The macrocyclic peptidyl phosphonate 1-L was designed on the basis of the conformation of an acyclic analogue (4) bound to the aspartic protease penicillopepsin. This material and the two acyclic comparison compounds 2-L and 3 were synthesized and evaluated as inhibitors; their binding affinity was found to be inversely related to the degree of conformational flexibility across the series : 3 (K-i = 110 mu M), 2-L (K-i = 7.6 mu M), 1-L (K-i = 0.80 mu M). NMR methods in conjunction with molecular modeling were used to assign the stereochemical configurations of the precursor 16-L and its diastereomer 16-D and to determine the solution conformations of the macrocyclic ring systems. The conformation of the peptide backbone in 1-L closely approximates that desired for a mimic of the lead inhibitor 4, and it appears that the low-energy conformation of 1-L can be accommodated in the pencillopepsin active site without significant distortion.