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Fluorescent cell surface receptor agonists allow visualization of processes that are set in motion by receptor activation. This study describes the synthesis of two fluorescent, low molecular weight ligands for the follicle-stimulating hormone receptor (FSHR), based on a dihydropyridine (DHP) agonist. We show that both BODIPY- and Cy5-conjugated DHP (m-DHP-BDP and m-DHP-Cy5) are potent FSHR agonists, able to activate receptor signalling with nanomolar potencies and to effect receptor internalisation at higher concentrations. FSHR-dependent uptake of m-DHP-Cy5 is in stark contrast to the cellular uptake of m-DHP-BDP which was efficiently internalised also in the absence of FSHR. Our results comprise a first-in-class fluorescent low molecular weight ligand for in situ FSHR imaging and pertain the potential means for targeted delivery of drugs into the endolysosomal pathway of FSHR-expressing cells., Discovery of a potent, small-molecule, fluorescent agonist of the follicle-stimulating hormone receptor (FSHR) for selective staining of FSHR-expressing cells.

The highly lipophilic outer barrier of mycobacteria, such as M. tuberculosis and M. leprae, is key to their virulence and intrinsic antibiotic resistance. Various components of this mycomembrane interact with the host immune system but many of these interactions remain ill-understood. This review covers several chemical syntheses of one of these components, mycobacterial phenolic glycolipids (PGLs), and outlines the interaction of these PGLs with the human immune system, as established using these well-defined pure compounds.

Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram‐positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d‐alanine and N‐acetyl‐d‐glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA‐modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well‐defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA‐fragments, incorporating all possible glycosylation modifications (α‐GlcNAc at the RboP C4; β‐GlcNAc at the RboP C4; β‐GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA‐type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA‐hexamers. Automated solid phase syntheses were used to assemble a WTA‐dodecamer and glycosylated WTA‐hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA‐fragments were used to probe binding of monoclonal antibodies using WTA‐functionalized magnetic beads, revealing the binding specificity of these WTA‐specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA‐chains., By using tailor‐made teichoic acid building blocks, a library of glycosylated ribitol phosphate Staphylococcus aureus wall teichoic acids (WTA) has been assembled, ranging in length, and varying in N‐acetyl glucosamine substitution pattern. The library of well‐defined WTAs has been used to develop a magnetic bead binding assay to interrogate monoclonal antibody binding, revealing the preference of the various antibodies for different glycosylation patterns.

Point‐of‐care (POC) diagnostic tests for the rapid detection of individuals infected with Mycobacterium leprae, the causative pathogen of leprosy, represent efficient tools to guide therapeutic and prophylactic treatment strategies in leprosy control programs, thus positively contributing to clinical outcome and reducing transmission of this infectious disease. Levels of antibodies directed against the M. leprae‐specific phenolic glycolipid I (PGL−I) closely correlate with an individual's bacterial load and a higher risk of developing leprosy. We describe herein the assembly of a set of PGL glycans carrying the characteristic phenol aglycon and featuring different methylation patterns. The PGL trisaccharides were applied to construct neoglycoproteins that were used to detect anti‐PGL IgM antibodies in leprosy patients. ELISAs and quantitative lateral‐flow assays based on up‐converting nanoparticles (UCP‐LFAs) showed that the generated PGL−I and PGL‐II trisaccharide neoglycoconjugates can be applied for the detection of anti M. leprae IgM antibodies in POC tests., Getting to the point of care: Trisaccharides of phenolic glycolipids (PGL) originating from M. leprae were evaluated for their diagnostic potential as neoglycoproteins in ELISA and an up‐converting particle lateral flow assay (UCP‐LFA). Two naturally occurring trisaccharides were found to be suitable for this purpose and can thus be used for future field studies in leprosy endemic areas.

Glycopolymers are found surrounding the outer layer of many bacterial species. The first uses as immunogenic component in vaccines are reported since the beginning of the XX century, but it is only in the last decades that glycoconjugate based vaccines have been effectively applied for controlling and preventing several infectious diseases, such as H. influenzae type b (Hib), N. meningitidis, S. pneumoniae or group B Streptococcus. Methicillin resistant S. aureus (MRSA) strains has been appointed by the WHO as one of those pathogens, for which new treatments are urgently needed. Herein we present an overview of the carbohydrate-based cell wall polymers associated with different S. aureus strains and the related affords to deliver well-defined fragments through synthetic chemistry.

The stereoselective construction of 1,2-cis-glycosidic linkages is key in the assembly of biologically relevant glycans, but remains a synthetic challenge. Reagent-controlled glycosylation methodologies, in which external nucleophiles are employed to modulate the reactivity of the glycosylation system, have become powerful means for the construction of 1,2-cis-glycosidic linkages. Here we establish that nucleophilic additives can support the construction of α-1,2-glucans, and apply our findings in the construction of a d-alanine kojibiose functionalized glycerol phosphate teichoic acid fragment. This latter molecule can be found in the cell wall of the opportunistic Gram-positive bacterium, Enterococcus faecalis and represents a structural element that can possibly be used in the development of therapeutic vaccines and diagnostic tools.

International audience; Dendritic cells (DCs) are important initiators of adaptive immunity, and they possess a multitude of Pattern Recognition Receptors (PRR) to generate an adequate T cell mediated immunity against invading pathogens. PRR ligands are frequently conjugated to tumor-associated antigens in a vaccination strategy to enhance the immune response toward such antigens. One of these PPRs, DC-SIGN, a member of the C-type lectin receptor (CLR) family, has been extensively targeted with Lewis structures and mannose glycans, often presented in multivalent fashion. We synthesized a library of well-defined mannosides (mono-, di-, and tri-mannosides), based on known "high mannose" structures, that we presented in a systematically increasing number of copies (n = 1, 2, 3, or 6), allowing us to simultaneously study the effect of mannoside configuration and multivalency on DC-SIGN binding via Surface Plasmon Resonance (SPR) and flow cytometry. Hexavalent presentation of the clusters showed the highest binding affinity, with the hexa-α1,2-di-mannoside being the most potent ligand. The four highest binding hexavalent mannoside structures were conjugated to a model melanoma gp100-peptide antigen and further equipped with a Toll-like receptor 7 (TLR7)-agonist as adjuvant for DC maturation, creating a trifunctional vaccine conjugate. Interestingly, DC-SIGN affinity of the mannoside clusters did not directly correlate with antigen presentation enhancing properties and the α1,2-di-mannoside cluster with the highest binding affinity in our library even hampered T cell activation. Overall, this systematic study has demonstrated that multivalent glycan presentation can improve DC-SIGN binding but enhanced binding cannot be directly translated into enhanced antigen presentation and the sole assessment of binding affinity is thus insufficient to determine further functional biological activity. Furthermore, we show that well-defined antigen conjugates combining two different PRR ligands can be generated in a modular fashion to increase the effectiveness of vaccine constructs.

Enhancing T cell responses against both viral and tumor Ags requires efficient costimulation and directed delivery of peptide Ags into APCs. Long peptide vaccines are considered favorable vaccine moieties from a clinical perspective, as they can harbor more than one immunogenic epitope enabling treatment of a broader target population. In addition, longer peptides are not extracellularly loaded on MHC class I; rather, they require intracellular processing and will thereby be presented to T cells mainly by professional APCs, thereby avoiding the risk of tolerance induction. The drawback of peptide vaccines regardless of peptide length is that naked peptides are not actively targeted to and taken up by APCs, and the standard nonconjugated adjuvant-peptide mixtures do not ensure cotargeting of the two to the same APC. We have identified a tetanus toxin–derived B cell epitope that can mediate the formation of immune complexes in the presence of circulating Abs. In this study, we show that these immune complexes improve both Ag uptake by APCs (blood monocytes and CD1c+ dendritic cells) and consequently improve CD8+ T cell recall responses in a human ex vivo blood loop system. The uptake of the peptide conjugate by blood monocytes is dependent on Abs and the complement component C1q. We envision that this strategy can be used to facilitate active uptake of Ags into APCs to improve T cell responses against pathogens or cancer.

Immune complexes are potent mediators of cellular immunity and have been extensively studied for their disease mediating properties in humans and for their role in anti-cancer immunity. However, a viable approach to use antibody-complexed antigen as vehicle for specific immunotherapy has not yet reached clinical use. Since virtually all people have endogenous antibodies against tetanus toxoid (TTd), such commonly occurring antibodies are promising candidates to utilize for immune modulation. As an initial proof-of-concept we investigated if anti-tetanus IgG could induce potent cross-presentation of a conjugate with SIINFEKL, a MHC class I presented epitope of ovalbumin (OVA), to TTd. This protein conjugate enhanced OVA-specific CD8+ T cell responses when administrated to seropositive mice. Since TTd is poorly defined, we next investigated whether a synthetic peptide-peptide conjugate, with a chemically defined linear B cell epitope of tetanus toxin (TTx) origin, could improve cellular immune responses. Herein we identify one linear B cell epitope, here after named MTTE thru a screening of overlapping peptides from the alpha and beta region of TTx, and by assessment of the binding of pooled IgG, or individual human IgG from high-titer TTd vaccinated donors, to these peptides. Subsequently, we developed a chemical protocol to synthesize defined conjugates containing multiple copies of MTTE covalently attached to one or more T cell epitopes of choice. To demonstrate the potential of the above approach we showed that immune complexes of anti-MTTE antibodies with MTTE-containing conjugates are able to induce DC and T cell activation using model antigens.

Tremendous progress has been made in the construction of oligosaccharides, and many impressive examples of large and complex oligosaccharide total syntheses have appeared over the years. The stability, lifetime, and reactivity of an oxocarbenium ion depend besides the nature of the counterion on the nature and orientation of the functional groups present on the carbohydrate ring. This chapter explores the role of oxocarbenium ions and contact or close ion pairs (CIPs) that features a glycosyl cation, in chemical glycosylation reactions. It deals with the stability, reactivity, and -conformational behavior of glycosyl oxocarbenium ions, and describes their intermediacy in the assembly of (complex) oligosaccharides. The chapter presents sophisticated and detailed DFT computational approaches to study glycosyl oxocarbenium ions. Where many anomeric triflates have been spectroscopically characterized, glycosyl oxocarbenium ions are too reactive to detect by straightforward NMR techniques. NMR spectroscopy has been used to characterize a multitude of covalent reactive intermediates such as anomeric triflates.

Mono-ADP-ribosylation is a dynamic posttranslational modification (PTM) with important roles in signaling. Mammalian proteins that recognize or hydrolyze mono-ADP-ribosylated proteins have been described. We report the synthesis of ADP-ribosylated peptides from the proteins histone H2B, RhoA and, HNP-1. An innovative procedure was applied that makes use of pre-phosphorylated amino acid building blocks. Binding assays revealed that the macrodomains of human MacroD2 and TARG1 exhibit distinct specificities for the different ADP-ribosylated peptides, thus showing that the sequence surrounding ADP-ribosylated residues affects the substrate selectivity of macrodomains.

Chirally pure R- and S-epimers of TLR2 ligand Pam3CysSK4 were prepared and separately conjugated to an OVA model epitope, in which lysine was replaced by azidonorleucine. The azide function in the conjugate permitted labelling with different fluorophores by use of strain-promoted 3+2 cycloaddition. The R-epimer of the labelled conjugates induced TLR2-dependent DC maturation, while S-epimer proved to be inactive. Combining the lipophilicity of Pam3CysSK4 ligand with fluorophores influenced the solubility of the resulting conjugates in an unpredictable way and only the conjugates labelled with Cy-5 were suitable for confocal fluorescence microscopy experiments. It was shown that both epimers of the Cy-5 labelled lipopeptides were internalized equally well, indicating TLR2-independent cellular uptake. The presented results demonstrate the usefulness of strain-promoted azide-alkyne cycloaddition in the labelling of highly lipophilic lipopeptides without disturbing the in vitro activity of these conjugates with respect to activation of TLR-2.

Tri-functional activity-based protein probes that encompass an electrophilic trap, a photo-reactive group and a bio-orthogonal ligation handle are described. With these, and in a three-step chemical proteomics approach, proteasomal catalytic sites are covalently and irreversibly modified, followed by photocrosslinking of these to flanking subunits and Staudinger-Bertozzi ligation for visualization and identification of the resulting conjugates.

Three fluorescent cathepsin inhibitor glycoconjugates have been designed, synthesized, and evaluated in terms of their cell internalization and cathepsin inhibitory properties. The conjugates are composed of a peptide epoxysuccinate, capable of covalent and irreversible binding to cysteine proteases, coupled to a fluorescent BODIPY dye and functionalized with a mono-, tri-, or heptamannoside. Mannose-receptor-dependent uptake of the probes in live dendritic cells is shown to depend on the type of carbohydrate attached. Where uptake of the monomannoside is poor and mannose-receptor-independent, the intracellular labeling of cathepsins by the probes equipped with a tri- or heptamannoside conjugate appeared concentration- and mannose-receptor-dependent.