Sarah-Jane Richards

Probing Bacterial-Toxin Inhibition with Synthetic Glycopolymers Prepared by Tandem Post-Polymerization Modification: Role of Linker Length and Carbohydrate Density†

Probing the depths: A tandem post-polymerization modification strategy was used to systematically probe the multivalent inhibition of a bacterial toxin as a function of linker length (see scheme), carbohydrate density, and glycopolymer chain length. Guided by structural-biology information, the binding-pocket depth of the toxin was probed and used as a means to specifically improve inhibition of the toxin by the glycopolymer.

A detailed study on understanding glycopolymer library and Con A interactions

Synthetic glycopolymers are important natural oligosaccharides mimics for many biological applications. To develop glycopolymeric drugs and therapeutic agents, factors that control the receptor-ligand interaction need to be investigated. A library of well-defined glycopolymers has been prepared by the combination of copper mediated living radical polymerization and CuAAC click reaction via post-functionalization of alkyne-containing precursor polymers with different sugar azides. Employing Concanavalin A as the model receptor, we explored the influence of the nature and densities of different sugars residues (mannose, galactose, and glucose) on the stoichiometry of the cluster, the rate of the cluster formation, the inhibitory potency of the glycopolymers, and the stability of the turbidity through quantitative precipitation assays, turbidimetry assays, inhibitory potency assays, and reversal aggregation assays. The diversities of binding properties contributed by different clustering parameters will make it possible to define the structures of the multivalent ligands and densities of binding epitopes tailor-made for specific functions in the lectin-ligand interaction.

Glycopolymers with secondary binding motifs mimic glycan branching and display bacterial lectin selectivity in addition to affinity

The application of synthetic glycopolymers to anti-adhesive therapies has so far been limited by their lack of lectin specificity. Here we employ a macromolecular engineering approach to mimic glycan architecture. A new, 3-step tandem post-polymerisation methodology was developed which afforded precise control over both chain length and carbohydrate (galactose)-polymer backbone linker distance. This route also allowed a secondary binding (branched) motif to be introduced onto the linker, increasing specificity and affinity towards bacterial toxins without the need for extensive carbohydrate or organic chemistry. Sequential variation of this motif was found to dramatically alter both the affinity and the specificity of the glycopolymers towards two lectins, CTx and PNA, by up to 20-fold either via direct binding, or increased steric constraints. Using this method, a glycopolymer that showed increased specificity towards CTx was identified.

Glycopolymer-coated gold nanorods synthesised by a one pot copper(0) catalyzed tandem RAFT/click reaction

Side-chain functionalized glycopolymers have been successfully prepared in a well-controlled manner via a copper(0)-catalyzed one-pot reaction combining living radical polymerisation and ‘click chemistry’. The polymerisation was confirmed by the first-order kinetic plots, the linear relationships between molecular weights and the monomer conversions while keeping relatively narrow polydispersity (<1.33). The click conversion and polymerisation conversion versus reaction time plots for a Cu(0)-catalyzed one-pot reaction elucidate that the rates of click reaction are significantly faster than the polymerisation rates. Modified gold nanorods (GNRs) with glycopolymeric coatings were prepared through the interaction of Au–S bonds and the glycopolymer substituted GNRs showed strong, specific molecular recognition abilities with lectin (PNA).

Poly(azlactone)s: versatile scaffolds for tandem post-polymerisation modification and glycopolymer synthesis

The synthesis of polymers with latent reactivity suitable for ‘click’ type modifications in a tandem post-polymerisation modification process starting with poly(azlactone) precursors is investigated. Poly(azlactones), obtained by copper(I) mediated radical polymerisation, were functionalised in a one-pot process with amines bearing functional groups which are incompatible with controlled radical polymerisation: alkynes, alkenes, furfuryl and phenol. The reaction is quantitative and 100% atom efficient presenting an efficient route to clickable scaffolds without the need for protecting group chemistry. Additionally, the poly(azlactones) were exploited to obtain synthetic glycopolymers. The ring opening procedure introduces a 5-atom spacer between glycan and backbone, which provides improved access to carbohydrate-binding proteins with deep binding pockets, such as the cholera toxin, for anti-adhesion applications.

Gold nanoparticle-linked analysis of carbohydrate–protein interactions, and polymeric inhibitors, using unlabelled proteins; easy measurements using a ‘simple’ digital camera

Traditional methods of measuring the affinity of lectins (or other carbohydrate-binding proteins) to their target carbohydrate ligand rely on the use of chemically/recombinantly modified proteins in sorbent assays, microarrays or the use of expensive label-free methods such as surface plasmon resonance spectrometry. In this work we exploit the extremely high extinction coefficient (i.e. colour) of gold nanoparticles as resolving agents in sorbent assays. The anionic nanoparticles adhere strongly to immobilized proteins, but not to the carbohydrate-surfaces allowing investigation of protein binding and screening of novel multivalent inhibitors. Furthermore, the use of a simple digital camera (or mobile phone) to obtain the data is shown, providing a simple ultra-low cost route to the detection of unmodified, carbohydrate-binding proteins.

Evaluation of the Antimicrobial Activity of Cationic Polymers against Mycobacteria: Toward Antitubercular Macromolecules

Antimicrobial resistance is a global healthcare problem with a dwindling arsenal of usable drugs. Tuberculosis, caused by Mycobacterium tuberculosis, requires long-term combination therapy and multi- and totally drug resistant strains have emerged. This study reports the antibacterial activity of cationic polymers against mycobacteria, which are distinguished from other Gram-positive bacteria by their unique cell wall comprising a covalently linked mycolic acid–arabinogalactan–peptidoglycan complex (mAGP), interspersed with additional complex lipids which helps them persist in their host. The present study finds that poly(dimethylaminoethyl methacrylate) has particularly potent antimycobacterial activity and high selectivity over two Gram-negative strains. Removal of the backbone methyl group (poly(dimethylaminoethyl acrylate)) decreased antimycobacterial activity, and poly(aminoethyl methacrylate) also had no activity against mycobacteria. Hemolysis assays revealed poly(dimethylaminoethyl methacrylate) did not disrupt red blood cell membranes. Interestingly, poly(dimethylaminoethyl methacrylate) was not found to permeabilize mycobacterial membranes, as judged by dye exclusion assays, suggesting the mode of action is not simple membrane disruption, supported by electron microscopy analysis. These results demonstrate that synthetic polycations, with the correctly tuned structure are useful tools against mycobacterial infections, for which new drugs are urgently required.

Multivalent Antimicrobial Polymer Nanoparticles Target Mycobacteria and Gram-Negative Bacteria by Distinct Mechanisms

Because of the emergence of antimicrobial resistance to traditional small-molecule drugs, cationic antimicrobial polymers are appealing targets. Mycobacterium tuberculosis is a particular problem, with multi- and total drug resistance spreading and more than a billion latent infections globally. This study reports nanoparticles bearing variable densities of poly(dimethylaminoethyl methacrylate) and the unexpected and distinct mechanisms of action this multivalent presentation imparts against Escherichia coli versus Mycobacterium smegmatis (model of M. tuberculosis), leading to killing or growth inhibition, respectively. A convergent “grafting to” synthetic strategy was used to assemble a 50-member nanoparticle library, and using a high-throughput screen identified that only the smallest (2 nm) particles were stable in both saline and complex cell media. Compared with the linear polymers, the nanoparticles displayed two- and eight-fold enhancements in antimicrobial activity against M. smegmatis and E. coli, respectively. Mechanistic studies demonstrated that the antimicrobial particles were bactericidal against E. coli due to rapid disruption of the cell membranes. Conversely, against M. smegmatis the particles did not lyse the cell membrane but rather had a bacteriostatic effect. These results demonstrate that to develop new polymeric antituberculars the widely assumed, broad spectrum, membrane-disrupting mechanism of polycations must be re-evaluated. It is clear that synthetic nanomaterials can engage in more complex interactions with mycobacteria, which we hypothesize is due to the unique cell envelope at the surface of these bacteria.

Multivalent Glycopolymer-Coated Gold Nanoparticles.

Glycosylated noble metal nanoparticles are a useful tool for probing biological binding events due to their aggregation-induced color changes, particularly for lectins that have multiple binding sites. To overcome the challenges of colloidal instability, which leads to false-positive results, it is essential to add polymeric coatings to these particles. Here we describe a versatile, and reliable, approach to enable coating of gold nanoparticles using well-defined polymers, with carbohydrate end groups. This produces multivalent nanoparticles that are both colloidally stable, but still retain their rapid colorimetric responses to lectin binding.

Photochemical “in-air” combinatorial discovery of antimicrobial co-polymers

Abstract There is an urgent need to identify new, non‐traditional antimicrobials. The discovery of new polymeric antimicrobials is limited by current low‐throughput synthetic tools, which means that limited chemical space has been explored. Herein, we employ photochemical “in‐air” reversible addition–fragmentation chain‐transfer (RAFT) polymerization with microwell plates, using liquid‐handling robots to assemble large libraries of cationic polymers, without the need for degassing or purification steps, facilitating transfer to screening. Several lead polymers were identified including a co‐polymer with propylene glycol side chains with significantly enhanced antimicrobial activity and increased therapeutic window. Mechanistic studies showed that this polymer was bacteriostatic, and surprisingly did not lyse the cell membranes, implying an alternative mode of action. This versatile method using simple robotics will help to develop new biomaterials with emergent properties.