Phenyl 2,3,4-tri-O-benzyl-1-thio-α-d-mannopyran­oside monohydrate

In the title compound, C33H34O5S·H2O, the mannopyran­oside ring adopts a chair conformation with the 2-α-thio­phenyl group occupying an axial position. One of the pendant benzyl groups is disordered over two sets of sites in a 0.5:0.5 ratio. In the crystal, the water mol­ecule makes two O—H⋯O hydrogen bonds to an adjacent sugar mol­ecule with the O atoms of the primary alcohol and ether groups acting as acceptors. At the same time, the OH group of the sugar makes a hydrogen bond to a water mol­ecule

Synthetic Receptors for the High-Affinity Recognition of O-GlcNAc Derivatives

The combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water-soluble carbohydrate receptors ("synthetic lectins"). Both systems show outstanding affinities for derivatives of N-acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside GlcNAc-β-OMe with Ka ≈20,000 m(-1), whereas the other one binds an O-GlcNAcylated peptide with Ka ≈70,000 m(-1). These values substantially exceed those usually measured for GlcNAc-binding lectins. Slow exchange on the NMR timescale enabled structural determinations for several complexes. As expected, the carbohydrate units are sandwiched between the pyrenes, with the alkoxy and NHAc groups emerging at the sides. The high affinity of the GlcNAcyl-peptide complex can be explained by extra-cavity interactions, raising the possibility of a family of complementary receptors for O-GlcNAc in different contexts

Site-Specific Multi-Functionalization of the Carrier Protein CRM197 by Disulfide Rebridging for Conjugate Vaccine Development

Conjugation of an antigen to a carrier protein is widely used for vaccine development. To develop the next generation of conjugate vaccines, we describe here a method for the controlled multi-functionalization of the widely employed carrier protein CRM197 with a carbohydrate-based antigen and an immune potentiator. The approach is based on the selective reduction of one of the disulfides of CRM197 followed by disulfide rebridging employing an appropriately functionalized dibromopyridazinedione. Efficient protein modification required that the reduction and functionalization with a dibromopyridazinedione was performed as a one-step procedure with control over the reaction temperature. Furthermore, ligations were most successful when dibromopyridazinediones were employed having a functional entity such as a TLR7/8 agonist and a cyclooctyne for further modification. Site-specific conjugation avoids modification of T-epitopes of the carrier protein and covalent attachment of an immune potentiator will ensure that cytokines are produced where the vaccine interacts with relevant immune cells resulting in efficient immune potentiation

Receptor Density-Dependent Motility of Influenza Virus Particles on Surface Gradients

Influenza viruses can move across the surface of host cells while interacting with their glycocalyx. This motility may assist in finding or forming locations for cell entry and thereby promote cellular uptake. Because the binding to and cleavage of cell surface receptors forms the driving force for the process, the surface-bound motility of influenza is expected to be dependent on the receptor density. Surface gradients with gradually varying receptor densities are thus a valuable tool to study binding and motility processes of influenza and can function as a mimic for local receptor density variations at the glycocalyx that may steer the directionality of a virus particle in finding the proper site of uptake. We have tracked individual influenza virus particles moving over surfaces with receptor density gradients. We analyzed the extracted virus tracks first at a general level to verify neuraminidase activity and subsequently with increasing detail to quantify the receptor density-dependent behavior on the level of individual virus particles. While a directional bias was not observed, most likely due to limitations of the steepness of the surface gradient, the surface mobility and the probability of sticking were found to be significantly dependent on receptor density. A combination of high surface mobility and high dissociation probability of influenza was observed at low receptor densities, while the opposite occurred at higher receptor densities. These properties result in an effective mechanism for finding high-receptor density patches, which are believed to be a key feature of potential locations for cell entry

Hierarchical Multivalent Effects Control Influenza Host Specificity

Understanding how emerging influenza viruses recognize host cells is critical in evaluating their zoonotic potential, pathogenicity, and transmissibility between humans. The surface of the influenza virus is covered with hemagglutinin (HA) proteins that can form multiple interactions with sialic acid-terminated glycans on the host cell surface. This multivalent binding affects the selectivity of the virus in ways that cannot be predicted from the individual receptor-ligand interactions alone. Here, we show that the intrinsic structural and energetic differences between the interactions of avian- or human-type receptors with influenza HA translate from individual site affinity and orientation through receptor length and density on the surface into virus avidity and specificity. We introduce a method to measure virus avidity using receptor density gradients. We found that influenza viruses attached stably to a surface at receptor densities that correspond to a minimum number of approximately 8 HA-glycan interactions, but more interactions were required if the receptors were short and human-type. Thus, the avidity and specificity of influenza viruses for a host cell depend not on the sialic acid linkage alone but on a combination of linkage and the length and density of receptors on the cell surface. Our findings suggest that threshold receptor densities play a key role in virus tropism, which is a predicting factor for both their virulence and zoonotic potential.Fil: Overeem, Nico J.. University of Twente; Países BajosFil: Hamming, P. H. Erik. University of Twente; Países BajosFil: Grant, Oliver C.. University of Georgia; Estados UnidosFil: Di Iorio, Daniele. University of Twente; Países BajosFil: Tieke, Malte. Utrecht University; Países BajosFil: Bertolino, María Candelaria. University of Twente; Países Bajos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Li, Zeshi. Utrecht University; Países BajosFil: Vos, Gaël. Utrecht University; Países BajosFil: de Vries, Robert P.. Utrecht University; Países BajosFil: Woods, Robert J.. University of Georgia; Estados UnidosFil: Tito, Nicholas B.. Electric Ant Laboratory; Países BajosFil: Boons, Geert-Jan P. H.. Utrecht University; Países BajosFil: van der Vries, Erhard. Utrecht University; Países BajosFil: Huskens, Jurriaan. University of Twente; Países Bajo

A novel knowledge repository to support industrial symbiosis

The development of tools and methods supporting the identification of Industrial Symbiosis opportunities is of utmost importance to unlock its full potential. Knowledge repositories have proven to be powerful tools in this sense, but often fail mainly due to poor contextualization of information and lack of general applicability (out of the boundaries of specific areas or projects). In this work, a novel approach to the design of knowledge repositories for Industrial Symbiosis is presented, based on the inclusion and categorization of tacit knowledge as well as on the combination of mimicking and input-output matching approaches. The results of a first usability test of the proposed tool are also illustrated

Functionality of the putative surface glycoproteins of the Wuhan spiny eel influenza virus

A panel of influenza virus-like sequences were recently documented in fish and amphibians. Of these, the Wuhan spiny eel influenza virus (WSEIV) was found to phylogenetically cluster with influenza B viruses as a sister clade. Influenza B viruses have been documented to circulate only in humans, with certain virus isolates found in harbor seals. It is therefore interesting that a similar virus was potentially found in fish. Here we characterize the putative hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins of the WSEIV. Functionally, we show that the WSEIV NA-like protein has sialidase activity comparable to B/Malaysia/2506/2004 influenza B virus NA, making it a bona fide neuraminidase that is sensitive to NA inhibitors. We tested the functionality of the HA by addressing the receptor specificity, stability, preferential airway protease cleavage, and fusogenicity. We show highly specific binding to monosialic ganglioside 2 (GM2) and fusogenicity at a range of different pH conditions. In addition, we found limited antigenic conservation of the WSEIV HA and NA relative to the B/Malaysia/2506/2004 virus HA and NA. In summary, we perform a functional and antigenic characterization of the glycoproteins of WSEIV to assess if it is indeed a bona fide influenza virus potentially circulating in ray-finned fish

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

Recent human H3N2 influenza A viruses have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by (humanized) Madin-Darby Canine Kidney cells, which are commonly employed to propagate influenza A virus, resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase 1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAcs), would result in improved A/H3N2 propagation. Stable overexpression of β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase 1 in Madin-Darby Canine Kidney and "humanized" Madin-Darby Canine Kidney cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the β-1,3-N-acetylglucosaminyltransferase and/or β-1,4-galactosyltransferase 1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on "humanized" Madin-Darby Canine Kidney-β-1,3-N-acetylglucosaminyltransferase cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 influenza A viruses require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.</p

Contemporary human H3N2 influenza a viruses require a low threshold of suitable glycan receptors for efficient infection

Recent human H3N2 influenza A viruses (IAV) have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by (humanized) Madin-Darby Canine Kidney cells (MDCK and hCK) which are commonly employed to propagate IAV, resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases B3GNT2 and B4GALT1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAc), would result in improved A/H3N2 propagation. Stable overexpression of B3GNT2 and B4GALT1 in MDCK and hCK cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the B3GNT2 and/or B4GALT1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on hCK-B3GNT2 cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 IAVs require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency

Well-Defined Heparin Mimetics Can Inhibit Binding of the Trimeric Spike of SARS-CoV-2 in a Length-Dependent Manner

The emergence of new SARS-CoV-2 variants and the dangers of long-covid necessitate the development of broad-acting therapeutics that can reduce viral burden. SARS-CoV-2 employs heparan sulfate (HS) as an initial cellular attachment factor, and therefore, there is interest in developing heparin as a therapeutic for SARS-CoV-2. Its use is, however, complicated by structural heterogeneity and the risk of causing bleeding and thrombocytopenia. Here, we describe the preparation of well-defined heparin mimetics by a controlled head-to-tail assembly of HS oligosaccharides having an alkyne or azide moiety by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Alkyne- and azide-containing sulfated oligosaccharides were prepared from a common precursor by modifying an anomeric linker with 4-pentynoic acid and by enzymatic extension with an N-acetyl-glucosamine having an azide moiety at C-6 (GlcNAc6N 3), respectively, followed by CuAAC. The process of enzymatic extension with GlcNAc6N 3 followed by CuAAC with the desired alkyne-containing oligosaccharides could be repeated to give compounds composed of 20 and 27 monosaccharides, respectively. The heparin mimetics could inhibit the binding of the SARS-CoV-2 spike or RBD to immobilized heparin or to Vero E6 cells. The inhibitory potency increased with increasing chain length, and a compound composed of four sulfated hexasaccharides linked by triazoles had a similar potency as unfractionated heparin. Sequence analysis and HS microarray binding studies with a wide range of RBDs of variants of concern indicate that they have maintained HS-binding capabilities and selectivities. The heparin mimetics exhibit no or reduced binding to antithrombin-III and platelet factor 4, respectively, which are associated with side effects