Determining the Role of the Aromatic Ring of N-Arylmethyl ent-conduramine F-1 in their Interactions with α-Glucosidases by Saturation Transfer Difference NMR Spectroscopy Experiments

Glucosidases are a group of enzymes responsible of the glycosidic bond cleavage with different specificities depending on the number of monosaccharides, the position of cleavage, and the configuration of the hydroxy groups in their substrates. In particular, inhibitors of a-1,4-glucosidases have generated great interest as potential therapeutic agents for the treatment of type II diabetes (e.g. , miglitol (N-2-hydroxyethyl-1-deoxynojirimycin), Glyset, Diastabol, Glucobay), 2] obesity, hepatitis B and C and other viral diseases, and cancer. Previously, we have shown that N-benzylation of (+)-entconduramine F-1 (1) significantly increases its inhibitory activity toward a-1,4-glucosidase from yeast. Because of their relatively high hydrophobicity, N-benzyl derivatives of 1 might represent a-1,4-glucosidase inhibitors with improved bioavailability and pharmacokinetics. So far, rational drug design has not been applied to inhibitors of a-1,4-glucosidases, because the structural information available is very scarce and only related to the free forms of the protein. The lack of structural information on the nature of the interactions between a-1,4-glucosidases and their inhibitors has made it a difficult task to discover good lead compounds. In this work, we have attempted to understand the role of the aryl moieties in improving the inhibitory activities of N-benzyl derivatives of 1 using saturation transfer difference (STD) NMR spectroscopy. The method allows the binding of a ligand to a receptor to be characterize by using small amounts of protein (micromolar range), and it is very useful for mapping the binding epitope of the ligand with atomic resolution. Ligand protons that are in close contact with the protein binding pocket experience a larger fraction of saturation transfer than protons further away. Thus, protons of ligand directly involved in binding show larger signal increments than other ligand protons in STD NMR spectra. a-1,4-Glucosidase from Saccharomyces cerevisiae was used in this study as a model system to evaluate the interactions of ligand 1 and its N-benzyl derivatives 2–4 with the enzyme by STD NMR spectroscopy (Figure 1). Significant STD effects were observed for all ligands in the presence of a-1,4-glucosidase,