Prevention of ischemia-reperfusion lung injury by sulfated Lewis(a) pentasaccharide. The Paris-Sud University Lung Transplantation Group.

Inhibition of polymorphonuclear neutrophil (PMN) adhesion to the pulmonary endothelium attenuates ischemia-reperfusion (I/R) lung injury. We hypothesized that 3'-sulfated Lewis(a) (SuLa), a potent ligand for the selectin adhesion molecules, may have a beneficial effect on I/R lung injury, as measured by the filtration coefficient (K(fc)), and reduce pulmonary sequestration of PMN as assessed by the lung myeloperoxidase (MPO) activity. Blood-perfused rat lungs were subjected to 30 min of perfusion, 60 min of warm ischemia, and 90 min of reperfusion after treatment with either SuLa (200 microg) or saline. Effects of SuLa on PMN adhesion to cultured human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor-alpha and calcium ionophore were also investigated. Compared with preischemia conditions, I/R induced a significant increase in K(fc), which was attenuated with SuLa (80 +/- 8 vs. 30 +/- 5%; P < 0.001). SuLa reduced lung MPO and PMN adhesion to stimulated HUVEC. These results indicate that SuLa reduces I/R-induced lung injury and PMN accumulation in lung. This protective effect might be related to inhibition of PMN adhesion to endothelial cells.

Synthesis of aryl D-gluco- and D-galacto-pyranosides and 1-O-acyl-D-gluco- and -D-galacto-pyranoses exploiting the Mitsunobu reaction. Influence of the pKa of the acid on the stereoselectivity of the reaction.

Glycosides (alpha- and beta-D-glucosides and -D-galactosides) derived from three pesticides, 2-tert-butyl-2,4-dinitrophenol,2,6-dibromo-4-cyanophenol, and 5-(2,4-dichlorophenoxy)-2-nitrobenzoic acid, were synthesized from 2,3,4,6-tetra-O-chloroacetyl-D-gluco- and -D-galactopyranose by use of the Mitsunobu reaction. It was shown that selectivity for the beta-D anomer increases with the pKa of the acid component.

Preparation of alpha -and beta-dienyl glycosides used as dienes in aqueous Diels-Alder reactions. Influence of the carbohydrate moiety on the thermodynamics of the reaction.

A series of 1,3-butadienyl glycosides (mono- and di-saccharides) have been prepared and the kinetics of their Diels-Alder reaction with buten-2-one in water have been studied. The activation parameters for these aqueous cycloadditions provide clues for the hydration structure of such glyco-organic compounds.

Improved synthesis of glycosylamines and a straightforward preparation of N-acylglycosylamines as carbohydrate-based detergents.

D-Glucose, D-galactose, lactose, cellobiose, and maltose yield quantitatively the corresponding glycosylamine when treated at 42 degrees C for 36 h with a commercial aqueous solution of ammonia in the presence of one equivalent of ammonium hydrogen carbonate. After lyophilisation, the residue (i.e., the pure glucosylamine) was dissolved in a mixture of ethanol and water, and treated with acyl chlorides to afford in a few minutes N-acylglucosylamines. Micellar properties of these amphiphilic derivatives were determined.

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

alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-D-GlcpNAc (2) and, alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-beta-D-GlcpNAcOMBn+ ++ were prepared on a large scale by the action of beta-D-Galp-(1----3)-D-GalpNAc (2----3)-alpha-sialyltransferase (partially purified from porcine liver) on beta-D-Galp-(1----3)-D-GlcpNAc and beta-D-Galp-(1----3)-beta-D-GlcpNAcOMBn, respectively. The trisaccharide 2 is the epitope of the tumor-associated carbohydrate antigen CA 50, highly expressed in human pancreatic adenocarcinoma.

Porcine liver (2-->3)-alpha-sialyltransferase: substrate specificity studies and application of the immobilized enzyme to the synthesis of various sialylated oligosaccharide sequences.

In search of substrate analogues for the porcine liver beta-D-Gal p-(1-->3)-D-Gal p-NAc: CMP-Neu5Ac-(2-->3')-alpha-sialyltransferase, three disaccharides beta-D-Gal p-(1-->3)-beta-D-Gal p-O-CH3 (5), beta-D-Gal p-(1-->3)-beta-D-(2-OAc)-Gal p-O-CH3 (7) and beta-D-Gal p-(1-->3)-beta-D-(2-OAc)-Gal p-O-Bn (11) were synthesized and tested with the enzyme. Disaccharide 7 turned out to be a very good substrate allowing a rapid access to the trisaccharide alpha-Neu5Ac-(2-->3)-beta-D-Gal p-(1-->3)-beta-D-(2-OAc)-Gal p-O-CH3 (13) on a preparative scale using the crude enzyme immobilized on cationic exchanger. Trisaccharide 13 was further exploited, first as a sialyl donor in Trypanosoma cruzi trans-sialidase catalyzed reaction and second through acetolysis reaction as a source for the synthon alpha-Neu5Ac-(2-->3)-D-Gal (16).

New access to C-disaccharide analogs of alpha, alpha-trehalose using an aqueous hetero Diels-Alder reaction.

C-disaccharide analogs of trehalose were prepared using an aqueous Diels-Alder reaction as a key step. The resulting major stereoisomer was shown by NMR spectroscopy analysis to have the correct (alpha, alpha') stereochemistry of trehalose.

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.

Binding properties and esterase activity of monoclonal antibodies elicited against sucrose 6-heptylphosphonate.

Various sugar phosphonates were prepared by a Mitsunobu condensation between phosphonic diacids and properly protected carbohydrates. 6'-O-p-Aminophenylsucrose 6-heptylphosphonate was coupled to Bovine Serum Albumin (BSA) and Keyhole Limpet Hemocyanin (KLH) and the KLH conjugate was used for generation of monoclonal antibodies. Binding properties of these antibodies were screened by competitive enzyme-linked immunosorbent assay (ELISA) using the BSA conjugate. A monoclonal antibody with good binding properties showed a regioselective esterase activity toward 6-octanoylsucrose compared with 6'-octanoylsucrose.

[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.

First synthesis of the 3'-sulfated Lewis(a) pentasaccharide, the most potent human E-selectin ligand so far.

Tri- and pentasaccharides of Lewis(a)-type, sulfated at position 3 of the outer galactose, have been prepared using the new 4-methoxybenzyl glycoside of N-acetylglucosamine 5 as starting material. The synthesis of the pentasaccharide 2 was achieved through a beta-stereoselective coupling of an alpha-trichloroacetimidate activated form of the N-acetamido protected trisaccharide 18 on to a 3',4'-unprotected lactose derivative.

Acquired B antigen: further studies using synthetic oligosaccharides.

The reactivity of acquired-B red cells with various antisera has been investigated by agglutination inhibition assays using four trisaccharides obtained by chemical synthesis. Two of these had the structure GalNAc alpha 1-3 (LFuc alpha 1-2). Gal and Gal alpha 1-3 (LFuc alpha 1-2) Gal which are characteristic of the A and B determinants respectively and were indeed strong inhibitors of human anti-A and -B antibodies. The other two sugars denoted B-OAc and B-NH2 are derivatives of the B-trisaccharide by substitution of the hydroxyl group on carbon-2 of the alpha-galactose residue with the O-acetyl group (B-OAc) or the amino group (B-NH2) respectively. We have shown that both B and B-NH2 trisaccharides inhibited strongly the agglutination of acquired B red cells by the anti-B reagents (crude or affinity purified) whereas sera containing "anti-acquired B" agglutinins were specifically inhibited by B-NH2 but not by the A, B or B-OAc structures. We have also shown that the agglutination of Tk-activated erythrocytes by the BS-II lectin is specifically inhibited by N-acetylglucosamine but not by B, B-OAc or B-NH2 structures. These results and the observation that anti-acquired B agglutinins cannot be adsorbed on Tk red cells suggest that (i) the "B-like" and the "acquired-B" determinants share a common structure best represented by B-NH2 and (ii) Tk and "acquired-B" antigens are not identical.

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.

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.

Conformational studies on the selectin and natural killer cell receptor ligands sulfo- and sialyl-lacto-N-fucopentaoses (SuLNFPII and SLNFPII) using NMR spectroscopy and molecular dynamics simulations. Comparisons with the nonacidic parent molecule LNFPII.

This investigation is focused on the conformational behavior of the blood group Lewisa (Le(a)-active pentasaccharide lacto-N-fucopentaose II (LNFPII) and its sulfated and sialylated analogs, SuLNFPII and SLNFPII. The latter two are more potent oligosaccharide ligands for the animal lectins, E- and L-selectin, and the natural killer cell receptor, NKR-P1, than are the shorter chain analogs based on the trisaccharide Le(a) domain. We report here that the three oligosaccharides based on the fucopentasaccharide have very similar average solution conformations as determined from NMR spectroscopical parameters, in particular 13C chemical shift differences. From restrained simulated annealing and restrained molecular dynamics (MD) simulations performed in order to determine the most probable conformational distributions around the glycosidic linkages we derive models for these oligosaccharides that are in good agreement with experimental parameters, such as rotating-frame Overhauser effects (ROE's) and long-range 1H,13C coupling constants across the glycosidic linkages. In these model structures the Le(a) domain at the non-reducing end of the longer chain oligosaccharides approximates the same rigid structure as in the shorter analogs. The Gal beta 1-4Glc linkage at the reducing end is also rather rigid, showing only little more flexibility than the Le(a) domain. However, the NeuAc alpha 2-3Gal linkage in SLNFPII, and the GlcNAc beta 1-3Gal linkage in all three oligosaccharides are flexible, in each case fluctuating mainly between two minimum energy structures: (phi = -81 degrees, psi = 8 degrees) and (phi = -160 degrees, psi = -20 degrees) for the NeuAc alpha 2-3Gal linkage, as reported previously for the isomeric sequence 3'-sialyl Le(x), and (phi = -25 degrees, psi = -26 degrees) and (phi = 20 degrees, psi = 24 degrees) for the GlcNAc beta 1-3Gal linkage. The flexibility of the latter linkage may allow the lactosyl domain at the reducing end to fit with little strain into extended carbohydrate binding sites on the recognition proteins, and, for the purposes of drug designs, it will be important to establish which conformational distribution is assumed for the GlcNAc beta 1-3Gal linkage in these longer chain oligosaccharides in the bound state.

Valency dependent patterns of binding of human L-selectin toward sialyl and sulfated oligosaccharides of Le(a) and Le(x) types: relevance to anti-adhesion therapeutics.

The human L-selectin is known to bind to immobilized 3'-sialyl-Le(x) and -Le(a) oligosaccharides both under static and physiological flow conditions. Here the reactivities toward 3'-sulfated and 3'-sialyl-Le(a) and -Le(x) pentasaccharides are compared by in-vitro binding and inhibition assays using preparations of human L-selectin-IgG-Fc chimera in which the selectin is predominantly in di- and tetrameric form (paucivalent) or in the form of a complex with anti-IgG (multivalent). Affinity for the sulfated ligands is marginally greater than for the sialyl ligands, as judged by concentrations required to give 50% inhibition of the multivalent selectin binding to the immobilized sulfated and sialyl ligands. There is a striking difference, however, in the avidities of binding of the two L-selectin forms toward the sulfated and sialyl ligands when these are immobilized in the clustered state: the paucivalent selectin gives detectable binding only to the sulfated ligands when these are immobilized as neoglycolipids on plastic microwells (up to 100 pmol immobilized per well) whereas the multivalent L-selectin binds well to both classes of ligand. Moreover, binding of the paucivalent selectin form is effectively inhibited only by the sulfated ligand, although binding of the multivalent selectin is inhibitable by both the sulfated and sialyl ligands. Such striking valency-dependent differences in ligand binding avidity and inhibitability may be manifest in vivo with the membrane-bound L-selectin, as marked variations occur in its density of expression on leukocytes. Thus, for the purpose of selecting inhibitors for development of therapeutic anti-inflammatory compounds, experimental designs based on the paucivalent L-selectin would more clearly single out compounds with broad spectrum anti-adhesive activities toward the both the high- and low-avidity interactions of the cell adhesion protein.

Further studies of the binding specificity of the leukocyte adhesion molecule, L-selectin, towards sulphated oligosaccharides--suggestion of a link between the selectin- and the integrin-mediated lymphocyte adhesion systems.

This communication is concerned with the binding specificity of the leukocyte-adhesion molecule L-selectin (leukocyte homing receptor) towards structurally defined sulphated oligosaccharides of the blood group Le(a) and Le(x) series, and of the glycosaminoglycan series heparin, chondroitin sulphate and keratan sulphate. The recombinant soluble form of the rat L-selectin (L-selectin-IgG Fc chimera) investigated here was shown previously to bind to lipid-linked oligosaccharides 3-O, 4-O and 6-O sulphated at galactose, such as sulphatides and a mixture of 3-sulphated Le(a)/Le(x) type tetrasaccharides isolated from ovarian cystadenoma, as well as to the HNK-1 glycolipid with 3-O sulphated glucuronic acid. In the present study, the L-selectin investigated in both chromatogram binding and plastic microwell binding experiments using neoglycolipids was found to bind to the individual 3-sulphated Le(a) and Le(x) sequences (penta-, tetra- and trisaccharides), and with somewhat lower intensities to their non-fucosylated analogues. Glycosaminoglycan disaccharides of keratan sulphate, heparin and chondroitin sulphate types were also bound by L-selectin in one or both assay systems, leading to the conclusion that clustered glycosaminoglycan oligosaccharides with 6-O sulphation of N-acetylgalactosamine, N-acetylglucosamine or glucosamine, 4-O sulphation of N-acetylgalactosamine, 2-O sulphation of uronic acid, N-sulphation of glucosamine and, to a lesser extent, the non-sulphated uronic acid-containing disaccharides, can support L-selectin adhesion. As inflammatory chemokines (short-range stimulators of lymphocyte migration which trigger integrin activation) are known to bind to endothelial glycosaminoglycans, we propose that the binding of the lymphocyte membrane L-selectin to endothelial glycosaminoglycans may provide a link between the selectin-mediated and integrin-mediated adhesion systems in leukocyte extravasation cascades. The possibility is also raised that lymphocyte L-selectin interactions with glycosaminoglycans may contribute to pathologies of glycosaminoglycan-rich tissues, e.g. cartilage loss in rheumatoid arthritis and inflammatory lesions of the cornea.