Fmoc-Val-OH-[15N]

A novel strategy for a more efficient synthesis of difficult sequence-containing peptides, the S-acyl isopeptide method, was developed and successfully applied. A model pentapeptide Ac-Val-Val-Cys-Val-Val-NH2 was synthesized via its water-soluble S-acyl isopeptide using an S-acyl isodipeptide unit, Boc-Cys(Fmoc-Val)-OH. An S-acyl isopeptide possessing excellent water solubility could be readily and quantitatively converted to the native peptide via an S--N intramolecular acyl migration reaction at pH 7.4. Thus, the S-acyl isopeptide method provides a useful tool in peptide chemistry.

A PEGA-resin was derivatized with a 3:1 mixture of hydroxymethyl benzoic acid and Fmoc-Lys(Boc)-OH and the fluorogenic substrate Ac-Y(NO2)KLRFSKQK(Abz)-PEGA was assembled on the lysine using the active ester approach. Following esterification of the hydroxymethyl benzoic acid with Fmoc-Val-OH a library XXX-k/r-XXXV containing approximately 200,000 beads was assembled by split synthesis. The resulting 'one bead, two peptides' library was subjected to extensive hydrolysis with cruzipain. One hundred darker beads were isolated and the 14 most persistently dark beads were collected and sequenced. The putative inhibitor peptides and several analogues were synthesized and found to be competitive microM to nM inhibitors of cruzipain in solution. The inhibitory activity was found to be unspecific to cruzipain when compared with cathepsins B and L and specific when compared with kallikrein. One of the inhibitors was docked into the active site of cathepsin B and was found most probably to bind to the enzyme cavity in an unusual manner, owing to the inserted D-amino acid residue.