Compound-Specific Nitrogen Isotope Analysis of d-Alanine, l-Alanine, and Valine: Application of Diastereomer Separation to δ¹⁵N and Microbial Peptidoglycan Studies

Abstract

We have developed an analytical method to determine the compound-specific nitrogen isotope compositions of individual amino acid enantiomers using gas chromatography/combustion/isotope ratio mass spectrometry. A novel derivatization of amino acid diastereomers by optically active (<i>R</i>)-(−)-2-butanol or (<i>S</i>)-(+)-2-butanol offers two advantages for nitrogen isotope analysis. First, chromatographic chiral separation can be achieved without the use of chiral stationary-phase columns. Second, the elution order of these compounds on the chromatogram can be switched by a designated esterification reaction. We applied the method to the compound-specific nitrogen isotope analysis of d- and l-alanine in a peptidoglycan derived from the cell walls of cultured bacteria (Firmicutes and Actinobacteria; <i></i><i>Enterococcus faecalis</i>, <i></i><i>Staphylococcus aureus</i>, <i></i><i>Staphylococcus staphylolyticus</i>, <i></i><i>Lactobacillus acidophilus</i>,<i></i> <i>Bacillus subtilis</i>, <i></i><i>Micrococcus luteus</i>, and <i>Streptomyces</i> sp.), natural whole bacterial cells (<i></i><i>Bacillus subtilis</i> var. <i>natto</i>), (pseudo)-peptidoglycan from archaea (<i>Methanobacterium</i> sp.), and cell wall from eukaryota (<i>Saccharomyces cerevisiae</i>). We observed statistically significant differences in nitrogen isotopic compositions; e.g., δ¹⁵N (‰ vs air) in <i>Staphylococcus staphylolyticus</i> for d-alanine (19.2 ± 0.5‰, <i>n</i> = 4) and l-alanine (21.3 ± 0.8‰, <i>n</i> = 4) and in <i>Bacillus subtilis</i> for d-alanine (6.2 ± 0.2‰, <i>n</i> = 3) and l-alanine (8.2 ± 0.4‰, <i>n</i> = 3). These results suggest that enzymatic reaction pathways, including the alanine racemase reaction, produce a nitrogen isotopic difference in amino acid enantiomers, resulting in ¹⁵N-depleted d-alanine. This method is expected to facilitate compound-specific nitrogen isotope studies of amino acid stereoisomers