Vinyl Ether Maleic Acid Polymers: Tunable Polymers for Self-Assembled Lipid Nanodiscs and Environments for Membrane Proteins.

Native lipid bilayer mimetics, including those that use amphiphilic polymers, are important for the effective study of membrane-bound peptides and proteins. Copolymers of vinyl ether monomers and maleic anhydride were developed with controlled molecular weights and hydrophobicity through reversible addition-fragmentation chain-transfer polymerization. After polymerization, the maleic anhydride units can be hydrolyzed, giving dicarboxylates. The vinyl ether and maleic anhydride copolymerized in a close to alternating manner, giving essentially alternating hydrophilic maleic acid units and hydrophobic vinyl ether units along the backbone after hydrolysis. The vinyl ether monomers and maleic acid polymers self-assembled with lipids, giving vinyl ether maleic acid lipid particles (VEMALPs) with tunable sizes controlled by either the vinyl ether hydrophobicity or the polymer molecular weight. These VEMALPs were able to support membrane-bound proteins and peptides, creating a new class of lipid bilayer mimetics.

Transient Covalent Polymers through Carbodiimide-Driven Assembly.

Biochemical systems make use of out-of-equilibrium polymers generated under kinetic control. Inspired by these systems, many abiotic supramolecular polymers driven by chemical fuel reactions have been reported. Conversely, polymers based on transient covalent bonds have received little attention, even though they have the potential to complement supramolecular systems by generating transient structures based on stronger bonds and by offering a straightforward tuning of reaction kinetics. In this study, we show that simple aqueous dicarboxylic acids give poly(anhydrides) when treated with the carbodiimide EDC. Transient covalent polymers with molecular weights exceeding 15,000 are generated which then decompose over the course of hours to weeks. Disassembly kinetics can be controlled using simple substituent effects in the monomer design. The impact of solvent polarity, carbodiimide concentration, temperature, pyridine concentration, and monomer concentration on polymer properties and lifetimes has been investigated. The results reveal substantial control over polymer assembly and disassembly kinetics, highlighting the potential for fine-tuned kinetic control in nonequilibrium polymerization systems.

Carbodiimide-Driven Toughening of Interpenetrated Polymer Networks.

Recent work has demonstrated that temporary crosslinks in polymer networks generated by chemical "fuels" afford materials with large, transient changes in their mechanical properties. This can be accomplished in carboxylic-acid-functionalized polymer hydrogels using carbodiimides, which generate anhydride crosslinks with lifetimes on the order of minutes to hours. Here, the impact of the polymer network architecture on the mechanical properties of transiently crosslinked materials was explored. Single networks (SNs) were compared to interpenetrated networks (IPNs). Notably, semi-IPN precursors that give IPNs on treatment with carbodiimide give much higher fracture energies (i.e., resistance to fracture) and superior resistance to compressive strain compared to other network architectures. A precursor semi-IPN material featuring acrylic acid in only the free polymer chains yields, on treatment with carbodiimide, an IPN with a fracture energy of 2400 J/m2, a fourfold increase compared to an analogous semi-IPN precursor that yields a SN. This resistance to fracture enables the formation of macroscopic complex cut patterns, even at high strain, underscoring the pivotal role of polymer architecture in mechanical performance.

Network Polymer Properties Engineered Through Polymer Backbone Dispersity and Structure.

Dispersity (Ð or Mw/Mn) is an important parameter in material design and as such can significantly impact the properties of polymers. Here, polymer networks with independent control over the molecular weight and dispersity of the linear chains that form the material are developed. Using a RAFT polymerization approach, a library of polymers with dispersity ranging from 1.2-1.9 for backbone chain-length (DP) 100, and 1.4-3.1 for backbone chain-length 200 were developed and transformed to networks through post-polymerization crosslinking to form disulfide linkers. The tensile, swelling, and adhesive properties were explored, finding that both at DP 100 and DP 200 the swelling ratio, tensile strength, and extensibility were superior at intermediate dispersity (1.3-1.5 for DP 100 and 1.6-2.1 for DP 200) compared to materials with either substantially higher or lower dispersity. Furthermore, adhesive properties for materials with chains of intermediate dispersity at DP 200 revealed enhanced performance compared to the very low or high dispersity chains.

Oxygen-Enhanced Atom Transfer Radical Polymerization through the Formation of a Copper Superoxido Complex.

In controlled radical polymerization, oxygen is typically regarded as an undesirable component resulting in terminated polymer chains, deactivated catalysts, and subsequent cessation of the polymerization. Here, we report an unusual atom transfer radical polymerization whereby oxygen favors the polymerization by triggering the in situ transformation of CuBr/L to reactive superoxido species at room temperature. Through a superoxido ARGET-ATRP mechanism, an order of magnitude faster polymerization rate and a rapid and complete initiator consumption can be achieved as opposed to when unoxidized CuBr/L was instead employed. Very high end-group fidelity has been demonstrated by mass-spectrometry and one-pot synthesis of block and multiblock copolymers while pushing the reactions to reach near-quantitative conversions in all steps. A high molecular weight polymer could also be targeted (DPn = 6400) without compromising the control over the molar mass distributions (D < 1.20), even at an extremely low copper concentration (4.5 ppm). The versatility of the technique was demonstrated by the polymerization of various monomers in a controlled fashion. Notably, the efficiency of our methodology is unaffected by the purity of the starting CuBr, and even a brown highly-oxidized 15-year-old CuBr reagent enabled a rapid and controlled polymerization with a final dispersity of 1.07, thus not only reducing associated costs but also omitting the need for rigorous catalyst purification prior to polymerization.

Chemically Fueled Reinforcement of Polymer Hydrogels.

Carbodiimide-fueled anhydride bond formation has been used to enhance the mechanical properties of permanently crosslinked polymer networks, giving materials that exhibit transitions from soft gels to covalently reinforced gels, eventually returning to the original soft gels. Temporary changes in mechanical properties result from a transient network of anhydride crosslinks, which eventually dissipate by hydrolysis. Over an order of magnitude increase in the storage modulus is possible through carbodiimide fueling. The time-dependent mechanical properties can be modulated by the concentration of carbodiimide, temperature, and primary chain architecture. Because the materials remain rheological solids, new material functions such as temporally controlled adhesion and rewritable spatial patterns of mechanical properties have been realized.

3D printable adhesive elastomers with dynamic covalent bond rearrangement.

Repairable adhesive elastomers are emerging materials employed in compelling applications such as soft robotics, biosensing, tissue regeneration, and wearable electronics. Facilitating adhesion requires strong interactions, while self-healing requires bond dynamicity. This contrast in desired bond characteristics presents a challenge in the design of healable adhesive elastomers. Furthermore, 3D printability of this novel class of materials has received limited attention, restricting the potential design space of as-built geometries. Here, we report a series of 3D-printable elastomeric materials with self-healing ability and adhesive properties. Repairability is obtained using Thiol-Michael dynamic crosslinkers incorporated into the polymer backbone, while adhesion is facilitated with acrylate monomers. Elastomeric materials with excellent elongation up to 2000%, self-healing stress recovery >95%, and strong adhesion with metallic and polymeric surfaces are demonstrated. Complex functional structures are successfully 3D printed using a commercial digital light processing (DLP) printer. Shape-selective lifting of low surface energy poly(tetrafluoroethylene) objects is achieved using soft robotic actuators with interchangeable 3D-printed adhesive end effectors, wherein tailored contour matching leads to increased adhesion and successful lifting capacity. The demonstrated utility of these adhesive elastomers provides unique capabilities to easily program soft robot functionality.

PET-RAFT Polymerization of Star Polymers with Folded ortho-Phenylene Cores.

ortho-Phenylenes are one of the simplest classes of aromatic foldamers, adopting helical geometries because of aromatic stacking interactions. The folding and misfolding of ortho-phenylenes are slow on the NMR timescale at or below room temperature, allowing detection of folding states using 1 H NMR spectroscopy. Herein, an ortho-phenylene hexamer is coupled with a RAFT chain transfer agent (CTA) on each repeat unit. A variety of acrylic monomers are polymerized onto the CTA-functionalized ortho-phenylene using PET-RAFT to yield functionalized star polymers with ortho-phenylene cores. The steric bulk of the acrylate monomer units as well as the chain length of each arm of the star polymer is varied. 1 H NMR spectroscopy shows that the folding of the ortho-phenylenes do not vary, providing a robust helical core for star polymer systems.

Polymer modification of SARS-CoV-2 spike protein impacts its ability to bind key receptor.

The global spread of SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) has caused the loss of many human lives and severe economic losses. SARS-CoV-2 mediates its infection in humans via the spike glycoprotein. The receptor binding domain of the SARS-CoV-2 spike protein binds to its cognate receptor, angiotensin converting enzyme-2 (ACE2) to initiate viral entry. In this study, we examine how polymer modification of the spike protein receptor binding domain impacts binding to ACE2. The horseradish peroxidase conjugated receptor binding domain was modified with a range of polymers including hydrophilic N,N-dimethylacrylamide, hydrophobic N-isopropylacrylamide, cationic 3-(N,N-dimethylamino)propylacrylamide, and anionic 2-acrylamido-2-methylpropane sulfonic acid polymers. The effect of polymer chain length was observed using N,N-dimethylacrylamide polymers with degrees of polymerization of 5, 10 and 25. Polymer conjugation of the receptor binding domain significantly reduced the interaction with ACE2 protein, as determined by an enzyme-linked immunosorbent assay. Stability analysis showed that these conjugates remained highly stable even after seven days incubation at physiological temperature. Hence, this study provides a detailed view of the effect specific type of modification using a library of polymers with different functionalities in interrupting RBD-ACE2 interaction.

Formation of styrene maleic acid lipid nanoparticles (SMALPs) using SMA thin film on a substrate.

Despite the important role of membrane proteins in biological function and physiology, studying them remains challenging because of limited biomimetic systems for the protein to remain in its native membrane environment. Cryo electron microscopy (Cryo-EM) is emerging as a powerful tool for analyzing the structure of membrane proteins. However, Cryo-EM and other membrane protein analyses are better studied in a native lipid bilayer. Although traditional, mimetic systems have disadvantages that limit their use in the study of membrane proteins. As an alternative, styrene-maleic acid copolymers are used to form nanoparticles with POPC:POPG lipids. Traditional characterization of these styrene maleic acid lipid nanoparticles (SMALPs) includes dynamic light scattering (DLS), electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and transmission electron microscopy (TEM). In this study a new method was developed that utilizes SMALPs using a styrene-maleic acid copolymer (SMA) thin film on a TEM grid, acting as a substrate. By directly adding POPC:POPG lipid vesicles to the SMA coated grid SMALPs can be formed, visualized, and characterized by TEM without the need to make them in solution prior to imaging. We envision these functionalized grids could aid in single particle specimen preparation, increasing the efficiency of structural biology and biophysical techniques such as Cryo-EM.

Investigating the Impact of Polymer Length, Attachment Site, and Charge on Enzymatic Activity and Stability of Cellulase.

The thermophilic cellulase Cel5a from Fervidobacterium nodosum (FnCel5a) was conjugated with neutral, cationic, and anionic polymers of increasing molecular weights. The enzymatic activity toward an anionic soluble cellulose derivative, thermal stability, and functional chemical stability of these bioconjugates were investigated. The results suggest that increasing polymer chain length for polymers compatible with the substrate enhances the positive impact of polymer conjugation on enzymatic activity. Activity enhancements of nearly 100% were observed for bioconjugates with N,N-dimethyl acrylamide (DMAm) and N,N-dimethyl acrylamide-2-(N,N-dimethylamino)ethyl methacrylate (DMAm/DMAEMA) due to proposed polymer-substrate compatibility enabled by potential noncovalent interactions. Double conjugation of two functionally distinct polymers to wild-type and mutated FnCel5a using two conjugation methods was achieved. These doubly conjugated bioconjugates exhibited similar thermal stability to the unmodified wild-type enzyme, although enzymatic activity initially gained from conjugation was lost, suggesting that chain length may be a better tool for bioconjugate activity modulation than double conjugation.

Dynamic Bonds: Adaptable Timescales for Responsive Materials.

Dynamic bonds introduce unique properties such as self-healing, recyclability, shape memory, and malleability to polymers. Significant efforts have been made to synthesize a variety of dynamic linkers, creating a diverse library of materials. In addition to the development of new dynamic chemistries, fine-tuning of dynamic bonds has emerged as a technique to modulate dynamic properties. This Review highlights approaches for controlling the timescales of dynamic bonds in polymers. Particularly, eight dynamic bonds are considered, including urea/urethanes, boronic esters, Thiol-Michael exchange, Diels-Alder adducts, transesterification, imine bonds, coordination bonds, and hydrogen bonding. This Review emphasizes how structural modifications and external factors have been used as tools to tune the dynamic character of materials. Finally, this Review proposes strategies for tailoring the timescales of dynamic bonds in polymer materials through both kinetic effects and modulating bond thermodynamics.

Ubiquitous Nature of Rate Retardation in Reversible Addition-Fragmentation Chain Transfer Polymerization.

Reversible addition-fragmentation chain transfer (RAFT) polymerization is one of the most powerful reversible deactivation radical polymerization (RDRP) processes. Rate retardation is prevalent in RAFT and occurs when polymerization rates deviate from ideal conventional radical polymerization kinetics. Herein, we explore beyond what was initially thought to be the culprit of rate retardation: dithiobenzoate chain transfer agents (CTA) with more active monomers (MAMs). Remarkably, polymerizations showed that rate retardation occurs in systems encompassing the use of trithiocarbonates and xanthates CTAs with varying monomeric activities. Both the simple slow fragmentation and intermediate radical termination models show that retardation of all these systems can be described by using a single relationship for a variety of monomer reactivity and CTAs, suggesting rate retardation is a universal phenomenon of varying severity, independent of CTA composition and monomeric activity level.

Hydrolytically Stable Maleimide-End-Functionalized Polymers for Site-Specific Protein Conjugation.

Site-specific conjugation to cysteines of proteins often uses ester groups to link maleimide or alkene groups to polymers. However, the ester group is susceptible to hydrolysis, potentially losing the benefits gained through bioconjugation. Here, we present a simple conjugation strategy that utilizes the amide bond stability of traditional 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide coupling while introducing site specificity. Hydrolytically stable maleimide-end-functionalized polymers for site-specific conjugation to free cysteines of proteins were synthesized using reversible addition-fragmentation chain-transfer (RAFT) polymerization. The alpha terminus of the polymers was amidated with a furan-protected aminoethyl maleimide using carbodiimide-based chemistry. Finally, the maleimide was exposed by a retro Diels-Alder reaction to yield the maleimide group, allowing for thiol-maleimide click chemistry for bioconjugation. A thermophilic cellulase from Fervidobacterium nodosum (FnCel5a) was conjugated using various strategies, including random 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling, site-specific hydroxyethyl maleimide (HEMI) end-functionalized coupling, hydroxyethyl acrylate (HEA) end-functionalized coupling, and amidoethyl maleimide (AEMI) end-functionalized coupling. Only the polymers conjugated by EDC and AEMI remained conjugated a week after attachment. This indicates that hydrolytically stable amide-based maleimides are an important bioconjugation strategy for conjugates that require long-term stability, while esters are better suited for systems that require debonding of polymers over time.

Polymer Modification of Lipases, Substrate Interactions, and Potential Inhibition.

An industrially important enzyme, Candida antarctica lipase B (CalB), was modified with a range of functional polymers including hydrophilic, hydrophobic, anionic, and cationic character using a "grafting to" approach. We determined the impact of polymer chain length on CalB activity by synthesizing biohybrids of CalB with each polymer at three different chain lengths, using reversible addition-fragmentation chain transfer (RAFT) polymerization. The activity of CalB in both aqueous and aqueous-organic media mixtures was significantly enhanced for acrylamide (Am) and N,N-dimethyl acrylamide (DMAm) conjugates, with activity remaining approximately constant in 25 and 50% ethanol solvent systems. Interestingly, the activity of N,N-dimethylaminopropyl-acrylamide (DMAPA) conjugates increased gradually with increasing organic solvent content in the system. Contrary to other literature reports, our study showed significantly diminished activity for hydrophobic polymer-protein conjugates. Functional thermal stability assays also displayed a considerable enhancement of retained activity of Am, DMAm, and DMAPA conjugates compared to the native CalB enzyme. Thus, this study provides an insight into possible advances in lipase production, which can lead to new improved lipase bioconjugates with increased activity and stability.

Heat- and Light-Responsive Materials Through Pairing Dynamic Thiol-Michael and Coumarin Chemistry.

Covalent adaptable networks (CANs) based on the thiol-Michael (TM) linkages can be thermal and pH responsive. Here, a new vinyl-sulfone-based thiol-Michael crosslinker is synthesized and incorporated into acrylate-based CANs to achieve stable materials with dynamic properties. Because of the reversible TM linkages, excellent temperature-responsive re-healing and malleability properties are achieved. In addition, for the first time, a photoresponsive coumarin moiety is incorporated with TM-based CANs to introduce light-mediated reconfigureability and postpolymerization crosslinking. Overall, these materials can be on demand dynamic in response to heat and light but can retain mechanical stability at ambient condition.

Toward Next-Generation Biohybrid Catalyst Design: Influence of Degree of Polymerization on Enzyme Activity.

Due to their capacity to conduct complex organic transformations, enzymes find extensive use in medical and industrial settings. Unfortunately, enzymes are limited by their poor stability when exposed to harsh non-native conditions. While a host of methods have been developed to stabilize enzymes in non-native conditions, recent research into the synthesis of polymer-enzyme biohybrids using reversible deactivation radical polymerization approaches has demonstrated the potential of increased enzymatic activity in both native and non-native environments. In this manuscript, we utilize the enzyme lipase, as a model system, to explore the impact that modulation of grafted polymer molecular weight has on enzyme activity in both aqueous and organic media. We studied the properties of these hybrids using both solution-phase enzyme activity methods and coarse-grain modeling to assess the impact of polymer grafting density and grafted polymer molecular weight on enzyme activity to gain a deeper insight into this understudied property of the biohybrid system.

Simple Derivatization of RAFT-Synthesized Styrene-Maleic Anhydride Copolymers for Lipid Disk Formulations.

Styrene-maleic acid copolymers have received significant attention because of their ability to interact with lipid bilayers and form styrene-maleic acid copolymer lipid nanoparticles (SMALPs). However, these SMALPs are limited in their chemical diversity, with only phenyl and carboxylic acid functional groups, resulting in limitations because of sensitivity to low pH and high concentrations of divalent metals. To address this limitation, various nucleophiles were reacted with the anhydride unit of well-defined styrene-maleic anhydride copolymers in order to assess the potential for a new lipid disk nanoparticle-forming species. These styrene-maleic anhydride copolymer derivatives (SMADs) can form styrene-maleic acid derivative lipid nanoparticles (SMADLPs) when they interact with lipid molecules. Polymers were synthesized, purified, characterized by Fourier-transform infrared spectroscopy, gel permeation chromatography, and nuclear magnetic resonance and then used to make disk-like SMADLPs, whose sizes were measured by dynamic light scattering (DLS). The SMADs form lipid nanoparticles, observable by DLS and transmission electron microscopy, and were used to reconstitute a spin-labeled transmembrane protein, KCNE1. The polymer method reported here is facile and scalable and results in functional and robust polymers capable of forming lipid nanodisks that are stable against a wide pH range and 100 mM magnesium.

Dynamic Covalent Bonds in Polymeric Materials.

Dynamic covalent bonds (DCBs) have received significant attention over the past decade. These are covalent bonds that are capable of exchanging or switching between several molecules. Particular focus has recently been on utilizing these DCBs in polymeric materials. Introduction of DCBs into a polymer material provides it with powerful properties including self-healing, shape-memory properties, increased toughness, and ability to relax stresses as well as to change from one macromolecular architecture to another. This Minireview summarizes commonly used powerful DCBs formed by simple, often "click" reactions, and highlights the powerful materials that can result. Challenges and potential future developments are also discussed.

In-situ Chemiluminescence-Driven Reversible Addition-Fragmentation Chain-Transfer Photopolymerization.

The power of chemical light generation (chemiluminescence) is used to drive polymerization reactions. A biphasic reaction is developed such that light-generating reactions are confined to the organic phase and photopolymerization occurs in the aqueous phase. Well-defined RAFT-capped polymers are synthesized and the kinetics are shown to be dictated by light generation.