The acceptor specificities of rat liver Gal(@ -4)GlcNAc a-2,6-sialyltransferase, recombinant fulllength human liver Gal(j31-4)GlcNAc cx-2,6-sialyltransferase, and a soluble form of recombinant rat liver Gal(/j1-3/4)GlcNAc a-2,3-sialyltransferase were studied with a panel of analogues of the trisaccharide Gal(j31-4)GlcNAc(pl-2)Man(al -O)(CH,),CH,. These analogues contain structural variants of D-galactose, modified at either C3, C4 or C5 by deoxygenation, fluorination, 0-methylation, epimerization, or by the introduction of an amino group. In addition, the enantiomer of D-galactose is included. The a-2,6-sialyltransferases tolerated most of the modifications at the galactose residue to some extent, whereas the a-2,3-sialyltransferase displayed a narrower specificity. Molecular dynamics simulations were performed in order to correlate enzymatic activity to three-dimensional structure. Ineffective acceptors for rat liver n-2,6-sialyltransferase were shown to be inhibitory towards the enzyme; likewise, the a-2,3-sialyltransferase was found to be inhibited by all non-substrates. Modified sialyloligosaccharides were obtained on a milligram scale by incubation of effective acceptors with one of each of the three enzymes, and characterized by 500-MHz 'H-NMR spectroscopy.Keywords: sialyltransferase ; substrate specificity ; enzymatic synthesis ; glycoprotein.Sialic acids that are located at the periphery of glycoconjugate glycans can be involved in a variety of biological phenomena, such as cell-cell and receptor-ligand interactions, cell differentiation, or tumor progression and metastasis [l -51. The various types of sialylation patterns found in either glycolipid or glycoprotein glycans are the result of the action of at least 12 sialyltransferases, that can be readily distinguished by their strict in vivo specificity for acceptor substrates, including the type of linkage formed in the product [l]. However, in patients suffering from a-mannosidosis, we found the unusual trisaccharide Neu5-Ac(a2-6)Man@1-4)GlcNAc [6], and it has been suggested that this compound is formed by transfer of Neu5Ac from CMPNeuSAc to H06' in the accumulated disaccharide Manwl-4)-GlcNAc. Additional studies by us 17, 81 and by others 191 have shown that the purified rat liver Gal(/j-4)GlcNAc a-2,6-sialyltransferase, though specific for the B-1,4-linkage in the Nacetyllactosamine epitope in N-glycans, tolerated modifications in the accepting terminal monosaccharide in vitro, producing varying yields of sialyloligosaccharides. These results indicated that some of the hydroxyl groups of the terminal monosaccha- Enzymes. CMP-NeuSAc :Gal@ -4)GlcNAc-R a-2,6-sialyltransferase (EC 2.4.99.1 ): CMP-Neu5Ac:Gal(/~l-3/4)GlcNAc-R rr-2,3-sialyltransferase (EC 2.4.99.6); orthophosphoric monoester phosphohydrolase, alkaline phosphatase (EC 3.1 3.1).ride are of minor importance for effective sialylation, at least by the applied a-2,6-sialyltransferase, and prompted us to a program aimed at the exploration of the specific topology required by sialyltransferases....