[Oligosaccharide sulfate inhibitors of selectin-sugar interactions in inflammatory processes]. / Oligosaccharides sulfatés inhibiteurs des interactions sélectines-sucres intervenant dans les processus inflammatoires.

Leucocyte migration into lymphatic tissues or inflammatory sites depends upon the expression of adhesion molecules. Among these molecules, the selectins expressed on endothelial cells (E- and P-selectins) and leucocytes (L-selectin) recognize carbohydrate ligands such as sialyl Lewis A or sialyl Lewis X oligosaccharides due to the same positioning of NeuAc, Gal and Fuc residues in both isomeric structures. We have shown that the sialic acid residue could be replaced by a sulfate group such as in the sulfated Lewis A pentasaccharide, one of the most potent monovalent ligand for human E-selectin, which was shown to be very active in the prevention of ischemia reperfusion lung injury. In the same way, we have prepared through chemoenzymatic syntheses, two disulfated Lewis X pentasaccharides, the sulfated analogs of carbohydrate ligands found on GLYCAM 1, the natural receptor of L-selectin. Finally, based on the double recognition of L-selectin with Lewis type and glycosaminoglycan structures, we tentatively introduced a possible link between the selectin- and the integrin-mediated lymphocyte adhesion systems.

L-selectin interactions with novel mono- and multisulfated Lewisx sequences in comparison with the potent ligand 3'-sulfated Lewisa.

The cell adhesion molecule L-selectin binds to 3'-sialyl-Lewis (Le)x and -Lea and to 3'-sulfo-Lex and -Lea sequences. The binding to 3'-sialyl-Lex is strongly affected by the presence of 6-O-sulfate as found on oligosaccharides of the counter receptor, GlyCAM-1; 6-O-sulfate on the N-acetylglucosamine (6-sulfation) enhances, whereas 6-O-sulfate on the galactose (6'-sulfation) virtually abolishes binding. To extend knowledge on the specificity of L-selectin, we have investigated interactions with novel sulfo-oligosaccharides based on the Lex pentasaccharide sequence. We observe that, also with 3'-sulfo-Lex, the 6-sulfation enhances and 6'-sulfation suppresses L-selectin binding. The 6'-sulfation without 3'-sialyl or 3'-sulfate gives no binding signal with L-selectin. Where the 6-sulfo,3'-sialyl-Lex is on an extended di-N-acetyllactosamine backbone, additional 6-O-sulfates on the inner galactose and inner N-acetylglucosamine do not influence the binding. Although binding to the 6,3'-sulfo-Lex and 6-sulfo, 3'-sialyl-Lex sequences is comparable, the former is a more effective inhibitor of L-selectin binding. This difference is most apparent when L-selectin is in paucivalent form (predominantly di- and tetramer) rather than multivalent. Indeed, as inhibitors of the paucivalent L-selectin, the 3'-sulfo-Lex series are more potent than the corresponding 3'-sialyl-Lex series. Thus, for synthetic strategies to design therapeutic oligosaccharide analogs as antagonists of L-selectin binding, those based on the simpler 3'-sulfo-Lex (and also the 3'-sulfo-Lea) would seem most appropriate.

Heterologous expression of an engineered truncated form of human Lewis fucosyltransferase (Fuc-TIII) by the methylotrophic yeast Pichia pastoris.

A stable GS115 Pichia pastoris recombinant strain was constructed to secrete a truncated form of the human alpha(1,3/4) fucosyltransferase (amino acids 45-361). Enzyme production resulted from a secretory pathway based on the pre-pro- alpha mating factor signal sequence of the yeast Saccharomyces cerevisiae . Following its transit through the Golgi apparatus, the enzyme accumulated in the periplasmic space before its release in the culture broth (about 30 mg/l). Cell-enclosed enzyme ( approximately 0.16%) proved to be fairly stable for many freezing and thawing cycles and could be used several times as an immobilized catalyst. Soluble enzyme (>99.8%) representing the main protein of the culture broth (10%) has been characterized by Western-blotting, substrate specificities and kinetic parameters. The two forms (cell-enclosed and soluble) of recombinant enzyme may be used for in vitro synthesis of Lewisadeterminants.

Chemoenzymatic synthesis of a 3IV,6III-disulfated Lewis(x) pentasaccharide, a candidate ligand for human L-selectin.

The disulfated pentasaccharide 3-O-SO3(-)-beta-D-Galp-(1-->4)-[alpha-L-Fucp-(1-->3)]-6-O-SO3(-)- beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-D-Glcp was prepared according to a chemoenzymatic approach, starting from 4-methoxybenzyl O-(4-O-acetyl-2,6-di-O-benzyl-beta- D-galactopyranosyl)-(1-->4)-O-2,3,6-tri-O-benzyl-beta-D-glucopyranoside, obtained in six steps from hepta-O-acetyl lactosyl bromide. Coupling of this lactose derivative with O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl) trichloracetimidate afforded, after dephthaloylation and re-N-acetylation, 4-methoxybenzyl O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1-->3)-O-(2,6-di-O-benzyl-beta-D-galactopyranosyl)-(1-- >4)- O-2,3,6-tri-O-benzyl-beta-D-glucopyranoside. Regioselective sulfation at the primary position of the glucosamine residue was then successfully achieved and the benzyl groups were removed. Enzymatic galactosylation of 4-methoxybenzyl O-(2-acetamido-2-deoxy-6-O-sulfo-beta-D- glucopyranosyl)-(1-->3)-O-beta-D-galactopyranosyl-(1-->4)-O-beta-D- glucopyranoside sodium salt, and subsequent regioselective sulfation at position 3 of the outer galactose residue through the stannylene procedure, led then to 4-methoxybenzyl O-(3-sulfo-beta-D- galactopyranosyl)-(1-->4)-O-(2-acetamido-2-deoxy-6-sulfo-beta-D- glucopyranosyl)-(1-->3)-O-beta-D-galactopyranosyl)-(1-->4)-O-beta-D- glucopyranoside disodium salt, which was finally fucosylated using human milk alpha-(1-->3/4)-fucosyltransferase affording, after anomeric deprotection, the target pentasaccharide.

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.