Microbe-focused glycan array screening platform.

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.

Improving vaccines against Streptococcus pneumoniae using synthetic glycans.

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.

Synthesis of Streptococcus pneumoniae serotype 9V oligosaccharide antigens.

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.

Nucleophile-Directed Stereocontrol Over Glycosylations Using Geminal-Difluorinated Nucleophiles.

The glycosylation reaction is the key transformation in oligosaccharide synthesis, but it is still difficult to control in many cases. Stereocontrol during cis-glycosidic linkage formation relies almost exclusively on tuning the glycosylating agent or the reaction conditions. Herein, we use nucleophile-directed stereocontrol to manipulate the stereoselectivity of glycosylation reactions. Placing two fluorine atoms in close proximity to the hydroxy group of an aliphatic amino alcohol lowers the oxygen nucleophilicity and reverses the stereoselectivity of glycosylations to preferentially form the desired cis-glycosides with a broad set of substrates. This concept was applied to the design of a cis-selective linker for automated glycan assembly. Fluorination of an amino alcohol linker does not impair glycan immobilization and lectin binding as illustrated by glycan microarray experiments. These fluorinated linkers enable the facile generation of α-terminating synthetic glycans for the formation of glycoconjugates.

Intramolecular cyclization manifolds of 4-alkylpyridines bearing ambiphilic side chains: construction of spirodihydropyridines or benzylic cyclization via anhydrobase intermediates.

4-Alkylpyridines possessing nucleophilic ß-dicarbonyl side chains have been converted to spirodihydropyridines upon treatment with ethyl chloroformate and sub-stoichiometric amounts of Ti(O(i)Pr)(4). Alternatively, inclusion of mild base in the reaction medium was found to facilitate generation of anhydrobase intermediates. Subsequent aldol-like condensations with electrophilic side chain moieties followed by hydrolysis delivered benzylically cyclized pyridines in good yield. In situ hydrogenation of cyclized anhydrobase intermediates afforded 4-substituted piperidines.

A semisynthetic glycoconjugate provides expanded cross-serotype protection against Streptococcus pneumoniae.

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.

Semisynthetic Glycoconjugate Vaccine Candidates against Escherichia coli O25B Induce Functional IgG Antibodies in Mice.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a major health concern due to emerging antibiotic resistance. Along with O1A, O2, and O6A, E. coli O25B is a major serotype within the ExPEC group, which expresses a unique O-antigen. Clinical studies with a glycoconjugate vaccine of the above-mentioned O-types revealed O25B as the least immunogenic component, inducing relatively weak IgG titers. To evaluate the immunological properties of semisynthetic glycoconjugate vaccine candidates against E. coli O25B, we here report the chemical synthesis of an initial set of five O25B glycan antigens differing in length, from one to three repeat units, and frameshifts of the repeat unit. The oligosaccharide antigens were conjugated to the carrier protein CRM197. The resulting semisynthetic glycoconjugates induced functional IgG antibodies in mice with opsonophagocytic activity against E. coli O25B. Three of the oligosaccharide-CRM197 conjugates elicited functional IgGs in the same order of magnitude as a conventional CRM197 glycoconjugate prepared with native O25B O-antigen and therefore represent promising vaccine candidates for further investigation. Binding studies with two monoclonal antibodies (mAbs) revealed nanomolar anti-O25B IgG responses with nanomolar K D values and with varying binding epitopes. The immunogenicity and mAb binding data now allow for the rational design of additional synthetic antigens for future preclinical studies, with expected further improvements in the functional antibody responses. Moreover, acetylation of a rhamnose residue was shown to be likely dispensable for immunogenicity, as a deacylated antigen was able to elicit strong functional IgG responses. Our findings strongly support the feasibility of a semisynthetic glycoconjugate vaccine against E. coli O25B.

Improved synthesis and biological evaluation of chelator-modified α-MSH analogs prepared by copper-free click chemistry.

Radionuclide chelators (DOTA, NOTA) functionalized with a monofluorocyclooctyne group were prepared. These materials reacted rapidly and in high yield with a fully deprotected azide-modified peptide via Cu-free click chemistry under mild reaction conditions (aqueous solution, room temperature). The resulting bioconjugates bind with high affinity and specificity to their cell-surface receptor targets in vitro and appear stable to degradation in mouse serum over 3h of incubation at 37°C.

A DOTA-peptide conjugate by copper-free click chemistry.

Attachment of DOTA to a novel monofluoro-cyclooctyne facilitates bioconjugation to an azide-modified peptide via Cu-free click chemistry. The resulting conjugate was radiolabeled with (111)In to afford a potential targeted molecular imaging agent with high specific activity and an excellent radiochemical purity.

A semisynthetic Streptococcus pneumoniae serotype 8 glycoconjugate vaccine.

Glycoconjugate vaccines based on capsular polysaccharides (CPSs) of pathogenic bacteria such as Streptococcus pneumoniae successfully protect from disease but suffer from incomplete coverage, are troublesome to manufacture from isolated CPSs, and lack efficacy against certain serotypes. Defined, synthetic oligosaccharides are an attractive alternative to isolated CPSs but require the identification of immunogenic and protective oligosaccharide antigens. We describe a medicinal chemistry strategy based on a combination of automated glycan assembly (AGA), glycan microarray-based monoclonal antibody (mAb) reverse engineering, and immunological evaluation in vivo to uncover a protective glycan epitope (glycotope) for S. pneumoniae serotype 8 (ST8). All four tetrasaccharide frameshifts of ST8 CPS were prepared by AGA and used in glycan microarray experiments to identify the glycotopes recognized by antibodies against ST8. One tetrasaccharide frameshift that was preferentially recognized by a protective, CPS-directed mAb was conjugated to the carrier protein CRM197. Immunization of mice with this semisynthetic glycoconjugate followed by generation and characterization of a protective mAb identified protective and nonprotective glycotopes. Immunization of rabbits with semisynthetic ST8 glycoconjugates containing protective glycotopes induced an antibacterial immune response. Coformulation of ST8 glycoconjugates with the marketed 13-valent glycoconjugate vaccine Prevnar 13 yielded a potent 14-valent S. pneumoniae vaccine. Our strategy presents a facile approach to develop efficient semisynthetic glycoconjugate vaccines.

Titanium-mediated spirocyclization reactions of 4-alkylpyridines.

Pyridines substituted at the 4-position with alkyl tethers containing beta-dicarbonyl moieties were converted to spirocyclic 4,4-disubstituted dihydropyridines. Optimal conditions for these transformations involved N-acylation of the pyridine substrate with a chloroformate electrophile in the presence of Ti(O(i)Pr)(4). Cyclization products could be easily converted into spiro-piperidine derivatives or elaborated into more complex heterocyclic frameworks via Au-catalyzed cycloisomerization.

Synthesis of a DOTA--biotin conjugate for radionuclide chelation via Cu-free click chemistry.

A strain-induced copper-free click reaction mediated by a new and easily prepared cyclooctyne derivative was used to efficiently assemble a DOTA-biotin adduct capable of radionuclide ((68)Ga) uptake. This synthetic strategy offers a potentially general and convenient means of preparing targeted radiolabeling and radiotherapeutic agents.