Correction: Fluorescent glycoconjugates and their applications.

Correction for 'Fluorescent glycoconjugates and their applications' by Baptiste Thomas et al., Chem. Soc. Rev., 2020, 49, 593-641, DOI: 10.1039/C8CS00118A.

Synthetic Analogues of Huwentoxin-IV Spider Peptide With Altered Human NaV1.7/NaV1.6 Selectivity Ratios.

Huwentoxin-IV (HwTx-IV), a peptide discovered in the venom of the Chinese bird spider Cyriopagopus schmidti, has been reported to be a potent antinociceptive compound due to its action on the genetically-validated NaV1.7 pain target. Using this peptide for antinociceptive applications in vivo suffers from one major drawback, namely its negative impact on the neuromuscular system. Although studied only recently, this effect appears to be due to an interaction between the peptide and the NaV1.6 channel subtype located at the presynaptic level. The aim of this work was to investigate how HwTx-IV could be modified in order to alter the original human (h) NaV1.7/NaV1.6 selectivity ratio of 23. Nineteen HwTx-IV analogues were chemically synthesized and tested for their blocking effects on the Na+ currents flowing through these two channel subtypes stably expressed in cell lines. Dose-response curves for these analogues were generated, thanks to the use of an automated patch-clamp system. Several key amino acid positions were targeted owing to the information provided by earlier structure-activity relationship (SAR) studies. Among the analogues tested, the potency of HwTx-IV E4K was significantly improved for hNaV1.6, leading to a decreased hNaV1.7/hNaV1.6 selectivity ratio (close to 1). Similar decreased selectivity ratios, but with increased potency for both subtypes, were observed for HwTx-IV analogues that combine a substitution at position 4 with a modification of amino acid 1 or 26 (HwTx-IV E1G/E4G and HwTx-IV E4K/R26Q). In contrast, increased selectivity ratios (>46) were obtained if the E4K mutation was combined to an additional double substitution (R 26A/Y33W) or simply by further substituting the C-terminal amidation of the peptide by a carboxylated motif, linked to a marked loss of potency on hNaV1.6 in this latter case. These results demonstrate that it is possible to significantly modulate the selectivity ratio for these two channel subtypes in order to improve the potency of a given analogue for hNaV1.6 and/or hNaV1.7 subtypes. In addition, selective analogues for hNaV1.7, possessing better safety profiles, were produced to limit neuromuscular impairments.

Glycan Array Technology.

Glycan (or carbohydrate) arrays have become an essential tool in glycomics, providing fast and high-throughput data on protein-carbohydrate interactions with small amounts of carbohydrate ligands. The general concepts of glycan arrays have been adopted from other microarray technologies such as those used for nucleic acid and proteins. However, carbohydrates have presented their own challenges, in particular in terms of access to glycan probes, linker attachment chemistries and analysis, which will be reviewed in this chapter. As more and more glycan probes have become available through chemical and enzymatic synthesis and robust linker chemistries have been developed, the applications of glycan arrays have dramatically increased over the past 10 years, which will be illustrated with recent examples.

Fluorescent glycoconjugates and their applications.

Glycoconjugates and their applications as lectin ligands in biology have been thoroughly investigated in the past decades. Meanwhile, the intrinsic properties of such multivalent molecules were limited essentially to their ability to bind to their receptors with high selectivity and/or avidity. The present review will focus on multivalent glycoconjugates displaying an additional capability such as fluorescence properties not only for applications toward imaging of cancer cells and detection of proteins or pathogens but also for drug delivery systems toward targeted cancer therapy. This review is a collection of research articles discussed in the context of the structural features of fluorescent glycoconjugates organized according to their fluorescent core scaffold and with their representative applications.

Heteroglycoclusters With Dual Nanomolar Affinities for the Lectins LecA and LecB From Pseudomonas aeruginosa.

Multivalent structures displaying different instead of similar sugar units, namely heteroglycoclusters (hGCs), are stimulating the efforts of glycochemists for developing compounds with new biological properties. Here we report a four-step strategy to synthesize hexadecavalent hGCs displaying eight copies of αFuc and ßGal. These compounds were tested for the binding to lectins LecA and LecB from Pseudomonas aeruginosa. While parent fucosylated (19) and galactosylated (20) homoclusters present nanomolar affinity with LecB and LecA, respectively, we observed that hGCs combining these sugars (11 and 13) maintain their binding potency with both lectins despite the presence of an unspecific sugar. The added multivalency is therefore not a barrier for efficient recognition by bacterial receptors and it opens the route for adding different sugars that can be selected for their immunomodulatory properties.

Characterisation of a Bacterial Galactokinase with High Activity and Broad Substrate Tolerance for Chemoenzymatic Synthesis of 6-Aminogalactose-1-Phosphate and Analogues.

Glycosyl phosphates are important intermediates in many metabolic pathways and are substrates for diverse carbohydrate-active enzymes. Thus, there is a need to develop libraries of structurally similar analogues that can be used as selective chemical probes in glycomics. Here, we explore chemoenzymatic cascades for the fast generation of glycosyl phosphate libraries without protecting-group strategies. The key enzyme is a new bacterial galactokinase (LgGalK) cloned from Leminorella grimontii, which was produced in Escherichia coli and shown to catalyse 1-phosphorylation of galactose. LgGalK displayed a broad substrate tolerance, being able to catalyse the 1-phosphorylation of a number of galactose analogues, including 3-deoxy-3-fluorogalactose and 4-deoxy-4-fluorogalactose, which were first reported to be substrates for wild-type galactokinase. LgGalK and galactose oxidase variant M1 were combined in a one-pot, two-step system to synthesise 6-oxogalactose-1-phosphate and 6-oxo-2-fluorogalactose-1-phosphate, which were subsequently used to produce a panel of 30 substituted 6-aminogalactose-1-phosphate derivatives by chemical reductive amination in a one-pot, three-step chemoenzymatic process.

Heterovalent Glycodendrimers as Epitope Carriers for Antitumor Synthetic Vaccines.

The large majority of TACA-based (TACA=Tumor-Associated Carbohydrate Antigens) antitumor vaccines target only one carbohydrate antigen, thereby often resulting in the incomplete destruction of cancer cells. However, the morphological heterogeneity of the tumor glycocalix, which is in constant evolution during malignant transformation, is a crucial point to consider in the design of vaccine candidates. In this paper, an efficient synthetic strategy based on orthogonal chemoselective ligations to prepare fully synthetic glycosylated cyclopeptide scaffolds grafted with both Tn and TF antigen analogues is reported. To evaluate their ability to be recognized as tumor antigens, direct interaction ELISA assays have been performed with the anti-Tn monoclonal antibody 9A7. Although both heterovalent structures showed binding capacities with 9A7, the presence of the second TF epitope did not interfere with the recognition of Tn except in one epitope arrangement. This heterovalent glycosylated structure thus represents an attractive epitope carrier to be further functionalized with T-cell peptide epitopes.

Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger.

Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.

Application of Biocatalysis to on-DNA Carbohydrate Library Synthesis.

DNA-encoded libraries are increasingly used for the discovery of bioactive lead compounds in high-throughput screening programs against specific biological targets. Although a number of libraries are now available, they cover limited chemical space due to bias in ease of synthesis and the lack of chemical reactions that are compatible with DNA tagging. For example, compound libraries rarely contain complex biomolecules such as carbohydrates with high levels of functionality, stereochemistry, and hydrophilicity. By using biocatalysis in combination with chemical methods, we aimed to significantly expand chemical space and generate generic libraries with potentially better biocompatibility. For DNA-encoded libraries, biocatalysis is particularly advantageous, as it is highly selective and can be performed in aqueous environments, which is an essential feature for this split-and-mix library technology. In this work, we demonstrated the application of biocatalysis for the on-DNA synthesis of carbohydrate-based libraries by using enzymatic oxidation and glycosylation in combination with traditional organic chemistry.

Divergent and convergent synthesis of GalNAc-conjugated dendrimers using dual orthogonal ligations.

The synthesis of glycodendrimers remains a challenging task. In this paper we propose a protocol based on both oxime ligation (OL) to combine cyclopeptide repeating units as the dendritic core and the copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC) to conjugate peripheral α and ß propargylated GalNAc. By contrast with the oxime-based iterative protocol reported in our group, our current strategy can be used in both divergent and convergent routes with similar efficiency and the resulting hexadecavalent glycodendrimers can be easily characterized compared to oxime-linked analogues. A series of glycoconjugates displaying four or sixteen copies of both α and ß GalNAc have been prepared and their ability to inhibit the adhesion of the soybean agglutinin (SBA) lectin to polymeric-GalNAc immobilized on microtiter plates has been evaluated. As was anticipated, the higher inhibitory effect (IC50 = 0.46 µM) was measured with the structure displaying αGalNAc with the higher valency (compound 13), which demonstrates that the binding properties of these glycoconjugates are strongly dependent on the orientation and distribution of the GalNAc units.

A multi-ligation strategy for the synthesis of heterofunctionalized glycosylated scaffolds.

Well-defined heterofunctionalized glycosylated scaffolds with unprecedented molecular combinations have been prepared using up to five different bioorthogonal ligations. This approach opens up chemical access to a diversity of biomolecular structures with high biological potential.

Multivalent glycocyclopeptides: toward nano-sized glycostructures.

Cyclopeptides have recently emerged as attractive molecular scaffolds for the multivalent presentation of carbohydrates in a well-defined constrained spatial orientation. This mini-review describes the last advances on the synthesis and the biological applications of these particular structures, going from low molecular weight glycoclusters to fully synthetic nano-sized glycodendrimers.

A cancer therapeutic vaccine based on clustered Tn-antigen mimetics induces strong antibody-mediated protective immunity.

Tumor-associated carbohydrate antigens (TACAs) are key components of cancer vaccines. A variety of vaccines based on native TACAs such as α-Tn have shown immunogenicity and protection in preclinical animal studies, however, their weak immunogenicity, low in vivo instability, and poor bioavailability, have discouraged their further evaluations in clinical studies. A new improved vaccine prototype is reported. It is composed of four clustered Tn-antigen mimetics and a immunogenic peptide epitope that are conjugated to a cyclopeptide carrier. The immunization of mice with this vaccine 1) was safe, 2) induced a strong and long-lasting Tn-specific response with IgM/IgG antibodies able to recognize native carbohydrate antigens; 3) produced high titers of IgG1, IgG2a, and IgG3 antibodies; and 4) produced a significant antibody-dependent regression of tumors and conferred protection. Altogether, these findings pave the way for the clinical development of safe and effective therapeutic vaccines against Tn-expressing cancers.

Orthogonal dual thiol-chloroacetyl and thiol-ene couplings for the sequential one-pot assembly of heteroglycoclusters.

We describe the first one-pot orthogonal strategy to prepare well-defined cyclopeptide-based heteroglycoclusters (hGCs) from glycosyl thiols. Both thiol-chloroactetyl coupling (TCC) and thiol-ene coupling (TEC) have been used to decorate cyclopeptides regioselectively with diverse combination of sugars. We demonstrate that the reaction sequence starting with TCC can be performed one-pot whereas the reverse sequence requires a purification step after the TEC reaction. The versatility of this orthogonal strategy has been demonstrated through the synthesis of diverse hGCs displaying alternating binary combinations of α-D-Man or ß-D-GlcNAc, thus providing rapid access to attractive heteroglycosylated platforms for diverse biological applications.

Expanding the scope of oxime ligation: facile synthesis of large cyclopeptide-based glycodendrimers.

A new series of 64-valent glycodendrimers was prepared in excellent yield and purity using a divergent and iterative strategy based on oxime ligation. Enzyme-linked lectin assays revealed the first nanomolar inhibitor for the fucose-specific lectin isolated from Ulex europaeus.

High affinity glycodendrimers for the lectin LecB from Pseudomonas aeruginosa.

Following an iterative oxime ligation procedure, cyclopeptide (R) and lysine-based dendron (D) were combined in all possible arrangements and successively functionalized with α-fucose and ß-fucose to provide a new series of hexadecavalent glycosylated scaffolds (i.e., scaffolds RD16, RR16, DR16, and DD16). These compounds and smaller analogs (tetra- and hexavalent scaffolds R4 and R6) were used to evaluate the influence of the ligand valency and architecture, and of the anomer configuration in the binding to the αFuc-specific lectin LecB from Pseudomonas aeruginosa . Competitive enzyme-linked lectin assays (ELLA) revealed that only the RD16 architecture displaying αFuc (9A) reaches strong binding improvement (IC50 of 0.6 nM) over αMeFuc, and increases the α-selectivity of LecB. Dissociation constant of 28 nM was measured by isothermal titration micorcalorimetry (ITC) for 9A, which represents the highest affinity ligand ever reported for LecB. ITC and molecular modeling suggested that the high affinity observed might be due to an aggregative chelate binding involving four sugar head groups and two lectins. Interestingly, unprecedented binding effects were observed with ß-fucosylated conjugates, albeit being less active than the corresponding ligands of the αFuc series. In particular, the more flexible lysine-based dendritic structures (15B and 18B) showed a slight inhibitory enhancement in comparison with those having cyclopeptide core.

Synthesis of heteroglycoclusters by using orthogonal chemoselective ligations.

Synthetic heteroglycoclusters are being subjected to increasing interest due to their potential to serve as selective ligands for carbohydrate-binding proteins. In this paper, we describe an expedient strategy to prepare cyclopeptides displaying well-defined distributions and combinations of carbohydrates. By using both oxime ligation and copper(I)-catalyzed alkyne-azide cycloaddition, two series of compounds bearing binary combinations of αMan, αFuc or ßLac in an overall tetravalent presentation, and either 2:2 or 3:1 relative proportions, have been prepared.