Incorporation of a biotinylated ruthenium tris(bipyridine) [Ru(bpy)₂(Biot-bpy)]²⁺ (1) in either avidin or streptavidin-(strept)avidin-can be conveniently followed by circular dichroism spectroscopy. To determine the stepwise association constants, cooperativity, and chiral discrimination properties, diastereopure (Λ and δ)-1 species were synthesized and incorporated in tetrameric (strept)avidin to afford (δ-[Ru(bpy)₂(Biot-bpy)]²⁺)x⊂avidin, (Λ- [Ru(bpy)₂(Biot-bpy)]²⁺)x⊂avidin, (δ-[Ru(bpy)₂(Biot- bpy)]²⁺)x⊂streptavidin, and (Λ-[Ru(bpy)₂(Biot-bpy)]²⁺) x⊂streptavidin (x = 1-4) For these four systems, the overall stability constants are log β₄ = 28.6, 30.3, 36.2, and 36.4, respectively. Critical analysis of the CD titrations data suggests a strong cooperativity between the first and the second binding event (x = 1, 2) and a pronounced difference in affinity between avidin and streptavidin for the dicationic guest 1 as well as modest enantiodiscrimination properties with avidin as host
