Abnormal elution behavior of ornitine derivatized with 1-fluoro-2,4-dinitrophenyl-5-leucinamide in advanced Marfey’s method

During the course of establishing the advanced Marfey’s method that has been developed to non-empirically determine the absolute configuration of constituent amino acids in a peptide using LC–MS, we encountered the “ornitine mystery” in the di-DLA (2,4-dinitrophenyl-5-leucinamide) derivative such that the elution order of ornitine (Orn) was opposite (d→l) in spite of their relatively long retention time. In order to resolve this problem, the elution behavior of several mixed DLA and DPEA (2,4-dinitrophenyl-5-phenylethylamine) derivatives with different absolute configurations was carefully observed during HPLC. The length of the methylene chain in basic amino acids was obviously critical for this behavior, because Dab (2,4-diamino-n-butyric acid) and lysine (Lys) did not exhibit this abnormality. The presence of the carboxyamide moiety at the ω position was also essential for this phenomenon, because it was never observed in the DPEA derivatives at the ω position. Furthermore, it was found that the following combination of absolute configurations of Orn and DLA at the ω position only induced this abnormality: d-Orn and l-DLA, and l-Orn and d-DLA. This suggested that the structural interaction such as hydrogen bonding between the carboxyamide of DLA at the ω position and carboxylic acid at the α position in these derivatives reduced their retention power on the reversed-phase column.