The Evolution of SNAP-Tag Labels.

The conjugation of proteins with synthetic molecules can be conducted in many different ways. In this Perspective, we focus on tag-based techniques and specifically on the SNAP-tag technology. The SNAP-tag technology makes use of a fusion protein between a protein of interest and an enzyme tag that enables the actual conjugation reaction. The SNAP-tag is based on the O6-alkylguanine-DNA alkyltransferase (AGT) enzyme and is optimized to react selectively with O6-benzylguanine (BG) substrates. BG-containing dye derivatives have frequently been used to introduce a fluorescent tag to a specific protein. We believe that the site-specific conjugation of polymers to proteins can significantly benefit from the SNAP-tag technology. Especially, polymers synthesized via reversible deactivation radical polymerization allow for the facile introduction of a BG end group to enable SNAP-tag conjugation.

Chemical Identity of Poly(N-vinylpyrrolidone) End Groups Impact Shape Evolution During the Synthesis of Ag Nanostructures.

Ag nanocubes (AgNCs) are predominantly synthesized by the polyol method, where the solvent (ethylene glycol) is considered the reducing agent and poly(N-vinylpyrrolidone) (PVP) the shape-directing agent. An experimental phase diagram for the formation of Ag nanocubes as a function of PVP monomer concentration (Cm) and molecular weight (Mw) demonstrated end groups of PVP impact the final Ag product. Measured rates of the initial Ag+ reduction at different PVP Cm and Mw confirmed the reducing effect originates from end-groups. PVP with well-defined aldehyde and hydroxyl end groups lead to the formation of Ag nanocubes and nanowires respectively, indicating the faster reducing agent formed kinetically preferred nanowires. We demonstrate PVP end-groups induce initial reduction of Ag+ to form seeds followed by autocatalytic reduction of Ag+ by ethylene glycol (and not solvent oxidation products) to form Ag nanostructures. The current study enabled a quantitative description of the role of PVP in nanoparticle shape-control and demonstrates a unique opportunity to design nanostructures by combining nanoparticle synthesis with polymer design to introduce specific physicochemical properties.

Influence of DIBMA Polymer Length on Lipid Nanodisc Formation and Membrane Protein Extraction.

Polymer-based lipid nanoparticles like styrene-maleic acid lipid particles have revolutionized the study of membrane proteins. More recently, alternative polymers such as poly(diisobutylene-alt-maleic acid) (DIBMA) have been used in this field. DIBMA is commonly synthesized via conventional radical copolymerization. In order to study the influence of its chain length on lipid nanodisc formation and membrane protein extraction, we synthesized DIBMA with molar masses varying from 1.2-12 kDa via RAFT-mediated polymerization. For molar masses in the range of 3-7 kDa, the rate of lipid nanodisc formation was the highest and similar to those of poly(styrene-co-maleic acid) (SMA) and commercially available DIBMA. ZipA solubilization efficiency was significantly higher than for commercially available DIBMA and similar to SMA (circa 75%). Furthermore, RAFT-made DIBMA with a molar mass of 1.2-3.9 kDa showed a much cleaner separation on SDS-PAGE, without the smearing that is typically seen for SMA and commercially available DIBMA.

Iterative RAFT-Mediated Copolymerization of Styrene and Maleic Anhydride toward Sequence- and Length-Controlled Copolymers and Their Applications for Solubilizing Lipid Membranes.

The use of poly(styrene-co-maleic acid) (SMA) for the solubilization of lipid membranes and membrane proteins is becoming more widespread, and with this, the need increases to better understand the chemical properties of the copolymer and how these translate into membrane solubilization properties. SMA comes in many different flavors that include the ratio of styrene to maleic acid, comonomer sequence distribution, average chain length, dispersity, and potential chemical modifications. In this work, the synthesis and membrane active properties are described for 2:1 (periodic) SMA copolymers with Mw varying from ∼1.4 to 6 kDa. The copolymers were obtained via an iterative RAFT-mediated radical polymerization. Characterization of these polymers showed that they represent a well-defined series in terms of chain length and overall composition (FMAnh ∼ 0.33), but that there is heterogeneity in comonomer sequence distribution (FMSS ∼ 0.50) and some dispersity in chain length (1.1 < D < 1.6), particularly for the larger copolymers. Investigation of the interaction of these polymers with phosphatidylcholine lipid self-assemblies showed that all copolymers inserted equally effectively into lipid monolayers, independent of the copolymer length. Nonetheless, smaller polymers were more effective at solubilizing lipid bilayers into nanodiscs, possibly because longer polymers are more prone to become intertwined with each other, thereby hampering their solubilization efficiency. Nanodisc sizes were independent of the copolymer length. However, nanodiscs formed with larger copolymers were found to undergo slower lipid exchange, indicating a higher stability. The results highlight the usefulness of having well-defined copolymers for systematic studies.

Poly(N-vinylpyrrolidone) Antimalaria Conjugates of Membrane-Disruptive Peptides.

The concepts of polymer-peptide conjugation and self-assembly were applied to antimicrobial peptides (AMPs) in the development of a targeted antimalaria drug delivery construct. This study describes the synthesis of α-acetal, ω-xanthate heterotelechelic poly(N-vinylpyrrolidone) (PVP) via reversible addition-fragmentation chain transfer (RAFT)-mediated polymerization, followed by postpolymerization deprotection to yield α-aldehyde, ω-thiol heterotelechelic PVP. A specific targeting peptide, GSRSKGT, for Plasmodium falciparum-infected erythrocytes was used to sparsely decorate the α-chain ends via reductive amination while cyclic decapeptides from the tyrocidine group were conjugated to the ω-chain end via thiol-ene Michael addition. The resultant constructs were self-assembled into micellar nanoaggregates whose sizes and morphologies were determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The in vitro activity and selectivity of the conjugates were evaluated against intraerythrocytic P. falciparum parasites.

Synthesis and Cell Interaction of Statistical l-Arginine-Glycine-l-Aspartic Acid Terpolypeptides.

Copolymerizations and terpolymerizations of N-carboxyanhydrides (NCAs) of glycine (Gly), Nδ-carbobenzyloxy-l-ornithine (Z-Orn), and ß-benzyl-l-aspartate (Bz-Asp) were investigated. In situ 1H NMR spectroscopy was used to monitor individual comonomer consumptions during binary and ternary copolymerizations. The six relevant reactivity ratios were determined from copolymerizations of the NCAs of amino acids via nonlinear least-squares curve fitting. The reactivity ratios were subsequently used to maximize the occurrence of the Asp-Gly-Orn ( DGR') sequence in the terpolymers. Terpolymers with variable probability of occurrence of DGR' were prepared in the lab. Subsequently, the ornithine residues on the terpolymers were converted to l-arginine (R) residues via guanidination reaction after removal of the protecting groups. The resulting DGR terpolymers translate to traditional peptides and proteins with variable RGD content, due to the convention in nomenclature that peptides are depicted from N- to C-terminus, whereas the NCA ring-opening polymerization is conducted from C- to N-terminus. The l-arginine containing terpolymers were evaluated for cell interaction, where it was found that neuronal cells display enhanced adhesion and process formation when plated in the presence of statistical DGR terpolymers.

Synthesis, Characterization, and Evaluation of Cytotoxicity of Poly(3-methylene-2-pyrrolidone).

The homo- and copolymerization of 3-methylene-2-pyrrolidone (3M2P) is introduced. 3M2P is readily polymerized via conventional free radical polymerization, and two reversible deactivation radical polymerization methods including reversible addition-fragmentation (chain) transfer and single-electron-transfer living radical polymerization. Poly(3M2P) has a high thermal stability and a very high glass transition temperature. Poly(3M2P) does not dissolve in most common organic solvents, but it has a high aqueous solubility. Cytotoxicity tests reveal that it is nontoxic to cells, even up to concentrations of 1 mg/mL. This adds poly(3M2P) to the family of water-soluble and biocompatible pyrrolidone-based vinyl polymers.

Templated hierarchical self-assembly of poly(p-aryltriazole) foldamers.

A biomimetic approach has been used for the templated self-assembly of a helical poly(para-aryltriazole) foldamer. The solvophobic folding process yields helices that further self-assemble into long nanotubes (see picture; scale bar: 100 nm). Constructs of controlled length and chirality can be generated by applying a poly(γ-benzyl-l-glutamate) scaffold at the appropriate assembly conditions, mimicking tobacco mosaic virus self-assembly.

Linear Dichroism Activity of Chiral Poly(p-Aryltriazole) Foldamers.

Controllable higher-order assembly is a central aim of macromolecular chemistry. An essential challenge to developing these molecules is improving our understanding of the structures they adopt under different conditions. Here, we demonstrate how flow linear dichroism (LD) spectroscopy is used to provide insights into the solution structure of a chiral, self-assembled fibrillar foldamer. Poly(para-aryltriazole)s fold into different structures depending on the monomer geometry and variables such as solvent and ionic strength. LD spectroscopy provides a simple route to determine chromophore alignment in solution and is generally used on natural molecules or molecular assemblies such as DNA and M13 bacteriophage. In this contribution, we show that LD spectroscopy is a powerful tool in the observation of self-assembly processes of synthetic foldamers when complemented by circular dichroism, absorbance spectroscopy, and microscopy. To that end, poly(para-aryltriazole)s were aligned in a flow field under different solvent conditions. The extended aromatic structures in the foldamer give rise to a strong LD signal that changes in sign and in intensity with varying solvent conditions. A key advantage of LD is that it only detects the large assemblies, thus removing background due to monomers and small oligomers.

Facile Route to Targeted, Biodegradable Polymeric Prodrugs for the Delivery of Combination Therapy for Malaria.

A facile synthetic methodology has been developed to prepare multifaceted polymeric prodrugs that are targeted, biodegradable, and nontoxic, and used for the delivery of combination therapy. This is the first instance of the delivery of the WHO recommended antimalarial combination of lumefantrine (LUM, drug 1) and artemether (AM, drug 2) via a polymeric prodrug. To achieve this, reversible addition-fragmentation chain transfer (RAFT)-mediated polymerization of N-vinylpyrrolidone (NVP) was conducted using a hydroxy-functional RAFT agent, and the resulting polymer was used as the macroinitiator in the ring-opening polymerization (ROP) of α-allylvalerolactone (AVL) to synthesize the biodegradable block copolymer of poly(N-vinylpyrrolidone) and poly(α-allylvalerolactone) (PVP-b-PAVL). The ω-end thiol group of PVP was protected using 2,2'-pyridyldisulfide prior to the ROP, and was conveniently used to bioconjugate a peptidic targeting ligand. To attach LUM, the allyl groups of PVP-b-PAVL underwent oxidation to introduce carboxylic acid groups, which were then esterified with ethylene glycol vinyl ether. Finally, LUM was conjugated to the block copolymer via an acid-labile acetal linkage in a "click"-type reaction, and AM was entrapped within the hydrophobic core of the self-assembled aggregates to render biodegradable multidrug-loaded micelles with targeting ability for combination therapy.