Photophysics of Ruthenium Complexes Bound to Double Helical DNA

Binding of the chiral metal complexes [Ru(bpy),]CI, (I), [ R ~ ( p h e n ) ~ ] C l ~ (11), and [Ru(DIP),]CI2 (111) to calf thymus DNA is examined by following changes in the photophysical properties of these probes with use of steady-state as well as time-resolved methods. Increasing luminescence is seen for the ruthenium complexes I1 and I11 with DNA addition whereas no enhanced luminescence is detectable for I. A biexponential decay in luminescence is found for I1 and I11 with emission lifetimes of the complexes bound to DNA appearing 3-5 times longer than those of the free complexes. Quenching of the luminescence by the ferrocyanide anion further amplifies the ability to distinguish bound forms. I* is quenched by ferrocyanide in the presence of DNA as efficiently as in its absence, indicating little or no binding. In contrast, biphasic Stern-Volmer plots are found for I1 and 111, indicating extensive protection of 11* and III* in the presence of DNA from ferrocyanide. Here emission quenching was found to be completely static as a result of counterion condensation at the DNA polyanion. Emission polarization measurements revealed that the binding of I1 and 111 to DNA is accompanied by significant increases in the steady-state polarization. The results are interpreted in terms of two binding modes: electrostatic, which is easily quenched by ferrocyanide and contributes no polarization in emission, and intercalative, which is protected from ferrocyanide quenching and, since rigidly bound, retains emission polarization. The distinction becomes more apparent for I11 where significant enantiomeric selectivity is observed on binding to DNA. Thus A-Ru(DIP),*+ binds to DNA both electrostatically and by intercalation; extensive curvature is seen in Stern-Volmer plots, and increases in polarization are observed. The b isomer, which gives strictly linear Stern-Volmer plots, binds only electrostatically. This chiral discrimination for intercalative binding is explained in terms of the helical asymmetry of a right-handed DNA structure which is matched by the asymmetry of the A isomer but precludes binding by the A isomer. The nature and dynamics of binding small molecules to biopolymers represents an area of active investigation. Studies directed toward the design of siteand conformation-specific reagents provide routes toward rational drug design as well as a means to develop sensitive chemical probes of polymer structure. A simple example is given by the intercalation of small heterocyclic dyes into DNA.'-4 This noncovalent binding mode where the dye stacks between adjacent base pairs of the D N A duplex is particularly favored by positively charged species possessing a planar aromatic moiety. Intercalators tend to be strongly mutagenic and some have shown promising chemotherapeutic a ~ t i v i t y . ~ Their carcinogenecity and antitumor activity furthermore correlate well with DNA binding affinity. Moreover, the photophysical properties of bound intercalators have provided useful information concerning nucleic acid structure. Ethidium is a common fluorescence probe for D N A and has recently been employed in examinations of the torsional rigidity of the double helix.6 Cationic metal complexes possessing planar aromatic ligands also may bind to D N A by intercalation.' Platinum complexes have been shown by X-ray diffraction methods to be valuable electron-dense probes of the intercalative process and generally of nucleic acid structure.s-10 Metallointercalators which cleave DNA," owing to the redox activity of the metal center, have furthermore been successfully employed in footprinting studies of drug binding and in the examination of higher-order chromatin s t r ~ c t u r e . ' ~ J ~ Chiral octahedral metal complexes containing aromatic ligands have been found recently to display enantiomeric selectivity in binding to double helical DNA.I4 Equilibrium dialysis of DNA with the racemic mixture of chiral metal complexes showed the optical enrichment of the less favored isomer in the dialysate. Absolute configuration assignments for tris(phenanthro1ine)ruthenium(I1) complexes revealed that it is the A isomer that binds preferentially to right-handed B-DNA.lS Ruthenium(I1) complexes have been particularly useful in monitoring stereoselective binding to DNA not only because of the stability of enantiomers but also because of the sensitivity of their photophysical properties to DNA binding.15 Luminescence enhancements and absorption hypochromism in the intense metal to ligand charge transfer band *To whom reprint requests should be sent. 0002-7863/85/ 1507-55 18$01.50/0 (MLCT) accompany DNA binding. Furthermore, enantiomers of tris(diphenylphenanthroline)ruthenium(II) have been shown to be useful chemical probes for helix handedness, since absorption decreases accompany binding of the A isomer but not of the A isomer to a right-handed helix, whereas spectrophotometric titrations indicate that both isomers bind equally to Z-form poly dGC.I6 In this report a detailed study of the photophysical properties of ruthenium(I1) complexes in the presence of DNA has been carried out. We were interested in determining how spectroscopic characteristics of the ruthenium(I1) complexes vary as a function of DNA binding, whether different modes of DNA binding might be distinguished by using these photophysical properties, and how best to detect chiral discrimination so as to optimize the sensitivity and utility of our spectroscopic probes for DNA handedness. The relatively long lifetimes of these complexes, their excellent, readily (1) Berman, M. H.; Young, P. R. Annu. Rev. Biophys. 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