Synthesis and Performance of <scp>l</scp>-Tryptophanamide and (<i>S</i>)-1-(Naphthalen-2′-yl)ethanamine-Based Marfey-Type Derivatives for Amino Acid Configurational Analysis: Diastereomeric Resolutions Directed by π–Cation Bonding

The configurational analysis of amino acids (AAs) in natural product peptides, often containing nonproteinogenic AAs, is mostly carried out by the venerable Marfey's method using a chiral derivatizing agent (CDA) 1-fluoro-2,4-dinitrophenyl-5-l-alaninamide (l-FDAA)─Marfey's reagent─which undergoes <i>S</i>_NAr reaction of the 1° amino group. The resulting AA-DAA derivatives are mostly well-separated by reversed-phase HPLC, but some DAA derivatives resist resolution. Here, we report the synthesis and characterization of two CDAs: l-FDTA (<b>4</b>) in which the l-alanine-derived auxiliary is replaced by l-tryptophanamide and (<i>S</i>)-FDNE (<b>3</b>) where the auxiliary is <i>S</i>-(6-methoxynaphth-2-yl)-1-ethylamine. Side-by-side comparisons of the two reagents were carried out by AA derivatization and reversed-phase HPLC analysis with variables such as organic solvent, additives, and the ionic strength of the mobile phase. l-DTA derivatives of l- and d-AAs were found to show superior HPLC performance and an improvement in resolutions. When incorporated into the mobile phase, the ammonium ion (NH₄⁺, 0-100 mM) showed a dramatic influence on differential retention times [Δ<i>t</i>_R = Δ<i>t</i>_Rd - Δ<i>t</i>_Rl] of several key AAs. We attributed the effect to π-cation interactions between the indole ring of DTA and the NH₄⁺ counterion in the analyte, a hypothesis supported by ¹H NMR titrations and DFT calculations.