title: Chymotrypsin inhibition induced by side chain-side chain intramolecular CH/pi interaction in D-Thr-L-Phe benzylamide.
abstract: The dipeptide benzyl amide H-D-Thr-Phe-NH-CH2-C6H5 was found to inhibit chymotrypsin strongly (K1 = 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, K1 = 9.1 x 10(-7) M). In conformational analysis by 400 mHz 1H-NMR, all dipeptides having D-Thr-Phe backbone structure showed large upfield 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 presence of shielding effects from the benzene ring. In addition, NOE enhancements between the D-Thr-gamma CH3 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/pi interaction. All these results support the idea that the amide-benzyl group binds at the chymotrypsin S1 site, while the hydrophobic core with CH/pi interaction binds at the S2 or S1' site.
authors.affiliation: Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka.
authors.forename: I
authors.initials: I
authors.lastname: Maeda
authors.forename: Y
authors.initials: Y
authors.lastname: Shimohigashi
authors.forename: K
authors.initials: K
authors.lastname: Ikesue
authors.forename: T
authors.initials: T
authors.lastname: Nose
authors.forename: Y
authors.initials: Y
authors.lastname: Ide
authors.forename: K
authors.initials: K
authors.lastname: Kawano
authors.forename: M
authors.initials: M
authors.lastname: Ohno
mesh_terms: D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D004151:Dipeptides / Q000737:chemistry* / Q000494:pharmacology*; D007536:Isomerism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D011487:Protein Conformation; D012995:Solubility; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004151:Dipeptides; D002918:Chymotrypsin