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Journal of Biochemistry Advance Access originally published online on October 30, 2007
Journal of Biochemistry 2008 143(1):21-30; doi:10.1093/jb/mvm200
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© 2007 The Japanese Biochemical Society.

Enzymatic Synthesis of Spacer-Linked Divalent Glycosides Carrying N-Acetylglucosamine and N-Acetyllactosamine: Analysis of Cross-Linking Activities with WGA

Yoshinori Misawa1, Takashi Akimoto2, Satoshi Amarume2, Takeomi Murata2 and Taichi Usui1,2,*

1Science of Biological Resource, The United Graduate School of Agricultural Science, Gifu University, Yanagido, Gifu 501-1193; and 2Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Ohya, Shizuoka 422-8529, Japan

*To whom correspondence should be addressed. Tel: +81-54-238-4965, Fax: +81-54-238-4873, E-mail: actusui{at}agr.shizuoka.ac.jp

Received July 16, 2007; Accepted September 22, 2007


  Abstract

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-β-D-glucopyranoside) (GlcNAc-Hx-GlcNAc) and 3,6-dioxaoct-1,8-diyl bis-(2-acetamido-2-deoxy-β-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-[β-D-galactopyranosyl-(1->4)-2-acetamido-2-deoxy-β-D-glucopyranoside] (LacNAc-Hx-LacNAc) and 6-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-hexyl β-D-galactopyranosyl-(1->4)-2-acetamido-2-deoxy-β-D-glucopyranoside (LacNAc-Hx-GlcNAc), and respective 3,6-dioxaoct-1,8-diyl bis-[β-D-galactopyranosyl-(1->4)-2-acetamido-2-deoxy-β-D-glucopyranoside] (LacNAc-Doo-LacNAc) and 8-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-3,6-dioxaoctyl β-D-galactopyranosyl-(1->4)-2-acetamido-2-deoxy-β-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.

Key Words: Amycolatopsis orientalis, cross-linking, divalent glycoside, enzymatic synthesis, WGA

Abbreviations: AP, alkaline phosphatase; Con A, concanavalin A; Doo, 3,6-dioxaocto-1,8-diyl; EDC, N-ethyl-N'-(dimethylaminopropyl)carbodiimide; Gal, D-Galactose; β-GalT, β1,4-galactosyltransferase; GlcNAc, N-acetyl-D-glucosamine; (GlcNAc)2, N, N'-diacetylchitobiose; (GlcNAc)3, N,N',N''-triacetylchitotriose; (GlcNAc)4, N,N',N'',N'''-tetraacetylchitotetraose; Hx, hexan-1,6-diyl; KD, dissociation constant; LacNAc, N-acetyllactosamine; MIC, minimum inhibitory concentrations; NHS, N-hydroxysuccimide; pNP, p-nitrophenyl; RU, resonance unit; SPR, surface plasmon resonance; TPS, sodium 3-(trimethylsilyl)-propionate; WGA, wheat germ (Triticum vulgaris) agglutinin


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