Efficient Methods for Synthesizing Oligosaccharide Units and Derivatives Utilizing Lysozyme and Cellulase

We descibe herewith an efficient enzymatic method for synthesizing useful oligosaccharides and derivatives utilizing lysozyme-mediated and cellulase-mediated transglycosylation.

I. Lysozyme-mediated transglycosylation
p-Nitorophenyl penta-N-acetyl--chitopentaoside (pNP--(GlcNAc)5), which is available as a substrate of lysozyme assay, was efficietly synthesized through hen egg-white lysozyme-mediated transglycosylatrion from penta-N-acetylchitopentaose (GlcNAc)5 to the OH-4 position of p-nitrophenyl -N-acetylglucosaminide (pNP--GlcNAc) in an aqueous solution containing dimethylsulfoxide (Me2SO) in a high concentration.1) It was obtained in a high yield of 46% based on the (GlcNAc)5 donor added. The desired compound insolubilized from the reaction mixture was directly obtained in a high state of purity by washing with aqueous methanol only without using any column chromatographic separation. As a result, the addition of Me2SO to the reaction system not only ensured the solubility of pNP--GlcNAc, but also resulted in a high yield of pNP--(GlcNAc)5.

Hen egg-white lysozyme also had GlcNAc transferase activity and was utilized to synthesize the 4-O--N-acetylglucosaminyl-mannnose (GlcNAc1-4Man) sequence present in N-linked glycan. The enzyme produced GlcNAc1-4Man through a regioselective transglycosylation from N,N’-diacetylchitobiose (GlcNAc)2 and Man in a yield of 20.9 % based on the donor added.2) It was capable of transferring an N-acetylglucosaminyl group from the donor exclusively to the OH-4 of the Man moiety. This was also the case for the synthesis of GlcNAc1-4Man-OC6H4NO2-p with p-nitorophenyl -D-mannopyranoside as an acceptor. It was obtained in a yield of 10.5 % based on the donor added.

II. Cellulase-mediated transglycosylation
Our interest was directed to an enzymatic approach involving a lactosyl unit -glycosidically linked to aliphatic groups as mimic units of glycosphingolipids. Aliphatic -lactosides were conveniently synthesized by -lactosyl transfer reaction from Lac-pNP donor to various 1-alkanols (n = 2~12) accetors, utilizing cellulase from Trichoderma reesei. With ethanol acceptor, the enzyme induced ethyl -lactoside in 18% yield based on the donor added in an aqueous system. However, the yield decreased as the alkyl chain of alkanol acceptors increased from C2 to C8. With octanol acceptor, octyl -lactoside was observed in a low yield (<1%). This problem was partially solved by using sodium cholate as the detergent. When 1-octanol and 1-dodecanol acceptors were incubated in the presence of sodium cholate, the transfer products octyl -lactoside and dodecyl -lactoside were obtained in 13 and 5% yields respectively based on the donor added. The addition of sodium cholate to the reaction system not only guaranteed solubility of the substrate, but also resulted in a remarkable increase in the yield. These enzyme reactions were efficient enough to allow one-pot preparation of the desired lactoside derivatives.3)

The enzyme also had LacNAc transferring activity and was utilized to synthesize part of the antennary structure of an N-linked sugar chain. Preparative scale syntheses were done utilizing Man and Glc as acceptors with a LacNAc-pNP donor. As a result, the enzyme transferred an N-acetyllactosamine unit from the donor exclusively to the OH-4 of Man and Glc moieties. The compounds Gal1-4GlcNAc1-4Man and Gal1-4GlcNAc1-4Glc were obtained in 13 and 9% yields respectively based on the donor added. These products were conveniently isolated by one-step chromatographic separation on a charcoal-Celite column.3)
Taichi Usui
(Faculty of Agriculture, Shizuoka University)
References (1) T, Usui : Regioselective synthesis of useful oligosaccharide by glycosidase, Trends in Glycosci. Glycotechnol. 4, 116-122, 1992
(2) T, Murata T, Usui : Enzymatic synthesis of important oligosaccharide units involved in N- and O-linked glycans, Trends in Glycosci. Glycotechnol. 12, 161-174, 2000
(3) K, Totani N, Yasutake H, Ohi T, Murata T, Usui : Enzymatic synthesis of aliphatic -lactosides as mimic units of glycosphingolipids by use of Trichoderma reesei cellulase, Arch. Biochem. Biophys. 385, 70-77, 2001
Mar.15, 2002

GlycoscienceNow INDEX Return to Top Page