Racing toward fast and effective ¹⁷O isotopic labeling and nuclear magnetic resonance spectroscopy of n-formyl-MLF-OH and associated building blocks

Solid-state ¹H, ¹³C, and ¹⁵N nuclear magnetic resonance (NMR) spectroscopy has been an essential analytical method in studying complex molecules and biomolecules for decades. While oxygen-17 (¹⁷O) NMR is an ideal and robust candidate to study hydrogen bonding within secondary and tertiary protein structures for example, it continues to elude many. We discuss an improved multiple-turnover labeling procedure to develop a fast and cost-effective method to ¹⁷O label fluoroenylmethyloxycarbonyl (Fmoc)-protected amino acid building blocks. This approach allows for inexpensive ($0.25 USD/mg) insertion of ¹⁷O labels, an important barrier to overcome for future biomolecular studies. The ¹⁷O NMR results of these building blocks and a site-specific strategy for labeled N-acetyl-MLF-OH and N-formyl-MLF-OH tripeptides are presented. We showcase growth in NMR development for maximizing sensitivity gains using emerging sensitivity enhancement techniques including population transfer, high-field dynamic nuclear polarization, and cross-polarization magic-angle spinning cryoprobes.