Thursday, September 11, 2008

J. Am. Chem. Soc., 130 (36), 11892–11900, 2008.

13C-Labeled N-Acetyl-neuraminic Acid in Aqueous Solution: Detection and Quantification of Acyclic Keto, Keto Hydrate, and Enol Forms by 13C NMR Spectroscopy
Thomas Klepach, Ian Carmichael, and Anthony S. Serianni

Aqueous solutions of N-acetyl-neuraminic acid (Neu5Ac, 1) labeled with 13C at C1, C2, and/or C3 were analyzed by 13C NMR spectroscopy to detect and quantitfy the acyclic forms (keto, keto hydrate, enol) present at varying pHs. In addition to pyranoses, solutions contained the keto form, based on the detection of C2 signals at ~198 ppm (~0.7% at pH 2). Spectra of [2-13C] and [3-13C] isotopomers contained signals arising from labeled carbons at ~143 and ~120 ppm, respectively, which were attributed to enol forms. Solution studies of [1,2,3-13C3]1 substantiated the presence of enol (~0.5% at pH 2). Enol was not detected at pH > 6.0. A C2 signal observed at ~94 ppm was identified as C2 of the keto hydrate (~1.9% at pH 2), based partly on its abundance as a function of solution pH. Density functional theory (DFT) calculations were used to study the effect of enol and hydrate structure on JCH and JCC values involving C2 and C3 of these forms. Solvated DFT calculations showed that 2JC2,H3 in cis and trans enols have similar magnitudes but opposite signs, making this J-coupling potentially useful to distinguish enol configurations. Solvent deuterium exchange studies of 1 showed rapid incorporation of 2H from 2H2O at H3axial in the pyranoses at p2H 8.0, followed by slower exchange at H3equatorial. The acyclic keto form, which presumably participates in this reaction, must assume a pseudo-cyclic conformation in solution in order to account for the exchange selectivity. Weak 13C signals arising from labeled species were also observed consistently and reproducibly in aqueous solutions of 13C-labeled 1, possibly arising from products of lactonization or intermolecular esterification.

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