4hJCHOCH Spin Coupling in a Lewisx Trisaccharide as Evidence of Inter-Residue C–H···O Hydrogen Bonding in Aqueous Solution

Prior studies of the solution conformation of the Lewis^x (Le^x) trisaccharide, αFuc-(1→3)[βGal-(1→4)]-βGlcNAc, suggest that nonclassical inter-residue C–H···O hydrogen bonding in aqueous solution contributes to the stabilization of its 3D structure and affects its biological properties. Experimental evidence for this hydrogen bond in aqueous solution has been reported in the form of a ^4hJ_CHOCH NMR spin-coupling constant between C5′Fuc and H1″Gal measured by 2D NMR methods in unlabeled samples. A methyl glycoside of Le^x (MeβLe^x) was prepared containing selective ¹³C-labeling at C5′Fuc, and the H1″Gal signal was examined in high-field ¹H NMR spectra for evidence of splitting or line-broadening caused by the ¹³C at C5′Fuc. High-resolution ¹H NMR spectra obtained at high field and at different temperatures using different FID processing parameters showed no resolved splitting of the H1″Gal signal or evidence of line-broadening. Spectral simulation showed that this splitting and/or line-broadening would be observable if the reported J-value (∼1.1 Hz) is correct. DFT calculations on MeβLe^x and a carbon analog (O5″Gal replaced by a CH₂ group) gave very small and nearly identical calculated ^4hJ_C5′,H1″ values, suggesting that the coupling is essentially zero. DFT calculations also showed that an alternate inter-residue ^3hJ_H5′,H1″ is small. Based on NMR analyses and DFT calculations, we found that ^4hJ_C5′,H1″ in MeβLe^x has an upper limit of ∼0.4 Hz and that the value could be lower, possibly zero, calling into question its value as experimental proof of persistent nonclassical hydrogen bonding in aqueous solutions of MeβLe^x and related structures.