Conformational equilibrium isotope effects in glucose by C-13 NMR spectroscopy and computational studies

Anomeric equilibrium isotope effects for dissolved sugars are required prel udes to understanding isotope effects for these molecules bound to enzymes. This paper presents a full molecule study of the alpha- and beta -anomeric forms of D-glucopyranose in water using deuterium conformational equilibri um isotope effects (CEIE). Using 1D C-13 NMR, we have found deuterium isoto pe effects of 1.043 +/- 0.004, 1.027 +/- 0.005, 1.027 +/- 0.004, 1.001 +/- 0.003, 1.036 +/- 0.004, and 0.998 +/- 0.004 on the equilibrium constant, K- H/D(beta/alpha), in [1-H-2]-, [2-H-2]-, [3-H-2]-, [4-H-2]-, [5-H-2]-, and [ 6,6'-H-2(2)]-labeled sugars, respectively. A computational study of the ano meric equilibrium in glucose using semiempirical and ab initio methods yiel ds values that correlate well with experiment. Natural bond orbital (NBO) a nalysis of glucose and dihedral rotational equilibrium isotope effects in 2 -propanol strongly imply a hyperconjugative mechanism for the isotope effec ts at H1 and H2. We conclude that the isotope effect at ill is due to n(p) - sigma* hyperconjugative transfer from O5 to the axial C1-H1 bond in beta -glucose, while this transfer makes no contribution to the isotope effect a t H5. The isotope effect at H2 is due to rotational restriction of OH2 at 1 60 degrees in the alpha form and 60 degrees in the beta -sugar, with concom itant differences in n --> sigma* hyperconjugative transfer from O2 to CH2. The isotope effects on H3 and H5 result primarily from syn-diaxial steric repulsion between these and the axial anomeric hydroxyl oxygen in a-glucose . Therefore, intramolecular effects play an important role in isotopic pert urbation of the anomeric equilibrium. The possible role of intermolecular e ffects is discussed in the context of recent molecular dynamics studies on aqueous glucose.