Chymotrypsin inhibition induced by side chain-side chain intramolecular CH/π interaction in D-Thr-L-Phe benzylamide

The dipeptide benzyl amide H-D-Thr-Phe-NH-CH₂-C₆H₅ was found to inhibit chymotrypsin strongly (K(i) = 4.5 x 10^-6 M) in a competitive manner. When a series of phenyl amides H-D-Thr-Phe-NH-(CH₂)(n)-C₆H₅ (n = 0-4) were tested, inhibitory potency peaked at n = 1 (benzyl amide). Incorporation of a methyl group into the benzyl methylene resulted in formation of stereoisomers, H-D-Thr-Phe-NH-(R or S)-CH(CH₃)-C₆H₅, with considerably different inhibitory potencies. The R-isomer was as active as the benzyl amide, while the S-isomer was about 30-fold less active than the benzyl amide. Furthermore, when a fluorine atom was introduced into the para-position of the amide-benzyl group, the resulting H-D-Thr-Phe-NH-CH₂-C₆H₄(p-F) showed considerably enhanced inhibitory activity (about 5-fold, K(i) = 9.1 x 10^-7 M). In conformational analysis by 400 mHz ¹H-NMR, all dipeptides having D-Thr-Phe backbone structure showed large upheld shifts of D-Thr-βOH (shifts in ppm, 0.09-0.17), D-Thr-βCH (0.23-0.32), and D-Thr-γCH₃ (0.38-0.53), indicating the presence of shielding effects from the benzene ring. In addition, NOE enhancements between the D-Thr-γCH₃ and Phe-phenyl groups were evidenced by measurements of two-dimensional NOESY spectra and NOE difference spectra. These observations demonstrated the spatial proximity of these side chains, which is due to side chain-side chain CH/π interaction. All these results support the idea that the amide-benzyl group binds at the chymotrypsin S₁ site, while the hydrophobic core with CH/π interaction binds at the S₂ or S₁' site.