Glycyl endopeptidase catalysed solid-to-solid synthesis can be carried out efficiently for the model peptides Z-Gly-Phe-NH2, Z-Gly-Leu-NH2, Z-Gly-Tyr-NH2 and Z-Gly-Tyr-OEt. A small excess of acyl donor Z-Gly improved both the initial rate and final yield, whereas an excess of nucleophile Phe-NH2 prevented reaction. The highest conversion was achieved when using a substrate molar ratio (Z-Gly:Phe-NH2) of 2:1. Including solid cysteine in the reaction mixture improved both initial rate and final conversion, probably by protecting the enzyme from oxidation. The reasons why conversion to Z-Gly-Phe-NH2 stopped at around 83% were investigated. Entrapment of residual solid starting material did not seem significant, while autolysis and inactivation of glycyl endopeptidase in the reaction mixture during catalysis was important. The role of chemical equilibrium position was less clear. (c) 2006 Elsevier Inc. All rights reserved.