| Abstract
| - N-Acetyl-neuraminic acid (Neu5Ac, 2) was prepared enzymatically containing single sites of 13C-enrichment at C1, C2, and C3. Aqueous solutions of the three 13C isotopomers were studied by 1H and 13C NMR spectroscopy at p2H 2 and pH 8 to obtain JCH and JCC values involving the labeled carbons. Experimental studies were complemented by DFT calculations of the same set of J-couplings in protonated and ionized structural mimics of 2 to determine how well theoretical predictions match the experimental findings in saccharides bearing ionizable functionality. Results show that: (a) 2JC2,H3ax/eq values in 2 depend on anomeric configuration, thus complementing 3JC1,H3ax/eq behavior, (b) JCH and JCC values involving C2 depend on anomeric configuration, the C1−C2 bond torsion, and solution pH, and (c) long-range 4JC2,H7 is sensitive to glycerol side-chain conformation. Intraring JHH and most 2JCH, 3JCH, 2JCC, and 3JCC involving C1−C3 of 2 appear largely unaffected by the ionization state of the carboxyl group. In vacuo and solvated DFT calculations of geminal and vicinal JCH and JCC values are similar and reproduce the experimental data well, but better agreement with experiment was observed for 1JC1,C2 in the solvated calculations. The present work provides new information for future treatments of trans-glycoside couplings involving Neu5Ac residues by (a) providing new standard values of intraring JCC for coupling pathways that mimic those for trans-glycoside JCC, (b) identifying potential effects of solution pH on trans-glycoside couplings inferred through the behavior of related intraring couplings, and (c) providing specific guidelines for more reliable DFT predictions of JCH and JCC values in ionizable saccharides.
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