Investigations of the Active Site of Microsomal Leucine Aminopeptidase by Probing with Ethylenediamine Derivatives

Abstract

The active site of porcine microsomal aminopeptidase was probed by studying the inhibition of the enzyme using derivatives of ethylenediamine and diaminopropionic acid. In addition, some amino acids, substituted hydroxamates and phosphates were also tested. In order to synthesize diaminopropionic acid derivatives, CBZ-amino acid p-nitrophenyl esters were reduced to the corresponding aldehydes by lithium tri-t-butoxy-aluminohydride. Through the Strecker synthesis, the aldehyde intermediates were converted to diaminopropionitrile analogues which were then hydrolysed in acid to the desired products. Unfortunately, these compounds were not potent inhibitors for this enzyme. α-Amino acids were found to be better inhibitors than their β-amino counterparts and the Kᵢ of α-leucine was about 7-fold lower than its B-analogue. The amino group position of the amino acids is therefore important for enzyme recognition. On the other hand, N-alkylation of ethylenediamine was observed to abolish its inhibition potential. Furthermore, another unexpected finding in this work is that N- or 0-methylation of the hydroxamate group hinders the ability of these inhibitors to act as a bidentate zinc ligand. Although some phosphate derivatives that we tested showed poor inhibitory potency, phosphonamidate, a potential transition state analogue, might serve as a powerful inhibitor. In summary, the relationship between the structure and inhibitory potency of some inhibitors was demonstrated.