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Divalent glycosides carrying N-acetyl-d-glucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. First, hexan-1,6-diyl bis-(2-acetamido-2-deoxy-beta-d-glucopyranoside) (GlcNAc-Hx-GlcNAc) and 3,6-dioxaoct-1,8-diyl bis-(2-acetamido-2-deoxy-beta-d-glucopyranoside) (GlcNAc-Doo-GlcNAc) were enzymatically synthesized by transglycosylation of an N,N'N'',N'''-tetraacetylchitotetraose [(GlcNAc)(4)] donor with a primary diol acceptor, utilizing a chitinolytic enzyme from Amycolatopsis orientalis. The resulting divalent glycosides were further converted to the respective hexan-1,6-diyl bis-[beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside] (LacNAc-Hx-LacNAc) and 6-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-hexyl beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside (LacNAc-Hx-GlcNAc), and respective 3,6-dioxaoct-1,8-diyl bis-[beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside] (LacNAc-Doo-LacNAc) and 8-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-3,6-dioxaoctyl beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside (LacNAc-Doo-GlcNAc) by galactosyltransferase. The interaction of wheat germ agglutinin (WGA) with a series of divalent glycosides and related compounds were studied using a biosensor based on surface plasmon resonance (SPR) and by precipitation analysis. Our results demonstrated that divalent glycosides carrying GlcNAc on both sides and GlcNAc and LacNAc on each side are capable of precipitating WGA as divalent ligands, but that the corresponding monovalent controls and divalent glycosides carrying LacNAc on both sides are unable to precipitate the lectin and bind as univalent ligands.

Novel chromogenic substrates for endo-b-galactosidase were designed on the basis of the structural features of keratan sulfate. Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-pNP (2), which consists of two repeating units of N-acetyllactosamine, was synthesized enzymatically by consecutive additions of GlcNAc and Gal residues to p-nitrophenyl b-N-acetyllactosaminide. In a similar manner, GlcNAcb1-3Galb1-4GlcNAcb-pNP (1), GlcNAcb1-3Galb1-4Glcb-pNP (3), Galb1-4GlcNAcb1-3Galb1-4Glcb-pNP (4), Galb1-3GlcNAcb1-3Galb1-4Glcb-pNP (5), and Galb1-6GlcNAcb1-3Galb1-4Glcb-pNP (6) were synthesized as analogues of 2. Endo-b-galactosidases released GlcNAcbpNP or Glcb-pNP in an endo-manner from each substrate. A colorimetric assay for endo-b-galactosidase was developed using the synthetic substrates on the basis of the determination of p-nitrophenol liberated from GlcNAcbpNP or Glcb-pNP formed by the enzyme through a coupled reaction involving b-N-acetylhexosaminidase (b-NAHase) or b-D-glucosidase. Kinetic analysis by this method showed that the value of V max /K m of 2 for Escherichia freundii endob-galactosidase was 1.7-times higher than that for keratan sulfate, indicating that 2 is very suitable as a sensitive substrate for analytical use in an endo-b-galactosidase assay. Compound 1 still acts as a fairly good substrate despite the absence of a Gal group in the terminal position. In addition, the hydrolytic action of the enzyme toward 2 was shown to be remarkably promoted compared to that of 4 by the presence of a 2-acetamide group adjacent to the p-nitrophenyl group. This was the same in the case of a comparison of 1 and 3. Furthermore, the enzyme also catalysed a transglycosylation on 1 and converted it into GlcNAc b1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-pNP (9) and GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-pNP (10) as the major products, which have N-acetyllactosamine repeating units.Keywords: endo-b-galactosidase; enzyme assay; kinetics; poly (N-acetyllactosamine); transglycosylation. [4,5], and Bacteroides fragilis [6]. E. freundii keratanase was found to have hydrolysing activity for a wide range of nonsulfated oligosaccharides isolated from human milk and carbohydrate moieties of glycoproteins and glycolipids [7][8][9][10]. The use of endo-b-galactosidase has been expanded to detection of poly (N-acetyllactosamine) chains in a variety of complex glycoconjugates in addition to keratan sulfate. Bacteroides fragilis endo-b-galactosidase has properties similar to those of E. freundii endo-b-galactosidase [11][12][13]. Therefore, the endo-b-galactosidases from E. freundii and B. fragilis have been widely used as tools for structural and functional analyses of glycans involved in glycoconjugates.An assay using keratan sulfate as a substrate has been widely used for estimation of endo-b-galactosidase activity. However, this method is not always reproducible because of lack of uniformity of the polymer. Methods using low molecular mass substrates have been preferred and recommended for accurate determination of activities of en...

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