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Flexible multivalent 3D nanosystems that can deform and adapt onto the virus surface via specific ligand–receptor multivalent interactions can efficiently block virus adhesion onto the cell. We here report on the synthesis of a 250 nm sized flexible sialylated nanogel that adapts onto the influenza A virus (IAV) surface via multivalent binding of its sialic acid (SA) residues with hemagglutinin spike proteins on the virus surface. We could demonstrate that the high flexibility of sialylated nanogel improves IAV inhibition by 400 times as compared to a rigid sialylated nanogel in the hemagglutination inhibition assay. The flexible sialylated nanogel efficiently inhibits the influenza A/X31 (H3N2) infection with IC50 values in low picomolar concentrations and also blocks the virus entry into MDCK‐II cells., Fighting influenza: Through the combination of flexibility and multivalent binding, a sialylated nanogel is able to bind and adapt onto the influenza A virus surface. The resulting flexible nanogel efficiently blocks the virus binding onto the cell and inhibits infection at low pm concentrations.

Two sugar PEG-based amphiphilic copolymers have been synthesized by Novozym®-435-catalyzed greener solvent free transesterification reaction of diastereomeric 2,6-anhydro-3,4,5-tri-O-benzyl-glucoheptitol and 2,6-anhydro-3,4,5-tri-O-benzyl-mannoheptitol with PEG-1000 diethyl ester in 80 and 76% yields, respectively. It has been observed that the configurational change at one of the carbons of diastereomeric 2,6-anhydro-heptitols has significant bearing on the degree of polymerization and characteristics of the synthesized polymers. Thus, the study of physico-chemical characteristics of synthesized anhydroglucoheptitol- and anhydromannoheptitol-PEG copolymers revealed that their aqueous solution forms spherical micelles of hydrodynamic diameters 11.2 and 6.4 nm, respectively. Micelles derived from both the amphiphilic copolymers have the potential to solubilize the hydrophobic dye, Nile red. Furthermore, a fluorescence microscopy study revealed that Nile red encapsulated micelles of synthesized amphiphilic copolymers uniformly internalized in A549 cells and thus can be used as a cargo carrier in biological systems. The viability of A549 cells was found to be very high even at a concentration of 500 μg mL−1 of copolymer indicating that they are cyto-compatible.

The development of multivalent sialic acid-based inhibitors active against a variety of influenza A virus (IAV) strains has been hampered by high genetic and structural variability of the targeted viral hemagglutinin (HA). Here, we addressed this challenge by employing sialylated polyglycerols (PGs). Efficacy of prototypic PGs was restricted to a narrow spectrum of IAV strains. To understand this restriction, we selected IAV mutants resistant to a prototypic multivalent sialylated PG by serial passaging. Resistance mutations mapped to the receptor binding site of HA, which was accompanied by altered receptor binding profiles of mutant viruses as detected by glycan array analysis. Specifying the inhibitor functionalization to 2,6-α-sialyllactose (SL) and adjusting the linker yielded a rationally designed inhibitor covering an extended spectrum of inhibited IAV strains. These results highlight the importance of integrating virological data with chemical synthesis and structural data for the development of sialylated PGs toward broad anti-influenza compounds.

Herein, the chemical synthesis and binding analysis of functionalizable rigid and flexible core trivalent sialosides bearing oligoethylene glycol (OEG) spacers interacting with spike proteins of influenza A virus (IAV) X31 is described. Although the flexible Tris‐based trivalent sialosides achieved micromolar binding constants, a trivalent binder based on a rigid adamantane core dominated flexible tripodal compounds with micromolar binding and hemagglutination inhibition constants. Simulation studies indicated increased conformational penalties for long OEG spacers. Using a systematic approach with molecular modeling and simulations as well as biophysical analysis, these findings emphasize on the importance of the scaffold rigidity and the challenges associated with the spacer length optimization.

The mucus layer is a hydrogel network that covers mucosal surfaces of the human body. Mucus has important protective properties that are related to its unique rheological properties, which are based on mucins being the main glycoprotein constituents. Mucin macromolecules entangle with one another and form a physical network that is instrumental for many important defense functions. Mucus derived from various human or animal sources is poorly defined and thus not suitable for many application purposes. Herein, a synthetic route is fabricated to afford a library of compositionally defined mucus-inspired hydrogels (MIHs). MIHs are synthesized by thiol oxidation to render disulfide bonds between the crosslinker ethoxylated trimethylolpropane tri(3-mercaptopropionate) (THIOCURE ETTMP 1300) and the linear precursors, dithiolated linear polyglycerol (LPG(SH)2 ) or polyethylene glycol (PEG(SH)2 ) of different molecular weights. The mixing ratio of linear polymers versus crosslinker and the length of the linear polymer are varied, thus delivering a library of compositionally defined mucin-inspired constructs. Their viscoelastic properties are determined by frequency sweeps at 25 and 37 °C and compared to the corresponding behavior of native human mucus. Here, MIHs composed of a 10:1 ratio of LPG(SH)2 and ETTMP 1300 are proved to be the best comparable to human airway mucus rheology.

Inhibition of respiratory viruses is one of the most urgent topics as underlined by different pandemics in the last two decades. This impels the development of new materials for binding and incapacitation of the viruses. In this work, we have demonstrated that an optimal deployment of influenza A virus (IAV) targeting ligand sialic acid (SA) on a flexible 2D platform enables its binding and wrapping around IAV particles. A series of 2D sialylated platforms consisting graphene and polyglycerol are prepared with different degrees of SA functionalization around 10%, 30%, and 90% named as G-PG-SAL, G-PG-SAM, and G-PG-SAH, respectively. The cryo-electron tomography (Cryo-ET) analysis has proved wrapping of IAV particles by G-PG-SAM. A confocal-based colocalization assay established for these materials has offered the comparison of binding potential of sialylated and non-sialylated nanoplatforms for IAV. With this method, we have estimated the binding potential of the G-PG-SAM and G-PG-SAH sheets for IAV particles around 50 and 20 times higher than the control sheets, respectively, whereas the low functionalized G-PG-SAL have not shown any significant colocalization value. Moreover, optimized G-PG-SAM exhibits high potency to block IAV from binding with the MDCK cells.

Indocyanine green (ICG) is a near-infrared dye for wide-ranging applications, but its utility for biological studies is limited due to poor aqueous stability and concentration-dependent aggregation. Aqueous polymeric micelles/micellar aggregates of amphiphilic copolymers could be used to protect indocyanine green (ICG) by encapsulation, which is used in laser-mediated photothermal therapy (PTT) and photodynamic therapy (PDT). The sugar–PEG-based amphiphilic copolymers were synthesized using Novozym 435-catalyzed transesterification reaction under bulk conditions for encapsulation of ICG. A dye loading study revealed that micelles derived from copolymers having decanoylated and myristoylated sugar moieties encapsulate 62% and 92% of the ICG, respectively, with a multi-fold increase in its aqueous stability. Furthermore, an efficient internalization of micelles of acylated amphiphilic copolymers in aqueous medium was demonstrated by incubating cells with Nile red-encapsulated amphiphiles.

Inhibition of influenza A virus infection by multivalent sialic acid inhibitors preventing viral hemagglutinin binding to host cells of the respiratory tract is a promising strategy. However, optimal geometry and optimal ligand presentation on multivalent scaffolds for efficient inhibition both in vitro and in vivo application are still unclear. Here, by comparing linear and dendritic polyglycerol sialosides (LPGSA and dPGSA) we identified architectural requirements and optimal ligand densities for an efficient multivalent inhibitor of influenza virus A/X31/1 (H3N2). Due to its large volume, the LPGSA at optimal ligand density sterically shielded the virus significantly better than the dendritic analog. A statistical mechanics model rationalizes the relevance of ligand density, morphology, and the size of multivalent scaffolds for the potential to inhibit virus-cell binding. Optimized LPGSA inhibited virus infection at IC50 in the low nanomolar nanoparticle concentration range and also showed potent antiviral activity against two avian influenza strains A/Mallard/439/2004 (H3N2) and A/turkey/Italy/472/1999 (H7N1) post infection. In vivo application of inhibitors clearly confirmed the higher inhibition potential of linear multivalent scaffolds to prevent infection. The optimized LPGSA did not show any acute toxicity, and was much more potent than the neuraminidase inhibitor oseltamivir carboxylate in vivo. Combined application of the LPGSA and oseltamivir carboxylate revealed a synergistic inhibitory effect and successfully prevented influenza virus infection in mice.

The influenza A virus infects target cells through multivalent interactions of its major spike proteins, hemagglutinin (HA) and neuraminidase (NA), with the cellular receptor sialic acid (SA). HA is known to mediate the attachment of the virion to the cell, whereas NA enables the release of newly formed virions by cleaving SA from the cell. Because both proteins target the same receptor but have antagonistic functions, virus infection depends on a properly tuned balance of the kinetics of HA and NA activities for viral entry to and release from the host cell. Here, dynamic single-molecule force spectroscopy, based on scanning force microscopy, was employed to determine these bond-specific kinetics, characterized by the off rate k(off), rupture length x(β) and on rate k(on), as well as the related free-energy barrier ΔG and the dissociation constant K(D). Measurements were conducted using surface-immobilized HA and NA of the influenza A virus strain A/California/04/2009 and a novel, to our knowledge, synthetic SA-displaying receptor for functionalization of the force probe. Single-molecule force spectroscopy at force loading rates between 100 and 50,000 pN/s revealed most probable rupture forces of the protein-SA bond in the range of 10–100 pN. Using an extension of the widely applied Bell-Evans formalism by Friddle, De Yoreo, and co-workers, it is shown that HA features a smaller x(β), a larger k(off) and a smaller ΔG than NA. Measurements of the binding probability at increasing contact time between the scanning force microscopy force probe and the surface allow an estimation of K(D), which is found to be three times as large for HA than for NA. This suggests a stronger interaction for NA-SA than for HA-SA. The biological implications in regard to virus binding to the host cell and the release of new virions from the host cell are discussed.

Sugar-PEG-based polymers were synthesized by enzymatic copolymerization of 4-C-hydroxymethyl-1,2-O-isopropylidene-β-L-threo-pentofuranose/4-C-hydroxymethyl-1,2-O-benzylidene-β-L-threo-pentofuranose/4-C-hydroxymethyl-1,2-O-isopropylidene-3-O-pentyl-β-L-threo-pentofuranose with PEG-600 dimethyl ester using Novozyme-435 (Candida antarctica lipase immobilized on polyacrylate). Carbohydrate monomers were obtained by the multistep synthesis starting from diacetone-D-glucose and PEG-600 dimethyl ester, which was in turn obtained by the esterification of the commercially available PEG-600 diacid. Aggregation studies on the copolymers revealed that in aqueous solution those polymers bearing the hydrophobic pentyl/benzylidene moiety spontaneously self-assembled into supramolecular aggregates. The critical aggregation concentration (CAC) of polymers was determined by surface tension measurements, and the precise size of the aggregates was obtained by dynamic light scattering. The polymeric aggregates were further explored for their drug encapsulation properties in buffered aqueous solution of pH 7.4 (37 °C) using nile red as a hydrophobic model compound by means of UV/vis and fluorescence spectroscopy. There was no significant encapsulation in polymer synthesized from 4-C-hydroxymethyl-1,2-O-isopropylidene-β-L-threo-pentofuranose because this sugar monomer does not contain a big hydrophobic moiety as the pentyl or the benzylidene moiety. Nile red release study was performed at pH 5.0 and 7.4 using fluorescence spectroscopy. The release of nile red from the polymer bearing benzylidene moiety and pentyl moiety was observed with a half life of 3.4 and 2.0 h, respectively at pH 5.0, whereas no release was found at pH 7.4.

Objectives To evaluate the potential of a novel dihydropyrimidinone, ethyl 4-(4′-heptanoyloxyphenyl)-6-methyl-3,4-dihydropyrimidin-2-one-5-carboxylate (H-DHPM), as a calcium channel blocker, endowed with the ability to inhibit platelet aggregation effectively. Methods In-vitro and in-vivo studies were conducted for the determination of antiplatelet activity using adenosine diphosphate (ADP), collagen or thrombin as inducers. Calcium channel blocking activity and nitric oxide synthase (NOS) activity were monitored. Lipopolysaccharide (LPS)-mediated prothrombotic conditions were developed in rats to study the efficacy of H-DHPM to suitably modulate the inflammatory mediators such as inducible NOS (iNOS) and tissue factor. The cGMP level and endothelial NOS (eNOS) expression were checked in aortic homogenate of LPS-challenged rats pretreated with H-DHPM. The effect of H-DHPM on FeCl3-induced thrombus formation in rats was examined. Key findings The concentrations of H-DHPM required to give 50% inhibition (IC50) of in-vitro platelet aggregation induced by ADP, collagen or thrombin were 98.2 ± 2.1, 74.5 ± 2.3 and 180.7 ± 3.4 µm, respectively. H-DHPM at a dose of 52.0 ± 0.02 mg/kg (133 µmol/kg) was found to optimally inhibit ADP-induced platelet aggregation in-vivo. The level of nitric oxide was found to be up to 9 ± 0.08-fold in H-DHPM-treated platelets in-vitro and 8.2 ± 0.05-fold in H-DHPM-pretreated rat platelets in-vivo compared with control. OH-DHPM, the parent compound was found to be ineffective both in-vitro and in-vivo. H-DHPM-pretreated rats were able to resist significantly the prothrombotic changes caused by LPS by blunting the expression of iNOS, tissue factor and diminishing the increased level of cGMP to normal. H-DHPM enhanced the eNOS expression in aorta of rats treated with LPS. H-DHPM displayed synergy with antiplatelet activity of aspirin even at lower doses. H-DHPM was found to inhibit the LPS-induced platelet aggregation in younger as well as older rats. H-DHPM exhibited the ability to markedly decrease FeCl3-induced thrombus formation in rats. Conclusions H-DHPM has the attributes of a promising potent antiplatelet candidate molecule that should attract further study. H-DHPM displayed antiplatelet activity both in vivo and in vitro, which was due partially by lowering the intraplatelet calcium concentration.

Regio- and enantioselective synthesis of (S)-(+)-3-arylamino-1-chloropropan-2-ols has been achieved by the epoxide ring opening of (±)-epichlorohydrin with different aromatic amines in the presence of Candida rugosa lipase. Activities of seven model (S)-(+)-3-arylamino-1-chloropropan-2-ols, out of 10 compounds synthesized, have been evaluated for the inhibition of tumor necrosis factor-α TNF-α) induced expression of intercellular adhesion molecule-1 (ICAM-1), which is one of the factors responsible for the modulation of inflammation in biological systems; (S)-(+)-1-chloro-3-(2'-chlorophenylamino)-propan-2-ol has been found to exhibit highest activity, that is, 86% inhibition of TNF-α induced expression of ICAM-1 at a concentration of 40 μg/ml.

A series of 3-(N-alkyl-N-phenylamino)propan-2-ol derivatives were synthesized from epichlorohydrine in a multi-step strategy and were evaluated as Src kinase inhibitors. First, epoxy ring opening of epichlorohydrine was carried out in the presence of N-alkylanilines to yield 3-(N-alkyl-N-phenylamino)-1-chloro-propan-2-ol derivatives using Ca(OTf)2 as catalyst based on our previous studies [1] . Second, ring closure was performed under basic conditions to afford N-epoxymethyl N-alkylaniline derivatives. Finally, the epoxide ring opening with four different secondary amines and three nucleobases afforded the final products, i.e., a series of β-amino alcohols. All compounds were screened for their inhibitory activity against Src kinase and anticancer activity on human breast carcinoma cells, BT-20 cell line. Among all compounds, 3-N-methyl-N-phenylamino-1-(pyrrolidin-1-yl)propan-2-ol (13b) exhibited the highest inhibitory potency (IC50 = 66.1 μM) against Src kinase. Structure-activity relationship studies suggested that the incorporation of bulky groups at position 1 and N-substitution with groups larger than methyl moiety, reduced the inhibitory potency of the compound significantly. Compounds 3-(N-ethyl-N-phenylamino-)-1-(4-methylpiperazin-1-yl)propan-2-ol (14c) and 3-(N-ethyl-N-phenylamino)-1-(thymine-1-yl)propan-2-ol (17) were found to inhibit the growth of breast carcinoma cells by approximately 45–49% at concentration of 50 μM.

Multivalency is a widespread phenomenon in many biological systems, and inevitable for specific surface-surface interactions. The influenza virus provides a suitable system for studying multivalent interactions as it is covered by a densely packed layer of hemagglutinin (HA), which provides the virus high affinity to the host cells glycocalyx by multivalent interactions. To prevent such interactions and thus, infection, one promising strategy is the use of inhibitors presenting multiple HA receptors. For efficient inhibition, we investigated the optimal spatial arrangement, and amount of ligands of multivalent structures, among others multivalent polyglycerols and DNA-PNA heteroduplexes. Natural receptors such as sialic acid or peptide fragments from HA antibodies, form the basis of our multivalent constructs. In order to determine exact distances between HA binding sites, we introduced bivalent DNA-PNA heteroduplexes, presenting sialic acid with distinct distances, and thus serving as molecular rulers. Inhibition of fluorescent virions to red blood cells by heteroduplexes was assessed by flow cytometry measurement. By this strategy, we determined the spatial arrangement of sialic acid for optimal binding to virus at angstrom resolution. Microscale thermophoresis (MST) technology served as an independent and complementary approach to assess binding. Here, we detected nanomolar dissociation constants to full virus particles free in solution. In some instances, we observed multiphasic binding kinetics of multivalent structures to influenza virions. These studies are not only highly relevant for inhibition of virus infection, but also to provide new insights on the fundamentals of multivalent interactions.