Synthetic Oligomers Mimicking Capsular Polysaccharide Diheteroglycan are Potential Vaccine Candidates against Encapsulated Enterococcal Infections.

Infections caused by Enterococcus spp. are a major concern in the clinical setting. In Enterococcus faecalis, the capsular polysaccharide diheteroglycan (DHG), composed of ß-d-galactofuranose-(1 → 3)-ß-d-glucopyranose repeats, has been described as an important virulence factor and as a potential vaccine candidate against encapsulated strains. Synthetic structures emulating immunogenic polysaccharides present many advantages over native polysaccharides for vaccine development. In this work, we described the synthesis of a library of DHG oligomers, differing in length and order of the monosaccharide constituents. Using suitably protected thioglycoside building blocks, oligosaccharides up to 8-mer in length built up from either Galf-Glcp or Glcp-Galf dimers were generated, and we evaluated their immunoreactivity with antibodies raised against DHG. After the screening, we selected two octasaccharides, having either a galactofuranose or glucopyranose terminus, which were conjugated to a carrier protein for the production of polyclonal antibodies. The resulting antibodies were specific toward the synthetic structures and mediated in vitro opsonophagocytic killing of different encapsulated E. feacalis strains. The evaluated oligosaccharides are the first synthetic structures described to elicit antibodies that target encapsulated E. faecalis strains and are, therefore, promising candidates for the development of a well-defined enterococcal glycoconjugate vaccine.

The influence of acceptor nucleophilicity on the glycosylation reaction mechanism.

A set of model nucleophiles of gradually changing nucleophilicity is used to probe the glycosylation reaction mechanism. Glycosylations of ethanol-based acceptors, bearing varying amounts of fluorine atoms, report on the dependency of the stereochemistry in condensation reactions on the nucleophilicity of the acceptor. Three different glycosylation systems were scrutinized, that differ in the reaction mechanism, that - putatively - prevails during the coupling reaction. It is revealed that the stereoselectivity in glycosylations of benzylidene protected glucose donors are very susceptible to acceptor nucleophilicity whereas condensations of benzylidene mannose and mannuronic acid donors represent more robust glycosylation systems in terms of diastereoselectivity. The change in stereoselectivity with decreasing acceptor nucleophilicity is related to a change in reaction mechanism shifting from the SN2 side to the SN1 side of the reactivity spectrum. Carbohydrate acceptors are examined and the reactivity-selectivity profile of these nucleophiles mirrored those of the model acceptors studied. The set of model ethanol acceptors thus provides a simple and effective "toolbox" to investigate glycosylation reaction mechanisms and report on the robustness of glycosylation protocols.

Simultaneous quantitation of sphingoid bases by UPLC-ESI-MS/MS with identical 13C-encoded internal standards.

Free sphingoid bases (lysosphingolipids) of primary storage sphingolipids are increased in tissues and plasma of several sphingolipidoses. As shown earlier by us, sphingoid bases can be accurately quantified using UPLC-ESI-MS/MS, particularly in combination with identical 13C-encoded internal standards. The feasibility of simultaneous quantitation of sphingoid bases in plasma specimens spiked with a mixture of such standards is here described. The sensitivity and linearity of detection is excellent for all examined sphingoid bases (sphingosine, sphinganine, hexosyl-sphingosine (glucosylsphingosine), hexosyl2-sphingosine (lactosylsphingosine), hexosyl3-sphingosine (globotriaosylsphingosine), phosphorylcholine-sphingosine) in the relevant concentration range and the measurements show very acceptable intra- and inter-assay variation (<10% average). Plasma samples of a series of male and female Gaucher Disease and Fabry Disease patients were analyzed with the multiplex assay. The obtained data compare well to those earlier determined for plasma globotriaosylsphingosine and glucosylsphingosine in GD and FD patients. The same approach can be also applied to measure sphingolipids in the same sample. Following extraction of sphingolipids from the same sample these can be converted to sphingoid bases by microwave exposure and subsequently quantified using 13C-encoded internal standards.

Multigram-scale synthesis of an orthogonally protected 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (AAT) building block.

Reported is the gram-scale synthesis of tert-butyldiphenylsilyl 4-(N-benzyloxycarbonyl)-amino-2-azido-2,4,6-trideoxy-ß-D-galactopyranoside, which represents an orthogonally protected 2,4-diamino-D-fucose building block, a common constituent of various zwitterionic polysaccharides. The building block has been synthesized from D-glucosamine in 19% overall yield over 14 steps, requiring 5 chromatographic purifications. The key step in the synthesis is the introduction of the C-4 amino substituent, which has been accomplished by a one-pot three step procedure, involving regioselective C-3-O-trichloroacetimidate formation, C-4-O-triflation, and intramolecular substitution. The building block can be used as an acceptor and is readily transformed into a donor glycoside.

Light fluorous synthesis of glucosylated glycerol teichoic acids.

We here describe the synthesis of glucosylated teichoic acid (TA) fragments using two complementary fluorous scaffolds. The use of a perfluorooctylpropylsulfonylethyl (F-Pse) linker in combination with (glucosyl)glycerol phosphoramidite building blocks allows for the assembly of TA fragments with a terminal phosphate mono-ester, whereas the use of a perfluorooctylsuccinyl spacer delivers TA oligomers featuring a terminal alcohol functionality. These complementary linker systems have been developed because the nature of the TA chain terminus can play a role in the biological activity of the synthetic TAs. A novel α-glucosylated glycerolphosphoramidite building block is introduced to allow for a robust light fluorous synthetic protocol.

Microreactors as tools in the hands of synthetic chemists.

Recent developments in the construction of microstructured reaction devices and their wide-ranging applications in many different areas of chemistry suggest that microreactors may significantly impact the way chemists conduct experiments. Miniaturizing reactions offers many advantages for the synthetic organic chemist: high-throughput scanning of reaction conditions, precise control of reaction variables, the use of small quantities of reagents, increased safety parameters, and ready scale-up of synthetic procedures. A wide range of single and multiphase reactions has been performed in microfluidic-based devices. Certainly, microreactors cannot be applied to all chemistries yet and microfluidic systems also have disadvantages. Limited reaction time ranges, high sensitivity to precipitating products, and analytical challenges have to be overcome. An overview of microfluidic devices available for chemical synthesis is provided and some specific examples, mainly from our laboratory, are discussed to illustrate the potential of microreactors.