Supplementary information Unusual mode of protein binding by a cytotoxic π-arene ruthenium(II) piano-stool compound containing an O,S–chelating ligand 5 Jana Hildebrandt, a Helmar Görls, a Norman Häfner, b Giarita Ferraro, c Matthias Dürst, b Ingo B. Runnebaum, b Wolfgang Weigand, a,* and Antonello Merlino c,d,* Materials and Methods 10 Synthesis and characterization of Compound 1 Crystal structure determination Cytotoxicity UV-Vis absorption spectroscopy Crystallization, X-ray diffraction data collection, structure resolution and refinement of RNase A-Compound 1 adduct 15 References Table S1. Crystal data and structure refinement for Compound 1 Table S2. Bond lengths [Å] and angles [°] for Compound 1 Table S3. Selected distances and angles of Compound 1 compared to L 20 Table S4. Data collection and refinement statistics of the RNase A-Compound 1 adduct Figure S1. UV-Vis spectra of 0.3 mM Compound 1 under different experimental conditions followed each 1 h over 24 h. (A) 100 % DMSO; (B) 50 % DMSO; 50 % PBS pH 7.4. (C) 10 mM sodium citrate pH 5.1; (D) 10 mM sodium citrate pH 5.1, protein:metallodrug ratio 1:3; (E) 10 mM sodium citrate pH 5.1, protein:imidazole ratio 1:3; UV/Vis spectra of Compound 1 in DMSO show an intense peak at 346 nm, a small peak at 296 nm and a shoulder at 457 nm. The 25 Compound experiences a slow red shift of its main band up to 354 nm, accompained by a progressive slow decrease in intensity of all bands of the spectrum. UV/Vis spectra of Compound 1 in PBS pH 7.4 show a similar behaviour when compared to the spectra of the compound in DMSO, although changes in the spectra are less pronounced. Peaks are observed at 295 nm and at 345 nm. Shoulder at 450 nm. UV/Vis spectra of Compound 1 in sodium citrate pH 5.1 show an intense peak at 342 nm, a small peak at 293 nm and a shoulder at 450 30 nm. Within 24 h, the complex experiences a red shift of its major band up to 346 nm and a blue shift of the band at 293 nm which disappears upon 24 h and of the shoulder to 431 nm. The observed spectral changes are different in the presence of the protein. In fact, Compound 1 spectra in the presence of RNase A show a major peak at 346 nm and a very small peak at 457 nm that decrease their intensity with time. The peak at 293 nm is overlapped with that of the protein at 280 nm. After 24 h a red shift of the band at 346 nm is observed also in this case. 35 Figure S2. UV-Vis spectra of 1 mM Compound 1 in 90% PBS at pH 7.4, 10% DMSO, 1M NaCl, followed each 1 h over 24 h. These spectra should be compared to those reported in Figure 2B. Figure S3. UV-Vis spectra of 1 mM Compound 1 followed each 1 h over 24 h in 90% PBS at pH 7.4, 10% DMSO in the presence of 5’- GMP Figure S4. Hydrolysis of yeast RNA (evaluated by measuring the variation of absorbance at 300 nm as function of time upon addition of 40 the protein to the yeast RNA sample) by RNase A (black) and its adducts with Compound 1. Catalytic activity of RNase A in the presence of Compound 1 at different protein to metal ratio was determined spectrophotometrically by using the Kunitz assay [19]. 0.5 mg x mL -1 of RNA and enzyme concetration =0.5 μg x mL -1 were used in 50 mM sodium citrate buffer pH 5.1, at 298 K. Spectrophotometric measurements were performed with a Jasco spectrophotometer. Experiments have been performed after 24 h of incubation. Protein remains well active in the presence of the compound. 45 Figure S5. Compound 1 binding site in RNase A-Compound 1 adduct showing the Ru centre bound to His105. Anomalous electron density map that allows the identification of Ru centre is shown at 4σ level. Figure S6. Details of Compound 1 binding site in molecule A of RNase A-Compound 1 adduct showing the Ru centre bound to His105. 2Fo-Fc electron density maps are contoured at 5σ (red) and 0.8σ (cyan) level. 50 Electronic Supplementary Material (ESI) for Dalton Transactions. This journal is © The Royal Society of Chemistry 2016