Synthesis and Performanceof l‑Tryptophanamideand (<i>S</i>)‑1-(Naphthalen-2′-yl)ethanamine-BasedMarfey-Type Derivatives for Amino Acid Configurational Analysis: DiastereomericResolutions 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.