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Interactions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein–glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans.

Streptococcus pneumoniae (SP) bacteria cause serious invasive diseases. SP bacteria are covered by a capsular polysaccharide (CPS) that is a virulence factor and the basis for SP polysaccharide and glycoconjugate vaccines. The serotype 9V is part of the currently marketed conjugate vaccine and contains an acetate modification. To better understand the importance of glycan modifications in general and acetylation in particular, defined oligosaccharide antigens are needed for serological and immunological studies. Here, we demonstrate a convergent [2 + 3] synthetic strategy to prepare the pentasaccharide repeating unit of 9V with and without an acetate group at the C-6 position of mannosamine.

The Gram-positive bacterium Streptococcus pneumoniae causes severe disease globally. Vaccines that prevent S. pneumoniae infections induce antibodies against epitopes within the bacterial capsular polysaccharide (CPS). A better immunological understanding of the epitopes that protect from bacterial infection requires defined oligosaccharides obtained by total synthesis. The key to the synthesis of the S. pneumoniae serotype 12F CPS hexasaccharide repeating unit that is not contained in currently used glycoconjugate vaccines is the assembly of the trisaccharide β-D-Gal p NAc-(1→4)-[α-D-Glc p -(1→3)]-β-D-Man p NAcA, in which the branching points are equipped with orthogonal protecting groups. A linear approach relying on the sequential assembly of monosaccharide building blocks proved superior to a convergent [3 + 3] strategy that was not successful due to steric constraints. The synthetic hexasaccharide is the starting point for further immunological investigations.

1,3-Dibromo-5,5-dimethylhydantoin (DBDMH), an inexpensive, non-toxic and stable reagent, is a competent activator of thioglycosides for glycosidic bond formation. Excellent yields were obtained when triflic acid (TfOH) or trimethylsilyl trifluoromethanesulfonate (TMSOTf) were employed as co-promoters in solution or automated glycan assembly on solid phase.

Vaccines against S. pneumoniae , one of the most prevalent bacterial infections causing severe disease, rely on isolated capsular polysaccharide (CPS) that are conjugated to proteins. Such isolates contain a heterogeneous oligosaccharide mixture of different chain lengths and frame shifts. Access to defined synthetic S. pneumoniae CPS structures is desirable. Known syntheses of S. pneumoniae serotype 3 CPS rely on a time-consuming and low-yielding late-stage oxidation step, or use disaccharide building blocks which limits variability. Herein, we report the first iterative automated glycan assembly (AGA) of a conjugation-ready S. pneumoniae serotype 3 CPS trisaccharide. This oligosaccharide was assembled using a novel glucuronic acid building block to circumvent the need for a late-stage oxidation. The introduction of a washing step with the activator prior to each glycosylation cycle greatly increased the yields by neutralizing any residual base from deprotection steps in the synthetic cycle. This process improvement is applicable to AGA of many other oligosaccharides.

Streptococcus pneumoniae remains a deadly disease in small children and the elderly even though conjugate and polysaccharide vaccines based on isolated capsular polysaccharides (CPS) are successful. The most common serotypes that cause infection are used in vaccines around the world, but differences in geographic and demographic serotype distribution compromises protection by leading vaccines. The medicinal chemistry approach to glycoconjugate vaccine development has helped to improve the stability and immunogenicity of synthetic vaccine candidates for several serotypes leading to the induction of higher levels of specific protective antibodies. Here, we show that marketed CPS-based glycoconjugate vaccines can be improved by adding synthetic glycoconjugates representing serotypes that are not covered by existing vaccines. Combination (coformulation) of synthetic glycoconjugates with the licensed vaccines Prevnar13 (13-valent) and Synflorix (10-valent) yields improved 15- and 13-valent conjugate vaccines, respectively, in rabbits. A pentavalent semisynthetic glycoconjugate vaccine containing five serotype antigens (sPCV5) elicits antibodies with strong in vitro opsonophagocytic activity. This study illustrates that synthetic oligosaccharides can be used in coformulation with both isolated polysaccharide glycoconjugates to expand protection from existing vaccines and each other to produce precisely defined multivalent conjugated vaccines.

Glycoconjugate vaccines based on isolated capsular polysaccharide (CPS) save millions of lives annually by preventing invasive pneumococcal disease caused by Streptococcus pneumoniae. Some components of the S. pneumoniae glycoconjugate vaccine Prevnar13 that contains CPS antigens from 13 serotypes undergo modifications or degradation during isolation and conjugation, resulting in production problems and lower efficacy. We illustrate how stable, synthetic oligosaccharide analogs of labile CPS induce a specific protective immune response against native CPS using S. pneumoniae serotype 5 (ST-5), a problematic CPS component of Prevnar13. The rare aminosugar L-PneuNAc and a branched L-FucNAc present in the natural repeating unit (RU) are essential for antibody recognition and avidity. The epitope responsible for specificity differs from the part of the antigen that is stabilized by chemical modification. Glycoconjugates containing stable, monovalent synthetic oligosaccharide analogs of ST-5 CPS RU induced long-term memory and protective immune responses in rabbits superior to those elicited by the ST-5 CPS component in multivalent Prevnar13.

Streptococcus pneumoniae (S. pneumoniae)infections are the leading cause of child mortality globally. Currentvaccines fail to induceaprotective immune response towards a conserved part of the pathogen,resulting in newserotypescausing disease. Therefore, new vaccinestrategies are urgently needed.Described is atwo-pronged approach combiningS. pneumoniaeproteins, pneumolysin (Ply) and pneumococcal surface protein A (PspA),with aprecisely defined synthetic oligosaccharide,wherebythe carrier protein actsas a serotype-independent antigen to provideadditional protection. Proof of concept in mice and swine modelsrevealed thatthe conjugatesinhibited colonization of the nasopharynx, decreased the bacterial load and reduced disease severity in the bacteria challenge model. Immunization of piglets provided the first evidence for the immunogenicity and protective potential of synthetic glycoconjugate vaccine in a large animal model.Acombination of synthetic oligosaccharides with proteins from the target pathogen opens the path to create broadly cross-protective (“universal”) pneumococcal vaccines.

Streptococcus pneumoniae infections are the leading cause of child mortality globally. Current vaccines fail to induce a protective immune response towards a conserved part of the pathogen, resulting in new serotypes causing disease. Therefore, new vaccine strategies are urgently needed. Described is a two-pronged approach combining S.pneumoniae proteins, pneumolysin and PspA, with a precisely defined synthetic oligosaccharide, whereby the carrier protein acts as a serotype-independent antigen to provide additional protection. Proof of concept in mice and swine models revealed that the conjugates inhibit colonization of the nasopharynx, decrease the bacterial load and reduce disease severity in the bacteria challenged model. Immunization of piglets provided the first evidence for the immunogenicity and protective potential of synthetic glycoconjugate vaccine in a large animal model. A combination of synthetic oligosaccharides with proteins from the target pathogen opens the path to create broadly cross-protective (\"universal\") pneumococcal vaccines. Competing Interest Statement European Patent Application No. EP 20 170 605.8; Synthetic Streptococcus pneumoniae saccharide conjugates to conserved membrane protein filed by the inventors P.H.S., L.E.S., P.K. and S.V.H. Glycoconjugates containing the synthetic glycan structures of Streptococcus pneumoniae serotypes serotype 2 and 3 are included in patent \"Pneumococcal oligosaccharide-protein conjugate composition\" no. EP 16 179 133.0 filed by the inventors P.H.S. and P.K.

Chapter 1. Total synthesis of Fumonisin B1: The first total synthesis of fumonisin B1, a sphingolipid biosynthesis inhibitor was achieved. The synthesis used the 2-oxonia-[3, 3]-sigmatropic rearrangement methodology developed in the McDonald laboratory, thereby demonstrating the utility of these rearrangements in total synthesis. The synthesis also confirmed the assigned stereochemistry and the biological profile of the natural product. Chapter 2. Synthesis of Unnatural Sphingolipids, 1-deoxy-5-hydroxysphinganine and its diastereomers: Utilizing 2-oxonia-[3, 3]-sigmatropic rearrangements, we synthesized cis and trans homoallylic alcohols which were converted to the epoxides. Epoxide opening using an azide nucleophile and azide reduction gave the respective 1-deoxy-5-hydroxysphinganine sphingolipid compounds. Chapter 3. Development of Methodology, Synthesis of cis- and trans-homoallylic alcohols: Diastereo- and enantioselective synthesis of branched homoallylic synthons were achieved. These were then used to synthesize the linear cis- and trans-homoallylic alcohols through a 2-oxonia-[3, 3]-sigmatropic rearrangement. Chapter 4. Human Milk Oligosaccharides (HMO), Synthesis of Natural and Unnatural oligomers: Natural HMO’s 2’-fucosyllactose, 3-fucosyllactose and unnatural HMO 2’-glucoseptanosyllactose were synthesized using a lactose derived acceptor and a fucosyl or septanosyl derived donor. The synthesis utilised a greener approach of using non-toxic reagents and milder reaction conditions for the preparation of mono- and disaccharide synthons.