Bacterial cell-surface polysaccharides are involved in various biological processes and have attracted widespread attention as potential targets for developing carbohydrate-based drugs. However, the accessibility to structurally well-defined polysaccharide or related active oligosaccharide domains remains challenging. Herein, we describe an efficiently stereocontrolled approach for the first total synthesis of a unique pentasaccharide repeating unit containing four difficult-to-construct 1,2-cis-glycosidic linkages from the cell wall polysaccharide of Cutibacterium acnes C7. The features of our approach include: 1) acceptor-reactivity-controlled glycosylation to stereoselectively construct two challenging rare 1,2-cis-ManA2,3(NAc)2 (β-2,3-diacetamido-2,3-dideoxymannuronic acid) linkages, 2) combination use of 6-O-tert-butyldiphenylsilyl (6-O-TBDPS)-mediated steric shielding effect and ether solvent effect to stereoselectively install a 1,2-cis-glucosidic linkage, 3) bulky 4,6-di-O-tert-butylsilylene (DTBS)-directed glycosylation to stereospecifically construct a 1,2-cis-galactosidic linkage, 4) stereoconvergent [2+2+1] and one-pot chemoselective glycosylation to rapidly assemble the target pentasaccharide. Immunological activity tests suggest that the pentasaccharide can induce the production of proinflammatory cytokine TNF-α in a dose-dependent manner.

Published work has shown that glycoconjugate vaccines, based on truncated detoxified lipopolysaccharides from Moraxella catarrhalis attached through their reducing end to a carrier protein, gave good protection for all three serotypes A, B, and C in mice immunisation experiments. The (from the non-reducing end) truncated LPS structures were obtained from bacterial glycosyl transferase knock-out mutants and contained the de-esterified Lipid A, two Kdo residues and five glucose moieties. This work describes the chemical synthesis of the same outer Moraxella LPS structures, spacer-equipped and further truncated from the reducing end, i.e., without the Lipid A part and containing four or five glucose moieties or four glucose moieties and one Kdo residue, and their subsequent conjugation to a carrier protein via a five‑carbon bifunctional spacer to form glycoconjugates. Immunisation experiments both in mice and rabbits of these gave a good antibody response, being 2-7 times that of pre-immune sera. However, the sera produced only recognized the immunizing glycan immunogens and failed to bind to native LPS or whole bacterial cells. Comparative molecular modelling of three alternative antigens shows that an additional (2 → 4)-linked Kdo residue, not present in the synthetic structures, has a significant impact on the shape and volume of the molecule, with implications for antigen binding and cross-reactivity.

A synthesis of 2-(2,2,2-trichloroethoxy)-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-galactopyrano)-[2,1-d]-2-oxazoline - a previously unknown 2-alkoxy glyco-[2,1-d]-2-oxazoline derivative with d-galacto configuration was carried out. Glycosylating activity of the obtained galactooxazoline has been studied and it has been shown that in the presence of a weak protic acid, such as sym-collidinium triflate, this substance exhibits properties of a reactive and 1,2-trans-stereoselective glycosyl donor. The homopolymerization reaction of oxazoline derivatives of sugars has been found to proceed under the same conditions, leading to the formation of pseudo-oligosaccharide products. It has been found that this undesirable side reaction could be suppressed by changing the acid catalyst concentration, resulting in the development of efficient methods for the synthesis of glycoside and oligosaccharide derivatives of β-d-galactosamine using the synthesized 2-(2,2,2-trichloroethoxy)-2-oxazoline glycosyl donor under very mild conditions.

The protozoan parasite Leishmania (Viannia) braziliensis is among Latin America\'s most widespread Leishmania species and is responsible for tegumentary leishmaniasis (TL). This disease has multiple clinical presentations, with cutaneous leishmaniasis (CL) being the most frequent. It manifests as one or a few localized skin ulcers, which can spread to other body areas. Hence, early diagnosis and treatment, typically with pentavalent antimonials, is critical. Traditional diagnostic methods, like parasite culture, microscopy, or the polymerase chain reaction (PCR) for detection of the parasite DNA, have limitations due to the uneven distribution of parasites in biopsy samples. Nonetheless, studies have revealed high levels of parasite-specific anti-α-Gal antibodies in L. (V.) braziliensis-infected patients. Previously, we demonstrated that the neoglycoprotein NGP28b, consisting of the L. (Leishmania) major type-2 glycoinositolphospholipid (GIPL)-3-derived trisaccharide Galpα1,6Galpα1,3Galfβ conjugated to bovine serum albumin (BSA) via a linker, acts as a reliable serological biomarker (BMK) for L. (V.) braziliensis infection in Brazil. This indicates the presence of GIPL-3 or a similar structure in this parasite, and its terminal trisaccharide either functions as or is part of an immunodominant glycotope. Here, we explored whether extending the trisaccharide with a mannose unit would enhance its efficacy as a biomarker for the serological detection of L. (V.) braziliensis. We synthesized the tetrasaccharide Galpα1,6Galpα1,3Galfβ1,3Manpα(CH2)3SH (G31SH) and conjugated it to maleimide-functionalized BSA to afford NGP31b. When we assessed the efficacy of NGP28b and NGP31b by chemiluminescent enzyme-linked immunosorbent assay on a cohort of CL patients with L. (V.) braziliensis infection from Bolivia and Argentina against a healthy control group, both NGPs exhibited similar or identical sensitivity, specificity, and accuracy. This finding implies that the mannose moiety at the reducing end is not part of the glycotope recognized by the parasite-specific anti-α-Gal antibodies in patients\' sera, nor does it exert a relevant influence on the terminal trisaccharide\'s conformation. Moreover, the mannose does not seem to inhibit glycan-antibody interactions. Therefore, NGP31b is a viable and dependable BMK for the serodiagnosis of CL caused by L. (V.) braziliensis.

Lipopolysaccharides from Bacteroides vulgatus represent interesting targets for the treatment of inflammatory bowel diseases. However, efficient access to long, branched and complex lipopolysaccharides remains challenging. Herein, we report the modular synthesis of a tridecasaccharide from Bacteroides vulgates through an orthogonal one-pot glycosylation strategy based on glycosyl ortho-(1-phenylvinyl)benzoates, which avoids the issues of thioglycoside-based one-pot synthesis. Our approach also features: 1) 5,7-O-di-tert-butylsilylene-directed glycosylation for stereoselective construction of the α-Kdo linkage; 2) hydrogen-bond-mediated aglycone delivery for the stereoselective formation of β-mannosidic bonds; 3) remote anchimeric assistance for stereoselective assembly of the α-fucosyl linkage; 4) several orthogonal one-pot synthetic steps and strategic use of orthogonal protecting groups to streamline oligosaccharide assembly; 5) convergent [1+6+6] one-pot synthesis of the target.

Chondroitin sulfate (CS), a linear acidic polysaccharide, exhibits numerous biological activities that are dependent on sulfation patterns. CS oligosaccharides comprise repeating disaccharide units with different (hetero)-type sulfation patterns and are common in nature. We herein report the synthesis of the following biotinylated CS tetrasaccharides: CS-AD [βGalNAc4S(1-4)βGlcA(1-3)βGalNAc6S(1-4)βGlcA2S] and CS-DA [βGalNAc6S(1-4)βGlcA2S(1-3)βGalNAc4S(1-4)βGlcA], in a stereo-controlled manner. We also demonstrated that the CS-d-specific monoclonal antibody MO-225 bound more strongly to CS-DA than to CS-DD or -AD.

A chemical synthesis of two novel phenyl glycosides of trisaccharides related to acetylarabinoxylan is described. The trisaccharides bear acetyl and arabinofuranosyl moieties at the non-reducing-end xylopyranosyl residue, which is substituted at positions 2 and 3. Both compounds were treated with various xylan deacetylases classified in different carbohydrate esterase (CE) families and significant differences between the families were found. While the arabinosylation hampers deacetylation by CE2-CE5 and CE12 family members, both epitopes are deesterified by CE1 and in particular CE6 enzymes. The 3-O-acetylated 2-O-arabinosylated compound is also processed by CE7 and majority of CE16 esterases, but not by a hitherto non-classified Flavobacterium johnsoniae acetylxylan esterase. The data suggests that a slow deesterification of the 2-O-acetylated 3-O-arabinosylated compound may be due to the acetyl group migration followed by deacetylation of this migration product.

Access to homogeneous high-mannose glycans in high-mg quantities is necessary for carbohydrate-based HIV vaccine development research. We have used directed evolution to design highly antigenic oligomannose clusters that are recognized in low-nM affinity by HIV antibodies. Herein we report an optimized large-scale synthesis of Man9GlcNAc2 including improved building block synthesis and a fully stereoselective 5 + 6 coupling, yielding 290 mg of glycan. We then use this glycan to study the effect of the GlcNAc2 core on the antigenicity of an evolved 2G12-binding glycopeptide, 10F2.

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi and affects 6-7 million people worldwide. The diagnosis is still challenging, due to extensive parasite diversity encompassing seven genotypes (TcI-VI and Tcbat) with diverse ecoepidemiological, biological, and pathological traits. Chemotherapeutic intervention is usually effective but associated with severe adverse events. The development of safer, more effective therapies is hampered by the lack of biomarker(s) (BMKs) for the early assessment of therapeutic outcomes. The mammal-dwelling trypomastigote parasite stage expresses glycosylphosphatidylinositol-anchored mucins (tGPI-MUC), whose O-glycans are mostly branched with terminal, nonreducing α-galactopyranosyl (α-Gal) glycotopes. These are absent in humans, and thus highly immunogenic and inducers of specific CD anti-α-Gal antibodies. In search for α-Gal-based BMKs, here we describe the synthesis of neoglycoprotein NGP11b, comprised of a carrier protein decorated with the branched trisaccharide Galα(1,2)[Galα(1,6)]Galβ. By chemiluminescent immunoassay using sera/plasma from chronic CD (CCD) patients from Venezuela and Mexico and healthy controls, NGP11b exhibited sensitivity and specificity similar to that of tGPI-MUC from genotype TcI, predominant in those countries. Preliminary evaluation of CCD patients subjected to chemotherapy showed a significant reduction in anti-α-Gal antibody reactivity to NGP11b. Our data indicated that NGP11b is a potential BMK for diagnosis and treatment assessment in CCD patients.

New methods for the synthesis of the title oxazoline 2 from the corresponding 2-deoxy-2-(2,2,2- trichloroethoxycarbonylamino)glucosyl bromide were developed. The target 2-(2,2,2-trichloroethoxy) gluco-[2,1-d]-2-oxazoline 2 can be synthesized under conditions of halide ion catalysis, using triethylamine as a base. The synthesized 2-(2,2,2-trichloroethoxy)-2-oxazoline glycosyl donor was used for stereo-, regio-, and chemoselective glycosylation reactions under extremely mild conditions. The undesirable side reaction of intermolecular aglycone transfer between an ethyl thioglycoside glycosyl acceptor and the 2-(2,2,2-trichloroethoxy)-2-oxazoline glycosyl donor occurred to a relatively small extent. Regio-, and chemoselectivity of the disaccharide synthesis with the oxazoline glycosyl donor depended on the reaction conditions.