The enzyme-bound conformation of C-lactose, an Escherichia coli β-galactosidase inhibitor has been determined by NMR spectroscopy. It is demonstrated that the enzyme selects a high-energy conformation of this closely related structural analogue of the natural substrate, lactose. In addition, a molecular modeling protocol has been performed in order to obtain a detailed three-dimensional structure of the complex that can explain, in structural terms, the role that the key amino acid residues play in the catalytic mechanism. The implications of the recognition of a high-energy conformation of the analogue are also outlined.

The conformational behavior of the synthetic glycosidase inhibitor C-lactose (1) has been studied in different solvents (water, N,N-dimethylformamide, dimethyl sulfoxide, pyridine) using NMR spectroscopy and molecular mechanics calculations. The obtained results have been compared to those previously obtained for its natural analogue, methyl α-lactoside (2). It is shown that the conformational behavior of C- and O-lactoses is only similar around the glycosidic bond, but not around the aglyconic bond. In addition, the extent of flexibility around the β(1→4) linkage is much larger for C-lactose (1) than for methyl α-lactoside, about 23% of the complete potential energy surface of 1 is appreciably populated, and several energy minima coexist in solution. The obtained results indicate that β-linked C-glycosides are fairly flexible compounds and that even variations of the solvent may heavily affect their conformational behavior. Finally, we report on the use of 2D transferred NOE experiments to study the recognition of C-lactose and its β-methyl derivative (3) by a galactose-binding protein, ricin-B. We also compare the obtained results to those reported for the complexation of regular lactose analogues. The experimental results unambiguosly indicate that ricin-B selects different conformers of C-lactose (anti conformer) and its O-analogue (2) (syn conformer).