INHIBITION OF THERMOLYSIN AND CARBOXYPEPTIDASE-A BY PHOSPHORAMIDATES

A series of N-phosphorylated dipeptides and amino acid derivatives have been synthesized. These compounds, which are analogues of phosphoramidon, are competitive inhibitors of thermolysin. The simple phosphoramidate P-Leu-NH2 was an excellent inhibitor with a K1, of 1.3 μM, while H-Leu-NH2 is ca. 103 times poorer. O-Methylphosphoryl-Leu-NH2 inhibits thermolysin 100 times poorer than P-Leu-NH2. O-Methyl-O-phenylphosphoryl-Leu-NH2 and N-ethylphosphoramidate do not show any inhibition at 3 mM. It is suggested that the N-phosphorylleucyl moiety plays the major role in the inhibition and accounts for at least 65% of the free energy of binding of phosphoramidon. The N-phosphorylated dipeptides P-Ile-Ala-OH, P-Leu-Phe-OH, and P-Leu-Trp-OH inhibited thermolysin up to 85-fold better than P-Leu-NH2. A hydrophobic P1 amino acid is required since P-Ala-Ala-OH binds 240-fold less tightly than P-Ile-Ala-OH. An aromatic residue at P2‘ also contributes to the binding since the K1 of P-Leu-Trp-OH or P-Leu-Phe-OH is ca. 200 times lower than that of P-Ile-Ala-OH. The chelation of the active site zinc atom by the phosphoryl group and a hydrophobic interaction between the side chain of the P1‘ amino acid residue and the S1‘ subsite of the enzyme are the primary factors responsible for the inhibition. Additional interactions with Tyr-153 and His-231 are proposed to account for the lower K1 values of phosphoramidates when compared with their monoalkyl esters. N-Phosphorylphenylalanine (P-Phe-OH) is a good inhibitor of carboxypeptidase A, and other N-phosphoryl derivatives, P-Leu-NH2, P-Leu-Trp-OH, and P-NH-Et, also show weaker inhibition. Phosphoramidate derivatives of the appropriate amino acids or peptides should be useful as reversible inhibitors or affinity ligands for other metalloproteases. © 1979, American Chemical Society. All rights reserved.