Synthesis of C-disaccharides via a hetero-Diels-Alder reaction and further stereocontrolled transformations.

The partial de novo synthesis of two new C-disaccharides containing D-glucosamine is described. The strategy is based on a hetero-Diels-Alder reaction leading to a 1:1 mixture of separable cycloadducts, which have been stereoselectively functionalized and converted into alpha-D-Galp-(1-->3)-C-D-GlcpNAc and alpha-l-Galp-(1-->3)-C-D-GlcpNAc.

New and general synthesis of beta-C-glycosylformaldehydes from easily available beta-C-glycosylpropanones.

A highly effective method for the introduction of a formyl group at the anomeric position of pyranosides was developed via enolisation of beta-C-D-glycopyranosylpropan-2-one using thermodynamic conditions then oxidative cleavage of the more substituted double bond. This sequence affords the desired aldehydes that are conveniently protected as aminals for purification and storage and easily regenerated using Dowex resin H+. In this paper, the syntheses of nine differently protected aldehydes derived from d-glucose, d-galactose, lactose and N-acetyl-d-glucosamine are presented. Our strategy proved to be very efficient in most cases excepted in the D-mannose series.

A new route to 2-C- and 4-C-branched sugars by palladium-indium bromide-mediated carbonyl allylation.

Palladium-catalysed carbonyl allylations can be effectively applied to the regio- and diastereoselective synthesis of 2-C- and 4-C-branched sugars from allylic esters or carbonates via the formation of pi-allylpalladium(II) intermediates and their reductive transmetalation with indium(I) bromide.

Heparan sulfate mimicry: a synthetic glycoconjugate that recognizes the heparin binding domain of interferon-gamma inhibits the cytokine activity.

Cell-associated heparan sulfate (HS) is endowed with the remarkable ability to bind numerous proteins. As such, it represents a unique system that integrates signaling from circulating ligands with cellular receptors. This polysaccharide is extraordinary complex, and examples that define the structure-function relationship of HS are limited. In particular, it remains difficult to understand the structures by which HS interact with proteins. Among them, interferon-gamma (IFNgamma), a dimeric cytokine, binds to a complex oligosaccharide motif encompassing a N-acetylated glucosamine-rich domain and two highly sulfated sequences, each of which binds to one IFNgamma monomer. Based on this template, we have synthesized a set of glycoconjugate mimetics and evaluated their ability to interact with IFNgamma. One of these molecules, composed of two authentic N-sulfated octasaccharides linked to each other through a 50-Angstroms-long spacer termed 2O(10), displays high affinity for the cytokine and inhibits IFNgamma-HS binding with an IC(50) of 35-40 nm. Interestingly, this molecule also inhibits the binding of IFNgamma to its cellular receptor. Thus, in addition to its ability to delocalize the cytokine from cell surface-associated HS, this compound has direct anti-IFNgamma activity. Altogether, our results represent the first synthetic HS-like molecule that targets a cytokine, strongly validating the HS structural determinants for IFNgamma recognition, providing a new strategy to inhibit IFNgamma in a number of diseases in which the cytokine has been identified as a target, and reinforcing the view that it is possible to create"tailor-made"sequences based on the HS template to isolate therapeutic activities.

Investigation of the aqueous transmetalation of pi-allylpalladium with indium salt: the use of the Pd(OAc)2-TPPTS catalyst.

pi-Allylpalladium complexes could be generated in water by the palladium(0) water soluble catalyst prepared in situ from palladium acetate and TPPTS. These complexes were transmetalated with indium to react with benzaldehyde. The aqueous solution of Pd(0)(TPPTS)(n) could be reused without deterioration of the catalyst in the first and second recycling. The system proved to be efficient with primary and secondary allylic substrates. The stereochemical outcome of the allylation through umpolung of allylpalladium, was also studied using models with a restraint conformation.

Indium-promoted Barbier-type allylations in aqueous media: a convenient approach to 4-C-branched monosaccharides and (1-->4)-C-disaccharides.

Starting from methyl 6-bromo-4,6-dideoxy-alpha-D-threo-4-enopyranoside, 4-C-branched sugars have been prepared through indium-promoted Barbier-type allylation of various aldehydes in aqueous media followed by hydroboration of the resulting double bond. The intermediate unsaturated monosaccharides were shown to rearrange in acidic media to give 4-C-acetyl-5-C-alkyl pyranose compounds. From beta-1-formyl sugars the corresponding beta-(1-->4)-C-disaccharides were obtained.

Synthesis of tailor-made glycoconjugate mimetics of heparan sulfate that bind IFN-gamma in the nanomolar range.

We have recently described the preparation of three building blocks for the combinatorial synthesis of heparan sulfate (HS) fragments. Herein we show that one of these building blocks (disaccharide 4) allows the preparation, in high yields and with total alpha stereoselectivity, of tetra-, hexa- and octasaccharides from the heparin (HP) regular region, by using 2+2, 2+4 and 4+4 glycosylation strategies, respectively. These oligosaccharides were processed into sulfated derivatives bearing an allyl moiety in the anomeric position. The UV-promoted conjugation of these compounds with alpha,omega-bis(thio)poly(ethylene glycol) spacers of three different lengths allowed us to prepare nine benzylated glycoconjugates. After final deprotection, the glycoconjugates 1 a-c, 2 a-c and 3 a-c were obtained and their ability to inhibit the interaction between IFN-gamma and HP was tested by using surface plasmon resonance detection. Compound 3 b, containing two HP octasaccharides linked by a 50-A linker was able to inhibit the IFN-gamma/HP interaction with an IC(50) value of approximately 35 nM. In addition, the nine glycoconjugates were perfect tools in the study to ascertain the topology of the IFN-gamma binding site on HS. Compounds 1 a-c, 2 a-c and 3 a-c, by mimicking the alternating sulfated and nonsulfated regions found in HS, thus comprise the first example of a library of synthetic HS mimetics giving access to the "second level of molecular diversity" found in HS.

One-step synthesis of beta-C-glycolipid derivatives from unprotected sugars.

Condensations of nonsymmetrical or symmetrical beta-diketones and unprotected sugars in aq NaHCO3 soln were explored. C-glucosyl and C-maltosyl derivatives bearing lipophilic residue of 8 or 11 carbon atoms were prepared efficiently using this one-step procedure. The amphiphilic properties of these compounds were demonstrated by measuring their CMC.

Recombinant (2-->3)-alpha-sialyltransferase immobilized on nickel-agarose for preparative synthesis of sialyl Lewis(x) and Lewis(a) precursor oligosaccharides.

The specificity of recombinant (2-->3)-alpha-sialyltransferase (ST3Gal-III), expressed in baculovirus-infected insect cells, has been determined with various oligosaccharide acceptors and sugar-nucleotide donors using a fluorescence based assay. Recombinant ST3Gal-III tagged with a polyhistidine tail was immobilized on Ni(2+)-NTA-Agarose as an active enzyme for use in the synthesis of three sialylated oligosaccharides: (i) the divalent molecule [alpha-Neu5Ac-(2-->3)-D-Galp-(1-->4)-beta-D-GlcpNAc-O-CH(2)](2)-C-(CH(2)OBn)(2) (12); (ii) the dansylated derivative, alpha-Neu5Ac-(2-->3)-D-Galp-(1-->3)-beta-D-GlcpNAc-O-(CH(2))(6)-NH-dansyl and; (iii) the tetrasacharide alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-O-CH(3). Compound 12 was itself prepared from the divalent N-acetyllactosamine molecule built on pentaerythritol by a chemo-enzymatic route.

Chemo-enzymatic synthesis of a divalent sialyl Lewis(x) ligand with restricted flexibility.

To study the influence of the entropic factor in cluster cooperative effects, a divalent sialyl Lewis(x) ligand with restricted flexilbility was chemo-enzymatically synthesized. First, a cyclized precursor with both glucosamine residues bridged together by a succinyl group was readily obtained in 42% yield by treatment of 2,2-bis(benzyloxymethyl)-1,3-bis(3,4,6-tri-O-acetyl-2-amino-2-deoxy-beta-D-glucopyranosyloxy)-propane with succinyl chloride. After deacetylation, this precursor was subjected to stepwise enzymatic elongation utilizing successively, soluble galactosyltransferase, then recombinant sialyltransferase and fucosyltransferase; the latter enzymes immobilized on Ni(2+)-Agarose, to afford, after debenzylation, a divalent sialyl Lewis(x) ligand of restricted flexibility, in 45% overall yield. Following the same enzymatic sequence, a totally flexible ligand, required as a reference compound for evaluation of inhibitory activity toward selectins, was also prepared from 2,2(bis-benzyloxymethyl)-1,3-bis(2-acetamido-2-deoxy-beta-D-glucopyranosyloxy)-propane, as well as both related divalent Lewis(x) molecules lacking the sialic acids, the rigid one and the flexible one.