Introduction: Therapeutic peptides are a significant class of drugs in the treatment of a wide range of diseases. To enhance their properties, such as stability or binding affinity, they are usually chemically modified. This includes, among other techniques, cyclization of the peptide chain by bridging, modifications to the backbone, and incorporation of unnatural amino acids. One approach previously established, is the use of halogenated aromatic amino acids. In principle, they are thereby enabled to form halogen bonds (XB). In this study, we focus on the -R-CF₂X moiety (R = O, NHCO; X = Cl, Br) as an uncommon halogen bond donor. These groups enable more spatial variability in protein–protein interactions. The chosen approach via Fmoc-protected building blocks allows for the incorporation of these modified amino acids in peptides using solid-phase peptide synthesis.
Results and Discussion: Using a competitive fluorescence polarization assay to monitor binding to Mdm4, we demonstrate that a p53-derived peptide with Lys24Nle(ϵNHCOCF₂X) exhibits an improved inhibition constant K_i compared to the unmodified peptide. Decreasing K_i values observed with the increasing XB capacity of the halogen atoms (F ≪ Cl < Br) indicates the formation of a halogen bond. By reducing the side chain length of Nle(ϵNHCOCF₂X) to Abu(γNHCOCF₂X) as control experiments and through quantum mechanical calculations, we suggest that the observed affinity enhancement is related to halogen bond-induced intramolecular stabilization of the α-helical binding mode of the peptide or a direct interaction with His54 in human Mdm4.
Keywords: halogen bonding, solid-phase peptide synthesis, unnatural amino acids, fluorescence polarization assay, oncological target, MdmX