J. Phys. Chem. B 2005, 109, 17327-17332
17327
Enantioselective Luminescence Quenching of DNA Light-Switch [Ru(phen)2dppz]2+ by Electron Transfer to Structural Homologue [Ru(phendione)2dppz]2+ Fredrik Westerlund,* Fre´ de´ ric Pierard,† Mattias P. Eng, Bengt Norde´ n, and Per Lincoln Department of Chemistry and Bioscience, Chalmers UniVersity of Technology, SE-41296 Gothenburg, Sweden ReceiVed: April 4, 2005; In Final Form: June 27, 2005
The quenching of the luminescence of [Ru(phen)2dppz]2+ by structural homologue [Ru(phendione)2dppz]2+, when both complexes are bound to DNA, has been studied for all four combinations of ∆ and Λ enantiomers. Flow linear dichroism spectroscopy (LD) indicates similar binding geometries for all the four compounds, with the dppz ligand fully intercalated between the DNA base pairs. A difference in the LD spectrum observed for the lowest-energy MLCT transition suggests that a transition, potentially related to the final localization of the excited electron to the dppz ligand in [Ru(phen)2dppz]2+, is overlaid by an orthogonally polarized transition in [Ru(phendione)2dppz]2+. This would be consistent with a low-lying LUMO of the phendione moiety of [Ru(phendione)2dppz]2+ that can accept the excited electron from [Ru(phen)2dppz]2+, thereby quenching the emission of the latter. The lifetime of excited ∆-[Ru(phen)2dppz]2+ is decreased moderately, from 664 to 427 ns, when bound simultaneously with the phendione complex to DNA. The 108 ns lifetime of opposite enantiomer, Λ-[Ru(phen)2dppz]2+, is only shortened to 94 ns. These results are consistent with an average rate constant for electron transfer of approximately 1‚106 s-1 between the phenanthroline- and phendione-ruthenium complexes. At binding ratios close to saturation of DNA, the total emission of the two enantiomers is lowered equally much, but for the Λ enantiomer, this is not paralleled by a decrease in luminescence lifetime. A binding isotherm simulation based on a generalized McGhee-von Hippel approach shows that the ∆ enantiomer binds approximately 3 times stronger to DNA both for [Ru(phendione)2dppz]2+ and [Ru(phen)2dppz]2+. This explains the similar decrease in total emission, without the parallel decrease in lifetime for the Λ enantiomer. The simulation also does not indicate any significant binding cooperativity, in contrast to the case when ∆-[Rh(phi)2bipy]3+ is used as quencher. The very slow electron transfer from [Ru(phen)2dppz]2+ to [Ru(phendione)2dppz]2+, compared to the case when [Rh(phi)2phen]3+ is the acceptor, can be explained by a much smaller driving free-energy difference.
Introduction Ruthenium complexes that bind noncovalently to DNA have been of great interest for the last 25 years, as this class of molecules has potential as DNA-conformational probes, anticancer drugs, or initiators for electron-transfer studies on DNA. The parent compound is the symmetric complex, [Ru(phen)3]2+,1-12 where the six-coordinated ruthenium(II) ion binds the three bidentate phenanthroline moieties to form a propeller-like structure, giving two possible conformations, the right-handed (∆) and the left-handed (Λ) enantiomer. Whether the two enantiomers of [Ru(phen)3]2+ bind in one of the grooves or are intercalated between the DNA base pairs has been the matter of intense discussion for many years; however, most data are consistent with a model where the complexes bind by semiintercalating one of the phenanthroline moieties between the base pairs of DNA.12 The DNA-binding mode is more distinct when one of the phenanthroline moieties is extended by two aromatic rings to a dipyridophenazine (dppz) ligand, i.e., [Ru(phen)2dppz]2+ (1 in Figure 1), which readily intercalates between the base pairs of DNA.13-26 The extension to a dppz unit not only results in a * Author to whom correspondence should be addressed. Email:
[email protected]. Telephone: +46-31-772 30 53. Fax: +46-31-772 38 58. † Current address: Organic Chemistry and Photochemistry, Free University of Brussels, B-1050 Brussels, Belgium.
Figure 1. [Ru(phen)2dppz]2+ (1) and [Ru(phendione)2dppz]2+ (2); phen ) 1,10-phenanthroline, dppz ) dipyrido[3,2-a:2′,3′-c]phenazine, phendione ) 1,10-phenanthroline-5,6-diketone.
stronger and more distinct DNA binding, but also in a complex with more interesting photophysical properties. Hydrogen bonding to the aza-nitrogens of the dppz moiety almost completely quenches the luminescence from 1 in aqueous solution. By contrast, when the dppz moiety intercalates between the DNA bases, the aza-nitrogens are protected from the hydrogenbonding solvent, leading to an increase in luminescence quantum yield by several orders of magnitude (>104). This large increase in luminescence quantum yield, referred to as the “light-switch” effect,18,27 makes the compound very useful in spectroscopic DNA-binding studies. When 1 is excited by light, an electron is transferred from the ruthenium(II) ion to the dppz-ligand, formally oxidizing the metal and reducing the ligand (metal-to-ligand charge transfer, MLCT). When the electron jumps back to the ruthenium(II)
10.1021/jp0517091 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/19/2005
17328 J. Phys. Chem. B, Vol. 109, No. 36, 2005 ion, light can be emitted, giving the complex its interesting photophysical properties. However, if the excited electron is trapped, and its energy dissipated by another process, the emission is quenched. The rhodium complex ∆-[Rh(phi)2bipy]3+ (phi ) phenanthrene-9,10-diimine, bipy ) 2,2′-bipyridyl) quenches the luminescence of ∆-1, by electron transfer, with extreme efficiency, when they are both bound to DNA.28-32 However, whether this observation is an effect of an unusually strong cooperative binding of the two complexes to DNA,33,34 or an ultra-efficient electron transfer through the DNA base stack,31 has been debated. [Ru(phendione)2dppz]2+ (2 in Figure 1), where the two phenanthroline moieties on 1 have been oxidized to phendiones, is nonluminescent, presumably because of a low-lying electronic state on the phendione moieties that trap the excited electron.35-38 It would, therefore, be interesting to see if 2 could accept an electron also intermolecularly, from a simultaneously DNAbound 1 complex, and to study the importance of the chirality for any such interactions between the two complexes. As expected, 2 is found to bind to calf thymus DNA (CTDNA) by intercalation, but the equilibrium constant is lower compared to 1. The quenching of the luminescence of 1 by 2 is not at all as efficient as the quenching by ∆-[Rh(phi)2bipy]3+. The longest lifetime of Λ-1 (108 ns) is lowered only about 13%, whereas the longest lifetime of ∆-1 (664 ns) is lowered about 33%. The lowering of the total emission is, however, similar for both ∆-1 and Λ-1 because of a lower binding constant and, thereby, larger competition from 2 for Λ-1. Materials and Methods Chemicals. All experiments were performed in aqueous buffers (10 mM NaCl, 1 mM cacodylate at pH 7.0). Calf thymus DNA (CT-DNA), obtained from Sigma, was dissolved in buffer and filtered three times through a 0.8-µm Millipore filter before use. The enantiomers of [Ru(phen)2dppz]Cl2 (1) were prepared as reported elsewhere.19 ∆- and Λ-[Ru(phendione)3](PF6)2 were obtained by oxidation of respective [Ru(phen)3](PF6)2 optical isomers12,39 and isolated as PF6- salts by using NH4PF6 instead of the perchlorate used in the literature method.40 ∆- and Λ-[Ru(phendione)2dppz]Cl2 (2) were synthesized from respective [Ru(phendione)3](PF6)2 enantiomers as described below. ∆-[Ru(phendione)2dppz]2+ (∆-2). A solution of 12 mg (0.11 mmol) o-phenylenediamine in 5.5 mL of acetic acid/H2O 1:10 was added dropwise with stirring to a solution of 153 mg (0.15 mmol) ∆-[Ru(phendione)3](PF6)2 in 270 mL acetic acid/H2O/ CH3CN 1:10:2.5. After 20 min, TLC (support SiO2, solvent CH3CN/KNO3 0.1 M aqueous solution 8:1) showed the reaction to be complete. The volume of the reaction mixture was reduced to 20 mL by rotary evaporation, resulting in precipitation. Addition of 5 mL of saturated KPF6 aqueous solution completed the precipitation of the crude product, which was collected on a filter and washed successively with water, absolute ethanol, and diethyl ether and then dried. The product was purified by column chromatography on silica gel with CH3CN/KNO3 0.1 M aqueous solution 8:1 as eluent. The two first fractions containing small amounts of the byproducts [Ru(dppz)3]2+ and [Ru(phendione)(dppz)2]2+ were discarded; the large third fraction gave ∆-2. After the solvent was removed from the desired fraction, the complex was redissolved in water, isolated as the PF6 salt by precipitation with aqueous KPF6, and washed twice with water. An additional recrystallization from acetonitrile/ diethyl ether yielded 38 mg (32%) of ∆-[Ru(phendione)2dppz](PF6)2. Conversion to dichloride salts was achieved through
Westerlund et al. anion-exchange chromatography (1:1 acetonitrile/water eluent) on Sephadex QAE-25 (Aldrich). The Λ enantiomer of 2 was prepared in the same way from Λ-[Ru(phendione)3](PF6)2. 1H NMR for ∆-2(PF6)2 (300 MHz, CD3CN, 298 K, see Figure 1 for proton numbering): δ ppm ) 9.74 (dd, 2H, 3J3′4′ ) 8.4 Hz, H4′), 8.61 (d, 2H, 3J34 ) 7.8 Hz, H4), 8.51 (d, 2H, 3J78 ) 7.8 Hz, H7), 8.50 (dd, 2H, 3Jab ) 6.6 Hz, Hb), 8.32 (dd, 2H, 4J2′4′ ) 1.2 Hz, H2′), 8.22 (d, 2H, 3J23 ) 5.7 Hz, H2), 8.16 (dd, 2H, 4J ) 3.6 Hz, H ), 8.00 (d, 2H, H ), 7.96 (dd, 2H, 3J ab a 9 2′3′ ) 5.4 Hz, H3′), 7.72 (dd, 2H, H3), 7.52 (dd, 2H, 3J89 ) 5.7 Hz, H8). ES-MS for ∆-2(PF6)2 (acetonitrile/water 1:1): m/e ) 402.2 (100%, calcd [M-2PF6-]2+ ) 402.0), 411.3 (100%, calcd [M-2PF6- + H2O]2+ ) 411.0), 420.2 (68%, calcd [M-2PF6+ 2H2O]2+ ) 420.0), 429.2 (10%, calcd [M-2PF6- + 3H2O]2+ ) 429.0). Hydration of the carbonyl groups in 1,10-phenanthroline-5,6-dione coordinated to metal cations has been previously reported in the literature.41-43 UV-vis for ∆-2(Cl)2 [water, λmax/nm, (/1000 M-1 cm-1)]: 432 (16.0), 372 (15.4, shoulder), 355 (17.5), 292 (57.1, shoulder), 278 (62.5). CD for ∆-2(Cl)2 [water, λextr/nm, (∆/M-1 cm-1)]: 482 (-11.0), 410 (+16.9), 349 (+2.0), 306 (-91.5), 298 (-77.0), 276 (-5.6), 258 (-14.5). (ee ) 0.99) The CD signals for Λ-2(Cl)2 were of the same amplitude and opposite signs (ee ) 0.96). The enantiomeric excess (ee) was estimated from CD measurements in water with a sample of ∆-[Ru(dppz)3][Cl2] prepared under similar conditions from the same starting material ∆-[Ru(phendione)3](PF6)2 and compared to the literature value for a reference sample of ∆-[Ru(dppz)3][Cl2].40 Sample Preparation. Samples were prepared by mixing equal volumes of ruthenium complex and DNA dissolved in buffer. The final sample concentrations were 10 µM of complex and 160 µM of DNA for LD measurements. Concentrations were determined on a Varian Cary 4B spectrophotometer. The extinction coefficients used were: 440nm ) 20 000 M-1 cm-1 for [Ru(phen)2dppz]2+, 432nm ) 16 000 M-1 cm-1 for [Ru(phendione)2dppz]2+, and 260nm ) 6600 M-1 cm-1 for ctDNA. Flow Linear Dichroism. Linear dichroism (LD)44 is defined as the difference in absorbance of linearly polarized light parallel and perpendicular to a macroscopic orientation axis (here the flow direction):
LD(λ) ) A|(λ) - A⊥(λ) Samples with ruthenium complex and DNA were oriented in a Couette flow cell with an outer rotating cylinder at a shear gradient of 3000 s-1. LD spectra were measured on a Jasco J-720 CD spectropolarimeter equipped with an Oxley prism to obtain linearly polarized light. All spectra were recorded between 220 and 650 nm and baseline-corrected by subtracting the spectrum recorded for the nonoriented sample. Steady-State Luminescence Measurements. Emission spectra were recorded on a xenon lamp equipped SPEX fluorolog τ-2 spectrofluorimeter (JY Horiba) between 420 and 800 nm using an excitation wavelength of 415 nm. Using a short optical path length for the excitation light, the inner filter effect due to excitation light absorption was kept below 10%. Transient Emission Measurements. The nanosecond emission decay measurements were performed on a setup where the exciting light is provided by a pulsed Nd:YAG laser (Continuum Surelite II-10, pulse width
