Glycoconjugate Journal (1994) 11:105-110 Enzymic synthesis of the trisaccharide core region of the carbohydrate chain of N-glycoprotein TAICHI USUI 1., MASAHIRO SUZUKI ~, TOSHINARI SATO 1, HIROKAZU KAWAGISHI 1, KYOKO ADACHI z and HIROSHI SANO 2 Faculty of Agriculture, Department of Applied Biological Chemistry, Shizuoka University, 836 Ohya, Shizuoka 422, Japan 2 Marine BiotechnoIogy Institute Co., Ltd, 1900 Sodeshi-cho, Shimizu 424, Japan Received 26 August 1993, revised 27 September 1993 Transmannosylation from mannotriose (Manfll-4Manfll-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue of N,N'-diacetylchitobiose was regioselectively induced through the use of fi-D-mannanase from Aspergillus niger. The enzyme formed the trisaccharide Manfll-4GlcNAcfll-4GlcNAc (3.7% of the enzyme-catalysed net decrease of N,N'-diacetylchitobiose) from mannotriose as a donor and N,N'-diacetylchitobiose as an acceptor. Mannobiose (Manfll-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis. Keywords: Manfil-4GlcNAcfil-4GlcNAc, fl-o-mannanase in carbohydrate synthesis, trisaccharide core of N-glycoprotein Abbreviations: Man, D-mannose; (M~) (n = 1-5), fl-linked n-mer of mannose; GlcNAc2; 2-acetamido-2-deoxy-fi- D-glucopyranosyl-(1-4)-2-acetamido-2-deoxy-o-glucose. Introduction There is at present great interest in developing synthetic routes to oligosaccharide involved in glycoconjugates. From a practical viewpoint, the use of glycosidases is very attractive in oligosaccharides synthesis, utilizing the trans- glycosylation reaction. Our interest was directed to an enzymic approach to the trisaccharide Manfll-4GlcNAcfil- 4GlcNAc (1), which is the common structural feature at the core region of most of the N-glycoproteins. This compound will be useful as an exogenous substrate for fl-D-mannosidase [1, 2], a probe for lectin [3], and a common synthetic intermediate of N-linked gtycan. Organic chemicaI methods for obtaining 1 have been developed [5-8], but are characterized by variously elaborated protection, glyco- sylation, and deprotection. Especially, site-selective glyco- sylation for the introduction of the fi-D-Man residue is known to be a key reaction of synthesizing compound 1 [7]. From a practical viewpoint, the use of glycosidases, which are commercially available, is very attractive in the synthesis of an oligosaccharide in glycoconjugates, utilizing transglycosylation. However, use of glycosidases in synthesis has been limited due to the preponderant formation of the (1.-6) linkage over of the (I-2), (1-3), and (1-4) linkages [9, 10]. This paper reports that fi-D-mannanase (1,4-fl-D- * To whom correspondence should be addressed. 0282-0080 © 1994 Chapman & Hall mannan mannohydrolase; EC 3.2.1.78) from A. niger regio- selectively forms 1 (Manfll-4GlcNAcfil-4GlcNAc) through a transglycosylation reaction from M3 (Manfil-4Manfll- 4Man) and GlcNAc z (GlcNAcfit-4GlcNAc). Materials and methods Materials Commercially available hemicellulase (AC, Shinnihon Kagaku Co., Ltd, Japan) prepared from the culture filtrates of A. niger was used as an enzyme source. Mannooligo- saccharides (d.p. 2-5) were prepared by the method of Kusakabe [11]. The charcoal-Celite column for the separ- ation of transglycosylation products was prepared as follows: equal parts by weight of dry charcoal and Celite were slurried in water and packed into a glass column. All other chemicals were obtained from commercial sources. Enzyme assays fi-D-Mannanase activity was assayed as follows. The mixture containing 0.5 mg of Konjac mannan in 0.1 ml, 20 mM sodium acetate buffer (pH 5.0) and enzyme solution in a total volume of 0.2 ml was incubated at 40 °C for 10 rain. The amount of reducing activity produced was determined by the Somogyi-Nelson method [12, 13J. One unit of activity was defined as the amount of enzyme