNA. The reported HOMO and NHOMO (i.e., HOMO 1) energies of DNA section model with base pairs are much higher ()1.27 and )1.33 eV) [26] than our calculated LUMO and NLUMO (i.e., LUMO + 1) energies ()7.2 eV) of the complexes [Ru(phen)2 (p-L)]2þ (L ¼ MOPIP, HPIP, and NPIP). We believe that such a trend in the relative energies to be kept in our DNA system, because the attraction of metal complex cations with high positive charges for electrons in MOs is much stronger than that of various DNA, and thus the electron must easily transfer from the HOMO of base pairs of DNA to the LUMO (or other virtual orbitals near LUMO) of the complex intercalating to DNA. From Table 2 and Fig. 4, we can see that eL ð1Þ > eL ð2Þ > eL ð3Þ in this series of complexes and this trend can be attributed to the electronic effect due to the order of the electron-withdrawing ability being –OR < – OH < –NO2 . This energy order suggests that the trend in DNA-binding constants (Kb ) should be Kb ð1Þ < Kb ð2Þ < Kb (3). It is satisfactorily in accordance with above experimental results, in which the DNA-binding constants (Kb ) are 0.2, 0.69, and 1.56 ( 105 M1 ) for the complexes 1, 2 and 3, respectively. From Fig. 5, we can also see that the max (k) singlet metal to ligand charge-transfer (1 MLCT) bands of the three complexes should correspond to the electron transitions from their NHOMOs to LUMOs, respec-
Table 2 Some frontier molecular orbital energies (ei /a.u.) and the related energy differences (Dei /a.u.) of Ru(II) complexes Compound
Occa
Occ
Occ
HOMO
LUMO
Virb
Vir
DeLH c
DeLNH
1 2 3
)0.3937 )0.3965 )0.4061
)0.3926 )0.3956 )0.4038
)0.3906 )0.3921 )0.3997
)0.3464 )0.3546 )0.3934
)0.2656 )0.2668 )0.2736
)0.2622 )0.2634 )0.2696
)0.2589 )0.2601 )0.2693
0.0808 0.0878 0.1198
0.1250 0.1253 0.1261
a
Occ: occupied molecular orbital; HOMO (or H): the highest Occ. Vir: virtual (unoccupied) molecular orbital; LUMO: the lowest Vir. c DeLH : the energy difference between LUMO and HOMO; DeLNH : the energy difference between LUMO and NHOMO. b
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ε/a.u. NL
-0.26
NL L
L
NL L
-0.28 -0.30 -0.32 -0.34
H
H
-0.36 -0.38
H NH
-0.40
NH
NH
-0.42 1.1
1.2
1
1.3
1.4
2
1.5
1.6
1.7
3
Fig. 4. Schematic diagram of some frontier MO energies and related 1 MLCT transitions of [Ru(phen)2 (p-L)]2þ .
291
tively, according to the molecular orbital components of the complexes. This assignation can be preliminarily firmed by the spectral experiments. According to the approximate correlation of reverse ratio of the difference between the LUMO and the HOMO (or NHOMO) energies (DeLH ) to experimental wave length (k), using the parent complex [Ru(bpy)3 ]2þ as a standard sample (experimental k ¼ 452 nm, DeLH ¼ 0:1239 a.u. [31]) and DeLH data of the complexes in Table 2, the computed max (k) 1 MLCT absorption spectra of this series of Ru(II) polypyridyl complexes are 448 nm near experimental wave length 456 nm. Table 1 also contains the wavelengths of complexes binding to calf thymus DNA (the third column). Since in absorption and emission spectra of the Ru(II) complexes, the corresponding energy change between the presence and absence of DNA is small, and no special
Fig. 5. Some related frontier MO contour plots of complexes [Ru(phen)2 (p-L)]2þ .
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pattern changes in the absorption or emission spectra of Ru(II) complexes in the presence of DNA have been detected, except an increase in the luminescence intensity and hypochromism (H%) in the absorption intensity, we consider that there is not a greater effect on the HOMO and LUMO and DeLH for the complexes binding to DNA. It further suggests that the interaction between the series of complexes and DNA should be a weak one, so that the max (k) electron transition band in these Ru(II) complexes binding to DNA can be still assigned to 1 MLCT. At the present time, we are not yet able to calculate the frontier molecular orbital energies of the whole super-molecule system formed from these complexes and DNA, therefore, the DFT studies on some trends in the electronic structures and related properties for such a kind of complexes should be significant.
5. Conclusion The electronic effects of a series of Ru(II) complexes [Ru(phen)2 (p-L)] (L ¼ MOPIP, HPIP, and NPIP) binding to DNA have been investigated with spectroscopic methods, viscosity measurements, and the DFT calculations. The experimental results show that all complexes 1, 2, and 3 can bind to DNA tightly in intercalation mode, and the DNA-binding affinity of these complexes is greatly influenced by electronic effects from their substituent properties. The intrinsic binding constants for 1, 2, and 3 are 0.20, 0.69, and 1.56 105 M1 , respectively, and this order is in accordance with that of the electron-withdrawing ability of substituent [–OR < –OH < –NO2 ]. Such a trend of electronic effects of Ru(II) complexes binding to DNA can be reasonably explained by the DFT calculations.
Acknowledgements We are grateful to the National Nature Science Foundation of China, the Nature Science Foundation of Guangdong Province, the Coordination Chemistry State Key Laboratory in Nanjing University, the State Key Laboratory of Bio-Organic and Natural Products Chemistry of Shanghai Institute of Organic Chemistry, and the Research Fund of Royal Society of Chemistry UK for their financial supports.
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Electronic effects on the interactions of complexes [Ru(phen)2(p-L)]2þ (L ¼ MOPIP, HPIP, and NPIP) with DNA Jie Liu, Wen J. Mei, Li J. Lin, Kang C. Zheng *, Hui Chao, Feng C. Yun, Liang N. Ji
*
School of Chemistry and Chemical Engineering, Zhongshan University, Guangzhou 510275, PR China Received 4 July 2003; accepted 12 July 2003
Abstract In order to explore the electronic effects of Ru(II) complexes binding to DNA, a series of Ru(II) complexes [Ru(phen)2 (pMOPIP)]2þ (1), [Ru(phen)2 (p-HPIP)]2þ (2), and [Ru(phen)2 (p-NPIP)]2þ (3) were synthesized and characterized by elementary, 1 H NMR, and ES-MS analysis. The binding properties of these complexes to CT-DNA were investigated with spectroscopic methods and viscosity experiments. Furthermore, the computations for these complexes applying the density functional theory (DFT) method have also been performed. The results show that all of these complexes can well bind to DNA in intercalation mode and DNA-binding affinity of these complexes is greatly influenced by electronic effects of intercalating ligands. The intrinsic binding constants for 1, 2, and 3 are 0.20, 0.69, and 1.56 105 M1 , respectively. This order is in accordance with that of the electronwithdrawing ability of substituent [–OR < –OH < –NO2 ]. Such a trend in electronic effects of Ru(II) complexes binding to DNA can be reasonably explained by the DFT calculations. Ó 2003 Elsevier B.V. All rights reserved. Keywords: Ru(II) complex; DNA; Intercalation mode; Density functional theory (DFT); Electronic effect
1. Introduction The interactions of transition metal complexes with DNA have attracted many attentions for many years, because of their potential utilities for DNA structure probes, DNA-dependent electron transfer (ET) probes, DNA footprinting and sequence-specific cleaving agents, etc. [1–12]. In general, Ru(II) complexes can bind to DNA with intercalating, groove binding, and/or electrostatic binding modes. However, the binding af-
Abbreviations: phen ¼ 1,10-phenanthroline; p-MOPIP ¼ 2-(4-methoxylphenyl)imidazo[4,5-f][1,10]phenanthroline; p-HPIP ¼ 2-(4-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline; p-NPIP ¼ 2-(4-nitrophenyl) imidazo[4,5-f][1,10]phenanthroline; phi ¼ 9,10-phenanthrenequinone diimine); dppz ¼ dipyridophenazine; ip ¼ imidazole[4,5-f][1,10] phenanthroline. * Corresponding authors. Tel.: +862084110696; fax: +862084035497. E-mail addresses: [email protected] (K.C. Zheng), cesjln@zsu. edu.cn (L.N. Ji). 0020-1693/$ - see front matter Ó 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0020-1693(03)00478-X
finity and binding sequence depend on not only the conformations of DNA, but also the structures of Ru(II) complexes and salts [13–16]. In particular, the structures of intercalating ligands play a very important role in the DNA-binding of complexes. It has been found that 2-phenylimidazo[4,5-f ][1,10]-phenanthroline (pip) and its substituted derivates are excellent intercalating ligands in DNA-binding [17–20]. Factors affecting the interactions of Ru(II) complexes with DNA have been experimentally studied and effects of planarity [20] and intramolecular hydrogen bond of intercalating ligands have also been reported [19]. However, studies on the electronic effects of Ru(II) complexes have been still untapped, and it is very important and significant to understand the electronic effects and modify substituents in order to design new complexes applied to the field of DNA-probes. In addition to the experimental studies, transition metal complexes, especially Ru(II) polypyridine complexes have attracted more and more attentions from theoretical chemists. Recently, many computations
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applying the density functional theory (DFT) method [21–24] on transition metal complexes have been reported [25–34]. Rillema and co-workers [25] suggested that the HOMO and LUMO distributions for Ru(II) two ring diimine complex cations from the DFT calculations support the idea that the lowest energy transition is a metal-to-ligand charge transfer and that the LUMO for the mixed ligand complexes is mainly located on the ligand with the lowest LUMO energy in the corresponding complex [RuL3 ]2þ . Kurita and Kobayashi [26] further reported density functional MO calculations for stacked DNA base-pairs with backbones. We have also reported the studies on the disubstitution effects, electron structures and related properties in some Ru(II) polypyridyl complexes with the DFT method [30–34]. These direct theoretical efforts on the level of molecular electronic structures of the complexes are very significant in guiding experimental works, and quantum chemical computations can offer important information on the electronic structures of complexes and DNA base-pairs with backbones, and help us to understand the electronic effects on the interaction of Ru(II) complexes with DNA. In order to explore the electronic effects of Ru(II) complexes binding to DNA experimentally and theoretically, a series of Ru(II) complexes [Ru(phen)2 (pMOPIP)]2þ (1), [Ru(phen)2 (p-HPIP)]2þ (2), and [Ru(phen)2 (p-NPIP)]2þ (3) (see Scheme 1) have been synthesized and characterized by elementary, 1 H NMR, and ES-MS analysis. The binding properties of these complexes to CT-DNA were investigated with spectroscopic methods and viscosity experiments. Furthermore, the theoretical computations for these complexes applying the DFT method have also been performed.
2. Experimental 2.1. Chemicals CT-DNA was purchased from the Sino-American Biotechnology Company. All reagents and solvents were purchased commercially and used without further purification unless otherwise noted, and doubly distilled water was used to prepare buffers. Solutions of DNA in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2) gave a ratio of UV absorbance at 260 and 280 nm of ca. 1.8–1.9:1, indicating that the DNA was sufficiently free of protein [35]. The concentration of calf thymus DNA was determined spectrophotometrically using the molar absorptivity 6600 mol1 cm1 (260 nm) [36]. Stock solutions were stored at 4 °C and used in no more than four days. 2.2. Synthesis and characteristics [Ru(phen)2 Cl2 ] 2H2 O was prepared following the literature and determined to be the identical compound [37]. 2.2.1. p-MOPIP (1a) The ligand 2-(4-methoxylphenyl)imidazo[4,5-f ] [1,10]phenanthroline (p-MOPIP) was prepared using the similar method to that as the literatures [38,39] with some modifications. A solution of phenanthraquinone (0.26 g, 1.2 mmol), p-anisaldehyde (0.24 g, 1.8 mmol), and ammonium acetate (1.9 g, 25 mmol) in 10 cm3 glacial acetic acid was refluxed for 2 h. The cooled deep red solution was diluted with 25 cm3 water and neutralized with ammonium hydroxide. Then the mixture was filtered and the
N
N N
N
N
NH
Ru N
N
N
N
NH
N
N
Ru
OMe N
OH
N
N
[Ru(phen) 2 (p-HPIP)]2+ (2)
[Ru(phen)2 (p-MOPIP)]2+ (1)
N N
N
NH
N
N
Ru N
NO2
N
[Ru(phen)2 (p-NPIP)]2+ (3)
Scheme 1. Molecular structural schemes and computational models of Ru(II) complexes.
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precipitates were washed with water and acetone, then dried, and purified by chromatography over 60–80 mesh SiO2 using methanol as an eluent, yields: 0.35 g (85%, calculated from phenanthraquinone). Calculated for C20 H14 N4 O H2 O (%): C, 69.8; H, 4.68; N, 16.3; Found (%): C, 69.3; H, 4.66; N, 16.2. ES-MS (in MeOH): m=z 327.0. 2.2.2. p-HPIP (2a) p-HPIP was synthesized by the same method as above, but with phenanthraquinone (0.26 g, 1.2 mmol) and 4-hydroxybenzaldehyde (0.22 g, 1.8 mmol), yield: 0.32 g (81%). Calculated for C19 H12 N4 O H2 O (%): C, 69.1; H, 4.27; N, 17.0; Found (%): C, 68.8; H, 4.22; N, 16.4. ES-MS (in MeOH)): m=z 312.3. 2.2.3. p-NPIP (3a) p-NPIP was synthesized by the same method as above, but with phenanthraquinone (0.26 g, 1.2 mmol) and 4-nitrobenzaldehyde (0.27 g, 1.8 mmol), yield: 0.37 g (86%). Calculated for C19 H11 N5 O2 H2 O (%): C, 63.5; H, 3.65; N, 19.5; Found (%): C, 63.6; H, 3.71; N, 19.4. ES-MS: (in MeOH): m=z 341.8. 2.2.4. [Ru(phen)2 (p-MOPIP)](PF6 )2 2H2 O (1) [Ru(phen)2 (p-MOPIP)]2þ was synthesized as the literatures [39,40] with some modifications. [Ru(phen)2 Cl2 ] 2H2 O (0.090 g, 0.17 mmol) and 1a (0.058 g, 0.17 mmol) were added to 10 cm3 ethylene glycol. The mixture was refluxed for 2 h under an argon atmosphere. The cooled reaction mixture was diluted with water (20 cm3 ) and filtered to remove solid impurities. The complex was then separated from soluble impurities by precipitation with NH4 PF6 . The precipitated complex was dried, dissolved in a small amount of acetonitrile, and purified by chromatography over alumina oxide using MeCN–toluene (2:1, v/v) as an eluent, yield: 0.16 g (84%, calculated from [Ru(phen)2 Cl2 ] 2H2 O). 1 H NMR (DMSO-d6 , d ppm): 9.04 (2H, d); 8.75 (4H, d); 8.38 (4H, s); 8.27 (2H, d); 8.11 (2H, s); 8.07 (2H, d), 8.00 (2H, d); 7.79 (6H, m); 7.23 (2H, d); 3.89 (3H, s); ES-MS of the PF6 salt in MeCN: m=z 933.0 (calc: 932.8); 394.3 (calc: 393.9). Resolution of the peak 394.3 shows that the species is double charged and the isotopic distribution corresponds to the calculated one. For absorption UV–Vis in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2), kmax (e=104 M1 cm1 ): 263 nm (8.6), 455 nm (1.7). Uncorrected emission maximum in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2):590 nm. 2.2.5. [Ru(phen)2 (p-HPIP)](PF6 )2 2H2 O (2) [Ru(phen)2 (p-HPIP)]2þ was prepared following the same method as above but with 2a (0.056 g, 0.17 mmol); yield: 0.15 g (81%). 1 H NMR (DMSO-d6 , d ppm): 10.00 (1H, s); 9.06 (2H, d); 8.78 (4H, d); 8.39 (4H, s); 8.16 (2H, d); 8.15 (2H, d); 8.13 (2H, d); 8.00 (2H, d), 7.78 (6H, m);
287
7.04 (2H, d); ES-MS of the PF6 salt in MeCN: m=z 919.0 (calc: 918.8); 773.3 (calc: 772.8); 387.3 (calc: 386.9). For absorption UV–Vis in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2), kmax (e=104 M1 cm1 ): 264(7.9), 456(1.7). Uncorrected emission maximum in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2): 588 nm. 2.2.6. [Ru(phen)2 (p-NPIP)](PF6 )2 2H2 O (3) [Ru(phen)2 (p-NPIP)]2þ was prepared following the same method as above but with 3a (0.061 g, 0.17 mmol); yield: 0.14 g (73%). 1 H NMR (DMSO-d6 , d ppm): 8.96 (2H, d); 8.77 (4H, d); 8.60 (2H, d); 8.38 (4H, s); 8.34 (2H, d); 8.12 (4H, 2d); 7.79 (6H, m), 7.65 (2H, m); ESMS of the PF6 salt in MeCN: m=z 947.9 (calc: 947.8); 802.3 (calc: 801.8); 401.8 (calc: 401.4). Resolution of the peak 401.8 shows that the species is double charged and the isotopic distribution corresponds to the calculated one. For absorption UV–Vis in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2), kmax (e=104 M1 cm1 ): 265 nm (6.1), 392 nm (1.8), 455 nm (1.8). Uncorrected emission maximum in 5 mM Tris–HCl and 50 mM NaCl buffer (pH 7.2): 589 nm. 2.3. Physical measurements Microanalysis was carried out on an Elementar Vario EL elemental analyser. Electrospray mass spectra (ESMS) were recorded on a LCQ system (Finnigan MAT, USA). The spray voltage, tube lens offset, capillary voltage, and capillary temperature were set at 4.50 kV, 30.00 V, 23.00 V, and 200 °C, respectively, and the quoted m=z values are for the major peaks in the isotope distribution. UV–Vis absorption spectra were recorded on a Shimadzu UVPC-3000 spectrophotometer and emission spectra on a Shimadzu RF-5000 spectrofluorophotometer. The intrinsic binding constants of Ru(II) complexes to DNA were obtained via electronic titration experiments. Equal small aliquots of DNA stock solution were added to both Ru(II) complex and reference solution to eliminate the effect of absorbance of complex. The binding constants Kb were calculated, according to the decay at MLCT absorption bands following Eq. (1) [41]: ½DNA ½DNA 1 ¼ þ ea ef eb ef Kb ðeb ef Þ
ð1Þ
where [DNA] is the concentration of DNA in base pairs. ea , ef , and eb are the apparent extinction coefficient (Aobsd =½M), the extinction coefficient for free ruthenium complex, and the extinction coefficient for the metal(M) complex in the fully bound form respectively. In plot of ½DNA=ðea ef Þ versus [DNA], Kb is given by the ratio of slope to intercept.
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Viscosity experiments were performed on an Ulbbelodhe viscometer, immersed in a thermostatted water-bath maintained at 30.0 0.1 °C. Data were presented as (g=g0 Þ1=3 versus the concentration of presence of [complex]/[DNA] [42]. Viscosity values were calculated from the observed flow time of DNAcontaining solutions (t > 100 s) corrected for the flow time of buffer alone (t0 ), g ¼ t t0 .
3. Theoretical section Each of the octahedral complexes [Ru(phen)2 (p-L)]2þ (L ¼ MOPIP, HPIP, and NPIP) forms from Ru(II) and one main ligand (L) or intercalated ligand and two called co-ligands(phen). There is not any symmetry in these complexes. The full geometry optimization computations were performed for these complexes applying the DFT-B3LYP method [21–24] and LanL2DZ basis set [43]. The structural models of the studied compounds were shown in Scheme 1 and the ground states of these complexes were regarded as a singlet state [44]. All computations were performed with the G98 quantum chemistry program-package [45]. In order to vividly depict the detail of the frontier molecular orbital interactions, the contour plots of some related frontier MOs of the complexes were drawn with the Molden v3.6 program [46] based on the obtained computational results.
4. Results and discussion 4.1. Electronic effects of [Ru(phen)2 L]2þ binding to DNA from experiments 4.1.1. Electronic absorption spectra and binding constants The electronic spectra are the most common ways to investigate the interactions of complexes with DNA. In the presence of DNA, the electronic absorption spectra of Ru(II) complexes are usually broadened and undergone apparent hypochromism and red shift [47,48]. The electronic spectra of Ru(II) complexes 1, 2, and 3 in the absence and in the presence of CT-DNA in 5
mM Tris–HCl and 50 mM NaCl buffer were listed in Table 1. The electronic absorption spectra of Ru(II) complex 1 were characterized by a metal to ligand charge-transfer (MLCT) transition band and intraligand (IL) absorption band at about 456 and 264 nm, respectively. For complex 2, the MLCT and IL absorption bands also exhibit at about 456 and 264 nm, respectively. For complex 3, the MLCT absorption band appears at about 441 nm, accompany with a shoulder at about 365 nm, and the IL absorption appears at 264 nm. Upon the addition of CTDNA, both the MLCT and IL absorptions of these Ru(II) complexes exhibit obviously hypochromisms (H) and red shifts (Dk), as shown in Table 1. The intrinsic binding constants for complexes 1, 2, and 3 were calculated according to the Eq. (1) at the MLCT absorption bands, and the data were listed in Table 1. These values are comparable to those observed for [Ru(phen)2 phi]2þ (4.7 104 M1 ) [49], but smaller than those observed for [Ru(bpy)2 dppz]2þ (>106 M1 ) [50] and [Ru(ip)2 dppz]2þ (2.1 107 M1 ) [51]. These results show that the DNA binding affinity of these complexes closely correlates to the electronic effects of their intercalating ligands. The electron-withdrawing substituent (–NO2 ) on intercalating ligand can improve the DNA-binding affinity of the complex, whereas the electron-pushing substituent (–OCH3 or –OH) is reverse. This trend suggests us that binding strength of the complex to DNA can be effectively controlled by the substituents. 4.1.2. Emission spectra To further clarify the interaction of these Ru(II) complexes with DNA, the emission spectra of 1, 2, and 3 have been measured in the absence and in the presence of CT-DNA, the results are shown in Fig. 1. When excited at the MLCT absorption, all of 1, 2, and 3 emit weak fluorescence in the range of 500–700 nm at room temperature with the maximum (I0 ) at ca. 590, 588, and 589 nm, respectively. However, when calf thymus DNA was added into the solution, the enhanced emission (I) for all of these complexes was observed (Fig. 1). It can be explained that the hydrophobic environment inside the DNA helix reduces the accessibility of water
Table 1 Electronic spectra, hypochromism (H%), and DNA-binding constants of Ru(II) complexes 1, 2, and 3 Compound
kmax (free) (nm)
k0max (bound) (nm)
Dk (nm)
H (%)
Kb (105 M1 )
1 (–OCH3 ) 2 (–OH) 3 (–NO2 )
456 264 456 264 456 264
464 266 466 266 465 266
8 2 10 2 9 2
)11 )34 )16 )34 )15 )20
0.20
[Ru] ¼ 20 lM, [DNA] ¼ 2.5 mM.
0.69 1.56
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16
289
10
14 8
Intensity (a.u.)
Intensity (a.u.)
12 10 8 6 4
6 4 2
2 0 500
0 500
550
600
650
700
750
550
600
650
700
Wavelength / nm
Wavelength / nm 1
2 1.0
Intensity (a.u.)
0.8 0.6 0.4 0.2 0.0 500
550
600
650
700
750
Wavelength / nm 3 Fig. 1. Emission spectra of 1, 2 and 3 in the absence and in the presence of increasing amount of CT-DNA in 5 mM Tris–HCl and 50 mM NaCl buffer (pH ¼ 7.2). [Ru] ¼ 10 lM, [DNA] ¼ 2.5 mM.
4.1.3. Viscosity properties The viscosity experiments were performed to investigate the binding mode of these complexes to DNA. Optical photophysical probes provide necessary but not sufficient clues to support a binding mode. Viscosity experiment is considered as one of the least ambiguous
5
4
3
I/I 0
molecules to the complex and the complex mobility is restricted at the binding site, leading to decrease of the vibrational modes of relaxation [52,53]. The emission intensity for complex 1 increases steadily to around 1.9 times than the original and saturates at a [DNA]/[Ru] ratio of ca. 13:1 (Fig. 2). However, for complexes 2 and 3, the emission intensities increase by about 2.3 and 4.5 times under the same conditions, respectively. These data are in agreement with that of previously reported within error [39]. These results also imply that the DNA binding affinities (A) of these complexes increase in the sequence: Að1Þ < Að2Þ < Að3Þ, indicating that the electronwithdrawing substituent on intercalating ligand can stabilize the DNA-complex system more strongly than the electron-pushing substituent.
2
1 0
4
8
12
[DNA]/[Ru] Fig. 2. Plots of relative emission intensity vs. [DNA]/[Ru] ratio for complexes 1 (j), 2 (d), and 3 (N). [Ru] ¼ 10 lM.
and the most critical tests of a binding mode in solution in the absence of crystallographic structure data [54]. In general, a classical intercalation mode may increase the relative viscosity of DNA, because the binding ligand
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Fig. 3. Effect of increasing amount of [Ru(bpy)3 ]2þ (.), 1 (j), 2 (d), and 3 (N) on the relative viscosity of CT-DNA at 30.0 0.1 °C. [DNA] ¼ 0.5 mM.
will separate base pairs of double helix DNA, and thus lengthen the DNA helix. On the contrary, a partial and/ or non-classical intercalation of complex will reduce the relative viscosity of DNA, since the binding ligand may bend (or kink) the DNA helix and reduce its effective length [54]. The relative viscosities of rod like CT-DNA in the presence of complex 1, 2, and 3, as well as [Ru(bpy)3 ]2þ , respectively, are shown in Fig. 3. The relative viscosity of CT-DNA is almost kept unchanged in the presence of [Ru(bpy)3 ]2þ , which is in agreement with an electrostatic binding mode. However, the relative viscosity of CTDNA increases apparently in the presence of complex 1, and more in the presence of complex 2 and the most in the presence of complex 3. These results indicate unambiguously that complexes 1, 2, and 3 behave as intercalators of DNA base pairs. 4.2. Theoretical explanation on the electronic effects of complexes binding to DNA The above-mentioned trend in the electronic effects of complexes binding to DNA can be well explained by our theoretical computations with DFT method. Some frontier molecular orbital energies, and the schematic diagram of energies and related 1 MLCT transitions of [Ru(phen)2 (p-L)]2þ , as well as molecular orbital contour
of [Ru(phen)2 (p-L)]2þ based on the computed results were given in Table 2, Fig. 4 and Fig. 5, respectively. As well-known, there are p–p interactions in the DNA-binding of these complexes in intercalation mode. According to the frontier molecular orbital theory [55– 57], for a reaction controlled by orbital interactions between reactant molecules, a higher HOMO energy of one reactant molecule and a lower LUMO energy of other one are more advantageous to the reaction between the two molecules, because electrons more easily transfer from the HOMO of one reactant to the LUMO of another one in the orbital interaction. A simple calculation model and computed results by the DFT method for stacked DNA base-pairs with backbones have been reported by Kruita and Kobayashi [26]. It should be a perfect simplified approximation model for DNA, and thus should be useful and feasible for discussion on some trends in interaction between the complexes and DNA. The reported HOMO and NHOMO (i.e., HOMO 1) energies of DNA section model with base pairs are much higher ()1.27 and )1.33 eV) [26] than our calculated LUMO and NLUMO (i.e., LUMO + 1) energies ()7.2 eV) of the complexes [Ru(phen)2 (p-L)]2þ (L ¼ MOPIP, HPIP, and NPIP). We believe that such a trend in the relative energies to be kept in our DNA system, because the attraction of metal complex cations with high positive charges for electrons in MOs is much stronger than that of various DNA, and thus the electron must easily transfer from the HOMO of base pairs of DNA to the LUMO (or other virtual orbitals near LUMO) of the complex intercalating to DNA. From Table 2 and Fig. 4, we can see that eL ð1Þ > eL ð2Þ > eL ð3Þ in this series of complexes and this trend can be attributed to the electronic effect due to the order of the electron-withdrawing ability being –OR < – OH < –NO2 . This energy order suggests that the trend in DNA-binding constants (Kb ) should be Kb ð1Þ < Kb ð2Þ < Kb (3). It is satisfactorily in accordance with above experimental results, in which the DNA-binding constants (Kb ) are 0.2, 0.69, and 1.56 ( 105 M1 ) for the complexes 1, 2 and 3, respectively. From Fig. 5, we can also see that the max (k) singlet metal to ligand charge-transfer (1 MLCT) bands of the three complexes should correspond to the electron transitions from their NHOMOs to LUMOs, respec-
Table 2 Some frontier molecular orbital energies (ei /a.u.) and the related energy differences (Dei /a.u.) of Ru(II) complexes Compound
Occa
Occ
Occ
HOMO
LUMO
Virb
Vir
DeLH c
DeLNH
1 2 3
)0.3937 )0.3965 )0.4061
)0.3926 )0.3956 )0.4038
)0.3906 )0.3921 )0.3997
)0.3464 )0.3546 )0.3934
)0.2656 )0.2668 )0.2736
)0.2622 )0.2634 )0.2696
)0.2589 )0.2601 )0.2693
0.0808 0.0878 0.1198
0.1250 0.1253 0.1261
a
Occ: occupied molecular orbital; HOMO (or H): the highest Occ. Vir: virtual (unoccupied) molecular orbital; LUMO: the lowest Vir. c DeLH : the energy difference between LUMO and HOMO; DeLNH : the energy difference between LUMO and NHOMO. b
J. Liu et al. / Inorganica Chimica Acta 357 (2004) 285–293
ε/a.u. NL
-0.26
NL L
L
NL L
-0.28 -0.30 -0.32 -0.34
H
H
-0.36 -0.38
H NH
-0.40
NH
NH
-0.42 1.1
1.2
1
1.3
1.4
2
1.5
1.6
1.7
3
Fig. 4. Schematic diagram of some frontier MO energies and related 1 MLCT transitions of [Ru(phen)2 (p-L)]2þ .
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tively, according to the molecular orbital components of the complexes. This assignation can be preliminarily firmed by the spectral experiments. According to the approximate correlation of reverse ratio of the difference between the LUMO and the HOMO (or NHOMO) energies (DeLH ) to experimental wave length (k), using the parent complex [Ru(bpy)3 ]2þ as a standard sample (experimental k ¼ 452 nm, DeLH ¼ 0:1239 a.u. [31]) and DeLH data of the complexes in Table 2, the computed max (k) 1 MLCT absorption spectra of this series of Ru(II) polypyridyl complexes are 448 nm near experimental wave length 456 nm. Table 1 also contains the wavelengths of complexes binding to calf thymus DNA (the third column). Since in absorption and emission spectra of the Ru(II) complexes, the corresponding energy change between the presence and absence of DNA is small, and no special
Fig. 5. Some related frontier MO contour plots of complexes [Ru(phen)2 (p-L)]2þ .
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pattern changes in the absorption or emission spectra of Ru(II) complexes in the presence of DNA have been detected, except an increase in the luminescence intensity and hypochromism (H%) in the absorption intensity, we consider that there is not a greater effect on the HOMO and LUMO and DeLH for the complexes binding to DNA. It further suggests that the interaction between the series of complexes and DNA should be a weak one, so that the max (k) electron transition band in these Ru(II) complexes binding to DNA can be still assigned to 1 MLCT. At the present time, we are not yet able to calculate the frontier molecular orbital energies of the whole super-molecule system formed from these complexes and DNA, therefore, the DFT studies on some trends in the electronic structures and related properties for such a kind of complexes should be significant.
5. Conclusion The electronic effects of a series of Ru(II) complexes [Ru(phen)2 (p-L)] (L ¼ MOPIP, HPIP, and NPIP) binding to DNA have been investigated with spectroscopic methods, viscosity measurements, and the DFT calculations. The experimental results show that all complexes 1, 2, and 3 can bind to DNA tightly in intercalation mode, and the DNA-binding affinity of these complexes is greatly influenced by electronic effects from their substituent properties. The intrinsic binding constants for 1, 2, and 3 are 0.20, 0.69, and 1.56 105 M1 , respectively, and this order is in accordance with that of the electron-withdrawing ability of substituent [–OR < –OH < –NO2 ]. Such a trend of electronic effects of Ru(II) complexes binding to DNA can be reasonably explained by the DFT calculations.
Acknowledgements We are grateful to the National Nature Science Foundation of China, the Nature Science Foundation of Guangdong Province, the Coordination Chemistry State Key Laboratory in Nanjing University, the State Key Laboratory of Bio-Organic and Natural Products Chemistry of Shanghai Institute of Organic Chemistry, and the Research Fund of Royal Society of Chemistry UK for their financial supports.
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