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.

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.

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.

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.

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.

Influence of the reducing-end anomeric configuration of the Man9 epitope on DC-SIGN recognition.

High-mannose (Man9GlcNAc2) is the main carbohydrate unit present in viral envelope glycoproteins such as gp120 of HIV and the GP1 of Ebola virus. This oligosaccharide comprises the Man9 epitope conjugated to two terminal N-acetylglucosamines by otherwise rarely-encountered ß-mannose glycosidic bond. Formation of this challenging linkage is the bottleneck of the few synthetic approaches described to prepare high mannose. Herein, we report the synthesis of the Man9 epitope with both alpha and beta configurations at the reducing end, and subsequent evaluation of the impact of this configuration on binding to natural receptor of high-mannose, DC-SIGN. Using fluorescence polarization assays, we demonstrate that both anomers bind to DC-SIGN with comparable affinity. These relevant results therefore indicate that the more synthetically-accesible Man9 alpha epitope may be deployed as ligand for DC-SIGN in both in vitro and in vivo biological assays.

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 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.

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.

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.

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.

Adaptation of targeted nanocarriers to changing requirements in antimalarial drug delivery.

The adaptation of existing antimalarial nanocarriers to new Plasmodium stages, drugs, targeting molecules, or encapsulating structures is a strategy that can provide new nanotechnology-based, cost-efficient therapies against malaria. We have explored the modification of different liposome prototypes that had been developed in our group for the targeted delivery of antimalarial drugs to Plasmodium-infected red blood cells (pRBCs). These new models include: (i) immunoliposome-mediated release of new lipid-based antimalarials; (ii) liposomes targeted to pRBCs with covalently linked heparin to reduce anticoagulation risks; (iii) adaptation of heparin to pRBC targeting of chitosan nanoparticles; (iv) use of heparin for the targeting of Plasmodium stages in the mosquito vector; and (v) use of the non-anticoagulant glycosaminoglycan chondroitin 4-sulfate as a heparin surrogate for pRBC targeting. The results presented indicate that the tuning of existing nanovessels to new malaria-related targets is a valid low-cost alternative to the de novo development of targeted nanosystems.

Chondroitin Sulfate Tetrasaccharides: Synthesis, Three-Dimensional Structure and Interaction with Midkine.

The biological activity of midkine, a cytokine implicated in neuro- and tumourigenesis, is regulated by its binding to glycosaminoglycans (GAGs), such as heparin and chondroitin sulfate (CS). To better understand the molecular recognition of GAG sequences by this growth factor, the interactions between synthetic chondroitin sulfate-like tetrasaccharides and midkine were studied by using different techniques. Firstly, a synthetic approach for the preparation of CS-like oligosaccharides in the sequence GalNAc-GlcA was developed. A fluorescence polarisation competition assay was then employed to analyse the relative binding affinities of the synthetic compounds and revealed that midkine interacted with CS-like tetrasaccharides in the micromolar range. The 3D structure of these tetramers was studied in detail by a combination of NMR spectroscopy experiments and molecular dynamics simulations. Saturation transfer difference (STD) NMR spectroscopy experiments indicate that the CS tetrasaccharides bind to midkine in an extended conformation, with similar saturation effects along the entire sugar chain. These results are compatible with docking studies that suggest an interaction of the tetrasaccharide with midkine in a folded structure. Overall, this study provides valuable information on the interaction between midkine and well-defined, chemically synthesised CS oligosaccharides and these data can be useful for the design of more active compounds that modulate the biological function of this protein.

Langerin-heparin interaction: two binding sites for small and large ligands as revealed by a combination of NMR spectroscopy and cross-linking mapping experiments.

Langerin is a C-type lectin present on Langerhans cells that mediates capture of pathogens in a carbohydrate-dependent manner, leading to subsequent internalization and elimination in the cellular organelles called Birbeck granules. This mechanism mediated by langerin was shown to constitute a natural barrier for HIV-1 particle transmission. Besides interacting specifically with high mannose and fucosylated neutral carbohydrate structures, langerin has the ability to bind sulfated carbohydrate ligands as 6-sulfated galactosides in the Ca(2+)-dependent binding site. Very recently langerin was demonstrated to interact with sulfated glycosaminoglycans (GAGs), in a Ca(2+)-independent way, resulting in the proposal of a new binding site for GAGs. On the basis of those results, we have conducted a structural study of the interactions of small heparin (HEP)-like oligosaccharides with langerin in solution. Heparin bead cross-linking experiments, an approach specifically designed to identify HEP/heparan sulfate binding sites in proteins were first carried out and experimentally validated the previously proposed model for the interaction of langerin extracellular domain with 6 kDa HEP. High-resolution NMR studies of a set of eight synthetic HEP-like trisaccharides harboring different sulfation patterns demonstrated that all of them bound to langerin in a Ca(2+)-dependent way. The binding epitopes were determined by saturation transfer difference NMR and the bound conformations by transferred NOESY experiments. These experimental data were combined with docking and molecular dynamics and resulted in the proposal of a binding mode characterized by the coordination of calcium by the two equatorial hydroxyl groups, OH3 and OH4, at the non-reducing end. The binding also includes the carboxylate group at the adjacent iduronate residue. This epitope is shared by all eight ligands, explaining the absence of any impact on binding from differences in their substitution patterns. Finally, in contrast to the small trisaccharides, we demonstrated that a longer HEP-like hexasaccharide, bearing an additional O-sulfate group at the non-reducing end, which precludes binding to the Ca(2+) site, interacts with langerin in the previously identified Ca(2+)-independent binding site.

Importance of the polarity of the glycosaminoglycan chain on the interaction with FGF-1.

Heparin-like saccharides play an essential role in binding to the fibroblast growth factor (FGF)-1 and to their membrane receptors fibroblast growth factor receptor forming a ternary complex that is responsible of the internalization of the signal, via the dimerization of the intracellular regions of the receptor. In this study, we report the binding affinities between five synthetic hexasaccharides with human FGF-1 obtained by surface plasmon resonance experiments, and compare with the induced mitogenic activity previously obtained. These five oligosaccharides differ in sulfation pattern and in sequence. We have previously demonstrated that all the five hexasaccharides have similar 3D structure of the backbone. Consequently, the differences in binding affinity should have their origin in the substitution pattern. Subsequently, the different capacity for induction of mitogenic activity can be, at least partially, explained from these binding affinities. Interestingly, one of the oligosaccharides lacking axially symmetry ( 3: ) was biologically inactive, whereas the other ( 2: ) was the most active. The difference between both compounds is the order of the FGF-binding motifs along the chain relative to the carbohydrate polarity. We can conclude that the directionality of the GAG chain is essential for the binding and subsequent activation. The relative biological activity of the compounds with regular substitution pattern can be inferred from their values of IC50. Remarkably, the sulfate in position 6 of d-glucosamine was essential for the mitogenic activity but not for the interaction with FGF-1.

Synthesis of hyaluronic acid oligosaccharides and exploration of a fluorous-assisted approach.

The synthesis of hyaluronic acid oligomers (tri- and tetrasaccharide) is described. We have followed a pre-glycosylation oxidation strategy. Glucuronic acid units were directly employed in coupling reactions with suitably protected glucosamine derivatives. In order to simplify the purification of synthetic intermediates, a fluorous-assisted strategy has been also explored. Using this approach, a hyaluronic acid trisaccharide was prepared.

3D structure of a heparin mimetic analogue of a FGF-1 activator. A NMR and molecular modelling study.

The motional behaviour of heparin oligosaccharides in solution is best described as a top rotor having two perpendicular rotation axes. This prevents an accurate extraction of interprotonic distances by NOESY/ROESY based methods. In this paper, we describe the solution structure of the hexasaccharide 1 calculated from high exactitude distance data obtained from off-resonance ROESY combined with a long MD simulation of 500 ns. In previous studies, we have found that two synthetic hexasaccharides having the sulphate groups directed towards one side of its central plane have an opposite biological activity, while 1 is unable to activate the FGF-1 signalling pathway, the other (2) is even more active than the regular region derived hexasaccharide (3) that mimics the natural active compound, heparin. From the structural analysis it was concluded that 1 has similar three-dimensional characteristics to 2 or 3 and therefore the differences in the activity should be due to the arrangement of the sulphate groups within the hexasaccharidic sequence.