Chymotrypsin inhibitory conformation of dipeptides constructed by side chain–side chain hydrophobic interactions

✍ Scribed by Hiroshi Sakamoto; Yasuyuki Shimohigashi; Iori Maeda; Takeru Nose; Kin-ichi Nakashima; Ichiro Nakamura; Tomoshisa Ogawa; Motonori Ohno; Keiichi Kawano

• Publisher: John Wiley and Sons
• Year: 1993
• Tongue: English
• Weight: 639 KB
• Volume: 6
• Category: Article
• ISSN: 0952-3499
• DOI: 10.1002/jmr.300060207

✦ Synopsis

Abstract

A complete series of configurationally isomers (L‐L, L‐D, D‐L AND D‐D) of a dipeptide Leu‐Phe benzyl ester have been synthesized and assayed for chymotrypsin. In the conformational analysis by 400 MMz ^1^H NMR, the L‐D and D‐L isomers, but not hte L‐L and D‐D isomers, showed fairly large up field shifts (0.2–0.4 ppm) of Leu‐βCH~2~ and γCH proton signals, indicating the presence of shielding effects from the benzene ring. In addition to distinct signal splitting of Phe‐βCH~2~, the NOE enhancement observed between Leu‐δCH~3~ and Phe‐phenyl groups revealed that these groups are in close proximity. These data indicated that L‐D and D‐L isomers from a hydrophobic core between side chains of adjacent Leu and Phe residues. When the dipeptides were examined for inhibition of chymotrypsin using Ac‐Try‐OEt as a substrate, the L‐L isomer showed no inhibition, itself becoming a substrate. However, the other three isomers inhibited chymotrypsin in a competitive manner, and the D‐L isomer was strongest with K~i~ of 2.2 × 10~−5~ M. It was found that the D‐L isomer was only slowly hydrolysed but the L(or D)‐D isomer was not. H‐D‐Phe‐L‐Leu‐OBzl with the inverse sequence of H‐D‐Leu‐L‐Pre‐OBzl inhibited chymotrypsin more strongly (K~i~ = 6.3 × 10^−6^ M). Since the free acid analogue of the D‐L isomer exhibited no inhibition, the benzyl ester moiety itself was thought to be involved in the enzyme inhibition. It is assumed that in the inhibitory conformation the ester‐benzyl group fits the S~1~ site of chymotrypsin, while the side chain‐side chain complexing hydrophobic core fits the S~2~ site.