Controlling the Surface Functionalization of Ultrasmall Gold Nanoparticles by Sequence-Defined Macromolecules.

Ultrasmall gold nanoparticles (diameter about 2 nm) were surface-functionalized with cysteine-carrying precision macromolecules. These consisted of sequence-defined oligo(amidoamine)s (OAAs) with either two or six cysteine molecules for binding to the gold surface and either with or without a PEG chain (3400 Da). They were characterized by 1 H NMR spectroscopy, 1 H NMR diffusion-ordered spectroscopy (DOSY), small-angle X-ray scattering (SAXS), and high-resolution transmission electron microscopy. The number of precision macromolecules per nanoparticle was determined after fluorescent labeling by UV spectroscopy and also by quantitative 1 H NMR spectroscopy. Each nanoparticle carried between 40 and 100 OAA ligands, depending on the number of cysteine units per OAA. The footprint of each ligand was about 0.074 nm2 per cysteine molecule. OAAs are well suited to stabilize ultrasmall gold nanoparticles by selective surface conjugation and can be used to selectively cover their surface. The presence of the PEG chain considerably increased the hydrodynamic diameter of both dissolved macromolecules and macromolecule-conjugated gold nanoparticles.

Correction: Sophia, B.; et al. Presenting Precision Glycomacromolecules on Gold Nanoparticles for Increased Lectin Binding. Polymers 2017, 9, 716.

In the original version of this Article, an error occurred in the "Results and Discussion" section, under the subheading "Preparation and Characterization of Glyco-AuNPs"[...].

Sequence-Defined Introduction of Hydrophobic Motifs and Effects in Lectin Binding of Precision Glycomacromolecules.

This study investigates the influence of an increasingly hydrophobic backbone of multivalent glycomimetics based on sequence-defined oligo(amidoamines) on their resulting affinity toward bacterial lectins. Glycomacromolecules are obtained by stepwise assembly of tailor-made building blocks on solid support, using both hydrophobic aliphatic and aromatic building blocks to enable a gradual change in hydrophobicity of the backbone. Their binding behavior toward model lectin Concanavalin A (ConA) is evaluated using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) showing higher affinities for glycomacromolecules with higher content of hydrophobic and aromatic moieties in the backbone. Finally, glycomacromolecules are tested in a bacterial adhesion inhibition study against Escherichia coli where more hydrophobic backbones yield higher inhibitory potentials most likely due to additional secondary interactions with hydrophobic regions of the protein receptor as well as a change in conformation exposing carbohydrate ligands for increased binding. Overall, the results highlight the influence and thereby importance of the polymer backbone itself on the resulting properties of polymeric biomimetics.

Multivalent Binding of Precision Glycooligomers on Soft Glycocalyx Mimicking Hydrogels.

We present a synthetic approach toward soft, glycooligomer-functionalized microgel particles mimicking carbohydrate presenting cell surfaces and analyze their specific binding to a model lectin (Concanavalin A, ConA). Focusing on multivalent presentation, a series of sequence-controlled glycooligomers with varying spacing and number of mannose units was synthesized and analyzed for the resulting glycooligomer-ConA affinity. Both direct binding and inhibition studies show a higher affinity with increasing the number of sugar moieties, but they level off for higher valent systems, indicating steric hindrance. Furthermore, the results suggest that increasing the scaffold length tends to decrease binding due to entropic repulsion, which could be compensated by larger scaffolds able to address multiple ConA binding sites. These findings were consistent in all assays (adhesion, fluorescence, and ITC) regardless of binding partner immobilization, demonstrating that flexible ligands exert similar binding modes in solution and when attached to polymer networks, which is relevant for designing glyco-functionalized materials.

Linear Precision Glycomacromolecules with Varying Interligand Spacing and Linker Functionalities Binding to Concanavalin A and the Bacterial Lectin FimH.

A series of precision glycomacromolecules is prepared following previously established solid phase synthesis allowing for controlled variations of interligand spacing and the overall number of carbohydrate ligands. In addition, now also different linkers are installed between the carbohydrate ligand and the macromolecular scaffold. The lectin binding behavior of these glycomacromolecules is then evaluated in isothermal titration calorimetry (ITC) and kinITC experiments using the lectin Concanavalin A (Con A) in its dimeric and tetrameric form. The results indicate that both sterical and statistical effects impact lectin binding of precision glycomacromolecules. Moreover, ITC results show that highest affinity toward Con A can be achieved with an ethyl phenyl linker, which parallels earlier findings with the bacterial lectin FimH. In this way, a first set of glycomacromolecule structures is selected for testing in a bacterial adhesion-inhibition study. Here, the findings point to a one-sugar binding mode mainly affected by sterical restraints of the nonbinding parts of the respective glycomacromolecule.

Sequence-Controlled Glycopolymers via Step-Growth Polymerization of Precision Glycomacromolecules for Lectin Receptor Clustering.

A versatile approach for the synthesis of sequence-controlled multiblock copolymers, using a combination of solid phase synthesis and step-growth polymerization by photoinduced thiol-ene coupling (TEC) is presented. Following this strategy, a series of sequence-controlled glycopolymers is derived from the polymerization of a hydrophilic spacer macromonomer and different glycomacromonomers bearing between one to five α-d-Mannose (Man) ligands. Through the solid phase assembly of the macromonomers, the number and positioning of spacer and sugar moieties is controlled and translates into the sequence-control of the final polymer. A maximum M̅n of 16 kDa, corresponding to a X̅n of 10, for the applied macromonomers is accessible with optimized polymerization conditions. The binding behavior of the resulting multiblock glycopolymers toward the model lectin Concanavalin A (ConA) is studied via turbidity assays and surface plasmon resonance (SPR) measurements, comparing the ability of precision glycomacromolecules and glycopolymers to bind to and cross-link ConA in dependence of the number of sugar moieties and overall molecular weight. The results show that there is a clear correlation between number of Man ligands and Con A binding and clustering, whereas the length of the glycooligomer- or polymer backbone seems to have no effect.

Presenting Precision Glycomacromolecules on Gold Nanoparticles for Increased Lectin Binding.

Glyco-functionalized gold nanoparticles have great potential as biosensors and as inhibitors due to their increased binding to carbohydrate-recognizing receptors such as the lectins. Here we apply previously developed solid phase polymer synthesis to obtain a series of precision glycomacromolecules that allows for straightforward variation of their chemical structure as well as functionalization of gold nanoparticles by ligand exchange. A novel building block is introduced allowing for the change of spacer building blocks within the macromolecular scaffold going from an ethylene glycol unit to an aliphatic spacer. Furthermore, the valency and overall length of the glycomacromolecule is varied. All glyco-functionalized gold nanoparticles show high degree of functionalization along with high stability in buffer solution. Therefore, a series of measurements applying UV-Vis spectroscopy, dynamic light scattering (DLS) and surface plasmon resonance (SPR) were performed studying the aggregation behavior of the glyco-functionalized gold nanoparticles in presence of model lectin Concanavalin A. While the multivalent presentation of glycomacromolecules on gold nanoparticles (AuNPs) showed a strong increase in binding compared to the free ligands, we also observed an influence of the chemical structure of the ligand such as its valency or hydrophobicity on the resulting lectin interactions. The straightforward variation of the chemical structure of the precision glycomacromolecule thus gives access to tailor-made glyco-gold nanoparticles (glyco-AuNPs) and fine-tuning of their lectin binding properties.

Development and optimization of a competitive binding assay for the galactophilic low affinity lectin LecA from Pseudomonas aeruginosa.

Infections with the Gram-negative bacterium Pseudomonas aeruginosa result in a high mortality among immunocompromised patients and those with cystic fibrosis. The pathogen can switch from planktonic life to biofilms, and thereby shields itself against antibiotic treatment and host immune defense to establish chronic infections. The bacterial protein LecA, a C-type lectin, is a virulence factor and an integral component for biofilm formation. Inhibition of LecA with its carbohydrate ligands results in reduced biofilm mass, a potential Achilles heel for treatment. Here, we report the development and optimization of a fluorescence polarization-based competitive binding assay with LecA for application in screening of potential inhibitors. As a consequence of the low affinity of d-galactose for LecA, the fluorescent ligand was optimized to reduce protein consumption in the assay. The assay was validated using a set of known inhibitors of LecA and IC50 values in good agreement with the known Kd values were obtained. Finally, we employed the optimized assay to screen sets of synthetic thio-galactosides and natural blood group antigens and report their structure-activity relationship. In addition, we evaluated a multivalent fluorescent assay probe for LecA and report its applicability in an inhibition assay.