Development and application of highly sensitive labeling reagents for amino acids

Peptide natural products produced by microorganisms have attracted considerable attention as ideal drug leads owing to their low toxicity and high specificity toward target proteins compared with small-sized molecules. These peptide drug leads possess unusual structural features that endow them with unique biological activities and ideal physicochemical properties. In particular, these peptides often have d-amino acids, and therefore the absolute configuration of the component amino acids must be determined during the structural elucidation of newly isolated peptide drug leads. Recently, we developed highly sensitive labeling reagents FDVDA and FDLDA for the structural determination of the component amino acids in peptides. In an LC-MS-based structural study of peptides, these reagents enabled us to detect infinitesimal amounts of amino acids derived from mild degradation of the samples; we named this method the highly sensitive-advanced Marfey's method (HS-advanced Marfey's method). Herein, we first report the synthesis of these reagents and the LC-MS protocols for highly sensitive analyses of amino acids. Second, we discuss applications of the design concept. Specifically, two other labeling reagents were synthesized and their performance in terms of detection sensitivity was evaluated. These investigations provide insights on the structure-property relationship of these labeling reagents and therefore facilitate future on-demand structural modifications of the reagents to enhance their hydrophobicity, stability, and affinity for use with specialized HPLC columns. Finally, we demonstrated the effectiveness of our highly sensitive labeling reagents by using them to detect component amino acids in peptide natural products.