As part of an ongoing project aimed at developing vaccine candidates against Cryptococcus neoformans the preparation of tri- and tetrasaccharide thioglycoside building blocks, to be used in construction of structurally defined part structures of C. neoformans GXM capsular polysaccharide, was investigated. Using a naphthalenylmethyl (NAP) ether as a temporary protecting group and trichloroacetimidate donors in optimized glycosylations the target building blocks, ethyl 6-O-acetyl-2,4-di-O-benzyl-3-O-(2-naphthalenylmethyl)-α-D-mannopyranosyl-(1→3)-[2,3,4-tri-O-benzyl-β-D-xylopyranosyl-(1→2)]-4,6-di-O-benzyl-1-thio-α-D-mannopyranoside (16) and ethyl 2,3,4-tri-O-benzyl-β-D-xylopyranosyl-(1→2)-4,6-di-O-benzyl-3-O-(2-naphthalenylmethyl)-α-D-mannopyranosyl-(1→3)-[2,3,4-tri-O-benzyl-β-D-xylopyra-nosyl-(1→2)]-6-O-acetyl-4-O-benzyl-1-thio-α-D-mannopyranoside (21), were efficiently prepared. These synthesized thiosaccharide building blocks were then used as donors in high-yielding (∼90%) DMTST promoted glycosylations to a spacer-containing acceptor to, after deprotection, afford GXM polysaccharide part structures ready for protein conjugation to give vaccine candidates. Also, the NAP groups in the building blocks were removed to obtain tri- and tetrasaccharide acceptors suitable for further elongation towards larger thiosaccharide building blocks.

•Glycosyl donor hydrolysis is discussed.•Glycosyl donors may undergo elimination reactions in competition with substitution.•The sulfur atom of thioglycosides may act as a nucleophile in glycosylation.•Glycosyl trichloroacetimidates can undergo rearrangement to amides.Chemical glycosylation is central to carbohydrate chemistry and is generally recognised as a challenging reaction. This review describes the most reoccurring side reactions of glycosyl donors in glycosylation and how scientists have attempted to explain their observations and in some cases succeeded in solving a particular encountered problem. The topics covered are donor hydrolysis, elimination to form glycals, intermolecular aglycon transfer of thioglycosides and glycosyl imidate rearrangement.

•The ‘nitrile effect’ with benzylated glucuronic acid donors has been investigated.•Alternative routes to benzyl protected β-linked glucuronides have been investigated.•Efficient β-selective glycosylations with benzylated GlcA donors are described.•Pathways developed are reproducible and can be performed on a large scale.In a project targeting the synthesis of large oligosaccharide structures corresponding to the Cryptococcus neoformans GXM capsular polysaccharide, an easy access to thiodisaccharide building blocks comprising a β-linked glucuronic acid moiety and a 6-O-acetyl group was required. Several pathways to such building blocks have been investigated, addressing the problem of constructing a β-linked glucuronic acid residue protected with groups that are orthogonal to a primary acetyl group. Two efficient routes have been developed, one using benzoylated glucosyl donors to form the β-linkage followed by a change of protecting groups to benzyls and subsequent introduction of the carboxyl function and the acetyl group. The second route explored the possibility to achieve β-selectivity using glucuronyl donors without acyl protecting groups. BF3-etherate promoted glycosylations with benzyl (2,3,4-tri-O-benzyl-α-d-glucupyranosyl)uronate trichloroacetimidate in the presence of nitrile solvents and at low temperatures reproducibly gave good yields of disaccharides with high β-selectivity. Furthermore, the use of recently reported glucuronyl thioglycoside donors protected with a cyclic 2,4-silylene acetal was found to represent another efficient and completely β-selective way to desired disaccharide building blocks.

For the investigation of glycosidases, and for the construction of glycan arrays the p-nitrophenyl- and p-aminophenyl glycosides of mucin O-glycan core structures 1–7 and the 2,6-ST-antigen have been chemically synthesized using d-galactose as a precursor for GalNAc residues. GlcNAc residues have partly been introduced using a 4,6-di-O-benzoyl-2,3-N,O-oxazolidinone-protected donor, which allowed deprotection of the formed di- and tri-saccharides in one step using sodium methoxide.

Blocking lectin/toxin binding to human cells by suitable inhibitors can therapeutically protect them from harmful effects. Clustered design of ligand presentation holds the promise of affinity increase relative to the free sugar and inherent selectivity among lectin targets. Using first a solid-phase assay with a glycoprotein presenting N-glycans as lectin-reactive probe, we assessed the inhibitory potency of bi- to tetravalent clusters on a plant toxin and three human adhesion/growth-regulatory lectins. Enhanced avidity relative to the free sugar was detected together with lectin-type selectivity. These effects were confirmed on the level of cells in vitro, also for two leguminous lectins. The lack of toxicity in cell proliferation assays excluded concerns to further work on these compounds. The given cluster design and the strategic combination of the two assay systems of increasing biorelevance will thus be helpful to take the next steps in drug development, e.g. tailoring the sugar headgroup.Keywords: Agglutinin; colon cancer; glycan branching; glycocluster; multivalency;

2-(N-Benzyloxycarbonyl)aminoethyl 7-O-acetyl-6-O-allyl-2-O-benzoyl-4-O-benzyl-3-O-chloroacetyl-l-glycero-α-d-manno-heptopyranosyl-(1→3)-[2,3,4,6-tetra-O-benzoyl-β-d-glucopyranosyl-(1→4)]-6,7-di-O-acetyl-2-O-benzyl-l-glycero-α-d-manno-heptopyranoside, a spacer-equipped protected derivative of the common 3,4-branched diheptoside trisaccharide structure of the lipopolysaccharide core of Neisseria meningitidis and Haemophilus influenzae has been synthesized. The protecting group pattern installed allows regioselective introduction of phosphoethanolamine residues in the 3- and 6-position of the second heptose unit in accordance with native structures. From this intermediate the 3-and 6-monophosphoethanolamine as well as the non-phosphorylated deprotected trisaccharides have been synthesized to be used in evaluation of antibody binding specificity and in investigation of the substrate specificity of glycosyl transferases involved in the biosynthesis of LPS core structures.

The recently described [Attolino, E.; Bonaccorsi, F.; Catelani, G.; D’Andrea, F. Carbohydr. Res. 2008, 343, 2545–2556.] β-d-MaNAcp-(1→4)-β-d-Glcp thiophenyl glycosyl donor 3 was used in α-glycosylation reactions of OH-2 and OH-3 of the suitably protected p-MeO-benzyl α-l-rhamnopyranoside acceptors 7 and 8. Glycosylation of the axial OH-2 of 7 took place in high yield (76%) and with acceptable stereoselectivity (α/β = 3.4) leading to the protected trisaccharide α-11, corresponding to the repeating unit of Streptococcus pneumoniae 19F. The same reaction on equatorial OH-3 of acceptor 8 gave the trisaccharide α-15, a constituent of the repeating unit of S. pneumoniae 19A, but in lower yield (41%) and without stereoselection (α/β = 1:1.3). Utilizing the introduced orthogonal protection of OH-1 and OH-4″, the trisaccharide α-11 was transformed into a trisaccharide building block suitable for the synthesis of its phosphorylated oligomers.

•Acetylated ethyl 2-azido-2-deoxy-1-thio-α-d-cellobioside was prepared on a multigram scale with no chromatography required.•The regioselective in various benzylation methods have been investigated on 4,6-acetals of this (deactylated) precursor.•A number of prepared mono-ols, diols, and triols have been further processed to give heparin building block intermediates.Crystalline acetylated ethyl 2-azido-2-deoxy-1-thio-α-d-cellobioside has been prepared on a multigram scale from cellobiose in an overall yield of 23% with no chromatography required and converted after deacetylation into the 4′,6′-O-benzylidene and 4′,6′-O-benzylidene-6-O-TBDMS protected derivatives. Applying a number of regioselective benzylation methods on these gave access to a variety of regioselectively protected derivatives, both mono-ols (2′- and 3-OH), diols (2′,6-, 2′,3-, and 3,6-di-OH), and triols (2′,3,6- and 2′,3′,3-tri-OH). A number of these derivatives were further processed by benzoylation followed by removal or opening of the benzylidene acetal and selective oxidation of the exposed primary alcohol to give heparin building block intermediates comprising a range of possible sulfation patterns.

As part of an ongoing project aimed at developing vaccine candidates against Cryptococcus neoformans the preparation of tri- and tetrasaccharide thioglycoside building blocks, to be used in construction of structurally defined part structures of C. neoformans GXM capsular polysaccharide, was investigated. Using a naphthalenylmethyl (NAP) ether as a temporary protecting group and trichloroacetimidate donors in optimized glycosylations the target building blocks, ethyl 6-O-acetyl-2,4-di-O-benzyl-3-O-(2-naphthalenylmethyl)-α-D-mannopyranosyl-(1→3)-[2,3,4-tri-O-benzyl-β-D-xylopyranosyl-(1→2)]-4,6-di-O-benzyl-1-thio-α-D-mannopyranoside (16) and ethyl 2,3,4-tri-O-benzyl-β-D-xylopyranosyl-(1→2)-4,6-di-O-benzyl-3-O-(2-naphthalenylmethyl)-α-D-mannopyranosyl-(1→3)-[2,3,4-tri-O-benzyl-β-D-xylopyra-nosyl-(1→2)]-6-O-acetyl-4-O-benzyl-1-thio-α-D-mannopyranoside (21), were efficiently prepared. These synthesized thiosaccharide building blocks were then used as donors in high-yielding (∼90%) DMTST promoted glycosylations to a spacer-containing acceptor to, after deprotection, afford GXM polysaccharide part structures ready for protein conjugation to give vaccine candidates. Also, the NAP groups in the building blocks were removed to obtain tri- and tetrasaccharide acceptors suitable for further elongation towards larger thiosaccharide building blocks.