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

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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 (R)-(−)-2-butanol or (S)-(+)-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; Enterococcus faecalis, Staphylococcus aureus, Staphylococcus staphylolyticus, Lactobacillus acidophilus,Bacillus subtilis, Micrococcus luteus, and Streptomyces sp.), natural whole bacterial cells (Bacillus subtilis var. natto), (pseudo)-peptidoglycan from archaea (Methanobacterium sp.), and cell wall from eukaryota (Saccharomyces cerevisiae). We observed statistically significant differences in nitrogen isotopic compositions; e.g., δ15N (‰ vs air) in Staphylococcus staphylolyticus for d-alanine (19.2 ± 0.5‰, n = 4) and l-alanine (21.3 ± 0.8‰, n = 4) and in Bacillus subtilis for d-alanine (6.2 ± 0.2‰, n = 3) and l-alanine (8.2 ± 0.4‰, n = 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.

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