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Astrid Zervosen, Veronika Nieder, Ricardo Gutiérrez Gallego, Johannis P. Kamerling, Johannes F.G. Vliegenthart, Lothar Elling
Synthesis of Nucleotide-Activated Oligosaccharides by ?-Galactosidase from Bacillus circulans
The enzymatic access to nucleotideactivated oligosaccharides by a glycosidasecatalyzed transglycosylation reaction was explored. The nucleotide sugars UDPGlcNAc and UDPGlc were tested as acceptor substrates for ?galactosidase from Bacillus circulans using lactose as donor substrate. The UDPdisaccharides Gal(?1-4)GlcNAc(?1-UDP) (UDPLacNAc) and Gal(?1 4) Glc(?1-UDP) (UDPLac) and the UDPtrisaccharides Gal(?1-4)Gal(?1-4)GlcNAc(?1- UDP and Gal(?1 4 ) Gal(?1 4 ) Glc(?1-UDP) were formed stereo and regioselectively. Their chemical structures were characterized by [1]H and [13]C NMR spectroscopy and fast atom bombardment mass spectrometry. The synthesis in frozen solution at -5 C instead of 30 C gave significantly higher product yields with respect to the acceptor substrates. This was due to a remarkably higher product stability in the small liquid phase of the frozen reaction mixture. Under optimized conditions, at 5 C and pH 4.5 with 500 mM lactose and 100 mM UDPGlcNAc, an overall yield of 8.2% (81.8 mol, 62.8 mg with 100% purity) for Gal(b1 4 ) GlcNAc(?1-UDP) and 3.6% (36.1 mol, 35 mg with 96% purity) for Gal(?1-4)Gal(?1-4)GlcNAc(?1- UDP) was obtained. UDPGlc as acceptor gave an overall yield of 5.0% (41.3 mol, 32.3 mg with 93% purity) for Gal(?1-4)Glc(?1-UDP) and 1.6% (13.0 mol, 12.2 mg with 95% purity) for Gal(?1-4)Gal(?1- 4)Glc(?1-UDP). The analysis of other nucleotide sugars revealed UDPGal, UDPGalNAc, UDPXyl and dTDP, CDP, ADP and GDPGlc as further acceptor substrates for ?galactosidase from Bacillus circulans.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 382, 03/2001
Pages: 299 - 311