Chymotrypsin inhibitory conformation induced by amino acid side chain-side chain intramolecular CH/π interaction

Dipeptide amides H-D-Leu-Phe-NH-R have been found to assume a conformation induced by the CH/π interaction and to inhibit chymotrypsin strongly. A series of benzyl amide derivatives H-D-Leu-Phe-NH-[CH₂]_n-C₆H₅ (n = 0-4) have been assayed for chymotrypsin. They inhibit the enzyme in a competitive manner and the highest inhibition is achieved by the amide of n = 1 (K_i = 3.6 × 10^-6 M). The activity enhancement is dependent upon the length of methylene chain, not upon the increase in molecular hydrophobicity, indicating the presence of an optimal distance between dipeptide backbone and C-terminal phenyl group for chymotrypsin inhibition. The C-terminal phenyl group has been found to interact with chymotrypsin stereospecifically. The R-isomer of H-D-Leu-Phe-NH-CH(CH₃)-C₆H₅ is as active as the benzyl amide, while the S-isomer is about twenty-fold less active. When the fluorine atom is introduced at a para-position of the C-terminal phenyl group, the resulting dipeptide H-D-Leu-Phe-NH-CH₂-C₆H₄F-p exhibits about six-times increased inhibitory activity (K_i = 6.1 × 107 M; this dipeptide is one of the most potent chymotrypsin inhibitors to date). ¹H NMR conformational analyses of these dipeptide amide derivatives show the CH/π interaction between D-Leu-isobutyl and Phe-phenyl as a key structural element for chymotrypsin inhibition. These structural examinations strongly suggest that in the inhibitory conformation the C-terminal phenyl group fits the chymotrypsin S₁ site, while the hydrophobic core constructed by D-Leu-Phe CH/π interaction fits the chymotrypsin S₂ or S₁′ site.