A thorough thermodynamic analysis by isothermal titration calorimetry of allosteric and chelate cooperativity effects in divalent crown ether/ammonium complexes is combined with DFT calculations including implicit solvent on the one hand and large-scale molecular dynamics simulations with explicit solvent molecules on the other. The complexes studied exhibit binding constants up to 2×10 m with large multivalent binding enhancements and thus strong chelate cooperativity effects. Slight structural changes in the spacers, that is, the exchange of two ether oxygen atoms by two isoelectronic methylene groups, cause significantly stronger binding and substantially increased chelate cooperativity. The analysis is complemented by the examination of solvent effects and allosteric cooperativity. Such a detailed understanding of the binding processes will help to efficiently design and construct larger supramolecular architectures with multiple multivalent building blocks.

Fluorescent binders of the estrogen receptor (ER) are used in binding assays and in detection or imaging studies. However, fluorescence labelling of ER ligands usually leads to substantial decreases in binding affinity. In this study, we describe the development of high affinity fluorescent ER ligands. Cyanine dyes of the MiDye series were directly attached to the SERMs 4‐hydroxytamoxifen (OHT) and raloxifene (Ral); linkers were deliberately omitted. This approach yielded conjugates with ERα binding affinities superior to the natural ligand estradiol. The OHT‐ and Ral‐MiDye conjugates emitted in the 600–800 nm range. First round staining experiments showed that the conjugates, but not the dyes alone, accumulate in cells expressing estrogen‐binding receptors.