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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., Depto. de Química Orgánica, Fac. de Ciencias Químicas, TRUE, pub

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.

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.

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 with 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. We have systematically characterized the uptake efficiency and the intracellular distribution of these glycopeptides by flow cytometry, confocal microscopy and in zebrafish animal models. The incorporation of these glycan residues into the peptide structure influenced the internalization efficiency and the cellular toxicity of the resulting glycopeptide hybrids in the different cell lines tested. The results reported here highlight the potential of the glycosylation of penetrating peptides to modulate their activity

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.

The straightforward preparation of glycosyl neoconjugates by oxime (or hydrazone) bond formation represents a key bioorthogonal tool in chemical biology. However, when this strategy is employed by reacting the reducing end of the glycan moiety, the configuration and the stereochemical information is lost due to partial (or complete) opening of the glycan cyclic hemiacetal and the formation of the corresponding opened tautomers. We have completed the synthesis of a library of glycosyl aldehydes to be used as scaffold for the synthesis of neoglycoconjugates via oxime bond formation. These glycosyl aldehydes constitute a simple and accessible alternative to avoid loss of chiral information when conjugating, by oxime (or hydrazone) bonds, the aldehyde functionality present at the reducing end of natural carbohydrates

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.

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.

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.

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 where 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.

The synthesis of a new highly symmetric hexakis adduct of C60 appended with 12 cyclooctyne moieties has been carried out. This compound has been used for the copper-free strain-promoted cycloaddition reaction to a series of azides with excellent yields.This strategy for the obtention of clicked adducts of [60]fullerene is of special interest for biological applications., Unión Europea. FP7, Ministerio de Economía y Competitividad (MINECO), Comunidad de Madrid, European Research Council (ERC), Depto. de Química Orgánica, Fac. de Ciencias Químicas, TRUE, pub

The synthesis of a new highly symmetric hexakis adduct of C60 appended with 12 cyclooctyne moieties has been carried out. This compound has been used for the copper-free strain-promoted cycloaddition reaction to a series of azides with excellent yields. This strategy for the obtention of clicked adducts of [60]fullerene is of special interest for biological applications

The synthesis of a new highly symmetric hexakis adduct of C60 appended with 12 cyclooctyne moieties has been carried out. This compound has been used for the copper-free strain-promoted cycloaddition reaction to a series of azides with excellent yields. This strategy for the obtention of clicked adducts of [60]fullerene is of special interest for biological applications

DC-SIGN is a dendritic cell-specific C-type lectin receptor that recognizes highly glycosylated ligands expressed on the surface of various pathogens. This receptor plays an important role in the early stages of many viral infections, including HIV, which makes it an interesting therapeutic target. Glycomimetic compounds are good drug candidates for DC-SIGN inhibition due to their high solubility, resistance to glycosidases, and nontoxicity. We studied the structural properties of the interaction of the tetrameric DC-SIGN extracellular domain (ECD), with two glycomimetic antagonists, a pseudomannobioside (1) and a linear pseudomannotrioside (2). Though the inhibitory potency of 2, as measured by SPR competition experiments, was 1 order of magnitude higher than that of 1, crystal structures of the complexes within the DC-SIGN carbohydrate recognition domain showed the same binding mode for both compounds. Moreover, when conjugated to multivalent scaffolds, the inhibitory potencies of these compounds became uniform. Combining isothermal titration microcalorimetry, analytical ultracentrifugation, and dynamic light scattering techniques to study DC-SIGN ECD interaction with these glycomimetics revealed that 2 is able, without any multivalent presentation, to cluster DC-SIGN tetramers leading to an artificially overestimated inhibitory potency. The use of multivalent scaffolds presenting 1 or 2 in HIV trans-infection inhibition assay confirms the loss of potency of 2 upon conjugation and the equal efficacy of chemically simpler compound 1. This study documents a unique case where, among two active compounds chemically derived, the compound with the lower apparent activity is the optimal lead for further drug development

DC-SIGN is a dendritic cell-specific C-type lectin receptor that recognizes highly glycosylated ligands expressed on the surface of various pathogens. This receptor plays an important role in the early stages of many viral infections, including HIV, which makes it an interesting therapeutic target. Glycomimetic compounds are good drug candidates for DC-SIGN inhibition due to their high solubility, resistance to glycosidases, and nontoxicity. We studied the structural properties of the interaction of the tetrameric DC-SIGN extracellular domain (ECD), with two glycomimetic antagonists, a pseudomannobioside (1) and a linear pseudomannotrioside (2). Though the inhibitory potency of 2, as measured by SPR competition experiments, was 1 order of magnitude higher than that of 1, crystal structures of the complexes within the DC-SIGN carbohydrate recognition domain showed the same binding mode for both compounds. Moreover, when conjugated to multivalent scaffolds, the inhibitory potencies of these compounds became uniform. Combining isothermal titration microcalorimetry, analytical ultracentrifugation, and dynamic light scattering techniques to study DC-SIGN ECD interaction with these glycomimetics revealed that 2 is able, without any multivalent presentation, to cluster DC-SIGN tetramers leading to an artificially overestimated inhibitory potency. The use of multivalent scaffolds presenting 1 or 2 in HIV trans-infection inhibition assay confirms the loss of potency of 2 upon conjugation and the equal efficacy of chemically simpler compound 1. This study documents a unique case where, among two active compounds chemically derived, the compound with the lower apparent activity is the optimal lead for further drug development

The key role of carbohydrates in many biological events has attracted the interest of the scientific community. This fact has demanded the access to new tools necessary to understand this role and the interaction of carbohydrates with their corresponding receptors, lectins. Glycodendrimers and glycodendritic structures in general, have demonstrated to be very efficient and interesting tools to intervene in those processes where carbohydrates participate. In this review, we discuss the different glycodendritic structures that have been used to interfere with DC-SIGN, a very attractive lectin involved in infection processes and in the regulation of the immune response.

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.

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 (LeX). 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. © 2011 The Royal Society of Chemistry.

[EN] The present invention finds application in the field of medicine and, in particular, it relates to new compounds for the treatment and/or prevention of HIV; Ebola, Dengue, Hepatitis C, SARS or tuberculosis infections., [FR] La présente invention concerne le domaine de la médecine et, notamment, de nouveaux composés pour le traitement et/ou la prévention d'infections par le VIH, le virus Ébola, la dengue, l'hépatite C, le SRAS ou la tuberculose.

Since the discovery of HIV at the beginning of the 1980s, numerous efforts have been devoted to the search of an efficient vaccine. There are at least 25 drugs available for HIV treatment, but no cure is available. The observation that therapy for HIV disease is life long and that these drugs are associated with a number of side effects underlines the need for approaches aimed at preventing rather than treating infection. Additionally, the economic burden of treatment for the HIV infection occupies an increasing share of healthcare expenditure, making current practices likely to become difficult to sustain in the long run. Unfortunately, no effective vaccine for this disease is foreseeable in the near future. Microbicides could be an alternate way to build preventative approaches to HIV infection. Strategies based on preventing the virus from reaching its target cells seem to have some room for development and application. In this review we explore the state-of-the-art of available microbicides, focusing on HIV entry inhibitors. In addition, we discuss new compounds that show anti-HIV activity, which could be effective candidates.

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.

Protein–carbohydrate recognition is of fundamental importance for a large number of biological processes; carbohydrate–aromatic stacking is a widespread, but poorly understood, structural motif in this recognition. We describe, for the first time, the measurement of carbohydrate–aromatic interactions from their contribution to the stability of a dangling-ended DNA model system. We observe clear differences in the energetics of the interactions of several monosaccharides with a benzene moiety depending on the number of hydroxy groups, the stereochemistry, and the presence of a methyl group in the pyranose ring. A fucose–benzene pair is the most stabilizing of the studied series (-0.4 Kcal mol-1) and this interaction can be placed in the same range as other more studied interactions with aromatic residues of proteins, such as Phe–Phe, Phe–Met, or Phe–His. The noncovalent forces involved seem to be dispersion forces and nonconventional hydrogen bonds, whereas hydrophobic effects do not seem to drive the interaction.

Protein–carbohydrate recognition is of fundamental importance for a large number of biological processes; carbohydrate–aromatic stacking is a widespread, but poorly understood, structural motif in this recognition. We describe, for the first time, the measurement of carbohydrate–aromatic interactions from their contribution to the stability of a dangling-ended DNA model system. We observe clear differences in the energetics of the interactions of several monosaccharides with a benzene moiety depending on the number of hydroxy groups, the stereochemistry, and the presence of a methyl group in the pyranose ring. A fucose–benzene pair is the most stabilizing of the studied series (−0.4 Kcal mol−1) and this interaction can be placed in the same range as other more studied interactions with aromatic residues of proteins, such as Phe–Phe, Phe–Met, or Phe–His. The noncovalent forces involved seem to be dispersion forces and nonconventional hydrogen bonds, whereas hydrophobic effects do not seem to drive the interaction.

DC-SIGN, a lectin, which presents at the surface of immature dendritic cells, constitutes nowadays a promising target for the design of new antiviral drugs. This lectin recognizes highly glycosylated proteins present at the surface of several pathogens such as HIV, Ebola virus, Candida albicans, Mycobacterium tuberculosis, etc. Understanding the binding mode of this lectin is a topic of tremendous interest and will permit a rational design of new and more selective ligands. Here, we present computational and experimental tools to study the interaction of di- and trisaccharides with DC-SIGN. Docking analysis of complexes involving mannosyl di- and trisaccharides and the carbohydrate recognition domain (CRD) of DC-SIGN have been performed. Trisaccharides Manalpha1,2[Manalpha1,6]Man 1 and Manalpha1,3[Manalpha1,6]Man 2 were synthesized from an orthogonally protected mannose as a common intermediate. Using these ligands and the soluble extracellular domain (ECD) of DC-SIGN, NMR experiments based on STD and transfer-NOE were performed providing additional information. Conformational analysis of the mannosyl ligands in the free and bound states was done. These studies have demonstrated that terminal mannoses at positions 2 or 3 in the trisaccharides are the most important moiety and present the strongest contact with the binding site of the lectin. Multiple binding modes could be proposed and therefore should be considered in the design of new ligands.

El objetivo principal de esta Tesis Doctoral el desarrollo de nuevos y mejores inhibidores de la infección y propagación de agentes patógenos que utilizan la proteína DC-SIGN como receptor de entrada celular. Para alcanzar dicho objetivo general se marcaron los siguientes objetivos concretos que constituyen el trabajo de la presente Tesis Doctoral: 1. Ampliar el espectro de inhibición a otros agentes de infección viral como el VIH. 2. Análisis de la interacción entre estos sistemas glicodendríticos y el receptor DC-SIGN utilizando biosensores con detección de Resonancia del Plasmón de Superficie (SPR). 3. Mejora de la actividad antiviral de los compuestos utilizando oligosacáridos de manosa más complejos (disacáridos y trisacáridos), similares a los que se encuentran en la estructura de high mannose. 4. Preparación de sistemas multivalentes de forma monodispersa por síntesis química, que faciliten la caracterización de los productos finales y la interpretación de los resultados biológicos.