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Expanding the utility of Marfey’s analysis by using HPLC-SPE-NMR to determine the Cβ configuration of threonine and isoleucine residues in natural peptides

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Abstract

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.

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Funding

The work of the authors is supported by Fundación MEDINA, a non-profit partnership between Merck Sharp and Dohme de España, the Regional Government of Andalusia and the University of Granada.

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Authors and Affiliations

  1. Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento 34, 18016, Armilla, Granada, Spain

    Ignacio Pérez-Victoria, Gloria Crespo & Fernando Reyes

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  1. Ignacio Pérez-Victoria
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  2. Gloria Crespo
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  3. Fernando Reyes
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Correspondence to Ignacio Pérez-Victoria.

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Pérez-Victoria, I., Crespo, G. & Reyes, F. Expanding the utility of Marfey’s analysis by using HPLC-SPE-NMR to determine the Cβ configuration of threonine and isoleucine residues in natural peptides. Anal Bioanal Chem 414, 8063–8070 (2022). https://doi.org/10.1007/s00216-022-04339-2

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  • DOI: https://doi.org/10.1007/s00216-022-04339-2

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