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Chapter 3
Aqueous Visible-Light RAFT Polymerizations and Applications C. Adrian Figg and Brent S. Sumerlin* George & Josephine Butler Polymer Research Laboratory, Center for Macromolecular Science & Engineering, Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, Florida 32611-7200, Unted States *E-mail:
[email protected].
Visible-light reversible addition-fragmentation chain transfer (RAFT) polymerizations facilitate synthesis under mild reactions conditions, while still affording the control over molecular weight, polydispersity, and end group functionality that are the hallmarks of reversible-deactivation radical polymerization (RDRP). Recent developments in aqueous RAFT polymerizations that rely on initiator-free, externallyinitiated, and photoinduced electron transfer conditions have provided alternative strategies to polymer-protein conjugates, nanoparticle synthesis, and heterogeneous catalysis scaffolds. This chapter seeks to provide insight into recent progress in these areas and to highlight the strengths and limitations of conducting RAFT photopolymerization in water employing low energy light sources in the visible range.
Introduction Visible light provides a low-energy irradiation source that has been exploited for a variety of reversible-deactivation radical polymerization (RDRP) techniques, including reversible addition-fragmentation chain transfer (RAFT) polymerization and atom-transfer radical polymerization (ATRP) (1, 2). Photopolymerizations typically use either photochemically active initiators or electron/energy transfer catalysts to introduce the radicals needed for initiation. However, the majority of © 2018 American Chemical Society
work in this area has involved polymerizations in organic solvents, with aqueous systems only recently having been considered. Aqueous systems allow synthesis in a green, benign solvent and provide significant advantages for polymerizations in biological contexts, which require low energy irradiation conditions to minimize or avoid damage to biomolecules (e.g., proteins, DNA, cells) that can occur during traditional UV-irradiated photopolymerizations. Moreover, aqueous polymerization conditions are also compatible with monomers/polymers that are not typically soluble in organic solvents (e.g., ionic monomers and polyelectrolytes) (3, 4). The combination of low energy irradiation and benign aqueous conditions makes aqueous visible-light RAFT (5) polymerizations ideal for many biological and materials applications.
Catalyst-Free RAFT Photopolymerizations Most thiocarbonylthio (TCT) compounds absorb in the visible light region between 380-525 nm due to the n→π* transition of the C=S group. This range of excitation wavelengths can be used to initiate polymerizations and mediate control via a photoiniferter mechanism (6). Initiation is proposed to occur via homolytic C-S bond cleavage to generate a carbon-centered radical capable of adding to monomer and a stable TCT radical that eventually deactivates growing chains via reversible termination (Figure 1a). Polymerizations were initially reported using low irradiation intensity (4.8 W) and required longer reaction times to reach high monomer conversions (>12 h) (7, 8). Increasing light intensity (from 26 W to 208 W at λ = 402 nm) has been shown to decrease reaction time without a significant loss in polymerization control that could result from increased irreversible termination events (9). For example, when polymerizations were held at 17 °C with 208 W of irradiation power, 95% monomer conversion was achieved in 20 min with good control over molecular weights and molecular weight distributions. However, maintaining the temperature at moderate levels was essential, as an analogous polymerization with the same irradiation intensity but without temperature control reached up to 80 °C as a result of the polymerization exotherm and high irradiation intensity and resulted in a large increase in polymer molar mass dispersity, most likely due to significant hydrolysis of the TCT at elevated temperatures. Initiator-free photopolymerizations are an attractive route to well-defined polymers since every polymer chain is initiated from the R-group of the CTA and no additional reagents are needed. However, higher-energy violet or blue light is typically used to target the peak of the C=S n→π* transition, which could be troublesome over long irradiation times or with high-intensity light sources. An alternative route to increase the rate of polymerization is via the addition of a tertiary amine (10). Tertiary amines are hypothesized to undergo a redox reaction with the excited state TCT to yield the TCT anion and a carbon-centered radical for initiation/propagation (Figure 1b). While adding tertiary amines can minimize reactions times to be more amenable for biological entities, high concentrations (i.e., stoichiometric amounts) of amine to CTA are often required. 44
Figure 1. Initiator-free photopolymerizations where initiation occurs via carbon-sulfur bond photolysis and subsequent addition to monomer to achieve a degenerative chain-transfer equilibrium between propagating chains and reversible termination with the stable sulfur-centered radical (a). The polymerization rate can be increased using a tertiary amine (NR3) that is proposed to undergo a redox reaction with the excited-state thiocarbonylthio moiety (b).
Externally-Initiated RAFT Photopolymerizations Analogous to conventional thermally-initiated RAFT polymerizations, visible-light aqueous polymerizations using an external initiating species that absorbs and fragments in the visible-light region are also possible (Figure 2). The water-soluble poly(ethylene glycol) acrylate (PEGA) monomer was first investigated using (2,4,6-trimethylbenzoyl)diphenylphosphine oxide (TPO), a classic photoinitiator that has absorbance in the visible-light region up to λ = 420 nm (11). PEGA was chosen as the monomer since it has been used extensively in polymer-protein engineering and shows a fast polymerization rate. An initialization period was observed for all polymerizations, an observation attributed to conversion of the CTA into oligomers prior to significant propagation, but well-defined polymers were prepared with up to 80% conversion of PEGA after 10 min. These results indicated that photolysis of the TPO initiator resulted in fast and well-controlled polymerizations at room temperature. The 45
polymerization conditions were also amenable to other acrylic monomers at various pH ranges (12). However, the CTA was found to be more stable under acidic pH (13, 14), while hydrolysis rates up to 3.3% were observed in solutions at pH 10.2. Interestingly, oxygen tolerance could be achieved for polymerizations initiated by TPO when the oxygen-scavenging enzyme glucose oxidase (GOx) and glucose were included (15).
Figure 2. Externally-initiated RAFT photopolymerization mechanism analogous to a thermally-initiated RAFT polymerization.
Compared to initiator-free systems where every chain is initiated from the CTA R-group, the overall polymer composition synthesized by RAFT in the presence of an external initiator contains more chain-end heterogeneity. Although only a small fraction of initiator relative to CTA is introduced into the system, that same fraction of chains are expected to either not contain the CTA-derived R-group at the α chain end (due to the initiating fragments) or the thiocarbonylthio at the ω chain end (due to irreversible radical-radical coupling or chain-transfer events). Photoinduced Electron/Energy Transfer RAFT Photopolymerizations Photoinduced electron/energy-transfer (PET) catalysts are widely used in organic synthesis to conduct single-electron redox reactions (16). Many photocatalysts (PCs) have been reported to efficiently mediate both ATRP (17, 18) and RAFT polymerization (19, 20) through a redox reaction with the halide or TCT, respectively. However, the most prevalent PCs 46
(tris[2-phenylpyridinato-C2, N)iridium(II) (Ir(ppy)3), phenothiazine, etc.) are poorly soluble in aqueous systems, precluding their use for polymerizations in water. Tris(2,2′-bipyridyl)ruthenium(II) chloride (Ru(bpy)3Cl2) was the first PC to be employed under aqueous conditions by Boyer and co-workers (21). The oxidative quenching pathway of the catalyst was used to activate the TCT RAFT compound and initiate RAFT polymerization of DMA (Figure 3a). This pathway worked exceptionally well in water, even at low catalyst loadings relative to CTA ([CTA] : [Ru(bpy)3Cl2] = 1 : 2×10-4). Additionally, this polymerization approach worked for other acrylamides, acrylates, and methacrylates. When switching the polymerization medium to fetal bovine serum (FBS), the resultant polymers showed a broad molecular weight distribution (Ð > 1.5) when the [CTA] : [Ru(bpy)3Cl2] ratio was 1 : 2×10-4. Upon increasing the [CTA] : [Ru(bpy)3Cl2] ratio tenfold to 1 : 2×10-3, much lower molar mass dispersities were obtained. Therefore, the FBS was proposed to interact with the catalyst to encumber polymerization when catalyst loading was too low. Polymerizations using Ru(bpy)3Cl2 in the presence of ascorbic acid as a reducing agent, inducing a reductive quenching mechanism of the excited-state PC (Figure 3b), reach 90% conversion in 30 min (22). These polymerization rates were especially effective to polymerize activated ester monomers in aqueous solutions while maintaining >85% of their hydrolytically sensitive ester moieties. An inexpensive and less-toxic alternative to Ru(bpy)3Cl2 is the water soluble zinc porphyrin (Zn(II) meso-tetra(4-sulfonatophenyl)porphyrin), which also works with a variety of monomer classes and can be activated or deactivated according to the pH of the solution (23, 24). Indeed, metal-based PCs work exceptionally well and facilitate fast polymerization rates in aqueous systems using low catalyst loadings and allow irradiation wavelengths from violet to red; however, the potential toxicity of Ru is a concern when using metal-based PET-RAFT polymerizations. Metal-free approaches to PET-RAFT polymerization have also been reported that employ inexpensive organic dyes as PCs to make polymerization conditions more amenable to biological applications. Eosin Y (EY) can be used in PET-RAFT polymerizations in organic media (25) and was the first organic dye used in aqueous polymerizations, as described by Hawker and coworkers (26). In this report, a tertiary amine co-catalyst reduced the excited triplet state of EY to the radical anion, which then readily reduced TCT compounds to liberate the carbon-centered radical that initiated polymerization and led to activation/propagation later in the polymerization. Since a reductive quenching mechanism was employed, oxygen tolerance was realized. Later mechanistic studies of EY under both blue and green irradiation wavelengths provided more insight into the use of this organocatalyst for RAFT polymerization (27). While the reductive catalyst pathway is more commonly reported, an oxidative pathway (i.e., polymerization in the absence of a tertiary amine) yields polymer molecular weights much closer to predicted molecular weights. The authors hypothesized that this results from the high concentration of radicals present in reducing conditions leading to reduced control during polymerization. The pH of the polymerization solutions also affected the apparent rate of polymerization, as at a lower pH, a slower kp, app was observed, which was attributed to the partial protonation/deactivation of the catalyst and/or tertiary amine reducing agent. 47
Figure 3. Photo-induced electron transfer RAFT polymerization using a photocatalyst (PC) via either an oxidative catalyst pathway (a) or a reductive catalyst pathway (b, NR3 can also be ascorbic acid).
PET-RAFT initiation relies on the PC activating the CTA to induce C-S bond cleavage, generally resulting in every polymer chain being initiated from the R-group. Oxidative quenching mechanisms of the PC show exceptional characteristics of RDPD. A reductive-quenching catalyst initiation mechanism also introduces oxygen tolerance to the polymerization via concurrent reduction of molecularly dissolved oxygen to yield peroxides, but other radical initiation species (e.g., peroxides, amine radical cations) may be introduced into the system causing some discrepancies in kinetics and molecular weight control. Overall, these characteristics yield well-defined polymer distributions with homogeneous end groups and bode well for biological applications, especially those that require low catalyst loadings, mild irradiation wavelengths, and aerobic polymerizations conditions. Amphiphilic Self-Assembled Nanoparticles Visible-light techniques have been used extensively to synthesize various nanoparticle morphologies (e.g., micelles, worms, vesicles) using catalyst-free, externally-initiated, and PET-RAFT polymerizations, since these methods all allow block copolymers to be precisely accessed. Control of nanoparticle morphology is commonly achieved through polymer dispersion parameters, 48
and in particular, recent advances in polymerization-induced self-assembly (PISA) (28–30) have led to targeting of specific self-assembled structures under relatively high concentrations. PISA provides a facile reaction set-up whereby a chain-extension polymerization is performed using a hydrophilic macro-chain transfer agent (macro-CTA) with a monomer that forms a hydrophobic polymer to eventually induce phase separation (i.e., self-assembly) during the polymerization. The nanoparticle morphology is dictated according to the hydrophilic-to-hydrophobic volume fractions of the block copolymers, which can be easily manipulated via either monomer feed ratio or monomer conversion.
Figure 4. (a) Polymerization scheme to achieve vesicles loaded with functional proteins; (b) cryo-TEM and fluorescence micrographs of empty vesicles (I, II) and vesicles loaded with green fluorescent protein (III, IV); (c) fluorescence absorption (dotted lines) and emission (solid lines) of untreated protein, the first supernatant after washing, the disassembled vesicles that contained green fluorescent protein after purification, and the second supernatant after washing. Adapted with permission from ref. (30). Copyright 2017 American Chemical Society. 49
Catalyst-free photo-PISA has been employed to prepare vesicle-based nanoreactors that encapsulated either green fluorescent protein (GFP), horseradish peroxidase (HRP), or glucose oxidase (GOx) (31). Using 405 nm light, a photoiniferter mechanism was accessed to polymerize N-2-hydroxypropyl methacrylate (HPMA) from a PEG-based macro-CTA to form vesicles. Passive encapsulation of the proteins yielded nanoreactors, which were shown to have a permeable membrane that would allow diffusion of reactants into the vesicle interior to undergo enzymatic reactions. Figure 4 shows the synthesis of GFP-loaded vesicles (Figure 4a), confirmation of vesicle formation and GFP loading (Figure 4b), and purification of the GFP-loaded vesicles (Figure 4c). Vesicle systems containing HRP or GOx retained activity following photo-PISA and purification, and a cascade reaction was successfully performed employing both HRP- and GOx-containing vesicles. Another report of protein encapsulation into PISA-derived vesicles was reported by Li and coworkers via photo-PISA of HPMA using a TPO-based photoinitiator (32). First, the polymerizations were characterized in the absence of protein and found to reach >95% conversion in 15 min, and a linear increase in molecular weight with conversion and low molar mass dispersities were observed. Importantly, various nanoparticles morphologies including spheres, lamella, vesicles, multi-lamellar vesicles, and worms could be accessed depending on the solids concentration or PHPMA degree of polymerization (Figure 5). Then, bovine serum albumin (BSA) was successfully encapsulated into the vesicles, and the enzyme retained activity after polymerization, demonstrating that the mild synthesis conditions did not affect protein integrity. Externally-initiated RAFT photopolymerizations have also elucidated the differences between photo-PISA and thermally-regulated PISA. During thermodesmic polymerizations, photo-PISA reactions using a high light intensity yielded kinetically trapped structures that rearranged into other morphologies after annealing (33). Conversely, using either lower-intensity light irradiation or a thermal initiation approach yielded stable aggregates, even after annealing. The authors were able to show that high-intensity irradiation at wavelengths where the CTA absorbs (405 nm) can lead to fragmentation of the C-S bond and decrease the end-group fidelity of the polymers, thereby affecting morphology. Furthermore, Tan et. al. demonstrated that low temperatures can lead to macroscopic precipitation or kinetically trapped structures, as the core blocks may not be mobile enough to rearrange (34). Few aqueous PET-RAFT PISA systems have been reported, but rely on either Ru (35, 36) or EY (37) catalysts. The Ru-based system was used to synthesize micrometer-sized vesicles (35, 36), contrary to the nanometer-sized vesicles typically synthesized. PEG-HPMA vesicles up to 10 microns in diameter were prepared, which, interestingly, enabled direct observation of the polymeric vesicles using light microscopy. The polymer morphology oscillated between vesicles and droplets during the polymerization due to the thermodynamic instability of the mesoparticles, but the structures could be “frozen” in the absence of irradiation. PISA experiments using EY and ascorbic acid under green-light irradiation (37) led to oxygen tolerance in low volumes (20 μL), while maintaining the expected morphology progression. The authors also 50
demonstrated this approach could be applied to different topological polymer architectures, including core-crosslinked stars. In another Ru-based system, the viscosity change of a solution transitioning from a spherical morphology to a worm-like morphology was used to precisely target worm phases during PISA in organic media (38). This approach should be analogous in aqueous solutions and provide facile access to the worm phase, which occupies a small area of PISA phase diagrams and can be difficult to observe.
Figure 5. Phase diagram of diblock copolymer nanoparticle morphologies achieved using photo-PISA to grow an N-2-hydroxpropyl methacrylamide block from a poly(ethylene glycol) macro-chain transfer agent. Reprinted with permission from ref. (31). Copyright 2015 American Chemical Society.
Polymer-Protein Conjugates The mild conditions of visible-light mediated polymerizations prove to be remarkably amenable to conditions that benefit and preserve sensitive biological materials. In particular, this fact facilitates the synthesis and engineering of hybrid bio-synthetic materials, since protein degradation can be avoided and the low energy irradiation even causes minimal damage to live cells. Chen and coworkers reported using an externally-initiated visible-light RAFT polymerization to graft-from E. coli inorganic pyrophosphatase (PPase) by site-specifically attaching a CTA near the active site of a mutated PPASE (39). Using sodium TPO as the initiator, polymerizations displayed increased molecular weights with conversion by GPC, 1H NMR spectroscopy, and SDS-PAGE, and on-off cycles demonstrated that the polymerization required light 51
to proceed. Since the polymer was close to the active site, upon the attachment of a thermoresponsive polymer (poly(N-isopropylacrylamide) (PNIPAM)), the activity of the enzyme could be regulated through the coil-to-globule transition of PNIPAM around the cloud point of the polymer.
Figure 6. Characterization of the PET-RAFT polymerization of N,N-dimethylacrylamide using eosin Y as a photocatalyst to graft-from lysozyme. (a) Pseudo-first order kinetics plot; (b) SDS-PAGE of kinetics time points; (c) GPC analysis of polymer-protein conjugate and cleaved polymer. Adapted with permission from ref. (38). Copyright 2017 American Chemical Society. Grafting-from proteins using PET-RAFT polymerization has been reported using either Ru(bpy)3Cl2 (21) or EY (40) as the photocatalyst. The first example of polymer-protein conjugates using PET-RAFT involved CTA attachment to BSA via disulfide conjugation, followed by PET-RAFT polymerization using Ru(bpy)3Cl2 as a PC to yield polymer-protein conjugates of either poly(N,N-dimethylacrylamide) (PDMA) or PPEGA. Following reduction of the disulfide, GPC analysis confirmed that well-defined polymers had been successfully polymerized. Polymers synthesized using EY as a PC were prepared by modifying lysozyme with a trithiocarbonate CTA via carbodiimide coupling (40). Polymerizations in the presence of a sacrificial tertiary amine co-catalyst yielded fast polymerizations at dilute monomer concentrations with good control 52
over polymer dispersity and high chain-end retention of the CTA (Figure 6). Acrylate and styrenic monomers were also polymerized, albeit both monomer classes required longer polymerization times due to the differences in monomer radical stability. Importantly, lysozyme activity was minimally affected by the polymerization conditions. Combining polymer-protein conjugates and dispersion polymerization, BSA was conjugated with CTAs using activated-ester chemistry (41). PET-RAFT polymerization of HPMA using a Ru catalyst was performed to grow multiple chains from the protein. The growing amphiphilicity of the polymers induced aggregation, leading to the formation of spherical nanoparticles (Figure 7). Although different morphologies were not attained (ascribed to BSA incorporating into the core of the nanoparticles), the size of the spheres increased with polymer molecular weight. In accordance with previous polymer-protein syntheses using photoinduced RAFT polymerization, the conjugates retained activity. Also, the amphiphilicity of the nanoparticles facilitated encapsulation of hydrophilic and hydrophobic drugs.
Figure 7. (a) GPC analysis of polymers following enzymatic degradation of BSA; (b) conversion and dispersity of polymer-protein conjugates; (c-e) TEM of polymer-protein conjugate aggregates showing increasing size. Scale bars = 500 nm. Adapted with permission from ref. (39). Copyright 2017 American Chemical Society. PET-RAFT polymerization has also been used to efficiently grow polymers from the surface of live yeast and Jurkat cells (26). EY and the co-catalyst triethanolamine (TEA) were used to rapidly grow PEG acrylamide (PEGAm), azide-functionalzied PEGAm (PEGAm-azide), and biotin-functionalized PEGAm (PEGAm-biotin) brushes from cell surfaces. For yeast cells, amine residues on membrane-bound proteins on the cell surface were derivatized 53
with dibenzylcyclooctyne (DBCO) groups and subsequently reacted with azide-functionalized CTAs in a metal-free azide-alkyne cycloaddition. Using high CTA to EY ratios (CTA:EY = 1:0.01) and additional low molecular weight CTA, well-controlled PEGAm and PEGAm-co-PEGAm-azide brushes were synthesized. Cell viability remained >95%, surface functionalization of the azide groups of PEGAm-azide with Alexafluor 647 DBCO proved successful, and cell aggregation was induced after using the polymers containing PEGAm-biotin to conjugate with streptavidin. Incorporating a degradable disulfide linker between the cell and growing polymer facilitated characterization of the grafted polymers and evaluation of CTA chain-end retention on the yeast cell surface. Interestingly, normal cell functions were not inhibited after polymer grafting, demonstrated through the observation that polymer was incorporated into daughter cells following proliferation. The Jukart cell studies relied on similar chemistry, though a non-covalent approach was used to immobilize the CTA to the cell surface by modifying a TTC with a lipophilic tail that could insert into the cell membrane. Heterogeneous Catalysis PET-RAFT polymerization has been demonstrated to provide ideal conditions for a variety of polymer engineering pursuits. However, a significant challenge is posed by the possible retention of catalyst in the polymer products following polymerization. For example, trace metals such as ruthenium can be toxic, and EY has a distinct pink color, even at low concentrations. One route to avoid polymer impurities after polymerization is to covalently attach the PET catalyst to a heterogeneous material. Shanmugam et. al. successfully attached EY to silica nanoparticles and showed that the catalyst remained active, even after successive recycles (42). EY is known to photobleach during polymerization due to bimolecular quenching mostly through the formation of the leuco form of the dye (27). By physically restricting the dye to limit bimolecular termination events, no photobleaching was observed. This technique worked with a variety of monomer classes and solvents, as well.
Conclusion Initiator-free, externally-initiated, and PET visible-light RAFT polymerization techniques in aqueous media provide access to multiple polymer classes and architectures. Each means of initiation possesses certain limitations; however, precise polymer synthesis is possible for all these strategies. Consequently, aqueous visible-light RAFT polymerizations are providing fundamental insights into light-initiated polymerizations and enabling the development of new synthetic routes to bio-derived and nanoparticle-based materials. Fundamentally, for example, the effects of temperature and light intensity or the effects of PET initiation mechanism on the polymerization outcome are being investigated. Additional elucidation into the effect of polymerization initiation/activation mechanism on end-group composition or termination events occurring during PET-RAFT would facilitate further 54
understanding of the radical processes operative during irradiation. The material applications provide perhaps the most direct and powerful way to synthetically alter biological entities using polymers (e.g., polymer-protein bioconjugates) or incorporate biological entities into polymeric materials. Nanoparticles are readily accessible using visible-light techniques, while derivatization with proteins provides an avenue to bio-based nanoparticles under conditions that do not disrupt protein function. Furthermore, oxygen-tolerant polymerization conditions introduce myriad opportunities for engineering on or in living species. Therefore, light irradiation provides control over polymerizations with temporal and spatial specificity during materials synthesis, while the mild polymerization conditions are resulting in significant advances in bio-synthetic polymer engineering.
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