Methyl [13C]glucopyranosiduronic acids: Effect of COOH ionization and exocyclic structure on NMR spin-couplings

Methyl α- and β-d-glucopyranuronides singly labeled with 13C at C1–C6 were prepared from the corresponding 13C-labeled methyl d-glucopyranosides, and multiple NMR J-couplings (JHH, JCH, and JCC) were measured in their protonated and ionized forms in aqueous (2H2O) solution. Solvated density functional theory (DFT) calculations of J-couplings in structurally related model compounds were performed to determine how well the calculated J-couplings matched the experimental values in saccharides bearing an ionizable substituent. Intraring JHH values in both uronide anomers, including 3JH4,H5, are unaffected by solution pD, and COOH ionization exerts little effect on JCH and JCC except for 1JC1,H1, 1JC4,H4, 1JC5,H5, 1JC5,C6, and 2JC3,C5, where changes of up to 5 Hz were observed. Some of these changes are associated with changes in bond lengths upon ionization; in general, better agreement between theory and experiment was observed for couplings less sensitive to exocyclic C–O bond conformation. Titration of 1H and 13C chemical shifts, and some J-couplings, yielded a COOH pKa of 3.0 ± 0.1 in both anomers. DFT calculations suggest that substituents proximal to the exocyclic COOH group (i.e., the C4–O4 bond) influence the activation barrier to C5–C6 bond rotation due to transient intramolecular H-bonding. A comparison of J-couplings in the glucopyranuronides to corresponding J-couplings in the glucopyranosides showed that more pervasive changes occur upon conversion from a COOH to a CH2OH substituent at C6 than from COOH ionization within the uronides. Twelve J-couplings are affected, with the largest being 1JC5,C6 (∼18 Hz larger in the uronides), followed by 2JC6,H5 (∼2.5 Hz more negative in the uronides).