Multivalent glycosystems for human lectins.

Human lectins are involved in a wide variety of biological processes, both physiological and pathological, which have attracted the interest of the scientific community working in the glycoscience field. Multivalent glycosystems have been employed as useful tools to understand carbohydrate-lectin binding processes as well as for biomedical applications. The review shows the different scaffolds designed for a multivalent presentation of sugars and their corresponding binding studies to lectins and in some cases, their biological activities. We summarise this research by organizing based on lectin types to highlight the progression in this active field. The paper provides an overall picture of how these contributions have furnished relevant information on this topic to help in understanding and participate in these carbohydrate-lectin interactions.

Fluorinated Man9 as a High Mannose Mimetic to Unravel Its Recognition by DC-SIGN Using NMR.

Lectins are capable of reading out the structural information contained in carbohydrates through specific recognition processes. Determining the binding epitope of the sugar is fundamental to understanding this recognition event. Nuclear magnetic resonance (NMR) is a powerful tool to obtain this structural information in solution; however, when the sugar involved is a complex oligosaccharide, such as high mannose, the signal overlap found in the NMR spectra precludes an accurate analysis of the interaction. The introduction of tags into these complex oligosaccharides could overcome these problems and facilitate NMR studies. Here, we show the preparation of the Man9 of high mannose with some fluorine tags and the study of the interaction with its receptor, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). This fluorinated ligand has allowed us to apply heteronuclear two-dimensional (2D) 1H,19F STD-TOCSYreF NMR experiments, using the initial slope approach, which has facilitated the analysis of the Man9/DC-SIGN interaction, unequivocally providing the binding epitope.

Carbon-based glyco-nanoplatforms: towards the next generation of glycan-based multivalent probes.

Cell surface carbohydrates mediate a wide range of carbohydrate-protein interactions key to healthy and disease mechanisms. Many of such interactions are multivalent in nature and in order to study these processes at a molecular level, many glycan-presenting platforms have been developed over the years. Among those, carbon nanoforms such as graphene and their derivatives, carbon nanotubes, carbon dots and fullerenes, have become very attractive as biocompatible platforms that can mimic the multivalent presentation of biologically relevant glycosides. The most recent examples of carbon-based nanoplatforms and their applications developed over the last few years to study carbohydrate-mediate interactions in the context of cancer, bacterial and viral infections, among others, are highlighted in this review.

Stability of Human Telomeric G-Quadruplexes Complexed with Photosensitive Ligands and Irradiated with Visible Light.

Guanine-rich DNA sequences can fold into non-canonical nucleic acid structures called G-quadruplexes (G4s). These nanostructures have strong implications in many fields, from medical science to bottom-up nanotechnologies. As a result, ligands interacting with G4s have attracted great attention as candidates in medical therapies, molecular probe applications, and biosensing. In recent years, the use of G4-ligand complexes as photopharmacological targets has shown significant promise for developing novel therapeutic strategies and nanodevices. Here, we studied the possibility of manipulating the secondary structure of a human telomeric G4 sequence through the interaction with two photosensitive ligands, DTE and TMPyP4, whose response to visible light is different. The effect of these two ligands on G4 thermal unfolding was also considered, revealing the occurrence of peculiar multi-step melting pathways and the different attitudes of the two molecules on the quadruplex stabilization.

Structure-activity relationship studies on divalent naphthalene diimide G quadruplex ligands with anticancer and antiparasitic activity.

Naphthalene diimide (NDI) is a central scaffold that has been commonly used in the design of G-quadruplex (G4) ligands. Previous work revealed notable anticancer activity of a disubstituted N-methylpiperazine propyl NDI G4 ligand. Here, we explored structure-activity relationship studies around ligand bis-N,N-2,7-(3-(4-methylpiperazin-1-yl)propyl)-1,4,5,8-naphthalenetetracarboxylic diimide, maintaining the central NDI core whilst modifying the spacer and the nature of the cationic groups. We prepared new disubstituted NDI derivatives of the original compound and examined their in vitro antiproliferative and antiparasitic activity. Several N-methylpiperazine propyl NDIs showed sub-micromolar activity against Trypanosoma brucei and Leishmania major parasites with up to 30 fold selectivity versus MRC-5 cells. The best compound was a dimorpholino NDI with an IC50 of 0.17 µM against T.brucei and 40 fold selectivity versus MRC-5 cells. However, no clear correlation between G4 binding of the new NDI derivatives and antiproliferative or antiparasitic activity was observed, indicating that other mechanisms of action may be responsible for the observed biological activity.

Topological and Multivalent Effects in Glycofullerene Oligomers as EBOLA Virus Inhibitors.

The synthesis of new biocompatible antiviral materials to fight against the development of multidrug resistance is being widely explored. Due to their unique globular structure and excellent properties, [60]fullerene-based antivirals are very promising bioconjugates. In this work, fullerene derivatives with different topologies and number of glycofullerene units were synthesized by using a SPAAC copper free strategy. This procedure allowed the synthesis of compounds 1-3, containing from 20 to 40 mannose units, in a very efficient manner and in short reaction times under MW irradiation. The glycoderivatives were studied in an infection assay by a pseudotyped viral particle with Ebola virus GP1. The results obtained show that these glycofullerene oligomers are efficient inhibitors of EBOV infection with IC50s in the nanomolar range. In particular, compound 3, with four glycofullerene moieties, presents an outstanding relative inhibitory potency (RIP). We propose that this high RIP value stems from the appropriate topological features that efficiently interact with DC-SIGN.

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.

A Vibration-Induced-Emission-Based Fluorescent Chemosensor for the Selective and Visual Recognition of Glucose.

The development of chemosensors to detect analytes in biologically relevant solutions is a challenging task. We report the synthesis of a fluorescent receptor that combines vibration-induced emission (VIE) and dynamic covalent chemistry for the detection of glucose in aqueous media. We show that the bis-2-(N-methylaminomethyl)phenylboronic acid-decorated N,N'-diphenyl-dihydrodibenzo[a,c]phenazine (DPAC) receptor 1 can detect glucose and discriminate between closely related monosaccharides including those commonly found in blood. Preliminary studies suggest monosaccharides bind to the DPAC-receptor with a 1:1 stoichiometry to produce pseudomacrocyclic complexes, which in turn leads to distinct optical changes in the fluorescent emission of the receptor for each host. Moreover, the complexation-induced change in emission can be detected visually and quantified in a ratiometric way. Our results highlight the potential of VIE-type receptors for the quantitative determination of saccharides in biological samples.

An Ultra-Long-Lived Triplet Excited State in Water at Room Temperature: Insights on the Molecular Design of Tridecafullerenes.

Suitably engineered molecular systems exhibiting triplet excited states with very long lifetimes are important for high-end applications in nonlinear optics, photocatalysis, or biomedicine. We report the finding of an ultra-long-lived triplet state with a mean lifetime of 93 ms in an aqueous phase at room temperature, measured for a globular tridecafullerene with a highly compact glycodendrimeric structure. A series of three tridecafullerenes bearing different glycodendrons and spacers to the C60 units have been synthesized and characterized. UV/Vis spectra and DLS experiments confirm their aggregation in water. Steady-state and time-resolved fluorescence experiments suggest a different degree of inner solvation of the multifullerenes depending on their molecular design. Efficient quenching of the triplet states by O2 but not by waterborne azide anions has been observed. Molecular modelling reveals dissimilar access of the aqueous phase to the internal structure of the tridecafullerenes, differently shielded by the glycodendrimeric shell.

Exploring Multi-Subsite Binding Pockets in Proteins: DEEP-STD NMR Fingerprinting and Molecular Dynamics Unveil a Cryptic Subsite at the GM1 Binding Pocket of Cholera Toxin†B.

Ligand-based NMR techniques to study protein-ligand interactions are potent tools in drug design. Saturation transfer difference (STD) NMR spectroscopy stands out as one of the most versatile techniques, allowing screening of fragments libraries and providing structural information on binding modes. Recently, it has been shown that a multi-frequency STD NMR approach, differential epitope mapping (DEEP)-STD NMR, can provide additional information on the orientation of small ligands within the binding pocket. Here, the approach is extended to a so-called DEEP-STD NMR fingerprinting technique to explore the binding subsites of cholera toxin subunit B (CTB). To that aim, the synthesis of a set of new ligands is presented, which have been subject to a thorough study of their interactions with CTB by weak affinity chromatography (WAC) and NMR spectroscopy. Remarkably, the combination of DEEP-STD NMR fingerprinting and Hamiltonian replica exchange molecular dynamics has proved to be an excellent approach to explore the geometry, flexibility, and ligand occupancy of multi-subsite binding pockets. In the particular case of CTB, it allowed the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket to be revealed, paving the way to the design of novel leads for inhibition of this relevant toxin.

Peptide Glycodendrimers as Potential Vaccines for Olive Pollen Allergy.

Olive pollen is one of the most important causes of respiratory allergy, with Ole e 1 being the most clinically relevant sensitizing allergen. Peptide-based vaccines represent promising therapeutic approaches, but the use of adjuvants is required to strengthen the weak immunogenicity of small peptides. We propose the use of dendrimeric scaffolds conjugated to the T cell immunodominant epitope of Ole e 1 (OE109-130) for the development of novel vaccines against olive pollen allergy. Four dendrimeric scaffolds containing an ester/ether with nine mannoses, an ester succinimidyl linker with nine N-acetyl-glucosamine units or nine ethylene glycol units conjugated to OE109-130 peptide were designed, and their cytotoxicity, internalization pattern, and immunomodulatory properties were analyzed in vitro. None of the dendrimers exhibited cytotoxicity in humanized rat basophil (RBL-2H3), human bronchial epithelial Calu-3, and human mast LAD2 cell lines. Confocal images indicated that mannosylated glycodendropeptides exhibited lower colocalization with a lysosomal marker. Moreover, mannosylated glycodendropeptides showed higher transport tendency through the epithelial barrier formed by Calu-3 cells cultured at the air-liquid interface. Finally, mannosylated glycodendropeptides promoted Treg and IL10+Treg proliferation and IL-10 secretion by peripheral blood mononuclear cells from allergic patients. Mannosylated dendrimers conjugated with OE109-130 peptide from Ole e 1 have been identified as suitable candidates for the development of novel vaccines of olive pollen allergy.

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.

Nanocarbon-Based Glycoconjugates as Multivalent Inhibitors of Ebola Virus Infection.

SWCNTs, MWCNTs, and SWCNHs have been employed as virus-mimicking nanocarbon platforms for the multivalent presentation of carbohydrates in an artificial Ebola virus infection model assay. These carbon nanoforms have been chemically modified by the covalent attachment of glycodendrons and glycofullerenes using the CuAAC "click chemistry" approach. This modification dramatically increases the water solubility of these structurally different nanocarbons. Their efficiency in blocking DC-SIGN-mediated viral infection by an artificial Ebola virus has been tested in a cellular experimental assay, finding that glycoconjugates based on MWCNTs functionalized with glycofullerenes are potent inhibitors of viral infection.

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.

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.

Probing the binding and antiparasitic efficacy of azobenzene G-quadruplex ligands to investigate G4 ligand design.

Novel strategies against parasitic infections are of great importance. Here, we describe a G4 DNA ligand with subnanomolar antiparasitic activity against T. brucei and a remarkable selectivity index (IC50 MRC-5/T. brucei) of 2285-fold. We also correlate the impact of small structural changes to G4 binding activity and antiparasitic activity.

DNA G-quadruplexes in the genome of Trypanosoma cruzi as potential therapeutic targets for Chagas disease: Dithienylethene ligands as effective antiparasitic agents.

Chagas disease is caused by the parasite Trypanosoma cruzi and affects over 7 million people worldwide. The two actual treatments, Benznidazole (Bzn) and Nifurtimox, cause serious side effects due to their high toxicity leading to treatment abandonment by the patients. In this work, we propose DNA G-quadruplexes (G4) as potential therapeutic targets for this infectious disease. We have found 174 PQS per 100,000 nucleotides in the genome of T. cruzi and confirmed G4 formation of three frequent motifs. We synthesized a family of 14 quadruplex ligands based in the dithienylethene (DTE) scaffold and demonstrated their binding to these identified G4 sequences. Several DTE derivatives exhibited micromolar activity against epimastigotes of four different strains of T. cruzi, in the same concentration range as Bzn. Compounds L3 and L4 presented remarkable activity against trypomastigotes, the active form in blood, of T. cruzi SOL strain (IC50 = 1.5-3.3 µM, SI = 25-40.9), being around 40 times more active than Bzn and displaying much better selectivity indexes.

Synthesis, biological evaluation, WAC and NMR studies of S-galactosides and non-carbohydrate ligands of cholera toxin based on polyhydroxyalkylfuroate moieties.

The synthesis of several non-carbohydrate ligands of cholera toxin based on polyhydroxyalkylfuroate moieties is reported. Some of them have been linked to D-galactose through a stable and well-tolerated S-glycosidic bond. They represent a novel type of non-hydrolyzable bidentate ligand featuring galactose and polyhydroxyalkylfuroic esters as pharmacophoric residues, thus mimicking the GM1 ganglioside. The affinity of the new compounds towards cholera toxin was measured by weak affinity chromatography (WAC). The interaction of the best candidates with this toxin was also studied by saturation transfer difference NMR experiments, which allowed identification of the binding epitopes of the ligands interacting with the protein. Interestingly, the highest affinity was shown by non-carbohydrate mimics based on a polyhydroxyalkylfuroic ester structure.