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

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

Automated summary

O-17 NMR spectroscopy is a powerful technique for studying biomolecules. This study presents cost-effective strategies for enriching amino acids and peptides with O-17 using mechanochemistry. The enriched samples showed high levels of enrichment with no loss of purity, making them suitable for future NMR studies.