Amide-sialoside protein conjugates as neomucin bioshields prevent influenza virus infection.

We report the preparation of multivalent amide-sialoside-decorated human serum albumin (HSA) and bovine serum albumin (BSA) as mimics of natural mucin and bioshields against influenza virus infection. Free sialic acid with an amine on C-2 was covalently attached to the protein scaffolds using di-(N-succinimidyl) adipate. Dynamic light scattering (DLS) showed that the synthetic neomucins were able to act as bioshields and aggregate the influenza virion particles. The dissociation constants (KD) of the interactions between the prepared glycoconjugates and three different viral strains were measured by isothermal titration calorimetry (ITC) indicating the multivalent presentation of sialyl ligands on the HSA and BSA backbones can dramatically enhance the adsorbent capability compared to the corresponding monomeric sialoside. Hemagglutinin inhibition (HAI) and neuraminidase inhibition (NAI) assays showed that the glycoconjugates acted as moderate HA and NA inhibitors, thus impeding viral infection. Moreover, the different binding affinities of the glycoproteins to HA and NA proteins from different influenza viruses demonstrated the importance of HA/NA balance in viral replication and evolution. These findings provide a foundation for the development of antiviral drugs and viral adsorbent materials based on mimicking the structure of mucin.

Direct immobilization of sugar probes on bovine serum albumin-coated gold substrate for the development of glycan biosensors.

Glycan biosensors based on surface plasmon resonance (SPR) spectroscopy have attracted a great deal of interest due to their potential applications in numerous biological and biomedical fields. Controlled immobilization of sugar probes on a gold substrate is believed to be critical for the performance of these SPR biosensors. In this regard, herein the authors report a direct coupling of mannose probes with bovine serum albumin (BSA) layer on the gold substrate via a squaric acid-mediated reaction under mild conditions, in which the BSA layer provides not only reactive amine groups but also a nonfouling surface property. SPR measurements show that the resultant biosensor with an appropriate amount of mannose probes exhibits high affinity to its corresponding lectin (i.e., concanavalin A) and at the same time could resist nonspecific adsorption of other lectins. The limit of detection of the current SPR biosensor is 1.9 nM. Thus, the squaric acid-mediated immobilization strategy appears to be effective and useful for the fabrication of bioanalytical devices.

BSA-Sugar Conjugates as Ideal Building Blocks for SPR-Based Glycan Biosensors.

Controlled immobilization of sugar probes is of key importance for the development of glycan biosensors. To this end, a series of BSA-sugar conjugates with different numbers of mannose units are prepared via the squaric acid-mediated coupling reaction. The conjugates can absorb directly on gold substrate without any derivation reactions, thus providing a simple and effective method for the construction of SPR-based glycan biosensors. SPR measurements show that the BSA-mannose conjugate with 11 mannoses exhibit the highest affinity to the lectin concanavalin A with a limit of detection of ca. 1.8 nM. Regeneration and specificity of the obtained glycan biosensors are also investigated.

Multivalent S-sialoside protein conjugates block influenza hemagglutinin and neuraminidase.

A new class of S-sialoside Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) conjugates were prepared to enhance the binding affinity to hemagglutinin (HA) and neuraminidase (NA). The valency of glycoconjugates was controlled by the reaction ratio of the S-sialoside monomer and protein. Hemagglutination inhibition assay showed that these synthetic glycoproteins have higher affinity to HA than the small clusters of sialosides with lower valency, due to multivalent effect and optimized three dimensional presentation of sialosides on the protein platform. The results of fluorescent NA inhibition assay showed that some of the conjugates have moderate NA inhibitory activity, in comparison to the monomer and low valent conjugates with weak or none inhibitory activity. These synthetic sialylated proteins were not cytotoxic with concentrations up to 100 µM, since the sialylation did not change the secondary structure of protein. This new kind of conjugates can be used as lead compounds for antiviral drug design and the construction of pseudo sialoside-protein conjugates library to investigate the carbohydrate-HA/NA recognition process and a platform for the influenza virus capturing.