MACROCYCLIC INHIBITORS OF PENICILLOPEPSIN - 2 - X-RAY CRYSTALLOGRAPHIC ANALYSES OF PENICILLOPEPSIN COMPLEXED WITH A P3-P1 MACROCYCLIC PEPTIDYL INHIBITOR AND WITH ITS 2 ACYCLIC ANALOGS

Macrocyclic inhibitor 1 )phosphinyloxyethyl]-1-naphthaleneacetamide]so dium salt} was designed according to the conformation of the acyclic a nalogue Iva-L-Val-L-Val-L-Leu(P)-(O)Phe-OMe [Leu(P) = the phosphinic a cid and analogue of L-leucine; (O)Phe = L-3-phenyllactic acid; OMe = m ethyl ester] (4) bound to penicillopepsin, by linking the P1 and P3 si de chains of the penicillopepsin inhibitor. This compound and its two acyclic derivatives, yl)ethyl)hydroxyphosphinyloxy]-3-phenylpropanoate , sodium salt} (2) and nomethyl)hydroxyphosphinyloxy]-3-phenylpropanoa te, sodium salt} (3), have been synthesized and evaluated as inhibitor s of penicillopepsin. Their binding affinity to the enzyme was found t o be inversely related to the predicted degree of conformational flexi bility across the series: 3 (K-i = 110 mu M), 2 (K-i = 7.6 mu M), 1 (K -i = 0.8 mu M). The X-ray crystallographic structures of penicillopeps in complexed with the macrocyclic peptidyl phosphonate 1 and with its two derivatives 2 and 3 have been determined and refined to crystallog raphic agreement factors R (=Sigma\\F-o\ - \F-c\\/Sigma\F-o\) of 15.9% , 16.0%, and 15.2% and R-free of 19.8%, 20.3%, and 21.4%, respectively . The intensity data for all complexes were collected to 1.5 Angstrom resolution. One 1.25 Angstrom resolution data set was obtained for the complex with 1 at 110 K; the structure was refined to an R factor of 15.0% (R-free of 19.7%) The binding interactions that 1 and 2 make wit h penicillopepsin are similar to those that have been observed for oth er transition-state mimics with aspartyl proteinases. On the other han d, the acyclic Linear inhibitor 3 exhibits distinctive binding to peni cillopepsin with the phosphonate group shifted similar to 3.0 Angstrom from the average position observed for the other complexes. These str uctural results show that the macrocyclic constraint of 1 enhances its binding affinity over those of the acyclic analogues but the differen ces in the observed bound dispositions mean that the effect has yet to be quantified.