The determination of amino acid chirality in natural peptides is typically addressed by Marfey's analysis. This approach relies on the complete hydrolysis of the peptide followed by the reaction of the resulting amino acid pool with Marfey's reagent, a chiral derivatizing agent which turns amino acid enantiomers into diastereomeric pairs which can be resolved by conventional reversed-phase HPLC. However, for certain amino acids possessing a second chiral centre at Cβ, the discrimination between the two possible epimers may still be challenging due to the lack of chromatographic resolution. Such is the case of isoleucine and threonine which can also be found in natural nonribosomal peptides as their allo-diastereomers. We describe a new approach based on the extension of Marfey's analysis using HPLC-SPE-NMR to sort out this challenge. Marfey's derivatives of these epimeric amino acids at Cβ can be differentiated by their distinct NMR spectra. Thus, simple comparison of the NMR spectra of trapped HPLC peaks with the corresponding spectra of standards enables the unambiguous assignment of the absolute configuration at the second chiral centre in such cases. The general applicability of this approach is showcased for two model cyclic peptides bearing L-Ile and L-Thr.
Keywords: Amino acids; Cβ configuration; HPLC-SPE-NMR; Marfey’s analysis; Natural peptides.
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