Crystal state and solution conformation of the B blood group trisaccharide alpha-L-Fucp-(1-->2)-[alpha-D-Galp]-(1-->3)]-beta-D-Galp-OCH3.

The crystal structure of the human B blood group related trisaccharide alpha-L-Fucp-(1-->2)-[alpha-D-Galp]-(1-->3)-beta-D-Galp-OCH3 (1) has been determined. The solution structure of 1 was studied by two-dimensional NMR techniques at 600 MHz in D2O solution and the conformational properties were analyzed in terms of the torsional angles phiH and psiH, derived from 3JCH coupling constants, and 10 inter-residue proton-proton distances. 3JCH could be accurately measured by a recently introduced two-dimensional heteronuclear correlation experiment (EXSIDE). The nuclear Overhauser enhancement-derived distances and the calculated torsion angles were compared with the same information available from the crystal structure. The agreement is excellent, indicating that the trisaccharide adopts a restricted conformation in solution, which was also predicted by the Hard Sphere Exo-Anomeric forcefield. The data of 1 are complemented by NMR studies of the closely related alpha-L-Fucp-(1-->2)-[6-deoxy-alpha-D-Galp]-(1-->3)-beta-D-Galp O-(CH2 )7CH3 trisaccharide (2).

Chemoenzymic synthesis of sialylated and fucosylated oligosaccharides having an N-acetyllactosaminyl core.

Several sialylated and fucosylated oligosaccharides, based upon the N-acetyllactosaminyl core structure, have been synthesized from a single trisaccharide glycoside, beta-D-GlcNAc-(1-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-OCH2(CH2)++ +7CO2CH3, by the sequential use of several glycosyltransferases and one sialidase. In these chemoenzymic syntheses, selective internal monofucosylation of a dimeric N-acetyl-lactosaminyl tetrasaccharide is achieved via two routes. It is demonstrated that the pentasaccharide beta-D-Gal-(1-->4)-beta-D-GlcNAc-(1-->3)-beta-D-Gal-(1-->4)-[alpha- L-Fuc-(1-->3)]-beta-D-GlcNAc-OCH2(CH2)7-CO2CH3 is an acceptor for the rat liver beta-D-Gal-(1-->3/4)-D-Glc-NAc alpha 2,3- and beta-D-Gal-(1-->4)-D-GlcNAc alpha 2,6-sialyltransferases. Among the structures obtained is the terminal hexasaccharide of the CD-65/VIM-2 epitope.

Use of human-milk fucosyltransferase in the chemoenzymic synthesis of analogues of the sialyl Lewis(a) and sialyl Lewis(x) tetrasaccharides modified at the C-2 position of the reducing unit.

Two series of trisaccharides, having the formulas alpha-Neu5Ac-(2-->3)-beta-D-Gal-(1-->4)-beta-D-GlcZ-OR and alpha-Neu5Ac-(2-->3)-beta-D-Gal-(1-->3)-beta-D-GlcZ-OR [R = (CH2)8CO2CH3] respectively, in which the 2-deoxy substituent Z is azido, amino, propionamido, or acetamido, were prepared by chemical synthesis. Both types of modified trisaccharides are acceptors for a fucosyltransferase preparation obtained from human milk. Preparative fucosylations using this enzyme provided analogues of the sialyl Lewis(x) and sialyl Lewis(a) tetrasaccharide structures, which have been proposed to be ligands for cell-adhesion molecules. These syntheses further demonstrate the utility of glycosyltransferases in the preparation of oligosaccharide analogues.

Determination of the specificities of rat liver Gal(beta 1-4)GlcNAc alpha 2,6-sialyltransferase and Gal(beta 1-3/4)GlcNAc alpha 2,3-sialyltransferase using synthetic modified acceptors.

Apparent kinetic parameters have been measured for the transfer of N-acetyl-D-neuraminic acid (Neu5Ac) from CMP-Neu5Ac to analogues of the Gal(beta 1-4)GlcNAc (type II) and Gal(beta 1-3)GlcNAc (type I) substrates by the rat liver Gal(beta 1-4)GlcNAc alpha 2,6-sialyltransferase and the Gal(beta 1-3/4)GlcNAc alpha 2,3-sialyltransferase. In these acceptor analogues, the substituents of the pyranose rings were modified, particularly by deoxygenation, to identify (i) the key polar groups required for efficient transfer and (ii) the substituents that can be removed or modified. A topography including the 6-hydroxyl of the beta Gal and the 2-acetamido of the GlcNAc unit is required for transfer to a terminal type II disaccharide by the alpha 2,6-sialyltransferase. The other hydroxyls can be replaced by hydrogen without a substantial decrease in activity. The alpha 2,3-sialyltransferase requires the 3-, 4-, and 6-hydroxyls of the terminal beta Gal and some contribution from the subterminal sugar. This may explain the cross-reactivity of this enzyme for the type I and type II acceptors. For both enzymes, an influence of the hydrophobic nature of the aglycone is noticed. The results allow an evaluation of the efficiency of the transfer of Neu5Ac to modified substrates.

Five specificity patterns of (1----3)-alpha-L-fucosyltransferase activity defined by use of synthetic oligosaccharide acceptors. Differential expression of the enzymes during human embryonic development and in adult tissues.

The use of synthetic trisaccharides as acceptors led to the definition of five main (1----3)-alpha-L-fucosyltransferase activity patterns in human adult tissues: (I). Myeliod cells, granulocytes, monocytes, and lymphoblasts, transfer an alpha-L-fucopyranosyl group to O-3 of a 2-acetamido-2-deoxy-D-glucosyl residue of H blood-group Type 2 oligosaccharide [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----4)-beta-D-GlcpNAc----R] with Mn2+ as activator. (II) Brain has the same acceptor specificity pattern as myeloid cells, but can also use Co2+ as activator. (III) Plasma and liver transfer an alpha-L-furopyranosyl group to H blood-group Type 2 and to sialyl-N-acetyllactosamine [alpha-NeuAc-(2----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc----R]. (IV) Intestine, gall bladder, kidney, and milk have the same activity as (III), but also transfer an alpha-L-fucopyranosyl group to O-4 of a 2-acetamido-2-deoxy-D-glucose residue of H blood-group Type 1 [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc----R] and sialyl Type 1 [alpha-NeuAc-(1----3)-beta-D-Galp-(1----3)-beta-D-GlcpNAc----R]. (V) Stomach mucosa is not able to use sialyl-N-acetyllactosamine, but can transfer an alpha-L-fucopyranosyl group to the other Type 1 and Type 2 acceptors. Unlike in adult tissue, a single myeloid-like pattern of (1----3)-alpha-L-fucosyltransferase activity was found at early stages of development in all tissues tested. This embryonic enzyme is later progressively replaced by enzymes or mixtures of enzymes having the corresponding adult patterns of enzyme expression. All lymphoblastoid cell lines and half of the tumor epithelial cell lines tested expressed the myeloid-like pattern of enzyme found in normal embryonic tissues. The remaining tumor epithelial cell lines expressed different forms of (1----3/4)-alpha-L-fucosyltransferase acceptor specificity patterns.

Enzymic synthesis of oligosaccharides terminating in the tumor-associated sialyl-Lewis-a determinant.

The isomeric sialyl-Lea-terminating pentasaccharide derivatives, alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-[alpha-L-Fucp-(1 ----4)]-beta- D-GlcpNAc-(1----3)-beta-D-Galp-O(CH2)8COOMe and alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-[alpha-L-Fucp-(1 ----4)]- beta-D-GlcpNAc-(1----6)-beta-D-Galp-O(CH2)8COOMe, have been prepared by the action in sequence of a porcine submaxillary (2----3)-alpha-sialyltransferase and a human-milk (1----3/4)-alpha-fucosyltransferase on the chemically synthesized trisaccharides beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)- and -(1----6)-beta-D-Galp- O(CH2)8COOMe, respectively.