Synthesis of 1,5-dideoxy-1,5-imino-d-arabinitol (5-nor-l-fuco-1-deoxynojirimycin) and its application for the affinity purification and characterisation of α-l-fucosidase

The title, 1,5-dideoxy-1,5-imino-d-arabinitol (2), was synthesized in seven steps from d-arabinose with 5-azido-5-deoxy-d-arabinofuranose as key intermediate and 40% overall yield. An affinity procedure employing the N-carboxypentyl derivative of 2 linked to aminohexyl agarose permitted the isolation of pure α-l-fucosidase from bovine kidney homogenate in two steps. Inhibition constants of 2 with the purified enzyme ranged from 39 μM (pH 5.0) to 3 μM (pH 7.0) which was ∼ 1,500-fold larger than KI for l-fuco-1-deoxynojirimycin (1) at these pH values. A comparably large contribution of the methyl group of 1 to the inhibition was also indicated by the K1 values for d-manno- and l-gulo-1-deoxynojirimycin. The pH-dependence of K1 for 1, 2, and other basic analogues was very similar to that of K1 for l-fucose (205 μM at pH 5.0 to 25 μM at pH 7.0). On the other hand, KM for p-nitrophenyl α-l-fucoside and K1 for methyl α-l-fucoside showed only small variations with pH. A higher affinity, of up to 100-fold, of the enzyme for l-fucose relative to methyl α-l-fucoside in conjunction with its different pH-dependence is tentatively explained by a hydrogen bond of the anomeric hydroxyl group as donor with an active site carboxylate with pKa 6.1 as acceptor. The inhibitory potency of 2 was greatly lowered by N,N-dimethylation (K1 3600 μM at pH 5.0 to 305 μM at pH 7.0).