Protein surface recognition using geometrically pure Ru(ii) tris(bipyridine) derivatives† †This article is part of the ‘Emerging Investigators’ themed issue for ChemComm ‡ ‡Electronic supplementary information (ESI) available: Synthetic procedures, characterization and fluorescence binding studies. 

Fac and mer isomers of functionalised ruthenium(ii) tris(bipyridine) derivatives are shown to have different recognition properties towards cytochrome c.


Deprotection of ruthenium complexes
Stirring of four isomers of 1 and of 2 in trifluoroacetic acid 90% and triisopropylsilane 1% in water for a period of 3 days to affoar following concentration the de-protected species.

UV-vis spectra
Stock solutions of receptors were prepared to a concentration of 1 mM in 5 mM phosphate buffer.
Once the receptors had been dissolved, the pH was adjusted to 7.4 via addition of 1 N sodium hydroxide. A series of stock dilutions (10 M -300 M) was prepared and the extinction coefficient determined by monitoring the absorbance at 467 nm.

Fluorescence Titrations
Horse Heart cytochrome c was purchased from Sigma (C2506). The concentration of horse heart cytochrome c and 60% acetylated horse heart cytochrome c was determined using the molar extinction coefficient at 550 nm of 2.95 x 10 4 dm -3 mol -1 cm -1 following reduction with excess

UV-Vis-monitored ascorbate reduction assay
A solution (5 mM sodium phosphate, pH 7.4) containing cytochrome c (15 M) and compound 1 was incubated for 10 min at room temperature with ruthenium complex or on its own in a quartz cuvette (pathlength of 1cm). 150 L of a 20 mM stock solution of ascorbate in 5 mM sodium phosphate buffer (pH 7.4) was then added. Upon addition the content of the cuvette was immediately mixed using a 1 mL pipette twice whilst starting the spectrophotometer. The instrument was set to measure the absorbance at 550 nm, every 0.5 seconds, for 2 min. To test the rate dependence on concentration of the receptor, five different solutions with molar ratios of 0 to 1.75 (receptor: Cyt c) were subjected to ascorbate reduction. The collected data was analysed based on the equation derived from a pseudo first order rate expression: Aobs = max(1-exp(-kobs*t)); where Aobs = Aobs t -Aobs t0 = 0, and t =time in seconds. Both max and k were floated, and were determined by regression analysis in the

Titrations with -chymotrypsin
Titrations were performed in a similar manner as for cytochrome c. Bovine -chymotrypsin was purchased from Sigma (C4129) and the concentration of stock solutions determined using the molar extinction coefficient at 280 nm of 5.06 x 10 4 dm -3 mol -1 cm -1 calculated using the online facility on the ExPAsy website. The concentration of protein in the stock solution was verified from the absorbance at 280 nm in 6 M GdmHCl, adjusted to pH 6.0 by addition of sodium hydroxide.