Through application of a thorough design and modeling protocol, the macrocyclic peptidyl phosphonate 5 was derived from the bound structure of an acyclic inhibitor of penicillopepsin, 4, by linking the P1＇ and P2 side chains : the acyclic analogues 6 and 7 were also synthesized and evaluated for comparison. As observed for a related set of inhibitors, 1-3 [Meyer, J.H.; Bartlett, P.A. J. Am. Chem. Soc. 1998, 120, xxxx], the binding affinity at pH 4.5 was found to be inversely related to the degree of conformational flexibility across the series, 7 (K-i = 1300 nM), 6 (K-i = 42 nM), and 5 (K-i = 0.10 nM), although the differences are much greater. The structure of the macrocyclic ring of 5 in solution was determined by a combined NMR and molecular modeling method and found to correspond closely to the designed structure and to the backbone conformation of the original target, 4, The binding enhancement conveyed by the macrocyclic constraint corresponds to 0.9-1.4 kcal/mol/deg of rotational freedom lost, indicating what can be gained from this structure, based design strategy.