DNA Binding DOI: 10.1002/ange.201206260 Temperature-Switched Binding of a Ru II (dppz)/DNA Light-Switch Complex** Michael G. Walker, Veronica Gonzalez, Elena Chekmeneva, and Jim AThomas* Dipyridylphenazine (dppz)-based ruthenium complexes are extremely well studied DNA binding substrates. The proto- typical system in such studies is the “DNA light-switch complex”, [Ru(N-N) 2 (dppz)] 2+ (N-N = 2,2’-bipyridyl, 1,10- phenanthroline; Figure 1). In aqueous solutions, the 3 MLCT-based emission of this chiral complex is only “switched on” when it intercalates into the duplex. [1] Numer- ous studies designed to identify the exact nature of the binding site of the complex, to outline the binding differences between its L and D enantiomers, and to delineate the photophysical details of its binding-induced optical response have been carried out. [1–3] In order to facilitate the construction of mixed-motif and oligonuclear DNA binding substrates based on the Ru II (dppz) moiety, we investigated the properties of achiral [Ru(tpy)(L)- (dppz)] 2+ complexes (tpy = tris(pyrazolyl)methane, L = monodentate N-donor ligand). [4–8] Recently, we demonstrated that the DNA binding proper- ties of such systems are highly dependent on small changes in the structure of L; for example, while complex 1 is a typical light-switch DNA intercalator with high binding affinities for duplexes (K b > 10 6 m 1 ), complex 2 shows reduced interaction with DNA. [7] Through experimental and computational studies, we ascribed this effect to unfavorable steric inter- actions involving the amino group of the pyridyl-based pyNH 2 ancillary ligand preventing intercalation of the dppz moiety. To explore this unusual behavior more fully, the thermody- namic interaction of 2 with calf thymus (CT)-DNA at 25 8C as well as 10 8C and 35 8C were initially investigated through isothermal calorimetry (ITC). To aid comparisons, the interaction of 1 with CT-DNA under the same conditions was also studied. Although we have previously reported an ITC study on the interaction of 1 with synthetic homopol- ymers, [4] this is the first study on genomic B-DNA. At all three temperatures, complex 1 binds to CT-DNA through an endothermic, entropically driven interaction (see Table 1 and the Supporting Information). This kind of thermodynamic signature was seen for several other Ru II - (dppz)-based metallointercalators; indeed the parameters at 25 8C are very similar to those reported for L-[Ru(phen) 2 - (dppz)] 2+ at this temperature (DH = 2.9 kcal M 1 , TDS = 11.4 kcal M 1 ). [9] In contrast, the temperature dependence for the interaction of 2 with CT-DNA is much more complex. Despite carrying out experiments at different concentra- tions of complex and/or DNA, the titration of 2 with CT-DNA at 25 8C only showed heat changes because of the dilution of the complex, thus indicating that the free energy of binding at this temperature is entirely due to favorable entropic changes. However, identical studies at other temperatures give con- trasting results. At 10 8C, 2 clearly interacts with CT-DNA through two binding modes (see the Supporting Information for ITC traces). The first few injections of 2 lead to a relatively high affinity interaction (DG = 7.18 kcal M 1 ; K b = 3.6  10 5 m 1 ), which is entirely enthalpically driven (DH = 9.96 kcal M 1 ) and actually displays an unfavorable entropic contribution (TDS = 2.38 kcal M 1 ). The thermodynamic signature for this interaction is reminiscent of that observed for the initial binding mode of 1 with A-tract duplexes; [4] a second similarity is that the site size for the initial interaction with CT-DNA is also very large (18.7 bp). It has been previously suggested that such interactions are due to binding at the fraying ends and/or gaps of duplexes [4, 10] and, given its thermodynamic signature, it seems likely that this mode involves the amino group of 2 Figure 1. Ru II (dppz)-based complexes. Table 1: ITC-derived CT-DNA binding parameters for complex 1 at selected temperatures. 10 8C 25 8C 35 8C DH [kcal m 1 ] 3.67 2.82 1.50 TDS [kcal m 1 ] 10.6 9.36 9.09 DG [kcal m 1 ] 6.93 6.54 7.60 S [bp] 2.3 4.9 3.8 K [m 1 ] 1.2  10 5 7.4  10 4 2.3  10 5 [*] M.G. Walker, Dr. V. Gonzalez, Dr. E. Chekmeneva, Dr. J.A. Thomas Department of Chemistry, University of Sheffield Sheffield, S3 7HF (UK) E-mail: james.thomas@sheffield.ac.uk Dr. V. Gonzalez School of Chemistry, University of Cardiff Cardiff (UK) [**] We are grateful to the EPSRC and the BBSRC for studentships (M.W. and V.G.) and the Royal Society Newton Fellowship Scheme (E.C.). dppz = Dipyridylphenazine. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201206260. A ngewandte Chemi e 12273 Angew. Chem. 2012, 124, 12273 –12276  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim