Specific inhibitory conformation of dipeptides for chymotrypsin

Based on the analyzed conformation of a chymotrypsin inhibitor H-{down triangle, open}Phe-Phe-OMe, we have designed a series of diastereomeric phenylalanylphenylalanine methyl esters and derivatives as possible inhibitors. Among the peptides synthesized, H-D-Phe-L-Phe-OMe was found to be very resistant to chymotrypsin in spite of its L-Phe-OMe structure at the C-terminus. It inhibited the enzyme fairly strongly and competitively with K_i = 9.0 × 10^-5 M in the assay using Ac-Tyr-OEt as a substrate. The measurements of the NOEs in high-resolution ¹H-NMR analyses indicated the presence of the hydrophobic core built by the intramolecular interaction between the D-Phe-phenyl and ester-methyl groups. It was suggested that this core interacts with the chymotrypsin S₂ site (Trp²¹⁵) and Phe² with the S₁ site. The backbone structure of this dipeptide was assumed to be in an inhibitory conformation that fits the active center of the enzyme.