Abstract
7-Amino-4-methylcoumarin (AMC) is a low molecular weight fluorescent probe that can be attached to a peptide to enable the detection of specific proteases, such as chymotrypsin, expressed in certain diseases. Because this detection depends on the specificity of the protease toward the peptidyl AMC, the development of specific substrates is required. To investigate the specificity of chymotrypsin, peptidyl AMC compounds incorporating four different amino acid residues were prepared by liquid-phase synthesis. Two unnatural amino acids, 2-amino-4-ethylhexanoic acid (AEH) and cyclohexylalanine (Cha), were used to investigate the substrate specificity as these amino acids have structures different from natural amino acids. AEH was synthesized using diethyl acetamidemalonate as a starting material. The substrate containing Cha had high hydrophobicity and showed a high reaction velocity with chymotrypsin. Although the AEH substrate with a branched side chain had high hydrophobicity, it showed a low reaction velocity. The substrate containing the aromatic amino acid phenylalanine was less hydrophobic than the Cha and AEH substrates, but chymotrypsin showed the highest specificity for this compound. These results demonstrated that the substrate specificity of chymotrypsin is not only affected by the hydrophobicity and aromaticity, but also by the structural expanse of amino acid residues in the substrate.
Highlights
The relative hydrophobicity of the peptide peptide substrates could be determined based on the retention times in the used substrates could be determined based on the retention times in the HPLC used for purificafor purification
The m value, of kcat /Km values of the Cha and AEH substrates with higher hydrophobicity than the Phe substrate 3 were lower than those of the Phe substrate 3. These results suggested that the specificity of chymotrypsin for a particular substrate may be because of the presence of an aromatic amino acid residue rather than a hydrophobic amino acid residue
The synthesized AEH was incorporated into a tripeptide as a novel peptide substrate
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Analysis of the interaction of proteases with substrates can assist in the development and analysis of the characteristics of protease inhibitors [14] Such analyses are frequently performed with fluorescent probes for analysis of the protease activity [15,16]. Substrates with natural amino acids are known to be hydrolyzed chymotrypsin [28]. Substrate peptides containing unnatural amino by chymotrypsin [28]. Substrate containing unnatural amino acids were synthesized, amino acid residues for peptides which chymotrypsin was highly speacids were synthesized, and amino acid residues for which chymotrypsin was highly cific were explored. For, which exceeds that of substrates containing natural amino acids
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