18 A DETERMINATION METHOD OF CONSTITUENT AMINO ACIDS IN PEPTIDE USING LC/MS : ADVANCED MARFEY'S METHOD

In 1984, Marfey reported that a mixture of D- and L-amino acid can be separated into each enantiomer by usual reversed phase HPLC after derivatization with FDAA (1-fluoro-2,4-dinitrophenyl-5-L-alanine-amide). The method has been refered to as "Marfey's method" and has an advantage that it can determine sterochemistry of an amino acid by simple operation. Although it has been widely used, it is hard to apply the method to non-proteinogenic amino acid, because it is difficult to obtain authentic sample. In such a case it would be effective to combine Marfey's method with an appropriate mass spectrometry. In this study we tried to establish a total method for determination of constituent amino acids including stereochemistry, even unusual amono acids, in peptides without authentic sample. In order to establish the total system shown in Fig. II, the following four problems have to be resolved: 1. Elucidation of limitation of Marfey's method and its separation mechanism 2. Optimization of various conditions for combination of Marfey's method and mass spectrometry 3. Collection of structural information by LC/MS and optimization of epimerization 4. Application of the advanced method to natural products As a result of extensive experiments Marfey's method proved to have a wide applicability except for a few basic amino acids. A separation mechanism was proposed based on NMR measurement of D,L-Val-FDAA derivative and elution behavior of various amino acids. According to this mechanism both isomers can be resolved due to the difference of their hydrophobicity which is derived from cis or trans configuration of two more hydrophobic substituents at both α-carbons of introduced amino acid and L-Ala-NH2, so that D-isomer interacts more strongly with ODS silica gel and has longer retention time. Although Frit-FAB and ESI were used as interfaces in the present study, derivatized amino acids with FDAA showed poor sensitivities. Since it was considered that this result may be due to poor hydrophobicity of the derivatives, a new derivatization reagent, FDLA (1-fluoro-2,4-dinitrophenyl-5-L-leucine-amide) was prepared. Derivatized amino acids with FDLA showed the almost same retention behavior as that with FDAA and much stronger sensitivity than that with FDAA by both ionization methods. The advanced Marfey's method was successfully applied to analysis of hydrolyzate of a known antibiotic bacitracin A composed of 12 amino acid residues and it was ensured that the combination method is much superior to the original method. The method is being applied to analysis of naturally occurring peptides from cyanobacteria and would contribute to progress of various researches in this field.