Investigations on the enzyme specificity of clostripain: A new efficient biocatalyst for the synthesis of peptide isosteres

To explore the ability of the cysteine protease clostripain as a biocatalys t for the synthesis of peptide isosteres, the S'-subsite specificity of thi s enzyme toward unnatural substrates was investigated. First, the function of clostripain for acylating aliphatic noncyclic and cyclic amines varying in chain length and ring size was analyzed using a standard acyl donor. Add itionally, this series was expanded by use of aromatic amines, amino alcoho ls, derivatives of non-a-amino carboxylic acids, and symmetric and asymmetr ic diamines, respectively. The results obtained give a detailed picture of the unique reactivity of clostripain toward synthetic substrates, allowing insights into the basic enzyme-substrate interactions. Furthermore, the dat a provide a guideline for the use of clostripain as a biocatalyst for synth esis of peptide isosteres. The study was completed by the utilization of a model substrate mimetic enabling clostripain to react with noncoded and non -amino acid-derived amines as well as nonspecific acyl moieties. The result s of this study indicate that this approach may extend the application rang e of clostripain as a biocatalyst outside of peptide synthesis.