bk-2013-1144.ch012

Chapter 12

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Biocatalytic ATRP: Controlled Radical Polymerizations Mediated by Enzymes Kasper Renggli, Mariana Spulber, Jonas Pollard, Martin Rother, and Nico Bruns* Department of Chemistry, University of Basel, Klingelbergstrasse 80, CH-4056 Basel, Switzerland *E-mail: [email protected]

The advent of controlled radical polymerizations has made polymer science a key discipline for the preparation of nano-, biomedical-, and high tech-materials. Atom transfer radical polymerization (ATRP) is one of the most widely applied controlled radical polymerization. However, an ongoing quest is to develop ATRP reaction conditions that allow reducing the amount of catalyst needed, or to replace the currently used transition metal complex catalysts with less toxic ones. Using enzymes as catalysts is a classic strategy in the green chemistry approach, and many enzymatic polymerizations are known. However, controlled radical polymerizations that are catalyzed by enzymes or proteins were not known until our discovery that the metalloproteins horseradish peroxidase and hemoglobin can polymerize vinyl-monomers under conditions of activators regenerated by electron transfer (ARGET) ATRP. In this book chapter, we review the emerging field of biocatalytic ATRP.

© 2013 American Chemical Society In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Introduction Radical polymerization is one of the most widely employed reactions to synthesize polymers on an industrial scale or in academic labs, due to its simplicity and tolerance of functional groups. Products like polystyrene (e.g. Styrofoam), polymethylmethacrylate (Plexiglass), and polyvinylchloride (PVC) are all synthesized on a scale of millions of tons worldwide by free radical polymerization, i.e. a chain growth polymerization in which the active, propagating species are radicals. These radicals are highly reactive and readily undergo termination and side reactions that interrupt the growth of a polymer chain (1). Due to the short life-span of the growing chains, they are active for about 1 s, chemical control of these reactions is poor, giving rise to ill-defined molecular weights and preventing end-group-functionalization of polymer chains (1). Poor control represents the main drawback of the method, especially when well-defined molecular weights, architectures, sequences of monomers, and functional chain end groups are desired. For example, block copolymers of a specific design can be used as emulsifiers, as drug-delivery systems, as building blocks for nanostructures, and as materials in solar cells and batteries (2–7). The formation of conjugates of polymers and proteins, e.g. for therapeutic applications, relies on the well-defined end-group chemistry of polymers (8, 9). If control of radical polymerization is achieved, some materials’ applications, e.g. self-healing plastics, can be implemented (10). Over the last two decades, synthesis techniques have been developed that allow controlling radical polymerization, such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, and nitroxide-mediated radical polymerization (NMP) (1, 11). Each of these methods has its specific strengths and drawbacks. ATRP is one of the most popular, controlled radical polymerizations because of its great versatility: It is applicable to most vinyl and styrene monomers, tolerates most functional groups, is compatible with proteins and other biomolecules and it results in halide-terminated polymer chains that can be easily converted into a multitude of other functional end-groups (1, 11–14). ATRP relies on the reversible deactivation of propagating radicals by transition metal complex catalysts, most often copper(I)-copper(II) redox couples, thus lowering the radical concentration in a reaction and therefore the chances of chain termination, while still producing reactive chain ends (1). Without a doubt, ATRP and other controlled radical polymerizations are amongst the most important recent developments in the field of polymer chemistry and have spurred thousands of scientific publications and several industrial processes (11). Polymers prepared by ATRP can be used, e.g., as sealants, lubricants, oil additives, wetting agents, blend compatibilizers, surfactants and pigment stabilizers (11). However, ATRP also suffers some limitations. The catalysts are tedious to remove from a polymer product, causing unwanted coloration, toxicity and environmental issues (12, 15). The problem of residual traces of transition metal or amine ligands in final products can hinder biomedical, food-grade, and electronic applications of the polymers. Several recent developments aim to make ATRP environmentally friendlier and the resulting polymers more compatible with biomedical, food grade and electronic requirements. Less toxic iron catalysts are investigated 164 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


as alternative to copper-based catalysts (13)(16), the ATRP catalyst can be scavenged during work-up of the polymerization (12, 15) and variations of the experimental protocol of ATRP, such as Activators ReGenerated by Electron Transfer (ARGET) and Initiators for Continuous Activator Regeneration (ICAR) ATRP lead to a significant reduction in concentration of catalysts required for this polymerization (11, 12). Radical polymerizations are not limited to man-made processes. Nature uses radical polymerization to produce a variety of biopolymers (17). The prime example is the synthesis of lignin by an enzyme-catalyzed coupling between aromatic compounds (18). Lignin’s key function is to strengthen wood, and it is the second most abundant polymer on earth (19). However, the manner in which Nature controls this polymerization is not fully elucidated and is a topic of ongoing scientific debate (18–20). Enzymes are environmentally friendly, sustainable, and non-toxic catalysts (21). They are derived from natural resources, are completely biodegradable (and even edible), and work under mild conditions such as ambient temperature, ambient pressure and in aqueous solution. Moreover, they are highly selective, allowing for desired regio-, stereo-, or chemo-selective transformations. Not surprisingly, enzymes have been extensively used in vitro to the benefit of synthetic chemists, and are often considered as “green” alternatives for conventional catalysts (21–25). Indeed, many enzymatic reactions have been exploited for the synthesis of polymers, including polycondensation, ring-opening polymerizations, free radical polymerizations of vinyl-type monomers and the polymerization of aromatic compounds by radical-induced oxidative coupling (22–25). However, controlled radical polymerizations catalyzed by enzymes remained unknown. Recently our group (26, 27) and di Lena and coworkers (28) discovered concurrently and independently that some metalloproteins can mediate ATRP. These findings represent the first reports of biocatalytic, controlled radical polymerization.

ATRPases While investigating conjugates of proteins and copper complexes as ATRP catalysts to confine radical polymerization into the cavity of the protein cage thermosome (29, 30), we discovered that the heme proteins horseradish peroxidase (HRP) and bovine hemoglobin (Hb) can catalyze the polymerizations of N-isopropylacrylamide (NIPAAm), polyethyleneglycol methylether acrylate (PEGA) and polyethyleneglycol methylether methacrylate (PEGMA) in aqueous solutions under ARGET ATRP conditions, i.e. in the presence of an excess of ascorbic acid or sodium ascorbate as reducing agents (Figure 1) (26, 31). The polmyerizations were not only catalyzed by pure enzymes, but also by fresh red blood cells. Investigation of PNIPAAm polymers with 1H COSY NMR and neutron activation analysis (NAA) revealed that the polymer chains carried the ATRP initiator and that the chains were bromine terminated (26). Thus, the activity of the enzymes in these reactions encompasses radical formation due to a homolytic cleavage of a C-Br bond of an organohalogen initiator, and reversible halogen 165 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


atom transfer from the initator to the enzyme and back to the polymer chain, giving rise to the same type of radical control as in conventional, transition-metal catalyzed ATRP. We therefore named this novel activity of enzymes and proteins ATRPase activity. While HRP can be used without further modification as a catalyst in these reactions, native Hb formed protein-polymer conjugates, due to radical chain transfer to a free cysteine residue on the surface of the protein. Blocking the cysteine with N-(2-hydroxyethyl) maleimide prior to polymerization (yielding Cys-blocked Hb) suppressed this side reaction.

Figure 1. Examples of ATRP catalyzed by hemoglobin (Cys-blocked Hb) or horseradish peroxidase (HRP). The polymerizations with HRP and Cys-blocked Hb followed first order kinetics at room temperature and yielded polymers with relatively low molecular weight distributions. Using 2-hydroxyethyl-2-bromoisobutyrate (HEBIB) as the initiator, HRP catalyzed the polymerization of NIPAAm to polyNIPAAm with a polydispersity index (PDI) down to 1.44 (26). With the same initiator, Cys-blocked Hb produced polyPEGA with PDIs between 1.14 – 1.42 and polyPEGMA with PDIs below 1.2 (31). As an example, the kinetics of the polymerization of PEGA using 2-bromopropionitrile (BPN) as the initiator are shown in Figure 2. After an induction period of approx. 30 min in which presuamably small conformational changes occured within the protein (see below), the reaction followed first order kinetics and the molecular weight increased linearly with conversion. The PDI was around 1.1. Taken together, these findings show that biocatalytic ATRP can result in controlled radical polymerizations and that biocatalysts have potential as environmentally benign catalysts for the synthesis of well-defined polymers. However, the degree of control, especially of NIPAAM polymerizations, was not perfect. Thus, there is still a need to optimize reaction parameters, to identify and suppress possible side reactions and to tailor the ratio of activation and deactivation rates in the ATRP equilibrium. 166 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Figure 2. (a) Kinetic plot and (b) evolution of molecular weight (■) and polydispersity index (○) with conversion in an ARGET ATRP of PEGA catalyzed by Cys-blocked Hb using BPN as initiator. (Reaction conditions: Molar ratio of BPN:PEGA:ascorbic acid:Hb 1:77:1:0.007; water, room temperature). (Data from reference (31)).

Enzymes are much more complex catalysts than the simple transition metal complexes usually used for ATRP. The performance of the biomolecules depends not only on the properties of the metal center, but is also a function of the whole macromolecular structure of the biomolecules. Therefore, our studies on ATRPase activity were flanked by extensive characterization of the biomolecules before, during and after the polymerizations with methods including gel electrophoresis, circular dichroism (CD) spectroscopy, UV/Vis spectroscopy and mass spectrometry. HRP and Cys-blocked Hb were stable under the reaction conditions and did not precipitate. Neither did they form conjugates (as revealed by size exclusion chromatography and mass spectrometry; see Figure 3 for the data of HRP). Moreover, the CD spectrum of HRP proved that the protein’s 167 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


structure was not altered during the reaction. Cys-blocked Hb showed some minor changes in the CD spectrum, indicating that it underwent slight conformational changes. Hb is a tetrameric protein consisiting of two α- and two β-subunits. UV/Vis measurements during the polymerization revealed that, under the chosen reaction conditions with ascorbate as the reducing agent, only the β-subunits participated in the redox reactions involved in ATRP. These results exemplify that it is essential to study the fundamentals of ATRPase activity, both from a polymer chemistry perspective and from a biochemistry perspective, in order to understand the role of the biomolecules in these reactions.

Figure 3. Characterization of HRP before and after polymerization of NIPAAm under ARGET ATRP conditions at pH 6.0 for 16 h. (a) circular dichroism spectra, (b) UV/Vis spectra, (c) native and SDS gel electrophoresis. (d) MALDI-TOF mass spectrum of recycled HRP shows the molecular ion peak of HRP+ at m/z 44000, HRP2+ at m/z 22000, HRP dimer+ at m/z 88000 and HRP trimer2+ weak at m/z 66000. (Reproduced with permission from reference (26). Copyright 2011 Wiley-VCH Verlag GmbH & Co. KGaA).

Independent of our results, Di Lena and coworkers reported free radical polymerizations that were initiated from an alkylbromide initiator by the copper enzyme laccase under reductive conditions (32). Polymerization of PEGMA was performed in homogeneous aqueous solution. Emulsion polymerization was used for the water-insoluble monomers methylmethacrylate, styrene and hydroxyethyl methacrylate. The reactions were free radical polymerizations with no control over the molecular weight. They yielded polymers of high molecular weight that were often insoluble. The addition of chain transfer agents and of RAFT 168 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


agents allowed controlling the reactions. In their next paper, the authors turned to the heme enzyme catalase and the monomer PEGA (28). Controlled radical polymerizations were achieved under aqueous ARGET ATRP conditions at 60 °C. The reactions resulted in polymers with low molecular weight distributions (polydispersity indices (PDI) between 1.2 and 1.7), and bromine-terminated chain ends. The molecular weight increased with conversion and the reaction followed first order kinetics. A limited set of reactions was also performed with the enzymes HRP and laccase, with similar results. In these two papers the ezymes were not characterized with biochemical methods during or after the polymerizations. Nevertheless, these reports underscore the fact that enzymes can catalyze ATRP, and therefore the reports from the two groups complement each other nicely.

Conclusion and Outlook In conclusion, the first examples of biocatalytic ATRP are promising approaches to alleviating environmental and toxicity issues that hamper conventional ATRP. The investigated enzymes are non-toxic and environmentally friendly catalysts. Moreover, enzymes are easy to remove from a polymerization mixture by methods well-established in biochemistry, such as precipitation with ammonium sulfate or affinity binding to microbeads. Some of the investigated enzymes, especially Hb, are cheap and abundantly available (Hb is a waste product of meat production), scale-up with these enzymes is thus feasible. In order to be able to compete with conventional ATRP catalysts in terms of performance, the biocatalysts will need to improve. Fortunately, a whole range of methods exist in biotechnology to enhance the catalytic performance of biocatalysts. The optimization of simple parameters such as pH, temperature, addition of cosolvents, or the concentration of salts is one possibility. More sophisticated methods include the use of enzymes in organic solvents, or genetic engineering methods. Enzymes potentially offer opportunities that cannot be realized with conventional ATRP catalysts. For example, some monomers are difficult to polymerize in ATRP. These include amine-containing monomers, as they tend to complex copper non-specifically, and acrylic acid, which protonates the amine-ligands of ATRP catalysts. These problems are unlikely to be encountered with enzymes, as the metal ions are firmly bound within the protein structure and the protonation of coordinating residues and cofactors within the active site is controlled (and therefore buffered) by the overall electrostatic potential of the enzyme. Thus, it is expected that the use of ATRPases can complement the monomer range of ATRP. Many interesting questions regarding the biochemical mechanism, the scope of monomers and accessible polymers, as well as applications of ATRPases in material and nano sciences, remain yet to be elucidated and will be the scope of future work.

169 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

Acknowledgments Generous financial support by the Swiss National Science Foundation, by the Holcim Stiftung Wissen, by the NCCR Nanosciences, by a Marie Curie Intra European Fellowship and a Marie Curie European Reintegration Grant within the 7th European Community Framework Programme is gratefully acknowledged. We thank Mark Inglin for editing and proofreading the manuscript.


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