High-Mannose Oligosaccharide Hemimimetics that Recapitulate the Conformation and Binding Mode to Concanavalin A, DC-SIGN and Langerin.

The "carbohydrate chemical mimicry" exhibited by sp2 -iminosugars has been utilized to develop practical syntheses for analogs of the branched high-mannose-type oligosaccharides (HMOs) Man3 and Man5 . In these compounds, the terminal nonreducing Man residues have been substituted with 5,6-oxomethylidenemannonojirimycin (OMJ) motifs. The resulting oligomannoside hemimimetic accurately reproduce the structure, configuration, and conformational behavior of the original mannooligosaccharides, as confirmed by NMR and computational techniques. Binding studies with mannose binding lectins, including concanavalin A, DC-SIGN, and langerin, by enzyme-linked lectin assay and surface plasmon resonance revealed significant variations in their ability to accommodate the OMJ unit in the mannose binding site. Intriguingly, OMJMan segments demonstrated "in line" heteromultivalent effects during binding to the three lectins. Similar to the mannobiose (Man2 ) branches in HMOs, the binding modes involving the external or internal monosaccharide unit at the carbohydrate binding-domain exist in equilibrium, facilitating sliding and recapture processes. This equilibrium, which influences the multivalent binding of HMOs, can be finely modulated upon incorporation of the OMJ sp2 -iminosugar caps. As a proof of concept, the affinity and selectivity towards DC-SIGN and langerin were adjustable by presenting the OMJMan epitope in platforms with diverse architectures and valencies.

Cell-free expression and characterization of multivalent rhamnose-binding lectins using bio-layer interferometry.

Lectins are important biological tools for binding glycans, but recombinant protein expression poses challenges for some lectin classes, limiting the pace of discovery and characterization. To discover and engineer lectins with new functions, workflows amenable to rapid expression and subsequent characterization are needed. Here, we present bacterial cell-free expression as a means for efficient, small-scale expression of multivalent, disulfide bond-rich, rhamnose-binding lectins. Furthermore, we demonstrate that the cell-free expressed lectins can be directly coupled with bio-layer interferometry analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables the determination of lectin substrate specificity and estimation of binding affinity. Overall, we believe that this method will enable high-throughput expression, screening, and characterization of new and engineered multivalent lectins for applications in synthetic glycobiology.

Advanced high-affinity glycoconjugate ligands of galectins.

Galectins are proteins of the family of human lectins. By binding terminal galactose units of cell surface glycans, they moderate biological and pathological processes such as cell signaling, cell adhesion, apoptosis, fibrosis, carcinogenesis, and metabolic disorders. The binding of monovalent glycans to galectins is usually relatively weak. Therefore, the presentation of carbohydrate ligands on multivalent scaffolds can efficiently increase and/or discriminate the affinity of the glycoconjugate to different galectins. A library of glycoclusters and glycodendrimers with various structural presentations of the common functionalized N-acetyllactosamine ligand was prepared to evaluate how the mode of presentation affects the affinity and selectivity to the two most abundant galectins, galectin-1 (Gal-1) and galectin-3 (Gal-3). In addition, the effect of a one- to two-unit carbohydrate spacer on the affinity of the glycoconjugates was determined. A new design of the biolayer interferometry (BLI) method with specific AVI-tagged constructs was used to determine the affinity to galectins, and compared with the gold-standard method of isothermal titration calorimetry (ITC). This study reveals new routes to low nanomolar glycoconjugate inhibitors of galectins of interest for biomedical research.

Multivalent glycocyclopeptides: conjugation methods and biological applications.

Click chemistry was extensively used to decorate synthetic multivalent scaffolds with glycans to mimic the cell surface glycocalyx and to develop applications in glycosciences. Conjugation methods such as oxime ligation, copper(I)-catalyzed alkyne-azide cycloaddition, thiol-ene coupling, squaramide coupling or Lansbury aspartylation proved particularly suitable to achieve this purpose. This review summarizes the synthetic strategies that can be used either in a stepwise manner or in an orthogonal one-pot approach, to conjugate multiple copies of identical or different glycans to cyclopeptide scaffolds (namely multivalent glycocyclopeptides) having different size, valency, geometry and molecular composition. The second part of this review will describe the potential of these structures to interact with various carbohydrate binding proteins or to stimulate immunity against tumor cells.

Characterization of the interaction of multivalent glycosylated ligands with bacterial lectins by biolayer interferometry.

The study of multivalent carbohydrate-protein interactions remains highly complicated and sometimes rendered impossible due to aggregation problems. Biolayer interferometry is emerging as a tool to monitor such complex interactions. In this study, various glycoclusters and dendrimers were prepared and evaluated as ligands for lectins produced by pathogenic bacteria Pseudomonas aeruginosa (LecA and Lec B) and Burkholderia ambifaria (BambL). Reliable kinetic and thermodynamic parameters could be measured, and immobilization of either lectin or ligands resulted in high quality data. The methods gave results in full agreement with previous isothermal titration calorimetry experiments, and presented strong advantages because they require less quantity and purity for the biomolecules.

Targeting Tn-Antigen-Positive Human Tumors with a Recombinant Human Macrophage Galactose C-Type Lectin.

Alterations in glycosylation cause the emergence of tumor-associated carbohydrate antigens (TACAs) during tumorigenesis. Truncation of O-glycans reveals the Thomsen nouveau (Tn) antigen, an N-acetylgalactosamine (GalNAc) frequently attached to serine or threonine amino acids, that is accessible on the surface of cancer cells but not on healthy cells. Interestingly, GalNac can be recognized by macrophage galactose lectin (MGL), a type C lectin receptor expressed in immune cells. In this study, recombinant MGL fragments were tested in vitro for their cancer cell-targeting efficiency by flow cytometry and confocal microscopy and in vivo after administration of fluorescent MGL to tumor-bearing mice. Our results demonstrate the ability of MGL to target Tn-positive human tumors without inducing toxicity. This outcome makes MGL, a fragment of a normal human protein, the first vector candidate for in vivo diagnosis and imaging of human tumors and, possibly, for therapeutic applications.

Multivalent Presentations of Glycomimetic Inhibitor of the Adhesion of Fungal Pathogen Candida albicans to Human Buccal Epithelial Cells.

Candida albicans causes some of the most prevalent hospital-acquired fungal infections, particularly threatening for immunocompromised patients. C. albicans strongly adheres to the surface of epithelial cells so that subsequent colonization and biofilm formation can take place. Divalent galactoside glycomimetic 1 was found to be a potent inhibitor of the adhesion of C. albicans to buccal epithelial cells. In this work, we explore the effect of multivalent presentations of glycomimetic 1 on its ability to inhibit yeast adhesion and biofilm formation. Tetra-, hexa-, and hexadecavalent displays of compound 1 were built on RAFT cyclopeptide- and polylysine-based scaffolds with a highly efficient and modular synthesis. Biological evaluation revealed that the scaffold choice significantly influences the activity of the lower valency conjugates, with compound 16, constructed on a tetravalent polylysine scaffold, found to inhibit the adhesion of C. albicans to human buccal epithelial cells more effectively than the glycomimetic 1; however, the latter performed better in the biofilm reduction assays. Interestingly, the higher valency glycoconjugates did not outperform the anti-adhesion activity of the original compound 1, and no significant effect of the core scaffold could be appreciated. SEM images of C. albicans cells treated with compounds 1, 14, and 16 revealed significant differences in the aggregation patterns of the yeast cells.

Clicked Bifunctional Dendrimeric and Cyclopeptidic Addressable Redox Scaffolds for the Functionalization of Carbon Nanotubes with Redox Molecules and Enzymes.

Carbon nanotube electrodes were modified with ferrocene and laccase using two different click reactions strategies and taking advantage of bifunctional dendrimers and cyclopeptides. Using diazonium functionalization and the efficiency of oxime ligation, the combination of both multiwalled carbon nanotube surfaces and modified dendrimers or cyclopeptides allows the access to a high surface coverage of ferrocene in the order of 50 nmol cm-2, a 50-fold increase compared to a classic click reaction without oxime ligation of these highly branched macromolecules. Furthermore, this original immobilization strategy allows the immobilization of mono- and bi-functionalized active multicopper enzymes, laccases, via copper(I)-catalyzed azide-alkyne cycloaddition. Electrochemical studies underline the high efficiency of the oxime-ligated dendrimers or cyclopeptides for the immobilization of redox entities on surfaces while being detrimental to electron tunneling with enzyme active sites despite controlled orientation.

Development of C-type lectin-oriented surfaces for high avidity glycoconjugates: towards mimicking multivalent interactions on the cell surface.

Multivalent interactions between complex carbohydrates and oligomeric C-type lectins govern a wide range of immune responses. Up to date, standard SPR (surface plasmon resonance) competitive assays have largely been to evaluate binding properties from monosaccharide units (low affinity, mM) to multivalent elemental antagonists (moderate affinity, µM). Herein, we report typical case-studies of SPR competitive assays showing that they underestimate the potency of glycoclusters to inhibit the interaction between DC-SIGN and immobilized glycoconjugates. This paper describes the design and implementation of a SPR direct interaction over DC-SIGN oriented surfaces, extendable to other C-type lectin surfaces as such Langerin. This setup provides an overview of intrinsic avidity generation emanating simultaneously from multivalent glycoclusters and from DC-SIGN tetramers organized in nanoclusters at the cell membrane. To do so, covalent biospecific capture of DC-SIGN via StreptagII/StrepTactin interaction preserves tetrameric DC-SIGN, accessibility and topology of its active sites, that would have been dissociated using standard EDC-NHS procedure under acidic conditions. From the tested glycoclusters libraries, we demonstrated that the scaffold architecture, the valency and the glycomimetic-based ligand are crucial to reach nanomolar affinities for DC-SIGN. The glycocluster 3·D illustrates the tightest binding partner in this set for a DC-SIGN surface (KD = 18 nM). Moreover, the selectivity at monovalent scale of glycomimetic D can be easily analyzed at multivalent scale comparing its binding over different C-type lectin immobilized surfaces. This approach may give rise to novel insights into the multivalent binding mechanisms responsible for avidity and make a major contribution to the full characterization of the binding potency of promising specific and multivalent immodulators.

Multifunctional Glycoconjugates for Recruiting Natural Antibodies against Cancer Cells.

We have developed a fully synthetic and multifunctional antibody-recruiting molecule (ARM) to guide natural antibodies already present in the blood stream against cancer cells without pre-immunization. Our ARM is composed of antibody and tumor binding modules (i.e., ABM and TBM) displaying clustered rhamnose and cyclo-RGD, respectively. By using a stepwise approach, we have first demonstrated the importance of multivalency for efficient recognition with naturel IgM and αv ß3 integrin expressing M21 tumor cell line. Once covalently conjugated by click chemistry, we confirmed by flow cytometry and confocal microscopy that the recognition properties of both the ABM and TBM are conserved, and more importantly, that the resulting ARM promotes the formation of a ternary complex between natural IgM and cancer cells, which is required for the stimulation of the cytotoxic immune response in vivo. Due to the efficiency of the synthetic process, a larger diversity of heterovalent ligands could be easily explored by using the same multivalent approach and could open new perspectives in this field.

Multifunctional Glycoconjugates for Recruiting Natural Antibodies against Cancer Cells.

Invited for the cover of this issue is Olivier Renaudet and co-workers at the Université Grenoble Alpes and funded by the European Research Council (CoG "LEGO'" no. 647938). The image illustrates a synthetic chemist playing with supramolecular structures to kill cancer cells by using natural antibodies present in the blood stream. Read the full text of the article at 10.1002/chem.201903327.

Aminooxylated Carbohydrates: Synthesis and Applications.

Among other classes of biomolecules, carbohydrates and glycoconjugates are widely involved in numerous biological functions. In addition to addressing the related synthetic challenges, glycochemists have invested intense efforts in providing access to structures that can be used to study, activate, or inhibit these biological processes. Over the past few decades, aminooxylated carbohydrates have been found to be key building blocks for achieving these goals. This review provides the first in-depth overview covering several aspects related to the syntheses and applications of aminooxylated carbohydrates. After a brief introduction to oxime bonds and their relative stabilities compared to related C═N functions, synthetic aspects of oxime ligation and methodologies for introducing the aminooxy functionality onto both glycofuranosyls and glycopyranosyls are described. The subsequent section focuses on biological applications involving aminooxylated carbohydrates as components for the construcion of diverse architectures. Mimetics of natural structures represent useful tools for better understanding the features that drive carbohydrate-receptor interaction, their biological output and they also represent interesting structures with improved stability and tunable properties. In the next section, multivalent structures such as glycoclusters and glycodendrimers obtained through oxime ligation are described in terms of synthetic design and their biological applications such as immunomodulators. The second-to-last section discusses miscellaneous applications of oxime-based glycoconjugates, such as enantioselective catalysis and glycosylated oligonucleotides, and conclusions and perspectives are provided in the last section.

Towards the preparation of synthetic outer membrane vesicle models with micromolar affinity to wheat germ agglutinin using a dialkyl thioglycoside.

A series of alkyl thioglycosides and alkyl thiodiglycosides bearing glucose and N-acetylglucosamine residues were prepared by thiol-ene coupling in moderate to good yields (40-85%). Their binding ability towards wheat germ agglutinin was measured by competitive enzyme-linked lectin assays. One of the synthetic compounds presenting two GlcNAc units showed the highest inhibitory effect of this study with an IC50 of 11 µM corresponding to a 3182-fold improvement compared to GlcNAc. These synthetic molecules were used to produce giant vesicles, alone or in mixture with phospholipids, mimicking bacterial outer membrane vesicles (OMV) with potential antiadhesive properties.

Multivalent Glycomimetics with Affinity and Selectivity toward Fucose-Binding Receptors from Emerging Pathogens.

Bacterial and fungal pathogens involved in lung infection in cystic fibrosis patients utilize a particular family of glycan-binding proteins, characterized by the presentation of six fucose-binding sites on a ring-shaped scaffold. These lectins are attractive targets for anti-infectious compounds that could interfere in the recognition of host tissues by pathogens. The design of a cyclopeptide-based hexavalent structure allowed for the presentation of six fucose residues. The synthetic hexavalent compound displays liable geometry resulting in high-avidity binding by lectins from Aspergillus fumigatus and Burkholderia ambifaria. Replacing the fucose residue with a conformationally constrained fucomimetic does not alter the affinity and provides fine specificity with no binding to other fucose-specific lectins.

The study of multivalent carbohydrate-protein interactions by bio-layer interferometry.

The study of complex multivalent carbohydrate-protein interactions remains highly complicated and sometimes rendered impossible due to aggregation problems. In this study, we demonstrate that bio-layer interferometry is an excellent complementary method to standard techniques such as SPR and ITC. Using tetra- and hexadecavalent GalNAc glycoconjugates and Helix pomatia agglutinin (HPA) as a model lectin, we were able to measure reliable kinetic and thermodynamic parameters of multivalent interactions going from the micro to the nanomolar range.

Chemobacterial Synthesis of a Sialyl-Tn Cyclopeptide Vaccine Candidate.

A conjugatable form of the tumour-associated carbohydrate antigen sialyl-Tn (Neu5Ac-α-2,6-GalNAc) was efficiently produced in Escherichia coli. Metabolically engineered E. coli strains overexpressing the 6-sialyltransferase gene of Photobacterium sp. and CMP-Neu5Ac synthetase genes of Neisseria meningitidis were cultivated at high density in the presence of GalNAc-α-propargyl as the exogenous acceptor. The target disaccharides, which were produced on the scale of several hundreds of milligrams, were then conjugated by using copper(I)-catalysed azide-alkyne cycloaddition click chemistry to a fully synthetic and immunogenic scaffold with the aim to create a candidate anticancer vaccine. Four sialyl-Tn epitopes were introduced on the upper face of an azido-functionalised multivalent cyclopeptide scaffold, the lower face of which was previously modified by an immunogenic polypeptide, PADRE. The ability of the resulting glycoconjugate to interact with oncofoetal sialyl-Tn monoclonal antibodies was confirmed in ELISA assays.

Heterovalent Glycodendrimers as Epitope Carriers for Antitumor Synthetic Vaccines.

The large majority of TACA-based (TACA=Tumor-Associated Carbohydrate Antigens) antitumor vaccines target only one carbohydrate antigen, thereby often resulting in the incomplete destruction of cancer cells. However, the morphological heterogeneity of the tumor glycocalix, which is in constant evolution during malignant transformation, is a crucial point to consider in the design of vaccine candidates. In this paper, an efficient synthetic strategy based on orthogonal chemoselective ligations to prepare fully synthetic glycosylated cyclopeptide scaffolds grafted with both Tn and TF antigen analogues is reported. To evaluate their ability to be recognized as tumor antigens, direct interaction ELISA assays have been performed with the anti-Tn monoclonal antibody 9A7. Although both heterovalent structures showed binding capacities with 9A7, the presence of the second TF epitope did not interfere with the recognition of Tn except in one epitope arrangement. This heterovalent glycosylated structure thus represents an attractive epitope carrier to be further functionalized with T-cell peptide epitopes.

Immunological characterization of a rigid α-Tn mimetic on murine iNKT and human NK cells.

The ability of a rigid α-Tn mimetic (compound 1) to activate murine invariant natural killer T (iNKT) and human natural killer (NK) cells, two subsets of lymphocytes involved in cancer immunesurveillance, was investigated. For this purpose, the mimetic 1 was properly conjugated to a stearic acid containing glycerol-based phospholipid (compound 5) to be presented, in the context of the conserved non polymorphic major histocompatibility complex class I-like molecules (CD1d), to iNKT cells. On the contrary, the mimetic 1 was conjugated to a multivalent peptide-based scaffold (compound 6) to induce NK cell activation.

An oxime-based glycocluster microarray.

Carbohydrate microarrays represent powerful tools to study and detect carbohydrate-binding proteins, pathogens or cells. In this paper, we report two original oxime-based methods to prepare surfaces displaying well-defined structures and valency in a given microspot with improved recognition potency with lectins. In a first "direct" approach, fully synthetic aminooxylated glycoclusters have been coated onto aldehyde-activated SiO2 (silicium substrate doped with 50 nm thermal oxide layer). To improve the preparation of the microarray in terms of rapidity and simplicity and to provide addressable surfaces on which sugars can be linked chemoselectively as clusters at defined plots, a second "indirect" strategy has been developed using successive oxime ligation steps. In both cases, binding assays with labelled lectins have revealed more potent and selective interaction due to the clustered presentation of sugars. The observed differences of interaction have been confirmed in solution by ITC.

Characterization of a high-affinity sialic acid-specific CBM40 from Clostridium perfringens and engineering of a divalent form.

CBMs (carbohydrate-binding modules) are a class of polypeptides usually associated with carbohydrate-active enzymatic sites. We have characterized a new member of the CBM40 family, coded from a section of the gene NanI from Clostridium perfringens Glycan arrays revealed its preference towards α(2,3)-linked sialosides, which was confirmed and quantified by calorimetric studies. The CBM40 binds to α(2,3)-sialyl-lactose with a Kd of ∼30 µM, the highest affinity value for this class of proteins. Inspired by lectins' structure and their arrangement as multimeric proteins, we have engineered a dimeric form of the CBM, and using SPR (surface plasmon resonance) we have observed 6-11-fold binding increases due to the avidity affect. The structures of the CBM, resolved by X-ray crystallography, in complex with α(2,3)- or α(2,6)-sialyl-lactose explain its binding specificity and unusually strong binding.