Differential Binding Energy: A Detailed Evaluation of the Influence of Hydrogen-Bonding and Hydrophobic Groups on the Inhibition of Thermolysin by Phosphorus-Containing Inhibitors

Abstract

Two series of phosphorus-containing peptide analogues, 3 (Cbz-Gly-ψ(PO2-CH2)Leu-Xaa) and 4 (Cbz-Gly-ψ-(PO2-NH)Leuψ[CO2]Xaa), have been synthesized and evaluated as inhibitors of the zinc endopeptidase thermolysin. In comparison with the previously reported phosphonamidates 1, the phosphinates 3 lose only 0.1 kcal/mol in binding affinity, whereas the depsipeptides 4 are bound 2.7 kcal/mol more weakly; these values are contrasted to the 4.0 kcal/moi reduction in binding affinity observed for the phosphonates 2 (Cbz-Gly-ψ(PO2-O)Leu-Xaa) in comparison to 1 (Cbz-Gly-ψ(PO2-NH)Leu-Xaa). The observed effects are interpreted through consideration of the differences in active-site and solvent interactions. For the comparison between the diamides 1 and the depsipeptides 4, a full accounting of the balance between these interactions can be approached. The arylphosphonates 5 (Aryl-ψ(PO2-O)Leu-Leu) were synthesized and evaluated to investigate the importance of phosphonate basicity on the overall binding affinity of these zinc-coordinating inhibitors; the inhibitor Ki values were found to be independent of phosphonate pKa, indicating that the basicity of the phosphonate moiety exerts counterbalancing effects on the energies of zinc coordination and solvation. For analysis of the influence of structural variations on observed affinity, the definition of “differential binding energy” is introduced as a practical alternative to the concept of “intrinsic binding energy”. © 1991, American Chemical Society. All rights reserved.