Astrid Zervosen

One-pot enzymatic synthesis of the Gal alpha 1-->3Gal beta 1-->4GlcNAc sequence with in situ UDP-Gal regeneration.

The trisaccharide Galα1→3Galβ1→4GlcNAcβ1→O-(CH2)8COOCH3 was enzymatically synthesized, within situ UDP-Gal regeneration. By combination in one pot of only four enzymes, namely, sucrose synthase, UDP-Glc 4′-epimerase, UDP-Gal:GlcNAc β4-galactosyltransferase and UDP-Gal:Galβ1→4GlcNAc α3-galactosyltransferase, Galα1→3Galβ1→4GlcNAcβ1→O-(CH2)8COOCH3 was formed in a 2.2 µmol ml−1 yield starting from the acceptor GlcNAcβ1→O-(CH2)8COOCH3. This is an efficient and convenient method for the synthesis of the Galα1→3Galβ1→4GlcNAc epitope which plays an important role in various biological and immunological processes.

Application of recombinant sucrose synthase-large scale synthesis of ADP-glucose

Abstract The synthesis of ADP-glucose with recombinant sucrose synthase from potato was combined with the synthesis of ADP from AMP and ATP catalysed by myokinase from rabbit muscle. By using the repetitive-batch-technique we were able to reach enzyme productivities (mg ADP-glucose/U enzyme) of 28 mg/U sucrose synthase and 140 mg/U myokinase yielding 2.8 g ADP-glucose (55% yield). After product isolation, 2.2 g ADP-glucose was obtained corresponding to 44% overall yield.

Combined enzymatic synthesis of nucleotide (deoxy) sugars from sucrose and nucleoside monophosphates

Abstract The synthesis of NDP-glucose 3a-d (Nue5fbA, C, U, dU) with sucrose synthase B was combined with the enzymatic synthesis of nucleoside diphosphates 2a-d from their corresponding nucleoside monophosphates 1a-d by different kinases A . Further combination with recombinant dTDP-glucose 4,6-dehydratase D enabled us to synthesize dUDP-6-deoxy-α-D- xylo -4-hexulose 5 from 1d on a preparative scale. By using the repetitive batch technique the enzymatic syntheses of nucleotide (deoxy) sugars 3a-d, 5 could be realized on a 0.1 – 0.5 g scale.

Synthesis of nucleotide-activated oligosaccharides by beta-galactosidase from Bacillus circulans.

Abstract 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.

UDP-N-Acetyl-alpha-D-glucosamine as acceptor substrate of beta-1,4-galactosyltransferase. Enzymatic synthesis of UDP-N-acetyllactosamine.

The capacity of UDP-N-acetyl-α-D-glucosamine (UDP-GlcNAc) as an in vitro acceptor substrate for β-1,4-galactosyltransferase (β4GalT1, EC 2.4.1.38) from human and bovine milk and for recombinant human β4GalT1, expressed in Saccharomyces cerevisiae, was evaluated. It turned out that each of the enzymes is capable to transfer Gal from UDP-α-D-galactose (UDP-Gal) to UDP-GlcNAc, affording Gal(β1-4)GlcNAc(α1-UDP (UDP-LacNAc). Using β4GalT1 from human milk, a preparative enzymatic synthesis of UDP-LacNAc was carried out, and the product was characterized by fast-atom bombardment mass spectrometry and 1H and 13C NMR spectroscopy. Studies with all three β4GalTs in the presence of α-lactalbumin showed that the UDP-LacNAc synthesis is inhibited and that UDP-α-D-glucose is not an acceptor substrate. This is the first reported synthesis of a nucleotide-activated disaccharide, employing a Leloir glycosyltransferase with a nucleotide-activated monosaccharide as acceptor substrate. Interestingly, in these studies β4GalT1 accepts an α-glycosidated GlcNAc derivative. The results imply that β4GalT1 may be responsible for the biosynthesis of UDP-LacNAc, previously isolated from human milk.