Elisabeth Kallin

Derivatization Procedures for Reducing Oligosaccharides, Part 3: Preparation of Oligosaccharide Glycosylamines, and Their Conversion Into Glycosaccharide - Acrylamide Copolymers

Abstract Reducing oligosaccharides were converted in good yields into the corresponding primary glycosylamines by treatment with aqueous ammonium bicarbonate. The glycosylamines were then acryloylated and the obtained oligosaccharide N-acryloyl glycosylamines were copolymerized with acrylamide. High molecular weight, linear copolymers were obtained, which were useful as antigens in immunoassays.

Specificity of human antibodies against galα1-3gal carbohydrate epitope and distinction from natural antibodies reacting with galα1-2gal or galα1-4 gal

Antibodies against galactosyl-α1-3-galactose epitopes were characterized in normal and patient sera by radioimmunoassay binding to mouse laminin and oligosaccharide inhibition. Binding was strictly dependent on α-linked galactose in a terminal position. Reduced affinities were observed for digalactoses with α(1-2)-, α(1-6)- and α(1-4)-linkages and for the blood group B epitope, Galα1-3(Fucα1-2)Gal. Conformational models of various active and inactive oligosaccharides provided a clearer picture of the epitope requirements for the observed antibody specificity. Some antibody heterogeneity was detected by comparing individual sera and by hapten elution from a laminin adsorbent. New assays were developed with synthetic Galα1-3Gal-albumin conjugates and these were shown to be more sensitive than assays with mouse laminin. Two more ubiquitous human antibodies could be detected with Galα1-2Gal and Galα1-4Gal conjugates. They were distinct from Galα1-3Gal-specific antibodies as shown by carbohydrate inhibition. This demonstrates a considerable diversity in the recognition of α-linked galactose epitopes by natural antibodies.

New derivatization and separation procedures for reducing oligosaccharides

Abstract4-Trifluoroacetamidoaniline was reacted with reducing oligosaccharides in the presence of sodium cyanoborohydride to give aminoalditol derivatives, useful for linkage to proteins or solid matrices. A mixture of reducing oligosaccharides, difficult to separate by HPLC, was treated in the same way. The resulting derivatives were easily separated by HPLC.

Derivatization Procedures for Reducing Oligosaccharides, Part 4: Use of Glycosylamines in a Reversible Derivatization of Oligosaccharides with the 9-Fluorenylmethoxycarbonyl Group, and Hplc Separations of the Derivatives

ABSTRACT A new, reversible derivatization procedure for reducing oligosaccharides was developed. Reaction of the model compound lacto-N-tetraose (β-D-Gal-(1→3)-β-D-GlcNAc-(1→3)-β-D-Gal-(1→4)-D-Glc) with aqueous ammonium bicarbonate gave the corresponding β-glycosylamine, which was reacted with fluorenylmethoxycarbonyl chloride to give an N-fluorenyl-methoxycarbonyl β-glycosylamine (FMOC derivative). The free oligosaccharide could be recovered in high yield from the FMOC derivative by brief treatment with 15% aqueous ammonia. Complex oligosaccharide mixtures from human milk were derivatized with the new procedure, and excellent separations of the derivatives on straight-phase silica HPLC columns were achieved.

Derivatization procedures for reducing oligosaccharides, part 2: Chemical transformation of 1-deoxy-1-(4-trifluoroacetamidophenyl)aminoalditols

Three chemical transformations of oligosaccharide 1-deoxy-1-(4-trifluoroacetamidophenyl)aminoalditols are described. 1) Oxidation with hydrogen peroxide to give the corresponding reducing oligosaccharides. 2) Oxidation with cerium ammonium nitrate to give the corresponding 1-amino-1-deoxyalditols. 3) Treatment with acetic anhydride to give the correspondingN-acetylated derivatives, which are more stable towards oxidation.

Preparation of oligosaccharide-polyacrylamide conjugates and their use as antigens in enzyme immunoassay (EIA)

Oligosaccharides derived fromSalmonella lipopolysaccharides or from human milk were converted to theirN-acetyl-N-(4-acrylamidophenyl)-1-amino-1-deoxyalditol derivatives. These derivatives were copolymerized with acrylamide to give linear, water-soluble polymers, which were used as coating antigens in EIA assays.

Glycosidations with thioglycosides activated by sulfuryl chloride/trifluoromethanesulfonic acid: synthesis of a human blood group B trisaccharide glycoside

The trisaccharide 2-(p-trifluoroacetamidophenyl)ethyl 2-O-(α-l-fucopyranosyl)-3-O-(α-d-galactopyranosyl)-β-d-galactopyranoside, corresponding to the human blood group B determinant, was synthesized. Thioglycosides activated by sulfuryl chloride/trifluoromethanesulfonic acid were used as glycosyl donors in the construction of the three glycosidic linkages.

Synthesis of p-Trifluoroacetamidophenylethyl O-(2-Acetamido-2-Deoxy-β-D-Galactopyranosyl)-(1→4)-O-β-D- Galactopyanosyl-(1→4)-O-β-D-Glucopyranoside, Containing the Trisaccharide Portion of the Asialo-GM2 Glycolipid

ABSTRACT The p-trifluoroacetamidophenylethyl β-glycoside 9 of the trisaccharide O-(2-acetamido-2-deoxy-β-D-galactopyranosyl)-(1→4)-O-β-D-galactopyranosyl-(1→4)-D-glucopyranose (gangliotriose, asialo-GM2) was synthesised. The key step was coupling of a suitably protected lactose derivative with a galactosamine thioglycoside derivative using sulfuryl chloride/trifluoromethanesulfonic acid activation.

Affinity purification of monoclonal antibodies, using a bifunctional oligosaccharide hapten

A bifunctional hapten was synthesized consisting of a blood group A active tetrasaccharide (A-tetra) and a blood group Lea active pentasaccharide. lacto-N-fucopentaose II (LNF II), linked to each other with a phenylaminothiourea spacer connecting the reducing ends (A-tetra-LNF II). The hapten was demonstrated to retain both blood group A and Lea activity and could be easily bound to both monoclonal anti-A and anti-Lea affinity columns. Due to the strong temperature dependence of the two antibodies in their binding to oligosaccharides, the bifunctional hapten could be utilized to achieve easy desorption in the final step of affinity purification of either monoclonal anti-Lea or anti-A. The system is postulated to have general applicability in affinity purification of any ligate that binds with an avidity too high to achieve non-denaturing desorption.

Synthesis of Oligosaccharides Representing the Chitobiosyl Link in Glycoproteins

The carbohydrate portion of the link region of N-glycosidically linked oligosaccharide chains in glycoproteins is composed of a chitobiosyl residue which sometimes has a 6-linked α-L-fucopyranosyl unit at the innermost 2-acetamido-2-deoxy-D-glucose residue.