N-Benzyl Residues as the P1′ Substituents in Phosphorus-Containing Extended Transition State Analog Inhibitors of Metalloaminopeptidases

Abstract

Peptidyl enzyme inhibitors containing an internal aminomethylphosphinic bond system (P(O)(OH)-CH2-NH) can be termed extended transition state analogs by similarity to the corresponding phosphonamidates (P(O)(OH)-NH). Phosphonamidate pseudopeptides are broadly recognized as competitive mechanism-based inhibitors of metalloenzymes, mainly hydrolases. Their practical use is, however, limited by hydrolytic instability, which is particularly restricting for dipeptide analogs. Extension of phosphonamidates by addition of the methylene group produces a P-C-N system fully resistant in water conditions. In the current work, we present a versatile synthetic approach to such modified dipeptides, based on the three-component phospha-Mannich condensation of phosphinic acids, formaldehyde, and N-benzylglycines. The last-mentioned component allowed for simple and versatile introduction of functionalized P1′ residues located on the tertiary amino group. The products demonstrated moderate inhibitory activity towards porcine and plant metalloaminopeptidases, while selected derivatives appeared very potent with human alanyl aminopeptidase (Ki = 102 nM for 6a). Analysis of ligand-protein complexes obtained by molecular modelling revealed canonical modes of interactions for mono-metallic alanyl aminopeptidases, and distorted modes for di-metallic leucine aminopeptidases (with C-terminal carboxylate, not phosphinate, involved in metal coordination). In general, the method can be dedicated to examine P1′-S1′ complementarity in searching for non-evident structures of specific residues as the key fragments of perspective ligands.

Phosphinotripeptidic Inhibitors of Leucylaminopeptidases

“…Moreover, Grembecka et al showed that the crystal structure of bovine lens LAP can be applicable to the design of new inhibitors of LAP from other organism tissues [ 4 ]. Furthermore, Wanat et al, as well as Janiszewska et al showed that the known structure of tomato LAP is a good model of the barley seeds LAP [ 22 , 24 ]. All the above-mentioned observations make the use of enzymes from two different organisms for biological testing and molecular modeling highly justified.…”

“…The optimization of the character of residues in positions P1 and P1’ in phosphinic dipeptides obtain the first most potent unnatural inhibitors of LAP [ 18 ]. The further investigation, focused on modification of side chains in the positions P1 and P1′, lead to numerous phosphinate and phosphinic dipeptidic inhibitors of LAP [ 19 , 20 , 21 , 22 , 23 , 24 ].…”