Methyl β-lactoside: 600-MHz 1H- and 75-MHz 13C-n.m.r. studies of 2H- and 13C-enriched compounds

Abstract

Using UDP- d-galactose : 2-acetamido-2-deoxy- d-glucose 4-β- d-galactosyltransferase (EC 2.4.1.22), several methyl β-lactosides have been prepared with 2H- and/or 13C-enrichment at specific sites to facilitate study by 13C (75 MHz) and 1H (600 MHz) n.m.r. spectroscopy. 13C-Chemical shift assignments were verified and the 1H-spectrum of β-lactoside was fully assigned. Sites of enrichment were selected to permit all of the potential three-bond C-C and C-H couplings through the glycosidic bond to be obtained. Replacement of H-3 of the d-glucose residue of methyl β-lactoside with 2H allowed resolution of C-1–H-4′ coupling in the 600-MHz 1H-spectrum. Single or multiple 13C-enrichment at C-1, C-2, C-3, C-1′, C-3′, and/or C-4′ in the disaccharide allowed observation of intra- and inter-residue couplings. 13C-Spin-lattice relaxation-times ( T 1) are interpreted in terms of molecular motion in solution. The data suggest that methyl β-lactoside has an extended conformation with little rotation about the glycosidic bond. Inter-residue couplings are best explained by tortion angles of φ ~ 40° and ψ ~ 15°, indicating that the conformations of β-lactoside in solution and in the crystal are similar.