Synthetic routes for the preparation of new sugar nitriles 8–10 derived from 2‐acetamido‐2‐deoxy‐β‐D‐glucopyranosides bearing a cyano group at the C‐5 or C‐6 position are presented. In an attempt to prepare the glycosyl azide 10 by treatment of tosylate 23 with KCN/DMF at 60 °C, an intramolecular 1,3‐dipolar cycloaddition reaction occurred to give the highly constrained nonisolable tetrazole 24, which was readily converted into the imino‐azido compound 25 through an azido‐tetrazole tautomerism. Compounds 8 and 10 werefound to be poorer substrates of fungal β‐N‐acetylhexosaminidases than compound 9 and none of these compounds was accepted as substrates of the nitrilase or nitrile hydratase. Docking of the nitriles 8–10 in the active site of the β‐N‐acetylhexosaminidase from Aspergillus oryzae gave interaction energies comparable with the natural substrate. Based on these data, which indicate strong binding of these compounds (8 > 9 > 10) to the active site, it has been proposed that some cyano derivatives may act as competitive inhibitors of β‐N‐acetylhexosaminidases. This hypothesis is consistent with enzyme inhibition experiments which showed strong inhibitory properties of compound 9 (KI = 0.37 mM) and in particular of compound 8 (KI = 7.6 μM). (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)