Understanding the binding mechanisms of hydroxy-chalcone-based 24-membered macrocyclic bis-epoxide with...

The lipocalin carrier protein, β-lactoglobulin (β-lg), stands out as a crucial protein in the food industry, known for its ability to effectively bind with hydrophobic small molecules. However, it was unclear how β-lg interacts with macrocyclic molecules. In this research, we focused on two key aspects. First, we synthesized a 24-membered macrocycle 4d by modifying a natural product chalcone to create a macrocycle by connecting two ortho-hydroxyl groups of each phenyl ring of two chalcone units with alkyl chains. To enhance solubility, we converted the chalcone CC bonds to epoxide rings. Second, we investigated the binding ability and mechanism of binding of the compound with the β-lg. The β-lg and 4d interaction shows an isoemissive point at 382 nm with K_b = 4.64 ± 0.02 × 10⁵ at 298 K, indicating the excellent protein binding ability of 4d. Remarkably, despite its size, 4d binds to the protein without altering its conformation, suggesting the availability of a spacious binding site on the protein where the molecule fits well. Molecular docking analysis confirmed the presence of such a site at the mouth of the calyx. Additionally, our 200 ns molecular dynamics simulation demonstrated that 4d adopts a conformation to interact with the hydrophobic amino acids of the binding site, ultimately stabilizing the protein.