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.

Synthesis and interaction with growth factors of sulfated oligosaccharides containing an anomeric fluorinated tail.

Compounds that mimic the biological properties of glycosaminoglycans (GAGs) and can be more easily prepared than the native GAG oligosaccharides are highly demanded. Here, we present the synthesis of sulfated oligosaccharides displaying a perfluorinated aliphatic tag at the reducing end as GAG mimetics. The preparation of these molecules was greatly facilitated by the presence of the fluorinated tail since the reaction intermediates were isolated by simple fluorous solid-phase extraction. Fluorescence polarization competition assays indicated that the synthesized oligosaccharides interacted with two heparin-binding growth factors, midkine (MK) and FGF-2, showing higher binding affinities than the natural oligosaccharides, and can be therefore considered as useful GAG mimetics. Moreover, NMR experiments showed that the 3D structure of these compounds is similar to that of the native sequences, in terms of sugar ring and glycosidic linkage conformations. Finally, we also demonstrated that these derivatives are able to block the MK-stimulating effect on NIH3T3 cells growth.

Glycosaminoglycans: What Remains To Be Deciphered?

Glycosaminoglycans (GAGs) are complex polysaccharides exhibiting a vast structural diversity and fulfilling various functions mediated by thousands of interactions in the extracellular matrix, at the cell surface, and within the cells where they have been detected in the nucleus. It is known that the chemical groups attached to GAGs and GAG conformations comprise "glycocodes" that are not yet fully deciphered. The molecular context also matters for GAG structures and functions, and the influence of the structure and functions of the proteoglycan core proteins on sulfated GAGs and vice versa warrants further investigation. The lack of dedicated bioinformatic tools for mining GAG data sets contributes to a partial characterization of the structural and functional landscape and interactions of GAGs. These pending issues will benefit from the development of new approaches reviewed here, namely (i) the synthesis of GAG oligosaccharides to build large and diverse GAG libraries, (ii) GAG analysis and sequencing by mass spectrometry (e.g., ion mobility-mass spectrometry), gas-phase infrared spectroscopy, recognition tunnelling nanopores, and molecular modeling to identify bioactive GAG sequences, biophysical methods to investigate binding interfaces, and to expand our knowledge and understanding of glycocodes governing GAG molecular recognition, and (iii) artificial intelligence for in-depth investigation of GAGomic data sets and their integration with proteomics.

Mannobioside biomimetics that trigger DC-SIGN binding selectivity.

Selective DC-SIGN targeting vs. langerin might lead to anti-infective agents, given their counteracting effects upon infection by some pathogens. Here we show that multivalent sp2-iminosugar-containing mannobioside analogs can achieve total DC-SIGN selectivity by levering the canonic binding mode towards high-mannose oligosaccharide ligands, behaving as factual biomimics.

Pleiotrophin Interaction with Synthetic Glycosaminoglycan Mimetics.

Chondroitin sulfate (CS) E is the natural ligand for pleiotrophin (PTN) in the central nervous system (CNS) of the embryo. Some structures of PTN in solution have been solved, but no precise location of the binding site has been reported yet. Using 15N-labelled PTN and HSQC NMR experiments, we studied the interactions with a synthetic CS-E tetrasaccharide corresponding to the minimum binding sequence. The results agree with the data for larger GAG (glycosaminoglycans) sequences and confirm our hypothesis that a synthetic tetrasaccharide is long enough to fully interact with PTN. We hypothesize that the central region of PTN is an intrinsically disordered region (IDR) and could modify its properties upon binding. The second tetrasaccharide has two benzyl groups and shows similar effects on PTN. Finally, the last measured compound aggregated but beforehand, showed a behavior compatible with a slow exchange in the NMR time scale. We propose the same binding site and mode for the tetrasaccharides with and without benzyl groups.

The Interaction between Chondroitin Sulfate and Dermatan Sulfate Tetrasaccharides and Pleiotrophin.

Pleiotrophin (PTN) is a neurotrophic factor that participates in the development of the embryonic central nervous system (CNS) and neural stem cell regulation by means of an interaction with sulfated glycosaminoglycans (GAGs). Chondroitin sulfate (CS) is the natural ligand in the CNS. We have previously studied the complexes between the tetrasaccharides used here and MK (Midkine) by ligand-observed NMR techniques. The present work describes the interactions between a tetrasaccharide library of synthetic models of CS-types and mimetics thereof with PTN using the same NMR transient techniques. We have concluded that: (1) global ligand structures do not change upon binding, (2) the introduction of lipophilic substituents in the structure of the ligand improves the strength of binding, (3) binding is weaker than for MK, (4) STD-NMR results are compatible with multiple binding modes, and (5) the replacement of GlcA for IdoA is not relevant for binding. Then we can conclude that the binding of CS derivatives to PTN and MK are similar and compatible with multiple binding modes of the same basic conformation.

Fluorous-Tag-Assisted Synthesis of GAG-Like Oligosaccharides.

The classic, solution-phase synthesis of glycosaminoglycan (GAG) oligosaccharides is hampered by the numerous, time-consuming chromatographic purifications required for the isolation of the glycosylation products after each coupling step between sugar building blocks. Here, we present a detailed experimental procedure for a glycosylation reaction involving a glycosyl acceptor unit that is equipped with a perfluorinated tag. The presence of this fluorous tail allows the quick purification of the desired glycosylation product by performing a simple fluorous solid-phase extraction (F-SPE). The described fluorous-tag-assisted glycosylation strategy greatly facilitates the assembly of building blocks, speeding up the preparation of biologically relevant GAG-like oligomers.

GAG Multivalent Systems to Interact with Langerin.

Langerin is a C-type Lectin expressed at the surface of Langerhans cells, which play a pivotal role protecting organisms against pathogen infections. To address this aim, Langerin presents at least two recognition sites, one Ca2+-dependent and another one independent, which are capable to recognize a variety of carbohydrate ligands. In contrast to other lectins, Langerin recognizes sulfated glycosaminoglycans (GAGs), a family of complex and heterogeneous polysaccharides present in the cell membrane and the extracellular matrix, at the interphase generated in the trimeric form of Langerin but absent in the monomeric form. The complexity of these oligosaccharides has impeded the development of welldefined monodisperse structures to study these interaction processes. However, in the last few decades, an improvement of synthetic developments to achieve the preparation of carbohydrate multivalent systems mimicking the GAGs has been described. Despite all these contributions, very few examples are reported where the GAG multivalent structures are used to evaluate the interaction with Langerin. These molecules should pave the way to explore these GAG-Langerin interactions.

Synthesis, self-assembly and Langerin recognition studies of a resorcinarene-based glycocluster exposing a hyaluronic acid thiodisaccharide mimetic.

Herein, we report the synthesis of an octavalent glycocluster exposing a thiodisaccharide mimetic of the repetitive unit of hyaluronic acid, ßSGlcA(1 → 3)ßSGlcNAc, constructed on a calix[4]resorcinarene scaffold by CuAAC reaction of suitable precursors. This glycocluster showed a strong tendency toward self-aggregation. DOSY-NMR and DLS experiments demonstrated the formation of spherical micelles of d ≅ 6.2 nm, in good agreement. TEM micrographs showed the presence of particles of different sizes, depending on the pH of the starting solution, thus evidencing that the negative charge on the micelle surface due to ionization of the GlcA residues plays an important role in the aggregation process. STD-NMR and DLS experiments provided evidence of the interaction between the synthetic glycocluster and Langerin, a relevant C-type lectin. This interaction was not observed in the STD-NMR experiments performed with the basic disaccharide, providing evidence of a multivalent effect.

Midkine Interaction with Chondroitin Sulfate Model Synthetic Tetrasaccharides and Their Mimetics: The Role of Aromatic Interactions.

Midkine (MK) is a neurotrophic factor that participates in the embryonic central nervous system (CNS) development and neural stem cell regulation, interacting with sulfated glycosaminoglycans (GAGs). Chondroitin sulfate (CS) is the natural ligand in the CNS. In this work, we describe the interactions between a library of synthetic models of CS-types and mimics. We did a structural study of this library by NMR and MD (Molecular Dynamics), concluding that the basic shape is controlled by similar geometry of the glycosidic linkages. Their 3D structures are a helix with four residues per turn, almost linear. We have studied the tetrasaccharide-midkine complexes by ligand observed NMR techniques and concluded that the shape of the ligands does not change upon binding. The ligand orientation into the complex is very variable. It is placed inside the central cavity of MK formed by the two structured beta-sheets domains linked by an intrinsically disordered region (IDR). Docking analysis confirmed the participation of aromatics residues from MK completed with electrostatic interactions. Finally, we test the biological activity by increasing the MK expression using CS tetrasaccharides and their capacity in enhancing the growth stimulation effect of MK in NIH3T3 cells.

Synthesis, structure and midkine binding of chondroitin sulfate oligosaccharide analogues.

The preparation of chondroitin sulfate (CS) oligosaccharide mimetics, more easily synthesized than natural sequences, is a highly interesting task because these compounds pave the way for modulation of the biological processes in which CS is involved. Herein, we report the synthesis of CS type E analogues which present easily accessible glucose units instead of glucuronic acid (GlcA) moieties. NMR experiments and molecular dynamics simulations showed that the 3D structure of these compounds is similar to the structure of the natural CS-E oligosaccharides. In addition, fluorescence polarization (FP) and saturation transfer difference NMR (STD-NMR) experiments revealed that the synthesized CS-like derivatives were able to interact with midkine, a model heparin-binding growth factor, suggesting that the presence of the GlcA carboxylate groups is not essential for the binding. Overall, our results indicate that the synthesized glucose-containing oligosaccharides can be considered as functional and structural CS mimetics.

Second-Generation Dendrimers with Chondroitin Sulfate Type-E Disaccharides as Multivalent Ligands for Langerin.

Chondroitin sulfate type-E (CS-E) is a sulfated polysaccharide that shows several interesting biological activities, such as modulation of the neuronal growth factor signaling and its interaction with langerin, a C-type lectin with a crucial role in the immunological system. However, applications of CS-E are hampered by the typical heterogeneous structure of the natural polysaccharide. Well-defined, homogeneous CS-E analogues are highly demanded. Here, we report the synthesis of monodispersed, structurally well-defined second-generation glycodendrimers displaying up to 18 CS-E disaccharide units. These complex multivalent systems have a molecular weight and a number of disaccharide repeating units comparable with those of the natural polysaccharides. In addition, surface plasmon resonance experiments revealed a calcium-independent interaction between these glycodendrimers and langerin, in the micromolar range, highlighting the utility of these compounds as CS-E mimetics.

Synthesis of (1→3) Thiodisaccharides of GlcNAc and the Serendipitous Formation of 2,3-Dideoxy-(1→2)-thiodisaccharides through a Vinyl Azide Intermediate.

The syntheses of ß-S-GlcA(1→3)GlcNAc and ß-S-Gal(1→3)GlcNAc thiodisaccharides, which can be considered mimetics of the repeating units of hyaluronan and keratan respectively, were achieved by SN2 displacement of a triflate group allocated at the 3-position of a convenient 2-azido-4,6-O-benzylidene-2-deoxy-ß-d-allopyranose precursor by the corresponding nucleophilic suitable protected thioaldoses derived from glucuronic acid (GlcA) and galactose (Gal). The study of the reaction led to the finding that the vinyl azide formed by competitive E2 reaction of the mentioned triflate was an interesting precursor of a new kind of 2,3-dideoxy-2-azido-(1→2) thiodisaccharides through an addition reaction. Determination of the stereochemistry of the new stereocenter at C-2 was achieved by NOESY experiments. Final protecting group manipulation of the (1→3) thiodisaccharides led to a family of derivatives that could be used as building blocks for the synthesis of complex glycomimetics.

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.

Unexpected loss of stereoselectivity in glycosylation reactions during the synthesis of chondroitin sulfate oligosaccharides.

Here, we present an exploratory study on the fluorous-assisted synthesis of chondroitin sulfate (CS) oligosaccharides. Following this approach, a CS tetrasaccharide was prepared. However, in contrast to our previous results, a significant loss of ß-selectivity was observed in [2 + 2] glycosylations involving N-trifluoroacetyl-protected D-galactosamine donors and D-glucuronic acid (GlcA) acceptors. These results, together with those obtained from experiments employing model monosaccharide building blocks, highlight the impact of the glycosyl acceptor structure on the stereoselectivity of glycosylation reactions. Our study provides useful data about the substitution pattern of GlcA units for the efficient synthesis of CS oligomers.

The Use of NMR to Study Transient Carbohydrate-Protein Interactions.

Carbohydrates are biologically ubiquitous and are essential to the existence of all known living organisms. Although they are better known for their role as energy sources (glucose/glycogen or starch) or structural elements (chitin or cellulose), carbohydrates also participate in the recognition events of molecular recognition processes. Such interactions with other biomolecules (nucleic acids, proteins, and lipids) are fundamental to life and disease. This review focuses on the application of NMR methods to understand at the atomic level the mechanisms by which sugar molecules can be recognized by proteins to form complexes, creating new entities with different properties to those of the individual component molecules. These processes have recently gained attention as new techniques have been developed, while at the same time old techniques have been reinvented and adapted to address newer emerging problems.

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.

Rational-Differential Design of Highly Specific Glycomimetic Ligands: Targeting DC-SIGN and Excluding Langerin Recognition.

At the surface of dendritic cells, C-type lectin receptors (CLRs) allow the recognition of carbohydrate-based PAMPS or DAMPS (pathogen- or danger-associated molecular patterns, respectively) and promote immune response regulation. However, some CLRs are hijacked by viral and bacterial pathogens. Thus, the design of ligands able to target specifically one CLR, to either modulate an immune response or to inhibit a given infection mechanism, has great potential value in therapeutic design. A case study is the selective blocking of DC-SIGN, involved notably in HIV trans-infection of T lymphocytes, without interfering with langerin-mediated HIV clearance. This is a challenging task due to their overlapping carbohydrate specificity. Toward the rational design of DC-SIGN selective ligands, we performed a comparative affinity study between DC-SIGN and langerin with natural ligands. We found that GlcNAc is recognized by both CLRs; however, selective sulfation are shown to increase the selectivity in favor of langerin. With the combination of site-directed mutagenesis and X-ray structural analysis of the langerin/GlcNS6S complex, we highlighted that 6-sulfation of the carbohydrate ligand induced langerin specificity. Additionally, the K313 residue from langerin was identified as a critical feature of its binding site. Using a rational and a differential approach in the study of CLR binding sites, we designed, synthesized, and characterized a new glycomimetic, which is highly specific for DC-SIGN vs langerin. STD NMR, SPR, and ITC characterizations show that compound 7 conserved the overall binding mode of the natural disaccharide while possessing an improved affinity and a strict specificity for DC-SIGN.

Interactions between a Heparin Trisaccharide Library and FGF-1 Analyzed by NMR Methods.

FGF-1 is a potent mitogen that, by interacting simultaneously with Heparan Sulfate Glycosaminoglycan HSGAG and the extracellular domains of its membrane receptor (FGFR), generates an intracellular signal that finally leads to cell division. The overall structure of the ternary complex Heparin:FGF-1:FGFR has been finally elucidated after some controversy and the interactions within the ternary complex have been deeply described. However, since the structure of the ternary complex was described, not much attention has been given to the molecular basis of the interaction between FGF-1 and the HSGAG. It is known that within the complex, the carbohydrate maintains the same helical structure of free heparin that leads to sulfate groups directed towards opposite directions along the molecular axis. The precise role of single individual interactions remains unclear, as sliding and/or rotating of the saccharide along the binding pocket are possibilities difficult to discard. The HSGAG binding pocket can be subdivided into two regions, the main one can accommodate a trisaccharide, while the other binds a disaccharide. We have studied and analyzed the interaction between FGF-1 and a library of trisaccharides by STD-NMR and selective longitudinal relaxation rates. The library of trisaccharides corresponds to the heparin backbone and it has been designed to interact with the main subsite of the protein.

Glycodendrimers as Chondroitin Sulfate Mimetics: Synthesis and Binding to Growth Factor Midkine.

Chondroitin sulfate (CS) is a member of the glycosaminoglycan (GAG) family, a class of polysaccharides implicated in relevant biological functions. The structural complexity of these carbohydrates demands the development of simple glycomimetics as useful tools to study the biological processes in which GAGs are involved. In this work we described the synthesis of the disaccharide unit of the CS-E (GlcA-GalNAc(4,6-di-OSO3 )), in a multivalent presentation. Using a fluorescence polarization competition assay we have demonstrated that a hexavalent dendrimer of this disaccharide interact with midkine, in the low micromolar range. This result highlights the potency of these disaccharide-displaying multivalent systems as interesting mimetics of longer and synthetically more complex GAG oligosaccharides.