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P Sinaÿ

Publications and source records attributed to P Sinaÿ.

At least 19 recordsLinked to original sources
PubMed2001

Synthesis of conformationally locked L-iduronic acid derivatives: direct evidence for a critical role of the skew-boat 2S0 conformer in the activation of antithrombin by heparin.

We have used organic synthesis to understand the role of L-iduronic acid conformational flexibility in the activation of antithrombin by heparin. Among known synthetic analogues of the genuine pentasaccharidic sequence representing the antithrombin binding site of heparin, we have selected as a reference compound the methylated anti-factor Xa pentasaccharide 1. As in the genuine original fragment, the single L-iduronic acid moiety of this molecule exists in water solution as an equilibrium between three conformers 1C4, 4C1 and 2S0. We have thus synthesized three analogues of 1, in which the L-iduronic acid unit is locked in one of these three fixed conformations. A covalent two atom bridge between carbon atoms two and five of L-iduronic acid was first introduced to lock the pseudorotational itinerary of the pyranoid ring around the 2S0 form. A key compound to achieve this connection was the D-glucose derivative 5 in which the H-5 hydrogen atom has been replaced by a vinyl group, which is a progenitor of the carboxylic acid. Selective manipulations of this molecule resulted in the 2S0-type pentasaccharide 23. Starting from the D-glucose derivative 28, a covalent two atom bridge was now built up between carbon atoms three and five to lock the L-iduronic acid moiety around the 1C4 chair form conformation, and the 1C4-type pentasaccharide 43 was synthesized. Finally the L-iduronic acid containing disaccharide 58 which, due to the presence of the methoxymethyl substituent at position five adopts a 4C1 conformation, was directly used to synthesize the 4C1-type pentasaccharide 61. The locked pentasaccharide 23 showed about the same activity as the reference compound 1 in an antithrombin-mediated anti-Xa assay, whereas the two pentasaccharides 43 and 61 displayed very low activity. These results clearly establish the critical importance of the 2S0 conformation of L-iduronic acid in the activation of antithrombin by heparin.

Antithrombins↗
PubMed2001

Ultraweak sugar-sugar interactions for transient cell adhesion.

Carbohydrate-carbohydrate interactions are rarely considered in biologically relevant situations such as cell recognition and adhesion. One Ca(2+)-mediated homotypic interaction between two Lewis(x) determinants (Le(x)) has been proposed to drive cell adhesion in murine embryogenesis. Here, we confirm the existence of this specific interaction by reporting the first direct quantitative measurements in an environment akin to that provided by membranes. The adhesion between giant vesicles functionalized with Le(x) was obtained by micropipette aspiration and contact angle measurements. This interaction is below the thermal energy, and cell-cell adhesion will require a large number of molecules, as illustrated by the Le(x) concentration peak observed at the cell membranes during the morula stage of the embryo. This adhesion is ultralow and therefore difficult to measure. Such small interactions explain why the concept of specific interactions between carbohydrates is often neglected.

Calcium↗
PubMed2000

Reconstitution of membranes simulating "glycosignaling domain" and their susceptibility to lyso-GM3.

GM3 ganglioside at the surface of mouse melanoma B16 cells is clustered and organized with signal transducer molecules c-Src, Rho A, and focal adhesion kinase (FAK) to form a membrane unit separable from caveolae, which are enriched in cholesterol and caveolin but do not contain GM3 or the above three signal transducers. The GM3-enriched membrane units are involved in GM3-dependent cell adhesion coupled with activation of c-Src, Rho A, and FAK and are termed the "glycosphingolipid signaling domain" or the "glycosignaling domain" (GSD). In order to assess the essential components that display GSD function, membranes with properties similar to those of GSD were reconstituted using GM3, sphingomyelin, and c-Src, with or without other lipid components. The reconstituted membrane thus prepared displayed GM3-dependent adhesion to plates coated with Gg3 or anti-GM3 antibody, resulting in enhanced c-Src phosphorylation (c-Src phosphorylation response). This response in reconstituted membrane depends on GM3 concentration and was not observed when GM3 was absent or replaced with other gangliosides GM1 or GD1a, or with LacCer. The GM3-dependent c-Src phosphorylation response was enhanced when cholesterol and phosphatidylcholine were added. Although GM3, sphingomyelin, and c-Src are essential for GSD function, a small quantity of cholesterol and phosphatidylcholine may act as an auxiliary factor to stabilize membrane. GSD function in terms of GM3-dependent adhesion and signaling was blocked in the presence of lyso-GM3 or its analogue but not psychosine, lactosyl-sphingosine, or lyso-phosphatidylcholine. Such susceptibility of reconstituted GSD to lyso-GM3 and other lyso compounds is the same as GSD of original B16 cells. Thus, functional organization of the reconstituted membrane closely simulates that of GSD in B16 cells, which is based on clustered GM3 organized with c-Src as the essential components.

Animals↗
PubMed1999

Expression of heparan sulfate D-glucosaminyl 3-O-sulfotransferase isoforms reveals novel substrate specificities.

The 3-O-sulfation of glucosamine residues is an important modification during the biosynthesis of heparan sulfate (HS). Our previous studies have led us to purify and molecularly clone the heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST-1), which is the key enzyme converting nonanticoagulant heparan sulfate (HSinact) to anticoagulant heparan sulfate (HSact). In this study, we expressed and characterized the full-length cDNAs of 3-OST-1 homologous genes, designated as 3-OST-2, 3-OST-3A, and 3-OST-3B as described in the accompanying paper (Shworak, N. W., Liu, J., Petros, L. M., Zhang, L., Kobayashi, M., Copeland, N. G., Jenkins, N. A., and Rosenberg, R. D. (1999) J. Biol. Chem. 274, 5170-5184). All these cDNAs were successfully expressed in COS-7 cells, and heparan sulfate sulfotransferase activities were found in the cell extracts. We demonstrated that 3-OST-2, 3-OST-3A, and 3-OST-3B are heparan sulfate D-glucosaminyl 3-O-sulfotransferases because the enzymes transfer sulfate from adenosine 3'-phosphophate 5'-phospho-[35S]sulfate ([35S]PAPS) to the 3-OH position of glucosamine. 3-OST-3A and 3-OST-3B sulfate an identical disaccharide. HSact conversion activity in the cell extract transfected by 3-OST-1 was shown to be 300-fold greater than that in the cell extracts transfected by 3-OST-2 and 3-OST-3A, suggesting that 3-OST-2 and 3-OST-3A do not make HSact. The results of the disaccharide analysis of the nitrous acid-degraded [35S]HS suggested that 3-OST-2 transfers sulfate to GlcA2S-GlcNS and IdoA2S-GlcNS; 3-OST-3A transfers sulfate to IdoA2S-GlcNS. Our results demonstrate that the 3-O-sulfation of glucosamine is generated by different isoforms depending on the saccharide structures around the modified glucosamine residue. This discovery has provided evidence for a new cellular mechanism for generating a defined saccharide sequence in structurally complex HS polysaccharide.

Animals↗
PubMed1999

NMR study of a Lewis(X) pentasaccharide derivative: solution structure and interaction with cations.

The structure and conformation of the synthetic pentasaccharide Gal(beta 1-4){Fuc(alpha 1-3)}GlcNAc(beta 1-3)Gal(beta 1-4)Glc-beta OMe of the Lewis(X) family has been determined by NMR spectroscopy in dimethyl sulfoxide and methanol. In these solvents, the binding constants with calcium have been evaluated as 9.5 and 29.6 M-1, respectively. Study of the interaction sites has been achieved through the use of paramagnetic divalent cations and distance triangulation methods. Two regions have been found, the first one in the vicinity of the fucose unit, the second one closer to the lactose part.

Calcium↗
PubMed1999

A synthetic heparan sulfate pentasaccharide, exclusively containing L-iduronic acid, displays higher affinity for FGF-2 than its D-glucuronic acid-containing isomers.

It has been suggested that the FGF-2 binding site on heparan sulfate chains is a trisulfated pentasaccharide containing three hexuronic acid units. The configuration at C-5 of two of them being undetermined, we have synthesized the four possible pentasaccharides, and have evaluated their FGF-2 binding affinity through in vitro biological assays. The pentasaccharide containing L-iduronic acid as the sole hexuronic acid showed higher affinity for FGF-2 than the other pentasaccharides, where one hexuronic acid unit at least is D-glucuronic acid.

Aorta↗
PubMed1999

Oligosaccharides corresponding to the regular sequence of heparin: chemical synthesis and interaction with FGF-2.

It has been proposed that oligosaccharides corresponding to the so-called regular region of heparin/heparan sulfate (HS) bind to fibroblast growth factor-2 (FGF-2). In order to explore the molecular basis of FGF/HS interaction, we describe here the chemical synthesis of a tetra and a hexasaccharide, prepared as methyl glycosides, corresponding to the regular sequence of heparin. The strategy relies on the efficient preparation of three building blocks: a seeding block, an elongating block and a capping block. The hexasaccharide inhibited the binding of FGF-2 on its receptor on human aorta vascular smooth muscle cells with an IC50 value (16+/-1.2 microg/mL) close to that of standard heparin (14.8+/-0.5 microg/mL) whereas the tetrasaccharide was much less potent (IC50 = 127+/-10.5 microg/mL). The hexasaccharide and heparin, inhibited in a dose-dependent manner FGF-2 (30 nM) induced proliferation (IC50 = 23.7+/-1.6 and 30.1+/-1.3 microg/mL, respectively). Under the same experimental conditions, the tetrasaccharide only slightly inhibited the mitogenic effect of FGF-2 (IC50 > 100 microg/mL).

Carbohydrate Conformation↗
PubMed1999

Synthesis of a highly hydrophobic dimeric Lewis X containing glycolipid: a model for the study of homotypic carbohydrate--carbohydrate interaction.

This paper describes the synthesis of an octasaccharidic glycolipid 1 based on a stereo- and regioselective glycosylation between a Lewis X trisaccharidic donor and a pentasaccharidic acceptor. A highly hydrophobic lipid moiety of a new type was selected, making the compound 1 a good candidate for the study of the interaction of Lewis X functionalised vesicles.

Carbohydrate Sequence↗
PubMed1998

Synthetic C-oligosaccharides mimic their natural, analogous immunodeterminants in binding to three monoclonal immunoglobulins.

The binding of three monoclonal antigalactan immunoglobulins, IgAs X24, J539 and X44 to their natural haptens methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside, and the corresponding tri- and tetrasaccharides, was compared to the binding of these immunoglobulins with the comparable C-linked oligosaccharide analogues 1-3. The near identity of affinities of the two sets of oligosaccharides indicated the absence of any hydrogen bond involvement by the intersaccharidic oxygen atoms in the natural immunodeterminants.

Antibodies, Monoclonal↗
PubMed1998

Introducing a C-interglycosidic bond in a biologically active pentasaccharide hardly affects its biological properties.

We describe here the synthesis and the biological activity of a 'C-pentasaccharide', a new analogue of the antithrombin III (AT III) binding region of heparin containing a methylene bridge in place of an interglycosidic oxygen atom. The affinity for AT III and the anti-factor Xa activity of this compound have been compared with that of the corresponding selected 'O-pentasaccharide'. Such a structural modification slightly decreased the affinity of this compound for AT III as well as its anti-factor Xa activity (880 +/- 40 anti-Xa units versus 1180 +/- 30 anti-Xa units for the C-pentasaccharide and the O-pentasaccharide, respectively). This compound therefore represents the first example of a new class of anti-factor Xa pentasaccharides containing a C-interglycosidic bond.

Antithrombin III↗
PubMed1998

Synthesis of a 3-deoxy-L-iduronic acid containing heparin pentasaccharide to probe the conformation of the antithrombin III binding sequence.

We report in this work the total synthesis of a close analogue of the pentasaccharide active site of heparin, in which the L-iduronic acid residue has been deoxygenated at position three. 1H NMR studies demonstrated that, as anticipated, such a modification induces a shift of the conformational equilibrium toward 1C4 (contribution to the conformational equilibrium rises from 37% to 65%) and a substantial decrease of the affinity for antithrombin III (Kd 0.154 microM versus 0.050 microM).

Antithrombin III↗
PubMed1997

Transfer reactions catalyzed by cyclodextrin glucosyltransferase using 4-thiomaltosyl and C-maltosyl fluorides as artificial donors.

Cyclodextrin glycosyltransferase enzyme from Bacillus circulans catalyzed the effective conversion of 4-thio-alpha-maltosyl fluoride into cyclo-alpha-(1-->4(2))-thiomalto -tetraoside, -pentaoside, -hexaoside and linear hemithiomaltooligosaccharides. However, under the same conditions, C-maltosyl fluoride afforded only linear modified maltotetraose, maltohexaose and maltooctaose in moderate yield.

Bacillus↗
PubMed1996

L-iduronic acid derivatives as glycosyl donors.

O-[Methyl (2-O-acetyl-3-O-benzyl-4-O-levulinyl-alpha, and beta-L-idopyranosid)uronate] trichloroacetimidate and the corresponding n-pentenyl glycosides are efficient L-iduronic acid glycosyl donors. Both have been used for the high-yielding synthesis of basic disaccharide blocks which are useful for the subsequent synthesis of complex oligosaccharides related to heparin/heparan sulfate, and dermatan sulfate. In contrast, the corresponding thioethyl glycosides, thiophenyl glycosides, and fluoride, did not yield the expected disaccharides.

Carbohydrate Sequence↗