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 bee n developed to non-empirically determine the absolute configuration of cons tituent amino acids in a peptide using LC-MS, we encountered the "ornitine mystery" in the di-DLA (2,4-dinitrophenyl-5-leucinamide) derivative such th at the elution order of ornitine (Orn) was opposite (D -->L) in spite of th eir relatively long retention time. In order to resolve this problem, the e lution behavior of several mixed DLA and DPEA (2,4-dinitrophenyl-5-phenylet hylamine) derivatives with different absolute configurations was carefully observed during HPLC. The length of the methylene chain in basic amino acid s was obviously critical for this behavior, because Dab (2,4-diamino-n-buty ric acid) and lysine (Lys) did not exhibit this abnormality. The presence o f the carboxyamide moiety at the omega position was also essential for this phenomenon, because it was never observed in the DPEA derivatives at the w position. Furthermore, it was found that the following combination of abso lute configurations of Orn and DLA at the w position only induced this abno rmality: D-Orn and L-DLA, and L-Orn and D-DLA. This suggested that the stru ctural interaction such as hydrogen bonding between the carboxyamide of DLA at the omega position and carboxylic acid at the alpha position in these d erivatives reduced their retention power on the reversed-phase column. (C) 2001 Elsevier Science BN. All rights reserved.