Fluorine Scan of Inhibitors of the Cysteine Protease Human Cathepsin L: Dipolar and Quadrupolar Effects in the π‐Stacking of Fluorinated Phenyl Rings on Peptide Amide Bonds

The π‐stacking of fluorinated benzene rings on protein backbone amide groups was investigated, using a dual approach comprising enzyme–ligand binding studies complemented by high‐level quantum chemical calculations. In the experimental study, the phenyl substituent of triazine nitrile inhibitors of human cathepsin L (hCatL), which stacks onto the peptide amide bond Gly67−Gly68 at the entrance of the S3 pocket, was systematically fluorinated, and differences in inhibitory potency were measured in a fluorimetric assay. Binding affinity is influenced by lipophilicity (clog P), the dipole and quadrupole moments of the fluorinated rings, but also by additional interactions of the introduced fluorine atoms with the local environment of the pocket. Generally, the higher the degree of fluorination, the better the binding affinities. Gas phase calculations strongly support the contributions of the molecular quadrupole moments of the fluorinated phenyl rings to the π‐stacking interaction with the peptide bond. These findings provide useful guidelines for enhancing π‐stacking on protein amide fragments.