The solution conformation of (D)Phe-Pro-containing peptides: implications on the activity of Ac-(D)Phe-Pro-boroArg-OH, a potent thrombin inhibitor.

Abstract

Ac-(D)Phe-Pro-boroArg-OH is a potent, competitive inhibitor of thrombin (Ki = 40 pM). 1H-NMR studies have shown that the peptide portion, -(D)Phe-Pro-, has secondary structure in aqueous solutions. This structure corresponds fairly closely to the structure of H-(D)Phe-Pro-ArgCH2Cl complexed to thrombin in the protein crystal structure (Bode, W.; et al. EMBO J. 1989, 193, 3467-3475.). These results indicate that, in addition to enthalpic interactions in the active site of the enzyme, there are significant entropic advantages in binding this molecule not previously recognized. We estimate that they contribute approximately 10-fold to binding. The structure we have observed can be explained by pi-pi interactions between the phenyl side chain of (D)Phe and the (D)Phe-Pro peptide bond. Assignment of structure is based first on the 0.8-1.2 ppm difference between the two Pro C delta protons. The magnitude of these chemical shifts are consistent with aromatic ring current-induced effects expected for distances in our structure. The structure was further defined by interproton distances and correlation times calculated by backtransformation and correction of the NOESY and ROESY data to the longitudinal and transverse cross relaxation rates. Analysis of the vicinal coupling constants show that Phe chi 1 is not fixed. Correlation times for the peptide side chains and backbone indicate that the phenyl ring and boroArg side chain possess various degrees of internal motion, and that the rest of the peptide has a fairly rigid conformation.