Claudine Augé

Synthesis with immobilized enzyme of the most important sialic acid.

Abstract Starting from a crude mixture of N-acetylmannosamine and N-acetylglucosamine, N-acetylneuraminic acid has been easily synthesized on 2.8 millimoles scale, with one immobilized enzyme.

Scope and limitations of the aldol condensation catalyzed by immobilized acylneuraminate pyruvate lyase

New substrates have been used in the aldol condensation catalyzed by immobilized acylneuraminate pyruvate lyase : hexoses 3, 4, 7, 8 and pentoses 2, 5, 6. Condensation of pyruvate with 2 –5, 7 and 8 led to pure compounds related to 3-deoxy-d-glycero-d-galacto-nonulosonic acid (KDN). Surprisingly, condensation with d-arabinose 6 gave a mixture of diastereomers, 3-deoxy-α-d-manno-2-octulosonic acid (KDO) and 4-epi KDO.

The use of immobilised glycosyltransferases in the synthesis of sialyloligosaccharides

The CMP-sialic acids, cytidine 5-(5-acetamido-3,5-dideoxy-beta-D-glycero-D-galacto-2-nonulopyranosy lonic acid monophosphate) (1) and cytidine 5-(5-acetamido-9-O-acetyl-3,5-dideoxy-beta-D-glycero-D-galacto-2- nonulopyranosylonic acid monophosphate) (2) were prepared from CMP, phosphoenolpyruvate, N-acetylneuraminic acid or its 9-acetate, and a catalytic amount of ATP in the presence of immobilised pyruvate kinase, nucleoside monophosphate kinase, inorganic pyrophosphatase, and CMP-sialic acid synthetase. CMP-NeuAc (1) was used as a donor of N-acetylneuraminic acid in the reaction catalysed by immobilised porcine liver beta-D-Galp-(1----4)-alpha-D-GlcpNAc-(2----6)-sialyltransferase, alpha-D-Neup5Ac-(2----6)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1--- -2)-alpha-D- Man-OMe (5) was obtained on a 0.1-mmol scale by enzymic sialylation of beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----2)-alpha-D-Man-OMe (4), prepared by enzymic galactosylation of beta-D-GlcpNAc-(1----2)-alpha-D-Man-OMe (3). Likewise, using 2, alpha-D-Neup-5,9Ac2-(2----6)-beta-D-Galp-(1----4)-D-GlcpNAc (7) was obtained from N-acetyl-lactosamine (6).

Synthesis with immobilized enzymes of two trisaccharides, one of them active as the determinant of a stage antigen.

Abstract Glycosidation of disaccharides I and III with a system of five immobilized enzymes gave excellent yields of trisaccharides β-D -Gal-(1→4)- β-D -GlcNAc-(1→6)- D -Gal ¦ the I(Ma) determinant ¦ and β-D -Gal-(1→4)- β-D -Glc-NAc-(1→3)- D -Gal, on the milimole scale.

Synthesis with an immobilized enzyme of N-acetyl-9-O-acetyl-neuraminic acid, a sugar reported as a component of embryonic and tumor antigens

Abstract Condensation of 2-acetamido-6- O -acetyl-2-deoxy-D-mannose with pyruvic acid in the presence of immobilized acylneuraminate pyruvate lyase (2.8 units) gave 5-acetamido-9- O -acetyl-3,5-dideoxy-D- glycero -D- galacto -2-nonulopyranos-1-onic acid ( N -acetyl-9- O -acetylneuraminic acid) on the 0.3 mmole scale.

The use of porcine liver (2→3)-α-sialyltransferase in the large-scale synthesis of α-Neup5Ac-(2→3)-β-D-Galp-(1→3)-D-GlcpNAc, the epitope of the tumor-associated carbohydrate antigen CA 50

Abstract α-Neu p 5Ac-(2 » 3)-β- d -Gal p (1 » 3)- d -Glc p NAc ( 2 ) and, α-Neu p 5Ac-(2 » 3)-β- d -Gal p -(1 » 3)-β- d - Glc p NAcOMBn were prepared on a large scale by the action of β- d -Gal p NAC (2 » 3)α-sialyltransferase (partially purified from porcine liver) on β- d -Gal p -(1 » 3)- d -Glc p NAc and β- d Gal p (1 » 3)-β- d -Glc p NAcOMBn, respectively. The trisaccharide 2 is the epitope of the tumor-associated carbohydrate antigen CA 50, highly expressed in human pancreatic adenocarcinoma.

Chemo-enzymatic synthesis of a selectin ligand using recombinant yeast cells

Abstract Functional soluble human α(1-3/4) fucosyltransferase was successfully expressed in Pichia pastoris cells. The recombinant protein was located in the periplasmic space; thus, incubation of the whole yeast cells with disulfated tetrasaccharide1 (SO3−3)Galβ1-4(SO3−-6)GlcNAcβ1-3Galβ1-4GlcβOMBn and GDP-fucose directly provided pentasaccharide 2 (SO3−3)Galβ1-4(Fucα1-3)(SO3−-6)GlcNAcβ1-3Galβ1-4GlcβOMBn in 70% yield.

Combined chemical and enzymatic synthesis of the sialylated non reducing terminal sequence of GM1b glycolylated ganglioside, a potential human tumor marker.

N-Glycolylglucosamine 8 was synthesized in 4 steps from anisal glucosamine, via the new crystalline monochloracetyl derivatives 3, 4 and 7. N-Glycolylneuraminic acid 10 was prepared in 59% yield starting from pyruvate and a mixture of 8 and its manno epimer 9 in a 2:3 ratio, with immobilized sialic acid aldolase. Neu5Gc 10 was converted into CMP-NeuGc 11 in the presence of immobilized calf brain CMP-sialate synthetase. Finally 11 was used as a donor in the transfer to the acceptor beta-D-Gal-(1-3)-beta-D-GalNAc-OBn 12 catalyzed by a preparation of porcine liver (2-3)-alpha-sialyltransferase, roughly purified by a chromatography on Cibacron Blue-agarose. alpha-Neu5Gc-(2-3)-beta-D-Gal-(1-3)-beta-D-GalNac-OBn 13 isolated in 56% yield was deprotected to give the non-reducing terminal sequence of GM1b glycolylated ganglioside, which might be expressed in human tumors.

Outstanding stability of immobilized recombinant α(1→3/4)-fucosyltransferases exploited in the synthesis of Lewis a and Lewis x trisaccharides

Recombinant human α(1→3/4)-fucosyltransferases n(FucT-III) expressed in CHO cells or baculovirus-infected insect cells, nimmobilized on Ni2+-agarose through a 6His tag, exhibit a marked nstability, which was exploited in the synthesis of Lewis a and Lewis x ntrisaccharides.

Synthesis of the Sialyloligosaccharide Gal-β(1→3)-[Neu5Ac-α(2→6)]-GalNAc, The Epitope of the Tumor-Associated Glycoprotein Tag 72

Abstract Benzyl 2-acetamido-2-deoxy-4,6-O-(4-methoxybenzylidene)-β-D-glucopyrano-side (3) was glycosylated with 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide (2) to give disaccharide 4. Reductive ring-opening of the acetal group provided as a major compound (64%) the 6-O-(4-methoxybenzyl) derivative (5) which was directly converted into benzyl 2-acetamido-3-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-4-O-benzoyl-2-deoxy-β-D-galactopyranoside (10) through triflate displacement by tetra-n-butylammonium benzoate, followed by removal of the 4-methoxybenzyl ether group. The minor compound (30%) (6) could be also converted into 10 through the 6-O-(tert-butyldimethylsilyl) ether 8 which was treated similarly to compound 5. Stannous triflate promoted condensation of methyl (5-acetamido 4,7,8,9-tetra-O-acetyl-2,3,5-trideoxy-D-glycero-β-D-galacto-2-nonulopyranosyl chloride)onate (12) with the glycosyl acceptor 10, in tetrahydrofuran, afforded a 3:1 mixture of a and β (2→6) linked trisaccharides 13 and 14 ...