A 3D Peptide/[60]Fullerene Hybrid for Multivalent Recognition.

Fully substituted peptide/[60]fullerene hexakis-adducts offer an excellent opportunity for multivalent protein recognition. In contrast to monofunctionalized fullerene hybrids, peptide/[60]fullerene hexakis-adducts display multiple copies of a peptide in close spatial proximity and in the three dimensions of space. High affinity peptide binders for almost any target can be currently identified by in vitro evolution techniques, often providing synthetically simpler alternatives to natural ligands. However, despite the potential of peptide/[60]fullerene hexakis-adducts, these promising conjugates have not been reported to date. Here we present a synthetic strategy for the construction of 3D multivalent hybrids that are able to bind with high affinity the E-selectin. The here synthesized fully substituted peptide/[60]fullerene hybrids and their multivalent recognition of natural receptors constitute a proof of principle for their future application as functional biocompatible materials.

Synthesis and Supramolecular Functional Assemblies of Ratiometric pH Probes.

Tracking pH with spatiotemporal resolution is a critical challenge for synthetic chemistry, chemical biology and beyond. Over the last decade, different small probes and supramolecular systems have emerged for in cellulo or in vivo pH tracking. However, pH reporting still presents critical limitations, such as background reduction, improved sensor stability, cell targeting, endosomal escape, near- and far-infrared ratiometric pH tracking and adaption to new imaging techniques (i.e., super-resolution). These challenges will require the combined efforts of synthetic and supramolecular chemistry working together to develop the next generation of smart materials that will resolve current limitations. Herein, recent advances in the synthesis of small fluorescent probes, together with new supramolecular functional systems employed for pH tracking, are described with an emphasis on ratiometric probes. The combination of organic synthesis and stimuli-responsive supramolecular functional materials will be essential to solve future challenges of pH tracking, such as improved signal to noise ratio, on target activation and microenvironment reporting.

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.

Synthesis of Highly Efficient Multivalent Disaccharide/[60]Fullerene Nanoballs for Emergent Viruses.

After the last epidemic of the Zika virus (ZIKV) in Brazil that peaked in 2016, growing evidence has been demonstrated of the link between this teratogenic flavivirus and microcephaly cases. However, no vaccine or antiviral drug has been approved yet. ZIKV and Dengue viruses (DENV) entry to the host cell takes place through several receptors, including dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), so that the blockade of this receptor through multivalent glycoconjugates supposes a promising biological target to inhibit the infection process. In order to get enhanced multivalency in biocompatible systems, tridecafullerenes appended with up to 360 1,2-mannobiosides have been synthesized using a strain-promoted cycloaddition of azides to alkynes (SPAAC) strategy. These systems have been tested against ZIKV and DENV infection, showing an outstanding activity in the picomolar range.

Glycosylated Cell-Penetrating Peptides (GCPPs).

The cell membrane regulates the exchange of molecules and information with the external environment. However, this control barrier hinders the delivery of exogenous bioactive molecules that can be applied to correct cellular malfunctions. Therefore, the traffic of macromolecules across the cell membrane represents a great challenge for the development of the next generation of therapies and diagnostic methods. Cell-penetrating peptides are short peptide sequences capable of delivering a broad range of biomacromolecules across the cellular membrane. However, penetrating peptides still suffer from limitations, mainly related to their lack of specificity and potential toxicity. Glycosylation has emerged as a potential promising strategy for the biological improvement of synthetic materials. In this work we have developed a new convergent strategy for the synthesis of penetrating peptides functionalized with glycan residues by an oxime bond connection. The uptake efficiency and intracellular distribution of these glycopeptides have been systematically characterized by means of flow cytometry and confocal microscopy and in zebrafish animal models. The incorporation of these glycan residues into the peptide structure influenced the internalization efficiency and cellular toxicity of the resulting glycopeptide hybrids in the different cell lines tested. The results reported herein highlight the potential of the glycosylation of penetrating peptides to modulate their activity.

Maleimide and Cyclooctyne-Based Hexakis-Adducts of Fullerene: Multivalent Scaffolds for Copper-Free Click Chemistry on Fullerenes.

The synthesis of multivalent systems based on hexakis-adducts of [60]fullerene employing a biocompatible copper-free click chemistry strategy has been accomplished. A symmetric hexakis-adduct of fullerene bearing 12 maleimide units (3) is reported, and it has been employed to carry out the thiol-maleimide Michael addition. To achieve orthogonal click addition, an asymmetric derivative bearing one maleimide and 10 cyclooctynes has been synthesized. The sequential and one-pot transformations of the two clickable groups have been explored, finding the best results in the case of the one-pot experiment. This route has been used to obtain a biocompatible hexakis-adduct appended with two different biomolecules, carbohydrates, and amino acids.

Topological Defects in Hyperbranched Glycopolymers Enhance Binding to Lectins.

Central scaffold topology and carbohydrate density are important features in determining the binding mechanism and potency of synthetic multivalent of poly- versus monodisperse carbohydrate systems against a model plant toxin (Ricinus communis agglutinin (RCA120 )). Lower densities of protein receptors favour the use of heterogeneous, polydisperse glycoconjugate presentations, as determined by surface plasmon resonance and dynamic light scattering.

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.

Straightforward synthesis of Man9, the relevant epitope of the high-mannose oligosaccharide.

The high-mannose oligosaccharide (or its corresponding Man9 epitope) is the most abundant structure present in pathogen envelope glycoproteins. These glycans play a key role in the pathogenesis of several pathogens and also in the communication with the immune system. Understanding the mechanism of action of these glycans requires the access to pure and chemically well-defined structures in reasonable amounts. The synthesis of these complex branched oligosaccharides is not trivial and few syntheses are reported in the literature with several synthetic and purification steps and low overall yields. In this work, we described a very efficient synthetic alternative to access this relevant Man9 epitope in a very straightforward manner.

Rapid and efficient synthesis of α(1-2)mannobiosides.

α(1,2)mannobiosides with different substituents at the reducing end have been synthesized by a common strategy using benzoyls as the permanent protecting groups and an acetyl as the orthogonal protecting group at position C2 of the glycosyl acceptor. The new synthetic strategy has been performed remarkably reducing the number of purification steps, the time of synthesis (less than 72 hours) and improving the overall yield at least three times with respect to the best procedure described in the literature at the moment. Additionally, this protecting group strategy is compatible with the presence of azido groups and the use of Cu catalyzed azide alkyne cycloaddition (CuAAC) also called "click chemistry" for conjugating the α(1-2)mannobiosides to different scaffolds for the preparation of mannosyl multivalent systems.

Comprehensive analysis of blood group antigen binding to classical and El Tor cholera toxin B-pentamers by NMR.

Cholera is a diarrheal disease responsible for the deaths of thousands, possibly even hundreds of thousands of people every year, and its impact is predicted to further increase with climate change. It has been known for decades that blood group O individuals suffer more severe symptoms of cholera compared with individuals with other blood groups (A, B and AB). The observed blood group dependence is likely to be caused by the major virulence factor of Vibrio cholerae, the cholera toxin (CT). Here, we investigate the binding of ABH blood group determinants to both classical and El Tor CTB-pentamers using saturation transfer difference NMR and show that all three blood group determinants bind to both toxin variants. Although the details of the interactions differ, we see no large differences between the two toxin genotypes and observe very similar binding constants. We also show that the blood group determinants bind to a site distinct from that of the primary receptor, GM1. Transferred NOESY data confirm that the conformations of the blood group determinants in complex with both toxin variants are similar to those of reported X-ray and solution structures. Taken together, this detailed analysis provides a framework for the interpretation of the epidemiological data linking the severity of cholera infection and an individual's blood group, and brings us one step closer to understanding the molecular basis of cholera blood group dependence.

Effective targeting of DC-SIGN by α-fucosylamide functionalized gold nanoparticles.

Dendritic Cells (DCs), the most potent antigen-presenting cells, play a critical role in the detection of invading pathogens, which are recognized also by multiple carbohydrate-specific receptors. Among them, DC-SIGN is one of the best characterized, with high-mannose and Lewis-type glycan specificity. In this study, we present a potent DC-SIGN targeting device developed using gold nanoparticles functionalized with α-fucosyl-ß-alanyl amide. The nanoparticles bound to cellular DC-SIGN and induced internalization as effectively as similar particles coated with comparable amounts of Lewis(X) oligosaccharide. They were found to be neutral toward dendritic cell maturation and IL-10 production, thus envisaging a possible use as targeted imaging tools and antigen delivery devices.

Targeting mannose-binding lectin confers long-lasting protection with a surprisingly wide therapeutic window in cerebral ischemia.

BACKGROUND: The involvement of the complement system in brain injury has been scarcely investigated. Here, we document the pivotal role of mannose-binding lectin (MBL), one of the recognition molecules of the lectin complement pathway, in brain ischemic injury. METHODS AND RESULTS: Focal cerebral ischemia was induced in mice (by permanent or transient middle cerebral artery occlusion) and rats (by 3-vessel occlusion). We first observed that MBL is deposited on ischemic vessels up to 48 hours after injury and that functional MBL/MBL-associated serine protease 2 complexes are increased. Next, we demonstrated that (1) MBL(-/-) mice are protected from both transient and permanent ischemic injury; (2) Polyman2, the newly synthesized mannosylated molecule selected for its binding to MBL, improves neurological deficits and infarct volume when given up to 24 hours after ischemia in mice; (3) anti-MBL-A antibody improves neurological deficits and infarct volume when given up to 18 hours after ischemia, as assessed after 28 days in rats. CONCLUSIONS: Our data show an important role for MBL in the pathogenesis of brain ischemic injury and provide a strong support to the concept that MBL inhibition may be a relevant therapeutic target in humans, one with a wide therapeutic window of application.

Both El Tor and classical cholera toxin bind blood group determinants.

Cholera is a disease which shows a clear blood group profile, with blood group O individuals experiencing the most severe symptoms. For a long time, the cholera toxin has been suspected to be the main culprit of this blood group dependence. Here, we show that both El Tor and classical cholera toxin B-pentamers do indeed bind blood group determinants (with equal affinities), using Surface Plasmon Resonance and NMR spectroscopy. Together with previous structural data, this confirms our earlier hypothesis as to the molecular basis of cholera blood group dependence, with an interesting twist: the shorter blood group H-determinant characteristic of blood group O individuals binds with similar binding affinity compared to the A-determinant, however, with different kinetics.

Highly polar carbohydrates stack onto DNA duplexes via CH/π interactions.

Carbohydrate-nucleic acid contacts are known to be a fundamental part of some drug-DNA recognition processes. Most of these interactions occur through the minor groove of DNA, such as in the calicheamicin or anthracycline families, or through both minor and major groove binders such as in the pluramycins. Here, we demonstrate that carbohydrate-DNA interactions are also possible through sugar capping of a DNA double helix. Highly polar mono- and disaccharides are capable of CH/π stacking onto the terminal DNA base pair of a duplex as shown by NMR spectroscopy. The energetics of the carbohydrate-DNA interactions vary depending on the stereochemistry, polarity, and contact surface of the sugar involved and also on the terminal base pair. These results reveal carbohydrate-DNA base stacking as a potential recognition motif to be used in drug design, supramolecular chemistry, or biobased nanomaterials.

Insights into molecular recognition of Lewis(X) mimics by DC-SIGN using NMR and molecular modelling.

In this work, we have studied in detail the binding of two α-fucosylamide-based mimics of Lewis(X) to DC-SIGN ECD (ECD = extracellular domain) using STD NMR and docking. We have concluded that the binding mode occurs mainly through the fucose moiety, in the same way as Lewis(X). Similarly to other mimics containing mannose or fucose previously studied, we have shown that both compounds bind to DC-SIGN ECD in a multimodal fashion. In this case, the main contact is the interaction of two hydroxyl groups one equatorial and the other one axial (O3 and O4) of the fucose with the Ca(2+) as Lewis(X) and similarly to mannose-containing mimics (in this case the interacting groups are both in the equatorial position). Finally, we have measured the K(D) of one mimic that was 0.4 mM. Competitive STD NMR experiments indicate that the aromatic moiety provides additional binding contacts that increase the affinity.

Second generation of fucose-based DC-SIGN ligands: affinity improvement and specificity versus Langerin.

DC-SIGN and Langerin are two C-type lectins involved in the initial steps of HIV infections: the former acts as a viral attachment factor and facilitates viral invasion of the immune system, the latter has a protective effect. Potential antiviral compounds targeted against DC-SIGN were synthesized using a common fucosylamide anchor. Their DC-SIGN affinity was tested by SPR and found to be similar to that of the natural ligand Lewis-X (Le(X)). The compounds were also found to be selective for DC-SIGN and to interact only weakly with Langerin. These molecules are potentially useful therapeutic tools against sexually transmitted HIV infection.

Synthesis and in vitro inhibition properties of siRNA conjugates carrying glucose and galactose with different presentations.

Oligoribonucleotide conjugates and the corresponding siRNA duplexes against tumor necrosis factor carrying one, two, or four glucose and galactose residues at the 5'-end have been prepared using phosphoramidite chemistry. Carbohydrate-modified siRNA duplexes have similar inhibitory properties than unmodified RNA duplexes in HeLa cells transfected with oligofectamine. When HeLa cells were treated with siRNA carrying one, two, or four glucose residues without oligofectamine, no inhibition was observed. The inhibitory properties of siRNA carrying galactose residues without transfecting agent were tested on HuH-7 cells that have abundant asialoglycoprotein receptors. In these cells siRNA carrying galactose residues have slight anti-TNF inhibitory properties (25% in the best case) that are eliminated if the receptors are blocked with a competitor. These results demonstrate receptor-mediated uptake of siRNA carrying galactose residues, although the efficacy of the process is not enough for efficient RNA interference experiments.

Synthesis, cell-surface binding, and cellular uptake of fluorescently labeled glucose-DNA conjugates with different carbohydrate presentation.

Oligonucleotide conjugates carrying carbohydrates at the 5'-end have been prepared. Glucose, fucose, and saccharides containing glucose at the nonreducing end were attached to DNA strands using the classical phosphoramidite chemistry. Two types of spacers and a dendron scaffold helped to obtain a diversity of sugar presentations in the DNA conjugates. Cellular surface adsorption and cellular uptake of carbohydrate oligonucleotide antisense sequences were measured using flow cytometric analysis. Conjugates with the glucose moiety linked through long spacers (15 to 18 atom distances) were internalized better than those with short linkers (4 atom distance) and than DNA control strands without sugar modification. Conjugates with tetravalent presentation of glucose did not improve cell uptake.

Mannose hyperbranched dendritic polymers interact with clustered organization of DC-SIGN and inhibit gp120 binding.

DC-SIGN (dendritic cell-specific ICAM-3 grabbing non-integrin) is a C-type lectin receptor of dendritic cells and is involved in the initial steps of numerous infectious diseases. Surface plasmon resonance has been used to study the affinity of a glycodendritic polymer with 32 mannoses, to DC-SIGN. This glycodendrimer binds to DC-SIGN surfaces in the submicromolar range. This binding depends on a clustered organization of DC-SIGN mimicking its natural organization as microdomain in the dendritic cells plasma membrane. Moreover, this compound inhibits DC-SIGN binding to the HIV glycoprotein gp120 with an IC50 in the micromolar range and therefore can be considered as a potential antiviral drug.