Flexibility in the donor substrate specificity of β 1,4-galactosyltransferase: application in the synthesis of complex carbohydrates

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Abstract
Biosynthetically, bovine N-acetylglucosainine β 1,4-galacto-syltransferase (GalT) catalyses the transfer of galactosyl residues from UDP-Gal to the 4-position of GlcNAc units, resulting in the production of N-acetyllactosamine sequences. UDP-Glc and UDP-GalNAc were also found to act as donors for this enzyme, allowing the preparation of βGlc(1→4)-βGlcNAc and βGalNAc(1→4)βGlcNAc terminating structures on the milligram scale. GalT could thus be used to add βGalNAc to βGlcNAc(1→2)αMan terminating structures, converting them to the βGalNAc(1→4)βGlcNAc(1→2)αMan sequences found on glycoprotein hormones. GalT did not transfer GlcNAc residues from UDP-GlcNAc, but it could utilize UDP-GlcNH2 as a donor. Synthesis of βGlcNAc(1→4)βGlcNAc sequences could therefore be accomplished by transfer of GlcNH2 from its UDP derivative, followed by N-acetylation of the product amino-disaccharide using acetic anhydride in methanol. The products of the enzymatic reactions were characterized by 1H-NMR-spectroscopy and fast-atom bombardment mass spectrometry. This work expands the scope of the combined chemical-enzymatic synthesis of complex carbohydrates, using glycosyltrans-ferases, to the production of oligosaccharides different from those for which these enzymes were designed. These unnatural reactions should find application in glycoprotein and glycolipid remodelling.

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