The kinetically controlled synthesis of N-benzyloxycarbonyl (Z)-dipeptides was investigated by the use of free amino acids as nucleophiles and a cysteine protease papain as catalyst. The coupling efficiency was significantly improved by the combined use of the carbamoylmethyl (Cam) ester of a Z-amino acid as acyl donor and frozen aqueous solution (ice, -16 or -24 degrees C) as reaction medium. The yield of peptide synthesis became high when both P-1- and P-1'-positions were occupied by small non-polar amino acids (Z-Gly-Gly-OH, 76%: Z-Gly-Ala-OH, 75%: Z-Ala-Ala-OH, 72%). Similar results were observed by the use of ficin as catalyst instead of papain. Furthermore, this strategy was applied to the papain-catalyzed incorporation of a D-configured amino acid such as D-alanine into the resulting peptides. Although the coupling in aqueous solution (30 degrees C) afforded the desired Z-dipeptides in low yields, the freezing of reaction medium reduced significantly unfavorable hydrolysis of the acyl donors, resulting in improvement of the coupling efficiency (Z-Gly-D-Ala-OH, 80%; Z-Ala-D-Ala-OH, 45%: Z-D-Ala-Ala-OH, 22%). (C) 2008 Elsevier Inc. All rights reserved.