SYNTHESIS OF 1,5-DIDEOXY-1,5-IMINO-D-ARABINITOL (5-NOR-L-FUCO-1-DEOXYNOJIRIMYCIN) AND ITS APPLICATION FOR THE AFFINITY PURIFICATION AND CHARACTERIZATION OF ALPHA-L-FUCOSIDASE

• Published in: Carbohydrate research Vol. 272; no. 1; pp. 17 - 30
• Main Authors: LEGLER, G, STUTZ, AE, IMMICH, H
• Format: Journal Article
• Language: English
• Published: OXFORD Elsevier 21.07.1995
• Subjects: Biochemistry & Molecular Biology; Chemistry; Chemistry, Applied; Chemistry, Organic; Life Sciences & Biomedicine; Physical Sciences; Science & Technology; ALPHA-L-FUCOSIDASE, CHARACTERIZATION OF ACTIVE SITE; ALPHA-L-FUCOSIDASE, AFFINITY PURIFICATION; D-ARABINITOL, 1,5-DIDEOXY,L,5-IMINO-, SYNTHESIS; ACTIVE-SITE; RING; D-ARABINITOL, 1,5-DIDEOXY-1,5-IMINO-, INHIBITION OF ALPHA-L-FUCOSIDASE; ALPHA-L-FUCOSIDASE, INHIBITION; INHIBITORS; CHROMATOGRAPHY
• ISSN: 0008-6215; 1873-426X
• DOI: 10.1016/0008-6215(95)00032-O

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 alpha-L-fucosidase from bovine kidney homogenate in two steps. Inhibition constants of 2 with the purified enzyme ranged from 39 mu M (pH 5.0) to 3 mu M (pH 7.0) which was similar to 1,500-fold larger than K-I 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 K-I values for D-manno- and L-gulo-1-deoxynojirimycin. The pH-dependence of K-I for 1, 2, and other basic analogues was very similar to that of K-I for L-fucose (205 mu M at pH 5.0 to 25 mu M at pH 7.0). On the other hand, K-M for p-nitrophenyl alpha-L-fucoside and K-I for methyl alpha-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 alpha-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 pK(a) 6.1 as acceptor. The inhibitory potency of 2 was greatly lowered by N,N-dimethylation (K-I 3600 mu M at pH 5.0 to 305 mu M at pH 7.0).