Tailored Branched Polymer-Protein Bioconjugates for Tunable Sieving Performance.

Protein-polymer conjugates combine the unique properties of both proteins and synthetic polymers, making them important materials for biomedical applications. In this work, we synthesized and characterized protein-branched polymer bioconjugates that were precisely designed to retain protein functionality while preventing unwanted interactions. Using chymotrypsin as a model protein, we employed a controlled radical branching polymerization (CRBP) technique utilizing a water-soluble inibramer, sodium 2-bromoacrylate. The green-light-induced atom transfer radical polymerization (ATRP) enabled the grafting of branched polymers directly from the protein surface in the open air. The resulting bioconjugates exhibited a predetermined molecular weight, well-defined architecture, and high branching density. Conformational analysis by SEC-MALS validated the controlled grafting of branched polymers. Furthermore, enzymatic assays revealed that densely grafted polymers prevented protein inhibitor penetration, and the resulting conjugates retained up to 90% of their enzymatic activity. This study demonstrates a promising strategy for designing protein-polymer bioconjugates with tunable sieving behavior, opening avenues for applications in drug delivery and biotechnology.

Visible-light-induced ATRP under high-pressure: synthesis of ultra-high-molecular-weight polymers.

In this study, a high-pressure-assisted photoinduced atom transfer radical polymerization (p ≤ 250 MPa) enabled the synthesis of ultra-high-molecular-weight polymers (UHMWPs) of up to 9 350 000 and low/moderate dispersity (1.10 < D < 1.46) in a co-solvent system (water/DMSO), without reaction mixture deoxygenation.

Biomass RNA for the Controlled Synthesis of Degradable Networks by Radical Polymerization.

Nucleic acids extracted from biomass have emerged as sustainable and environmentally friendly building blocks for the fabrication of multifunctional materials. Until recently, the fabrication of biomass nucleic acid-based structures has been facilitated through simple crosslinking of biomass nucleic acids, which limits the possibility of material properties engineering. This study presents an approach to convert biomass RNA into an acrylic crosslinker through acyl imidazole chemistry. The number of acrylic moieties on RNA was engineered by varying the acylation conditions. The resulting RNA crosslinker can undergo radical copolymerization with various acrylic monomers, thereby offering a versatile route for creating materials with tunable properties (e.g., stiffness and hydrophobic characteristics). Further, reversible-deactivation radical polymerization methods, such as atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer (RAFT), were also explored as additional approaches to engineer the hydrogel properties. The study also demonstrated the metallization of the biomass RNA-based material, thereby offering potential applications in enhancing electrical conductivity. Overall, this research expands the opportunities in biomass-based biomaterial fabrication, which allows tailored properties for diverse applications.

Red-Light-Driven Atom Transfer Radical Polymerization for High-Throughput Polymer Synthesis in Open Air.

Photoinduced reversible-deactivation radical polymerization (photo-RDRP) techniques offer exceptional control over polymerization, providing access to well-defined polymers and hybrid materials with complex architectures. However, most photo-RDRP methods rely on UV/visible light or photoredox catalysts (PCs), which require complex multistep synthesis. Herein, we present the first example of fully oxygen-tolerant red/NIR-light-mediated photoinduced atom transfer radical polymerization (photo-ATRP) in a high-throughput manner under biologically relevant conditions. The method uses commercially available methylene blue (MB+) as the PC and [X-CuII/TPMA]+ (TPMA = tris(2-pyridylmethyl)amine) complex as the deactivator. The mechanistic study revealed that MB+ undergoes a reductive quenching cycle in the presence of the TPMA ligand used in excess. The formed semireduced MB (MB•) sustains polymerization by regenerating the [CuI/TPMA]+ activator and together with [X-CuII/TPMA]+ provides control over the polymerization. This dual catalytic system exhibited excellent oxygen tolerance, enabling polymerizations with high monomer conversions (>90%) in less than 60 min at low volumes (50-250 µL) and high-throughput synthesis of a library of well-defined polymers and DNA-polymer bioconjugates with narrow molecular weight distributions (D < 1.30) in an open-air 96-well plate. In addition, the broad absorption spectrum of MB+ allowed ATRP to be triggered under UV to NIR irradiation (395-730 nm). This opens avenues for the integration of orthogonal photoinduced reactions. Finally, the MB+/Cu catalysis showed good biocompatibility during polymerization in the presence of cells, which expands the potential applications of this method.

Reactivity Prediction of Cu-Catalyzed Halogen Atom Transfer Reactions Using Data-Driven Techniques.

In catalysis, linear free energy relationships (LFERs) are commonly used to identify reaction descriptors that enable the prediction of outcomes and the design of more effective catalysts. Herein, LFERs are established for the reductive cleavage of the C(sp3)-X bond in alkyl halides (RX) by Cu complexes. This reaction represents the activation step in atom transfer radical polymerization and atom transfer radical addition/cyclization. The values of the activation rate constant, kact, for 107 Cu complex/RX couples in 5 different solvents spanning over 13 orders of magnitude were effectively interpolated by the equation: log kact = sC(I + C + S), where I, C, and S are, respectively, the initiator, catalyst, and solvent parameters, and sC is the catalyst-specific sensitivity parameter. Furthermore, each of these parameters was correlated to relevant descriptors, which included the bond dissociation free energy of RX and its Tolman cone angle θ, the electron affinity of X, the radical stabilization energy, the standard reduction potential of the Cu complex, the polarizability parameter π* of the solvent, and the distortion energy of the complex in its transition state. This set of descriptors establishes the fundamental properties of Cu complexes and RX that determine their reactivity and that need to be considered when designing novel systems for atom transfer radical reactions. Finally, a multivariate linear regression (MLR) approach was adopted to develop an objective model that surpassed the predictive capability of the LFER equation. Thus, the MLR model was employed to predict kact values for >2000 Cu complex/RX pairs.

Synthesis of RNA-Amphiphiles via Atom Transfer Radical Polymerization in the Organic Phase.

The combination of hydrophobic polymers with nucleic acids is a fascinating way to engineer the self-assembly behavior of nucleic acids into diverse nanostructures such as micelles, vesicles, nanosheets, and worms. Here we developed a robust route to synthesize a RNA macroinitiator with protecting groups on the 2'-hydroxyl groups in the solid phase using an oligonucleotide synthesizer. The protecting groups successfully solubilized the RNA macroinitiator, enabling atom transfer radical polymerization (ATRP) of hydrophobic monomers. As a result, the RNA-polymer hybrids obtained by ATRP exhibited enhanced chemical stability by suppressing cleavage. In addition, we demonstrated evidence of controlled polymerization behavior as well as control over the molecular weight of the hydrophobic polymers grown from RNA. We envision that this methodology will expand the field of RNA-polymer conjugates while vastly enhancing the possibility to alter and engineer the properties of RNA-based polymeric materials.

RNA-Polymer Hybrids via Direct and Site-Selective Acylation with the ATRP Initiator and Photoinduced Polymerization.

Combining synthetic polymers with RNA paves the way for creating RNA-based materials with non-canonical functions. We have developed an acylation reagent that allows for direct incorporation of the atom transfer radical polymerization (ATRP) initiator into both short synthetic oligoribonucleotides and natural biomass RNA extracted from torula yeast. The acylation was performed in a quantitative yield. The resulting initiator-functionalized RNAs were used for grafting polymer chains from the RNA by photoinduced ATRP, resulting in RNA-polymer hybrids with narrow molecular weight distributions. The RNA initiator was used for the polymerization of oligo(ethylene oxide) methyl ether methacrylate, poly(ethylene glycol) dimethacrylate, and N-isopropylacrylamide monomers, resulting in RNA bottlebrushes, hydrogels, and stimuli-responsive materials. This approach, readily applicable to both post-synthetic and nature-derived RNA, can be used to engineer the properties of a variety of RNA-based macromolecular hybrids and assemblies providing access to a wide variety of RNA-polymer hybrids.

Fully Oxygen-Tolerant Visible-Light-Induced ATRP of Acrylates in Water: Toward Synthesis of Protein-Polymer Hybrids.

Over the last decade, photoinduced ATRP techniques have been developed to harness the energy of light to generate radicals. Most of these methods require the use of UV light to initiate polymerization. However, UV light has several disadvantages: it can degrade proteins, damage DNA, cause undesirable side reactions, and has low penetration depth in reaction media. Recently, we demonstrated green-light-induced ATRP with dual catalysis, where eosin Y (EYH2) was used as an organic photoredox catalyst in conjunction with a copper complex. This dual catalysis proved to be highly efficient, allowing rapid and well-controlled aqueous polymerization of oligo(ethylene oxide) methyl ether methacrylate without the need for deoxygenation. Herein, we expanded this system to synthesize polyacrylates under biologically relevant conditions using CuII/Me6TREN (Me6TREN = tris[2-(dimethylamino)ethyl]amine) and EYH2 at ppm levels. Water-soluble oligo(ethylene oxide) methyl ether acrylate (average M n = 480, OEOA480) was polymerized in open reaction vessels under green light irradiation (520 nm). Despite continuous oxygen diffusion, high monomer conversions were achieved within 40 min, yielding polymers with narrow molecular weight distributions (1.17 ≤ D̵ ≤ 1.23) for a wide targeted DP range (50-800). In situ chain extension and block copolymerization confirmed the preserved chain end functionality. In addition, polymerization was triggered/halted by turning on/off a green light, showing temporal control. The optimized conditions also enabled controlled polymerization of various hydrophilic acrylate monomers, such as 2-hydroxyethyl acrylate, 2-(methylsulfinyl)ethyl acrylate), and zwitterionic carboxy betaine acrylate. Notably, the method allowed the synthesis of well-defined acrylate-based protein-polymer hybrids using a straightforward reaction setup without rigorous deoxygenation.

Visible-Light-Mediated Controlled Radical Branching Polymerization in Water.

Hyperbranched polymethacrylates were synthesized by green-light-induced atom transfer radical polymerization (ATRP) under biologically relevant conditions in the open air. Sodium 2-bromoacrylate (SBA) was prepared in situ from commercially available 2-bromoacrylic acid and used as a water-soluble inibramer to induce branching during the copolymerization of methacrylate monomers. As a result, well-defined branched polymethacrylates were obtained in less than 30 min with predetermined molecular weights (36 000

Open-air green-light-driven ATRP enabled by dual photoredox/copper catalysis.

Photoinduced atom transfer radical polymerization (photo-ATRP) has risen to the forefront of modern polymer chemistry as a powerful tool giving access to well-defined materials with complex architecture. However, most photo-ATRP systems can only generate radicals under biocidal UV light and are oxygen-sensitive, hindering their practical use in the synthesis of polymer biohybrids. Herein, inspired by the photoinduced electron transfer-reversible addition-fragmentation chain transfer (PET-RAFT) polymerization, we demonstrate a dual photoredox/copper catalysis that allows open-air ATRP under green light irradiation. Eosin Y was used as an organic photoredox catalyst (PC) in combination with a copper complex (X-CuII/L). The role of PC was to trigger and drive the polymerization, while X-CuII/L acted as a deactivator, providing a well-controlled polymerization. The excited PC was oxidatively quenched by X-CuII/L, generating CuI/L activator and PC˙+. The ATRP ligand (L) used in excess then reduced the PC˙+, closing the photocatalytic cycle. The continuous reduction of X-CuII/L back to CuI/L by excited PC provided high oxygen tolerance. As a result, a well-controlled and rapid ATRP could proceed even in an open vessel despite continuous oxygen diffusion. This method allowed the synthesis of polymers with narrow molecular weight distributions and controlled molecular weights using Cu catalyst and PC at ppm levels in both aqueous and organic media. A detailed comparison of photo-ATRP with PET-RAFT polymerization revealed the superiority of dual photoredox/copper catalysis under biologically relevant conditions. The kinetic studies and fluorescence measurements indicated that in the absence of the X-CuII/L complex, green light irradiation caused faster photobleaching of eosin Y, leading to inhibition of PET-RAFT polymerization. Importantly, PET-RAFT polymerizations showed significantly higher dispersity values (1.14 ≤ D ≤ 4.01) in contrast to photo-ATRP (1.15 ≤ D ≤ 1.22) under identical conditions.

Alternating Methyl Methacrylate/n-Butyl Acrylate Copolymer Prepared by Atom Transfer Radical Polymerization.

Poly(methyl methacrylate/n-butyl acrylate) [P(MMA/BA)] copolymer with an alternating structure was synthesized via an activator regenerated by electron transfer (ARGET) atom transfer radical (co)polymerization (ATRP) of 2-ethylfenchyl methacrylate (EFMA) and n-butyl acrylate (BA) with subsequent postpolymerization modifications (PPM). Due to the steric hindrance of the bulky pendant group of EFMA, as well as the low reactivity ratio of BA in copolymerization with methacrylates, copolymerization of EFMA and BA generated a copolymer with a high content of alternating dyads. A subsequent PPM procedure of the alternating EFMA/BA copolymer was comprised of the hydrolysis of a tertiary ester by trifluoroacetic acid and methylation by (trimethylsilyl)diazomethane. After the modifications, the architecture of the obtained alternating MMA/BA copolymers was compared with gradient and statistical copolymers with overall similar compositions, molecular weights, and dispersities. 13C NMR indicated the absence of either MMA/MMA/MMA or BA/BA/BA sequences, in contrast to an abundance of homotriads in either the statistical or especially in the gradient copolymer. All three copolymers had similar glass transition temperatures, as measured by differential scanning calorimetry (DSC), but the alternating copolymer had the narrowest range of glass transition.

Sulfoxide-Containing Polyacrylamides Prepared by PICAR ATRP for Biohybrid Materials.

Water-soluble and biocompatible polymers are of interest in biomedicine as the search for alternatives to PEG-based materials becomes more important. In this work, the synthesis of a new sulfoxide-containing monomer, 2-(methylsulfinyl)ethyl acrylamide (MSEAM), is reported. Well-defined polymers were prepared by photoinduced initiators for continuous activator regeneration atom transfer radical polymerization (PICAR ATRP). The polymerizations were performed in water under biologically relevant conditions in a small volume without degassing the reaction mixture. DNA-PMSEAM and protein-PMSEAM hybrids were also synthesized. The lower critical solution temperature (LCST) of PMSEAM was estimated to be approximately 170 °C by extrapolating the LCST for a series of copolymers with variable content of N-isopropylacrylamide. The cytotoxicity studies showed excellent biocompatibility of PMSEAM, even at concentrations up to 2.5 mg/mL. Furthermore, the MSEAM monomer exhibited relatively lower toxicity than similar (meth)acrylate-based monomers at comparable concentrations.

Red-Light-Induced, Copper-Catalyzed Atom Transfer Radical Polymerization.

Despite advances in photochemical atom transfer radical polymerization (photoATRP), these systems often rely on the use of UV light for the activation/generation of the copper-based catalytic species. To circumvent the problems associated with the UV light, we developed a dual photoredox catalytic system to mediate photoinduced ATRP under red-light irradiation. The catalytic system is comprised of a Cu catalyst to control the polymerization via ATRP equilibrium and a photocatalyst, such as zinc(II) tetraphenylporphine or zinc(II) phthalocyanine, to generate the activator CuI species under red-light irradiation. In addition, this system showed oxygen tolerance due to the consumption of oxygen in the photoredox reactions, yielding well-controlled polymerizations without the need for deoxygenation processes.

Controlled Release of Exosomes Using Atom Transfer Radical Polymerization-Based Hydrogels.

Exosomes are 30-200 nm sized extracellular vesicles that are increasingly recognized as potential drug delivery vehicles. However, exogenous exosomes are rapidly cleared from the blood upon intravenous delivery, which limits their therapeutic potential. Here, we report bioactive exosome-tethered poly(ethylene oxide)-based hydrogels for the localized delivery of therapeutic exosomes. Using cholesterol-modified DNA tethers, the lipid membrane of exosomes was functionalized with initiators to graft polymers in the presence of additional initiators and crosslinker using photoinduced atom transfer radical polymerization (ATRP). This strategy of tethering exosomes within the hydrogel network allowed their controlled release over a period of 1 month, which was much longer than physically entrapped exosomes. Exosome release profile was tuned by varying the crosslinking density of the polymer network and the use of photocleavable tethers allowed stimuli-responsive release of exosomes. The therapeutic potential of the hydrogels was assessed by evaluating the osteogenic potential of bone morphogenetic protein 2-loaded exosomes on C2C12 and MC3T3-E1 cells. Thus, ATRP-based exosome-tethered hydrogels represent a tunable platform with improved efficacy and an extended-release profile.

Biocompatible photoinduced CuAAC using sodium pyruvate.

Sodium pyruvate, a natural intermediate produced during cellular metabolism, is commonly used in buffer solutions and media for biochemical applications. Here we show the use of sodium pyruvate (SP) as a reducing agent in a biocompatible aqueous photoinduced azide-alkyne cycloaddition (CuAAC) reaction. This copper(I)-catalyzed 1,3-dipolar cycloaddition is triggered by SP under UV light irradiation, exhibits oxygen tolerance and temporal control, and provides a convenient alternative to current CuAAC systems, particularly for biomolecular conjugations.

Conjugated Cross-linked Phenothiazines as Green or Red Light Heterogeneous Photocatalysts for Copper-Catalyzed Atom Transfer Radical Polymerization.

Using the power of light to drive controlled radical polymerizations has provided significant advances in synthesis of well-defined polymers. Photoinduced atom transfer radical polymerization (ATRP) systems often employ UV light to regenerate copper activator species to mediate the polymerization. Taking full advantage of long-wavelength visible light for ATRP would require developing appropriate photocatalytic systems that engage in photoinduced electron transfer processes with the ATRP components to generate activating species. Herein, we developed conjugated microporous polymers (CMP) as heterogeneous photocatalysts to exploit the power of visible light in promoting copper-catalyzed ATRP. The photocatalyst was designed by cross-linking phenothiazine (PTZ) as a photoactive core in the presence of dimethoxybenzene as a cross-linker via the Friedel-Crafts reaction. The resulting PTZ-CMP network showed photoactivity in the visible region due to the extended conjugation throughout the network because of the aromatic groups connecting the PTZ units. Therefore, photoinduced copper-catalyzed ATRP was performed with CMPs that regenerated activator species under green or red light irradiation to start the ATRP process. This resulted in efficient polymerization of acrylate and methacrylate monomers with high conversion and well-controlled molecular weight. The heterogeneous nature of the photocatalyst enabled easy separation and efficient reusability in subsequent polymerizations.

Making ATRP More Practical: Oxygen Tolerance.

Atom-transfer radical polymerization (ATRP) is a well-known technique for the controlled polymerization of vinyl monomers under mild conditions. However, as with any other radical polymerization, ATRP typically requires rigorous oxygen exclusion, making it time-consuming and challenging to use by nonexperts. In this Account, we discuss various approaches to achieving oxygen tolerance in ATRP, presenting the overall progress in the field.Copper-mediated ATRP, which we first discovered in the late 1990s, uses a CuI/L activator that reversibly reacts with the dormant C(sp3)-X polymer chain end, forming a X-CuII/L deactivator and a propagating radical. Oxygen interferes with activation and chain propagation by quenching the radicals and oxidizing the activator. At ATRP equilibrium, the activator is present at a much higher concentration than the propagating radicals. Thus, oxidation of the activator is the dominant inhibition pathway. In conventional ATRP, this reaction is irreversible, so oxygen must be strictly excluded to achieve good results.Over the last two decades, our group has developed several ATRP techniques based on the concept of regenerating the activator. When the oxidized activator is continuously converted back to its active reduced form, then the catalytic system itself can act as an oxygen scavenger. Regeneration can be accomplished by reducing agents and photo-, electro-, and mechanochemical stimuli. This family of methods offers a degree of oxygen tolerance, but most of them can tolerate only a limited amount of oxygen and do not allow polymerization in an open vessel.More recently, we discovered that enzymes can be used in auxiliary catalytic systems that directly deoxygenate the reaction medium and protect the polymerization process. We developed a method that uses glucose oxidase (GOx), glucose, and sodium pyruvate to very effectively scavenge oxygen and enable open-vessel ATRP. By adding a second enzyme, horseradish peroxidase (HPR), we managed to extend the role of the auxiliary enzymatic system to generating carbon-based radicals and changed ATRP from an oxygen-sensitive to an oxygen-fueled reaction.While performing control experiments for the enzymatic methods, we noticed that using sodium pyruvate under UV irradiation triggers polymerization without the presence of GOx. This serendipitous discovery allowed us to develop the first oxygen-proof, small-molecule-based, photoinduced ATRP system. It has oxygen tolerance similar to that of the enzymatic methods, exhibits superior compatibility with both aqueous media and organic solvents, and avoids problems associated with purifying polymers from enzymes. The system was able to rapidly polymerize N-isopropylacrylamide, a challenging monomer, with a high degree of control.These contributions have substantially simplified the use of ATRP, making it more practical and accessible to everyone.

Reflection on the Matyjaszewski Lab Webinar Series and the Rise of Webinars in Polymer Chemistry.

Webinar series are helping our community of polymer scientists to stay engaged and connected, despite the cancellation of in-person meetings and the periodic closure of laboratories to contain the spread of the coronavirus pandemic. The sustainable and inclusive character of these virtual events make them valuable learning and networking opportunities. As organizers of the Matyjaszewski Lab Webinar Series, we share herein our experience, highlighting the benefits of virtual meetings and providing a short guide for webinar organizers. Researchers, particularly young scientists, are encouraged to organize such virtual events to broaden their skills and strengthen their professional network.

p-Substituted Tris(2-pyridylmethyl)amines as Ligands for Highly Active ATRP Catalysts: Facile Synthesis and Characterization.

A facile and efficient two-step synthesis of p-substituted tris(2-pyridylmethyl)amine (TPMA) ligands to form Cu complexes with the highest activity to date in atom transfer radical polymerization (ATRP) is presented. In the divergent synthesis, p-Cl substituents in tris(4-chloro-2-pyridylmethyl)amine (TPMA3Cl ) were replaced in one step and high yield by electron-donating cyclic amines (pyrrolidine (TPMAPYR ), piperidine (TPMAPIP ), and morpholine (TPMAMOR )) by nucleophilic aromatic substitution. The [CuII (TPMANR2 )Br]+ complexes exhibited larger energy gaps between frontier molecular orbitals and >0.2 V more negative reduction potentials than [CuII (TPMA)Br]+ , indicating >3 orders of magnitude higher ATRP activity. [CuI (TPMAPYR )]+ exhibited the highest reported activity for Br-capped acrylate chain ends in DMF, and moderate activity toward C-F bonds at room temperature. ATRP of n-butyl acrylate using only 10-25 part per million loadings of [CuII (TPMANR2 )Br]+ exhibited excellent control.

Fully oxygen-tolerant atom transfer radical polymerization triggered by sodium pyruvate.

ATRP (atom transfer radical polymerization) is one of the most robust reversible deactivation radical polymerization (RDRP) systems. However, the limited oxygen tolerance of conventional ATRP impedes its practical use in an ambient atmosphere. In this work, we developed a fully oxygen-tolerant PICAR (photoinduced initiators for continuous activator regeneration) ATRP process occurring in both water and organic solvents in an open reaction vessel. Continuous regeneration of the oxidized form of the copper catalyst with sodium pyruvate through UV excitation allowed the chemical removal of oxygen from the reaction mixture while maintaining a well-controlled polymerization of N-isopropylacrylamide (NIPAM) or methyl acrylate (MA) monomers. The polymerizations of NIPAM were conducted with 250 ppm (with respect to the monomer) or lower concentrations of CuBr2 and a tris[2-(dimethylamino)ethyl]amine ligand. The polymers were synthesized to nearly quantitative monomer conversions (>99%), high molecular weights (M n > 270 000), and low dispersities (1.16 < D < 1.44) in less than 30 min under biologically relevant conditions. The reported method provided a well-controlled ATRP (D = 1.16) of MA in dimethyl sulfoxide despite oxygen diffusion from the atmosphere into the reaction system.