CHYMOTRYPSIN INHIBITION INDUCED BY SIDE CHAIN-SIDE CHAIN INTRAMOLECULAR CH PI INTERACTION IN D-THR-L-PHE BENZYLAMIDE/

The dipeptide benzyl amide H-D-Thr-Phe-NH-CH2-C6H5 was found to inhibi t chymotrypsin strongly (K-i=4.5X10(-6) M) in a competitive manner. Wh en 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). Incorporatio n 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 considerabl y different inhibitory potencies. The R-isomer was as active as the be nzyl amide, while the S-isomer was about 30-fold less active than the benzyl amide. Furthermore, when a fluorine atom was introduced into th e para-position of the amide-benzyl group, the resulting H-D-Thr-Phe-N H-CH2-C6H4(p-F) showed considerably enhanced inhibitory activity (abou t 5-fold, K-i=9.1X10(-7) M). In conformational analysis by 400 MHz H-1 -NMR, all dipeptides having D-Thr-Phe backbone structure showed large upheld shifts of D-Thr-beta OH (shifts in ppm, 0.09-0.17), D-Thr-beta CH (0.23-0.32), and D-Thr-gamma CH3 (0.38-0.53), indicating the presen ce of shielding effects from the benzene ring. In addition, NOE enhanc ements between the D-Thr-gamma CH3, and Phe-phenyl groups were evidenc ed by measurements of two-dimensional NOESY spectra and NOE difference spectra. These observations demonstrated the spatial proximity of the se side chains, which is due to side chain-side chain CH/pi interactio n. All these results support the idea that the amide-benzyl group bind s at the chymotrypsin S-1 site, while the hydrophobic core with CH/pi interaction binds at the S-2 or S-1 site.