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Investigating the Mechanism of Horseradish Peroxidase As a RAFT-Initiase

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Investigating the Mechanism of Horseradish Peroxidase As a RAFT-Initiase polymers Article Investigating the Mechanism of Horseradish Peroxidase as a RAFT-Initiase Alex P. Danielson 1, Dylan Bailey Van-Kuren 1, Joshua P. Bornstein 1, Caleb T. Kozuszek 1, Jason A. Berberich 2, Richard C. Page 1 ID and Dominik Konkolewicz 1,* ID 1 Department of Chemistry and Biochemistry Miami University 651 E High St, Oxford, OH 45056, USA; [email protected] (A.P.D.); [email protected] (D.B.V.-K.); [email protected] (J.P.B.); [email protected] (C.T.K.); [email protected] (R.C.P.) 2 Department of Chemical, Paper and Biomedical Engineering Miami University 650 E High St, Oxford, OH 45056, USA; [email protected] * Correspondence: [email protected] Received: 20 June 2018; Accepted: 3 July 2018; Published: 5 July 2018 Abstract: A detailed mechanistic and kinetic study of enzymatically initiated RAFT polymerization is performed by combining enzymatic assays and polymerization kinetics analysis. Horseradish peroxidase (HRP) initiated RAFT polymerization of dimethylacrylamide (DMAm) was studied. This polymerization was controlled by 2-(propionic acid)ylethyl trithiocarbonate (PAETC) in the presence of H2O2 as a substrate and acetylacetone (ACAC) as a mediator. In general, well controlled polymers with narrow molecular weight distributions and good agreement between theoretical and measured molecular weights are consistently obtained by this method. Kinetic and enzymatic assay analyses show that HRP loading accelerates the reaction, with a critical concentration of ACAC needed to effectively generate polymerization initiating radicals. The PAETC RAFT agent is required to control the reaction, although the RAFT agent also has an inhibitory effect on enzymatic performance and polymerization. Interestingly, although H2O2 is the substrate for HRP there is an optimal concentration near 1 mM, under the conditions studies, with higher or lower concentrations leading to lower polymerization rates and poorer enzymatic activity. This is explained through a competition between the H2O2 acting as a substrate, but also an inhibitor of HRP at high concentrations. Keywords: RAFT polymerization; enzymatic polymerization; reaction kinetics; horseradish peroxidase; polymerization mechanism 1. Introduction Enzymes are fundamental to biological processes due to their ability to efficiently catalyze reactions [1]. These same catalytic properties have applications in chemistry and biochemistry and can be used to synthesize complex molecules and materials [2,3]. Of particular interest is the concept of using enzymes to catalyze polymerization reactions and allow for polymer synthesis at faster reaction rates and under mild conditions [4–7]. This takes advantage of the efficiency of enzymes albeit for a new, non-native function, such as synthetic chemistry. The discovery of new enzyme applications for polymerization and the optimization of these processes through mechanistic and kinetic studies offer potential environmental and economic benefits, which makes them significant areas of interest [8]. Free radical polymerization is a commonly used polymerization technique that allows for the synthesis of a broad range of materials [9,10]. This type of polymerization involves a radical-producing initiation step, which can come from various sources including thermal initiators, photochemical processes, and can be catalyzed by a variety of enzymes such as horseradish peroxidase [11–23]. However, free radical polymerization has certain limitations, such as poor control over polymer Polymers 2018, 10, 741; doi:10.3390/polym10070741 www.mdpi.com/journal/polymers Polymers 2018, 10, 741 2 of 14 Polymers 2018, 10, x FOR PEER REVIEW 2 of 14 microstructurephotochemical and processes, broad molecularand can be weight catalyzed distributions by a variety [ 24of]. enzymes Reversible-deactivation such as horseradish radical polymerizationperoxidase (RDRP)[11–23]. representsHowever, free an alternativeradical polymerization type of polymerization has certain thatlimitations, allow forsuch the as synthesis poor of well-definedcontrol polymersover polymer and is compatiblemicrostructure with aand wide broad range ofmolecular functional weight groups distributions [25,26]. RDRP [24]. methods includeReversible-deactivation nitroxide-mediated radicalradical polymerization polymerization (NMP), (RDRP) atom-transfer represents radicalan alternative polymerization type (ATRP),of and reversiblepolymerization addition-fragmentation that allow for the synthesis polymerization of well-defined (RAFT) polymers [27– 30and]. is Metalloenzymes compatible with a have wide been range of functional groups [25,26]. RDRP methods include nitroxide-mediated radical demonstrated as efficient initiators for RDRP processes, especially those that follow the ATRP-like polymerization (NMP), atom-transfer radical polymerization (ATRP), and reversible mechanism [17,31–35]. This is in addition to the deoxygenation processes facilitated by enzymes such as addition-fragmentation polymerization (RAFT) [27–30]. Metalloenzymes have been demonstrated glucoseas efficient oxidase initiators to promote for polymerizationRDRP processes, underespecially simple those conditions that follow [36 the–39 ATRP-like]. mechanism RAFT[17,31–35]. is a This prominent is in addition RDRP to the variant deoxygenation which offers processes distinct facilitated advantages by enzymes such such as as compatibility glucose withoxidase a wide to range promote of functionalpolymerization groups under and simple the conditions ability to [36–39]. be run under simple and near ambient conditionsRAFT [40]. is Horseradisha prominent RDRP peroxidase variant which (HRP) offers has beendistinct shown advantages as an such effective as compatibility initiator forwith RAFT polymerizationa wide range [39 of,41 functional–44], making groups HRP and a RAFT-initiasethe ability to be or run an enzymeunder simple capable and of near initiating ambient RAFT reactions.conditions Horseradish [40]. Horseradish peroxidase pero catalyzesxidase (HRP) the generation has been ofshown free radicalsas an effective from hydrogen initiator for peroxide RAFT [45]. Acetylacetonepolymerization (ACAC) [39,41–44], is used making as a radical HRP mediator,a RAFT-initiase which or transfers an enzyme the radicalcapable toof theinitiating monomer, RAFT which reactions. Horseradish peroxidase catalyzes the generation of free radicals from hydrogen peroxide can then enter the RAFT equilibrium. Unlike ATRP processes that use HRP and other metalloproteins as [45]. Acetylacetone (ACAC) is used as a radical mediator, which transfers the radical to the catalystsmonomer, [31–35 which], RAFT can has then a potential enter the advantageRAFT equilib inrium. enzymatic Unlike RDRP. ATRP Thisprocesses is because that use a well-controlled HRP and RAFTother process metalloproteins requires the as enzymecatalysts to[31–35], catalyze RAFT radical has a generation,potential advantage but not in radical enzymatic deactivation RDRP. This back to the dormantis because state, a well-controlled since control RAFT is attained process through requires the the RAFT enzyme degenerative to catalyze transfer radical generation, equilibrium but [46 ,47]. In contrast,not radical enzymatic deactivation ATRP back processes to the dormant require state, the enzymesince control to be is responsibleattained through for both the RAFT chain-end activationdegenerative and radical transfer deactivation. equilibrium In [46,47]. the RAFT In processescontrast, enzyma initiatedtic usingATRP an processes enzyme, require a chain the transfer agentenzyme (CTA) to such be responsible as (2-propionic for both acid)yl chain-end ethyl ac trithiocarbonatetivation and radical is useddeactivation. to facilitate In the the RAFT uniform propagationprocesses of initiated polymers using as an the enzyme, monomers a chain are transfer added agent to the(CTA) living such chainsas (2-propionic in a controlled acid)yl ethyl fashion. HRPtrithiocarbonate initiated RAFT hasis used been to demonstrated facilitate the uniform as a rapid propagation and versatile of polymers polymerization as the monomers technique are capable added to the living chains in a controlled fashion. HRP initiated RAFT has been demonstrated as a of synthesizing well-defined homopolymers and complex architecture such as block copolymers, rapid and versatile polymerization technique capable of synthesizing well-defined homopolymers protein-polymerand complex conjugates.architecture such Polymerization as block copolyme synthesisrs, protein-polymer was capable conjugates. of exceeding Polymerization 90% monomer ◦ conversionsynthesis in 30was min. capable at a reactionof exceeding temperature 90% monomer of 25 converC, whichsion in demonstrates 30 min. at a reaction it as a rapid temperature technique of [44]. This25 process °C, which of HRP demonstrates initiated it RAFTas a rapid is shown technique in [44]. Scheme This1 process, with theof HRP enzymatic initiated radicalRAFT is generationshown shownin inScheme the top 1, ofwith the the scheme, enzymatic and radical the RAFT generati degenerativeon shown transferin the top used of the to controlscheme, theand reaction the RAFT shown in thedegenerative bottom of thetransfer scheme. used to control the reaction shown in the bottom of the scheme. O O M O OH Mj H O HRP H O 2 2 2 M M S S S S M S S M M M M j + Mk j + k j k Z Z Z Scheme 1. Top: enzymatic carbon centered radical generation and initiation of polymerization, and Scheme 1. Top: enzymatic carbon centered radical generation and
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