Fast and Cost-Efficient ¹⁷ O-Isotopic Labeling of Carboxylic Groups in Biomolecules: From Free Amino Acids to Peptide Chains

¹⁷ O NMR spectroscopy is a powerful technique, which can provide unique information regarding the structure and reactivity of biomolecules. However, the low natural abundance of ¹⁷ O (0.04 %) generally requires working with enriched samples, which are not easily accessible. Here, we present simple, fast and cost-efficient ¹⁷ O-enrichment strategies for amino acids and peptides by using mechanochemistry. First, five unprotected amino acids were enriched under ambient conditions, consuming only microliter amounts of costly labeled water, and producing pure molecules with enrichment levels up to ∼40 %, yields ∼60-85 %, and no loss of optical purity. Subsequently, ¹⁷ O-enriched Fmoc/tBu-protected amino acids were produced on a 1 g/day scale with high enrichment levels. Lastly, a site-selective ¹⁷ O-labeling of carboxylic functions in peptide side-chains was achieved for RGD and GRGDS peptides, with ∼28 % enrichment level. For all molecules, ¹⁷ O ssNMR spectra were recorded at 14.1 T in reasonable times, making this an important step forward for future NMR studies of biomolecules.