Expanding the synthesis of a library of potent glucuronic acid glycodendrons for Dengue virus inhibition.

Multivalent glycodendrons are valuable tools to mimic many structural and functional features of cell-surface glycoconjugates and its focal position scaffolds represent important components to increase specificity and affinity. Previous work in our group described the preparation of a tetravalent glucuronic acid dendron that binds with good affinity to Dengue virus envelope protein (KD = 22 µM). Herein, the chemical synthesis and binding analysis of a new library of potent glucuronic acid dendrons bearing different functional group at the focal position and different level of multivalency are described. Their chemical synthesis was performed sequentially in three stages and with good yields. Namely a) the chemical synthesis of the oligo and polyalkynyl scaffolds, b) assembling with fully protected glucuronic acid-based azide units by using a microwave assisted copper-catalysed azide-alkyne cycloaddition reaction and c) sequential deprotection of hydroxyl and carboxylic acid groups. Surface Plasmon Resonance studies have demonstrated that the valency and the focal position functional group exert influence on the interaction with Dengue virus envelope protein. Molecular modelling studies were carried out in order to understand the binding observed. This work reports an efficient glycodendrons chemical synthesis that provides appropriate focal position functional group and multivalence, that offer an easy and versatile strategy to find new active compounds against Dengue virus.

A Versatile Urea Type Linker for Functionalizing Natural Glycans and Its Validation in Glycan Arrays.

The isolation from organisms and readily available glycoproteins has become an increasingly convenient source of N-glycans for multiple applications including glycan microarrays, as reference standards in glycan analysis or as reagents that improve bioavailability of protein and peptide therapeutics through conjugation. A problematic step in the isolation process on a preparative scale can be the attachment of a linker for the improved purification, separation, immobilization and quantification of the glycan structures. Addressing this issue, we firstly aimed for the development of an UV active linker for a fast and reliable attachment to anomeric glycosylamines via urea bond formation. Secondly, we validated the new linker on glycan arrays in a comparative study with a collection of N-glycans which were screened against various lectins. In total, we coupled four structurally varied N-glycans to four different linkers, immobilized all constructs on a microarray and compared their binding affinities to four plant and fungal lectins of widely described specificity. Our study shows that the urea type linker showed an overall superior performance for lectin binding and once more, highlights the often neglected influence of the choice of linker on lectin recognition.

Interactions of marine sulfated glycans with antithrombin and platelet factor 4.

The molecular interactions of sulfated glycans, such as heparin, with antithrombin (AT) and platelet factor 4 (PF4) are essential for certain biological events such as anticoagulation and heparin induced thrombocytopenia (HIT). In this study, a library including 84 sulfated glycans (polymers and oligomers) extracted from marine algae along with several animal-originated polysaccharides were subjected to a structure-activity relationship (SAR) study regarding their specific molecular interactions with AT and PF4 using surface plasmon resonance. In this SAR study, multiple characteristics were considered including different algal species, different methods of extraction, molecular weight, monosaccharide composition, sulfate content and pattern and branching vs. linear chains. These factors were found to influence the binding affinity of the studied glycans with AT. Many polysaccharides showed stronger binding than the low molecular weight heparin (e.g., enoxaparin). Fourteen polysaccharides with strong AT-binding affinities were selected to further investigate their binding affinity with PF4. Eleven of these polysaccharides showed strong binding to PF4. It was observed that the types of monosaccharides, molecular weight and branching are not very essential particularly when these polysaccharides are oversulfated. The sulfation levels and sulfation patterns are, on the other hand, the primary contribution to strong AT and PF4 interaction.

Binding of Glycans to the SARS CoV-2 Spike Protein, an Open Question: NMR Data on Binding Site Localization, Affinity, and Selectivity.

We have used NMR experiments to explore the binding of selected glycans and glycomimetics to the SARS CoV-2 spike glycoprotein (S-protein) and to its receptor binding domain (RBD). STD NMR experiments confirm the binding of sialoglycans to the S-protein of the prototypic Wuhan strain virus and yield dissociation constants in the millimolar range. The absence of STD effects for sialoglycans in the presence of the Omicron/BA.1 S-protein reflects a loss of binding as a result of S-protein evolution. Likewise, no STD effects are observed for the deletion mutant Δ143-145 of the Wuhan S-protein, thus supporting localization of the binding site in the N-terminal domain (NTD). The glycomimetics Oseltamivir and Zanamivir bind weakly to the S-protein of both virus strains. Binding of blood group antigens to the Wuhan S-protein cannot be confirmed by STD NMR. Using 1 H,15 N TROSY HSQC-based chemical shift perturbation (CSP) experiments, we excluded binding of any of the ligands studied to the RBD of the Wuhan S-protein. Our results put reported data on glycan binding into perspective and shed new light on the potential role of glycan-binding to the S-protein.

Structural Considerations for Building Synthetic Glycoconjugates as Inhibitors for Pseudomonas aeruginosa Lectins.

Pseudomonas aeruginosa is a pathogenic bacterium, responsible for a large portion of nosocomial infections globally and designated as critical priority by the World Health Organisation. Its characteristic carbohydrate-binding proteins LecA and LecB, which play a role in biofilm-formation and lung-infection, can be targeted by glycoconjugates. Here we review the wide range of inhibitors for these proteins (136 references), highlighting structural features and which impact binding affinity and/or therapeutic effects, including carbohydrate selection; linker length and rigidity; and scaffold topology, particularly for multivalent candidates. We also discuss emerging therapeutic strategies, which build on targeting of LecA and LecB, such as anti-biofilm activity, anti-adhesion and drug-delivery, with promising prospects for medicinal chemistry.

Druggable Allosteric Sites in ß-Propeller Lectins.

Carbohydrate-binding proteins (lectins) are auspicious targets in drug discovery to combat antimicrobial resistance; however, their non-carbohydrate drug-like inhibitors are still unavailable. Here, we present a druggable pocket in a ß-propeller lectin BambL from Burkholderia ambifaria as a potential target for allosteric inhibitors. This site was identified employing 19 F NMR fragment screening and a computational pocket prediction algorithm SiteMap. The structure-activity relationship study revealed the most promising fragment with a dissociation constant of 0.3±0.1 mM and a ligand efficiency of 0.3 kcal mol-1 HA-1 that affected the orthosteric site. This effect was substantiated by site-directed mutagenesis in the orthosteric and secondary pockets. Future drug-discovery campaigns that aim to develop small molecule inhibitors can benefit from allosteric sites in lectins as a new therapeutic approach against antibiotic-resistant pathogens.

Carbohydrate-Protein Interactions: Advances and Challenges.

A carbohydrate, also called saccharide in biochemistry, is a biomolecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms. For example, sugars are low molecular-weight carbohydrates, and starches are high molecular-weight carbohydrates. Carbohydrates are the most abundant organic substances in nature and essential constituents of all living things. Protein-carbohydrate interactions play important roles in many biological processes, such as cell growth, differentiation, and aggregation. They also have broad applications in pharmaceutical drug design. In this review, we will summarize the characteristic features of protein-carbohydrate interactions and review the computational methods for structure prediction, energy calculations, and kinetic studies of protein-carbohydrate complexes. Finally, we will discuss the challenges in this field.

Synthesis of Glycodendrimers with Antiviral and Antibacterial Activity.

Glycodendrimers are an important class of synthetic macromolecules that can be used to mimic many structural and functional features of cell-surface glycoconjugates. Their carbohydrate moieties perform key important functions in bacterial and viral infections, often regulated by carbohydrate-protein interactions. Several studies have shown that the molecular structure, valency and spatial organisation of carbohydrate epitopes in glycoconjugates are key factors in the specificity and avidity of carbohydrate-protein interactions. Choosing the right glycodendrimers almost always helps to interfere with such interactions and blocks bacterial or viral adhesion and entry into host cells as an effective strategy to inhibit bacterial or viral infections. Herein, the state of the art in the design and synthesis of glycodendrimers employed for the development of anti-adhesion therapy against bacterial and viral infections is described.

Crystal structures of peanut lectin in the presence of synthetic ß-N- and ß-S-galactosides disclose evidence for the recognition of different glycomimetic ligands.

Carbohydrate-lectin interactions are involved in important cellular recognition processes, including viral and bacterial infections, inflammation and tumor metastasis. Hence, structural studies of lectin-synthetic glycan complexes are essential for understanding lectin-recognition processes and for the further design of promising chemotherapeutics that interfere with sugar-lectin interactions. Plant lectins are excellent models for the study of the molecular-recognition process. Among them, peanut lectin (PNA) is highly relevant in the field of glycobiology because of its specificity for ß-galactosides, showing high affinity towards the Thomsen-Friedenreich antigen, a well known tumor-associated carbohydrate antigen. Given this specificity, PNA is one of the most frequently used molecular probes for the recognition of tumor cell-surface O-glycans. Thus, it has been extensively used in glycobiology for inhibition studies with a variety of ß-galactoside and ß-lactoside ligands. Here, crystal structures of PNA are reported in complex with six novel synthetic hydrolytically stable ß-N- and ß-S-galactosides. These complexes disclosed key molecular-binding interactions of the different sugars with PNA at the atomic level, revealing the roles of specific water molecules in protein-ligand recognition. Furthermore, binding-affinity studies by isothermal titration calorimetry showed dissociation-constant values in the micromolar range, as well as a positive multivalency effect in terms of affinity in the case of the divalent compounds. Taken together, this work provides a qualitative structural rationale for the upcoming synthesis of optimized glycoclusters designed for the study of lectin-mediated biological processes. The understanding of the recognition of ß-N- and ß-S-galactosides by PNA represents a benchmark in protein-carbohydrate interactions since they are novel synthetic ligands that do not belong to the family of O-linked glycosides.

Saturation Transfer Difference in Characterization of Glycosaminoglycan-Protein Interactions.

Novel methods in nuclear magnetic resonance (NMR) spectroscopy have recently been developed to investigate the binding properties of intermolecular complexes endowed with biomedical functions. Among these methods is the saturation transfer difference (STD), which enables the mapping of specific binding motifs of functional ligands. STD can efficiently uncover the specific and preferential binding sites of these ligands in their intermolecular complexes. This is particularly useful in the case of glycosaminoglycans (GAGs), a group of sulfated polysaccharides that play pivotal roles in various biological and pathological processes. The activity of GAGs is ultimately mediated through molecular interactions with key functional proteins, namely, GAG-binding proteins (GBPs). The quality of the GAG-GBP interactions depends on sulfation patterns, oligosaccharide length, and the composing monosaccharides of GAGs. Through STD NMR, information about the atoms of the GAG ligands involved in the complexes is provided. Here we highlight the latest achievements of the literature using STD NMR on GAG oligosaccharide-GBP complexes. Interestingly, most of the GBPs studied so far by STD NMR belong to one of the three major classes: coagulation factors, growth factors, or chemokine/cytokines. Unveiling the structural requirements of GAG ligands in bindings with their protein partners is a crucial step to understand the biochemical and medical actions of GAGs. This process is also a requirement in GAG-based drug discovery and development.

Polyproline Tri-Helix Macrocycles as Nanosized Scaffolds to Control Ligand Patterns for Selective Protein Oligomer Interactions.

Multivalent ligand-receptor interactions play essential roles in biological recognition and signaling. As the receptor arrangement on the cell surface can alter the outcome of cell signaling and also provide spatial specificity for ligand binding, controlling the presentation of ligands has become a promising strategy to manipulate or selectively target protein receptors. The lack of adjustable universal tools to control ligand positions at the size of a few nanometers has prompted the development of polyproline tri-helix macrocycles as scaffolds to present ligands in designated patterns. Model lectin Helix pomatia agglutinin has shown selectivity toward the matching GalNAc ligand pattern matching its binding sites arrangement. The GalNAc pattern selectivity is also observed on intact asialoglycoprotein receptor oligomer on human hepatoma cells showing the pattern-selective interaction can be achieved not only on isolated protein oligomers but also the receptors arranged on the cell surface. As the scaffold design allows convenient creation of versatile ligand patterns, it can be expected as a promising tool to probe the arrangement of receptors on the cell surface and as nanomedicine to manipulate signaling or cell recognition.

Synthesis of a Fluorous-Tagged Hexasaccharide and Interaction with Growth Factors Using Sugar-Coated Microplates.

Here, we report the synthesis of a sulfated, fully protected hexasaccharide as a glycosaminoglycan mimetic and the study of its interactions with different growth factors: midkine, basic fibroblast growth factor (FGF-2) and nerve growth factor (NGF). Following a fluorous-assisted approach, monosaccharide building blocks were successfully assembled and the target oligosaccharide was prepared in excellent yield. The use of more acid stable 4,6-O-silylidene protected glucosamine units was crucial for the efficiency of this strategy because harsh reaction conditions were needed in the glycosylations to avoid the formation of orthoester side products. Fluorescence polarization experiments demonstrated the strong interactions between the synthesized hexamer, and midkine and FGF-2. In addition, we have developed an alternative assay to analyse these molecular recognition events. The prepared oligosaccharide was non-covalently attached to a fluorous-functionalized microplate and the direct binding of the protein to the sugar-immobilized surface was measured, affording the corresponding KD,surf value.

Carbohydrate-protein interactions and multivalency: implications for the inhibition of influenza A virus infections.

INTRODUCTION: Protein-carbohydrate interactions play a very important role in many biological processes. A single interaction between a protein and a carbohydrate is usually weak, but multivalent ligands can compensate for this deficiency by binding multiple binding sites to one biological entity simultaneously. Over the past few years, numerous efforts have been made for the design and synthesis of carbohydrate-based multivalent ligands thereby serving as potent inhibitors for pathogens such as the influenza A virus. Areas covered: In this review, the authors cover a variety of multivalent systems from small to large molecules which showed a potent inhibitory effect against several pathogens. Expert opinion: Scaffold structure, linker type, and ligand density are important parameters that need to be optimized for potent multivalent inhibitors. The challenges of multivalent glycodrugs include issues such as bioavailability, pharmacokinetics, and immunogenicity which greatly depend on where the compounds are used in the body. Anti-flu (influenza) applications in the lungs using multivalent carbohydrates particularly has potential because of the high binding affinities. With much more research focusing on Influenza A virus inhibition, therapeutic applications may be achieved in the near future.

Fluorescent Neoglycoprotein Gold Nanoclusters: Synthesis and Applications in Plant Lectin Sensing and Cell Imaging.

Carbohydrate-protein interactions mediate fundamental biological processes, such as fertilization, cell signaling, or host-pathogen communication. However, because of the enormous complexity of glycan recognition events, new tools enabling their analysis or applications emerge in recent years. Here, we describe the first preparation of neoglycoprotein functionalized fluorescent gold nanoclusters, containing a biantennary N-glycan G0 as targeting molecule, ovalbumin as carrier/model antigen, and a fluorescent gold core as imaging probe (G0-OVA-AuNCs). Subsequently, we demonstrate the utility of generated G0-OVA-AuNCs for specific sensing of plant lectins and in vitro imaging of dendritic cells.

The neoglycolipid (NGL) technology-based microarrays and future prospects.

The neoglycolipid (NGL) technology is the basis of a state-of-the-art oligosaccharide microarray system, which we offer for screening analyses to the broad scientific community. We review here the sequential development of the technology and its power in pinpointing and isolating naturally occurring ligands for glycan-binding proteins (GBPs) within glycan populations. We highlight our Designer Array approach and Beam Search Array approach for generating natural glycome arrays to identify novel ligands of biological relevance. These two microarray approaches have been applied for assignments of ligands or antigens on glucan polysaccharides for effector proteins of the immune system (Dectin-1, DC-SIGN and DC-SIGNR) and carbohydrate-binding modules (CBMs) on bacterial hydrolases. We also discuss here the more recent applications to elucidate the structure of a prostate cancer- associated antigen F77 and identify ligands for adhesins of two rotaviruses, P[10] and P[19], expressed on an epithelial mucin glycoprotein.

Fluorous-tag assisted synthesis of a glycosaminoglycan mimetic tetrasaccharide as a high-affinity FGF-2 and midkine ligand.

Here, we present the preparation of a sulfated, fully protected tetrasaccharide derivative following the glycosaminoglycan (GAG)-related sequence GlcNAc-ß(1 → 4)-Glc-ß(1 → 3). The tetramer was efficiently assembled via an iterative glycosylation strategy using monosaccharide building blocks. A fluorous tag was attached at position 6 of the reducing end unit enabling the purification of reaction intermediates by simple fluorous solid phase extraction. Fluorescence polarization competition experiments revealed that the synthesized tetrasaccharide strongly interacts with two heparin-binding growth factors, midkine and FGF-2 (IC50 of 270 nM and 2.4 µM, respectively). Our data indicate that this type of oligosaccharide derivatives, displaying sulfates, hydrophobic protecting groups and a fluorinated tail can be considered as interesting GAG mimetics for the regulation of relevant carbohydrate-protein interactions.

CH/π Interactions in Carbohydrate Recognition.

Many carbohydrate-binding proteins contain aromatic amino acid residues in their binding sites. These residues interact with carbohydrates in a stacking geometry via CH/π interactions. These interactions can be found in carbohydrate-binding proteins, including lectins, enzymes and carbohydrate transporters. Besides this, many non-protein aromatic molecules (natural as well as artificial) can bind saccharides using these interactions. Recent computational and experimental studies have shown that carbohydrate-aromatic CH/π interactions are dispersion interactions, tuned by electrostatics and partially stabilized by a hydrophobic effect in solvated systems.

Highly Ordered Self-Assembly of Native Proteins into 1D, 2D, and 3D Structures Modulated by the Tether Length of Assembly-Inducing Ligands.

In nature, proteins self-assemble into various structures with different dimensions. To construct these nanostructures in laboratories, normally proteins with different symmetries are selected. However, most of these approaches are engineering-intensive and highly dependent on the accuracy of the protein design. Herein, we report that a simple native protein LecA assembles into one-dimensional nanoribbons and nanowires, two-dimensional nanosheets, and three-dimensional layered structures controlled mainly by small-molecule assembly-inducing ligands RnG (n=1, 2, 3, 4, 5) with varying numbers of ethylene oxide repeating units. To understand the formation mechanism of the different morphologies controlled by the small-molecule structure, molecular simulations were performed from microscopic and mesoscopic view, which presented a clear relationship between the molecular structure of the ligands and the assembled patterns. These results introduce an easy strategy to control the assembly structure and dimension, which could shed light on controlled protein assembly.

Chemoselective Reactions for the Synthesis of Glycoconjugates from Unprotected Carbohydrates.

Glycobiology is the comprehensive biological investigation of carbohydrates. The study of the role and function of complex carbohydrates often requires the attachment of carbohydrates to surfaces, their tagging with fluorophores, or their conversion into natural or non-natural glycoconjugates, such as glycopeptides or glycolipids. Glycobiology and its "omics", glycomics, require easy and robust chemical methods for the construction of these glycoconjugates. This review gives an overview of the rapidly expanding field of chemical reactions that selectively convert unprotected carbohydrates into glycoconjugates through the anomeric position. The discussion is divided in terms of the anomeric bond type of the newly formed glycoconjugates, including O-, N-, S-, and C-glycosides.

Fabrication of Carbohydrate Microarrays by Boronate Formation.

The interactions between soluble carbohydrates and/or surface displayed glycans and protein receptors are essential to many biological processes and cellular recognition events. Carbohydrate microarrays provide opportunities for high-throughput quantitative analysis of carbohydrate-protein interactions. Over the past decade, various techniques have been implemented for immobilizing glycans on solid surfaces in a microarray format. Herein, we describe a detailed protocol for fabricating carbohydrate microarrays that capitalizes on the intrinsic reactivity of boronic acid toward carbohydrates to form stable boronate diesters. A large variety of unprotected carbohydrates ranging in structure from simple disaccharides and trisaccharides to considerably more complex human milk and blood group (oligo)saccharides have been covalently immobilized in a single step on glass slides, which were derivatized with high-affinity boronic acid ligands. The immobilized ligands in these microarrays maintain the receptor-binding activities including those of lectins and antibodies according to the structures of their pendant carbohydrates for rapid analysis of a number of carbohydrate-recognition events within 30 h. This method facilitates the direct construction of otherwise difficult to obtain carbohydrate microarrays from underivatized glycans.