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As the first step in establishing our proposed method, the advanced Marfey's method, which is planned for the simultaneous determination of the absolute configuration of amino acids in a peptide, we applied Marfey's method to commercially available amino acids, and the separation behavior was examined in detail. Although good resolution of the diastereomeric pair of an individual amino acid was obtained for all amino acids tested and the applicability of the method was confirmed, the (1-fluoro-2,4-dinitrophenyl)-5-l-alaninamide (FDAA) derivative of the l-amino acid was not always eluted prior to its corresponding d-amino acid derivative. Because this proposed method relies on the elution order of a derivatized amino acid with FDAA to determine its absolute configuration, its separation mechanism was carefully investigated using UV and NMR spectral techniques. The results suggested that the resulting conformations of the l- and d-amino acid derivatives are stable and that the resolution between the l- and d-amino acid derivatives is due to the difference in their hydrophobicity, which is derived from the cis- or trans-type arrangement of two more hydrophobic substituents at both α-carbons of an amino acid and l-alanine amide, so that the FDAA derivative of the cis (Z)-type arrangement interacts more strongly with ODS silica gel and has a longer retention time than that of the trans (E)-type arrangement. Therefore, the l-amino acid derivative is usually eluted from the column before its corresponding d-amino acid derivative in Marfey's method. According to this separation mechanism, the elution order of a desired amino acid can be elucidated from the average retention time of the l- and d-amino acid derivatives, and the dl-serine and -asparagine derivatives are critical for Marfey's method.

Microcystins LR, YR, and RR, cyclic heptapeptide hepatotoxins produced by cyanobacteria, were synthetically converted into glutathione (GSH) and cysteine (Cys) conjugates. Fast atom bombardment mass spectra showed [M + H]+ ions corresponding to GSH and Cys conjugates of microcystins LR, YR, and RR for the obtained compounds. 1H NMR spectral analyses revealed that two singlet signals of olefinic protons of N-methyldehydroalanine (Mdha) in microcystins disappeared in the conjugates, confirming that thiols of GSH and Cys added nucleophilically to the alpha, beta-unsaturated carbonyl of the Mdha moiety. On examination of the 50% lethal dose (LD50) with intravenous injection using mice, both GSH and Cys conjugates showed reduction in toxicity compared with microcystins, but their toxicity still remained. Microcystin LR and its GSH conjugate were separated and identified in a standard mixture by using a frit-fast atom bombardment liquid chromatography/mass spectrometry (Frit-FAB LC/MS) method. Obtained conjugates in the present study would be important compounds as the standard samples for study of metabolism of microcystins, and the Frit-FAB LC/MS method would be applicable to mass spectrometric identification of metabolites of microcystins.

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Total structures of 13 minor components of bacitracin (BC) were proposed, and their antimicrobial activities were investigated. The components of BCincluding bacitracins A (BC-A) and F (BC-F) were isolated by preparative HPLCand were hydrolyzed under acidic conditions.The resulting amino acids were derivatized with l-fluoro-2,4-dinitrophenyl-5-L-alanineamide and were separated by HPLCto determine their absolute configurations. It was found that the TV-terminal amino acids of BC-Aand its related components were epimerized during the hydrolysis to yield their enantiomers. The formation of these artifactual amino acids suggests that our previously proposed structures of the BCminor componentsare incorrect; therefore, the structures were corrected based on these results. The structures of the BCminor componentswere the same as that of BCs-A and -F except that one to three of the L-isoleucines, including the TV-terminal one, were replaced by L-valines. These structures were confirmed by tandem mass spectrometry under fast atom bombardment (FAB) conditions and Frit-FAB liquid chromatography/mass spectrometry. Based on the UVspectra of the BC components determined by photodiode array detection-HPLC analysis, a new systematic nomenclature was proposed for the minor components. The isolated components were also used for the determination of their minimal inhibition concentrations and it was found that BC-Ais 2~8 times more potent than the other minor componentsagainst strains of Micrococcus luteus and Staphylocoecusaureus.

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