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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Walsh CT, O’Brien RV, Khosla C. Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid polyketide scaffolds. Angew Chem Int Ed. 2013;52(28):7098–124.
Hedges JB, Ryan KS. Biosynthetic pathways to nonproteinogenic α-amino acids. Chem Rev. 2020;120(6):3161–209.
Götze S, Stallforth P. Structure elucidation of bacterial nonribosomal lipopeptides. Org Biomol Chem. 2020;18(9):1710–27.
Marfey P. Determination of D-amino acids. II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res Commun. 1984;49(6):591.
Bhushan R, Brückner H. Marfey’s reagent for chiral amino acid analysis: a review. Amino Acids. 2004;27(3–4):231–47.
Bhushan R, Brückner H. Use of Marfey’s reagent and analogs for chiral amino acid analysis: assessment and applications to natural products and biological systems. J Chromatogr B. 2011;879(29):3148–61.
Sethi S, Martens J, Bhushan R. Assessment and application of Marfey’s reagent and analogs in enantioseparation: a decade’s perspective. Biomed Chromatogr. 2021;35(1):e4990.
Fujii K, Ikai Y, Mayumi T, Oka H, Suzuki M, Harada K-i. 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. Anal Chem. 1997;69(16):3346–52.
Fujii K, Ikai Y, Oka H, Suzuki M, Harada K-i. A nonempirical method using LC/MS for determination of the absolute configuration of constituent amino acids in a peptide: combination of Marfey’s method with mass spectrometry and its practical application. Anal Chem. 1997;69(24):5146–51.
Arrault A, Witczak-Legrand A, Gonzalez P, Bontemps-Subielos N, Banaigs B. Structure and total synthesis of cyclodidemnamide B, a cycloheptapeptide from the ascidian Didemnum molle. Tetrahedron Lett. 2002;43(22):4041–4.
Capon RJ, Skene C, Stewart M, Ford J, O’Hair RAJ, Williams L, et al. Aspergillicins A-E: five novel depsipeptides from the marine-derived fungus Aspergillus carneus. Org Biomol Chem. 2003;1(11):1856–62.
Hess S, Gustafson KR, Milanowski DJ, Alvira E, Lipton MA, Pannell LK. Chirality determination of unusual amino acids using precolumn derivatization and liquid chromatography–electrospray ionization mass spectrometry. J Chromatogr A. 2004;1035(2):211–9.
Ratnayake R, Fremlin LJ, Lacey E, Gill JH, Capon RJ. Acremolides A−D, lipodepsipeptides from an Australian marine-derived fungus, Acremonium sp.⊥. J Nat Prod. 2008;71(3):403–8.
Vijayasarathy S, Prasad P, Fremlin LJ, Ratnayake R, Salim AA, Khalil Z, et al. C3 and 2D C3 Marfey’s methods for amino acid analysis in natural products. J Nat Prod. 2016;79(2):421–7.
Zhou T, Katsuragawa M, Xing T, Fukaya K, Okuda T, Tokiwa T, et al. Cyclopeptides from the mushroom pathogen fungus Cladobotryum varium. J Nat Prod. 2021;84(2):327–38.
Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, et al. A new antibiotic kills pathogens without detectable resistance. Nature. 2015;517(7535):455–9.
Pérez-Victoria I, Martín J, González-Menéndez V, de Pedro N, El Aouad N, Ortiz-López FJ, et al. Isolation and structural elucidation of cyclic tetrapeptides from Onychocola sclerotica. J Nat Prod. 2012;75(6):1210–4.
Balkovec JM, Hughes DL, Masurekar PS, Sable CA, Schwartz RE, Singh SB. Discovery and development of first in class antifungal caspofungin (CANCIDAS®)—a case study. Nat Prod Rep. 2014;31(1):15–34.
Schmidt JS, Lauridsen MB, Dragsted LO, Nielsen J, Staerk D. Development of a bioassay-coupled HPLC-SPE-ttNMR platform for identification of α-glucosidase inhibitors in apple peel (Malus × domestica Borkh.). Food Chem. 2012;135(3):1692–9.
Zhang S, De Leon Rodriguez LM, Lacey E, Piggott AM, Leung IKH, Brimble MA. Cyclization of linear tetrapeptides containing N-methylated amino acids by using 1-propanephosphonic acid anhydride. Eur J Org Chem. 2017;2017(1):149–58.
Leonard WR, Belyk KM, Conlon DA, Bender DR, DiMichele LM, Liu J, et al. Synthesis of the antifungal β-1,3-glucan synthase inhibitor CANCIDAS (caspofungin acetate) from pneumocandin B0. J Org Chem. 2007;72(7):2335–43.
Mizutani K, Hirasawa Y, Sugita-Konishi Y, Mochizuki N, Morita H. Structural and conformational analysis of hydroxycyclochlorotine and cyclochlorotine, chlorinated cyclic peptides from Penicillium islandicum. J Nat Prod. 2008;71(7):1297–300.
Bewley CA, He H, Williams DH, Faulkner DJ. Aciculitins A−C: cytotoxic and antifungal cyclic peptides from the lithistid sponge Aciculites orientalis. J Am Chem Soc. 1996;118(18):4314–21.
Seger C, Godejohann M, Spraul M, Stuppner H, Hadacek F. Reaction product analysis by high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance: application to the absolute configuration determination of naturally occurring polyyne alcohols. J Chromatogr A. 2006;1136(1):82–8.
Molinski TF. NMR of natural products at the ‘nanomole-scale.’ Nat Prod Rep. 2010;27(3):321–9.
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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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