A Nonempirical Method Using LC/MS for Determination of the Absolute Configuration of Constituent Amino Acids in a Peptide:  Elucidation of Limitations of Marfey's Method and of Its Separation Mechanism

Abstract

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.

A new anti-MRSA antibiotic complex, WAP-8294A II. Structure characterization of minor components by ESI LCMS and MS/MS

“…Their absolute configurations were determined by amino acid analysis using the advanced Marfey's method. [14][15][16] The mass chromatograms of the amino acid derivatives from 1 and 4 using ESI-LC/MS are shown in Figures 2A and B Table 1.…”

“…14,15 Each 500 mg sample was hydrolyzed at 110 1C for 1 h with 500 ml of 6 M HCl. This solution was divided into two portions, and each portion was derivatized with L-or D-FDLA.…”

Production of Secondary Metabolites by Freshwater Cyanobacteria

“…This method is named the "advanced Marfey's methods". 16,17) First of all, a peptide is subjected to the usual acidic hydrolysis to obtain each constituent amino acid. The reaction product is divided into two portions (samples 1 and 2), one of which is derivatized with L-FDLA (1-fluoro-2,4-dinitrophenylleucinamide) and another is derivatized with D-FDLA.…”

“…Although there are a few exceptions, we have already established a separation mechanism based on conformational analysis. 16) Therefore, this method can be applied to any amino acid even if a standard sample is not available.…”

Clonostachysins A and B, New Anti-dinoflagellate Cyclic Peptides from a Marine-derived Fungus

“…The connections of nine amino acid residues were mainly determined by the HMBC signals between the methyl protons of the a -amino groups and carbonyl carbons, and between the methyl protons of the a -amino groups and neighboring a-protons of amino acids ( Figure 2). [6,7]. This rule has been shown to be applicable to the FDLA derivatives of Nmethylated amino acid and O-methylated Tyr [9].…”