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

Abstract

17O NMR spectroscopy is a powerful technique, which can provide unique information regarding the structure and reactivity of biomolecules. However, the low natural abundance of 17O (0.04 %) generally requires working with enriched samples, which are not easily accessible. Here, we present simple, fast and cost‐efficient 17O‐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, 17O‐enriched Fmoc/tBu‐protected amino acids were produced on a 1 g/day scale with high enrichment levels. Lastly, a site‐selective 17O‐labeling of carboxylic functions in peptide side‐chains was achieved for RGD and GRGDS peptides, with ∼28 % enrichment level. For all molecules, 17O ssNMR spectra were recorded at 14.1 T in reasonable times, making this an important step forward for future NMR studies of biomolecules.

High-Resolution 17O Solid-State NMR as a Unique Probe for Investigating Oxalate Binding Modes in Materials: The Case Study of Calcium Oxalate Biominerals

“…The LT- 13 C/ 17 O-COM phase was synthesized starting from 99% 13 C-labeled diethyl oxalate (enriched on both carboxyl carbon atoms) and using ∼90% H 2 17 O for the mechanochemical saponification step, leading to an average 17 O-enrichment level ca. 40–45% on the oxalate oxygen atoms (as estimated from our previous experience on such labeling reactions). , First, the 1D 13 C NMR spectrum was recorded (Figure a, top). Due to the very high 13 C-enrichment level in LT- 13 C/ 17 O-COM, the spectrum is significantly broadened compared to the nonenriched COM phase (Figure a, bottom) due to the J coupling and n ∼ 0 rotational resonance of the dipolar coupling between two 13 C with close isotropic shifts, but different CSA tensor orientations.…”

“…The enrichment of oxalate oxygens was achieved through mechanochemical saponification of an ester derivative of oxalic acid (Scheme ), by adapting experimental procedures previously reported to prepare 17 O/ 18 O-enriched fatty acids and amino acids. , The labeled hydroxy ions (H*O – ) are produced in situ by reaction of EtONa with H 2 *O, and react with the ester derivative of the carboxylic acid. Ball-milling (BM) experiments were performed on a Retsch Mixer Mill MM400 apparatus, using milling jars and beads predried under vacuum before the syntheses.…”

High-resolution 17O solid-state NMR as a unique probe for investigating oxalate binding modes in materials: The case study of calcium oxalate biominerals

“…The enrichment of oxalate oxygens was achieved through mechanochemical saponification of an esterderivative of oxalic acid (Scheme 1), by adapting experimental procedures previously reported to prepare 17 O/ 18 O-enriched fatty acids and amino acids. 38,40 The labeled hydroxy ions (H*O -) are produced in situ by reaction of EtONa with H2*O, and react with the ester derivative of the carboxylic acid. Ballmilling (BM) experiments were performed on a Retsch Mixer Mill MM400 apparatus, using milling jars and beads pre-dried under vacuum before the syntheses.…”

“…Based on our recent developments of 17 O-labeling protocols for carboxylic groups using mechanochemistry, which enabled the efficient labeling of fatty acids and amino acids using only microliter quantities of H2 17 O, [38][39][40] we decided (i) to test this enrichment methodology to the label oxalate oxygen atoms, and (ii) to demonstrate how such labeled ligands can then be used to probe via advanced 17 O ssNMR analyses the structure and reactivity of biominerals involved in pathological calcifications.…”

Ex Situ Generation of 18O2 and 17O2 from Endoperoxides for *O-Labeling and Mechanistic Studies of Oxidations by Dioxygen

“…Indeed, 18 O can be easily differentiated by mass analysis from the predominant 16 O (99.759%), and recent advances in NMR instrumentation allow efficient detection of 17 O (chemical shifts ranging from −30 to +1000 ppm) . Due to the extremely low natural abundance of 18 O and 17 O (0.204% and 0.037%, respectively), the use of isotopically enriched compounds is essential, and synthetic methodologies for the incorporation of labeled oxygen (*O) have been extensively studied. − They generally rely on one of the cheapest isotope precursors, [*O]­H 2 O, but often require harsh conditions limiting their use to simple synthons and/or involve reversible isotopic exchange yielding moderate isotopic enrichments. Advanced isotopic oxygen sources ([*O]­CO 2 , [*O]­CH 3 OH, ...) are compatible with more specific reactions but are extremely costly due to their low availability.…”