Recombinant (2→3)-α-sialyltransferase immobilized on nickel-Agarose for preparative synthesis of sialyl Lewisx and Lewisa precursor oligosaccharides

Abstract The specificity of recombinant (2→3)-α-sialyltransferase (ST3Gal-III), expressed in baculovirus-infected insect cells, has been determined with various oligosaccharide acceptors and sugar-nucleotide donors using a fluorescence based assay. Recombinant ST3Gal-III tagged with a polyhistidine tail was immobilized on Ni 2+ -NTA-Agarose as an active enzyme for use in the synthesis of three sialylated oligosaccharides: (i) the divalent molecule [α-Neu5Ac-(2→3)- d -Gal p -(1→4)-β- d -Glc p NAc-O-CH 2 ] 2 -C-(CH 2 OBn) 2 ( 12 ); (ii) the dansylated derivative, α-Neu5Ac-(2→3)- d -Gal p -(1→3)-β- d -Glc p NAc-O-(CH 2 ) 6 -NH-dansyl and; (iii) the tetrasacharide α-Neu5Ac-(2→3)-β- d -Gal p -(1→4)-β- d -Glc p NAc-(1→2)-α- d -Man p -O-CH 3 . Compound 12 was itself prepared from the divalent N -acetyllactosamine molecule built on pentaerythritol by a chemo-enzymatic route.