Methyl alpha- and beta-pyranosides of d-glucose and d-galactose 1-4 were prepared containing single sites of (13)C-enrichment at C4, C5, and C6 (12 compounds), and (1)H and (13)C[(1)H] NMR spectra were obtained to determine a complete set of J-couplings ((1)J, (2)J, and (3)J) involving the labeled carbon and nearby protons and carbons within the exocyclic hydroxymethyl group (CH(2)OH) of each compound. In parallel theoretical studies, the dependencies of (1)J, (2)J, and (3)J involving (1)H and (13)C on the C5-C6 (omega) and C6-O6 (theta;) torsion angles in aldohexopyranoside model compounds were computed using density functional theory (DFT) and a special basis set designed to reliably recover the Fermi contact contribution to the coupling. Complete hypersurfaces for (1)J(C5,C6), (2)J(C5,H6)(R), (2)J(C5,H6)(S), (2)J(C6,H5), (2)J(C4,C6), (3)J(C4,H6)(R), (3)J(C4,H6)(S), and (3)J(C6,H4), as well as (2)J(H6)(R)(,H6)(S), (3)J(H5,H6)(R), and (3)J(H5,H6)(S), were obtained and used to parametrize new equations correlating these couplings to omega and/or theta;. DFT-computed couplings were also tested for accuracy by measuring J-couplings in (13)C-labeled 4,6-O-ethylidene derivatives of d-glucose and d-galactose in which values of omega and theta; were constrained. Using a new computer program, Chymesa, designed to utilize multiple J-couplings sensitive to exocyclic CH(2)OH conformation, the ensemble of experimental couplings observed in 1-4 were analyzed to yield preferred rotamer populations about omega and theta;. Importantly, due to the sensitivity of some couplings, most notably (2)J(H6)(R)(,H6)(S), (2)J(C5,H6)(R), and (2)J(C5,H6)(S), to both omega and theta;, unique information on correlated conformation about both torsion angles was obtained. The latter treatment represents a means of evaluating correlated conformation in 1,6-linked oligosaccharides, since psi and theta; are redundant in these linkages. In the latter regard, multiple, redundant scalar couplings originating from both sides of the glycosidic linkage can be used collectively to evaluate conformational correlations between psi/theta; and C5-C6 bond rotamers.

Two-bond (13)C-(1)H NMR spin-spin coupling constants ((2)J(CCH)) between C2 and H1 of aldopyranosyl rings depend not only on the relative orientation of electronegative substituents on the C1-C2 fragment but also on the C-O torsions involving the same carbons. The latter dependencies were elucidated theoretically using density functional theory and appropriate model pyranosyl rings representing the four relative configurations at C1 and C2, and a 2-deoxy derivative, to probe the relationship between (2)J(C2,H1) magnitude and sign and the C1-O1 (phi, phi) and C2-O2 (alpha) torsion angles. Related calculations were also conducted for the reverse coupling pathway, (2)J(C1,H2). Computed J-couplings were validated by comparison to experimentally measured couplings. (2)J(CCH) displays a primary dependence on the C-O torsion involving the carbon bearing the coupled proton and a secondary dependence on the C-O torsion involving the coupled carbon. These dependencies appear to be caused mainly by the effects of oxygen lone pairs on the C-H and C-C bond lengths along the C-C-H coupling pathway. New parameterized equations are proposed to interpret (2)J(C1,H2) and (2)J(C2,H1) in aldopyranosyl rings. The equation for (2)J(C2,H1) has particular value as a potential NMR structure constraint for the C1-O1 torsion angle (phi) comprising the glycosidic linkages of oligosaccharides.

13C-13C Spin-spin coupling constants (JCC) have been measured in a group of aldohexopyranoses and methyl aldopyranosides singly labeled with 13C at different sites to confirm and extend prior correlations between JCC magnitude and sign and saccharide structure. Structural correlations for 2JC1,C3, 2JC2,C4, 2JC4,C6, and 2JC1,C5 have been confirmed using density functional theory calculations to test empirical predictions. These geminal couplings depend highly on the orientation of C-O bonds appended to the terminal coupled carbons, but new evidence suggests that 2JCCC values are also affected by intervening carbon structure and C-O bond rotation. 3JC1,C6 and 3JC3,C6 values show Karplus-like dependences but also are affected by in-plane terminal hydroxyl substituents. In both cases, rotation about the C5-C6 bond modulates the coupling due to the alternating in-plane and out-of-plane O6. 3JC3,C6 is also affected by C4 configuration. Both 3JC1,C6 and 3JC3,C6 are subject to remote effects involving the structure at C3 and C1, respectively. New structural correlations have been determined for 2JC3,C5, which, like 3JC3,C6, shows a remote dependence on anomeric configuration. Investigations of dual pathway 13C-13C couplings, 3+3JC1,C4 and 3+3JC2,C5, revealed an important additional internal electronegative substituent effect on 3JCC in saccharides, a structural factor undocumented previously and one of importance to the interpretation of trans-glycoside 3JCOCC in oligosaccharides.

Density functional theory (DFT) is a powerful computational tool to enable structural interpretations of NMR spin-spin coupling constants ( J-couplings) in saccharides, including the abundant (1)H-(1)H ( JHH), (13)C-(1)H ( JCH), and (13)C-(13)C ( JCC) values that exist for coupling pathways comprised of 1-4 bonds. The multiple hydroxyl groups in saccharides, with their attendant lone-pair orbitals, exert significant effects on J-couplings that can be difficult to decipher and quantify without input from theory. Oxygen substituent effects are configurational and conformational in origin (e.g., axial/equatorial orientation of an OH group in an aldopyranosyl ring; C-O bond conformation involving an exocyclic OH group). DFT studies shed light on these effects, and if conducted properly, yield quantitative relationships between a specific J-coupling and one or more conformational elements in the target molecule. These relationships assist studies of saccharide structure and conformation in solution, which are often challenged by the presence of conformational averaging. Redundant J-couplings, defined as an ensemble of J-couplings sensitive to the same conformational element, are particularly helpful when the element is flexible in solution (i.e., samples multiple conformational states on the NMR time scale), provided that algorithms are available to convert redundant J-values into meaningful conformational models. If the latter conversion is achievable, the data can serve as a means of testing, validating, and refining theoretical methods like molecular dynamics (MD) simulations, which are currently relied upon heavily to assign conformational models of saccharides in solution despite a paucity of experimental data needed to independently validate the method.